Cancer Therapy Volume 3 Issue B

CANCER THERAPY

Volume 3 Number 2 December, 2005

CANCER THERAPY FREE ACCESS www.cancer-therapy.org

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Editor

Teni Boulikas Ph. D., CEO Regulon Inc. 715 North Shoreline Blvd.

Mountain View, California, 94043 USA Tel: 650-968-1129 Fax: 650-567-9082 E-mail: teni@regulon.org

Teni Boulikas Ph. D., CEO, Regulon AE. Gregoriou Afxentiou 7 Alimos, Athens, 17455 Greece Tel: +30-210-9853849 Fax: +30-210-9858453 E-mail: teni@regulon.org

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Assistant to the Editor Maria Vougiouka B.Sc., Gregoriou Afxentiou 7 Alimos, Athens, 17455 Greece Tel: +30-210-9858454 Fax: +30-210-9858453 E-mail: maria@cancer-therapy.org

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Editorial Board

Ablin, Richard J., Ph.D., Arizona Cancer Center, University of Arizona, USA Armand, Jean Pierre, M.D. Ph.D., European Organization for Research and Treatment of Cancer (EORTC), Belgium Aurelian, Laure, Ph.D., University of Maryland School of Medicine, USA Berdel, Wolfgang E, M.D., University Hospitals, Germany Bertino, Joseph R., M.D., Cancer Institute of New Jersey, USA Beyan Cengiz, M.D.,!Gulhane Military Medical Academy, Turkey Bottomley, Andrew, Ph.D., European Organization for Research and Treatment of Cancer Data Center (EORTC), Belgium Bouros, Demosthenes, M.D., University Hospital of Alexandroupolis. Greece Cabanillas, Fernando, M.D, The University of Texas M. D. Anderson Cancer Center, USA Castiglione, Monica, MHA, SIAK/IBCSG Coordinating Center, Switzerland Chou, Kuo-Chen, Ph.D., D.Sc., Pharmacia Upjohn, USA Chu, Kent-Man, M.D., University of Hong Kong Medical Center, Queen Mary Hospital, Hong Kong, China Chung, Leland W.K, Ph.D., Winship Cancer Institute,

USA Coukos, George, M.D., Ph.D., Hospital of the University of Pennsylvania, USA Darzynkiewicz, Zbigniew, M.D., Ph.D., New York Medical College, USA Devarajan, Prasad M.D., Cincinnati Children's Hospital, USA Der Channing, J. Ph.D, Lineberger Comprehensive Cancer Center, USA Dritschilo, Anatoly, M.D., Georgetown University Hospital, USA Duesberg, Peter H., Ph.D, University of California at Berkeley, USA El-Deiry, Wafik S. M.D., Ph.D., Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, USA Federico, Massimo, M.D. Università di Modena e Reggio Emilia, Italy Fiebig, Heiner H, Albert-Ludwigs-Universität, Germany Fine, Howard A., M.D., National Cancer Institute, USA Frustaci, Sergio, M.D., Centro di Riferimento Oncologico di Aviano, Italy Georgoulias, Vassilis, M.D., Ph.D., University General Hospital of Heraklion, Greece Giordano, Antonio, M.D., Ph.D., Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, USA Greene, Frederick Leslie, M.D., Carolinas Medical

Center, USA Gridelli, Cesare M.D., Azienda Ospedaliera, "S.G.Moscati", Italy Hengge, Ulrich, M.D., Heinrich-Heine-University Duesseldorf, Germany Huber, Christian M.D., Johannes-GutenbergUniversity, Germany Hunt, Kelly, M.D., The University of Texas M. D. Anderson Cancer Center, USA Kamen, Barton A., M.D. Ph.D, Cancer Institute of New Jersey, USA Kaptan, Kursat, M.D., Gulhane Military Medicine Academy, Turkey Kazuma, Ohyashiki, M.D., Ph.D., Tokyo Medical University, Japan Kinsella, Timothy J. M.D., The research Institute of University Hospitals in Cleveland, USA Kmiec, Eric B, Ph.D., University of Delaware, USA Kosmidis Paris, M.D., "Hygeia" Hospital, Athens, Greece Koukourakis Michael, M.D., Democritus University of Thrace, Greece Kroemer, Guido, M.D. Ph.D., Institut Gustave Roussy, France Kurzrock, Razelle, M.D., F.A.C.P., M. D. Anderson Cancer Center, USA Leung, Thomas Wai-Tong M.D., Chinese University of Hong Kong, China Levin, Mark M.D., Sister Regina Lynch Regional Cancer Center, Holy Name Hospital, USA Lichtor, Terry M.D., Ph.D., Rush Medical College, USA Liebermann, Dan A., Ph.D., Temple Univ. School of Medicine, USA Lipps, Hans J, Ph.D., Universität Witten/Herdecke, Germany Lokeshwar, Balakrishna L., Ph.D., University of Miami School of Medicine, USA Mackiewicz, Andrzej, M.D., Ph.D., University School of Medical Sciences (USOMS) at Great Poland Cancer Center, Poland Marin, Jose J. G., Ph.D., University of Salamanca, Spain McMasters, Kelly M., M.D., Ph.D., University of Louisville, J. Graham Brown Cancer Center, USA Morishita, Ryuichi, M.D., Ph.D., Osaka University, Japan Mukhtar, Hasan Ph.D., University of Wisconsin, USA Norris, James Scott, Ph.D., Medical University of South Carolina, USA Palu, Giorgio, M.D., University of Padova, Medical School, Italy

Park, Jae-Gahb, M.D., Ph.D., Seoul National University College of Medicine, Korea Perez-Soler, Roman M.D., The Albert Einstein Cancer Center, USA Peters, Godefridus J., Ph.D., VU University Medical Center (VUMC), The Netherlands Poon, Ronnie Tung-Ping, M.D., Queen Mary Hospital, Hong Kong, China Possinger, Kurt-Werner, M.D., Humboldt University, Germany Rainov G Nikolai M.D., D.Sc., The University of Liverpool. UK Randall, E Harris, M.D., Ph.D., The Ohio State University, USA Ravaioli Alberto, M.D. Ospedale Infermi, Italy Remick, Scot, C. M.D., University Hospitals of Cleveland, USA Rhim, Johng S M.D., Uniformed Services University of Health Sciences, USA Schadendorf, Dirk, M.D., Universitäts-Hautklinik Mannheim, Germany Schmitt, Manfred, Ph.D., Universität München, Klinikum rechts der Isar, Germany Schuller, Hildegard M., D.V.M., Ph.D., University of Tennessee, USA Slaga, Thomas J., Ph.D., AMC Cancer Research Center (UICC International Directory of Cancer Institutes and Organisations), USA Soloway, Mark S., M.D., University of Miami School of Medicine, USA Srivastava, Sudhir, Ph.D., MPH, MS, Division of Cancer Prevention, National Cancer Institute, USA Stefanadis, Christodoulos, M.D., University of Athens, Medical School, Greece, Stein, Gary S Ph.D., University Of Massachusetts, USA Tirelli, Umberto, National Cancer Institute, Italy Todo, Tomoki, M.D., Ph.D., The University of Tokyo, Japan van der Burg, Sjoerd H, Leiden University Medical Center, The Netherlands Wadhwa Renu, Ph. D., Nat. Inst. of Advan. Indust. Sci. and Technol. (AIST), Japan Waldman, Scott A. M.D., Ph.D., USA Walker, Todd Ph.D., Charles Sturt University, Australia Watson, Dennis K. Ph.D., Medical University of South Carolina, Hollings Cancer Center, USA Waxman, David J., Ph.D., Boston University, USA Weinstein, Bernard I., M.D., D.Sci (Hon.), Columbia University, USA

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Associate Board Members

Chen, Zhong, M.D, Ph.D, National Institute of Deafness and other Communication Disorders, National Institutes of Health, USA Dietrich Pierre Yves, Hopitaux Universitaires de GenFve Switzerland Jeschke Marc G, M.D., Ph.D. Universität Erlangen-Nürnberg. Germany Limacher Jean-Marc, MD Hôpitaux Universitaires de Strasbourg, France Los Marek J, M.D., Ph.D. University of Manitoba, USA Mazda Osam, M.D., Ph.D. Kyoto Prefectural University of Medicine, Japan Merlin Jean-Louis, Ph.D Centre Alexis Vautrin, National Cancer Institute University Henri Poincaré France Okada Takashi, M.D., Ph.D. Jichi Medical School Japan Pisa Pavel, M.D, Ph.D. Karolinska Hospital, Sweden

Squiban Patrick, MD Transgene SA France Tsuchida Masanori, M.D, Ph.D Niigata University Graduate School of Medical and Dental Sciences Japan Ulutin, Cuneyt, M.D., Gulhane Military Medicine Academy, Turkey Xu Ruian, Ph.D., The University of Hong Kong, Hong Kong

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For submission of manuscripts and inquiries: Editorial Office Teni Boulikas, Ph.D./ Maria Vougiouka, B.Sc. Gregoriou Afxentiou 7 Alimos, Athens 17455 Greece Tel: +30-210-985-8454 Fax: +30-210-985-8453 and electronically to maria@cancer-therapy.org

Instructions to authors: Cancer Therapy FREE ACCESS www.cancer-therapy.org

Scope This journal, bridging various fields is one of the most rapid with free access at www.cancer-therapy.org.

The scope of Cancer Therapy is to rapidly publish original and in-depth review articles on cancer embracing all fields from molecular mechanisms to results on clinical trials. Articles (both invited and submitted) review or report novel findings of importance to a general audience in cancer therapy, molecular medicine, gene discovery, and molecular biology with emphasis to molecular mechanisms and clinical applications. The journal will accept papers on all aspects of cancer, at the clinical, preclinical or cell culture stage on chemotherapy and new experimental drugs, gene discovery, cancer immunotherapy, DNA vaccines, use of DNA regulatory elements in gene transfer, cell therapy and drug discovery related to cancer therapy. The authors are encouraged to elaborate on the molecular mechanisms that govern a cancer therapy approach. To make the publication attractive authors are encouraged to include color figures.

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Buschle M, Schmidt W, Berger M, Schaffner G, Kurzbauer R, Killisch I, Tiedemann J-K, Trska B, Kirlappos H, Mechtler K, Schilcher F, Gabler C, and Birnstiel ML (1998) Chemically defined, cell-free cancer vaccines: use of tumor antigen-derived peptides or polyepitope proteins for vaccination. Gene Ther Mol Biol 1, 309-321.

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Editorial Office Teni Boulikas, Ph.D./ Maria Vougiouka, B.Sc. Gregoriou Afxentiou 7 Alimos, Athens 17455 Greece Tel: +30-210-985-8454 Fax: +30-210-985-8453 and electronically to maria@cancer-therapy.org The free electronic access to articles published in "Cancer Therapy" to a big general audience, the attractive journal title, the speed of the reviewing process, the no-charges for page numbers or color figure reproduction, the 25 complimentary reprints, the rapid electronic publication, the embracing of many fields in cancer, the anticipated high quality in depth reviews and first rate research articles and most important, the eminent members of the Editorial Board being assembled are prognostic factors of a big success for the newly established journal.

Table of contents Cancer Therapy Vol 3 Number 2, December 2005

Pages

Type of Article

Article title

Authors (corresponding author is in boldface)

365-378

Review Article

Laurence Delacroix, Douglas Vernimmen, Dominique Begon Pascale Jackers, Rosita Winkler

379-382

Case Report

Mechanisms responsible for ERBB2 gene overexpression in human breast and non-breast cancer cells. The role of AP-2 transcription factors Pancreatic cancer palliation using radiofrequency ablation. A new technique

383-396

Review Article

397-400

Case Report

401-406

Review Article

407-418

Research Article

419-434

Review Article

435-442

Research Article

443-460

Review Article

461-470

Research Article

471-476

Research Article

Neuroendocrine differentiation in prostate cancer Spontaneous ovarian hyperstimulation syndrome caused by hypothyroidism

Progress in prostate cancer research: a focus on bone health Synergistic augmentation of vincristine-induced cytotoxicity by phosphatidylinositol 3-kinase inhibitor in human malignant glioma cells: evidence for the involvement of p38 and ERK signaling pathways Implications of HPV infection in uterine cervical cancer Aggressive work-up is needed for menopausal women with atypical glandular cells of uncertain significance (AGUS) Pap smear Epithelial to mesenchymal transition, cell surface receptors activation and intracellular communications in cancer metastasis Adequacy of anticipatory anxiety in women receiving chemotherapy for breast cancer Ki-67 index and skin carcinomas with skull base invasion: a case窶田ontrol study

John D Spiliotis, Anastasios C Datsis, Panagiotis Chatzikostas, Spyros P. Kekelos, Athina N. Christopoulou, Athanasios G. Rogdakis, P Symeonides Siamak Daneshmand, Marcus L. Quek, Jacek Pinski Azam Sadat Mousavi, Nadereh Behtash, Malihe Hasanzadeh, Mitra Modares Gilani, Fatemeh Ghaemmaghami, Encie Shahroch, Tehrani Nejad Susan Doyle-Lindrud, and Robert S. DiPaola Daniel R. Premkumar, Beth Arnold, John Mathas, and Ian F. Pollack

Hoenil Jo, Jae Weon Kim Dong Ock Lee, Hoenil Jo, Youn-kyung Chung, Jae Weon Kim, Noh-Hyun Park, Yong-Sang Song, Soon-Beom Kang, Hyo-Pyo Lee Wael M. ElShamy

Michael Trimmel, Christina Semrad, Ernst Kubista, Gﾃｼnther Steger, Christoph Zielinski Claudio R. Cernea, Alberto R. Ferraz, Inﾃｪs V. de Castro, Miriam N. Sotto, Angela F. Logullo, Andrﾃｩ S. Potenza, Carlos E. Bacchi

477-488

Review Article

489-494

Case Report

495-510

Review Article

511-514

Research Article

515-524

Research Article

525-530

Research Article

531-542

Research Article

543-550 551-554 555-564

Novel biomarkers for the early prediction of acute kidney injury Peritoneal carcinomatosis versus peritoneal tuberculosis: a rare diagnostic dilemma in ovarian masses Cellular senescence–an integrated perspective Cellular senescence–an integrated perspective Combination treatment of unresectable hepatomas with chemotherapy, octreotide and antioestrogens: A preliminary study Growth inhibition of head and neck squamous cell carcinoma by imatinib mesylate (Gleevec) Incidence of gastrointestinal toxicity during estramustine phosphate therapy for prostate cancer is associated with the single-nucleotide polymorphisms in the cytochrome P450 1A1 (CYP1A1) gene

Cytokine up-regulation of IL13R-"2 in GBM cells leads to an increased potency of recombinant IL13 Cytotoxin Review 17#-hydroxysteroid dehydrogenases Article and breast cancer Case report Successful treatment of gestational trophoblastic neoplasm metastatic to the colon Review Future of gene therapies in high grade Article gliomas

565-578

Review Article

T cell-based strategies for immunotherapy of prostate cancer

579-588

Research Article

The Protein Kinase C$ inhibitor Rottlerin modulates bortezomibinduced apoptosis of B-cell chronic lymphocytic leukemia cells

Prasad Devarajan Konstantinos Vagenas, Christos Stratis, Charalambos Spyropoulos, John Spiliotis, John Petrochilos, Helen Kourea, Dionisios Karavias Ola Larsson

Panagiotis Ginopoulos Athina Christopoulou, John Spiliotis

Hyung Ro Chu Weg M. Ongkeko, Amilcar Diaz, Xabier Altuna, Joe Aguilera, Robert A. Weisman and Jessica Wang-Rodriguez Mohammed Rafiqul Islam Mamun Motofumi Suzuki Satoru Takahashi Kazuo Hara, Takeshi Ozeki, Yasuhiko Yamada, Takashi Kadowaki, Yoshitsugu Yanagihara, Shuji Kameyama, Yoichi Minagawa Ito, Takumi Takeuchi and Tadaichi Kitamura Nianping Hu, Denise M. Gibo and Waldemar Debinski

Pirkko Vihko, and Veli Isomaa Fatemeh Ghaemmaghami, Farnaz Sohrabvand, Haleh Ayatollahi, Mitra Modarres Deepak Kumar Gupta, Mattei Tobias Alecio, Ashok Kumar Mahapatra, Ramina Ricardo Marc Schmitz, Andrea Kiessling, Bernd Weigle, Susanne Fuessel, Axel Meye, Rebekka Wehner, Achim Temme, Michael Bachmann, Manfred P. Wirth, E. Peter Rieber Markus Düchler, Rainer Hubmann, Dieter Mitteregger, Martin Hilgarth, Josef D. Schwarzmeier and Medhat Shehata

Cancer Therapy Vol 3, page 365 Cancer Therapy Vol 3, 365-378, 2005

Mechanisms responsible for ERBB2 gene overexpression in human breast and non-breast cancer cells. The role of AP-2 transcription factors Review Article

Laurence Delacroix1, Douglas Vernimmen2, Dominique Begon1 Pascale Jackers1, Rosita Winkler1,* 1

Laboratory of Molecular Oncology, CBIG, Experimental Cancer Research Center, University of Liege Sart Tilman, Tour de Pathologie B23, B4000 Liege, Belgium; 2 MRC Molecular Haematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3, 3DS, United Kingdom.

__________________________________________________________________________________ *Correspondence: Rosita Winkler, Laboratory of Molecular Oncology, CBIG, Experimental Cancer Research Center, University of Liege Sart Tilman, Tour de Pathologie B23, B4000 Liege, Belgium; Tel: 324366250; Fax: 003243662922; email: rwinkler@ulg.ac.be Key words: the ERBB network, ERBB2 overexpression, transcriptional elements , Molecular mechanisms , breast cancers , rodent Neu promoter, Distant regulatory regions, endogenous ERBB2 gene expression, Transgenic overexpression of AP-2, mice mammary gland Abbreviations: AP-2 binding sites, (AP2BS); chromatin immunoprecipitation, (ChIP); ductal carcinoma in situ, (DCIS); ETS binding site, (EBS); immunohistochemistry, (IHC); initiator like region, (Inr); invasive carcinoma, (IC) Received: 21 June 2005; Accepted: 28 June 2005; electronically published: July 2005

Summary The ERBB2 gene codes for p185erbB2, a transmembrane protein with intrinsic tyrosine kinase activity. P185erbB2 is a member of the EGFR family of growth factor receptors. The gene is expressed in embryonic and adult cells and its function is necessary throughout the entire life. ERBB2 is overexpressed in a significant proportion of human breast cancers where it is correlated to poor prognosis for the patient. The gene is also overexpressed in non-breast cancers but researchers disagree on the prognostic significance of the overexpression in these cancers. Gene amplification and increased transcription rates account for the very high levels of p185erbB2 accumulated in breast cancer cells. In a proportion of breast cancer cells a moderate increase in p185erbB2 level is due to transcriptional deregulation alone.

The mechanisms responsible for ERBB2 gene overexpression in non-breast cancer cells is not well understood. The molecular mechanisms responsible for ERBB2 gene overexpression have been investigated mostly in breast cancers.

In this paper we review the data from the literature and our own results on the involvement of the AP-2 transcription factors family in ERBB2 gene overexpression in breast cancer cells. We conclude that AP-2 family of transcription factors contribute to the ERBB2 overexpression in a fraction of breast cancers. In contrast, AP-2 factors are not responsible for increased ERBB2 expression in the non-breast cancer cells we have analyzed. activity of the ERBB3 gene product is impaired by mutation in the tyrosine kinase domain. P185erbB2 and the ERBB3 gene product are activated by hetero-dimerisation with another active ligand-bound receptor (reviewed in Brennan et al, 2000; Harari and Yarden, 2000; Olayioye et al, 2000; Yarden and Sliwkowski, 2001; Citri et al, 2003; Casalini et al, 2004).

The ErbB receptors together with the EGF family of ligands form the ErbB signaling network. In the healthy tissues the ligands are secreted by stromal cells and bind receptors present on the surface of epithelial cells (Burden and Yarden, 1997).

I. Introduction: the ERBB network The ERBB2 gene (also known as HER2 or Neu) encodes a 185 kDa transmembrane protein, p185erbB2, with intrinsic tyrosine kinase activity. P185erbB2 belongs to the EGF receptor (ErbB1) family of tyrosine kinase receptors, along with the products of EGFR, ERBB3 and ERBB4 genes. ErbB -1, -3 and -4 are recognized by more than 20 growth factors belonging to the EGF family. Ligand bound receptors form homodimers and/or heterodimers composed of two different receptors of the EGFR family.

No soluble growth factor recognizing p185erbB2 with high affinity has been identified so far and p185erbB2 is thus considered as an “orphan receptor�. The enzymatic 365

Delacroix et al: ERBB2 gene overexpression and the role of AP-2 transcription factors in cell types expressing p185erbB2 and expression levels were observed. Variations in p185erbB2 levels during the menstrual cycle in the adult human breast were described.

The protein was more abundant during the luteal than during the follicular phase of the cycle (Gompel et al, 1996).

Once activated, tyrosine residues at the carboxyl-end of the receptors are phosphorylated, creating docking sites for cytoplasmic signalling molecules. This triggers several signalling cascades, resulting in differentiation, survival, migration, depending on the growth factor, the composition of the dimer and the signalling molecules present in the cell.

P185erbB2 is the preferred dimerisation partner for the three other receptors of the ErbB family. The heterodimers containing p185erbB2 last longer and are more active than all the other homo- or heterodimers. High levels of p185erbB2, such as those measured in cancerous cells overexpressing the gene stimulate proliferation, inhibit apoptosis, induce migration and modify the response to chemo- and hormone- therapy (reviewed in Harari and Yarden, 2000; Olayioye et al, 2000; Yarden and Sliwkowski, 2001).

Here we summarize first some data on the normal ERBB2 gene expression and functions. The following section presents an overview of ERBB2 gene overexpression in breast and non-breast cancers. The molecular mechanisms leading to ERBB2 overexpression are the main topic of this paper. We present our data on ERBB2 overexpression in breast cancer cells. We discuss our results and the data from the literature concerning the role of AP-2 transcription factors on ERBB2 gene overexpression. Our results on the overexpression in nonbreast cancers are summarised in the last section of this paper.

III. ERBB2 overexpression in cancers Not surprisingly given the importance of the cellular processes it regulates, the ErbB signalling network plays a central role in the development of numerous human cancers. Indeed, shortly after the discovery of the ErbB2 gene (Neu) as an oncogene in chemically induced rat brain cancers, Slamon and co-workers described the amplification and overexpression of the corresponding human gene in breast and ovary cancers. Moreover, the overexpression was associated with a poor prognosis (reviewed by DiGiovanna, 1999). This initial observation has been confirmed since by numerous studies and lead to the development of p185erbB2 targeted therapies for breast cancer (Ross et al, 2003). Interestingly, ErbB2 overexpression was reported recently in spontaneous canine (de la Mulas et al, 2003) and feline (De Maria et al, 2005) mammary cancers. In both species this was a poor prognostic factor.

ERBB2 gene overexpression was also observed in non-breast human cancers, such as Wilm’s tumours, bladder, pancreas, colon, lung and prostate cancers. The prognostic significance of ERBB2 gene overexpression in non-breast tumours is debated (Klapper et al, 2000; Menard et al, 2001).

Twenty to forty percent of ovary cancers were reported to overexpress ERBB2 (Kupryjanczyk et al, 2004). The overexpression was most frequent in metastatic ovary carcinoma specimen and cancer cell lines (HellstrĂśm et al, 2001). In a subset of ovarian cancers the gene was amplified but not overexpressed (Wu et al, 2004). ERBB2 overexpression was shown to be necessary for the growth of the ovarian cancer cells (Juhl et al, 1997; Hsieh et al, 2000) and might condition the response to chemotherapy (Abuharbeid et al, 2004).

ERBB2 status in other types of human cancer is controversial, mostly because of methodological problems.

Maurer et al, (1998) reported co-expression of ERBB2 and ERBB3 genes in a high proportion of primary colorectal cancers. This was interesting since in breast cancers the erbB2/erbB3 dimer was shown to be the most tumorigenic (Harari and Yarden, 2000). Vadlamudi and co-workers (1999) further showed that these receptors were constitutively phosphorylated in colon cancer cells.

However, other investigators detected ERBB2 gene amplification and overexpression only in a small proportion of primary human colon cancers (Nathanson el al, 2003; Half et al, 2004).

Increased p185erbB2 levels were detected by IHC in ductal pancreatic adenocarcinoma (Apple et al, 1999; work cited by Hruban et al, 2000). In contrast, Koeppen et al, (2001) did not observe a significant increase in p185erbB2 levels in pancreatic cancers.

According to Craft et al, (1999) and to Signoretti et al. (2000) ERBB2 expression was increased in prostate

II. ERBB2 expression in healthy tissues Low levels of membranous p185 erbB2 were detected in epithelial cells of a variety of normal human tissues such as those of the gastro-intestinal, respiratory, reproductive and urinary tracti, as well as in skin, breast and placenta (Press et al, 1990; King et al, 1992; Camp et al, 2003).

An intact ErbB signaling network is required during embryonic development and throughout the entire life of an animal. Mice embryos carrying knocked-out ErbB2 gene died as a consequence of defects in the development of the heart and the nervous system. Animals where the ErbB2 gene was inactivated specifically in the heart after birth developed dilated cardiomyopathy. Formation of neuromuscular synapses, development of muscle spindles, Schwann cell function and survival of motor neurons were impaired in ErbB2 deficient mice (reviewed by Garratt et al, 2003; Holbro and Hynes, 2004). ErbB2 gene expressed in mice colon epithelial cells ensured the survival of enteric neurons and glia (Crone et al, 2003). In the inner ear, ErbB2 expressed by supporting cells ensured the survival of spiral ganglion neurons (Stankovic et al, 2004).

The differentiating virgin mouse mammary glands express and activate ErbB2. The receptor drives the alveolar differentiation during pregnancy (reviewed by Troyer and Lee, 2001; Stern, 2003).

P185erbB2 levels are physiologically modulated in healthy tissues and in non-cancerous pathologies. Erbb2 expression was increased in the regenerating mice intestine after small bowel resection (Falcone et al, 1999).

During the maturation of the mice (Schroeder and Lee, 1998) and rat (Darcy et al, 2000) mammary gland changes 366

Cancer Therapy Vol 3, page 367 cancer cells after androgen ablation and in hormone independent cancers. Some authors confirmed these observations (Osman et al, 2001; Shi et al, 2001). P185 erbB2 overexpression might thus contribute to progression toward androgen independence. The high p185erbB2 levels detected in a significant proportion of circulating prostate cancer cells supported a role for the oncogene in prostate cancer progression (Ady et al, 2004; Carles et al, 2004).

However, no consensus has been reached yet as to the role of ERBB2 in the progression of hormone independent prostate cancers, since other investigators did not observe a correlation between ERBB2 expression and hormone sensitivity (Reese et al, 2001; Savinainen et al, 2002; Calvo et al, 2003). The discrepancy between the results is probably due to methodological problems (Sanchez et al, 2002).

In summary, the involvement of ERBB2 gene overexpression in breast and ovary cancers progression is generally accepted. Breast cancer cells contain very high amounts of p185erbB2 as a consequence of gene amplification combined with increased transcription rates.

P185erbB2 targeted therapies are developed for the treatment of breast cancer patients with ERBB2 overexpression.

Whether ERBB2 gene overexpression is significantly involved in non-breast tumours progression is less clear.

When present, the increase in p185erbB2 levels in most of these tumours was moderate and the methodological problems concerning the detection of ERBB2 gene overexpression are not solved.

To the best of our knowledge no mutations of the ERBB2 promoter have been reported in the cancerous cells overexpressing the gene. In contrast, overexpression and activation of transcription factors have been involved in ERBB2 overexpression in breast cancer cells.

We have analyzed the transcriptional activity of a 6kb fragment of the ERBB2 promoter. After describing the general transcriptional elements on the promoter, we focus on our data and the results from the literature concerning the role of increased levels of AP-2 transcription factors on ERBB2 overexpression.

A. General transcriptional elements on the human ERBB2 promoter Three independent transcription start sites have been mapped on the ERBB2 gene promoter (Figure 1A). First, a TATA (-22 to -26) and a CAAT (-71 to -75) box direct transcription initiation at the site marked +1. This site will be referred to as the main transcription start site. An initiator like region (Inr), consisting of six GGA polypurine / polypyrimidine repeats, located from -65 to 45 base pairs upstream of the major transcription start site directs a second set of transcription start sites. The TATA box and the Inr independently govern the initiation of transcription (Mizuguchi et al, 1994). A third set of transcription initiation sites has been identified recently 12kb upstream from the main transcription start site. The mRNA initiated at this upstream site was present in low amounts in all the tested cells. It has an original 5’ untranslated region and encodes a protein that is identical to the one translated from the major transcripts (Nezu et al, 1999).

The polypurine (GGA)-polypyrimidine (TTC) rich region forms an internal triplex structure (H-DNA) that represses ERBB2 gene transcription (Scott et al, 2000).

An approximately 500bp sequence at the 5’ end of the 6kb fragment contains multiple AA, TA and CA dinucleotide repeats. These features are characteristic of DNA sequences associated with the nuclear matrix and mediate the attachment of chromatin loops to the nuclear matrix (Laemmli et al, 1992). These Matrix Attachment Regions (MAR) have been involved in important cellular processes such as transcription activation (Bode et al, 2000) or insulation of genes from position effects (Allen et al, 2000).

We analyzed the sequence of a 20kb fragment of the human ERBB2 promoter with the MAR-finder program (Singh et al, 1997) to find out if the region containing the repeats has indeed the characteristics of a MAR. A maximal MAR potential was revealed in the region located between the positions -6/-5.6kb upstream from the main transcription start site (Figure 1B). This region of the ERBB2 promoter could thus be implicated in the regulation of ERBB2 transcription by organizing the chromatin domain containing the gene. Interestingly, a DNase hypersensitive site has been previously localized around position -5.5kb, indicating that this chromatin region is accessible to DNA binding factors (Vernimmen, unpublished). The accessibility is essential for MAR sequences that often co-localize with DNaseI hypersensitive sites (Bode et al, 2000).

IV. Molecular mechanisms leading to ERBB2 overexpression in breast cancers The mechanisms responsible for ERBB2 gene overexpression were investigated almost exclusively in breast cancers. Gene amplification and increased transcription rates lead to the very high increase in erbB2 transcript and protein levels in breast cancers. Moderate overexpression was often the consequence of transcriptional deregulation alone (Jimenez et al, 2000; Pauletti et al, 2000; Ménard et al, 2001; Hammock et al, 2003; Merkelbach-Bruse et al, 2003; Owens et al, 2004).

We did show by run-on experiments, increased transcription rates in breast cancer cells overexpressing ERBB2 (Pasleau et al, 1993).

Several teams, including our own, are interested in unravelling the mechanisms of deregulated ERBB2 transcription in breast cancer cells.

Briefly, transcription rates are controlled by binding of transcription factors to specific enhancer sequences on the promoter. Transcription factors bound to regulatory sequences (enhancers or silencers) interact directly or indirectly with general transcription factors which recruit RNA polymerase II to the core promoter. Transcription rates can be increased by different mechanisms: increased levels or activity of transcription factors; mutations in the promoter creating binding sequences for a new activator or disrupting the binding site for a repressor. Recently, epigenetic mechanisms – DNA and histone methylation, histone acetylation – were involved in gene expression levels modulations.

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Delacroix et al: ERBB2 gene overexpression and the role of AP-2 transcription factors Three binding sites for transcription factors implicated in the overexpression of the gene have been localized on the proximal promoter. An ETS binding site (EBS) was located immediately upstream the TATA box (Scott et al, 2000) and two AP-2 binding sites (AP2BS) were located 213 (Bosher et al, 1996) and 495bp upstream from the transcription start site (Grooteclaes et al, 1994; 1999; Vernimmen et al, 2003a) (Figure 1C).

B. Regulated transcriptional elements of the ERBB2 promoter 1. The proximal promoter The transcriptional activity of the proximal 500bp of the ERBB2 promoter was in good agreement with the level of expression of the endogenous gene in different breast cancer cell lines (Hollywood and Hurst, 1993; Grooteclaes et al, 1994; Scott et al, 1994).

Figure 1. A. General transcriptional elements on 12kb of the human ERBB2 promoter. The broken arrows indicate the transcription start sites. The start site at position +1 is considered as the main transcription start site of the human gene. GGA: region containing the GGA repeats corresponding to the Initiator element (Inr). The gray box indicates the region containing the dinucleotide repeats. B. MAR potential analysis of the ERBB2 promoter. Positions are given relative to the major transcription start site. The sequence of an 18kb fragment of the human ERBB2 promoter was reconstituted from Z13970 (Hudson et al, 1990), X56495 (Grooteclaes et al, 1994) and AB025285 (Nezu et al, 1999) sequences. C. Regulated transcriptional elements of the ERBB2 promoter. Broken arrows indicate the transcription start sites. Positions of the EBS and AP2BS associated with ERBB2 overexpression are indicated. The arrows point to the extremities of the promoter fragments which have been tested for activity. The plus signs indicate the promoter fragments active in the breast cancer cells which overexpress ERBB2. The minus signs indicate the repressing fragments. Âą indicates a fragment which has different transcriptional activity according to the cell line.

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Cancer Therapy Vol 3, page 369 The identity of the Ets transcription factor responsible for ERBB2 overexpression is not precised yet.

Several members of this vast family of transcription factors, such as ESX, are overexpressed and/or activated in breast cancer cells which overexpress ERBB2 (Chang et al, 1997).

The AP-2 family of transcription factors includes five members: AP-2 -!, -Ă&#x;, -" (Bosher et al, 1996), -# (Cheng et al, 2002) and -$ (Tummala et al, 2003). Breast cancer cell lines overexpressing ERBB2 contain high amounts of AP-2 -! and -" factors (Bosher et al, 1995; 1996; Grooteclaes et al, 1999).

We assessed the contribution of the -495 AP2BS to the promoter activity. For this purpose, we used a reporter vector containing the luciferase cDNA under the control of the proximal 750bp of the ERBB2 promoter. New vectors were derived from the initial one where each one or both AP2BS were inactivated by site-directed mutagenesis.

Mutating the AP-2 sites, either individually or in combination, reduced the activity of the promoter to one fifth the activity of the wild type promoter. Thus, both AP2 sites must be present for full promoter activity (Vernimmen et al, 2003a).

C. The rodent Neu promoter The promoters of the Neu genes, the rat and mice orthologs of the human ERBB2 gene, have been sequenced and the sequences were compared to those of the human promoter. The sequences of proximal promoters, extending 200bp upstream from the human main transcription start site are well conserved in the three promoters (White et al, 1992). However, there are some important differences between these sequences, indicating that the regulation of the human and the rodent genes expression might differ. For instance, the Neu promoters lack the TATA box and there is no initiation of transcription at the sites marked +1 on the human promoter. Moreover, some regulatory sites mapped on the rodent proximal promoters have not been found to regulate the activity of the human promoter (reviewed by Barnes and Hurst, 1997). Interestingly, as shown in Figure 2A, the two proximal AP-2 binding sites are not conserved in the rodent promoters.

A multalin alignment of the regions extending upstream the proximal 200bp revealed very limited sequence identity between the human, mouse and rat ERBB2 promoters (not shown). We also analyzed the mouse and rat Neu gene promoter sequences with the MAR-finder program. Indeed, several experimentally identified MARs are present at similar positions in the promoters of orthologous genes (Avramova et al, 1998; Greally et al, 1999). Despite limited sequence conservation, the potential of MAR occurrence on Neu promoters was very high for a region overlapping the position located 6kb upstream from the transcription start sites (Figure 2B and C). This conserved putative MAR sequence of ERBB2 distal promoter might thus be a general regulator of ERBB2 gene expression. Noteworthy, the effect of such a MAR cannot be detected in reporter vectors experiments, since they are believed to act by chromatin remodeling. Transgenic animal models or stable transfection experiments have to be used to further study the contribution of the distal MAR to the expression of ERBB2 gene.

2. Distant regulatory regions We have investigated the transcriptional activity of promoter regions located upstream the proximal promoter (Figure 1C). The 3.5kb fragment preceding the proximal promoter repressed its activity in most cells. The mechanism of repression remains to be precised. The further upstream 2.2kb fragment reversed the repression specifically in breast cancer cells overexpressing ERBB2 (Grooteclaes et al, 1994; Delacroix et al, in press).

The distal promoter region contains two AP-2 binding sites (Figure 1C). Binding of the factors to these sites contributed to the activity of the fragment. Indeed, the region of the distal activating fragment containing the two distal AP2BS was able to stimulate transcription from a heterologous TK promoter (Delacroix et al, in press).

Thus, the ERBB2 promoter contains at least four AP2 binding sites, which contribute to the overexpression of the gene in breast cancer cells (Figure 1C). To evaluate the contribution of AP-2 factors to the transcriptional activity of the entire 6kb fragment of the ERBB2 promoter, we expressed an amino-terminal truncated AP-2 protein with dominant negative activity (DN-AP2). DN-AP2 has conserved the dimerisation and DNA binding domains, but lacks the transactivation domain (Williams and Tjian, 1991). BT-474 and ZR-75-1 breast cancer cells, overexpressing ERBB2 and containing high amounts of endogenous AP-! and -", were co-transfected with a constant amount of a reporter vector containing the 6kb promoter fragment and increasing amounts of the DN-AP2 expression vector. We measured a dose dependent decrease in the activity of the ERBB2 promoter, reaching half of the activity measured in the absence of the inhibitor. This shows that AP-2 factors contribute significantly to the activity of the ERBB2 promoter (Delacroix et al, in press).

V. AP-2 transcription factors and ERBB2 expression in breast cancer cells AP-2 factors activate the ERBB2 promoter in reporter vectors. These results initiated new research to find out if these transcription factors do indeed play a role in the overexpression of the endogenous ERBB2 gene in breast cancer cells. Three methodologies were used to address this question. First, AP-2 binding to the endogenous ERBB2 promoter and the consequences of AP-2 downregulation on ERBB2 expression were analyzed in cultured cells. Second, AP-2 and p185erbB2 levels, visualized by immunohistochemistry (IHC) on primary breast cancer sections, were compared. Third, Neu expression levels were investigated in transgenic mice overexpressing AP-2 in the mammary gland.

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Figure 2. A. Alignment of the human ERBB2 (-590/+183bp relative to the main transcription start site), the mouse (-652/+6bp relative to the ATG translation initiation codon) and rat Neu (683/+15bp relative to the ATG translation initiation codon) proximal promoters. The black nucleotides are conserved, whereas the grey nucleotides are not conserved between the three species. The broken line indicates the main transcription start site on the human promoter.

The sequences corresponding to the human EBS and AP2BS are boxed.

B. The mouse Neu promoter sequence from -13/+2.9kb (relative to translation start site) isolated from mouse BAC clone AL591390 was analyzed for the presence of MAR. Classically, the ATG, which is conserved in the human sequence, is designated as position +1 of the mouse promoter and the transcriptional start site lies 211bp upstream. C. The rat Neu promoter sequence comprised between positions -9.3 and +3kb, extracted from the rat supercontig NW47339.1 (Rat genome resource, NCBI), was subjected to MAR analysis. In the rat promoter the ATG is located 9bp upstream the human and mouse ATG and defines classically the position +1.

The transcriptional start site lies at position - 203bp.

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A. Modulation of the endogenous ERBB2 gene expression by AP-2 in breast cancer cell lines

B. Correlation between AP-2 and p185erbB2 levels in primary breast cancers.

A first study using two antibodies recognizing specifically AP-2! and AP-2" reported a positive correlation between the levels of p185erbB2 on one hand and the presence of both AP-2! and AP-2" on the other hand (Turner et al, 1998).

A second study using a single antibody recognizing both AP-2! and AP-2% found a negative correlation between the receptor and the transcription factor levels (Gee et al, 1999).

A third report addressed the same question using a commercial anti AP-2! antibody. In only a proportion of tumours overexpressing ERBB2 were the AP-2 levels also increased. The overall survival was shorter in patients whose tumours overexpressed p185erbB2 but not AP-2 (Pellikainen et al, 2004). Interestingly, we observed that in ERBB2 overexpressing MDA-MB-453 cells, AP-2 did not bind efficiently to DNA. ERBB2 overexpression in these cells might not be dependent on AP-2 factors (Grooteclaes et al, 1999).

An additional, thorough immunohistochemical analysis localized AP-2! and AP-2" in healthy breast tissues, in ductal carcinoma in situ (DCIS) and in invasive carcinoma (IC). Higher expression levels were detected in the healthy breast and in DCIS than in IC. Moreover, the two isoforms were expressed in distinct cell types.

Glandular epithelial cells expressed AP-2!, while myoepithelial cells expressed AP-2". AP-2! and ERBB2 levels were weakly but significantly correlated. In undifferentiated invasive carcinoma occasional coexpression of the two AP-2 isoforms was noted (Friedrichs et al, 2005). Interestingly, most breast cancer cell lines which overexpress ERBB2, overexpress both AP-2! and AP-2".

Finally, the relation between AP-2! gene methylation and expression was analyzed in breast cancer cells and a panel of normal breast, DCIS and IC samples (Douglas et al, 2004). AP-2! gene was unmethylated in most normal mammary epithelial cells and DCIS. AP-2! protein was detected in the nuclei of both these types of cells by IHC, with a tendency for overexpression in DCIS. In contrast, in 75% of invasive carcinoma AP-2! was hypermethylated and the protein was undetectable. These observations do not exclude an association between AP-2 and ERBB2 overexpression during breast cancer progression. Indeed, ERBB2 was overexpressed in a high proportion of DCIS (van de Vijver et al, 1988), while less than 30% of IC overexpress the gene.

In summary, AP-2 isoforms are expressed in healthy human breast cells, possibly in different cell types. These cells express low levels of p185erbB2. AP-2 and ERBB2 expression was increased in DCIS. Three out of four IHC analysis observed a tendency for a correlation AP-2 and p185erbB2 levels in invasive carcinomas.

Binding of a transcription factor to the endogenous chromatin embedded promoter region is the sine-qua-non requirement for its activity. We thus checked for AP2 association to breast cancer cells endogenous ERBB2 promoter by chromatin immunoprecipitation (ChIP). The chromatin from BT-474 cells was cross-linked, sonicated and immunoprecipitated with an AP-2 specific antibody.

The AP-2 bound DNA fragments were PCR amplified with primers amplifying the region containing the proximal and the two distant AP-2 binding sites. The results show that AP-2 factors were bound to the proximal and the distal AP2BS (Begon et al, in press; Delacroix et al, in press). Thus, AP-2 factors are associated to the chromatin on the ERBB2 gene promoter in the cells expressing the gene. However, association to the promoter is not sufficient to prove that the factor is active.

To prove that AP-2 factors do contribute to ERBB2 overexpression, we measured ErbB2 mRNA levels in breast cancer cells where AP-2 -! and -" were downregulated by siRNA. BT-474 breast cancer cells, which overexpress ERBB2, were transfected with AP-2! and AP2" siRNA, independently and in combination. After two to four days of treatment, AP-2!, AP-2", ErbB2 and VEGF (an AP-2 target gene) mRNAs were quantitated by realtime RT-PCR. The results are presented in Figure 3. AP2! siRNA induced a rapid down-regulation of the corresponding protein, which became undetectable 2 days after the treatment, while AP-2" levels were unmodified.

In contrast AP-2" was greatly reduced in cells treated with the specific siRNA, without significant changes in AP-2! content. Three days after treatment with both AP-2-! and " siRNAs both factors became undetectable (Figure 3A).

The evolution of ErbB2 and VEGF transcript levels was measured in the cells transfected with the AP-2 directed siRNAs. Transfection with AP-2! siRNA inhibited AP-2! mRNA but did not modify significantly either ErbB2 or VEGF transcript levels (Figure 3B). Comparable results were obtained in cells transfected with AP-2-" siRNAs (Figure 3C). In contrast, transfection of both AP-2-! and " siRNAs induced a transitory but significant reduction in ErbB2 and VEGF mRNA levels (Figure 3D).

In conclusion, the association of AP-2 factors with the endogenous gene promoter and the inhibition of expression by the down-regulation of AP-2! and AP-2" are strong indications that AP-2 factors do contribute to ERBB2 overexpression in breast cancer cells. Our results further indicate that both AP-2! and AP-2" are necessary for ERBB2 overexpression. However, clearly other transcription factors are involved in the increased ERBB2 gene expression observed in breast cancer cells (Delacroix et al, unpublished).

Cell lines present the advantage of being easily manipulated, however they might not reflect the properties of primary tumours. In the next sections we summarize results obtained on primary human breast cancers and in transgenic mouse models.

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Figure 3. Suppression of AP-2! and AP-2" expression downregulates ERBB2 transcript level in BT-474 cells. Cells cultured in 6-well dishes were transfected on day 0 and 3 by 150nM siRNA directed against AP-2! and/ or AP-2" transcripts. Control cells were transfected with siRNA directed against luciferase mRNA. RNA and proteins were extracted after 2, 3 or 4 days of treatment. A. Detection by western blotting of AP-2! and AP-2" levels in 20mg of total proteins from cells transfected with the siRNAs. Ku protein served as control for the protein amount charged on the gel. B. Real time RT-PCR for AP-2!, AP-2", ErbB2, VEGF (AP-2 target gene) and B2M (standard gene) transcripts was performed on 1mg of total RNA from cells transfected with AP-2! siRNA. The standardized transcript levels were reported to the values obtained in control cells transfected with the luciferase siRNA. C. Same experiments performed on cells transfected with AP-2" siRNA. D. Same experiments performed on cells transfected with AP-2! and AP-2" siRNA.

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Cancer Therapy Vol 3, page 373 and in HepG2 liver cancer cells were comparable to those of ZR-75-1 breast cancer cells, which overexpress ERBB2 with a normal diploid set of genes.

The expression levels differed significantly between cells derived from the same cancer type (Table 2). So, ErbB2 mRNA levels were about seven times higher in LNCaP than in PC-3 prostate cancer cells. The difference between ErbB2 mRNA content in COLO320 and HTm29 colon cancer cells was thirteen fold. Pancreas cancer cells presented a 40-fold difference between the cells expressing the highest and the lowest amounts of the transcripts.

P185erbB2 was detected by western blot in the majority of the analyzed cells. Protein and mRNA levels were in good agreement in the non-breast cancer cells, with the exception of colon cancer cells (Table 2 ) (Vernimmen et al, 2003b).

Next we compared AP-2 and p185erbB2 levels in the non-breast cancer cell lines to find out if the overexpression of the transcription factors contributes to ERBB2 overexpression. Contrary to breast cancer cells, AP-2 and p185erbB2 levels were not correlated in the nonbreast cancer cells. Strikingly, HepG2 cells expressed fair amounts of erbB2 mRNA cells but were devoid of AP-2 (Vernimmen et al, 2003b).

In order to identify the factor(s) responsible for the increased ERBB2 expression in non-breast cancer cells, we compared the activity of ERBB2 promoter fragments in couples of cells of the same origin expressing low and high levels of the transcript. This approach lead to the identification of AP-2 and ETS factors involvement in ERBB2 overexpression in breast cancer cells. We could carry out these experiments only in ovary and colon cancer cells, because of very low transfection efficiencies of the other cell types. Figures 4 B-E summarize our results (Vernimmen et al, 2003b). The differences in promoter fragments activities between breast and non-breast cancer cells are striking. In ERBB2 overexpressing breast cancer cells, the activity of the promoter fragments from vectors 2 and 4 was maximal, when compared to minimal promoter from vector 1. This activating potential is specific to ERBB2 overexpression since the activity of all four promoter fragments were similar in breast cancer cell lines expressing low levels of the gene. Thus, the activating potential of promoter fragments contained in vectors 2 and 4 reflect the endogenous ERBB2 expression level in breast cancer cells (Figure 4B). In contrast, all promoter fragments displayed comparable activities in HCT116 (Figure 4C) and in COLO320 cells (Figure 4D), in spite of the 3 fold difference in their ErbB2 mRNA content. In the ovary cancer cells the activity of the 6kb promoter fragment was higher in OVCAR-3 (Figure 4E) than in SKOV-3 cells (Figure 4F), while only the latter overexpress ERBB2.

The low AP-2 levels might explain the low activity of the p716-LUC vector in colon and ovary cancer cells since AP-2 binding drives the activity of this fragment.

These results indicate that different mechanisms lead to ErbB2 mRNA upregulation in cancerous cells of different origins.

C. Transgenic overexpression of AP-2 in mice mammary gland Mammary targeted overexpression of AP-2! inhibited the development of the gland. The expression level of Neu, the mice ERBB2 ortholog, was not modified

by AP-2! overexpression (Zhang et al, 2003). Transgenic overexpression of AP-2" elicited hyper-proliferation of the epithelial cells, which was counterbalanced by increased apoptosis, the sum of these effects leading to hypoplesia of the gland. In these mice the transgene did induce a slight increase in Neu expression (J채ger et al, 2003).

However, the failure of AP-2 to stimulate Neu gene expression in mice mammary gland does not imply that the human ERBB2 is not regulated by these transcription factors. Indeed, as we have mentioned above, the AP2BS are missing from the mice Neu promoter (Figure 2).

The results of the experiments on the role of AP-2 transcription factors on ERBB2 gene overexpression in breast cancer cells are summarized in Table 1. In breast cancer cell lines AP-2 factors stimulate ERBB2 promoter activity. The factors are bound to the endogenous ERBB2 promoter and their down regulation inhibits the expression of the endogenous gene. Thus, in breast cancer cell lines, experimental evidence clearly indicates that AP-2 factors stimulate ERBB2 gene transcription. With one exception, a positive correlation was also reported between AP-2 and p185erbB2 levels in primary breast cancers. As discussed above, the null effect of AP-2 overexpression on Neu expression in transgenic mice was probably due to the absence of AP2BS in the rodent promoter. These results indicate that AP-2 factors do contribute to ERBB2 gene overexpression in some human breast cancers.

VI. ERBB2 overexpression in nonbreast cancer cells The transcriptional mechanisms responsible for the increased ERBB2 expression in cancers others than the breast are poorly understood. We decided to use our knowledge on breast cancer cells to understand the mechanisms leading to ERBB2 gene overexpression in non-breast human cancer cells. We used three prostate, two ovary, five colon and seven pancreas cancer cell lines.

To start, we compared the transcript and protein levels with the gene copy number. Next, we compared ErbB2 transcript and protein levels with those of AP-2 protein levels and DNA binding. Finally, we analyzed the activity of the ERBB2 promoter fragments which have been previously characterized in breast cancers (Vernimmen et al, 2003b).

We compared ERBB2 gene copy number, mRNA and protein levels in the non-breast cancer cells with those of well characterised breast cancer cells. ERBB2 gene amplification was detected only in SKOV-3 ovary cancer cells, where the amplification has been described previously (King et al, 1992). These cells contained the highest amounts of ErbB2 transcripts, comparable with those measured in breast cancer cells with a similar degree of gene amplification. Variable amounts of ErbB2 mRNA were detected in the other cells we have analyzed. The ErbB2 transcript levels in one colon cancer (COLO320) 373

Delacroix et al: ERBB2 gene overexpression and the role of AP-2 transcription factors The promoter fragments we have analyzed do not contain the sequences responsible for increased ERBB2 gene expression in colon and ovary cancer cells. It is possible that the transcription factors responsible for the differences in transcription levels recognize sequences outside the fragments we have studied.

Another possibility is that, contrary to breast cancer cells, post-transcriptional mechanisms might be responsible for the increased ErbB2 mRNA and protein levels in the ovary and colon cancer cells we have analyzed. Indeed SKOV-3 ovary carcinoma cells express a variant ErbB2 mRNA with an extended half-life (Doherty et al, 1999). These mechanisms will have to be taken into account for the understanding of ERBB2 gene expression regulation in non-breast cancer cells.

of gene amplification and increased transcription rates. In other tumours, increased protein levels, in the highest range of the levels measured in breast cancers without gene amplification, might be sufficient for cancer progression, probably in cooperation with other oncogenic signalling pathways. Our results indicate that different mechanisms are responsible for increased receptor content in non-breast cancer cells and in breast cancer cells. These mechanisms are not known at present.

Recent data indicate that non-breast cancer cells might become resistant to chemotherapy by upregulating ERBB2 expression. Thus, understanding the mechanisms responsible for the increase in p185erbB2 levels in different cancer cells is important for the development of more efficient therapeutic strategies for cancerous and non cancerous diseases involving this protein.

VII. Conclusions P185erbB2 contributes to mammary carcinogenesis if present in very high amounts, reached by the combination Table 1. Summary of the data relating AP-2 transcription factors to ERBB2 overexpression in breast cancers.

Experiment Cell lines Expression Promoter activity

Results References Increased AP-2-! and -" levels in cells Bosher et al, 1995 overexpressing ERBB2 AP-2-! and -" stimulate ERBB2 promoter Bosher et al, 1995; Grooteclaes et al, 1999; Vernimmen et al, 2003a fragments containing AP-2 binding sites Mutation of AP-2 binding sites inhibits Bosher et al, 1995, 1996; Vernimmen ERBB2 promoter activity et al, 2003a DN-AP2 inhibits ERBB2 promoter activity Delacroix et al, 2005 Endogenous ERBB2 promoter AP-2 binding endogenous promoter proven Begon et al, 2005

by ChIP Delacroix et al, 2005 AP-2-! and -" directed siRNAs downDelacroix in preparation regulate ERBB2 expression Immunohistochemistry on primary human breast cancer sections p185erbB2, AP-2! and AP-2" Positive correlation between AP-2! and Tumer et al,1998 AP-2" levels and p185erbB2 levels p185erbB2/ AP-2!

Positive correlation in a fraction of the Pellikainen et al, 2004 tumors p185erbB2 AP-2!

Week correlation between AP-2! and Friedrichs et al, 2005 p185erbB2 levels p185erbB2 and AP-2! + AP-2" Negative correlation Gee et al,1999 AP-2 overexpression in the mammary gland of transgenic mice AP-2!

The development of the gland is inhibited Zhang et al, 2003 AP-2" Increased proliferation and apoptosis J채ger et al, 2003 Table 2. Differences in ErbB2 mRNA and protein levels in human cancer cell lines of different origins. The transcript levels were measured by quantitative RT-PCR, while the protein levels were estimated by western blotting (adapted from Vernimmen et al, 2003b). For each cancer type two cell lines are presented, one containing the lowest the second the highest amounts of ErbB2 mRNA and protein. For each cancer type, the lowest transcript and protein amounts were considered as equal to one. The relative increase in expression was calculated by dividing the highest values by the smallest values measured in cells from the same cancer type. The asterisks indicate an underestimated value for SKOV-3 protein level, because of autoradiograph saturation.

Origin Cell line mRNA Protein

Ovary Ovcar-3 SKOV-3 1 1

60 13**

Prostate Low High (PC-3) (LNCaP) 1 7.3 1 7.5

Colon Low (HTm29) 1 1

374

High (COLO320) 13 0.5

Pancreas Low High (SU.86.86) (Capan-2) 1 42.5 1 23

Cancer Therapy Vol 3, page 375

Figure 4. ERBB2 promoter activity in human cancer cells of different origins. A. Reporter vectors used in this study containing the luciferase (LUC) cDNA under the transcriptional control of 215 (1), 716 (2), 3798 (3) and 6007 (4) bp fragments of the human ERBB2 promoter. B. Relative luciferase activities induced by reporter vectors 2, 3 and 4 transfected into BT-474 breast cancer cells overexpressing ERBB2, reported to the activity induced by vector 1 considered as equal to one (Delacroix et al, in press). C. Relative luciferase activities induced by reporter vectors 2, 3 and 4 transfected into HCT116 colon cancer cells reported to the activity induced by vector 1 considered as equal to one. D. Relative luciferase activities induced by reporter vectors 2, 3 and 4 transfected into COLO 320 colon cancer cells, reported to the activity induced by vector 1 considered as equal to one. E. Relative luciferase activities induced by reporter vectors 2, 3 and 4 transfected into OVCAR-3 ovary cancer cells reported to the activity induced by vector 1 considered as equal to one. F. Relative luciferase activities induced by reporter vectors 2, 3 and 4 transfected into SKOV-3 ovary cancer cells overexpressing ERBB2 reported to the activity induced by vector 1 considered as equal to one (Vernimmen et al, 2003b).

Research Associate (FNRS).

Aknowledgements Our work was supported by the FNRS, the Belgian Federation against Cancer, the Centre Anticancereux près l’Université de Liège. LD and DV were recipients of Televie grants from the FNRS; BD was a recipient of FRIA fellowship and Televie grant; RW is Senior

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Pancreatic cancer palliation using radiofrequency ablation. A new technique Case Report

John D Spiliotis1, Anastasios C Datsis1, Panagiotis Chatzikostas2, Spyros P. Kekelos1, Athina N. Christopoulou3, Athanasios G. Rogdakis1, P Symeonides2 1

Department of Surgery, “Hatzikostas” General Hospital of Messologi, Messologi, Greece Department of Surgery, Nicosia General Hospital, Cyprus 3 Department of Internal Medicine, Section of Oncology, “St Andrews” General Hospital, Patras, Greece 2

__________________________________________________________________________________ *Correspondence: John Spiliotis, M.D., Director of the Department of Surgery, “Hatzikostas” General Hospital of Messologi, 73 Pente Pigadion Str, GR-26441 Patras, Greece; Tel: +30-2610-278356; E-mail: jspil@in.gr Key words: radiofrequency ablation, pancreatic adenocarcinoma, palliative treatment of cancer Abbreviations: radiofrequency ablation, (RFA) Received: 6 June 2005; Revised: 16 June 2005 Accepted: 27 June 2005; electronically published: August 2005

Summary We present the use of a new radiofrequency ablation (RFA) system, in five patients, with inoperable pancreatic cancer. In the current literature these cases are the only treated by this RFA device. We performed RFA in five patients with advanced pancreatic cancers. Four of them had obstructive jaundice and the other had gastric outlet obstruction. The pancreatic cancers considered as unresectable during the laparatomy, due to advanced local disease in all patients. We used the newer Cool-tipTM RFA system (Radionics), with the cooled electrode. The electrode circulates water internally to cool the tissue adjacent to this, maximizing energy deposition. Especially, in the patient with the huge tumor in the pancreatic body we used the Cool-tipTM Cluster electrode (Radionics) to increase the coagulation volume. None of our patients developed a significant complication from the treatment, such as pancreatitis or bleeding. RFA of unresectable pancreatic cancer is a safe palliative procedure according to our preliminary results in five patients. Probably in some cases RFA may slow tumor growth resulting in long-term survival, as in one of our patients, who lives 15 months after surgery without evidence of disease progression. have been published in the medical literature (Goldberg et al, 1999; Yoichi et al, 2000; Elias et al, 2004). On the other hand there is an extensive experience with the radiofrequency ablation (RFA) in the treatment of unresectable liver tumors and promising results have also been obtained in tumors of the kidney, breast, lung, bones and prostate (Mirza et al, 2001).

This article present the use of a RFA system (CooltipTM, Radionics), in five patients, with inoperable pancreatic adenocarcinoma. In the current literature these cases are the only treated by this RFA device.

I. Introduction Pancreatic cancer is the fifth cause of cancer death in the United States and one of the leading causes of cancer death in the ‘western’ countries becoming so a major worldwide public health problem. The radical surgical resection represents the only chance for cure but, unfortunately is possible in only 15% of patients. Even at experienced centers the 5-year survival rates for the most favorable patients who undergo resection and adjuvant therapy are less than 20% (White et al, 2003).

Treatment options in the advanced unresectable pancreatic cancer are very limited. Palliation involves either biliary stenting or surgical bypass. Combined chemoradiation has been associated with improvements in pain, wasting and obstructive symptoms over chemotherapy alone and offers some benefits in these patients (Fisher et al, 1999). The ablation of unresectable pancreatic cancers with the use of radiofrequency devices is a relatively new treatment option. Only a few papers

II. Cases report In our departments the last year, we performed radiofrequency ablation in five patients with advanced pancreatic cancers. Four of the patients had obstructive jaundice due to pancreatic head tumors and the other had gastric outlet obstruction from a huge tumor (10X7cm) in the body of the pancreas. The treatments were performed

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Spiliotis et al: Pancreatic cancer palliation using radiofrequency ablation from February 2004 to March 2005. Table 1, summarizes patients characteristics.

Herein, we describe the technical details of the procedure in a particular patient (we followed the same procedure in all of our patients). A 65 years old man, with progressive painful obstructive jaundice was admitted to the surgical department, Messologi General Hospital. A radiologic investigation with ultrasound and computed tomography demonstrated a tumor of the head of the pancreas (4,5 cm) with local invasion of the superior mesenteric vein. A laparotomy was performed in which the tumor was confirmed (positive tumor FNAC/ frozen

section positive). There was not evidence of lymph node (lymph node of hepatoduodenal – frozen section negative) or liver metastasis. After an extensive dissection of the pancreatic head the tumor was considered unresectable due to infiltration of the superior mesenteric vein. Due to the above we decided for palliative operation and radiofrequency ablation of the tumor. We performed two ablations, one at the anterior and the other at the posterior surface of the pancreatic head (Figures 1 and 2 respectively), for 6 and 7 minutes each, under direct vision of the duodenum to avoid burn damage to it.

Table 1. Characteristics of the patients: response to treatment and outcome Patient Symptoms

_, 65y Painless obstructive jaundice (POJ) Tumor 3 cm (head), CT vessel infiltration Criteria of Superior mesenteric inoperability vein obstruction – Positive tumor cytology Operation Biliary-Gastric bypass + RFA (B-G bypass+RFA) RFA device and Cool-tipTM, two technique ablations 6 and 7 minutes Postoperative complications related to RFA Follow - up Outcome

None 15 months Alive without evidence of disease progression (locally or distant)

_, 74y POJ

_, 79y POJ

_, 66y _, 64y Gastric outlet POJ obstruction Tumor 3 cm (head) Tumor 4,5 cm Tumor 10 cm (body) Tumor 3 cm (head), (head) vessel infiltration Hepatoduodenal Positive cyotology, Locally advanced Superior mesenteric lymph node positive locally advanced disease – gastric vein obstruction in frozen section tumor outlet obstruction Positive tumor cytology B-G bypass+RFA B-G bypass+RFA Gastric bypass + B-G bypass+RFA RFA Cool-tipTM, one Cool-tipTM, three Cool-tipTM Cluster Cool-tipTM, two ablation,7 minutes ablations, 8, 2 and 4 electrode, two ablations 6 minutes minutes ablation, 7 minutes each each None None None None 8 months Liver metastases Died

14 months Alive without any problem (diabetes, diarrhea, or jaundice)

Figure 1. RFA of the tumor at the anterior surface of the pancreatic head.

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9 months Alive

2 months Alive

Cancer Therapy Vol 3, page 381 Also continuous infusion/perfusion of the area of the head of pancreas with cold normal saline was done during the radiofrequency ablation (Figure 3).

We used the newer Cool-tipTM RF ablation system (Radionics), with the cooled electrode (a 17-gauge, 20 cm with a 3 cm exposure length for rapid tumor destruction).

The “biopsy needle� design allowed the accurate placement decreasing the potential injury of surrounding vital structures (common bile duct, duodenum, vessels).

The electrode circulates water internally to cool the tissue adjacent to this, maximizing energy deposition. We completed the operation with a double anastomosis operation (a common bile duct-jejunostomy plus a gastrojejunostomy). A drainage tube was left in the area of the

ablated tumor. We used the same technique (ablation under direct vision and palpation of the tumor) for all patients. Post-operatively the patients were covered with subcutaneous octreotide (Sandostatin) and antibiotics. We had not any postoperative complication related to the tumor ablation (pancreatitis, bleeding, hyperamylasemia) in anyone of our five patients.

III. Discussion Radiofrequency energy has been used in the last decades for the destruction of solid tumors. Unresectable liver tumors, mainly metastases from colon and rectal cancer, is the primary indication for the method.

Figure 2. RFA of the tumor at the posterior surface of the pancreatic head.

Figure 3. Infusion/perfusion of the area of the head of pancreas with cold normal saline during the RFA.

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Spiliotis et al: Pancreatic cancer palliation using radiofrequency ablation Promising results have also been reported for many other tumors as early stage breast cancer, osteoid osteoma, osseous metastases, solid renal tumors, pulmonary malignancies, brain and prostate tumors.

Proximity of the target tumor to a fragile structure (bowel, nerve, bile duct, vessel) is considered as a relative contraindication for the radiofrequency ablation (Elias et al, 2004). For the above reason (fragility of pancreatic parenchyma and fear of postoperative complications) the use of the RFA in the treatment of advanced pancreatic tumors is not so usual in the surgical routine. A recently published paper reports two patients with multiple metastases from renal cancer in the pancreas, who were treated with RF destruction. The authors used a high local temperature (>900 C) for the ablation, resulting in the effective tumor destruction. However, the two patients presented postoperatively severe necrotizing pancreatitis (Elias et al, 2004). The authors ascribed the complication to the lack of adequate device or to inadequate use of the existing device to perform intrapancreatic RFA. They used for the first patient a monopolar small 10-gauge needle electrode (1 cm tip) with the ElektrotomTM perfused system (Berchtold, Tuttigen, Germany) and for the other a bipolar device with two small 10-gauge needle electrodes (1cm tip) placed parallel each side of the tumor. In both cases a high temperature (>90 0 C) was used. Conclusively, the authors do not recommend the RFA of pancreatic tumors because of severe compications (Elias et al, 2004).

A decade ago, Goldberg et al, used the RF energy for the ablation of normal pancreatic tissue of 13 pigs. They used a modified electrode (19-gauge) without internal cooling maintained an electrode tip temperature of 900 C. They noticed foci of coagulation necrosis from 8 to 12 mm in diameter. Only one animal (13%) from the eight pigs that were not immediately sacrificed, presented a focal pancreatitis (Goldberg et al, 1999).

In our patients, we used the newer Cool-tipTM RF ablation system (Radionics), with the cooled electrode for rapid tumor destruction. The electrode is a 17-gauge, 20 cm with a 3 cm exposure length. The entire electrode can easily imaged on the intraoperative ultrasound.

Furthermore it is easy to reposition allowing coagulation of varying lesion sizes. The “biopsy needle” design allows the accurate placement and decreasing the potential injury of surrounding vital structures (common bile duct, duodenum, vessels). The electrode circulates water internally to cool the tissue adjacent to this, maximizing energy deposition. So, the result is reduced treatment time (8-12 min) and maximal ablation zone. The hyperthermia was maintained for 7 min, twice, with different directions at a controlled temperature of 800- 900 C in the RF field.

The temperature of the surrounding tissue was maintained at < 350 C with continuous cooling with perfusion, infusion of cool normal saline solution. The patients were subsequently monitored by computed tomography (CT) scanning. None of our patients developed a significant complication from the treatment, such as pancreatitis or bleeding. The levels of serum amylase were within normal limits in all of the cases.

Our excellent results are very similar to these of the pioneer of the pancreatic cancer ablation. From September

1994 to February 1999, Yoishi and coworkers performed RF ablation in 20 patients with pancreatic adenocarcinomas which were judged to be unresectable based on the presence of distal metastases and/or local invasion into major blood vessels. The authors used a completely different RF ablation system than ours (OMRON Co. Ltd. Kyoto, Japan). The electrodes consisted of 4 needles, which were 2 cm long and 0,8 cm in diameter and which were positioned in a square array at intervals of 2 cm. They had only two serious complications (one patient with a cyst formation who required percutaneous drainage and another who developed an abscess in the peritoneal cavity). Both patients died 23 and 21 days after treatment respectively (Yoichi et al, 2000).

Our preliminary results, together with the results of Yoishi et al and Goldberg et al, indicate that the different RF ablation devices aren’t responsible for the appearance of the post treatment severe pancreatitis observed in Elias et al cases. It seems more logical that the ablation of multiple pancreatic tumors (as the multiple pancreatic metastases in Elias et al cases) resulted in extensive destruction of the pancreatic parenchyma and it was the reason for the development of severe post treatment complications. So, based on our preliminary result we can recommend the use of RF ablation in solitary unresectable pancreatic tumors.

References Elias D, Baton O, Sideris L, Lasser P, Pocard M (2004) Necrotizing pancreatitis after radiofrequency destruction of pancreatic tumors. Eur J Surg Oncol 30, 85-87.

Fisher BJ, Perera FF, Kocha W, Tomiak A, Taylor M, Vincent M, Bauman GS (1999) Analysis of the clinical benefit of 5fluorouracil and radiation treatment in locally advanced pancreatic cancer. Int J Rasiat Oncol Biol Phys 45, 291295.

Goldberg SN, Mallery S, Gazelle S, Brugge WR (1999) EUSguided radiofrequency ablation in the pancreas: results in a porcine model. Gastrointest Endosc 50, 392-401.

Mirza AN, Fornage BD, Sneige N, Kuerer HM, Newman LA, Ames FC, Singletary SE (2001) Radiofrequency ablation of solid tumors. Cancer J 7, 95-102.

White RR, Shah SA, Tyler SD (2003) Pancreatic cancer since Halsted. How far have we come and where are we going?

Ann Surg 238, S132-S144.

Yoichi M, Akihilo N, Yasuo K, Koji Y, Nobuo K, Yuzo N (2000) Selective thermocoagulation of unresectable pancreatic cancers by using radiofrequency capacitive heating. Pancreas 20, 14-20.

John D Spiliotis

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Neuroendocrine differentiation in prostate cancer Review Article

Siamak Daneshmand1, Marcus L. Quek2, Jacek Pinski3,* 1

Division of Urology, Oregon Health and Science University, Portland, Oregon Department of Urology 3 Division of Medical Oncology, Keck School of Medicine at the University of Southern California, Los Angeles, California 2

__________________________________________________________________________________ *Correspondence: Jacek Pinski, M.D., Ph.D., Assistant Professor, Division of Medical Oncology, USC/Norris Comprehensive Cancer Center, 1441 Eastlake Avenue, Suite 3449, Los Angeles, CA 90089, USA; Tel: (323) 865-3929; Fax: (323) 865-0061; E-mail: pinski_j@ccnt.hsc.usc.edu Key words: Neuroendocrine differentiation, prostate cancer, hormone refractory prostate cancer, Small cell (neuroendocrine) carcinoma of the prostate Abbreviations: androgen receptor, (AR); benign prostatic hyperplasia, (BPH); human chorionic gonadotropin, (hCG); immunohistochemical, (IHC); interleukin-6, (IL-6); lymphocyte conditioned medium, (LCM); Neuroendocrine, (NE); neuron-specific enolase, (NSE); parathyroid hormone-related protein, (PTHrP); prostate-specific antigen, (PSA); prostatic intraepithelial neoplasia, (PIN); serotonin, (5-HT), neuron-specific enolase, (NSE); short-interfering RNA, (siRNA); somatostatin, (SST); thyroid-stimulating-like peptide, (TSH) Received: 19 May 2005; Accepted: 6 June 2005; electronically published: July 2005

Summary Neuroendocrine (NE) cells likely play a role in both normal development and pathologic conditions of the prostate.

Neuroendocrine expression has attracted increasing attention in prostate cancer research as a potential mechanism for regulation of growth and differentiation. This review discusses the role of NE cells in normal and malignant prostatic tissue and examines the current literature on the topic. NE cells are thought to originate from basal stem cells and are known to produce a number of secretory factors that may act through endocrine, paracrine, and autocrine mechanisms. Virtually all benign prostatic tissue and prostatic adenocarcinomas show some degree of NE differentiation, which appears to vary with age and ethnic background. Clinical studies suggest that the extent of NE differentiation increases with tumor progression and the development of androgen insensitivity; however, there is controversy regarding its prognostic significance. NE cells lack the androgen receptor and appear to increase in response to androgen ablation. In vitro studies have shown that they can derive through a process of transdifferentiation from tumor cells. Some studies suggest that serum chromogranin A measurements in prostate cancer patients correlate with tissue expression and may provide prognostic information. Recent work from our laboratory has shown that NE cells may in fact have an inhibitory effect on prostate cancer cells. Lastly, we discuss small cell neuroendocrine carcinomas of the prostate and their clinical features. The current review underscores the need to improve our understanding of neuroendocrine cells, their regulatory products and their influence in prostate carcinogenesis. Further studies are needed to elucidate the contribution and significance of NE cells to prostate cancer growth and progression. embryogenesis or from a common precursor for NE and prostatic epithelial cells. The first hypothesis is supported by the finding that chromogranin A-positive cells are observed in the urethral epithelium and the surrounding mesenchyme of the fetus at very early stages of gestation (Abrahamsson, 1999a). On the other hand, the stem cell theory is supported by the presence of intermediate cells expressing both endocrine and exocrine markers (basal cell-specific cytokeratins and prostate-specific antigen [PSA]), which occur frequently in prostate cancer with NE differentiation (Bonkhoff, 1998). Moreover, NE cells have been shown to manifest simultaneous expression of

I. Introduction The epithelial cells of the human prostate are composed of three principal cell types: secretory cells, basal cells, and neuroendocrine (NE) cells (Hansson and Abrahamsson, 2001). The NE cells are thought to be involved in cell regulation through the release of numerous secretory products that may act in an endocrine, paracrine or autocrine manner (di Sant'Agnese, 1992a).

NE cells likely play a role in both normal as well as pathologic conditions of the prostate. It is controversial whether NE cells originate from the neural crest during

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Daneshmand et al: Neuroendocrine differentiation in prostate cancer chromogranin A and basal cell-specific cytokeratins (Bonkhoff, 1998; Rumpold et al, 2002).

Based on morphology, di Sant’Agnese (1992b) described two types of NE cells the “open” type, which resembles an open flask-shaped form with long slender luminal extensions, and the “closed” type, which lacks these extensions. Both types characteristically have irregular dendritic processes extending between adjacent epithelial cells (Figure 1). Despite these morphologic classifications, the different functional roles for these cells remain unclear. NE cells produce a variety of neurosecretory products that regulate cellular growth and differentiation, while also providing useful markers for their identification. Most studies have focused on growth stimulatory factors such as serotonin, calcitonin, and parathyroid hormone-related peptides (di Sant'Agnese, 1992a, 1998). Some of these regulatory peptides, such as bombesin, calcitonin, and parathyroid hormone-related protein, have been shown to stimulate tumor cell proliferation in vitro (Bologna et al, 1989; Iwamura et al, 1994b; Shah et al, 1994). It has been suggested that NE cells may also produce and secrete inhibitory factors based on in vitro co-culture experiments with prostate cancer cell lines (Wang et al, 2004b). Whatever their functional role, several of these markers, including chromogranin A, neuron-specific enolase, synaptophysin, and serotonin, provide useful marker proteins for immunohistochemical localization in both human and animal models (Angelsen

et al, 1997a; Rodriguez et al, 2003). Furthermore, heterogeneity and variation in the neurosecretory products expressed by these cells suggest that there are actually several populations of prostatic NE cells, each with its own set of secretory factors (Abrahamsson, 1999a).

The origin of NE cells in the prostate remains a subject of controversy. A general stem cell theory postulates that NE cells share a common origin with other epithelial cell types from pluripotent stem cells within the basal layer (Bonkhoff, 1998). This concept is based on the finding of epithelial cells of intermediate differentiation based on morphologic and immunohistochemical marker analysis. Furthermore, these cells do not show proliferative activity (lack the proliferation-associated antigens, Ki-67 and MIB-1), as would be expected of postmitotic terminally-differentiated cells. Aumuller et al (1999) proposed that NE cells originate from the neural crest during embryogenesis. This hypothesis is supported by the finding of NE cells in the urogenital epithelium early in gestation, and by studies demonstrating a preponderance of NE cells in the periurethral and ductal regions. Cohen et al, (1990) raise the possibility of 2 functionally distinct populations of NE cells with potentially different embryonic origins, with peripheral NE cells deriving from a pluripotent stem cell and periurethral NE cells originating from the neural crest.

Still another theory suggests that the increased NE cell

Figure 1. Typical neuroendocrine cell with its dendritic-like process found in the epithelial cell layer of the prostate.

(Immunohistochemical staining for chromogranin A, 1:1000 dilution, mouse monoclonal antibody from Dako, Denmark, in a benign prostate sample taken from a cystoprostatectomy specimen, developed with diaminobenzidine tetrahydrochloride solution and counterstained with hematoxylin; original magnification X 400).

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Cancer Therapy Vol 3, page 385 expression seen in prostatic malignancies, may be a result of a process of transdifferentiation from prostatic adenocarcinoma cells in response to various cytokines (Wang et al, 2004b).

Several studies report that NE cells lack expression of the androgen receptor (Bonkhoff et al, 1993; Krijnen et al, 1993; Abrahamsson, 1999a), while others suggest that a separate subpopulation of NE cells exist that may be responsive to androgens (Cohen et al, 1990; Nakada et al, 1993). In either case, the effects of hormonal manipulation on NE cell expression are not clearly understood.

Angelsen et al. (1999) demonstrated that testosterone administration during the pubertal period results in an increased number of NE cells in proliferative lesions in the dorsal lobe of the rat prostate. On the other hand, other studies looking at both dog and human prostate tissue have shown promotion of NE differentiation under androgen ablative conditions (Ismail et al, 2002). In the dog model, the administration of androgens after castration restores NE cell density to normal levels, implying that NE differentiation is hormonally repressed and potentially reversible. Other studies looking at radical prostatectomy specimens following neoadjuvant hormonal ablation have also noted increased NE cell expression (Jiborn et al, 1998; Ahlgren et al, 2000). Collectively, these studies suggest that the hormonal milieu may affect NE differentiation in the human and animal prostate, and that further systematic studies are needed to establish the exact nature of the relationship.

Though their functional role remains largely unknown, these cells likely affect cellular growth and differentiation and exocrine secretions of the prostate through the local release of various neurosecretory products. Commonly found secretory products include bombesin, serotonin (5-HT), neuron-specific enolase (NSE), a thyroid-stimulating-like peptide (TSH), somatostatin (SST), parathyroid hormone-related protein (PTHrP), calcitonin as well as other members of the calcitonin family (di Sant'Agnese et al, 1985; Hansson and Abrahamsson, 2001). More products likely exist which have yet to be characterized. These cell products have the potential to regulate the growth, differentiation and homeostasis of normal as well as pathologic prostatic conditions. They are seen in both benign as well as malignant prostatic tissue (Figure 2). Increasing attention has focused on the potential clinical and prognostic implications of NE differentiation in prostate cancer.

However, in order to appreciate the impact of NE expression in prostatic malignancies, it is imperative that we first understand its role in the development and function in normal benign prostate tissue.

of highest proliferation, lacked NE staining. Although there was great variability in the number of NE cells in prenatal prostates, the ratio of NE cells to epithelial cell area appeared to be relatively constant through adult life.

Another autopsy series by Cohen et al, (1993) looked at the distribution of NE cells in different prostatic structures from infancy to elder adulthood. The periurethral and ductal areas had the highest number of NE cells, while the peripheral acini had the least. Interestingly, this group noted age-dependent NE expression in the peripheral acini, such that NE cells were noted in this area during the first few months of life, conspicuously absent between 4 and 13 years, then reappearing at approximately 14 years and through adult life. This was in contrast to the relatively constant expression in the periurethral and ductal areas in all age groups. The authors suggest that 2 functionally distinct subpopulations of NE cells exist during normal development, with the ones in the peripheral zone being hormonally-responsive.

Regional differences in the distribution of NE cells noted in adult prostates may be associated with the predilection for particular areas to develop pathologic processes. Santamaria and colleagues, (2002) evaluated the distribution of NE cells in various prostatic zones. A predominance of NE cells was noted in the transition zone, scarce involvement in the central zone, and intermediate in the peripheral zone. This group hypothesized that the NE cells noted in the transition zone could play a stimulatory role in the development of benign prostatic hyperplasia (BPH) often noted in this area, while peripheral zone NE cells could potentially induce androgen-independent growth of prostate cancer. Similarly, Islam et al, (2002) noted NE cell density to be greater in the verumontanum and main prostatic ducts than in the acini of the peripheral zone, regardless of age. Taken together, these studies agree that more NE cells are found in glandular structures closer to the urethra and less prominent in the periphery of the gland. Others studies have suggested a causal link between NE cell expression and BPH. Although most adenomatous nodules lack significant amounts of NE cells, Cockett and others, (1993) distinguished between proliferating foci in smaller BPH nodules with numerous NE cells and larger â&#x20AC;&#x153;matureâ&#x20AC;? nodules that lacked NE cells, suggesting that NE cells provided mitogenic stimuli for areas of active hyperplasia.

There appears to be a difference in the distribution of NE cells among different ethnic backgrounds. We determined the relative distribution of NE cells in the benign prostate tissue of men from four different ethnic backgrounds to determine whether NE expression levels mirror the degree to which incidence and mortality vary across racial groups.

We observed a 6-8 fold decrease in the mean number of NE cells in the prostates from African American men when compared to other races (Figure 3). There was a trend toward higher NE expression in Asians as compared to Caucasians and Hispanics, however, this difference did not reach statistical significance (Daneshmand et al, 2005).

Given their potential role in regulation of growth and carcinogenesis, it is conceivable that decreased NE cell expression in the prostates of African American men may

II. Neuroendocrine cells in normal prostatic tissue NE cells have been detected immunohistochemically as early as 13 weeks gestation, and in nearly all prostates by 21 weeks (Cohen et al, 1993). Xue et al, (2000) evaluated the number and distribution of NE cells in autopsy-collected prostates from fetuses, prepubertal males and young adults. NE cells were found primarily in the acinus/ductal regions, while the budding tips, the areas 385

Daneshmand et al: Neuroendocrine differentiation in prostate cancer

Figure 2. Neuroendocrine differentiation seen in benign prostatic hyperplasia from a cystoprostatectomy specimen (A) and primary Gleason pattern 4 prostatic adenocarcinoma from a radical prostatectomy specimen (B). (Immunostaining for chromogranin A was performed as described in Figure 1; original magnification X 400).

Figure 3. Distribution of neuroendocrine cells in benign prostates taken from cystoprostatectomy specimens from four different ethnic backgrounds: Asian (n=15), Caucasian (n=16), Hispanic (n=13), and African American (n=15).

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Cancer Therapy Vol 3, page 387 have a significant influence on the higher incidence of prostate cancer observed in this population.

patients. The short course of androgen ablation significantly increased the extent of NE differentiation in the benign glands of the human prostate. Similarly in a prostate cancer xenograft model, short-term androgen withdrawal resulted in an increased number of NE cells. A time course experiment with these PC-295 tumor bearing mice provided evidence that this increase occurred by induction of NE differentiation rather than by rapid proliferation and subsequent differentiation (Noordzij et al, 1996). Other investigators have shown that the androgen receptor (AR) is responsible for the repression of NE transdifferentiation. In vitro experiments have shown that AR silencing through short-interfering RNA (siRNA) directed against AR induces NE transdifferentiation.

Furthermore, AR silencing inhibits the growth of LNCaP cells in vitro (Wright et al, 2003). This confirms that the AR actively represses the NE transdifferentiation process in prostate cancer cells.

NE cells produce a number of hormonal growth factors such as serotonin, bombesin-related peptides, PTHrP, neurotensin, and calcitonin, that may act through endocrine, paracrine, and autocrine mechanisms (di Sant'Agnese, 1998). Several of these growth factors including bombesin and neurotensin, have been shown to stimulate the proliferation of some prostate cancer cell lines in vitro (Burchardt et al, 1999; Cox et al, 1999; Cox et al, 2000). Although NE tumor cells themselves do not proliferate, they have been implicated in the progression of prostate cancer through the production of mitogenic factors that maintain cell proliferation in adjacent tumor cells through a paracrine mechanism. Extensive and multifocal NE differentiation in prostatic tumors has been reported to be more aggressive and resistant to hormonal therapy (Abrahamsson, 1996; Bonkhoff, 1998).

The origin of NE cells in prostate cancer is unknown.

The androgen sensitive human prostate cancer cell line, LNCaP, has been documented to transdifferentiate into a NE phenotype in response to the cytokine interleukin-6 (IL-6), increasing levels of cyclic AMP, or following culture in steroid-deprived medium (Abrahamsson, 1999b; Burchardt et al, 1999; Cox et al, 1999; Spiotto and Chung, 2000; Zelivianski et al, 2001). The observation that conditions associated with transformation of prostate cancer cells in vitro, such as androgen deprivation or exposure to IL-6 also increases NE differentiation in prostatic tumors in vivo, suggest that NE cells may be derived from adenocarcinoma cells by a process of transdifferentiation in response to changes in the hormonal and growth factor milieu of the microenvironment (Jiborn et al, 1998; Wang et al, 2004a,b).

The implication of NE cells in the progression of prostate cancer is largely based on the fact they produce growth factors which stimulate the proliferation of prostate cancer cell lines in vitro (Jongsma et al, 2000; Amorino and Parsons, 2004). Most studies describing the products of prostatic NE cells have been derived from prostatic carcinoma. NE cells have also been implicated in the upregulation of Bcl-2, which is known to have antiapoptotic activity (Segal et al, 1994). However, recent work from our laboratory has shown that NE cells may actually have an inhibitory effect on prostate cancer cells

III. Neuroendocrine differentiation in prostate cancer In 1984 di Sant'Agnese and De Mesy Jensen, in 1984 described the endocrine-paracrine cells of the prostate and suggested that they play a role in various pathologic conditions. Since then, more than 300 articles have been published on the topic. Recent studies suggest that prostatic NE cells may be involved in carcinogenesis, however the influence of NE cells in the regulation and progression of prostate cancer is not well understood.

Virtually all prostate carcinomas contain at least focal areas of NE cells believed to be quiescent, nonproliferative cells that do not stain with the human mitotic indicator antibodies Ki-67 or MIB1 (Cox et al, 1999). NE cells in prostate cancer are easily identified using immunohistochemical markers.

Serotonin and chromogranin A appear to be the best markers for identifying NE cells in formalin-fixed sections of the prostate (Bostwick et al, 2002). NE differentiation can also be identified in 25% of prostate cancer needle biopsies though it does not appear to provide any useful prognostic information (Casella et al, 1998).

Clinical studies to date have suggested that the extent of NE differentiation increases with tumor progression and the development of androgen insensitivity. Hirano et al, (2004) examined 72 prostate cancer specimens obtained at radical prostatectomy (38 from patients with no neoadjuvant therapy and 34 patients with neoadjuvant therapy for 3-6 months) and 21 prostate cancer autopsy specimens from patients who died from hormone refractory prostate cancer after androgen deprivation therapy for more than one year. They found that NE differentiation increased with longer duration of hormone therapy, but the study could not attribute the increase in NE differentiation to the condition of hormone refractoriness. Whether NE cells have any prognostic significance in primary prostate adenocarcinomas or lymph node metastases is controversial. Several studies have reported a significant correlation between NE differentiation and pathologic stage or survival (Bostwick et al, 2002). Conversely, other studies have found no significant association between neuroendocrine differentiation and patient survival or prostate cancer progression (Noordzij et al, 1995).

