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CANCER THERAPY

Volume 4 Number 1 June, 2006

CANCER THERAPY FREE ACCESS www.cancer-therapy.org

!!!!!!!!!!!!!!!!!!!!!!!! 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

!!!!!!!!!!!!!!!!!!!!!!!! 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

!!!!!!!!!!!!!!!!!!!!!!!! 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-Gutenberg-University, 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, Kürsat, M.D., Gülhane 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., Universit채t 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., Universit채ts-Hautklinik Mannheim, Germany Schmitt, Manfred, Ph.D., Universit채t M체nchen, 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

!!!!!!!!!!!!!!!!!!!!!!!! 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

!!!!!!!!!!!!!!!!!!!!!!!! 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.cancertherapy.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. Type of articles Both review articles and original research articles will be considered. Original research articles should contain a generous introduction in addition to experimental data. The articles contain information important to a general audience as the volume is addressed to researches outside the field. There is no limit on the length of the articles provided that the subject is interesting to a general audience and covers exhaustively a field. The typical length of each manuscript is 12-60 manuscript pages (approximately 4-20 printed pages) plus Figures and Tables. Free of Charge publication, Complimentary reprints & Subscriptions There are no charges for color figures or page numbers. Corresponding authors get a one-year free subscription (hard copy) plus 25 reprints free of charge. The free subscription can be renewed for additional years by having one paper per year accepted for publication. Sections of the manuscript Each manuscript should have a Title, Authors, Affiliation, Corresponding Author (with Tel, Fax, and E-mail), Summary, and Introduction; review articles are subdivided into headings I, II, III, etc. (starting with I. Introduction) and subdivided into A, B, C, etc. You can further subdivide into 1, 2, 3, etc. Research articles are divided into Summary; I. Introduction; II. Results; III Discussion; Acknowledgments IV. Materials and Methods and References. Please include in your text citations the name of authors and year in parenthesis; for three or more authors use: (name of first author et al, with year); for two authors please use both names. Please delete hidden text for references. In the reference list, please, type references with year and Journal in boldface and provide full title of the article such as: 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. Please use Microsoft Word, font “Times” (Mac users) or “Times New Roman” (PC users) and insert Greek or other characters using the “Insert/Symbol” function in the Microsoft Word rather than simple conversion to font “Symbol”. Please boldface Figure 1, 2, 3 etc. as well as Table 1, 2, etc. throughout the text. Please provide the highest quality of prints of your Figures; whenever possible, please provide in addition an electronic version of your figures (optional). Corresponding authors are kindly requested to provide a color (or black/white) head photo of themselves (preferably 4x5 cm or any size), as we shall include these in the publication. Submission and reviewing Peer reviewing is by members of the Editorial Board and external referees. Please suggest 2-3 reviewers providing their electronic addresses, mailing addresses and telephone/fax numbers. Authors are being sent page proofs. Cancer Therapy (Volume 1, 2003) is published on high quality paper with excellent reproduction of color figures and electronically. Reviewing is completed within 5-15 days from receiving the manuscript. Articles accepted without revisions (i.e., review articles) will be published online (www.cancertherapy.org) in approximately 1 month following submission. Please submit an electronic version of full text and figures preferably in jpeg format. The electronic version of the figures will be used for the rapid reviewing process. High quality prints or photograph of the figures and the original with one copy should be sent via express mail to the Editorial Office. Citation in MedLine Articles accepted for publication by GTMB or Cancer Therapy can be included in MedLine (PubMed) as full articles upon the request of authors provided that the authors have completed their published work under a government grant by NIH (or EU/Japan government grant). If this is you case, please consult the NIH Manuscript Submission System http://www.nihms.nih.gov/. 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.

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Table of contents Cancer Therapy Vol 4 Number 1, June 2006

Pages

Type of Article

Article title

Authors (corresponding author is in boldface)

1-12

Review Article

Payal R. Sheth and Stanley J. Watowich

13-26

Review Article

27-34

Review Article

35-46

Review Article

47-72

Review Article

73-80

Review Article

81-98

Review Article

Biochemical ground-rules regulating c-MET !receptor tyrosine kinase activation and signaling! Overcoming tumor resistance to immunotherapy! The role of IDO in immune system evasion of !malignancy: Another piece to the tolerance puzzle! Vitamin E analogs as anti-cancer agents: The role of !modulation of apoptosis signalling pathways ! Sensitivity and resistance of human cancer cells to !TRAIL: mechanisms and therapeutical perspectives! Selenium and prostate cancer: biological pathways and biochemical nuances Cancer immunotherapy

99-124

Review Article

125-134

Research Article

135-142

Research Article

143-152

Review Article

Molecular basis for androgen independency in prostate cancer

153-162

Research Article

Nutritional patterns and lung cancer risk in Uruguayan men

Role of platelet derived endothelial cell growth factor / thymidine phosphorylase in health and disease The protective effect of vitamin C on Azathioprine induced seminiferous tubular structural changes and cytogenetic toxicity in albino rats The usefulness of oral TS-1 treatment for potentially curable gastric cancer patients with intraperitoneal free cancer cells

Lana Y. Schumacher and Antoni Ribas Jeannine A. Villella, Kunle Odunsi, Shashikant Lele Lan-Feng Dong, Xiu-Fang Wang, Yan Zhao, Marco Tomasetti, Kun Wu, J! iri Neuzil Luca Pasquini, Eleonora Petrucci, Roberta Riccioni, Alessia Petronelli and Ugo !Testa Vasundara Venkateswaran

Carole L. Berger, Joshua Shofner, Juan Gabriel Vasquez, Kavita Mariwalla and Richard L. Edelson Michiel de Bruin, Olaf H. Temmink, Klaas Hoekman, Herbert M. Pinedo, Godefridus J. Peters Fardous S. Karawya and Abeer F. ElNahas

Yutaka Yonemura, Yoshio Endou, Etsurou Bando, Taiichi Kawamura, Gorou Tsukiyama, Shouji Takahashi, Naoko Sakamoto, Kiyoshi Tone, Kimihide Kusafuka, Ichirou Itoh, Masashi Kimura, Masakazu Fukushima, Takuma Sasaki, Narikazu Boku Jesús Gil Eduardo De Stefani, Alvaro L. Ronco, Paolo Boffetta, Hugo DeneoPellegrini, Pelayo Correa, Giselle Acosta, Luis Piñeyro Gutiérrez and María Mendilaharsu

163-170

Research Article

Advantages of a unique DNA-based vaccine in comparison to paclitaxel in treatment of an established intracerebral breast cancer in mice

Terry Lichtor, Roberta P Glick, Henry Lin, Amla Chopra, InSug OSullivan and Edward P Cohen

Cancer Therapy Vol 3, page 1 Cancer Therapy Vol 3, 1-12, 2005

Biochemical ground-rules regulating c-MET receptor tyrosine kinase activation and signaling Review Article

Payal R. Sheth and Stanley J. Watowich* Department of Biochemistry and Molecular Biology and the Sealy Center for Structural Biology, University of Texas Medical Branch at Galveston, TX 77555-0645

__________________________________________________________________________________ *Correspondence: Stanley J. Watowich; Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, TX 77555-0647, USA; Tel. 1 409 747 4748; Fax. 1-409 747 4745; E-mail. watowich@xray.utmb.edu Keywords: c-MET, receptor tyrosine kinase, dimerization, phosphorylation, signaling, autophosphorylation, dephosphorylation Abbreviations: activation loop, (AL); adenosine triphosphate, (ATP) epidermal growth factor receptor, (EGFR); epidermal growth factor, (EGF); fibroblast growth factor receptor, (FGFR); fibroblast growth factor, (FGF); Grb2-associated binder 1, (Gab1); hepatocytegrowth factor, (HGF); leukocyte common antigen-related, (LAR); phosphatidylinositol 3-kinase, (PI3K); phospholipase C, (PLC); PI3K protein kinase B, (PKB); platelet-derived growth factor, (PDGF); protein tyrosine binding, (PTP); protein-tyrosine phosphatase, (PTP); receptor tyrosine kinase, (RTK); regulator of kinase, (Crk); scatter factor, (SF); vascular endothelial growth factor receptor, (VEGFR) Received: 7 November 2005; Accepted: 9 January 2006; electronically published: January 2006

Summary c-MET receptor tyrosine kinase-mediated signaling governs numerous important cellular responses including cellular proliferation, differentiation, migration and apoptosis. Deregulation of these signals results in malignant behaviors, often leading to cancers. While the identity of many signaling molecules that are activated following hepatocyte-growth factor (HGF)-induced activation of c-MET have been established, little is known about the mechanism of activation of c-MET. From a therapeutic perspective, it is necessary to understand the detailed molecular mechanisms regulating c-MET activation to selectively target these molecules. Classically it has been believed that the sole role of ligand-induced dimerization was to autophosphorylate the receptor, thereby activating receptor’s kinase catalytic function. However, recent studies have shown that dimerization-induced changes in the kinetic, thermodynamic and dephosphorylation properties of c-MET work synergistically to selectively induce specific signaling from the dimeric and not the monomeric receptor. In this review, we highlight biochemical studies of c-MET and related RTKs that are consistent with a dynamic equilibrium mechanism of c-MET activation. Although, the proposed mechanism differs from the traditional view of the RTK activation, it successfully explains all the relevant experimental data in the literature.

I. Introduction

II. c-MET receptor tyrosine kinase (RTK) and hepatocyte growth factor (HGF)/scatter factor (SF)

The strength and duration of numerous intracellular signaling responses are dependent on c-MET activation, defined as sustained c-MET phosphorylation and subsequent downstream signaling. c-MET activation is a critical and tightly regulated process in normal functioning of cells; aberrant signaling has been linked to pathological conditions including tumorigenesis and metastasis (Birchmeier et al, 2003). Thus, blocking c-MET activation may be an effective strategy to control these conditions. In this review, we highlight some of the significant advances towards understanding c-MET signaling, with particular emphasis on the structural and biochemical basis of cMET activation.

A. c-MET structure c-MET, the receptor tyrosine kinase (RTK) for hepatocyte growth factor (HGF)/scatter factor (SF), was first identified as an oncogene mediating the chemically induced transformation of a human osteogenic sarcoma cell line (Cooper et al, 1984). Cellular physiological functions of c-MET include, but are not limited to, proliferation, differentiation, motility and survival. c-MET is single-pass transmembrane glycoproteins that consist of an extracellular region that possesses the specificity for the ligands, and a cytoplasmic region that harbors the tyrosine kinase catalytic activity (Ullrich et al, 1990; Pazin et al,

1

Sheth and watowich: Biochemical regulation of c-MET RTK activation and signaling 1992; Hubbard et al, 2000). Related members of the RTK family include receptors for epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and insulin. While the intracellular domains of different RTKs are highly conserved, the extracellular domains contain multiple structurally distinct regions that form the basis of further classification of RTKs (Hubbardet al, 2000; Blume-Jensen et al, 2001). c-MET sub-family of RTKs includes MET, RON, and SEA. These receptors have a short !-chain and a longer "-chain linked together by a disulfide bond (Huff et al, 1993; Ronsin et al, 1993). This heterodimeric structure results from furin cleavage of a single-chain precursor. The mature form of c-MET constitutes an extracellular 45kDa !-chain and a 190kDa membrane-spanning "chain. The "-chain consists of an extracellular region, a membrane-spanning region and a cytoplasmic region containing the signaling elements - the juxtamembrane element, the catalytic domain and the carboxy-terminal tail (Figure 1). The extracellular region contains a sema domain with marked structural similarity to extracellular domains of semaphorins and plexins (Winberg et al, 1998), a small cysteine rich PSI domain similar to those found in plexins, semaphorins and integrins (Bork et al, 1999) and four IgG-like domains with similarity to the IgG domains present in plexins and transcription factors (Ohta K, et al, 1995). Structural and biochemical studies have shown that c-MET sema domain forms a high-affinity binding site for HGF (Gherardi et al, 2003; Stamos et al, 2004). The residues critical for HGF binding in c-MET have been mapped and are contained within the !-chain and 212 residues of the "-chain (Gherardi et al, 2003). Recent structural studies have revealed the domain architecture of the c-MET extracellular region, and have shed light on the mechanism of HGF-c-MET interactions (Kozlov et al, 2004; Stamos et al, 2004).

The cytoplasmic catalytic domain of c-MET is highly conserved amongst RTKs. Several structures of this domain in an unphosphorylated and phosphorylated state have been determined (Hubbard et al, 1994; Mohammadi et al, 1996, 1998; Hubbard, 1997; Schiering et al, 2003). The catalytic domain folds into distinct N-terminal and Cterminal lobes,connected via a flexible polypeptide linker. The N-terminal and C-terminal lobes are formed predominantly by "-sheets and !-helices respectively. The ATP substrate binds within a cleft formed between the two lobes, and the peptide substrate binds to the C-terminal lobe. Crystal structure of insulin receptor (IR) catalytic domain in the phosphorylated and unphosphorylated state provided a structural basis of the observed increase in the catalytic activity upon activation loop (AL) phosphorylation. The AL is a segment of amino acids within the catalytic domain that contains one or more tyrosine residues that upon autophosphorylation increase the kinase catalytic activity. Within apo IR, the AL traverses the cleft between the N-terminal and the C terminal lobes, thus obstructing the ATP binding site (Hubbard et al, 1994). The AL conformation in the unphosphorylated and phosphorylated forms in IR, showed dramatic differences, which impact the ability of ATP to access the kinase catalytic site (Hubbard, 1997). In FGFR, autoinhibition occurs via a different mechanism: a proline residue in the AL together with flanking residues occlude the substrate binding site (Mohammadi et al, 1996). Crystal structures of apo and inhibitor-bound forms of the c-MET catalytic domain showed the characteristic kinase bilobal structure described above (Schiering et al, 2003). The AL loop residues in the apo c-MET were disordered. However, the structure of the c-MET: inhibitor binary complex showed

Figure 1 . Domain map of c-MET. c-MET is composed of an ! and " chain linked together via a single disulfide bond. The " chain includes the extracellular, transmembrane (TM), and the cytoplasmic regions. The cytoplasmic region contains the signaling apparatus consisting of the juxtamembrane region (JM), the kinase domain (KD) and carboxy-terminal tail (CT). HGF binds to the extracellular region of c-MET, and signal transduction is mediated by in part by phosphorylation of residues in the multifunctional docking site (Y1349, Y1356 and Y1365). c-MET catalytic activity is positively regulated by the phosphorylation of tyrosine residues 1231,1234 and 1235 in the catalytic domain. Phosphorylation of juxtamembrane residue Y1003 is important for c-MET degradation. The important effectors of c-MET are also shown.

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Cancer Therapy Vol 3, page 3 a unique AL conformation. This conformation may represent a quasi-stable intermediate state along the transition pathway between phosphorylated and unphosphorylated AL conformations, although it is possible this conformation is an artifact arising from mutations to the conserved residues within the AL.

the binding stoichiometry of EGF to receptor was 1:1, and the dimeric complex was stabilized solely through receptor-receptor interactions formed upon ligand binding (Ogiso et al, 2002). The mechanism utilized by HGF to induce c-MET dimerization remained largely elusive until recently, when the crystal structure of the HGF "-chain and c-MET sema domain highlighted a possible dimer interface between the ligand-receptor pair, and suggested a potential 2:2 HGF:c-MET complex (Stamos et al, 2004). Future structural studies on intact HGF and c-MET ectodomain would shed light on the structural-basis of recruitment of HGF by c-MET, and subsequent c-MET dimerization induced by HGF. Interestingly, cross-linking c-MET receptors by specific antibodies to the extracellular domain can trigger c-MET signaling implying that dimerization is sufficient to activate c-MET (Prat et al, 1998). Irrespective of its mode of dimerization, autophosphorylation of c-MET tyrosines necessary for signaling occurs after dimerization and presumably, by transphosphorylation between the catalytic domains of dimeric c-MET. The biochemical events regulating c-MET signaling have been recently elucidated (as discussed below), although, the structural basis for c-MET autophosphorylation upon HGF binding remains largely unclear. Tyrosines Y1231, Y1234 and Y1235 in the AL of the c-MET catalytic domain have been shown to be phosphorylated in response to HGF-induced c-MET dimerization (Figure 1). The presence of three phosphotyrosine sites in the AL is also a characteristic of the insulin receptor, a disulfide-linked constitutive dimer. While the phosphorylation of AL tyrosines is important for increased c-MET kinase activity (Rodrigues et al, 1994), the phosphorylation of carboxy-terminal tail tyrosines Y1349, Y1356 and Y1365 (Birchmeier et al, 2003) is required for the recruitment of cytoplasmic signaling proteins with Src homology-2 (SH2) and protein tyrosine binding (PTP) domains. Phenylalanine substitution at residues Y1349 and Y1356 render c-MET functionally impaired in its ability to induce proliferation, motility, differentiation and survival (Weidner et al, 1995). In addition, phosphorylation of Y1003 within the juxtamembrane region appears to be critical for receptor degradation (Peschard et al, 2001, 2004).

B. HGF/SF structure The ligand for c-MET was independently identified by two different laboratories as a mitogen for hepatocytes, HGF and a SF in fibroblasts (Stoker et al, 1986, 1987; Nakamuraet al, 1989). Since its discovery, HGF has been shown to elicit plietropic cellular responses including mitogenesis, motility and morphogenesis. HGF/SF is synthesized as an inactive single-chain precursor that is proteolytically cleaved to form an active disulfide-linked heterodimer. Both the single-chain precursor as well as disulfide-linked heterodimer appear to bind c-MET with high affinity, however, c-MET activation occurs only by the cleaved mature form of the ligand (Lokker et al, 1992). HGF shows sequence homology to the plasminogenrelated growth factor family: these proteins have a similar cleavage-mediated activation mechanism. The 69 kDa !chain of HGF consists of an N-terminal domain (N), followed by four kringle domains (K1-K4) (Lokker et al, 1992). The 34 kDa "-chain forms a conserved proteaselike domain; this domain is inactive due to substitution of required active site serine and histidine residues. The HGF residues that form the receptor binding site are unknown, although a number of studies indicate that the !- and "chains have distinct roles in c-MET binding, and subsequent dimerization (Ultsch et al, 1998; Lietha et al, 2001; Gherardi et al, 2003; Stamos et al, 2004). Several truncated forms of the !-chain region, including NK1, bind c-MET with high affinity. HGF is also a high-affinity ligand for heparan sulfate proteoglycans. However, unlike fibroblast growth factor receptor (FGFR), this interaction does not appear to be critical for c-MET activation (Lokker et al, 1992; DiGabriele et al, 1998; Hartmannet al, 1998). The "-chain of HGF, which harbors the serineprotease-like catalytic domain, has also been shown to bind the sema domain within c-MET, albeit with relatively lower affinity (Stamos et al, 2004).

C. HGF-induced c-MET dimerization and activation

III. c-MET functions A. c-MET signaling

Normal signaling by RTKs requires ligand-induced receptor oligomerization and tyrosine phosphorylation of the cytoplasmic domains of the receptor. Although ligandmediated receptor dimerization appears to be a common event preceding RTK activation, structures of several receptor ectodomains bound to their cognate ligands showed RTKs used different binding modes to accomplish dimerization. In vascular endothelial growth factor receptor (VEGFR), dimerization was induced by binding of dimeric ligand (Wiesmann et al, 1997), whereas FGFR bound monomeric FGF and the dimeric complex was stabilized by heparin cofactors (Schlessinger et al, 2000; Mohammadi et al, 2005). Recent crystallographic studies of epidermal growth factor receptor (EGFR) showed that

Functional genetic studies of c-MET and HGF have conclusively revealed an indispensable role of these molecules in mammalian development. HGF -/- and cMET -/- mice die in utero after incurring severe placental and live defects, along with disruption in the migration of myogenic precursors into the limb bud (Bladt et al, 1995; Schmidt et al, 1995; Uehara et al, 1995). Furthermore, in adults, c-MET and HGF are widely expressed, and c-MET signaling has been shown to be important for tissue repair and organ regeneration (Michalopoulos et al, 1997; Matsumoto et al, 2001). In the recent years, extensive studies have been conducted to elucidate the mechanism by which HGF/c-MET regulate such diverse physiological responses. 3

Sheth and watowich: Biochemical regulation of c-MET RTK activation and signaling HGF-activated c-MET recruits cytoplasmic signaling molecules such as Grb2-associated binder 1 (Gab1), growth factor receptor-bound protein (Grb2), phosphatidylinositol 3-kinase (PI3K), SH2-containing protein (Shc) via a unique multisubstrate docking site that is conserved in the c-MET family of RTKs (Ponzetto et al, 1994). This docking site encompasses phosphorylated Y1349, Y1356 and adjacent residues. Y1365 has also been implicated in the c-MET-initiated morphogenesis, although the signaling molecules that interact with this cMET site are largely unknown (Weidner et al, 1995). Recruitment of the signaling molecules results in the activation of specific signaling pathways that regulate multiple cellular processes including proliferation, disruption of intracellular junctions, migration and survival. Furthermore, c-MET signaling is also involved in complex processes such as cellular differentiation and formation of branching tubules. Some of the wellcharacterized signaling pathways activated by c-MET are Ras-MAPK, PI3K, Src and Stat3 (Bertotti et al, 2003; Birchmeier et al, 2003). Although several researchers have tried to link individual effector molecules and/or specific signaling pathways to a particular cellular response, it is becoming increasingly apparent that HGF-induced c-MET signaling is complex and branches into distinct but interacting cascades. The multiadapter Gab1 plays a critical role in mediating c-MET signaling by providing a scaffold for simultaneous binding several signaling molecules. The central role of Gab1 in c-MET signaling is evident from the phenotype of Gab1 -/- mice, which show the characteristic placental, liver and muscle defects seen in cMET null mice (Sachs et al, 2000). Upon HGF stimulation, Gab1 directly interacts with phosphorylated cMET, via a unique Met-binding domain, which is not present in other members of the Gab family, and indirectly interacts with phosphorylated c-MET via Grb2 (Lock et al, 2000). The c-MET-Gab1 interaction appears to be critical for stimulating branching morphogenesis (Maroun et al, 1999). Phosphorylation of specific Gab1 tyrosines creates sites for binding the SH2 domain of Shp2, a proteintyrosine phosphatase (PTP) (Gu et al, 2003). The Shp2Gab1 interaction plays an important role inactivating the Erk/MAPK pathway (Gu et al, 2003; Schaeper et al, 2000). Mutations that disrupt Gab1-Shp2 binding result in a phenotype incapable of activating the Erk/MAPK pathway. Although Shp2 is believed to act upstream of Ras and Raf, the direct effectors of Shp2 are currently unknown. Interestingly, recent studies show that Gab1 can directly interact with Erk1/2 via its Met-binding domain and this interaction is critical for transporting Erk1/2 to the nucleus (Osawa et al, 2004). However, the significance of this interaction for c-MET signaling is unclear. Upon HGF stimulation, Gab1 also interacts with CT10 regulator of kinase (Crk), phospholipase C (PLC), PI3K and Shc. Signaling from Gab1 and Crk appears to be important for motility (Schaeper et al, 2000), whereas the Gab1-PLC and Gab1-Shp2 interactions have been shown to be important for branching morphogenesis (Gual et al, 2000; Maroun et al, 2000).

Another important adapter molecule for c-MET is Grb2, which possesses a SH2 domain and multiple SH3 domains. Grb2 constitutively associates with Sos, a Rasspecific guanine nucleotide exchange factor. Grb2 binds phosphorylated RTKs via its SH2 domain, thereby shuttling the Sos to the plasma membrane, where Ras is localized (Ponzetto et al, 1994). This sequence of events activates Ras, which then activates the Raf1 serine threonine kinase. Raf1 activates the MAPK signaling pathway by phosphorylating MEK, which in turn phosphorylatesthe MAP kinase (Campbell et al, 1998). As mentioned earlier, Grb2 also provides a high-affinity binding site for Gab1. Grb2 has been implicated in cMET-mediated proliferation, transformation and motility. Upon its phosphorylation Grb2 is also able to interact with Shc, which can also directly bind c-MET (Pelicci et al, 1995). The SH2 domain of the effector protein PI3K has been shown to bind phosphorylated c-MET (Ponzetto et al, 1994). In addition, PI3K indirectly interacts with c-MET via Gab1 (Holgado-Madruga et al, 1996). Several studies have concluded that PI3K mediates most of the METinduced signaling responses namely- mitogenesis, motility, and morphogenesis (Royal et al, 1995, 1997; Khwaja et al, 1998; Potempa et al, 1998). Furthermore the PI3K protein kinase B (PKB)/Akt pathway, which mediates MET-induced scattering and branching morphogenesis (Royal et al, 1997), is also the main mediator for cell survival (Xiao et al, 2001). Other proteins reportedly recruited to c-MET phosphotyrosine docking sites include Shc, Src and Stat3. Shc and Src are involved in cellular proliferation and motility, Stat3 is involved in branching morphogenesis, and Stat3 and Src are also involved in cellular transformation (Ponzetto et al, 1993; Pelicci et al, 1995; Boccaccio et al, 1998; Rahimi et al, 1998; Zhang et al, 2002). Phosphorylation of c-MET Y1003 is important for recruitment of c-Cbl, a member of the E3 ubiquitin ligase family (Preschard et al, 2001). c-Cbl has also shown to be recruited indirectly to the MET-signaling complex via interactions with Grb2. The c-Cbl-c-MET interaction appears to be critical for MET ubiquitination and degradation. Finally, several transmembrane proteins namely !6"4 integrin (Trusolino et al, 2001), Plexin B1 (Giordano et al, 2002; Basile et al, 2005), and CD44 (Orian-Rousseau et al, 2002) have also been shown to associate with c-MET, although the significance of these interactions for c-MET signaling in vivo is unclear. Thus HGF-activated c-MET triggers complex cellular responses by activating interacting signaling pathways.

B. Aberrant c-MET regulation and human malignancies Aberrant regulation of c-MET signaling has emerged as a likely causative element for a number of human malignancies. Abnormal activation of c-MET can occur via different mechanisms, some of the reported mechanisms include c-MET or HGF overexpression, and c-MET mutations (Figure 2). c-MET activation and signaling is clearly deregulated in several osteosarcomas, glioblastomas and melanoma, where c-MET and HGF 4

Cancer Therapy Vol 3, page 5 have been observed to be constitutively overexpressed (Koochekpouret al, 1997; Fukuda et al, 1998; Hendrix et al, 1998; Birchmeier et al, 2003). These observations are further strengthened by the evidence of c-MET and/or HGF expression in carcinomas, and other types of human solid tumors and their metastasis (Birchmeieret al, 2003). Furthermore, mouse and human cells that ectopically overexpress HGF or c-MET become tumorigenic and metastatic in athymic nude mice (Rong et al, 1994). A large number of sporadic and germline mutations of cMET have been identified in human renal papillary carcinomas (Danilkovitch-Miagkova et al, 2002) and homologous c-MET mutations produce distinct tumor profiles in mice (Graveel et al, 2004). These mutations occur within the c-MET kinase domain, often making it capable of constitutive signaling. Mutations in the c-MET juxtamembrane domain, a region important for c-Cbl binding have been observed in gastric and lung cancers (Lee et al, 2000; Ma et al, 2003). Recently, mutations in extracellular semaphorin domain that is important for HGF binding, were identified in lung cancers (Ma et al, 2003; Ma et al, 2005). The role of c-MET in physiological processes such as proliferation, survival, invasion and angiogenesis could point to its involvement in corresponding stages during tumor progression. c-MET signaling has also repeatedly emerged as a pathway that is exploited by several pathogens including Listeria monocytogenes, Plasmodium spp. and Helicobacter pylori (Figure 2) (Shen et al, 2000; Carrolo et al, 2003; Churin et al, 2003). InlB, a listerial protein was identified as a bacterial agonist for c-MET and shown to mimic HGF-induced c-MET activation, endocytosis (Ireton et al, 1999; Li et al, 2005) and signaling (Shen et al, 2000). H. pylori CagA protein also activated c-MET, although by a distinct mechanism. The CagA- induced cMET signaling could be important for H. pylori-induced cancer onset and tumor progression (Churinet al, 2003). Contrary to Listeria and H pylori, Plasmodium, the

causative agent for malaria, did not directly interact with c-MET, but exploited HGF-c-MET signaling to make the host cell more susceptible to infection (Carrollo et al, 2003).

IV. Regulating activity

c-MET

enzymatic

HGF-mediated dimerization facilitates c-MET autophosphorylation. The kinetics of c-MET autophosphorylation has not been extensively studied, although the phosphotyrosine sites and their role in c-MET signaling is well-characterized (as reviewed above). Phosphorylation of Y1231, Y1234 and Y1235 in the kinase domain AL has been reported to modulate c-MET catalytic activity (Rodrigues et al, 1994). The correlation between AL phosphorylation and increased kinase catalytic activity has been extensively documented in a number of RTKs (Cobbet et al, 1989; Parast et al, 1998; Murray et al, 2001). In IR, where insulin binding induces receptor activation of a constitutive dimeric receptor, autophosphorylation kinetics were observed to follow a two phase model where the ligand activated receptor had a prolonged fast phase compared to the non-ligand stimulated receptor (Kohanski, 1993). Murray et al, determined the kinetic parameters for phosphorylated and unphosphorylated Tie2 cytoplasmic kinase domain and showed that phosphorylation resulted in a 2-5-fold decrease in substrate KM relative to the unphosphorylated kinase (Murray et al, 2001). Parast et al also showed an order of magnitude increase in the catalytic activity of the phosphorylated VEGFR2 tyrosine kinase domain versus unphosphorylated receptor (Parast et al, 1998). The crystal structure of IR in its phosphorylated and unphosphorylated forms provided a structural interpretation for how AL phosphorylation might modulate kinase activity (Hubbard et al, 1994; Hubbard, 1997). In the phosphorylated state, the AL adopted a

Figure 2. c-MET signaling and function. HGF-mediated c-MET signaling is important for several physiological processes including cell proliferation, differentiation and survival. Aberrant regulation of c-MET signaling by HGF/c-MET overexpression or c-MET mutations is associated with tumorigenesis and metastasis. Furthermore, c-MET signaling has been shown to be exploited by several pathogens, including Listeria monocytogenes, Helicobacter pylori and malarianparasite Plasmodium spp for tissue invasion and pathogen dissemination.