These studies however do not elucidate or reflect the role of NE cells in carcinogenesis. The increase in NE cells seen during androgen deprivation is not necessarily attributable to the hormone refractory state of the tumor.

Ismail et al, (2002) studied NE differentiation in the dog and human prostate following androgen ablation. They found that castration induced NE differentiation in the normal prostates of dogs and that this was reversible with the addition of androgen or estrogen supplements. To verify whether similar changes are seen in humans, the investigators examined NE differentiation in radical prostatectomy specimens obtained from patients who received neo-adjuvant androgen ablative therapy prior to surgery and compared them to a similar group of untreated 387

Daneshmand et al: Neuroendocrine differentiation in prostate cancer (Wang et al, 2004b). In cell culture studies, the proliferation of 3 prostate cancer cell lines (LNCaP, PC-3 and DU-145) was significantly inhibited when exposed to IL-6-induced NE cell conditioned medium or NE cell coculture. These results imply that NE cells may be releasing inhibitory factors, which could dominate the mitogenic effects of growth factors characteristically associated with NE cells.

A number of publications have presented evidence, which not only questions the presumed mitogenic influence of NE cells, but suggests that they may potentially protect the prostate from carcinogenesis. For example, Algaba et al, (1995) noted that with advancing age there is a gradual decrease in the number of NE cells in the peripheral zone of the prostate, the area that is most susceptible to carcinogenesis. Others have noted that as BPH development progresses, the NE cells are greatly diminished in number or completely lost from most adenoma nodules (Islam et al, 2002). Bostwick et al, (1994) reported that the number of NE cells is decreased around areas containing high-grade prostatic intraepithelial neoplasia (PIN), a premalignant lesion. The same investigators also found that benign prostatic epithelium and primary prostate cancer express a significantly greater number of NE cells than lymph node metastases, suggesting that decreased expression of NE cells may be involved in progression of prostate cancer (Bostwick et al, 2002). Autopsy studies have also revealed that in the developing fetus, NE cells are found only in the acinous/ductal compartment of the prostate and conspicuously absent in the budding tips, an area of active growth (Xue et al, 2000).

The association between neuroendocrine elements in relapsed prostate cancer and sensitivity to chemotherapy has also been studied. In a study by Steineck et al, (2002), about one-half of progressive metastatic androgenindependent prostate cancers showed measurable response to cytotoxic therapy regardless of degree of neuroendocrine differentiation. The exact role of NE cells and contribution to the progression of prostate cancer is still unclear. Although more NE cells are seen in the androgen-independent prostate carcinomas, whether these cells have the potential to induce androgen-independent cell growth is not known.

expression in PIN compared with normal cells and carcinoma. Algaba et al. (1995) also found lower numbers of neuroendocrine cells in patients with foci of PIN and carcinoma. They suggested that the decrease in neuroendocrine cells make the prostate more susceptible to carcinogenic factors. di Sant'Agnese (1996) also agreed that the degree of neuroendocrine differentiation in PIN is intermediate between normal prostate (containing the most number of cells with neuroendocrine differentiation) and carcinoma.

V. Neuroendocrine serum markers in prostate cancer

tumor

The various secretory products from NE cells not only function at the local tissue level of the prostate, but may also be secreted in the serum. Serum detection of NE proteins may provide some prognostic information, and may actually constitute a more representative indicator of overall neuroendocrine differentiation since it accounts for the entire tumor cell population (Abrahamsson, 1999b).

Studies have correlated immunohistochemical tissue staining intensity for chromogranin A with serum levels of chromogranin A, suggesting that serum levels may be useful markers for NE differentiation in prostatic tumors (Angelsen et al, 1997b). Early studies correlated elevated serum chromogranin A and neuron-specific enolase (NSE) levels to hormone therapy resistance and poor prognosis (Kadmon et al, 1991; Deftos et al, 1996; Kimura et al, 1997; Wu et al, 1998; Tarle, 1999). In a review by Berruti and colleagues, (2001), serum chromogranin A levels were found to be higher in prostate cancer patients than in patients with benign or pre-malignant diseases, and correlated with advanced stage or hormone refractoriness.

There was no apparent relationship to serum PSA levels, however supranormal levels of chromogranin A or NSE were correlated with decreased survival in the hormone refractory cases. Isshiki et al, (2002) also suggested a potential diagnostic or prognostic role for chromogranin A in patients with advanced stage or hormone-refractory disease independent of PSA. They found that poorly differentiated adenocarcinoma was associated with higher chromogranin A levels. The stage D cases with higher chromogranin A had a poorer prognosis than those with lower chromogranin A, however this only held true for those with a median PSA of 172 ng/ml. or less. Cussenot et al (1996) observed that plasma chromogranin A and neuron-specific enolase levels were elevated in 55% and 30% of patients with hormone independent prostate cancer. In patients with stage D3 disease, patients with elevated chromogranin A levels had statistically worse survival. Pre-treatment serum NSE levels have also been shown on multivariate analysis to be an independent prognostic factor for survival in metastatic prostate cancer on androgen ablative therapy (Kamiya et al, 2003), hormone-resistant prostate cancer (Hvamstad et al, 2003), as well as localized prostate cancer treated with external beam radiation (Lilleby et al, 2001).

The role of serum NE markers may prove to be a more useful indicator of prostatic NE differentiation than its corresponding tissue expression. Further studies are needed to accurately determine the diagnostic and

IV. Neuroendocrine differentiation in PIN Neuroendocrine differentiation is also present in PIN, a precursor of prostatic carcinoma. Bostwick et al, (1994) determined the extent of neuroendocrine differentiation in high-grade PIN by examining the immunohistochemical expression of 10 markers in 26 radical prostatectomy specimens with PIN and adenocarcinoma. At least one of the markers was present in 88% of cases of PIN and 92% of carcinoma. Serotonin, NSE, chromogranin, and human chorionic gonadotropin (hCG) were expressed in all nonneoplastic epithelial cells with levels significantly greater than in PIN and cancer. They concluded that neuroendocrine differentiation is down regulated in prostatic carcinogenesis, with intermediate levels of

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Cancer Therapy Vol 3, page 389 prognostic significance of these markers during various stages of the disease and its response to different therapeutic modalities.

VI. Prognostic significance neuroendocrine differentiation

Recently a consensus panel of the College of American Pathologists determined that there is no demonstrated clinical usefulness of evaluating neuroendocrine differentiation in prostate adenocarcinoma (Bostwick et al, 2000).

of

VII. Neuroendocrine differentiation in hormone refractory prostate cancer

There have been conflicting reports regarding the predictive value of neuroendocrine differentiation in prostate cancer. Although it appears that neuroendocrine differentiation is present at least focally in all cases of prostatic adenocarcinoma, reports of the percent of tumors containing NE differentiation vary from 24% to 99% in radical prostatectomy specimens (Aprikian et al, 1993; Theodorescu et al, 1997; Abrahamsson, 1999b; Bostwick et al, 2002). Bostwick et al, (2002) comprehensively reviewed published reports of the predictive value of NE differentiation by immunohistochemistry in patients with adenocarcinoma of the prostate. According to most reports neuroendocrine cells have no apparent clinical or prognostic significance in benign epithelium, primary prostate cancer or lymph node metastases. Allen et al (1995) examined 120 prostate tumors from different stages and reported no association with cancer grade, stage or survival. Noordzij et al (1995) studied NE differentiation in 90 patients who underwent radical prostatectomy (stages T2-T4) for prostate cancer. They found NE differentiation was not associated with Gleason sum, pathological stage or cancer specific death. Abrahamson et al, (1998) evaluated 87 patients with clinically localized prostate cancer who underwent radical prostatectomy and found no correlation between NE differentiation and disease progression with a mean follow-up of 4.2 years.

Bostwick et al, (2002) determined the expression of chromogranin A and serotonin in 196 patients with lymph node positive prostate cancer. They found the greatest number of neuroendocrine cell in benign prostate epithelium with less expression in primary cancer and lymph node metastases. There was no significant association of chromogranin A expression with cancer specific or all cause survival, while serotonin expression was associated with cancer specific but not all cause survival. Aprikian et al, (1993) found no correlation of neuroendocrine differentiation with pathological stage or metastases.

Conversely Weinstein et al, (1996) studied 104 patients with clinically localized prostate cancer with a mean follow-up of 8 years and found that neuroendocrine differentiation was associated with patient survival. Cohen et al, (1990) found that neuroendocrine cells were an independent prognostic variable in prostate cancer.

However, they examined only a small number of cases, and did not control for other predictive factors.

Theodorescu et al, (1997) showed that neuroendocrine differentiation predicted patient survival in 71 patients with stages T1-2 prostate cancer but only on a univariate or multivariate analysis of 1 variable. Krijnen et al, (1997) reported that neuroendocrine cell density along with Gleason score was an independent prognostic factor for cancer progression in 72 transurethral prostate resection detected cancers followed by androgen deprivation.

Androgen deprivation has been shown to increase the number of NE cells in prostate tissue (Krijnen et al, 1997; Ahlgren et al, 2000; Hvamstad et al, 2003). A number of independent studies have shown that the NE cells are androgen receptor negative(Krijnen et al, 1993), thus androgen ablation therapy may lead to preferential growth of these cells, which would account for their increased concentration in hormone-refractory tumors. NE cells themselves are non-proliferative and exert their effects through secretion of neuropeptides. It has been suggested that NE cells contribute to the development of androgenindependent prostate cancers through the paracrine effects of growth factors (Abrahamsson, 1999b). This has led some investigators to conclude that NE cells promote aggressive, hormone-independent growth of prostate cancer, however sound evidence to support this theory is lacking.

It is well documented that withdrawal of androgen causes androgen-dependent prostate cancer cells to transdifferentiate into the NE phenotype in vitro and in vivo (Ismail et al, 2002; Wright et al, 2003). Ismail et al, (2002) showed that NE cell densities were within the same range in normal and hyperplastic dog prostates but that it significantly increased following castration. This transdifferentiation was reversible with the addition of androgens and estrogens. A number of studies have shown an induction of NE differentiation following neoadjuvant hormonal treatment of patients undergoing radical prostatectomy or transurethral resection of the prostate (Iwamura et al, 1994a; Van de Voorde et al, 1994; Guate et al, 1997; Pruneri et al, 1998). Ahlgren et al, (2000) performed immunohistochemical (IHC) analysis on 103 specimens of patients on a clinical protocol who were randomized to neoadjuvant LH-RH analogue versus radical prostatectomy alone. While NE cells were statistically more abundant in hormone-manipulated prostates, this was not associated with a difference in the degree of cancer regression or tumor-cell proliferation in response to treatment.

Wright et al, (2003) suggested that activation of the NE transdifferentiation process represents an early response to androgen receptor inactivation induced by androgen withdrawal. They showed that AR silencing induces a NE phenotype in both androgen-dependent LNCaP and androgen-independent LNCaP-AI human prostate cancer cells. Neuroendocrine differentiation in prostate cancers appears to increase with time during androgen withdrawal therapy (Jiborn et al, 1998; Hirano et al, 2004) although this has not been proven in a prospective manner in vivo. In the androgen-dependent human prostate cancer xenograft PC-310 cell line, androgen deprivation causes time-dependent NE differentiation (Jongsma et al, 2002). In the CWR22 389

Daneshmand et al: Neuroendocrine differentiation in prostate cancer human prostate cancer model, androgen deprivation induces a significant increase in tumor-associated NE cells and this precedes the increase in tumor cell proliferation signaling a recurrence (Huss et al, 2004). This has implications on the selection of androgen-independent tumor cells and subsequent progression of prostate cancer.

It is as yet unclear whether the increase in NE differentiation can be attributed to the hormone refractory nature of the tumor or just long-term androgen deprivation.

Some published evidence suggests a suppressive effect of NE cells on prostate cancer cells. Bostwick et al, (2002) found only 37.5% of lymph node metastases had any cells which expressed NE markers, with an average of 2.2% of total cells staining positive, compared with 98.5% of primary tumors, in which an average of 6% of cells stained positive. Similarly, Roudier et al, (2003) analyzed multiple bone metastases of 14 prostate cancer patients and found that in the majority of bone metastases, fewer than 1% of cells expressed chromogranin A. This suggests that loss of NE differentiation may facilitate metastasis of prostate cancer cells.

relationship between NE cells and androgen-independent prostate cancer.

Recent work from our laboratory has shown that NE cells may in fact have an inhibitory effect on PCA cells. In our cell culture studies, the proliferation of 3 PCA cell lines tested (LNCaP, PC-3 and DU-145) was significantly inhibited when exposed to IL-6-induced NE cell conditioned medium or NE cell co-culture. These results imply that NE cells may be releasing inhibitory factors, which could dominate the mitogenic effects of growth factors characteristically associated with NE cells, such as bombesin or neurotensin (Wang et al, 2004b). In advanced PCA, increased NE differentiation might reflect a response of some cancer cells to changes in the microenvironment, such as increased release of IL-6 from osteoblasts, resulting in tumor growth inhibition rather than tumor progression. IL-6 treatment causes G0 arrest through the induction of the cyclin-dependent kinase inhibitor p27.

The paradox that these patients will eventually die due to progression of this disease despite enhanced NE differentiation, suggests that the suppressive effect of NE cells on the proliferation of surrounding PCA cells might delay advancement of PCA but is not potent enough to completely prevent tumor expansion and cancer spread.

Additional support for this hypothesis would be derived by documenting an increased amount of apoptosis in areas of prostate cancer with high-density NE expression compared to areas with few NE cells. To our knowledge, such estimation has not as yet been reported.

Fixemer et al, (2002) reported that in 18 radical prostatectomy specimens apoptosis in NE cells was an extremely rare event, and the overall number of NE cells in a specimen did not correlate with the overall number of apoptotic cells, however they did not evaluate tumor cells in the immediate vicinity of NE cells for apoptosis, which is imperative given the likelihood of paracrine rather than endocrine activity of pro-apoptotic signals (Aprikian et al, 1993).

Other experiments have suggested that select compounds exert their inhibition of prostate cancer proliferative activity through NE transdifferentiation.

Melatonin, the main secretory product of the pineal gland has been shown to dramatically reduce the number of prostate cancer cells and stop cell cycle progression in both LNCaP and PC3 cells and promote neuroendocrine differentiation (Sainz et al, 2004). Jolkinolide B, a compound found in Euphorbia fischeriana, a Chinese herbal medicine reported to possess chemotherapeutic effects has potent anti-proliferative activity in LNCaP cells in part by inducing G1 arrest and neuroendocrine differentiation (Liu et al, 2002).

The majority of investigators argue that NE differentiation in prostate carcinoma correlates with poor prognosis, tumor progression, and androgen-independence (Abrahamsson, 1999a; di Sant'Agnese, 2001; Bonkhoff and Fixemer, 2004). NE differentiation exclusively occurs in the G0 phase of the cell cycle and thus NE cells are resistant to radiation therapy and cytotoxic drugs (Bonkhoff and Fixemer, 2004). In addition, NE tumor cells are also resistant to apoptosis (Vanoverberghe et al, 2004). NE cells themselves do not proliferate and

VIII. In vitro models of neuroendocrine differentiation Although the origin of the neuroendocrine cells is uncertain, a number of recent publications have demonstrated that prostate cancer cells can transdifferentiate into a neuroendocrine phenotype in vitro (Bang et al, 1994; Qiu et al, 1998; Burchardt et al, 1999; Zelivianski et al, 2001; Horiatis et al, 2004). The androgen sensitive human prostate cancer cell line, LNCaP, has been documented to transdifferentiate into a NE phenotype in response to IL-6, increasing levels of intracellular cyclic AMP, or following culture in steroid-deprived medium (Abrahamsson, 1999b; Cox et al, 1999; Cox et al, 2000; Spiotto and Chung, 2000). This transformation is reversible within a few hours of treatment with cAMPinducing agents, such as forskolin and epinephrine.

However, treatment with 7-10 days of continuous IL-6 leads to permanent NE transdifferentiation (Wang et al, 2004b). The observation that conditions associated with transformation of PCA cells in vitro, such as androgen deprivation or exposure to IL-6 also increase NE differentiation in prostatic tumors in vivo (Jongsma et al, 1999, 2002; Wang et al, 2004b), suggest that NE cells might be derived from adenocarcinoma cells by a process of transdifferentiation.

NE cells may in fact represent terminal differentiation of hormone refractory prostate cancer cells to a less malignant form. Hsieh et al (1995) studied the growth of LNCaP cells when cultured in lymphocyte conditioned medium (LCM). Prostate cancer cells in LCM acquired NE differentiation and their growth slowed, with cells halted in G1 rather than progressing further in the cell cycle into S phase. The induced differentiation was associated with ultimate termination of cells, rather than proliferation, even though androgen receptor expression was down regulated and PSA secretion decreased. These provocative results suggest there is more to learn about the

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Cancer Therapy Vol 3, page 391 represent and immortal cell population within the prostate and are thought to exert their mitogenic effects through paracrine interactions. In vitro systems may not accurately reflect the behavior of prostate cancer cells in vivo and animal models are necessary to elucidate the role of neuroendocrine differentiation in prostatic tumor progression.

significantly higher after castration. Androgens as well as estrogens given after castration restored NE cell density back to normal values. In human, the density of serotoninpositive NE cells was also significantly higher in benign glands after androgen ablation in prostate cancer patients subjected to androgen ablation prior to prostatectomy.

More animal models are needed to investigate the role of NE differentiation in prostate cancer. Experiments aimed at inducing NE differentiation in an animal model can be used to test the influence of these cells in a prostate carcinogenesis model.

IX. In vivo models of neuroendocrine differentiation Much of the uncertainty about the pathogenic significance of NE differentiation derives from a paucity of reliable animal models. These cells are not common in the normal human prostatic epithelium and even rarer in the mouse prostate. In 1996, Noordzij et al established 2 prostate cancer xenograft models that contained NE cells.

They showed that short-term androgen withdrawal resulted in a rapidly increased number of NE cells and suggested that this increase occurred by induction of NE differentiation rather than by rapid proliferation and subsequent differentiation or selective persistence. Since then other cell line xenograft and transgenic mouse models for neuroendocrine prostatic carcinoma have been described (di Sant'Agnese, 1998). Uchida et al, (2004) have established an immortalized cell line designated NECS, developed from an NE mouse prostate allograft (NE10) that has characteristics of NE cells in vitro. When inoculated subcutaneously into athymic mice the NE-CS cells formed tumors with the NE phenotype and exhibited accelerated growth compared to the original NE-10 allograft. Other investigators have found that in castrated mice bearing both LNCaP and NE-10 tumors, LNCaP tumors continue to grow, and have increased levels of nuclear AR, as opposed to the decrease in growth seen after castration in mice bearing LNCaP tumors alone (Jin et al, 2004). Hu et al, (2004) have established prostate NE cancer cell lines from CR2-TAg prostate tumors and metastases and have used GeneChip analyses to reveal factors that enhance survival by inhibiting apoptosis.

Although these are useful models to understand the molecular mechanisms of NE tumors, they may not accurately reflect the role of benign NE cells seen in prostate cancers.

Conversely we have recently shown that IL-6 can significantly inhibit the growth of LNCaP xenografts in nude mice by the process of NE differentiation. IL-6 treatment results in G0 arrest in over 90% of cells and NE differentiation with augmentation of neuron-specific enolase and Ă&#x;III tubulin. In prostate cancer, clones of IL-6 producing cells could induce paracrine NE differentiation of some surrounding cancer cells. Thus, enhanced tumor NE differentiation may not only indicate the presence of aggressive cancer cells but also reflect a suppressive and protective response of IL-6 transformable cancer cells to an aggressive tumor behavior.

Transdifferentiation into an NE phenotype does not necessarily require the presence of carcinoma. In an experiment to verify the presence of NE cells in the dog prostate and test their hormonal regulation, Ismail et al, (2002) showed that NE cell densities were within the same range in normal and hyperplastic dog prostates but

X. Small cell (neuroendocrine) carcinoma of the prostate Primary small cell carcinoma of the prostate is uncommon and is usually discovered incidentally coexisting with adenocarcinomas. Pure small cell carcinoma of the prostate is an extremely rare occurrence and is a highly aggressive tumor. There is typically no associated elevation of PSA, making early diagnosis difficult (Ro et al, 1987; Nadig et al, 2001). Several theories have been proposed to describe their origin. One theory suggests that small cell carcinomas of the prostate arise from amine precursor uptake decarboxylation cells of local endodermal origin. Another theory proposes that they arise from dedifferentiation of prostatic adenocarcinomas, suggesting that small cell carcinomas are part of a spectrum of prostatic adenocarcinomas rather than a separate disease entity (Sandhu et al, 1997; Yashi et al, 2002). Because of the histologic similarities between prostate and lung small cell carcinomas and the occurrence of similar neuroendocrine paraneoplastic syndromes, the most widely accepted view is that prostatic small cell carcinomas arise from totipotential stem cells of the prostate, which have the ability to differentiate into either epithelial or neuroendocrine type carcinomas (Rubenstein et al, 1997).

Whereas mixed small cell carcinomas and adenocarcinomas usually are aggressive recurrences of a primary adenocarcinoma, pure small cell carcinoma of the prostate often is associated with early metastatic disease because of its aggressive nature. Like adenocarcinomas, small cell prostate cancers arise in the periphery of the prostate gland and hence can occur without urinary symptoms. The disease has a propensity to metastasize to visceral organs, including the liver, bone, lungs, central nervous system, and pericardium, and regionally to the pelvic lymph nodes, rectum, and bladder. In addition, small cell prostate cancers have been reported to produce paraneoplastic syndromes associated with the production of adrenocorticotrophic and antidiuretic hormones. (Tetu et al, 1989; Kawai et al, 2003) Despite treatment with chemotherapy, the prognosis of small cell prostate cancer is extremely poor, and the median survival is 7 months (Rubenstein et al, 1997).

Because of the rarity of the condition, no standard therapeutic regime has been developed. Small cell carcinomas of the prostate are generally unresponsive to hormone therapy. Reported cases have generally been managed by chemotherapeutic regimens similar to those recommended for small cell lung cancer. Small case 391

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Bonkhoff H, Stein U and Remberger K (1993) Androgen receptor status in endocrine-paracrine cell types of the normal, hyperplastic and neoplastic human prostate.

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Bostwick DG, Dousa MK, Crawford BG and Wollan PC (1994) Neuroendocrine differentiation in prostatic intraepithelial neoplasia and adenocarcinoma. Am J Surg Pathol 18, 12406.

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Bostwick DG, Qian J, Pacelli A, Zincke H, Blute M, Bergstralh EJ, Slezak JM and Cheng L (2002) Neuroendocrine expression in node positive prostate cancer: correlation with systemic progression and patient survival. J Urol 168, 120411.

Burchardt T, Burchardt M, Chen MW, Cao Y, de la Taille A, Shabsigh A, Hayek O, Dorai T and Buttyan R (1999) Transdifferentiation of prostate cancer cells to a neuroendocrine cell phenotype in vitro and in vivo. J Urol 162, 1800-5.

Casella R, Bubendorf L, Sauter G, Moch H, Mihatsch MJ and Gasser TC (1998) Focal neuroendocrine differentiation lacks prognostic significance in prostate core needle biopsies. J Urol 160, 406-10.

Cockett AT, di Sant'Agnese PA, Gopinath P, Schoen SR and Abrahamsson PA (1993) Relationship of neuroendocrine cells of prostate and serotonin to benign prostatic hyperplasia. Urology 42, 512-9.

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Cox ME, Deeble PD, Bissonette EA and Parsons SJ (2000) Activated 3',5'-cyclic AMP-dependent protein kinase is sufficient to induce neuroendocrine-like differentiation of the LNCaP prostate tumor cell line. J Biol Chem 275, 13812-8.

Cox ME, Deeble PD, Lakhani S and Parsons SJ (1999) Acquisition of neuroendocrine characteristics by prostate tumor cells is reversible: implications for prostate cancer progression. Cancer Res 59, 3821-30.

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reports have shown poor responses to etoposide and cisplatin or cyclophosphamide (Debras et al, 1994; Steineck et al, 2002).

XII. Conclusion Although much has been learned about the distribution and expression of NE cells in the prostate, we are still far from understanding the precise role of these cells in carcinogenesis. Studies have delineated the molecular pathways leading to neuroendocrine differentiation and many of the factors produced by the NE cell have been well characterized. More animal models are needed to clarify the role of these cells within the prostate and the effect of the released factors on neighboring cells. There is some controversy regarding the role of these cells in carcinogenesis and studies should focus on the mechanisms by which these cells interact with normal and/or malignant prostate cells.

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Jacek Pinski

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Spontaneous ovarian hyperstimulation syndrome caused by hypothyroidism Case Report

Azam Sadat Mousavi*, Nadereh Behtash, Malihe Hasanzadeh, Mitra Modares Gilani, Fatemeh Ghaemmaghami, Encie Shahroch, Tehrani Nejad Department of Gynecology Oncology, Reproductive Health Research center, Vali-e-Asr Hospital, Tehran University of Medical Sciences, Tehran, Iran.

__________________________________________________________________________________ *Correspondence: Azam Sadat Mousavi, Gynecology-Oncology Department, Vali-e-Asr Hospital, Imam Khomeini Hospital, Keshavarz Blvd., Tehran 14194, Iran.; Phone: # 98-21-723430, 773330, 6937766, 6930666; Fax: # 98-21-7880161, 8504404, 6937321, 6937766; E-mail: a3064@sina.tums.ac.ir, valrec2@yahoo.com Key words: Abbreviations: ovarian hyperstimulation syndrome, (OHSS); vascular endothelial growth factor, (VEGF) Received: 16 May 2005; Revised: 10 June 2005 Accepted: 13 June 2005; electronically published: July 2005

Summary Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication of assisted reproductive technology conception of unknown pathogenesis. Spontaneous ovarian hyperstimulation has been reported in women with hypothyroidism, polycystic ovary syndrome, pregnancy, gonadotroph pituitary adenoma. To our knowledge only four cases of spontaneous OHSS have been described in non pregnant women with primary hypothyroidism. A 26year-old primiparous presented with abdominal pain, myxedematous facies and bilateral multiseptated ovarian cysts. Thyroid stimulating hormone was elevated and total thyroxin and T3 and free thyroxin index and T3 resin uptake were low. Hypothyroidism associated with spontaneous ovarian hyperstimulation was confirmed. She had sever abdominal pain. We performed aspiration of cysts. Abdominal pain disappeared. We also started levothyroxin. After six months, follow up serial sonography is normal. Hypothyroidism can be associated with ovarian hyperstimulation. We must treat these patients with conservative management.

I. Introduction

II. Case report

Ovarian hyperstimulation syndrome usually occurs in association with ovulation induction, but the physiopathologic mechanisms are understood poorly (Mcelhinney and Mcclure, 2000). Spontaneous ovarian hyperstimulation has been reported in women with hypothyroidism (Rotmonsch and Scommegna, 1989; Van Voorhis et al, 1994; Hansen et al, 1997; Nappi et al, 1998; Cardoso et al, 1999), polycystic ovary syndrome and pregnancy (Zalel et al, 1995), gonadotroph pituitary adenoma and pregnancy (Shimon et al, 2001). A med line search using the key words spontaneous ovarian stimulation, hypothyroidism revealed only four cases, one of whom had Down syndrome (Cardoso et al, 1999).

The causal relationship between hypothyroidism and ovarian hyperstimulation is suggested by the consistent regression of the ovarian cysts after the institution of thyroid hormone replacement therapy (Taher et al, 2004).

This paper presents one new cases of OHSS and primary hypothyroidism. In Feb 2004, a 26-year-old woman (primiparous) was referred to Gynecology oncology unit in Vali-e-Asr hospital, Tehran, Iran. Upon her visit in our clinic, she had abdominal pain in lower quadrant. In physical exam, there were ascitis and bilateral adenexal, tender masses. Pelvic and abdominal sonography (Figure 1) showed bilateral multiseptated ovarian masses: the left measuring 66x63x99mm and the right 69x63x96 mm. Liver, spleen, pancreas and both kidneys were normal. There was no para-aortic lymph node enlargement and a normal urinary bladder and uterus.

Bilateral pleural effusion (Right more than left) were noted on chest x-ray, CA 125 was 81 Iu/ml (normal<35u/ml), another tumor markers were negative.

Primary diagnosis was ovarian cancer. In past medical history, she had hypothyroidism, nine years ago. She interrupted her treatment six years ago. Hormonal studies

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confirmed hypothyroidism with TSH>50 miu/lit (0.2-5.1 miu/lit), total T4 0.3 mcg/dl (4.5-12.5 mcg/dl), T3 resin uptake 20% (25-35), free thyroxin index 0.1 (1.4-4.4), T3 0.4 ng/ml (0.7-2.1 ng/ml).

She was started on levothyroxin 100 mcg per day. Due to abdominal pain, aspiration of ovarian cysts was done under ultrasonographic guidance. Cytology of specimen was negative.

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Cancer Therapy Vol 3, page 399 Six months after starting thyroxin replacement and aspiration, serial follow up sonography show normal size her ovaries.

Transvaginal aspiration of follicular structures should be considered to interfere with the intra ovarian mechanism responsible for the clinical picture. laparatomy should be avoided in these precarious patients.

The key point is that the hyper stimulation syndrome will undergo gradual resolution within several months.

III. Discussion Our patient presented with acute severe abdominal pain and was found to have bilateral large complex ovarian masses, mimicking ovarian cancer. CA125 was elevated, she was also found to have pleural effusion.

Early diagnosis was ovarian cancer. Due to past medical history of hypothyroidism, hormonal studies performed.

Thyroid hormonal studies confirmed primary hypothyroidism.

The exact mechanism by which ovarian hyperstimulation might occur in hypothyroid patients is not understood clearly. A possible explanation was suggested by Rotmensch and Scommegna, on the basis of preferential formation of estriol in hypothyroid patients, Estriol is a weaker suppressor of gonadotropin release than estradiol and there is excessive gonadotropin release (Rotmonsch and Scommegna, 1989).

Grumbach explained another explanation, on the basis of low levels of thyroid hormone. It might activate the release of FSH and LH besides the targeted activation of TSH release (Grumbach and Styne, 1998). Another explanation of this rare association is that TSH has weak FSH activity on FSH receptors causing gonadal stimulation (Anasti et al, 1995).

It has been suggested that follicular aspiration offers partial protection against the hyperstimulation syndrome (Speroff et al, 1999).

Abu-louz reported one case of hyperstimulation syndrome was associated with pregnancy, aspiration of large superficial ovarian cyst was done under ultrasonographic guidance and laparatomy avoided (AbuLouz et al, 1997).

Ovarian hyper stimulation can be life-threateniy. The ovaries are tremendously enlarged with multiple follicular cysts, stromal edema, and many corpora lutea. Because of this enlargement, torsion of the adnexa is a relatively Common complication of this Syndrome. It might be expected that the mild type would be relatively common.

The genesis of the ascites is unclear. The very high level of estrogen secretion by the ovaries may be the primary factor, inducing increased local capillary permeability and leakage of fluid from the peritoneal capillaries as well as the ovaries.

A growing body of evidence implicates vascular endothelial growth factor (VEGF) in the pathophysiology of the hyper stimulation syndrome. The origin of VEGF is presumed to be the ovarian follicle, and increased capillary permeability and the severity of the syndrome are correlated with circulatory levels of VEGF. Other cytokines, especially the interlukin family is also believed to be involved in the permeability changes, and it is the hypothesized that these agents effect the nitric oxide system.

Aspiration of ascites can also be accomplished transvaginally (with ultrasound guidance).

IV. Conclusion This case suggests a possible relationship between spontaneous ovarian hyperstimulation and primary hypothyroidism. Thyroid hormone replacement seems to be the best therapeutic approach in such patients; thyroid function should be measured in women with spontaneous hyperstimulation ovaries. We must mention that t the hyperstimulation syndrome will undergo gradual resolution with times and laparatomy should be avoided in these patients. Follicular aspiration is one option of conservative management in acute condition.

The key point is that theovarian hyperstimulation syndrome in hypothyroid patients may mimic ovarian tumors.

References Abu-Louz SK, Ahmad AA, Swan RW (1997) Spontaneous ovarian hyperstimulation syndrome with pregnancy. AM J Obstet Gynecol 177, 476-7.

Anasti J, Flack M, Froehlich L, Nelson M, Nisula B (1995) A potential novel mechanism for precocious puberty in juvenile hypothyroidism. J Clin Endocrinol Metab 80, 276-9.

Cardoso CG, Graca LM, Dias T, Clode N, Soares L (1999) Spontaneous ovarian hyperstimulation and primary hypothyroidism with a naturally conceived pregnancy.

Obstet Gynecol 93, 809-11 .

Grumbach M, Styne D (1998) Williams text book of endocrinology. Wilson JD, Foster DW, Kronenberg HM, Larsen PR, editors. London: saunders, p:1593.

Hansen KA, Tho SP, Hanly M, Moretuzzo RW, Mcdonogh PG (1997) Massive ovarian enlargement in primary hypothriodism. Fertile Steril 67, 196-71.

Mcelhinney B, Mcclure N (2000) Ovarian hyperstimulation syndrome. Baillieres Best Pract Res Clin Obstet Gynaecol 14, 103-122.

Nappi RG, Dinaro E, Dâ&#x20AC;&#x2122;Aries AP, Nappi L (1998) Natural pregnancy in hypothriod woman complicated by spontaneous ovarian hyperstimulation syndrome. AM J Obstet Gynecol 178, 610-1.

Rotmonsch S, Scommegna A (1989) Spontaneous ovarian hyperstimulation syndrome associated with hypothyroidism.

Am J Obstet Gynecol 160, 1220-2.

Shimon I, Rubinek T,Bar-Hava I, Nass D, Hadani M, Amsterdom A, et al (2001) Ovarian hyperstimulation without elevated serum estradiol associated with pure Folliclestimulating hormone-secreting pituitary adenoma. J Clin Endocrinol Metab 86, 3635-40.

Speroff L, Glass RH, Kase NG (1999) Clinical Gynecologic endocrinology and infertility. Sixth edition. Lippincott Williams and Wilkins, Philadelphia, 30, 1115-1116.

Taher BM, Ghariabeh RA, Jarrah NS, Hadidy AM, Radaideh AM, Ajlouni KM (2004) Spontaneous ovarian hyperstimulation syndrome caused by hypothyroidism in an adult. Eur J Obstet Gynecol Reprod Biol 112, 107-109.

Van Voorhis BJ, Neff TW, Syrop CH, Chapler FK (1994) Primary hypothyroidism associated with multicystic ovaries and ovarian torsion in an adult. Obstet Gynecol 83, 885-7.

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Mousavi et al: Spontaneous OHSS Zalel Y, Orvieto R, Ben â&#x20AC;&#x201C;Rafael Z, Homburg R, Fisher O, Insler V (1995) Recurrent spontaneous ovarian hyperstimulation

syndrome associated with polycystic ovary syndrome.

Gynecol Endocrinol 9, 313-5.

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Cancer Therapy Vol 3, page 401 Cancer Therapy Vol 3, 401-406, 2005

Progress in prostate cancer research: a focus on bone health Review Article

Susan Doyle-Lindrud1,* and Robert S. DiPaola1,2 1

Department of Medicine, University of Medicine and Dentistry- Robert Wood Johnson Medical School, New Brunswick New Jersey, USA. 2 The Dean and Betty Gallo Prostate Cancer Center at The Cancer Institute of New Jersey, New Brunswick New Jersey, USA

__________________________________________________________________________________ *Correspondence: Susan Doyle-Lindrud, MS, NPC, The Dean and Betty Gallo Prostate Cancer Center, The Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, New Jersey 08901; Phone: (732) 235-6988; Fax: (732) 235-8095; E-mail: Lindrusm@umdnj.edu Key words: prostate cancer, Clinical studies of bisphosphonates, Potential recommendations, Abbreviations: androgen deprivation therapy, (ADT); bone mineral density, (BMD); bone progression free survival, (BPFS); confidence interval, (CI); dual energy x-ray absorptiometry, (DEXA); hazard ratio, (HR); hormone refractory prostate cancer, (HRPC); quantitative computed tomography, (QCT); World Health Organization, (WHO) Received: 15 March 2005; Revised: 24 June 2005 Accepted: 27 June 2005; electronically published: July 2005

Summary One in six men will be diagnosed with prostate cancer in their lifetime; in this year, approximately 200,000 will be diagnosed with, and 30,000 will die of prostate cancer (Hemminki et al; Jemal et al, 2004). Recent advances reported this past year in the therapy of prostate cancer include data demonstrating the survival benefit of chemotherapy in metastatic hormone refractory prostate cancer (HRPC) (Gulley and Dahut, 2004). Despite these successes, many questions about the management of prostate cancer need to be answered. For example, further studies are needed to determine the value of chemotherapy earlier in the progression of disease, the value of chemopreventive approaches, and efforts to decrease morbidity of the disease and current therapies. This review will focus on recent and ongoing studies with bisphosphonates in prostate cancer. prostate cancer may increase the risk of fracture (Shahinian et al, 2005). Recent data also demonstrates that treatment with bisphosphonates may improve bone density in men without metastatic disease and decrease skeletal related events in men with hormone refractory bone metastasis (Saad, 2002; Smith et al, 2003). Despite possible benefits of bisphosphonates, specific guidelines on the use of bisphosphonates in patients with nonmetastatic disease, to decrease bone loss, or metastatic disease, to decrease skeletal events are unclear.

I. Introduction Recent data supports a potential role of bisphosphonates to decrease bone complications of androgen ablation therapy in patients with non-metastatic prostate cancer and to decrease skeletal problems in patients with metastatic prostate cancer. The primary treatment for metastatic prostate cancer and locally advanced non-metastatic prostate cancer is androgen deprivation therapy (ADT). This is usually obtained through an orchiectomy or by treatment with a gonadotropin-releasing hormone agonist. Studies reveal that adjuvant androgen deprivation therapy improves survival for men with locally advanced prostate cancer treated with radiation therapy and for men with lymph node positive prostate cancer treated with radical prostatectomy and pelvic lymphadenectomy (Bolla et al, 2002; Messing et al, 2002). An unfortunate complication of such therapy is a decrease in bone mineral density. A recent study by Shahinian, et al in The NEJM demonstrated that androgen-deprivation therapy for

II. Clinical studies of bisphosphonates in non-metastatic prostate cancer Multiple studies have demonstrated that androgen ablation therapy represents an important risk factor for osteoporosis in men (Barrass et al, 2004). This effect to decrease bone density in men on androgen ablation therapy has occurred within 6 months (Daniell et al, 2000).

The significance of bone loss in patients on androgen

401

Doyle-Lindrudan and DiPaola: Progress in prostate cancer research in New Jersey ablation therapy, without metastasis, has also been documented. Increased skeletal fractures have been associated with the use of androgen ablation therapy and decreased bone mineral density (BMD) (Melton et al, 2003; Diamond et al, 2004; Krupski et al, 2004).

Shahinian, et al reviewed the records of 50,613 men who were linked in the database of the Surveillance, Epidemiology, and End Results program and Medicare as having received a diagnosis of prostate cancer between 1992 through 1997 and being at least 66 years of age.

Comparisons were limited to men who received at least one dose of a gonadotropin-releasing hormone agonist or underwent an orchiectomy within 6 months after receiving the diagnosis with those with prostate cancer who received neither type of treatment at any time after diagnosis. The primary outcomes were the occurrence of any fracture and the occurrence of a fracture resulting in hospitalization.

The review demonstrated that 19.4 percent of those who received androgen deprivation therapy (ADT) had a fracture, as compared with 12.6 percent of those not receiving ADT (P<0.001) (Shahinian et al, 2005).

Multiple studies have now demonstrated improved bone mineral density with bisphosphonate therapy. For example, a study by Diamond et al. (1998) looked at markers of bone turnover and bone mineral density in men with disseminated prostate cancer treated with combined androgen blockade prior to and after 6 months of intermittent cyclic etidronate and calcium supplementation. The results of this study show that after treatment with etidronate, a significant increase in BMD was observed in the femoral neck and lumbar spine, concluding that adjuvant therapy with intermittent cyclic etidronate may prevent the high bone turnover and decrease the risk of spinal fractures. A second study involving patients with locally advanced, lymph node positive or recurrent prostate cancer and no bone metastases were randomly assigned to treatment with either a 22.5mg IM depot leuprolide injection and 60mg i.v. pamidronate every 12 weeks versus 22.5mg IM depot leuprolide injection alone (Smith et al, 2001). The results revealed that the men receiving the depot leuprolide injection alone had a decrease in bone mineral density in the lumbar spine and hip. In contrast, the bone mineral density did not change significantly in the men treated with depot leuprolide and i.v. pamidronate.

A recent study looked at a third generation bisphosphonate, zoledronic acid in the prostate cancer population (Smith et al, 2003). This study involved a randomized controlled trial of zoledronic acid to prevent bone loss in men receiving androgen deprivation for nonmetastatic prostate cancer. Zoledronic acid at a dose of 4mg i.v. was given every 3 months for one year. Results demonstrated that men receiving zoledronic acid had an increase in mean bone mineral density in the lumbar spine by 5.6% as compared to a decrease by 2.2% in those given placebo. Mean bone mineral density of the femoral neck, trochanter and total hip also increased in the zoledronic acid group and decreased in the placebo group. In summary, these data suggest that bisphosphonates may reduce the bone loss associated with ADT in men without metastasis, but the effect of bisphosphonates to decrease

rates of fracture is unclear and needs further study.

III. Clinical studies of bisphosphonates in metastatic prostate cancer Clinical studies have also assessed the role of bisphosphonates in men with metastatic prostate cancer to bone; randomized phase III studies have included the assessment of clodronate, pamidronate and zoledronic acid. For example, a double blind, placebo controlled, randomized trial was completed (Dearnaley et al, 2003) to determine whether the first generation bisphosphonate sodium clodronate improved bone progression-free survival times among men with bone metastases from prostate cancer. Between 1994 and 1998, 311 men who were started or responding to first line hormone therapy for bone metastases were randomly assigned to receive oral sodium clodronate or placebo for a maximum of three years. The primary endpoint was symptomatic bone progression free survival (BPFS). Secondary endpoints included overall survival, treatment toxicity and change in World Health Organization (WHO) performance status.

After a median follow up of 59 months, the sodium clodronate group was reported to have a better symptomatic BPFS, but this was not statistically significant (hazard ratio (HR) =0.79, 95% confidence interval (CI)=0.61 to 1.02; P=.066). Patients in the clodronate group were less likely to have worsened WHO performance status (HR+ 0.71, 96% CI= 0.56 to 0.92; P=.008). The clodronate group did experience more gastrointestinal problems and increased LDH levels and required more frequent modification of the trial drug dose.

Results of subgroup analyses suggested that clodronate might be more effective if started earlier after diagnosis of metastatic bone disease. These results suggest that further studies are needed to determine a benefit of bisphosphonates on BPFS in men with metastatic prostate cancer that are responding to ADT. In contrast to studies on this group of patients with metastasis responding to ADT, studies of patients with metastatic disease and progression on ADT have more definitive conclusions. For example, a study randomly assigned patients with HRPC and metastasis to treatment with i.v. zoledronic acid at 4mg, zoledronic acid at 8mg (subsequently reduced to 4mg) or placebo every 3 weeks for 15 months. Skeletal related events, time to first SRE, skeletal morbidity rate, pain and analgesic scores, disease progression and safety were assessed. SRE was defined as pathologic bone fracture (vertebral or nonvertebral), spinal cord compression, surgery to bone, radiation to bone (including radioisotopes) or a change of antineoplastic therapy to treat bone. The study demonstrated that a greater proportion of patients who received placebo had an SRE than those who received zoledronic acid at 4mg (44.2 % versus 33.2%) (Saad, 2002; Saad et al, 2002). These data, therefore, demonstrated a benefit to zoledronic acid in patients with metastatic prostate cancer, with hormone refractory disease. This study however does not assess the bone density status of the men and therefore does not clearly differentiate between malignant or osteoporotic 402

Cancer Therapy Vol 3, page 403 fractures. This leaves the question as to whether the decreased risk of fracture was related to osteoporosis or metastatic disease.

IV. Bisphosphonates palliation

and

using a 100-mm visual analog scale(VAS). Patients with prostate cancer experienced a reduction in pain scores from baseline at visit 2 only, although there was never any increase in pain noted on the pain score assessment (Vogel et al, 2004)

pain

V. Antitumor effects

In addition to the important benefit of bisphosphonates on SRE in the prostate cancer population, is the potential benefit of pain palliation from the osseous metastatic disease. Studies thus far have been both positive and negative and further studies will be needed to understand the role of bisphosphonates for pain palliation.

Eastham. et al (2005) looked at the effect of zoledronic acid on bone pain and skeletal morbidity in patients with advanced prostate cancer. 422 patients were enrolled. Zoledronic acid 4mg or placebo was given intravenously every three weeks. Bone pain was assessed using the Brief Pain Inventory (BPI) at 6 week intervals.

Of the 371 evaluable patients, 73% reported a baseline BPI score of 2.8 (range 0-10) in both groups. Among the patients receiving zoledronic acid, mean baseline pain scores of -10%, -4% and â&#x20AC;&#x201C;1% at months 3, 6 and 9 respectively were reported compared with increases of 6%, 9% and 13% from baseline in the placebo group (P=.021 at month 3).

After month 12, both groups had increases in pain scores, although the zoledronic acid treated patients had smaller increases (range of 1% to 6%) compared to placebo (15% to 25%) (Eastham et al, 2005).

Studies not revealing benefit include an analysis of two multicenter, double-blind, randomized, placebocontrolled trials involving patients with bone pain due to metastatic prostate cancer, with disease progression after first-line hormonal therapy. Intravenous pamidronate disodium (90 mg) or placebo was administered every 3 weeks for 27 weeks. Efficacy was measured via selfreported pain score (Brief Pain Inventory), and analgesic use. The results of the two trials were pooled. There were no sustained significant differences between the pamidronate and placebo groups in self-reported pain measurements and analgesic use. The conclusion of this analysis, with inherent limitations of a pooled analysis, was that Pamidronate disodium failed to demonstrate a significant overall treatment benefit compared with placebo in palliation of bone pain (Wong, 2004).

An open-label study conducted in community centers assessed the safety of zoledronic acid 4 mg intravenously over 15 minutes every 3â&#x20AC;&#x201C;4 weeks for a planned six infusions as treatment of bone metastases in patients with multiple myeloma, breast cancer, or prostate cancer with and without previous bisphosphonate exposure. Adverse events (AEs), pain, and quality-of-life (QOL) scores were recorded. Of 638 patients, 415 patients (65%) had received prior bisphosphonate therapy. 102 prostate cancer patients were enrolled. The change from baseline pain score was analyzed using paired t-tests.

Kaplan-Meier estimates were used to assess time to development of pain for patients who reported no pain upon study entry. Pain assessments were conducted at baseline, before each infusion, and at the final study visit

Recent clinical studies have looked at whether bisphosphonates interfere with the growth and survival of metastatic cancer cells in the bone. A study by Lee, et al (2001) looked at the effect of pamidronate and zoledronic acid on the growth and survival of prostate cancer cell lines in vitro. Treatment of PC3, DU145 and LNCaP cells with pamidronate or zoledronic acid significantly reduced the growth of all three cell lines. Pamidronate was shown to induce cell death in all three lines studied (Lee et al, 2001).

A second study by Dumon et al looked at the biological effects of bisphosphonates on cell survival. The study compared four bisphosphonates; clodronate, pamidronate, ibandronate and zoledronic acid. Cell cycle phases were analyzed and apoptotic effects were assessed.

The results revealed that the clodronate exhibited only a slight inhibitory effect on cell growth. In contrast, the aminobisphosphonates decreased cell growth in a time and dose dependent manner (Dumon et al, 2004).

A clinical phase III, double blind study assessing the development of bone metastases from prostate cancer (Mason, ASCO 2004) looked at patients with stage T2-T4 prostate cancer with no evidence of metastatic bone disease. The primary endpoint was time to the development of symptomatic bone metastases or death from prostate cancer. 508 patients were randomized and followed over 3.5 years. Patients were either given oral clodronate or placebo. Results revealed no difference in time to symptomatic bone metastases, death from prostate cancer or overall survival (Mason, 2004).

In summary, the findings on antitumor effect have been evaluated in vitro and will need to be confirmed in clinical trials. This is an area that needs further study and may translate into additional therapeutic regimens in the future.

VI. Ongoing studies Although there is not enough information at the present time to give definitive guidelines for the use of bisphosphonates in all of the stages of prostate cancer, there are ongoing clinical trials. One ongoing study is a CALGB protocol 90202 for metastatic prostate cancer patients, initiating androgen ablation within 3 months of study. It is a randomized, double blind phase III study comparing zoledronic acid 4 mg i.v. every 4 weeks, versus placebo i.v. every 4 weeks. The objectives of this study are to determine whether treatment with zoledronic acid, at the time of initiating androgen ablation therapy for metastatic prostate cancer will delay time to first skeletal related event, and to determine whether treatment with zoledronic acid will decrease the proportion of men with one or more vertebral fractures at two years compared to placebo in

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Doyle-Lindrudan and DiPaola: Progress in prostate cancer research in New Jersey men receiving androgen ablation therapy for metastatic prostate cancer.

A second study, involving the same population, metastatic prostate cancer to bone, commencing androgen ablation is being conducted by the Hoosier Oncology Group. It is a Phase III, Randomized, Double Blind, Placebo Controlled Trial evaluating the ability of risedronate to prevent skeletal related events. Patients were randomized to either daily oral risedronate combined with androgen deprivation or daily oral placebo combined with androgen deprivation. The primary objective of this study was to evaluate a daily oral dose of 30mg risedronate as compared to placebo to prevent skeletal complications in patients undergoing androgen deprivation for metastatic prostate cancer by measuring the time to a skeletal related event. The MRC trial entitled, Systemic Therapy in Advancing or Metastatic Prostate Cancer Evaluation of Drug Efficacy (STAMPEDE) trial is a multicentre international, randomized study, that will assess the safety and efficacy of three drugs, Zoledronic Acid, docetaxel and a cox 2 inhibitor in combination and alone along with ADT and a control arm of ADT. Further studies will also be needed to better define toxicities associated with short or long term use of therapy. For example, a poorly understood complication which may be associated with the use of the intravenously administered bisphosphonates pamidronate and zoledronate is osteonecrosis of the jaw (Greenberg, 2004).

In 2003, Marx described 36 cases of necrotic jaw bone seen in patients receiving intravenous bisphosphonates as part of their cancer therapy. 78% of the cases occurred after dental extractions and 22% were spontaneous. These patients were also receiving chemotherapy drugs and corticosteroids which put into question whether these factors were the cause of the osteonecrosis (Marx, 2003).

In June 2004, Ruggiero, et al performed a retrospective chart review of patients who presented for Oral Surgery service between February 2001 and November 2003 with the diagnosis of refractory osteomyelitis and a history of chronic bisphosphonate therapy. This review revealed 63 cases of osteonecrosis of the jaw; 56% had received an intravenous bisphosphonate for at least 6 months as part of cancer therapy and six were receiving long term oral bisphosphonate therapy for osteoporosis. Most occurred after dental extractions, but some were spontaneous (Ruggiero et al, 2004). Clearly, these retrospective associations need further study.

cancer are less clear at this point and further studies are needed. Likewise, further studies are needed to better understand other potential short term or long term toxicities. Accrual to clinical trials will be critical, and should be encouraged, to better define the use of bisphosphonate therapy in prostate cancer.

References Barrass BJ, Thurairaja R and Persad RA (2004) More should be done to prevent the harmful effects of long-term androgen ablation therapy in prostate cancer. BJU Int 93, 1175-1176.

Bolla M, Collette L, Blank L, Warde P, Dubois JB, Mirimanoff RO, Storme G, Bernier J, Kuten A, Sternberg C, Mattelaer J, Lopez Torecilla J, Pfeffer JR, Lino Cutajar C, Zurlo A and Pierart M (2002) Long-term results with immediate androgen suppression and external irradiation in patients with locally advanced prostate cancer (an EORTC study): a phase III randomised trial. Lancet 360, 103-106.

Daniell HW, Dunn SR, Ferguson DW, Lomas G, Niazi Z and Stratte PT (2000) Progressive osteoporosis during androgen deprivation therapy for prostate cancer. J Urol 163, 181-186.

Dearnaley DP, Sydes MR, Mason MD, Stott M, Powell CS, Robinson AC, Thompson PM, Moffat LE, Naylor SL and Parmar MK (2003) A double-blind placebo-controlled randomized trial of oral sodium clodronate for metastatic prostate cancer (MRC PR05 Trial). J Natl Cancer Inst 95, 1300-1311.

Diamond TH, Campbell J, Bryant C and Lynch W (1998) The effect of combined androgen blockade on bone turnover and bone mineral densities in men treated for prostate carcinoma, longitudinal evaluation and response to intermittent cyclic etidronate therapy. Cancer 83, 1561-1566.

Diamond TH, Bucci J, Kersley JH, Aslan P, Lynch WB and Bryant C (2004a) Osteoporosis and spinal fractures in men with prostate cancer, risk factors and effects of androgen deprivation therapy. J Urol 172, 529-532.

Dumon, JC, Journe, F, Kheddoumi, N, Lagneaux, L, Body, JJ (2004) Cytostatic and apoptotic effects of bisphosphonates on prostate cancer cells. Eur Urol 45, 521-528; discussion 528-529.

Eastham J, McKiernan J, Gleason D, Zheng M, Saad F (2005) Effect of zoledronic acid on bone pain and skeletal morbidity in patients with advanced prostate cancer; analysis by baseline pain. ASCO Abstract No. 4561, Greenberg MS (2004) Intravenous bisphosphonates and osteonecrosis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 98, 259-260.

Gulley J and Dahut WL (2004) Chemotherapy for prostate cancer, finally an advance! Am J Ther 11, 288-294.

Hemminki K, Rawal R and Bermejo JL (2004) Prostate cancer screening changing age-specific incidence trends and implications on familial risk. Int J Cancer 113, 312-5.

Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E, Feuer EJ and Thun MJ (2004) Cancer statistics 2004. CA Cancer J Clin 54, 8-29.

Krupski TL, Smith MR, Chan Lee W, Pashos CL, Brandman J, Wang Q, Botteman M and Litwin MS (2004) Natural history of bone complications in men with prostate carcinoma initiating androgen deprivation therapy. Cancer 101, 541549.

Lee, MV, Fong, EM, Singer, FR, Guenette, RS (2001) Bisphosphonate treatment inhibits the growth of prostate cancer cells. Cancer Res 61, 2602-2608.

Marx RE (2003) Pamidronate (Aredia) and zoledronate (Zometa) induced avascular necrosis of the jaws, a growing epidemic.

J Oral Maxillofac Surg 61, 1115-1117.

VII. Conclusions Skeletal complications in men with prostate cancer are an under-recognized problem. Men undergoing treatment with androgen ablation for their prostate cancer, even without metastasis, are at increased risk of bone loss, and men with bone metastasis are at risk for adverse skeletal related events. Recent studies have demonstrated a benefit of bisphosphonates to improve bone density in men without bone metastasis receiving ADT, and to decrease skeletal related events in men with hormone refractory metastatic prostate cancer. The benefit of bisphosphonates in other stages of progression of prostate

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Cancer Therapy Vol 3, page 405 Mason, MD (2004) Development of bone metastases from prostate cancer: first results of the MRC PR04 randomised controlled trial (ISCRTN61384873). J Clin Oncol 22, 384 Melton LJ, 3rd Alothman KI, Khosla S, Achenbach SJ, Oberg AL and Zincke H (2003) Fracture risk following bilateral orchiectomy. J Urol 169, 1747-1750.

Messing EM, Manola J, Sarosdy M, Wilding G, Crawford ED and Trump D (1999) Immediate hormonal therapy compared with observation after radical prostatectomy and pelvic lymphadenectomy in men with node-positive prostate cancer.

N Engl J Med 341, 1781-1788.

Ruggiero SL, Mehrotra B, Rosenberg TJ and Engroff SL (2004) Osteonecrosis of the jaws associated with the use of bisphosphonates, a review of 63 cases. J Oral Maxillofac Surg 62, 527-534.

Saad F (2002) Zoledronic acid significantly reduces pathologic fractures in patients with advanced-stage prostate cancer metastatic to bone. Clin Prostate Cancer 1, 145-152.

Saad F, Gleason DM, Murray R, Tchekmedyian S, Venner P, Lacombe L, Chin JL, Vinholes JJ, Goas JA and Chen B (2002) A randomized placebo-controlled trial of zoledronic acid in patients with hormone-refractory metastatic prostate carcinoma. J Natl Cancer Inst 94, 1458-1468.

Shahinian VB, Kuo YF, Freeman JL, Goodwin JS ( 2005) Risk of fracture after androgen deprivation for prostate cancer. N Engl J Med 352, 154-164.

Smith MR, Eastham J, Gleason DM, Shasha D, Tchekmedyian S and Zinner N (2003) Randomized controlled trial of zoledronic acid to prevent bone loss in men receiving androgen deprivation therapy for nonmetastatic prostate cancer. J Urol 169, 2008-2012.

Smith MR, McGovern FJ, Zietman AL, Fallon MA, Hayden DL, Schoenfeld DA, Kantoff PW and Finkelstein JS (2001) Pamidronate to prevent bone loss during androgendeprivation therapy for prostate cancer. N Engl J Med 345, 948-955.

Vogel CL, Yanagihara RH, Wood AJ, Schnell FM, Henderson C, Kaplan BH, Purdy MH, Orlowski R, Decker JL, Lacerna L, Hohneker JA ( 2004) Safety and pain palliation of zoledronic acid in patients with breast cancer, prostate cancer, or multiple myeloma who previously received bisphosphonate therapy. Oncologist 9, 687-695.

Wong R (2004) No difference between pamidronate disodium and placebo in relieving bone pain in men with advanced prostate cancer. Cancer Treat Rev 30, 395-400.

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Synergistic augmentation of vincristine-induced cytotoxicity by phosphatidylinositol 3-kinase inhibitor in human malignant glioma cells: evidence for the involvement of p38 and ERK signaling pathways Research Article

Daniel R. Premkumar, Beth Arnold, John Mathas, and Ian F. Pollack* Department of Neurosurgery, University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute Brain Tumor Center Pittsburgh, Pennsylvania 15213, USA

__________________________________________________________________________________ *Correspondence: Ian F. Pollack, M.D., F.A.C.S., F.A.A.P., Department of Neurosurgery, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA15213, USA.; Phone: 412-692-5881; Fax: 412-692-5921; E-mail: ian.pollack@chp.edu Key words: Synergy, vincristine, microtubule inhibiting agents, PI3K, glioma, MAPK, apoptosis, cell cycle Abbreviations: 3-[4,5-dimethylthiazol-2yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H Tetrazolium, (MTS); bovine serum albumin, (BSA); c-Jun NH2-terminal kinase/stress activated protein kinase, (JNK/SAPK); cyclin dependent kinase, (CDK); dimethyl sulfoxide, (DMSO); extracellular signal-regulated kinase kinase, (MEK); extracellular signal-regulated kinase, (ERK); fetal bovine serum, (FBS); fraction affected, (Fa); microtubule inhibiting agent, (MIA); mitogen activated protein kinase, (MAPK); phenazine methosulfate, (PMS); phenyl methylsuphonyl fluoride, (PMSF); phosphatase and tensin homologue deleted on chromosome ten, (PTEN); phosphate-buffered saline, (PBS); phosphatidylinositol 3’-kinase, (PI3K); poly (adenosine diphophate-ribose) polymerase, (PARP); sodium dodecyl sulfate-poly acrylamide gel electrophoresis, (SDS-PAGE)

This work was supported by NIH grant P01NS40923 and a grant from the Wichmann Foundation.

Received: 23 March 2005; Revised: 20 May 2005 Accepted: 06 July 2005; electronically published: July 2005

Summary Microtubule-interfering agents, such as vincristine, are widely used for the treatment of cancer, and are included in many treatment regimens for childhood brain tumors. Anticancer properties of vincristine have been attributed in part to interference with microtubule assembly, impairment of mitosis, and cytoskeletal changes, with additional effects on mitogen-activated protein kinase signaling and caspase activation. Because malignant gliomas commonly have dysregulation of PI3K/Akt signaling, which can promote cell survival and potentially limit the activity of conventional chemotherapeutic agents, we questioned whether phosphatidylinositol 3’-kinase inhibitor (PI3K) inhibition with LY294002 could potentiate the efficacy of vincristine in a panel of glioma cell lines versus normal astrocytes. We therefore examined the effects of vincristine and the PI3K inhibitor, LY294002, alone and in combination, on cell survival, signal transduction and apoptosis. Simultaneous exposure to these inhibitors significantly induced cell death, and inhibited proliferation and clonogenicity of a series of glioma cell lines at concentrations that had little or no independent activity. Quantitative analysis revealed that enhancement by LY294002 of vincristine-induced cytotoxicity was synergistic, leading to pronounced caspase activation and increased sub-G 1 peak on cell cycle analysis at concentrations that had no significant effects on non-neoplastic cells.

The enhanced cytotoxicity of this combination was associated with significant activation of p38 MAPK signaling.

Pre-treatment with either SB203580 or z-VAD.fmk, selective inhibitors of p38 MAPK and caspase signaling, respectively, abrogated the apoptotic response to the combination of LY294002 and vincristine. Taken together, these findings demonstrate that PI3K/Akt inhibition can potentiate the effects of vincristine, and that the combination of molecularly targeted therapies and conventional agents could provide a potent strategy to treat patients with malignant gliomas.

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I. Introduction

II. Materials and Methods A. Cell Culture

Glioblastoma multiforme (GBM) are highly malignant tumors of the central nervous system. They are characterized by rapid growth, extensive vascularization and poor prognosis (Nagane et al, 1997; Hanahan and Weinberg 2000; Maher et al, 2001). Even with radiation therapy and chemotherapy, the prognosis remains poor, with survival usually less than 1 year from the time of diagnosis. There is considerable evidence demonstrating a role for phosphatidylinositol 3-kinase (PI3K) signaling in oncogenic transformation, cancer progression, and resistance of cancer cells to cytotoxic therapies. The tumor suppressor gene PTEN, possessing a lipid phosphatase activity, negatively regulates PI3-kinase activity by dephosphorylation of PtdIns P3 leading to reduction in Akt activity. Genetic and biochemical evidence suggests that activation of PI3K or inactivation of PTEN by mutation or deletion play a major role in glial tumorigenesis (Maehama and Dixon 1999; Tamura et al, 1999). Deregulated PI3K signaling also provides an attractive target for therapy and pharmacological inhibitors of this pathway are already in early clinical trials (Di Cristofano and Pandolfi 2000).

Microtubule-interfering agents (MIAs) are widely used for the treatment of cancer (Joel 1996; Wall 1998; Gidding et al, 1999). The anticancer properties of MIAs have been attributed in part to interference with microtubule assembly, impairment of mitosis, and cytoskeletal changes (Wang et al, 1999). There is growing evidence that MIAs have multiple cellular targets. For example, MIAs elicit differential effects on mitogen activated protein kinase (MAPK) family signaling pathways (Stone and Chambers 2000; Mabuchi et al, 2002) and influence gene expression (Subbaramaiah et al, 2000). Furthermore, MIAs cause growth arrest, and induce caspase activation, degradation of PARP, and apoptosis in neoplastic cells (Stone and Chambers 2000). Vincristine is an MIA that is widely used to treat patients with malignant disease, including those with brain tumors (Kellie et al, 2004). However, many tumor cell lines are resistant to clinically achievable concentrations of this agent, which may in part reflect a resistance to undergoing apoptosis, as a result of dysregulated PI3K/Akt signaling. Accordingly, a potential strategy for enhancing the antitumor efficacy of vincristine involves combining this agent with targeted disruption of PI3K/Akt-mediated survival pathways.

Previous studies by Shingu et al (2003) have suggested the potential synergy of microtubule inhibiting agents, such as vincristine, and PI3kinase inhibition. In this report, we demonstrated the differential efficacy of this approach in a panel of glioma cell lines versus normal astrocytes and fibroblasts and identified the involvement of p38 and ERK in mediating the synergistic effects of combining vincristine and PI3K inhibition with LY294002. Our results suggest that multiple pathways are important for cell survival in malignant glioma cells and identify a role for activation of p38 and inhibition of ERK in the augmentation of vincristine-induced cytotoxicity by PI3K inhibition.

The established malignant glioma cell lines U87, T98G, A172, and human pulmonary fibroblasts were obtained from the American Type Culture Collection. Human astrocytes and human cerebellar astrocytes were obtained from ScienCell Research Laboratories, San Diego, CA. LN18, LN-Z308, and LN-Z428 were generously provided by Dr. Nicolas de Tribolet. U87, T98G and human pulmonary fibroblasts were cultured in growth medium composed of minimum essential medium supplemented with sodium pyruvate and non-essential amino acids; A172, LN18, LN-Z308, and LN-Z428 in ·-minimal essential medium supplemented with L-glutamine; human astrocytes in Astrocyte Growth Medium (ScienCell Research Laboratories). All growth media contain 10% fetal calf serum, L-glutamine, ribonucleosides, deoxynucleosides, 100 IU/ml penicillin, 100mg/ml streptomycin and 0.25 mg/ml amphotericin (Life Technologies, Inc., Bethesda, MD). Cells were grown in 75-cm2 flasks at 37oC in a humidified atmosphere with 5% carbon dioxide and were subcultured every 4 to 7 days by treatment with 0.25% trypsin in Hanks’ balanced salt solution (Life Technologies, Inc.).

B. Cell proliferation and cytotoxicity assay Cells (5 X 103/well) were plated in 96-well microtiter plates (Costar, Cambridge, MA) in 100 µl of growth medium.

After an overnight attachment period, cells were exposed for 3 days to varying concentrations of vincristine with or without 5µM LY294002. Control cells received vehicle alone (DMSO).

All studies were performed in triplicate and repeated at least three times independently. After the 3-day treatment period, cells were washed in inhibitor-free medium and the number of viable cells was determined using a colorimetric cell proliferation assay (CellTiter96 Aqueous Non-Radioactive Cell Proliferation Assay; Promega, Madison, WI), which measures the bioreduction of the tetrazolium compound MTS (3-[4,5-dimethylthiazol-2yl]-5-[3carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H tetrazolium) by dehydrogenase enzymes of metabolically active cells into a soluble formazan product, in the presence of the electron coupling reagent PMS (phenazine methosulfate) (Riss TL 1992).

To perform the assay, 20µl of combined MTS/PMS solution containing 2mg/ml MTS and 150µmol/L PMS in buffer (0.2g/L KCL, 8.0g/L NaCl, 0.2g/L KH2PO, 1.15g/L Na2HPO4, 133 mg/ml CaCl2.2H20, 100 mg/ml MgCl2.6H20, pH7.35) was added to each well and then after 1 h of incubation at 37oC in a humidified 5% CO2 atmosphere, absorbance was measured at 490nM in a microplate reader. Triplicate wells with predetermined cell numbers were subjected to the above assay in parallel with the test samples to normalize the absorbance readings; this also provided internal confirmation that the assay was linear over the range of absorbance and cell numbers measured.

To assess cellular toxicity, 2.5 X 105 cells were seeded in 60-mm Petri dishes and on the following day, treated with selected concentrations of inhibitors or vehicle. Cells were harvested, stained with trypan blue, and counted using a hemacytometer. All samples were tested in triplicate. Viable (trypan blue-excluding) and dead cell numbers were plotted as a function of inhibitor concentration.

C. Clonogenic growth assay A more direct assessment of the effect of different inhibitor concentrations on cell viability was performed using a clonogenic assay. For these studies, 250 cells were plated in 6well trays in growth medium and, after an overnight attachment period, were exposed to selected inhibitor concentrations or vehicle for 24h. The medium was aspirated and cells were

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III. Results A. Inhibition of cell proliferation by vincristine

D. Western blotting analysis

To characterize the interaction between the chemotherapeutic agent vincristine and the PI3-kinase inhibitor LY294002, a panel of human glioma and normal cells were cotreated with varying concentrations of vincristine and 5µM LY294002, a concentration well below IC50 in human glioma cell lines (Premkumar et al manuscript submitted), and then cell proliferation was measured using an MTS assay. As shown in Figure 1A, vincristine inhibited the growth of glioma cells in a dosedependent manner. At the concentrations examined, there were no significant effects on the normal cells, such as human astrocytes, human cerebellar astrocytes, and human fibroblasts (data not shown). The observed inhibitory effect of vincristine was further potentiated by LY294002.

To determine whether this potentiation was due to additive or synergistic interactions, we performed concentrationeffect and isobologram analyses. The data were then applied to determine the combination index (CI) which provides a semiquantitative assessment of the presence of additive, synergistic or antagonistic interactions at different effect levels (Chou and Talalay 1984). The combination index is 1 for additive interactions, greater than 1 for antagonistic interactions, and less than 1 for synergistic interactions. Figure 1B illustrates plots of the CI versus fraction affected (Fa) for vincristine and LY294002. The combination of vincristine and LY294002 produced a synergistic inhibition, based on the observation that the CI was substantially less than 1.

In order to confirm the specificity towards tumor cells, we compared the effect of vincristine and LY294002 alone or in combination on human glioma (U87 and T98G) and normal cells (human cerebellar astrocytes and human fibroblast). Cells were treated with 1nM vincristine or 5µM LY294002 or the combination of both for 3 days and cell proliferation was assessed by MTS assay.

Exposure to 5µM LY294002 alone was minimally toxic to these cells (about 10% reduction of cell proliferation versus control), whereas 1nM vincristine had no significant growth inhibitory effect in U87, human cerebellar astrocytes and human fibroblasts (Figure 2A).

However, the combination of vincristine and LY294002 significantly reduced cell proliferation in glioma cells (by 60 and 70% compared to control in U87 and T98G, respectively), whereas the combination had little effect on normal cells (25 and 15% reduction from control in human cerebellar astrocytes and human fibroblasts, respectively).

These results indicate that combination of vincristine and LY294002 works very efficiently in tumor cells compared with non-tumorigenic human astrocytes and fibroblasts.

The cytotoxic effect of vincristine and LY294002 was further confirmed using a clonogenic assay. U87 and T98G cells were treated with varying concentrations of vincristine for 1 day, medium was aspirated, and cells were washed with inhibitor-free medium. Cells were allowed to grow for an additional 2-week period. There was a dose-dependent decrease in colony forming ability

Treated and untreated cells were washed once in cold phosphate-buffered saline (PBS) and lysed in buffer containing 30mM Hepes, 10%glycerol, 1%Triton X-100, 100mM NaCl, 10mM MgCl2, 5mM EDTA, 2mM Na3VO4, 2mM !glycerophosphate, 1mM PMSF, 1mM AEBSF, 0.8µM Aprotinin, 50µM Bestatin, 15µM E-64, 20µM Leupeptin, 10µM Pepstatin A. After lysing on ice for 15 minutes, protein samples were collected from the supernatant after centrifugation of the samples at 12,000 X g for 15 min, and protein was quantified using Protein Assay Reagent (Pierce, Rockford, IL). Equal amounts of protein were separated by SDS-PAGE and electrotransferred onto a nylon membrane (Invitrogen, Carlsbad, CA). The blots were blocked with 2% BSA in Tris-buffered saline (TBS)-Tween 20 (0.1%) at room temperature for 1h and probed with the appropriate dilution of primary antibody overnight at 4oC. The blots washed three times in TBS-Tween 20 for 15 min and then incubated with a 1:1500 dilution of horseradish peroxidaseconjugated secondary antibody (Cell Signaling Technology, Beverly, MA) in TBS-Tween 20 at room temperature for 1h.

After washing three times in TBS-Tween 20 for 15 min, the proteins were visualized by Western Blot Chemiluminescence Reagent (Cell Signaling Technology). Where indicated, the blots were reprobed with antibodies against !-actin (Sigma, St.Louis, MO) to ensure equal loading and transfer of proteins.

The primary antibodies, such as extracellular signal related kinase 1/2 (ERK 1/2) and phospho-p44/42 ERK (Thr202/Tyr204), p38 and phospho-p38 MAPK (Thr180.Tyr182), JNK and phosphoSAPK/JNK (Thr183/Tyr185), Akt and phospho Akt (Ser 473), CDK4, CDK6, Cyclin D1, Cyclin D3, p27Kip, phospho-Cdc2, cleaved caspase 3, cleaved PARP, phospho-Bcl-2, Bcl-2-xL, and Bax were obtained from Cell Signaling Technologies.

E. Inhibition of p38 kinase and caspases T98G cells were seeded 24 h before treatment. Cells were pretreated with z-VAD.fmk (a broad spectrum caspase inhibitor) or SB203580 (a specific p38 kinase inhibitor) in the culture media 60 min prior to treatment with vincristine or LY294002 or the combination of both for 9 h. The cells, after washing twice with phosphate buffered saline, were lysed and Western immunoblot analysis was performed using cleaved caspase 3 specific antibody as described above.

F. Cell cycle analysis Analysis of DNA content of cells by flow cytometry was performed as described elsewhere (Gesbert et al, 2000). Briefly, T98G cells grown exponentially to 40-50% confluency were exposed to vincristine and/or LY294002, or vehicle (DMSO), harvested at the indicated time, washed briefly in ice-cold PBS, and fixed in 70% ethanol. DNA was stained by incubating the cells in PBS containing propidium iodide (50 µg/ml) and RNase A (mg/ml) for 60 min at room temperature, and fluorescence was measured and analyzed using a Becton Dickinson FACScan and the Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, CA).

G. Statistical analysis To define IC50 concentrations and to characterize synergistic effects between the agents, a commercially available software program was used (Calcusyn; Biosoft, Ferguson) (Chou and Talalay 1984).

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71, 10, and 18% G1, S and G2/M fractions, respectively after 72h.

Apoptotic cells undergo DNA fragmentation and therefore display a sub-G1 (<2N) DNA content. Although neither vincristine nor LY294002 independently induced a substantial sub-G1 fraction at the above concentrations, the combination of vincristine and LY294002 displayed a significant increase in cell death, revealed by the presence of a pronounced sub-G1 fraction on flow cytometry. T98G cells were shown to exhibit 32, 4 and 6% G1, S and G2/M phase fractions, respectively after 72 h (Figure 3B) when the cells were exposed concomitantly to vincristine (1nM) and LY294002 (5µM), whereas the fraction of cells with <2N DNA content (sub-G1) was 55%. This indicates that the decreased cell proliferation observed after treating with vincristine and LY294002 is at least partly due to induction of apoptosis.

Given the striking combinatorial effects of vincristine and PI3K inhibition on cell proliferation and colony forming ability of glioma cells, we questioned whether this

B. Vincristine and LY294002 cooperate to induce a sub-G1 fraction on cell cycle analysis of human glioma cell lines As our previous results indicated that human glioma cells are sensitive to the antiproliferative properties of vincristine, we performed a more detailed analysis of the effect of the drug on the cell cycle. We performed a time course of this effect by analyzing the DNA profile in T98G cells exposed to 1 nM of vincristine using propidium iodide staining and flow cytometry. As shown in Figure 3A, as early as 12h after treating the cells with 1nM of vincristine the percentage of cells in the G1 phase had increased to 61%, versus 39% in untreated control cells (Figure 3A). 5µM LY294002 was shown to induce

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Figure 1. Growth inhibition of human glioma cell lines by vincristine and PI3K inhibitor. (A) Logarithmically growing glioma cell lines were incubated with or without varying concentrations of vincristine with or without 5µM LY294002 for 3 days. The relationship between the compounds and cell numbers was assessed semiquantitatively by spectrophotometric measurement of MTS bioreduction in T98G, A172, LN308 and LN428, established malignant human glioma cell lines. Points represent the mean of three measurements ± standard deviation. There was a dose-dependent reduction in cell growth and addition of 5µM LY294002 potentiated the vincristineinduced toxicity. No significant inhibition was detected in control cells treated with equivalent concentrations of vehicle (DMSO) or 5 µM LY294002 alone. (B) Graphs showing concentration-response plots of inhibition (expressed as fraction affected) versus combination index. Four established human glioma cell lines (A172, LN308, LN18 and LN428) were exposed to varying concentrations of vincristine and LY294002 at a fixed molar ratio (1:3000) for 3 days. Each point was derived from triplicate measurements of cell numbers as assessed by an MTS-based colorimetric assay. The data were then used to calculate the combination index (CI) using commercially available software (Calcusyn; Biosoft), which provides a semiquantitative assessment of the presence of additive, synergistic, or antagonistic interactions at different effect levels. The CI is substantially less than 1 for the combination of vincristine and LY294002, indicating synergistic interactions.

combination would have comparable effects on cell cycle regulatory proteins. Accordingly, we examined the effects of these agents, alone and in combination, on several intermediates that play critical roles in glioma cell cycle progression. T98G cells were therefore seeded at subconfluency, treated with vincristine, LY294002, or the combination of both, and the effects on protein expression levels were assessed. Results from Western blot analysis showed that the combination of vincristine and LY294002 had no significant effect on phospho-cdc2, CDK4, CDK6, Cyclin D1, p21WAF and p27Kip compared to controls or each agent individually. However, coadministration of vincristine and LY294002 resulted in a modest but discernible reduction in Cyclin D3 (Figure 3C), suggesting that a reduction in Cyclin D3 expression could be responsible for the observed G1 arrest and increased apoptosis.

C. Vincristine down regulates ERK, Akt and activates p38 MAP kinase To establish whether vincristine induced selective effects on different signaling pathways, the phosphorylation status of ERK, p38, JNK/SAPK and Akt was evaluated in control and vincristine-treated T98G cells

by Western immunoblot analysis using respective phosphospecific antibodies. A vincristine concentration (50 nM) well above the IC50 was used to optimally demonstrate the time course of the effects observed. High levels of the phosphorylated forms of both ERK1 and 2 and Akt were detected in untreated control cells and treatment with 50nM vincristine produced a gradual decrease in these levels in a time-dependent manner (Figure 4A). Conversely, cells exposed to vincristine exhibited a time-dependent increase in phosphorylated p38. Quantification of the results revealed that phospho p38 was maximally stimulated to about 9 fold at 9 h and reduced to basal level after 24h (Figure 4A).

Phosphorylated JNK/SAPK was not detected in the treated cells (data not shown).

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Figure 2. Vincristine and LY294002 preferentially inhibit growth and colony formation of glioma cell lines. (A) Logarithmically growing glioma cell lines (T98G and U87), normal cells human cerebellar astrocytes (HAC), and human fibroblasts (HF) were incubated in media (C) with 1nM vincristine (V) or 5_M LY294002 (LY) or the combination of vincristine and LY294002 (V+LY) for 3 days and cell numbers were assessed by MTS assay. Points represent the mean of three measurements ± standard deviation. The combination of vincristine and LY294002 significantly reduced cell proliferation of glioma cells compared to normal cells. (B). Graph showing the relationship between colony counts (± standard deviation) and concentration of the inhibitors. Human glioma cell lines, U87 and T98G were exposed to varying concentrations of vincristine with or without LY294002 (5µM) for 24 h. On the following day, the media was changed and complete media was added and cells were grown for additional 14 days in the absence of inhibitors. Colonies were then counted. Points represent the mean of two experiments ± standard deviation.

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Figure 3. Cell cycle analysis of vincristine-treated T98G cells. (A) Exponentially growing T98G cells were exposed to 1nM vincristine for the indicated times, harvested, fixed, and DNA-stained with propidium iodide. DNA content of the cells was obtained by flow cytometry, and representative histograms for the indicated times are shown with values for each phase of the cell cycle. (B) Asynchronous T98G cells, grown to 40-50% confluency, were exposed to vincristine (1nM) or LY294002 (5µM) or the combination of both for 72h. Control cells received DMSO. DNA content (%) of cells was obtained by flow cytometry. (C) Cell lysates were obtained from T98G cells treated with vincristine (1nM) or LY294002 (5µM) or the combination of both for 72h, and 50µg of total protein from the cell lysates were resolved in polyacrylamide-SDS gels. Proteins were analyzed by Western immunoblotting using indicated antibodies as described in “Materials and Methods” and detected by enhanced chemiluminescence. Control cells received DMSO. Levels of ß-actin are indicated and served as a control to ensure equal protein loading per lane.

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Figure 4. Vincristine downregulates ERK and upregulates p38 MAPK. Logarithmically growing T98G cells were incubated for designated intervals in the presence of 50nM vincristine (A) or in vincristine (1nM) or LY294002 (5µM) or the combination of both (B).

The cells were lysed, and proteins were separated by SDS-PAGE and probed with a phosphospecific ERK 1, 2 antibody, which recognizes phosphorylated (Thr202 and Tyr204) ERK MAP kinase. Activation of p38 and Akt was assessed using phosphospecific p38 (Thr180 and Tyr 182) and phosphospecific Akt (Ser473) antibodies, respectively; then the blots were stripped and reprobed with total ERK, p38 or Akt.

To assess potential interactions between vincristine and LY294002, T98G cells were exposed to each of these agents alone or in combination for varying durations and the cell lysates were probed with phosphospecific ERK, Akt and p38 antibodies. Vincristine (1nM) or LY294002 (5µM) each had very little effect on phosphorylated ERK1/2 even after 3 days of exposure (Figure 4B), whereas combined exposure to these agents resulted in a significant (as early as 3h) to complete (after 24h) inhibition of phosphorylated ERK. Combined exposure to these agents resulted in significant to complete inactivation of pAkt after 24h. To determine the impact on the p38 activation, T98G cells were exposed to each agent individually or in combination. Incubation with 1nM vincristine induced phosphorylation of p38 at 6h and this was abolished after 24h, whereas coincubation with LY294002 further potentiated the p38 activation, which persisted over the ensuing 72h.

varying durations and the apoptotic cleavage of caspase 8, caspase 9, caspase 3 and PARP was assessed by Western analysis using specific antibodies. Cells incubated with vincristine or LY294002 did not show any activation of caspases or PARP cleavage (Figure 5A), whereas the combination of vincristine and LY294002 significantly increased the expression of the active forms of caspases 8, 9, 3 and PARP (Figure 5A, B). Activation of cleaved caspase 3 and PARP were seen as early as 6h after treatment and increased with longer durations of exposure.

The degree of caspase activation observed with the combination of 1 nM vincristine and 5 µM LY294002 was comparable to that observed with 10-fold higher concentrations of vincristine administered alone (Figure 6C). This effect was not associated with changes in expression of Bcl-2, Bcl-xL, or Bax (Figure 5C).

Importantly, non-neoplastic human astrocytes showed no activation of caspase 3 or PARP even after 3 days of treatment (Figure 5D).

D. Vincristine and LY294002-induced activation of caspases and PARP. Caspases are

E. Vincristine and LY294002-induced Activation of caspase 3 is inhibited by SB203580 and z-VAD.fmk

aspartate-specific cysteine proteases activated by cleavage of their inactive pro-caspase forms, which function as important intermediates in apoptotic signaling. We examined the involvement of caspases and the DNA repair enzyme poly (ADP-ribose) polymerase (PARP), a target of caspase cleavage in the apoptotic effects of vincristine and LY294002. T98G cells were treated with vincristine (1nM), LY294002 (5µM) or the combination of both for

To confirm the role of p38 signaling and caspase activation in mediating the glioma-specific cytotoxicity of the combination of vincristine and LY294002, we examined the effect of selective inhibition of these pathways, using SB203580, which prevents p38 activation, and z-VAD.fmk, a broad-spectrum caspase

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Figure 5. Vincristine and LY294002 induces caspases and PARP activation in T98G cells. Logarithmically growing T98G cells were incubated for designated intervals in the presence of 1nM vincristine with or without LY294002 (5µM). The cells were lysed, and proteins were separated by SDS-PAGE and probed with specific antibodies which recognize the cleaved products of caspase-3 (C. caspase) or PARP (C. PARP) (A) as described in “Materials and Methods”. B. T98G glioma cell line (B and C) and normal human astrocytes (D) were treated as above for 3 days and the proteins were separated and probed with indicated antibodies, analyzed by Western immunoblot and detected by enhanced chemiluminescence. Control cells received DMSO.

inhibitor. Cells were pretreated with caspase and p38 inhibitors in the culture media 60 min prior to treatment with vincristine or LY294002 or the combination of both for 9 h. Western immunoblot analysis was performed using cleaved caspase 3 specific antibody and cell viability was assessed by trypan blue exclusion analysis in parallel.

Vincristine induced caspase 3 activation which was further potentiated when vincristine was combined with LY294002 (Figure 6A, B). On the other hand active caspase 3 was not detected in the cells pretreated with SB203580 or z-VAD.fmk. Neither LY294002 nor SB203580 had a significant effect on cell survival. In contrast, exposure to 10nM vincristine resulted in 31% cell death and combination of 10µM LY294002 and 10nM vincristine resulted in a further increase in cell death (to about 60%). Pretreating the cells with p38 inhibitor, SB203580 or z-VAD.fmk significantly reduced the vincristine and LY294002-induced cell death (to 12 and 10% respectively). This suggests that the inhibition of p38 activation by vincristine and LY294002 protects the cells by preventing caspase 3 activation and progression towards apoptosis.

in mitosis by triggering the mitotic checkpoint, a series of biochemical reactions that ensure proper attachment of chromosomes to the mitotic spindle before cells enter anaphase (Rudner and Murray 1996; Amon 1999; Burke 2000). In the present study, we have shown that LY294002, an inhibitor of PI3K/Akt kinase, interacts synergistically with low concentrations of vincristine, a chemotherapeutic MIA to induce both G1 arrest and apoptosis. Cells treated with vincristine demonstrated G1 arrest at low drug concentrations, whereas coadministration of LY294002 produced a substantial sub-G1 fraction, suggesting that the combination treatment induced apoptosis at concentrations of vincristine and LY294002 that produced minimal independent cytotoxicity.

The sensitivity of cells to apoptosis-inducing stimuli appears to be dependent on the balance between apoptosisinducing signals and survival signals (Berra et al, 1998; Murillo et al, 2001; Shingu et al, 2003). Several lines of evidence implicate an important role for PI3K/Akt pathways in tumorigenesis and suppression of apoptosis (Datta et al, 1997; Datta et al, 1999; Katso et al, 2001).

Akt represents a major downstream target of PI3K and is linked to a wide variety of antiapoptotic functions (Datta et al, 1999; Nicholson and Anderson 2002). Active Akt prevents apoptosis by a variety of mechanisms, including phosphorylation of Bad, caspase-9, Forkhead transcription factors and I"B kinase(Datta et al, 1997; Cardone et al, 1998; Vanhaesebroeck and Alessi 2000; Gelfanov et al, 2001).

IV. Discussion Cancer progression has been suggested to involve the loss of cell cycle checkpoint controls that regulate the passage through cell cycle. Checkpoints are control mechanisms that ensure the proper timing of cell cycle events and monitor the integrity of the DNA (Hartwell and Weinert 1989). At high concentrations, MIAs arrest cells 415

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Figure 6. Vincristine-induced toxicity is blocked by the inhibition of p38 MAPK. Logarithmically growing T98G cells were incubated in the presence of SB203580 (10µM) or z-VAD.fmk (100µM) 60 min prior to vincristine (10nM) or LY294002 (10µM) or the combination of both for 9h. Equal amounts of proteins (50µg) were separated by SDS-PAGE and probed with caspase-3 antibody which recognizes the cleaved products of caspase-3 (A) as described in “Materials and Methods”, and detected by enhanced chemiluminescence. Control cells received DMSO. (B) In parallel, at the end of the incubation period, the dead and viable cell numbers were determined by trypan-blue exclusion assay. For each analysis, at least 1000 cells were evaluated. The values represent the mean ± standard deviation for 2 separate experiments performed in triplicate.

Conversely, inhibition of Akt signaling may potentiate apoptosis in response to conventional chemotherapeutic agents as well as other growth signaling inhibitors (Ng et al, 2000; O'Gorman et al, 2000; Hu et al, 2002; Shingu et al, 2003). Microtubule stabilizing agents such as paclitaxel and docetaxel and microtubule-disrupting drugs such as vincristine, vinblastine and colchicines, interfere with the microtubule-related functions in signaling and gene expression (Kumar 1981; McNally 1996; Haldar et al, 1997; Saunders and Limbird 1997; Jordan and Wilson 1998; Subbaramaiah et al, 2000), and have antimitotic and apoptosis-inducing activity (Donaldson et al, 1994).

Blagasklonny et al (Blagosklonny et al, 1997) and Poruchynsky et al (Poruchynsky et al, 1998) showed that exposure to MIAs may promote apoptosis by phosphorylating Bcl-xL, Bcl-2 and related family members or by post-translational modifications in the proteins that interfere with anti-apoptotic functions. These findings led to the concept that Bcl-2/Bcl-xL phosphorylations represent key steps in the cell death pathway induced by microtubule disruption. Although we found that vincristine as a single agent or in combination with LY294002 produced no change in expression of Bcl-2, Bcl-xL, and Bax in glioma cell lines, we used substantially lower concentrations (1nM versus 100 nM or more) (Poruchynsky et al, 1998; Srivastava et al, 1998).

Nonetheless, our observations suggest that alternate mechanisms operate to enhance apoptosis in glioma cells, and that the augmentation of vincristine-induced cytotoxicity by LY294002 was not due to increased expression of Bax or decreased expression of antiapoptotic regulatory proteins like Bcl-2 or Bcl-xL. Previously, MIAs have been observed to induce p38, JNK and ERK MAPK activities in various cell lines (Lee et al, 1998; Schmid-Alliana et al, 1998; Wang et al, 1998; Moos et al, 1999). In this report, we demonstrate that the combination of PI3 kinase inhibition and vincristine results in concomitant down-regulation of ERK and activation of p38 MAP kinase in glioma cells, with minimal effects on JNK activity. This contrasts with observations in other tumor cell lines that MIAs activate JNK/SAP kinase (Wang et al, 1998; Shtil et al, 1999; Stone and Chambers 2000), although is consistent with recent findings that the microtubule stabilizer taxol activates p38 kinase in MCF-7 cells (Shtil et al, 1999).

These disparate findings suggest that there may be differential regulation of various MAP kinase family members in response to microtubule inhibition in different human tumor cell lines. Thus, the synergistic augmentation of vincristine-induced cytotoxicity by LY294002 and the ability of a cell to die or survive may

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dictated by a critical balance between ERK and the p38 pathway.

Taken together, our observations support and extend the observations of Shingu et al (2003), which suggested the potential synergy of microtubule inhibiting agents, such as vincristine, and PI3kinase inhibition in glioma cells. In this report, we demonstrated the differential efficacy of this approach in a more extensive panel of glioma cell lines versus normal astrocytes and fibroblasts, and identified the involvement of p38 and ERK in mediating the synergistic effects of combining vincristine and PI3K inhibition with LY294002. These results highlight the involvement of several signaling molecules in determining the susceptibility of human glioma cells to vincristine-induced apoptosis. The observation that inhibition of a major mediator of survival signals, PI3 kinase, can notably sensitize cells to cytotoxic druginduced apoptosis, is a finding which may be of significant therapeutic importance, given the current interest in developing selective inhibitors of this target. We conclude that the combination of molecularly targeted therapies and conventional agents may provide a potent strategy to treat patients with malignant gliomas.

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Ian F. Pollack

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Cancer Therapy Vol 3, page 429 Table 2. HPV vaccines currently being studied Target HPV 16 L1 VLP HPV 18 L1 VLP HPV 6 L1 VLP HPV 11 L1 VLP HPV 16 L1 VLP HPV 18 L1 VLP HPV 16 E6, 7 HPV 18 E6, 7

Vaccine Cervarix

Phase Phase II

Quadrivalent vaccine

Phase III

TA-HPV-Vaccina virus encoding

Phase I, II

HPV protein

TA-CIN/TA-HPV

Phase II

HPV-16 E7

HSP-E7 â&#x20AC;&#x201C; protein and/or peptide based vaccine ZYC101

Phase II

HPV DNA

Phase I, II

Results 100% protection against persistent HPV 16/ 18 infection, well tolerated 90% decrease in Combined incidence of persistent infection or disease with HPV 6, 11, 16, or 18, well tolerated

Company Glaxo-Smith Kline

Clinical responses in women with longstanding high-risk HPV positive VAIN or VIN, well tolerated Induction of HPV 16-specific T-cell and/or serological responses in HPV positive VIN patients, clinical response.

CD-8-dependent and CD-4-independent regression of HPV-16 E-7 expressing tumors in mice Currently in human trials Some responses observed in cervical dysplasia patients, well tolerated

Xenova

VII. Future prospects

Merck

Xenova StressGen Zycos

Acknowledgement

During the past few decades, the molecular mechanisms underlying the development and progression of HPV-associated cervical neoplasms have been extensively studied, and as a result a new appreciation for these mechanisms of cervical neoplasia development has emerged. Moreover, it has been established that HPV oncogenes are not only indispensable for malignant transformation, but that they are also important functional regulators of the various key genes involved in cervical carcinogenesis.

However, although various molecular interrelationships involved in HPV-associated-cervical carcinogenesis have been identified, the essential molecular genetic pathway remains to be elucidated.

Future studies should be directed at determining the following: (1) the roles of other HPV early proteins (such as E1, E2 and E4); (2) the oncogenicity of specific HPV variants, (3) the host control mechanism against HPV infection; (4) the mechanism of apoptosis modulation by HPV oncoprotein; (5) the interaction between HPV oncogene and co-factors; (6) the nature of cervical cancer risk genes; (7) the correlation between viral load and severity of disease; (8) the identities of additional vaccine targets, such as L1 and L2 and (9) those of the biomarkers of cervical neoplasm progression, and finally (9) inexpensive, low-technology HPV diagnostics should be developed.

Cervical cancer is a multifactorial and dynamic event in which numerous alterations contribute to disease development. Thus it is hoped that a better understanding of the pathogenic roles of HPV oncoprotein will help advance mechanism-based screening tools and therapies for the prevention and treatment of cervical cancer, and provide an insight of the fundamental rules of cervical carcinogenesis.

Korean Health 21 R&D Project, Korean Ministry of Health &Welfare; Grant number: 0412-CR01-0704-0001.

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Cancer Therapy Vol 3, page 419 Cancer Therapy Vol 3, 419-434, 2005

Implications of HPV infection in uterine cervical cancer Review Article

Hoenil Jo, Jae Weon Kim* Department of Obstetrics and Gynecology, Cancer Research Institute, Seoul National University, Seoul, Korea

__________________________________________________________________________________ *Correspondence: Jae Weon Kim, Department of Obstetrics and Gynecology and Cancer Research Institute, Seoul National University Hospital, 28 Yongon-dong, Seoul, Korea; Tel: 82 2 2072 3511; Fax: 82 2 762 3599; e-mail: kjwksh@snu.ac.kr Key words: Human papillomavirus, Cervical cancer, Molecular pathophysiology, Co-factors, HPV vaccine Abbreviations: 21-kDa protein, (p21); activating protein 1, (AP 1); arginine, (ARG); cyclin/cyclin dependent kinase, (cdk); double minute 2 gene, (MDM2); E6-associated protein, (E6AP); early, (E); late, (L); long control region, (LCR); nuclear factor 1, (NF-I); proline, (PRO); retinoblastoma protein, (pRb); Single nucleotide polymorphisms, (SNPs); upper regulatory region, (URR); virus-like particles, (VLP) Received: 31 May 2005; Accepted: 22 June 2005; electronically published: July 2005

Summary High-risk human papillomavirus (HPV) are the causative agent of uterine cervical carcinomas. Cervical carcinoma is initiated by infection with a high-risk human papillomavirus (HPV), usually HPV type 16 (HPV16) or HPV18. It has been theorized that integration of HPV DNA into the human genome, possibly at the E2 site, leading to persistent expression of the E6 and E7 genes. Moreover, the effects of E6 and E7 protein on the cell cycle are mediated by the inhibition of antioncogenes (primarily p53 and retinoblastoma) and by interference with the functions of cyclins and cyclin dependent kinases. In particular, E6 protein induces the degradation of tumor suppressor protein, p53, and E7 disrupts complex formation between pRB and the cellular transcription factor E2F. Specific genetic abnormalities other than those that affect p53 and Rb, might also play an important role in the carcinogenesis and aggressiveness of cervical cancer. In addition, c-myc oncogene, the ras family (K-ras, H-ras and N-ras), cyclin dependent kinases, cyclins, p16, p21, and p27 may also have a role in the pathogenesis of cervical cancer. Each of these genes has been reported to be overexpressed in cervical cancers and several have been associated with poor prognosis. In addition, SNPs, such as the p53 polymorphism, allelic variations, or CpG island hypermethylation may play roles in the development of cervical cancer. As the sensitivity and specificity of HPV DNA testing have improved, it has become increasingly apparent that most of the risk factors (e.g., age at first coitus, number of partners, socioeconomic status) merely reflect the probability of HPV exposure and acquisition.

Various cofactors have been investigated as potential contributors to disease progression. However, more recent epidemiologic studies have consistently identified smoking and HIV infection as independent co-factors that are likely to influence the risk of cervical cancer, after controlling for HPV infection. Taken together, molecular and epidemiologic data provide compelling evidence that HPV infection plays a central role in the development of cervical cancer. Based on the findings of a number of epidemiologic and laboratory studies that HPV infection is the major etiologic factor in the development of cervical cancer, new strategies against cervical neoplasia have evolved.

Various types of vaccines are currently being tested designed to prevent and treat HPV. The results of both preclinical and early clinical studies are promising and we now look forward to continuous advancements in the prevention and treatment of cervical cancer.

HPV infections and cervical cancer was first demonstrated in the early 1980s by Harold zur Hausen (Gissmann et al, 1980; de Villiers et al, 1981) and a number of molecular and epidemiologic studies have since demonstrated a strong co-relation between human papillomavirus (HPV) infection and this disease.

The HPV is a member of the Papovaviridae family and contains a double-stranded DNA virus. The

I. Introduction Cervical cancer is the second most common cancer among women worldwide, second only to breast cancer. In developing countries, cervical cancer is often the most common cancer in women and may constitute up to 25% of all female cancers (Burd et al, 2003).

The association between HPV infection and cervical neoplasm was established after the link between genital

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Jo and Kim: Implications of HPV infection in uterine cervical cancer papillomaviruses are a diverse group and have been detected in a wide variety of animals as well as in humans.

The virus contains a double-stranded, circular DNA genome containing 7800~7900 base pairs, a nonenveloped virion, and an icosahedral capsid. Because of the clinical importance, human papillomaviruses have been extensively studied, and at present approximately 118 different subtypes with limited DNA homologies have been identified.

Based on their association with cervical cancer and precursor lesions, HPVs can be divided into high-risk, intermediate-risk, and low-risk subtypes. Low risk subtypes are associated with venereal warts (condyloma acuminate), whereas intermediate and high-risk subtypes are associated with cervical dysplasia and invasive carcinoma. A recent worldwide review of HPV typing demonstrated that 87% of squamous cell carcinomas contain an identifiable HPV genome associated with the tumor, as compared with 76.4% of adenocarcinomas.

viral capsid proteins, which are encoded in the late region.

The L1 and L2 genes in the late region encode the major and minor capsid proteins, both of which are required late in the viral life cycle to encapsulate the virus. Table 1 summarizes the major function of each of the proteins encoded by E1, E2 and E4-7.

B. Life cycle of HPV HPV are strictly host-specific and also show distinct tropism for squamous epithelial cells. HPV can infect basal epithelial cells of the skin or the inner lining of tissues and are frequently found in the episomal state, that is, as genetic particles of virus in the host cell, in low- and high-grade squamous intraepithelial lesions. Initial HPV infection requires access to cells in the basal layer by infectious particles, which for some HPV types are thought to require a mild abrasion or microtrauma in stratified epithelium. For high-risk mucosal viruses, such as HPV16, the formation of cervical lesions may be facilitated by the infection of columnar cells, which can subsequently form a basal layer of transformed stratified epithelium. The nature of the cell surface receptor used for viral attachment is not known, although heparin sulphate and stabilizing proteoglycans have been suggested to be epithelial cell receptors for HPV (Giroglou et al, 2001).

Once in a host cell, the life cycle of HPV can be separated into two stages, i.e., nonproductive and productive. In the nonproductive stage, the virus maintains its genome as a low copy number episome by using the hostâ&#x20AC;&#x2122;s DNA replication machinery to synthesize its DNA in basal layer of the epithelium (Flores et al, 1997).The pattern of viral gene expression in these cells is not well defined, but it is generally believed that the viral E1 and E2 proteins are expressed in order to maintain the viral DNA as an episome (Wilson et al, 2002) and to facilitate the correct segregation of genomes during cell division (You et al, 2004).

II. HPV A. Morphology HPV is a relatively small (55nm diameter) nonenveloped virus. It has an icosahedral capsid composed of 72 capsomers, which contain at least two capsid proteins, L1 and L2. The HPV genome can be divided into three regions (Figure 1), the noncoding long control region (LCR), or the upper regulatory region (URR), and the early (E) and late (L) gene region (protein encoding). The long control region of 400 to 1,000 bp contains overlapping binding sites for many different transcriptional activators and repressors, including activating protein 1 (AP 1), and nuclear factor 1 (NF-I).

The LCR regulates transcription from the early and late regions, and therefore controls the production of viral proteins and particles. The early region is downstream of the LCR and contains six open reading frames, E1, E2 and E4â&#x20AC;&#x201C;E7, and is involved in viral replication and oncogenesis. These encode all viral proteins except for the

Figure 1. Schematic representation of HPV genome.

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Table 1. The functions of the products of HPV early region open reading frames Early region E1 E2 E4 E5 E6 E7

Protein functions Unwinds the DNA strands working with E2 protein Modulate the transcription activity of the E2 protein Enables E1 protein to bind to the viral origin of replication located within the LCR Encodes a LCR-binding protein that regulates transcription of the early region Encodes a protein that interacts with cytokeratin Expressed in later stages of infection, when complete virions are being assembled Augment cellular proliferation and DNA synthesis in a context of cell membrane receptors, such as EGF and PDGF Induces an increase in mitogen-activated protein kinase activity Binds to p53 and targets it for rapid degradation via a cellular ubiquitin ligase Induces telomerase activation Binds to the hypophosphorylated Rb proteins and liberate E2F, which results in S phase entry Interacts with inhibitors of cyclin dependent kinases Induces abnormal centrosome duplication resulting in aneuploidy

The productive stage of the viral life cycle occurs in the terminally differentiating suprabasal layers of the epithelium. In these cells, the virus switches to a rollingcircle mode of DNA replication and amplifies its genome to higher copy number, expresses late genes encoding capsid proteins, and produces viral progeny (Flores et al, 1999).

As a rule, in benign warts and pre-neoplastic lesions, the HPV genome is not integrated into the host cell genome (it is maintained in episomal form). However, in true neoplasia, it is wholly integrated into the host genome, although some authors have shown the coexistence of episomal and integrated forms in cervical cancer (Kristiansen et al, 1994).

The site at which the viral DNA is opened during this process of integration is fairly constant, and occurs within the E1/E2 open reading frame of the viral genome. The E2 protein can function as either an activator or repressor of viral gene transcription depending upon the location of the E2-binding sites within the promoter region of the viral genome (Bernard et al, 1989; Phelps et al, 1987).

However, as the E2 region of viral DNA normally represses the transcription of the E6 and E7 early viral genes, this interruption causes E6 and E7 protein overexpression (Finzer et al, 2002). After all, while HPV integration means the end of the viral life cycle due to the functional inactivation of large parts of the viral genome, it leads to de-regulated expression of the viral oncogenes E6 and E7.

Many experimental studies have investigated genomic HPV integration sites, and although integrated HPV genomes have been observed to show preferences for relatively few loci, no general integration hot spot has been identified. A recent review of integration sites confirmed that HPV integration sites are randomly distributed over the whole genome with a clear predilection for ‘fragile’ sites. No evidence supporting the targeted disruption or functional alteration of critical cellular genes by the integrated viral sequences has been unearthed.

III. Molecular pathophysiology in cervical cancer In normal epithelium, basal cells are sequestrated from the cell cycle following migration into the suprabasal cell layers and are committed to terminal differentiation.

During HPV infection, E7 and E6 are expressed in these cells, and abolish cell cycle progression restraints and delay normal terminal differentiation (Sherman et al, 1997). The effects of E6 and E7 on p53 and pRB and on many other cellular proteins have been extensively investigated, and significant alterations in cell cycle regulation can be attributed to the biochemical interactions between these two viral oncogenes and their respective cellular binding partners (Munger et al, 2001). Moreover, recently it was demonstrated that the E6 and E7 cooperatively disturb the mechanisms of chromosome duplication and segregation during mitosis, and thereby induce severe chromosomal instability (Duensing et al, 2001).

A. HPV E7 and retinoblastoma protein (pRb) The critical role of HPV E7 protein in the malignant transformation of cervical epithelial cells is attributed to its effects on pRb, a member of the ‘pocket protein’ family, which also includes p107 and p130 (Vogelstein et al, 1993). The proliferation of normal human cells follows an orderly progression through the cell cycle under the influence of cyclin/cyclin dependent kinase (cdk) complexes. Each cyclin/cdk complex control a specific cell cycle transition, the key downstream targets of the G1 phase cyclin/cdk complexes are members of the pRb family, i.e., Rb, p107, and p103. During G1 progression, Rb is sequentially phosphorylated by cyclin D1/cdk4 and 6 and cyclin E/cdk2 complexes, whereas hypophosphorylated pRB represents the active form and inhibits S phase entry, thus the sequential phosphorylation of Rb inhibits the repressor activity of pRb. The repressor 421

Jo and Kim: Implications of HPV infection in uterine cervical cancer activities of pRB and of the related pocket proteins p107 and p130, are mediated by members of the E2F family of transcription factors. In G0/G1 hypophosphorylated pRB is bound to E2F. Since pRB encodes a transcriptional repressor domain, pRB/E2F complexes function as transcriptional repressors. Following phosphorylation by cdk in G1, pRB/E2F complexes dissociate and E2F acts as a transcriptional activator, which results in S phase entry (Weinberg, 1995) (Figure 2). However, the virally encoded protein, E7, binds to hypophosphorylated pRb and displaces the E2F transcription factor from pRb. Thus, binding to Rb by E7 is essentially for E7-induced cells transformation, because E7 proteins, which are associated with HPV strains with a low cervical cancer risk, show little or no affinity for pRb23.

As for the E7/pRB interaction, E7 targets pRb for ubiquitin-mediated degradation by the proteasome (Boyer et al, 1996). Moreover, it was suggested that the cellular transformation activity of E7 tightly correlates with its ability to degrade pRB (Jones and Münger, 1997). It appears that pRB degradation, not solely binding, is important for the E7-induced inactivation of pRb (Gonzalez et al, 2001).

In addition to regulation by phosphorylation and dephosphorylation, cdks are regulated by a group of functionally related proteins called cdk inhibitors. In differentiating epithelial cells, high levels of cdk inhibitors (p21cip1 and p27kip1) can lead to the formation of inactive complexes consists of E7, cyclinE/cdk2 and either p21 or p27. As a result, it appears that during HPV infection, the ability of E7 to stimulate S-phase entry is limited to a subset of cells with low levels of p21/p27, or to cells which express high enough levels of E7 to subvert the block to S-phase entry. However, recent studies suggest that E7 can also interfere with the activity of the cyclindependent kinase inhibitors p21cip1 and p27kip1 and thus override normal G1 checkpoint control (Jones et al, 1997; Zerfass-Thome et al, 1996). In addition, to cyclin/cdk complexes and cdk inhibitors, E7 can also associate with

other proteins involved in cell proliferation, including histone deacetylases (Antinore et al, 1996) and components of the AP-1 transcription complex (Longworth et al, 2004), through p21 upregulation.

B. HPV E6 and p53 Although, E7 protein can independently immortalize various human cell types in tissue culture, efficiency is increased when E7 and E6 are coexpressed (Munger et al, 1989). Thus, E6 protein is believed to complement the role of E7 protein, and prevent apoptotic induction in response to unscheduled S-phase entry mediated by E7. However, the importance of HPV E6 in cancer appears to be primarily due to its effects on the cellular tumor suppressor gene, p53. The most commonly found alterations to p53 in cancers, such as, colon, breast, and lung cancer, are deletion, insertion, and point mutation (Bartek et al, 1990; Rodrigues et al, 1990; Takahashi et al, 1991). The p53 gene negatively regulates the cell cycle and “loss of function” mutation in p53 is required for tumor formation (Hollstein et al, 1991; Levine et al, 1991).

Up to 99% of invasive cervical carcinomas have been found to contain HPV 16 or 18 DNA and in these few are found without evidence of HPV p53 mutations (Crook et al, 1992).

Normally p53 is transiently upregulated after DNA damage, which leads to cell cycle arrest in the G1 phase and apoptosis. This arrest allows time for DNA repair, and if repair is not possible, cells are committed to apoptotic death. p53 acts through downstream regulators, such as p21, which leads to cdk inhibition and the eventual blockade of Rb gene phosphorylation, thus preventing cell cycle progression. However, virally encoded E6 binds to a cellular ubiquitin/protein ligase, E6-AP, and simultaneously to p53, which results in the ubiquitination of p53 and its subsequent proteolytic degradation (Ferenczy et al, 2002) (Figure 3).

Figure 2.

Sequential phosporylation of Rb by cyclin/cdk complex inhibits the repressor activity of pRb. The HPV E7 binds to the hypophosphorylated form of the pRb proteins. This binding disrupts the complex between pRB and the cellular transcription factor E2F, resulting in the liberation of E2F, which allows the cell to enter the S phase of the cell cycle.

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Cancer Therapy Vol 3, page 423 Figure 3. DNA damage induces p53 activation, leading to either cell cycle arrest or apoptosis. The HPV E6 binds to E6-AP and redirects it to p53, which results in the E6-APmediated ubiquitination and rapid proteasomal degradation of p53.

E6 proteins of high oncogenic risk HPVs, i.e., HPV 16 and 18, have a higher affinity for p53 than lower oncogenic risk types (HPV 6 and 11) (Crook et al, 1991; Lechner et al, 1994). Moreover, although the association between E6 and p53, and the inactivation of p53-mediated growth suppression and/or apoptosis have been well documented, this association can also lead to apoptosis via changes in the expressional levels of Bax and Bcl-2 family members (Selvakumaran et al, 1994). Consequently, the presence of E6 is considered to predispose the development of HPVassociated cancers, by allowing the accumulation of chance errors in host cell DNA to go unchecked.

Moreover, the E6 protein of high-risk HPV types can also stimulate cell proliferation independently of E7 through its C-terminal PDZ-ligand domain (Thomas et al, 2002) Both the p53 and Rb proteins interact with the double minute 2 gene (MDM2). P53 acts as a transcriptional activator of MDM2, whereas MDM2 acts in an autoregulatory fashion by providing negative feedback to p53 transcription. MDM2 can also interact with Rb and restrain its action. One study found that MDM2 was overexpressed in up to 35% of cervical tumors, although this was not found to be correlated with HPV infection (Momand et al, 1992).

pro-apoptotic species (Bax, Bad and Bid). Thus, Bax promotes apoptosis whereas Bcl-2 represses apoptosis.

E6 has been shown to prevent apoptosis in both a p53-dependent and a p53-independent manner (Pan et al, 1995). p53 is one of the best known inducers of apoptosis, but its activity in HPV-infected cells is countered by viral E6 protein. Moreover, E6 can damage p53 by targeting it for ubiquitin-mediated proteolysis. However, E6 has been shown to prevent apoptosis in both a p53-independent and dependent manner. Recent research into HPV E6 oncogenic properties found that pro-apoptotic effect of Bak is a target of anogenital HPV E6 protein, and that this proceeds in a p53-independent manner (Thomas et al, 1998). E6 proteins from HPV-18, HPV-16 and HPV-11, can all bind Bak in vitro and stimulate its degradation in vivo, and this Bak downregulation was found to induce apoptosis via a E6AP-dependent process (Thomas et al, 1998). In fact, Bak was found to bind E6-associated protein (E6AP) in the absence of E6 unlike p53 (zur Hausen, 2000).

D. HPV and telomerase Normal DNA replication leads to the erosion of chromosomal telomere termini, which leads to chromosomal instability and finally to cellular senescence.

Senescence arises mainly as a result of telomere shortening (Horikawa et al, 2003).

Telomerase, a ribonucleoprotein that prevents telomere erosion, is expressed in certain cell types that undergo repetitive cell division. Moreover, telomerase activity is present in immortalized and cancer cells but not in normal cells. These relations indicate that telomerase activation is critically required for immortalization and malignant transformation. The loss of telomerase activity

C. HPV E6 interaction with Bak Apoptosis, or programmed cell death, triggers a series of events that lead to the expeditious elimination of unwanted cells. In actively proliferating tissues, intrinsic apoptotic signaling is controlled by members of the Bcl-2 gene family, which are critical regulators of mitochondrial integrity and of mitochondria-initiated apoptosis. This family contains both anti-apoptotic (Bcl-2/Bcl-XL) and

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Jo and Kim: Implications of HPV infection in uterine cervical cancer in normal cells results in gradual decrease in telomere length with successive cell cycle rounds (Veldman et al, 2001).

Telomerase activity is regulated at the level of telomerase reverse transcriptase (hTERT), the catalytic telomerase subunit. The ectopic expression of hTERT in cancer cells was found to cause cellular immortalization (Blasco et al, 2003). Recently it was shown that cervical carcinoma cells expressing integrated copies of the HPV genome show high telomerase activity (Baege et al, 2002; Singh et al, 2004). Moreover, it was demonstrated that expression of telomerase correlated significantly with the histological severity of the cervical disease (Park et al, 2003).

Multiple factors have been shown to up-regulate expression hTERT, including c-myc and viral oncoproteins (Greenberg et al, 1999; Wu et al, 1999).

High-risk E6 is believed to induce telomerase activity during progression to malignancy (Klingelhutz et al, 1996), but the underlying mechanism remains elusive.

However, it has been postulated that E6 interacts with cmyc and the c-myc/E9 complex synergistically to promote E6-mediated hTERT induction (Oh et al, 2001). Direct stimulation of hTERT promoter and prevention of the inhibitory effects of p53 have been suggested as an alternative mechanism of telomerase maintenance in cervical cancer cells (Seo et al, 2004).

ras oncogene to transform epithelial cells (Storey et al, 1993; Phelps et al, 1988). However, research data in literature remain contradictory on the role of ras and HPV oncogenes and their probable co-operation in the pathogenesis of cervical neoplasm. In a recent study, any point mutation in codons 12 and 13 of K-ras gene was not identified in tissue from high-grade cervical dysplasia and invasive cervical carcinoma (Pochylski et al, 2003).

Downregulated c-Myc (a protooncogene) expression, accelerates cell proliferation and cell transformation, and occurs frequently in human tumors (Brenna et al, 2002). In normal cells, Myc is required for cell cycle entry, and its overexpression results in apoptosis after growth arrest has been induced by an external insult. On the other hand, in cancers its overexpression cause cell cycle re-entry and acts as an angiogenic switch. The gene encoding c-Myc protein is located on chromosome 8q24, the locus within which the HPV 16 sequence is integrated, which suggests that HPV integration into the fragile c-Myc region may be important element of HPV-induced oncogenesis.

Moreover, amplification and/or overexpression of the cmyc gene were frequently observed in advanced-stage cervical cancers, and these were shown to be associated with tumor progression (Covington et al, 1987). Moreover, c-myc overexpression was found to be related to a higher risk of distant metastases, which suggests that the activation of this proto-oncogene may lead to metastatic ability.

Alteration of the cell cycle regulatory gene CDKN2A also seems to be involved in HPV-associated carcinogenesis. The CDKN2A locus on chromosome 9p21 codes two proteins with different functions, the cyclindependent kinase inhibitor (CDKI) p16INK4a and p14ARF. While p16INK4a prevents S-phase entry by inhibiting CDK4/6-mediated phosphorylation of retinoblastoma (RB), p14ARF is a key trigger of p53 stabilization in response to oncogenic signaling (Serrano, 1993; Zhang, 1999). Although alterations of p16INK4A and p14ARF have been reported in some tumor types (Sharpless, 1999) the role of p14ARF/p16INK4a alteration in cervical cancer is less understood. Contradictory results have been reported about the expression of p16INK4a in cervical cancer. If, on the one hand, reduced expression of p16INK4a in cervical cancer has been reported in some studies (Nakashima et al, 1999), on the other hand, other studies have shown the overexpression of p16 INK4a in cervical cancer (Murphy et al, 2003; Sano et al; 1998). As for p14ARF, expression level is poorly investigated in cervical cancer. Because p14ARF expression is positively regulated by the E2F transcription factor and negatively regulated by p53 at the transcription level, HPV E6/E7 oncoprotein can be postulated to upregulate p14ARF expression. In a recent study, it has been shown that p14ARF and p16INK4A were overexpressed in HPV-positive cervical cancers as a consequence of HPV E6 and E7 expression (Kanao et al, 2004).

E. Other oncogenes Although, the over-expressions of E6 and E7 appear pivotal in the development of cancer, their expressing is not enough either for the immortalization of cultured human cells, or for malignant conversion. Rather, other specific genetic abnormalities are important during cervical carcinogenesis and the aggressiveness of cervical tumors. In addition to p53 and Rb, the ras family genes (K-ras, H-ras and N-ras) and c-myc oncogene might also have a role in the pathogenesis of cervical cancer (Baker et al, 1998; Bourhis et al, 1990; Dokianakis et al, 1998; Garzetti et al, 1998; Riou et al, 1987). Each of these genes has been reported to be overexpressed in cervical cancer and several of them have been associated with a poor prognosis.

The H-ras, K-ras, and N-ras genes are localized to chromosomes 11, 12, and 1, respectively, in humans. The three genes have a common structure and all encode 21kDa (p21) protein, 189 amino acids long, with GTPase activity, which participate in cellular signal transduction.

The activation of ras oncogenes by point mutations has been suggested to play an important role in the multistep process of carcinogenesis. The most frequent ras alterations in human cancer are mutations in codons 12, 13 and 61, which abolish p21 GTPase activity, thus rendering p21 constitutively activated. The over-expressions of ras genes has been reported in several human cancers, including those of the breast, colon, head and neck, bladder, and lung, and these have been associated with disease development. Elevated ras p21 protein expression was also reported in cervical tumors as opposed to benign or premalignant lesions. In in vitro studies, it was demonstrated that E6 and E7 can co-operate with activated

F. E6 and E7 oncoprotein and chromosomal instability Growing evidence indicates that a significant proportion of solid tumors show unstable aneuploidy, alteration in the number of chromosomes. It is shown that 424

Cancer Therapy Vol 3, page 425 aneuploidy in cervical dysplasia is associated with the presence of high-risk HPVs (Rihet et al, 1996; Kashyap et al, 1998). Furthermore, a number of chromosome aberrations have already been involved in pre-invasive high-risk HPV-associated cervical lesions (Steinbeck, 1997; Bulten et al, 1998). In particular, aneuploid cervical intraepithelial lesions have a significantly higher risk for carcinogenic progression, which strongly supports the concept of genomic instability as a hallmark for cervical carcinogenesis (Bibbo et al, 1989) The precise mechanisms underlying aneuploidy remain unclear, but it is believed to stem from an imbalance in chromosomal segregation (Pihan et al, 1998), which results from the unusual amplification of centrosomes (Chial et al, 1999) and/or the dysfunction of centromeres/kinetochores. In addition, certain viral oncoproteins have been implicated in the induction of chromosome copy number changes. In cervical cancer, it has been shown that expression of HPV E7 alone can be sufficient to induce a moderate level of aneuploidy, whereas the E6 oncoprotein renders cells prone to structural chromosomal changes (White et al, 1994). When both oncoproteins were co-expressed, an elevated level of aneuploid cells was found, indicating that HPV-16 E7 induces centrosome-related mitotic disturbances that are potentiated by HPV-16 E6 (Duensing S et al, 2000). Riley et al also reported that both E6 and E7 increased centrosome copy number and created invasive cancer when it acts in combination (Riley et al, 2003). In a more recent study, Schaeffer et al demonstrated that expression of either E6 or E7 interferes independently with the centrosome cycle, resulting in centrosome aberrations (Schaeffer et al, 2004).

the DQB1 03 alleles have an increased risk of HPV infection and cervical cancer (Maciag et al, 2000; Odunsi et al, 1997), and this association was observed for preinvasive and invasive disease and was found to be valid regardless of ethnicity. Moreover, increased risks of cervical cancer and CIN were observed for the DRB115 allele and the related DRB1 1501-DQB1 0602 haplotype among Hispanic and Swedish patients (Apple et al, 1994; Sanjeevi et al, 1996). Conversely, evidence suggests that DRB1*13 and/or DQB1*0603 in German, American, and French populations (Breitburd et al, 1996; Hildesheim et al, 1998; Madeleine et al, 2002) are likely to protect against the development of cervical cancer However, despite the relative consistency of findings supporting a role for HLA in cervical carcinogenesis, several inconsistencies remain. The reasons of these inconsistencies are attributable to variations in regional HPV types, HPV subtypes, ethnic HLA allele patterns, and differences in HLA typing techniques. It is also possible that HLA alleles reportedly associated with cervical cancer are located in genes that are in linkage disequilibrium with the actual gene or genes responsible for this additional risk. Variations in linkage would be expected between populations, and would be expected to result in the discrepancies described above. Finally, susceptibility may depend on interactions between immune response genes, and the specific alleles identified to date may represent only a portion of an extended haplotype that regulates immune response to HPV.

B. Single (SNPs)

nucleotide

polymorphisms

The p53 codon 72 polymorphism, which codes for either arginine (ARG) or proline (PRO), was first demonstrated by Storey et al. to be associated with cervical cancer and this has since been confirmed by numerous other investigators (Storey et al, 1998). The ARG/ARG genotype was found to confer an elevated risk for cervical cancer compared with the ARG/PRO genotype, by enhancing the binding and degradation of p53 by oncogenic HPV E6. Individuals with the homozygous arginine form were found to be seven times more susceptible to HPV-related carcinogenesis than heterozygotes (Storey et al, 1998). However, subsequent reports failed to corroborate these findings, and others were inconclusive. These observed discrepancies could be related to the ethnicities of the populations studied, as this polymorphism is known to differ by geographic region.

In a meta-analysis by Koushik and colleagues (2004), P53Arg homozygosity was not associated with an increased risk of cervical neoplasia (OR=1.1; 95% CI, 0.9 to 1.3) as compared with P53Pro homozygosity and P53Arg/Pro heterozygosity. This association was found to be statistically significant for invasive lesions but not for preinvasive lesions, and was also found valid to a lesser extent in squamous cell carcinoma (OR=1.5; 95% CI, 1.2 to 1.9) and adenocarcinoma (OR=1.7; 95% CI, 1.0 to 2.7).

However, reports published after this meta-analysis continued to be contradictory. Positive associations have been reported between cervical cancer and P53Arg homozygosity in Chile (Ojeda et al, 2003) and Mexico

IV. Genetic components in cervical cancer A. Allelic variation Certain HLA alleles or haplotypes seem to be involved in susceptibility to HPV infection and cervical neoplasia, probably by modulating immune response against HPV infection, and ultimately interfering in the establishment of productive persistent infections and cervical lesions. To date, the majority of HLA and cervical neoplasia studies have focused on the HLA Class II genes, since HLA II molecules are known to be responsible for the presentation of foreign antigens to the immune system.

The strongest associations have been found for genes in the HLA class II region (Coleman et al, 1994; Evans et al, 2001; Maciag et al, 2000). In particular, the class II DQ allele shows evidence of allelic association with cervical neoplasia in HPV-positive patients. Three groups of alleles/haplotypes known to be associated with cervical neoplasia have been extensively studied and include (1) DQB1*03 alleles (including DQB1*0301, DQB1*0302, and DQB1*0303); (2) DRB1*1501 and DQB1*0602 alleles; and (3) DRB1*13 (consisting of DRB1*1301-/5 alleles) and DQB1*0603). Among these, DQB1*03 alleles, and DRB1 1501 and DQB1 0602 alleles appear to increase the risk of cervical disease (Hildesheim et al, 2002). The most consistent finding is that individuals with

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Jo and Kim: Implications of HPV infection in uterine cervical cancer (Sifuentes Alvarez et al, 2003), whereas no associations were found in Argentina (Abba et al, 2003), central Italy (Cenci et al, 2003), or in Korea (Kim et al, 2001).

infections. Although age dependent, the prevalence of HPV infection among sexually active young women is in the range of 5-40% (Ho et al, 1998; IARC Working Group, 1995; Melkert et al, 1991). Further, most HPV infections are transient. It is estimated that newly diagnosed HPV infections will clear within 12â&#x20AC;&#x201C;18 months in approximately 80% of women, as the humoral immune system is brought to bear on the virus. Even low-grade precancerous lesions do not always progress into highgrade lesions. Instead, a cytotoxic T cell response is elicited against HPV-infected keratinocytes in the majority of cases. This suggests that other factors are involved in the carcinogenesis of cervical cancer. Smoking, high parity, and the long-term use of oral contraceptives are considered proven co-factors. Others are being scrutinized by ongoing research. Figure 3 depicts a multifactorial model of cervical cancer etiology.

C. Epigenetic changes Mutations (both point mutations and deletions) are not the only way in which a tumor suppressor gene can be inactivated, and in recent years the importance of epigenetic changes in the establishment of the malignant phenotype has been illuminated. In malignancies, some tumor suppressor genes are not transcribed because their promoter regions are methylated. Common examples of tumor suppressor genes that are inactivated by promoter region hypermethylation are provided by INK4A locus and RASSF1A (Das et al, 2004).

Of the hypermethylation events studied in association with carcinogenesis, promoter CpG island hypermethylation has been frequently investigated in many human cancers, including cervical cancer (Esteller et al, 2001; Muller et al, 1998; Virmani et al, 2001; Yang et al, 2004). CpG islands are often associated with promoter regions, and hypermethylation of these regions, which is probably the best characterized epigenetic change, is associated with transcriptional silencing of the associated gene, and thus provides a DNA-based surrogate marker of expression status. Moreover, it has been increasingly recognized over the past 4â&#x20AC;&#x201C;5 years that the CpG islands of a large number of genes, which are unmethylated in normal tissue, are methylated to varying extents in many human cancers, and that these methylations are a potential means of tumor suppressor gene inactivation (Kang et al, 2005).

At present, there is some evidence that increased rates of hypermethylation of various genes may be associated with cervical cancer. Dong et al, (2001) showed that promoter hypermethylation of at least one of the genes p16, DAPK, MGMT, APC, HIC-1, and E-cadherin occurred in 79% of cervical cancer tissues and in none of normal cervical tissues from 24 hysterectomy specimens.

Virmani et al, (2001) detected aberrant methylation of at least one of the genes p16, RARĂ&#x;, FHIT, GSTP1, MGMT, and hMLH1 in 14 of 19 cervical cancer tissue samples. In addition its implications in cervical tumorigenesis, DNA promoter hypermethylation are being investigated as a novel diagnostic target based on methylation-sensitive PCR techniques. Recently, Feng et al reported similar promoter methylation patterns in genes from exfoliated cell samples and corresponding biopsy specimens.

Furthermore, the frequency of hypermethylation increased statistically significantly with increasing severity of neoplasia present in the cervical biopsy. (Feng et al, 2005).

A. Smoking Smoking has long been associated with cervical cancer risk after Winkelstein first proposed the hypothesis that smoking is a risk factor of cervical cancer (Winkelstein Jr, 1977). This hypothesis has been supported by subsequent epidemiological studies (Castellsague et al, 2003), although it has been found difficult to rule out residual confounders, chiefly arising from sexual habits known to be related to both smoking and cervical cancer.

Various mechanisms have been proposed to explain the association between smoking and cervical cancer.

Tobacco is able to induce its carcinogenic effect in sites not directly exposed to cigarette smoke, as in pancreatic, kidney and bladder cancer (IARC Working Group, 1986).

In the cervix, it is possible to detect nicotine derivatives like nicotinine and tobacco specific nitrosamines. In addition, DNA adducts and other evidence of genotoxic damage are detectable in exfoliated cervical cells (Szarewski et al, 1998). It has been shown that smoking affects the ability of the host to mount an effective local immune response against viral infections in the cervix, and smokers show reductions in the number of Langerhans cells and in other markers of immune function (Poppe et al, 1995). Another possibility concerns the systemic effect of smoking, whereby the metabolisms of female hormones are altered.

Despite the consistency of the association between smoking and cervical cancer after adjusting for sexual behavior, it is not generally agreed that confounding can be ruled out as an explanation for this finding, since sexual behavior is evidently associated with transmission. Now that HPV has been identified as the principal cause of cervical cancer, it should be possible to resolve controversies over smoking. However, relatively few smoking targeted studies have incorporated adjustment for HPV infection A recent review of the relation between smoking and cervical cancer found consistent associations after adjusting for HPV-DNA or restricting analysis to HPVpositive women (Szarewski et al, 1998). These findings among HPV-positive women concur with subsequent studies (Deacon et al, 2000; Hildesheim et al, 2001;

V. Co-factors of cervical cancer It has been well established that a persistent infection in combination with high-risk HPV is the main risk factor of cervical cancer. Although HPV infection is necessary for the genesis of cervical cancer and its precursors, HPV infection alone is by no means sufficient cause.

Subclinical, clinical, and latent HPV infections are considered the most common sexually transmitted

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Cancer Therapy Vol 3, page 427 Plummer et al, 2003). Interestingly, in a multi-center case–control study of cervical adenocarcinoma, Lancey et al. reported a negative association between smoking and cervical adenocarcinoma (i.e., current smokers: OR = 0.6, 95% CI 0.3±1.1) and a marginal positive association between smoking and the risk of squamous cell carcinoma (e.g. current: OR = 1.6, 95% CI 0.9±2.9) (Lacey Jr et al, 2001).

Although a number of studies show that smoking is associated with an increased cervical cancer risk, further research using prospective designs that are well-controlled for HPV markers, confounding factors, and histologic types are needed to determine the nature of the relationship between smoking, HPV infection, and cervical cancer risk.

(Jay et al, 2000; Conley et al, 2002). In addition HIVpositive women have been reported to have higher rates of HPV infection (40% to 95%) and CIN lesions (10% to 36%) than HIV-negative women (23% to 55% and 1% to 12%, respectively) (Ellerbrock et al, 2000; Moscicki et al, 2000; Ferenczy et al, 2003) A meta-analysis by Mandelbaltt and colleagues concluded that HIV is a cofactor of HPV-related cervical carcinogenesis, and that this association seems to vary with immune function level (Mandelblatt et al, 1999).

Although the biologic mechanism for this interaction is not well understood, it is explained as being due to the effect of HIV infection on the immune system and a molecular interaction between HIV and HPV.

C. Oral contraceptives

B. Sexually transmitted diseases

Steroid contraceptive hormones have been identified to be a cofactor of HPV-related cervical carcinogenesis in many, but not all, epidemiological studies. However, although some epidemiologic studies have produced inconsistent results, the majority of studies have found that prolonged used of these agents increases the risk of cervical cancer (Moreno et al, 2002; Castellsague et al, 2003).

Little data is available about the mechanisms by which OCs increase the risks of acquiring or progressing HPV infection to cervical cancer. Two possible mechanisms have been proposed, i.e., increased exposure of the transformation zone to potential carcinogens and increased cell proliferation and transcription. An increased incidence of cervical ectropion has been reported among OC users, and this would increase the likelihood of transformation zone exposure to HPV and other potential carcinogens. Moreover, the hypothesis concerning the stimulation of cell proliferation and HPV transcription by estrogens and progesterone is gaining support. Steroids are believed to bind to specific DNA sequences within transcriptional regulatory regions on HPV DNA, to either increase or suppress the transcriptions of various genes (de Villiers et al, 2004; Moodley et al, 2003).

Results from epidemiologic studies, in which HPV status was controlled for, demonstrate, in most cases, positive correlations between OC use and cervical cancer risk. Kruger-Kjaer et al reported a pattern of decreasing risks of ASCUS, LSIL and HSIL with years with OC use among HPV DNA positive women (Kruger-Kjaer et al, 1998). Recently, the IARC’s pooled analysis of eight casecontrol studies reported that the odds ratio of cervical cancer resulting from the use of oral contraceptives was 2.82 (95% CI 1.46–5.42) for 5–9 years, and 4.03 (2.09–8.02) for use for 10 years or longer (Moreno et al, 2002). In another recent review article, Smith et al. concluded that the relative risk of cervical cancer increases with oral contraceptive use duration (Smith et al, 2003).

These findings are consistent for HPV positive women and after adjusting for HPV status. However, confounding must also be considered because women using contraception are more likely to be sexually active.

Further, barrier methods of contraception have been shown to protect against cervical intraepithelial neoplasms and cervical cancer. Moreover, detection bias may also

HSV-2 has been found to be carcinogenic in both in vitro and in vivo studies. Several possible mechanisms for the role of HSV-2 in cervical cancer have been suggested.

It was hypothesized that HSV-2 and HPV may act synergistically with HSV-2 to initiate mutations and carcinogenesis in HPV-infected cervical cells (de Sanjose, 1994; Zur Hausen, 1982). However, because of a lack of consistency in detecting HSV-2 DNA in cervical cells, it has been postulated that a “hit-and-run” mechanism may play a role in the initiation of cervical cancer (Galloway et al, 1983).

Serologic studies showed a higher prevalence of HSV-Ab in women with cervical neoplasia than in controls (de Sanjose et al, 1994; Dillner et al, 1994). The results of a pooled analysis of case-control studies conducted by IARC support the role of HSV-2 as a cofactor of HPV infection in cervical cancer (Smith et al, 2002a). In this study, HSV-2 seropositivity was found to be significantly higher among women with invasive squamous cell carcinoma (44.4%) and adeno- or adenosquamous carcinoma (43.8%) than in control women (25.6%); this association was observed after adjusting for potential confounders.

A consistent but modest association between the presence of serum Ig G antibodies to C. trachomatis and cervical cancer has been reported in epidemiological studies (de Sanjose et al, 1994; Dillner et al, 1994).

However, in other studies, infection with Chlamydia trachomatis was not found to be associated with the presence of HPV (Burger et al, 1996). In a IACR multicenter study, C. trachomatis seropositivity increased the risk for cervical cancer among HPV-positive women by 2.1-fold (Smith et al, 2002). In all, it appears that the lack of consistency shown by studies suggests that residual confounding due to HPV may have affected the finding of a positive association between cervical cancer and C. trachomatis.

Numerous studies have addressed the association between HIV and cervical neoplasia (Boyle et al, 1999), and the Center for Disease Control and Prevention included invasive cervical cancer in its definition of AIDS. Moreover, HIV-positive women have been reported to have higher rate of cervical abnormalities, larger lesions, and a higher recurrence rate than HIV-negative women 427

Jo and Kim: Implications of HPV infection in uterine cervical cancer affect these results, because women using oral contraceptives have more frequent gynecologic visits than non- users, and therefore, precancerous lesions are more likely to be detected and treated.

HPV VLPs in mice was found to induce systemic virusneutralizing antibodies (Rose et al, 1999).

Other important issues must also be resolved, such as, age at time of administration, booster shot timing, and whether or not to vaccinate men. Finally, given that cervical cancer does not develop in the vast majority of women infected with HPV, economic benefit should also be taken into consideration. In addition, because cervical cancer remains important public health problem in lowincome countries, the cost of the vaccine for developing countries should also be consideration.

VI. HPV vaccine A. Prophylactic HPV vaccine DNA-free virus-like particles (VLP) synthesized by the self-assembled viral particles of the main structural HPV proteins, L1 protein (or L1 and L2 protein), induce strong humoral responses from neutralizing antibodies.

VLPs are thus the best candidate immunogens currently available for HPV vaccine trials. These VLPs are morphologically indistinguishable from the authentic virion, are non-infectious, and lack any oncogenic DNA. Several studies in animals have demonstrated that the parenteral injection of these VLPs, or even of the pentameric L1 capsomer, elicits high titers of serumneutralizing antibodies and protection (Breitburd et al, 1995; Kirnbauer et al, 1996) .

As for human studies, early phase I/II clinical trials using HPV L1 VLP delivered intramuscularly have demonstrated the immunogenicity and safety of this vaccine (Evans et al, 2001; Harro et al, 2001). Importantly, Koutsky et al recently reported on a clinical trial of HPV 16 L1 VLPs, and indicated for the first time that a vaccine strategy can be implemented in humans to prevent HPV16 infections and HPV-16â&#x20AC;&#x201C;associated premalignant lesions (Koutsky et al, 2002). In another clinical trial reported by Harper et al, HPV-16, 18 L1 VLP vaccines proved 100% effective at preventing the acquisition of persistent HPV infection (Harper et al, 2004).

Several important issues require careful consideration before anti-HPV vaccines are made available for mass immunization programs. Humoral immunity to VLP-based vaccines is not only species specific but also type specific. Therefore, the number of HPV types to be included as immunogens is a key issue in HPV vaccine development, although single-type-specific VLP vaccines have produced encouraging results. Data from a recent overview of information collated from several case-control studies indicated that a pentavalent vaccine with VLPs of HPV types 16, 18, 45, 31, and 33 could potentially prevent 83% of all cervical carcinomas (Munoz et al, 2004). However, it is evident that the gains achieved by type coverage rapidly diminish for vaccines containing more than four types. A quadrivalent HPV VLP vaccine (types 6, 11, 16, and 18) produced by Merck is currently undergoing clinical trial; preliminary results show that the vaccine is well tolerated and generates adequate neutralizing antibody titers (Brown et al, 2001).

In addition to the type of HPVs covered by vaccines, the route of delivery is also an issue. Although VLP vaccination provides immunity from experimental inoculation, protection against the sexual transmission of HPV requires neutralizing antibodies acting at mucosal surfaces. The nasal instillation of VLPs was found to be efficient at generating specific antibodies, including IgG in serum and IgG and IgA in the mucosal secretions of mice (Balmelli et al, 1998). More recently, oral vaccination with

B. Therapeutic HPV vaccine Although vaccination with prophylactic HPV vaccines can generate high titers of serum-neutralizing antibodies in animals and humans, this form of immunization may not be able to generate the therapeutic effects required to counter established or breakthrough HPV infections that have escaped antibody-mediated neutralization. Preexisting HPV infection is highly prevalent and is responsible for considerable morbidity and mortality.

The life cycle of a HPV infection is characteristically intracellular, noncytopathic, and nonlytic. Therefore, the goal of therapeutic vaccination is to induce specific cellmediated immunity targeting preexisting lesions or even malignant tumors. In the case of cervical cancer, viral peptides derived from high-risk HPV oncoproteins are tumor-specific antigens, because viral genes are selectively expressed during the malignant progressions of virally induced neoplastic lesions. As a result early viral antigens (i.e., E1, E2, E5, E6, and E7) could be candidate targets for therapeutic vaccine antigens. However, most HPV-associated cancers only express E6 and E7, and E5 shows limited immunogenicity, and thus has not been extensively studied as a vaccine antigen. Likewise, E4 and the L1 and L2 capsid proteins are unlikely to be suitable targets for therapeutic vaccine development, because these proteins are not detectably expressed in the basal epithelial cells of benign lesions or in the abnormal proliferative cells of premalignant and malignant lesions (Stoler et al, 1992). Furthermore, because E6 and E7 are required for the induction and maintenance of the malignant phenotype of cancer cells, cervical cancer cells are unlikely to evade an immune response through antigen loss. Thus, the majority of investigations on therapeutic vaccines are directed toward E6 and E7 antigens.

The various categories of therapeutic vaccines are; vector-based, peptide-based, protein-based, DNA-based, chimeric VLP-based, and cell-based. However, most studies have focused on E7, because it is more abundantly expressed and better characterized immunologically, and because its sequence is more conserved than that of E6 (Zehbe et al, 1998). A summary of prophylactic and therapeutic vaccines currently being studied is presented in Table 2.

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Cancer Therapy Vol 3, page 435 Cancer Therapy Vol 3, 435-442, 2005

Aggressive work-up is needed for menopausal women with atypical glandular cells of uncertain significance (AGUS) Pap smear Research Article

Dong Ock Lee1, Hoenil Jo1,2, Youn-kyung Chung1, Jae Weon Kim1,2*, Noh-Hyun Park1,2, Yong-Sang Song1,2, Soon-Beom Kang1,2, Hyo-Pyo Lee1,2 1

Department of Obstetrics and Gynecology, Cancer Research Institute, College of Medicine, Seoul National University, Seoul, Republic of Korea

2

__________________________________________________________________________________ *Correspondence: Jae Weon Kim, M.D., Department of Obstetrics and Gynecology and Cancer Research Institute, Seoul National University, 28 Yungun-Dong, Chongno-Ku, Seoul, 110-744, Korea. Tel: +82-2-760-3511. Fax: +82-2-762-3599. E-mail address: kjwksh@snu.ac.kr.

Key words: Atypical glandular cells of undetermined significance, AGUS, Pap smear Abbreviations: American Society for Colposcopy and Cervical Pathology, (ASCCP); Atypical Glandular Cells of Undetermined Significance, (AGUS); atypical glandular cells, (AGCs); cervical intraepithelial neoplasia, (CIN); computed tomographic, (CT); endocervical curettage, (ECC); endometrial biopsy, (EMBx); Loop Electrosurgical Excision Procedure, (LEEP); M. D. Anderson Cancer Center, (MDACC); National Comprehensive Cancer Network, (NCCN); Not Otherwise Specified, (NOS); punch biopsy, (p-Bx)

The first two authors contributed equally to this work.

Received: 25 May 2005; Accepted: 16 August 2005; electronically published: August 2005

Summary This study was performed to evaluate the clinical significance of AGUS Pap smear and to review recent studies on the histological outcomes of AGUS Pap smear. From April 1998 to March 2003, a total of 78,072 Pap smears were performed at Seoul National University Hospital. Among these, 87 were classified as AGUS, an incidence of 0.11%.

We reviewed the charts of these patients retrospectively to identify patients’ characteristics, symptoms, and followup and management methods. In addition, to comparing our data with others, we searched for studies on histological outcomes of AGUS Pap smear in Korea. Of the 87 AGUS cases, clinically significant diseases were diagnosed in 18 cases, which were; 7 endometrial cancers, 6 cervical adenocarcinomas, 3 squamous intraepithelial lesions, 1 ovary cancer, and 1 metastatic adenocarcinoma. The incidence of AGUS Pap smear was similar to that found in other Korean hospitals (0.09%). At other Korean hospitals, histologically significant diagnoses were reported with frequencies ranging from 9.3% to 80.5%, but the overall mean rate (34.1%) was similar to our data (31.6%). In an analysis of demographic characteristics, menopausal women and women with vaginal bleeding showed histologically significant disease more frequently, but age was not a statistically significant factor. Our study indicates that all patients with an AGUS Pap smear should undergo an aggressive work up. In particular, menopausal women and women with vaginal bleeding should undergo work up for underlying cervical or endometrial disease. The determination of a follow up method according to age does not appear appropriate.

Glandular Cells of Undetermined Significance). AGUS was defined as glandular cells exhibiting changes beyond reactive / reparative changes, but lacking the unequivocal features of invasive adenocarcinoma. In this system, AGUS was sub-divided into endocervical, endometrial, and not otherwise specified (Lundberg, 1989). The 1991 Bethesda System recommended that AGUS should be further sub-divided into; ‘reactive’, a ‘premalignant /

I. Introduction The 1988 Bethesda System was introduced as a guideline for cytologic cervical and vaginal specimen reports. It was devised to provide effective communication between cytopathologists and referring physicians and to facilitate cytological-histologic correlation. Atypical cells were classified as either ASCUS (Atypical Squamous Cells of Undetermined Significance) or AGUS (Atypical 435

Lee et al: Aggressive work-up for menopausal women with (AGUS) Pap smear the analysis. We reviewed the charts of these cases retrospectively and recorded age, obstetrical characteristics, symptoms, menopausal status, use of hormone replacement therapy, methods for evaluation, and final histologic outcomes.

No Pap smear case showed concurrent ASCUS or SIL and no case had a history of a previous hysterectomy.

All subjects were Korean, and Pap smears were collected using a Cytobrush by clinicians. All cervicovaginal smears was prepared on one slide and fixed immediately with 95% alcohol.

There was no recommended follow-up method for AGUS Pap smear, so follow-up methods were decided upon by gynecologists. In 11 cases, Pap smear was performed repeatedly every 3 to 6 months. In other cases, colposcopy directed punch biopsy (p-Bx), endocervical curettage (ECC), endometrial biopsy (EMBx), Loop Electrosurgical Excision Procedure (LEEP), or hysterectomy was performed separately or in combination. We reviewed the final histologic outcomes of these cases.

To compare data from our center with other hospitals’ data, we searched for studies on the histologic outcomes of AGUS Pap smear at other single institutions using the KoreaMed Search System, which provides access to medical journals published in Korea. Seven studies conducted from 1998 to 2002 that reported histologic outcomes of AGUS were reviewed, and 6 among these were found suitable for statistical analysis. All studies used the Bethesda system, but subclassification was not always performed. All patients with a preexisting gynecologic malignancy were excluded. Each clinician decided on appropriate methods for the histologic diagnosis of patients with AGUS Pap smear. The incidence of AGUS Pap smear, the age distribution of patients, and histologic outcomes were reviewed and compared with our data.

We regarded both preinvasive and invasive gynecologic neoplasms as clinically significant lesions. These included squamous intraepithelial lesions, cervical adenocarcinoma in situ, endometrial hyperplasia with atypia, cervical squamous cell carcinoma, cervical adenocarcinoma, endometrial adenocarcinoma, and cancers of other sites.

Statistical analysis was performed using chi square test of the standard error of the difference between percentages.

Statistical calculations were performed using SPSS software.

malignant process is favored’ or ‘Not Otherwise Specified (NOS)’ (Broder, 1992) However, AGUS subclassifications were not helpful in terms of allowing clinicians to rule out underlying pathology. Up to 10~15% of patients with AGUS-reactive process favored and up to 24~40% of patients with AGUS-NOS had significant findings on follow-up. Patients with AGUS on cervical smears need a thorough evaluation, regardless of subtyping (Veljovich et al, 1998; Reuss et al, 2001). In the 2001 Bethesda System, the classification of glandular abnormalities was significantly revised, reflecting a reappraisal of the strengths and weaknesses of cytology to assess these findings. The term ‘Atypical Glandular Cells of Undetermined Significance’ (AGUS) has been eliminated to avoid confusion with ASCUS, and the term ‘Atypical Glandular Cells’ (AGCs) used instead. The finding of AGCs was considered important clinically because the percentage of cases associated with underlying high-grade disease is higher than for ASCUS. Based on this the qualifier ‘reactive process favored’ was considered misleading and was eliminated, and AGUS was subclassified into ‘AGC’ and ‘AGC, favor neoplastic’ (Solomon et al, 2002) The incidence of AGUS (AGC) is relatively infrequent with reported frequencies ranging from 0.1% to 0.63%. Although infrequent, AGUS is commonly associated with cervical and endometrial abnormalities in the reported range 29% to 60.9% (Gary et al, 1997; Zweizig et al, 1997; Veljovich et al, 1998; Chin et al, 2000; Chhieng et al, 2001b; Reuss et al, 2001; Hammoud et al, 2002). These studies recommended that all patients with AGUS (AGC) on Pap smear, regardless of their subclassification, should undergo a prompt and aggressive work up because of the high risk of a final pathogenic diagnosis. However, the management of AGUS on Pap smears has not been clearly defined. The American Society for Colposcopy and Cervical Pathology (ASCCP), the National Comprehensive Cancer Network (NCCN), and the M. D. Anderson Cancer Center (MDACC) proposed guidelines for the management of women with atypical glandular cells, but their recommendations differ somewhat.

The purpose of this study was to evaluate the clinical significance of AGUS on Pap smears using data from a single institution and to compare this with similar data on the histologic outcomes of AGUS on Pap smears at other institutions in Korea, and thus, to determine the effects of patient factors such as age or menopausal status or symptoms of vaginal bleeding with respect to the risk of histologically significant findings.

III. Results From April 1998 to March 2003, the incidence of AGUS Pap smear was 0.11% at Seoul National University Hospital. Among the 87 subjects with an AGUS Pap smear, 8 subjects with history of a preexisting gynecologic malignancy were excluded.

For the remaining 79 cases the mean age was 48.2 years (range from 31 to 77), and mean gravity and parity were 4.4±2.9 and 2.8±1.8 respectively. 38 cases had a postmenopausal status (48.1%) and 11 cases were receiving hormone replacement therapy (13.9%). The age distribution of the study subjects is shown in Figure 1.

Eleven (13.9%) were receiving hormonal replacement therapy at the time of diagnosis. Among 53 cases (67.1%) had no clinical symptoms, 18 (22.8%) had a presenting symptom of vaginal bleeding, and 3 of these 18 had complained of low abdominal pain, 1 of primary infertility, 1 of dysmenorrhea, 1 of leukorrhea, 1 of uterine prolapse, and 1 of vulvar discomfort. Of the 79 patients, 11 were lost to follow-up, and of the remaining 68, 11 underwent only repetitive Pap smear every 3~6 months.

And their follow-up Pap smear results were all within normal limits or showed benign cellular changes.

II. Materials and methods From April 1998 to March 2003, a total of 78,072 Pap smears were obtained at Seoul National University Hospital.

Among them 87 Pap smears were classified as AGUS by the Bethesda system. Subclassifications of AGUS were not always used, and thus patient subclassifications were not identified.

Cytologic preparations were not submitted for a second review during our study.

Patients with a previous history of gynecologic malignancy were excluded, but all remaining 79 cases were. Cases with a history of squamous intraepithelial lesions were included from

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Cancer Therapy Vol 3, page 437 Figure 1. Age distribution of women with an AGUS Pap smear.

Biopsies for histologic diagnoses were done in 57 patients (83.8%). Three of these had a history of LEEP due to cervical squamous intraepithelial neoplasia and 3 were in a postpartum state. For histologic diagnosis, we performed colposcopy directed p-Bx, ECC, EMBx, the LEEP, or hysterectomy separately or in combination. The types of initial procedures performed in patients that underwent histologic evaluation are shown in Table 1, and according to the result of these initial procedures, additional procedures or operations were performed. Two cases underwent an operation without another histologic evaluation after AGUS Pap smear. One of these underwent hysterectomy due to a fibroid of the uterus and the histologic result revealed a normal endometrium and cervix. The other case underwent a Pap smear during diagnostic work up, and a CT scan showed a lesion with a

high probability of malignancy; she underwent explorative laparotomy, and was diagnosed as ACUP (Adenocarcinoma of Unknown Primary).

The histologic outcomes of patients with an AGUS Pap smear are shown in Table 2. Of the 57 women with a histologic follow-up, 18 (31.6%) had clinically significant findings. Seven cases (12.3%) had endometrial cancer, 6 cases (10.5%) cervical adenocarcinoma, 3 cases (5.3%) a squamous intraepithelial lesion, 1 case (1.8%) ovary cancer, and 1 case (1.8%) metastatic adenocarcinoma.

Lesions by site were as follows: cervix, 50% (9/18); endometrium, 38.9% (7/18); ovary, 5.6% (1/18); and other, 5.6% (1/18). Of the 18 clinically significant findings, 3 (16.7%) were of squamous origin and 15 (83.3%) were of glandular origin.

Table 1. Methods of histologic evaluation Methods

No. of patients (%)

p-Bx* + EMBx** + ECC*** + LEEP**** p-Bx + EMBx + ECC p-Bx + EMBx + LEEP p-Bx + EMBx p-Bx + ECC EMBx + ECC p-Bx only EM Bx only ECC only LEEP only Operation Total *p-Bx: colposcopy directed punch biopsy **EMBx: endometrial biopsy ***ECC: endocervical biopsy ****LEEP: loop electrosurgical excision procedure

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3 (5.3%) 21 (36.8%) 1 (1.8%) 8 (14.0%) 3 (5.3%) 4 (7.0%) 7 (12.3%) 6 (10.5%) 1 (1.8%) 1 (1.8%) 2 (3.5%) 57 (100%)

Lee et al: Aggressive work-up for menopausal women with (AGUS) Pap smear Table 2. The histologic outcomes of patients with an AGUS Pap smear Histologic Finding

No of patients

Frequency (%)

SIL

3

5.3%

Cervix squamous cell carcinoma (Cx SCCA)

0

0%

Cervix adenocarcinoma (Cx adenoCa)

6

10.5%

Endometrial adenocarcinoma (EM adenoCa)

7

12.3%

Ovary cancer

1

1.8%

Other cancer*

1

1.8%

Total histologically significant cases

18

31.6%

Negative/benign

39

68.4%

Total

57

100%

*Other cancer: ACUP (Adenocarcinoma of Unknown Primary) with metastatic adenocarcinoma

To compare this data with data from other hospitals in Korea, we reviewed 6 studies with respect to the histologic outcomes of AGUS Pap smears. The outcomes as determined by the other hospitals are shown Table 3.

The mean incidence of AGUS Pap smear was 0.09% ranging from 0.08% to 0.3%, and mean age was 44.6 years ranging from 47.0 years to 50.5 years. Histologically significant diagnoses were found in 34.1% of the patients ranging from 9.3% to 78.2%. And this result was similar to ours. These studies cite a histologic evaluation rate of

83.1% ranging from 39.1% to 100%. In our study, a histologic evaluation was performed in 83.8%.

To evaluate demographic variables, we analyzed the relationships between some demographic variables and histologic results. The results are shown in Table 4. This analysis showed statistically significant differences for menopausal status and vaginal bleeding which were associated with a greater risk of histologically significant disease (p=0.006 and p=0.001). But no statistically significant difference was found with respect to age.

Table 3. Incidence and histologic outcome of AGUS Pap smear in other Korean hospitals

Incidence

Mean age No. of patients with biopsy

Kim et al, 1998

Kim et al, 2000

Song et al, 2000

Park et al, 2001

Seok et al, 2002

Suh et al, 2002

Total

0. 08%

0.13%

0.3%

0.1%

0.09%

0.15%

0.09%

(326/407,451)

(87/67,730)

(47/17,744)

(23/19,400)

(104/115,555)

(16/10,807)

(603/638,687)

43.0

45.8

47.0

40.9

50.5

42.6

44.6

268 (82.2%) 61/268

87 (100%) 30/87

43 (91.5%) 4/43

9 (39.1%) 3/9

87 (83.7%) 68/87

7 (43.8%) 5/7

501 (83.1%) 171/501

Histologically significant diagnoses Negative/Benign

(22.8%)

(34.5%)

(9.3%)

(33.3%)

(78.2%)

(71.4%)

(34.1%)

207(77.2%)

57(65.5%)

39(90.7%)

6(66.7%)

19(21.8%)

2(28.6%)

330(65.9%)

SIL

28(10.4%)

7(8.0%)

2(4.7%)

2(22.2%)

9(10.3%)

2(28.6%)

50(10.0%)

AIS

5(1.9%)

0

0

0

9(10.3%)

2(28.6%)

320(63.9%)

Cervical glandular atypia/dysplasia Cervix SCCA

8(3%)

0

0

0

0

0

8(1.6%)

0

2(2.3%)

0

0

10(11.5%)

0

12(2.4%)

Cervix adenoCa

7(2.6%)

14(16.1%)

1(2.3%)

0

21(24.1%)

0

43(8.6%)

Endometrial adenoCa Ovary cancer

11(4.1%)

7(8.0%)

1(2.3%)

1(11.1%)

15(17.2%)

1(14.3%)

36(7.2%)

0

0

0

0

3(3.4%)

1(14.3%)

4(0.8%)

Other cancer

2(0.7%)*

0

0

0

1(1.1%)**

0

3(0.6%)

*One case of MMMT (Malignant mixed Mullerian tumor) and one case of metastatic adenocarcinoma ** One case of colon cancer

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Cancer Therapy Vol 3, page 439 Table 4. The relationships between demographic variables and histologically significant disease

Menopausal status Premenopausal Postmenopausal Vaginal bleeding With vaginal bleeding Without vaginal bleeding Age ! 35 _ 35 ! 40 _ 40

No of patients

Histologically significant diseases (Rate)

Statistical significance

28/57 29/57

4/28 (14.3%) 14/29 (48.3%)

p=0.006

15/57 42/57

10/15 (66.7%) 8/42 (19.0%)

p=0.001

9/57 48/57 15/57 42/57

2/9 (22.2%) 16/48 (33.3%) 3/15 (20%) 15/42 (35.7%)

p=0.510 p=0.261

(Lee, 1993). Three patients in our study were in a postpartum state; on postpartum days 20, 47, and 50. We performed colposcopy and directed biopsy, endocervical curettage, and endometrial biopsy on these patients, and obtained a benign result in all. This result may be explained by an outgrowth of the endocervix during pregnancy that often causes downgrowth of the endocervical mucosa beyond the external os, where it may be readily sampled (Ostegard, 1979). Chhieng et al. reported that the incidence of AGUS among pregnant and postpartum women was 0.26%, and that the frequency of a significant pathology by biopsy following an AGUS Pap smear in pregnant and postpartum women was 29.4%, which is similar to that in the general population. They recommended women with an AGUS Pap smear should be followed closely with an aggressive work up (Chhieng et al, 2001a).

Stratification of AGUS by subclassification for the assessment of underlying pathologic conditions was attempted, but no impact on the need for working up was found, as the benign subgroup had a 26.3~30% risk of a pathologic condition (Gary et al, 1997; Veljovich et al, 1998). Thus, we did not perform a subclassification of AGUS. Zweizig et al, evaluated multiple demographic variables and failed to identify any subset of women with AGUS who were at increased risk of cancer or of its precursors. Age, menopause, abnormal bleeding, and current hormone replacement therapy were not found to be correlated with a poorer pathologic outcome. However, no woman <35 years old had endometrial pathologic findings and thus they recommended an endometrial biopsy for the subset > 35 years old (Zweizig et al, 1997). Chin et al. concluded that postmenopausal status and abnormal vaginal bleeding are associated with endometrial or glandular disease. However, they agreed that an evaluation of the endometrium may be warranted only in patients >35 years old, because the majority of glandular lesions of endometrial origin occurred in postmenopausal women (Chin et al, 2000). Meath et al. recommended that an evaluation of the endometrium in all women with AGUS on Pap smears is warranted, until the safety of excluding certain patient groups is established, because more than

IV. Discussion The purpose of our study was to evaluate the clinical significance of AGUS Pap smear results from our data and to compare our results with those of others. The incidence of an AGUS Pap smear was 0.11%, which is similar to the overall incidence at other hospitals in Korea (0.09%), but somewhat lower than that in the United States (0.17%~1.8%). This may be explained by the different incidences of endometrial and cervical cancer, which is much lower in Korea than in the United States, although, the rate of cervical adenocarcinoma in Korea (9.2%) is similar to that in the United States (KSGO, 2003).

The rate of pathologically significant diagnoses in patients with an AGUS Pap smear was 31.6% at our center, and 34.1% at other hospitals in Korea. So we concluded that although the incidence of AGUS Pap smear in Korea differs from that of the United States, the meaning of AGUS Pap smear serves to provide the same warning of the possible presence of a gynecologic malignancy or premalignant lesion.

On the basis of our current review, those with an AGUS Pap smear should be considered for aggressive work up. Several studies have reported high rates of premalignant and malignant lesions in patients with an AGUS Pap smear, and authors agree that colposcopy and directed biopsy with endocervical curettage should be performed in patients with an AGUS Pap smear (Chin et al, 2000; Hammond et al, 2002). The question as to whether an endometrial biopsy should be used routinely is controversial, but it is generally recommended in older women (Veljovich et al, 1998). In our data, patients underwent various combinations of follow-up procedures because of the absence of a confirmed follow-up protocol for patients with an AGUS Pap smear. In Korea, procedures for histologic diagnosis are relatively inexpensive. For example, it takes only about 16~42 U.S. dollar for EMBx and 1~3 U.S. dollar for p-Bx. Thus physicians are free to choose procedures for a histologic diagnosis without undue burden.

In the present study, three patients with history of LEEP due to cervical intraepithelial neoplasia (CIN) were included. Their pathologic outcomes were all benign lesion. This may have been due to an alteration of the microscopic anatomy of the endocervix following LEEP 439

Lee et al: Aggressive work-up for menopausal women with (AGUS) Pap smear half of the malignancies identified in their review were of endometrial origin (Meath et al, 2002).

Chhieng et al. reported that the incidence of AGUS in postmenopausal women was 0.51% in their study, a finding that is similar to that in the general patient population (0.56%). The incidence of clinically significant lesions in postmenopausal patients with AGUS Pap smears was 33%, which is similar to that in the general population (29%) (P>0.05) (Chhieng et al, 2001b).

In our data, post-menopausal women and women with vaginal bleeding showed significantly higher rates of an abnormal histologic outcome, but age did not show a statistically significant difference.

At present, no formal recommendation has been issued by the American College of Obstetricians and Gynecologist for the standard management of AGUS Pap smear (Chin et al, 2000). The ASCCP recommended that all patients with an AGUS Pap smear, except those with atypical endometrial cells, should undergo colposcopy with endocervical sampling. The majority of patients with an initial diagnosis of AGUS will have a squamous lesion, CIN 2 or 3, so the initial recommended work-up for all patients should include colposcopy and endocervical curettage (ECC) (Levine et al, 2003). If patients are >35 years old or have a history of abnormal bleeding, endometrial sampling should be added to the work up.

Women with atypical endometrial cells should undergo endometrial sampling. Then if the first histologic work up reveals no invasive disease, 4 repeat cytologies at 4~6 month intervals are recommended. And if the initial Pap is â&#x20AC;&#x153;favor neoplasiaâ&#x20AC;? and the first histopathologic work up reveals no invasive disease, a diagnostic excisional procedure is recommended.

Other cancers, including those which arise from the fallopian tube, ovary, peritoneum, colon, and pancreas, have been found in those with an AGC Pap smear. Due to the low incidence of these cancers, there is no consensus for the further work-up of women diagnosed with AGC favor neoplasia and with no identifiable lesion in vagina, cervix, or endometrium. Pelvic ultrasound, dilatation and curettage, or hysteroscopy with the addition of the serum tumor markers like CA-125 may be applied in these cases, and a computed tomographic (CT) scan of the abdomen and pelvis can be beneficial in selected patients. The addition of colonoscopy and mammography might be reasonable based on personal and family history (Levine et al, 2003).

Our study supports the opinion that all the patients with an AGUS Pap smear should undergo an aggressive work-up. In particular, menopausal women and women with vaginal bleeding should undergo work up for underlying cervical or endometrial disease. A determination of the follow up method according to age does not seem appropriate.

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Acknowledgement This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (0412-CR01-0704-0001).

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Jae Weon Kim

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Cancer Therapy Vol 3, page 443 Cancer Therapy Vol 3, 443-460, 2005

Epithelial to mesenchymal transition, cell surface receptors activation and intracellular communications in cancer metastasis Review Article

Wael M. ElShamy Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA

__________________________________________________________________________________ *Correspondence: Wael M. ElShamy, Ph.D., Dana-Farber Cancer Institute and Harvard Medical School, 44 Binney St., SM816, Boston, MA 02115, USA; Phone: 617-6324376; Fax: 617-6324381; e-mail: wael_elshamy@dfci.harvard.edu Key words: Metastasis, epithelial to mesenchymal transition, breast cancer Abbreviations: amphiregulin, (AR); epiregulin, (EPR); epithelial-to-mesenchymal transition, (EMT); farnesyltransferase inhibitors, (FTIs); heparin-binding EGF, (HB-EGF); immunoglobulin, (Ig); insulin receptor, (IR); integrin-linked kinase, (ILK); IR substrate 1, (IRS-1); mitogen-activated protein kinase, (MAPK); phosphatidylinositol 3- kinase, (PI3`K); phospholipase C!, (PLC!); PI-3 kinase, (PI-3K); Rho-kinase, (ROCK); signal transducer and activator of transcription 3, (STAT3); signal transducers and activators of transcription, (STATs); Src family kinases, (SFKs); transforming growth factor-", (TGF- "); transmembrane serine/threonine kinases, the type I and type II receptors, (T#RI and T#RII) Received: 12 July 2005; Revised: 29 July 2005 Accepted: 22 August 2005; electronically published: August 2005

Summary During the developmental cycle of a mammary gland, many properties that are associated with breast cancer are also displayed. The stromal factors necessary for mammary growth will either promote or protect against breast cancer. The epithelial-to-mesenchymal transition (EMT) is a developmental mechanism of crucial importance because it establishes the body plan in many multicellular organisms. Several transduction pathways controlling the various steps of this morphological transition have been identified by molecular analyses in both cell lines and in vivo. The newly formed mesenchymal cells can exhibit locomotory and invasive phenotypes, suggesting that EMTs may contribute to the progression of carcinomas. The genetic basis of tumorigenesis varies greatly between cancer types; however, the cellular and molecular steps required for metastasis are similar in all cancer types. This review will explore the connection between the EMT process and its involvement in the initiation and progression of breast cancer. Recently, there have been advances made in the understanding of molecular mechanisms that govern this lethal metastatic progression. New therapeutic approaches show promise because they target this process; however, before drug development can become successful, significant gaps in the basic knowledge of EMT processes and its molecular mechanisms must be met.

systems, arrest in distant organs, extravasation and growth into metastatic foci (Herlyn and Malkowicz, 1991; Woodhouse et al, 1997). Most if not all of these events require plasticity from tumor cells that is demonstrated by their ability to adopt a variety of phenotypes (Sood et al, 2001, 2002).

I. Introduction A. The clinical dilemma Most human tumors are of epithelial origin (carcinomas) and metastasis from such tumors lead to >80% of all cancer deaths (Hanahan and Weinberg, 2000; Greenlee et al, 2001). Unfortunately, the mechanisms involved in local invasion and metastasis are not fully understood. Thus, prognosis for a patient who is diagnosed with advanced invasive or metastatic disease has not improved in the past few decades (Sporn, 1997). The classical metastatic cascade encompasses intravasation by tumor cells, their circulation in lymph and blood vascular

B. EMT in normal development and cancer Normal tissue homeostasis is maintained between epithelial cells and their microenvironment that may include fibroblasts, endothelial and immunocompetent

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ElShamy: Cell treatments for breast cancer metastasis cells and the extracellular matrix (Nieto et al, 1994; Nieto, 2001). During malignant transformation and progression, there are (however deregulated) reciprocal and conspirational interactions between the neoplastic cells and the adjacent stromal cells (Bissell and Radisky, 2001; Wiseman and Werb, 2002). Although the genetic basis of tumorigenesis may vary greatly between different cancer types, the cellular and molecular steps required for metastasis are generally similar for all solid tumor cells (Woodhouse et al, 1997; Liotta and Kohn, 2003). Not surprisingly, the molecular mechanisms that propel invasive growth and metastasis are also found in embryonic development, but to a different extent. The perception of cancer has changed from viewing it just in its connection to genes to seeing it as a complex tissue resulting from disrupted organ homeostasis (Bogenrieder and Herlyn, 2002; Wiseman and Werb, 2002).

During development of even the most primitive species, remodeling of a simple epithelium by delamination, intercalation, invagination, evagination, branching, or cavitation of an aggregate of cells generates two or more layered epithelium (Hay et al, 1995; Markwald et al, 1996; Thiery, 2002). Some of these processes require reversible or in some instances irreversible convertion of epithelial cells into mesenchymal cells (EMT) (Birchmeier et al, 1996). EMT is defined as the acquisition of epithelium cells of fibroblastoid, migratory phenotype accompanied by profound changes in gene expression towards a mesenchymal program. It also includes the cell’s ability to digest and transmigrate through basement membranes and heterologous tissues (Rudland et al, 1985). Parallels between EMT in development and in breast and other tumors progression have already been described. For instrance, intra-tumor and inter-tumor heterogeneity of human breast cancer is considered as differentiation repertoires available to the neoplastic cells in response to the tumor microenvironment, including reversion to a ‘normal’ phenotype (Bissell et al, 1999) and not a consequence of phenotypic drifting due to genetic instability.

The EMT concept provides a new way to identify genes that are seen in the progression of carcinoma towards dedifferentiated and more malignant states (Gilles and Thompson 1996; Petersen et al, 1998; Thiery and Chopin, 1999; Boyer et al, 2000). In examining the cellular and developmental biology of EMTs, researches may gain insight into the mechanisms of tumor progression (Rønnov-Jessen et al, 1996). Several signaling pathways have been discovered to have a connection with EMTs. A more widespread knowledge of these pathways and the genes involved may be of great value to improve our understanding of breast cancer. This may allow doctors to provide a more reliable prognosis/therapies for their patients. Before an analysis of the data relevant to EMT can be examined, the major pathway initiating and maintaing it “the adherins pathway”, must first be explained.

forming adhesive bonds between one or several immunoglobulin (Ig) domains in its extracellular region and by them connecting to actin microfilaments indirectly via "- and #-catenin in the cytoplasm (Stockinger et al, 2001). The de novo production of E-cadherin in normal and transformed mesenchymal cells can induce the formation of stable cell-cell contacts and the development of adherns junctions to promote the formation of desmosomes (Bircheier and Behrens, 1994). Clinically, loss of heterozygosity at 16q22.1 is relatively frequent in breast carcinoma, implicating E-cadherin as a breast tumor suppressor gene. Numerous studies have reported a partial or complete loss of E-cadherin during carcinoma progression, which is connected to an unfavorable prognosis (Yoshiura et al, 1995; Perl et al, 1998; Cheng et al, 2001). This confirms that E-cadherin is a caretaker of the epithelial state. In vitro, lack of E-cadherin production correlates directly to the loss of epithelial phenotype (Birchmeier and Behrens, 1994; Cheng et al, 2001; Ilyas et al, 2000). While in vivo, E-cadherin is down regulated specifically at sites of EMT, such as gastrulation in Drosophila and in several vertebrates including the mouse (Craver et al, 2001).

In contrast, expression of N-cadherin de novo in breast carcinoma cells induces EMT (Hay, 1995; Hazan et al, 2000). Surprisingly, in these cellular contexts, Ncadherin behaves as a weak intercellular adhesion system.

The mechanism by which N-cadherin can overcome the maintenance of the epithelial state by E-cadherin is unknown; however, a domain in N-cadherin that is essential for this effect has been identified (Khoury et al, 2005). Furthermore, the process in which up-regulation of N-cadherin upon EMT initiation occurs is also unidentified. On the other hand, the mechanisms by which these epithelial lose E-cadherin expression have been extensively described. For instance, Snail can downregulate transcription of the E-cadherin gene through its interaction with E boxes in the proximal region of the promoter (Blanco et al, 2002). Snail is expressed mostly in dedifferentiated breast tumors and is correlated with grading (Yokoyama et al, 2001). In heterogeneous breast tumors, Snail is expressed in carcinoma cell islands devoid of E-cadherin and is found in all ductal invasive carcinomas with lymph node involvement (Batlle et al, 2000; Cano et al, 2000). Slug can also bind to the same region of the promoter and downregulate E-cadherin expression, although with lower affinity (Hajra et al, 1999; 2002). Other transcription factors also inhibit the transcription of E-cadherin genes: an example is the zinc finger protein SIP1, a downstream target gene in the TGF#-mediated induction of EMT in many cell lines (Xiao et al, 1999; Comijn et al, 2001).

A. Can we target E-cadherin pathway therapeutically?

Restoration of E-cadherin-mediated cell adhesion is a process that may prevent EMT in cancer. Several pathways may directly affect the adhesive properties of Ecadherin; these pathways include tyrosine kinases and tyrosine phosphatases, such as PTP-LAR (Levea et al, 2000). Blocking the transcriptional repressors such as

II. The adherins pathway E-cadherin mediate homophilic interactions by 444

Cancer Therapy Vol 3, page 445 Snail, SIP1 and E2A (Cano et al, 2000) might also make it possible to restore E-cadherin production (Perez-Moreno et al, 2001). In this context, the Rho and Rac pathways must be further investigated because they interfere with the stability of adherens junctions. Candidate genes that encode the receptors and ligands of the ephrin and semaphorin superfamilies, (Tamagnone et al, 1999; Tamagnone and Comoglio, 2000) both of which are involved in mapping the routes of motile cells, might also be involved in the EMT of cancer cells. These proteins may represent new therapeutic targets. In studying the mechanisms that induce nuclear translocation of #-catenin, researchers may develop new targets. For example, the integrin-mediated activation of the integrin-linked kinase (ILK) has been implicated in EMT in a colon cell line.