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Sheth and watowich: Biochemical regulation of c-MET RTK activation and signaling conformation that was more amenable to binding ATP and tyrosine-containing peptide substrate (Hubbard, 1997). These studies demonstrated the importance of the phosphorylation state of RTKs in modulating their kinase activity. However, these studies but did not address whether oligomerization could impact these parameters, although Hubbard et al, hypothesized that dimer formation could stabilize the “flipped out” activation loop conformation in a catalysis favorable position (Hubbard, 1997). The mechanism of autophosphorylation within the oligomeric RTK is still elusive, although evidence for intramolecular (i.e. cis) (Weber et al, 1984; Bertics et al, 1985; Biswas et al, 1985; Villalba et al, 1989), intermolecular (i.e. trans) mechanism (Yarden et al, 1987; Cobb et al, 1989; Treadway et al, 1991; Sherrill, 1997) as well as sequential cis/trans mechanisms (Iwasaki et al, 1997) exist. Structural studies support a trans mechanism of AL autophosphorylation within IR. In this system the AL tyrosine is believed to bind to the kinase catalytic site in a cis fashion, but cannot be phosphorylated due to steric constraints that prevent simultaneous binding of MgATP when the tyrosine is bound to the IR active site (Hubbard et al, 1994). The kinetic properties of several RTKs have been characterized and the reaction model is dependent on the purification process and the constructs used. Both the EGF receptor (Posner et al, 1992; Ward et al, 1994) and the IR (Walker et al, 1987; Yuan et al, 1990) were consistent with a rapid equilibrium random order mechanism, while the TrkA receptor showed an ordered sequential scheme (Angeles et al, 1998). In contrast, kinetic studies on the Rous sarcoma virus pp60src supported a steady state ordered bi-bi mechanism with ATP binding occurring first (Wong et al, 1984). The VEGFR was characterized as a hybrid of the rapid equilibrium random order and sequential mechanisms (Parast et al, 1998). Unfortunately, in some studies, an isolated kinase domain was used and in other studies, an immunoprecipitated whole receptor or kinase domain was used, making the direct comparison between these studies difficult. This difficulty was highlighted by Cheng and Koland, who showed that the binding properties of the EGF receptor were dependent on the form of the receptor studied, as the whole cytoplasmic domain had 10-fold greater affinity for ATP relative to the isolated kinase domain (Cheng et al, 1996). PTPs catalyze dephosphorylation of ligandstimulated and unstimulated c-MET (Villa-Moruzzi et al, 1993; Sheth et al, 2005). Recent studies have shown that RTK phosphorylation was dynamically regulated by competing autophosphorylation and dephosphorylation rates (Posner et al, 1994; Bohmer et al, 1995; Baxter et al, 1998; Shethet al, 2005). Regulation of c-MET by PTPs is poorly understood, although studies using substrate trapping mutants, antisense RNA, and phosphotyrosine peptides have proposed DEP-1 (CD148/PTP-#), PTP-S and leukocyte common antigen-related (LAR) to be potentially involved in c-MET dephosphorylation (Kulas et al, 1996; Villa-Moruzzi et al, 1998; Palka et al, 2003). Moreover, DEP-1 was observed to preferentially dephosphorylate the carboxyl-terminal Y1349 and Y1365

in c-MET, suggesting that phosphatase site-specific preferences might be an additional mechanism for regulating receptor signaling (Palka et al, 2003). Detailed animal model or cell culture studies have yet to substantiate a role of these putative PTPs in c-MET signaling. The classical RTK activation model consists of dimerization-mediated RTK autophosphorylation, which in turn activates kinase activity of the receptor. Thus, activation of the kinase activity of RTKs has been synonymously used for RTK activation. However, in vitro studies using isolated kinase domains and ex vivo studies using phosphatase inhibitors have shown conclusively that monomeric receptors can be rapidly phosphorylated on tyrosine residues involved in intracellular signal propagation (Posner et al, 1994; Baxter et al, 1998; Sheth et al, 2005). Thus, it is clear that ligand-induced receptor oligomerization is not necessary for kinase activity. Furthermore our previous studies have shown that oligomerization modifies the thermodynamic and kinetic properties of the MET receptor independent of the autophosphorylation reaction, such that dimeric phosphorylated MET more efficiently phosphorylates substrate molecules than the similarly phosphorylated monomeric MET (Figure 3) (Hays et al, 2003, 2004). Given the conformational flexibility observed in kinase structures, it is reasonable to postulate that observed biochemical differences between monomeric and dimeric MET result from dimerization-induced conformational changes, although structural data to unequivocally support this hypothesis has not been obtained. Kinase activity is additionally regulated by receptor phosphorylation levels, in particular the phosphorylation state of tyrosines within the receptor activation loop (Parast et al, 1998; Murray et al, 2001). Moreover, the extent of receptor phosphorylation is regulated by competing autophosphorylation and dephosphorylation reactions which in turn are modulated by the receptor’s oligomeric state (Kohanski, 1993; Baer et al, 2001; Shimizu et al, 2001; Sheth et al, 2005). Thus, receptor oligomerization can directly modulate kinase activity and can indirectly modulate kinase activity by modulating autophosphorylation and dephosphorylation rates which impact receptor phosphorylation levels and in turn affect kinase activity (Figure 4). There exists a complex feedforward loop between phosphorylation state, oligomerization state, and kinase activity which can effectively work to amplify and sharpen the separation between inactive and active c-MET states. A model that includes these oligomerization-dependent changes is necessary to provide a complete understanding of the molecular events that control c-MET (or RTK) activation, where activation is defined as receptor phosphorylation and subsequent downstream signaling. We have recently built a mathematical model that incorporates the above mentioned synergistic feed-forward reactions for RTK activation. Although, the details of the model will be described elsewhere (Sheth et al, 2005), the model was described based on several intermediates occurring in the c-MET activation process including the monomeric and dimeric c-MET in their phosphorylated

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Cancer Therapy Vol 3, page 7

Figure 3. Biochemical parameters for monomeric and dimeric c-MET. The kinetic and thermodynamic parameters that regulate c-MET phosphorylation (MET) and subsequent phosphorylation of substrate molecules (S) by c-MET are tabulated. The rate constants (k), catalytic efficiency (kcat) and substrate affinity (K D) for c-MET autophosphorylation and substrate phosphorylation reactions are shown. Dephosphorylation negatively regulates c-MET phosphorylation. The kinetics of PTP "-catalyzed dephosphorylation of c-MET monomer and dimer substrates has been studied, and the corresponding KM and VMAX for this reaction are shown.

Figure 4. Feed-forward mechanism of oligomerization-mediated c-MET activation. Ligand induced c-MET oligomerization increases kinase activity of the receptor, which results in build up of phosphorylated RTK by autophosphorylation. The levels of phosphorylated receptor generated are negatively regulated by the action of cellular phosphatases. Ligand-induced oligomerization reduces the c-MET’s susceptibility to dephosphorylation and thus affects the buildup of phosphorylated receptor. Thus, oligomerization amplifies the buildup of phosphorylated RTK via two independent mechanisms - by increasing the kinase catalytic activity and reducing c-MET’s susceptibility to dephosphorylation. The increased kinase catalytic efficiency also impacts substrate phosphorylation rates, which regulates the buildup of phosphorylated substrate. The phosphorylated receptor and substrate build up are critical determinants of RTK signaling potential.

7

Sheth and watowich: Biochemical regulation of c-MET RTK activation and signaling and dephosphorylated states. This kinetic model was built on a differential equation framework with key biochemical parameters derived from our in vitro studies on MET (Figure 3). Our model provides a robust quantitative description of c-MET activation, and provides a tool for analyzing the biochemical parameters critical for c-MET activation. It is clear from the biochemical studies that the ligand-stimulated c-MET is a highly competent signaling unit, which is sensitive to changes in phosphorylation and dephosphorylation rates. Unstimulated monomeric cMET, on the other hand, is an active kinase, but is repressed in its ability to sustain autophosphorylation and signal due to its signal its higher susceptibility to dephosphorylation, lower catalytic efficiency, and reduced substrate binding properties relative to dimeric c-MET (Figure 3). If these kinetic and thermodynamic properties are altered, for example by increasing the monomer kinase catalytic kcat by 10-fold, and decreasing the monomer’s susceptibility to dephosphorylation by 10-fold, then the buildup of phosphorylated monomer and phosphorylated exogenous substrate could occur without extracellular ligand stimulation. Thus, the term “inactive” for

monomeric unstimulated c-MET is inaccurate and can often be misleading. The “activation” that occurs in response to extracellular ligand results from the accumulation of dimeric receptors that can sustain a phosphorylated state and signal due to synergistic effects of their increased kcat for autophosphorylation and substrate phosphorylation, higher affinities for substrate, and reduced susceptibility to dephosphorylation relative to the monomeric receptor. A unified activation mechanism based on existing experimental data and modeling predictions is depicted in Figure 5. In this model, in absence of extracellular ligand, the monomeric c-MET exists predominantly in an inactive unphosphorylated state under steady-state conditions. The signaling from monomeric c-MET is repressed due to the inefficient catalytic properties and high dephosphorylation susceptibility associated with this state. In presence of extracellular ligand stimulation, the equilibrium of the system is shifted, favoring the buildup of dimeric c-MET. Signaling from dimeric c-MET occurs due to the increased kinase catalytic efficiency and substrate binding, and

Figure 5. Model for c-MET activation. This figure of c-MET activation integrates current experimental and modeling data. In absence of ligand stimulus, signaling from monomeric c-MET is repressed due to synergistic combination of decreased catalytic efficiency, reduced substrate binding and increased susceptibility to dephosphorylation, relative to oligomeric c-MET. Thus, monomeric c-MET exists predominantly as a dephosphorylated inactive receptor under steady-state conditions. Following extracellular ligand stimulus, c-MET dimerizes, which increases its kinase catalytic efficiency and substrate binding, and decreases its susceptibility to phosphatases, relative to monomeric receptor. Thus, dimeric c-MET exists predominantly as a phosphorylated active receptor under steady-state conditions.

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Cancer Therapy Vol 3, page 9 Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF (2003) Met, metastasis, motility and more. Nat Rev Mol Cell Biol 4, 915-925. Biswas R, Basu M, Sen-Majumdar A, Das M (1985) Intrapeptide autophosphorylation of the epidermal growth factor receptor: regulation of kinase catalytic function by receptor dimerization. Biochemistry 24, 3795-802. Bladt F, Riethmacher D, Isenmann S, Aguzzi A, Birchmeier C (1995) Essential role for the c- MET receptor in the migration of myogenic precursor cells into the limb bud. Nature 376, 768-771. Blume-Jensen P, Hunter T (2001) Oncogenic kinase signaling. Nature 411, 355-65. Boccaccio C, Ando M, Tamagnone L, Bardelli A, Michieli P, Battistini C, Comoglio PM (1998) Induction of epithelial tubules by growth factor HGF depends on the STAT pathway. Nature 391, 285-288. Bohmer FD, Bohmer A, Obermeier A, Ullrich A (1995) Use of selective tyrosine kinase blockers to monitor growth factor receptor dephosphorylation in intact cells. Anal Biochem 228, 267-273. Bork P, Doerks T, Springer TA, Snel B (1999) Domains in plexins: links to integrins and transcription factors. Trends Biochem Sci 24, 261-63. Campbell SL, Khosravi-Far R, Rossman KL, Clark GJ, Der CJ (1998) Increasing complexity of Ras signaling. Oncogene 17, 1395-1413. Carrolo M, Giordano S, Cabrita-Santos L, Corso S, Vigario AM, Silva S, Leiriao P, Carapau D, Armas-Portela R, Comoglio PM, Rodriguez A, Mota MM (2003) Hepatocyte growth factor and its receptor are required for malaria infection. Nat Med 9, 1363-1369. Cheng K, Koland JG (1996) Nucleotide binding by the epidermal growth factor receptor proteintyrosine kinase. Trinitrophenyl-ATP as a spectroscopic probe. J Biol Chem 271, 311-8. Christensen JG, Burrows J, Salgia R (2005) c-MET as a target for human cancer and characterization of inhibitors for therapeutic intervention. Cancer Lett 225, 1-26. Churin Y, Al-Ghoul L, Kepp O, Meyer TF, Birchmeier W, Naumann M (2003) Helicobacter pylori CagA protein targets the c-MET receptor and enhances the motogenic response. J Cell Biol 161, 249-55. Cobb MH, Sang BC, Gonzalez R, Goldsmith E, Ellis L (1989) Autophosphorylation activates the soluble cytoplasmic domain of the insulin receptor in an intermolecular reaction. J Biol Chem 264, 18701-18706. Cooper CS, Park M, Blair DG, Tainsky MA, Huebner K, Croce CM, Vande Woude GF (1984) Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature 311, 29-33. Corso S, Comoglio PM, Giordano S (2005) Cancer therapy: can the challenge be MET? Trends Mol Med 11, 284-92. Danilkovitch-Miagkova A, Zbar B (2002) Dysregulation of Met receptor tyrosine kinase activity in invasive tumors. J Clin Invest 109, 863-867. DiGabriele AD, Lax I, Chen DI, Svahn CM, Jaye M, Schlessinger J, Hendrickson WA (1998) Structure of a heparin-linked biologically active dimer of fibroblast growth factor. Nature 393, 812-817. Fukuda T, Ichimura E, Shinozaki T, Sano T, Kashiwabara K, Oyama T, Nakajima T, Nakamura T (1998) Coexpression of HGF and c-MET/HGF receptor in human bone and soft tissue tumors. Pathol Int 48, 757-762. Gherardi E, Youles ME, Miguel RN, Blundell TL, Iamele L, Gough J, Bandyopadhyay A, Hartmann G, Butler PJ (2003) Functional map and domain structure of MET, the product of the c-met protooncogene and receptor for hepatocyte growth

decreased susceptibility to dephosphorylation relative to the monomeric receptor. Thus, dimeric c-MET exists predominantly in a phosphorylated active state under steady-state conditions. Our model clearly shows that modulating catalytic activity of c-MET is not the only parameter dictating c-MET activation.

V. Conclusions Given the importance of c-MET in physiological processes such as cell proliferation, motility, and survival, it is not surprising that dysregulated c-MET signaling can result in the onset, progression and/or spread of tumors. cMET signaling is also involved in other pathologies such as malaria and listeriosis. Thus, targeting c-MET signaling has significant clinical implications for treating multiple pathological conditions. A prerequisite for the development of new diagnostic and therapeutic strategies is a detailed knowledge of the activation process of cMET. Structural, biochemical and genetic studies continue to reveal the precise molecular mechanisms underlying cMET activation, although developing effective methods to interfere with altered c-MET remains a significant challenge. Several approaches for targeting c-MET signaling have been described (Christensen et al, 2005; Corso et al, 2005) including targeting the catalytic domain with small molecule inhibitors. Acquiring high-resolution structures of c-MET in its activated (phosphorylated dimeric) state would aid the rationale design of inhibitors capableof interfering with the catalytic activity of the activated receptor and modulating dysregulated c-MET signaling.

Acknowledgements This work was supported by grant 4952-052 (S.W) from the Texas Higher Education Coordinating Board, Sealy Center for Structural Biology (University of Texas Medical Branch) and by a McLaughlin Predoctoral Fellowship (P.S).

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Stanley J. Watowich

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Cancer Therapy Vol 4, page 13 Cancer Therapy Vol 4, 13-26, 2006

Overcoming tumor resistance to immunotherapy Review Article

Lana Y. Schumacher1,* and Antoni Ribas2 1

Department of Surgery, Stanford University Hospital, Stanford, CA Departments of Medicine and Surgery and the Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, CA 2

__________________________________________________________________________________ *Correspondence: Lana Y. Schumacher, M.D. Department of Surgery, Stanford University Hospital, Stanford, CA. Division of General Surgery, Department of Surgery, SUMC, 300 Pasteur Drive, Rm H3691. Stanford, CA 94305-5641; Fax: (650) 724-9806; Email: lanas@stanford.edu Key words: tumor resistance, immunotherapy, immune surveillance, apoptosis, Death receptors, Antiapotosis, Immunosensitization, tumor antigens Abbreviations: 5-aza-2’-deoxycytidine, (DAC); B7 homolog-1, (B7-H1); B-cell lymphoma-2, (Bcl-2); cellular FLICE/caspase-8inhibitory protein, (cFLIP); class II transactivator, (CIITA); cytotoxic T lymphocytes, (CTLs); death-inducing signaling complex, (DISC); histone deacetylase inhibitors, (HDACi); human leukocyte antigens, (HLA); Inhibitor of apoptosis proteins, (IAPs); interferons, (IFNs); MHC class I chain-related, (MIC); mitogen-activated protein kinase, (MAPK); small cell lung cancer, (SCLC); TNF-receptor associated factors, (TRAFs); Transforming growth factor-beta, (TGF-!); transporter associated with antigen processing, (TAP); tumor infiltrating lymphocytes, (TILs); Vascular endothelial derived growth factor, (VEGF) Received: 27 September 2005; Accepted: 6 December 2005; electronically published: January 2006

Summary The field of cancer immunotherapy has expanded significantly over the last decade, and great progress has been made in understanding the relationship of the immune system and cancer. However, even with enhanced tumor specific immune responses, clinical response rates are low and cancer immunotherapy is limited. The lack of clinical response has generated more focus on analyzing the tumor resistance to the immune system and the escape mechanisms of tumor cells with hopes of being able to develop targeted therapy to overcome this resistance. This review discusses the many mechanisms of tumor escape and resistance that we have begun to understand such as escape from immune surveillance, tumor release of immunosuppressive factors and decreased sensitivity of tumor cells to immune-mediated apoptosis. There has also been an expansion in the development of small molecules inhibitors that generate targeted therapies that sensitize the tumor to immune mediated apoptosis. These targeted therapies have been tested on numerous cancer cells lines and have demonstrated an enhancement in apoptotic activity both in vitro and in vivo. Cancer cells resistant to chemotherapy have been shown to become sensitized with some of the targeted therapies discussed below. As we better understand our limitations with immunotherapy, we may be able to enhance our therapy against “resistant� tumor cells with small molecule inhibitors with the hopes of reversing this resistance to a cell capable of undergoing cell-mediated apoptosis.

1998 #2; Parmiani, 2002 #11} (Kim et al, 1997). It has been reported that tumor cells have numerous immune surveillance escape mechanisms as well as means of resistance to apoptosis/T-cell mediated cytotoxicity (Marincola et al, 2000). The increasing understanding of these pathways together with the development of specific inhibitors for critical molecules responsible for tumor resistance can facilitate the reversal of tumor escape from the immune system, a term described as immunosensitization (Ng and Bonavida, 2002). In this review we will discuss tumor cell resistance to apoptosis mediated cell death and the means to over this tumor resistance.

I. Introduction Over the past few decades there has been much excitement about applying immunotherapy to the treatment of malignant diseases. However results of immunotherapy trials, whether they be cytokine, or T-cell and dendritic cell based therapy, have been suboptimal (Rosenberg et al, 2004). It is of general consensus that T cells play a major role in tumor growth control and much emphasis has been directed towards understanding tumor antigen specific immune responses of T cells (Rosenberg et al, 2004). It has also been concerning that in many clinical trials tumor antigen-specific immune responses in patients do not correlate with clinical response {Nestle,

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Schumacher and Ribas: Overcoming tumor resistance to immunotherapy

II. Tumor escape surveillance (Table 1)

from

molecules are expressed in all somatic cells and are recognized by CD8+ CTL. Additionally, class I molecules are major negative ligands for NK cell function. Defects in processing and presentation of MHC class I antigens have been frequently reported in malignant cells (Garrido et al, 1993; Algarra et al, 1997), {Hicklin, 1999 #8} (Campoli et al, 2002). Monoclonal antibodies to MHC class I allospecificities have been used to study the phenotype of malignant lesions. These studies have revealed multiple selective losses or downregulation of one MHC class I allospecificity, loss of allospecificities encoded by one haplotype and down regulation of gene products of one locus 13. The frequency of selective MHC class I allospecificity loss and downregulation has been reported to be anywhere from 15-51% depending on the type of malignancy (Algarra et al, 2000). Seven major altered MHC class I phenotypes have been defined in different tumor tissues: 1) MHC class I total loss, 2) HLA haplotype loss (tumors can lose one of two HLA haplotypes in the tumor cell), 3) HLA A, B or C locus product downregulation, 4) HLA allelic loss, 5) HLA compound phenotypes, 6) unresponsiveness to interferons (tumor cells have lost the capacity of upregulating HLA molecules in response to different cytokines especially and " interferons, and 7) downregulation of HLA-A, B, C molecules and appearance of non-classical (class Ib) HLA-E molecules (Garrido et al, 1997). Defects in the processing of HLA class I antigens have also been observed with variable frequencies throughout tumor types. The mechanisms leading to total or partial loss of HLA expression can occur at any step required for HLA synthesis, assembly, transport and expression on the cell surface (Garrido et al, 1997). The presence of MHC and B2m gene mutations has been well described (D'Urso et al, 1991; Browning et al, 1996; Benitez et al, 1998; Perez et al, 1999). Alterations in glycosylation and transport (Cromme et al, 1994; Johnsen et al, 1999) and HLA gene deletions and loss of heterozygosity (Torres et al, 1996). The frequency of LMP2, LMP7 (proteasome subunits critical for the generation of antigenic peptides) downregulation has been reported as 37-55% in primary lesions of melanoma and 19-55% in metastatic melanoma lesions (Seliger et al, 2000).

immune

It has long been established that the immune system plays a significant role in preventing tumor development. Ehrlich proposed the role for immunity in the defense against spontaneous tumors in 1909. The immune surveillance hypothesis was further expanded in 1959 by Thomas and in 1970 by Burnet (Garcia-Lora et al, 2003). The seminal work of Robert D. Schreiber at the Washington University, St. Louis, M.O. and Lloyd J. Old at the Ludwig Institute for Cancer Research, N.Y. and colleagues demonstrated that experimental tumors develop more frequently in immune suppressed hosts, incriminating a central role of immune surveillance in the pathogenesis of cancer (Shankaran et al, 2001). Likewise, human subjects with congenital or acquired immune suppression have an increased frequency of cancer (Dunn et al, 2002). In addition, tumors have long been noted to contain infiltrating cells. Woglom in 1929 described these cells infiltrating the tumors as “small round cells”.These cells were subsequently established as lymphocytes, predominantly T cells but also present were B cells and NK cells. The mechanisms of the interplay between tumor infiltrating lymphocytes (TILs) and tumor cells was further elucidated by the identification of tumor antigens recognized by T lymphocytes (Boon et al, 1994) and the description of the molecular mechanisms of NK cell function (Lopez-Botet et al, 1996) (Figure 1). In brief, cytotoxic T lymphocytes (CTLs) can recognize tumor specific antigens restricted by MHC molecules and kill tumor cells. In addition, tumor cells that lack the expression of one or more major histocompatibility complex (MHC) class I alleles become targets for NKmediated cell lysis (Karre, 2002). Although we have such complex mechanisms for tumor surveillance by the immune systems, tumor cells continue to grow, invade and metastasize. In order to possibly overcome the tumor resistance to the immune systems, we need to better understand the tumor “escape” mechanisms. A major obstacle to the induction of an endogenous tumor-specific CTL response is the inefficient presentation of MHC class I molecules to professional antigenpresenting cells. Peripheral solid tumors developing outside the lymphoid organs are often ignored by the immune system and in other situations; CTLs may be tolerized to certain lymphohematopoietic tumors. Here we review the several mechanisms of altered MHC expression by tumor cells.

B. TAP The transporter associated with antigen processing (TAP) is a crucial component to MHC class I peptide presentation. TAP is composed of two subunits, TAP1 and TAP2, and selects peptides of certain lengths and specific sequences to be translocated to the endoplasmic reticulum for subsequent antigen processing and presentation (Seliger et al, 2000). In comparison with corresponding normal tissues, TAP1 downregulation or loss has been found in all tumor types analyzed, with a frequency ranging from 10-84% (Keating et al, 1995; Hicklin et al, 1999). TAP2 expression has also been analyzed however with less frequency than TAP1. The frequency of TAP2 downregulation appears to parallel that of TAP1 in

A. Loss or down-regulation of MHC class I molecules MHC class I and II molecules are cell surface glycoproteins which play a fundamental role in antigen processing and presentation required for immune cell recognition. Human MHC molecules are called human leukocyte antigens (HLA). MHC class II molecules are restricted to professional antigen presenting cells (B cells, macrophages and dendritic cells) and present antigens to CD4+ T helper cells. Unlike class II molecules, class I

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Cancer Therapy Vol 4, page 15 Table 1. Mechanisms of tumor evasion. Overview of Mechanisms of Tumor Evasion Escape From Immune Surveillance

Immune Suppression

Resistance to Apoptosis

Altered Expression of Molecules MHC class I TAP MIC MHC class II IL-10 COX TGF-! VEGF Increased Antiapoptotic Molecules: XIAP, Akt, c-FLIP, Bcl-2, Bcl-XL, Mcl-1 Decreased Proapoptotic Molecules: Bax, Bak

Abbreviations: MHC (major histocompatability complex), TAP (transporter associated with antigen processing), MIC (MHC class I chain-related molecules), IL-10 (interleukin 10), COX (cycloxygenase), TGF-! (transforming growth factor !), XIAP (X chromosome encoded inhibitor of apoptosis), c-FLIP (cellular FLICE/caspase-8 inhibitor protein)

Figure 1. Detailed schematic of the interplay between innate and adaptive immune response against tumor cells. Tumor cells elicit an immune response in both arms of the immune system, innate and adaptive. Tumor cells commonly lack MHC molecules thereby, activating the natural killer cells in the innate immune response. There are also other specific receptors that can directly activate or inhibit the innate immunity, CD36, MIC A/B respectively. The adaptive immunity is activated after the phagocytosis of a tumor cell by an antigen presenting cell. Tumor antigens are then presented on MHC I or II molecules of the APC to either CD8 or CD 4 T cells. Once these cells are activated there is further cytoxicity and secretion of cytokines to potentiate the immune response toward the tumor cells.

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Schumacher and Ribas: Overcoming tumor resistance to immunotherapy melanoma and breast carcinoma lesions analyzed (Vitale et al, 1998; Kageshita et al, 1999). TAP downregualtion in breast carcinoma, small cell lung cancer (SCLC), cervical carcinoma and melanoma lesions have been found to be associated with disease progression (Keating et al, 1995; Vitale et al, 1998; Hicklin et al, 1999). Tapasin which plays an important role in the assembly of MHC class I molecules with peptides in the endoplasmic reticulum, is also downregulated in high frequencies and associated with progressive disease in melanoma (Dissemond et al, 2003).

receptors and eliminate those cells with downregulated or loss of MHC class I (Moretta et al, 1996). Because most tumor cells have little to no MHC class I expression, NK cells have a significant function in the antitumor response. Data from mutant mouse models lacking distinct immune cell populations develop spontaneous tumors (Dunn et al, 2002). With respect to the tumors, many studies have demonstrated that MHC class I deficient transplantable tumor cell lines are rejected by NK cell (Ljunggren and Karre, 1985; Piontek et al, 1985; Taniguchi et al, 1985), and restoration of MHC class I expression reversed the effect (Franksson et al, 1993). In addition, tumor cells frequently express families of stress-related genes such as MICA and MICB which function as ligands for NKG2D receptors expressed by NK cells and other cytotoxic lymphocytes and phagocytes (Diefenbach and Raulet, 2002). Even though downregulation or loss of MHC class I cell surface expression should make tumor cells more susceptible to NK immune effector mechanisms, tumor cells continue to proliferate and invade. Patients with cancer have exhibited ex vivo impaired NK-cell function as determined by reduced proliferation, response to interferons (IFNs) and cytotoxicity (Whiteside and Goldfarb, 1994; Trapani et al, 2000). One possible mechanism by which tumor cells evade NK cell killing is by the continued expression of appropriate MHC class I ligands that engage inhibitory receptors on NK cells (Tajima et al, 2004). NK cells are also affected by the tumor cells secretion of immunosuppressive factors (discussed below).

C. !2m In addition there appears to be a role in selective immune pressures in the generation of HLA I mutations and loss of HLA I. In vitro, it has been described that tumor cells with HLA I defect will out grow normal HLA I antigen presenting tumor cells when exposed to CTL recognizing the allospecific HLA I antigen. On the contrary, when these same tumor cell population are grown in an immunologically na誰ve environment, cell grow appears similar. These findings support the clinical findings of increased frequency of HLA I losses in malignant lesions of patients treated with T cell-based immunotherapy (Restifo et al, 1996).

D. MHC class I Chain-related Molecules (MIC) Epithelial tumors that shed MHC class I chainrelated (MIC) molecules escape from NK and T cell recognition (Diefenbach and Raulet, 2002). MICs are ligands for the activating receptors NKG2D on both NK and T cells. It has been reported that elevated serum levels of soluble MIC in colorectal cancer are responsible for the down-modulation of NKG2D as well as chemokine CXCR1. In vitro, internalization of NKG2D and CXCR1 occurs within 4 and 24 h, respectively, of incubating normal NK cells with sMIC-containing serum and the addition of anit-MIC-Ab lead to an up-regulated expression of NKG2D, CXCR1 and CCR7. These results suggest that circulating sMIC in patients with cancer deactivates NK immunity by down-modulating important activating and chemokine receptors (Groh et al, 2002; Doubrovina et al, 2003; Lanier, 2003).

III. Tumor release immunosuppressive factors

of

It has been well known but not thoroughly understood that lymphocytes recovered from both the peripheral blood, as well as the tumors themselves, in patients with malignancies are functionally compromised. These lymphocytes can also be further characterized to have tumor-antigen specific responses although do not induce apoptosis or tumor cell death. Tumor cells create a microenvironment by expressing or secreting several different cytokines and growth factors to induce immune suppression.

A. IL-10

E. MHC Class II molecules

IL-10 has been described to be secreted by tumor cells to cause an effective immunosuppression of infiltrating T cells. IL-10 exerts an indirect effect on the immune system by inhibiting the secretion of proinflammatory cytokines like IL-1, IL-6, IL-8 and TNF# (Tsuruma et al, 1999), free oxygen radicals, nitric oxide derivatives from type 1 helper T cells, monocytes, macrophages and neutrophils, and inhibiting the secretion of IFN" by NK cells (Moore et al, 1993). Furthermore, IL10 downregulates MHC class I, II and B7 molecules (Matsuda et al, 1994; Salazar-Onfray et al, 1997), all of which are important for the induction of the antigenpresenting capacity of macrophages and dendritic cells and activation of T cells. There have been many reports of IL-

Although not as predominantly expressed as MHC class I molecules, MHC class II molecules may be expressed by tumor and elicit a CD4+ Tcell antitumor immune response via a professional APC (Wang, 2001). Alterations in the presentation of peptides on MHC class II may affect the generation of effective CD4+ T helper cell antitumor response. Defects have been noted at the AIR locus, which encodes the transcription factor and class II transactivator (CIITA) thus effecting expression of MHC class II antigens.

F. Escape from NK immune surveillance The role of NK cells is to monitor cells for their expression of self-MHC molecules via their specific KIR

16

Cancer Therapy Vol 4, page 17 10 production of a variety of solid tumors such as carcinomas of the colon, lung and skin (Gastl et al, 1993; Huang et al, 1995). Increased levels of tumor derived IL10 has been reported in plasma of patients with NSCLC and correlated with decreased survival (Neuner et al, 2001).

interacts with T cells to efficiently block their IL-2 production and proliferation (Chen et al, 2003). In addition, it interferes with the generation of CTL, inactivates NK cell and lymphokine activated killer cell cytotoxicity most likely by the inhibition of TNF-# and ! secretion (Gray et al, 1994, 1998; Ebert et al, 1999). Dendritic cell maturation and antigen presentation is also impaired by TGF-! (Geissmann et al, 1999), and TGF-! produced by tumors significantly reduced the potency of DC/tumor fusion vaccines (Kao et al, 2003). Furthermore, TGF-! may have a pivotal role in inducing immune suppression by CD4+/CD25+ regulatory T cells (Wahl et al, 2004). The mean concentration of IL-10 and TGF-! were evaluated in patients with pancreatic ductal adenocarcinoma and noted to be considerably higher than in control serum. In that same study TILs were analyzed and shown to have a severe loss of CD3 $ chain correlating to the increased levels of IL-10 and TGF-!. Killing of tumor cells by potentially cytotoxic TILs appeared to be suppressed by the prevention of a direct TIL/tumor cell contact and the inactivation of TILs, as shown by a loss of CD3 $ (von Bernstorff et al, 2001).