ILK inactivates glycogen synthase kinase-3#, an important controller of #-catenin in the WNT pathway (Wu and Dedhar, 2001).

Finally, regarding the EMT seen in breast cancer, it might be a good idea to explor the benefits of MTA3 overexpression to restore E-cadherin expression, especially in ER-positive breast cancers (Fujita et al, 2003)

their capacity to bind ErbB3 and ErbB4 (NRG-1 and NRG-2) or only ErbB4 (NRG-3 and NRG-4) (Harari et al, 1999). Each of the ligands has a different preference for stabilizing distinct receptor dimers; each receptor dimer has a different set of tyrosine autophosphorylation sites, which serve as a docking site for specific SH2-containing proteins and recruit different combinations of signaling molecules (Di Fiore et al, 1990; Olayioye et al, 2000).

Conversely, ErbB2 is activated only by heterodimerization with another ligand bound ErbB family member (Alimandi et al, 1995; Beerli et al, 1995). At least nine different homo- and heterodimers of ErbB proteins exist, but their formation displays a distinct hierarchy. In this network, ErbB2 plays a major coordinating role because each receptor with a specific ligand appears to prefer ErbB2 as its heterodimeric partner (Tzahar et al, 1996; Graus-Porta et al, 1997). This preference is further biased upon overexpression of ErbB2, as seen in many types of human cancer cells. ErbB2-containing heterodimers are characterized by extremely high signaling potency because ErbB2 dramatically reduces the rate of ligand dissociation. This allows strong and prolonged activation of downstream signaling pathways (Alimandi et al, 1995; Beerli et al, 1995; Graus-Porta et al, 1995; Holbro et al, 2003).

ErbB2 over expression is implicated in both breast cancer cell invasion and poor prognosis in Src- (Tan et al, 2005), mitogen activated protein kinase (MAPK), (Olayioye et al, 2000) as well as phosphatidylinositol- 3 kinase (PI-3K)-dependent manner (Fedi et al, 1994; Basso et al, 2002; Nicholson et al, 2003) (Figure 1).

Furthermore, ErbB2 cooperates with other RTKs, such as c-Met to disrupt epithelial morphogenesis and stimulate the breakdown of cell-cell junctions, dispersal and invasion of single cells (Niemann et al, 1998). Effects that are closely correlated with decrease in junctional proteins like claudin-1 and E-cadherin, in addition to the internalization of the tight junction protein ZO-1, implicate ErbB2 in the induction of invasion/metastasis through induction of EMT. Moreover, ErbB1 over expression correlates with metastasis in a variety of carcinomas, including breast (Lu et al, 2003). Finally, the expression of the mesenchymal protein, vimentin in highgrade breast tumors (grade 3) with invasiveness and chemoresistance was positively correlated with the over expression of EGFR or ErbB2 (Wade et al, 1982; Sommers et al, 1992; Korsching et al, 2005, Radovic et al, 2005). It is evident that induction in metastatsis by the ErbB family members over expression or deregulation at least in part occurs through their ability to induce EMT (Matthay et al, 1993).

III. The receptor kinase pathway The flow of information from the extracellular environment into the cell is at the core of a functional biological system. Receptor tyrosine kinases (RTKs) are primary mediators of many of these signals and thus determine whether the cell grows, differentiates, migrates, or dies. RTKs are cell surface allosteric enzymes consisting of a single transmembrane domain that separates an intracellular kinase domain from an extracellular ligand-binding domain. Ligand binding induces, in many instances, receptor homo- or heterodimerization, which is essential for activation of the tyrosine kinase and subsequent recruitment of target proteins. This initiates a complex signaling cascade that leads into distinct transcriptional programs (for instance, fos, jun, myc, Sp1, Egr1, as well as Ets family members) (Schaeffer et al, 1998). Many of the known tyrosine kinase receptors have been implicated in breast cancer invasion and metastasis. In many of the cases, EMT also played a role. However, more evidence is necessary to link tyrosine kinase and EMT to breast cancer.

A. ErbB receptors The ErbB family of RTKs consists of four receptors: epidermal growth factor receptor (EGFR or ErbB1), ErbB2, ErbB3 and ErbB4. Extensive receptor-receptor interactions and the existence of a wide group of ligands underlie the enormous potential for diversification of biological messages mediated by the ErbB family. There are several ErbB-specific ligands, EGF, amphiregulin (AR) and transforming growth factor-" (TGF-"), which bind specifically to ErbB1, #cellulin (BTC), heparinbinding EGF (HB-EGF) and epiregulin (EPR) (Shelly, 1998), which exhibit dual specificity for ErbB1 and ErbB4. A third group is composed of the neuregulins (NRG, also called Neu differentiation factors, NDFs, or heregulins, HRG) and includes two subgroups based on

1.Targeting the ErbB family members Several approaches have been utilized to target the ErbB family, but the most promising progress has been achieved in two areas: humanized antibodies against the receptor extracellular domains and small-molecule tyrosine kinase inhibitors. i. Antibodies In general, antibodies bind to the extracellular

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ElShamy: Cell treatments for breast cancer metastasis domain of the receptors, inhibiting their activation by ligand and promoting receptor internalization and down regulation. At present, the most advanced of this drug type against EGFR is a chimeric antibody-IMC-C225-which is developed by ImClone Systems and Bristol-Myers Squibb (Cetuximab, Erbitux). The other important anti-receptor drug is trastuzumab (Herceptin), which was developed by Genentech. This humanized monoclonal antibody against ErbB2 has proven to be effective against breast carcinomas in which ErbB2 is highly expressed, which accounts for 20-30% of cases of metastatic breast cancer (Figure 2).

I, IGF-II and insulin at supraphysiological doses), while # subunits transmit ligand-induced signal. The # subunits contain three major domains: the juxtamembrane, the tyrosine kinase and the C-terminus domains. Binding of ligands to IGF-IR induces its autophosphorylation and tyrosine phosphorylation of IGF-IR substrates, especially the IR substrate 1 (IRS-1) and Src and collagen-homology (Shc) protein. Tyrosine-phosphorylated IRS-1 and Shc bind different effector proteins (enzymes and/or adapters) inducing multiple signaling cascades, among them several interconnection pathways controlling cell survival and proliferation (Surmacz, 2000) (Figure 1).

The critical survival pathway activated by IGF-I stems from IRS-1. IRS-1 recruits and stimulates the PI-3 kinase (PI-3K), which then transmits a signal to the serine/threonine kinase Akt (Akt). Activated Akt phosphorylates and blocks a variety of proapoptotic proteins, including BAD, caspase-9, forkhead transcription factors and the GSK-3# kinase. Furthermore, Akt induces the expression of antiapoptotic proteins, such as Bcl-2 (Dews et al, 2000). Other mitogenic/survival IGF-IR pathways involve signal transducers and activators of transcription (STATs) that are phosphorylated and activated by IGF-I through JAK1/2 and PI-3K/Akt pathways (Nguyen et al, 2002; Yu et al, 2002). While antiapoptotic and growth pathways of IGF-IR have been extensively studied, the signals controlling nonmitogenic functions of IGF-IR, such as cell-substrate adhesion, migration, invasion, or intracellular interactions are less understood.

ii. Small molecules In general, small molecules competitively inhibit ATP binding to the receptor, thereby hindering autophosphorylation and kinase activation. Such molecules are the reversible small-molecule inhibitors of EGFR, ZD1839 (gefitinib, Iressa; AstraZeneca) and OSI774 (erlotinib, Tarceva; OSI Pharmaceuticals). Other EGFR directed small-molecule tyrosine kinase inhibitors in early stage trials include PKI116 (Novartis), GW2016 (GlaxoSmithKline), EKB-569 (Genetics Institute/ WyethAyerst) and CI-1033 (Pfizer) (Figure 2).

B. IGF receptors IGF-IR is an evolutionary conserved, ubiquitous transmembrane tyrosine kinase, structurally similar to the insulin receptor (IR) (Ullrich et al, 1986). IGF-IR is composed of two extracellular " subunits and two intracellular # subunits. The " subunits bind ligands (IGF-

Figure 1. Schematic representation of the possible signal cascades involved in HER, c-Met, IGF, and TGF-# receptors activation.

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Cancer Therapy Vol 3, page 447 Increasing evidence demonstrates that IGF-IR pathways interconnect with integrin and cadherins signaling systems (Guvakova and Surmacz, 1999; Mauro et al, 2003; Vuori and Ruoslahti, 1994). In some experimental models, IGF-IR has been shown to mediate metastasis, possibly through enhanced migration (Doerr and Jones, 1996), reduced cell-cell adhesion (Morford et al, 1997, Valentinis et al, 1999; Mauro et al, 2003) and upregulation of plasminogen activator uPA and matrix metalloproteinases (Mira et al, 1999; Zhang and Brodt, 2003). These molecular events correlate well with EMT and thus link IGF-IR signaling with cell-cell adhesion at the molecular level. In fact, IGF-IR can regulate cell aggregation and intercellular adhesion mediated by cadherins and cadherin-associated proteins. Furthermore, IGF-IR-mediated cell-cell adhesion was blocked with an anti-E-cadherin antibody and was not observed in Ecadherin-negative MDA-MB-231 breast cancer cells (Guvakova and Surmacz 1999). Even though the available data do not suggest a firm implication of IGFRs in the EMT process leading to invasion/metastasis of breast cancer, many studies support a strong inclination that it does.

i. Antibodies The mouse mAb "-IR-3 raised against the " domain of IGF-IR (Jacobs et al, 1986) inhibited IGF-IR activation and IGF-IR-dependent mitogenicity in several cell types in vitro, including breast carcinoma (Arteaga et al, 1992; Kalebic et al, 1994). However, in some cases "-IR-3 was ineffective in blocking IGF-I-sensitive tumors in animal models (Arteaga, 1992; De Leon et al, 1992; Hailey et al, 2002). Several other mouse anti-IGF-IR mAbs were described. One of them, mAb 1H7, which blocks IGFIR/IGF-I binding and IGF-IR-dependent DNA synthesis (Li et al, 2000; Sachdev et al, 2003) (Figure 2). ii. Small molecule The first described IGF-IR inhibitors, tyrphostins AG 538 and I-OMeAG, were modeled on the IR tyrosine kinase.

The compounds inactivated the IGF-IR tyrosine kinase by blocking the substrate-binding site; however, cross reactivity with the IR tyrosine kinase was reported. Recent advances in the characterization of the three-dimensional structures of IGF-IR and IR greatly facilitated the design of specific IGF-IR inhibitors (De Meyts and Whittaker, 2002). Most importantly, crystallographic studies reveal conformational differences in the phosphorylated forms of IGF-IR and IR kinases, the feature allowing the development of selective therapeutics (Favelyukis et al, 2001). Several new compounds with enhanced specificity towards IGF-IR and low cross

1. Targeting IGF-IR The greatest challenge in targeting IGF-IR is designing strategies that would specifically inhibit IGF-IR without blocking IR and producing diabetogenic effects (Ullrich et al, 1986). Inhibition of either IGF-IR/ligand binding, IGF-IR expression, or IGF-I signaling can exert antitumor effects (Figure 2).

Figure 2. Strategies to inactivate ErbB, c-Met, IGF-I and TGF receptors and their down-srream singaling molecules. (a) receptors function can be blocked with inactivating Abs. Binding of Abs to receptors prevents ligand binding an induces receptor degradation. (b) Receptors tyrosine kinase activity can be abolished with small-molecule inhibitors. (c) Small molecules can also be used to inactivate Receptors down-stream signaling molecules.

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ElShamy: Cell treatments for breast cancer metastasis reactivity with IR entered into preclinical studies. Specific small inhibitors of IGF-IR are likely to become anti-IGFIR drugs (Pietrzkowski et al, 1992, 1993).

expression of dominant-active Smad2 in squamous cancer cells result in enhanced tumor cell motility and metastasis dissemination (Oft et al, 2002). This evidence further supports that the tumor-promoting role of autocrine TGF#, expression of dominant-negative T#RII in metastasis cancer cells, prevents EMT while inhibiting motility, tumorigenicity and metastases (Dumont and Arteaga, 2003). This data suggests that TGF# may select for more metastatic cancers (Cui et al, 1996). Recently, over expression of active TGF#1 or activated T#RI in the mammary gland of transgenic mice has been shown to accelerate metastases derived from neu-induced primary mammary tumors (Watanabe et al, 2001; Siegel et al, 2003), suggesting that loss of autocrine TGF# signaling may limit systemic metastases.

TGF#-family members cooperate with either RTKs or their downstream signal transducers (Iascone et al, 1999).

This cooperation can overcome the well-known tumor suppressive effects of TGF# signaling (cell cycle arrest, apoptosis induction, Herzer et al, 2005). The same cooperation also allows TGF# signaling to modulate epithelial plasticity and migration/motility, a process crucial for tumor progression and metastasis (Blanco et al, 2002). Importantly, TGF#R and RTK signaling converge at the level of transcriptional regulation (Janda et al, 2002; Siegel et al, 2003). This cooperation may involve separate activation of different transcription factors with similar or opposing actions. These factors may regulate cell cycle progression (cdk inhibitors, D cyclins), apoptosis (pro- vs. antiapoptotic proteins), migration (Net1, Rho-A, PI3`K, ERK/MAPK) and epithelial adhesion/plasticity (Ecadherin repressors, snail, EF-1, EMT-genes and genes of the #-catenin signaling pathway) (Eger et al, 2000; Hemavathy et al, 2000; Keller et al, 1999; Reichmann et al, 1992).

C. TGF# receptor The transforming growth factor # (TGF#) family comprises a superfamily of ligands that include the TGF#s, activins and bone morphogenetic proteins (BMPs). There are three mammalian TGF# isoforms, TGF#1 (Dickson et al, 1995; Shull et al, 1992). TGF#2 (Sanford et al, 1997) and TGF#3 (Kaartinen et al, 1995; Proetzel et al, 1995), which, in general, exhibit similar function in vitro is most notable on cell growth regulation, extracellular matrix production and immune modulation (Massague, 1998; Miettinen et al, 1994). During development, TGF-#2 is a candidate inducer of EMT in the atrioventricular canal of the embryonic heart, whereas TGF-#3 is responsible for EMT following palate fusion (Kaartinen et al, 1995). In EMT in the chick heart, TGF#2 induces Slug, one of the key transcription factors in EMT (Romano et al, 2000). Similarly, mouse mammary epithelial NMuMG cells that have been made autocrine for TGF-# signalling become invasive and metastatic through EMT in a p38 MAPK activation/integrin signalingdependent fashion (Bhowmick et al, 2001b). Activation of the small GTPase RhoA, or its downstream target Rhokinase, appeared to be more significant in this model (Bhowmick et al, 2001a) (Figure 1).

The TGF#s bind to a heteromeric complex of transmembrane serine/threonine kinases, the type I and type II receptors (T#RI and T#RII) (Wrana et al, 1994).

Following ligand binding to T#RII, T#RI is recruited to ligand receptor complex, allowing the constitutive activation of T#RII kinase, which in turn transphosphorylate and activate the T#RI kinase (Wrana et al, 1994). Activated T#RI phosphorylates the receptorregulated Smad2 and Smad3. Finally, Smad2 and Smad3 then associate with the common mediator Smad4 and move to the nucleus where they regulate gene transcription (Massague et al, 2000). By contrast, the inhibitory Smad7 can interact with T#RI and prevent the phosphorylation of effector Smads (Hayashi et al, 1997). In addition to Smads, other signaling pathways have been implicated in TGF# actions. These include the Erk, JNK and p38, PI3`K and Rho GTPases (Derynck et al, 2001; Wakefield et al, 2002). The roles of these non-Smad pathways in mediating the cellular effects of TGF# remain to be fully characterized. Several reports support a causal association between an excess of endogenous or exogenous TGF# and breast tumor progression (Arteaga et al, 1993; Siegel et al, 2003). There is also evidence that high production and/or activation of TGF# in tumors can enhance cancer progression by autocrine and/or paracrine mechanisms (Dumont and Arteaga, 2000; Derynck et al, 2001; Wakefield et al, 2002) (Figure 1).

Evidences support the idea that TGF# induces EMT in tumor and in non-tumor epithelial cells (Miettinen et al, 1994; Oft et al, 1996). For example, expression of T#RII in colon cancer cells (with low invasive potential) restores tumor cell invasiveness (Oft et al, 1996). Forced

1. Targeting TGF#R signaling The improved outcome of patients who bear cancers with T#RII mutations supports an argument in favor of blocking autocrine TGF# which includes a therapeutic intent (Anbazhagan et al, 1999; Tian et al, 2004). An additional rationale can be inferred from the paracrine effects of tumor TGF#s on angiogenesis, stromal formation and remodeling and on immunosuppression.

These observations suggest that by blocking TGF# function, one can interrupt multiple events that are necessary for tumor maintenance. Indeed, preclinical studies support the principle that inhibition of TGF# affects these tumor-permissive autocrine and paracrine mechanisms (Dumont et al, 2003) (Figure 2). i. Antibodies Blocking ligand access to TGF# receptors using mAb is one way to effectively disrupt this signaling pathway. Two humanized monoclonal antibodies: CAT192, specific to TGF#1 and CAT-152, against TGF#2, are in early clinical development. The expression of multiple TGF# isoforms in tumors suggest that a pan-TGF# antibody might be more effective than isoform-specific antibodies. Two pan-TGF# monoclonal antibodies, 1D11

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Cancer Therapy Vol 3, page 449 and 2G7, have been reported. The 2G7 pan-TGF# neutralizing IgG2 suppresses the establishment of MDAMB231 tumors and lung metastasis in athymic mice and prevents the inhibition of host natural killer cell function induced by tumor inoculation. The antibody produced no effect against MDA-MB231 cells in vitro, nor did it exhibit an antitumor effect in natural killer-deficient mice.

This suggests that antibody mediated TGF# blockade is effective in disrupting tumor-host immunosuppressive interactions that are essential for tumor establishment and metastatic progression (Figure 2).

and maintenance of normal organ architecture such as muscle development, nervous system formation, hematopoietic differentiation, bone remodeling and angiogenesis (Birchmeier and Gherardi 1999). Moreover, Met activation plays a crucial role in the EMT that takes place during acute injury repair (Otonkoski et al, 1996; Yu and Merlino, 2002; Lengyel et al, 2005). Moreover, HGFactivated Met receptor stimulates tyrosine phosphorylation of FAK and induces actin reorganization during migration (Weidner et al, 1993; Comoglio and Boccaccio, 2001; Zhang and Vande Woude, 2003; Wasenius et al, 2005).

Met cooperates with different cell surface molecules.

For example, c-Met associates with CD44, which is a major component of the extra cellular matrix. It also correlates well as integrin "6#4, where the activity of the integrin is independent from its adhesive role because it forms an additional signaling platform necessary for the complete promotion of Met-induced invasive growth (Lengyel et al, 2005). Met also associates with all three members of class B plexins (transmembrane receptors for semaphorins), (Comoglio et al, 2002; Conrotto et al, 2004). These interactions have functional roles; over expression or stimulation of B plexins by their ligands induces scatter factor receptor activation and promotes the invasive-growth program (Giordano et al, 2002).

Furthermore, c-Met interacts with other surface RTKs, physically in the case of EGF receptor (Jafri et al, 2003), or through synergisim with intercellular signaling in the case of ErbB2 in promoting a malignant phenotype (Muller and Park, 2005; Saucier et al, 2004; Swiercz et al, 2004).

Until recently, RTK activity was believed to be modulated in different tissues on the basis of ligand availability, expression levels of the receptors and in the presence of a different panel of intracellular transducers.

With these cross-talk interactions, the activity of some receptors such as Met could depend on the simultaneous expression and/or activation of other membrane receptors.

Thus unveiling a new possibility for RTK control.

Activated c-Met recruits several SH2-domain-containing proteins, including adaptor proteins (such as Grb2, Shc, Gab1 and Cbl) and effector proteins (such as phosphatidylinositol 3- kinase (PI3`K), the tyrosine kinase Src, phospholipase C! (PLC!), the protein tyrosine phosphatase Shp2 and the transcription factor signal transducer and activator of transcription 3 (Stat3, Laird et al, 2003). Gab1, which amplifies the Met response, stimulates branching morphogenesis in vitro by activating Shp2 and PLC! in a sustained manner (Fixman et al, 1996).

ii. Small molecules A second group of strategies is aimed at directly blocking a receptorâ&#x20AC;&#x2122;s catalytic activity. SBI-14352, NPC 30345 and LY364947 are ATP competitive inhibitors of the ATP binding site of the T#RI kinase. This approach spares the T#RII kinase and, therefore, may not inhibit TGF# function completely (Wojtowicz-Praga 2003). If complete inhibition of TGF# was required for antitumor action, this selectivity could compromise anticancer activity while at the same time improve potential toxicities. These two possibilities are strictly theoretical because there are no known T#RII functions that do not require ThRI. Nonetheless, the development of bifunctional T#R kinase inhibitors would perhaps resolve these questions (Figure 2).

D. c-Met receptor signaling Met, which was discovered as an oncogene two decades ago (Zhang and Vande Woude 2003; Gao and Vande Woude 2005), encodes for a disulphide-linked heterodimer RTK that binds to and is activated by the growth and motility factor HGF (aka, scatter factor 1), (Comoglio and Boccaccio, 2001; Zhang and Vande Woude, 2003). Phosphorylation of the S985 located in the intracellular portion of the receptor by PCK or Ca2+/calmodulindependent kinases (DanilkovitchMiagkova and Zbar 2002) has an inhibitory function.

Phosphorylartion on the tyrosine residue (Y1003) allows binding to the E3-ubiquitin ligase Cbl, which promotes receptor ubiquitination, endocytosis and degradation (Zhang and Vande Woude 2003). However, the receptor C-terminal tail is a unique docking site for a wide spectrum of downstream signaling molecules. These molecules include PI3`K, the GRB2-SOS complex, the Src, the transcription factor signal transducer and activator of transcription 3 (STAT3) and the adaptors Shc and Gab1, which provide additional docking sites for many signaling molecules (Longati et al, 2001; Zhang and Vande Woude 2003) (Figure 1).

In both in vitro and in vivo, Met activation evokes pleiotropic biological responses often referred to as â&#x20AC;&#x153;invasive growthâ&#x20AC;? (Nakamura et al, 1989; Naldini et al, 1991). In vivo, Met is expressed on epithelial cells of many organs (Kamalati et al, 1999), both during embryogenesis and in adulthood; its function is essential for embryo development (knockout mice for either Met or Hgf are embryonic ally lethal; Birchmeier and Gherardi 1998). Under physiological conditions, Met contributes to the establishment of normal tissue patterns and the onset

1. Targeting c-Met signaling Approaches to block ligand-dependent activation have been developed.

Met

i. Antibodies A process to neutralize anti-HGF antibodies exists; however, it has been shown that, with available reagents, a minimum of three antibodies (each one with its own pharmacodynamic features) against different HGF epitopes are required to completely inhibit Met activation 449

ElShamy: Cell treatments for breast cancer metastasis (Jiang et al, 2005). Unless new antibodies are developed, these results raise concerns about the feasibility of this approach (Figure 2).

(Hahn et al, 2001; Baum and Kirschmeier, 2003) (Figure 2).

B. PI3`K/AKT signal transduction Phosphatidylinositol-3 kinases, PI3`Ks constitute a lipid kinase family characterized by their ability to phosphorylate inositol ring 30-OH group in inositol phospholipids to generate the second messenger phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) (Carpenter and Cantley, 1996). RTK activation results in PI(3,4,5)P3 and PI(3,4)P2 production by PI3`K at the inner side of the plasma membrane. Akt interacts with these phospholipids, causing its translocation to the inner membrane, where it is phosphorylated and activated by PDK1 and PDK2 (Anderson et al, 1998; Toker and Newton, 2000). Activated Akt modulates the function of numerous substrates involved in the regulation of cell survival, cell cycle progression and cellular growth. In recent years, it has been shown that PI3`K/Akt signaling pathway components are frequently altered in human cancers. For example, amplification and activation mutations have been detected in the PI3`K gene. The gene encodes the p110" catalytic subunit of PI3`K and is located in the chromosome 3q26, a region that is frequently amplified in several human cancers, including breast, ovarian and cervix cancer. No modifications or mutations in the akt gene have been found in mammals.

Nonetheless, various studies have found akt amplifications in human cancers, such as Akt2 gene amplifications in ovarian, pancreas, breast and stomach tumors (Bellacosa et al, 1995; Ruggeri et al, 1998; Chau and Ashcroft, 2004).

Survival signals induced by several receptors are mediated mainly by PI3`K/Akt, hence this pathway may play a major role in drug resistance appearance. In fact, there is convincing evidence from recent research suggesting that PI3`K/Akt pathway activation is related to tumor cell resistance to both chemotherapy and radiation.

Moreover, it has been suggested that activation of Akt1 by ErbB2/PI3`K plays an important role in mediating multidrug resistance in human breast cancer cells (Mills et al, 2003) and that Akt may therefore be a novel molecular target for therapies that would improve the outcome of patients with breast cancer (Kelland, 2005). In ovarian cancer, aberrant Akt expression or activation in different cell lines has been able to confer paclitaxel resistance (VanderWeele et al, 2004). It has also been reported that PI3`K inhibition increases paclitaxel efficiency in in vivo and in vitro ovarian cancer models (Hu et al, 2002).

Moreover, it has been shown that integrin-mediated protection to paclitaxel- and vincristine-induced apoptosis is dependant on PI3`K/Akt signaling pathway activation (Aoudjit and Vuori, 2001).

ii. Small molecules Alternatively, strategies that directly target the receptor can block both HGF-dependent and HGFindependent Met activation. The inhibition of Met kinase activity has been achieved through small ATP competitors (Sawyer et al, 2004). The molecules present a problem as a result of their selectivity. The available Met inhibitors are not specific; possible side effects raise concern for patients. Another approach is to interfere with HGF binding. The most thoroughly characterized HGF competitor is NK4 (Heideman et al, 2004), a molecule composed of the N-terminal hairpin and the four-kringle domain of HGF. NK4 binds to Met without inducing receptor activation and thus behaves as a full antagonist (Heideman et al, 2004) (Figure 2).

IV. The cytoplasmic transduction pathway

signal

EMT can also be induced in vitro in several epithelial cell lines by over-activation of cytoplasmic signal transduction pathways, e.g., Ras/mitogen-activated protein kinase (MAPK), PI3`K, Src and Rho/Rac all have an effect on particular aspects of EMT.

A. Ras/MAPK signal transduction Evidence suggests that Ras plays an essential role in the induction and maintainence of EMT during breast cancer progression (Rodenhuis, 1992). By using specific inhibitors and effector-specific Ras mutants, several research groups were able to show that hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT (Oft et al, 1996; 2002; Janda et al, 2002; Xie et al, 2004).

1. Targeting Ras signaling The attachment of the farnesyl isoprenoid group to the H-Ras, K-Ras and N-Ras proteins is essential for the biological activity of Ras. Therefore, the design of new rational therapies against the Ras pathway is in development. A large number of highly effective farnesyltransferase inhibitors (FTIs) have been identified (Cesario et al, 2005; Frassanito et al, 2005). These were shown to efficiently inhibit the farnesylation of H-Ras in cells in culture, which led to high expectations of being effective against the 20% of human tumors that have activating mutations in Ras genes (Frassanito et al, 2005).

Unfortunately, this early potential has not been realized.

The mode of action of FTIs has become increasingly unclear and the initial spectacular successes that were achieved in mouse models have not been reported in human patients. Despite uncertainty about their mechanism of function, FTIs do have marked effects on the growth and survival of some tumor cell lines in vitro and on xenografts in nude mice, although not necessarily those expressing activated Ras. The effects of FTIs in these pre-clinical systems have been reviewed extensively

1. Targeting PI3`K/Akt signaling pathway Wortmannin is a fungal metabolite and a potent inhibitor of type I PI3`K. Wortmannin has antitumor activity in vitro and in vivo studies, with an IC50 range for inhibition of PI3`K from 2 to 4 nM (Wymann aet al, 1996; Mills et al, 2003). Based upon the potent inhibitory effect of wortmannin in vitro assays, additional studies in animal models have been conducted to test the efficacy of wortmannin in inhibiting tumor growth in vivo (Davol et 450

Cancer Therapy Vol 3, page 451 al, 1999). Although these studies suggest that blocking the PI3`K/Akt pathway with wortmannin might be a valuable approach to treat cancer, one disadvantage of the use of wortmannin is its instability in an aqueous environment.

Wortmannin is soluble in organic solvents, which may limit its use in clinical trials. Currently, water-soluble wortmannin conjugates are being developed to circumvent this issue (Okaichi et al, 2002).

The flavonoid derivative, LY294002, is a competitive and reversible inhibitor of the ATP binding site of PI3`K. Several in vitro studies have shown that LY294002 alone has antiproliferative and proapoptotic activities (Uddin et al, 2005). Relatively, few in vivo studies have been conducted to demonstrate the efficacy of LY294002 on the inhibition of tumor growth, but these studies show that the administration of LY294002 in human cancer xenografts inhibit tumor growth and induced apoptosis (Semba et al, 2002). Similar to wortmannin, the combination of LY294002 with various cytotoxic drugs or radiation enhances the effectiveness of these treatments and highlights the therapeutic potential of targeting this pathway (Semba et al, 2002). Furthermore, an AKT inhibitor such as KP372-1 was found to suppress AKT activity and cell proliferation and induce apoptosis in thyroid cancer cells (Mandal et al, 2005) (Figure 2).

Src and that confer a migratory and invasive phenotype.

Compelling evidence that endogenous (Src family kinases (SFKs)) play a significant role in cell migration is provided by the impaired integrin-dependent migration of cells derived from mouse embryos that lack c-Src, Fyn and Yes (Matsuyoshi et al, 1992; Klinghoffer et al, 1999). In addition, loss of c-Src results in the strengthening of links between integrins and the force-generating cytoskeleton, which indicates that the normal role of c-Src or FAK is to weaken, or disrupt, such links (Ilic et al, 1995; Fincham et al, 2000).

1. Targeting Src pathway A novel Src homology (SH)-2 inhibitors incorporating non-hydrolyzable phosphotyrosine mimics (AP-22408, Ariad Pharmaceuticals) has been evaluated (Shakespeare et al, 2003). Another approach is the ATPbased Src kinase inhibitors (AP-23236, Ariad Pharmaceuticals, Shakespeare et al, 2003). The two compounds differ mechanistically by virtue of blocking Src-dependent non-catalytic or catalytic activities in osteoclasts. This process provides the framework for the next-generation molecules that have further advanced, in terms of preclinical studies, for the treatment of osteoporosis and related bone diseases, including osteolytic bone metastases (Figure 2).

C. Src signal tranduction D. Rho and Rac family

The viral src gene encoded by Rous sarcoma virus (RSV) was the first defined oncogene and encodes the first recognized tyrosine kinase, v-Src. Its cellular counterpart is c-Src (Hunter and Sefton, 1980; Martin, 2001; Boyer et al, 2000). Cells that are transformed by RSV lack bundled actin filaments and a reduction in the number and size of cell-substrate adhesions (focal adhesions) into which actin filaments are tethered. This results in conversion from a well-spread morphology to a more refractile, elongated cell shape reminiscent of EMT. Furthermore, gain-offunction mutation in the v-Src SH3 domain stabilizes a complex of Src and FAK and its localization to integrinassociated invadopodia (Kellie et al, 1986; Brunton et al, 2001). This, in turn, depends on the effects of some small GTPases on cytoskeletal modeling. For example, RhoA targets Src to focal adhesions, Rac1 targets it to focal complexes along lamellipodia and Cdc42 targets it to focal complexes along filopodia (Fincham et al, 1996).

V-Src and c-Src also target cortactin, an F-actin bundling protein that localizes to podosomes and lamellipodia (Wojakowski et al, 1993; Hiura et al, 1995; Weed et al, 1998, 2000; Boyer et al, 2001; Weaver et al, 2001). Cortactin associates with and activates, Arp2/3, a protein complex that is required to nucleate the formation of actin-filament networks. This interaction occurs through an amino-terminal acidic region of cortactin that has analogous functions to similar regions in the Wiskott Aldrich Syndrome protein (WASP) family (Mizutani et al, 2002). Specifically, cortactin functions to stabilize Arp2/3induced actin filament assembly at the cell periphery and may play a role in podosome formation (Weed et al, 2000). These observations indicate that actin regulators have a crucial function in mediating the assembly of dynamically regulated podosomes that are induced by v-

The importance of the Rho-GTPases in cancer progression, particularly in the area of breast cancer metastasis, is becoming increasingly evident. All aspects of cellular motility and invasion, including cellular polarity, cytoskeletal organization and transduction of signals from the outside environment are controlled through interplays between the Rho-GTPases (Itoh et al, 1999; Price et al, 2001; Ridley et al, 2001; EtienneManneville and Hall, 2002). Rho family consists of RhoA, B and C and their homologs Cdc42, Rac1 and 2 (Nobes and Hall, 1995). Like Ras, Rho proteins are localized to the inner plasma membrane by a C-terminal lipid modification (Hall, 1990; 1998) and are able to bind GDP/GTP and hydrolyze GTP and lead to activation of downstream effector molecules, which will lead to a cellular response (Kjoller and Hall 1999; Mareel and Leroy, 2003). Interestingly, some Rho family members such as Rnd and RhoH appear to lack intrinsic GTPase activity (Dallery-Prudhomme et al, 1997; Nobes et al, 1998). Ras constitutes 5% of human breast tumors and carries an identifiable Ras mutation, which renders the GTPase incapable of hydrolyzing bound GTP, thus remaining constitutively active (Rochlitz et al, 1989). No mutation in any of the Rho proteins has been identified in human tumors. Rather, over expression of Rho proteins, particularly RhoA and RhoC, appears to be the rule in human cancers (Moscow et al, 1994; Fritz et al, 1999; Imamura et al, 1999; Clark et al, 2000; van Golen, 2000).

Rac1 forms the leading lamellipodial edge of the cell (Evers et al, 2000a and b; Mareel and Leroy, 2003).

Furthermore, in cancers with weakened adherens junctions through for EGF or HGF initiated pathways, Rac is required to promote cell migration and invasion (Ridley et 451

ElShamy: Cell treatments for breast cancer metastasis al, 1995; Lamorte et al, 2002). Cdc42 forms the "ruffles" or "microspikes" known as filopodia, which redistribute the cell membranes to lamellipodium extension as the cell migrates and RhoA redistributes the actin stress fibers contracting the cell body in the direction of cell movement (Hall, 1990; 1998; Ridley 1994; Cussac et al, 1996; Price and Collard, 2001; Evers et al, 2000a and b). The ability to grow under anchorage-independent conditions signals by Rho family is through the PI3`K pathway. Motility and invasion signal through the Erk, JNK/SAPK and p38 MAPK pathways (Bouzahzah et al, 2001; Ridley, 2001; Rihet et al, 2001; Jo et al, 2002) and the production of angiogenic factors signals through p38.

Although Rho-kinase (ROCK) has been suggested to be a downstream target for both RhoA and RhoC, treatment of the cells with the pharmacological ROCK inhibitor, Y-27632, does not affect the RhoC-induced phenotype. Elucidating the mechanisms that result in Rhoover expression and activation are key in understanding the role of these proteins in breast cancer progression and metastasis. In normal cells, a fine balance maintains equilibrium between Rho GTPases in active and inactive states. Thus, perturbation of any of the Rho regulatory proteins, either through mutation, growth factor receptor dysregulation or oncogene expression can lead to aberrant Rho activation, increased motility, invasion and possibly metastasis (Kheradmand et al, 1998; Cho et al, 2000; Ozanne et al, 2000; Zhuge et al, 2001; Soon et al, 2003).

With the large number of RhoGDIs, RhoGEFs and RhoGAPs thus far identified and more being continuously added to the list, the main challenge will

regulated through three factors: signal integration, crosstalk and feedback control. However, death receptor, integrin, hedgehog, or Notch signaling pathways may at least in some cases, contribute to EMT. Therefore, during tumor progression, this complex signaling network is eventually dysregulated.

Reprogramming of gene expression towards mesenchymal traits may induce growth factor secretion and upregulate their receptors. This could cause hyperactivation of intracellular signaling, which may contribute to EMT, local invasion and metastasis. It is unclear whether EMT occurs in metastic carcinomas. A deeper understanding of EMT will lead to the development of better therapeutic approaches for patients.

Despite recent advances, researchers do not know how relevant or how frequent EMT and its induction is in human tumors. Several genetic mouse tumor models support EMT as a general mechanism in metastasis. An important aim for future research is to study the stages of EMT, both in embryos and in mouse models of carcinogenesis. In the future, candidate genes will be assessed for their contribution to EMT in human tumors by, for example, examining their function in tumors transplanted in immunodeficient mice or in transgenic mouse models. The main challenge is to discover how growth factors, scatter factors and ECM components cooperate to induce EMT. An extensive understanding of the processes that trigger EMT will lead researchers to develop ways to prevent it. This therapeutic strategy has the potential to block metastases. This could possibly prevent cancer recurrence because micrometastases often remains after conventional surgery, radiotherapy and/or chemotherapy.

E. Targeting Rho GTPase pathways Several drugs that block or decrease signaling by the Rho GTPases have now been shown to alter breast cancer morphology, cell growth and/or apoptosis. FTIs, originally designed to inhibit Ras lipid modification, also modify Rho proteins (for review, see Prendergast, 2001, Cesario et al, 2005) including RhoB. FTI treatment increases geranylgeranylated RhoB, which induces apoptosis selectively in cancer cells (Du and Prendergast, 1999).

Other promising approaches involve selective inhibition of certain signaling pathways downstream of Rho GTPases.

A specific PAK inhibitor decreased growth of Ras transformed cells (Nheu et al, 2002), but has yet to be tested in breast cancer cells. The ROCK effector kinase blocked metastasis of Hepatoma and may also prove effective in breast cancer cells (Itoh et al, 1999) (Figure 2).

Acknowledgements The author would like to thank Mrs. Lisa Luongo for excellent editing of this review article.

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Adequacy of anticipatory anxiety in receiving chemotherapy for breast cancer

women

Research Article

Michael Trimmel1,*, Christina Semrad1, Ernst Kubista6, GĂźnther Steger5, Christoph Zielinski2,4,5 1

Institute of Environmental Health, Unit for Public Health at the Medical University of Vienna, Austria Chair for Medical Experimental Oncology, University Hospital for Internal Medicine I, Medical University of Vienna, Austria 3 University Hospital for Internal Medicine I, Medical University of Vienna, Austria 4 Ludwig Boltzmann Institute for Clinical Experimental Oncology, Vienna, Austria 5 Clinical Division of Oncology, University Hospital for Internal Medicine I, Medical University of Vienna, Austria 6 Department of Special Gynecology, Medical University of Vienna, Austria.

2

__________________________________________________________________________________ *Correspondence: Prof. Michael Trimmel, Institute of Environmental Health, Medical University of Vienna, Kinderspitalgasse 15, A 1095 Vienna, Austria. Tel.: ++43/ 1 4277-64701, Fax: ++43/ 1 4277-9647, E-mail: michael.trimmel@univie.ac.at Key words: Sufficiency of information, occurrence of symptoms, Source of information, Education, Age, Expectations, Trait anxiety, ACE-19-scores, APT-scores, Anxiety scores, Abbreviations: Adequacy of Pre-Treatment Anxieties, (APT); Anxiety of 19 Chemotherapeutic Side Effects, (ACE-19) Received: 15 March 2005; Revised: 26 May 2005 Accepted: 16 August 2005; electronically published: August 2005

Summary Anxiety of potential side effects prior to cancer chemotherapy as well as retrospective judgements of these assessments as being appropriate or inappropriate were investigated. Forty-two breast cancer patients were asked to complete self-report questionnaires on their first and last day of cytotoxic treatment respectively. Results from 31 patients who completed the study indicate that trait anxiety and expectations of occurrence of side effects contribute to pre-treatment anxieties. In the retrospective view, younger patients were more likely to regard anticipatory anxiety levels as exaggerated. Subjective and objective amount of knowledge and its source were mainly unrelated to prospective and retrospective anxiety appraisals. Post-treatment rating showed that anxiety from physical consequences was highly overestimated at the beginning of the treatment. These findings suggest that patients should be informed as desired to avoid inappropriate and overwhelming anxiety. or worsening the response to the medication respectively (Walker et al, 1999).

On the other hand, very low anxiety might not be optimal either. According to Janis (1958), such people tend to minimize the impact of the medical intervention and are therefore not adequately motivated to examine and prepare for the treatment in advance. In line with this, Andersen et al, (1984) found out that while gynecologic cancer patients with low pre-treatment anxiety significantly increased in state anxiety during an interactive radiation therapy, whereas the high pretreatment anxiety group revealed a significant anxiety reduction. After completion of the radiation therapy, the two groups thereby did not differ in state anxiety any longer.

I. Introduction Chemotherapeutic treatment for breast cancer is associated with several short term and long term side effects such as nausea and vomiting, fatigue and impairment of cognitive ability. Patients who face chemotherapy were reported to be emotionally distressed and many experience significant levels of anxiety especially before commencement of chemotherapy (Cella, 1989; Vant Spijker 1997). The patientsâ&#x20AC;&#x2122; understanding of the illness and treatment can influence their coping ability and adjustment to the treatment regime. Exaggerated levels of anxiety before the beginning of treatment might be maladaptive for the coping process and might even lead to postponed chemotherapy or refusal (Gilbar and DeNour, 1989). Moreover, excessive anxiety could pose a problem by intensifying side effects (Palmer et al, 1980) 461

Trimmel et al: Adequacy of anticipatory anxiety in women receiving chemotherapy for breast cancer In contrast to this, patients who reported moderate anxiety prior to a medical intervention managed to adapt to the new situation more easily. By showing a medium level of anxiety, â&#x20AC;&#x153;work of worryâ&#x20AC;? is initiated, which encourages the person to attend to information that is required for successful coping (Andersen, 1990).

Adequate preparation of the patients prior to chemotherapy seems crucial to enable patients to correctly anticipate the consequences of the treatment and thereby optimize their coping skills and adjustment process (Burishet al, 1991; Tierney et al, 1991). Although several authors argue that full information can include certain risks for the cancer patients (Simes et al, 1986; Olver et al, 1995), the majority of studies suggest that extensive knowledge benefits individuals undergoing chemotherapy through correcting misbelieves or reducing anxiety of unexpected events.

The present longitudinal study had two main objects.

Firstly, to investigate the influence of pretreatment anxiety, expectations and information levels of breast cancer patients on the occurrence and evaluation of chemotherapy related side effects. Secondly, to identify aspects which influence a patients perception of adequacy of pretreatment anxiety levels. With reference to previous research it was expected that the source of information on chemotherapy affected whether pre-treatment anxiety was overestimated or underestimated, or adequately rated. In

addition, it was expected that demographic variables, trait anxiety and expectations of side effects (and their occurrence) will affect prospective and retrospective anxiety ratings.

II. Materials and methods A. Patients Participants were women who were scheduled to receive neoadjuvant or adjuvant chemotherapy at the general hospital in Vienna. Eligibility criteria were: diagnosis of breast cancer, no previous administration of chemotherapy and fluency in German.

Of the 50 patients asked to participate, 42 (84%) consented. Of these, 31 women (74%) could again be contacted after completion of their treatment. Two patients had dropped out of chemotherapy at their own request, one woman had developed metastases, and eight participants could not be attained for administrative reasons. The 31 patients who concluded the whole course of the study did not differ from the remaining 11 in terms of demographic and medical variables and state anxiety (T = -.771, df = 40, p = .445) or trait anxiety (T = -2.41, df = 40, p = .811). Demographic characteristics of the sample are presented in Table 1.

B. Procedure Patients were recruited during the waiting period right before their first infusion. After having signed a consent, the women announced demographic information and completed the

Table 1. Demographic and medical characteristics of participants who entered the study (total) and of those who completed the whole course (sub-sample).

Age group

Marital status

Highest education

Chemotherapy Regimen

Menopausal status

Total (N = 42) N % 1 2.4% 7 16.7% 8 19.0% 14 33.3% 8 19.0% 4 9.5% 3 7.1% 3 7.1% 23 54.8% 6 14.3% 7 16.7% 25 59.5% 4 9.5% 9 21.4% 4 9.5% 6 14.3% 36 85.7% 16 38.1% 2 4.8% 8 19.0% 14 33.3% 2 4.8% 16 38.1% 26 61.9%

< 30 30-39 40-49 50-59 60-69 > 70 single community of life married separated / divorced Widowed 9-year elementary school vocational / business school secondary school university / college neoadjuvant Adjuvant CMF ACMF ATCMF AC FAC non-menopausal menopausal

Abbreviations: C = cyclophosphamide; M = methotrexate; F = 5-fluorouracil; A = doxorubicin; T = taxotere.

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Sub-sample (N = 31) N % 1 3.2% 4 12.9% 6 19.4% 11 35.5% 6 19.4% 3 9.7% 2 6.5% 1 3.2% 17 54.8% 6 19.4% 5 16.1% 19 61.3% 4 12.9% 4 12.9% 4 12.9% 3 9.7% 28 90.3% 11 35.5% 2 6.4% 8 25.8% 10 32.3% 0 0% 11 35.5% 20 64.5%

Cancer Therapy Vol 3, page 463 Spielberger State-Trait Anxiety Scale (STAI) (Laux, Glanzmann, Schaffner and Spielberger, 1981). Moreover, several questionnaires designed for this study were filled out. The first contained a list of 15 physical side effects intended to be rated in regard of the expectations of their occurrence (on a scale from 1 “I am certain I will not have this” - to 5 - ”I am certain I will have this”). Furthermore, the patients assessed their anxiety of several physical and psychosocial symptoms on a four-point scale by means of the questionnaire “Anxiety of 19 Chemotherapeutic Side Effects” (ACE-19). In addition, the women were asked to check off the respective source of information about the possible side effects on an eight-item list: “physician / nurses”, “informative pamphlet from the hospital”, “other informative pamphlets”, “cancer patients”, “experience from professional life”, “media (for example newspapers, television)”, “I do not know” or “I did not receive any information about this”. Finally, patients were asked to indicate whether they regarded the obtained knowledge to be sufficient, and – if not – what was their favorite source of information in order to make further facts available. Medical information was obtained from the clinical records.

Patients who completed the whole study once again completed the STAI state form on the day of their last cycle of chemotherapy. In addition, the questionnaire “Adequacy of PreTreatment Anxieties” (APT), which contained the ACE-19 scores appraised by the women themselves on their first day of treatment, was administered. Subsequently, patients were asked to assess if the pre-treatment anxiety was appropriate, considering their actual experiences with the cytostatic therapy, or whether more or less anxiety would have been appropriate.

Finally, the same 15 physical side effects were rated in respect of the subjective probability of occurrence in order to state their incidence during chemotherapy on a scale from 0 (“not at all”) to 4 (“very severe”).

III. Results A. Clusters of anxiety and appropriateness In order to identify homogeneous groups of pretreatment anxieties, a hierarchical cluster analysis of ACE19 scores (“no anxiety” versus “low”, “moderate” or “high anxiety”) was performed. By using Ward’s method, four congruent clusters were identified: C1) anxiety of loss of independence and self-control (emotional), C2) anxiety of serious physical consequences (physical), C3) anxiety of physical damage with cognitive relevance (cognitive), C4) anxiety of crossing of life plans (existential). The comparison of the average intensities per cluster revealed that physical anxieties (C2) were more prevalent than emotional (p = .050), cognitive (p = .000) or existential ones (p = .001). Furthermore, emotional anxieties (C1) turned out to be more common than existential ones (p = .032) (Figure 1).

An analysis of the mean APT ratings per cluster showed that in this case, physical compared to existential anxieties were more often regarded as exaggerated (p = .013). Figure 2 illustrates the differences between these retrospective assessments.

B. Anxiety scores Mean STAI scores for state anxiety were 45.48 (SD = 15.05) (sub-sample, N = 31, mean = 46.5, SD = 15.12) prior to the first and 40.81 (SD = 12.51) at the last cycle of chemotherapy, having significantly declined in the course of the cytotoxic treatment (T = 2.447, df = 30, p = .010).

Figure 1. Means and standard errors of means of anxiety scores per cluster (0 = “no anxiety”, 1 = “high anxiety”).

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Trimmel et al: Adequacy of anticipatory anxiety in women receiving chemotherapy for breast cancer

Figure 2. Means and 2 standard errors of rated appropriateness of anxiety (1 = “less would be more appropriate”, 2 = “appropriate”, 3 = “more would be justified”) in the 4 clusters.

In comparison to previous research, these scores were fairly high. For example, Cassileth et al. (1986) found a decrease from an average of 39.5 to 35.5 in breast cancer patients randomized to chemotherapy.

As shown in Table 2, the present study indicates that several potential side effects are associated with considerable distress. Especially possible loss of hair led to high anxiety in about half of the sample. In contrast, most of the non-physical attendant phenomena did not mean distress at all for the majority of women.

In conclusion, final anxiety levels considered as appropriate in retrospective view were computed by combining the intensity of pre-treatment anxieties with the opinions given after the experience. Judgements stating that anticipatory anxiety was not fitting contributed to suitable alternatives in equal parts then. Hence, if “high anxiety” was judged exaggerated in pre-treatment, for instance, one third was added to “no anxiety” as well as to “low anxiety” and “moderate anxiety”. “Appropriate” anxiety levels remained constant. Table 3 demonstrates the results of this transformation.

Accordingly, compared to pre-treatment anxiety levels (Table 2), a high percentage of women (45%) reckoned self-assessed anxiety of hair loss as overstated – although this side effect still caused most distress after all.

Besides, 33% of patients thought that less anxiety of vomiting was better-suited, and between one fourth and one third of participants considered anxiety of the infusion, of nausea, pain and of impairment of everyday life exaggerated. On the whole, the majority of breast cancer patients felt their assessments to correspond with

experience though. Only anxiety of fatigue was regarded as understated by more than one fifth of women (23%).

C. Associations with ACE-19-scores 1. Trait anxiety Patients’ scores on the STAI trait form were examined for their associations with anxiety of concomitant phenomena due to chemotherapy. As shown in table 4, anxiety as a person’s attribute proved to be a predictor of pre-treatment anxiety of most of the symptoms (except anxiety of sterility, loss of employment and being a guinea-pig).

2. Expectations Anticipating the occurrence of physical symptoms turned out to predict anxiety of these likewise. Merely anxiety of hair loss and weakness did not correlate with the subjective possibility of their incidence at the significance level of five per cent (Table 4).

3. Age Kruskal-Wallis H / Mann-Whitney U tests were performed in order to explore differences in anticipatory anxiety between four age groups (<40, 40-49, 50-59, >59).

Only anxiety of impairment of family life was significantly more intense in both groups under 50 years of age than in the older patients (!2= 12.212, df = 3, p = .007). Further analysis revealed that these differences existed solely in women without a partner (single, separated / divorced or widowed) (!2= 9.894, df = 3, p = .006), whereas in cancer patients who were married or

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Cancer Therapy Vol 3, page 465 lived in a cohabitation, the result again failed significance (!2= 4.689, df = 3, p = .203).

effects showed that this matter had no relation to the extent of the respective anxiety. Within those women who had been informed by one of the six subjects listed, a Kruskal-Wallis analysis of variance along with a MannWhitney U test resulted in just one significant finding: those patients who had obtained their knowledge about

4. Source of information A comparison between patients who could quote any source for their knowledge and those who did not receive information about the physical or psychological side

Table 2. ACE-19 scores: Pre-treatment anxiety of attendant symptoms (total and subsample) C#1 C1 C2 C2 C2 C2 C1 C1 C2 C2 C3 C3 C3 C4 C1 C1 C3 C1 C1 C2

Anxiety of ...

Infusion Nausea Vomiting Hair loss Injury to immune system Weakness Fatigue Injury to blood counts Pain Loss of appetite / loss of weight Heart problems Memory disturbances Sterility Impairment of everyday life Impairment of family life Loss of employment Being helpless Being a guinea-pig Ineffectiveness of treatment

No anxiety N = 42 (N = 31) 36% (27%) 13% (20%) 16% ( 9%) 16% (36%) 16% (9%) 26% (36%) 16% (55%) 13% (18%) 36% (55%) 61% (46%) 45% (46%) 55% (64%) 82% (60%) 29% (36%) 57% (82%) 80% (86%) 58% (55%) 77% (73%) 48% (27%)

Low anxiety N = 42 (N = 31) 23% (46%) 30% (30%) 32% (36%) 16% (9%) 19% (27%) 39% (9%) 55% (0%) 23% (27%) 32% (9%) 26% (27%) 29% (27%) 23% (9%) 9% (20%) 23% (18%) 13% (9%) 10% (0%) 13% (36%) 16% (18%) 13% (46%)

Moderate anxiety N = 42 (N = 31) 29% (27%) 33% (30%) 26% (36%) 19% (0%) 45% (36%) 32% (46%) 29% (36%) 42% (27%) 29% (18%) 7% (27%) 19% (27%) 19% (9%) 0% (20%) 45% (18%) 30% (0%) 5% (14%) 13% (0%) 3% (9%) 29% (18%)

High anxiety N = 42 (N = 31) 13% (0%) 23% (20%) 26% (18%) 48% (55%) 19% (27%) 3% (9%) 0% (9%) 23% (27%) 3% (18%) 7% (0%) 7% (0%) 3% (18%) 9% (0%) 3% (27%) 0% (9%) 5% (0%) 16% (9%) 3% (0%) 10% (9%)

Low anxiety 29% 23% 23% 22% 21% 35% 36% 20% 25% 18% 23% 16% 9% 22% 18% 3% 23% 13% 17%

Moderate anxiety 15% 30% 18% 17% 31% 30% 24% 37% 15% 10% 20% 15% 0% 28% 16% 3% 9% 5% 33%

High anxiety 5% 20% 23% 28% 24% 7% 15% 24% 4% 9% 6% 7% 9% 12% 6% 8% 9% 4% 8%

1

Assignment of items to four clusters (C1, C2, C3, C4)

Table 3. Appropriate anxiety of attendant symptoms1 (N = 31) No C#1 Anxiety of ... anxiety C1 Infusion 52% C2 Nausea 27% C2 Vomiting 36% C2 Hair loss 33% C2 Injury to immune system 23% C1 Weakness 29% C1 Fatigue 26% C2 Injury to blood counts 19% C2 Pain 56% C3 Loss of appetite / loss of weight 64% C3 Heart problems 51% C3 Memory disturbances 62% C4 Sterility 82% C1 Impairment of everyday life 39% C1 Impairment of family life 60% C3 Loss of employment 88% C1 Being helpless 60% C1 Being a guinea-pig 77% C2 Ineffectiveness of treatment 43% 1

Computed by combining pre-treatment anxiety levels (ACE-19) and retrospective judgements (APT) (comments see text). 2 Assignment of items to four clusters (C1, C2, C3, C4)

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Trimmel et al: Adequacy of anticipatory anxiety in women receiving chemotherapy for breast cancer Table 4. Results of correlational analysis between self-assessed anxiety and STAI trait score / expectation of side effects

C#2 C1 C2 C2 C2 C2 C1 C1 C2 C2 C3 C3 C3 C4 C1 C1 C3 C1 C1 C2

Trait anxiety r .374** .510*** .516*** .490*** .337* .392** .440** .379** .404** .259* .383** .437** .210 .656*** .434** .322 .341* .204 .318*

Anxiety of...

Infusion Nausea Vomiting Hair loss Injury to immune system Weakness Fatigue Injury to blood counts Pain Loss of appetite / loss of weight Heart problems Memory disturbances Sterility Impairment of everyday life Impairment of family life Loss of employment Being helpless Being a guinea-pig Ineffectiveness of treatment

Expectations r 1 ***

.601 .612*** .217 .542*** .162 .350* .363** .740*** .405** .550*** .389** 1 1 1 1 1 1 1

1

Could not be computed because independent variable has not been collected.

Assignment of items to four clusters (C1, C2, C3, C4) * p " .05. ** p " .01. *** p " .001.

2

vomiting (!2 = 5.35, p = .011), impairment of everyday life (!2 = 4.89, p = .014), nausea (!2 = 4.16, p = .021), ineffectiveness of treatment (!2 = 3.52, p = .031), pain (!2 = 3.47, p = .032), impairment of family life (!2 = 2.82, p = .047) and weakness (!2 = 2.73, p = .050) – items of the clusters C1 (emotional) and C2 (physical) without exception. A partial correlation controlling for the occurrence of the physical symptoms led the associations concerning weakness to miss the one-tailed probability of five per cent (r = .27, p = .074), whereas those respecting nausea, vomiting and pain still remained significant.

nausea from the physician or nurses displayed less intense anxiety of this symptom than those who had been informed by other cancer patients (!2=11.291, df=5, p=0.046).

5. Sufficiency of information The examination for associations between the request for further information and the intensity of anticipatory anxiety revealed no significant results. This suggested that anxiety levels of patients who were content with the amount of facts given did in no case differ from those of women who announced shortage of information.

3. Education Having concluded school successfully turned out to have no connection with feeling anticipatory anxieties to correspond with experiences more frequently in retrospective view. Accordingly, women without schoolleaving examinations did not prove to be more likely to regard their pre-treatment anxieties as over- or understated than graduated ones.

D. Associations with APT-scores 1. Trait anxiety By means of chi-square tests, possible connections between low or high scores on the STAI trait form and the individual APT ratings were studied. The results indicated that anxiety of memory disturbances was more often considered inappropriate by patients high in trait anxiety ( ! 2 = 9.31, df = 2, p = .010). However, no significant tendency towards over- or understated pre-treatment anxiety assessments was observed.

4. Source of information The results of Yates’ chi-square tests indicated that breast cancer patients who did not receive information about the respective attendant circumstances of chemotherapy did not differ from informed participants at the dichotomized judgements of their anxieties as appropriate or inadequate (i. e. over- or understated). In the sub-sample of women who mentioned any source of information, a statistically significant difference was found only with reference to a potential impairment of everyday life (!2 = 9.47, df = 4, p = .050). As Figure 3 illustrates, in

2. Age In order to explore differences between the four age groups in considering the extent of anticipatory anxieties to be appropriate or over- / understated, MantelHaenszel’s chi-square tests were performed. Thus, the hypothesis (derived from previous investigations) that younger patients are more likely to reckon their anxieties as exaggerated has been confirmed in regard to anxiety of 466

Cancer Therapy Vol 3, page 467 that case, other cancer patients were able to prepare for the actual consequences on workaday routine best, whereas the informative pamphlet from the hospital as well as media caused unfitting anxiety levels without exception. On the other hand, an analysis of the ordinal (three-point) judgements again did not produce significant results, so that no source of information differed from the others in evoking inappropriately low or high anxiety.

were performed in order to explore associations with the incidence of the respective physical symptom. As shown in Table 6, “appropriate” anxiety of injury to blood counts (p = .120), hair loss (p = .457), injury to immune system (p = .811) and sterility (p = .423) did not statistically significant correlate with the actual occurrence of these side effects – all non-significant items belong to the clusters of physical or existential anxieties. Concerning anxiety of injury to immune system and in particular of sterility, even negative (non-significant) correlational coefficients emerged. As it was hypothesized that for the latter, age acted as a co-variable, a partial correlation was conducted. Although still non-significant, this analysis resulted in a positive Spearman coefficient then (r = .104, p = .403).

5. Sufficiency of information Being content with the quantity of information given turned out to have no effect on anticipatory anxiety.

Anxiety was not related to the source of information about possible side effects either.

6. Expectations and occurrence of symptoms

IV. Discussion

An exploration of the retrospective anxiety ratings of patients with or without correspondence of anticipation and occurrence of physical side effects revealed that expectations that came true mostly did not result in considering pre-treatment anxiety to be more suitable.

Accordingly, only patients who did not suffer from nausea or heart problems regarded their pre-treatment anxieties more often as appropriate if they had not expected these symptoms (Table 5; p = .023 in both cases).

E. Associations anxiety levels

with

This study aimed at identifying variables that contribute to an appropriate anticipation of physical and psychosocial symptoms due to cytotoxic treatment. 19 side effects were explored and assigned to one of the following clusters: C1) anxiety of loss of independence and selfcontrol (emotional), C2) anxiety of serious physical consequences (physical), C3) anxiety of physical damage with cognitive relevance (cognitive), C4) anxiety of crossing of life plans (existential). The results indicate that younger breast cancer patients show greater tendencies of experiencing exaggerated pre-treatment anxiety of physical and everyday lifestyle consequences than the elder patients. Since the initial intensities of the mentioned anxieties did not differ from those of the elder women, it is supposed that younger patients are able to cope with these side effects in a more effective way than do people advanced in years. In previous researches it has been

“appropriate”

1. Occurrence of side effects After having determined the final intensity of the respective anxiety which patients considered appropriate after having undergone chemotherapy (computed as explained before by combining pre-treatment anxiety levels with retrospective judgements), correlation analyses

Figure 3. Retrospective ratings of anxiety of impairment of everyday life by source of information

467

Trimmel et al: Adequacy of anticipatory anxiety in women receiving chemotherapy for breast cancer Table 5. Judgements of pre-treatment anxiety levels as being appropriate or not by symptom expectations1 and actual occurrence of side effects2 (N = 31) Variable

Nausea Vomiting Hair loss Injury to immune system Weakness Fatigue Injury to blood counts Pain Loss of appetite Heart problems Memory disturbances 1 2

not expected expected not expected expected not expected expected not expected

Not experienced Not appropriate 0 2 1 3 1 2 1

not expected expected not expected expected not expected expected not expected

Experienced Not appropriate 0 5 1 0 2 6 4

Appropriate 3 0 4 1 0 0 5

Appropriate 3 5 3 3 4 6 8

expected not expected expected not expected expected not expected

1 0 1 0 0 0

2 0 0 0 0 4

expected not expected expected not expected expected not expected

0 1 4 1 8 3

4 7 5 4 7 3

expected not expected expected not expected

0 4 1 0

2 8 0 4

expected not expected expected not expected

2 1 0 1

3 6 1 6

expected not expected

0 2

1 12

expected not expected

2 1

4 5

expected not expected

1 0

0 10

expected not expected

0 6

1 5

expected

0

0

expected

2

0

“not expected” = 1 or 2 on expectation scale; “expected” = 4 or 5 on expectation scale “not experienced” = 0 on experience scale; “experienced” = 1-4 on experience scale

Table 6. Results of correlational analysis between appropriate anxiety levels1 and occurrence of side effects

#C C2 C2 C2 C2 C1 C1 C2 C2 C3 C3 C3 C4

Occurrence of side effect r .667*** .546** .144 -.045 .522** .389* .290 .603*** .559** .507** .411* -.306

Appropriate anxiety of...

Nausea Vomiting Hair loss Injury to immune system Weakness Fatigue Injury to blood counts Pain Loss of appetite / loss of weight Heart problems Memory disturbances Sterility

1

Computed by combining pre-treatment anxiety levels and retrospective judgements. p " .05. ** p " .01. *** p " .001.

*

468

Cancer Therapy Vol 3, page 469 argued that younger patients tend to more pessimistic views by expressing increased perceived vulnerability and dramatizing chemotherapeutic treatment consequences and outcomes (Jacobsen, Bovbjerg and Redd, 1993; Mor, Allen and Malin, 1994). Our results stand in contradiction to this notion, since they suggest that during chemotherapy preparation, younger individuals were more tolerant of the previously mentioned concomitant phenomena than previously described. In light of this, unnecessarily high levels of anxiety of symptoms could perhaps be prevented.

It was also found that pre-treatment anxiety of impairment of family life was overestimated in young women. In that case anxiety turned out to be already more intense prior to the first infusion. Such findings imply that perhaps professionals should pay more attention to family background of young patients, and show ability in identifying high level of anxiety, thereby strengthening confidence in family acceptance.

Trait anxiety was found to predict anxiety levels of most of the side effects. The only exceptions were anxiety of sterility, loss of employment and being a guinea-pig. In agreement with previous investigations (Jacobsen et al, 1993), high trait anxiety could therefore be seen as a characteristic feature that represents propensity to feeling anxious in view of cytotoxic interventions. Since retrospective judgements except those concerning memory disturbances did not differ between patients with low or high scores on the STAI trait form, it can be concluded that for the latter, increased anxiety levels basically commensurate with experiences more truly, so that in this case, successful adaptation to treatment is thereby not impeded. On the other hand, patients high in trait anxiety felt anxiety of memory disturbances to be inadequate more frequently. As no significant trend towards the direction of the inadequacy appeared, these results indicate that high trait anxiety is likely to prevent appropriate anticipation of a cognitive side effect, whereas the remaining symptoms are affected to a smaller extent.

An examination of the relation between expectations and anxiety assessments revealed that anticipation of the majority of side effects entailed heightened distress.

Neither Cassileth et al. (1985) nor Jacobsen et al. (1993) could find an association between symptom expectation and anticipatory anxiety. However, in the present investigation, anxiety correlated with patients’ conviction.

Previous studies suggest that, while weakness barely caused considerable concerns it led to a lot of distress (Hasenbring et al, 1993; Nerenz et al, 1982). On the other hand, possible hair loss burdened even patients who did not expect to experience this side effect, which indicates that this symptom is subject to substantial anxiety in any case.

An amazing result emerged when retrospective ratings of anxiety levels were analyzed with reference to the point whether speculations about experiencing physical symptoms were fulfilled or not. Contrary to expectations, patients who turned out to comprise correspondence between anticipation and occurrence of secondary effects did hardly ever consider pre-treatment anxiety appropriate more often. With respect to this finding, it is suggested that patients’ outlook is very

important. An optimistic approach would lead to low expectations that rather reflect hope than conviction, a phenomenon that had already been observed by Andrykowski and Gregg (1992), whereas adjustment processes in the case of symptom occurrence are not influenced. The possible assumption that one had the fortune to be unaffected by a certain secondary result by way of exception could explain the incoherence between absence of anticipated side effects and anxiety judgements.

According to earlier investigations, it is argued that education influences subsequent ratings of anxiety levels by modifying the understanding and retaining of provided information (Muss et al, 1979; Olver et al, 1995) and, through that, the use of coping mechanisms. In the present study, however, patients’ educational level had no effect on appropriateness of anxiety. A possible explanation is that knowledge does not entail the ability to anticipate appropriate anxiety more accurately. This notion is supported by the comparison of patients who had obtained knowledge about the concomitant symptoms and those who did not receive any information. Although several preceding studies indicate that extensive knowledge facilitates adjustment to cancer treatment, it reduces the discrepancy between what patients anticipate and what actually occurs (Buick, 1997; Langer et al, 1989). No significant differences concerning retrospective judgements of self-assessed anxiety were found in this study and also acquired information to be satisfactory or insufficient did not affect anxiety ratings. While contradictory results have been found in past studies, including both intensification and diminution of anxiety in patients who had been informed completely, the present findings suggest that neither the subjective nor the objective state of knowledge influences anxiety levels.

Furthermore, the individual sources of information mostly did not entail significantly different pro- or retrospective anxiety assessments – contrary to expectations which were based upon previous studies that ascribed for instance to the mass media contents which are often outdated or dramatized (Knobf et al, 1998). Applied to clinical practice, this means that medical staff should comply with cancer patients’ demand for detailed information.

According to the women’s statements about their favorite source of information, especially the physicians and nurses should undertake this function and, in addition, to some extent also informative pamphlets. Similarly, the request for not receiving any information should also be respected.

Finally, it is intended that with the help of the present study, a contribution is made to improve the understanding for the psychical situation of breast cancer patients, so that their needs could be dealt with to a better extent.

Additional research, in particular dealing with coping strategies of people undergoing chemotherapy, could discover supplemental findings that are necessary to sustain quality of life during cytotoxic treatment.

Acknowledgement The authors want to thank Karin Trimmel for helping to prepare the manuscript.

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Warner N, King M, Langlands A, Kenny P and Boyages J ( 1999) Symptoms 2 weeks 3 months and 12 months after treatment of early breast cancer: the patients' perspectives. Breast 8, 273–277.

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Ki-67 index and skin carcinomas with skull base invasion: a case–control study Research Article

Claudio R. Cernea1,*, Alberto R. Ferraz1, Inês V. de Castro2, Miriam N. Sotto2, Ângela F. Logullo3, André S. Potenza1, Carlos E. Bacchi4 1

Department of Head and Neck Surgery, University of São Paulo Medical School (São Paulo, Brazil) Department of Pathology, University of São Paulo Medical School (São Paulo, Brazil) 3 Department of Pathology, Federal University of São Paulo (São Paulo, Brazil), 4 Department of Pathology, Albert Einstein Jewish Hospital (São Paulo, Brazil) 2

__________________________________________________________________________________ *Correspondence: Claudio R. Cernea, MD, Alameda Franca, 267, cjto. 21, CEP 01422-000, São Paulo, Brazil; Phone/Fax: 55-1132850058; e-mail: cerneamd@uol.com.br Key words: Ki-67; basal cell carcinoma; squamous cell carcinoma; base of skull; immunohistochemistry; cell proliferation Abbreviations: Basal cell carcinoma, (BCC); Squamous cell carcinoma, (SCC)

Presented at the Sixth Research Workshop on the Biology, Prevention and Treatment of Head and Neck Cancer, October 9-13, 2002, McLean, Virginia Received: 31 May 2005; Accepted: 31 August 2005; electronically published: September 2005

Summary Skin carcinomas may be very aggressive. Cell proliferation, measured by expression of Ki-67 antigen, has been associated with tumor aggressiveness, but controversy still persists. In this study, the Ki-67 index in basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) with skull base invasion was compared with tumors with good outcome. Expression of Ki-67 was graded as mild (if present in <30% of tumor cells), moderate (between 30% and 50%) and intense (more than 50%), in 24 BCCs and 11 SCCs with skull base invasion. Control group included 23 BCCs and 10 SCCs. Intense expression of Ki-67 was noted in 37.50% of BCCs with skull base invasion, compared to 13.04% in the control group (p=0.155). Regarding SCCs, intense expression of Ki-67 was found in 72.73% of aggressive tumors, compared to 20.00% in the control group (p=0.050). Ki-67 index was higher among skin cancers with skull base invasion, compared to controls with good outcome. However, this difference reached statistical significance only in SCCs. 1998). As a matter of fact, skin carcinomas with skull base involvement represent the main histological types of tumors in some series of oncological base of skull operations (Medina dos Santos et al, 1994; Cernea et al, 1997; Dias et al, 1997).

Ki-67 is a nuclear protein expressed during active phases of cell cycle (G1, S, G2 and M), being absent in G0 “resting” phase (Kanitakis et al, 1997). The precise mechanism of its action is still unknown, but its presence is essential for cell proliferation (Healy et al, 1995).

Immunohistochemical detection of Ki-67 expression allows a quantitative measure of proliferation potential of a particular neoplasia (Gonzalez-moles et al, 1996).

Hence, less proliferative tumors exhibit Ki-67 expression in a percentage lower than 30% of its cells. In contrast, more aggressive cancers have Ki-67 expression in more than 50% of cells (Bacchi and Gown, 1993).

Some authors have analyzed the prognostic value of

I. Introduction Basal cell carcinoma (BCC) of the skin is the most common human malignant tumor (Lang and Maize, 1991).

Squamous cell carcinoma (SCC) is the second most frequent type. Both occur more frequently in caucasians after the sixth decade, who had experienced prolonged exposure to sunlight (Freeman, 1976). Several factors have been associated with increased aggressiveness of these tumors: histopathological subtype, differentiation, depth of invasion and perineural invasion, among many others (Ruhoy et al, 2001). In addition, some authors have showed the relationship between some biological factors and cancer behavior (Cernea et al, 2004).

Sometimes, these skin carcinomas may be extremely aggressive, and despite adequate treatment, they may recur and invade fascia, muscle and bone. Due to the deformity, some authors call them horrifying tumors (Jackson and Adams, 1973; Bianchini and Wolter, 1984; Horlock et al, 471

Cernea et al: Ki-67 index and skin carcinomas with skull base invasion incubated in a buffered citrate solution at pH 6.4 for 15 minutes (Gown et al, 1993). Then, slides were incubated with specific primary antibody against Ki-67 (clone MIB-1), diluted 1:50, for 12 hours, at temperature of 4°C. After washing with buffered saline solution, slides were incubated for 60 minutes with biotinilated antibodies anti-IgG (Vector Corp., USA). Then, they were incubated for 45 minutes with ABC Elite! complex (Vector Corp., USA). Finally, slides were treated with 3.3’ diaminodibenzidine (Sigma Chemical Company, USA) and with peroxide 0.1% (Sigma Chemical Company, USA). Counter staining was performed with methylated green for 5 minutes. The immunohistochemical positive control was a lymph node with lymphoid hyperplasia.

Ki-67 as a tumor marker, and suggest an important prognostic role in different types of cancer, but results have been somewhat controversial (Lavertu et al, 2001; Fumic-Dunkic et al, 2003; Liu et al, 2003; Koch and Sidransky, 2004).

Regarding BCC, Baum et al, 1993 noted intense Ki67 expression index in 32.90% of 62 BCCs. Abdelsayed et al, 2000 found a 51% increased expression rate in a group of 20 cancers. Healy et al, 1995 compared three groups of BCC surgical specimens: 17 non-recurrent tumors, 17 initial specimens of cancers that recurred later and their corresponding specimens of the recurrences. They found a statistically significant higher Ki-67 expression among the recurrent tumors. However, Horlock et al, 1998 compared Ki-67 expression of 81 BCCs with 22 horrifying cancers, with no statistical difference. Interestingly, this group reported an increased frequency of expression among aggressive histological subtypes (mainly morphea-like), and this finding was confirmed by other authors (Barrett et al, 1997).

Kerschmann et al, 1994 noted Ki-67 expression in 46% of 20 patients with SCC. Mansoor et al, 1996studied Ki-67 expression in a series of 175 SCCs, finding a positive statistical correlation with differentiation, thickness and depth of invasion; however, they could not demonstrate any statistical relationship with recurrence.

Similarly, Kanitakis et al, 1997 compared Ki-67 expression between 14 aggressive and 28 non-aggressive SCCs, with no difference.

The objective of this study was to analyze the proliferation index, using immunohistochemical evaluation of Ki-67 with antibody the monoclonal antibody MIB-1, in a consecutive series of very aggressive skin carcinomas with skull base invasion submitted to combined craniofacial oncological operations. In addition, these findings were compared with skin carcinomas with good outcome, treated in the same Institution within the same time frame, in a case-control study.

C. Criteria for interpretation immunohistochemistry staining for Ki-67

of

Grading was semi-quantitative. Immunoreactivity was considered positive when nuclear staining in cells of tumor was found. The intensity was graded as mild, when positive in less than 30% of tumor cells (Figure 2), moderate, when positive between 30% and 50% of tumor cells (Figure 3), and intense, when positive in more than 50% of tumor cells (Figure 4). All slides were blindly graded by two co-authors (Angela F. Logullo, Carlos E. Bacchi), with good correlation scores between them.

D. Statistical analysis Data were collected in a databank, using a software Excell (Microsoft Corp., USA), in a personal computer Pentium II 400 MHz (LG Electronics, South Korea). For the statistical analysis, either the chi-square test or the Fisher exact test were employed, to a significance level of 0.050.

II. Materials and Methods A. Patients A retrospective review the cases with very advanced BCC or SCC with skull base involvement treated at the Department of Head and Neck Surgery of the University of São Paulo Medical School, Brazil, was undertaken. Only cases with enough tumor tissue in the paraffin-embedded blocks to harvest slides for immunohistochemistry were included. Thirty-five patients constituted the two study groups: Group 1: 24 BCCs (Figure 1) and Group 2: 11 SCCs. Seventeen patients (71%) in patients of Group 1 and five patients (50%) in Group 2 had recurrent tumors, after surgery and/or radiotherapy. One patient (4.17%) in Group 1 and four patients (36.36%) in Group 2 had lymph node metastasis. Two control groups included patients with BCCs and SCCs located on the head and neck area, treated at the Dermatology Department of the same Institution, with no recurrence for a minimum follow-up of 24 months (median follow-up period: 32.2 months): Group 3: 23 BCCs and Group 4: 10 SCCs.

B. Immunohistochemical analysis The procedure described by Hsu et al, in 1981 was employed. Representative slides obtained from the tumors were washed with a buffered saline solution at pH 7.4. They were then

Figure 1. Extensive SCC, involving left orbit, frontal region and anterior skull base, with parotid and cervical metastases.

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Cancer Therapy Vol 3, page 473 Figure 2. Immunostaining showing Ki-67 nuclear expression in less than 30% of cancer cells (ABC technique; counter-staining with methylated green; original magnification: 400X).

Figure 3. Immunostaining showing Ki-67 nuclear expression in between 30% and 50% of cancer cells (ABC technique; counter-staining with methylated green; original magnification: 400X).

Figure 4. Immunostaining showing Ki-67 nuclear expression in more than 50% of cancer cells (ABC technique; counter-staining with methylated green; original magnification: 400X).

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Cernea et al: Ki-67 index and skin carcinomas with skull base invasion compared with head and neck skin carcinomas with good outcome, with no evidence of disease at a median 36.2month follow-up, treated in the same Institution within the same time frame, in a case-control study.

In Group 1, 37.50% of BCCs had intense Ki-67 expression, similar to 32.90% reported by Baum et al 1993, but inferior to 51% observed by Abdelsayed et al, 2000. The frequency of intense Ki-67 expression was only 13.04% in Group 3; despite the absence of statistical significance, a clear trend towards an increased proliferation status was suggested.

In Group 2, intense Ki-67 expression was noted in 72.73% of the tumors, statistically higher than 20% encountered in Group 4; and also superior to 46% reported

by Kerschmann et al, 1994. Our data did not confirm the findings of Kanitakis et al, 1997 who observes no difference in Ki-67 expression comparing 14 aggressive SCCs with 28 non-aggressive tumors. It is noteworthy the low incidence of this expression in both groups in their series (15.0% and 17.5%, respectively). Our findings confirm the experience of other authors (Kerschmann et al, 1994; Healy et al, 1995), indicating a status of enhanced cell proliferation among these extremely aggressive skin tumors with skull base invasion.

In conclusion, an increased prevalence of intense proliferation among BCCs and SCCs with skull base invasion was noted. This observation could stimulate the analysis of the role of anti-proliferation strategies in the therapy of these tumors. Clearly, the preliminary findings of this study need to be confirmed with larger series.

III. Results A. Ki-67 expression in BCCs Intense Ki-67 expression was observed in 37.50% of BCCs of Group 1 (Table 1). Despite the fact that this prevalence was more than three times higher than in Group 3 (13.04%), the difference was not statistically significant (p=0.155).

B. Ki-67 expression in SCCs Most cancers in Group 2 (72.73%) showed intense Ki-67 expression, compared to only 20.00% of tumors in Group 4 (Table 2), and this difference was statistically significant (p=0.050).

IV. Discussion Sometimes, skin carcinomas are extremely aggressive, and are called by some authors horrifying tumors (Jackson and Adams, 1973; Bianchini and Wolter, 1984; Horlock et al, 1998). Deep anatomical planes may be reached, with skull base involvement. Indeed, these cancers represent the main histological types of tumors in some series of oncological craniofacial operations (Medina dos Santos et al, 1994; Cernea et al, 1997; Dias et al, 1997). In these series, many BCCs and SCCs actually invaded duramater and brain.

Immunohistochemical expression of Ki-67 reflects the cell proliferation status of a neoplasia. When it is present in more than 50% of tumor cells, it is usually associated with increased aggressiveness. However, findings are somewhat controversial.

To our knowledge, this is the first study on proliferation status of a consecutive series of skin carcinomas with skull base invasion. The objective of this study was to analyze this proliferation status, using immunohistochemical evaluation of Ki-67, in a consecutive series of very aggressive skin carcinomas with skull base invasion submitted to combined craniofacial oncological operations. In addition, these findings were

References Abdelsayed RA, Guijarro-Rojas M, Ibrahim NA, et al (2000) Immunohistochemical evaluation of basal cell carcinoma and trichoepithelioma using Bcl-2, Ki67, PCNA and P53. J Cutan Pathol 27, 169-75.

Bacchi CE, Gown AM (1993) Detection of cell proliferation in tissue sections. Braz J Med Biol Res 26, 677-87.

Table 1. Expression of Ki-67 in BCC Group 1 3

Positive in less than 30% of cancer cells 8 (33.33%) 10 (43.48%)

Positive in between 30% and 50% of cancer cells 7 (29.17%) 10 (43.48%)

Positive in more than 50% of cancer cells 9 (37.50%) 3 (13.04%)

Positive in between 30% and 50% of cancer cells 1 (9.09%) 5 (50.00%)

Positive in more than 50% of cancer cells 8 (72.73%) 2 (20.00%)

p=0.155 Table 2. Expression of Ki-67 in SCC Group 2 4

Positive in less than 30% of cancer cells 2 (18.18%) 3 (30.00%)

p=0.050

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Cancer Therapy Vol 3, page 475 Barrett TL, Smith KJ, Hodge JJ (1997) Immunohistochemical nuclear staining for p53, PCNA and Ki67 in different histologic variants of basal cell carcinoma. J Am Acad Dermatol 37, 430-7.

Baum HP, Meurer I, Unteregger G (1993) Ki-67 antigen expression and growth pattern of basal cell carcinomas. Arch Dermatol Res 285, 291-5.

Bianchini R and Wolter M (1984) Fatal outcome in a metatypical, giant, horrifying basal cell carcinoma. J Dermatol Surg Oncol 13, 556-557.

Cernea CR, Ferraz AR, de Castro IV, et al (2004) Angiogenesis and skin carcinomas with skull base invasion, a case-control study. Head Neck 26, 396-400.

Cernea CR, Teixeira GV, Medina dos Santos LR, et al (1997) Indications for, contraindications to, and interruption of craniofacial procedures. Ann Otol Rhinol Laryngol 106, 927-933.

Dias FL, Sรก GM, Kligerman J, et al (1997) Prognostic factors and outcome in craniofacial surgery for malignant cutaneous tumors involving the anterior skull base. Arch Otolaryngol Head Neck Surg 123, 738-742.

Freeman RG (1976) Histopathologic considerations in the management of skin cancer. J Dermatol Surg 2, 215-219.

Fumic-Dunkic L, Katic V, Janjanin S, et al (2003) Retrospective analysis of Ki-67 antigen expression in paraffin tissue blocks of laryngeal squamous cell carcinoma. Am J Otolaryngol 24, 106-10.

Gonzalez-moles MA, Caballero R, Rodriguea-Archilla A, et al (1996) Prognostic value of expression of Ki-67 for squamous cell carcinoma of the oral cavity. Acta Stomatol Belg 93, 159-65.

Gown AM, de Wever N, Battifora H (1993) Microwave-based antigenic unmasking, a revolutionary new technique for routine immunohistochemistry.

Appl Immunohistochemistry 1, 256 Healy E, Angus B, Lawrence CM, et al (1995) Prognostic value of Ki-67 antigen expression in basal cell carcinoma. Br J Dermatol 133, 737-741.

Horlock NM, Wilson GD, Daley FM (1998) Cellular proliferation characteristics do not account for the behaviour of horrifying basal cell carcinoma, a comparison of the growth fraction of horrifying and non horrifying tumours. Br J Plast Surg 51, 59-66.

Hsu SM, Raine L (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques, a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29, 577-580.

Jackson R and Adams RH (1973) Horrifying basal cell carcinoma, a study of 33 cases and a comparison with nonhorror cases and a report on four metastatic cases. J Surg Oncol 5, 431-463.

Kanitakis J, Naravaez D, Euvrards S, et al (1997) Proliferation markers Ki-67 and PCNA in cutaneous squamous cell carcinomas, lack of prognostic value. Br J Dermatol 136, 633-53.

Kerschmann RL, McCalmont TH, Leboit PE, et al (1994) p53 oncoprotein and proliferation index in keratoacanthoma and squamous cell carcinoma. Arch Dermatol 130, 181-6.

Koch W, Sidransky D (2004) Molecular markers of radiation effectiveness in head and neck squamous cell carcinoma.

Semin Radiat Oncol 14, 130-8.

Lang PG and Maize JC (1991) Basal cell carcinoma. In, Friedman RJ, Rigel DS, Kopf AW, Harris MN, Baker D, editors. Cancer of the skin. Philadelphia, W.B. Saunders Company. p. 35-73.

Lavertu P, Adelstein DJ, Myles J, et al (2001) P53 and Ki-67 as outcome predictors for advanced squamous cell cancers of the head and neck treated with chemoradiotherapy.

Laryngoscope 111, 1878-92.

Liu M, Lawson G, Delos M, et al (2003) Predictive value of the fraction of cancer cells immunolabeled for proliferating cell nuclear antigen or Ki-67 in biopsies of head and neck carcinomas to identify lymph node metastasis, comparison with clinical and radiologic examinations. Head Neck 25, 280-8.

Mansoor A, McKee PH, Simpson JA, et al (1996) Prognostic significance of Ki-67 and p53 immunoreactivity in cutaneous squamous cell carcinomas. Am J Dermatopathol 18, 351357.

Medina dos Santos LR, Cernea CR, et al (1994) Results and prognostic factors in skull base surgery. Am J Surg 168, 481-484.

Ruhoy SM, Flynn KJ, DeGuzman MJ, et al (2001) Pathology of selected skin lesions of the Head and Neck. In, Barnes L, editor. Surgical Pathology of the Head and Neck, 2 nd ed. New York, Marcel Dekker, Inc. p. 1793-1875.

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Novel biomarkers for the early prediction of acute kidney injury Review Article

Prasad Devarajan Nephrology and Hypertension, Cincinnati Childrenâ&#x20AC;&#x2122;s Hospital Medical Center, University of Cincinnati, Cincinnati, OH

__________________________________________________________________________________ *Correspondence: Dr. Prasad Devarajan, Nephrology & Hypertension, MLC 7022, Cincinnati Childrenâ&#x20AC;&#x2122;s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. Phone: (513) 636-4531. FAX: (513) 984-9770. E-mail: prasad.devarajan@cchmc.org Key words: Acute renal failure, nephrotoxicity, biomarkers, proteomics, microarray Abbreviations: Acute renal failure (ARF); glomerular filtration rate (GFR); Acute tubular necrosis (ATN); acute kidney injury (AKI); neutrophil gelatinase-associated lipocalin (NGAL); cardiopulmonary bypass (CPB); kidney injury molecule-1 (KIM-1); sodium hydrogen exchanger isoform 3 (NHE3); Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDITOF-MS)

Contributed by Prasad Devarajan Received: 3 June 2005; Accepted: 19 September 2005; electronically published: September 2005

Summary Acute renal failure (ARF) remains a common problem in hospitalized patients with cancer. Despite technical advances in supportive care, the associated mortality and morbidity have remained unacceptably high. Although pre-clinical studies in animals have identified successful therapeutic interventions, translational clinical trials in humans have yielded disappointing results. The reasons for this are the lack of a consensus operational definition for acute renal failure, and the paucity of predictive early biomarkers. In this review, we first propose a consensus definition for acute kidney injury that encompasses the entire spectrum of ARF, from sub-clinical injury to minimal elevation in serum creatinine to anuric renal failure. The clinical significance of ARF, especially in the setting of cancer, is reviewed, to illustrate the urgent need for identifying novel methods for the early diagnosis of acute kidney injury. The impact of modern enabling technologies such as microarrays and proteomics on the biomarker discovery process is outlined, followed by an update on emerging biomarkers. Specifically, the utility of NGAL as a novel, sensitive, specific, highly predictive early biomarkers for human AKI is examined, and the role of urinary proteomic biomarker patterns for the early diagnosis of AKI is explored.

In this review, we will first propose a consensus definition for acute kidney injury that encompasses the entire spectrum of ARF, and illustrate the urgent need for identifying novel biomarkers for the early prediction of acute renal injury. The impact of modern enabling technologies such as microarrays and proteomics on the biomarker discovery process will then be outlined, followed by an update on emerging biomarkers.

I. Introduction Acute renal failure (ARF) remains a common and vexing problem in hospitalized patients with cancer.

Despite technical advances in supportive care, the associated mortality and morbidity have remained unacceptably high, and have not changed appreciably in the last four decades. Although basic research and preclinical studies in animals have identified successful therapeutic interventions, translational clinical trials in humans have yielded disappointing results. One reason for this is the lack of a consensus operational definition for acute renal failure. This has resulted in non-uniformity in the criteria for initiating therapies, and confusion in the interpretation and comparison of existing trials. A second reason is the paucity of predictive early biomarkers. This has hindered our ability to institute potentially effective preventive and therapeutic measures in a timely manner.

II. A consensus definition for acute kidney injury Acute renal failure (ARF) has traditionally been defined as an abrupt reduction in glomerular filtration rate (GFR), leading to accumulation of waste products such as BUN and creatinine. A major quandary with this definition is the primary reliance on serum creatinine measurements for the diagnosis. Over 30 different definitions have been 477

Devarajan: Novel biomarkers for the early predictionof acute kidney injury used in the clinical literature, ranging widely from minimal changes in serum creatinine (0.3 mg/dl or 20% increase above baseline) to severe ARF requiring dialysis (Mehta and Chertow, 2003; Bellomo et al, 2004). This lack of consensus definition, combined with the inherent shortcomings of serum creatinine measurements in ARF (see below), have seriously jeopardized the interpretation and comparison of existing clinical trials.

Acute tubular necrosis (ATN) is the most common manifestation of ischemic or nephrotoxic ARF, and the two terms have frequently been used synonymously.

However, ATN remains a pathologic diagnosis, and cannot be easily quantified since biopsy specimens are seldom obtained in patients with ARF. Furthermore, ATN is a misnomer, because frank necrosis is rarely encountered in human ARF (Devarajan, 2005). Thus, the use of the term ATN is unsuitable for translational studies.

The term acute kidney injury (AKI) has recently been proposed by the American Society of Nephrology Steering Committee on Acute Renal Failure. This definition denotes a complex disorder comprising multiple etiological factors with varied clinical manifestations ranging from sub-clinical injury to minimal elevation in serum creatinine to anuric renal failure. AKI represents a paradigm shift that incorporates the entire spectrum of ARF, and appropriately encompasses even minimal degrees of injury. However, the inability to identify subclinical and early AKI prior to rise in serum creatinine continues to represent an unresolved challenge. The designations ARF and AKI are used interchangeably in this review.

critical illnesses. In addition, ARF can represent a primary complication of the malignancy itself (such as obstruction and infiltration), or more commonly a complication of cancer therapy (such as nephrotoxicity and sepsis). Major etiologies of ARF in cancer patients are listed in Table 1, but it should be emphasized that ARF in this patient population is usually multi-factorial in origin.

Nephrotoxic AKI, by itself or in combination with other mechanisms, represents one of the most frequently encountered causes of ARF in malignancy. Nephrotoxicity can result from a number of agents as listed in Table 2. major culprit is cisplatin, one of the most widely used and effective chemotherapeutic agents for the treatment of several human malignancies (Arany and Safirstein, 2003; Hanigan and Devarajan, 2003).

Table 1. ARF in cancer patients PRERENAL CAUSES Vomiting, diarrhea, dehydration Sepsis Hypotension Bleeding Congestive heart failure Hepatorenal syndrome RENAL CAUSES Prolonged prerenal factors Nephrotoxic injury Ischemic injury Tumor lysis syndrome Hemolytic uremic syndrome Thrombotic thrombocytopenic purpura Hypercalcemia Myeloma kidney Lymphomatous infiltration POSTRENAL CAUSES Bladder outlet obstruction Bilateral upper tract obstruction

III. Clinical significance of acute kidney injury ARF due to ischemic and nephrotoxic injuries continues to represent a very significant and potentially devastating problem in clinical medicine (Bonventre and Weinberg, 2003; Molitoris, 2003; Rabb, 2003; Siegel and Shah, 2003; Star, 1998; Herget-Rosenthal et al, 2004; Hewitt et al, 2004; Schrier, 2004; Schrier et al, 2004; Lameire et al, 2005b). The incidence of ARF varies from 5% of all hospitalized patients to 30-50% of patients in intensive care units, and there is now substantial evidence that this incidence is rising. Despite significant technical improvements in dialytic therapy, the mortality and morbidity associated with ARF remain dismally high and have not appreciably improved during the last four decades. The mortality rate among dialyzed patients in intensive care units exceeds 80%. Even among survivors, long term consequences are frequent, with about 50% of patients being inflicted with chronic renal insufficiency and about 15% progressing inexorably to end stage renal disease within 3 years of an ARF episode.

ARF is a particularly frequent complication in cancer patients, and a major source of mortality and morbidity (Lameire et al, 2005a). ARF in patients with malignancies not only limits our ability to deliver effective therapy, but has also emerged as a major risk factor for the development of non-renal complications. The mechanisms of ARF in cancer are similar to those encountered in other

Table 2. Nephrotoxic ARF in cancer patients ANTIBACTERIALS Aminoglycosides Vancomycin Polymyxins ANTIFUNGALS Amphotericin ANTIVIRALS Foscarnet Cidofovir Acyclovir ANTINEOPLASTICS Cisplatin Carboplatin Nitrosoureas Methotrexate Cytosine arabinoside 5-Fluorouracil Mitomycin C Ifosfamide

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Cancer Therapy Vol 3, page 479 anti-neoplastic efficacy. Nephrotoxicity following cisplatin treatment may manifest after a single dose with ARF or may present with a chronic syndrome of renal electrolyte wasting. Despite various hydration protocols A The efficacy of cisplatin is dose-dependent, but the significant risk of nephrotoxicity frequently hinders the use of higher doses to maximize its designed to minimize the nephrotoxicity, approximately one-third of patients who receive cisplatin develop evidence for ARF. This can have major consequences in terms of mortality and morbidity, especially in the presence of other co-morbid conditions related to the primary malignancy or its treatment.

Bone marrow transplantation is being increasingly used for the treatment of malignancies and non-malignant conditions, and ARF is a frequent complication (Hahn et al, 2003; Patzer et al, 2003). The incidence of severe ARF varies from 10-25%, but occurs to a milder degree in over 90% of hematopoietic cell transplants (Letourneau et al, 2002; Parikh et al, 2002; Schrier, 2002). ARF in this population most commonly occurs during the period of post-transplant neutropenia, and is frequently due to a combination of nephrotoxins, sepsis and other factors.

Several contributory etiologies have been identified, and may be classified according to the timing of the ARF in relation to the bone marrow transplant, as shown in Table 3. ARF requiring dialysis after bone marrow transplantation is associated with a very poor prognosis and a mortality rate of greater than 90% (Letourneau et al, 2002; Parikh et al, 2002; Schrier, 2002).

Schrier, 2004; Schrier et al, 2004; Lameire et al, 2005b).

In current clinical practice, ARF is typically diagnosed by measuring serum creatinine. Unfortunately, creatinine is an unreliable and delayed indicator during acute changes in kidney function (Bellomo et al, 2004). First, serum creatinine levels vary widely with age, gender, diet, muscle mass, medications, and hydration status. Second, serum creatinine levels are insensitive to the small changes in GFR that are characteristic of early reversible forms of AKI. Serum creatinine concentrations may not change until about 50% of kidney function has already been lost.

Third, serum creatinine does not accurately depict kidney function until a steady state has been reached. Changes in serum creatinine may lag behind alterations in GFR by several days, during decline as well as recovery of renal function. However, animal studies have shown that while AKI can be prevented and/or treated by several maneuvers, these must be instituted very early after the insult, in the initiation phase of the injury. The lack of early biomarkers for AKI in humans has hitherto crippled our ability to launch potentially effective therapies in a timely manner. Indeed, several human investigations have now established that the earlier the intervention, the better the chance of ameliorating the renal dysfunction (Schrier, 2004). Conversely, the longer the duration of ARF, the greater is the mortality rate (Schrier, 2004). Thus, there clearly exists an urgent need to identify novel methods for the early diagnosis of human AKI.

V. Genomic approaches to acute kidney injury

IV. Urgent need for biomarkers of acute kidney injury

Attempts at unraveling the molecular basis of complex biologic processes such as AKI have been markedly facilitated by recent advances in functional genomics that have yielded new tools for genome-wide analysis (Schena et al, 1995; Eisen et al, 1998; Golub et al, 1999; Lockhart and Winzeler, 2000; King and Sinha, 2001; Kurella et al, 2001; Yoshida et al, 2002a, b; Supavekin et al, 2003). The cDNA microarray methodologies provide parallel and quantitative expression profiles of thousands of genes, which when combined with stringent bioinformatic tools can identify genes in a biologic pathway, characterize the function of novel genes, and detect disease subclasses. We have utilized the cDNA microarray technology and extensive statistical analysis to define global changes in renal gene expression during the early reperfusion periods following ischemic injury in an established mouse model (Supavekin et al, 2003). We have screened for changes in expression of 9000 sequence-verified mouse genes at various early points (3, 12, and 24 hours) following ischemic AKI. We chose to examine the immediate and early responses because the protein products of these genes may represent early biomarkers that have hitherto eluded discovery. We identified several transcripts that were known to be overexpressed or repressed following ischemic injury, thereby validating this technique (Supavekin et al, 2003).

Surprisingly, several of the transcripts that were maximally induced after ischemic AKI were novel to the field. We have focused primarily on a subset of seven

Outstanding advances in basic research have illuminated the pathogenesis of AKI and have paved the way for successful therapeutic approaches in animal models of ischemic and nephrotoxic injuries. However, translational research efforts in humans have yielded disappointing results. A major reason for this is the lack of early markers for AKI, akin to troponins in acute myocardial disease, and hence an unacceptable delay in initiating therapy (Bonventre and Weinberg, 2003; Molitoris, 2003; Rabb, 2003; Siegel and Shah, 2003; Star, 1998; Herget-Rosenthal et al, 2004; Hewitt et al, 2004; Table 3. ARF following bone marrow transplantation FIRST 10 DAYS Tumor lysis syndrome Hemoglobinuria from infusate Sepsis FROM 10-21 DAYS Veno-occlusive disease Hepatorenal syndrome Sepsis AFTER 21 DAYS Cyclosporine Amphotericin Hemolytic uremic syndrome Irradiation

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Devarajan: Novel biomarkers for the early predictionof acute kidney injury genes whose expression is upregulated more than 10 fold within the first few hours following ischemic renal injury in the mouse model. One of these transcripts, cysteine rich protein 61, has recently been confirmed to be markedly upregulated following renal ischemia in animal models, and may represent a novel biomarker for AKI (Muramatsu et al, 2002). In recent studies (Devarajan et al, 2003; Mishra et al, 2003; Mishra et al, 2004a, b, 2005; Mori et al, 2005), we have further characterized one of these previously unrecognized genes, namely neutrophil gelatinase-associated lipocalin (NGAL). We confirmed the marked upregulation of NGAL mRNA by semiquantitative RT-PCR and protein levels by Western analysis in the early post-ischemic mouse kidney (both greater than 10-fold). NGAL protein expression was detected predominantly in PCNA-positive proximal tubule cells that were undergoing proliferation and regeneration.

These findings strongly implicate a role for this maximally induced gene and protein in the repair process following AKI. Other recent studies have also suggested that NGAL enhances the epithelial phenotype. During nephrogenesis, NGAL is expressed by the penetrating ureteric bud, and triggers nephrogenesis by stimulating the conversion of mesenchymal cells into kidney epithelia (Yang et al, 2002). These findings are especially pertinent to the mature kidney, in which one of the well-documented responses to AKI is the remarkable appearance of dedifferentiated epithelial cells lining the proximal tubules (Witzgall et al, 1994). An important aspect of renal regeneration and repair after injury involves the reacquisition of the epithelial phenotype, a process that recapitulates several aspects of normal development (Hammerman, 2000). This suggests that NGAL may be expressed by the damaged tubule in order to induce reepithelialization. Support for this notion derives from the recent identification of NGAL as a regulator of epithelial morphogenesis in cultured kidney tubule cells (Gwira et al, 2005), and as an iron transporting protein that is complementary to transferrin during nephrogenesis (Yang et al, 2002). It is well known that the delivery of iron into cells is crucial for cell growth and development, and this is presumably critical to post-ischemic renal regeneration just as it is during ontogeny. Since NGAL appears to bind and transport iron, it is also likely that NGAL may serve as a sink for iron that is shed from damaged proximal tubule epithelial cells. Because NGAL can be endocytosed by the proximal tubule, the protein could potentially recycle iron into viable cells. This might stimulate growth and development, as well as remove iron, a reactive molecule, from the site of tissue injury, thereby limiting iron-mediated cytotoxicity. Indeed, our recent findings indicate that exogenously administered NGAL ameliorates AKI in animal models by tilting the balance of tubule cell fate towards proliferation and survival (Mishra et al, 2004b; Mori et al, 2005). Importantly, we have also found that NGAL is easily detected in the urine very early following AKI in both animal and human models of ARF (Muramatsu et al, 2002). In recent studies (Devarajan et al, 2003; Mishra et al, 2003; Mishra et al, 2004a, b; Mishra et al, 2005; Mori et al, 2005). These results are detailed in

the next section. Thus, NGAL has rapidly emerged from the discovery phase using cDNA microarrays, to potentially occupying center-stage in the AKI field, not only as a novel biomarker but also as an innovative therapy.

It is important to recognize that one of the limitations to using genomic approaches is the fact that alterations in gene expression are not always predictive of downstream functional and/or pathophysiologic pathways. Although this method can suggest activation of biologic pathways at the mRNA level, additional post-transcriptional and/or post-translational events may be required to fully implicate the identified factors. Thus, the cDNA microarray results provide a stepping stone, and in the case of AKI it will be important in future studies to fully characterize the biology of genes with altered expression profiles in order to better understand their role.

VI. NGAL: a novel biomarker of acute kidney injury In follow up studies to our initial biomarker discovery experiments by gene expression profiling, we found NGAL protein to be markedly induced in kidney tubule cells after both ischemic (Mishra et al, 2003) and nephrotoxic (Mishra et al, 2004a) AKI in animal models.

NGAL protein in these tubule cells occupied a punctate cytoplasmic distribution that partially co-localized with endosomal markers, suggestive of a secreted and/or endocytosed protein (Figure 1). Since NGAL is known to represent a small secreted polypeptide that is protease resistant, we tested the hypothesis that it may be excreted in the urine. Indeed, we have found by Western blotting that NGAL is easily detected in the urine very early following ischemic kidney injury in both mouse and rat models of AKI after ischemia (Devarajan et al, 2003; Mishra et al, 2003) and cisplatin nephrotoxicity (Mishra et al, 2004a).

Figure 1. Mice treated with intraperitoneal cisplatin (20 mg/kg) show a rapid induction (within 3 hours) of NGAL protein in tubule cells by immunohistochemistry, in a punctate cytoplasmic distribution. NGAL staining is virtually undetectable in untreated animals (not shown). Magnification is 100X.

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Cancer Therapy Vol 3, page 481 The appearance of NGAL in the urine is closely related to the dose and duration of renal ischemia, and precedes by far the appearance of other known urinary markers such as NAG and !2-microglobulin. Our results indicate that NGAL represents an early, sensitive, non-invasive urinary biomarker for ischemic renal injury that compares very favorably with other biomarkers that have been described in animal studies. One of the best-studied examples is KIM-1, a putative adhesion molecule involved in renal regeneration that was also first detected as a result of genomic analysis (Ichimura et al, 1998; Bailly et al, 2002).

In a rat model of ischemia-reperfusion injury, KIM-1 was found to be up-regulated 24-48 hours after the initial insult, rendering it a reliable but somewhat late marker of tubular cell damage. In another recent example, Cyr61 was found to be a secreted cysteine-rich protein that is detectable in the urine 3-6 hours after ischemic renal injury in rats (Muramatsu et al, 2002). However, this detection required a bioaffinity purification step with heparin-sepharose beads, and even after such purification several cross-reacting peptides were apparent. In contrast, our studies demonstrate that NGAL was easily and rapidly detected as a clean immunoreactive peptide in Western blots in the very first unprocessed urine output following AKI due to both ischemia and nephrotoxicity. In addition, urinary NGAL was evident even after very mild â&#x20AC;&#x153;subclinicalâ&#x20AC;? renal ischemia, in spite of normal serum creatinine levels.

These findings prompted us to test the hypothesis that NGAL represents a novel early biomarker of ischemic renal injury in a representative human population, namely

patients undergoing CPB. It is well known that over 700,000 CPB procedures are performed each year in the US alone. AKI occurs in 10-40% of patients after CPB, with 1-5% requiring dialysis in whom the mortality rate approaches 80% (Chertow et al, 1997; Fortescue et al, 2000; Tuttle et al, 2003). A variety of clinical algorithms have been proposed for prediction of dialysis-requiring ARF based on pre-operative risk factors (Chertow et al, 1997; Fortescue et al, 2000; Eriksen et al, 2003; Tuttle et al, 2003; Thakar et al, 2005), but no tools were available for the early diagnosis of lesser degrees of renal injury.

We therefore prospectively studied children undergoing CPB. Exclusion criteria included pre-existing renal insufficiency, diabetes mellitus, peripheral vascular disease, and the use of nephrotoxic agents before or during the study period. Thus, we recruited a homogeneous population of patients with no confounding variables in whom the only conceivable renal insult would most likely be the result of ischemia-reperfusion injury following CPB. Serial urine and blood samples were analyzed by Western blots and a newly designed ELISA for NGAL expression. The primary outcome variable was AKI, defined as a 50% or greater increase in serum creatinine from baseline. Twenty eight percent of patients in our study cohort developed acute renal injury, but the diagnosis using serum creatinine was possible only 2-3 days after CPB. In contrast, urine NGAL in these patients rose more than 10-fold at 2 hours after CPB, as shown in Figure 2. Serum NGAL similarly increased 6-fold at 2

Figure 2. Panel shows urine NGAL (in ng/ml) at various times after CPB in patients who subsequently developed ARF (blue) versus those who did not (red), determined by ELISA. The green bar represents the time when the initial rise in serum creatinine was detected.

At all post CPB time points examined, urine NGAL was significantly greater in subjects who developed ARF, as defined by a 50% increase in serum creatinine over baseline. Adapted from reference 38.

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Devarajan: Novel biomarkers for the early predictionof acute kidney injury hours after CPB (Mishra et al, 2005). These results were similar when analyzed by either Western blotting or by ELISA. Univariate analysis showed a significant correlation between acute renal injury and the following: 2 hour urine NGAL, 2 hour serum NGAL, and CPB time.

By multivariate analysis, the urine NGAL at 2 hours post CPB emerged as the most powerful independent predictor of acute renal injury. A ROC curve for the 2 hour urine NGAL revealed an area under the curve of 0.998, and a sensitivity of 1.00 and specificity of 0.98 for a cutoff value of 50 ng/ml. For the 2 hour serum NGAL, the area under the curve was 0.91, the sensitivity 0.95 and the specificity 1.00 for a cutoff value of 50 ng/ml.

Our NGAL results compare favorably with or surpass those obtained for several other biomarkers for human AKI (Rabb, 2003; Herget-Rosenthal et al, 2004; Hewitt et al, 2004). The majority of human studies reported thus far have been retrospective, have examined biomarkers in the established phase of ARF, and have been restricted to only the urine and to only one method of detection. Several tubular proteins have been measured in the urine, with conflicting and unsatisfactory results (Han et al, 2002; Westhuyzen et al, 2003; Herget-Rosenthal et al, 2004). KIM-1 is detectable by ELISA in the urine of patients with established acute tubular necrosis (Han et al, 2002). Also, the sodium hydrogen exchanger isoform 3 (NHE3) has been shown by Western blots to be increased in the membrane fractions of urine from subjects with established ARF (du Cheyron et al, 2003). However, the sensitivity and specificity of these biomarkers for the detection of renal injury have not been reported. Of the inflammatory cytokines involved in ARF, elevated levels of urinary IL-6, IL-8 and IL-18 have been demonstrated in patients with delayed graft function following cadaveric kidney transplants (Kwon et al, 2003; Parikh et al, 2003).

With the exception of NGAL, none of the biomarkers have been examined prospectively for appearance in the urine during the evolution of ischemic ARF. A recent prospective study has demonstrated that an increase in serum cystatin C precedes the increase in serum creatinine in a select patient population at high risk to develop ARF (Herget-Rosenthal et al, 2004). However, the ARF in these subjects was multifactorial, due to a combination of ischemic, prerenal, nephrotoxic, and septic etiologies.

Furthermore, since cystatin C is primarily a marker of GFR, it can be inferred that serum cystatin C levels will rise only after the GFR begins to fall. On the other hand, NGAL is rapidly induced in the kidney tubule cells in response to ischemic injury, and its early appearance in the urine and serum is independent of the GFR, but is highly predictive of a fall in GFR that may occur several days later.

We conclude that urine and serum NGAL represent novel, sensitive, specific, highly predictive early biomarkers for AKI following CPB (Mishra et al, 2005). A limitation to this study is that it represents a single center analysis involving children with congenital heart disease, with predominantly ischemic kidney injury. While this cohort was intentionally chosen to eliminate common confounding variables and co-morbid conditions, it is acknowledged that ARF is frequently multifactorial, and

our results will need to be validated in a larger population in whom additional mechanisms of renal injury may be invoked. Examination of urine and serum NGAL in other human conditions that predispose to AKI (including cisplatin nephrotoxicity, kidney and bone marrow transplantation, contrast nephropathy, and sepsis) is currently in progress.

VII. Proteomic approaches to acute kidney injury Proteomics may be defined as the systematic analysis of proteins for their identity, quantity, and function (Peng and Gygi, 2001). This is a rapidly expanding field that offers several distinct advantages over microarray analysis, since it provides the technology to (a) simultaneously analyze all proteins, the primary mediators of function, within a cell or tissue of interest, (b) examine body fluids such as urine which are generally devoid of functional nucleic acids, and (c) account for posttranscriptional regulatory mechanisms that modulate protein structure and function. Recent advances in the field of clinical proteomics have greatly accelerated the discovery of novel protein biomarkers for renal diseases (Knepper, 2002; Clarke et al, 2003; Cutillas et al, 2004; Han and Bonventre, 2004; Hewitt et al, 2004; Klein and Thongboonkerd, 2004; Schaub et al, 2004a, b; Thongboonkerd, 2004; Thongboonkerd et al, 2004a, b). Of the various methods available, Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS) technology has emerged as the preferred platform for urinary protein profiling (Clarke et al, 2003; Schaub et al, 2004a, b). This approach allows for rapid high throughput profiling of multiple urine samples, detects low molecular weight biomarkers that are typically missed by other platforms, and even uncovers proteins bound to albumin.

We have tested the hypothesis that urinary proteomic analysis may identify novel early biomarker patterns for AKI in a representative human population, namely CPB. Urine samples were obtained at baseline and at 2 hours post CPB, and analyzed by SELDI-TOF-MS. The primary outcome variable was ARF, defined as a 50% or greater increase in serum creatinine. We have now completed a preliminary analysis of 30 patients (15 with ARF and 15 age-matched controls without ARF). SELDI-TOF-MS analysis of urine from the ARF group at baseline versus at 2 hours post-CPB consistently showed a marked and statistically significant enhancement of protein biomarkers with m/z of 6.4, 28.5, 33, 43 and 66 kDa, as shown in Figures 3, 4, and 5. The same biomarkers were also enhanced when comparing control versus ARF groups at 2 hours post-CPB. The specific identity of these biomarkers is currently unknown. However, it is likely that the 28 kDa biomarker species revealed in the present study may represent NGAL, since Western blot analysis of the same urine samples with a monoclonal antibody to NGAL identified an abundant immunoreactive peptide at the 28 kDa range (not shown). Our preliminary results indicate that SELDI-TOF-MS is a novel, non-invasive, sensitive, reproducible, highly predictive, rapid (with a turnaround 482

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Figure 3. Representative SELDI-TOF-MS spectra of urine obtained at baseline and 2 hours post-CPB from patients in the Control or ARF group. Figure shows proteins in the 5500-7500 kDa range. Marked enhancement of a 6.4 kDa species is noted in the ARF group at 2 hours post-CPB.

Figure 4. Representative SELDI-TOF-MS spectra of urine obtained at baseline and 2 hours post-CPB from patients in the Control or ARF group.

Figure shows proteins in the 20,000-70,000 kDa range. Marked enhancement of 28.5, 33, 43, and 66 kDa species is noted in the ARF group at 2 hours postCPB.

Figure 5 . Overlay of representative SELDI-TOF-MS spectra of urine obtained at baseline and 2 hours post-CPB from patients in the ARF group. Marked enhancement of 28.5, 33, 43, and 66 kDa species is noted in the ARF group at 2 hours post-CPB, as highlighted by the arrows. The specific identity of these biomarkers is currently unknown. However, it is likely that the 28 kDa species may represent NGAL.

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Devarajan: Novel biomarkers for the early predictionof acute kidney injury time of only 90 minutes), and non-invasive (requiring only microliter quantities of urine) method for the prediction of acute renal injury following CPB. These results will need to be validated in a larger population of susceptible patients. It will also be important in future studies to confirm the identity of the biomarkers for AKI uncovered by this study, and to determine their individual and collective robustness for the prediction of AKI.

Table 4. Traditional urinary biomarkers for AKI LOW MOLECULAR WEIGHT PROTEINS Retinol binding protein (67) !2-microglobulin (67) "1-microglobulin (68) TUBULE BRUSH BORDER PROTEINS Adenosine deaminase binding protein (67) Carbonic anhydrase (69) URINARY ENZYMES N-acetyl-!-D-glucosamine (67) Alanine aminopeptidase (67) Neutral endopeptidase (70) #-glutamyltransferase (71) Alkaline phosphatase (72) Lactate dehydrogenase (72) !-glucosidase (72) Cathepsin B (73)

VIII. Other emerging biomarkers of acute kidney injury The quest for easily measured and reliable biomarkers of AKI is and has been an area of intense research interest. Many urinary proteins have been evaluated in the past as noninvasive indicators of human AKI (Han and Bonventre, 2004). Traditional urinary biomarkers include low molecular weight proteins such as retinol binding protein and !2-microglobulin, brush border proteins such as carbonic anhydrase, and a variety of urinary enzymes, as shown in Table 4 (Taniguchi et al, 1979; Stonard et al, 1987; Tolkoff-Rubin et al, 1988; Olbricht et al, 1994; Nortier et al, 1997; Donadio et al, 1998; Bazzi et al, 2001). In general, these markers lack specificity, reproducibility, validation, and standardized assays (Wedeen et al, 1999). The utility of these biomarkers in human AKI is currently limited, although they are still commonly employed in pre-clinical studies.

Fortunately, modern enabling technologies for screening the genome and proteome have yielded promising new urinary biomarkers for human AKI (Table 5). In general, an ideal biomarker for AKI should be noninvasive, accurate, reproducible, measured using standardized assays, and adaptable to point-of-care testing.

The potential for NGAL to satisfy all these requirements has already been alluded to. The current status of other emerging biomarkers is reviewed below.

Kidney Injury Molecule-1 (KIM-1) is an adhesion molecule that is up-regulated in tubule cells in humans and rodents after ischemic or nephrotoxic injury (Ichimura et al, 1998; Bailly et al, 2002). In a small human study, a soluble form of the cleaved protein has been detected in the urine about 12 hours after an ischemic insult (Han et al, 2002). Attractive aspects of KIM-1 as a biomarker include the fact that its expression is specific to the kidney, and that it can be measured in a standardized fashion using ELISA. However, prospective human studies, with better definition of temporal sequence of appearance, sensitivities, specificities, and predictive values are lacking.

The NHE3, the most abundant apical membrane sodium transporter, has been detected in the membrane fractions of urine from patients with ATN and post-renal ARF (du Cheyron et al, 2003). However, this detection requires isolation of membrane fractions followed by Western blotting, which are cumbersome and not easy to quantify. Additional studies with assay standardization, validation, time course and biomarker statistics are required.

Pro-inflammatory cytokines such as IL-6 (Kwon et al, 2003), IL-8 (Kwon et al, 2003) and IL-18 (Parikh et al,

Table 5. Emerging urinary biomarkers for AKI BIOMARKERTYPE OF INJURY ASSAY REF NGALIschemic, NephrotoxicELISA33-38 KIM-1Ischemic, NephrotoxicELISA52 NHE3Ischemic, Post-renalWestern53 IL-6, IL-8Delayed graft functionELISA54 ActinDelayed graft functionWestern54 IL-18Delayed graft functionELISA55 "-GSTProximal tubule injuryELISA75 $-GSTDistal tubule injuryELISA75 Cystatin CAcute tubular necrosisNephelometry51, 76 Cyr61Ischemic (animals)Western32 2003) have been shown to be up-regulated within 24 hours following kidney transplantation in the urine of patients who subsequently developed delayed graft function (Kwon et al, 2003; Parikh et al, 2003). Actin is an abundant component of tubule epithelial cells, and its urinary excretion follows a similar pattern in delayed graft function (Kwon et al, 2003). The commercial availability of standardized ELISA assays for the cytokines render them attractive AKI biomarker candidates. Additional studies are needed to validate their utility in various forms of AKI, and to further define the timing of their appearance in the urine. However, urinary actin measurement is currently dependent on Western blotting methods that are inherently difficult to quantify and standardize.

Glutathione S-transferases are cytosolic proteins that are released from proximal ("-GST) or distal ($-GST) tubule cells following AKI. In one human study of kidney transplant patients with dysfunction, urinary levels of $GST were elevated in acute rejection, concentrations of "GST were increased in nephrotoxic injury secondary to cyclosporine, and both isoforms were increased in ATN (Sundberg et al, 1994). However, in a more recent study of patients with ATN from various etiologies, urinary excretion of "-GST was not found to be predictive of an unfavorable outcome (Herget-Rosenthal et al, 2004).

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Gwira JA, Wei F, Ishibe S, Ueland JM, Barasch J, Cantley LG (2005) Expression of Ngal regulates epithelial morphogenesis in vitro. J Biol Chem 280, 7875-7882.

Hahn T, Rondeau C, Shaukat A, Jupudy V, Miller A, Alam AR, Baer MR, Bambach B, Bernstein Z, Chanan-Khan AA, Czuczman MS, Slack J, Wetzler M, Mookerjee BK, Silva J, McCarthy PL Jr (2003) Acute renal failure requiring dialysis after allogeneic blood and marrow transplantation identifies very poor prognosis patients. Bone Marrow Transplant 32, 405-10.

Hammerman MR (2000) Recapitulation of phylogeny by ontogeny in nephrology. Kidney Int 57, 742-755.

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Additional studies are required to examine the utility of urinary GST measurements in various forms of AKI. Serum cystatin C has recently emerged as an encouraging marker of GFR in ATN that precedes a rise in serum creatinine (Herget-Rosenthal et al, 2004), but the utility of urinary cystatin C is less clear. The ratio of urinary cystatin C to urinary creatinine was shown to be a sensitive measure of decreased GFR in patients with diverse chronic renal diseases (Hellerstein et al, 2004), but prospective measurements in AKI are lacking.

IX. Summary and future directions In this review, we have redefined ARF as AKI to encompass sub-clinical injury and the initiation phase of ARF, which represents the window of opportunity for potentially effective preventive and therapeutic interventions. We have recognized the urgent need for early diagnosis of AKI prior to the rise in serum creatinine. We have reviewed the current status of promising early urinary biomarkers for AKI. It will be important in future studies to evaluate multiple potential AKI biomarkers in prospective studies of susceptible individuals. It is likely that not any one biomarker but a collection of strategically selected proteins may provide the hitherto elusive “ARF Panel” for the early and rapid diagnosis of acute renal injury. The most promising biomarkers will need to be cross-validated within a network of laboratories. We will need to partner with industry to design point-of-care kits and platforms that will enable the early diagnosis of AKI by the bedside.

Such tools would be indispensable for the timely institution of potentially effective therapies in human ARF, a common clinical condition still associated with a dismal prognosis where early intervention is desperately needed.

Acknowledgements Dr. Devarajan is supported by grants from the NIH/NIDDK (RO1-DK53289, P50-DK52612, R21DK070163), a Grant-in-Aid from the American Heart Association Ohio Valley Affiliate, and a Translational Research Initiative Grant from Cincinnati Children’s Hospital Medical Center.

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Cancer Therapy Vol 3, page 489 Cancer Therapy Vol 3, 489-494, 2005

Peritoneal carcinomatosis versus peritoneal tuberculosis: a rare diagnostic dilemma in ovarian masses Case Reports

Konstantinos Vagenas1,*, Christos Stratis1, Charalambos Spyropoulos1, John Spiliotis3, John Petrochilos1, Helen Kourea2, Dionisios Karavias1 1

Department of Surgery, Department of Pathology, University of Patras, Medical School of Patras, Greece, 3 Department of Surgery, â&#x20AC;&#x153;Hatzikostaâ&#x20AC;? Hospital, Mesolongi, Greece 2

__________________________________________________________________________________ *Correspondence: Konstantinos Vagenas, Associate Professor of Surgery, Aou Street 16, 26442, Patras (Exo Aghia), Greece; Tel. 0032610455635 / Fax 0032610993984; E-mail: kvagenas@hotmail.com Key words: Tuberculosis, Tuberculous Peritonitis, Ovarian Cancer Received: 28 June 2005; Accepted: 11 July 2005; electronically published: August 2005

Summary The incidence of tuberculosis is rising resulting in a simultaneous increase in the risk of TB peritonitis in surgical practice. This type of disease is fatal if it goes untreated. In women the symptoms may mimic ovarian carcinoma.

We present, retrospectively, five cases during the period 1998-2002 from three hospitals in SW Greece, which presented with elevated CA 125 and vague symptoms as ovarian cancer. Five women 23-76 years old. In all five cases the patient revealed the presence of ascetic fluid and elevated CA 125. The initial diagnosis was ovarian cancer, but the final histological diagnosis confirms the TB peritonitis. We expose our experience in five cases of tuberculous peritonitis and we discuss the problems in differential diagnosis and treatment of this disease, the role of surgery and the impact of antituberculous chemotherapy upon the disease. women with abdominal pain, ascites, obstruction or peritonitis, the diagnosis is ovarian cancer (Gitt S et al 1992). In this article, we describe five case of peritoneal tuberculosis in Southwestern Greece, mimicking ovarian cancer. We expose our experience in diagnosis and treatment of this disease, with particular regard to the role of surgery and the impact of antituberculous chemotherapy upon the disease.

I. Introduction Incidence of tuberculosis is sharply rising in the developing as well as in the developed countries and tuberculous peritonitis is often diagnosed late in the course of the disease, resulting increased patient morbidity and mortality. Despite the widespread impression that tuberculous peritonitis is rare today, the disease appears regularly on the surgical services worldwide. Its symptoms are insidious and non specific and often simulate symptoms of carcinomatous peritonitis (Lisehora et al 1996). It constitutes the third most common etiologic factor for ascites, after hepatic cirrhosis and neoplasm and it is the sixth most frequent cause of extra pulmonary tuberculosis in the USA, following lymphatics, genitourinary tract, bone and joint, miliary and meningeal tuberculosis. It may be associated with Human Immunodeficiency Virus, although the pattern of presentation seems to differ (Mehta et al 1991). It is often not considered in the differential diagnosis of abdominal pain and it is left untreated. Most often, especially in

II. Case Reports A. 1st Case A 65 year-old female presented to the hospital with progressive symptoms of abdominal bloating and mild pain persisting for two months, a low grade fever of 37.537.8 oC every afternoon, weight loss of 3 kg, loss of appetite and night sweating. The patient had been exposed to Mycobacterium Tuberculosis continuously for few months preceding her admission, through her husband who was suffering active pulmonary TB without knowing

489

Vagenas et al: Peritoneal carcinomatosis vs peritoneal tuberculosis it (his diagnosis was made after the diagnosis of TB peritonitis was made in the female patient). During the physical examination, arterial blood pressure and pulse rate were normal and the body temperature was 37.2oC. There was no confirmation of lymphadenopathy. Ascites was present, but there was no edema of the extremities.

Results of blood analysis on hospital admission were: Hct 39,6%, Hbg 13.1 g/dl, WBC 5,110/mm3 (PMN: 66%, LYM: 26%, MON: 8%) and a platelet count of 386,000/mm3. The Westergen sedimentation rate was 59 mm in one hour. Liver biochemistries included SGOT 33U/l, SGPT 16U/l and LDH 206U/l. Renal function laboratories and urine analysis were normal. CA-125 was highly elevated (465U/ml). CA-19 9 was 441 U/ml and aFP was 2,721 U/ml. The chest radiograph was normal and the abdominal ultrasound and CT scan revealed fluid without organomegaly or peritoneal masses. Finally, the PPD skin test, which was made without clinical suspicion of TB infection, was negative. A preliminary diagnosis of peritoneal carcinomatosis due to ovarian cancer was made, based on the physical examination, the patientâ&#x20AC;&#x2122;s history and the laboratory data. According to this diagnosis, the patient underwent laparoscopy on the fifth hospital day.

During the procedure, several litters of ascetic fluid were drained, showing the peritoneum studded with nodules along with adhesion of the omentum, small intestine and tranverse colon. The ovaries and uterus were covered with the same lesions. A bilateral oophorectomy was performed with simultaneous partial resection of the omentum and peritoneum along with suspicious lesions. The frozen section of the sample from the ovaries was negative for malignancy; instead it revealed granular lesions with necrosis. The cytology exam of the fluid was negative for tumor cells, as well the Gram and Ziehl-Neelsen stains.

The patient had an uncomplicated postoperative course and on the sixth day, she began anti-tuberculosis treatment with daily doses of 300 mg INH, 600 mg rifampycin, 1,500 mg pyrazinamide and 10 mg B6. Four weeks after surgery, the ascetic fluid culture was negative for mycobacterium while the abdominal bloating and the sweating completely resolved during the second week. At twelve weeks of therapy, CA-125 level was within normal range. Ten months after the beginning of anti-tuberculosis treatment, the serum CA-125 level was still within normal range and the abdominal ultrasound was negative for ascetic fluid.

level was elevated (1,25U/ml) and CA 15-3 was highly elevated (415 U/ml). The chest radiograph was normal and the abdominal CT scan revealed a large abdominal mass in proximity to the anatomic site of the uterus and ascites. An initial diagnosis of peritoneal carcinomatosis from ovarian cancer was made and the patient underwent laparotomy.

During the procedure, granules implanted in the surface of the parietal peritoneum and small bowel, were found.

Microscopic examination of these lesions revealed tuberculous granulation tissue while the pelvic mass proved to be uterine leiomyoma, arising from the remainder of the uterus. The ovary was free of malignant disease. The patient had an uncomplicated postoperative course and on the 10th day, she began anti-tuberculosis treatment with INH, rifampycin for at least 10 months. She is well having until now.

C. 3rd Case A 76 year old female patient presented with anemia, fatigue and abdominal meteorism for two weeks. Physical examination revealed distended abdomen with ascites.

Laboratory data demonstrated: Hct 27%, WBC 7,100/mm3. The ESR was 102 mm in the first hour. The serum CA-125 level was highly elevated (5,354 U/ml).

The chest X-ray was normal and the abdominal CT scan revealed ascites with lymph nodes enlargement without any mention of peritoneal masses. An exploratory laparotomy was performed under general anesthesia.

During the operation, several litters of ascetic fluid were evacuated. Besides the ascites, the operative findings included nodules and adhesions implanted to the peritoneum, uterus, ovaries, small and large intestine. An ovarian carcinoma was speculated and a bilateral oophorectomy was performed, including the uterus and part of the omentum. Unfortunately, the patient died the 4 th postoperative day, due to pulmonary embolism. The histological examination of the specimen was negative for malignancy and the final diagnosis was peritoneal tuberculosis.

D. 4th Case A 23 year old female patient was admitted to the hospital complaining acute abdominal pain and high fever of 38-38.8oC. The physical examination revealed acute abdomen. Laboratory data at presentation included Hct 29%, WBC 12,000/mm3 (PMN: 70%, LYM: 20%, MON: 8%) and a platelet count of 420,000/mm3. ESR was 73 mm in the first hour. Liver and renal function analyses were within normal range. The tumor marker CA-125 was moderately elevated (200 U/ml). The chest X-ray was normal and the abdominal ultrasound and CT scan revealed peritoneal fluid with evidence of peritoneal masses. The patient underwent a broad spectrum antibiotic therapy but the fever persisted high, even on the 4th day of treatment. Based on the physical findings and the resistant fever, a preliminary diagnosis of intraabdominal infection or peritoneal carcinomatosis from ovarian cancer was made and the patient was subjected to exploratory laparotomy. The surgical findings and the frozen section of a sample from the ovaries and the peritoneum,

B. 2nd Case A 67 year-old female was admitted to the hospital with facial edema, loss of appetite and muscular cramps.

Physical examination revealed distended abdomen with drillness, indicative of ascites. Her personal history was significant for a partial hysterectomy and unilateral oophorectomy 20 years ago for non-malignant disease, as well as a history of suspicious pleural collection more than forty years ago. Laboratory data at presentation included the following: Hct 33%, WBC 7,200/mm3 (PMN: 74%, LYM: 18%, MONO: 8%), PLT: 450,000/mm3 and ESR was 97mm in one hour. Liver biochemistry included SGOT 55U/l, SGPT 82 U/l, LDH 180 U/l. Renal function analysis was within normal range. The serum CA-125 490

Cancer Therapy Vol 3, page 491 confirmed the diagnosis of peritoneal tuberculosis. The patient had an excellent postoperative course and on the 5 th postoperative day, she began anti-TBC therapy, INH and rifampycin, for twelve months. The patient completed the treatment course of 1 year anti TB therapy and she is well having until now.

accounts for 25-30% of the disease in the tropics and perhaps even more in immigrant patients living in developing communities. Large series have estimated the frequency of tuberculous peritonitis to range from 0.1 to 0.7% of all cases of tuberculosis, while the frequency in females is approximately twice than that found in males. It can be encountered, not only in the lowest socioeconomic classes, but also in any socioeconomic class and age, even without the coexistence of pulmonary findings of the disease (al-Quorain et al 1993; Hasanzadeh et al 2005).

Abdominal tuberculosis has four major clinical presentations: mesenteric lymphadenopathy, ileocecal disease, peritonitis, colonic and anorectal disease. On the other hand there are three forms of tuberculous peritonitis: the “wet” (with ascites), the “dry” (peritoneal involvement without ascites) and the “fibroid” type (with profound omental thickening and extensive adhesion formation) (Groutz et al 1998; Bilgin et al 2001). In our cases, all five patients were presented with the “wet” type of tuberculous peritonitis; it represents the most common type and is attended by usually non specific clinical manifestations and may suggest an occult malignancy, especially ovarian carcinoma, or cirrhosis with ascites. (Table 1) (Lisehora et al 1996). The infection route of the peritoneum by the tubercular bacterium varies. Infection may occur by reactivation of a long-latent TB focus in the peritoneum from a primary focus in the lung or elsewhere, infected mesenteric lymph nodes, contamination from tuberculous enteritis or expansion of a tuberculous salpingitis in the female. (Sochocky 1967). The disease is insidious with mild symptoms and no specific signs and it can mimic ovarian cancer in women. If a patient presents with abdominal bloating and pain, low fever and weight loss persisting for more than a few weeks, then TB peritonitis must be added to the differential diagnosis.

E. 5th Case A 58 year old female patient was admitted to the hospital complaining colon obstruction. The physical examination revealed distended abdomen with ascites. Her personal history was significant for meningial tuberculosis in the age of twenty four, as well as “pneumonia” in the age of thirty two. Laboratory data at presentation included the following: Hct 31%, WBC 14,000/mm3 (PMN: 74%, LYM: 18%, MON: 8%) and a platelet count of 300,000/mm3. Liver biochemistries included SGOT 75U/l, SGPT 44U/l and LDH 187 U/l. Renal function analysis was within normal range. The serum CA-125 level was elevated (810 U/ml) and CA 15-3 was elevated (70 U/ml).

The chest radiograph was normal and the abdominal CT scan revealed an abdominal mass in the pelvis, proximally to the anatomic site of the left colon and the presence of ascetic fluid. An initial diagnosis of ovarian cancer with colon invasion was made and the patient underwent laparotomy. The surgical findings and the frozen section of a sample from the mesenterium and the peritoneum, confirmed the diagnosis of peritoneal tuberculosis. The patient had an excellent postoperative course and he received anti-tuberculosis treatment (starting the 7th postoperative day) with INH and rifampycin for nine months.

III. Discussion The incidence of abdominal tuberculosis is extremely low in developed countries, but tuberculous peritonitis

Table 1. Main patients characteristics with peritoneal TB mimicking ovarian cancer AGE

Symptoms

Exposed to TB +

Tumor Markers CA-125: 465 CA 19-9: 441

CT Scan Fluid No Mass

Initial Diagnosis Ovarian Cancer

Histological Diagnosis Peritoneal TB

Case 1

65

Ascites Weight Loss Low Fever

Case 2

67

Anemia Edema Loss of Appetite Fever Anemia Fatigue

+/-

CA-125: 185 CA 19-9: 415

Fluid Abdominal Mass

S. Sjögren Ovarian Cancer

Peritoneal TB

Case 3

76

-

CA-125: 5354

Fluid Lymph Nodes

Ovarian Cancer

Peritoneal TB

Case 4

23

High Fever Abdominal Pain

-

CA-125: 200

Fluid

Peritoneal TB

Bowel Obstruction

+

CA-125: 810 CA 19-9: 70

Fluid Mass

Abdominal Infection or Ovarian Cancer Ovarian Cancer

Case 5

58

491

Peritoneal TB

Follow-Up Anti-TB treatment Alive until now Anti-TB treatment Alive until now Death due to pulmonary embolism at 4th postoperative day Anti-TB treatment Alive until now Alive

Vagenas et al: Peritoneal carcinomatosis vs peritoneal tuberculosis negative examination), while in two cases revealed additionally the presence of pelvic mass. The extent and distribution of the implants often seen in TB peritonitis may lead the physician to the wrong diagnosis of a terminal stage ovarian cancer. Histological evaluation however may reveal mycobacilli and granulomas with epithelioid cells and giant cells and infiltration of lymphocytes in the periphery. Such implants were observed in the peritoneum of all our patients, frozen section of whom detected granulomas with central necrosis. The histological examination is very helpful in the setting of differential diagnosis from other forms of granulomatous peritonitis such as schistosomiasis or ascariasis that require a different therapeutic approach.

Different studies demonstrate that adenosine deaminase activity levels of greater than 32.3 U/l in ascetic fluid are as high as 100% sensitive and 96% specific for the diagnosis of tuberculous peritonitis (Kadayifci et al 1996).

It is important to establish the diagnosis of TB peritonitis at the operating table, in order to begin immediately the therapeutic measure, while waiting for the culture results to indicate sensitivity. Specific sensitivity to various medications must be known, in order to avoid the formation of resistant stain of mycobacilli, by using the correct regimen. The onset of therapy in our patients was delayed for a few days, waiting for histological examination that documented the diagnosis. In conclusion, unclear abdominal symptoms in a patient from a risk group should alert the surgeon to the possibility of abdominal tuberculosis.

Even more important, the disease must be considered when an elderly female patient presents with ascites and elevated serum CA-125 levels. In our cases, all patients presented with abdominal pain, fever and weight loss. The serum CA- 125 levels were highly elevated in all patients, misleading the dominant initial diagnosis to the suggestion of an ovarian cancer in four of the five patients, preoperatively (Table 1). However a patient presented with symptoms of acute abdomen. In this case, the diagnosis first suspected only at laparotomy. In one autopsy series, a primary focus of the disease was demonstrated in 97% of patients (Simsek et al 1997; Straughn et al 2000). In our study, the evidence that the patient had been exposed to TB mycobacterium was clear in two cases, suspicious in another one and negative in two cases. The prognosis of the disease depends primarily on early suspicion and diagnosis, followed by immediate onset of the proper therapy. Since the introduction of antituberculosis chemotherapy, the disease mortality decreased dramatically, from 49 to 7% (U-Bayramicli et al 2003). Diagnosis of tuberculous peritonitis is extremely difficult. Determination of serum CA- 125 levels, serves as a useful marker for the diagnosis, the disease activity and also in evaluating the response to therapy in patients with peritoneal tuberculosis. In our study, CA-125 level, although high before the induction of TB chemotherapy, was measured within normal range as soon as the treatment course was completed, confirming the role of this marker in the clinical monitoring of the disease.

However, serum CA-125 levels can also be raised in patients with ovarian cancer and other malignancies, chronic liver diseases and peritonitis. High CA-125 levels can be found in patients with diseases affecting the peritoneum and/or ascites, as well as in women during pregnancy or in benign diseases, e.g. endometriosis. In our cases, pelvic inflammation caused by tuberculous peritonitis produced raised CA-125 levels, which fell after anti-TB therapy took place. (Simsek et al 1996; Kucok, 1998) The correct diagnosis of tuberculous peritonitis depends primarily on the appreciation of the manifold clinical manifestation of the disease and on bacteriologic proof of tuberculosis somewhere in the body. TB infection cannot be ruled out based solely on sputum analysis, chest fluid, ascetic fluid or the gastric fluid analysis. It should be also noted that cultures of the ascetic fluid for tuberculous bacilli and the tuberculin skin tests often prove negative in patients with peritoneal TB. CT scan seems to be the most sensitive imaging modality. If there is a large amount of ascetic fluid (according to CT scan results), at least a litter should be aspired in order to perform a Ziehl-Neelsen stain, culture for mycobacterium tuberculosis and cytological examination. If there is a minimum ascites on CT scanning or the stain for acid- resistant bacteria is negative, then peritoneal biopsy via laparotomy or, in selected cases, laparasoscopy must be planned. (Rodriguez et al 1996; Vazquez Munoz et al 2004) Laparoscopy is probably contraindicated in advanced TB peritonitis, where the peritoneum may be more than 1 cm thick and is tightly adherent to underlying friable bowel. In our cases the abdominal CT scanning revealed only the presence of ascetic fluid and no intraperitoneal implants (false

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Cancer Therapy Vol 3, page 495 Cancer Therapy Vol 3, 495-510, 2005

Cellular senescenceâ&#x20AC;&#x201C;an integrated perspective Review Article

Ola Larsson University of Minnesota, Department of Medicine, MMC 276, Minneapolis MN55455, USA

__________________________________________________________________________________ *Correspondence: Ola Larsson, University of Minnesota, Department of Medicine, MMC 276, Minneapolis MN55455, USA; e-mail: larss004@tc.umn.edu Key words: Senescence pathways, p16/Rb pathway, p53/p21 pathway, Replicative senescence, Oncogene-induced senescence, apoptosis Abbreviations: cyclin dependent kinases, (CDK); cyclin-dependent kinase inhibitors, (CDKI); first gap phase, (G1); Mouse embryonic fibroblasts, (MEF); senescence associated heterochromatin foci, (SAHF); senescence associated-! galactosidase, (SA-!GAL); short interfering RNAs, (siRNAs); Simian virus 40, (SV40) Received: 10 June 2005; Accepted: 23 June 2005; electronically published: September 2005

Summary Replicative senescence is the principal phenomena that restricts the proliferative potential of all human primary cells. Cellular senescence can be induced by a variety of stimuli including oxidative stress and activated oncogenes.

Intense research during the last years have led to an increased understanding of the processes that lead to senescence. For example, we now know that there is extensive specie and cell type specificity in pathways that induce senescence. This review focuses on replicative senescence and oncogene-induced senescence from an integrated cellular perspective. Further, the current data relating senescence to cancer and aging, the two main biological processes suggested to involve senescence, are discussed. three commonly used criteria that should be fulfilled, at the same time. The first is irreversible growth arrest.

Although it seems like a criterion that is easy to define there are some uncertainties. In many studies one gene is overexpressed and shown to induce growth arrest. Is this irreversibility or should the overexpression be released to see if the growth arrest is dependent on continuous overexpression? Also there is data accumulating from traditional senescence models where the irreversible growth arrest can be reversed artificially (Gire and Wynford-Thomas, 1998; Beausejour et al, 2003). The second criterion is based on phenotypic changes including, morphology (Cristofalo et al, 2004); staining with a biochemical marker for senescence (senescence associated-! galactosidase (SA-!GAL)) (Dimri et al, 1995); and changes in gene expression that are inappropriate for that specific cell type. However, both the classical large flat morphology as well as staining of SA!GAL can be reversed in human fibroblasts when the media is changed to low serum media (Satyanarayana et al, 2004). Also, several pathways/genes have been discovered that mediate some of the morphological changes without contributing to the growth arrest. This indicates that the morphological changes may not be directly coupled to the growth arrest but result from the culture conditions (Dulic et al, 2000; Alexander et al, 2004; Wang et al, 2004b). The last criterion is apoptosis-

I. History When Hayflick found that his fibroblasts were unable to propagate in culture it contradicted the current theories of the day. However, instead of accepting the dogma he challenged it. Two experiments convinced him that he was able to maintain the cells under proper conditions and that a normal cell has a finite life span. In the first experiment he mixed young female and old male cells in a culture while maintaining the original cultures as well (Figure 1) (Hayflick and Moorhead, 1961; Hayflick, 1965). When the old male culture had stopped dividing he went back to the mixed culture and was only able to find female cells. He thereby concluded that there was no external factor that caused the growth arrest. In the second experiment, cells were freezed, stored for some time and thawed with the discovery that it is the time that the cells had been cultured that determines when they stop growing and not the cumulative time. He named the state when cells had stopped growing phase III and it was later called the Hayflick limit, cellular senescence or replicative senescence. His findings led to the understanding that in contrast to normal cells, cancer cells are essentially immortal.

II. Definitions The senescence field still struggles to clearly define the processes involved in cellular senescence. There are 495

Larsson: Cellular senescence–an integrated perspective resistance. Although this is commonly accepted, the mechanisms for the apoptosis resistance are poorly defined and the incidence of resistance is not well established.

Senescent cells have been shown to be resistant to serum withdrawal (Wang, 1995) and p53 mediated apoptosis (Seluanov et al, 2001) but at least some cell types have increased sensitivity towards TNF-" (DeJesus et al, 2002).

Some cell types spontaneously die by apoptosis after prolonged maintenance in vitro although they are senescent by most definitions (Zhang et al, 2002). It therefore appears that the “anti-apoptosis” dogma requires further investigation. In summary it is currently unclear what should be considered as senescence but the term is typically accepted if <5% of the cells proliferate and flat cellular morphology together with SA-!GAL staining, can be demonstrated.

regulated transcriptionally by several proteins and seems to be a sensor for cellular stress (Figure 2).

There is extensive evidence for an important role for the p16/Rb pathway during the induction of senescence.

Overexpression of p16 induces features of senescence including growth arrest (McConnell et al, 1998) while knock-down of p16 using short interfering RNAs (siRNAs) inhibited RAS-induced senescence in epithelial cells (Bond et al, 2004). Re-expression of Rb in a cancer cell line (Xu et al, 1997) or inhibition of E2F (Maehara et al, 2005) also induces senescence, indicating that the p16/Rb pathway can induce senescence under several conditions.

B. The p53/p21 pathway p53 has been named the “guardian of the genome” and is mutated in 50% of all tumors. It acts as an integrator for various signals and can mediate cell cycle arrest, apoptosis and differentiation. There are several mechanisms that regulate the activity of p53. The DNAdamage-ATM/ATR-Chk1/Chk2 pathway activate p53 by phosphorylation (Sancar et al, 2004) leading to displacement of the cellular protein MDM2, which relocates p53 from the nucleus to the cytoplasm and targets it for degradation (Sherr and McCormick, 2002).

MDM2 can also be regulated by p19ARF, which inactivates MDM2 leading to an increased activity of p53 (Weber et al, 1999). Many other proteins e.g. SUMO-1 (Gostissa et al, 1999) and Parc (Nikolaev et al, 2003) can modulate p53 activity and the p53 activity can further be modulated by protein modifications (e.g. acetylation) (Sancar et al, 2004; Sherr and McCormick, 2002). Once activated, p53 induces transcription of many genes involved with cell cycle arrest and apoptosis (Giaccia and Kastan, 1998; Zhao et al, 2000). One of the activated proteins that mediate the cell cycle arrest downstream of p53 is p21. p21 is a member of the “Cip/Kip” family of cyclindependent kinase inhibitors (CDKI) that inhibits CDK2/cyclin-E (Sherr and McCormick, 2002) and to a lesser extent CDK4/cyclin-D (Giaccia and Kastan, 1998). p21 is believed to be the main target for cell cycle arrest downstream of p53 (Figure 2).

III. Senescence pathways Several pathways can trigger senescence in various cell types and under a variety of different conditions. The most common pathways described in relation to senescence are the p16/Rb and p53/p21 pathways.

A. The p16/Rb pathway Rb mediates regulation of the cell cycle at the transition from first gap phase (G1) to DNA synthesis phase (S phase). Rb is hypophosphorylated during G1/G0 and is bound to E2F whereby the activity of E2F is inhibited. When Rb is phosphorylated it releases E2F and this occurs before the G1/S transition and through S-phase.

E2F mediates transcription of a variety of genes necessary for G1 to S progression and replication including cyclin-E, cyclin-A and thymidine kinase (Sherr and McCormick, 2002). Phosphorylation of Rb is mediated by cyclin dependent kinases (CDK) bound to cyclins (cyclinD1/CDK4-6 and cyclin-E/CDK2). CDK4/cyclin-D is activated by mitogenic signaling through the RAS pathway by transcriptional induction of cyclin-D (Sherr and McCormick, 2002). There are proteins called cyclin dependent kinase inhibitors that can inhibit the CDKs. One of them is p16 which inhibits phosphorylation of Rb and thereby G1 to S progression by inhibiting CDK4/cyclin-D (Sherr and McCormick, 2002). p16 can in turn be

Figure 1. Hayflicks experiment to establish that senescence did not occur because of culture conditions. Female and male cells of different culture ages were mixed and surveyed later for gender.

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Figure 2. Regulation of the cell cycle. G1 to S transition.

The p53/p21 pathway has clear role during induction of senescence. Mouse embryonic fibroblasts (MEF) lacking either p53 (Harvey and Levine, 1991) or p19 (Kamijo et al, 1997, 1999) do not senesce. Human cells bypass senescence when both the p53 and the Rb pathway is inhibited by e.g. Simian virus 40 (SV40) large T antigen (Stein et al, 1990). Furthermore, senescence induced by inactivation of SV40 large T antigen can be inhibited by introduction of a dominant negative p53 in some cell types (Fujii et al, 1999). Interestingly, inhibition of the p53 pathway in cells already senescent can reverse the phenotype as injection of anti-p53 antibodies (Gire and Wynford-Thomas, 1998) or a SV40 large T antigen that only binds and inactivates p53 (Beausejour et al, 2003), can reinitiate DNA synthesis at least in some cell types.

Also, overexpression of p53 can induce senescence in some tumor cell lines (Wang et al, 1998). The downstream target of p53, p21 can induce senescence in tumor cell lines independent of p53 status (Fang et al, 1999; Wang et al, 1999; Chang et al, 2000a), and human but not mouse fibroblasts lacking p21 bypass senescence (Brown et al, 1997; Pantoja and Serrano, 1999; Wei et al, 2001). This indicates an important role of p21 for induction of senescence in human cells and further indicates that induction of senescence differs between species. Reactive oxygen species (ROS) are possible mediators of the senescence response downstream of p53/p21 (Polyak et al, 1997). In support for an involvement of ROS, both p53-

and p21-induced senescence has been shown to be at least partly dependent on ROS (Macip et al, 2002, 2003).

IV. Replicative senescence The telomere is a structure located at the end of each chromosome. It consists of a repeated DNA sequence (TTAGGG.) and associates with several binding proteins.

There are species variations in telomere biology, for example humans have shorter telomeres compared to mice (Kipling and Cooke, 1990; Wright and Shay, 2000).

Telomerase, a reverse transcriptase, can extend the telomere DNA by using a nucleus-encoded RNA as a template for its RNA-dependent DNA polymerization (Blackburn, 1992). Each telomere ends with a 3# single stranded sequence of about 200 nucleotides that folds back to the double stranded telomere sequence to form a loop structure called the t-loop (Griffith et al, 1999; Wright et al, 1997). The t-loop together with the telomere binding proteins are believed to protect and hide the end of the telomere to avoid a DNA damage signal and/or regulate the length of the telomere.

Telomere-induced senescence is thought to result from the â&#x20AC;&#x153;end-replication problemâ&#x20AC;?. The replication machinery can not start at the absolute end of the chromosome and therefore a piece of the telomere is lost following each round of replication (Levy et al, 1992).

Recently, t-loop sized deletions of the telomere have been detected in primary cells indicating that other mechanisms

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Larsson: Cellular senescence–an integrated perspective could contribute to telomere shortening (Wang et al, 2004a). The rate of telomere shortening is suggested to be related to the length of the single stranded telomere DNA. However, this may differ between cell lines, where telomere shortening can be proportional to the length of the single stranded DNA, and primary cells from different donors, where no correlation between lengths of the single stranded DNA and telomere shortening was found (Huffman et al, 2000; Keys et al, 2004).

The main experimental evidence that the telomere drives replicative senescence reflects experiments where overexpression of telomerase in several cell types allows those cells to bypass senescence (Bodnar et al, 1998; Vaziri and Benchimol, 1998; Yang et al, 1999; Zhu et al, 1999; Ouellette et al, 2000b; Steinert et al, 2000; Rufer et al, 2001; Wood et al, 2001; Harada et al, 2003). However, in some cell types telomerase fails to overcome senescence as these cells senesce for other reasons then telomere shortening (see below) (Kiyono et al, 1998; Dickson et al, 2000; Farwell et al, 2000). Initially it was uncertain what overexpression of telomerase really achieved as cells with telomerase (that escaped senescence) showed shorter telomeres than senescent cells (Zhu et al, 1999; Ouellette et al, 2000a). A reasonable explanation for this paradox was that it is not the average telomere length that determines when a cell enters senescence, rather senescence is triggered by the shortest telomere within each cell (Hemann et al, 2001; Baird et al, 2003; der-Sarkissian et al, 2004). Interestingly, telomerase was initially reported to have no other role beyond maintaining the telomere (Jiang et al, 1999; Morales et al, 1999). However, it is now clear that this is not true as telomerase can contribute to tumorigenesis e.g. by mechanisms unrelated to telomere elongation (Stewart et al, 2002), stimulate proliferation (Smith et al, 2003) and change the response to TGF-! (Stampfer et al, 2001). It seems plausible that these other activities of telomerase could be important for the escape from senescence or the role telomerase has in tumor development (90% of all tumors overexpress telomerase, see Senescence and cancer).

A critical question is what happens to the telomere when it becomes shorter and shorter. Currently there are two main theories, and although they do not seem to rule each other out, it has led to a lot of controversy within the field. The first theory postulates that, upon shortening of the telomere, the proteins that usually cap the telomere are no longer able to protect and a DNA damage response is initiated. Inherited with the model is the assumption that the proteins that bind to the telomere suddenly are unable to do it, presumably because the telomere is too short. The second theory states that the single stranded telomere DNA is degraded or eroded; and that this makes that telomeric structure unstable and induces senescence. The telomere erosion theory does not imply that the telomere has to be particularly short, just that the single stranded overhang is lost. The erosion theory was initially supported by the finding that the single stranded DNA is lost during senescence (Stewart et al, 2003). However, recently this finding has been challenged, as no loss of the single stranded DNA was found in senescent cells (Chai et

al, 2005). This clearly questions the basis for the erosion theory. The difference between the two studies did not reflect the selected cell types, but may be related to the different methods used to measure single stranded telomere DNA lengths. The main evidence supporting that senescence is induced when the telomere proteins fail to protect the telomere structure, comes from experiments with TRF2 which binds to the telomere. Overexpression of TRF2 leads to shortening of the telomere but a delayed senescence which indicates that excess TRF2 can maintain a proper telomere structure of short telomeres and that this is a key event that regulates onset of senescence (Karlseder et al, 2002). Also, experiments with a dominant negative TRF2 lead to induction of senescence without loss of telomere DNA (Stansel et al, 2001).

In the last two years significant progress has been made through identification of the signaling pathways that are activated by the telomere and induce senescence.

Several groups have identified a DNA damage “response” specifically originating from the telomere structure in senescent cells (Bakkenist et al, 2004; d'Adda di Fagagna et al, 2003; Takai et al, 2003). All studies detected e.g. a phosphorylated form of histone H2AX and several DNA damage related proteins. It therefore seems likely that the telomere structure is identified as a double strand break that signals through ATM, which can phosphorylate H2AX as well as CHK2 (Gire et al, 2004). If telomere senescence is initiated through the ATM pathway, one might ask what maintains the senescent state. Previously it has been shown that it is possible to reverse the “irreversible” growth arrest, at least in some cell types, by inhibiting p53 (Beausejour et al, 2003; Gire and WynfordThomas, 1998), and it may therefore be plausible that the DNA damage signals from the telomere still persists in the fully senescent cell (d'Adda di Fagagna et al, 2003).

However in another cell type, the DNA damage signals disappeared once the cell became fully senescent (Bakkenist et al, 2004). The cells that maintained an active DNA damage response also demonstrate reversal of senescence by inhibition of p53 (Beausejour et al, 2003).

The cells that did not maintain the DNA damage signals have not yet been assessed for the irreversibility phenotype, but it seems possible that there might be cell type differences in this characteristic. Another group has challenged whether the DNA damage signals originate from the telomere and claim that they are randomly distributed upon induction of senescence (Sedelnikova et al, 2004). However the data presented by d'Adda di Fagagna et al. included several methods to assess where the signals originate from (d'Adda di Fagagna et al, 2003).

Based on current data, a likely model for senescence downstream of the telomere could be as follows: (i) the telomere structure and/or function is compromised as a result of telomere shortening, (ii) the DNA ends of the chromosome become exposed and trigger a DNA damage response through the ATM pathway, (iii) depending on the cell type this DNA damage is un-repairable and the ATM signalling persists and maintains the cell in a non-dividing state or the DNA damage is repaired but other pathways have been activated, and the cell maintains an irreversible growth arrest.

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Cancer Therapy Vol 3, page 499 What is downstream p53/p21 activation? Several microarray studies have looked at the transcriptome induced by p53/p21 or used bioinformatics approaches to identify p53 responsive genes (Giaccia and Kastan, 1998; Chang et al, 2000b; Zhao et al, 2000; Hoh et al, 2002; Wells et al, 2003; Zhang et al, 2003, 2004; Larsson et al, 2004). New method-developments have enabled large functional screens for genes essential for senescence using siRNAs. Using a reversed genetics approach in the temperature sensitive Simian virus 40 large T antigen model, five genes were identified as being essential for senescence (Berns et al, 2004). Although the identity of several of them was surprising, it indicates that senescence relies on large changes in basic cellular mechanisms.

Another protein (Smurf 2) has been identified in a microarray study of replicative senescence, and seems to be specifically induced during telomere senescence compared to hydrogen peroxide induced senescence.

Smurf2 can induce a senescent arrest through either the p53/p21 or the p16/Rb pathway but seems unlikely to be essential (Zhang and Cohen, 2004).

type and experimental condition specific. In support for a non essential role of p53 is RAS-induced senescence, fibroblasts lacking p21 entered senescence following RAS overexpression similarly to p53 negative cells (Wei et al, 2001).

Unexpectedly the stress activated kinase p38 seems to have an important role in human RAS-induced senescence. p38 is activated as a consequence of RAS expression and inhibition of p38 by a small molecule inhibitor (for isoforms " and !) bypassed RAS-induced senescence (Wang et al, 2002). There is data indicating that the activation of p38 could be related to an accumulation of reactive oxygen species as human fibroblasts overexpressing RAS did not senesce at low oxygen levels or in the presence of scavengers (Lee et al, 1999). It is also possible that overexpression of RAS changes the metabolic balance in the cells which activates p38. The later suggestion comes from a study where overexpression of an enzyme needed for glycolysis bypassed RAS-induced senescence in MEFs (Kondoh et al, 2005). The contribution of cell stress to RAS-induced senescence is further supported by a recent report where cells that showed lower stress levels (measured by p16 activity) before addition of RAS, did not senesce after RAS overexpression (Benanti and Galloway, 2004).

Together these data favor a model in human cells where overexpression of RAS leads to an accumulation of ROS and increased p38 activity which could activate the p16 pathway; and induction of senescence. It is also possible that both p16 and p38 are activated by ROS in a parallel pathway and that the activity of both is necessary to induce senescence. In support of this model, fibroblasts from a melanoma prone family with p16 deficiency, but functional p19, did not senesce when RAS was overexpressed (Brookes et al, 2002); and fibroblasts from a patient with bialelic mutations of the INK4a/ARF locus showed resistance towards RAS-induced senescence (Huot et al, 2002).

What then is the link between RAS/stress and activation of p16? Several transcription factors has been suggested to be mediators of RAS-induced senescence as they regulate transcription of p16, either positively or negatively. The first class represses transcription of p16 and would be expected to delay senescence when senescence is mediated by p16. Accordingly, the Id proteins have been shown to delay senescence in cell types where senescence is mainly p16 dependent (Alani et al, 1999; Nickoloff et al, 2000; Tang et al, 2002). Similar functions have been established for BMI-1 which inhibits both senescence and apoptosis induced by p19 (Jacobs et al, 1999a; Jacobs et al, 1999b), TBX2 (Jacobs et al, 2000; Lingbeek et al, 2002) although it also has activities on the p21 promotor (Prince et al, 2004) and CBX7 (Gil et al, 2004). Importantly, only the stress induced senescence can be inhibited by all of these p16 repressors while hTERT expression is needed for full immortalization. The second class includes activators of p16 transcription, i.e. mainly the Ets proteins. The Ets proteins are direct targets of RasRAF-MEK signaling and can directly activate transcription of p16 (Ohtani et al, 2001). They therefore provide interesting candidates for RAS-induced

V. Oncogene-induced senescence Activation of oncogenes renders the cell self sufficient in growth signaling and is important for cancer progression (Hanahan and Weinberg, 2000). Interestingly, overexpression of several oncogenes also induce senescence in vitro and this may be an important strategy to avoid cancer progression. RAS (Serrano et al, 1997) and its downstream targets RAF (Lin et al, 1998) and MEK (Zhu et al, 1998), as well as ERBB2 (Trost et al, 2005), eIF4e (Ruggero et al, 2004) and E2F in some cellular contexts (Dimri et al, 2000; Lomazzi et al, 2002) can all induce senescence. This indicates that senescence could be a general defence against activated oncogenes. RASinduced senescence is best described in the literature but is species specific.

RAS-induced senescence in MEFs is dependent on both p16/RB and p53 (Serrano et al, 1997) and p53 activity alone is not sufficient to induce a full senescent phenotype in MEFs (Ferbeyre et al, 2002). Further, in MEFs with functional p53, disruption of both Rb and the Rb-family member p107 was necessary to avoid senescence but not sufficient to induce transformation (Peeper et al, 2001). The activation of p53 and induction of senescence downstream of RAS in MEFs is dependent on Dmp1, which activates p19 through a Ets site in the INK4A promotor (Sreeramaneni et al, 2005). Therefore, it seems like both a functional p53 and Rb pathway is necessary to induce senescence in MEFs when RAS is overexpressed.

In human cells, RAS does not seem to be dependent on p53 to induce senescence as it was either unchanged (Benanti and Galloway, 2004; Takaoka et al, 2004) or was upregulated but not essential for RAS-induced senescence (Serrano et al, 1997; Wei et al, 2001). Some of the discrepancies of p53 induction during RAS-induced senescence may be related to the dual action of RAS on p53 status as RAS activates both MDM2 and p19 which inhibits and activates p53 respectively (Ries et al, 2000).

The outcome of RAS on p53 status may therefore be cell 499

Larsson: Cellular senescenceâ&#x20AC;&#x201C;an integrated perspective senescence but can currently not explain the dependence of ROS. Several attempts have been made to identify genes that repress senescence downstream of RAS either when overexpressed or inhibited. PLM was identified as upregulated in a microarray study of RAS-induced senescence and was shown to be sufficient for induction of senescence in the absence of RAS in both human and mouse cells (Bischof et al, 2002; de Stanchina et al, 2004; Ferbeyre et al, 2000; Fukuyo et al, 2004). PML can modulate the activity of both the p53 and the RB pathway (Ferbeyre et al, 2000; Pearson et al, 2000). Similarly to RAS-induced senescence, PML-induced senescence is dependent on Rb (Mallette et al, 2004) and Rb mutants that are less efficient in binding to E2F can induce senescence through induction of PML-nuclear bodies (PML-NB). This indicates that PML recaptures some of the characteristics of RAS-induced senescence and that PML might be one additional target of Rb during senescence (Fang et al, 2002).

A genetic screen for genes that overcome RASinduced senescence in MEFs, identified hDRIL, an E2F binding protein. Both the p53/p21 and the p16 pathways were activated during rescue from RAS-induced senescence mediated by hDRIL (Peeper et al, 2002) and it was postulated that the anti RAS-induced senescence action of hDRIL was through release of E2F from Rb. It was later established that the effect may also relate to PML as hDRIL disintegrate the PML-NBs which further indicates that RAS-induced senescence is somehow connected to the PML-NBs (Fukuyo et al, 2004). However overexpression of hDRIL in human cells induced senescence probably in an oncogene manner similar to E2F and inhibition of hDRIL lead to an accumulation of PML-NB and PML induced senescence (Peeper et al, 2002). hDRIL can therefore not be used in human cells to understand the impact of PML on RAS-induced senescence and seems unlikely to be a key mediator of RAS-induced senescence although it can modulate the pathways.

A reverse genetics approach to identify genes that mediate RAS-induced senescence in rat embryo fibroblasts identified Seladin-1 (Wu et al, 2004).

Originally described as a metabolic enzyme, Seladin-1 is activated by both RAS overexpression and hydrogen peroxide indicating that it could act as an oxidative stress sensor (Wu et al, 2004). Downregulation of Seladin-1 was further shown to allow bypass of senescence both in mouse and human fibroblasts (Wu et al, 2004). However, Seladin-1 activates the p53 pathway by releasing p53 from MDM2, which is surprising as the p53 pathway is not necessary for RAS-induced senescence in human cells (Wu et al, 2004). Future studies may demonstrate that Seladin-1 acts on the p16/Rb pathway and how it relates to the stress kinase p38. Regardless, these data further indicate that senescence downstream if RAS is stress dependent as it was activated by both RAS and hydrogen peroxide.

In summary there is some evidence that RAS-RAFMEK-Ets plays a role during induction of senescence downstream of RAS but questions remain as inhibition of

ROS, p38 or Seladin-1 also inhibits RAS-induced senescence. RAS-induced senescence seems likely to occur when p16 levels reach a critical level (Benanti and Galloway, 2004). This level could be achieved when both the direct RAS-RAF-MEK-Ets pathway is activated as well as the stress pathway mediated by ROS/p38/Seladin1/p16. The PML protein is likely to act as an amplifier, but may not be necessary if the stress level is high enough.

The kinetics of RAS-induced senescence supports this theory. While RAS expression is induced immediately, p38 signaling is activated together with p16 after four days (the design of the study did not allow a separation of p38 and p16 activation) and the senescent phenotype appears after seven days (Wang et al, 2002). Interestingly, overexpression of a constitutively active downstream target of p38, MKK6EE, induced senescence after 4 days (Haq et al, 2002). These data indicate that protein synthesis is needed at each step and that there is time for accumulation of ROS damage or a shift in the metabolic balance to activate p38/p16 which then needs further time to manifest the full senescent phenotype.

Regardless of the mechanism for RAS-induced senescence, the critical question is whether oncogeneinduced senescence is an in vitro artifact or if it occurs in vivo. A recent report indicates that senescence is induced by activated E2F3 in vivo and that this process likely leads to inhibited tumor formation in vivo in mice (Denchi et al, 2005). However, given the results described above where human oncogene-induced senescence seems to be dependent on artificially high oxygen level (as RASinduced senescence was inhibited at low oxygen levels), it is unsure if oncogene-induced senescence is an in vivo response in humans as well.

VI. Why did you senesce?

All primary human cells enter senescence after a certain number of cell divisions (Hayflick and Moorhead, 1961; Hayflick, 1965). However, senescence is defined by a set of shared characteristics, not by a common pathway.

Initially, cell hybrid studies of immortal cell lines indicated that there were four different complementation groups based on the occurrence of recessive genes that could induce senescence (Tominaga et al, 2002). The chromosomes carrying three of the recessive genes responsible for three of the groups have been identified but only one gene, MORF4 has been validated as responsible for the complementation phenotype (for a detailed review see (Tominaga et al, 2002)). During the last couple of years it has become clear that primary cells from different species and cell types differ in how they induce senescence. From a simple perspective, each human cell can be described as entering senescence with p16/Rb or p53/p21 activation. This view of senescence comes from a few studies of senescence in single cells. The initial finding was that there was no gradual increase in p21 expression in human fibroblasts as reported previously (Stein et al, 1999), rather the increase was abrupt in the single cell (Herbig et al, 2003). The gradual increase is clearly a function of heterogeneous telomere lengths in a multi- cellular population, that results in slightly different replicative potential of different cells (Martin-Ruiz et al, 500

Cancer Therapy Vol 3, page 501 2004). Similarly it was described that although senescing fibroblasts can show an increase in both p16 and p53/p21 activity, these pathways are typically not active in the same cell and telomeres induce senescence exclusively by activating the p53/p21 pathway (Herbig et al, 2004).

However, all data do not agree with this view as e.g. cells with a mutant TRF2, that generate telomere dysfunction before any stress induced senescence should be active, entered senescence with elevated p16 and p53 activity and abrogation of senescence was only possible with both E6 and E7 expression (Smogorzewska and de Lange, 2002).

However, it can not be excluded that the TRF mutant model simultaneously induced a stress response and hence the need for both Rb and p53 inactivation, or that the appearance of stress induced senescent cells occurs very early.

The single cell studies indicate that senescence resulting from the telomere pathway and from culture stress can coexist in a population of cells. Therefore, one can define each cell type based on whether they are more likely to enter senescence as a result of culture stress or telomere erosion. The culture stress senescence appears to be similar to the senescence response driven by RAS and is characterized by p16 overexpression. Human epithelial cells (Brenner et al, 1998; Jarrard et al, 1999; Romanov et al, 2001; Schwarze et al, 2001) and keratinocytes (Munro et al, 1999) senesce with high levels of p16 but with long telomeres, and telomerase did not overcome senescence (Kiyono et al, 1998). In contrast, human fibroblasts senesce mainly because of telomere shortening with p53/p21 induction. This probably reflects that human fibroblasts are more resistant to culture stress and

therefore reach the telomere restriction point and activation of ATM-p53-p21 signaling (Brown et al, 1997; Wei et al, 2001, 2003; Herbig et al, 2004). However, some fibroblast strains are also sensitive to culture stress and show a substantial stress induced senescence with p16 induction (Stein et al, 1999; Itahana et al, 2003; Bond et al, 2004; Brookes et al, 2004; Taylor et al, 2004). Further, human fibroblasts can be forced to senescence from stress (Munro et al, 2001) and can also enter a senescence state characterized by an increase in p16 expression if the telomere driven senescence is inhibited (Bond et al, 1999).

These data indicate that human fibroblasts can enter stress induced senescence similar to epithelial cells under some conditions, but normally senescence from telomere signaling.

Mouse cells do not senescence because of telomere erosion as they have substantially longer telomeres (Kipling and Cooke, 1990) and can grow indefinitely in low oxygen (Busuttil et al, 2003; Parrinello et al, 2003) or low serum conditions (Woo and Poon, 2004). In contrast to human cells that senesce from culture shock, both p53 and the p16/Rb pathway are necessary, but not p21. The TRF2 mutant senescence model support that the basic mechanisms differ between mouse and human cells as mouse senescence induced by telomere damage was not dependent of p16 expression whereas human senescence was (Smogorzewska and de Lange, 2002). In summary, depending on cell type and species, the mechanisms for induction of senescence varies but importantly the end point is similar in terms of phenotypic characteristics and gene expression signatures (Larsson et al, 2004) (Figure 3).

Figure 3. A summary of all senescence pathways described. Dotted lines are suggested/likely interactions/mechanisms and contious lines are experimentally validated. Bold lines show species differences.

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Larsson: Cellular senescenceâ&#x20AC;&#x201C;an integrated perspective that induces senescence in vitro is also needed for extensive proliferation in vivo; the detailed mechanisms in vitro and in vivo may not be the same. However, an interesting p53 mutant that is unable to induce apoptosis while still able to induce cell cycle arrest (at an intermediate level between wild type and p53-null), has provided some indications that senescence could operate in vivo and restrict cancer progression. Double mutants for this p53 allele did not develop early tumors compared to a p53 null mice and the tumors that eventually occurred were diploid showing that cell cycle arrest (and possibly senescence) is occurring in vivo (Liu et al, 2004). This remains the strongest principal proof for senescence being a natural mechanism that counteracts tumor progression.

There are indications that senescence is a response to ongoing chemotherapy treatment, as most cell lines are able to respond to doxorubicin by induction of senescence (Chang et al, 1999) and senescence can be an in vivo response to chemotherapy in mice (Chang et al, 1999; Schmitt et al, 2002).

Could induction of senescence be a reasonable strategy for cancer treatment? There is data supporting this idea: Inhibition of telomerase by expression of a mutated telomerase RNA-component, inhibited proliferation of human cancer cells (Kim et al, 2001). Induction of senescence was also achieved by adding single stranded oligo-nucleotides (telomere repeat) to the medium of tumor cells (Li et al, 2003), although a similar treatment has also been described to induce apoptosis in another cell type (Eller et al, 2002). The mechanism involves disruption of the telomere structure, possibly by titration of some of the telomere binding proteins, and induction of a DNA damage response (Eller et al, 2003; Li et al, 2004).

Interestingly the effect is specific for the telomeric repeat sequence (Li et al, 2003) and dependent on both the p53 and the Rb pathway, indicating that it probably mimics a normal replicative senescence response as well as a general stress response (Li et al, 2004). A compound was recently reported to reduce the levels of telomerase indirectly, induce senescence in vitro and showed in vivo effects in a mouse model (Incles et al, 2004; Burger et al, 2005).

While limiting cancer cell growth by induction of senescence sounds like a good strategy, it is not entirely clear how beneficial this would be in vivo. Indeed it has been discovered that senescent cells can actually promote tumor cell growth, in vitro and in vivo, in a model with pre-malignant epithelial cells (Krtolica et al, 2001; Parrinello et al, 2005). Similar promotion of cancer progression, by one cell type influencing another, has been shown in a system where fibroblasts deficient in TGF-! signaling were able to promote cancer progression in adjacent epithelial cells (Bhowmick et al, 2004). The mechanisms were described to be mediated both by cellcell interactions as well as paracrine stimulation (Bhowmick et al, 2004; Krtolica et al, 2001). Also, senescent cells have previously been described to secrete growth factors; and media from senescent cells can be mitogenic and anti-apoptogenic (Chang et al, 2000b). In that sense the ability to enter senescence may be beneficial for overall tumor survival. An increased knowledge of

VII. Why is senescence sometimes irreversible?

There seems to be a fundamental difference in the degree of irreversibility depending on which pathway that triggers senescence. Human fibroblasts that senesce with p16 activity do not reenter the cell cycle after microinjection of SV40 large T antigen whereas cells that senesced with p53/p21 activity do (Beausejour et al, 2003). This effect could be a result of an establishment of senescence associated heterochromatin foci (SAHF) that was described in senescent cells with an active p16/Rb pathway (Narita et al, 2003). SAHF leads to a stable repression of E2F target genes such as cyclin-A and cyclin-E (Narita et al, 2003). The mechanism could include BRG1, HDAC1, SUV39H1 and/or a transcriptionally repressive form of the histone protein H2A called macroH2A. All these proteins mediate changes on chromatin structure and have been linked to formation of SAHA or to the growth restrictive activities of Rb. BRG1 is a component of the SWI/SNF chromatin remodeling complex that can induce senescence in cells with functional Rb (Dunaief et al, 1994) and seems important for Rb mediated growth arrest (Strobeck et al, 2000) although some recent data indicate that the effects may not be directly through the physical interaction with Rb as BRG1 can induce p21 as well (Kang et al, 2004).

Similarly, HDAC1 was found in complex with Rb and is also important for Rb mediated repression of cyclin-E but not necessary for Rb/SWI/SNF mediated repression of cyclin-A (Zhang et al, 2000). SUV39H1 associates with RB and corporate to repress cyclin-E probably through methylation of histone H3 followed by binding of HP1 to the chromatin and establishment of heterochromatin (Nielsen et al, 2001). MacroH2A is enriched in SAHF and could affect the chromatin structure by removing the chromatin modifications (Zhang et al, 2005). Two chaperone proteins that can assemble macroH2A onto DNA (HIRA and Asf1a) are sufficient and necessary for establishment of SAHF and senescence at least in some cell types (Zhang et al, 2005). Interestingly there could be a role of the PML-NB as the proteins that localized to SAHF first associate with the PML-NB (Zhang et al, 2005). It is likely that some of these factors contribute to stably repress E2F target genes and thereby mediate the genetic death that is characteristic of some forms of senescence. An interesting question is why only the p16/Rb pathway leads to irreversibility and not the p53/p21 pathway. It could be related to the phosphorylation pattern of Rb which will differ if mainly CDK4/cyclin-D or CDK2/cyclin-E is inhibited by p16 or p21 respectively.