B. COX COX (cyclooxygenase or PG endoperoxidase) is a rate-limiting enzyme involved in the production of prostaglandins and thromboxanes from free arachidonic acid. Two forms of this enzyme have been described: COX-1 which is constitutively expressed in most cells and tissues, and COX-2 which is induced by cytokines, growth factors and other stimuli (Herschman, 1996). COX-2 is constitutively overexpressed in a variety of malignancies (Soslow et al, 2000) and has been to enhance tumor resistance to apoptosis (Tsujii and DuBois, 1995), increase angiogenesis, invasion and metastasis (Leahy et al, 2000; Dohadwala et al, 2001) and impair host immunity {Huang, 1998 #34} (Stolina et al, 2000). In regards to the immune systems, the overexpression of COX-2 has been reported to significantly enhance a PGE2-dependent IL 10 production by macrophages, dendritic cells and lymphocytes as well as decrease IL-12 production. In addition, tumor COX-2 lead to a suppression of dendritic cell function by decreasing the dendritic cell capacity to process and present antigens and induce alloreactivity as well as alter the dendritic cell phenotype to an immature phenotype with decreased expression of CD11c, CD80, CD86, MHC class I and II (Sharma et al, 2003). When T cells and dendritic cells from patients with breast cancer were evaluated, their function directly correlated with COX-2 and PGE2 overexpression. T cells demonstrated a decreased proliferation in response to CD3 antibody stimulation, reduced production of interferon ", TNF-#, IL-12, IL-2 and increased levels of IL-10 and IL-4. Dendritic cells revealed a reduced expression of costimulatory molecules, reduced phagocytic ability and reduced antigen presentation as well (Pockaj et al, 2004).

D. B7-H1 In addition, tumors can actively inhibit immune responses by expressing a B7 homolog-1 (B7-H1) which is known as a programmed death ligand 1. B7-H1 is expressed on many tumors including carcinomas of the breast, lung, ovary and colon, whereas normal tissues do not express B7-H1. Their expression can interact with the CD28 receptor on CTLs and promote CTL death via induction of Fas Ligand and IL-10 {Dong, 2003 #6}.

E. VEGF Vascular endothelial derived growth factor (VEGF) may be produced by tumor cells, which promotes tumor angiogenesis but inhibits the immune cell function by impairing both the effector function and early stages of hematopoiesis. VEGF receptors are present on early hematopoetic progenitor cells (Ferrara, 1996). Almost all tumor cells produce VEGF; elevated levels are frequently detected in the sera of cancer patients and these elevated levels are associated with a poor prognosis (Dikov et al, 2001). VEGF causes a defect in the maturation of dendritic cells from early hematopoetic progenitor cells (Gabrilovich et al, 1998). In addition, mice treated with continuous infusion of recombinant VEGF have a decreased number of T cells and a decreased T-to-B-cell ratio in their lymph nodes and spleen (Ohm and Carbone, 2002; Ohm et al, 2003).

C. TGF-! Transforming growth factor-beta (TGF-!) is overexpressed by numerous malignant tumors such as breast cancer, prostate cancer, small and non-small lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, bladder cancer, melanoma and malignant gliomas (Wojtowicz-Praga, 2003). TGF-! is a potent immunosuppressor, helping tumor cells evade the immune system. A second role of TGF-! is to stimulate angiogenesis further promoting tumor growth. TGF-! is known to activate cytostatic gene responses at any point the cell cycle, especially G1 as well as repress growthpromoting transcription factors. Although TGF-! can induce apoptosis in hematopoetic cells (Siegel and Massague, 2003), tumor cells become refractory to TGF!-mediated growth arrest by either the loss of TGF-! receptors, mutation in the receptors or due to dysregulation in TGF-! signaling pathways whereas immune cells remain sensitive (Wojtowicz-Praga, 2003). TGF-!

IV. Decreased sensitivity of tumor cells to immune-mediated apoptosis It is well known that human cancer cells have dysfunctional apoptotic pathways leading to the resistance of these cells to apoptosis by therapeutic agents. In addition, impairment of the apoptotic signaling pathway plays an important role in the initiation and progression of normal cells into cancer cells (LaCasse et al, 1998; Reed, 17

Schumacher and Ribas: Overcoming tumor resistance to immunotherapy 1999; Hickman, 2002). In the last several years we have gained much incite on the individual cellular factors involved in apoptosis as well as their roles in the apoptosis pathways. The two main pathways involved in apoptosis are the extrinsic pathway and the intrinsic pathway. In brief, the extrinsic pathway of apoptosis is initiated by the interaction of cellular surface death receptors with their successive ligands and the intrinsic pathway is dependent on the leakage of cytochrome c from the mitochondria, which is prompted from the change or loss of mitochondrial membrane potential. Triggering either of these pathways leads to a downstream activation of a cascade of caspase proteolysis reactions. The initiator caspase group includes caspase-8 and caspase-10 for the extrinsic pathway and caspase-2 and caspase-9 for the intrinsic pathway. These caspases bind to adapter molecules forming a death-inducing signaling complex (DISC). Once the DISC is formed and the initiator caspases have been activated, they in turn activate a series of downstream caspases known as effector caspases (caspase-3, 6, 7) which are similar for both pathways. These effector caspases subsequently cleave numerous structural and regulatory proteins leading to apoptosis of the cell (Liu et al, 1997; Budihardjo et al, 1999). With such involved pathways, cancer cells have evolved to resist apoptosis by many mechanisms such as downregulating death receptor expression and overexpressing inhibitors of apoptosis.

function in atypical or malignant melanocytic lesions as well as melanoma cell lines. Von Reyher and colleagues determined that Fas is expressed in every colonocyte of normal colon mucosa, but it is downregulated or lost in the majority of colon carcinomas (von Reyher et al, 1998). Hughes and colleagues demonstrated that 69.5% of esophageal adenocarcinomas evaluated were negative for Fas (Hughes et al, 1997). We are now starting to develop a better understanding to the mechanisms of escape to Fas or TRAIL receptor engagement. A mutant p53 gain of function has been shown to repress Fas gene expression (Zalcenstein et al, 2003). Many tumor cells have been described to overexpress or constitutively express Fas which, in turn, downregulates Fas expression. Defects in the Fas-signaling pathway have also been described. The cellular FLICE/caspase-8-inhibitory protein (cFLIP) can interfere with Fas-mediated cell death and therefore favor tumor immune escape (French and Tschopp, 2002). cFLIP has been reported to be constitutively expressed in all human HCC cell lines, more so than in normal non-tumor liver tissues (Okano et al, 2003). cFLIP expression was undetectable in all but one benign melanocytic lesion (31/32, 97%). In contrast, cFLIP was strongly expressed in most melanomas (24/29 = 83%). Overexpression of cFLIP by transfection in a Fas- and TRAIL-sensitive human melanoma cell line rendered this cell line more resistant to death mediated by both TRAIL and FasL. Selective expression of FLIP by human melanomas may confer in vivo resistance to FasL and TRAIL, thus representing an additional mechanism by which melanoma cells escape immune destruction (Bullani et al, 2001). Furthermore, loss of FADD protein expression, as well as loss of caspase 8 expression, is a means for tumor resistance to the death-receptor induced apoptosis pathway (Teitz et al, 2000; Tourneur et al, 2003).

A. Death receptors and tumor resistance Like chemotherapy and irradiation, immune effector cells (both T and NK cells) kill targets by activating apoptosis. This is achieved by either releasing perforin and granzyme B, which activate caspases directly, or by expressing or releasing death receptor ligands that interact with death receptors on the surface of the target cells. The death receptor pathway is an important aspect of perforinindependent cytotoxicity. Death receptors are a subgroup of TNF-receptor family members that can trigger caspase8 activation and apoptosis upon interaction with their respective ligands. Eight human death receptors - Fas (CD95), TNF-R1, TRAMP (WSL-1/Apo-3/DR3/LARD), TRAIL-R1 (DR-4), TRAIL-R2 (DR-5), DR-6, EDA-R and NGF-R - have been identified to date (Ashkenazi and Dixit, 1998, 1999). All are type I membrane proteins containing two-four cysteine-rich extracellular domains and a cytoplasmic "death domain". This cytoplasmic death domain couples to receptors to trigger the caspase casade to induce apoptosis. Tumor cells, however, have numerous mechanisms to resist this apoptosis driven pathway by altering expression of these receptors or their downstream effector molecules. One of the death receptors, Fas (CD95), and its ligand, both critically involved in immune homeostasis and effector function, are also the major pathway of cytolytic T-cell -mediated immunity involved in specific killing of tumor cells. It has been well described that many different tumor cells downregulate or lose of Fas expression on their surface (Shin et al, 1999) {Lee, 2000 #37}. Bullani and colleagues, 2002 demonstrated the reduction in Fas expression and/or death signaling

B. Antiapotosis molecules/inhibitors of apoptosis

signaling

Many antiapoptotic molecules are involved in the resistance of tumor cells to apoptosis. Inhibitor of apoptosis proteins (IAPs) consist of at least six family members (Deveraux and Reed, 1999; Deveraux et al, 1999) IAP1,2, XIAP, ML-IAP (Vucic et al, 2000), Livin (Kasof and Gomes, 2001), Bruce and Surivin (Verhagen et al, 2001). XIAP is widely distributed throughout the cytosol in both normal and cancer cells and inhibit caspase-3, 6 and 7. IAP1 and 2 interact with TNF-receptor associated factors (TRAFs) in the membrane and perinuclear areas to inhibit caspase-3 and 7 (Verhagen et al, 2001). ML-IAP inhibits caspase-3, 7 and 9 in the nucleus and filamentous structures in the cytoplasm (Vucic et al, 2000). There are molecules that inhibit the binding of IAPs to caspases. These proteins are released from the mitochondria into the cytosol during changes of mitochondrial membrane potential permeability (Verhagen et al, 2000). They are known as second mitocondria derived activator of caspases (Smac/Diablo) and are located in the intermembrane space of the mitochondria. They compete for binding sites on XIAPs thus releasing 18

Cancer Therapy Vol 4, page 19 caspase 3,7 and 9. Omi is another protein released from the mitochondria which binds to IAP and facilitates apoptosis by freeing caspase 3 and 7 (Hedge and Williams, 2002). Members of the B-cell lymphoma-2 (Bcl-2) protein family have important roles in the regulation of cellular apoptosis for which they elicit anti- or proapoptotic functions. Bcl-2, BclXl, Mcl-1, Bcl-w, Bfl-1/a1, Bcl-b and Bcl-2-L-10 are antiapoptotic molecules whereas Bax, Bak, Bad, Bid, Bcl-Xs, Mcl-1S, Bok/Mtd and Bik/Nbk are proapoptotic. There is a general understanding that the proapoptotic molecules bind and neutralize antiapoptotic molecules (Cheng et al, 2001) thus allowing apoptosis. Many cancers, both solid and hematogenous, demonstrate an overexpression of Bcl-2 such as melanoma (Vlaykova et al, 2002), breast (Silvestrini et al, 1994; Wu et al, 2000), prostate (McDonnell et al, 1992; Colombel et al, 1993), small cell lung carcinoma (Jiang et al, 1995; Dingemans et al, 1999), colorectal carcinoma (Sinicrope et al, 1996; Ilyas et al, 1998), transitional cell carcinoma (Pollack et al, 1997) and solitary fibrous tumors (Hasegawa et al, 1998). This overexpression of Bcl-2 correlates with worse prognosis. High levels of Bcl-Xl were detected in bladder transitional cell carcinoma (Kirsh et al, 1998), squamous cell cancer of the oropharynx, (Aebersold et al, 2001) and pancreatic cancer. These molecules also contribute to tumor initiation, progression and resistance to therapy and additionally associated with a worse prognosis (Friess et al, 1998; Amundson et al, 2000). The pro-apoptotic molecules, Bax and Bak, are required in order to achieve apoptosis (Wei et al, 2001). Both Bax and Bak exist in inactive conformations and are activated in response to various apoptotic stimuli (Wolter et al, 1997; Gross et al, 1998). Bax is located in the cytosol and translocates to the outer mitochondrial membrane upon activation. Bak is located in active and inactive forms on the outer mitochondrial membrane (Griffiths et al, 1999). Once activated, both Bax and Bak form multimeric complexes. Mouse embryo fibroblasts deficient for Bax and Bak were refractory to apoptosis after induction by various agents causing cell and mitochondrial stress (Wei et al, 2001). Apoptosis induced by chemotherapeutic agents are dependent on Bax. (Bellosillo et al, 2002, Deng et al, 2002). Furthermore, epithelial cancer cells lacking Bax were resistant to apoptosis (Theodorakis et al, 2002).

(Eisenmann et al, 2003).

C. Regulation of apoptosis MEK/ERK pathway

A. Blocking immune suppressive soluble factors

D. Regulation Akt/PkB pathway

of

apoptosis

by

the

Numerous studies have shown the relevance of the Akt pathway in promoting cell survival. Akt is constitutively activated in many carcinomas such as prostate, breast, ovary, lung and liver (Vivanco and Sawyers, 2002). It is thought that the activation of Akt leads to downstream inhibition of apotosis. It has been suggested that Akt inhibits TRAIL induced apoptosis by blocking Bid cleavage (Kandasamy and Srivastava, 2002). Akt also inhibited Fas-mediated apoptosis by reducing recruitment of caspase 8 to the DISC, reduced activation of caspase 8 and Bid (Jones et al, 2002). In addition, Akt activation was reported to up-regulate c-FLIP in numerous cancer cell lines (Panka et al, 2001). The down-regulation of pro-apoptotic molecules and upregulation of antiapoptotic molecules mediated by Akt/PkB has been suggested in several studies (Hayakawa et al, 2000).

V. Immunosensitization The increasing understanding of the pathways by which tumor cells resist apoptosis, together with the development of specific inhibitors for critical molecules responsible for tumor resistance, may facilitate the reversal of tumor escape from the immune system (Frost et al, 2001; Ng and Bonavida 2002). Modern immunestimulating interventions resulting from a more detailed knowledge on how immune effector cells are activated and regulated have resulted in unprecedented expansion of circulating antigen-specific T cells. Dendritic cell-based vaccines, repeated peptide-based immunizations, viralvector-mediated genetic immunization and adoptive transfer of activated antigen-specific T cells have the ability to expand tumor-antigen specific T cells to levels similar to those able to protect from viral infections. However, the magnitude of tumor-antigen-specific T cell expansion has not been unequivocally correlated with clinical responses. Therefore, it is likely that a limiting step is that target cells can escape from death signals delivered by adequately activated T cells. The knowledge of how target cells escape immune-cell-mediated killing provides several means of intervention to revert sensitivity to the immune system.

As we have described earlier, there are several wellknown immune suppressive soluble factors that are secreted by most tumor cells. It has been the focus of many to block these factors to reverse the immune suppression.

The RAS-RAF-MEK-ERK (extracellular signalregulated protein kinase) -MAPK (mitogen-activated protein kinase) pathway regulates several key growth factors, cytokines and proto-oncogenes to promote cell growth and differentiation. RAS is mutated to an oncogenic form in about 15% of human cancers leading to an overexpression or constitutive activation of this pathway (Davies et al, 2002). This pathway regulates antiapoptotic molecules like Bcl-2, Bcl-XL and Mcl-1 leading to MAPK-dependent tumor-specific survival signals in cancer cells, especially pancreatic carcinoma cells (Boucher et al, 2000) and melanoma cells

1. TGF-! There has been evidence of suppressing or blocking TGF-! signaling can suppress tumor progression making this molecule an attractive target. There are several agents targeting TGF-! that are in early stages of development such as anti- TGF-! antibodies, small molecule inhibitors 19

Schumacher and Ribas: Overcoming tumor resistance to immunotherapy silencing 141. Demethylation of tumor antigen promotors responsible for gene silencing can lead to a reactivation of their expression thus increasing the sensitivity of tumor cells to immune surveillance. This can be achieved by two classes of drugs, demethylating agents and histone deacetylase inhibitors (HDACi). The MAGE genes (also known as cancer testis antigens), expressed not only in melanomas but also in many other tumors, are activated by promoter demethylation (De Smet et al, 1996; Coral et al, 1999). Furthermore, the activation of MAGE expression on melanoma cells with 5-aza-2’-deoxycytidine (DAC) elicits a MAGE-specific CTL response (Serrano et al, 2001). Other tumor antigens, RAGE-1, GAGE 1-6 and NY-ESO-1, in renal cell carcinoma are also regulated by DNA methylation and expression of these antigens can be induced by DAC. In addition, de novo expression of NYESO-1 was noted in renal cell carcinoma which elicited a NY-ESO-1 specific CTL-mediated lysis (Coral et al, 2002). In addition, these drugs have been shown to generate a pro-apoptotic cell milieu by modulating the expression of several pro and anti-apoptotic genes (Egger et al, 2004). The use of demethylating agents as an adjunct to immunotherapy may be a possibility to enhance MHC expression of tumor antigens and increase immune recognition and subsequent destruction of tumor cells.

of TGF-!, Smad inhibitors (downstream target of TGF-!) and antisense gene therapy. Stable transduction of breast and glioma tumor cells with antisense TGF-! 1 and TGF-! 2 retroviruses restored their immunogenicity and induced partial rejection of unmodified established tumors (Fakhrai et al, 1996; Dumont and Arteaga, 2003). Reversal of NK inhibition induced by tumor inoculation in athymic mice was noted after the use of TGF-! neutralizing antibodies (Arteaga et al, 1993). Furthermore, antisense oligonucleotides targeting TGF-!2 DNA or mRNA inhibited malignant mesothelioma growth in vitro and in vivo (Marzo et al, 1997) as well as inhibited hepatocellular carcinoma growth in vivo and increased CTL lytic activity twofold in vitro (Maggard et al, 2001). Blocking TGF-! also improved dendritic cell vaccines in vivo after using a combined TGF-! gene transfer plus TGF-! neutralizing antibody in established TGF-!-secreting 4T1 mammary tumors. The combined therapy with the dendritic cell vaccine resulted in tumor regression in 40% of established tumors 135.

2. COX-2 and prostaglandins COX-2 inhibitors have recently been intensively evaluated for their ability to treat and prevent cancers. COX-2 inhibitors are being used in combination with other anti-cancer drugs or irradiation to treat solid tumors, and have shown efficacy as a single agent for the prevention of colorectal cancer in patients with familial adenomatous polyposis (Xu, 2002). It has been shown that by inhibiting COX-2 tumor expression either genetically or pharmacologically, the immunosuppressive effects on dendritic cells can be reversed and dendritic cell phenotype and function can be restored (Sharma et al, 2003). COX-2 inhibition also reversed tumor-induced suppression of macrophage function (Duff et al, 2003). COX-2 inhibitors also induce apoptosis via activation of caspase-3, caspase-9 and cytochrome c release in tumor cells expressing COX-2, and block cell cycle regardless of their COX-2 expression (Maier et al, 2004). In vivo treatment of established Lewis lung cancer tumors with COX-2 inhibitor reduced tumor growth in C57BL/6 mice (Williams et al, 2000).

C. Interference molecules

with

anti-apoptotic

Because of the up-regulation of anti-apoptotic molecules is a frequent observation in cancer cells, specific therapy to target anti-apoptotic molecules may potentially immunosensitize tumor cells by increasing their ability to undergo apoptosis. Many of these specific therapies are currently in clinical trials, including an antisense to Bcl-2 construct (G3139). A Bcl-2 inhibitor, ABT-737 was designed to be a potent inhibitor of antiapoptotic proteins Bcl-2, Bcl-Xl and BCL-w and has been shown to enhance the effects of death signals and display synergistic toxiticities with chemotherapies and radiation. As a single agent, it was therapeutic in the cytotoxicity to lymphoma and small-cell lung carcinoma cell lines in both patient derived cells and in animal models (Oltersdorf et al, 2005). New molecules have been studied to specifically disrupt the MAPK cascade leading to an overall decrease in activity of the Bcl-2 family of anti-apoptotic molecules. Antitumor activity has been seen in preclinical models with CI-1040, an orally active inhibitor of MEK1/2, for pancreas colon, breast cancer (Allen et al, 2003) and melanoma (Collisson et al, 2003). In addition ERK inhibitors PD98059 and U0126, when used in conjunction with docetaxel, increased apoptosis and inactivated Bcl-2 in human prostate cancer cells (Zelivianski et al, 2003). The use of XIAP antagonists have recently demonstrated efficacy in inducing apoptosis in cancer cells on top of sensitizing cancer cells to chemotherapy (Yang et al, 2003). XIAP antagonists have been designed creating a SMAC peptide complexed with the BIR3 domain of XIAP, which bind to the BIR3 domian of XIAP and promote cell death in several human cancer cell lines and has inhibited growth of tumors in a xenograft breast

3. VEGF VEGF has several immunosuppressive effects that promote tumor growth and invasion. Blocking negative effects of VEGF on dendritic cell maturation by a neutralizing antibody reverses the negative effects on dendritic cells in vitro (Gabrilovich et al, 1999). It was then subsequently shown that antibodies to VEGF can enhance the efficacy of cancer immunotherapy by improving dendritic cell function in vivo. In this study, tumor growth in mice was delayed and survival was prolonged by the addition of VEGF antibody to a p53 peptide pulsed vaccine (Gabrilovich et al, 1999).

B. Re-expression of tumor antigens DNA methylation is known to be an important mechanism of gene regulation. It has also been noted to control tumor antigen expression and lead to gene

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Cancer Therapy Vol 4, page 21 Algarra I, Cabrera T, Garrido F (2000) The HLA crossroad in tumor immunology. Hum Immunol 61, 65-73. Algarra I, Collado A, Garrido F (1997) Altered MHC class I antigens in tumors. Int J Clin Lab Res 27, 95-102. Allen LF, Sebolt-Leopold J, Meyer MB (2003) CI-1040 (PD184352), a targeted signal transduction inhibitor of MEK (MAPKK). Semin Oncol 30, 105-16. Amundson SA, Myers TG, Scudiero D, Kitada S, Reed JC, Fornace AJ Jr (2000) An informatics approach identifying markers of chemosensitivity in human cancer cell lines. Cancer Res 60, 6101-10. Arteaga CL, Hurd SD, Winnier AR, Johnson MD, Fendly BM, Forbes JT (1993) Anti-transforming growth factor (TGF)-! antibodies inhibit breast cancer cell tumorigenicity and increase mouse spleen natural killer cell activity. Implications for a possible role of tumor cell/host TGF-! interactions in human breast cancer progression. J Clin Invest 92, 2569-76. Ashkenazi A, Dixit VM (1998) Death receptors: signaling and modulation. Science 281, 1305-8. Ashkenazi A, Dixit VM (1999) Apoptosis control by death and decoy receptors. Curr Opin Cell Biol 11, 255-60. Bellosillo B, Villamor N, Lopez-Guillermo A, Marce S, Bosch F, Campo E, Montserrat E, Colomer D (2002) Spontaneous and drug-induced apoptosis is mediated by conformational changes of Bax and Bak in B-cell chronic lymphocytic leukemia. Blood 100, 1810-6. Benitez R, Godelaine D, Lopez-Nevot MA, Brasseur F, Jimenez P, Marchand M, Oliva MR, van Baren N, Cabrera T, Andry G, Landry C, Ruiz-Cabello F, Boon T, Garrido F (1998) Mutations of the !2-microglobulin gene result in a lack of HLA class I molecules on melanoma cells of two patients immunized with MAGE peptides. Tissue Antigens 52, 5209. Boon T, Cerottini JC, Van den Eynde B, van der Bruggen P, Van Pel A (1994) Tumor antigens recognized by T lymphocytes. Annu Rev Immunol 12, 337-65. Boucher MJ, Morisset J, Vachon PH, Reed JC, Laine J, Rivard N (2000) MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-X(L), and Mcl-1 and promotes survival of human pancreatic cancer cells. J Cell Biochem 79, 355-69. Browning M, Petronzelli F, Bicknell D, et Browning M, Petronzelli F, Bicknell D, Krausa P, Rowan A, Tonks S, Murray N, Bodmer J, Bodmer W.47, 364-71. Budihardjo I, Oliver H, Lutter M, Luo X, Wang X (1999) Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 15, 269-90. Bullani RR, Huard B, Viard-Leveugle I, Byers HR, Irmler M, Saurat JH, Tschopp J, French LE (2001) Selective expression of FLIP in malignant melanocytic skin lesions. J Invest Dermatol 117, 360-4. Bullani RR, Wehrli P, Viard-Leveugle I, Rimoldi D, Cerottini JC, Saurat JH, Tschopp J, French LE (2002) Frequent downregulation of Fas (CD95) expression and function in melanoma. Melanoma Res 12, 263-70. Campoli M, Chang CC, Ferrone S (2002) HLA class I antigen loss, tumor immune escape and immune selection. Vaccine 20(suppl4), A40-5. Chen CH, Seguin-Devaux C, Burke NA, Oriss TB, Watkins SC, Clipstone N, Ray A (2003) Transforming growth factor ! blocks Tec kinase phosphorylation, Ca2+ influx, and NFATc translocation causing inhibition of T cell differentiation. J Exp Med 197, 1689-99. Cheng EH, Wei MC, Weiler S, Flavell RA, Mak TW, Lindsten T, Korsmeyer SJ (2001) BCL-2, BCL-XL sequester BH3 domain-only molecules preventing BAX- and BAKmediated mitochondrial apoptosis. Mol Cell 8, 705-11.

cancer model in mice (Oost et al, 2004). Additionally, new classes of polyphenylureas with XIAP-inhibitory activity have been shown to overcome XIAP mediated suppression of caspase 3 and 7, stimulate increased caspase activity and directly induced apoptosis in many types of tumor cell lines in culture. These polyphenylurea compounds also suppressed growth of established tumors in xeongraft models in mice and sensitized cancer cells to chemotherapeutic drugs (Schimmer et al, 2004).

D. Enhancement of pro-apoptotic receptors and molecule expression Anti-apoptotic family members heterodimerize with pro-apoptotic family members and antagonize their function to resist cell death. Synthetic peptide sequences derived from the BH3 domain of pro-apoptotic Bcl-2 family members such as Bax and Bak have recently been generated to block this heterodimerization thus allowing apoptosis. The usage of these synthetic peptides increased apoptosis by 40% in prostate cells (Finnegan et al, 2001). The use of an inducible recombinant Bax adenovirus was effective in enhancing apoptotic cell death in both ovarian cancer cell lines and patient-derived primary cancer cells and may provide a means to overcome the heterogeneous nature of tumors (Xiang et al, 2000). A small molecule mimic of Smac, a pro-apoptotic protein that functions by relieving the IAP-mediated suppression of caspase activity, has been synthesized to overcome IAP antagonist. This molecule also synergizes with both TNF-# and TRAIL and is a potential new therapy for cancer (Li et al, 2004).

VI. Conclusions In the past decade we have developed a remarkable appreciation of the adaptive immunity and its role in detecting cancer cells and impairing cancer growth, which have lead to numerous immunotherapy trials. However, our immunotherapy strategies have not clinically demonstrated effective control over tumor growth. We are now trying to focus our attention on comprehending the potential reasons for failure by understanding both the cellular and molecular pathways that interfere with the immune system’s own ability to develop powerful immunologic responses against the tumor cells. By exploring this knowledge of how tumor cells escape immune surveillance and resist immune-mediated killing, we may be able to target therapy and recreate an immune “sensitive” environment. With the advent of many newly developed small molecules inhibitors and specific antibodies it may be possible to direct our therapy to specific apoptosis pathways and reverse immune resistance, becoming an effective adjuvant to immunotherapy.

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Schumacher and Ribas: Overcoming tumor resistance to immunotherapy Zelivianski S, Spellman M, Kellerman M, Kakitelashvilli V, Zhou XW, Lugo E, Lee MS, Taylor R, Davis TL, Hauke R, Lin MF ( 2003) ERK inhibitor PD98059 enhances docetaxel-

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Cancer Therapy Vol 4, page 27 Cancer Therapy Vol 4, 27-34, 2006

The role of IDO in immune system evasion of malignancy: Another piece to the tolerance puzzle Review Article

Jeannine A. Villella, Kunle Odunsi, Shashikant Lele* Roswell Park Cancer Institute, Department of Surgery, Division of Gynecologic Oncology

__________________________________________________________________________________ *Correspondence: Shashikant B. Lele, MD, Chair, Gynecologic Oncology, Department of Surgery, Roswell Park Cancer Institute, School of Medicine & Biomedical Sciences, University at Buffalo, Elm & Carlton Streets, Buffalo, NY 14209, USA; e-mail: Shashikant.Lele@RoswellPark.org Key words: Immune Response, immune tolerance, indolamine 2, 3 dioxygenase Abbreviations: Indoleamine 2, 3-dioxygenase, (IDO); Lewis lung carcinoma, (LLC); lipopolysaccharide, (LPS); major histocompatibility complex, (MHC); tumor draining lymph nodes, (TDLN); tumor necrosis factor, (TNF) Received: 10 October 2005; Accepted: 25 October 2005; electronically published: January 2006

Summary Mechanisms of immune system evasion have tremendous implications in health and disease including inflammation, autoimmune disease, organ transplantation, pregnancy and carcinogenesis. The immune system discriminates based on stimuli that either provokes immune response or prevents response, resulting in immune tolerance. A delicate balance is essential for normal immune function and homeostasis. Factors involved in this process are complex, and the players somewhat elusive, making the pathophysiology of immune surveillance so intriguing. Breakdown of this fundamental homeostasis can tip the scale allowing disease to prevail. This article describes one piece to the puzzle: the role of the enzyme indolamine 2, 3- dioxygenase in the anti-tumor immune response. protects us from tumor development, but also sculpts the tumor phenotype (Dunn et al, 2004). The final phase of cancer immunoediting is immune escape from the forces of immunologic control, and supports the notion that tumor cell-specific neo-antigens could cause regression of developing cancers (Burnet, 1957; 1964). This concept of immune tolerance emerged and was described as a state of specific unresponsiveness that is antigen specific. There is an overall lack of attack on self antigens that is a normal part of the immune response. Since failure of self-tolerance leads to autoimmune disease, a delicate balance is imperative for normal immune function. It is to be expected that no single mechanism can completely explain the discrimination of the immune system and regulation of immune surveillance in circumstances such as pregnancy, auto immune disease, carcinogenesis and cancer progression. One aspect of the immunoregulatory process that has gained much attention in tumor immunology over the last several years is the role of the enzyme indolamine 2, 3- dioxygenase in modulating T cell responses through the essential amino acid catabolism of tryptophan. In this review article, we will discuss the mechanism and consequence of tryptophan metabolism in T-cell-mediated immune response, and the effect it has on cancer initiation and progression. We have

I. Introduction The immune system protects from pathogens that have the potential to be lethal. In the case of tumors, they usually grow despite an intact immune system; seemingly with apparent disregard for immune system control. It is intuitive to think that the immune system would recognize tumor cells as foreign and destroy them. Unfortunately this is not always the case. In 1909, Paul Ehrlich proposed the cancer immunosurveillance hypothesis and predicted that the immune system repressed the growth of cancers that would otherwise occur with greater frequency (Ehrlich, 1909). This hypothesis was embraced by tumor immunologists until the 1970s when Stutman showed no increased frequency of cancers in nude mice (Stutman, 1974). For many decades thereafter, immunologists focused on immunologic roles in organ transplantation and pregnancy (Thomas, 1959). Nobel Prize winners F. Macfarlane Burnet and Peter Brian Metawar focused on immunologic tolerance and its role in allograft rejection, and ultimately resurrected the concept of natural immune defense against cancer. Recently, there has been a resurgence in the concept of cancer immunosurveillance, and it is more appropriately named “cancer immunoediting� (Dunn et al, 2004). Cancer immunoediting holds that the immune system not only

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Villella et al: The role of IDO in immune system evasion of malignancy summarized the evidence supporting this role in immune tolerance including the role of regulatory T cells. Lastly, we discuss the clinical implications of IDO inhibition as an adjuvant to potentiate cancer vaccine therapy.