VIII. Senescence and cancer Cancer cells proliferate beyond the normal point of replicative senescence and thus need to maintain telomere lengths to continue to divide. 90% of all tumors maintain stable telomeres by overexpression of telomerase while the remaining 10% use an alternative mechanism of telomere maintenance that involves recombination, called ALT (Shay, 1997). Although this suggests that a mechanism

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Cancer Therapy Vol 3, page 503 senescence may therefore lead to a reevaluation of the potential for senescence as a treatment strategy and possibly show that specific inhibition of senescence, with retained apoptosis induction, during treatment with standard chemotherapy is the way forward.

(Dimri et al, 1995) could not be confirmed (Severino et al, 2000). Interestingly both humans (Cawthon et al, 2003) and worms (Joeng et al, 2004) with longer telomeres show extended life span. The human telomeres were measured from blood samples and the extended life span may at least partly reflect the immune system, as the increased mortality with short telomeres was attributed by the authors to death in infectious disease and increased heart disease. The extended life span in worms was somehow related to the main survival pathway in C. elegans controlled by DAF-16.

The best link between senescence and aging comes from a premature aging syndrome called Werner syndrome. Werner syndrome patients die at an median age of 47 with myocardial infarctions and cancer and show several signs of accelerated aging (Martin, 2005). The Werner syndrome arises as a consequence of mutations of the Werner protein which is a RecQ DNA helicase (Gray et al, 1997). Interestingly, primary fibroblasts from Werner patients senesce early and show similar expression patterns as normal senescent cells indicating that the early senescence may drive an accelerated aging phenotype (Ly et al, 2000). Several lines of evidence indicate that the early senescent phenotype is related to an inability to maintain the correct telomere structure/length and that this causes the aging ohenotype as the Werner cells can be rescued from senescence by overexpression of telomerase (Wyllie et al, 2000); a third generation telomerase activity deficient mouse with a Werner mutation shows an accelerated aging phenotype (Chang et al, 2004); the Werner protein is associated with the telomere and can bind to TRF2 (Opresko et al, 2002, 2004); and cells with the Werner mutation lose their lagging strand telomere at a high rate (Crabbe et al, 2004). It has also been reported that the average telomere length in Werner cells is not different from normal cells (Schulz et al, 1996) but that could be explained by the observation that some lagging strand telomeres are very short while others are normal (Crabbe et al, 2004). Several other functions of the Werner protein has been reported including transcription (Balajee et al, 1999) but it seems likely that it is the function related to the telomere structure that drives the early senescent phenotype and possibly also the early aging phenotype.

There is no data suggesting that Werner patients have more senescent cells in their tissues but given the functions of the Werner protein the effects could be in stem cell compartments that would be depleted from replicatively competent cells.

In summary there is no substantial data showing that senescence drives aging or is accumulated as a function of age in humans. Senescent cells have been detected during aging as well as in an accelerated aging phenotype and some other conditions in mice, indicating that they could exist. Interestingly gene expression data supports this species difference as the senescence transcriptome was found to be similar to that of mouse but not human aging (Wennmalm et al, in preparation).

IX. Senescence and aging When Hayflick discovered that primary cells in vitro have a finite life span and enter senescence, one of the first theories that arose was that cells in a tissue would behave similarly and cause aging (Hayflick and Moorhead, 1961; Hayflick, 1965). According to the theory, these senescent cells have lost their original function and impair organ function. The aging phenotype was therefore the sum of all malfunctions in all organs that the senescent cells cause. While plausible and accepted among many researchers, the data is not convincing: The theory dictates that the number of senescent cells increases with age in tissues and several attempts have been made to detect such an increase. Initial studies established cultures of primary cells from differently aged donors and measured their replicative life span. Some studies managed to find a decreased replicative lifespan from older subjects while others did not (Martin et al, 1970; Cristofalo et al, 1998). However this approach may not be valid as there will be a clonal expansion of the cells with the longest telomeres and although there are senescent or close to senescent cells in the population, these could be difficult to detect. Another approach is to look for a decrease in telomere lengths as a function of life span and take this as an indication of a replicative decline in the tissue. When combining all such efforts the conclusion was that although the main differences in telomere lengths depend on the individual, there is a gradual decrease of the telomeres with age in some organs (Takubo et al, 2002).

Neither of these approaches demonstrates that the senescent cells actually accumulate in a tissue. Therefore, several attempts have been made to identify an increase in senescent cells with age but the main setback of this approach has been the lack of markers for senescence, except the commonly used SA-!GAL. An increase in SA!GAL staining cells with age has been detected in human skin (Dimri et al, 1995) and in mouse kidney (Krishnamurthy et al, 2004). In the human study only cells close to the hair follicle were stained, while the whole kidney stained blue in the mouse study, which would indicate that almost all cells in the kidney were senescent.

An alternative explanation in both these studies is a lack of specificity of SA-!GAL that has been described (Severino et al, 2000). SA-!GAL staining cells have also been observed in mice after chemotherapy treatment (Schmitt et al, 2002), after liver hepactomy in third generation telomerase-activity deficient mice (Satyanarayana et al, 2003) and in a knock-out mouse for Bub1 that shows an accelerated aging phenotype (Baker et al, 2004).

Therefore, if one believes that SA-!GAL is a valid marker for senescence in vivo there seems to be evidence for senescent cells in vivo of the mouse but not humans as the age dependent increase of SA-!GAL cells in skin

X. Senescence vs. apoptosis As described above, replicate senescence involves a DNA damage response that is also capable of promoting 503

Larsson: Cellular senescenceâ&#x20AC;&#x201C;an integrated perspective apoptosis; so why is senescence the outcome of telomere instability? The first thing to point out is that there is little evidence of senescence in human tissues, so far senescent cells have only been detected in skin of elderly people (Dimri et al, 1995), a finding that could not be repeated (Severino et al, 2000).

One obvious mechanism that could regulate the choice between senescence and apoptosis is if the apoptotic process was inhibited and senescence occurred instead as a default mechanism. The mitochondrial antiapoptotic protein Bcl-2 has been proposed to represent such a mechanism. Unexpectedly, overexpression of Bcl-2 has been shown to induce senescence, judged by SA!GAL staining, yet this may more resemble quiescence as p27 was overexpressed (Crescenzi et al, 2003). Bcl-2 can also accelerate RAS-induced senescence to some extent (Tombor et al, 2003). In support of the hypothesis, Bcl-2 has been described to shift the response from apoptosis to senescence when artificially overexpressed in rat cells (Rincheval et al, 2002). In the report describing a shift from apoptosis to senescence upon Bcl-2 overexpression, p21 was found to be overexpressed. In fact, this could be the reason for the shift from apoptosis to senescence as p21 expression after DNA damage lead to senescence while absence of p21 induction after DNA damage lead to apoptosis (Seoane et al, 2002). Similarly, apoptosis was associated with low p21 levels whereas senescence was associated with high p21 levels in a cancer cells treated with interferon-$ (Detjen et al, 2003). If p21 decides if the response, downstream of p53induction, will be senescence or apoptosis, then an important question is why p53 sometimes induces p21 expression and sometimes not.

Some of the regulation could be a result of the convergence of several pathways that directly regulate p21. For example both Miz-1 and CUGBP have been described to affect the transcription and translation of p21 respectively (Iakova et al, 2004; Seoane et al, 2002). It is also possible that the decision, of whether or not to induce p21, occurs at the level of p53 activation. Interestingly, the phosphorylation patterns of p53 during induction of senescence and after a DNA damage treatment leading to apoptosis seems to differ (Chehab et al, 1999; Webley et al, 2000). The question would then be what regulates the differential phosphorylation of p53 during senescence and apoptosis. Interestingly, there are some indications of how this could be achieved. It appears that a large DNA damage response leads to apoptosis while a low but persistent activation of p53 induces senescence. For example, upon hydrogen peroxide treatment both senescence and apoptosis are possible outcomes; apoptosis was associated with higher levels of p53 and low levels of p21 while senescence was associated with lower levels of p53 and higher levels of p21 (Chen et al, 2000). Similarly a TRF2 mutant that cause telomere dysfunction induced apoptosis or senescence, depending on the expression level and thereby the extent of telomere damage (Lechel et al, 2005); and substantial overexpression of p53 induced apoptosis while lower overexpression induced senescence (Macip et al, 2003). In summary, it seems like p21 and the nature of the p53 response is the major determinant whether the p53 response will induce apoptosis or

senescence. The differential regulation of p21 needs to be further clarified.

Acknowledgements I would like to thank Dr James A Timmons, Dr Christina Karlsson-Rosenthal and Dr Claes Wahlestedt for reading the manuscript.

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Cancer Therapy Vol 3, page 511 Cancer Therapy Vol 3, 511-514, 2005

Combination treatment of unresectable hepatomas with chemotherapy, octreotide and antioestrogens: A preliminary study Research Article

Panagiotis Ginopoulos1, Athina Christopoulou1,*, John Spiliotis2 1

Department of Medical Oncology “St. Andrews” General Hospital of Patras, Patras Greece Department of Surgery, “Chatzikosta” General Hospital of Mesolongi, Mesolongi Greece.

2

__________________________________________________________________________________ *Correspondence: Athina Christopoulou MD, PhD, Votsi 31 – 33, Patras Greece; Tel: +30- 6942402626; E-mail: athinachris@in.gr Key words: unresectable hepatomas with chemotherapy, octreotide and antioestrogens, Kaplan Meier survival, Drugs tolerate, Quality of life Abbreviations: Complete response, (CR); Hepatocellular carcinoma, (HCC); median survival rate, (MSR); Partial response, (PR); Progressive disease, (PD) Received: 30 March 2005; Revised: 20 May 2005 Accepted: 25 May 2005; electronically published: October 2005

Summary Hepatocellular carcinoma (HHC) is a highly malignant tumor with a very high morbidity and mortality, carrying a poor prognosis and presenting considerable management problems. The aim of the study was to estimate if and how much the administration of two drugs regimens chemotherapy together with Sandostatin LAR 30 and tamoxifen improves survival and quality of life in patients with inoperable HCC comparing the results with these of nothing received group patients.15 patients with HCC were included in the treatment group between 2002 – 2004 (Group A). All patients received: Caelyx 25 mgr/m 2 – day 1, Gemsar 1000 mgr/m 2 (d1 + d8), with repeat cycle every 21 days for 6 moths, Sandostatin LAR 30 once a month until progression of disease (PD) and Nolvadex 20 mgr/day. 7 patients with HCC, who refused to received any treatment remain as a control group (Group B). These patients have received medical treatment for pain, nausea or local effects of the disease. and Bruix, 2000). No regimen has proven to be curative in an analysis, no single drug or combination of drugs showed a reproducible response rate of more than 20% (Okuda, 1997).

In this study, a preliminary report is presented of a combination treatment with two chemotherapeutic drugs: liposomal doxorubicn (Caelyx) + gemcitabine (Gemsar) in combination with somatostatin analog (Octreotide) and an antioestrogen (Tamoxifen) with some interesting and promising results.

I. Introduction Hepatocellular carcinoma (HCC) is the most common primary epithelial malignancy occurring in the liver and is characterized by an extremely poor prognosis, and presenting considerable management problems (Johnson, 1996; Alsowmely and Hodgson, 2002).

Surgical resection – either as partial hepatectomy or by orthotopic liver grafting – has traditionally been regarded as the first-choice treatment and the only realistic chance for the cure of HCC. However only 10% are suitable for curative resections due to many factors: multicentric tumors, vascular invasion, advanced liver cirrhosis, extrahepatic metastases and comorbidities (Hodgson, 1983; Rasmussen and Garden, 1996; Liu and Fan, 1997).

Recently some interetsing results have been reported with anti-oestrogenic, -anti-androgenic or somatostatin analogues treatment (Farineti et al, 1992; Martinez Cerero et al, 1994; Cascinu et al, 1995; Kouroumalis, 2001) have shown some activity. On the other hand HCC is generally one of the most resistant tumor to chemotherapy (Llovet

II. Patients and Methods Fifteen patients, ten males and five females, median age 70years old (range 62y – 80y) with HCC were included in the therapeutic trial between 2002 and 2004 (Group A). Inclusion criteria were liver biopsy or FNA B diagnosis of HCC, increased levels of a fetoprotein (AFP) with compatible liver ultrasound and CT scan (Table 1).

Seven patients, five males and two females with HCC were included as a control group between 2002 and 2004 (Group B).

All of these patient with confirmation of HHC with FNA B or liver biopsy refused to received the therapeutic trial and remain

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Ginopoulos et al: Hepatomas treatment with chemotherapy, octreotide and antioestrogens with medical treatment for pain, nausea or local effect of the disease (Table 2).

Treatment responses are usually evaluated according to the WHO and response evaluation criteria in solid tumors group criteria (Therasse et al, 2000).

Complete response (CR) is defined as the complete disappearance of all target lesions for more than 4 weeks. In our study two patients (13.3%) presented CR and they are still alive 20 and 17 months retrospectively with median survival rate (MSR) of 18,5 months.

Partial response (PR) is defined as at least 30% reduction in the sum of the longest diameters of target lesions lasting for more than 4 weeks. In our study five patients (33.3%) presented P.R with a MSR of 11,6 months.

Progressive disease (PD) is defined as at least 20% increase in the sum of the longest diameters of target lesions or the appearance of one or more new lesions.

All patients had a monthly follow-up with routine liver biochemical tests and a FP concentrations, liver ultrasound was performed over three months.

III. Results A. Kaplan Meier survival At the time of the final analysis (May 2004) only two patients (both from the treatment group) were alive.

The overall mean survival was 11.2 months for group A vs 4.3 months (p < 0.003) for group B. There was a significant difference of survival in six months (80% vs 28.5) and in one year (26.6% vs 0%). The treatment schedule was well tolerated with mild complications due to chemotherapeutic drugs. Complete and partial response rates were observed in 46.6% of our patients and all patients reported improvement in the feeling well being.

Figure 1 shows the overall survival between the two groups. There is a significant survival difference (p < 0.003) between those who under went treatment and those who did not.

Table 3 shows the percentage of patients surviving three, six, nine, twelve and eighteen months.

A. Treatment schedule and follow-up All the patients of Group A received the follow therapeutic schedule: Liposomal Doxorunicn (Caelyx) 30mg/m2 â&#x20AC;&#x201C; day 1 Gemcitabine (Gemzar) 1000mg/m2 â&#x20AC;&#x201C; day 1 + 8 With repeat cycle every 21 days for 6 cycles Sandostatin LAR 30 once a month until PD Tamoxifen (Nolvadex) 20mg/day

Table 1. Patient Characteristics Group A No

Gender

Age

Tumor diameter

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Male Female Male Male Male Female Female Male Male Male Female Female Male Male Male

62 64 78 74 80 65 64 74 78 80 64 62 70 67 66

7 cm 4 cm Multiple 7 cm Diffuse 5 cm 9 cm 6 cm 4 cm Diffuse Diffuse 5 cm 5 cm 9 cm 9 cm

Perf. Status (Karnofsky) 70 % 60 % 40 % 70 % 60 % 50 % 50 % 50 % 70 % 40 % 50 % 60 % 60 % 50 % 50 %

Child

Perf. Status (Karnofsky) 50 % 60 % 40 % 30 % 50 % 70 % 50 %

Child

A B A A B A C A A B B B C A A

Table 2. Patient Characteristics Group B No

Gender

Age

Tumor diameter

1 2 3 4 5 6 7

Female Male Male Female Male Male Male

67 78 82 80 73 76 77

9 cm 5 cm Diffuse Diffuse 7 cm 8 cm 6 cm

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A A C B B B A

Cancer Therapy Vol 3, page 513

Figure 1. Kaplan-Meier survival

Table 3. Cumulative survival in 3 / 6 / 9 / 12 / 18 months

Characteristics Group A Group B

3 months % surv.

100% (15/15) 71.4% (5/7)

Survival 6 months 9 months 80% (12/15) 28.5% (2/7)

66.6% (10/15) 0

B. Drugs tolerate

12 months

18 months

26.6% 4/15) 0

13.3% (2/15) 0

V. Discussion

The treatment schedule was well tolerated.

Hematologic toxicity with anemia and Neutropenia was observed in nine patients (60%) and they were treated with G-CSF and EPO. Diffuse abdominal pain and mild diarrhea was observed in four patients (27%) due to caelyx administration.

In two patients a Hand-foot-syndrome was observed and alopecia was observed in five patients (34%).

HCC is generally one of the most resistant tumors to chemotherapy (Llovet and Bruix, 2000). A wide variety of chemotherapeutic agents have been tried and are in use.

No regimen has proven to be curative. Often the response rate and prolongation of survival are minimal and there is a significant morbidity associated with poor treatment effects (Okuda, 1997).

On the other hand recently, promising results for the treatment of HCC have been reported with somatostatin analog actreotide (Raderer et al, 1999). In addition Kouroumalis et al, have reported the first study to demonstrate a survival benefit with application of octreotide in patients with advanced HCC as compared to untreated controls (Kouroumalis et al, 1998). All this outcome together with the controversial beneficial effect of anti-oestrogens (tamoxifen) suggesting our group to use the combination of chemotherapy and hormonal manipulations with octreotide and tamoxifen in patients with advanced HCC. There are important large randomized studies with chemembolization-radiotherapy or intra arterial radiotherapy (Kaneto et al, 2000; Lygidakis et al, 2000) providing really promising results of advanced hepatocellular carcinoma.

C. Quality of life In this study two patients (13.3%) presented CR and they arestill alive 20 and 17 months rertrospectively with MSR of 18.5 months, seven patients (33.3%) presented with PR with a MSR of 11.6 months and seven patients (46.6%) presented PD with a MSR of 6.3 months.

Appetite, body weight pain and the general feeling as well being were used as criteria of quality of life. An increase in appetite was reported in twelve patients (80%).

All patients reported improvement in the feeling of well being and four patients (40%) gained weight.

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Ginopoulos et al: Hepatomas treatment with chemotherapy, octreotide and antioestrogens Kouroumalis ET (2001) Octreotide for cancer of liver and biliary tree Chemotherapy 47(Sup2), 150-161.

Liu CL, Fan ST (1997) Non resectional therapies for hepatocellural carcinoma. Am J Surg 173, 358-364.

Llovet J, Bruix J (2000) Early diagnosis and treatment of HCC. Baillieres Best Pract Res Clin Gastroenterol 14, 991-1008.

Lygidakis NJ, Singh G, Bardaxoglou E, Sgourakis G, Pedonomou M, Nestoridis J, Maliotakis A, Dedemadi G, Solomou EK, Alamani M (2004) New frontiers in the management of advanced hepatocellular carcinoma. "A new look to an old problem". Hepatogastroenterology 51. 62-7.

Martinez Cerero FJ, Tomas A, Donoso L, et al (1994) Controlled trial of tamoxifen in patients with advanced hepatocellular carcinoma. J. Hepat 20, 702-706.

Okuda S (1997) Chemotherapy for HCC In Okuda K, Tabo E, eds, Cancer New York: Churchill Livigston 441-447.

Raderer M, Hejna M, Kurtaran A, et al (1999) Succesful treatment of advanced hepatocellular carcinoma with the long acting somatostatin analog, Lanreotide. AJG 94, 278279.

Rasmussen JB, Garden J (1996) The management of liver cell cancer. Eur J Gastr Hepat 8, 861-866.

Samonakis D, Moschandreas J, Arnaoutis T, et al (2002) Treatment of hepatocellular carcinoma with long acting somatostatin analogs. Oncol Rep 9, 903-907.

Therasse P, Arbuck SG, Eisenhauer EA et al (2000) New guidelines to evaluate the response to treatment solid tumor.

J Natl Cancer Inst 92, 205-206.

Our results confirms a prolongation of survival 80% in six months as compared with 28.7% to untreated controls, and a 27% one year survival in treatment group vs 0% of untreated patients. The results of our study were similar with some others recently publlcated studies using octreotide only and more promising with some others using tamoxifen only (Elba et al, 1994; Samonakis et al, 2002) The treatment schedule with combination of systemic chemotherapy and octreotide and tamoxifen improves survival of patients with HCC and is an alternative for inoperable HCC. On the other hand before embracing this warm recommendation it seems prudent in the future to requiring confirmation of this out-come with large-scale trials as the drugs although expensive, are well tolerated.

References Alsowmely AM, Hodgson HF (2002) Non-surgical treatment of hepatocellular carcinoma. Alimentary Pharm Therap 16, 115.

Cascinu S, Del Ferro E, Catalano G (1995) A rando mised trial of octreotide vs supportive care only in advanced gastrointestinal cancer patients refractory to chemotherapy.

Br J Cancer 71, 97-101.

Elba S, Giannuzzi V, Misciagna G, Manghisio G (1994) Randomized frial of tamoxifen versus placebo in inoperable hepatocellular carcinoma. Ital J Gastroenterol 24, 66-68.

Farineti F, De Maria N, Fornasiero A, et al (1992) Prospective controlled trial with antiestrogen drug tamoxifen in patients with unrespectable HCC. Dig Dis Sci 37, 659-662.

Hodgson HJF (1983) Primary hepatocellular carcinoma. Br J Hosp. Med 29, 240-255.

Johnson PJ (1996) The epidemiology of hepatocellurar carcinoma. Eur J Gastr Hepat 8, 845-849.

Kaneko T, Nakao A, Takagi H (2000) Experimental studies of new embolizing material for portal vein embolization.

Hepatogastroenterology 47, 790-4.

Kouroumalis E, Skordilis P, Thermos K, et al (1998) Treatment of hepatocellular carcinoma with octreotide, a randomised controlled study. Gut 42, 442-447.

Athina Christopoulou

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Cancer Therapy Vol 3, page 515 Cancer Therapy Vol 3, 515-524, 2005

Growth inhibition of head and neck squamous cell carcinoma by imatinib mesylate (Gleevec) Research Article

Hyung Ro Chu 1, Weg M. Ongkeko2, Amilcar Diaz 3, Xabier Altuna 4, Joe Aguilera 5, Robert A. Weisman2 and Jessica Wang-Rodriguez6 1

Department of Otolaryngology-HNS, Kangnam Sacred Heart Hospital, Hallym University, Seoul, Korea Department of Surgery, Division of Head and Neck Surgery, University of California, San Diego, California 3 Department of Pathology, University of California, San Diego, California 4 Servicio de Otorrinolaringología, Hospital Donostia. San Sebastián, Spain 5 University of California, San Diego, Moore’s Cancer Center 6 Department of Pathology, University of California, San Diego and VA San Diego Healthcare System, San Diego, California 2

__________________________________________________________________________________ *Correspondence: Jessica Wang-Rodriguez, M.D., Department of Pathology, University of California, San Diego, VA San Diego Healthcare System, 3350 La Jolla Village Dr. (113), San Diego, CA 92161, USA; Telephone: (858) 552-8585 ext. 7734; Fax: (858) 5524370; Email: jwrodriguez@ucsd.edu Key words: Gleevec, Imatinib mesylate, Akt, tyrosine kinase, serine threonine kinase, head and neck cancer Abbreviations: acute myelogenous leukemia, (AML); gastrointestinal stromal tumors, (GIST); head and neck squamous cell carcinoma, (HNSCC); phophoinositide 3-kinases, (PI3Ks); phosphoinositides, (PIs); phosphorylated Akt, (p-Akt); platelet-derived growth factor receptor, (PDGFR); pleckstrin homology, (PH); protein tyrosine kinases, (PTKs); receptor tyrosine kinases, (RTKs); small-cell lung cancer, (SCLC); tyrosine kinases, (TKs) Received: 4 October 2005; Accepted: 13 October 2005; electronically published: October 2005

Summary Imatinib mesylate (Gleevec) is known to exert anti-growth effects through many protein tyrosine kinases (PTKs) such as platelet-derived growth factor receptor (PDGFR), c-Kit, and c-abl. The serine/threonine kinase Akt, or protein kinase B (PKB), was described to be the downstream effector of many PTKs and inhibits apoptosis in cancer cells. The purpose of this study was to examine the expression of these PTKs and Akt in head and neck squamous cell carcinoma (HNSCC) after exposure to Imatinib and to determine if this drug will inhibit the growth of HNSCC cells at clinically relevant doses. Imatinib was introduced into cultures of the squamous cell lines UMSCC10B, HN12 and HN30 at clinically used concentrations. Protein tyrosine kinases, PDGFR, c-Kit, and c-Abl, were evaluated by Western blot. Cell viability was assessed by clonogenic survival analysis. HNSCC tissue samples were stained for PDGFR, c-Kit and phosphorylated Akt (p-Akt). Akt kinase activity was measured in the presence or absence of Imatinib. In addition, Akt phosphorylation following Imatinib treatment was assessed using Western blot. Akt siRNA was used as the positive control for complete inhibition of Akt. Colony forming efficiency decreased with an increase in concentration of Imatinib. Three µM of Imatinib completely reduced cell viability in HN12 and HN30 and 10 µM in UMSCC10B. Immunohistochemistry confirmed high expression of PDGFR, c-Kit, and p-Akt in human HNSCC tissues. Expression of PDGFR, c-Kit, and c-Abl in the HNSCC cell lines did not change after Imatinib treatment. Akt kinase activity was significantly inhibited with increasing concentration of Imatinib in HNSCC cells, and near complete dephosphorylation of Akt was observed at 6 µM of Imatinib in the UMSCC10B and HN30 cell lines. Akt SiRNA alone, however, significantly reduced the cell viability by approximately 1/3.

Imatinib at clinically relevant concentrations caused a dose dependent decrease in HNSCC survival with a complete inhibition of growth at the highest concentrations tested. The decreased cell survival may be related to the inhibition of PTKs and a reduction of Akt kinase activity but was not due to inhibition of Akt alone. today (Jemal et al, 2004). The overall 5-year survival rate for patients is less than 50%, among the lowest of the major cancer types, and has not improved during the last

I. Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignant neoplasm in the world

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Chu et al: Imatinib mesylate (Gleevec) inhibits head and neck squamous cell carcinoma decade (Jemal et al, 2004). The lack of progress in head and neck oncology emphasizes the importance of molecular genetic studies that correlate with tumor biology. The molecular alterations observed in HNSCC are mainly due to oncogene activation and tumor suppressor gene inactivation, leading to deregulation of cell proliferation. Understanding the molecular mechanisms of the tumor biology will aid in identifying targets for treatment.

Protein-tyrosine kinases (PTKs) are important regulators of intracellular signal-transduction pathways mediating cell development, growth, and multicellular communication. Their activity is normally tightly controlled and regulated. Perturbation of PTK signaling by mutations and other genetic alterations results in deregulated kinase activity and malignant transformation (Blume-Jensen and Hunter, 2001). Protein kinases were thought to be poor therapeutic targets because of their ubiquitous nature and crucial role in many normal physiologic processes.

The advent of Gleevec (STI-571, imatinib, CGP 57148; Novartis Pharmaceuticals, Basel, Switzerland) demonstrated that kinases could be clinically useful drug targets for treating certain types of cancer. Imatinib is a PTK inhibitor, which selectively suppresses the activity of c-Abl, Bcr-Abl, platelet-derived growth factor receptor (PDGFR), and c-Kit (Buchdunger et al, 1996; Druker et al, 1996; Heinrich et al, 2000; Capdeville et al, 2002; Roskoski, 2003). This compound is now standard treatment for chronic myelogenous leukemia (CML) (Druker et al, 2001; Kantarjian et al, 2002; Kurzrock et al, 2003). Human CML and some forms of Philadelphia chromosome-positive acute lymphocytic leukemia are characterized by reciprocal translocation between chromosomes 9 and 22. This translocation results in the fusion of two cellular genes, Abl and Bcr, resulting in the formation of a chimeric gene termed Bcr-Abl (Shore et al, 1990). Imatinib occupies the nucleotide-binding pocket of Bcr-Abl protein and blocks access to ATP thereby preventing phosphorylation of any substrate (Druker et al, 1996; Kurzrock et al, 2003) and inducing apoptosis (Druker et al, 1996; Kantarjian et al, 2002; Kurzrock et al, 2003).

Imatinib is not specific for Bcr-Abl, and its action extends to c-Kit and PDGFR (Heinrich et al, 2000; Pietras et al, 2001, 2002). The c-Kit proto-oncogene is a 145 kD transmembrane glycoprotein and a number of the receptor PTK subclass III family that includes receptors for PDGF, macrophage colony-stimulating factor, and flt3 ligand (Yarden et al, 1987, Small et al, 1994). Its ligand, alternatively known as stem cell factor, mast cell growth factor, or steel factor, is an early growth factor that supports the growth and differentiation of multiple hematopoietic lineages (Besmer, 1991; Ashman, 1999). In addition to its importance in normal cellular physiologic activities, c-Kit plays a role in biologic aspects of certain human cancers, including germ cell tumors, mast cell tumors, gastrointestinal stromal tumors (GIST), small-cell lung cancer (SCLC), melanoma, breast cancer, acute myelogenous leukemia (AML), and neuroblastoma (Besmer, 1991; Hibi et al, 1991; Beck et al, 1995; DiPaola

et al, 1997; Hirota et al, 1998; Ashman, 1999; Tian et al, 1999; Heinrich et al, 2000). Imatinib has been recently approved for the treatment of c-Kit-positive advanced and/or surgically unresectable GISTs and is undergoing trials for the treatment of SCLC. Autocrine PDGFR stimulation is found in several human tumor types, including dermatofibroma protuberans, giant cell fibroblastoma, and glioblastoma, each of which responds to Imatinib with inhibition of growth and apoptosis in vitro and in xenograft models (Buchdunger et al, 2000; Sjoblom et al, 2001).

A significant advantage of Imatinib is that it is effective when administered orally. Many anticancer drugs are effective only when injected intravenously. In contrast to other chemotherapy drugs, side effects from Imatinib are mild. The most common side effects are mild nausea, edema, myalgias, arthralgias, diarrhea, and skin rash which occur in about 10% of patients (Druker et al, 2001; Kurzrock et al, 2003).

In a preliminary study, we found that PDFGR, c-Kit, and c-Abl were highly expressed in cultured squamous cell lines by Western blot analysis. For stimuli that induce PTK activity in cells almost invariably induce subsequent activity of proteins with Src homology domains, such as phophoinositide 3-kinases (PI3Ks) which in turn generate inositol phospholipids (Stephens et al, 1993; Vanhaesebroeck and Alessi, 2000). These lipids and the protein kinase are mostly likely activated by the protein kinase Akt (also known as PKB), and trigger a cascade of responses, from cell growth and proliferation to survival and motility that lead to tumor progression (Vanhaesebroeck and Alessi, 2000; Blume-Jensen and Hunter, 2001; Amornphimoltham et al, 2004). Most of the growth factors activate Akt through a PI3K signaling pathway-dependent mechanism (Cross et al, 1995; Franke et al, 1995), but a few PI3K-independent mechanisms also have been reported (Moule et al, 1997; Filippa et al, 1999).

Because these mechanisms may be involved in the action of Imatinib against cancer cells, we designed a study to investigate the effect of Imatinib in human HNSCC where PTKs such as PDGFR and c-Kit are expressed, and to examine its effect on the activation of Akt as a probable mechanism of action.

II. Materials and Methods A. Reagents Imatinib (Novartis, Basel, Switzerland) was dissolved in Me2SO4 (DMSO) to a stock concentration of 3 mM and stored at -20°C. Dilutions for all experiments were freshly made before use.

B. Cell lines and tissue culture Human HNSCC cell lines UMSCC10B, HN12, and HN30, were used in this study. UMSCC10B was supplied by Dr. Tom Carey form University of Michigan. HN12 and HN30 cell lines were obtained form Dr. J.S. Gutkind, Oral and Pharyngeal Cancer Branch, National Institute of Craniofacial and Dental Research, National Institutes of Health, Bethesda, MD. All cell lines were grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum, 10,000 unit/ml penicillin G sodium, 10 mg/ml streptomycin sulfate (Invitrogen

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Cancer Therapy Vol 3, page 517 incubation of goat serum (1:20 dilution) for 60 minutes. Primary antibodies were placed on slides and incubated for 1 hour at room temperature in the case of PDGFR and c-Kit and overnight at room temperature in the case of p-Akt. Secondary antibodies conjugated with Strepavidine/HRP (LSAB2, DAKO) were used.

The slides were washed and antibody complex visualized by 3,3’-diaminobenzidine (DAB, DAKO). The nuclei were counterstained by Gill’s II hematoxylin. Immunoactivity in the tissues was estimated by counting the number of positive cells per 1,000 tumor cells. Cases were considered positive if more than 20% of the tumor cells were staining.

Co., Carlsbad, CA), and L-glutamine (Invitrogen Co.) at 37°C in 5% CO2.

C. Western blot analysis for PDGFR, c-Kit, and c-Abl After twenty-four hours of achieving cell confluence, 5 ml of 6 µM of Imatinib was added to each plate in duplicate for 24 hours at 37 °C, and 5 ml of DMSO was added to the control plate. Cells were lysed in lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM !-glycerophosphate, 1 mM Na3VO4, and 1 µg/ml leupeptin. Cell lysates were separated on 10% NuPAGE® Novex Bis-Tris Gels (Invitrogen Co.) and transferred electrophoretically to a nitrocellulose membrane (0.45 µm; Bio-Rad Laboratories, Hercules, CA). Membranes were incubated for one hour at room temperature with primary antibodies in the following concentrations: anti-PDGFR-" antibody (1:200 dilution), anti-PDGFR-! antibody (1:200), antic-Kit antibody (1:200), and anti-c-Abl antibody (1:100; all from Santa Cruz Biotechnology, Santa Cruz, CA). Appropriate secondary antibodies (1:2,000 dilution anti-rabbit antibodies for PDGFR-", PDGFR-!, and c-Kit; 1:2,000 antimouse for c-Abl) were added. Immunoreactive proteins were visualized with the enhanced chemiluminescence detection system (Pierce, Rockford, IL).

F. Akt kinase activity assay Equal numbers of cells were seeded into 3 cell culture plates (100 mm). After allowing cells to attach overnight, Imatinib or DMSO was added at concentrations of 0, 5, and 10 µM. Cells were harvested after 24 hours for Akt kinase assay (nonradioactive, Cell Signaling Technology). The harvested cell pellet was lysed with 1 mM phenylmethylsufonyl fluoride (PMSF) and 0.5 ml of cell lysis buffer. Cell lysates (supernatant) were prepared after sonication and centrifugation for 10 minutes at 4°C. The total protein of each cell line was standardized using a BCA protein assay reagent kit (Pierce, Rockford, IL).

Immobilized Akt monoclonal antibody (Cell Signaling Technology, Beverly, MA) was added to an equal amount (200 µl) of each cell lysate and incubated overnight at 4°C in rocker.

The immunoprecipitate was washed twice each with cell lysis buffer and kinase buffer containing 25 mM Tris (pH 7.5), 5 mM !-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2. The pellet was re-suspended in 50 µl of kinase buffer, 10 mM of ATP, and 1 µg of GSK-3 fusion protein. After incubation for 30 minutes at 30°C, the reaction was terminated with 25 µl 3% SDS buffer. Samples were loaded on 10% NuPAGE® Novex Bis-Tris Gels (Invitrogen Co.) and transferred electrophoretically to a nitrocellulose membrane. The volume of sample per well on gel was adjusted appropriately according to the total protein amount of each sample that was determined by BCA protein assay. The membrane was incubated for 2 hours with blocking buffer that contained PBS and 5% nonfat dry milk. GSK-3 phosphorylation was detected using phosphopho-GSK-3"/!

(Ser21/9) antibody (1:1,000 dilution) and horseradish peroxidase-conjugated anti-rabbit antibody by Western blot. To assess the level of expression of p-GSK, SeeBlue Plus2® (Invitrogen Co.) was used as a standard ladder.

D. Clonogenic survival analysis Cultures were trypsinized to generate a single cell suspension, which was seeded onto 50 mm tissue culture plates at a density of 700 cells/plate in triplicate. To assess the effect of Imatinib on cell proliferation, the HNSCC cell lines were incubated for a total of 8 days in various concentrations (0, 0.5, 1, 3, 6, and 10 µM) of Imatinib which was renewed on day 4.

Control plates received DMSO only. Eight days after seeding, colonies were stained with crystal violet, and the number of colonies containing at least 50 cells was counted using AlphaImager 2200 and 1220 software (Alpha Innotech Co., San Leandro, CA). All of the assays were performed in triplicate and were repeated at least twice.

In a separate experiment, Akt siRNA (SignalSilenceTM siRNA kit; Cell Signaling Technology) at a concentration of 100 nM was added to UMSCC10B without the addition of Imatinib and incubated for 10 days. A repeat dose of Akt siRNA was added on day 4. Colonies were counted using the above method at the end of the 10th day.

E. HNSCC specimens immunohistochemical analysis

G. Akt siRNA as a positive control

and

To investigate the mechanism of action of Imatinib on Akt, we compared the expression of Akt and p-Akt after exposure to Imatinib, using Akt siRNA (SignalSilenceTM siRNA kit; Cell Signaling Technology) as the positive control. The three HNSCC cell lines were cultured in three 50 mm culture plates and allowed to grow to less than 50% confluence. Then the cells were incubated with either Akt siRNA or 6 µM of Imatinib for 24 hours. The expression was compared to the control samples that were exposed to Transfection reagent (SignalSilenceTM siRNA kit component; Cell Signaling Technology).

For cells incubated with Akt siRNA, two sets of 1 ml serum-free media were prepared in sterile microfuge tubes. 20 µl of Transfection reagent were added to the each tube, followed by incubation for 10 minutes in room temperature. Akt siRNA was added at a concentration of 100 nM and was incubated for another 10 minutes at room temperature. This mixture was added to one set of the culture plates and agitated for 30 seconds. The contents of the other microfuge tube were added to another plate as a control. The three cell lines were exposed to either Akt

Immunohistochemical studies on archived, formalin fixed, paraffin embedded HNSCC tissue samples were performed using polyclonal rabbit antibodies against PDGFR (Santa Cruz Biotechnology) and c-Kit (Santa Cruz Biotechnology). A mouse monoclonal antibody against p-Akt (Ser 473) (Cell Signaling Technology, Beverly, MA) was used for phosphorylated Akt. Negative control slides were from the same tissue but without the addition of the primary antibodies. As normal controls, 5 specimens each of normal tonsils, uvula, and laryngeal mucosa were included.

The tissue sections were deparaffinized in xylene and rehydrated in ethanol. Antigen retrieval was performed by steam heating with 1x DAKO Target Retrieval solution. The sections were then allowed to cool to room temperature in the solution.

The endogenous peroxidase was removed by 3% H2O2. Nonspecific binding of biotin and avidin was abolished by blocking solution for 30 minutes (Protein Block Serum-Free, DAKO, Carpinteria, CA). The background staining was reduced with

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Chu et al: Imatinib mesylate (Gleevec) inhibits head and neck squamous cell carcinoma siRNA or Imatinib at 6ÌM for 24 hours. Scramble siRNA was used as a negative control.

After incubation with Imatinib or Akt siRNA, cells were harvested and centrifuged at 2,000 rpm. Protein was extracted from cells using lysis buffer containing protease inhibitor and was separated using 10% NuPAGE® Novex Bis-Tris Gels and blotted onto a nitrocellulose membrane. The membrane was probed using anti-p-Akt antibody (1:100; Cell signaling Technology), anti-Akt1 antibody (1:100; Santa Cruz Biotechnology), and anti-!-actin antibody (1:5,000; SigmaAldrich Inc.), followed by 1-hour room temperature incubation with the following secondary antibodies: anti-mouse antibody (1:5,000) for p-Akt; anti-goat antibody (1:10,000) for Akt1; and anti-mouse antibody for actin (1:2,000). Positive protein signals were visualized with the enhanced chemiluminescence detection system.

By Western Blot analysis, human HNSCC cell lines, UMSCC10B, HN12, and HN30, were shown to express PDGFR-", PDGFR-!, c-Kit, and c-Abl (Figure 1).

Exposure of HNSCC cells to 6 µM Imatinib for 24 hours has no apparent effect on the quantitative expressions of these PTKs.

B. Inhibition of cell growth by Imatinib at clinically relevant concentrations The effect of Imatinib on three HNSCC cell lines was measured by means of clonogenic survival analysis. A near-complete growth inhibition in HN12 and HN30 cells was observed when treated with 3 µM Imatinib, whereas UMSCC10B cells exhibited a similar response at a concentration of 10 µM (Figure 2).

III. Results A. Expression of TKs in HNSCC cell lines

Figure 1. Western blot analysis of PDGFR-", PDGFR-!, c-Kit, and cAbl in UMSCC10B, HN12, and HN30 cells. The protein tyrosine kinases were present in all three cell lines, but their expression was not changed after 24 hours of Imatinib treatment at 6 µM.

Figure 2. Clonogenic assay in three head and neck squamous cell carcinoma (HNSCC) cell lines. Each cells line was exposed to control (DMSO) or increasing concentration of Imatinib (0, 0.5, 1, 3, 6, and 10 µM) and stained eight days after seeding. The darker staining spots represented viable cells. The viability of UMSCC10B cell ( A) was grossly inhibited only at the dose of 10 µM Imatinib. Cell viability of HN12 cells (B) and HN30 (C) cells were markedly inhibited at the dose of 3 µM Imatinib. Each assay condition was performed in triplicate.

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Cancer Therapy Vol 3, page 519 The concentration of 3 to 6 µM can be achieved by once daily administration of Imatinib orally in patients (4).

Consistent with the results from viability staining assays, the quantitative analyses of colony forming efficiency demonstrated that Imatinib exerted a significant inhibitory effect on cell growth of HNSCC cell lines (Figure 3).

Akt siRNA alone at a concentration of 100 nM for 10 days achieved approximately 33% inhibitory effect on the cell colony. The number of colonies in the control cells was 459+/-31. Cells treated with Akt SiRNA had a reduction of colonies to 300+/-54 (p=0.01, Figure 4).

D. Effect of Imatinib on Akt kinase activity HNSCC cells in the presence of Imatinib inhibited Akt kinase activity in a dose-dependent fashion (Figure 6). Imatinib at a concentration of 5 µM inhibited the Akt kinase by approximately 50% in these cell lines. At a concentration of 10 µM, all three cell lines revealed further reduction, but not complete absence of Akt kinase activity.

E. Akt SiRNA vs. Imatinib effect on phosphorylated Akt

C. Immunohistochemical study

Figure 7 showed the presence of Akt1 and phosphorylated Akt (p-Akt) in cells after exposure to 6 uM of Imatinib vs. Akt SiRNA for 24 hours. Both Akt siRNA and Imatinib caused a near-complete dephosphorylation of Akt, as evidenced by a significant reduction in p-Akt expression, in all three cell lines, to levels that were comparable to those seen after exposure to Akt SiRNA in UMSCC10B cells. The expression of Akt1 remained present after Imatinib treatment, although there appeared be a reduction of Akt1 in UMSCC10B after exposure to Imatinib. In contrast, Akt siRNA caused marked reduction of both Akt1 and the p-Akt.

Immunohistochemistry was performed on 37 HNSCC tumors and 5 controls. Figure 5 shows a representative example of immunohistochemical staining for (A) PDGFR, (B) c-Kit, and (C) p-Akt. Positive staining is located in the cytoplasm of PDGFR and c-Kit, and in the cytoplasm and nucleus for the phospho-Akt. PDGFR was positive in 36 of 37 cancer samples (97.3%) as well as all 5 control specimens. The proportion of positive staining for c-Kit and p-Akt were 70.3% (26 of 37) and 67.6% (25 of 37), respectively. In contrast, the control cases showed no expression in p-Akt and c-Kit (p=0.07).

Figure 3. Effect of Imatinib on tumor cell viability. Colony-forming efficiency was determined 8 days later. The colonies were counted if they had equal or greater than 50 cells. Each value represented the mean ± standard deviation for three independent experiments.

Figure 4. Clonogenic assay on cell UMSCC10B after 10 days of incubation with 100 nM Akt SiRNA. The number of colonies counted in control cells was significantly higher than that treated with Akt SiRNA (p=0.01).

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Chu et al: Imatinib mesylate (Gleevec) inhibits head and neck squamous cell carcinoma Figure 5. Immunohistochemical detection for PDGFR (A), c-Kit (B), and phospho-Akt (C) and negative control (D). Positive staining indicated the presence of p-Akt. Positive staining was observed mainly in the cytoplasm and evenly distributed within the tumor. p-Akt is a phosphorylated form of Akt. All images were photographed at 400X magnification..

Figure 6. Analysis of Akt kinase activity in Imatinib-treated head neck squamous cell carcinoma cell lines. Cells were exposed to 0, 5, and 10 µM of Imatinib for 24 hours and lysed for immunoblot analysis.

Kinase assay was performed using GSK-3 fusion protein as a substrate.

The protein levels of phosphorylated GSK reflect the Akt kinase activity.

Akt kinase was reduced by approximately 50% at 5µM of Imatinib in all three cell lines, and approximately by 75% at 10µM in UMSCC10B and HN30 cells.

Figure 7. Imatinib or Akt SiRNAdependant suppression in Akt expression. Western blots showed phosphorylated Akt (p-Akt), Akt1, and !-actin from three HNSCC cell lines. The three cell lines were treated with Imatinib at 6 µM for 24 hours and separate UMSCC10B cells were treated with 100 nM Akt SiRNA for 24 hours as positive control. The expressions of Akt1 were not significantly altered with Imatinib treatment, with the exception of UMSCC10B, that showed approximately 50% reduction in Akt1 expression. The presence of p-Akt was severely reduced to a level similar to the effect of Akt SiRNA.

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Cancer Therapy Vol 3, page 521 tumors with a pattern of expression and localization correlating with the progression of lesions. Grille et al.

(Grille et al, 2003) reported that activation of the Akt pathway led to epithelial-mesenchymal transition (EMT) and invasion in HNSCC cells. In this study, we also found a high percentage of p-Akt expression in human HNSCC by immunohistochemistry.

Previous studies have demonstrated that Imatinib activity is mediated in part through direct inhibition of the Akt signaling pathway (Matei et al, 2004; Appel et al, 2005). In this study, we also demonstrated the inhibitory effect of Imatinib on Akt in the HNSCC cells, mainly in the phosphorylated, comparable to that of Akt siRNA. When used Akt siRNA as a positive control to completely block Akt, we observed that Imatinib at 6Ì remarkably inhibits phospho-Akt in all three cells to a similar level as to when cells were exposed to Akt siRNA. However, the near absence of p-Akt did not correlate with the growth inhibition in UMSCC 10B cells, which required a higher dose (10µM) of Imatinib. We concluded that Imatinibinduced growth inhibition was not due to the blocking of Akt phosphorylation alone. Furthermore, different cell lines with different genomic instability may have unpredictable sensitivity to Imatinib.

In addition, directly incubating the UMSCC10B cells with the Akt siRNA showed significant growth inhibition, but failed to demonstrate the growth inhibitory effect similar to that of Imatinib. Therefore, inactivation of Akt alone did not appear to account for the action of Imatinib.

This growth inhibition may involve other PTKs, perhaps in combination with Akt or Akt independent pathways.

However, in the absence of additional information on other signal transduction pathways mediating cell proliferation and multicellular communication, it is difficult to present an exact molecular mechanism of growth inhibition of HNSCC by Imatinib. We expect that Imatinib can inhibit cancer cell proliferation at more than one level. We are actively investigating other TKs that maybe modulated by Imatinib.

In contrast to CML, in which one gene mutation drives cancer progression (Sawyers, 1999), most solid tumors are thought to be the result of several genetic alterations (Hanahan and Weinberg, 2000). Our study shows that a high percentage of human HNSCC tissue expressed PDGFR, c-Kit, and p-Akt. Drugs like Imatinib, which target PTKs and possibly Akt in cancers that overexpress them, may prove to be useful as single agent, or in combination with other types of therapy, especially since their toxicity profile is very acceptable.

In summary, we have demonstrated that at concentrations achieved in serum by oral administration of standard therapeutic doses, Imatinib caused significant inhibition of cell proliferation in three HNSCC cell lines.

The mechanism of this inhibition may involve many PTKs with or without inactivation of Akt. This study serves as the first step in exploring the role of Imatinib in the treatment of HNSCC. The marked inhibition of Imatinib on proliferation and survival of HNSCC cell lines justifies a similar study in animal models. Because of the low toxicity of Imatinib, the data from this study can also support preliminary clinical trials of this drug as an

IV. Discussion In the current study, we found that HNSCC cell lines as well as human HNSCC’s expressed high levels of PDGFR, c-Kit, and c-Abl tyrosine kinases (TKs). The receptor tyrosine kinases (RTKs), PDGFR and c-Kit, and a non-receptor thyrosine kinase, c-Abl, are the main targets for Imatinib (Buchdunger et al, 2000; Vlahovic and Crawford, 2003). Since Imatinib has proven to be an effective inhibitor for various tyrosine kinases, we hypothesized that this tyrosine kinase inhibitor could also inhibit the proliferation of HNSCC cells.

The clonogenic analysis showed a significant inhibition of cell viability in HN12 and HN30 cells at a concentration of 3 µM Imatinib, which was the serum level achieved by the typical dosage of 300mg Imatinib taken orally in the once daily for CML treatment (Roskoski, 2003).

Though the activity of these target kinases are presumably lowered by Imatinib, the protein levels of PDGFR, c-Abl and c-Kit expression in HNSCC cells were not altered following Imatinib treatment. Because of its critical role in the downstream-signaling pathway of PTKs such as PDGFR and c-Kit, we proceeded to measure Akt kinase activity following the treatment of Imatinib. We hypothesized that Imatinib would inhibit cancer cell proliferation either through inhibition of PI3K/Akt or PI3K-independent Akt pathway. Akt is a 57 kD serine/threonine kinase with pleckstrin homology (PH) domain that preferentially binds phophatidylinositol (Blume-Jensen and Hunter, 2001; Roskoski, 2003) diphosphate (PtdIns(3,4)P 2) and PtdIns(3,4,5)P 3 over other phosphoinositides (PIs) (James et al, 1996; Stephens et al, 1998; Vanhaesebroeck and Alessi, 2000). Stimuli that induce PTK activity in cells almost invariably lead to the generation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (Stephens et al, 1993).

Akt itself has a pivotal role in cell cycle progression, differentiation of smooth muscle cells, angiogenesis, inhibition of apoptosis, and cell growth (Franke et al, 1995; Brennan et al, 1997; Muise-Helmericks et al, 1998; Hayashi et al, 1999; Ozes et al, 1999; Verdu et al, 1999; Okano et al, 2000). Akt also plays a key role in cancer progression by stimulating cell proliferation and inhibiting apoptosis (Blume-Jensen and Hunter, 2001; Vivanco and Sawyers, 2002). Akt promotes cell survival through downstream signaling. Phosphorylation of Akt contributes to the activation of Akt (Kohn et al, 1996). To date, there are at least seven signaling pathways downstream of Akt (McCormick, 2004). Proteins that encourage apoptosis, such as BAD, caspase-9, are among the first targets of Akt to be identified. In addition, Akt triggers other cell-death regulators such as IKK", the forkhead transcription factors, Mdm2 and Yap (Basu et al, 2003; Wendel et al, 2004). Such association has been shown in several cancer types such as breast, pancreatic, and ovarian cancer (Cheng et al, 1992, 1996; Ballacosa et al, 1995).

The involvement of Akt in human HNSCC has been demonstrated previously in a number of in vitro and in vivo studies. For example, Amornphimoltham et al.

(Amornphimoltham et al, 2004) demonstrated that activation of Akt could be detected in head and neck 521

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Cancer Therapy Vol 3, page 525 Cancer Therapy Vol 3, 525-530, 2005

Incidence of gastrointestinal toxicity during estramustine phosphate therapy for prostate cancer is associated with the single-nucleotide polymorphisms in the cytochrome P450 1A1 (CYP1A1) gene Research Article

Mohammed Rafiqul Islam Mamun 1, Motofumi Suzuki1, Satoru Takahashi1, Kazuo Hara2, Takeshi Ozeki3, Yasuhiko Yamada3, Takashi Kadowaki4, Yoshitsugu Yanagihara5, Shuji Kameyama6, Yoichi Minagawa Ito7, Takumi Takeuchi1 and Tadaichi Kitamura1,* 1

Department of Urology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 1138655, Japan 2 Department of Clinical Bioinformatics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan 3 Department of Clinical Evaluation of Drug Efficacy, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan 4 Department of Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan 5 Department of Pharmacy, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan 6 Department of Urology, Kanto Medical Center NTT EC, 5-9-22 Higashigotanda, Shinagawa, Tokyo 141-8625, Japan 7 Department of Biostatistics / Epidemiology and Preventive Health Sciences, School of Health Sciences and Nursing, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

__________________________________________________________________________________ *Correspondence: Tadaichi Kitamura, M.D., Ph.D., Professor and Chairman, Department of Urology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; Phone: +81-3-5800-8662, Fax: +81-3-5800-8917; E-mail: KITAMURA-DIS@h.u-tokyo.ac.jp Key words: Estramustine phosphate, CYP1A1, Gastrointestinal toxicity, Prostate cancer, Tailor-made medication Abbreviations: confidence interval, (CI); Cytochrome P450, (CYP); Estramustine phosphate, (EMP); estramustine, (EaM); estromustine, (EoM); Gastrointestinal toxicity, (GIT); intervening sequence, (IVS); luteinizing-hormone releasing-hormone agonist, (LH-RHa); odds ratio, (OR); polymerase chain reaction, (PCR); single nucleotide polymorphisms, (SNPs)

Mohammed Rafiqul Islam Mamun and Motofumi Suzuki had equal contribution to this study.

Received: 25 August 2005; Accepted: 4 October 2005; electronically published: November 2005

Summary Gastrointestinal toxicity (GIT) is observed frequently during estramustine phosphate (EMP) therapy in prostate cancer patients. This adverse effect often deteriorates the patientsâ&#x20AC;&#x2122; compliance and quality of life, which results in drug discontinuation. The CYP1A1 gene is polymorphic and involves in the metabolism of EMP. Polymorphisms of the CYP1A1 gene might have a role to modulate the metabolism of EMP and convert patientsâ&#x20AC;&#x2122; ability to comply with the drug toxicities. We performed genotyping of the CYP1A1 gene to reveal interindividual difference of GIT associated with EMP therapy. The study enrolled 126 patients with untreated advanced prostate cancer. Low-dose of EMP was administered orally. Genotyping of m1, m2 and IVS1-728 polymorphisms of the CYP1A1 gene was

525

Manum et al: Gastrointestinal toxicity during estramustine phosphate therapy with P450 1A1 (CYP1A1) gene performed by PCR-based direct sequencing method. In the multivariate analysis, GIT risk was increased significantly in the major allele homozygous genotypes of m1 and IVS1-728 SNPs compared with the heterozygous and minor allele homozygous genotypes (T/T genotype of m1, odds ratio (OR), 2.69; 95% CI, 1.24 to 5.96; P = 0.01; and G/G genotype of IVS1-728, OR, 3.31; 95% CI, 1.52 to 7.47; P < 0.01). Haplotype study showed that the risk of GIT was enhanced approximately 12 times higher when ‘T’ nucleotide in m1, ‘A’ nucleotide in m2 and ‘G’ nucleotide in IVS1-728 locus are present in a same allele (OR, 11.86; 95% CI, 3.61 to 39.02; P < 0.01) compared with the other allelic combinations. This study demonstrated that m1, m2 and IVS1-728 polymorphisms in the CYP1A1 gene were significantly associated with GIT during EMP therapy in prostate cancer patients. Genotyping of these polymorphisms prior to commence the EMP therapy will be a useful method to select the patients with a risk of GIT and to improve patients’ compliance and quality of life.

CYP1A1 gene might play a role in modulating the metabolism of EMP and act as one of the determining factors for the inter-individual variations in adverse effects of EMP therapy. To find out the link between genotypes encoding the enzymes of EMP metabolism and its adverse effects, we evaluated m1, m2, and IVS1-728 polymorphisms in the CYP1A1 gene among the prostate cancer patients treated with EMP.

I. Introduction Estramustine phosphate (EMP) is a chemoendocrine agent that was applied for the treatment of prostate cancer in the 1970s. Nowadays, usually it is used for the treatment of hormone refractory prostate cancer. Recently, synergy between EMP and other antimicrotubule agents (i.e. paclitaxel and docetaxel) in the treatment of androgen-independent prostate cancer has been assessed and the clinical efficacy of EMP has been re-evaluated (Vaishampayan et al, 2002; Oudard, 2005).

Gastrointestinal toxicity (GIT) is a frequent adverse effect during EMP therapy in prostate cancer patients. GIT includes anorexia, heartburn, nausea and vomiting.

Recently, we conducted a clinical trial of low-dose EMP monotherapy for previously untreated prostate cancer. The object is to achieve the goal of lowering the incidence of adverse effects without compromising the therapeutic efficacy. We found 93.4% of prostate specific antigen (PSA) response rate (Kitamura, 2002), which was comparable with that of conventional dose EMP therapy.

However, incidence of the toxicity remained rather high as 39.5% and discontinuation rate was 32.1%. Individual susceptibility to the adverse effects perhaps linked with the polymorphisms of the enzymes encoding genes that are related to the metabolism of this drug.

Figure 1 is a schematic diagram showing the metabolic pathway of EMP. EMP consists of 17!-estradiol bound to nor-nitrogen mustard. After ingestion, EMP undergoes rapid dephosphorylation to yield estramustine (EaM), and EaM is oxidized by 17!-hydroxysteroid dehydrogenases to estromustine (EoM). EaM and EoM then yield 17!-estradiol and estrone, respectively, by hydrolysis (Gunnarsson et al, 1984).

Furthermore, 17!-estradiol is hydroxylated by Cytochrome P450 (CYP) 1A1, 1A2 and 3A4 to yield 2hydroxyestradiol (Lakhani et al, 2003). 2-hydroxyestardiol can compete with dopamine receptor effectively (Schaeffer et al, 1979). It was also reported that 2hydroxyestradiol behaves like an antagonist for dopaminergic receptors in striatum and pituitary (Paden et al, 1982).

The m1 (3801T>C) and intervening sequence (IVS) 1-728 (G>A) are intronic single nucleotide polymorphisms (SNPs), and m2 (2455A>G) is an exonic SNP of the CYP1A1 gene. The minor genotypes of m1 and m2 polymorphisms have been reported to increase its enzymatic activity (Cosma et al, 1993; Crofs et al, 1994), and reflect the mass of catechol estrogen transformation from 17!-estradiol and estrone. Thus, the SNPs of the

II. Materials and methods A. Study design and treatment plan A total of 126 patients with untreated advanced prostate cancer were enrolled in this study (Table 1). Age range was 48 to 89 years (mean 72.5 years). Patients with significant active concurrent medical illness, other malignancy, or cardiovascular diseases were excluded from this study. The Ethics Committee of the University of Tokyo approved this study and prior to study written informed consent was obtained from each patient. The treatment regimens were oral EMP alone (280 mg/day) for 42 patients, oral EMP (280 mg/day) plus luteinizinghormone releasing-hormone agonist (LH-RHa) or surgical castration for 13 patients, and oral EMP (140 mg/day) plus LH-RHa for 71 patients. Physical condition of the patients and incidence of the adverse effects were assessed monthly on the basis of the National Cancer InstituteCommon Toxicity Criteria (Version 2.0, Jan. 30, 1998).

According to their medical reports, 42 of 126 (33.3%) patients suffered from GIT during the low-dose EMP therapy. Then, we compared the genotypes of SNPs in the CYP1A1 gene among the patients with or without GIT.

B. Genotyping assay Genotyping was performed by polymerase chain reaction (PCR) based-direct sequencing method. Genomic DNA was extracted from the peripheral blood lymphocytes by standard method. To use it in PCR solution, concentration of genomic DNA was adjusted to 100 ng/µL. Primers used for DNA amplification are forward 5'-CAG TGA AGA GGT GTA GCC GCT-3' and reverse 5'-TAG GAG TCT TGT CTC ATG CCT-3' for m1; forward 5'AGT GGC ACG CTG AAT TCC A-3' and reverse 5'-CCC CTG ATG GTG CTA TCG AC-3' for m2; forward 5'-TGT TCT CAG GGG AAT TAG GG-3' and reverse 5'-AAG CAA TGT GGT TTG GGA AG-3' for IVS1-728. Melting temperature were 60°C for m1 and IVS1-728, 58°C for m2. Cyclic thermal conditions were 95°C for 10 minutes for one cycle; 94°C for 30 seconds, melting temperature for each set of primers, for 30 seconds, 72°C for 3 minutes for 37 cycles; followed by a cycle of 72°C for 10 minutes. The amplification reactions of each SNP were performed in 50 µL solution containing 100 ng of genomic DNA, 5 µL of 10xPCR Gold Buffer, 1.5 mmol MgCl2 solution, 0.2

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Cancer Therapy Vol 3, page 527 mmol dNTPs, 1.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Branchburg, NJ, USA), and 0.5 µM of each specific primer (synthesized by Fasmac, Atsugi, Kanagawa, Japan). GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) was used to perform PCR. All PCR amplicons were purified with the Montage PCRµ96 Plate (Millipore Corporation, Bedford, MA, USA) to remove deoxynucleotide triphosphates and excess primers. All sequencing reactions were performed by dye terminator chemistry (ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit; Applied Biosystems, Warrington, UK) with each sequencing primer, and the products were purified with the MultiScreen filter plates (Millipore Corporation, Bedford, MA, USA) with Sephadex G-50 Superfine (Amersham Biosciences, Uppsala, Sweden). Purified samples were applied to an ABI Prism 3700 DNA Analyzer (Applied Biosystems, Foster City, CA, USA), and sites of polymorphisms were identified with Sequencher software (version 4.1.2; Gene Codes Corporation, Ann Arbor, MI, USA).

Table 1. Patient characteristics.

Characteristics Number Age (mean ± SD) 72.5 ± 8.8 years Baseline PSA (mean ± SE) 418.3 ± 106.7 ng/mL Performance status 0 93 1 26 2 7 Clinical stage C 60 D 66 Gleason score 2 to 7 71 8 to 10 55 Dose of EMP 140 mg/day 71 280 mg/day 55

C. Statistical analysis The allele frequency of each SNP was estimated by direct count and assessed for deviation from the Hardy-Weinberg equilibrium by using "2 tests. Univariate and multivariate analyses were conducted by nominal logistic regression analysis.

In the multivariate analysis, OR, 95% CI and P value were obtained after adjustment for age, baseline PSA level, performance status, clinical stage, Gleason score and dose of EMP. EH program (version 1.20) was used for haplotype analyses. These statistical analyses were conducted by JMP software, version 5.1.2 (SAS, Cary, NC) and the P value was considered as significant when it was less than 0.05.

SD, standard deviation; SE, standard error; PSA, prostate specific antigen.

Figure 1. Metabolic pathway of estramustine phosphate (EMP). EMP is dephosphorylated to yield estramustine (EaM). EaM is oxidized by 17!-hydroxysteroid dehydrogenases (HSD17Bs) to estromustine (EoM). EaM and EoM then yield 17!-estradiol and estrone, respectively, by hydrolysis. Furthermore, 17!-estradiol is hydroxylated by Cytochrome P450 (CYP) 1A1, 1A2 and 3A4 to yield 2hydroxyestradiol.

527

Manum et al: Gastrointestinal toxicity during estramustine phosphate therapy with P450 1A1 (CYP1A1) gene homozygous genotypes of m1 and IVS1-728 are frequent among the patients suffered from GIT. The associations between genotypes of m1, m2, and IVS1-728 polymorphisms with GIT are shown in Table 5. The major alleles homozygous genotypes of m1 and IVS1-728 polymorphisms are significantly frequent among the patients suffered from GIT. Univariate analysis shows that the frequency of T/T genotype of m1 and G/G genotype of IVS1-728 are significantly associated with GIT (T/T genotype of m1, OR 2.78; 95% CI, 1.31 to 6.07; P < 0.01; and G/G genotype of IVS1-728, OR 3.41; 95% CI, 1.59 to 7.56; P < 0.01). Multivariate analysis demonstrated an independent significant relationship between m1, IVS1728 polymorphisms and GIT (T/T genotype of m1, OR2.69; 95% CI, 1.24 to 5.96; P = 0.01; and G/G genotype of IVS1-728, OR 3.31; 95% CI, 1.52 to 7.47; P < 0.01).

III. Results Of the 126 patients, 42 (33.3%) suffered from GIT during low-dose EMP therapy. Seventy-one patients were administered EMP at a dose of 140 mg/day and 21 (29.6%) of them suffered from GIT. Fifty-five patients were administered EMP at a dose of 280 mg/day and 21 (38.2%) of them suffered from GIT. Incidence of GIT was more frequent in the group of EMP 280 mg/day, however, it was not statistically significant ("2 = 1.03, P = 0.31).

Table 2 shows grade and symptoms of GIT. Approximately 70% of total GIT were grade 1 and the most common GIT was anorexia (45.2%). One patient developed hematemesis associated with grade 3 gastric ulcer.

We performed genotyping assay for 3 SNPs in the CYP1A1 gene and frequency of each genotype is shown in Table 3 and 4. The distribution of genotypes of these SNPs did not deviate from the Hardy-Weinberg equilibrium. It was observed that the major allele Table 2. GIT observed during EMP therapy (n = 42).

GIT grade of the NCI-CTC Grade 1

Grade 2

Grade 3

Total

n (%)

n (%)

n (%)

n (%)

Anorexia

14 (33.3)

5 (11.9)

0

19 (45.2)

Heartburn

11 (26.2)

3 (7.1)

0

14 (33.3)

Nausea and Vomiting

5 (11.9)

3 (7.1)

0

8 (19.0)

Gastric ulcer Total, n (%)

0 0 30 (71.4) 11 (26.2)

1 (2.4) 1 ( 2.4)

1 (2.4) 42 (100)

GIT, gastrointestinal toxicity; NCI-CTC, National Cancer Institute-Common Toxicity Criteria.

Table 3. Allele frequency of m1, m2, and IVS1-728 polymorphisms with Hardy-Weinberg equilibrium test. dbSNP ID Trivial name Position Allele frequency HWE P value

Major allele Minor allele "2

rs4646903 m1 3801T>C

rs1048943 m2 2455A>G

rs4646421 – IVS1-728G>A

T: 0.66 C: 0.34 0.44 NS

A: 0.79 G: 0.21 2.05 NS

G: 0.66 A: 0.34 0.61 NS

–, not applicable; HWE, Hardy-Weinberg equilibrium; NS, not significant.

Table 4. Distribution of genotypes in m1, m2, and IVS1-728 loci with or without GIT.

m1 GIT (+), GIT (–), Genotypes n=42 n=84 C/C 4( 9.5)* 12(14.3) T/C 12 (28.6) 41 (48.8) T/T 26 (61.9) 31 (36.9)

Locus m2 GIT (+), GIT (–), Genotypes n=42 n=84 G/G 1 (2.4) 7(8.3) A/G 10 (23.8) 26 (31.0) A/A 31 (73.8) 51 (60.7)

* No. of patients (%), GIT, gastrointestinal toxicity; IVS, intervening sequence.

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IVS1-728 GIT (+), Genotypes n=42 A/A 4 ( 9.5) G/A 11 (26.2) G/G 27 (64.3)

GIT (–), n=84 13(15.5) 42(50.0) 29(34.5)

Cancer Therapy Vol 3, page 529 The results of haplotype analyses are shown in Table 6. It is recognized from haplotype analyses that the chance of GIT is over two times higher when ‘A’ nucleotide in m2 and ‘G’ nucleotide in IVS1-728 loci are present in a same allele compared with other combinations (OR, 2.33; 95% CI, 1.00 to 5.41; P = 0.05). Furthermore, when the patients represent ‘T’ nucleotide in m1, ‘A’ nucleotide in m2 and ‘G’ nucleotide in IVS1-728 locations in a same allele, the risk of GIT was enhanced approximately 12 times higher than other allelic combinations (m1+m2+IVS1-728 = T+A+G, OR, 11.86; 95% CI, 3.61 to 39.01; P < 0.01).

the patients. Also additional treatments and measures are required to manage the patients for adverse effects.

Therefore, selection of suitable patients prior to commence the therapy might be beneficial for survival of patients as well as medical economy.

GIT is one of the major adverse effects of EMP therapy. About 25.9% patients suffered from GIT during EMP therapy (Kitamura, 2001 and 2002). To overcome this adverse effect is crucial, however, the mechanism of GIT during EMP therapy is not well known.

The allele frequencies of these three polymorphisms are shown in the database of National Cancer Institute (NCI) (http://snp500cancer.nci.nih.gov/snp.cfm). Allelic frequencies of m1 and IVS1-728 in our patients are very similar to that of NCI data. However, minor allelic frequency of m2 among the patients appeared higher than

IV. Discussion Withdrawal of chemotherapy due to drug toxicities may allow disease progression and worsen the survival of

Table 5. Relation between polymorphisms of the CYP1A1 gene and risk of GIT (univariate and multivariate analyses).

Factors OR ml T/C & C/C T/T m2 A/G &G/G A/A IVS1-728 G/A &A/A G/G Age < 73 # 73 Baseline PSA level < 418 ng/mL $ 418 ng/mL Peformance status 0 and 1 2 Clinical stage C D Gleason score 2 to 7 8 to 10 Dose of EMP 140 mg/day 280 mg/day

Univariate 95% CI

P value

OR†

Multivariate 95% CI†

P value

1.0* 2.78

1.31 to 6.07

< 0.01

1.0* 2.69

1.24 to 5.96

0.01

1.0* 1.82

0.82 to 4.25

NS

1.83

0.81 to 4.39

NS

1.0* 3.41

1.59 to 7.56

< 0.01

1.0* 3.314

1.52 to 7.47

< 0.01

1.0* 0.68

0.32 to 1.43

NS

1.0* 0.80

0.37 to 1.74

NS

1.0* 1.09

0.38 to 2.92

NS

1.0* 1.13

0.35 to 3.46

NS

1.0* 0.32

0.02 to 1.94

NS

1.0* 0.25

0.01 to 1.69

NS

1.0* 0.87

0.41 to 1.82

NS

1.0* 0.71

0.30 to 1.64

NS

NS

1.0* 1.35

0.59 to 3.09

NS

NS

1.0* 1.72

0.78 to 3.81

NS

1.0* 1.27 1.0* 1.47

0.60 to 2.69

0.70 to 3.11

IVS, intervening sequence; EMP, estramustine phosphate; PSA, prostate-specific antigen; OR, odds ratio; CI, confidence interval; NS, not significant. *Reference †OR, 95% CI and P value were obtained after adjustment for age, baseline PSA level, performance status, clinical stage, Gleason score, and dose of EMP.

529

Manum et al: Gastrointestinal toxicity during estramustine phosphate therapy with P450 1A1 (CYP1A1) gene Table 6. Results of haplotype analyses between m1, m2, and IVS1-728 polymorphisms.

Allelic combination m1+m2 = T+A others m1+IVS1-728 = T+G others m2+IVS1-728 = A+G others m1+m2+IVS1-728 = T+A+G others

Number of patients GIT (+) GIT (â&#x20AC;&#x201C;) 32.00 50.91 10.00 33.09 32.00 50.00 10.00 34.00 32.50 50.00 9.50 34.00 38.62 41.18 3.39 42.82

OR (95% CI)

P value

2.08 (0.90 to 4.79)

NS

2.18 (0.95 to 5.01)

NS

2.33 (1.00 to 5.41)

0.05

11.86 (3.61 to 39.02)

< 0.01

IVS, intervening sequence; GIT, gastrointestinal toxicity; OR, odds ratio; CI, confidence interval; NS, not significant.

that of NCI data (our data, A: G = 0.79: 0.21; NCI data, A: G=0.88: 0.12). Perhaps this variation is due to racial differences and the factors related to the disease. Further studies are needed to clarify the cause of this variation.

Our data demonstrated an association between genetic polymorphisms of m1, m2 and IVS1-728 in the CYP1A1 gene and GIT during the EMP therapy.

Therefore, genotyping of these polymorphisms prior to EMP therapy will be useful to estimate the risk of GIT. At the present, we could not compare the serum 2hydroxyestradiol levels on the basis of genotypes.

However, the mechanisms of emesis and comparative study of serum 2-hydroxyestradiol levels in different genotypes of these three SNPs during EMP therapy are remaining for future study. Our results are novel and the association was statistically significant, however, much larger population is needed to confirm our data. Because, in the haplotype analyses, one allelic subgroup had less than five subjects.

From the results reported herein, genotyping of these three SNPs could be a useful tool to find out the risk group of patients for GIT prior to commence the EMP therapy in prostate cancer. Finally, using a tailor-made medication by detecting the risk group, the drug compliance of EMP could be increased markedly.

References Cosma G, Crofts F, Taioli E, Toniolo P, and Garte S (1993) Relatationship between genotype and function of the human CYP1A1 gene. J Toxicol Envion Health 40, 309-316.

Crofts F, Taioli E, Trachman J, Cosma GN, Currie D, Toniolo P, and Garte SJ (1994) Functional significance of different human CYP1A1 genotypes. Carcinogenesis 15, 2961-2963.

Gunnarsson PO and Forshell GP (1984) Clinical pharmacokinetics of estramustine phosphate. Urology 23, 22-27.

Kitamura T (2001) Necessity of reevaluation of estramustine phosphate sodium (EMP) as a treatment option for first line monotherapy in advanced prostate cancer. Int J Urol 8, 3336.

Kitamura T, Nishimatsu H, Hamamoto T, Tomita K, Takeuchi T, Ohta N (2002) EMP combination chemotherapy and lowdose monotherapy in advanced prostate cancer. Expert Rev Anticancer Ther 2, 59-71.

Lakhani NJ, Venitz J, Figg WD, and Sparreboom A (2003) Pharmacogenetics of estrogen metabolism and transport in relation to cancer. Curr Drug Metab 4, 505-513.

Oudard S, Banu E, Beuzeboc P, Voog E, Dourthe LM, HardyBessard AC, Linassier C, Scotte F, Banu A, Coscas Y, Guinet F, Poupon MF, and Andrieu JM (2005) Multicenter randomized phase II study of two schedules of docetaxel, estramustine, and prednisone versus mitoxantrone plus prednisone in patients with metastatic hormone-refractory prostate cancer. J Clin Oncol 23, 3343-3351.

Paden CM, McEwen BS, Fishman J, Snyder L, and DeGroff V (1982) Competition by estrogens for catecholamine receptor binding in vitro. J Neurochem 39, 512-520.

Schaeffer JM and Hsueh AJW (1979) 2-Hydroxyestradiol interactions with dopamine receptor binding in rat anterior pituitary. J Biol Chem 254, 5606-5608.

Vaishampayan U, Fontana J, Du W, and Hussain M (2002) An active regimen of weekly paclitaxel and estramustine in metastatic androgen-independent prostate cancer. Urology 60, 1050-1054.

Acknowledgements We would like to give special thanks to A. Hirose and E. Tanaka for their excellent technical assistance. This study was supported by a Grant-in-aid from the Japanese Ministry of Education, Science, Sports and Culture (Project No. 15390485) and a grant from Yamaguchi Endocrine Research Association.

Mohammed Rafiqul Islam Mamun, Motofumi Suzuki, and Tadaichi Kitamura 530

Cancer Therapy Vol 3, page 533 glial (SVGp12) and human umbilical vein endothelial cells (HUVEC-C) were used as normal cell controls. The A-172 MG and HaCat cells were cultured in Dulbecco's Modified Eagle's Minimal Essential Medium (DMEM) with 10% Fetal Bovine Serum (FBS) (Sigma); G48a cells were grown in RPMI 1640, 100 µg/ml L-Cystine (Sigma), 20 µg/ml L-Proline (Sigma), 100 µg/ml Sodium Pyruvate (GIBCO), 1X HT Supplement (GIBCO), 10% FBS; SNB19 cells were cultured in RPMI 1640, 0.1 mM non-essential amino acids (NEAA) (Invitrogen), 100 µg/ml Sodium Pyruvate, 10% FBS; U-251 MG cells were cultured in DMEM, 0.1mM NEAA, 10% FBS; SVGp12 cells were cultured in Eagle's MEM, 0.1 mM NEAA, 100 µg/ml Sodium Pyruvate, 10% FBS; HUVEC cells were cultured in F-12 Kaighn’s, 100µg/ml Heparin (Sigma), 30 µg/ml Endothelial Cell Growth Supplement (ECGS) (Sigma), 10% FBS. Cells were cultured in regular or serum-free medium supplemented with EGF, TNF!, IL4 and TNF! + IL4. Serum-free groups started treatment after 24h serum starvation.

following the outlined protocol, without the addition of primary antibodies.

E. Cell proliferation assay Glioblastoma cells (U-251 MG and G48a) were plated into 96-well culture plates and incubated overnight. Then, 20 or 100 ng/ml of EGF were added to cells and incubated for 24 h at 37 ° C. After the incubation, increasing concentrations of hIL13.

E13K-PE38QQR were added (0.01-100 ng/ml final concentration) and the cells we re incubated for 2 days. The rate of proliferation of the cells was determined by a colorimetric MTS [3-(4, 5-dimethylthiazol-2-yl) -5(3carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt]/PMS (phenazine methosulfate) cell proliferation assay (Promega). The cell samples were incubated with the dye for 2 h and then their absorbance at 490 nm was recorded for each well using a micro-plate reader. The wells with cells treated with high concentrations of cycloheximide served as background for the assay.

B. Western blot analyses

III. Results

For Western blot, cells were seeded onto 100-mm cell culture dishes and allowed to reach 80% confluence. Cultures either in normal or in 24-h serum-starved media were stimulated with EGF, TNF!, IL4 and TNF! +IL4 for 8, 24 and 48 h, then the cells were harvested in RIPA buffer. The cell lysates were electrophoresed on 12 or 15% SDS-PAGE and transferred to polyvinylidene difluo ride memb rane, blocked with 5% blotto in phosphate-buffered saline (PBS/0.05% Tween) for 1 h at room temperature. The polyclonal goat anti-human IL13R-!2 antibody was diluted into blotto and incubated at 4 °C overnight. The membrane was washed three times for 5 min each in 0.05% Tween 20/PBS and was incubated for 1h in 5% blotto containing anti-goat conjugated to horseradish peroxidase. Subsequently, the membrane was washed three times with 0.05% Tween 20/PBS..

The membrane was stripped by 0.5 N NaOH for 15 min, then reprobbed for "-Actin as internal standard. The proteins were detected on the film by enhanced chemiluminescent substrate (ECL) detection system. The density of the immunoreactive bands was measured using “Scion Image Software” from Scion Corporation (Frederick, Maryland).

Using gene expression analysis, radio-receptor binding assays and autoradiography, we found IL13R-!2 to be highly over-expressed in a vast majority of GBM cell lines and tissue specimens (Debinski et al, 1999a, c; Debinski and Gibo, 2000; Mintz et al, 2002). We now demonstrate an immunoreactive IL13R-!2 using Western blotting (Figure 1). The A-172 MG, G48a, SNB-19, U-87 MG and U-251 MG GBM cells demonstrate highly upregulated receptor protein (Figure 1). The only exception among the high-grade glioma cell lines was the T-98G cells, which contain less than 500 IL13 binding sites per cell (Debinski et al, 1995c). In sharp contrast, normal human cells, such as HaCat and HUVEC and the transformed human glial cells, SVGp12 exhibited faint immunoreactive bands of IL13R-!2 (Figure 1).

We thus began analyzing the regulation of IL13R-!2 in glioma cells by monitoring immunoreactive protein.

The serum-starved G48a GBM cells were treated with either 5 or 20 ng/ml of EGF and the addition of EGF caused a time- and dose-dependent increase of IL13R-!2 (Figure 2A). Of interest, the levels of IL13R-!2 in cells treated with EGF were up to five-fold higher than the background levels of the receptor, irrelevant of the serum content in the media (Figure 2A). TNF!, IL4 or their combination also increased the intensity of the IL13R!2 immunoreactive band in cells deprived of serum; however, contrary to the EGF effect, they were unable to elevate IL13R-!2 to its background level detected at normal culture conditions (data not shown).

Next, we examined whether under normal cell culture conditions the levels of IL13R!2 could be further increased in response to various cytokines in G48a cells.

We used higher concentrations of EGF, TNF! and IL4 (100, 50 and 100 ng/ml, respectively), in the presence of serum. We found that EGF was able to further up-regulate IL13R!2 from its basal levels, while TNF! was deprived of such an effect (data not shown) and IL4 was less potent than EGF in elevating the receptor. The combination of TNF! and IL4 did not produce any larger effect from that of TNF! or IL4 alone in G48a cells.

We further analyzed the localization of immunoreactive IL13R-!2 when up-regulated by the cytokines in glioma cells.

We performed

C. EGFR pathway block assay The G48a and U251-MG cells were seeded onto 100-mm dishes and allowed to reach 80% confluence. The cells were pretreated with AG1478 (20 µM), SB203580 (10 µM), Wortmannin (100 ng/ml), PD 98059 (20 µM), SP 600125 (40 µM), and LY 294002 (10 µM) for 1 h, then 100 ng/ml of EGF was added for 5 or 15 min, or 24 h, dependent on the target for the inhibitors. Cell lysates were harvested and analyzed for IL13R!2 expression by Western blot.

D. Immunofluorescence

Expression of IL13R!2 in GBM cells or control cells was assessed by immunofluorescence. Cells were seeded onto 8-well chamber slides and grown to 80% confluency. Following washing twice for 10 min with PBS, cells were fixed in acetone for 2 min at -20 °C. The slides were washed twice 10 min each in PBS and either used immediately or air dried and stored at -80 °C until assayed. The slides were blocked in PBS with 10% (V/V) normal horse serum (NHS) for 30 min. Primary antibody diluted with PBS/1.5% NHS was added to cells for 1 h at room temperature. Cells were washed three times in PBS for 5 min and incubated with the secondary antibody (1:200) and DAPI diluted with PBS/1.5% NHS for 45 min in the dark. After washing the cells three times in PBS for 5 min, a cover-slip was mounted using gel mount (Biomeda). Fluorescent staining was digitally captured with a Zeiss Axiocam using a FITC filter set. Images were obtained from the same experiment at the same exposure.

Non-specific binding of the secondary antibody was examined by

533

Hu et al: Cytokine regulation of IL13R-!2 in malignant glioma cells immunofluorescence in G48a cells under normal serum conditions after treating with either 20 or 100 ng/ml of EGF for various periods of time. We found that 100 ng/ml of EGF produced a large increase in immunoreactive

IL13R!2 that localized mainly to the plasma membrane of G48a cells (Figure 2B).

Figure 1. Western blot of IL13R-!2 in various human GBM and normal (SVGp12, HaCat and HUVEC-C) cell.

Figure 2. A. Immunoreactive IL13R-!2 in G48a GBM cells maintained under serum-free conditions. The cells were treated with EGF for 12, 24 and 36 hr. B. Immunofluorescent staining of IL13R-!2 of human G48a GBM cells in serum-containing media treated with either 20 ng/ml or 100 ng/ml of EGF for 8, 24 and 48 hr.

We subsequently monitored the immunoreactive IL13R-!2 in three other GBM cell lines in order to verify whether the potential regulation of this receptor by various cytokines is a general phenomenon in glioma cells. A-172

MG, SNB-19 and U-251 MG GBM cells were examined under serum-free and normal culture conditions and treated the same way as the G48a cells with EGF, TNF!, IL4 or the combination of the latter. In the A-172 MG

534

Cancer Therapy Vol 3, page 535 cells, serum starvation caused a substantial decrease in IL13R-!2, but EGF raised the levels of the receptor even above its basal levels, as found in G48a cells (Figure 3A).

Both TNF! and IL4 had a more prominent effect on IL13R-!2 in the A-172 MG cells than that seen in serumfree G48a cells (data not shown). EGF upregulated IL13R!2 in A-172 MG cells, in serum containing media (Figure 3B). TNF! and IL4 and their combination were as effective as EGF alone, or more effective, in these glioma cells, but at different time intervals (Figure 3C). The newly induced IL13R!2 was detected readily by

immunofluorescence and localized to the cell membranes (data not shown).

Similar responses to the cytokines were seen in the U-251 MG cells, which were good responders to EGF, but not the other cytokines (Figure 4A). EGF caused IL13R!2 to increase under both serum-free and normal serum conditions.

Immunofluorescent IL13R-!2 also prominently increased in response to the EGF treatment in the U-251 MG cells under normal serum conditions. The biggest increase was found 24 hr post EGF addition and IL13R-!2 localized primarily to the plasma membrane of these cells, as was seen with G48a cells (Figure 4B).

Figure 3. A. Immunoreactive IL13R-!2 in A-172 MG GBM cells maintained under serum-free conditions. The cells were treated with EGF for 12, 24 and 36 hr. B. Immunoreactive IL13R-!2 in Western blot performed on lysates from cells maintained in normal media.

The A-172 MG cells were treated with EGF (100 ng/ml) or TNF ! (50 ng/ml). C. A-172 MG cells were treated with TNF! + IL4 or IL4 (100 ng/ml) . S+, serum containing media; S-, no serum.

535

Hu et al: Cytokine regulation of IL13R-!2 in malignant glioma cells

Figure 4. A. Immunoreactive IL13R-!2 in U-251 MG GBM cells maintained under serum-free conditions. The cells were treated with EGF for 12, 24 and 36 hr. B. Immunofluorescence of IL13R!2 staining of human U-251 MG GBM cells in serum-containing media treated with either 20 ng/ml or 100 ng/ml of EGF.

In addition, we used a glioma cell line, T-98G expressing low number of the restricted binding sites for IL13 when compared with other GBM cells (Figure 1).

We examined the response of T-98G cells to the cytokines similarly to the four other GBM cell lines over-expressing IL13R-!2. The levels of immunoreactive IL13R-!2 remained low independent of the cytokine added and culture conditions used (data not shown).

We next examined how IL13R-!2 responds to the cytokines in normal human cells. IL13R-!2 gene expression is poor in normal organs and on average normal cells contain little if any of this receptor protein (Debinski and Gibo, 2000; Debinski et al, 1995c). We used normal human endothelial cells, HUVEC-C and immortalized normal human glial cells, SVGp12 that show minute amounts of immunoreactive IL13R-!2 when compared with the studied GBM cell lines (Figure 1).