(Yuan et al, 1998; Munn et al, 1999). Human and murine T cells enter the cell cycle, but progression terminates midway through the G1 phase before the commencement of S phase when tryptophan is withheld from the culture media (Munn et al, 1999). Growth arrested T cells fail to acquire cytolytic effector functions. This may imply that T cells possess a tryptophan sensitive checkpoint in the cell cycle that determines whether or not they proliferate (Mellor et al, 2001). Thus, tryptophan is important for T cell proliferation, as in its absence T cells arrest in G1 phase of the cell cycle (Mellor et al, 2003). This enzyme is expressed by human macrophages. When present in the tumor microenvironment, it suppress T cell responses locally by limiting tryptophan availability (Mellor and Munn, 2003). IDO is important in maintaining maternal tolerance toward the fetus during pregnancy, as well as in suppressing T cell response to MHC-mismatched allografts (Mellor and Munn, 2004). Dendritic cells are key regulators of immune outcomes as they are the most efficient antigen presenting cells in the body. They are capable of promoting or suppressing T cell responses depending on the circumstances (Moser, 2003). They function as the foreman of the immune system, whereby they integrate incoming signals and direct and appropriate T cell response: immune activation or tolerance (Mosmann and Livingstone, 2004).

II. What is IDO? Tryptophan is an amino acid required for protein synthesis and other metabolic functions. Tryptophan is synthesized from molecules such as phosphoenolpyruvate in bacteria, fungi and plants, and these organisms fuel the tryptophan flux through the food chain (Moffett and Namboodiri, 2003). Animals are incapable of synthesizing tryptophan, so they ingest it in protein form, which is then hydrolyzed into the constituted amino acids. It is delivered through the hepatic portal system, and that portion which is not used for protein synthesis in the liver can either be distributed through the blood stream for protein synthesis or it can be degraded in the liver by the kynurenine pathway (Moffett and Namboodiri, 2003). There is a clear association between tryptophan catabolism and inflammatory reactions, which may be occurring in the immune system rather than in the liver (Moffett and Namboodiri, 2003). Indoleamine 2, 3-dioxygenase (IDO) is a hemecontaining enzyme that catabolizes the first and ratelimiting step in oxidative degradation of tryptophan. IDO is encoded by a single gene and is transcribed in response to inflammatory mediators such as interferon-! (Taylor and Feng, 1991). In the late 1970s, Hayashi et al discovered that de novo synthesis of IDO was induced by immune stimulating agents such as lipopolysaccharide (Hayaishi and Yoshida, 1978; Yoshida and Hayaishi, 1978); and later interferon was found to be one of the primary inducers of IDO synthesis (Yoshida et al, 1986). Tryptophan depletion by IDO was then thought to be limited to areas of inflammation where leukocytes were actively producing inflammatory mediators. In 1984, it was shown that growth of Toxoplasmosis gondii could be inhibited by IFN-mediated IDO induction, and could be controlled by the amount of tryptophan concentrations in the culture media (Pfefferkorn, 1984). Tryptophan depletion by IDO was the principal mechanism responsible for inhibiting parasitic growth. Numerous human cell lines express IDO when exposed to interferon! while it is scarcely detected in freshly isolated tissues (Burke et al, 1995; Varga et al, 1996; Munn et al, 1999; Hwu et al, 2000). Other cytokines such as LPS, TNF" and IL1 act in a synergistic fashion with interferon-! to further increase IDO expression in human dendritic cells (Babcock and Carlin, 2000; Hwu et al, 2000). Antiinflammatory cytokines such as IL4, IL10 and TGF# inhibit IDO expression (Yuan et al, 1998; MacKenzie et al, 1999).

IV. Regulation of IDO gene expression The IDO enzyme is encoded by a single gene with 10 exons spread over approximately 15 kb of DNA located in a syntenic region of human and mouse chromosome 8 (Mellor and Munn, 2004). Transcription is controlled by responding to inflammatory mediators like IFN-"# and IFN-! interferons (Dai and Gupta, 1990; Taylor and Feng, 1991; Hassanain et al, 1993; Mellor and Munn, 1999). Myeloid-lineage cells such as Dendritic cells and macrophages, fibroblasts, endothelial cells and tumor-cell lines express IDO after exposure to IFN-! (Taylor and Feng, 1991; Burke et al, 1995; Varga et al, 1996; Munn et al, 1999; Hwu et al, 2000). Signal transducer and activator of transcription and IFN-regulatory factor 1 function cooperatively to mediate the induction of IDO expression by IFN-!, and mice that lack either IFN-! or IRF1 are deficient in IDO expression during infection (Silva et al, 2002). Due to the synergistic actions of lipopolysaccharide (LPS) and the inflammatory cytokine IL-1 and tumor necrosis factor (TNF), they enhance IDO expression in vitro (Babcock and Carlin, 2000; Robinson et al, 2003). However, in vivo, responsiveness to LPS depends on TNF, but does not require IFN-! indicating an IFN-! independent pathway for IDO expression (Fujigaki et al, 2001). There may be some cell specific inflammatory mediators of transcription, further modulating IDO expression (Yuan et al, 1998; MacKenzie et al, 1999). IDO is functionally an intracellular enzyme. This enzyme is known to be found constitutively at the maternal-fetal interface by human extra-villous trophoblast cells (Kudo and Boyd, 2000; Kudo et al, 2001, 2004;

III. How does Tryptophan deprivation thwart immune response? We know that IDO is released by stimuli such as interferon-!. Tryptophanyl tRNA synthetase is the only amino-acyl tRNA synthetase whose expression is enhanced by inflammatory mediators such as interferon-! 28

Cancer Therapy Vol 4, page 29 Honig et al, 2004). Also at this interface, trophoblast giant cells of fetal origin also express IDO (Baban et al, 2004). Functional IDO expression can be seen in lymphoid organs such as epididymis, colon and ileum, lymph nodes, spleen and thymus (Takikawa et al, 1986). Protein expression may be present without functional activity. Although human DCs constitutively express immunoreactive IDO protein, it does not have enzymatic activity until IFN-! and CD80/CD86 cells activate (Munn et al, 2004). Incorporation of the heme group into the active site is required for IDO activity, and inhibitors of heme biosynthesis inhibit functional activity without affecting protein levels (Thomas et al, 2001).

al, 2001). When the fetal trophoblasts invade the uterine tissue at the site of implantation with human leukocyte antigen G, it inhibits maternal natural killer cell activation (Aluvihare et al, 2004). This produces an anatomic barrier, thereby insulating the fetus from maternal immune system (Mellor and Munn, 2000). This may support one of Medawar’s initial three hypotheses for preventing fetal rejection that was later rebuked: anatomic separation.

VI. The role of IDO in the anti-tumor immune response Indolamine 2,3 dioxygenase has been detected in various tumors, including gynecological malignancies (Sedlmayr et al, 2002; Schroecksnadel et al, 2005) and in tumor draining lymph nodes (Mellor and Munn, 2004). Recent evidence suggests that IDO plays an important role in suppressing anti-tumor immunity (Uyttenhove et al, 2003). Mellor and Munn propose that one mechanism by which IDO exerts suppressive activity involves the generation of regulatory T cells (Mellor and Munn, 2004). Specifically, they suggest that IDO-arrested T cells can adopt a regulatory T cell phonotype. Thus, individuals with higher IDO activity might also have greater regulatory T cells activity; due to the acquired regulatory T cell phenotype of IDO arrested cells. These regulatory T cells have the ability to induce expression of IDO by dendritic cells and thus mediate the inhibitory effects of regulatory T cells. This concept of IDO competent dendritic cells is combined with concept of immunogenic versus tolerogenic signal integration (Mellor and Munn, 2004). The IDO mediated immune regulation would require an immature dendritic cell that would express IDO and perhaps during maturation, the IDO competent cell can receive conditioning signal that lead to different signals. For instance, the tolerogenic signals such as CD80/CD86 ligation by CTLA4+ regulatory T cells would induce IDO expression, thereby eliciting the functionally suppressive regulatory T cell phenotype (Mellor and Munn, 2004). Conversely, immunogenic signals such as CD40 ligation by T helper cells would promote a nonsuppressive phenotype and down regulate IDO expression (Grohmann et al, 2000). It is believed that both pathways lead to competent and mature APCs, specialized for opposing functions. Several studies have demonstrated that IDO expressing dendritic cells can suppress potent T cell responses in vivo and promote systemic tolerance (Grohmann et al, 2001a, b) One mechanism by which this may occur is by recruitment of regulatory T cell development. When mice are exposed to immunomodulatory agent CTLA4-Ig, IDO may serve as a downstream suppressor mechanism used by certain Tregs (Mellor et al, 2003), these cells may promote acquired regulatory T cells that arise extra-thymically and are responsible for acquired peripheral tolerance and antigenspecific anergy (Bluestone and Abbas, 2003). Research in tumor immunology has clarified that tumors can induce tolerance to their own antigens, and thereby evade immune destruction despite the presence of cytotoxic T cells in the circulation (Pardoll, 2003). IDO

V. Why doesn’t a mother reject her fetus? The immunologic paradox of fetal survival was investigated by Medawar in 1953. The phenomenon allowing allogenic mammalian fetus to survive in the maternal circulation contradicted the recent concepts involved in organ transplantation and rejection. Fetuses have paternally encoded genes that are foreign to the maternal immune system. The three possible mechanisms explored were: anatomic separation, antigenic immature fetus, immunologic inertness of the mother. Since the maternal T cells are aware of the paternally inherited major histocompatibility complex (MHC) class I alloantigens during pregnancy (Tafuri et al, 1995; Munn et al, 1998; Jiang and Vacchio, 1998), only the latter seemed plausible. The enzyme IDO was found in syncytiotrophoblasts (Kamimura et al, 1991), thus in a mouse model, they exposed syngeneic or allogeneic fetuses to 1-methyl-tryptophan that competitively inhibits the IDO enzyme activity. PCR product specific for IDO was found in both syngeneic and allogeneic conceptuses by post conception day 7.5, and by day 9.5, all the allogenic fetuses were deteriorating. When the experiment was repeated using female mice carrying a defective RAG1 gene thus preventing lymphocyte development, the conceptuses were normal. The data demonstrated that inhibition of tryptophan catabolism during pregnancy allows maternal lymphocytes to mediate fetal rejection (Munn et al, 1998). Also, IDO protected the fetus by suppressing T cell driven local inflammatory response to fetal alloantigens. Thus, IDO did not act alone in fetal tolerance. Another interesting question about the catabolism of tryptophan by IDO is that tryptophan is an essential amino acid required to nurture fetal growth. It has been shown that cultured human IDO+ macrophages express an inducible high affinity tryptophan transporter activity (Munn et al, 1999). This particular positioning of a tryptophan transporter at the point of contact between T cells and APCs would provide an effective way for APCs to remove free tryptophan from T cells rapidly without necessarily depleting tryptophan from the surrounding tissue milieu (Mellor and Munn, 2001). Recently, it has been discovered that fetal tissues deplete tryptophan at the maternal-fetal interface, thus inhibiting T cell proliferation (Munn et al, 1998; Mellor et

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Villella et al: The role of IDO in immune system evasion of malignancy may be involved in this process by preventing T cell proliferation at the tumor microenvironment. Transfecting tumor cells with IDO rendered a normally immunogenic tumor cell line resistant to immune rejection in primed hosts that were fully protected against their untransfected tumors (Uyttenhove et al, 2003). Tumor cells have been shown to express IDO in vivo (Curiel et al, 2004; Schroecksnadel et al, 2005). This may be a mechanism at which tumor cells exert their anti-proliferative effect on T cells, thus enhancing increased tumor survival. Ubiquitous expression of IDO has been observed in a population of host APCs in tumor-draining lymph nodes of both humans and mice (Munn et al, 2004). In this scenario, presentation of tumor antigens by host APCs allows na誰ve T cells to be familiarized with tumor derived antigens (Munn et al, 2004). Dendritic cells either can be activating or tolerizing. Expression by certain cells of the immune system allows them to inhibit T cell proliferation (Munn et al, 1999, 2002; Hwu et al, 2000). In recent publications, the downstream molecular mechanisms that IDO utilizes to regulate T cell function have been explored. Using a mouse model, IDO expressing plasmacytoid DCs were shown to activate the GCN2 kinase pathway in responding T cells. In GCN2-knockout T cells were not inhibited by IDO expressing DCs from tumor-draining lymph nodes, thus indicating that GCN2 acts as a molecular sensor in T cells that promotes proliferative arrest and anergy induction in response to IDO (Munn et al, 2004). This mechanism indicates the role of IDO in stress-related immune response such as malignancy. IDO expression has been demonstrated in many tumors and cell lines including hepatocellular carcinoma (Ishio et al, 2004), gynecologic cancer cell lines such as cervical, vulva, breast and ovarian (Leung et al, 1992). IDO may be exploited by tumor cells as a mechanism of tumor evasion (Munn and Mellor, 2004). Transfection of IDO in tumor cell lines confers the ability to inhibit antigen-specific T cell responses in vitro (Mellor et al, 2002). These tumors are able to grow as a result of local immunosuppression within the tumor microenvironment. If this is true, in order for APCs to present the tumor antigens, it must take place at tumor draining lymph nodes (TDLN) (Munn et al, 2004). In a murine model, TDLN had more IDO+ cells versus few in the lymph nodes that were negative for tumor (Munn et al, 2004). These same TDLNs had a population of suppressive DCs, whereas the negative lymph nodes had excellent stimulatory DCs (Munn et al, 2004). Thus, the population of immunoregulatory DCs in TDLN is capable of mediating active immunosuppression in vivo, rather than their having a defective ability to stimulate T cells (Almand et al, 2000; Vicari et al, 2002; Yang et al, 2003 Furumoto et al, 2004). This underscores the localized nature of the immune suppression that lends support to the cell type specific function of IDO. One critical barrier of cancer therapy is mechanisms of drug resistance in tumor cells. Although much is still unknown, multi-drug resistance genes have been identified in many tumors. The mechanisms of chemoresistance may not be ubiquitous for all malignancies, and thus tumor

specific identification is necessary. Recently, IDO has been identified as a mechanism of chemoresistance in ovarian cancer (Okamoto et al, 2005). IDO was identified as one of 17 genes responsible for chemoresistance in ovarian cancer using gene chip analysis. Real-time quantitative PCR was performed on chemoresistant cell lines and tumor from refractory patients, but not in those that were chemosensitive (Okamoto et al, 2005). Immunohistochemical analysis of IDO protein showed differences in those tumors with poor versus good prognosis. All patients without relapse had tumors negative for IDO This single, small study demonstrates another tumor specific mechanism of tumor survival (Okamoto et al, 2005). Malignant tumors may exploit the mechanism of Tcell response inhibition by recruiting IDO-expressing APCs to the tumor-draining lymph nodes. Abnormal accumulations of IDO-positive cells with a monocytoid or plasmacytoid morphology were identified in the perisinusoidal regions of draining lymph nodes in 45% of nodes studied (Lee et al, 2003). Recruitment of IDOpositive cells was seen in nodes with and without malignancy. It is possible that these IDO-positive APCs may contribute mechanistically to acquired tolerance to tumor antigens. Immunostaining of tumor-draining lymph nodes for abnormal accumulation of IDO-expressing cells might thus constitute an adverse prognostic factor (Lee et al, 2003).

VII. Cancer vaccines Vaccinations against cancer aim to induce tumor specific effector T cells that can reduce the tumor mass, as well as tumor specific memory T cells that can control tumor relapse (Banchereau and Palucka, 2005). Dendritic cells are often used as adjuvants for vaccination because of their ability to regulate T cell immunity by antigen presentation. Dendritic cells can induce and maintain immune tolerance (Steinman et al, 2003). Central tolerance depends on mature thymic DCs, which are essential for the deletion of newly generated T cells that have a receptor that recognizes self components (Brocker, 1999). This may not be sufficient for all antigens, and those antigens expressed locally will not have access to them. Thus, peripheral tolerance, which occurs in lymphoid organs, may be important for antigen presentation. Peripheral tolerance requires immature DCs to present tissue antigens to T cells in the absence of appropriate co-stimulation leading to T cell anergy or deletion (Brocker, 1999), or to the development of IL-10 secreting inducible regulatory T cells (Jonuleit et al, 2000; Dhodapkar et al, 2001). Mature DCs may contribute to peripheral tolerance by promoting the clonal expansion of naturally occurring regulatory T cells (Banchereau and Palucka, 2005). In order to make the antigen presentation stimulate a more robust immune response, these mechanisms of T cell activation, regulatory T cell function provides a basis for a potential revolution in cancer immunotherapy. By administering an IDO inhibitor such as 1-methyl tryptophan, it may be possible to break one barrier that allows tumor escape and improve the antitumor immune response. 30

Cancer Therapy Vol 4, page 31

VII. Preclinical studies of inhibition and tumor immunity

Aluvihare VR, Kallikourdis M, Betz AG (2004) Regulatory T cells mediate maternal tolerance to the fetus. Nat Immunol 5, 266-71. Baban B, Chandler P, McCool D, Marshall B, Munn DH, Mellor AL (2004) Indoleamine 2,3-dioxygenase expression is restricted to fetal trophoblast giant cells during murine gestation and is maternal genome specific. J Reprod Immunol 61, 67-77. Babcock TA, Carlin JM (2000) Transcriptional activation of indoleamine dioxygenase by interleukin 1 and tumor necrosis factor " in interferon-treated epithelial cells. Cytokine 12, 588-94. Banchereau J, Palucka AK (2005) Dendritic cells as therapeutic vaccines against cancer. Nat Rev Immunol 5, 296-306. Bluestone JA, Abbas AK (2003) Natural versus adaptive regulatory T cells. Nat Rev Immunol 3, 253-7. Brocker T (1999) The role of dendritic cells in T cell selection and survival. J Leukoc Biol 66, 331-5. Burke F, Knowles RG, East N, Balkwill FR (1995) The role of indoleamine 2,3-dioxygenase in the anti-tumour activity of human interferon-! in vivo. Int J Cancer 60, 115-22. Burnet M (1957) Cancer; a biological approach. I. The processes of control. Br Med J 5022, 779-86. Burnet M (1964) Immunological Factors in the Process of Carcinogenesis. Br Med Bull 20, 154-8. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, Evdemon-Hogan M, Conejo-Garcia JR, Zhang L, Burow M, Zhu Y, Wei S, Kryczek I, Daniel B, Gordon A, Myers L, Lackner A, Disis ML, Knutson KL, Chen L, Zou W (2004) Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10, 942-9. Dai W, Gupta SL (1990) Regulation of indoleamine 2,3dioxygenase gene expression in human fibroblasts by interferon-!. Upstream control region discriminates between interferon-! and interferon-". J Biol Chem 265, 19871-7. Dhodapkar MV, Steinman RM, Krasovsky J, Munz C, Bhardwaj N (2001) Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med 193, 233-8. Dunn GP, Old LJ, Schreiber RD (2004) The immunobiology of cancer immunosurveillance and immunoediting. Immunity 21, 137-48. Ehrlich P (1909) Ueber den jetzigen Stand der Karziomforschung. Ned Tijdschr Geneeskd 5, 273-90. Friberg M, Jennings R, Alsarraj M, Dessureault S, Cantor A, Extermann M, Mellor AL, Munn DH, Antonia SJ (2002) Indoleamine 2,3-dioxygenase contributes to tumor cell evasion of T cell-mediated rejection. Int J Cancer 101, 1515. Fujigaki S, Saito K, Sekikawa K, et al (2001) Lipopolysaccharide induction of indoleamine 2,3-dioxygenase is mediated dominantly by an IFN-!-independent mechanism. Eur J Immunol 31, 2313-8. Furumoto K, Soares L, Engleman EG, Merad M (2004) Induction of potent antitumor immunity by in situ targeting of intratumoral DCs. J Clin Invest 113, 774-83. Grohmann U, Bianchi R, Belladonna ML, Silla S, Fallarino F, Fioretti MC, Puccetti P (2000) IFN-! inhibits presentation of a tumor/self peptide by CD8"- dendritic cells via potentiation of the CD8"+ subset. J Immunol 165, 1357-63. Grohmann U, Fallarino F, Bianchi R, Belladonna ML, Vacca C, Orabona C, Uyttenhove C, Fioretti MC, Puccetti P (2001a) IL-6 inhibits the tolerogenic function of CD8"+ dendritic cells expressing indoleamine 2,3-dioxygenase. J Immunol 167, 708-14. Grohmann U, Fallarino F, Silla S, Bianchi R, Belladonna ML, Vacca C, Micheletti A, Fioretti MC, Puccetti P (2001b)

IDO

The mechanism by which IDO interferes with antitumor immunity is of interest. Preclinical studied of IDO inhibition in efforts to improve anti-tumor immune responses have been done in mouse models. IDO is under genetic control of Bin1, which is attenuated in many human malignancies (Muller et al, 2005; Schroecksnadel et al, 2005). Mouse knockout studies indicate that Bin1 loss elevates the STAT1- and NF-$B-dependent expression of IDO, driving escape of oncogenically transformed cells from T cell-dependent anti-tumor immunity (Muller et al, 2005). In an established breast cancer mouse model, small-molecule inhibitors of IDO cooperate with cytotoxic agents to elicit regression of established tumors refractory to single-agent therapy. This finding suggests that Bin1 loss promotes immune escape in cancer by deregulating IDO and that IDO inhibitors may improve responses to cancer chemotherapy. In melanoma studies, when mice were injected with IDO inhibitor 1-methy-dl-tryptophan, the cytotoxic activity of the mice NK cells was reduced in a dose dependent fashion (Kai et al, 2003). IDO acts as an immunosuppressive enzyme, and when expressed by mononuclear cells that invade tumors and tumor-draining lymph nodes, is one mechanism that may account for this function. Lewis lung carcinoma (LLC) cells stimulated a more robust allogeneic T cell response in vitro in the presence of a competitive inhibitor of IDO, 1-methyl tryptophan (Friberg et al, 2002). When administered in vivo this inhibitor also resulted in delayed LLC tumor growth in syngeneic mice. The function of IDO as an inhibitor of cytotoxic activity of NK cells in melanoma has been demonstrated in a dose-dependent manner when an 1-methyltryptophan is given (Kai et al, 2003). In conclusion, these results indicated that IDO plays an important role in anti-tumor immunity by regulating cytotoxic activity of NK cells.

VIII. directions

Conclusions

and

future

The mechanism by which the immune system of a tumor-bearing host acquires tolerance toward tumor antigens is still elusive. Antigen-presenting cells (APCs) are critical regulators of the decision between immune response and tolerance. Recent evidence suggests that the immunosuppressive effect of IDO may allow tumors to escape immune surveillance. To date, there are no human clinical studies utilizing IDO inhibitors to counteract the tolerogenic effects of IDO in the tumor microenvironment. The manipulation of this enzyme and the modification of its effects may enhance the efficacy of immunotherapeutic strategies designed to generate durable anti-tumor immunity.

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Jeannine A. Villella

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Cancer Therapy Vol 4, page 35 Cancer Therapy Vol 4, 35-46, 2006

Vitamin E analogs as anti-cancer agents: The role of modulation of apoptosis signalling pathways Review Article

Lan-Feng Dong1, Xiu-Fang Wang1, Yan Zhao2, Marco Tomasetti3, Kun Wu2, Jiri Neuzil1,4,* 1

Apoptosis Research Group, School of Medical Science, Griffith University, Southport, Qld, Australia Department of Nutrition, Harbin Medical University, Harbin, Heilongjiang Province, China 3 Department of Molecular Pathology and Innovative Therapies, Polytechnic University of Marche, Ancona, Italy 4 Laboratory of Cell Signalling and Apoptosis, Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czech Republic 2

__________________________________________________________________________________ *Correspondence: Jiri Neuzil, Apoptosis Research Group, Heart Foundation Research Centre, School of Medical Science, Griffith University Gold Coast Campus, Southport, Qld, Australia; phone: +61-7-55529109; fax: +61-7-55528444; email: jneuzil@griffith.edu.au Key words: Vitamin E analogs, pro-apoptotic activity, anti-cancer drugs, destabilization of mitochondria, Mitochondrial apoptogenic pathways, apoptosis, c-Jun pathway, pro-survival pathways, Sensitisation of cancer cells, TRAIL, signaling pathways, !-tocopheryl succinate Abbreviations: !-tocopheryl maleate, (!-TOM); !-tocopheryl succinate, (!-TOS); 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)-chroman-6-yloxyacetic acid, (!-TEA); apoptosis-inducing factor, (AIF); c-Jun NH2-terminal kinase, (JNK); cyclin-dependent kinases, (CDK); death domain, (DD); death-inducing signalling complex, (DISC); decoy receptors, (DcRs); dominant-negative, (DN); extra-cellular signal-regulated kinases, (ERKs); Fas-associated death domain, (FADD); FLICE-like inhibitory protein, (FLIP); inhibitors of apoptosis proteins, (IAPs); malignant mesothelioma, (MM); nuclear factor-"B, (NF"B); protein phosphatase 2A, (PP2A); sphingomyelinase, (SMase); tumor necrosis factor-!, (TNF-!); tumor necrosis factor-related apoptosis-inducing ligand, (TRAIL/Apo2L) Received: 16 January 2006; Accepted: 31 January 2006; electronically published: February 2006

Summary Recently, considerable decrease in a number of previously fatal pathologies has been achieved, largely due to the advancement in molecular medicine and due to modern approaches to treatment. In spite of this success, neoplastic disease remains a serious problem for several reasons. These include an exceedingly high variability of cancer cells even within the same type of tumor. Cancer cells, albeit of clonal origin, mutate so that they escape established treatments, resulting in the fatal outcome of current therapies. Moreover, there are types of cancer, such as mesotheliomas, that cannot be treated at present. A novel group of clinically interesting anti-cancer drugs has been a recent focus in the literature that hold substantial promise as selective anti-cancer agents. These compounds, epitomized by !-tocopheryl succinate, comprise redox-silent analogs of vitamin E that have been shown to suppress several types of cancer in animal models, including breast, colon and lung cancer as well as mesotheliomas and melanomas, while being non-toxic to normal cells and tissues. It is now proven that the strong anti-cancer effect of vitamin E analogs stems from their propensity to induce selective apoptosis in malignant cells. The results point to the novel group of vitamin E analogs as promising agents applicable to different types of tumors.

other hand others are on the increase or, even worse, beyond treatment at this stage. Therefore, new strategies and approaches are needed to successfully manage the multitude of neoplasias. These should also encompass one of the most coveted for feature of anti-cancer agents: selectivity for malignant cells. We and others have been studying over the last five or so years a novel group of anti-cancer agents that befit

I. Introduction Neoplastic disease is a complex pathology with multiple facets and with exceeding promiscuity in terms of mutations of relevant genes, necessary for execution of the anti-tumor activity of established drugs. The clonal origin of cancer cells and their constant mutations make efficient treatment of malignancies an unrelenting challenge. On one hand, some types of cancer are being curbed, on the 35

Dong et al: Vitamin E analogues as anti-cancer agents the above scenario, i.e. analogs of vitamin E (Prasad et al, 2003, Neuzil et al, 2004). These intriguing compounds have been shown to be highly efficient against a variety of malignancies, including the fatal mesotheliomas. The most studied member of these drugs, !-tocopheryl succinate (!TOS), exerts its pro-apoptotic activity by triggering a massive apoptogenic response in a variety of cancer cells of different origin as well as by arresting the cell cycle and inhibiting proliferation of cancer cells by disrupting autocrine signaling pathways. The agent has also been shown to be highly selective for malignant cells, being largely non-toxic to normal cells and tissues. Thus, !-TOS and relative compounds may represent the long sought after drugs of choice for treatment of multiple malignancies (Neuzil et al, 2004). This review summarises our current knowledge of the mechanism of action of vitamin E analogs in the context of their anti-cancer activity. The paper focuses on their structure-function relationship and the major pathways that they initiate/modulate, which translates into efficient inhibition of cancer. Future perspectives of these intriguing compounds are suggested.

The term vitamin E refers to eight naturally occurring, structurally related agents, four tocopherols (!TOH, #-TOH, $-TOH, and %-TOH) and the four corresponding tocotrienols (!-T3H, #-T3H, $-T3H, %T3H) (Figure 1). Structural features, consisting of Domain I, II and III, play essential roles in activities of vitamin E and its analogs (Neuzil et al, 2004). Domain I, also referred to as a Functional Domain, makes vitamin E an antioxidant due to the redox-active hydroxyl group. !TOH, present at the highest concentration in serum and in dietary supplements, is proved to be biologically the most active isoform of vitamin E. To date, experimental and epidemiological evidence for the association of !-TOH with anti-cancer effect has been weak and controversial in the past decades (Woodson et al, 1999; Malila et al, 2002). The situation, however, is greatly different when some variations are made in vitamin E domains. The apoptogenic vitamin E analogs have been the focus of anticarcinogenesis research in recent years. In the case of !TOS, hydroxyl group within Domain I is esterfied with a succinyl moiety that makes the analogue redox-silent and endows it with strong apoptogenic activity. Furthermore, !-tocopheryl maleate (!-TOM), a maleyl monoester analog of vitamin E, exhibits nearly 20-fold greater apoptogenic activity than does !-TOS (Birringer et al, 2001). A large number of in vitro and in vivo data reveal that !-TOS displays apparently pro-apoptotic propensity towards malignant cells. Typical morphological and biochemical alterations, characterized by chromatin condensation, chromatin crescent formation and/or margination, DNA fragmentation and apoptotic body formation, occur when apoptosis is triggered by !-TOS in a variety of types of tumor cells. In fact, !-TOS has shown high levels of apoptosis in at least 50 types of cancer cell lines tested thus far, including different origin of the species (human, murine and avian) and tissue type (breast, prostate, lung, stomach, ovary, monocyte, colon and even mesothelium) (Israel et al, 2000; Bang et al, 2001; Neuzil et al, 2001, 2003, 2004; Yu et al, 2001, 2003;

II. Vitamin E analogs as anti-cancer drugs and adjuvants – relation to their pro-apoptotic activity Vitamin E analogs are lipid-soluble micronutrients consumed on regular bases and their dosage can be increased by food supplement without secondary deleterious effects. The potential use of vitamin E analogs as anti-cancer drugs and adjuvants has been intriguing for years because they show evident redox activities and function as scavengers of free radicals. There has been interest in their anti-cancer effects, largely via induction of apoptosis. Apoptosis, or programmed cell death, is one of the major mechanisms to regulate homeostasis through elimination of malignant or unwanted cells in metazoic organisms. Understanding of its molecular details has provided novel strategies for cancer therapy (Sun et al, 2004; Ghobrial et al, 2005).

Figure 1. Scheme of major domains in !-tocopherol and !-tocopheryl succinate. Both !-TOH and !-TOS comprize three major domains. Domain III (Hydrophobic Domain) is responsible for docking the agents in circulating lipoproteins and in biological membranes. Domain II (Signaling Domain) is involved in modulation of signaling pathways, such as the protein phosphatase 2A/protein kinase C pathway. Domain I (Functional Domain) provides the analogs with their major biological activity. In case of !-TOH, it is the hydroxyl group that gives it its redox activity, while in !-TOS, the succinyl moiety provides the agent with strong apoptogenic efficacy.