Treatment with the cytokines (EGF, TNF! or IL4) caused sporadic changes in the levels of the receptor, but they were minimal and of magnitudes lower when compared with the background levels of IL13R-!2 in GBM cells, such as G48a (Figure 5A, HUVEC and Figure 5B, SVGp12 cells; EGF and TNF! treatment shown).

Being that EGF was the most potent cytokine in the induction of IL13R-!2 in GBM cells, we attempted to document (i) a direct involvement of the EGFR receptor in

transducing the signal for the IL13 receptor expression and (ii) which of the major pathways of EGFR stimulation that are operational in cancer cells is responsible for this phenomenon. We thus employed three different inhibitors of the EGFR signaling pathway in GBM cell lines. The EGF tyrosine kinase inhibitor, AG1478, either had a minimal effect on the basal levels of IL13R-!2 (G48a cells) or significantly reduced the levels of the receptor under baseline conditions (U-251 MG cells) (Figure 6A).

However, AG1478 prevented completely an up-regulation of the receptor in response to EGF in all these cells (Figure 6A and data not shown). This directly demonstrates that the activation of the EGFR is indeed transducing stimulatory signals for the IL13R-!2 expression in GBM cells. These stimulatory signals can be carried out by either PI3-K or MAPK in cancer cells (Cuadrado et al, 2003). SB203580, an inhibitor of P38 MAPK changed moderately the basal levels of IL13R-!2 in glioma cells, but contrary to AG1478, it did not prevent an increase in IL13R-!2 in response to EGF (Figure 6A).

On the other hand, an inhibitor of PI3-K, Wortmannin, exerted a similar to AG1478, but to a smaller extent, effect in neutralizing EGF-induced up-regulation of IL13R-!2 in GBM cells (Figure 6A).

536

Cancer Therapy Vol 3, page 537

Figure 5. A. Immunoreactive IL13R-!2 in HUVEC-C cells maintained in normal media. The cells, were treated with EGF (100 ng/ml) or TNF! (50 ng/ml) for 8, 24 and 48 hr. B. Immunoreactive IL13R-!2 in Western blot performed on lysates from SVGp12 cells maintained in serum-free media. The cells were treated with EGF (20 ng/ml) and TNF! (5 ng/ml) for 8, 24 and 48 hr. S+, serum containing media; S-, no serum. The lysate of G48a GBM cells was loaded for comparison.

Figure 6. A. The effect of inhibitors of the EGFR and its signaling on immunoreactive IL13R-!2 in U-251 MG cells. Cells were pretreated with AG1478 (10 µM), SB203580 (10 µM) and Wortmannin (100 ng/ml) 1h before the addition of EGF (100 ng/ml) for 24 hr. B. Cells were pre-treated with inhibitors of TK (AG 1478; 20 µM), ERK (PD 98059; 20 µM), JNK (SP 600125; 40 µM), p38 (SB 203580; 10 µM) and PI3-K (LY 294002; 10 µM) inhibitors 1h before the addition of EGF (100 ng/ml) for 24 hr.

537

Hu et al: Cytokine regulation of IL13R-!2 in malignant glioma cells In a further attempt to dissect more precisely which intracellular signaling pathways take part in the IL13R-!2 up-regulation in response to EGF, we treated the U-251 MG cells with 100 ng/ml EGF in the presence of TK, ERK, JNK, p38 and PI3-K inhibitors, and measured immunoreactive IL13R-!2 by Western blot. In these experiments, we used more specific inhibitors of PI3-K (LY 294002), JNK (SP 600125) and ERK (PD 98059). We found that an increase in immunoreactive IL13R-!2 due to the treatment with EGF was best prevented by the TK, PI3-K and ERK inhibitors (Figure 6B). In order to demonstrate that PD 98059 blocked its target activation, we measured phosphorylated ERK in the presence or absence of the inhibitor (Figure 7A). Phosphorylated ERK increased by more than forty times after 5 and 15 min of

treatment with EGF and PD 98059 prevented the rise in activated ERK almost completely in the U-251 MG cells (Figure 7A). Furthermore, in order to document that the PI3-K inhibitor, LY 294002, exerted its specific function in GBM cells, we measured phosphorylated AKT and observed a potent inhibition of both baseline and EGFinduced levels of the activated AKT (Figure 7B).

IL13R-!2 is a molecular target for IL13-based recombinant cytotoxin candidate drugs (Debinski et al, 1999a). Even though it is over-expressed in GBM cells, its levels can be further increased by PI3-K or ERK pathways activation (Figure 6A and 6B). We have thus tested whether this increase in IL13R-!2 may lead to further sensitization of GBM cells toward IL13-based cytotoxins.

Figure 7. A. The effect of an ERK inhibitor (PD98059) on EGF-induced phosphorylated ERK (P-ERK) and immunoreactive ERK proteins (T-ERK) in U-251 MG cells. Cells were pre-treated with PD98059 (10 ÂľM) for 1 h before the addition of EGF (100 ng/ml) for 5 or 15 min. A quantitation of the density of the immunoreactive bands is shown for P-ERK. B. The effect of PI3-K inhibitor ( ) on EGFinduced immunoreactive phosphorylated AKT (P-AKT) and immunoreactive AKT protein (T-AKT) in U-251 MG cells. Cells were pretreated with LY 294002 (10 ÂľM) for 1 h before the addition of EGF (100 ng/ml) for 5 min or 24 h.

Thus, the G48a and U-251 MG glioma cells that over-

express IL13R-!2 were pre-treated with EGF (20 or100 538

Cancer Therapy Vol 3, page 539 ng/ml) for 24 h before the addition of IL13.E13KPE38QQR. The pre-treatment with EGF made the G48a and U-251 MG cells more susceptible to the cytotoxin; which was seen more pronounced at a higher concentration of EGF (Figure 8A). The cytotoxin could achieve its IC50 even at two logs lower concentration when cells were pre-treated with EGF compared to not pretreated cells. However, when we used T-98G GBM cells that do not over-express IL13R-!2 and are non-responders to IL13.E13K-PE38QQR, pre-treatment with EGF did not result in making these cells any more susceptible to the cytotoxin (Figure 8B). The same was observed with mouse G-26 V2 glioma cells transfected with an empty vector (Figure 8B) (Mintz et al, 2003). Also, mouse G-26 H2 glioma cells that carry the IL13R-!2 transgene (Mintz

et al, 2003) did not become more responsive to the cytotoxin when pre-treated with EGF (Figure 8B). In a final set of experiments, we examined whether EGF alone has any influence on the glioma cells proliferation under the conditions shown in Figure 8A and B. We found no significant effect of EGF (up to 100 ng/ml) on either human or mouse glioma cells (Figure 8C).

IV. Discussion

We have previously found that IL13R-!2 is up-regulated in GBM cells (Debinski et al, 1999a; Mintz et al, 2002). In the current work, we have found that the level of protein significantly decreases in some serum-starved GBM cells.

However, the protein can be brought back to

Figure 8. A. The effect of EGFR suprastimulation on the responsiveness of GBM cells to an IL13.E13K cytotoxin (IL13.E13K-PE38QQR). The G48a and U-251 MG cells were treated with EGF (20 or 100 ng/ml) 24 hr prior to the addition of recombinant cytotoxin. B. The effect of EGFR supra-stimulation on the responsiveness of GBM cells to IL13.E13K-PE38QQR in T-98G, low IL13R-!2 levels expressing GBM cells, G-26 mouse glioma cells not expressing IL13R-!2 (G-26 V2) or expressing a transgene of human IL13R-!2 (G-26 H2). The cells were pre-treated with EGF prior to the addition of recombinant cytotoxin as in A. C. The effect of EGFR supra-stimulation on proliferation of G48a, SNB-19, and U-251 MG GBM cells and that of G-26 mouse glioma cells with (H2) or without human IL13R!2 (V2).

the original levels, or even higher, by treating cells with

AP-1 and/or STAT-6 activators. EGF, by activating also

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Hu et al: Cytokine regulation of IL13R-!2 in malignant glioma cells the AP-1 pathway, among others, was the most potent in inducing this phenomenon, while TNF! and IL4 demonstrate variable ability to further up-regulate IL13R!2 in glioma cells. Cytokine-induced IL13R-!2 immunoreactivity localizes primarily to cell membranes, i.e. the receptor is produced and placed into the plasma membrane compartment of glioma cells. Under normal cell culture conditions, supra-physiologic amounts of EGF and that of TNF!/IL4 evoked even further increase in an already elevated immunoreactive IL13R-!2. This indicates that the IL13R-!2 is expressed in a nonconstitutive manner in glioma tumor cells. We have also demonstrated that an increase in IL13R-!2 in response to EGF is mediated through the activation of the EGFR signaling pathways. In addition, we have documented that the PI3-K and ERK pathways, rather than P38 and JNK MAPK pathways, of EGFR activation appears to take part in regulating the restricted IL13 receptor in glioma cells.

Finally, glioma cells pre-treated with EGF became more susceptible to the killing by an IL13 mutant-based recombinant anti-cancer cytotoxin. Thus, IL13R-!2 can be regulated in brain tumor cells and this can be potentially utilized to increase the responsiveness of cancer cells to anti-cancer drug candidates.

EGF was found to be the most potent among the studied cytokines in up-regulating IL13R-!2 in glioma cells. EGF activates the AP-1 pathway potently in glioma cells (Debinski et al, 2001; Debinski and Gibo, 2005).

This action is mediated through the EGF receptor tyrosine kinase first and we demonstrated that blocking this step by using AG1478 EGFR kinase inhibitor prevents EGF from up-regulating IL13R-!2. There are at least two major pathways for EGFR signaling in cancer cells that involve PI3-K and MAPK (Cuadrado et al, 2003). Using respective inhibitors of these routes of signaling, we found that the PI3-K and ERK pathways appear to be more important for EGF-induced IL13R-!2 up-regulation than the MAPK pathway. In support of this contention others found that P38 and JNK are EGF signaling-independent MAPK (Dong et al, 2004). We are continuing the search for other factors related to the PI3-K and ERK that might be important for IL13R-!2 expression in glioma cells.

Recombinant anti-cancer cytotoxin killing potency is roughly proportionate to the number of targeted receptors expressed on cells (Debinski et al, 1995c; Mintz et al, 2003). It would be thus desirable to increase the expression levels of IL13R-!2 on GBM cells, in order to make them even more susceptible to the killing by the IL13 cytotoxins and/or making more GBM cells responsive to the cytotoxin in general. Our current work documents that STAT-6 and AP-1 activators influence the levels of IL13R-!2 expression in glioma cells. This increase in immunoreactive protein levels is most likely responsible for an increased sensitivity of glioma cells to the killing by the recombinant IL13-based cytotoxin that we observed. This phenomenon is specific to the presence of endogenous regulated IL13R-!2 in cells and it requires that the receptor is over-expressed to start with. This finding may offer an attractive approach in which a pretreatment with the compounds of neutral or even anticancer activity on their own would lead to an upregulation of IL13R-!2 and subsequent better efficacy of the IL13 cytotoxins. Being that normal cells which have

little if any of IL13R-!2 do not respond to various cytokines by up-regulating the receptor not even near to the levels found in GBM cells, this approach further delineated the specificity of the use of recombinant cytotoxins as anti-cancer drugs.

Recent intense interest in the inhibitors of the EGFR in cancer treatment (Yang et al, 2004) would prompt one to use such inhibitors in combination with the IL13-based cytotoxins. Unfortunately, our study suggests that by inhibiting the signaling through the EGFR the expression of IL13R-!2, a target for the recombinant cytotoxins, is significantly diminished. Thus, less of a target is available for the binding of targeted cytotoxins. However, novel anti-cancer drugs have been recently identified that appear to work through a supra-stimulation of the EGFR leading to cancer cell death (Cuadrado et al, 2003) or they are superagonist of the EGFR themselves (Monticello, 2003).

It is plausible that such a drug(s) would amplify the efficacy of the IL13R-!2 targeted recombinant cytotoxins through further up-regulation of the receptor in GBM cells.

In summary, IL13R-!2, a glioma-associated receptor is expressed non-constitutively in glioma cell; its levels can be further up-regulated by growth factors/ cytokines.

The PI3-K and ERK pathways appear to be primarily involved in the regulation of IL13R-!2 in glioma cells. A possibility of regulation of the receptor levels in cancer cells may offer a therapeutic advantage while using IL13R-!2-targeted agents.

Acknowledgements

This work was supported by the NIH/NCI grant R01 CA 74145.

References

Aman MJ, Tayebi N, Obiri NI, Puri RK, Modi WS and Leonard WJ (1996) cDNA cloning and characterization of the human interleukin 13 receptor ! chain. J Biol Chem 271, 2926529270.

Cuadrado A, Garcia-Fenrandez LF, Gozales L, Suarez Y, Losada A, Alcaide V, Martinez, T, Fernandez-Sousa JM, SancehzPuelles JM and Munoz A (2003) Aplidin induces apoptosis in human cancer cells via glutathione depletion and sustained activation of the epidermal growth factor receptor, Src, JNK and p38 MAPK. J Biol Chem 278, 241-250.

David MD, Bertoglio J and Pierre J (2003) Functional characterization of IL13 receptor a2 gene promoter: a critical role of the transcription factor STAT6 for regulated expression. Oncogene 22, 3386-3394.

Debinski W and Gibo DM (2000) Molecular expression analysis of restrictive receptor for interleukin 13, a brain tumorassociated cancer/testis antigen. Mol Med 6, 440-449.

Debinski W and Gibo DM (2005) Fra-1 modulates malignant features of glioma cells. Mol Cancer Res 3, 237-249.

Debinski W, Gibo DM and Mintz A (2003) Epigenetics in highgrade astrocytomas: Opportunities for prevention and detection of brain tumors. Ann NY Acad Sci 983, 232-242.

Debinski W, Gibo DM, Hulet SW, Connor JR and Gillespie GY (1999a) Receptor for interleukin 13 is a marker and therapeutic target for human high grade gliomas. Clin Cancer Res 5, 985-990.

Debinski W, Obiri NI, Pastan I and Puri RK (1995b) A novel chimeric protein composed of interleukin 13 and Pseudomonas exotoxin is highly cytotoxic to human

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Cytokine up-regulation of IL13R-!2 in GBM cells leads to an increased potency of recombinant IL13 Cytotoxin Research Article

Nianping Hu, Denise M. Gibo and Waldemar Debinski* The Brain Tumor Center of Excellence, Wake Forest University School of Medicine Comprehensive Cancer Center, Departments of Neurosurgery, Radiation Oncology and Cancer Biology

__________________________________________________________________________________ *Correspondence: Waldemar Debinski, M.D, Ph.D., Director of Brain Tumor Center of Excellence, Comprehensive Cancer Center of Wake Forest University, Departments of Neurosurgery, Radiation Oncology and Cancer Biology, Wake Forest University School of Medicine, Medical Center Boulevard Winston-Salem, NC 27157; Phone: (336) 716-9712; Fax: (336) 713-7639; E-mail: debinski@wfubmc.edu Key words: Interleukin 13 receptor !2, Cytotoxin, EGFR signaling, Gliomas, Immunofluorescence Abbreviations: activating protein-1, (AP-1); protein kinase B (AKT); American Type Culture Collection, (ATCC); cancer/testes tumor antigen, (CTA); Dulbecco's Modified Eagle's Minimal Essential Medium, (DMEM); epidermal growth factor (EGF); enhanced chemiluminescent substrate, (ECL); extracellular signal-regulated kinase (ERK); Fetal Bovine Serum, (FBS); Fos-related antigen 1, (Fra-1); glioblastoma multiforme, (GBM); human endothelial cells, (HUVEC-C); interleukin 13, (IL13); c-Jun N-terminal kinase (JNK); mitogen-activated protein kinase (MAPK); non-essential amino acids, (NEAA); normal horse serum, (NHS); phosphatidylinositol-3kinase (PI3-K); phenazine methosulfate, (PMS); tumor necrosis factor !, (TNF!); tyrosine kinase, (TK) Received: 23 May 2005; Revised: 27 September 2005 Accepted: 26 October 2005; electronically published: November 2005

Summary We documented that a receptor for interleukin13, IL13R-!2, is an attractive target for glioma (brain tumor) molecular therapies. Little is known whether and how the expression of this receptor is regulated in glioma cells.

Recent studies suggested that this receptor might be under AP-11 and STAT-6 control. Thus, IL13R-!2 was examined in a variety of cancer and normal cells that had been activated by AP-1 and STAT-6 stimulatory cytokines. We found that serum-starved glioma cell lines had the levels of IL13R-!2 significantly diminished. The addition of EGF, TNF! or IL4 increased IL13R-!2, the extent of which was cytokine-, cell line- and timedependent. We also found that primarily EGF could further increase the levels of the receptor in glioma cells under normal conditions. As expected, IL13R-!2 was found low in normal cells when compared with brain tumor cells and neither AP-1 nor STAT-6 activation produced any large increase in the receptor levels in those cells. Blocking the PI3-K and ERK pathways, but not that of P38 and JNK MAPKs, resulted in the neutralization of EGF effects on IL13R-!2. Furthermore, EGF pre-treatment caused higher sensitivity of glioma cells to cell killing with very low doses of IL13-based recombinant cytotoxin, which was not found in cells with either low levels or transgene-induced expression of IL13R-!2. EGF alone did not have any significant effect on GBM cells proliferation. Thus, the expression of IL13R-!2 appears to be non-constitutive. PI3-K and ERK play important roles in activating mechanisms responsible for the EGF-dependent expression of the receptor. Also, up-regulating the levels of IL13R!2 produces higher susceptibility to the killing by a recombinant cytotoxin molecularly targeted to IL13R-!2.

restricted from the one found in many vital organs; the latter is shared between IL13 and IL4. A monomeric plasma membrane protein, IL13R-!2, has been identified as a molecular entity responsible for the restricted binding of IL13 to GBM (Debinski et al, 1995c; Debinski and Gibo, 2000; Mintz and Debinski, 2000; Mintz et al, 2002).

On the other hand, the physiological form of the IL13R is

I. Introduction

We have demonstrated that a vast majority of patients with glioblastoma multiforme (GBM), high-grade glioma (grade IV), over-express binding sites for interleukin 13 (IL13) (Debinski et al, 1999a, 2000). In principle, the measurable binding of IL13 to GBM is independent of its homologue, IL4 and thus, more 531

Hu et al: Cytokine regulation of IL13R-!2 in malignant glioma cells a 140-kDa chain of the IL4R, termed IL4R!, which dimerizes with a 45-50 kDa IL13-responsive subunit (Zurawski et al, 1993, 1995; Vita et al, 1995; Hilton et al, 1996; Miloux et al, 1997; Kelly-Welch et al, 2003). The IL13 binding protein of the IL13/4R, termed IL13R-!1, binds IL13 at a low affinity (Hilton et al, 1996). Only in the presence of IL4R! does the 140-kDa IL4 binding protein site become one of high affinity (Kd~ 30 pM) (Aman et al, 1996; Hilton et al, 1996; Miloux et al, 1997).

We have characterized IL13R!2 as a cancer/testes tumor antigen (CTA)-like factor (Debinski and Gibo, 2000). This restricted receptor is one of the first three factors ever documented to be expressed in a majority of patients with GBM and not in normal brain (Murphy et al, 1995; Rich et al, 1996). This finding has marked the start of uncovering molecular denominators in a disease as heterogeneous as GBM. Our laboratory conceived and generated IL13-based, bacterial toxin-containing recombinant cytotoxic chimera fusion proteins that have been shown to be very potent anti-glioma cytotoxins in vitro and in vivo (Debinski et al, 1999a, c). The first generation of IL13-based cytotoxin, hIL13-PE38QQR that was produced more than a decade ago, entered Phase III clinical trials in 2004. hIL13PE38QQR was generated as a counterpart to an IL4-based cytotoxin without intent of targeting brain tumors (Debinski et al, 1995b) and it does not have the inherent specificity for the targeting of IL13R!2 while sparing the normal tissue receptor shared with IL4. However, several therapeutic approaches targeting specifically IL13R-!2 are being developed: vaccines (Mintz et al, 2002; Okano et al, 2002), targeted viruses (Zhou et al, 2002), re-targeted cytotoxic T cells (Kahlon et al, 2004) and new IL13-based cytotoxins (Mintz et al, 2003). Thus, IL13R!2 is a truly attractive molecular target in the treatment of brain cancer.

We have been intrigued by the fact that the IL13R!2 gene resides on Chromosome X and the fact that numerous cancer/testes tumor antigen have their genes on the same chromosome. We have discussed hypothetical implications for the presence of IL13R-!2 in malignancy in the context of X-linkage and suggested that a significant component responsible for the restricted IL13 receptor over-expression in GBM might be the DNA methylation status and subsequent aberrant gene expression regulation (Mintz and Debinski, 2000). Epigenetic mechanisms have been implicated previously in the appearance of other CTAs (Lethe et al, 1998). In support of this contention, other investigators demonstrated an 8-fold increase in the expression of IL13R-!2 in bladder cancer cells in response to a DNA de-methylating agent, 5-aza-2â&#x20AC;&#x2122;deoxycytidine; this was not seen in the corresponding normal cells (Liang et al, 2002). We treated GBM cells with 5-aza-2â&#x20AC;&#x2122;-deoxycytidine and found a 3-fold increase in IL13R-!2 gene expression against the background of already elevated levels of the receptor in these cells (Debinski et al, 2003). Thus, in two independent studies the IL13R-!2 expression has been increased in response to a DNA de-methylating agent. Our study in GBM cells also suggested that the expression of IL13R-!2 might not be constitutive.

We have outlined several lines of evidence suggestive of epigenetic involvement in the up-regulation of activating protein-1 (AP-1) activity (Debinski et al,

2003) and we have concomitantly documented an increase in AP-1 in gliomas (Debinski et al, 2001; Debinski and Gibo, 2005). For example, a Fos-related antigen 1 (Fra-1), an AP-1 factor is highly up-regulated in GBM (Debinski et al, 2001; Debinski and Gibo, 2005). Accordingly, recent data demonstrate that the IL13R-!2 gene promoter region contains an AP-1 binding site that might be important for receptor expression (Wu et al, 2003). The promoter region of the IL13R-!2 gene, besides containing an AP-1 binding site, has putative binding sites for the STAT6, NFkB and C/EBPb transcription factors (David et al, 2003). IL4 and IL13 increased IL13R-!2 at both gene and protein levels (David et al, 2003). Tumor necrosis factor ! (TNF!), when combined with either IL13 or IL4, synergized upregulation of IL13R-!2 in one cell type (David et al, 2003).

Initially, we have demonstrated the restricted binding of IL13 to the HGG in in vitro and in situ by using autoradiography with iodo-labeled IL13 as a ligand for the receptor (Debinski et al, 1999a, 2000). The first commercially available antibody against IL13R!2 works only in cell sorting assays (Mintz et al, 2002). We were able to produce limited amounts of a polyclonal antibody against IL13R-!2 that showed positive staining of GBM tissue specimens (Mintz et al, 2002). Recently, antibodies have become available that perform in Western blots and immunocytochemistry. The results by others confirmed the presence and prevalence of IL13R!2 in GBM specimens to a degree similar to that seen with autoradiography (Liu et al, 2003). Being that AP-1 and STAT-6 appear to be involved in IL13R-!2 regulation in some cells (David et al, 2003), we examined the regulation of immunoreactive IL13R-!2 expression in brain tumor cells.

II. Materials and Methods

A-172 MG, SNB-19, U-251 MG, normal human endothelial cells (HUVEC-C) and SVGp12 transformed normal glial cell lines were obtained from the American Type Culture Collection (ATCC) (Rockville, MD), G48a cell line was isolated in our laboratory (Debinski and Gibo, 2005), HaCat cells were kindly provided by Dr. Abby Maizel. Media were obtained f rom Invitrogen. Tissue culture ware was from Corning Glass (Corning, NY). SDS-PAGE supplies were from Bio-Rad. Antibodies, including anti-IL13R-!2 antibody, EGF and TNF! were purchased from the R & D System, Inc (Minneapolis, MN).

Antibodies against ERK, phosphorylated ERK, JNK, phosphorylated JNK, p38, phosphorylated p38, AKT and phosphorylated AKT, were purchased from Cell Signaling (Beverly, MA). IL4 was produced by our laboratory. EGFR tyrosine kinase inhibitor (AG1478), MAPK inhibitor (SB203580), and PI-3K inhibitor (Wortmannin) were purchased from Calbiochem (La Jolla, CA). The inhibitors of ERK (PD 98059) and PI3-K (LY 294002) were obtained from Sigma (St. Louis, MO). SP 600125 inhibitor of JNK was purchased from A.G. Scientific, Inc. (San Diego, CA). SuperSignal Substrate ECL for chemiluminescent detection was purchased from Amersham Biotech. Chamber slides were purchased from Nalge Nunc (Naperville, IL) and Alexa Fluor 488 donkey anti-goat IgG was from Molecular Probes Inc (Eugene, OR). DAPI was from Sigma. Recombinant IL13.E13K-PE38QQR was produced and purified in our laboratory (Mintz et al, 2003).

A. Cell culture Several GBM cell lines, such as A-172 MG, G48a, Snb19 and U-251 MG were grown in the appropriate media. Normal

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17!-hydroxysteroid dehydrogenases and breast cancer Review Article

Pirkko Vihko1,2 and Veli Isomaa1 1

Research Center for Molecular Endocrinology and WHO Collaborating Centre, Biocenter Oulu, P.O. Box 5000, FI-90014 University of Oulu, Finland 2 Department of Environmental Sciences, Division of Biochemistry, FI-00014 University of Helsinki, Finland

__________________________________________________________________________________ *Correspondence: Professor Pirkko Vihko, M.D., Ph.D., Research Center for Molecular Endocrinology and WHO Collaborating Centre, Biocenter Oulu, P.O. Box 5000, FI-90014 University of Oulu, Finland; Tel: +358-40-5431734; FAX: +358-8-3155631; e-mail: pirkko.vihko@oulu.fi Key words: 17!-hydroxysteroid dehydrogenases, breast cancer, hormonal treatment Abbreviations: 17!-hydroxysteroid dehydrogenases, (17HSDs); dihydrotestosterone, (DHT); estrogen receptor ", (ER"); estrogen receptor !, (ER!); progesterone receptor, (PR); Tumor-Node-Metastasis, (TNM) Received: 9 August 2005; Accepted: 20 October 2005; electronically published: October 2005

Summary Experimental data suggest that ovarian sex steroids, particularly estrogens but also progesterone, have important roles in the development of breast cancer. The biological activity of female sex steroids in target tissues such as the breast is regulated by several enzymes, including 17!-hydroxysteroid dehydrogenases (17HSDs). Changes in the expression patterns of these enzymes may significantly modulate the intracellular steroid hormone activity and concentration and, therefore, play a pathophysiological role in malignant transformation. Estrogen influence is one important focus of breast cancer therapies. We have previously shown that estradiol-producing 17HSD type 1 and estradiol-inactivating type 2 are present in normal breast tissue, breast cancer and breast cancer cell lines. To further clarify the role of 17HSDs in breast cancer, we recently analyzed the mRNA expressions of the 17HSD type 1, 2 and 5 enzymes in 794 breast carcinoma specimens. Both 17HSD type 1 and 2 mRNAs were detected in normal breast tissue from premenopausal women but not in specimens from postmenopausal women. 17HSD type 5 mRNA was present in the normal breast of both pre- and postmenopausal women. Of the breast cancer specimens, 16 % showed signals for 17HSD type 1 mRNA, 25 % for type 2 and 65 % for type 5. No association between the 17HSD type 1, type 2 and type 5 expressions was detected. The patients with tumors expressing 17HSD type 1 mRNA or protein had significantly shorter overall and disease-free survival than the other patients. The expression of 17HSD type 5 was significantly higher in breast tumor specimens than in normal tissue. Cox multivariate analyses showed that tumor size, 17HSD type 1 mRNA and estrogen receptor " (ER") had independent prognostic significance. Our data show that 17HSD type 1 expression is associated with a poor prognosis and support the idea that inhibition of this enzyme could be a beneficial therapy for breast cancer patients. non-pregnant breast epithelial proliferation is maximal about one week after ovulation (Anderson et al, 1982). At this stage of the menstrual cycle both estradiol and progesterone concentrations are high. Further, both estrogen and progesterone increase the proliferation of breast cancer cells in in vitro assays (Lippman et al, 1976; Cullen and Lippman 1989; Musgrove et al, 1991).

In premenopausal women, the ovary is the main source of estrogens, but estrogenic steroids are also formed in peripheral tissues. After menopause, all estrogens are formed locally. The peripheral tissues are not

I. Introduction Breast cancer is the most common malignant disease in the western countries and its incidence is increasing (Greenlee et al, 2001). Exposure to elevated concentrations of estrogens is associated with the etiology of this hormone-associated cancer. Epidemiological and endocrine evidence indicates that estrogens have a proliferative effect on breast epithelium, but the exact mechanism in the development and progression of breast cancer is not known. The apparent role of progesterone in the control of the cell cycle is not known, but in adult, 543

Vihko and Isomaa: 17!-hydroxysteroid dehydrogenases and breast cancer capable of performing de novo steroid synthesis but contain the enzymes needed for the formation of active androgens and estrogens form adrenal-derived C19 steroids, such as dehydroepiandrosterone, its sulphate and androstenedione, which are abundantly secreted into the circulation in humans (Labrie et al, 1997). These steroids serve as substrates in peripheral tissues and locally produced active steroids exert their action in the intracrine manner on the same cell in which they are synthesized without diffusion into the circulation and, therefore, effectively regulate sex steroid influence at the prereceptor level in the target cells. The enzymes modulating sex steroid metabolism and, consequently, the concentration of active steroids in peripheral tissues include steroid sulphatases, 3!-hydroxysteroid dehydrogenases, 3"-hydroxysteroid dehydrogenases, aromatase, 17!-hydroxysteroid dehydrogenases and 5"reductases.

We have focused our studies on 17!-hydroxysteroid dehydrogenases (17HSDs), which regulate the biological activity of sex steroid hormones by catalyzing the interconversions between highly active steroid hormones, e.g. estradiol and testosterone and corresponding less active hormones, estrone and androstenedione (Figure 1).

Up till now, at least nine different 17HSD isoenzymes, namely the types 1-5, 7-8 and 10-11 (Peltoketo et al, 2003; Mindnich et al, 2004), have been characterized in humans.

In intact cells, the types 1, 3, 5 and 7 mainly catalyze reductive reactions, whereas the other types are considered as oxidative enzymes. The presence of a series of 17HSD enzymes with cell-specific expression patterns and differential substrate specificities point to the important role of 17HSDs in intracrine steroid formation.

Human 17HSD type 1 catalyzes the reduction of estrone to estradiol (Puranen et al, 1997), preferring the phosphorylated form of nicotinamide-adenine dinucleotide, NADPH, as a cofactor. In cultured cells, the human type 1 enzyme is also capable of reducing androstenedione and 5"-androstanedione to some extent, but it clearly gives preference to phenolic substrates over androgens. 17HSD type 1 is essential for estradiol production and it is most abundantly expressed in the granulosa cells of the ovary (Ghersevich et al, 1994; Sawetawan et al, 1994) and the syncytiotrophoblasts of the placenta (Fournet-Dulguerov et al, 1987; M채entausta et al, 1990), which secrete estradiol into the circulation. In addition, the type 1 enzyme contributes to the estrogen response by converting estrone to estradiol locally in certain targets of estrogen action, such as normal and malignant breast tissue (Poutanen et al, 1995; Miettinen et al, 1996a).

17HSD type 2 is involved in the inactivation of estradiol, testosterone and dihydrotestosterone (DHT) and the activation of progesterone. 17HSD type 2 is expressed in a wide variety of tissues, such as breast, uterus, prostate, placenta, liver, intestine and kidney (Peltoketo et al, 1999; 2003). Typically, the type 2 enzyme is expressed in epithelial cells, such as the surface epithelial cells of the gastrointestinal and urinary tracts (Mustonen et al, 1998a; Oduwole et al, 2003). The type 2 enzyme may restrict the access of active sex steroids into the circulation and it may protect the target tissues of hormonal action against excessive sex hormone influence by catalyzing the conversion of androgens and estrogens into less active forms. In the placenta, the type 2 enzyme may limit the access of fetal androgens into the maternal tissue and the access of maternal estrogen into the fetus, acting as a barrier between the fetus and the mother (Mustonen et al, 1998b, Li et al, 2005).

II. Enzymatic characteristics of human 17HSD type 1, type 2, type 5 and type 7

Figure 1. The key reactions in the metabolism of estrogens and androgens.

A-dione = androstenedione; 17HSD1, 2 etc. refer to different 17HSD-enzymes; P450arom = P450 aromatase enzyme.

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Cancer Therapy Vol 3, page 545 Unlike other 17HSDs, which belong to the shortchain dehydrogenase/reductase superfamily, 17HSD type 5, a reductive 17HSD, is a member of the aldo-keto reductase superfamily (Dufort et al, 1999; Luu-The et al, 2001). 17HSD5 has a broad tissue distribution and it recognizes several different substrates in in vitro assays (Luu-The et al, 2001; Penning et al, 2001). 17HSD5 has some activity as a reductase of 3-keto and 17-keto and as an oxidase of 3"- and 17!-hydroxysteroids (Penning et al, 2001). Human 17HSD5 also has a high level of 20"hydroxysteroid dehydrogenase activity, which inactivates progesterone to 20"-OH-progesterone (Luu-The et al, 2001). In myeloid leukemia cell lines, this enzyme possesses marked 11-ketoreductase activity, converting prostaglandin D2 to PGF2" and functions to regulate cell differentiation (Desmond et al, 2003). So far, little is known about the role of 17HSD5 in breast tissue, but it may be involved in the metabolism of female sex steroids.

In the prostate, 17HSD5 catalyzes the formation of testosterone and the inactivation of DHT (Dufort et al, 1999).

Human 17HSD type 7 is a membrane-associated reductive enzyme converting estrone to estradiol and DHT to an estrogenic metabolite, 5"-androstane-3!, 17!-diol, thereby catalyzing the reduction of the keto group in either the 17- or the 3-position of the substrate. Minor 3!-HSDlike activity towards progesterone and 20"hydroxyprogesterone, leading to inactivation of progesterone by 17HSD type 7, has also been detected (Tรถrn et al, 2003). Human 17HSD type 7 is expressed in steroidogenic cells and several peripheral tissues, such as liver, lung and thymus. Its function is not known, but it may be responsible for the local production of estrogenic metabolites in peripheral tissue (Tรถrn et al, 2003). 17HSD type 7 has also other substrates apart from sex steroids (Breitling et al, 2001). It was shown that the enzyme acts as a 3-ketosteroid reductase in cholesterol biosynthesis, converting zymosterone to zymosterol (Marijanovic et al, 2003).

etiology (Vihko and Apter 1989). Premature loss of ovarian function greatly reduces the breast cancer risk, supporting the idea that ovarian hormones are important factors in breast carcinogenesis. While the exact mechanisms of estrogen action in breast cancer development remain to be elucidated, it has been shown that estrogens induce and promote mammary cancer in rodents (Nandi et al, 1995) and exert proliferative effects on cultured human breast cancer cells (Cullen and Lippman 1989). A positive correlation between the plasma estrogen concentration and the breast cancer risk has been observed in postmenopausal women (Tonilo et al, 1995; Berrino et al, 1996; Hankinson et al, 1998). The majority of breast cancers, however, are detected during the postmenopausal period, when the ovaries have ceased to produce estrogens. Despite the low circulating estrogen concentrations in these patients, the tissue concentrations of estrogens in the breast are higher and, further, the estradiol concentration has been shown to be significantly higher in breast tumors than in normal breast tissue (Vermeulen et al, 1986; Pasqualini and Chetrite 2002).

The proliferation of breast epithelial cells is maximal during the second half of the menstrual cycle, at the time when both estradiol and progesterone are secreted (Anderson et al, 1982) suggesting that also progesterone may have a role in the development of breast cancer.

Both 17HSD type 1 and type 2 are expressed in the epithelium of normal breast tissue in premenopausal women (Miettinen et al, 1999) and oxidative activity seems to be the dominant form in non-tumorous cells (Miettinen et al, 1999, Spiers et al, 1998). The type 1 enzyme is expressed in the epithelial cells of ducts and alveoli throughout the menstrual cycle. Breast cancer cell lines have been shown to express 17HSD type 1, 17HSD type 2, or both enzymes (Miettinen et al, 1996b).

We recently analyzed the mRNA expressions of the 17HSD type 1, type 2 and type 5 enzymes were analyzed in 794 breast carcinoma specimens by using tissue microarrays and normal histological sections. The results were correlated with ER" and ER !, progesterone receptor (PR), Ki67 and c-erbB-2 expressions analyzed by immunohistochemical techniques and the the TumorNode-Metastasis (TNM) classification, tumor grade, disease-free interval and survival of the patients (Oduwole et al, 2004).

Both 17HSD type 1 and type 2 mRNAs were detected in the normal breast tissue of premenopausal women, but no expression of 17HSD type 1 or 17HSD type 2 mRNA was observed in the normal tissue specimens from postmenopausal women. The mRNAs were localized in the ductal or lobular epithelial cells (Oduwole et al, 2004).

In malignant breast lesions, variable expression patterns for 17HSD type 1 and type 2 mRNA were observed (Figure 2). There were 16 % 17HSD type 1 mRNA and 25 % type 2 mRNA positive cases. No significant differences were observed for the 17HSD type 1 enzyme in malignant tissue between the pre- and postmenopausal groups. In contrast, the number of cases showing signals for 17HSD type 2 mRNA was higher in the premenopausal than the postmenopausal patients.

III. 17HSDs in breast cancer Estrogens are essential for the growth and differentiation of the mammary gland. The female mammary gland undergoes a surge of cell divisions during puberty. There is also cyclic proliferation and involution in the breast during the menstrual cycle throughout adult life (Russo et al, 1999). Multiplication of normal breast cells cultured in vitro is increased by the addition of estradiol to the culture (Mauvais-Jarvis et al, 1986). Further, the phenotypes of estrogen receptor " (ER") and ! (ER!) knockout mice indicate that ER" is important for the growth and development of the mammary gland, whereas ER! is involved in the terminal differentiation of glandular epithelium (Couse and Korach 1999; Foster et al, 2002).

Breast cancer is the most common malignant neoplastic disease in the female. The majority of breast carcinomas are invasive ductal or lobular carcinomas.

Several endocrine and reproductive factors, such as early age at menarche, nulliparity, or delayed first childbirth, late age at menopause and obesity, are associated with its

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Vihko and Isomaa: 17!-hydroxysteroid dehydrogenases and breast cancer There was a moderate agreement between the in situ hybridization results and immunohistochemistry of 17HSD type 1. The 17HSD type 1 positive breast cancer specimens accounted for 20 % of all cases in immunohistochemical analysis and for 16 % in in situ hybridization (Oduwole et al, 2004).

17HSD type 5 mRNA expression was detected in the epithelial cells of normal and malignant breast tissue specimens from both pre- and postmenopausal women.

However, the expression of 17HSD type 5 was significantly higher in breast cancer specimens than in normal breast tissue (Figure 3). Altogether 65% of the breast cancer specimens were positive for 17HSD type 5 expression. Our data showed no correlation between 17HSD type 1 and type 2 and/or type 5 (Oduwole et al, 2004).

No association was found between 17HSD type 1, type 2 and type 5 mRNA expression and tumor grade.

17HSD type 1 mRNA expression did not associate with TNM status. Similar observations were made for 17HSD type 2 mRNA. The overexpression of 17HSD type 5 was associated with N but not with T or M status.

A significant association was observed between ER" and ER! expression. There was also a significant inverse association between ER" and 17HSD type 1 as well as ER" and 17HSD type 5. Tumors expressing 17HSD type 1, type 2 and/or type 5 mRNA did not associate with the expression of Ki67 or with c-erb-b2 status (Oduwole et al, 2004).

In benign breast tissue and breast cancer, variable amounts of 17HSD type 1 and 17HSD type 2 enzyme proteins have been reported in previous studies (Poutanen et al, 1992; Sasano et al, 1996; Suzuki et al, 2000; Gunnarsson et al, 2001). While we detected 17HSD type 1 mRNA in about one fifth of our specimens, some other studies have reported the presence of 17HSD type 1 enzyme protein in about half of the specimens.

Gunnarsson et al, (2001), using RT-PCR, detected 17HSD type 1 in all of the 84 archival breast cancer specimens they studied. The reason for the discrepancies is not known, but it may be partially explained by the different methodologies used and by the relatively small patient series in most previous studies.

Figure 2. In situ hybridization of 17HSD1 in breast lesions from a pre- and a postmenopausal woman. Strong signals for 17HSD1 mRNA (white arrows) in a ductal invasive carcinoma in a premenopausal woman (A) Corresponding (H&E) stain with arrows indicating the margin of the tumor area (B) Another case of ductal invasive carcinoma from a postmenopausal woman showing strong signals for 17HSD1 mRNA (C) A corresponding H&E-stained slide of the same area (D) The arrows indicate tumor cell islands among a heavy inflammatory infiltrate. Magnification: x 100. Reproduced from Oduwole et al, 2004 with kind permission from Cancer Research.

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Cancer Therapy Vol 3, page 547

Figure 3. In situ hybridization of 17HSD5 in normal and malignant breast tissue. Strong signals of 17HSD5 mRNA (arrows) in breast tumor cells (A). Negative control using sense probe in the tumor area (B) Magnification: x 400. Low signals of 17HSD5 mRNA in normal breast cells (C) Negative control using sense probe (D) Magnification: x 200. Reproduced from Oduwole et al, 2004 with kind permission from Cancer Research.

Our study also revealed that patients with tumors expressing 17HSD type 1 mRNA had significantly worse survival and a shorter disease-free interval than the other patients (Figure 4). No such association was found for tumors expressing 17HSD type 2 mRNA. The group with 17HSD type 5 overexpression had worse survival than the groups with lower or no expression. Patients with ER"positive breast cancer had better survival than those without. Multivariate Cox analysis (forward stepwise regression) was used to determine the possible independent prognostic significance of the following parameters: tumor size, presence of nodal and distant metastases, tumor grade, ER", ERâ&#x20AC;&#x161;, PR, 17HSD type 1, 17HSD type 2, 17HSD type 5, Ki67 and c-erb-b2. Since nodal status and the presence of metastasis showed an association with tumor size, they were not included in the model. According to the analysis, tumor size, 17HSD type 1 and ER " had independent prognostic value (Oduwole et al, 2004).

production is quantitatively the most important, systemic treatments are needed particularly in postmenopausal women. Adjuvant systemic therapies, such as hormonal therapy before and after surgery, have greatly improved the prognosis of breast cancer. Two strategies have been widely used to decrease estrogen influence. One is the use of antiestrogens, i.e. compounds that interact with the estrogen receptor, such as tamoxifen. The efficacy of tamoxifen in the treatment of breast cancer was first reported by Cole et al, 1971 and it has become the most widely used endocrine therapy for breast cancer.

Tamoxifen increases overall survival, reduces recurrence and has minimal side effects (Early Breast Cancer Trialistsâ&#x20AC;&#x2122; Collaborative Group, 1998).

Another strategy has been the use of aromatase inhibitors, i.e. compounds inhibiting the conversion of androgens to estrogens (Brodie, 2002). The use of aromatase inhibitors began in the 1980s and several inhibitors, including both steroidal and nonsteroidal compounds, are in clinical use.

High estradiol concentrations have been detected in breast cancer tissue. In addition to aromatase, other enzymes, particularly 17HSD type 1, are involved in the production of active estradiol in breast tissue. Since estrone sulfate is quantitatively the most important circulating estrogen in pre- and postmenopausal women, the combined activities of estrone sulfatase and 17HSD type 1 may be more important in local estradiol production

IV. Hormonal treatment of breast cancer As stated above, estrogens are known to be important for the growth of breast cancer in both pre- and postmenopausal women. Therefore, disruption of the estrogen-signaling pathway is an important treatment strategy for breast cancer. Since peripheral estrogen

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Vihko and Isomaa: 17!-hydroxysteroid dehydrogenases and breast cancer than aromatase activity in breast cancer tissue. Our data demonstrating that 17HSD type 1 is an independent prognostic factor in breast cancer support this suggestion.

Since high 17HSD type 1 expression is associated with a poor prognosis, it is clear that inhibition of this enzyme could be a beneficial therapy for breast cancer patients selected based on the expression of 17HSD type 1 in tissue specimens.

Acknowledgements This work was supported by the Research Council for Health of the Academy of Finland (Project Nos. 47630 and 51618), the Finnish Cancer Foundation and the Sigrid Juselius Foundation.

Figure 4. A, Kaplan-Meier curve showing the survival of patients with breast carcinoma in relation to 17HSD1. Patients with tumors expressing 17HSD1 mRNA had a significantly worse prognosis (p=0.0010, log rank). B, Kaplan-Meier curve showing the disease-free interval of breast carcinoma patients in relation to 17HSD1. Patients with 17HSD1 mRNA expressing tumors has a significantly shorter disease-free interval than the other patient. Reproduced from Oduwole et al, 2004 with kind permission from Cancer Research.

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Cancer Therapy Vol 3, page 549 Labrie F, Luu-The V, Lin SX, Labrie C, Simard J, Breton R and Labrie F (1997) The key role of 17!-hydroxysteroid dehydrogenases in sex steroid biology. Steroids 62, 148-158.

Li Y, Isomaa V, Pulkka A, Herva R, Peltoketo H and Vihko P (2005) Expression of 3!-hydroxysteroid dehydrogenase type 1, P450 aromatase and 17!-hydroxysteroid dehydrogenase types 1, 2, 5 and 7 mRNAs in human early and mid-gestation placentas. Placenta 26, 387-392.

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Marijanovic Z, Laubner D, Möller G, Gege C, Hausen B, Adamski J and Breitling R (2003) Closing the gap: Identification of human 3-ketosteroid reductase, the last unknown enzyme of mammalian cholesterol biosynthesis.

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Mauvais-Jarvis P, Kuttenn F and Compel A (1986) Estradiol/progesterone interaction in normal and pathologic breast cells. Ann NY Acad Sci 464, 152-167.

Miettinen MM, Poutanen MH and Vihko RK (1996a) Characterization of estrogen-dependent growth of cultured MCF-7 human breast-cancer cells expressing 17!hydroxysteroid dehydrogenase type 1. Int J Cancer 68, 600604.

Miettinen MM, Mustonen MV, Poutanen MH, Isomaa VV and Vihko RK (1996b) Human 17_-hydroxysteroid dehydrogenase type 1 and type 2 isoenzymes have opposite activities in cultured cells and characteristic cell- and tissuespecific expression. Biochem J 314, 839-845.

Miettinen M, Mustonen M, Poutanen M, Isomaa V, Wickman M, Söderqvist G, Vihko R and Vihko P (1999) 17!Hydroxysteroid dehydrogenase in normal human mammary epithelial cells and breast tissue. Breast Cancer Res Treat 57, 175-182.

Mindnich R, Möller G and Adamski J (2004) The role of 17!hydroxysteroid dehydrogenases. Mol Cell Endocrinol 218, 7-20.

Musgrove EA, Lee CS and Sutherland RL (1991) Progestins both stimulate and inhibit breast cancer cell cycle progression while increasing expression of transforming growth factor alpha, epidermal growth factor receptor, c-foe and c-myc genes. Mol Cell Biol 11, 5032-5043.

Mustonen MV, Poutanen MH, Kellokumpu S, de Launoit Y, Isomaa VV, Vihko RK and Vihko PT (1998a) Mouse 17!hydroxysteroid dehydrogenase type 2 mRNA is predominantly expressed in hepatocytes and in surface epithelial cells of the gastrointestinal and urinary tracts. J Mol Endocrinol 20, 67-74.

Mustonen MV, Isomaa VV, Vaskivuo T, Tapanainen J, Poutanen MH, Stenbäck F, Vihko RK and Vihko PT (1998b) Human 17!-hydroxysteroid dehydrogenase type 2 messenger ribonucleic acid expression and localization in term placenta and in endometrium during the menstrual cycle. J Clin Endocrinol Metab 83, 1319-1324.

Mäentausta O, Peltoketo H, Isomaa V, Jouppila P and Vihko R (1990) Immunological measurement of human 17!hydroxysteroid dehydrogenase. J Steroid Biochem 36, 673680.

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Breitling R, Krazeisen A, Möller G and Adamski J (2001) 17!hydroxysteroid dehydrogenase type 7 – an ancient 3ketosteroid reductase of cholesterogenesis. Mol Cell Endocrinol 171, 199-204.

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Desmond JC, Mountford JC, Drayson MT, Walker EA, Hewison M, Ride JP, Luong QT, Hayuden RE, Vanin EF and Bunce CM (2003) The aldo-keto reductase AKR1C3 is a novel suppressor of cell differentiation that provides a plausible target for the non-cyclooxygenase-dependent antineoplastic actions of nonsteroidal anti-inflammatory drugs. Cancer Res 63, 505-512.

Dufort I, Rheault P, Huang XF, Soucy P and Luu-The V (1999) Characteristics of a highly labile human type 5 17!hydroxysteroid dehydrogenase. Endocrinology 140, 568574.

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Foster C, Mäkelä S, Wärri A, Kietz S, Becker D, Hultenby K, Warner M and Gustafsson J_ (2002) Involvement of estrogen receptor ! in terminal differentiation of mammary gland epithelium. Proc Natl Acad Sci USA 99, 15578-15583.

Fournet-Dulguerov N, MacLusky NJ, Leranth CZ, Todd R, Mendelson CR, Simpson ER and Naftolin F (1987) Immunohistochemical localization of aromatase cytochrome P-450 and estradiol dehydrogenase in the syncytiotrophoblast of the human placenta. J Clin Endocrinol Metab 65, 757764.

Ghersevich SA, Poutanen MH, Martikainen HK and Vihko RK (1994) Expression of 17!-hydroxysteroid dehydrogenase type 1 in human granulosa cells: correlation with follicular size, cytochrome P450 aromatase activity and oestradiol production. J Endocrinol 143, 139-150.

Greenlee RT, Hill-Hermon MB, Murray T and Thun M (2001) Cancer statistics. CA Cancer J Clin 51, 15-36.

Gunnarsson C, Olson BM, St_l O and Members of the Southeast Sweden Breast Cancer Group. (2001) Abnormal expression of 17!-hydroxysteroid dehydrogenases in breast cancer predicts late recurrence. Cancer Res 61, 8448-8451.

Hankinson SE, Willett WC, Manson JE, Colditz GA, Hunter DJ, Spiegelman D, Barbieri RL and Speizer FE (1998) Plasma sex steroid hormone levels and risk of breast cancer in postmenopausal women. J Natl Cancer Inst 90, 1292-1299.

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Oduwole OO, Li Y, Isomaa VV, Mäntyniemi A, Pulkka AE, Soini Y and Vihko PT (2004) 17!-hydroxysteroid dehydrogenase is an independent prognostic marker in breast cancer. Cancer Res 64, 7604-7609.

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Peltoketo H, Isomaa V, Ghosh D and Vihko P (2003) Estrogen metabolism genes: HSD17B1 and HSD17B2. In: B.E. Henderson, B. Bonner, R.K. Ross (Eds.), Hormones, Genes and Cancer, Oxford University Press, pp. 181-198.

Peltoketo H, Vihko P and Vihko R (1999) Regulation of estrogen action: Role of 17!-hydroxysteroid dehydrogenases. Vitam Horm 55, 353-398.

Penning TM, Burczynski ME, Jez JM, Lin H-K, Ma H, Moore M, Ratnam K and Palackal N (2001) Structure-function aspects and inhibitor design of type 5 17!-hydroxysteroid dehydrogenase (AKR1C3). Mol Cell Endocrinol 171, 137149.

Poutanen M, Isomaa V, Lehto VP and Vihko (1992) Immunological analysis of 17!-hydroxysteroid dehydrogenase in benign and malignant human breast tissue.

Int J Cancer 50, 386-390.

Poutanen M, Isomaa V, Peltoketo H and Vihko R (1995) Role of 17!-hydroxysteroid dehydrogenase type 1 in endocrine and intracrine estradiol biosynthesis. J Steroid Biochem Mol Biol 55, 525-532.

Puranen T, Poutanen M, Ghosh D, Vihko R and Vihko P (1997) Origin of substrate specificity of human and rat 17_hydroxysteroid dehydrogenase type 1, using chimeric enzymes and site-directed substitutions. Endocrinology 138, 3532-3539.

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receptor in relation to proliferating cells in the mammary gland. Breast Cancer Res Threat 53, 217-227.

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Sawetawan C, Milewich L, Word A, Carr BC and Rainey WE (1994) Compartmentalization of type 1 17_-hydroxysteroid oxidoreductase in the human ovary. Mol Cell Endocrinol 99, 161-168.

Speirs V, Green AR and Atkin SL (1998) Activity and gene expression of 17!-hydroxysteroid dehydrogenase type I in primary cultures of epithelial and stromal cells derived from normal and tumourous human breast tissue: the role of IL-8.

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Suzuki T, Moriya T, Ariga N, Kaneko C, Kanazawa M and Sasano H, (2000) 17 !-hydroxysteroid dehydrogenase type 1 and type 2 in human breast carcinoma: correlation to clinicopathological parameters. Brit J Cancer 82, 518-523.

Toniolo PG, Levitz M, Zeleniuch-Jacquotte A, Bajernee S, Koenig KL, Shore RE, Strax P and Pasternack BS (1995) A prospective study of endogenous estrogens and breast cancer in postmenopausal women. J Natl Cancer Inst 87, 190-197.

TĂśrn S, Nokelainen P, Kurkela R, Pulkka A, Menjivar M, Ghosh S, Coca-Prados M, Peltoketo H, Isomaa V and Vihko P (2003) Production, purification and functional analysis of recombinant human and mouse 17!-hydroxysteroid dehydrogenase type 7. Biochem Biophys Res Commun 305, 37-45.

Vermeulen A, Deslypere JP, Paridaens R, Leclercq G, Roy F and Heuson C (1986) Aromatase, 17!-hydroxysteroid dehydrogenase and intratissular sex hormone concentrations in cancerous and normalglandular breast tissue in postmenopausal women. Eur J Cancer Clin Oncol 22, 515525.

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First row (in the back) from left to right: Riitta Kurkela, Annakaisa Herrala, Toni Luokkala, Mirja Makelainen, Riikka Wirkkala, Ileana Quintero, Svea Torn, Marja Kantola and Veli Isomaa.

Second row (in the middle) from left to right: Helena Kaija, Anitta Pulkka, Anna Ronka, Airi Vesala and Paivi Harkonen.

In the front: Pirkko Vihko

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Cancer Therapy Vol 3, page 551 Cancer Therapy Vol 3, 551-554, 2005

Successful treatment of gestational trophoblastic neoplasm metastatic to the colon Case report

Fatemeh Ghaemmaghami*, Farnaz Sohrabvand, Haleh Ayatollahi, Mitra Modarres Gynecology & Reproductive Medicine, Tehran University of Medical Sciences

__________________________________________________________________________________ *Correspondence: Fatemeh Ghaemmaghami, Associate Professor, Gynecologist Oncologist, Tehran University of Medical Sciences.

2nd Floor, Department of Gynecology Oncology, Vali-e-Asr Hospital, Keshavarz Blvd.,Tehran 14194, Iran; Phone: 0098-21-6937766; Fax: 0098-21-6937321; E-mail: valrec2@yahoo.com, ftghaemmagh@yahoo.com Key words: choriocarcinoma, colon metastasis, colostomy, GTN, EMA-EP Abbreviations: arteriovenous malformations, (AVM); Federation of Gynecology and Obstetrics (FIGO); Gestational trophoblastic neoplasm, (GTN) Received: 31 January 2005; Accepted: 21 February 2005; electronically published: November 2005

Summary Gestational trophoblastic neoplasm (GTN) normally spreads to the lungs and brain; metastasis to intra-abdominal organs such as the gastrointestinal tract is rare. We present a 41-year-old patient with locally invasive GTN who underwent two urgent laparotomies because of internal hemorrhage. First, a total hysterectomy was performed to manage perforation of the uterus; one month later, due to rectal bleeding emergency colon resection with colostomy was performed because of colon metastasis. Although the most appropriate management of GTN is chemotherapy, surgical intervention may be needed due to bleeding that may require resection of the involved structures. gastrointestinal tract may present with symptoms of acute abdomen (Balagopal et al, 2003). According to various case reports, metastasis to the gastrointestinal tract may manifest as small bowel perforation, metastasis to the colon may present as pseudoobstruction (Bandy et al, 1985), and metastasis to the stomach may present as upper gastrointestinal bleeding (Galloway et al, 2001).

In this report, we describe a case of GTN metastatic to the colon to demonstrate that surgery may be needed in the management of gastrointestinal complications.

I. Introduction Gestational trophoblastic neoplasm (GTN) represents a spectrum of pathologic and clinical alterations, ranging from molar pregnancy to metastatic gestational trophoblastic neoplasm. Locally invasive GTN develops in 15% of patients after the evacuation of a molar pregnancy; it infrequently develops after normal pregnancies, ectopic pregnancies, and spontaneous or therapeutic abortions.

Metastatic tumors develop after a complete molar pregnancy in 4% of patients, but they are more common after nonmolar pregnancies (Berkowitz and Goldstein, 1993).

Gestational trophoblastic neoplasm is suspected with persistent or irregular uterine hemorrhage. Trophoblastic neoplasm may perforate the myometrium or erode uterine vessels, causing intraperitoneal and vaginal bleeding, respectively (Berek and Hacker, 2000). Trophoblastic tumors are highly vascular and prone to severe hemorrhage, either spontaneously or during biopsy. The most common sites of metastasis are the lungs (80%), vagina (30%), pelvis (20%), liver (10%), and brain (10%) (Berkowitz and Goldstein, 1996). Less than 5% of the cases of metastatic GTN involve the intra-abdominal organs (Newlands, 2003). The gastrointestinal tract is a rare site of metastasis; patients with metastasis to the

II. Case Report We present the case of a 41-year-old female patient gravida 3, para 3, ab 1, post-miscarriage (7 years), and not sexually active. The patient also had multiple sclerosis and was being treated with corticosteroids. In September 2002, she was admitted to the general surgery ward of Vali-eAsr Hospital with abdominal pain, nausea, vomiting, and epigastric tenderness. Her hemoglobin value was 9.7g/dl.

Because of an acute abdomen and suspicion of internal hemorrhage she underwent an emergency laparotomy. Exploration of the abdomen during laparatomy showed uterine perforation and severe, intractable hemorrhage (about 1500 ml blood). The patient then underwent a total hysterectomy and unilateral

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Ghaemmaghami et al: Treatment of gestational trophoblastic neoplasm metastatic to the colon salpingo-oophorectomy; she had a mass which was similar to a submucous myoma that was 5cm in diameter.

A histologic examination showed invasive gestational trophoblastic neoplasm (Figure 1). The patient was referred to the gynecologic oncology ward for further management in October 2002. The patientâ&#x20AC;&#x2122;s HCG level was 39500 mIU/ml. A radiologic exam showed multiple sclerosis lesions in the brain, but there was no evidence of brain metastasis. A chest x-ray revealed multiple lung metastases.

An ultrasound of the abdomen showed normal abdominal and pelvic findings. Therefore, according to the International Federation of Gynecology and Obstetrics (FIGO) 2000 staging/scoring, the patient had stage III GTN with a total score of 11.

Since the patient was in a high-risk category, a combined chemotherapy regimen consisting of etoposide, methotrexate, actinomycin, etoposide, and cisplatin (EMA-EP) was administered. After the first course of chemotherapy, the patient had a sudden episode of severe rectal hemorrhage and her hemoglobin level fell to 8g/dl.

Therefore, she received a blood transfusion. A second emergent laparotomy was performed, to explore the intraabdominal organs. A lesion was discovered at the splenic flexure of the colon, ascending to the transverse colon.

Because we suspected metastatic gestational trophoblastic neoplasm to the colon, the lesion was resected and the patient underwent an emergent colostomy because the bowel was not prepped for anastomosis. A histologic examination confirmed metastatic GTN to the colon (Figure 2). Metastasis to the colon changed the patientâ&#x20AC;&#x2122;s

GTN to stage IV (distant metastases) with a total score of 13. The patient received four courses of EMA-EP, followed by weekly HCG measurements. This was followed by three more courses of chemotherapy, after which the HCG titer reached normal levels. Another three courses of chemotherapy were administered, and the colostomy was repaired. At a follow-up of 30 month, there were no signs of relapse and the patient was well.

III. Discussion We have described a rare case of metastatic GTN with an unusual presentation and response to treatment.

GTN is extremely responsive to chemotherapy, even in its metastatic forms (Berkowitz and Goldstein, 1996).

Metastases from GTN tend to be highly vascular and have a tendency toward central necrosis and hemorrhage; therefore, surgical intervention may be necessary (Newlands, 2003). Indeed, uterine perforation, due to rapid tumor growth may cause intraperitoneal hemorrhage which would require immediate surgery (Balagopal et al, 2003). A hysterectomy may be required in patients with metastatic GTN in order to control uterine hemorrhage or sepsis. Furthermore, in patients with extensive uterine tumors, a hysterectomy may substantially reduce the trophoblastic tumor and limit the need for multiple courses of chemotherapy (Berek and Hacker, 2000).

With the development of advanced interventional radiology techniques, selective angiographic localization and embolization techniques have been used to conservatively manage hemorrhage from active sites of

Figure 1. Choriocarcinoma invading the myometrium. The right half of the micrograph shows the tumor with a necrotic area and the left half shows deep myometrium (200X).

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Cancer Therapy Vol 3, page 553

Figure 2. Choriocarcinoma with large bowel metastasis. The tumor (right upper portion of the picture) and large intestinal mucosa (left lower portion) are seen in a background of extravasated red blood cells. (400X)

metastatic GTN and to treat intrauterine arteriovenous malformations (AVM) that can occasionally develop after the treatment of GTN. Vaginal metastases are most often treated with selective angiographic embolization after simple packing or suturing techniques have failed to control hemorrhage (Newlands 2003).

Angiographic abnormalities can persist for many months after the evacuation of a hydatidiform mole or the treatment of malignant GTN. However, intractable bleeding from intrauterine AVM after successful treatment for GTN is a relatively rare complication. Lim et al. studied 14 patients with selective angiographic embolization over 20 years; hemorrhage was controlled in 11 patients (79%), 6 patients (43%) required a second immobilization for recurrent bleeding, and only 2 patients (14%) required a hysterectomy (Lim et al. 2002).

Successful full term pregnancies have been reported after this procedure.

GTN of the gastrointestinal tract can most often be managed with chemotherapy alone, but some patients will experience bleeding which would first require resection of the involved structures in order to stop internal hemorrhage. Liver metastases, while often producing catastrophic intra-abdominal hemorrhage, are less likely to be successfully controlled with surgical resection.

Selective angiographical embolization techniques should be considered as an option (Hammond et al, 1980).

High-risk patients and patients with stage IV disease should be treated with primary combination chemotherapy. We used EMA-EP regimen as the first-line chemotherapy in this high-risk patient; our previous studies have shown that this regimen produces acceptable results for high-risk patients (Ghaemmaghami et al, 2004).

Patients generally respond to three to four cycles of chemotherapy, but we administered three more cycles of chemotherapy our high-risk patient had a â&#x20AC;&#x161;HCG (Ghaemmaghami et al, 2004; Matsui et al, 2000).

Alternating EMA-EP chemotherapy with etoposide, methotrexate, actinomycin, cyclophosphamide, and vincristine (EMA-CO) chemotherapy is common for highrisk patients.

In conclusion, although combination chemotherapy is the main treatment regimen used in patients with highrisk GTN, surgical intervention may occasionally be needed to control hemorrhage.

References Balagopal P, Pandey M, Chandramohan K, Somanathan T, Kumar A (2003) Unusual presentation of choriocarcinoma.

World Surg Oncol 1, 4.

Bandy LC, Clarke-Pearson PC, Hamment CB (1985) Pseudoobstruction of the colon complicating choriocarcinoma. Gynecol Oncol 20, 402-407.

Berek JS, Hacker NE (2000) Gestational trophoblastic neoplasia.

In, Practical Gynecologic Oncology. 3rd edition. William & Wilkins; p. 615-638.

Berkowitz RS, Goldstein DP (1996) Chorionic tumors. N Engl J Med 335, 1740-1748.

Berkowitz RS, Goldstein DP (1997) Presentation and management of molar pregnancy. In, Hancock BW, Newlands ES. Berkowitz RS, editors. Gestational trophoblastic disease. London, Chapman and Hall; p. 127146.

Galloway SW, Yeung EC, Lan JY, et al (2001) Laparoscopic gastric resection for bleeding metastatic choriocarcinoma.

Surg Endo 15, 100.

Ghaemmaghami F, Behtash N, Soleimani K, Hanjani P (2004) Management of patients with metastatic gestational trophoblastic tumor. Gynecol Oncol 94, 187-190.

Ghaemmaghami F, Modarres M, Arab M, et al (2004) EMA-EP regimen, as first line multiple agent chemotherapy in highrisk GTT patients (stage II-IV). Int J Gynecol Cancer 14, 360-365.

Hammond CB,Weed JC, Currie JL (1980) The role of operation in the current therapy of gestational trophoblastic disease.

Am J Obstet Gynecol 136, 844-858.

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Ghaemmaghami et al: Treatment of gestational trophoblastic neoplasm metastatic to the colon Lim AK, Agrwal R, Secki MJ, et al (2002) Embolization of bleeding residual uterine vascular malformations in patients with treated gestational trophoblastic tumors. Radiology 222, 640-644.

Matsui H, Suzuka K, Iitsuka Y, et al (2000) Combination chemotherapy with Methotrexate, Etoposide and Actinomycin-D for high risk gestational trophoblastic tumors. Gynecol Oncol 78, 28-31.

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Cancer Therapy Vol 3, page 555 Cancer Therapy Vol 3, 555-564, 2005

Future of gene therapies in high grade gliomas Research Article

Deepak Kumar Gupta1,*, Mattei Tobias Alecio2, Ashok Kumar Mahapatra1, Ramina Ricardo2 1

Department of Neurosurgery, All India Institute of Medical Sciences,Neurosciences Centre, New Delhi, India Department of Neurosurgery, Sao Paulo Medical School, Brazil

2

__________________________________________________________________________________ *Correspondence: Mahapatra, Ashok Kumar, Department of Neurosurgery Neurosciences Center, All India Institute of Medical Sciences Ansari Nagar, New Delhi 110029 (India); Tel. +91 11 6864851 (ext 4915); Fax +91 11 6862663; E-mail: akmahapatra_22000@yahoo.com Key words: Genetic transduction, genetic vectors, history, recurrent malignant gliomas, transfection, convection enhanced delivery system,antisense oligonucleotide Abbreviations: 5-fluorocytosine, (5-FC); 5-fluorouracil, (5-FU); Adenovirus, (Ad); anaplastic astrocytoma, (AA); antisense phosphorothioate oligonucleotides, (S-ODN); blood-brain barrier, (BBB); carcinoembryonic antigen, (CEA); central nervous system, (CNS); Convection-enhanced delivery, (CED); cytosine deaminase, (CD); Cytotoxic T lymphocytes, (CTL); epidermal growth factor receptor, (EGFR); early growth response 1, (Egr1); ganciclovir, (GCV); glial fibrillary acidic protein gene, (GFAP); glioblastoma multiforme, (GBM); herpes simplex virus thymidine kinase, (HSV-tk); Hypoxia-inducible factor, (HIF); hypoxia-responsive elements, (HRE); interferon-alpha, (IFN)-!; interleukin 12, (IL-12); ionizing radiation, (IR); Lymphokine activated killer, (LAK); radiotherapy, (RT); retroviral, (RV); Semliki forest virus vector carrying the human interleukin 12 encapsulated in cationic liposomes, (LSFV-IL12); Semliki forest virus vector, (SFV); transferring receptors, (TfR); transferring, (Tf); Transforming growth factor beta, (TGF"2); Tumor infiltrating lymphocytes, (TIL); tumor necrosis factor-related apoptosis-inducing ligand, (TRAIL)

Received: 29 August 2005; Accepted: 15 September 2005; electronically published: November 2005

Summary High-grade gliomas are relatively frequent in adults, and consist of the most malignant kind of primary brain tumor. Being resistant to standard treatment modalities such as surgery, radiation, and chemotherapy, it is fatal within 1 to 2 years of onset of symptoms. Although several gene therapy systems proved to be efficient in controlling or eradicating these tumors in animal models, the clinical studies performed so far are not equally successful. Most clinical studies showed that methodologies that increase tumor infection/transduction and, consequently confer more permanent activity against the tumor, would lead to enhanced therapeutic results.Most gliomas are incurable despite improvements in surgical techniques, radiotherapy, and chemotherapy. The therapeutic challenge is partially a result of diffuse tumor infiltration into surrounding normal brain tissue having an intact blood-brain barrier (BBB). Along with the development of novel antineoplastic therapies with improved tumor specificity, innovative ways of delivering these agents to the brain tumor are also under investigation.Although survival in patients with malignant gliomas remains limited, there is renewed optimism with the emergence of novel treatment strategies. Cytotoxic agents such as temozolomide and CPT-11 have shown promising clinical activity. Biological treatments for brain tumors, including antisense oligonucleotides, gene therapy, and angiogenesis inhibitors, are also being evaluated in clinical trials.Delivery strategies have been developed to overcome challenges presented by the blood-brain barrier. These noteworthy treatments, alone or in combination, may ultimately prolong survival and enhance quality of life in this group of patients. weeks; and by the addition of chemotherapy, 40-50 weeks.In spite of the application of multimodal therapies, 94% of glioma patients still die within 24 months after initial diagnosis, this outcome hasnot improved considerably during the last two decades despite technical advances in neurosurgery, radiotherapy and the evaluation of novel anticancer chemotherapeutical agents (Davis et al, 1998). Numerous experimental therapies based on

I. Introduction Malignant gliomas (glioblastoma multiforme (GBM) and anaplastic astrocytoma (AA)) comprise the most common types of primary central nervous system (CNS) tumors and have a combined incidence of 5-8/100,000 population. The median survival of patients with malignant gliomas treated conservatively is 14 weeks; by surgical resection alone, 20 weeks; by surgery and radiation, 36 555

Mohapatra and Gupta: Future of gene therapies in high grade gliomas augmentation of antitumor host immune response by local and/or systemic application of Lymphokine activated killer cells (LAK), Tumor infiltrating lymphocytes (TIL) and Cytotoxic T lymphocytes (CTL) and various immunostimulants (e.g. cytokines) did not prolong patient survival in the past (Mahaley et al, 1988). Although survival for GBM has not changed significantly over the past three decades, the emergence of novel treatment strategies for these tumors has led to heightened interest and optimism among oncologists. In adults, gliomas are devastating diseases and the best available treatments, such as surgical resection and radiotherapy, have been only temporarily successful. This happens because with the post-resection tumor residues, which are almost always present, it becomes fatal within 1 to 2 years of the first onset of symptoms. The two factors that promote the use of gene therapy for gliomas are the failure and toxicity of conventional therapies, as well as the identification of genetic abnormalities, which contribute to the malignancy

of gliomas. Uncontrolled cellular proliferation, lack of apoptosis, invasion, and angiogenesis are among the biological processes that make these tumors both aggressive and difficult to treat (Phuong et al, 2003).

A. Genetic therapies in gliomas During the malignant progression of gliomas, several tumor suppressor genes are inactivated, and numerous growth factors and oncogenes are overexpressed progressively. Consequently, gliomas’ gene therapy may aim at molecular interference with ‘gain of function’ genes (oncogenes) or replacement of ‘loss of function’ genes (tumor suppressor genes). Such approaches require transgene expression in entire tumor cell populations (if other mechanisms do not come into play), which cannot be achieved with current vector systems. Hence, other strategies have been pursued that may be independent of the genes actually involved in tumor genesis (Tables 1-4).

Table 1. List of different molecular strategies and the related genes for treating gliomas Anti-angiogenic factors Angiostatin. Endostatin, IFN-!, Platelet factor 4 (sPF4), p16 Apoptosis-related genes p-53, Rb, gas-i, TRAILFas, FADD, NF-#B. gas-i Bcl-2.

Bcl-X(L), #B. p73!. Bax, Apaf-1 caspase-3, caspase-8 and caspase-9 Prodrug Activation Systems HSV-tk. Na+/l - symporter (hNlS) cytochrome P450 2B1 (Cyclophospharnide) FolylpolygIutamyl synthetase Immunogenes IL-12, IL-6, IL-4, lL-18, GM-CSF, IFN-$, TNF-!, B7-2, TGF-", LIF and LT Chemosensitization genes WAF1/Cip1 Cytosine deaminase/5-fluorocytosine Radiosensitization genes IR-responsive Egr1, p53, Cytokines (GM-CSF, IL-4, IL-12) Other genes Urokinase-type plasminogen activator, CEAFusogenic Membrane GIycoprotein Linamarase Table 2. List of possible vectors and their modified techniques Adenovirus Fiber-mutant (F/K20) adenovirus Pretreatment with protease Single-chain antib6dies targeting GFAP targeting Hypoxic tumor targeting Herpes simplex virus-1 Vectors producing cells Encapsulation %FasL microporous membranes) Parvovi rus HVJ-liposonies Semliki forest virus. Measles virus Epstein-Barr virus, Newcastle disease virus Mumps virus. Vesicular Stomatitis virus Influenza virus, Reovirus and Poliovirus 556

Cancer Therapy Vol 3, page 557 Table 3. Various targeted viral and nonviral therapies in brain tumors Vehicle

Immunogenicity Advantages

Disadvantages

Nonviral !Polynucleotide !Plasmid !Cellular (pro/eukaryotic)

Low

Unstable/transient/difficult delivery

!

No replication or expression in target

!

Non immunogenic

!

Viral non integrating High !Adenovirus !

Herpes

!

Large insert capacity. !Clinical expenence,relative stability !Efficient transduction of dividing and nondividing cells. !

Toxicity esp.I.m.use !Production capacity limited !Recombination common and sequencing impractical !Persistence limited by immune response !

Risk of insertional mutagene 515 !Safety concerns !No clinical experience !Production capacity limited. !

Viral intergrating !Onco retroviral !Lentivirus !AAV

Low

Moderate insert capacity !Persistence !Transduce dividing and non dividing cells. !Persistence !

Table 4. Phase trials involving targeted receptor therapies for brain tumors Targeted receptor EGFR tyrosine kinase inhibitors

PDGFR, EGFR BGFR VBGF tyrosine VBDF/PDGF kinase inhibitors VEGFR-2 Farnesyl transferase K-ras inhibitors K-ras, Av"3 Integrin antagonists Av"3/5 Bfgf/vegf Bndothelin receptor antagonists ET-A Metalloproteinase inhibitors MMP-1,2,7,9 Phosphoinositide 3 kinase inhibitors m-Tor Cyclooxygenase 2 inhibitors Cox-2 Proteosome inhibitors 26sproteosome

Microbial genes (e.g. herpes simplex virus thymidine kinase) may be transferred into the tumors allowing prodrug activation (e.g. ganciclovir). Furthermore, cytokines or other immunomodulatory genes may be used for vaccination purposes, which frequently involves ex vivo transfection of autologous tumor cells with such genes.

Malignant gliomas were chosen for the first clinical study on new gene therapy approaches because these

Phase

Company

I/II I/II I I/II I II II II I II/III I/II III I/II I/II

Novartis, Basel, Switz Astra, Wilmington OSI, CA Pfizer Novartis Johnson & Johnson Schering-Plough Merck, Germany Celegene,NZ Celegene,NZ Abbott lab Schering Wyeth, PA Pfizer, NY Millenium, Cambridge

tumors are non-metastatic and develop on the largely postmitotic background of normal glial and neuronal tissue.

Several molecular strategies have been tested, either in animal models or clinical trials: prodrug activating systems, introduction of tumor suppressor or cell-cyclerelated genes, inhibition of growth factors and/or their receptors, inhibition of neovascularization, immunomodulatory maneuvers, oncolytic viruses, inhibition of matrix metalloproteinases, induction of toxic

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Mohapatra and Gupta: Future of gene therapies in high grade gliomas agents and sensitization of tumorsof local expression to chemotheapeutic agents and radiotherapy (Germano et al, 2003).

There are different physical methods for vector delivery to malignant primary brain tumors in experimental or clinical settings: stereotactic or direct intratumoral injection or convection-enhanced bulk-flow interstitial delivery; intrathecal and intraventricular injection; intravascular infusion with or without modification of the blood-tumor-barrier; and direct intratumoral delivery of anti-sense oligonucleotides (Rainov and Kramm, 2001). Critical evaluation of gene transfer and therapy studies has led to the conclusion that even using identical vectors, the anatomical route of the vector can dramatically affect both the efficiency of tumor transduction and its spatial distribution, as well as the extent of intratumoral and intracerebral transgene expression. The safety and efficiency of these therapeutic systems in humans has been confirmed by several controlled pre-clinicaland clinical therapeutic trials (Ren et al, 2003).

slice cultures. The mechanisms of this anti-tumor effect were most likely caused by the major anti-angiogenic action of the cytokine, because IFN-! (Davis et al, 1998) expression provoked a pronounced decrease in blood vessel density, which was accompanied by extensive necrosis in the tumorsâ&#x20AC;&#x2122; body mass (De Bouard et al, 2003).

E. p16 gene Loss of p16 is a frequent event in the progression of malignant gliomas. High-grade gliomas are distinguished from low-grade gliomas by intense angiogenesis in addition to their frequent loss of p16. Infection with a recombinant replication-defective adenovirus vector containing the cDNA of wild-type p16, significantly reduced the expression of vascular endothelial growth factor, which is thought to be a pivotal mediator of tumor angiogenesis, in p16-deleted glioma cells. Restoring wildtype p16 expression into p16-deleted glioma cells markedly inhibited angiogenesis induced by tumor cells in vivo.

Furthermore, wild-type p16 inhibited neovascularization more potently than did wild-type p53 transfer (Harada et al, 1999).

B. Genes Prodrug Activating System HSV-TK Ganciclovir: Tumor cell transduction with the herpes simplex virus thymidine kinase (HSV-TK) gene and treatment with GCV is the most widely studied cancer gene therapy (Nafe et al, 2003). HSV-TK converts the prodrug GCV into a toxic nucleotide analogue, whose incorporation into cellular DNA blocks cell proliferation. Following repetitive ganciclovir (GCV) intraperitoneal or intravenous injection, effective killing of glioma cells in mouse brain is observed. There are several techniques described in the literature for the HSV-TK/GCV therapy with variations depending on the vector utilized, and the concentration of injection solution.

II. Apoptosis related genes p-53 gene:The p53 gene is thought to function abnormally in the majority of malignant gliomas, although it has been demonstrated to be mutated in only approximately 30%. This has led to studies in which adenoviral transduction with wild-type human p53 has been investigated in an attempt to slow tumor cell growth.

Some authors demonstrated that multiple gene replacements with simultaneous exposure to adenoviruscontaining p53 gene can produce additive effects in the treatment of glioma cell lines (Kim et al, 2002).

A. Rb gene Retinoblastoma tumor suppressor gene abnormalities are found in the majority of cancers, including, at least, 30% of malignant gliomas (Fueyo et al, 1998). These findings provide direct evidence that inactivation of the retinoblastoma protein is a critical event in gliomas and suggest that the restoration of wild-type retinoblastoma activity in these tumors through vector delivery gene therapy may have great therapeutic utility.

Other apoptosis-related genes: Some studies suggest that adenoviral vector-mediated delivery of other apoptosis-related genes may also be potentially useful in the gene therapy approach towards the treatment of human brain gliomas. The apoptosis-related genes already studied are: Fas/Fas ligand, caspase-8, p33ING1, p73!, Bax, Apaf-1, caspase-9, I-#BdN, NF-#B, caspase-3, gas-1, Bcl2, and Bcl-X (L) (Shinoura et al, 2003).

C. Anti angiogenic factors Inhibition of angiogenesis has been considered among the most promising approaches to treat highly vascularized solid tumors, such as high-grade gliomas.

However, chronic systemic delivery of therapeutic proteins, such as inhibitors of angiogenesis, presents several difficult pharmacological challenges. The concept that targeted anti-angiogenesis, using virally mediated gene transfer, represents a promising strategy for delivering this anti-angiogenicfactors (Tanaka et al, 1997).

D. Angiostatin, Endostatin, and IFN-!

(Davis et al, 1998) Some researchers evaluated the effects of local production of three endogenous inhibitors of angiogenesis (angiostatin, endostatin, and interferon (IFN)-! (Davis et al, 1998)), using a stably transfected rat (9L) and human (GL15) glioblastoma cells, on tumor vascularization and growth in an in vitro assay system based on the implantation of tumor cells into organotypic brain slice cultures. Although all the three genes showed angiogenesis inhibitory effect, IFN-!demonstrated the most potent anti-angiogenic effect in organotypic brain

III. Immunogenes Cancer immunogene therapy is based on vaccination with radiated, autologous tumor cells transduced with immunostimulatory genes. It has been demonstrated that high-grade gliomas produce immunosuppressive factors, like TGF-", which reduce the anti-tumor response by peripheral blood effector cells. These immunosuppressive 558

Cancer Therapy Vol 3, page 559 factors could be neutralized to improve anti-tumor response (Ashley et al, 1998).

Vaccination treatment using genetically modified tumor cells to express certain cytokines consists in the following steps: First, glioma cells are cultured primarily from the patientsâ&#x20AC;&#x2122; surgically resected tumor tissues.

Afterwards, in vitro infection with a recombinant virus vector containing the gene of the cytokine is procede, and afterwards, the transduced cells are re-injected in the patient.

Vaccination therapy induces specific activation of cytotoxic T lymphocytes measured by cell-mediated cytotoxicity assay, suggesting the generation of a specific anti-tumor response and the potential for systemic immunity. This immunization results in the regression of the implanted cells, as well as the original brain tumor.Several cytokines have been studied: IL-12, IL-6, IL-4, IL-18, IFN-$, TGF-", TNF-!, GM-CSF, B7-2, TNF!, LIF and LT (Herrlinger et al, 2000).

cells has been proposed. The results of in vivo experiments showed that apoptotic death may be enhanced by the combination of the treatment with Ad-containing Bax gene (Ad-Bax) and RT. Ad/Bax synergistically radiosensitizes glioma, with a seemingly favorable therapeutic index (Yamanaka et al, 2002b).

A. Radiation-responsive gene promoters Synthetic gene promoters, responsive to clinical doses of ionizing radiation (IR), have been developed for use in suicide gene therapy vectors. The crucial DNA sequences utilized are units with the consensus motif CC(A/T)(6)GG, known as CarG elements, derived from the IR-responsive Egr1 gene. These elements had their sequences incorporated into a synthetic gene promoter and assayed for the ability to induce expression of a downstream reporter gene following irradiation. Exposure of cells to ionizing radiation resulted in the activation of these specific transcriptional control elements within the early growth response 1 (Egr1) gene promoter, leading to increased gene expression. Studies revealed that increasing the number of CarG elements up to a certain level, increases promoter radiation-response; specific alteration of the core A/T sequences caused an even greater positive response. (Inoue et al, 2004). These enhancers can be used to drive suicide gene expression from vectors delivered to a tumor within an irradiated field. These results demonstrate that the synthetic promoter is responsive to low doses of ionizing radiation, and therefore, isolated CarG elements function as radiation-mediated transcriptional enhancers outside their normal sequence context.

IV. Chemosensitizing fenes A. Cell cycle regulator WAF1/Cip1: Studies have shown that negative cell cycle regulator WAF1/Cip1 is often overexpressed in human gliomas and that WAF1/Cip1â&#x20AC;&#x2122;s overexpression makes glioma cells resistant to chemotherapy agents.These results show that the attenuation of WAF1/Cip1 expression initiated glioma cell death and sensitized glioma cells to apoptosis induced by 1,3-bis(2chloroethyl)-1-nitrosourea and cisplatin. Thus, blocking WAF1/Cip1 production may serve as a useful chemosensitization regimen for treating glioma (Yamanaka et al, 2002a).

B. p53 gene Adenoviral vector-mediated expression of human wild-type p53 not only slows tumor cell growth but also enhances the radiosensitivity of malignant glioma cells that express native wild-type p53.RT2 tumor cells express native rat wild-type p53 before the transduction, and markedly overexpress human p53 following adenoviral p53 transduction. The combination of p53 transduction followed by radiation results in marked decreases of RT2 cell survival and increases in apoptosis at radiation doses from 2 to 6 Gy. The results support a new perspective in the p53 genetic therapy, showing the ability to enhance the radiosensitivity of malignant glioma cells that express wild-type p53 by using adenoviral transduction to induce overexpression of p53 and offer new hope for the p53 viral-mediated genetic therapy as a successful therapeutic strategy, not only in human gliomas that express mutant p53, but also in those that express wild-type p53 (Ruan et al, 1999).

B. Cytosine deaminase/5-fluorocytosine Adenovirus (Ad) vector-mediated cytosine deaminase (CD)/5-fluorocytosine (5-FC) gene therapy has been proposed as a potential technique to overcome pharmacokinetic issues associated with systemic 5-FU and is particularly well suited to use with tumors in which local control is paramount, such as malignant gliomas.The bacterial enzyme CD catalyzes the conversion of 5-FC to the lethal 5-fluorouracil (5-FU). Cloning the CD gene from Escherichia coli and expression in human tumor cell lines enabled these cells to convert 3H-labeled 5-FC into 3H-5-FU. Glioblastoma cell line T1115 became 200-fold more sensitive to 5-FC than the non-expressing parental cell lines. At least 90% of the cells are killed within 7 days.

CD-expressing cells are able to kill non-expressing cells when grown in the same culture flask (bystander effect).

The results of clinical studies in human patients with highgrade gliomas confirm the previous findings in rat models, demonstrating the potential clinical utility of Ad 5-FC gene therapy for gliomas. (Yamanaka et al, 2002a)

C. Cytokines Some cytokine vaccination (GM-CSF, IL-4, IL-12) therapies have not only primary immunity generation against the tumor but also an important radiosensitization effect. In some studies with tumors treated with vaccination therapy and posteriorly, irradiation, about 80100% of the glioma-bearing mice were cured.