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Cancer Therapy Vol 4, page 37 Weber et al, 2002; Wu et al, 2002, 2004a; Prasad et al, 2003; Anderson et al, 2004; Hrzenjak et al, 2004; Kline et al, 2004; Stapelberg et al, 2005). Diverse types of malignant cells also show different susceptibilities in response to !-TOS. Neuzil et al, (2001) demonstrated that apoptotic rate induced by exposure to !-TOS at 50 µM for 12 h varied from 30% to 60% in different malignant cells. About 50% of apoptosis was induced by !-TOS treatment at 20 µg/ml (equivalent to 38 µM) for 48 h in MDA-MB435 human breast cancer cells (Yu et al, 2003). Exposure to !-TOS at 20 µg/ml for 24 and 48 h triggered 14 and 90% of SGC-7901 human stomach cancer cells to undergo apoptosis, respectively, but !-TOH at the same dosage did not show any apoptotic effect (Wu et al, 2002, 2004a). Importantly, !-TOS is not harmful toward normal cells and tissues with apoptotic rate less than 5% (Neuzil et al, 2001). In summary, !-TOS is a potent apoptosis inducer highly selective for malignant cells. Recently published in vivo results have supported the hypothesis that the non-antioxidant analogs of vitamin E strongly suppress cancer cell growth as well. Such inhibition is observed in athymic mice with tumor xenografts including human neuroblastoma, breast cancer, colon carcinoma, peritoneal mesotheliomas and murine melanoma (Malafa et al, 2000, 2002; Barnett et al, 2002; Weber et al, 2002; Stapelberg et al, 2005) and in the benzo(a)pyrene-induced fore stomach carcinoma in female thymus-bearing mice (Wu et al, 2001) after intraperitoneal administration of !-TOS. Colon and mammary tumor metastases are reduced by !-TOS (Barnett et al, 2002; Lawson et al, 2004). !-TOS may also enhance sensitization of resistant cells to other inducers of apoptosis, such as the immunological TNF-related apoptosis-inducing ligand (Weber et al, 2002). The above results further strengthen and extend the prospects for !TOS, an efficient apoptosis-triggering agent, as a promising anti-cancer drug. !-TOS is effective only when given intraperitoneally, not by oral administration in the in vivo experiments, because the ester bond linking the succinyl moiety to tocopherol is subject to hydrolysis by nonspecific esterases upon intestinal uptake of the drug. The recently synthesized ether analogues of vitamin E, !tocopheryloxybutyrate and 2,5,7,8-tetramethyl-2R(4R,8R,12-trimethyltridecyl)-chroman-6-yloxyacetic acid (!-TEA) have improved this aspect to a great extent. In the two compounds, butyryl and malonyl groups are attached to the Functional Domain of the analog, respectively, via an ether bond that is resistant to esterasemodulated hydrolysis, endowing the compounds with superior stability to their ester counterparts. It has been reported that !-tocopheryloxybutyrate exerts comparable apoptotic activity to that of !-TOS in leukaemia cell lines (Fariss et al, 1994) or even more potency in prostate cancer cells (Wu et al, 2004b). !-TEA exerts similar anticancer and apoptogenic properties as does !-TOS in human breast, prostate, colon, lung and endometrium cancer cells, and is more efficient than !-TOS in apoptosis induction in human ovarian and cervical cancer cells and mouse mammary tumor cells regardless of the

administration method (Anderson et al, 2004; Lawson et al, 2004). These new vitamin E analogs plus !-TOM and !-TOS may epitomize new approaches to development and establishment of anti-cancer drugs of the broad vitamin E group of compounds. Taken together, vitamin E analogs with potent apoptogenic activity show efficient anti-cancer activity in vitro and in vivo using experimental animal models. However, the exact mechanisms of triggering apoptosis are still unclear at this stage. Most of the recent advances have shed some light on the characterization of the effector mechanisms. Two such mechanisms, associated with the caspase cascade, have been intensively investigated, viz. the intrinsic or mitochondria-mediated mechanism and the extrinsic or death receptor-mediated mechanism. Some aspects of these signaling pathways, in relation to apoptosis induction in cancer cells by vitamin E analogs, are provided below.

III. Mitochondrial apoptogenic pathways as a target for pro-apoptotic activity of vitamin E analogs Mitochondria are membrane-enclosed organelles distributed through the cytosol of most eukaryotic cells. They have an outer membrane that defines their structure and an inner membrane (also known as cristae) that encloses a fluid-filled matrix. The outer membrane contains complexes of integral membrane proteins that form channels through which a variety of molecules and ions move in and out of the mitochondrion. The inner membrane contains 5 complexes of integral membrane proteins: NADH dehydrogenase, succinate dehydrogenase, cytochrome c reductase (also known as the cytochrome bc1 complex), cytochrome c oxidase and ATP synthase. Mitochondria are essential for optimal life of most eukaryotic cells by mediating energy generation in the form of ATP. Paradoxically, recent research demonstrated that mitochondria also play an important role in programmed cell death (Green and Kroemer, 2004), and the role of mitochondria has also been demonstrated for apoptosis induced by vitamin E analogs (Neuzil et al, 2004). Thus, vitamin E analogs belong to the class of ‘mitocans’, agents that initiate cell death and potentially suppress cancer by targeting mitochondria (Ralph et al, 2006).

A. Initiation of apoptotic pathways leading to destabilization of mitochondria How could vitamin E analogs affect mitochondria and trigger the initial apoptotic signals? The first event observed upon exposure of cells to !-TOS is the activation of sphingomyelinase (SMase), an enzyme that converts sphingomyelin, which is a relatively rare constituent of the plasma membrane, to a lipid second messenger ceramide, a well-known strong inducer of apoptosis (Ogretmen and Hannun, 2004). We showed that treatment of Jurkat cells resulted in activation of SMase within 15-30 min and this was not suppressed by a pan-caspase inhibitor, zVADfmk, suggesting that SMase is a caspase-in-dependent, possibly a direct target of the vitamin E analog (Weber et 37

Dong et al: Vitamin E analogues as anti-cancer agents al, 2003). It is also plausible that the activation is due to a change in the plasma membrane fluidity upon incorporation of the lipophilic !-TOS, consistent with a recently suggested mechanism (Dimanche-Boitrel et al, 2005). Generation of the lipid second messenger ceramide in cancer cells as a very early response to !-TOS may also provide an explanation for activation of protein phosphatase 2A (PP2A) and the ensuing hypophosphorylation of protein kinase C-! in cells exposed to !-TOS, since the drug does not directly target PP2A (Neuzil et al, 2001c). This hypothesis is in agreement with the previous finding that long-chain ceramides are activators of PP2A (Ruvolo et al, 1999). There is evidence, however, that treatment of cells with !-TOS causes generation of reactive oxygen species (ROS) (Ottino and Duncan, 1997; Kogure et al, 2001, 2002; Weber et al, 2003; Wang et al, 2005; Stapelberg et al, 2005; Swettenham et al, 2005). Generation of radicals appears to be a relatively early event in cells as a response to vitamin E analogs, since we observed substantial accumulation of ROS in Jurkat cells after one hour of !TOS challenge. The major form of ROS generated by cells in response to !-TOS appears to be superoxide, because addition of superoxide dismutase removed the radicals and also inhibited apoptosis (Kogure et al, 2001; Wang et al, 2005). Moreover, the site of superoxide generation as well as the target of ROS are very likely mitochondria, as suggested by experiments, in which mitochondrially targeted coenzyme Q (Kelso et al, 2001) suppressed radical generation and inhibited apoptosis induced by !TOS in cancer cells (Alleva et al, 2001; Weber et al, 2003; Wang et al, 2005). Also, it has been reported that !-TOSinduced apoptosis was more pronounced in cancer cells with low efficacy of the antioxidant machinery (Kogure et al, 2002). It is not clear at present, whether the initiation of apoptotic pathways leading to mitochondria-dependent events, is a direct response to the challenge of !-TOS or whether this is mediated via ceramide formation, which, in both cases, results in destabilization of the mitochondrial membrane. This process is either a direct consequence of ROS or is amplified by the oxyradicals, which are generated as a response to !-TOS challenge (Ottino et al, 1997; Kogure et al, 2001).

leading to auto-activation of the initiator caspase-9 with ensuing activation of the effector caspase-3, -6 or -7. At this stage, the cell enters the ‘point of no return’, i.e. the irreversible phase of the apoptotic pathway (Yamamoto et al, 2000; Neuzil et al, 2001a; Weber et al, 2003). It is now clear that this particular pathway is critically important in apoptosis induced by !-TOS in a variety of cancer cells (Neuzil et al, 2004). Smac/Diablo is an important agonist of the caspasedependent apoptotic signaling, since it antagonises the caspase-inhibitory members of the family of inhibitors of apoptosis proteins (IAPs), including c-IAP1, c-IAP2 and X-IAP (Du et al, 2000; Verhagen et al, 2002). The expression of IAPs is under control of the transcriptional factor nuclear factor-"B (NF"B), whose activity is inhibited by !-TOS (Erl et al, 1997; Neuzil et al, 2001a; Dalen and Neuzil, 2003). Thus, cytosolic translocation of Smac/Diablo may promote inhibition of the survival pathways in apoptosis induced by !-TOS, which could maximize the apoptogenic potential in resistant cells (Neuzil et al, 2003; Wang et al, 2005). Another mitochondrial protein amplifying apoptosis in cells exposed to vitamin E analogs is AIF (Weber et al, 2003) that translocates directly into the nuclei, thereby bypassing the caspase activation cascade (Susin et al, 1999). AIF, upon translocation to the nucleus, triggers cleavage of chromatin in a caspase-independent manner (Cande et al, 2002). AIF can thus avoid mutations in the caspase-dependent signaling or situations where IAPs are over-expressed, that can render the cancer cell resistant, and may mediate !-TOS-induced apoptosis in cells resistant to conventional anti-cancer drugs that rely solely on caspase activation (Neuzil et al, 2004). The mitochondrial pro- and anti-apoptotic proteins, including Bax, Bcl-2, Mcl-1 and Bcl-xL, are important factors related to mitochondrial apoptotic signaling pathways (Cory et al, 2003). Generation of the mitochondrial permeability transition pore has also been suggested in cells exposed to !-TOS (Yamamoto et al, 2000). It is likely that this is modulated by a cross talk between the mitochondrial pro- and anti-apoptotic proteins (Yamamoto et al, 2000; Weber et al, 2003). Overexpression of Bax sensitized cells to !-TOS-induced apoptosis (Weber et al, 2003; Yu et al, 2003), whereas over-expression of Bcl-2 or Bcl-xL protected them from the vitamin E analog. This was not observed when truncated proteins lacking the mitochondrial-targeting terminus were used for transfection of the cells (Weber et al, 2003). Similarly, down-regulation of Bcl-2 by antisense oligodoxynucleotide treatment sensitized cells to the vitamin E analog (Neuzil et al, 2001b, c; Weber et al, 2003). Finally, transfection with a gain-of-function mutant of Bcl-2 protected from while a loss-of-function mutant of the protein sensitized cancer cells to !-TOS (Neuzil et al, 2001c): in these mutant versions of Bcl-2, serine 70 was replaced with glutamine and alanine, respectively. This observation can be explained by PKC-dependent phosphorylation of S70 that plays a role in mitochondrial docking of Bcl-2 (Ruvolo et al, 1998). A compelling evidence for mitochondria as major transmitters of apoptotic signaling induced by vitamin E

B. Apoptotic signaling down-stream of mitochondria and their culmination in the commitment phase of apoptosis While the evidence of the initial triggers in apoptosis induced by vitamin E analogs is not very clear, the events in apoptosis induced by vitamin E analogs down-stream of mitochondria are known in more detail. Mitochondria are sites of mediators of apoptosis, whose re-localization relays further the up-stream proapoptotic signals. In apoptosis induced by vitamin E analogs, such down-stream events following mitochondrial destabilization comprise mobilization of apoptotic mediators, which include cytochrome c, the apoptosis-inducing factor (AIF) and Smac/Diablo (Neuzil et al, 2004). Cytochrome c, upon cytosolic translocation, forms a ternary complex with Apaf-1 and pro-caspase-9,

38

Cancer Therapy Vol 4, page 39 analogs follows from experiments, in which mtDNAdeficient (&째) cells were found to be resistant to !-TOS when compared to their wild-type and revertant counterparts (Weber et al, 2003; Wang et al, 2005). It has also been observed that transfection of cancer cells with dominant-negative (DN) caspase-9 suppressed apoptosis induced by !-TOS (Weber et al, 2002). We found that cancer cells lacking mtDNA, resistant to apoptosis (Dey et al, 2000), failed to translocate cytochrome c when challenged with !-TOS, unlike the apoptosis-sensitive parental and revertant cells, and this resistance also included low levels of phosphatidyl serine externalization and caspase-3 activation (Weber et al, 2003). Similar resistance of &째 cells has been found for other inducers of apoptosis, such as tumor necrosis factor-! (TNF-!) (Higuchi et al, 1997). Thus, mitochondria are indisputably the major intracellular organelles that relay the initial apoptotic signals down-stream to the stage at which the cell enters the apoptosis commitment stage. It needs to be emphasized though, that other organelles may also be involved in the process of apoptosis induced by vitamin E analogs, such as lysosomes, as shown in the literature (Neuzil et al, 1999, 2002). Notwithstanding, mitochondria are obligatory for transmission of the early apoptogenic events in cells, probably amplified by mediators released from organelles like lysosomes or the endoplasmatic reticulum. The major pathways of apoptosis induction by vitamin E

analogs are suggested in Figure 2.

IV. Modulation of signaling pathways by vitamin E analogs and its role in apoptosis induction Although mitochondria play a major role in apoptosis triggered by vitamin E analogs, there are other signaling pathways that parallel and/or amplify the intrinsic apoptogenic pathway (Neuzil et al, 1999; Yamamoto et al, 2000; Weber et al, 2003; Yu et al, 2003). The mitochondrial pathway is initiated by cytosolic translocation of the mediators cytochrome c (Weber et al, 2003; Wang et al, 2005), AIF (Weber et al, 2003; Neuzil et al, 2001) or Smac/Diablo (Wang et al, 2005), all of which may occur as a response to exposure of cancer cells to vitamin E analogs, as shown primarily for !-TOS. Mobilization of these modulators of apoptosis results in either caspase-dependent (cytochrome c) or caspaseindependent apoptosis (AIF), or in secondary modulation of other pathways regulating the ultimate outcome of proapoptotic signaling routes (Smac/Diablo). The cytochrome c- and AIF-dependent pathways were discussed in the previous chapter, the role of Smac/Diablo is also covered below in more detail, as well as are signaling pathways modulated by vitamin E analogs in apoptosis induction/amplification in cancer cells.

Figure 2. Possible pathways in apoptosis induction by !-TOS. 1.Upstream apoptosis signaling from mitochondria: !-TOS translocates to the cell, activates SMase and possibly causes the destabilization of lysosomes, giving rise to the formation of the lipid second message ceramide, leading to the destabilization of the mitochondrial membrane. !-TOS directly and/or via ceramide formation destabilizes mitochondrial membrane, and the ROS generation may amplify this process. 2. Down-stream apoptosis signaling from mitochondria: Mitochondrial membrane destabilization, likely promoted by leakage by lysosomal proteases, leads to cytosolic relocalization of pro-apoptotic factors (such as Cyt c, Smac/Diablo or AIF) that can be regulated by Bcl-2 family proteins (including Bcl-2, Bcl-xL or Mcl-1, which can be compromized by another Bcl-2-related protein Bax, probably mobilized to mitochondria after cleavage of Bid to its pro-apoptotic form.). Cyt c, Apaf-1 and pro-caspase-9 form a ternary complex, leading to the activation of the initiator caspase9, that in turn leads to the activation of the effector caspases. Smac/Diablo may amplify this process by suppressing the caspaseinhibitory activity of IAP family proteins, while IAP is supposed to transmit the mitochondrial destabilization to nuclear apoptotic events.

39

Dong et al: Vitamin E analogues as anti-cancer agents treatment, with accumulation of apoptotic cells in sub-G1. In MG63 cells, !-TOS induced cell accumulation in the S/G2 phase, and this was associated with disappearance of cells in G1, similarly as observed in SAOS cells. In order to evaluate the molecular mechanism involved in the cell cycle arrest caused by !-TOS, expression of the cell cycle regulatory proteins that control the S/G2 progression was examined. Treatment of SAOS and U2OS cells with !-TOS did not affect the expression of cyclin A and cyclin E, which was similar in the untreated cells for up to 72 h of incubation. Conversely, treatment of MG63 cells with !-TOS caused a reduction in both cyclin A and cyclin E protein levels. The above results showed that vitamin E analogs, epitomized by !-TOS, exert a potent modulatory activity towards cell cycle progression and that different cell types respond differently to the agent, in particularly as shown by arrest in different stages of cell cycle. Although these differences point to possibly different targets for !-TOS in various cells, the vitamin E analog does affect the cell cycle progression, and this in its own right inhibits cell proliferation resulting in suppression of tumor growth, and/or amplifies the apoptogenic signaling pathways.

A. Inhibition of cell cycle progression by vitamin E analogs Several reports implicated inhibition of the cell cycle progression as a means by which vitamin E analogs may induce apoptosis or inhibit proliferation of cancer cells and/or sensitize them to other anti-cancer drugs. Ni et al, (2003) showed that !-TOS inhibits proliferation of prostate cancer cells by down-regulating expression of several critical cyclins and the cognate cyclin-dependent kinases (CDK), resulting in hypo-phosphorylation of the Rb protein and a G1/S arrest. Cell cycle arrest and apoptosis were also induced by !-TOS in osteosarcoma cells via activation of p53 and reduced expression of the transcription factor E2F1, critical for the G1/S transition (Alleva et al, 2006). Further, exposure of osteosarcoma cells to !-TOS promoted a prolonged arrest at the S/G2 border, sensitizing the cells to methotrexate-induced apoptosis (Alleva et al, 2005). These findings can be reconciled with an earlier report, in which !-TOS suppressed proliferation of breast cancer cells by inhibiting the E2F1-dependent trans-activation via increased binding of cyclin A (Turley et al, 1997). Apoptosis induction and inhibition of proliferation by !-TOS have been shown for malignant mesothelioma cells (Tomasetti et al, 2004), the latter paradigm being due to selective disruption of the FGF-FGFR autocrine signaling loop, most likely affected by modulation of the E2F1 and egr-1 trans-activation activity (Stapelberg et al, 2004, 2005). These are exciting results, since malignant mesotheliomas cannot be treated at this stage and since we found that !-TOS shows a strong anti-mesothelioma effect in animal models (Tomasetti et al, 2004; Stapelberg et al, 2005). Thus, proliferation and apoptosis are intimately coupled, and cell cycle modulators can influence both cell division and apoptosis (Vermeulen et al, 2003). The cell cycle is coordinately controlled by CDKs and their cyclin partners, whose levels fluctuate throughout the cell cycle. The pRb pathway plays a central role in cell proliferation by modulating the activity of the transcription factor E2F (Dimova and Dyson, 2005). E2F1 can signal p53 phosphorylation that is coincident with p53 accumulation and apoptosis (Rogoff et al, 2002). The p53 gene is frequently lost or mutated in many cancers, and lack of functional p53 is accompanied by elevated rates of genomic instability, rapid tumor progression and resistance to anti-cancer drugs and radiation (Weller et al, 1998). Alleva et al, (2006) used three human osteosarcoma cell lines, the SAOS and U2OS cells carrying the wildtype p53 gene, and the mutant p53 cell line MG63. They showed that !-TOS markedly inhibited cell proliferation in MG63 cells without affecting cell growth in both SAOS and U2OS cells. In SAOS cells, !-TOS induced cell accumulation in S/G2 phase coincident with a decrease of cells in G1, which was observed after 24 h of treatment. However, the highest !-TOS concentration was able to induce cell death at prolonged times of drug exposure. The U2OS cell line responded to !-TOS treatment by a transient accumulation of cells in the G1 phase. Higher concentration of !-TOS induced cell death after 48h of

B. The c-Jun pathway as a target for apoptosis induced by vitamin E analogues Several signaling pathways that have been shown to play a role in modulation of apoptotic signaling appear to be affected by vitamin E analogs. Of these the c-Jun pathway has been investigated in more detail due to its tight association with modulation of apoptotic pathways (Liu and Lin, 2005). The effect of !-TOS on the activity of the c-Jun NH2-terminal kinase (JNK) pathway up-stream components was investigated (Zu et al, 2005). The vitamin E analog markedly increased the level of expression of the Ask1, GADD45, Sek1, and phospho-Sek1 proteins, of which Ask1 and GADD45 are associated with the cell membrane. Consistent with these findings, the phosphorylated form of JNK was also noticeably increased, although the expression level of total JNK was not affected. Activated Ask1 and GADD45 phosphorylate the Sek1 protein that then leads to phosphorylation of JNK itself. In relation to this effect, the protein Bim, that is normally in the cytosol, tranlocates during apoptosis to the mitochondrial membrane, where it binds to Bcl-2 and BclxL. Prior to this tranlocation, Bim is phosphorylated by JNK (Kirschnek et al, 2005). Thus, JNK activation leads to antagonization of the anti-apoptotic function of proteins like Bcl-2 and Bcl-xL, a mechanism permitting cytosolic translocation of mitochondrial mediators of apoptosis. Activation of c-Jun NH2-terminal kinase by !-TOS has also been shown for gastric cancer cells (Wu et al. 2004a), and it may amplify the mitochondrial apoptosis signaling pathway, as shown for prostate cancer cells (Zu et al. 2005).

C. Akt, NF"B and other pro-survival pathways as a target for vitamin E analoginduced apoptosis 40

Cancer Therapy Vol 4, page 41 Of the signaling pathways modulated by vitamin E analogs, some are implicated in high levels of malignancy and resistance of cancers to established drugs. One problem encountered in pathologies like breast carcinomas stems from over-expression of erbB2, a receptor tyrosine kinase proto-oncogene. ErbB2 is a member of the epithelial growth factor receptor super-family and a product of the c-neu gene (Roskoski, 2004; Slamon et al, 1989). This tyrosine kinase-linked trans-membrane protein is over-expressed in >30% breast cancers. The major complication associated with erbB2 over-expression is linked to activation of Akt via the phos-phatidylinositol 3kinase pathway (Zhou and Hung, 2003; Vivanco and Sawyers, 2002). Akt is a serine/threonine kinase that promotes cellular survival (Dudek et al, 1997). Once activated, Akt exerts anti-apoptotic effects through phosphorylation of several proteins, including Bad (Datta et al, 1997) or caspase-9 (Cardone et al, 1998). Moreover, Akt causes activation of the transcriptional factor NF"B (Kane et al, 1999) that controls expression of pro-survival genes, such as members of the IAP family (LaCasse et al, 1998). In most non-transformed cells, NF"B complexes (a heterotrimer composed of p50 and p65 subunits bound to an inhibitor subunit I"B) are largely cytoplasmic. Activation of NF"B results in its translocation to the nucleus and binding to promoter regions of specific prosurvival genes, such as those coding for IAPs, the caspase8 inhibitor FLIP, or the TRAIL decoy receptor DcR1. One possibility by which !-TOS may suppress NF"Bdependent transcription of pro-survival genes is activation of caspase-3 that cleaves the NF"B subunit p65 (Neuzil et al, 2001a), as also documented for growth factor-starved cells (Levkau et al, 1999). We showed that vitamin E analogs induced apoptosis at comparable levels in mouse and human breast cancer cells, regardless of their erbB2 status. One plausible mechanism is that these agents induce re-localization of Smac/Diablo from mitochondria to the cytosol (Wang et al, 2005), where Smac/Diablo binds to IAPs so that caspase-3 is librated to execute its apoptotic function (Du et al, 2000). In another report, it has been shown that !tocopheryloxybutyric acid, a compound analogous to !TOS, induced apoptosis in the erbB2-over-expressing human breast cancer cells MDA-MB-453 by simultaneously inhibiting activation (phosphorylation) of erbB2 and ensuing activation of p38 MAP kinase (Akazawa et al, 2002). Several other papers showed modulation of the MAP kinase pathway by vitamin E analogs as a way by which the agents induce apoptosis. Interestingly, Kline’s group reported that extra-cellular signal-regulated kinases (ERKs) and JNK, but not the p38 MAP kinase, were involved in !-TOS-induced apoptosis in the human breast cancer cells MDA-MB-435 cells, and this activated the down-stream transcription factors c-Jun and ATF-2 (Yu et al, 2001). It is possible that this pathway is targeted by !-TOS in the erbB2-low MDA-MB-435 cells while the erbB2-high MDA-MB-453 cells activate their apoptotic machinery by concerted deregulation of the erbB2/Akt and p38 pathways when challenged with vitamin E analogs. One of the intriguing targets of vitamin E analogs is

the pro-survival transcription factor NF"B (see above). Inhibition of activation of NF"B by !-TOS was first documented in the context of cardiovascular diseases (Erl et al, 1997). One possibility is that the vitamin E analogs trigger apoptosis resulting in activation of caspase-3 that cleaves the obligatory NF"B subunit p65, rendering it inactive (Levkau et al, 1999). We have shown that !-TOS initiates a ‘sub-apoptotic’ phenotype, under which cells activate their effector caspase but do not enter the commitment phase (Neuzil et al. 2001a), probably because this requires efficient activation of specific cyclindependent kinases (Harvey et al, 2000). Regardless of the precise mechanism, inhibition of NF"B activation by vitamin E analogs has an anti-survival effect, i.e. is proapoptotic, and can also be implicated in adjuvant cancer therapy, such as shown for the T lymphoma Jurkat cells, whose treatment with !-TOS sensitized them to TRAILdependent killing (Dalen and Neuzil, 2003).

V. Sensitization of cancer cells to TRAIL by modulation of signaling pathways by vitamin E analogs The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has attracted much interest because of its potential selectively in triggering apoptosis in tumor cells rather than in their normal counterparts (Pitti et al, 1996; Ashkenazi et al, 1999; Shi et al, 2003). TRAIL has been proven relatively safe in “in vivo” studies of rodents and primates compared with other death receptor ligands, TNF and Fas, which induce significant inflammation and tissue injury (Ashkenazi et al, 1999; Walczak et al, 1999). Thus, two unique characteristics of TRAIL have been identified: firstly, TRAIL can selectively induce apoptosis in tumorigenic or transformed cells, but not in normal cells, highlighting its potential application in cancer treatment. Second, in contrast to other member of TNF family, whose expression is tightly regulated and which are often only transiently expressed on activated cells, TRAIL mRNA is expressed continuously in a wide range of tissues (Wiley et al, 1995).

A. TRAIL-induced apoptosis

signaling

and

TRAIL is a type II membrane protein or secreted in soluble form which binds to its cognate DRs, DR4 and DR5, inducing their trimerization and intracellular recruitment of the adaptor protein Fas-associated death domain (FADD) (Schneider et al, 1997). The death domain (DD), in turn, recruits pro-caspase-8 into a deathinducing signaling complex (DISC) that triggers autocatalytic cleavage and activation of caspase-8, which then leads to activation of effector caspase-3 (type I pathway). Alternatively, the death pathway can be further amplified by involvement of the mitochondrial signaling (type II pathway) (Scaffidi et al, 1998). TRAIL-activated caspase-8 can generate truncated Bid, which triggers the release of cytochrome c from mitochondria, leading to the assembly of the apoptosome (cytochrome c, Apaf-1, procaspase-9). Formation of apoptosome activates caspase-9, which then activates effector caspases (Zou et al, 1999). 41

Dong et al: Vitamin E analogues as anti-cancer agents Type I cells exert DISC-activated caspase-8, which activates down-stream effector caspases and triggers execution of apoptosis. However, in the majority of cells (Type II), TRAIL-induced activation of caspase-8 is insufficient to kill without recruiting the mitochondrial apoptotic program. A number of proteins are involved in regulation of the TRAIL apoptotic pathways. The FLICE-like inhibitory protein (FLIP) contains two DDs that can bind to DDs of FADD and inhibit recruitment of pro-caspase-8 to the DISC) (Irmler et al, 1997). Inhibitor of apoptosis proteins (IAPs) are characterized by the presence of one to three baculoviral IAP repeated (BIR) domains that bind to caspases (Verhagen et al, 2001). Binding of IAPs to caspases can be inhibited by several proteins released from mitochondria. The second mitochondria-derived activator of caspases (Smac, also referred to as Diablo), which directly binds IAPs, is located in the inter-membrane space of mitochondria and is released into the cytosol upon changes in the mitochondrial membrane permeability (Verhangen et al, 2000). Smac/Diablo facilitates apoptosis by liberating caspase-3 or -7 from inhibition mediated by IAPs.

B. Resistance apoptosis

to

A synergistic and cooperative effect was observed when TRAIL was combined with a derivative of vitamin E, !-TOS, in malignant mesothelioma (MM) cells and the effect was selective for cancer cells (Tomasetti et al, 2004b). MM is a fatal type of neoplasia with poor therapeutic prognosis, largely due to resistance to apoptosis. Impaired apoptotic pathways render MM cells rather resistant to TRAIL-induced apoptosis. Sub-lethal doses of !-TOS significantly decreased the high IC50 values for TRAIL by a factor of ~10-100 (Tomasetti et al, 2004b). The observation that !-TOS and TRAIL synergize in p53wt MM but not in the p53null cells suggests a role of p53 in trans-activation of the pro-apoptotic genes involved in drug synergism (Tomasetti et al, 2006). The p53 protein is a key component of the cellular ‘emergency-response’ mechanism (Levin, 1997; Sionov and Haupt, 1999). A variety of stress-associated signals activate p53 that induces growth arrest or apoptosis, thereby eliminating damaged and potentially dangerous cells (Lane, 1992). The p53 apoptotic target genes can be divided into two groups; the first group encodes proteins that act through receptor-mediated signaling, the second group codes for proteins involved in regulation of the apoptotic effector proteins. !-TOS has the propensity to induce apoptosis in a p53-independent manner (Weber et al, 2002). However, at low concentrations the vitamin E analog induces expression and activation of p53. The p53 induction was concomitant with the enhancement of both DR4 and DR5 expression. Notably, such expression of DRs does not occur in the p53null MM cells. Studies using siRNA directed at p53 revealed that the p53 protein contributes significantly to the expression of TRAIL’s DRs. Thus, p53-dependent up-regulation of DR4 or DR5 is a basis for sensitization of MM cells to TRAIL. Additionally, the presence of a redox environment efficiently contributes to enhanced expression of DR4 and DR5 via p53 when MM cells are treated with the vitamin E analog (Tomasetti et al, 2006). Regulation of activity of many transcription factors by redox modulators was previously described (Sun and Oberley, 1996). Thus, a novel mode of action for !-TOS has been described: reduction of the redox-sensitive amino acid residues on the p53 protein leads to an increase in the efficiency of TRAIL’s DR expression, sensitizing MM cells to the immunological apoptogen. MM cells express both DR4 and DR5 on the surface and their up-regulation by !-TOS could facilitate activation of caspase-8 and cleavage of Bid. Kinetic analysis of TRAIL-induced signaling revealed a transient activation of caspase-8, which resulted in induction, albeit low, of apoptosis. Caspase-8 activation was less pronounced in the presence of TRAIL plus !-TOS. Under this setting, activation of the mitochondria-dependent apoptotic pathway, including Bid cleavage, cytochrome c cytosolic mobilization and, finally, caspase-9 activation, was observed (Tomasetti et al, 2004). Bid cleavage may lead to mitochondrial translocation of Bax, as shown for !-TOS in other cancer models (Weber et al, 2003, Yu et al, 2003). Thus, the elevation of p53 in response to !-TOS could facilitate TRAIL-induced apoptosis by releasing both Bid and Bax from their sequestration by Bcl-xL, promoting mitochondria-dependent apoptosis (Fig. 2).

TRAIL-induced

Although TRAIL is a potent anti-cancer agent in preclinical models, it is known that some tumor cells possess intrinsic or acquired resistance to TRAIL. Tumor cells can acquire resistance to apoptosis through interference with either intrinsic (Type II pathway) or extrinsic (Type I pathway) apoptotic signaling pathways. Mutation of the pro-apoptotic Bcl-2 family member Bax confers resistance to TRAIL-induced apoptosis in HCT116 cells (LeBlanc et al, 2002). Over-expression of FLIP suppresses DRinduced apoptosis in malignant mesothelioma (MM) cells (Rippo et al, 2004). Tumor cells may avoid TRAIL-mediated killing by down-regulation of DRs (extrinsic resistance). Besides the importance of the balance of DRs and decoy receptors (DcRs), which lack the functional cytoplasmic death domain, the ratio between DR4 and DR5 plays a role in determining sensitivity to TRAIL. In addition, in order to induce apoptosis, DR4 and DR5 have distinct crosslinking requirements. DR4 equally responds to crosslinked TRAIL (membrane bound) and non cross-linked TRAIL (soluble), whereas DR5 signals only in response to non cross-liked soluble TRAIL (Wajant et al, 2001). It was observed that low expression of DRs on the cell surface is responsible for cellular resistance to TRAIL-induced cytotoxicity in human colon cancer cells (Jin et al, 2004). Anticancer drugs have been shown to sensitize TRAIL receptor-negative cells to TRAIL-mediated apoptosis by inducing expression of DRs on the cell surface (Arizono et al, 2003). Thus, combination of TRAIL with other drugs resulted in a cooperative or synergist effect (LeBlanc et al, 2002; Wang and El-Deiry, 2003).

C. Modulation of TRAIL sensitivity by !TOS 42

Cancer Therapy Vol 4, page 43 A cooperative pro-apoptotic effect of !-TOS with immunological apoptogens has been also observed in breast cancer (Yu et al, 1999) and colon cancer cells and in an animal model (Weber et al, 2002). The former report showed that !-TOS converted Fas-resistant cells to Fassensitive ones via mobilization of the Fas receptor from the cytosol to the plasma membrane. Moreover, !-TOS enhanced sensitivity of Jurkat T lymphoma cells to the induction of apoptosis by TRAIL and the effect was not observed in the presence of !-TOH (Dalen and Neuzil, 2003). In this report, a transient NF"B activation was found when the cells were exposed to TRAIL. Control of transcription by NF"B proteins can be of relevance to the function of TRAIL by induction of the anti-apoptotic NF"B-dependent genes that determine cellular susceptibility toward apoptosis induction via DR4 and/or DR5. It is known that NF"B controls expression of pro-survival genes, including FLIP (Kreuz et al, 2001) and IAPs (Degli-Esposti et al, 1997). !-TOS, by inhibiting the TRAIL-induced transient NF"B activation, which in turn inhibits expression of pro-survival proteins that confer resistance of cells to TRAIL-induced apoptosis, may have a role in adjuvant therapy of TRAIL-resistant cancers. In conclusion, there is evidence that !-TOS can be used in combination with immunological inducers of apoptosis. It can also be used alone, since it is expected to sensitize cancer cells to endogenously produced immunological inducers of apoptosis by cells of the immune system, whereby potentiating the natural tumor surveillance.

VI. Conclusions perspectives

and

malignant mesothelioma patients. The results will be available later in 2006. We believe that the multifaceted activity and selectivity of vitamin E analogs provide a substantial promise of these agents to become drugs of choice against multiple malignancies. It can be expected that during 2006 or 2007, we will, hopefully, witness the advent of the use of vitamin E analogs, like !-TOS, in clinical experiments, culminating in application of the agent as an anti-cancer drug. This will require a better knowledge of the molecular mechanism underlying the multiple activity of vitamin E analogs, which have been a subject of intensive research within the recent years.

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further

This paper summarizes some of the major mechanisms by which redox-silent vitamin E analogs, epitomized by !-TOS, induce apoptosis in cancer cells. These intriguing agents, as shown primarily for !-TOS, exert their anti-cancer effect by inducing, inhibiting or modulating a variety of cellular processes and intracellular pathways. Some of them are highlighted in this review. All of these activities separately or in combination contribute to the overall efficacy with which vitamin E analogs suppress cancer progression, including the metastatic process. Studies in pre-clinical settings, using experimental animals, clearly emphasize the potential of agents like !-TOS to become wide-spectrum anti-cancer drugs. Thus far, there is very little, if anything, known about the effect of vitamin E analogs on cancer in case of human patients. One of the problems encountered is the process of administration of the agents. Most analogs of vitamin E with anti-cancer activity are hydrophobic esters that are completely hydrolyzed upon intestinal uptake. Thus, other means have to be used to deliver the drugs to the bloodstream. The intraperitoneal or intravenous injection used in animal experiments is not readily applicable to humans. A plausible delivery of !-TOS and other analogues of vitamin E with anti-cancer activity may be achieved by trans-dermal application. We are pursuing this option in a limited perspective clinical trial with post-chemotherapy 43

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Cancer Therapy Vol 4, page 47 Cancer Therapy Vol 4, 47-72, 2006

Sensitivity and resistance of human cancer cells to TRAIL: mechanisms and therapeutical perspectives Review Article

Luca Pasquini, Eleonora Petrucci, Roberta Riccioni, Alessia Petronelli and Ugo Testa* Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di SanitĂ , Viale Regina Elena 299, 00161 Rome, Italy

__________________________________________________________________________________ *Correspondence: Dr Ugo Testa, Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di SanitĂ , Viale Regina Elena 299, 00161 Rome, Italy; e-mail: u.testa@iss.it Key words: apoptosis, cancer, TRAIL, receptors, monoclonal antibodies, resistance, sensitivity Abbreviations: 2-ciano-3,12-dioxooleana-1,9(11)-dien-28-oic acid, (CDDO); acute lymphoblastic leukaemia, (ALL); Acute Myeloid Leukaemia, (AML); Apoptosis-Inducing-Factor, (AIF); Apoptotic Protease Activating Factor 1, (APAF-1); B-Chronic Lymphocytic Leukemia,, (B-CLL); cellular FLICE inhibitory proteins, (c-FLIP); Chronic Myeloid Leukemia, (CML); c-Jun kinase, (JNK); death domains, (DD); death-induced signalling complex, (DISC); endoplasmic reticulum, (ER); hepatocarcinoma cancer cells, (HCC); Histone acetyltransferases and histone deacetylases, (HDAC); histone deacetylase inhibitors, (HDACi); imidazole derivative of CDDO, (CDDOIm); inhibitor of apoptosis proteins, (IAPs); interferon-!, (IFN-!); interferon-alpha, (IFN-"); Multiple Myeloma, (MM); non-small cell lung cancer, (NSCLC); osteoprotegerin, (OPG); renal cell carcinoma, (RCC); Tumor necrosis factor, (TNF); Tumor Necrosis FactorRelated Apoptosis-Inducing Ligand, (TRAIL); ultraviolet B radiations, (UVB) Received: 22 September 2005; Accepted: 3 February 2006; electronically published: February 2006

Summary Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines, able to induce apoptotic cell death in a variety of tumor cells by engaging the death receptors TRAIL-R1 and TRAIL-R2, while sparing most normal cells. Endogenously expressed TRAIL plays an important role in immunosurveillance of developing and metastatic tumors. The majority of studies aiming to evaluate the proapoptotic effects of TRAIL on tumor cells have been carried out on cancer cell lines and showed variable levels of sensitivity of these cells to TRAIL-induced apoptosis, while the few studies carried out on primary tumor cells have almost invariably shown low sensitivity or resistance of tumor cells to TRAIL-induced apoptosis. The mechanisms of resistance of tumor cells to TRAIL are related to increased expression or activity in these cells of anti-apoptotic molecules, mainly c-FLIP or one of the IAPs, or to the defective expression of molecules such as FADD or caspases8, involved in the transduction of the apoptotic signal originated from TRAIL-Rs. However, many studies have shown several strategies able to induce a high sensitivity of tumor cells to TRAIL, based on the combined administration with TRAIL of different drugs, including triterpenoids, proteasome inhibitors, histone deacetylase inhibitors, interferon-! or anti-cancer cytotoxic drugs: these various agents either decrease the expression of antiapoptotic molecules or increase the expression of molecules involved in TRAIL-Rs signalling, thus restoring a high level of sensitivity of tumor cells to TRAIL. On the other hand, preclinical studies in mice and non-human primates have shown the potential utility of recombinant soluble TRAIL and, mostly, of agonistic anti-TRAIL-R1 or -R2 antibodies for cancer therapy. Two anti-TRAIL-R antibodies are under evaluation in phase I/II clinical studies.

treatment-resistant tumor cells. Given these limitations there is the absolute need for the development of alternative therapeutic strategies based on new drugs endowed with a different mechanism of action, specifically targeting to the tumor cells and with more acceptable toxicity profiles. In this context, particularly promising is the area of biologic therapies, based on

I. Introduction Chemotherapy, surgical resection and radiotherapy represent the standard therapeutic approaches in the treatment of cancer. These therapeutic strategies, however, are often non curative and associated with considerable secondary and toxic effects, limiting the treatment and result frequently in the selection of highly malignant

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Pasquini et al: Sensitivity and resistance of human cancer cells to TRAIL agents able to selectively kill tumor cells, sparing normal cells, display limited in vivo toxicity, and able to bypass or circumvent acquired tumor resistance against conventional treatments. Among new biologic agents, the death receptor ligand Apo2 Ligand/Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (Apo2L/TRAIL) seems to offer a promising anti-tumor therapeutic potential, based on its capacity to selectively induce the killing of a wide variety of cancer cells, sparing normal cells. Importantly, unlike conventional anti-cancer drugs, death receptor ligands kill tumor cells through a molecular mechanism completely independent on the p53 tumor suppressor gene, very frequently mutated in human tumors. In this article we provide an overview of the Apo2L/TRAIL molecule and its receptors, of the sensitivity and resistance of the tumor cells to this death receptor ligand and of the possible therapeutic use of Apo2L/TRAIL for the treatment of cancer used alone or in combination with other drugs.

The understanding of the mechanisms responsible for the resistance of tumor cells to programmed cell death has a fundamental importance not only for a better understanding of cancer biology, but also for the development of new cancer therapies focused on devising ways to overcome this resistance and to induce apoptosis of cancer cells.

B. Apoptotic pathways The most common and well defined form of programmed cell death is represented by apoptosis, a physiological process of cellular suicides required for the maintenance of cell homeostasis, embryonic development and for the differentiation and function of haematopoietic and lymphoid cells. A common feature of the apoptotic process is the constant involvement of caspases, a family of intracellular cysteine proteases (Cysteine Aspartyl-specific Proteases). These enzymes are present as inactive zymogens in all animal cells, but can be triggered to assume an active state, usually trough their proteolytic processing at conserved aspartic acids residues. Pro-caspases contain three domains: a NH2-terminal domain, a large subunit (about 20 kDa) and a small subunit (about 10 kDa). During activation the pro-caspase is cleaved to generate large and small subunits; the active enzymes are heterotetramers composed of two large and two small subunits. It is important to note that active caspases cleave their substrates at the Asp residues and are themselves also activated by proteolytic cleavage at Asp residues. This unique property implies that these enzymes make part of proteolytic cascades, where caspases activated themselves and each other. Based on their level of action caspases are distinguished in “initiatior” caspases and “effector” caspases: the first ones act at the origin of the apoptotic process, while the latter ones at the late steps of the apoptotic process. Initiator caspases possess long Nterminal prodomains that contain recognizable proteinprotein interaction motifs, while effector caspases have short or no prodomains. The activation of an effector caspase is mediated by an initiator caspase through cleavage at internal Asp residues that separate the large (p20) and small (p10) subunits. p10 and p20 subunits associate to form a monomer. In contrast, initiator caspases are auto-activated. The process of activation of initiator caspases is tightly controlled (Reidel et al, 2004). There are two pathways by which caspases activation is induced, the extrinsic and the intrinsic apoptotic pathways. Both the pathways converge on activation of effector caspases, but require different initiator caspases to start the process. The extrinsic pathway is activated by engagement of death receptors on the cell membrane. Binding of ligands, such as FasL, TNF and TRAIL to their respective membrane receptors Fas, TNF-R and TRAILRs induces the formation of the death-induced signalling complex (DISC). DISC in turn promotes caspases-8 recruitment and promotes a cascade of caspases activation that culminates with cell death.

A. Apoptosis and cancer Defects in DNA repair lead to genomic instability and predispose to cancer development. This genetic instability represents the mechanism through which normal cells can accumulate a sufficient number of mutations to become malignant (Hanahn and Weimberg, 2000). The cells, however, possess important mechanisms of protection against this genomic instability, mainly orchestrated by the tumor suppressor protein p53, which acts as “a guardian of the genome” protecting cells against cancer (i.e. by inducing the death of cells that have accumulated genetic defects) (Brown and Attardi, 2005). The p53 is the most frequently mutated gene in human cancers and its inactivation by mutation certainly plays an important role in tumor development (Brown and Attardi, 2005). There is now evidence that in addition to a p53 inactivation tumor cells exhibit multiple defects in cell death pathways. Resistance to cell death and particularly to apoptotic cell death plays a key role both in tumor development and in the mechanism of resistance to anticancer drugs (Okada and Mak, 2004). The resistance to programmed cell death plays a major role in several pathogenetic mechanisms of tumors, allowing tumor cells to abnormally survive beyond their normal life, reducing the need for exogenous growth factors, providing protection from hypoxia and oxidative stress, increasing the time and therefore the opportunity for the development of additional genetics abnormalities altering cell proliferation, interfering with cell differentiation, promoting angiogenesis and increasing cell motility and invasiveness during tumor progression. Furthermore, standard anticancer treatments, chemotherapy and radiotherapy, must be administered in relatively small doses to allow the normal tissues to receive from sub-lethal radiation or cytotoxic damage between treatments. However, surviving cancer cells proliferate during the intervals between treatments and this process of repopulation is an important cause of treatment failure (Kim and Tannock, 2005).

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Cancer Therapy Vol 4, page 49 The activation of caspase-8 is antagonized by cellular FLICE inhibitory proteins (c-FLIP), an enzymaticallyinactive relative of caspase-8 and -10 that binds to DISC. The knockdown of c-FLIP augments DISC recruitment, activation and processing of caspases-8, thereby enhancing effector-caspase stimulation and apoptosis. The intrinsic pathway is triggered by various extracellular and intracellular stresses, including growth factor deprivation, DNA damage, oncogene induction, hypoxia and cytotoxic drugs. Cellular signals originated by various mechanisms by these different stresses converge on a cellular target represented by mitochondria. A series of biochemical events is then induced leading to damage of the outer mitochondrial membrane, the release of cytochrome c and other pro-apoptotic molecules, with consequent formation of the apoptosome, a large molecular complex formed by cytochrome C, Apoptotic Protease Activating Factor 1 (APAF-1) and caspase-9, and caspase activation. The release of cytochrome c is essential for caspase-9 activation (Hao et al 2005). Activated caspase-9 in turn activates the downstream effector caspase-3 and -7, which rapidly cleave intracellular substrates. Other released mitochondrial proteins facilitate caspase activation through inactivation of endogenous inhibitors, the inhibitor of apoptosis proteins (IAPs). The permeabilization of the outer mitochondrial membrane is inhibited by antiapoptotic molecules pertaining to the Bcl-2 family. Activated executioner caspases kill cells via apoptosis; however, inhibition of these caspases only transiently protect cells since, once the mitochondrial permeability is achieved, death will proceed regardless of caspase activation, either due to toxic mediators released from the mitochondria or eventual loss of essential mitochondrial functions (Chipuk and Green, 2005). The apoptotic intrinsic pathway is also controlled by other mitochondrial proteins. Thus, Apoptosis-InducingFactor (AIF) and endonuclease G may induce cell death independently of caspases activation. Furthermore, Smac/DIABLO and HTRA2 promote caspases activation by counteracting the activity of Inhibitors of Apoptosis (IAP): these proteins exert an inhibitory activity on caspases activation. A third apoptotic pathway originated by stress occurring at the level of the endoplasmic reticulum (ER), the cellular structure involved in the assembly and routing of proteins. In this pathway, caspases-12, localized in outer membrane of the ER acts as an initiator caspase. In addition to caspase-dependent cell death, some cell death processes occur in a caspase-independent manner. Basically, four caspase-independent cell death processes have been described: autophagy, paraptosis, slow cell death and mitotic catastrophe (Broken et al, 2005). The autophagy is a cell death process characterized by sequestration of a part of cell cytoplasm, including organelles, in autophagic vesicles and their delivery to and subsequent degradation following fusion with the cellular lysosomes. Paraptosis involves cytoplasmic vacuolation and mitochondrial swelling in the absence of caspase activation and nuclear changes typical of apoptosis. Mitotic catastrophe is a caspase-independent cell death

process originated by mitotic failure due to defective cell cycle checkpoints and the generation of aneuploid cells. Finally, slow cell death corresponds to a process od a delayed programmed cell death occurring when caspases are asent or inhibited.

C. TRAIL and its receptors Death receptors are members of the TNF receptor gene superfamily, composed by more than 20 proteins exhibiting a broad range of biological activities. Particularly, some of them play a key role in the control of cell death and survival. Members of the TNF receptors superfamily share some common, structural features consisting in a cysteyne-rich extracellular domain and in a cytoplasmic domain of about 80 aminoacids, called death domain which plays a key role in the transmission of the death signal triggered by the interaction of a death ligand with its receptors. The typical examples of this receptor superfamily are represented by CD95 (also known as Fas), TNF-R1, TRAIL-R1 and TRAIL-R2. The corresponding ligands of the death receptors are members of the TNF superfamily, which comprise ligands such as FasL, TNF, TRAIL and TWEAK. These ligands are type II transmembrane proteins, which exists also as soluble proteins released after their cleavage by metalloproteases present in the microenvironment. TRAIL is a type II membrane bound ligand of the TNF family of about 33-35 KD, displaying 28% aminoacid sequence identity with FasL and 23% identity with TNF (Kimberly and Screaton 2004; Kelley and Screaton, 2004). The native form of the ligand is present as a transmembrane protein with the C-terminal located at the extracellular side and the N-terminal at the cytoplasmic side. The molecule is released in the microenvironment in a vesicle-associated form, or is cleaved and exists as a soluble homotrimeric ligand stabilized by a zinc moiety. The extracellular domain of the molecule can be cleaved proteolytycally to release a soluble ligand. Crystallographic analysis has shown that TRAIL, like other TNF ligands, exists as a homotrimeric molecule, stabilized by an internal Zn2+ atom that is coordinated at the level of the single impaired cysteine residue (Cys 230) of each monomer. This zinc atom is essential for structural integrity of TRAIL. At variance with other death receptor ligands, TRAIL expression is constitutively observed in many tissues, with the exception of liver, testis and brain. The receptor system responsible for the binding and transduction of a cell signalling of TRAIL is complex in that it involves five specific membrane receptors. (Figure 1) Two of these receptors, TRAIL-R1 (also known as DR4) and TRAIL-R2 (also known as DR5) are true death receptors in that they possess death domains in their cytoplasmic tail and are able to transduce a death signal and to engage the apoptosis machinery (Figure 1). In contrast TRAIL-R3 to TRAIL-R5 are antagonistic decoy receptors, able to bind TRAIL, but not to transmit a death signal (Figure 1); therefore, they compete with TRAIL-R1 and -R2 for TRAIL binding.

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Pasquini et al: Sensitivity and resistance of human cancer cells to TRAIL TRAIL-R3 is a glycolphosphatidylinositol GPIanchored cell surface protein, which lacks a cytoplasmic tail; TRAIL-R4 is a transmembrane cell receptor harbouring a truncated cytoplasmic death domain. As a consequence of these structural features TRAIL-R3 and TRAIL-R4 upon binding of TRAIL are unable to induce a death signal. In addition to these four canonical receptors, there is also TRAIL-R5, known as Osteoprotegerin, a soluble decoy receptor exhibiting low affinity for TRAIL, which is involved in the regulation of osteoclastogenesis. TRAIL-R2 is a membrane protein non-glycosylated exhibiting 58% homology to TRAIL-R1. TRAIL-R2 is a type I trans-membrane protein, of 411 amino acids with a very large signal sequence (51 amino acids), a 132 amino

acid extra-cellular region, a 22 amino acid transmembrane domain and a 206 amino acid cytoplasmic domain (Figure 1). This receptor possesses two cysteine rich domains in its extracellular domain. Two different isoforms of TRAIL-R2 are generated (a long and a short, differing for 23 amino acids at the level of the extracellular domain) by a differential RNA splicing event. The two isoforms are equally active in mediating cell death. Recent studies indicate that the TRAIL-R2 is more active than the TRAIL-R1 in mediating TRAIL-induced cell death (Kelley et al, 2005). TRAIL-R2 is widely expressed in human tissues and its expression is upregulated in activated T lymphocytes and in interferon-treated monocytes/ macrophages.

Figure 1. Human TRAIL receptors. A trimeric human TRAIL molecule is able to induce apoptosis through binding to TRAIL-R1 or TRAIL-R2. In fact, both these receptors possess a death domain in their cytoplasmic tail (shown as a black box). In the cytoplasmic region of TRAIL-R2 is present also a TRAF binding motif (shown as red box) responsible for the capacity of this receptor to activate the NF-#B transcription factor. TRAIL-R3 and TRAIL-R4 do not signal apoptosis and act as decoy receptors: TRAIL-R3 is anchored to the cell membrane through a GPI anchor (shown as a red circle) and TRAIL-R4 possesses a truncated death domain in its cytoplasmic tail (shown as a black box) and TRAF binding motifs (shown as red boxes). OPG is a soluble decoy receptor.

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Cancer Therapy Vol 4, page 51 The structure of TRAIL-R1 is highly comparable to that of the TRAIL-R2, as well as its pattern of expression and regulation. TRAIL-R1 is a 468 amino acid type I transmembrane protein that contains a 23 amino acid signal sequence, a 226 amino acid extra-cellular region, a 19 amino acid cytoplasmic region (Figure 1). The cytoplasmic domain of TRAIL-R1 contains a death domain. Although the TRAIL-R1 is able, as well as the TRAIL-R2, to induce a death signal, studies with TRAIL mutants suggest a major role of TRAIL-R2 and not TRAIL-R1 in mediating TRAIL-mediated anti-tumor activity (Kelley et al, 2005). TRAIL-R3 (also known as DcR1) is a 299 amino acid protein with a 23 amino acid signal sequence, a 217 amino acid extracellular region and a 19 amino acid transmembrane domain (Figure 1). Lacking a cytoplasmic domain, TRAIL-R3 is linked to the cell membrane through a GPI linker. TRAIL-R3 seems to act as an antagonizing decoy receptor: its overexpression induces resistance to TRAIL, while its removal from the cell surface by phosphatidylinositol phospholipase C augments the sensitivity of the cells to the apoptotic effect of TRAIL. However, the impact of TRAIL-R3 in the normal TRAIL/TRAIL-Rs system is limited due to scarce expression of this receptor in normal tissues. TRAIL-R4 (also known as DcR2) contains a truncated death domain and is therefore unable to convey an apoptotic signal. TRAIL-R4 acts, like TRAIL-R3, as an inhibitor of TRAIL-induced apoptosis acting through mechanisms similar to those reported for TRAIL-R3 and probably also activating the anti-apoptotic NF-#B pathway. TRAIL-R4 may have a greater importance than TRAIL-R3 in the physiology of the TRAIL/TRAIL-R system in that it is widely expressed in normal tissues. The fifth TRAIL receptor is represented by osteoprotegerin (OPG). The most characterized function of this soluble receptor consists in the inhibition of RANKLstimulated osteoclast formation. OPG binds to RANKL, preventing its interaction with RANK; however, it is also able to bind with low affinity to TRAIL. The role of OPG in the normal physiology of the TRAIL/TRAIL-R system is unclear. However, in some pathologic conditions, such as prostate cancer and multiple myeloma, OPG could act in a paracrine/autocrine way by binding TRAIL and promoting tumor growth. The TRAIL-R1 and TRAIL-R2 activate two overlapping signalling pathways: the extrinsic cell death pathway and a cell survival pathway mainly involving the activation of NF-#B transcription factor (Figure 2). The TRAIL homotrimer induces the trimerization of TRAIL-R1 or TRAIL-R2 on the surface of target cells, which determines the formation of DISC. Briefly, the trimerization of TRAIL-R1 or TRAIL-R2 results in the recruitment of an adaptor molecule FADD through the interaction of their respective death domains (DD). Next, FADD recruits DED-containing initiator pro-caspase-8 through DED/DED (death effector domain) interactions, thus forming the DISC. Control of FADD recruitment to the DISC is mediated by DED-containing c-FLIPs: these proteins exert their inhibitory activity on TRAIL-Rs

signalling through their binding to the DED of FADD, thus blocking pro-caspase-8 activation (Figure 2). In some cells (type I cells) the activation of caspase-8 is sufficient to trigger the activation of the effector caspase-3 and to execute cellular apoptosis (Figure 2). In other cell types (type II cells), the amplification of the apoptotic cascade through the mitochondrial pathway is required for the induction of cellular apoptosis: in this pathway Bid is cleaved by caspase-8 and, in turn, the truncated Bid activates Bax- and Bak-mediated release of cytochrome C and Smac/DIABLO from mitochondria with subsequent induction of cell apoptosis (Figure 2). In type I cells, death receptorinduced death cannot be blocked by overexpression of anti-apoptotic Bcl-2 family members. In type II cells, overexpression of antiapoptotic Bcl-2 family members blocks death receptor-induced cell death. Interestingly, the mode and efficiency of death receptor signalling was correlated with the submembrane localization of these receptors: in type I cells a portion of death receptors resides constitutively in lipid rafts (membrane liquid-ordered microdomains enriched in sphingolipids and cholesterol that constitute a distinct biophysical compartment, serving a signalling platform for membrane receptors), while in type II cells, these receptors are excluded from lipid rafts during early signalling(Muppidi and Siegel, 2004). The presence of a death receptor in lipid rafts allow type I cells to undergo apoptosis in response to stimulation with the corresponding death ligand, which cannot induce apoptosis in type II cells (Muppidi et al, 2004). Disruption of lipid raft structure by cholesterol depletion reduces death receptor signalling efficiency in type I cells, but not in type II cells (Legembre et al, 2006). This signalling pathway for TRAIL-R1 and TRAILR2 was now firmly established (Yagita et al, 2004). In addition to the death signalling pathway, TRAILR1 and TRAIL-R2 are also able to activate survival signals via the transcription factor NF-#B (Figure 2), which can up-modulate antiapoptotic genes (Schneider et al, 1997). However, it is important to note that NF-#B activation via TRAIL-Rs is much weaker than that via TNF-Rs and that the activation of NF-#B alone is not sufficient to inhibit apoptosis triggered via TRAIL-R2. Inhibition of NF-#B activation attenuates TRAIL resistance of tumor cells (Jeremias et al, 1998). TRAILR1/TRAIL-R2-induced NF-#B activation is mediated by a TRAF2-NIK-I!B kinase alpha/beta signalling cascade (Hu et al, 1999). TRAIL induces NF-#B in two phases: an early caspases independent phase and a late caspases-8, but not caspases-3, dependent phase; this last phase leads to the caspases-mediated cleavage of I#B" (Rathore et al, 2004). TRAIL has been shown to activate also c-Jun NH2terminal kinase (JNK) and the induction of the JNK pathway was shown to have an amplifying effect on TRAIL-induced apoptosis. Several studies suggest that TRAIL and its receptors may play an important role in the control of development of tumors, acting at the level of several important steps in the development of cancers.

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Pasquini et al: Sensitivity and resistance of human cancer cells to TRAIL

Figure 2. Outline of the signalling cascade triggered by TRAIL-R2. The binding of one homotrimeric TRAIL molecule to the TRAILR2 determines its trimerization with consequent binding of a FADD molecule to its cytoplasmic tail through a DD/DD interaction. FADD through its DED domain recruits pro-caspase-8. Caspase-8 activation is controlled by DED-containing FLICE inhibitory proteins (c-FLIPs) that exert an inhibitory activity on this process. In type I cells the activation of caspase-8 is sufficient to induce the activation of caspase-3 with subsequent induction of apoptosis. In type II cells, the amplification of the apoptotic signal through the mitochondrial pathway is required for induction of apoptosis: in this pathway Bid is cleaved by caspase-8; in turn, truncated Bid activates Bax and Bakmediated release of cytochrome C and SMAC/DIABLO from mitochondria with consequent induction of apoptosis. In addition to death signalling, TRAIL-R2 is also able to activate survival signals via the TRADD-mediated activation of the NF-#B transcription factor. However, it is important to note that usually the TRAIL-R2-mediated activation of NF-#B is weak and not able to counteract the death signalling activity induced by the activation of this receptor.

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Cancer Therapy Vol 4, page 53 The role of TRAIL in cancer development is supported by several lines of evidence: • TRAIL-R1 and TRAIL-R2 are mutated in some tumors, including breast cancer, ovarian cancer, nonHodgkin lymphomas, non small cell lung cancer and neck cancer. • Tumor cells may express TRAIL allowing them to kill T cells and thus to suppress the immune system. • Mice made deficient in TRAIL expression by gene targeting have a clearly increased tendency to develop tumors thus suggesting a physiologic role for TRAIL in immune surveillance against tumor development (Cretney et al, 2002, Takeda et al, 2002). On the other hand, TRAIL-R2 knockout mice exhibit a clear compromission in radiation-induced apoptosis (Finnberg et al, 2005). TRAIL-R1 or TRAIL-R2 are silenced in some tumors through epigenetic mechanisms, mainly related to gene hypermethylation (Horak et al, 2005). The analysis of TRAIL and TRAIL-Rs expression in normal tissues at protein level showed that: (i) TRAIL expression was found to be limited mostly to smooth muscle in lung and spleen as well as glial cells in the cerebellum and follicular cells in the thyroid gland; (ii) TRAIL decoy receptors are rarely expressed in normal tissues; (iii) TRAIL-R1 and TRAIL-R2 are expressed in many tissues and particularly on smooth muscle cells in all tissues, around blood vessels, on neuronal cells in cerebellum, on hepatocytes, follicular cells in the thyroid, monocytes and macrophages present in various tissues (Daniles et al, 2005). The level of TRAIL-Rs may be modulated by various agents. Agents that induce DNA damage, such as anti-tumor chemotherapies, UV irradiation or ! ray radiation have been shown to up-regulate TRAIL-R1 and TRAIL-R2 through p53- and p63- dependent mechanisms (Liu et al, 2004; Gressner et al, 2005). For TRAIL-R2 a direct p53-dependent transactivation mechanism via a p53 DNA binding motif within intron-1, has been shown. In contrast, the upregulation of TRAIL-R2 by interferon-! and by glucocorticoids is independent of p53 activation.

main steps of TRAIL resistance are outlined in Figure 3 and are represented by a single or by a combination of these mechanisms: defective TRAIL-R1 and/or TRAIL-R2 expression, predominant expression of decoy TRAIL-Rs, defective expression of caspases-8 or of FADD, increased expression of c-FLIP or of IAPs (Figure 4).