V. Radiosensitizing genes Bax gene The hypothesis that Ad-mediated transfection of proapoptotic Bax gene could enhance the cytotoxicity of radiotherapy (RT) in RT-refractory glioma

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Mohapatra and Gupta: Future of gene therapies in high grade gliomas constructed, in which the HSV-TK gene is driven by a 2.2 kb DNA promoter which controls expression for the encoding glial fibrillary acidic protein gene (GFAP), an intermediate filament protein expressed primarily in astrocytes (Martinet et al, 2003).

Hypoxic Tumor Targeting: New therapy targeting the hypoxic fraction of tumors is very useful as this population of cells is the most resistant to radio- and chemotherapies.

Hypoxia-inducible factor (HIF) mediates transcriptional responses to hypoxia by binding to hypoxia-responsive elements (HRE) in target genes.Although this approach needs more experimental studies, the results suggest that it could be used to treat solid tumors that develop hypoxia, including the category of more malignant gliomas (Dupont et al, 2000).

D. Other genes Other genes which have been evaluated as candidates for genetic therapy in gliomas are: folylpolyglutamyl synthetase gene, growth arrest-specific genes (gas1), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), cell cycle regulator WAF1/Cip1 gene, cytosine deaminase/5-fluorocytosine gene, Bax gene, carcinoembryonic antigen (CEA) gene, urokinase-type plasminogen activator gene and fusogenic membrane glycoprotein gene (Chiocca et al, 2003).

VI. Vectors Viruses (Table 2) have emerged on the genetic therapy scene and gained attention due to their ability to play essentially two roles: first, as vectors for therapeutic gene delivery and second, as engineered infectious agents capable of selectively lysing tumor cells. Oncolytic viruses have shown promising results in solid tumor treatment, including gliomas, but their potency must be improved if their full clinical potential is to be realized (Fu et al, 2003).

2. Herpes Simplex Virus-1 The HSV-1 vectors are particularly useful because they can be genetically engineered to replicate and spread highly selectively in dividing tumor cells and can also express multiple foreign transgenes. If the viruses are directly injected into the brain, they might not be inactivated. The HSV-1 vectors have been recently utilized as oncolytic vectors instead of replicationdefective vectors. The oncolytic HSV-1 have demonstrated cytopathic effect in ratâ&#x20AC;&#x2122;s glioma models without damaging normal tissues, providing amplified gene delivery within the tumor, and inducing specific antitumor immunity. Different approaches are currently undertaken to improve the efficacy of oncolytic HSV-1 therapy which include: development of new generation vectors via further genetic engineering of existing safe vectors, combination with immune gene therapy, and combination with conventional therapies (Visted et al, 2000).

A. Adenovirus The local, intratumoral injection of adenovirus is an especially suitable strategy for gliomas because these tumors, although infiltrative, rarely metastasize. Two approaches have been used to generate tumor-selective replicative adenoviruses: use of tumor-specific promoters to regulate the expression of viral genes, and the deletion of the viral functions required for the cell cycle activation.Since normal cells surrounding gliomas are quiescent, the second strategy is particularly attractive to develop new treatments for brain tumors.Trials have showed that adenoviruses are more efficient than retroviruses in achieving in vivo gene transfer (Puumalainen et al, 1998).

3. Other viruses

B. Targeted adenovirus

Other viral vectors, like parvovirus, hemagglutinating virus of Japan, Semliki forest virus, Measles virus, Epstein-Barr virus, Newcastle disease virus, Mumps virus, Vesicular Stomatitis virus, Influenza virus, Reovirus and Poliovirus have been tested in genetic therapy experiments in vivo and in vitro (Shah et al, 2003).

However, they lack more investigation of their specific role in glioma therapy.

The application of adenoviral vectors in cancer gene therapy is hampered by low receptor expression on tumor cells and high receptor expression on normal epithelial cells. Targeted techniques with adenoviral vectors seem to be a promising tool for cancer gene therapy; they could provide an improved therapeutic index with efficient tumor transduction and effective protection of normal tissue.

4. Clinical trials

C. Single-chain antibodies targeting

In malignant glioma, standard gene therapy approaches employing non-replicating virus vectors failed to demonstrate significant benefit in clinical studies.

Therapy with oncolytic viruses seems to hold more promise in early clinical trials than gene therapy with nonreplicating virus vectors (Nestler et al, 2004).

The most studied candidates for gene therapy, which are in advanced stages of clinical trials include: the prodrug activating system HSVtk/GCV, utilizing either retrovirus vector producer cells or adenovirus vectors; the adenovirus-mediated p53 gene transfer; the adenovirusmediated IFN-" gene transfer and studies with oncolytic therapy with herpes virus or adenovirus vectors. The other

Some authors proposed specific tumoral targeting by the use of doubly ablated adenoviral vectors, lacking coxsackie virus-adenovirus receptor and !(v) integrin binding capacities, together with bispecific single-chain antibodies targeted toward human epidermal growth factor receptor (EGFR) or the epithelial cell adhesion molecule.

These vectors efficiently and selectively targeted both alternative receptors on the surface of human cancer cells (Kuriyama et al, 2000).

1. GFAP targeting In an attempt to limit the toxic effects on normal tissues, a recombinant adenoviral vector has been 560

Cancer Therapy Vol 3, page 561 vectors and genes previously discussed are still in cell or animal protocols investigation stage (Thomas et al, 2000).

There is an ongoing Phase I/II clinical study in adult patients with recurrent GBM which is aimed at evaluating biological safety, maximum tolerated dose, and anti-tumor efficacy of a cytokine vaccination model, using a genetically modified replication-disabled Semliki forest virus vector (SFV) carrying the human interleukin 12 (IL12) gene and encapsulated in cationic liposomes (LSFVIL12) (Sandmair et al, 2000). Several other Phase I and II clinical studies in patients with recurrent malignant glioma have shown a favorable safety profile and some efficacy of retroviruses (RV)-mediated gene therapy 3. More than 300 patients with glioma have already been treated in clinical trials with oncolytic viruses, and in most cases, the virus was administered directly into the tumor.

On the other hand, a prospective randomized Phase III clinical study of retroviral (RV) gene therapy in primary malignant glioma failed to demonstrate significant extension of the progression-free or overall survival times in RV-treated patients. (Puumalainen et al, 1998). The failure of this RV gene therapy study may be due to the low tumor cell transduction rate observed in vivo.

Biological effects of the treatment may heavily depend on the choice of transgene/prodrug system and on the vector delivery methods.

RV clinical trials in malignant glioma have, nevertheless, produced a substantial amount of data and have contributed towards the identification of serious shortcomings of the non-replicating virus vector gene therapy strategy. New types of therapeutic virus vector systems are currently being designed, and new clinical protocols are being created based on the lessons learned from the RV gene therapy trials in patients with malignant brain tumors.

The long-term consequences of adenovirus-mediated conditional cytotoxic gene therapy for gliomas remain uncharacterized. Some studies reported detection of active brain inflammation 3 months after successful inhibition of syngeneic glioma growth. The inflammatory infiltrate consisted of activated macrophages/microglia and astrocytes, and T lymphocytes positive for leucosyalin, CD3 and CD8, and included secondary demyelination.

(Mattei et al, 2005).

malignant solid tumors. This antigen provides an abundant, insoluble, nondiffusible anchor for the Mab. Once localized to necrotic regions of the tumor, Cotara delivers a cytotoxic dose of 131I radiation to the adjacent living tumor cells .The intact BBB may not allow passage of Cotara, a high-molecular-mass protein (Mr 150â&#x20AC;&#x201C;170 kD), from the vascular compartment into the interstitium of tumor-infiltrated brain. Convection-enhanced delivery (CED) (U.S. Patent No. 5,720,720) provides one method of bypassing the BBB for regional delivery of large macromolecules, such as Cotara, into the interstitium of the brain tumor and infiltrated brain. CED was originally used as a tool for delivering other therapeutic modalities, specifically gene therapies. The technique was first used clinically to deliver a ransferring receptor-based diphtheria toxin, Tf-CRM-107, into recurrent primary and metastatic brain tumors in a Phase 1 study. Tf-CRM-107 was subsequently used via CED in a Phase 2 multicenter trial, the results of which have been presented in a preliminary report. Forty-four patients with recurrent or progressive anaplastic astrocytoma or glioblastoma multiforme were treated, and responses were seen in 21 patients. Eight of the 44 patients had symptomatic progressive cerebral edema, which was responsive to medical treatment. Newonset seizures occurred in 3 of the 44 patients treated.

CED has similarly been used to deliver interleukin4â&#x20AC;&#x201C;Pseudomonas exotoxin chimeric fusion protein. A limited number of patients have been treated thus far. The dose-limiting toxicity is cerebral edema, which is treatable with medical or surgical methods, but the treatment protocol is still undergoing modification. Other CEDdeliverable gene therapies under clinical trial include transforming growth factor-(") and Pseudomonas exotoxin and interleukin-13 receptor-directed cytotoxin. CED uses a motor-driven pumping device to drive the flow of an infusate through a catheter tip that is stereotactically placed at the target site within the brain. The resulting pressure gradient drives the fluid through the interstitial space. Experimental studies show that CED can achieve a local drug concentration 10,000-fold greater than that achieved by intravenous drug administration without causing significant systemic exposure. The infused drug permeates the targeted region at a final concentration governed by such variables as infusion parameters, the flow resistance or hydraulic conductivity of the tissue, and the duration of treatment. CED can therefore effectively bypass the blocking effects of the BBB and deliver antitumoral compounds and agents to specific locations Limited clinical studies using CED have been reported in humans. Pilot studies using this technique have been used to deliver tumor-targeting immunotoxin conjugates (e.g., TF-CRM107 and IL4-Pseudomonas exotoxin (NBI3001)), chemotherapeutic agents (e.g., paclitaxel), and antiglioblastoma gene therapy (e.g., HSV-1-tk) to cancer patients (Patel Sunil et al, 2005).

Transforming growth factor (TGF"2) in tumor progression and immunosuppression of malignant glioma: Patients suffering from malignant glioma show a profound state of cellular immunodeficiency. The most important cause seems to be an increased release of the subtype TGF-" by the glioma cells.Kjellman suggested that the

IV. Targeted toxin therapies and drug delivery systems/convection enhanced delivery Most gliomas are incurable despite improvements in surgical techniques, radiotherapy, and chemotherapy. The therapeutic challenge is partially a result of diffuse tumor infiltration into surrounding brain tissue having an intact BBB. Along with the development of novel antineoplastic therapies with improved tumor specificity, innovative ways of delivering these agents to the brain tumor are also under investigation.Cotara (Peregrine Pharmaceuticals, Inc., Tustin, CA) is a 131I-labeled chimeric monoclonal antibody (131I-chTNT-1/B Mab) specific for a universal intracellular antigen (i.e., histone H1 complexed to deoxyribonucleic acid) exposed in the necrotic core of

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Mohapatra and Gupta: Future of gene therapies in high grade gliomas TGF "2 is specifically important in the later stages of malignancy, while TGF "1 and TGF "3 may be important during the earliest stages of tumor development (Kjellman et al, 2000). TGF "1 and TGF "2 were shown to have a negative growth regulating effect in low grade, near diploid gliomas. On the other hand, the majority of high grade tumors are either unresponsive or growth stimulated.Two additional pathomechanisms, in which tumor derived TGF "2 plays a role, may be associated with poor clinical prognosis (de Visser and Kast, 1999).

(Figure 1) These include: increased production of extracellular matrices supporting invasive growth and infiltration of non affected tissue, and neovascularization, meaning denovo production of new blood vessels supplying tumor tissue. A knowledge of these 4 pathomechanisms of malignant glioma progression results in therapeutic strategies that counteract TGF "2 activities.

A novel treatment approach has been developed based on specific inhibition of TGF "2 synthesis by antisense phosphorothioate oligonucleotides (S-ODN). Tumor cells are known to be an important source of TGF-" production there is evidence that TGF-" is an important promoter of malignant cell growth. Tumor growth in glioma seems to be due an increased release of TGF-"2 by the glioma tumor cells.

TGF-"2 is the most potent immunosuppressant known.

TGF-"2 production may represent a significant tumor escape mechanism from host immunosurveillance.

AP 12009 is a synthetic 18-mer antisense oligonucleotide targeted against TGF " (short strings of DNA/RNA down regulate gene expression by interfering with translation of encoded protein at mRNA level. A multinational multicentric open label, active controlled, randomized parallel group dose finding study â&#x20AC;&#x201C;phase IIb study of which the senior author is one of the principal investigators) to evaluate the efficacy and safety of two

doses of antisense protein in adult patients with recurrent high grade glioma, administered intratumorally as continuous high flow microperfusion over a 7 day period every other week for 6 months, is currently carried out in 6 different countries. The preliminary results are quiet encouraging though final results are still awaited.

The poor prognosis of central nervous system malignancy is in part related to a lack of potent agents with adequate tumor specificity. Targeting to specific cell receptors provides the possibility of creating novel therapeutic agents with greater tumor specificity than conventional chemotherapy. Monoclonal antibodies against tumor associated antigens and other binding moieties, which provide tumor selectivity, have been conjugated with radionuclides and with various toxins.

Investigators at National Institute 0f Health (NIH) studied a targeted protein toxin, which uses the physiological binding of human transferring (Tf) to transferring receptors (TfR) expressed on metabolically active cells to achieve tumor specificity (Greenfield et al, 1987). This targeted protein toxin is transferring-CRM-107, a conjugate of human Tf and diphtheria toxin with a point mutation which inactivates the nonspecific binding to mammalian cells. Another phase III multicentre study of intratumoral/interstitial therapy with Transmid (a conjugate of modified diphtheria toxin (CRM107) and human Tf joined by a stable, nonreducible thioether bond) compared to best standard of care in patients with progressive and/or recurrent, nonresectable glioblastoma multiforme patients is being carried out, the results are still awaited.TfRs transport iron into cells and are over expressed on rapidly dividing cells most notably on hematopoeitic cells and various tumor cells, including glioblastoma cells. Studies have demonstrated higher expression of these receptors on gliolastoma and

Figure 1. Schematic picture showing block of protein expression by antisense molecule.

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Cancer Therapy Vol 3, page 563 medulloblastoma tumor cells lines in comparison to human erythroleukemia cell line K 562. In contrast to this, TfRs in normal brain tissue are sparse and are largely restricted to the luminal surface of brain capillaries.

TranMID is being developed as a potential treatment for malignant brain tumors. A phase II clinical study of intratumoral infusions of TransMID in 44 patients with refractory and progressive GBM or Anaplastic Astrocytoma has been conducted in USA (drug delivered continuously over a period of 5-7 days via two catheters implanted in the tumors, given as two separate treatments between 4-10 weeks apart and good response was noted in 48% of patients (complete response in 11%, partial response in 16%, 21% had stable disease), it was concluded from these studies that the benefits of treatment of recurrent brain tumors with TransMID exceeds the risks and phase III trial is currently on its way for this drug (Laske and Rossi, 2002).

We would like to give special thanks to A. Hirose and E. Tanaka for their excellent technical assistance. This study was supported by a Grant-in-aid from the Japanese Ministry of Education, Science, Sports and Culture (Project No. 15390485) and a grant from Yamaguchi Endocrine Research Association.

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Dupont F, Avalosse B, Karim A, Mine N, Bosseler M, Maron A et al (2000) Tumour-selective gene transduction and cell killing with an oncotropic autonomous parvovirus-based vector. Gene Ther 7, 790-6.

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Fueyo J, Gomez-Manzano C, Yung WK, Liu TJ, Alemany R, Bruner JM, et al (1998) Suppression of human glioma growth by adenovirus-mediated Rb gene transfer. Neurology 50, 1307-15.

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Harada H, Nakagawa K, Iwata S, Saito M, Kumon Y, Sakaki S et al (1999) Restoration of wild-type p16 down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in human gliomas. Cancer Res 59, 3783-9.

Herrlinger U, Jacobs A, Quinones A, Woiciechowsky C, SenaEsteves M, Rainov NG et al (2000) Helper virus-free herpes simplex virus type 1 amplicon vectors for granulocytemacrophage colony-stimulating factor-enhanced vaccination therapy for experimental glioma. Hum Gene Ther 11, 142938.

Inoue R, Moghaddam KA, Ranasinghe M, Saeki Y, Chiocca EA, Wade-Martins R (2004) Infectious delivery of the 132 kb CDKN2A/CDKN2B genomic DNA region results in correctly spliced gene expression and growth suppression in glioma cells. Gene Ther 11, 1195-204.

Kim SK, Wang KC, Cho BK, Chung HT, Kim YY, Lim SY et al (2002) Interaction between p53 and p16 expressed by adenoviral vectors in human malignant glioma cell lines J Neurosurg, 97, 143-50.

Kjellman C, Olofsson SP, Hansson O, von Schantz T, Lindvall M, Nilsson I, Salford LG, Sjogren H, Widegren B (2000) Expression of TGF" isoforms, TGF" receptors, and smad

V. Conclusions Malignant gliomas remain a poorly understood form of cancer associated with high rates of morbidity and mortality. New treatment strategies are emerging that target steps in the molecular pathogenesis of these tumors.

Antiangiogenesis agents, antisense oligonucleotides, and signal transduction inhibitors are all examples of such therapies now entering clinical trials. Future treatment strategies for malignant gliomas will likely involve synergistic combinations of agents aimed at different pathways in the molecular pathogenesis of this type of cancer. Major steps to improve gene transfer into the central nervous system and the efficacy of gene therapy for malignant brain tumors include: 1) the design of more effective vector systems; 2) the development of new or improved prodrug/suicide systems, gene replacement approaches, or strategies targeting the immune response or tumor angiogenesis; 3) the study of new techniques to enhance delivery of genetic vectors into brain tumors and for monitoring gene delivery into tumors. Further major advancements in virus designs, application modalities, and understanding of the interactions of the hostâ&#x20AC;&#x2122;s immune system with the virus, are clearly needed before oncolytic virus therapy of malignant brain tumors can be introduced to clinical practice. Finally, strategies to circumvent the BBB (polymers, bradykinin analogues, gene therapy) are important advances that have also shown efficacy in early clinical trials. With the present results, it is clear that gene therapy strategies for gliomas are quite promising but more critical research is required, mainly in the vector field. The ultimate molecular therapy will probably involve the application of multiple simultaneous (combinatorial) therapeutic modalities. The pace and breadth of discovery in molecular biology promise a steady supply of novel agents as well as refinements of existing ones. One of the important challenges for the future is the development and implementation of sound clinical research methods that will enable investigators to identify active treatment regimens.

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Deepak Kumar Gupta and Ashok Kumar Mahapatra

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Cancer Therapy Vol 3, page 565 Cancer Therapy Vol 3, 565-578, 2005

T cell-based strategies for immunotherapy of prostate cancer Review Article

Marc Schmitz1,*, Andrea Kiessling1, Bernd Weigle 1, Susanne Fuessel2, Axel Meye2, Rebekka Wehner1, Achim Temme1, Michael Bachmann1, Manfred P. Wirth2, E. Peter Rieber1 1

Institute of Immunology, Medical Faculty, Technical University of Dresden, Fetscherstr. 74, 01307 Dresden, Germany Department of Urology, Medical Faculty, Technical University of Dresden, Fetscherstr. 74, 01307 Dresden, Germany

2

__________________________________________________________________________________ *Correspondence: Marc Schmitz, MD, Institute of Immunology, Medical Faculty, Technical University of Dresden, Fetscherstr. 74, 01307 Dresden, Germany. Tel: +49-351-4586501; Fax: +49-351-4586316; e-mail: mschmitz@rcs.urz.tu-dresden.de Key Words: T cells, dendritic cells, immunotherapy, prostate cancer Abbreviations: amino acid, (aa); antigen-presenting cells, (APCs); cytotoxic T cells, (CTLs); dendritic cells, (DCs); enzyme-linked immunospot, (ELISPOT); granulocyte-macrophage colony-stimulating factor, (GM-CSF); human leukocyte antigen, (HLA); hormonerefractory prostate cancer, (HRPC); human telomerase reverse transcriptase, (hTERT); interleukin, (IL); major histocompatibility complex, (MHC); parathyroid hormone-related protein, (PTH-rp); peripheral blood mononuclear cells, (PBMCs); prostate-specific antigen, (PSA); prostate-specific membrane antigen, (PSMA); transient receptor potential, (trp); tumor-associated antigens, (TAAs) Received: 08 November 2005; Accepted: 14 November 2005; electronically published: November 2005

Summary Prostate cancer is the most common noncutaneous cancer diagnosis and the second leading cause of cancer-related deaths among American men. The absence of effective curative therapies for advanced metastatic prostate cancer has entailed an intensive search for novel treatment modalities. T cells provide a powerful compartment of the adaptive immune system comprising important functions in antitumor immunity. Thus, CD8+ cytotoxic T lymphocytes (CTLs) are capable of efficient recognition and destruction of tumor cells. CD4+ T cells enhance the antigen-presenting capacity of dendritic cells (DCs) and provide help for the maintenance and expansion of tumorreactive CTLs. Consequently, much attention has been payed to the identification of tumor-associated antigens that may serve as target structures for a T cell-based immunotherapeutic strategy. In this context, several prostate cancer-related proteins have been described which are capable of inducing antigen-specific and/or tumor-reactive T cells in vitro. Following the identification of suitable prostate cancer-associated antigens, several clinical trials were conducted which were based on the administration of selected peptides, recombinant proteins or DNA. In addition, prostate cancer patients were immunized with peptide-, protein-, or RNA-loaded DCs which display an unique capacity for the induction of primary T cell responses. These clinical trials provide evidence that the different immunotherapeutic strategies represent safe and feasible concepts for the induction of immunological and clinical responses in prostate cancer patients.

et al, 2004). Androgen ablation with either surgical orchiectomy or application of luteinizing hormonereleasing hormone agonists with or without antiandrogens represents an effective initial treatment modality for recurrent disease (Miyamoto et al, 2004; Sharifi et al, 2005). However, within several years, most patients develop androgen-independent prostate cancer (Feldman and Feldman, 2001). Recent clinical trials of docetaxelbased chemotherapy in patients with metastatic hormonerefractory prostate cancer (HRPC) have demonstrated a decrease of serum prostate-specific antigen (PSA) level, a

I. Introduction Prostate cancer represents the most common noncutaneous cancer among American men with an estimated incidence of 232,090 cases in 2005 (Jemal et al, 2005). In addition, it is the second leading cause of cancerrelated deaths in American men with an estimated number of 30,350 deaths in 2005 (Jemal et al, 2005). Although the majority of patients are diagnosed with localized prostate cancer and are treated with radical prostatectomy or radiation therapy, 20-40% of patients will develop recurrent disease (Coen et al, 2002; Han et al, 2003; Roehl

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Schmitz et al: T cell-based strategies for immunotherapy of prostate cancer activated CD4+ T cells (Bennett et al, 1998; Ridge et al, 1998; Schoenberger et al, 1998). CD4+ T cells also provide help for the maintenance and expansion of CTLs by secreting cytokines such as interleukin (IL)-2. Additional functions of CD4+ T cells were documented by several studies indicating that these cells can eradicate tumors and can contribute to the inhibition of angiogenesis (Mumberg et al, 1999; Qin and Blankenstein, 2000).

Since effector T cells play a major role in the elimination of tumor cells, much attention has been payed on the identification of tumor-associated proteins that may provide targets of tumor-reactive T cells and on the definition of concrete peptide motifs within these proteins serving as T cell epitopes when presented by HLA molecules (Stevanovic, 2002). In prostate cancer, most of the target molecules for T cell-mediated immunotherapy are differentation antigens that are specifically expressed by normal and malignant prostate tissue. This group includes PSA, prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostate stem cell antigen (PSCA), prostein and transient receptor potential (trp)-p8. Some other potential target proteins as parathyroid hormone-related protein (PTH-rp), human telomerase reverse transcriptase (hTERT) and survivin are overexpressed in prostate cancer as well as in other tumors. A list of the so far identified T cell epitopes is given in Table 1.

reduction in pain, an improvement of the quality of life and, for the first time, a prolonged survival (Petrylak et al, 2004; Tannock et al, 2004). Although promising palliative benefit and modest but real prolongation of survival have been achieved, additional treatment strategies are needed to prevent progression from localized to advanced disease and to further improve survival outcomes for patients with metastatic prostate cancer.

II. Prostate cancer-associated antigens recognized by T cells Immunotherapy of tumors has advanced with the observation that CD8+ cytotoxic T cells (CTLs) provide a high capability to recognize and destroy tumor cells which expose peptides derived from tumor-associated antigens (TAAs) and bound to human leukocyte antigen (HLA) class I molecules (Rosenberg, 1997). In addition, clinical studies focussing on the adoptive transfer of cytotoxic effector cells revealed tumor regression in cancer patients (Dudley et al, 2002; Yee et al, 2002; Dudley and Rosenberg, 2003; Dudley et al, 2005; Vignard et al, 2005).

CD4+ T cells recognizing peptide motives in the context of HLA class II molecules also play an important role in antitumor immunity (Pardoll and Topalian, 1998; Toes et al, 1999; Wang, 2001). Thus, CD4+ T cells improve the capacity of dendritic cells (DCs) to induce CTLs by the interaction between CD40 on DCs and CD40 ligand on

Table 1. CD8+ and CD4+ T cell epitopes of prostate cancer-associated antigens Antigen

HLA restriction element

Peptide position

Amino acid sequence

Reference

PSA

HLA-A2

146-154

KLQCVDLHV

141-150

FLTPKKLQCV

154-163

VISNDVCAQV

154-163 (1Y)a 162-170 152-160

YISNDVCAQV QVHPQKVTK CYASGWGSI

248-257 68-77b 49-63 (6M, 10M)b, c 64-78b

HYRKWIKDTI VSHSFPHPLY

Xue et al, 1997; Perambakam et al, 2002 Correale et al, 1997; Correale et al, 1998 Correale et al, 1997; Correale et al, 1998; Heiser et al, 2000 Terasawa et al, 2002 Correale et al, 1998 Gotoh et al, 2002; Harada et al, 2003 Harada et al, 2003 Corman et al, 1998

ILLGRMSLFMPEDTG QVFQVSHSFPHPLYD

Corman et al, 1998 Corman et al, 1998

171-190 221-240 4-12b 711-719 27-35 441-450 178-186 227-235 624-632 334-348

LQCVDLHVISNDVCAQVHPQ GVLQGITSWGSEPCALPERP LLHETDSAV ALFDIESKV VLAGGFFLL LLQERGVAYI NYARTEDFF LYSDPADYF TSYVSFDSL TGNFSTQKVKMHIHS

Klyushnenkova et al, 2005 Klyushnenkova et al, 2005 Tjoa et al, 1996 Murphy et al, 1996 Lu and Celis, 2002 Harada et al, 2004 Horiguchi et al, 2002 Horiguchi et al, 2002 Kobayashi et al, 2003a Kobayashi et al, 2003b

687-701 730-744

DPQSGAAVVHEIVRS RQIYVAAFTVQAAAE

Kobayashi et al, 2003b Kobayashi et al, 2003b

HLA-A3 HLA-A24 HLA-A1 HLA-DR4 HLA-DR B1*1501 PSMA

HLA-A2

HLA-A24 HLA-DR4 HLA-DR9/ HLA-DR53 HLA-DR53

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Cancer Therapy Vol 3, page 567 PAP

HLA-A2

PSCA

HLA-A*2404 HLA class II HLA class II HLA-A*0201 HLA-A2

Prostein

Trp-p8 PTH-rp

HLA-A24 HLA-A*0201 HLA-B*5101 HLA-Cw*0501 HLA-A*0201 HLA-A*0201 HLA-A2 HLA-A24

hTERT

HLA-A*0201

HLA-A3 HLA-A24 HLA-A1 HLA-DR1/ HLA-DR7/ HLA-DR15

Survivin

HLA-DR4/ HLA-DR11/ HLA-DR15 HLA-A*0201

HLA-A2 HLA-A1

HLA-A3 HLA-A11

299-307 112-120 213-221 199-213 228-242 14-22

LLFGYPVYV TLMSAMTNL LYCESVHNF GQDLFGIWSKVYDPL TEDTMTKLRELSELS ALQPGTALL

Peshwa et al, 1998 Harada et al, 2004 Inoue et al, 2001 McNeel et al, 2001 McNeel et al, 2001 Dannull et al, 2000; Kiessling et al, 2002 Kiessling et al, 2002 Matsueda et al, 2004a Matsueda et al, 2004a Matsueda et al, 2004b Kiessling et al, 2004 Friedman et al, 2004 Friedman et al, 2004 Friedman et al, 2004 Kiessling et al, 2003 Francini et al, 2002 Francini et al, 2002 Yao et al, 2005 Yao et al, 2005 Yao et al, 2004 Yao et al, 2004 Vonderheide et al, 1999; Minev et al, 2000 Minev et al, 2000 Hernandez et al, 2002 Hernandez et al, 2002 Vonderheide et al, 2001 Arai et al, 2001 Arai et al, 2001 Schreurs et al, 2005

105-113 7-15 21-30 76-84 31- 39 464-472 292-300 464-473 187-195 59-68 165-173 59-67 42-51 36-44 102-111 540-548

AILALLPAL ALLMAGLAL LLCYSCKAQV DYYVGKKNI CLAAGITYV SACDVSVRV YTDFVGEGL SACDVSVRVV GLMKYIGEV FLHHLIAEIH TSTTSLEDL FLHHLIAEI QLLHDKGKSI RAVSEHQLL RYLTQETNKV ILAKFLHWL

865-873 572-580 572-580 (1Y)d 973-981 324-332 461-469 325-333

RLVDDFLLV RLFFYRKSV YLFFYRKSV KLFGVLRLK VYAETKHFL VYGFVRACL YAETKHFLY

672-686

RPGLLGASVLGLDDI

Schroers et al, 2002; Schroers et al, 2003

766-780 95-104

LTDLQPYMRQFVAHL ELTLGEFLKL

5-14

TLPPAWQPFL

96-104 (2M)e

LLLGEFKLK

18-28 92-101 38-46 (9Y)f 93-101 (2T) f 47-56 (10Y) f 18-27 (10K)f 53-62

RISTFKNWPFL QFEELTLGEF MAEAGFIHY FTELTLGEF PTENEPDLAY RISTFKNWPK DLAQCFFCFK

Schroers et al, 2003 Schmitz et al, 2000; Andersen et al, 2001a Schmitz et al, 2000; Siegel et al, 2004 Andersen et al, 2001a; Anderson et al, 2001b Reker et al, 2004 Reker et al, 2004 Reker et al, 2004 Reker et al, 2004 Reker et al, 2004 Reker et al, 2004 Reker et al, 2004

a

Agonist peptide in which valine at the first position was replaced by tyrosine.

Natural generation and presentation of this epitope by prostate cancer cells was not analyzed. c The methionine residues in the positions 6 and 10 were substituted in place of histidines. d The arginine residue in position 1 was replaced by tyrosine to increase immunogenicity. e The natural threonine at position 2 was changed to a methionine residue. f As compared to the native survivin protein sequence, cysteine was substituted by tyrosine at position 9 in peptide 38-46, glutamic acid by threonine at position 2 in peptide 93-101, glutamine by tyrosine at position 10 in peptide 47-56 and phenylalanine by lysine at position 10 in peptide 18-27, respectively. b

used serum marker for diagnosis and monitoring of prostate cancer and is nearly exclusively expressed by epithelial cells of the prostate (Balk et al, 2003).

Furthermore, it is found in the majority of prostate cancer

A. Prostate-specific antigen (PSA) PSA is a kallikrein-like serin-protease showing a high degree of homology with human pancreatic kallikrein (Lundwell and Lilija, 1989). It represents the most widely 567

Schmitz et al: T cell-based strategies for immunotherapy of prostate cancer tissues and can be detected in the cytoplasmic portion of these cells by immunoperoxidase staining (Oesterling, 1991).

Among prostate differentiation antigens, the T cellmediated immune response to PSA has been studied most thoroughly to date. Xue et al, 1997 identified an HLA-A2compatible peptide corresponding to amino acid (aa) residues 146-154 of PSA that was successfully used for the in vitro-stimulation of peptide-specific CTLs from a healthy donor by autologous peptide-pulsed peripheral blood mononuclear cells (PBMCs). In a complementary study, CTLs recognizing PSA peptide 146-154 were shown to specifically lyse HLA-A2-positive tumor cells endogenously expressing the PSA protein (Perambakam et al, 2002). Applying a similar stimulation protocol, two other HLA-A2-binding PSA-derived peptides consisting of aa 141-150 and 154-163 were defined as CD8+ T cell epitopes capable of inducing CTLs that were reactive against an HLA-A2- and PSA-positive prostate cancer cell line (Correale et al, 1997). PSA peptide 154-163 was additionally verified as a target structure of PSA-reactive CD8+ T effector cells from HLA-A2-positive donors that were generated by stimulation with PSA RNA-transfected autologous DCs (Heiser et al, 2000). In another study, modification of this PSA peptide by replacing the valine residue in the first position by a tyrosine led to a strong agonist peptide which markedly increased the efficiency to induce prostate cancer-reactive CTLs (Terasawa et al, 2002). Correale et al, 1998 developed a strategy to simultaneously induce PSA-restricted CTL activities to multiple epitopes. The authors constructed a 30-mer oligopeptide corresponding to aa 141-171 of the PSA protein that contained two immunogenic HLA-A2-binding peptides described previously (aa 141-150 and 154-163) and an additional HLA-A3-fitting peptide (aa 162-170).

CD8+ T cell lines from HLA-A2- and HLA-A3-positive donors that were generated by stimulation with autologous PBMCs loaded with the oligopeptide reacted against target cells pulsed with the nonamer or decamer peptides and expressing the respective HLA molecule.

Two HLA-A24-binding PSA peptides were reported to generate peptide-specific CTLs. Gotoh et al, 2002 revealed that the PSA peptide spanning the aa 152-160 is immunogenic in HLA-A*2402/Kb-transgenic mice.

Immunization with this peptide resulted in the induction of peptide-specific and HLA-A*2402-restricted CTLs. The same peptide as well as another one (aa 248-257) were demonstrated to function as HLA-A24-restricted CD8+ T cell epitopes by in vitro -activation of specific CTLs from HLA-A24-positive prostate cancer patients after stimulation with peptide-loaded PBMCs (Harada et al, 2003). Corman et al, 1998 described an HLA-A1-binding PSA-derived peptide (aa 68-77) with the capacity to induce CTLs specifically recognizing peptide-pulsed target cells. However, the endogenous generation and presentation of these motifs by prostate cancer cells was not analyzed by the authors.

T helper cell epitopes were defined by Corman et al, 1998 who identified HLA-DR4-binding peptides within the PSA protein (aa 49-63 with modifications in two positions and 64-78). Recently, two immunogenic HLA-

DRB1*1501-restricted 20-mer peptides (corresponding to aa 171-190 and 221-240) were found by immunization of HLA-DRB1*1501-transgenic mice with human PSA and subsequent screening a library of overlapping 20-mer peptides spanning the entire PSA protein for peptidespecific in vitro-proliferation (Klyushnenkova et al, 2005).

These peptides led to the in vitro-generation of specific CD4+ T cell lines from HLA-DRB1*1501-positive patients with granulomatous prostatitis or prostate cancer when presented by autologous antigen-presenting cells (APCs). In addition, the peptide-specific CD4+ T cells responded to APCs pulsed with the whole PSA protein.

B. Prostate-specific membrane antigen (PSMA) PSMA, an integral membrane glycoprotein that functions as protease and folate hydrolase, was identified using the monoclonal antibody 7E11.C5 (Israeli et al, 1993; Carter et al, 1996). Immunhistochemical findings indicate that PSMA is a marker of normal epithelial cells of the prostate (Murphy et al, 1998). In addition, its expression is increased in most prostate tumors, particularly in undifferentiated, metastatic and hormoneresistant cancer (Kawakami and Nakayama, 1997).

A number of studies has demonstrated the suitability of PSMA for T cell-based immunotherapy by the identification of immunogenic peptide epitopes. Tjoa et al, 1996 described an HLA-A2-binding peptide spanning the aa 4-12 that induced peptide-specific CTLs when PBMCs of prostate cancer patients were stimulated with peptidepulsed DCs. Furthermore, Murphy and co-workers revealed that the HLA-A2-binding peptide comprising aa 711-719 had the potential to decrease the levels of PSA in prostate cancer patients following administration of peptide-pulsed DCs (Murphy et al, 1996). An additional HLA-A*0201-restricted PSMA peptide (aa 27-35) proved to be effective in triggering antitumoral CTL responses as demonstrated by the capacity of peptide-induced CTLs to lyse an HLA-A*0201-positive prostate cancer cell line (Lu and Celis, 2002). Recently, the HLA-A2-restricted peptide comprising the aa 441-450 of PSMA protein has not only been shown to induce HLA-A2-restricted and prostate cancer-reactive CTLs but was described to serve as target of humoral immune responses in prostate cancer patients (Harada et al, 2004). By the same stategy, Kobayashi et al, 2003a identified an immunogenic HLA-A24-restricted PSMA peptide (aa 624-632). Furthermore, the in vitrostimulation of CD8+ T cells from a healthy HLA-A24positive donor using DCs loaded with predicted HLAA24-matching peptides revealed two additional peptides (aa 178-186 and 227-235) which originate from intracellular processing of PSMA protein in tumor cells (Horiguchi et al, 2002).

Recent approaches to identify PSMA-derived CD4+ T cell epitopes demonstrated that the peptide sequences comprising the aa positions 334-348, 687-701 and 730744 were restricted to HLA-DR4, HLA-DR9 or HLADR53 and HLA-DR53, respectively and induced antigenspecific T cells which were capable of reacting with naturally processed antigen (Kobayashi et al, 2003b).

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Cancer Therapy Vol 3, page 569 prostate cancer patients that recognize the PSCA-derived HLA-A*0201-restricted peptides with the aa positions 1422 and 105-113 (Kiessling et al, 2002). Moreover, these peptides had the capacity to induce peptide-specific and tumor-reactive CTLs from prostate cancer patients when loaded on autologous DCs for repetitive stimulations of CD8+ T cell cultures. Matsueda and colleagues identified two additional HLA-A2-restricted peptides (aa positions 7-15 and 21-30) and an HLA-A24-presented peptide (aa position 76-78) that effectively stimulated CTLs from prostate cancer patients (Matsueda et al, 2004a and 2004b).

C. Prostatic acid phosphatase (PAP) PAP was described as an isoenzyme of the heterogenous group of acid phosphatases specifically secreted by prostate cells (Gutman et al, 1936). The cDNA isolated by screening cDNA libraries with polyclonal antisera encodes a 386 aa protein which includes a 32 aa signal sequence (Yeh et al, 1987; Vihko et al, 1988). PAP expression was shown to be restricted to the prostate by RNA dot blot analysis (Solin et al, 1990) and by immunohistochemical staining with monoclonal antibodies (Kuciel et al, 1988; Lam et al, 1989).

Peshwa et al, 1998 identified an HLA-A2-restricted CTL epitope (aa position 299-307) within the PAP protein by stimulation of T cells from healthy donors with peptide-pulsed autologous DCs. Recently, an additional immunogenic HLA-A2-binding peptide (aa 112-120) activating peptide-specific and tumor-lysing CTLs from prostate cancer patients in vitro was defined (Harada et al, 2004). Inoue et al, 2001 revealed a PAP-derived, HLAA*2402-binding peptide (aa position 213-221) that induced tumor-reactive CTLs from prostate cancer patients and healthy donors. In addition, two peptides (aa positions 199-213 and 228-242) were described as potential CD4 + T cell epitopes, although the HLA class II restriction elements were not determined (McNeel et al, 2001).

E. Prostein Prostein was identified by a combination of cDNA substraction and microarray screening as a novel protein with a unique specificity for malignant and normal prostate tissues (Xu et al, 2001). Prostein is a protein of 553 aa that is predicted to contain eleven transmembrane domains and a cleavable signal sequence at the amino terminus. Xu et al, 2001 demonstrated the highly prostaterestricted expression pattern in normal human tissues by quantitative reverse-transcription PCR, Northern blot and cDNA microarray analyses as well as by immunhistochemical analysis. Determining the prostein mRNA level in paired samples of malignant and nonmalignant prostate tissue from prostate cancer patients by real-time PCR our group found abundant expression in all tested samples (Kiessling et al, 2004). In addition, the transcript levels were maintained or even elevated in 87% of the primary tumors when compared to prostein expression in the autologous non-malignant tissue samples. In a recent study, a prostein-specific monoclonal antibody was used to determine prostein expression at the protein level in a high number of tumorous and nontumorous human tissues of diverse histological origin (Kalos et al, 2004). In this study, prostein was detected in 94% of all non-malignant and maligant prostate samples including metastases, but in none of 4635 non-prostatic normal and tumor tissues. The tissue-specific expression profile of this molecule and the abundant expression in the great majority of prostate tumors are promising prerequisites for the use of this protein as target structure for specific immunotherapeutic strategies in prostate cancer.

To identify immunogenic CD8+ T cell epitopes from prostein, we selected six nonamer and decamer peptides from the aa sequence of prostein that were predicted to bind to HLA-A*0201 by a computer-based algorithm and verified the binding affinity to HLA-A*0201 by a competition assay (Kiessling et al, 2004). Using these peptides, exogenously loaded on DCs, for repetitive in vitro-stimulations of autologous CD8+ T lymphocytes from prostate cancer patients and healthy donors, we were able to activate cytotoxic T effector cells specifically recognizing a peptide comprising the aa positions 31-39 in the prostein protein. The peptide-specific CTLs that were raised from all T cell cultures stimulated with this peptide also efficiently lysed prostate tumor cells expressing both HLA-A*0201 und prostein. Recently, another group identified prostein-derived peptides, one of them presented

D. Prostate stem cell antigen (PSCA) PSCA was identified by a PCR-based subtractive hybridization strategy as a gene specifically expressed in the prostate (Reiter et al, 1998). The encoded protein belongs to the Thy-1/Ly-6 family of glycosylphosphatidylinositol-anchored cell surface glycoproteins and its aa sequence shares 30% identity with stem cell antigen 2. By mRNA in situ-hybridization and immunohistochemistry, PSCA expression was detected in more than 80% of primary prostate carcinomas and in all bone metastases analyzed (Reiter el al, 1998; Gu et al, 2000). Its increased expression level in both androgendependent and -independent prostate tumors when compared to the corresponding normal prostate tissues and its upregulation in carcinomas of high stages und Gleason Scores make PSCA a promising target structure for the immunotherapy of hormone-refractory tumors. In addition, PSCA may also provide a candidate for the immunotherapy of tumors with different histological origin, as PSCA expression has also been found in transitional cell carcinomas of the bladder (Amara et al, 2001) and pancreatic cancer (Argani et al, 2001).

Different studies have pointed out the suitability of PSCA as a target antigen of CTL-mediated immunotherapy. An HLA-A*0201-restricted PSCA peptide comprising the aa 14-22 was reported to be capable of generating a peptide-specific and tumorreactive CTL response from a patient with metastatic prostate cancer by an in vitro-stimulation protocol employing irradiated peptide-loaded PBMCs as APCs (Dannull et al, 2000). By enzyme-linked immunospot (ELISPOT) analyses, we detected increased frequencies of CD8+ T cells in the blood of HLA-A*0201-positive 569

Schmitz et al: T cell-based strategies for immunotherapy of prostate cancer

by HLA-B*5101 (aa position 464-472) and two presented

by HLA-Cw*0501 (aa positions 292-300 and 464-473), that are recognized by tumor-reactive CTLs (Friedman et al, 2004). The authors used APCs infected with a prosteinexpressing adenovirus for the stimulation of CD8+ T lymphocytes from two healthy donors and identified immunogenic peptides by the use of target cells expressing truncated prostein constructs or pulsed with synthetic prostein-derived peptides.

tumor-reactive CTLs when loaded on PBMCs from prostate cancer patients (Yao et al, 2004).

H. Human telomerase transcriptase (hTERT)

reverse

Whereas hTERT cannot be detected in most nontransformed somatic cells it is expressed in the majority of tumors of different histological origins including prostate cancer, (Kim et al, 1994) and is responsible for the protection of tumor cells from telomere erosion (Blasco and Hahn, 2003). Consequently, hTERT provides an attractive candidate for T cell-based immunotherapies of many tumors.

An immunogenic HLA-A*0201-restricted peptide comprising the aa 540-548 that is capable of inducing peptide-specific and tumor-reactive CTLs from healthy donors and prostate cancer patients was described by several groups (Vonderheide et al, 1999; Minev et al, 2000). Moreover, this peptide and an additional immunogenic HLA-A*0201-binding peptide (aa 865-873) were shown to induce peptide-specific CTLs in HLAA*0201 transgenic mice (Minev et al, 2000). Hernandez et al, 2002 identified a third HLA-A*0201-matching peptide spanning aa 572-580 whose immunogenicity was markedly increased by substitution of the arginine residue at position one by tyrosine. Furthermore, an HLA-A3fitting motif corresponding to aa 973-981 (Vonderheide et al, 2001), two HLA-A24-binding peptides (aa 324-332 and 461-469) (Arai et al, 2001) and an HLA-A1-restricted peptide (aa 325-333) (Schreurs et al, 2005) effectively inducing peptide-specific and tumor-lysing CTLs in vitro were described so far.

Schoers et al, 2002 identified an immunogenic HLA class II-restricted epitope (aa 672-686) by examining human T cell responses against synthetic peptides that had been selected by a prediction software. These authors demonstrated that the identified peptide is presented by HLA-DR7 molecules and derived from natural processing of hTERT in prostate cancer and other tumor cells. In a further study, the previously defined peptide comprising the aa 672-686 was demonstrated to be promiscuous and capable of inducing CD4+ T cell responses in the context of the HLA class II molecules HLA-DR1, HLA-DR7 and HLA-DR15 (Schroers et al, 2003). Moreover, these authors identified another CD4+ T cell epitope (aa 766780) that is efficiently presented by HLA-DR4, HLADR11 and HLA-DR15 molecules, naturally generated by tumor cells and elicited antigen-specific CD4+ T cell responses when used for immunization of HLA-DR4 transgenic mice.

F. Transient receptor potential (trp)-p8 The gene trp-p8 was recently identified by screening a prostate-specific substracted cDNA library (Tsavaler et al, 2001). It encodes a protein of 1104 aa with seven putative transmembrane domains that shows significant homology to a family of Ca 2+ channel proteins. By dot blot and Northern blot analyses as well as reverse transcription PCR, it has been demonstrated that trp-p8-mRNA expression in non-malignant human tissues is mainly restricted to the prostate (Tsavaler et al, 2001; Cunha et al, 2005). In addition, trp-p8 transcipts were detected in all 16 analyzed prostate cancer specimens by in situhybridization (Tsavaler et al, 2001). Quantitative RT-PCR analyses of matched samples of malignant and nonmalignant prostate tissues derived from prostatectomized patients revealed an abundant expression of the trp-p8 mRNA in all specimens and a marked level of overexpression in tumors of early stages and low grades when compared to the corresponding normal prostate tissue (Kiessling et al, 2003).

In an approach to determine the potential of trp-p8 as a target structure of specific CTLs, we used DCs pulsed with five HLA-A*0201-binding, trp-p8-specific peptides for the stimulation of autologous CD8+ T cells from prostate cancer patients (Kiessling et al, 2003). A peptide comprising the aa 187-195 was found to effectively induce CTLs and was demonstrated to be autochthonously presented on the surface of prostate cancer cells.

G. Parathyroid hormone-related protein (PTH-rp) PTH-rp is an autocrine or paracrine factor that binds to receptors on osteoblasts and induces bone formation and reabsorption. It is highly overexpressed in prostate cancer and other cancers of epithelial origin and is considered to be involved in the development of bone metastases (Guise, 1997; Francini et al, 2002). Therefore, it might represent a promising immunotherapeutical target for prostate cancer patients with bone metastases.

Two HLA-A*0201-restricted peptides (aa 59-68 and 165-173) have been identified by in vitro-stimulation protocols using autologous peptide-pulsed PBMCs from healthy donors as APCs (Francini et al, 2002). The induced peptide-specific CTLs were able to kill PTH-rpund HLA-A*0201-positive tumor cells. Recently, two other HLA-A2-fitting epitopes (aa 59-67 and 42-51) were defined inducing peptide-specific CTL responses in prostate cancer patients (Yao et al, 2005).

Furthermore, HLA-A24-binding peptides comprising the aa positions 36-44 and 102-111 were proved to be immunogenic in the activation of peptide-specific and

I. Survivin Survivin is a member of the inhibitor of apoptosis protein family and is highly overexpressed in most human tumors of epithelial and hematopoietic origin including prostate cancer (Ambrosini et al, 1997; Altieri, 2003).

Additionally, survivin expression correlates with poor prognosis of tumor disease (Swana et al, 1999). The wide expression in cancer and the almost complete absence of expression in differentiated adult tissues together with the functional role for the survival of tumor cells make 570

Cancer Therapy Vol 3, page 571 survivin an interesting target for the development of T cell-based immunotherapies.

Our group identified two HLA-A*0201-restricted peptides (aa 5-14 and 95-104) that induced peptidespecific CTL responses in vitro when presented by autologous DCs and one of these peptides (aa 95-104) was shown to evolve from intracellular processing of the protein as the CTLs effectively recognized Epstein-Barr virus-immortalized B cells transfected with survivin cDNA (Schmitz et al, 2000). By another group, the peptide spanning aa 5-14 was verified as target for immunotherapy by the lysis of survivin- and HLAA*0201-positive tumor cells by peptide-specific CTLs (Siegel et al, 2004). Using ELISPOT assay to detect survivin-specific CD8+ T cells in the blood of tumor patients Anderson et al, 2001a found in vivo reactivities against one of the previously defined peptides (aa 95-104) and a modified peptide (aa 96-104) in which the native threonine at position 2 was replaced by the better anchor residue methionine. In an additional study, multimeric complexes of this modified peptide and HLA-A2 molecules were used to isolate CD8+ T lymphocytes from a melanoma-infiltrated lymph node that specifically recognized the native peptide as well as survivin- and HLA-A2-expressing tumor cells (Anderson et al, 2001b).

A number of additional CD8+ T cell epitopes restricted to HLA-A1, HLA-A2, HLA-A3 and HLA-A11 were defined

by Reker et al, 2004 based on spontaneous peptidespecific CTL responses of tumor-infiltrating lymphocytes determined by ELISPOT assay. The positions and sequences of these peptides can be learned from Table 1.

emulsified in mineral oil. Whereas two patients had PSAreactive T cells before vaccination eight of 10 patients showed detectable PSA-reactive T cells after vaccination.

However, the frequency of PSA-reactive T cells in the circulation of patients was low. In a follow up report, 10 patients treated with JBT1001 plus GM-CSF and eight additional patients receiving JBT1001 emulsified in mineral oil were tested for !-chain expression in circulating T cells and spontaneous IL-10 secretion by PBMCs before and after vaccination (Meidenbauer et al, 2002). Prior to therapy, patients had lower !-chain expression in circulating CD3+ T cells, a higher percentage of !-chain negative CD3+ and CD4+ T cells and PBMCs producing more IL-10 than normal subjects. After vaccination, recovery of !-chain expression was observed in 50% of all patients and IL-10 secretion decreased in patients treated with JBT1001 and GM-CSF. Other clinical studies were conducted to evaluate the potential of a recombinant vaccinia virus expressing human PSA. Sanda and colleagues initiated a phase I clinical trial to determine the safety and biologic effects of recombinant vaccinia-PSA (rV-PSA) administered to six patients with recurrence of prostate cancer after radical prostatectomy (Sanda et al, 1999). Patients were treated with luteinizing hormone-releasing hormone agonist therapy until an undetectable PSA nadir was achieved and then vaccinated with rV-PSA. Treatment was well tolerated and one of six patients showed undetectable serum PSA for more than eight months after testosterone restoration. In another clinical trial, administration of rV-PSA led to stabilization of serum PSA levels in 14 of 33 prostate cancer patients for at least six months (Eder et al, 2000). Increases of at least twofold in the number of PSA-reactive T cells could be detected in five of seven evaluated patients. More recently, Gulley and colleagues administered rV-PSA to patients with metastatic androgen-independent prostate cancer (Gulley et al, 2002).

Six of 42 patients had stable disease and three of five analyzed patients showed a vaccine-induced increase of PSA-specific T lymphocytes. Furthermore, in vitrogenerated PSA-specific CTL lines of three patients were able to lyse PSA peptide-loaded APCs and prostate cancer cells.

Kaufman et al, 2004 conducted a clinical phase II study evaluating a heterologous prime/boost vaccination protocol with vaccinia and fowlpox viruses expressing PSA in prostate cancer patients with biochemical progression after local therapy. Of the eligible patients, 45.3% remained free of PSA progression at 19.1 months and 78.1% demonstrated clinical progression-free survival.

An increase in PSA-specific T cells was found in 46% of patients. Gulley et al, 2005 reported on another phase II clinical trial administering an admixture of rV-PSA plus recombinant vaccinia virus expressing the T cell costimulatory molecule B7.1/CD80 followed by booster vaccinations with fowlpox virus containing PSA in combination with standard radiotherapy. Thirteen of 17 evaluated patients with localized prostate cancer treated by the combination therapy showed an increase in PSAspecific T cells of at least threefold.

III. Vaccination of prostate cancer patients with TAA-derived peptides, proteins or DNA Following the identification of prostate cancerassociated proteins that may be suitable targets of tumorreactive T cells several clinical trials were conducted.

Noguchi et al, 2005 performed a clinical phase I/II study to determine the feasibility, toxicity, immunological and clinical responses to individualized peptide vaccination in combination with estramustine phosphate for HRPC patients. The selection of the administered peptides derived from several prostate cancer-related and epithelial cancer-related antigens was based on the measurement of peptide-specific CD8+ T cells in the blood of patients before vaccination.

Patients were immunized subcutaneously with only those peptides to which preexisting CD8 + T cells could be detected. Vaccination was well tolerated and augmentation of peptide-specific CD8+ T cells was observed. All 13 patients treated with the combination therapy showed a decrease of serum PSA levels, including six patients with a decrease of more than 50%.

Meidenbauer et al, 2000 reported on a clinical trial enrolling 10 prostate cancer patients which was based on JBT1001, a vaccine consisting of recombinant PSA with lipid A formulated in liposomes. Patients were vaccinated with JBT1001 either in combination with granulocytemacrophage colony-stimulating factor (GM-CSF) or

571

Schmitz et al: T cell-based strategies for immunotherapy of prostate cancer Other immunotherapeutic treatment modalities which were based on so-called â&#x20AC;&#x153;nakedâ&#x20AC;? DNA have also been explored. In a phase I/II clinical trial 26 prostate cancer patients with different stages of disease were immunized intradermally with varying combinations of separate DNA plasmids encoding either the extracellular domain of PSMA or the costimulatory molecule B7.2/CD86, a combined PSMA/CD86 plasmid and a replication deficient adenoviral vector expressing PSMA and GMCSF (Mincheff et al, 2000). Treatment was well tolerated.

Delayed-type hypersensitivity reactions against the PSMA plasmid were found in several patients including all patients that were initially vaccinated with the adenoviral vector expressing PSMA. Six out of 12 patients who received immunotherapy only were regarded as responders. More recently, a phase I study investigating the administration of a DNA plasmid encoding PSA in combination with GM-CSF and IL-2 to HRPC patients was conducted (Pavlenko et al, 2004). Two of three patients who received the highest dose developed a significant PSA-specific cellular immune response and a decrease in the slope of serum PSA.

PSMA-derived peptides to HRPC patients (Murphy et al, 1996; Tjoa et al, 1997). Treatment was well tolerated by all 51 patients and favourable antigen-specific cellular immune responses were observed in seven partial responders based on National Prostate Cancer Project criteria and a 50% reduction of PSA level. Following the phase I study, the same group initiated a phase II clinical trial to investigate the therapeutic efficiency of infused DCs loaded with two HLA-A*0201-restricted PSMAderived peptides. Nine partial responders were identified in a group of 33 HRPC patients which were already participants in the previous phase I study and were subsequently enrolled in the phase II trial (Tjoa et al, 1998). In addition, two complete and six partial responders were observed in a group of 25 evaluated patients with no previous immunotherapy experience (Murphy et al, 1999a). Furthermore, one complete and 10 partial responders were identified from 37 patients with presumed local recurrence of prostate cancer after primary treatment failure (Murphy et al, 1999b).

An additional clinical phase I trial which was based on the administration of peptide-loaded DCs to patients with metastatic HRPC was performed (Vonderheide et al, 2004). Five patients were vaccinated with DCs pulsed with an HLA-A*0201-restricted hTERT-derived peptide and keyhole limpet hemocyanin. No significant side effects were observed. T cells reactive against the hTERT-derived peptide were induced in two patients after vaccination. All four evaluable patients had stabilization of disease. More recently, we conducted a phase I clinical trial to evaluate the potential of DCs loaded with a cocktail consisting of HLA-A*0201-restricted peptides derived from PSA, PSMA, survivin, prostein and trp-p8 (unpublished data).

No severe side effects were noted. Four out of eight patients had a temporary decrease or stabilization of serum PSA level. In addition, three out of these four PSA responders exhibited antigen-specific T cell responses against prostein, survivin or PSMA. Small et al, 2000reported on a clinical phase I/II trial including 31 patients with HRPC. Patients were treated with enriched DC precursors preexposed in vitro to PA2024, a fusion protein consisting of human GM-CSF and PAP. Treatment was well tolerated. All patients developed immune responses to the fusion protein and 38% displayed immune responses to PAP. Six patients showed a decline in PSA level. Burch and colleagues also administered PA2024-loaded DCs to HRPC patients.

These infusions were followed by subcutaneous applications of PAP2024 without cells. Treatment was safe, induced antigen-specific cellular immunity and resulted in PSA level reduction in three out of 12 evaluated patiens (Burch et al, 2000). A subsequent phase II study demonstrated a decline in PSA level in three out of 19 evaluated patients (Burch et al, 2004).

Another clinical trial including patients with metastatic prostate cancer was based on the administration of DCs loaded with recombinant murine PAP. Minimal treatment-associated side effects were observed. All patients developed T cell immunity to mouse PAP and 11 of 21 patients to the homologous self antigen. Six of 21 patients had evidence of clinical stabilization of their

IV. Vaccination of prostate cancer patients with dendritic cells pulsed with prostate cancer-associated antigens DCs are professional APCs which display an extraordinary capacity to induce, sustain and regulate T cell responses (Banchereau and Steinman, 1998; Banchereau et al, 2000; Steinman, 2003). DCs circulate through the blood and become resident in peripheral tissues, where they continuously monitor invading pathogens. These immature DCs are particularly efficient in antigen capture but are rather ineffective in antigenprocessing and in stimulating antigen-specific T cells. DC maturation is induced by pathogens or proinflammatory cytokines. Its hallmark is the acquisition of the capacity to efficiently process and present antigens. During maturation DCs migrate from the peripheral tissues to the T cell-rich areas of secondary lymphoid organs, where they initiate antigen-specific T cell responses. Owing to their unique ability to activate naive T cells DCs evolved as promising candidates for vaccination protocols in cancer therapy (Fong and Engleman, 2000; Banchereau and Palucka, 2005; Nestle et al, 2005). The ability of TAA-loaded DCs to induce both protective and therapeutic antitumor responses has been documented in animal models (Mayordomo et al, 1995; Celluzzi et al, 1996; Nair et al, 2000). Also in human, clinical trials revealed promising immunologic and clinical effects of antigen-loaded DCs administered as a vaccine against cancer (Hsu et al, 1996; Nestle et al, 1998; Thurner et al, 1999).

In the setting of prostate cancer, clinical trials have shown that DCs pulsed with TAA-derived peptide, protein or mRNA were well tolerated, efficiently augmented antigen-specific T cell responses and exhibited partial or complete clinical effects. Thus, Murphy and colleagues conducted a phase I trial to determine the safe administration of DCs and HLA-A*0201-restricted

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previously progressing prostate cancer as determined by PSA level monitoring, computerized tomography and bone scans (Fong et al, 2001).

Barrou et al, 2004 performed a clinical trial enrolling prostate cancer patients in biochemical relapse after radical prostatectomy to assess the feasibility, safety and immunogenicity of vaccination with DCs pulsed with human recombinant PSA. Twenty-four patients received nine administrations of PSA-loaded DCs by combined intravenous, subcutaneous and intradermal routes. No severe side effects were observed, PSA-specific T cells were detected and 11 patients exhibited a transient PSA decrease.

Two other clinical plase I studies were conducted to evaluate the potential of DCs transfected with mRNA encoding TAAs. In the first trial, 13 patients with metastatic prostate cancer received PSA mRNAtransfected DCs (Heiser et al, 2002). Vaccination was well tolerated and PSA-specific T cells were detected in all patients. Six of seven evaluated patients had a significant decrease of PSA and three patients exhibited a transient molecular clearance of circulating tumor cells. In the second trial, hTERT mRNA-transfected DCs were administered to 20 patients with metastatic prostate cancer (Su et al, 2005). Expansion of hTERT-specific T cells was detected in 19 of 20 patients. Vaccination was associated with a reduction of PSA velocity and molecular clearance of circulating tumor cells.

V. Conclusion Current therapeutic approaches revealed only modest impact on survival outcomes for patients with metastatic prostate cancer. Recent advances in the identification of TAA-derived T cell epitopes and in the successful activation of tumor-reactive CTLs and CD4+ T cells paved the way for new treatment modalities for prostate cancer.

Clinical trials which were based on the in vivo-stimulation of effector T cells by the administration of peptides, proteins, DNA or TAA-pulsed DCs provide evidence that these concepts were safe and feasible. In addition, they led to the induction of antigen-specific T cells as well as clinical responses in prostate cancer patients. However, further improvement of prostate cancer therapy is required and may be achieved by combining T cell-based vaccination strategies with radio-, hormone-, chemo-, antibody- or anti-angiogenic therapy.

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The Protein Kinase C! inhibitor Rottlerin modulates bortezomib-induced apoptosis of B-cell chronic lymphocytic leukemia cells Research Article

Markus D端chler*, Rainer Hubmann, Dieter Mitteregger, Martin Hilgarth, Josef D. Schwarzmeier and Medhat Shehata Ludwig Boltzmann Institute for Cytokine Research, Medical University Vienna, Waehringer Guertel 18-20, Vienna, Austria

__________________________________________________________________________________ *Correspondence: Markus D端chler, Ludwig Boltzmann Institute for Cytokine Research, Medical University Vienna, Waehringer Guertel 18-20, Vienna, Austria; Tel. +43 1 40400 4414; Fax +43 1 40400 4461; e-mail: markus.duechler@univie.ac.at Key words: B-cell chronic lymphocytic leukemia, B-CLL, bortezomib, proteasome inhibitor, protein kinase C!, Rottlerin, apoptosis Abbreviations: Protein kinase C, (PKC); B-cell chronic lymphocytic leukemia, (B-CLL); proteasome inhibitor, (PI); 12-Otetradecanoylphorbol-13-acetate, (TPA); peripheral blood mononuclear cells, (PBMC); phosphate buffered saline, (PBS); bovine serum albumin, (BSA); Dimethylsulfoxid, (DMSO); 4',6-diamidino-2-phenylindole, (DAPI); interferon, (IFN); Diacylglycerol, (DAG) Received: 23 August 2005; Revised: 4 October 2005 Accepted: 21 November 2005; electronically published: November 2005

Summary Proteasome inhibitors (PI) have attracted interest as novel anticancer agents in B-cell chronic lymphocytic leukemia (B-CLL), since they efficiently induce apoptosis in these malignant cells in vitro . However, limited responses were observed when one of them, bortezomib, was used as monotherapy in first clinical trials. Therefore, combining bortezomib with other drugs might improve its therapeutic potential. The PKC! inhibitor Rottlerin has also been described to selectively induce apoptosis in B-CLL cells in vitro by interrupting survival signals mediated by PKC!.

In the present study, we investigated induction of apoptosis by the combined application of bortezomib and Rottlerin in vitro. Surprisingly, an inhibitory effect of Rottlerin on bortezomib-mediated apoptosis was observed in B-CLL cells. Rottlerin despite of its own pro-apoptotic activity caused a delay of the apoptotic process induced by bortezomib. Activation of PKC! by low concentrations of TPA, on the other hand, increased the apoptotic rate caused by bortezomib in B-CLL cells. In contrast, apoptosis induced by bortezomib was enhanced by Rottlerin when PBMC from healthy donors or the BL41 B-cell line were used. Our results suggest that in B-CLL cells PKC! critically contributes to their major apoptotic response to bortezomib.

Ciechanover, 2005). Due to its general function the proteasome is involved in all major cellular programs such as cell cycle, differentiation and apoptosis (Naujokat and Hoffmann, 2002). Inhibition of the proteasome by specific proteasome inhibitors has been shown to efficiently kill tumour cells in various types of leukemia and other malignancies including those which are resistant to conventional chemotherapy (Gillessen et al, 2002; Voorhees et al, 2003; Adams, 2004; Richardson et al, 2004; Rajkumar et al, 2005).

Based on the promising in vitro activity, the peptide boronic acid proteasome inhibitor bortezomib (VELCADE速, PS-341) (Adams and Kaufman, 2004) was introduced to various phase I and II clinical trials including multiple myeloma (Richardson et al, 2003; Jagannath et al, 2004), acute leukemias (Cortes et al,

I. Introduction B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the progressive accumulation of monoclonal well-differentiated B-cells exhibiting a defect in initiating apoptosis (Jewell, 2002; Guipaud et al, 2003; Chiorazzi et al, 2005). B-CLL is currently incurable, however, there is now a large number of new drugs and antibodies that may have significant activities in the treatment of this disease (Nabhan et al, 2004; Robak, 2005). Recently, proteasome inhibitors (PI) have been demonstrated to act as potent inducers of apoptosis in BCLL cells in vitro (Masdehors et al, 2000; Pahler et al, 2003; Kelley et al, 2004). Proteasome inhibitors block the ubiquitin-proteasome pathway, the major extra-lysosomal machinery for protein degradation via ubiquitylation (Fang and Weissmann, 2004; Roos-Mattjus and Sistonen, 2004; 579

Düchler et al: PKC! modulates bortezomib-induced apoptosis of BCLL cells 2004), and B-CLL (Orlowski et al, 2002; O’Connor, 2004; Goy et al, 2005). Recently, owing to high response rates to this agent reported in patients with relapsed and refractory multiple myeloma, bortezomib has been approved for this indication (Bross et al, 2004). First clinical studies with bortezomib as a monotherapy for B-CLL have shown only limited responses (O’Connor et al, 2005). This therapeutic approach might be improved by combining bortezomib with other chemotherapeutical or biological agents (An et al, 2003; Chauhan et al, 2004; Orlowski et al, 2004; Pei et al, 2004; Nikrad et al, 2005). The mode of action of proteasome inhibitors seems to be different from all pathways described for the action of other drugs making PI an ideal tool for combinatorial approaches (Goy and Gilles, 2004). Such combinations might enhance the efficacy of the treatment and simultaneously reduce toxic side effects. In the present study, combinations of bortezomib and Rottlerin, an inhibitor of PKC! were applied to B-CLL cells in vitro.

Protein kinase C (PKC) is a family of serine/threonine protein kinases which is involved in the regulation of a variety of cellular processes including proliferation, differentiation and cell death (Newton, 1995; Dempsey et al, 2000; Musashi et al, 2000; Liu et al, 2003).

At least 11 isoforms of PKC exist that differ in substrate specificities, and cell and tissue specific expression. Based on the requirements for its activation PKC! belongs to the group of the novel isoforms which are Ca2+ insensitive but are activated by diacylglycerol (DAG). PKC molecules consist of a catalytic and a regulatory domain. In their inactive state access to the catalytic domain is blocked by the regulatory domain. DAG or phorbol esters like TPA (12-O-tetradecanoylphorbol-13-acetate) bind to the regulatory domain of PKCs and induce a conformational change unmasking the catalytic domain. Alternatively, PKCs can be activated by proteolytic cleavage which separates the regulatory from the catalytic domain.

Activated PKC translocates from the cytosol to various organelles (DeVries et al, 2002; Brodie and Blumberg, 2003; Liu et al, 2003; Schmidmaier et al, 2004;). TPA mediated activation of PKC is transient, and prolonged exposure to TPA triggers ubiquitylation and degradation of PKC proteins (Shih and Floyd-Smith, 1996; Lu et al, 1998). The PKC! isoform exerts pro-apoptotic function in many cell types (Meinhardt et al, 2000; Jackson and Foster, 2004) and it is thought that the tumour promoting effect of TPA is due to PKC! depletion after activation.

In B cells PKC! fulfils specific functions (Guo et al, 2004). PKC! acts as a negative regulator of normal B cell activation and is critically involved in the development of B cell tolerance (Mecklenbrauker et al, 2002). In PKC!deficient mice B cell anergy is abrogated leading to hyperproliferation of self-reactive B cells (Miyamoto et al, 2002). In B-CLL cells, PKC! is highly expressed and is activated upon cytokine stimulation in a PI3-Kinase- and PDK1-dependent pathway (Ringshausen et al, 2002). The PKC inhibitor Rottlerin, which selectively inhibits the PKC! isoform, is able to induce apoptosis in B-CLL cells.

PKC! also controls the activity of several transcription factors. For example, the constitutive phosphorylation of STAT1 that is typical for B-CLL has

been connected to a high activity of PKC! in B-CLL cells (Battle and Frank, 2003; DeVries et al, 2004). We have shown recently, that B-CLL cells express a constitutively active form of Notch2, which might critically contribute to the malignant phenotype of B-CLL (Hubmannn et al, 2002; Schwarzmeier et al, 2005). The transcriptional activity of Notch2 is also regulated by PKC! (Hubmann et al, manuscript in preparation), and is lost in response to bortezomib treatment (Duechler et al, 2005a).

In the present work, the combined treatment of BCLL cells with bortezomib and Rottlerin was studied in vitro. Our results suggest that PKC! is involved in the execution of bortezomib mediated apoptosis.

II. Materials and methods A. Cell culture After obtaining informed consent from all patients, peripheral blood mononuclear cells (PBMC) from B-CLL patients (B-CLL cells) were isolated using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) centrifugation. Lymphoid cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (all reagents were obtained from Gibco, Life Technologies Inc., Paisley, UK) in a humidified atmosphere containing 5% CO2 at 37°C. Drug incubations were done in complete tissue culture medium. Bortezomib (VELCADE®, formerly PS-341, Millennium Pharmaceuticals, Boston, MA) was dissolved in DMSO at 10 mM, and for all incubations control samples were done containing equal amounts of DMSO. Rottlerin was purchased from Santa Cruz Biotechnology (Heidelberg, Germany) and dissolved in DMSO at 10 mM.

B. Flow cytometry Flow cytometry was performed on a FACScan" using CellQuest Pro" software (Becton Dickinson, San Jose, CA).

Annexin V and propidium iodide staining was performed to estimate the percentage of cells undergoing apoptosis using a kit from Alexis Biochemicals (Montreal, Canada) according to the suppliers instructions. All cells which were not stained by Annexin V and propidium iodide were counted as viable cells.

C. Western Blot B-CLL cells were washed with ice-cold PBS and cell lysates were prepared in 10mM Tris/HCl pH8.0; 1mM EDTA; 150mM NaCl; 0.65% NP40; supplemented with protease inhibitors. Protein concentration was determined using Bradford Assay (BioRad, Hercules, CA, USA). 20 µg total protein were separated by denaturing SDS-PAGE and transferred to PVDF membranes (BioRad). After protein transfer non-specific binding sites on the membranes were blocked with 2% non-fat milk in TBS-T (100mM Tris-HCl, 100mM NaCl, 0,1% Tween-20). The polyclonal antibody recognizing PKC! (0.2ng/ml) was applied in 1% milk/TBS-T, followed by a horseradish-peroxidase-linked goat anti-rabbit antibody (Pierce, Rockford, IL, USA).

Immunoreactive bands were visualized with SuperSignal Chemiluminescent System (Pierce).

D. Immunofluorescence staining For the visualization of mitochondria, MitoTracker Red 580 dye (Molecular Probes, Eugene, Oregon, USA) was added to the cell culture at 50nM final concentration 20 min before harvesting the cells. B-CLL cells were centrifuged to microscope glass slides at 200xg (cytospin). Cells were washed in PBS, fixed

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Cancer Therapy Vol 3, page 581 for 5 min in 1% paraformaldehyde/PBS, and permeabilized with 0.2% Triton-X100/PBS. For the detection of PKC! a polyclonal rabbit antibody from Santa Cruz Biotechnology, (Santa Cruz, Heidelberg, Germany) was used.

Antibodies were diluted in PBS/3%BSA, incubation time was 45 min and was followed by several washing steps with PBS. The cells were stained with anti-PKC! polyclonal antibody at 75ng/ml final concentration, followed by a FITC-labeled goat anti-rabbit IgG secondary antibody. To visualize cell nuclei, a fluorescent groove binding probe for DNA, 4',6-diamidino-2phenylindole (DAPI; Sigma), was added to the secondary antibody solution at 100 ng/ml. Slides were washed in PBS and mounted with mounting fluid (DAKO, Carpinteria, CA).

concentration dependent manner (Figure 1). Rottlerin was used at a constant concentration of 5µM to inhibit PKC! but not the classical PKC isoforms. Rottlerin induced apoptosis in B-CLL cells when applied as a single drug (compare “control” points of the curves in Figure 1) and increased the percentage of AnnexinV positive cells by 5% to 25% (mean 18% +/- 13%). The presence of Rottlerin reduced the apoptotic rates at 10nM and 20nM bortezomib as compared to incubation with bortezomib alone (Figure 1a). This effect was observed in all cases tested (Figure 1b).

B. Inhibition of the classical PKC isoforms has no influence on bortezomibmediated apoptosis

III. Results A. Bortezomib induced apoptosis in BCLL cells is counteracted by Rottlerin

To test whether the classical PKC isoforms also contribute to reduce bortezomib mediated apoptosis, Gö6976 was used to specifically inhibit PKC-# and -$. As shown in Figure 2, incubation of B-CLL cells with Gö6976 alone had no influence on cell viability of B-CLL cells after 24 hours in cell culture

PBMC were isolated from 20 B-CLL patients and incubated in cell culture with increasing concentrations (2.5-20nM) of bortezomib. After 24 hours cell viability was determined by Annexin V/propidium iodide staining and flowcytometric analysis. Bortezomib induced apoptosis in all B-CLL cell samples in vitro in a

Figure 1. Rottlerin counteracts bortezomib-induced apoptosis of B-CLL cells. a) One typical example showing the concentration dependence of apoptosis induction by bortezomib (filled symbols), as well as the protective effect of simultaneously applied Rottlerin (open symbols). Cells which remained un-stained by AnnexinV and propidium iodide are shown as percent viable cells. b) A collection of 15 B-CLL cell samples showing the effect of 5µM Rottlerin, 10nM bortezomib, or the combination of both on cell viability.

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Figure 2. Inhibition of the classical PKC isoforms by Gö6976 has no influence on bortezomib mediated apoptosis. B-CLL cells were incubated in vitro with increasing concentrations of bortezomib in the presence (open symbols) or absence (filled symbols) of Gö6976 and the amount of apoptotic cells was determined by annexin-V/PI staining and FACS analysis. One typical example is shown.

(first point of the graphs in Figure 2, "control").

Furthermore, no significant changes of the bortezomibinduced apoptotic rate was observed in the presence of Gö6976 indicating that the classical isoforms were not involved in PI-mediated cell death.

higher concentrations of bortezomib were necessary to obtain similar cytotoxic rates in healthy donor cells like in B-CLL cells. In contrast to the effects observed in B-CLL cells, Rottlerin increased the apoptotic rate when combined with bortezomib in PBMC from healthy donors (Figure 4), as well as in BL41, a Burkitt’s lymphoma cell line cell line (not shown). Stimulation of PKCs with TPA, on the other hand, protected healthy donor PBMC and BL41 cells from bortezomib-induced apoptosis (not shown). These results suggest that the inhibitory effect of Rottlerin on bortezomib mediated apoptosis is specific for B-CLL cells.

C. PKC stimulation increases the apoptotic rate of B-CLL cells treated with proteasome inhibitors As inhibition of PKC! suppressed bortezomibinduced apoptosis, we next investigated whether stimulation of PKC! could enhance cell death rates. TPA at concentrations of 10ng/ml or higher reduced the total amount of PKC! as shown by Western blotting (Figure 3a, lane 3). In contrast, at a TPA concentration of 1ng/ml the total amount of the protein appeared unchanged and a faster migrating band of about 32 kDa became visible, corresponding to the catalytically active fragment presumably resulting from caspase 3 cleavage (Figure 3a, lane 2). Treatment of B-CLL cell cultures with TPA alone resulted in improved cell viability. It is a well-known fact that TPA protects B-CLL cells from spontaneous and drug induced apoptosis. However, in combination with bortezomib TPA (1ng/ml) increased apoptotic rates of BCLL cells (Figure 3b). This effect was observed in 7 out of 10 B-CLL samples.

E. The inhibition of bortezomib-mediated apoptosis by Rottlerin is transient Next we asked whether the inhibition of PKC! resulted in a permanent protection against bortezomibmediated apoptosis in the fraction of B-CLL cells which survived Rottlerin treatment. B-CLL cells were incubated with a combination of bortezomib and Rottlerin for 24 and 48 hours and cell viability was determined. After 24 hours Rottlerin reduced bortezomib-induced apoptosis by about 15% relative to the treatment with bortezomib alone (Figure 5). When the drug treatment was continued during the next 24 hours similar apoptotic rates were reached in the presence or absence of Rottlerin indicating that inhibition of PKC! delays rather than prevents cell death induced by bortezomib.

D. Rottlerin increases bortezomibinduced apoptosis in PBMC from healthy donors and in BL41 cells

F. Translocation of PKC! to the mitochondrial compartment upon treatment of B-CLL cells with bortezomib is not influenced by Rottlerin

PBMC from healthy donors (n=3) were isolated and treated in cell culture with bortezomib, Rottlerin, TPA, and the drug combinations. In agreement with previously published data (Masdehors et al, 2000), about 10 times

PKC activation is accompanied by its translocation

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Cancer Therapy Vol 3, page 583 from the cytosolic to the particulate fraction. PKC! has been described to exert its pro-apoptotic function in the nucleus (DeVries et al, 2002), the endoplasmatic reticulum (Brodie and Blumberg, 2003), or at the mitochondrial membrane (Durrant et al, 2004). We visualized PKC! by immunofluorescence staining in B-CLL cells after 24 hours of cell culture. PKC! showed a broad subcellular

distribution in untreated control cells, but upon incubation with bortezomib it mainly co-localized with the mitochondrial compartment (Figure 6). Interestingly, the same translocation was found also in B-CLL cells treated with Rottlerin alone, as well as with the drug combination, showing that Rottlerin did not prevent the association of activated PKC! with the mitochondrial compartment.

Figure 3. a) Western blot showing PKC! in B-CLL cell lysates. B-CLL cells highly express PKC! (lane 1). Treatment with 1ng/ml TPA resulted in the appearance of a cleavage fragment (CF) of about 32 kDa (lane 2). At a TPA concentration of 10ng/ml total PKC! protein levels were clearly reduced (lane 3). b) TPA enhances bortezomib-induced apoptosis. One typical example showing the decrease in the percentage of viable cells with increasing concentrations of bortezomib (filled symbols) and the additional increase in cell death rates by simultaneous application of TPA (open symbols).

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Figure 4. PBMC from healthy donors (n=3) were incubated with increasing concentrations of bortezomib in the absence (filled symbols) of presence (open symbols) of 5µM Rottlerin. In contrast to the effects observed in B-CLL cells, Rottlerin increased the apoptotic rate when combined with bortezomib.

Figure 5. Rottlerin causes a delay in bortezomib induced apoptosis. B-CLL cells were either left untreated (grey bars) or were treated with 10nM bortezomib (black bars) for 24 and 48 hours in the presence or absence of Rottlerin at a concentration of 5µM as indicated.

The amount of viable cells was determined by annexin-V/PI staining.

induced apoptosis in B-CLL cells in vitro it was suggested that activated PKC! which has been detected in freshly isolated B-CLL cells, might contribute to the defect of programmed cell death in B-CLL (Ringshausen, 2002).

Surprisingly, the simultaneous application of bortezomib and Rottlerin clearly reduced the apoptotic response of BCLL cells in comparison to the application of bortezomib alone. A similar observation has been published recently

by Durrant et al, who showed that the co-incubation of bortezomib with Rottlerin suppressed apoptosis in U937 cells (Durrant et al, 2004). Consequently, when PKC! was stimulated with a low concentration of TPA the cytotoxic effect caused by bortezomib was increased. TPA was used at a concentration of 1ng/ml which protected B-CLL cells from spontaneous apoptosis but did not cause degradation of PKC! which was observed at higher TPA

IV. Discussion Proteasome inhibitors induce high rates of apoptosis in B-CLL cells in vitro, but first clinical trials with bortezomib as a single drug application have shown only limited response (O'Connor et al, 2005). Several studies suggested that combinations of proteasome inhibitors with other cytotoxic drugs increase their efficiency to induce apoptosis in leukemic cells (Chauhan et al, 2004; Pei et al, 2004). In B-CLL cells, the combination of bortezomib with purine nucleoside analogues Cladribine or Fludarabine resulted in a clear additive cytotoxicity (Duechler et al, 2005b).

In this study we combined bortezomib and Rottlerin with the aim to possibly multiply their cytotoxicity to BCLL cells. As the PKC! inhibitor Rottlerin selectively

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Figure 6 . Immunofluorescence staining of PKC! in B-CLL cells after 24 hours of cell culture. Mitochondria were loaded with MitoTracker fluorescent dye and are shown in red. PKC!, shown in green, localized to the mitochondrial compartment after incubation with bortezomib (5nM), Rottlerin (5ÂľM), or the combination of both. Nuclei are shown in blue after staining with DAPI.

concentrations. Inhibition of the classical PKC isoforms

by GĂś6976 had no influence on bortezomib-mediated apoptosis indicating that the PKC# and PKC$ isoforms did not contribute to the apoptotic response of B-CLL cells to bortezomib.

Rottlerin has been described to inhibit not only PKD! but other kinases as well (Davies et al, 2000).

Based on our data we cannot rule out the possibility that the effects observed after application of Rottlerin in BCLL cells are due to inhibition of other kinases. Several facts argue against this possibility. Active PKC! has been isolated from B-CLL cells (Ringshausen et al, 2002) and activation-induced translocation of PKC! to mitochondrial membranes have been shown in the current work (Figure 6). In addition, stimulation of PKC! with 1ng/ml TPA resulted in the appearance of the catalytically active fragment (Figure 3a) and an enhancement of bortezomib induced apoptosis. Both, TPA and Rottlerin act on PKC! thus strongly indicating involvement of this kinase. We therefore think that the effects observed after Rottlerin treatment mainly depend on inhibition of PKC! but not on other kinases. Specific downregulation of PKC! e.g. by anti-sense strategies should finally clarify this point.

Rottlerin induced apoptosis but at the same time reduced the apoptotic response mediated by bortezomib.

How is it possible that PKC! exerts pro- and antiapoptotic activities in the same cells? We think that the differences between an undisturbed and an apoptotic B-

CLL cell might explain this somehow confusing observation. In the non-apoptotic B-CLL cell PKC! is required for survival, and blocking its activity causes cell death. As soon as apoptosis is triggered e.g. by bortezomib, PKC! exerts another, pro-apoptotic function.

A similar dual function for PKC! has been described in HeLa cells (Basu et al, 2002), where its activity prevented receptor-mediated apoptosis but was required for the cell death program induced by DNA damage. Differential phosphorylation of the PKC! molecule has been suggested to determine its diverse functions (Jackson and Foster, 2004).

Our observations are consistent with a similar dual role of PKC! in B-CLL cells. Consequently one could ask whether PKC! activity in non-apoptotic B-CLL cells is required to prevent receptor-mediated cell death which has been already initiated? Some characteristics of B-CLL cells indeed support this view. B-CLL cells from most patients die in vitro by spontaneous apoptosis, but can be rescued by various stimuli like BCR engagement (Bernal et al, 2001), addition of BAFF/APRIL (He et al, 2004; Kern et al, 2004), or IFN-% (Zaki et al, 2000; Deb et al, 2003), all of which - among other signaling molecules also activate PKC!.

In vivo, activated PKC! might allow B-CLL cells to survive but in vitro due to the lack of appropriate signals to stimulate PKC! spontaneous apoptosis might occur.

These considerations together with our observations 585

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suggest that PKC! is directly involved in the defect in initiating apoptosis in B-CLL cells.

In contrast to B-CLL cells, the cytotoxic effect caused by bortezomib was increased by Rottlerin in PBMC isolated from healthy donors or BL41 cells. Again PKC! appears to be a key factor in the aberrant signaling in B-CLL cells.

Proteasome inhibitors exert their pro-apoptotic activity mainly via the mitochondrial pathway (Almond et al, 2001). In the present study we show that PKC! colocalizes with mitochondria in bortezomib-treated B-CLL cells. In apoptotic cells PKC! is proteolytically activated by caspase 3 and has been shown to facilitate cytochrome C release from mitochondrial membranes thereby promoting apoptosis (Durrant et al, 2004). In addition, PKC! has been linked to activation of caspase-3, suggesting that PKC! together with caspase 3 forms an amplification loop which accelerates apoptosis (Meinhardt et al, 2000). Inhibition of PKC! by Rottlerin might disrupt this amplification loop, prevent full activation of caspase 3 and consequently decrease the rate of cell death.

Interestingly, Rottlerin induced a similar translocation of PKC! to mitochondria like bortezomib, which might indicate a stress response common to both substances (Konishi et al, 1997).

In conclusion, the available data suggest that in nonapoptotic B-CLL cells PKC! critically supports cell survival, as inhibition of PKC! induces apoptosis in BCLL cells. When apoptosis is induced by bortezomib, PKC! might be involved in the execution of the cell death programme. Rottlerin did not completely block, but rather caused a delay of bortezomib-induced apoptosis. Whether this transient inhibitory effect of Rottlerin observed in cell culture also would occur in vivo is an important question especially in regard to possible clinical applications of this drug combination and needs further investigation.

Acknowledgements This work was supported by research grants from the Austrian “Hochschuljubiläumsstiftung”, Nr. H1232/2003, and the “Fellinger Krebsforschungsförderung”, Vienna, Austria. The proteasome inhibitor bortezomib was kindly provided by Millennium Pharmaceuticals, Boston, MA.

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