A. Brain tumors The sensitivity of neuroblastoma cells to TRAIL was investigated in detail. Initial studies showed that neuroblastoma non-invasive stromal-adherent cell lines were highly sensitive to TRAIL, while invasive cell lines are resistant (Hopkins-Donaldson et al, 2000). The absence of TRAIL-sensitivity correlated with loss of caspase-8 expression. This last finding was correlated with TRAIL resistance in a second study carried out in 18 neuroblastoma cell lines (Eggert at al, 2001). The caspase8 gene is silenced in neuroblastomas with amplification of the oncogene MYCN through DNA methylation (frequently) as well as through gene deletion (rarely) (Teitz et al, 2000). Analysis of the pattern of TRAIL-R expression in primary tumors showed that the large majority of them do not express TRAIL-R1 and TRAILR2, thus suggesting that loss of TRAIL-R expression may represent an additional mechanism of TRAIL resistance in neuroblastoma (Yang et al, 2003). TRAIL efficiently triggers apoptosis in some medulloblastoma cell lines (Nakamura et al, 2000). Analysis of the pattern of TRAIL-Rs expression by RTPCR showed that primary medulloblastoma cells expressed TRAIL-R2; however, a loss of caspase-8 expression was frequently (52% of cases) observed (Zuzak et al, 2002). Primary medulloblastoma cells did not express TRAIL, while TRAIL expression was present in reactive peri-tumoral astrocytes (Nakamura et al, 2000). Similarly, the resistance to TRAIL-induced apoptosis in primitive neuroectodermal brain tumors cell lines correlates with a loss of caspase-8 expression (Grotzer et al, 2000). Importantly, the loss of caspase-8 expression and the lack of TRAIL sensitivity in these tumors correlate with unfavourable survival outcome (Pingoud-Meyer et al, 2003). The majority of glioma cell lines are resistant or are scarcely sensitive to TRAIL-mediated apoptosis (Chunhai et al, 2001). These preliminary observations have been confirmed through the analysis of primary tumors derived from 21 glioblastoma patients, showing that only 4/21 of them are sensitive to TRAIL-induced apoptosis (Jeremias et al, 2004). The analysis of the mechanisms of TRAILresistance in glioma cells suggest a possible role of caspase inhibitors, like XIAP, that are constitutively expressed in these cells (Wagenknecht et al, 1999). Analysis of a panel of glioma cell lines showed that 1 glioma line, U373, was resistant to death ligand-induced apoptosis because it expressed very low levels of caspase8 (Knight et al, 2001). Analysis of primary glioma cells showed that a high proportion of them have low or undetectable caspase-8 levels (Ashley et al, 2005). In line with this finding, Smac agonists (that counteracts the antiapoptotic effects of IAPs) sensitize for

II. Sensitivity of human tumors to TRAIL Many studies have been carried out to investigate the sensitivity of human tumors to TRAIL. We focused our attention to the literature concerning the brain, lung, prostate, gynaecologic, gastro-enterologic, skin and hematologic tumors. A major limitation derives from the fact that the majority of these studies have been performed on tumor cell lines, that do not reflect entirely the properties of primary tumor cells. In spite these limitations, these studies clearly indicate that in the majority of cases tumor cells are resistant to the proapoptotic effects of TRAIL. The analysis of the molecular mechanisms responsible for TRAIL resistance of tumor cells has lead to the identification of some important defects in the apoptotic machinery of cancer cells. The

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Pasquini et al: Sensitivity and resistance of human cancer cells to TRAIL TRAIL and induce regression of malignant glioma in tumor xenograft models (Fulda et al, 2002).

The mechanism of prostate cancer resistance to TRAIL was explored only in cancer cell lines. The majority of these studies have been carried out in PC-3, DU-145 and LNCaP cell lines. The TRAIL-apoptotic signalling pathway is subjected to several levels of inhibitory regulation. It was shown that TRAIL-resistance in prostate cancer cells can be associated with: (i) a surface expression of decoy receptors (i.e. TRAIL-R3/DcR1, TRAIL-R4/DcR2 and OPG); (ii) the elevated expression of c-FLIP, a dominant negative form of caspase-8 that lacks the caspase catalytic site (Zhang et al, 2004); (iii) increased level of Bcl-2 antiapoptotic members (Sonneman et al, 2004).

B. Prostate cancer No data are available about the sensitivity of primary prostate cancer cells to TRAIL. However, several studies have been performed on a panel of prostate cancer cell lines. The response of prostate cancer cells lines to TRAIL depends on the cell type. For example the PC-3, PC-3M, DU-145, ALVA-31 cell lines were sensitive or semisensitive to TRAIL (Zhang et al, 2004; Sonneman et al, 2004; Holen et al, 2002) while LNCaP, PC-3-TR, CL-1 cell lines were resistant (Zhang et al, 2004; Ng & Bonavida, 2002).

Figure 3. Cell death apoptotic signalling and mechanisms of resistance to TRAIL-induced apoptosis. The cell death signalling apoptotic pathway induced by TRAIL-R1/-R2 is outlined. This cell death pathway may be inhibited at various levels: at receptor level by the decoy TRAIL receptors that act by competing with TRAIL-R1 and TRAIL-R2 for TRAIL binding; at post-receptor level, at the step of DISC formation by c-FLIP that acts by preventing the recruitment of caspase-8; at mitochondrial level by Bcl-2 and Bcl-X L that act by suppressing the Bax/Bak-mediated release of cytochrome-C and SMAC/DIABLO from mitochondria; at the level of caspase-3 and -9 activity by IAPs attenuating the activity of these caspases (the inhibitory activity of IAPs is counteracted by SMAC/DIABLO). Many of these inhibitory mechanisms are activated in cancer cells.

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Cancer Therapy Vol 4, page 55

Figure 4. Schematic representation of the structure of FADD and FLIP proteins. Left, top: the structure of FADD protein, with two boxes indicating one DED domain and one DD domain, is shown. The numbers indicate the amino acid residue. Within the DED domain, nuclear export sequence (NES) and nuclear localization sequence (NLS) have been identified: they determine the nuclear localization of FADD either in the nucleus or in the cytoplasm. In the COOH terminal site two serine residues (Ser 191 and Ser 194), essential for FADD function, are indicated. Right, top: the structure of the three c-FLIP isoforms, FLIP L, FLIPS and FLIPR with their structural domains is shown. Left, bottom: signalling along the extrinsic pathway based on FADD and caspase-8 activation with consequent induction of the effector apoptotic machinery. Right, bottom: block of the apoptotic signalling along the extrinsic pathway when c-FLIP interacts with FADD, hampering the subsequent caspase-8 recruitment and activation.

overexpression of c-FLIP might shift the responsiveness towards the resistant status, c-FLIP could be a prognostic marker for death receptor-sensitivity in immune therapy (Jonsson et al, 2003). Interestingly, TRAIL resistance in some bladder cancer cells was related to a rapid receptor downregulation (Steele et al, 2006). Histone deacetylase inhibitors clearly enhanced the sensitivity to TRAIL in bladder cancer cells resistant to this death ligand, via a mechanism involving both TRAILR2 upmodulation and loss of mitochondrial membrane potential (Earel et al, 2006).

C. Bladder cancer Bladder tumor cells show a spread resistance to TRAIL-mediated apoptosis. This conclusion is based on studies carried out on bladder cancer cell lines (Mizutani et al, 2001; Jonsson et al, 2003; Papageorgiou et al, 2004). Some bladder cancer cell lines displayed a significant sensitivity to TRAILinduced apoptosis (Steele et al, 2006). The induction of apoptosis in these cells is dependent on both TRAIL-R1 or TRAIL-R2-induced signalling (Steele et al, 2006). The level of TRAIL sensitivity of bladder cancer cells did not depend on the level of TRAIL-R1 or TRAIL-R2 expression. Normal urothelial cells exhibited a moderate sensitivity to TRAIL-mediated apoptosis (Steele et al, 2006). The resistance to TRAIL-induced apoptosis in bladder cancer seems to be mediated by a high c-FLIP expression. In fact the ratio between the c-FLIP and caspase-8 was directly correlated to resistance to antiCD95 or TRAIL-mediated apoptosis. Since

D Breast cancer The pro-apoptotic activity of TRAIL on breast cancer has been evaluated only in tumor cell lines and not in primary tumor cells. The majority of breast cancer cell lines are resistant or semisensitive (T47D, MCF-7, MDAMB-453, MDA-MB-468, MDA-MB-157, SKBr-3, ZR75); only one nontransformed (MCF10A) and one breast cancer cell line (MDA-MB-231) are significantly sensitive 55

Pasquini et al: Sensitivity and resistance of human cancer cells to TRAIL to TRAIL-induced apoptosis (Keane et al, 1999; Singh et al, 2003). No difference in sensitivity was found between normal and malignant (resistant) cell lines. A large part of breast cancer cell lines express TRAIL-R1 and TRAIL-R2 and at least one of the two decoy receptors (Singh et al, 2003). Recently, TRAIL-R1 and TRAIL-R2 expression was explored by immunohistochemistry and quantitated by automated image quantitative analysis, providing evidence about an heterogeneous expression of these two receptors on breast cancer cells (McCarthy et al, 2005). TRAIL-R1 expression was not associated with survival, while high TRAIL-R2 was clearly associated with decreased survival (McCarthy et al, 2005). To explain the association between high TRAIL-R2 expression and poor survival it was suggested that high TRAIL-R2 expression may be a marker of tumor cells that have increased activation of NF-#B (Biswas et al, 2004). Several studies were performed on a panel of cell lines (MCF-7, MDA-MB-453, MDA-MB-231, MDA-MB468, MDA-MB-361, T-47D, CAMA-1, AU565) to explore the mechanisms which confer TRAIL-resistance to breast cancer. One of the putative mechanisms of TRAILresistance is related to the expression of decoy receptors, in particular TRAIL-R4 (Sanlioglu et al, 2005) and OPG (Neville-Webbe et al, 2004). OPG produced by bone marrow stromal cells protects breast cancer cells from TRAIL-induced apoptosis (Neville-Webbe et al, 2004). It was shown that expression at high level of Bcl-2 and Survivin and XIAP, two members of the IAP protein family, can render breast cancer cells resistant to TRAILinduced apoptosis (Ruiz de Almod!var et al, 2001; Yang et al, 2003). In line with this observation, Smac-mimic compounds act to induce apoptosis alone, as well as sensitize breast cancer cells to TRAIL or etoposideinduced apoptosis via caspase-3 activation (Backbrader et al, 2005). Recent studies show that TRAIL-resistance is correlated with the action of the Small Heat Shock Protein "B-crystallin, a caspase-3 inhibitor (Kamradt et al, 2005), and by the high expression of FLIP (Hyer et al, 2005).

and by the antiapoptotic effect of NFkB, Survivin and Bcl2 molecules (Nam et al, 2003; Yang et al, 2004). One additional mechanism of TRAIL resistance could be represented by OPG, whose elevated expression is associated with poor outcome in gastric carcinoma (Ito et al, 2003). The ensemble of these observations suggest that targeting of TRAIL-R1 and TRAIL-R2 by agonistic monoclonal antibodies may be of value in the treatment of the highly chemoresistant gastro-esophageal carcinomas.

F. Pancreas cancer The evaluation of TRAIL’s effects has been carried out for the majority in pancreatic cancer cell lines; only one work performed on patient pancreatic adenocarcinomas grown in SCID mice model show heterogeneity of tumor response to TRAIL (Hylander et al, 2005). Contrasting data are available in pancreatic cancer cell lines about TRAIL-sensitivity. Recent studies performed on a large panel of pancreatic cancer cell lines showed no apoptosis upon stimulation by TRAIL (Matsuzaki et al, 2001; Ozawa et al, 2001; Bai et al, 2005). An high expression of DR4 and DR5 was found in TRAIL-sensitive pancreatic cancer cell lines (Ozawa et al, 2001), while an overexpression of decoy receptors DcR1 and DcR2 was detected specially in resistant cells (Bai et al, 2005). The mechanisms through which pancreas cancer cells evade TRAIL-mediated apoptosis is related to the action of antiapoptotic molecules like NFkB, Bcl-XL and c-FLIP (Thomas et al, 2002; Bai et al, 2005).

G. Colon cancer Several studies have investigated the effects of TRAIL mainly on colon cancer cell lines and rarely in primary cells. Using patient colon carcinoma-SCID mouse model has been possible to show a high heterogeneity in TRAIL sensitivity that, probably, reflects inherent patient-topatient differences (Naka et al, 2002). Recent studies performed in colon cancer cell lines have demonstrated a broad range in sensitivity of cell lines to TRAIL, in particular an increased TRAIL-sensitivity was found in colon carcinoma more than colon adenoma cells. (Hargue et al, 2005; Vasilevskaya & O’Dwyer, 2005). The mechanisms responsible for TRAIL resistance of colon cancer cells have been investigated in detail (reviewed by Van Geelen et al, 2005). Loss of function of TRAIL receptor genes by mutations or epigenetic changes is not frequently observed in colon cancer (Arai et al, 1998). Alterations of caspase-8 either due to inactivating mutations (Kim et al, 2003) or to decreased expression due to increased degradation (McDonald and El Deiry, 2004) are involved in TRAIL resistance of colon cancer cells. The majority of colon carcinoma and adenoma cell lines express TRAIL-R1 and TRAIL-R2, but the expression pattern of TRAIL receptors did not correlate

E. Gastric cancer Few studies were performed on primary gastric cancer cells and no data are available about TRAILsensitivity of primary cells. The majority of these works had shown that TRAIL and TRAIL receptors are highly coexpressed in primary and metastatic gastric carcinoma cells (Sheikh et al, 1999; Koyama et al, 2002). Similarly, the large majority of esophageal adenocarcinomas express functional TRAIL-R1 and TRAIL-R2 (Younes et al, 2006). TRAIL cytotoxicity was examined in a set of tumor cell lines: SNU-668 and MKN28, highly sensitive to TRAIL-induced cell death; SNU-601, SNU-719, SNU-1, SNU-5, moderately sensitive and SNU-216, MKN45, AGS and SGC-7901 almost completely resistant (Nam et al, 2003; Yang et al, 2004). All gastric cancer cell lines expressed DR4, DR5 and DcR2 indicating that TRAIL-sensitivity is regulated at the intracellular level: in fact, the resistance to TRAIL can be associated with the Akt activity that upregulates c-FLIPs 56

Cancer Therapy Vol 4, page 57 with TRAIL sensitivity of these cell lines (Hargue et al, 2005). The expression of antiapoptotic molecules (Bcl-2, XIAP, c-FLIP) can be correlated with TRAIL-resistance (Sinicrope et al, 2004; Vasilevskaya & O’Dwyer, 2005). Hepatocarcinoma cells could be sensitized to TRAIL with proteasome inhibitors (MG132 or PS-341), while this treatment did not modify TRAIL sensitivity of normal hepatocytes (Ganten et al, 2005).

expression (Brooks and Sayers, 2005) or to the low level of TRAIL-R1/ TRAIL-R2 expression in combination with high survivin expression (Griffith et al, 2002) or to the constitutive Akt phosphorylation (Asakuma et al, 2003) or the constitutive NF-#B activation (Oya et al, 2001).

J. Lung cancer Several in vitro studies have shown that some nonsmall cell lung cancer (NSCLC) cell lines are sensitive to apoptosis induction by recombinant TRAIL (Sun et al, 2000; Kagawa et al, 2001). No data are available about the sensitivity of primary NSCLC to TRAIL-mediated apoptosis. Analysis of the expression of TRAIL and TRAIL-Rs in NSCLC patients showed that TRAIL-R1, TRAIL-R2 and TRAIL were expressed in 99%, 82% and 91% of the tumors, respectively (Spierings et al, 2003). The level of TRAIL-R2 positivity was associated with increased risk of death (Spierings et al, 2003). Mutations of the ectodomain of TRAIL-R1 (Fisher et al, 2001) and of the death domain of TRAIL-R2 (Lee et al, 1999) have been observed in 35% and 11% of NSCLC patients. Some NSCLC cell lines are resistant to TRAIL through mechanisms related to Bcl-2 overexpression in these cells (Jiang et al, 1995; Ziegler et al, 1997), to mutations at the level of TRAIL-R1 and/or TRAIL-R2 (Fischer et al, 2001; Spierings et al, 2003), to decreased caspase-8 expression, to low DAP-kinase activity to promoter hypermethylation (Tang et al, 2004) due to promoter hypermethylation (Hopkins-Donaldson et al, 2003), and to spontaneous Akt activation (Kandasamy and Srivastava, 2002).

H. Liver cancer Few data on primary cells reveal that hepatocarcinoma cancer cells (HCC) show lower levels of TRAIL-R1 and -R2 expressions compared to the nonmalignant liver tissues. Moreover, Fas expression was found to be lower in the HCC tissues than in the normal ones (Shin et al, 2002). The expression of survivin was investigated in 38 cases of HCC tissues and survivin protein was detected in 23 (60,5%) of 38 HCCs. The expression of survivin seems to be related to the metastasis of HCC. Survivin might be considered then a very useful marker for evaluation of metastatic potential and prognosis of HCC (Zhu et al, 2005). Studies performed on cell lines show a broad spectrum of responses. HepG2 cells undergo TRAILinduce apoptosis in a dose-dependent manner. In contrast, the treatment of HepG2TR (TRAIL resistant variant of HepG2) and Hep3b cells with TRAIL did not result in a significant level of apoptosis induction (Ganten et al, 2004). 7 of 10 HCC cell lines showed resistance to TRAILinduced apoptosis and 5 of 7 TRAIL resistant cell lines become sensitive to TRAIL by co-treatment with cycloheximide and cisplatin (Shin et al, 2002). A recent study showed that human hepatocarcinoma tissues usually express membrane-bound TRAIL that enables these tumor cells to evade immune surveillance by inducing apoptosis of activated lymphocytes (Shiraki et al, 2005). TRAIL expression in hepatocarcinoma cells is enhanced by some chemotherapeutic agents, such as doxorubicin (Shiraki et al, 2005). Since upregulation of TRAILRs or downregulation of DcR1 and DcR2 (decoy receptors) could be a way to overcome resistance studies performed with TRAIL and 5FU have been performed. TRAIL-R1 upregulation potentially contributes to increase sensitivity of HepG2TR cells to TRAIL-induced apoptosis upon 5 FU treatment.

K. Ovarian cancer TRAIL represents a potentially interesting molecule for ovarian cancer therapy. In several studies, the action of TRAIL was evaluated in primary tumor cells and in ovarian cancer cell lines. Only few studies were carried out on primary tumor cells, showing a variable sensibility to TRAIL-induced cytotoxicity. For example an analysis done on four primary samples showed that two of them were highly resistant to TRAIL-induced cell death, whereas the other two were sensitive (Lane et al, 2004). There were no more data that could show the effects of TRAIL on primary tumor cells. Anyway, in another work the expression of TRAIL in ovarian cancer cells was analyzed: ovarian cancer cells exhibit 10-fold higher mean TRAIL expression than normal ovarian epithelial samples. This high TRAIL expression measured by RT-PCR was associated with prolonged survival (Lancaster et al, 2003). Similar studies on TRAIL resistance were performed on ovarian cancer cell lines. Upon treatment with TRAIL, cell lines were distinguished in TRAIL-sensitive (OVCAR3, CAOV3, MZ-26, ES-2, IGROV-1) and TRAIL-resistant cell lines (SKOV3, UCI-101, OV-4, A2780, A2780ADR, MZ-15) (Vignati et al, 2002; Lane et al, 2004; Tomek et al, 2004,) The expression of c-FLIP represents one of the most important mechanisms of resistance to TRAIL in ovarian

I. Kidney cancer Treatments of freshly-derived renal cell carcinoma (RCC) cells with TRAIL as well as treatments of Caki-1 cell line with TRAIL showed resistance. Only combination with 5-FU resulted in a synergistic cytotoxic effect (Mizutani et al, 2002). RCC cell lines displayed a great heterogeneity in their sensitivity to TRAIL-mediated apoptosis (Brooks and Sayers, 2005, Griffith et al, 2002, Asakuma et al, 2003). Their resistance to TRAIL was related either to c-FLIP

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Pasquini et al: Sensitivity and resistance of human cancer cells to TRAIL cancer cells. TRAIL-resistant ovarian cancer cell lines express elevated levels of c-FLIP (Tomek et al, 2004). One study has demonstrated that cisplatin-induced apoptosis in ovarian cancer cells is associated with decreased FLIP protein content and with activation of caspase-8 and caspase-3. Moreover, these results have been observed in sensitive but not in resistant cell lines. In fact, the hypothesis was that the overexpression of FLIP induces resistance of ovarian cancer cells to cisplatin and that the downregulation of FLIP sensitizes chemoresistant ovarian cancer cells to cisplatin. These findings suggest that FLIP may play an important role in regulating the sensitivity of ovarian cancer cells to cisplatin treatment (Abedini et al, 2004). Another study suggest that the inhibitory protein cFLIP is involved also in resistance to CD95-mediated apoptosis in ovarian carcinoma cells with wild type p53 (Mezzanzanica et al, 2004). It is unclear whether the expression of TRAIL decoy receptor could be involved in the genesis of TRAIL resistance in ovarian cancer cells.

positive leukaemia cell lines. Effectively, TRAIL induced apoptosis in Ph1-positive leukaemia cells and it was mostly correlated with the cell-surface expression levels of DR4 and DR5. Notably, TRAIL was also effective against leukaemia cells that were refractary to the BCR-ABL kinase inhibitor imatinib mesylate (STI571) (Kanako Unok et al, 2003). Regarding TRAIL sensitivity in leukaemic blasts from CML-BC, only one case was evaluated and showed resistance. FADD and caspase-8, component of DISC, as well as c-FLIP a negative regulator of caspase-8, are expressed ubiquitously in Ph1-positive leukaemia cell lines irrespectively of their differential sensitivities to TRAIL.

2. ALL (Acute Lymphoblastic Leukemia) The activity of TRAIL was investigated in 29 primary precursor B-cell acute lymphoblastic leukaemia (ALL) samples. TRAIL was found to have a modest activity as it killed a maximum of 29% of ALL cells within 18h compared with a high rate of killing (75%) of Jurkat cells (T-cell lymphoblastic origin). This differential effect may indicate that malignant precursor T-cells (Jurkat) may be more sensitive to TRAIL than B-cells. However preliminary data in three primary precursor Tlymphoblastic leukaemia cases do not support this hypotesis as all T-cell ALL cases were also resistant to TRAIL. TRAIL insensitivity of ALL is not related to the overexpression of the decoy R3 or R4 receptors, nor to the overexpression of the antiapoptotic protein c-FLIP. It is possible that the lack of sensitivity is simply related to the low levels of functional R1 and R2 receptors observed in ALL cells (Clodi et al, 2000). Primary malignant cells of haematological origin frequently express TRAIL transcripts and proteins which induce cell death of target cell lines that are known to be sensitive to TRAIL (Jurkat and HL60). This killing effect is dose-dependent and it is TRAIL-specific because anti-TRAIL antibody reversed this effect. Based on these observation, it was suggested that the functional expression of TRAIL by tumor cells could protect them from cytotoxic T cells (Zhao et al, 1999).

L. Melanoma Melanoma is a cancer characterized by a high metastatic potential as well as by a great apoptosis resistance, both highly contributing to immune escape mechanisms and resistance to chemotherapy. Melanoma cell lines displayed a differential sensitivity to TRAIL-mediated apoptosis (Griffith et al, 1998; Zhang et al, 1999). The sensitivity of these cell lines to TRAIL was correlated with the level of TRAIL-R1 and, particularly, of TRAIL-R2, while the expression of TRAIL decoy receptors does not correlate with TRAIL resistance (Griffith et al, 1998; Zhang et al, 1999). However, fresh isolates of melanoma are usually resistant to TRAILmediated apoptosis, and this phenomenon is associated with low TRAIL-R2 expression (Nguyen et al, 2001). The mechanisms of TRAIL resistance by melanoma cells are complex and involve the activation of several anti-apoptotic mechanisms, mainly represented by c-FLIP, survivin and Bcl-2 overexpression (reviewed in Hersey and Zhang, 2001). Downregulation of the expression of these anti-apoptotic proteins considerably potentiates the sensitivity of melanoma cells to TRAIL (Chawla-Sarkar et al, 2004). However, a recent study re-evaluated TRAIL-R expression in melanoma sections showing TRAIL-R1 and TRAIL-R2 in the majority of cases (Kurbanov et al, 2005). Furtehrmore, it was shown that both in melanoma cell lines and primary tumors both TRAIL-R1 and TRAIL-R2 are very frequently expressed and TRAIL-R1 signaling was able to induce apoptois of melanoma cells (Kurbanov et al, 2005). These observations suggest a therapeutic potential of TRAIL-R1 targeting in melanoma.

3. B-CLL Leukemia)

(B-Chronic

Lymphocytic

Primary B-CLL cells from patients are generally not sensitive to either TRAIL or anti-CD95, whereas three tumor cell lines, Jurkat, SKW, MC116 are sensitive. Even at high TRAIL concentration (200 ng) B-CLL are resistant to apoptosis. Lower levels of TRAIL death receceptors expression (TRAIL-R1, TRAIL-R2) are generally observed compared to the cell lines, with no detectable expression of TRAIL-R3 or TRAIL-R4. Thus, in agreement with other studies the resistance of the B-CLL cells to TRAIL-induced apoptosis is not probably due to increased cell surfarce expression of ‘decoy’ receptors (Griffith et al, 1998; Leverkus et al, 2000). The resistance of B-CLL cells to TRAIL may be due partly to low surface expression of the death receptors resulting in low levels of DISC formation and also to the high ratio of c-FLIP L

M. Haematological tumors 1. CML (Chronic Myeloid Leukemia) Given the IFN-" inducibile expression of TRAIL on human T cells, TRAIL may participate in the process of antileukemic effects against Ph1-positive leukaemias. The apoptotic effect of TRAIL was first investigated in Ph1

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Cancer Therapy Vol 4, page 59 (long form) to caspase-8 within the DISC, which would prevent further activation of caspase-8 (MacFarlane et al, 2002). More recently it has been shown that low concentrations of histone deacetylase inhibitors (HDACi) sensitize CLL cells to TRAIL-induced apoptosis by facilitating increased formation of the TRAIL DISC. Peripheral blood lymphocytes from 28 patients with CLL at different clinical stages were exposed to different forms of TRAIL plus HDACi. No increase in the spontaneous level of apoptosis was observed in cells from these different patients exposed to TRAIL alone. Moreover, pretretment with depsipeptide (VPA 2mM) at low concentrations sensitized CLL cells to TRAIL-induced apoptosis by facilitating increased formation of the TRAIL DISC via TRAIL-R1, although most studies suggested that TRAIL-R2 is the primary TRAIL receptor leading to cell death (MacFarlan et al, 2005). It is also known that B-CLL express at higher level TRAIL ligand respect to unfractioned lymphocytes, and the addiction in culture of recombinant TRAIL increased leukaemic cell survival. Thus an aberrant expression of TRAIL might contribute to the phatogenesis of B-CLL by promoting the survival in a subset of B-CLL cells (Secchiero et al, 2005). Primary lymphoma cells are scarcely moderately sensitive to the pro-apoptotic effects elicited either by recombinant TRAIL or by agonistic monoclonal antibodies to TRAIL-R1 (HGS-ETR-1) and TRAIL-R2 (HGS-ETR2) (Georgakis et al 2005). A defective TRAIL-R1 and TRAIL-R2 expression is frequently observed also in non-Hodgkin lymphomas, due to 8p21.3 chromosomal deletions (Rubio-Mascardo et al, 2005). The chromosomal deletions result in deletion of one or the two alleles encoding TRAIL-R1 and TRAILR2, determining a reduced expression of these receptors on lymphoma cells and a reduced sensitivity to TRAIL of these tumor cells (Rubio-Moscardo et al, 2005). Burkitt’s lymphomas are resistant to TRAILmediated apoptosis due to a high c-FLIP expression (Djerbi et al, 1999). The high c-FLIP expression was found highly related to a poor prognosis, characterized by a chemoresistant disease, resulting in a high death rate within the first year of diagnosis (Valnet-Rabier et al, 2005). Interestingly, all c-FLIP positive cases exhibit the presence of an active nuclear factor NF-#B.

study showed that a high proportion of AML blasts exhibit low or absent levels of FADD (Tourneur et al, 2004). Some reports have suggested a sensitivity of acute promyelocytic cells to TRAIL induced by retinoic acid treatment (Altucci et al, 2001). These findings, however, were not confirmed by other authors (Riccioni et al, 2001). The resistance of AML blasts to TRAIL may be bypassed by co-treatment with histone-deacetylase inhibitors (Insinga et al, 2005). Paradoxically, leukaemia cells resistant to TRAIL may be stimulated to proliferate by this death ligand, via a mechanism involving NF-#B activation (Baader et al, 2005).

5. MM (Multiple Myeloma) Primary myeloma cells display a low sensitivity to pro-apoptotic effects of TRAIL (Lincz et al, 2001). In parallel studies on myeloma cell lines showed a variable sensitivity to TRAIL (Lincz et al, 2001). TRAIL resistance of myeloma multiple cells was related to high expression in these cells of the anti-apoptotic proteins c-FLIP and cIAP-2 (Mitsiades et al, 2002). Interestingly, interferonalpha (IFN-") sensitize myeloma cells to both TRAIL (Crowder et al, 2005) and Fas L (Dimberg et al, 2005) via induction of the promyelocytic gene PML that plays an important role in the control of apoptosis.

III. Therapy based on TRAIL and monoclonal antibodies anti-TRAIL receptors The selective cytotoxic effect of TRAIL against cancer cells, sparing normal cells, stimulated many studies focused to evaluate the potential use of this death receptor ligand, as well as of agonistic anti TRAIL-R1 antibodies as anticancer drugs. Previous studies on two other death receptor ligands, TNF-" and Fas Ligand, were considerably hampered either by the pro-inflammatory properties (TNF-" or by the induction of fulminant hepatic necrosis (FasL) after in vivo administration. Both these ligands are therefore considered unsuitable as potential anti-cancer drugs, at least when administered by a systemic way. In contrast, the studies on TRAIL and agonistic antiTRAIL-R monoclonal antibodies indicate their potential use as anti-cancer drugs. A part of these studies were focused to evaluate the anti-tumor properties and the toxicity profile of recombinant preparations of human TRAIL. Although many concerns have been raised about a possible toxicity of TRAIL, carefull studies have shown that some toxicities against normal cells attributed to this death receptor ligand are in reality related to aberrant structural and biochemical properties of the recombinant variants of the protein. Four different recombinant versions of the human have been generated and molecularly characterized. A first molecular form contains TRAIL amino acids 114-281 fused to an amino-terminal polyhistidine tag (Pitti et al, 1996). A second variant contains TRAIL amino acids 95-281 fused to an amino-terminal leucine zipper, promoting the trimerization of the ligand (Walczak et al,

4. AML (Acute Myeloid Leukaemia) Studies based on the analysis of primary cells derived from a large set of AML patients pertaining to different FAB subtypes, provided evidence that they are invariably resistant to TRAIL-mediated apoptosis (Riccioni et al, 2005, Jones et al, 2003). Similarly, pediatric acute leukaemias are frequently resistant or scarcely sensitive to TRAIL (Baader et al, 2005). The mechanisms of TRAIL-resistance of AML blasts have been only in part explored. In this context the frequent expression of TRAIL-R3, and TRAIL-R4 on the surface of AML blasts has lead to suggest that these decoy receptors may be responsible for TRAIL resistance of these cells (Riccioni et al, 2005). Furthermore, a recent

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Pasquini et al: Sensitivity and resistance of human cancer cells to TRAIL 1999). A third version contains TRAIL amino acids 95281 fused to an amino-terminal Flag: the crosslinking of this tagged protein with anti-flag antibodies enhanced antitumor activity (Bodmer et al, 2000). A fourth version of the molecule is composed by TRAIL amino acids 114-281 without any additional exogenous sequences (Ashkenazi 2002). The preparation of this lost form of recombinant human TRAIL is improved by addition of zinc and reducing agents to the cell culture medium and extraction buffers and maintaining neutral pH in the buffers (Kelley 2001, Lawrence et al, 2001). Soluble untagged TRAIL displayed in vivo no toxicity and, particularly, no hepatic toxicity (Ashkenazi et al, 1999), while the His-tagged TRAIL preparation displayed a marked hepatotoxicity (Jo et al, 2000). The membrane bound form of TRAIL induced hepatic toxicity in mice (Ichikawa et al, 2001). The analysis of the toxicity of the different TRAIL recombinants versions confirmed the observations made on hepatocytes. Thus, His-tagged TRAIL 114-281 as well as L2-TRAIL, induced apoptosis on normal keratinocytic cells (Leverkus et al, 2000; Quin et al, 2001), the non-tagged TRAIL showed non apoptotic effect on these cells (Quin et al, 2001). Similar observations have been raised for normal epithelial prostate cells (Nesterov et al, 2002). The problem of a potential hepatotoxicity of TRAIL for normal hepatocytes was recently reinvestigated using in vitro studies on human hepatocytes in culture or in vivo in chimeric mice harbouring human hepatocytes, providing definitive evidence that non-tagged soluble TRAIL is not toxic for normal human hepatocytes (Hao et al, 2004). Normal human hepatocytes do not express TRAIL and possess very low levels of TRAIL-R2 (Hao et al, 2004). The analysis of some structural properties of these different versions of human TRAIL may provide an explanation for their differential in vivo toxicity. In fact, the tagged version of recombinant TRAIL are not optimized for zinc content and due to this limitation they have lower solubility levels and spontaneously aggregate: this TRAIL aggregation could induce TRAIL-R multimerization inducing strong receptor signalling bypassing the high threshold for apoptosis activation existing in normal cells (Kelley and Ashkenazi 2004). In contrast, the non-tagged recombinant human TRAIL is highly stable trimer inducing only the formation of trimeric TRAIL-Rs in normal cells, not sufficient to induce apoptosis of these cells (Kelley and Ashkenazi 2004). In vivo studies in animal models have provided that non-tagged TRAIL/Apo-2L exhibits potent anti-tumor activity and induces little toxic effects in immunodeficient mice xenograft models implanted with several human tumor cell lines (Ashkenazi et al, 2004). However, the in vivo half-life of the recombinant non-tagged TRAIL was very short (< 4minutes), thus suggesting that agonistic anti-TRAIL-R1 or -TRAIL-R2 could have a better pharmacologic impact (Kelley 2001). Initial studies have shown that TRA-8 an agonistic anti-human TRAIL-R2 monoclonal antibody exert in vivo a potent anti-tumor activity against a wide spectrum of human tumors, without

affecting the viability of normal cells, and particularly of hepatocytes (Ichikawa et al, 2001). Agonistic TRAIL-R1 or TRAIL-R2 antibodies may have enhanced therapeutic potential due to a prolonged half-life in vivo compared to TRAIL ligand (Kelley 2001). Another additional advantage of agonistic TRAIL-R1 or TRAIL-R2 antibodies is that they, at variance with TRAIL ligand, do not bind to TRAIL decoy receptors, TRAIL-R3 and TRAIL-R4 often present on the membrane of tumor cells. Two anti-TRAIL-Rs have been developed for clinical use. One of these two antibodies is called HGSETR1 and is a fully human agonistic antibody with high affinity and specificity for TRAIL-R1 (Pulac et al, 2005). This antibody induce cell killing of tumor cell lines through activation of both extrinsic and intrinsic apoptotic pathway. Importantly, HGS-ETR1 was shown to have in vivo a long half-life (7-9 days in mouse) and suppressed the growth of several tumors in xenografts models in athymic mice (Pulac et al, 2005). Finally, EGS-ETR1 potentiated the anti-tumor efficacy of several chemotherapeutic drugs (Pulac et al, 2005). These observations have clearly indicated that HGS-ETR1 has significant potential as a cancer therapeutic agent. The HGS-ETR1 antibody (Human Genome Sciences) was evaluated in Phase I/II clinical trial in patients with advanced solid or haematological tumors, revealing little toxicity (Pulac et al, 2005). A second fully human antibody to TRAIL-R2, KTMTR2, was recently reported (Motoki et al, 2005). This antibody through its binding to the TRAIL R2 induces the death of tumor cells: no crosslinking is required for induction of cell apoptosis (Motoki et al, 2005). Phase I and phase II trials in patients with advanced solid tumors, non-small-cell lung cancer, colon cancer and NHL are in progress. The antibodies are being studied as single agents or in combination with cytotoxic chemotherapy (De Bono et al, 2004, Hotte et al, 2004, Cohen et al, 2004, Tolcher et al, 2004). The use of fusion proteins composed by recombinant human TRAIL fused to a monoclonal antibody against a membrane antigen can be used to induce target antigenrestricted apoptosis. An example of this fusion protein is given by scFv CD7:sTRAIL, composed by the TRAIL genetically linked to an scFv antibody fragment specific for the T-cell surface antigen CD7: this fusion protein induces cytotoxicity of primary acute T lymphoblastic leukemia cells and potentiates the cytotoxic effect of the antitumor drug vincristine (Bremer et al, 2005). Additional examples of these fusion proteins are given by scFv425:sTRAIL (composed by the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL) (Bremer et al, 2005) or by scFvEGP2:sTRAIL fusion protein (composed by the anti-pancarcinomaassociated antigen EGP2 genetically fused to soluble TRAIL) (Bremer et al, 2004).

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Cancer Therapy Vol 4, page 61 cancer cell lines and, through this mechanism, sensitize these cells to the pro-apoptotic effects of TRAIL. Experiments on a battery of renal carcinoma cell lines have shown that Bortezomib may either increase TRAILR1 and/or TRAIL-R2 expression or decrease c-FLIP levels, but in all istances it sensitize these cells to the apoptogenic effects of TRAIL (Sayers and Murphy, 2005). The increased TRAIL-R2 expression induced by proteasome inhibitors is related to a transcriptional mechanism dependent upon an enhanced promoter activity, mediated by the binding of the CHOP transcription factor (Yoshida et al, 2005). Since TRAIL binding to its receptors induces NF-#B activation, that can induce several anti-apoptotic genes, it was suggested that proteasome inhibitors may sensitize the cells to TRAIL-mediated apoptosis by blocking NF-#B activity. However, several studies have shown that NF-#B blocking by proteasome inhibition is not essential for the sensitization of several tumor cell types to TRAIL (Sayers et al, 2003). In some tumor cells, NF-#B inhibition may represent an important event contributing to the sensitization to TRAIL (Luo et al, 2004). Interestingly, the combined addition of proteasome inhibitors and TRAIL resulted in a cytotoxic effect in chemoresistant Bcl-2-overexpressing cells that are otherwise resistant to TRAIL or cytotoxic drugs or proteasome inhibitors alone (Nencioni et al, 2005). The combination of TRAIL and antiblastic drugs was unable to induce the apoptosis of these cells (Nencioni et al, 2005). These observations indicate that proteasome inhibitors plus TRAIL induce mitochondrial dysfunction irrespective of upregulated Bcl-2. The molecular mechanisms through which proteasome inhibitors induce a decrease of c-FLIP levels and upmodulate TRAIL-R1 and TRAIL-R2 are largely unknown. In this context, it is important to note that several components of the apoptotic machinery, including some members of the Bcl-2 family, IAP family proteins, I#B and p53, are ubiquitinated (Zhang et al, 2004). The decrease in c-FLIP observed in cells treated with proteasome inhibitors is surprising in that c-FLIP was reported to be degraded by proteasome in some cells (Sayers et al, 2003). One explanation could be related to the effect of the proteasome inhibitor on cell cycle: cell cycle arrest in S-G2/M phase could result in c-FLIP decrease because during the normal cell cycle peak levels of this protein are observed in G1, with a marked decrease in G2/S phase. In contrast, the increase of c-FLIP levels induced by proteasome inhibitors observed in other tumor cells may be related to a reduced degradation of c-FLIP protein (Ganten et al, 2005). Recent studies carried out in bladder and prostate cancer cells have shown that the cell cycle regulatory protein p21, whose levels are greatly enhanced by Bortezomib+TRAIL, plays a key role in the mechanism through which the proteasome inhibitor allows to bypass TRAIL resistance (Lashinger et al, 2005). The increased p21 levels are required for optimal caspase-8 processing (Lashinger et al, 2005).

IV. Strategies to overcome TRAIL resistance As outlined in the previous chapters, very frequently primary cancer cells are resistant to TRAIL-mediated apoptosis. This observation had stimulated many studies focused to develop strategies to circumvent TRAIL resistance by cancer cells. The philosophy of these various strategies is based on the combination of TRAIL with another drug: the role of the other drug consists in sensitizing tumor cells to the apoptotic effects of TRAIL.

A. TRAIL and proteasome inhibitors The 26S is a multicatalytic enzyme present in the cytoplasm and in the nucleus of virtually all eukaryotic cells, involved in the degradation of proteins targeted by ubiquitin conjugation. The proteasome plays a key role in the control of cell homeostasis in that it regulates the halflife of cellular proteins essential for the life of the cell, such as transcription factors, tumor suppressors, oncogenes and proteins involved in cell cycle control. These observations have suggested that the proteasome may represent an important therapeutic target in cancer. Several proteasome inhibitors have been synthesized and one of them, Bortezomib (known also as VELCADE or PS-341), was approved by the Food and Drug Aministration for cancer treatment (Rajikumar et al, 2005). Bortezomib, a peptide boronate, selectively inhibits the chymotrypsic-like activity of the proteasome at nanomolar concentrations (Rajikumar et al, 2005). In vitro studies have shown that the effect of Bortezomib on cell lines mainly consists on cell cycle inhibition and induction of apoptosis (Rajikumar et al, 2005). Treatment of tumor cells with Bortezomib results in multiple biological effects, including inhibition of cell cycling, inhibition of NF-#B acivity, changes in cell adherence and increased apoptosis. Among these various effects it was initially observed that Bortezomib treatment may considerably increase the sensitivity of myeloma cells to TRAIL (Mitsiades et al, 2001). In the absence of the proteasome inhibitor, myeloma cells are resistant to TRAIL-mediated apoptosis. Subsequent studies have confirmed these initial observations in other tumor cell types and have explored the potential molecular mechanisms responsible for this phenomenon. Thus, Bortezomib was found to sensitize mouse myeloid leukaemia C1498 cells to TRAIL-mediated apoptosis through a mechanism related to a decreased expression on the anti-apoptotic protein c-FLIP, without significant decrease of Bcl-2 anti-apoptotic members or of the various IAP members (Sayers et al, 2003). In contrast, other studies carried out on different tumor cells, like hepatocellular carcinoma cells, have shown that the proteasome inhibitor MG-132 clearly increased c-FLIP levels; in spite the c-FLIP increase, MG-132-pretreated cells became more sensitive to TRAIL-mediated apoptosis (Ganten et al, 2005). Other studies have shown that Bortezomib (Johnson et al, 2003) or MG-132, another proteasome inhibitor, (He et al, 2004) induce a marked increase of TRAIL-R1 and TRAIL-R2 expression in prostate, colon and bladder

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Pasquini et al: Sensitivity and resistance of human cancer cells to TRAIL tumors in addition to hematologic malignancies. Several inhibitors of HDAC, which inhibit tumor growth both in vivo and in vitro have entered clinical trials. HDAC inhibitors exert their anti-tumor effects due to their ability to induce growth arrest, differentiation and apoptosis. Initial studies describing the induction of apoptosis by HDAC inhibitors have suggested that they induce apoptosis via the intrinsic pathway (Insinga et al, 2005). However, many recent observations, carried out in different tumor models, including primary tumor cells, clearly indicate that HDAC inhibitors potentiate deathreceptor induced apoptosis. These observations have been performed in leukemic cell lines and primary leukemic cells (McFarlane et al, 2005; Nebbioso et al, 2005), in chronic B lymphocytic leukaemia cells (Inoue et al, 2004; McFarlane et al, 2005), melanoma cell lines (Facchetti et al, 2004). The molecular mechanisms underlying this synergism between TRAIL and HDAC inhibitors is mainly related to a decreased expression of anti-apoptotic proteins (c-FLIP, XIAP, survivin) and an increased expression of TRAIL-R1 and/or TRAIL-R2 (Rosato et al, 2003; Guo et al, 2004; Inoue et al, 2004; Nakata et al, 2004; McFarlane et al, 2005). Particularly, TRAILresistant glioma cell lines became sensitive to TRAILmediated apoptosis when this cytokine is added together with sodium butyrate, a HDAC inhibitor, that induces a marked downregulation of surviving and XIAP (Kim HS et al, 2005).

B. Triterpenoids enhance the sensitivity of tumor cells to TRAIL Derivatives of naturally occurring triterpenoids have pronounced antiproliferative and anticarcinogenic activities (Kim et al, 2002). Particularly, two compounds of this chemical family, 2-ciano-3,12-dioxooleana-1,9(11)dien-28-oic acid (CDDO) and its imidazole derivative (CDDO-Im) exert a pronounced anti-tumor activity. In vitro studies have shown that these compounds are able to induce differentiation, to inhibit proliferation and to stimulate apoptosis of different types of cancer cells (Place et al, 2003). The apoptogenic effects of these compounds were relatively weak. However, both CDDO and CDDO-Im, the latter one being more potent than the former one, were able to sensitize leukemic cells to the pro-apoptotic effects of TRAIL (Konopleva et al, 2002). This effect seems to be largely related to the induction of a reduced c-FLIP expression (Konopleva et al, 2002). This last effect seems to be due to the stimulation of a proteolytic pathway involved in c-FLIP degradation (Kim et al, 2002). Studies carried out in breast cancer cell lines confirmed the findings obtained in leukemic lines showing that CDDO and CDDO-Im markedly enhanced the sensitivity of these cells to the pro-apoptotic effects of TRAIL, via a mechanism involving both a decrease of cFLIP and an enhanced expression of both TRAIL-R1 and TRAIL-R2 (Hyer et al, 2005). These effects were also observed in vivo in a xenograft model based on the implantation of breast cancer cells to nude mice (Hyer et al, 2005). The addition of CDDO or CDDO-Im to lung cancer cell lines resulted only in a weak pro-apoptotic effect, greatly enhanced by the concomitant addition of TRAIL: the sensitization to TRAIL was related to a selective upmodulation of TRAIL-R2 expression, mediated by JNK, whose activity is clearly stimulated by triterpenoids (Zou et al, 2004). In addition to triterpenoids, also some flavonoids and particularly luteolin, a compound found in many fruits, vegetables and medicinal plants, sensitizes TRAILinduced apoptosis in various human cancer cells, through a mechanism involving XIAP downmodulation mediated via protein kinase C inhibition (Shi et al, 2005).

D. Ionizing radiations, UV radiations and TRAIL Part of the signalling cascade of death receptor signalling is also utilized during irradiation-induced apoptosis. Ionizing radiations were shown to trigger death receptor-independent processing and activation of procaspase-8 that occurs through a caspase-3 dependent mechanism. Several studies indicate that ionizing radiations and TRAIL may cooperate in inducing the killing of tumor cells. Initial studies have shown that the combined administration of ionizing radiations and TRAIL was able to induce the death of lymphoma (Belka et al, 2001), breast cancer (Chinnayan et al, 2000) and renal cell carcinoma (Ramp et al, 2003). In animal models of nonsmall cell lung cancer the combination of TRAIL and radiotherapy significantly increased apoptosis in vivo, inhibited tumor growth, and significantly prolonged survival in mice bearing human tumors (Zhang et al, 2005). The analysis of the possible mechanisms underlying this synergism showed that: (i) the most active schedule in eliciting tumor cell killing consisted in the sequential administration first of ionizing radiations and then of TRAIL (Marini et al, 2005); (ii) pre-irradiation did not sensitize normal tissues to TRAIL; (iii) in the majority of tumor cell lines, ionizing radiations induced a clear upmodulation of TRAIL-R2 expression; (iv) the sequential treatment with ionizing radiations and then with TRAIL inhibits tumor growth in vivo and induces apoptosis of breast cancer xenografts in nude mice (Shankar et al, 2004). An intact Bax activity is strictly required to mediate

C. Histone deacetylase inhibitors act as modulators of the sensitivity of tumor cells to TRAIL Histone acetyltransferases and histone deacetylases (HDAC) catalyze the acetylation and deacetylation of lysine residues in the core nucleosomal histone tails, respectively, thus regulating the affinity of the nonhistone proteins transcriptional complexes with DNA and then controls the rate of transcription (Dockmanovic and Marks, 2005). Recently, HDACs have been shown to be involved in leukemogenesis for their capacity to complex with a variety of oncoproteins found in leukaemia and, through this mechanism, to aberrantly suppress the expression of genes required for cell differentiation and growth control. Furthermore, altered histone acetyltransferase or HDAC has been observed in many

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Cancer Therapy Vol 4, page 63 synergism between ionizing radiations and TRAIL (Wendt et al, 2005). The ensemble of these observations clearly indicate that in several neoplasias the sequential treatment with irradiation followed by TRAIL provides an approach to enhance therapeutic potential of TRAIL. Interestingly, a possible role for TRAIL and its receptors in the mechanism of cytotoxicity mediated by ionizing radiations is suggested by the phenotypic analysis of TRAIL-R2 knockout mice (Finnberg et al, 2005). These animals, in fact, exhibit reduced levels of apoptosis when exposed to ionizing radiations, compared to the corresponding tissues of normal animals (Finnberg et al, 2005). Studies on TRAIL-resistant melanoma cells showed that these tumor cells acquire the sensitivity to this death ligand after exposure to ultraviolet B radiations (UVB), through a molecular mechanism mainly involving c-FLIP downregulation (Zeise et al, 2004).

E. Interferon-! and other enhancing caspase-8 expression

mitochondrial pathway to drug-induced apoptosis was matter of controversy and there is evidence that both ways, extrinsic and intrinsic, are involved (Debatin and Krammer, 2004). Studies on several types of cancer cell lines and primary tumors cells have shown that treatment with anticancer drugs determines an increase of FasL expression, which acts as a triggering stimulus for the death receptors pathway in an autocrine or paracrine manner by binding to its receptor Fas (reviewed in Debatin and Krammer 2004). Many studies have clearly shown that several anticancer drugs, including etoposide, CPT-11, doxorubicin, 5-FU, carboplatin, topotecan, taxol and fludarabine greatly augment TRAIL-induced apoptosis of epithelial cancer cells (Gliniak and Le 1999; Kean et al, 1999; Kim et al, 2000; Nagane et al, 2000; Komdeur et al, 2004; Tomek et al, 2004; Schmeltz et al, 2004), acute myeloid leukaemia (Johnston et al, 2003) and chronic lymphocytic leukaemia (Wen et al, 2000). These findings were confirmed also in vivo animal models using xenografts of primary tumor cells (Hylander et al, 2005). In this context, particularly interesting are the results obtained in non-small-lung cancer cells that in xenografts models are scarcely sensitive to anticancer drugs (taxol and carboplatin) or to TRAIL. However, the combined administration of TRAIL and taxol or carboplatin resulted in a curative effect in the large proportion of tumorbearing mice (Jim et al, 2004). Importantly, this treatment was associated with low toxicity. Interestingly, similar observations have been made for prostate cancer xenograft models using a treatment strategy based on the sequential administration first of cytotoxic chemotherapeutic drugs and then of TRAIL (Shamker et al, 2005). Finally, the administration of irinotecan and TRAIL resulted in the elimination of hepatic metastases of colon cancer cells via a p53-indipendent mechanisms (Ravi et al, 2004). Because chemotherapeutic agents and TRAIL use different BH3-domain-containing proteins to activate BAX and BAK, the simultaneous delivery of both death signals may converge to promote apoptosis of tumor cells. The mechanisms of synergic effect between TRAIL and the chemotherapeutic drugs consists either in the upregulation of pro-apoptotic molecules or the downregulation of anti-apoptotic molecules. Among the effects on the upregulation of proapoptotic molecules a key event is represented by the upmodulation of TRAIL-R1 and TRAIL-R2 exerted by many anticancer drugs (Wen et al, 2000; Jhonston et al, 2003; Komdeur et al, 2004; Schmelz et al, 2004; Tomek et al, 2004). However, the acquisition of an increased TRAIL sensitivity by cancer cells seems to be related to multiple mechanism, involving both TRAIL-R1/ R2 upmodulation and a decrease expression of anti-apoptotic molecules. Interestingly, recent studies have shown that also anticancer drugs, such like cyclooxygenase 2 inhibitors, that do not act like the cytotoxic anticancer drugs, are able to induce apoptosis of tumor cells through upmodulation of TRAIL-R1 and TRAIL-R2 and greatly potentiate the pro-apoptotic effect of TRAIL of non-small-lung cancer cells (Liu et al, 2005).

agents

As mentioned in Section II, several types of cancers, including some brain tumors, colon cancer, retinoblastoma and Ewing sarcoma, display a reduced sensitivity to TRAIL due to a decreased or absent expression of caspase-8. Given this defect in the apoptotic response, attempts have been made in these tumors to reconstitute a sensitivity to the pro-apoptotic effects of TRAIL by pretreatment or co-treatment of tumor cells with agents that enhance caspase-8 expression, such as interferon-! (IFN!). The addition of IFN-! together with TRAIL is based on previous studies in mice showing that TRAIL plays a key role in IFN-!-dependent tumor surveillance (Takeda et al, 2002). Furthermore, IFN-! is known to enhance caspase-8 (Fulda et al, 2002), to facilitate DISC-mediated caspase-8 processing (Siegmund et al, 2005) and activate Sta-1 (Fulda et al, 2002) and IRF-1 (Park et al, 2004) and, through these mechanisms it greatly enhances the sensitivity of tumor cells to TRAIL, including those tumors with low caspase-8 expression due to promoter hypermethylation. Thus, the simultaneous treatment with IFN-! and TRAIL resulted in a consistent pro-apoptotic effect in Ewingâ&#x20AC;&#x2122;s sarcoma (Kotny et al, 2001; Marchant et al, 2004) hepatoma (Shin et al, 2001) colon carcinoma (Langaas et al, 2001; Van Gleen et al, 2004) and neuroblastoma (Yang et al, 2003) cell lines. Interestingly, the co-administration of IFN-! and TRAIL was efficacious only in a part of TRAIL-resistant neuroblastoma cell lines; in the remaining lines a significant level of apoptosis was achieved only when chemotherapic drugs were added to TRAIL and IFN-! (Yang et al, 2003). IFN-! stimulates TRAIL-induced apoptosis also in thyroid cancer cells, but through a peculiar mechanism involving BAK upmodulation (Wang et al, 2004).

F. Chemotherapeutic agents and TRAIL Killing of tumor cells by cytotoxic therapies such as chemotherapy, is predominantly mediated by triggering apoptosis. The relative contribution of the receptor and 63

Pasquini et al: Sensitivity and resistance of human cancer cells to TRAIL Arai T, Akiyama Y, Okabe S, Saito K, Iwai T, Yausa Y (1998) Genomic organization and mutataion analyses of the DR5/TRAIL Receptor 2 gene in colorectal carcinomas. Cancer Lett 33, 197-204. Asakuma J, Sumitomo M, Asano T, Asano T, Hayakawa M (2003) Selective Akt inactivation and tumor necrosis factorrelated apoptosis-inducing ligand sensitization of renal cancer cells by low concentrations of paclitaxel Canc Res 63, 1365-1370. Ashkenazi A (2002) Targeting death and decoy receptors of the tumor-necrosis factor superfamily. Nature Rev Cancer 2, 420-430. Ashkenazi A, Pai RC, Fong S (1999) Safety and antitumor activity of recombinant soluble Apo2 Ligand. J Clin Invest 104, 155-162. Ashley DM, Riffkin CD, Muscat AM, Knight M, Kaye A, Novak V, Hawkins CJ (2005) Caspase 8 is absent or low in many ex vivo gliomas. Cancer, in press. Baader E, Toloczko A, Fuchs U, Schmid I, Beltinger C, Ehrhardt H, Debetin KM, Jeremias I (2005) Tumor Necrosis FactorRelated Apoptosis-Inducing Ligand-Mediated Proliferation of Tumor Cells with Receptor-Proximal Apoptosis Defects. Cancer Res 65, 7888-95. Bai J, Sui J, Demirijian A, Vollmer CMJr., Marasco W, Callery MP (2005) Predominant Bcl-XL knockdown disables antiapoptotic mechanisms, Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-based triple chemotherapy overcomes chemoresistance in pancreatic cancer cells in vitro. Cancer Res 65(6), 2344-2352. Belka C, Schmid B, Marini P, Durand E, Rudner J, Faltin H, Bamberg M, Schultze-Osthoff K, Budach W (2001) Sensitization of resistant lymphoma cells to irradiationinduced apoptosis by the death ligand TRAIL. Oncogene 20, 2190-2196. Biswas DK, Shi Q, Baily S (2004) NF- B activation in human breast cancer specimens and its role in cell proliferation and apoptosis. Proc Natl Acad Sci USA 101, 10137-10142. Bockbrader KM, Tan M, Sun Y (2005) A small molecule Smacmimic compound induces apoptosis and sensitizes TRAILand etoposide-induced apoptosis in breast cancer cells. Oncogene, in press. Bodmer JL, Meier P, Tschopp J and Schneider P (2000) Cysteine 230 is essential fort the structure and activity of the cytotoxic ligand TRAIL. J Biol Chem 275, 20632-29637. Bremer E, Kuijlen J, Samploniud D, Walczak H, De Leij L, Helfrich W (2004) Target cell-restricted and-enhanced apoptosis induction by a scFv:sTRAIL fusion protein with specificity for the pancarcinoma associated antigen EGP2. Int J Cancer 109, 281-290. Bremer E, Samplonius DF, Peipp M, Van Genne L, Kroesen BJ, Fey GH, Gramatzki M, De Leig L, Helfrich W (2005) Target cell-restricted apoptosis induction of acute leukemic T cells by a recombinant tumor necrosis factor-related apoptosisinducing ligand fusion protein with specificity for human CD7. Cancer Res 65, 3380-3388. Bremer E, Samplonius DF, Van Genne L, Dijkstra MH, Kroesen BJ, Leij L, Helfrich W (2005) Simultaneous inhibition of epidermal growth factor receptor (EGFR) signaling and enhanced activation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated apoptosis induction by an scFv:sTRAIL fusion protein with specificity for human EGFR. J Biol Chem 280, 1002510033. Broker LE, Kruyt FA, Giaccone G. (2005) Cell death independent of caspases: a review. Clin Cancer Res 11, 3155-3162. Brooks AD, Saysers TJ (2005) Reduction of the antiapoptotic protein c-FLIP enhances the susceptibility of human renal

V. Conclusions The induction of apoptosis in response to cell damage generally requires the function of the tumor suppressor p53, which engages the intrinsic signalling pathway of apoptosis. Conventional anticancer treatment likely selects for tumor cells displaying inactivated p53, however, resulting in chemoresistance. Since death receptor activation can induce cell death of malignant cells through a mechanism independent of p53, targeting the TRAIL receptors with TRAIL-targeting agents (either recombinant TRAIL, or fully human agonistic monoclonal antibodies anti-TRAIL-R1 or anti-TRAIL-R2) is a rational therapeutic strategy to treat cancer. TRAIL may represent an ideal therapeutic agent for cancer treatment because it has been shown to be a potent apoptosis inducer in a wide variety of cancer and transformed cell lines without damaging most normal cells. However, the potential application of TRAIL in cancer therapy is limited since the majority of primary cancer cells are found to be resistant to TRAIL-induced apoptosis. The resitance may be due to mutations of TRAIL-R1 or TRAIL-R2, or peferential expression of the TRAIL decoy receptors, TRAIL-R3 and/or TRAIL-R4, low expression of pro-apoptotic molecules involved in TRAIL signalling (caspase-8 or FADD) or high expression of anti-apoptotic molecules FLIP, IAP, Survivin, Bcl-2. Thus, combination of TRAIL with other agents has been a promising strategy to potentiate the citotoxicity of TRAIL and its therapeutic applications. In this context, particularly promising seems to be the use of newly developped agonistic monoclonal antibodies anti-TRAILR1 or anti-TRAIL-R2, able to induce apoptisis, like recombinant TRAIL, but exhibiting an in vivo much longer half-life than the death ligand. The design of specific phase II and III clinical studies aiming to evaluate whether or nor either agonistic monoclonal antibodies to TRAIL-R1 and TRAIL-R2 or recombinant TRAIL and other pharmaceutical agents targeting TRAIL may represent an important step in the treatment of any specific malignancy and will represent one of the main objectives in the future developments in this area. The identification of the ideal tumor types to select for early proof of activity will represent one immediate goal. In this context, particularly useful will be the observations obtained in vitro on the corresponding primary tumor cells to select the tumor to be treated and the pharmaceutic association to be made. However, one of the major limitations could derive from the inadequacy of of these preclinical models to really evaluate the overall complexity of genetic alterations occurring in human tumors.

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Cancer Therapy Vol 4, page 71 regulation of death receptor 5 contributes to induction of apoptosis by the novel synthetic triterpenoid methyl-2-cyano3,12-dioxooleana-1, 9-Dien-28-oate in human lung cancer cells. Cancer Res 64, 7570-7578.

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Cancer Therapy Vol 4, page 73 Cancer Therapy Vol 4, 73-80, 2006

Selenium and prostate cancer: biological pathways and biochemical nuances Review Article

Vasundara Venkateswaran Division of Urology, Sunnybrook & Womenâ&#x20AC;&#x2122;s College Health Sciences Centre, Toronto, Ontario, Canada

__________________________________________________________________________________ *Correspondence: Dr. Vasundara Venkateswaran, Assistant Professor, Division of Urology, S-118B, Sunnybrook and Womenâ&#x20AC;&#x2122;s College Health Sciences Centre, 2075 Bayview Avenue, Toronto, Ontario M4N3M5, Canada; Tel: 416-480-6100 x 3127; Fax: 416-4805737; E-mail: vasundara.venkateswaran@sw.ca Key words: Selenium, Prostate Cancer, Prevention, Mechanisms Abbreviations: Human selenium binding protein, (hsp56); Methylseleninic acid, (MSA); Nuclear Factor kappa Beta, (NF!B); Prostate cancer, (PCa); reactive oxygen species, (ROS); Glutathione, (GSH); Glutathione Oxidase (GSSG); Androgen receptor (AR); Prostate Specific Antigen (PSA) Received: 18 January 2006; Accepted: 7 March 2006; electronically published: March 2006

Summary Prostate cancer is a complex multifactorial disease involving both genetic and environmental factors. Although epidemiology suggest an association between dietary constituents and the likelihood of developing PCa, a direct causal relationship between a specific dietary constituent and PCa has not yet been proven. As the etiology of PCa remains unknown, it is not feasible to develop primary intervention strategies to remove the causative agents from the environment. However, secondary intervention strategies with selenium and other agents represent a viable option to reduce the morbidity and mortality of PCa. Selenium is one of the most promising chemopreventive agents currently used in prostate cancer prevention. It is an essential trace element involved in several key metabolic activities via selenoproteins, enzymes that are essential to protect against oxidative damage and to regulate immune functions. Although the need for selenium in human and animals nutrition is well recognized, the questions concerning the proper form for supplemental use is being debatable. This article aims to review the diverse aspects of selenium biology, biochemistry, bioavailability, metabolism and toxicity. The in vitro and in vivo efficacy of selenium, mechanism of anti-carcinogenic action and synergy with micronutrients including its possible function in chemoprevention are also discussed.

I. Prostate cancer: selenium and cancer risk

There are several criteria for developing a successful chemoprevention strategy. These include a rational mechanism of action, acceptable level of side effects, demonstrable activity in PCa models and humans, availability of biomarkers and surrogate endpoints to monitor drug activity and efficacy (Ansari et al, 2002). Selenium was recognized about 4 decades ago as an important trace element and is one of the most promising agents being evaluated for PCa prevention. Selenium occurs in organic and inorganic forms (Klein, 2004). The organic forms are found predominantly in grains, fish, meat, poultry, eggs and dietary products. Historically, selenium intake was dependent on the soil selenium content within a region, this effect has been somewhat negated by modern food distribution systems. Japan has the lowest incidence of PCa and their average selenium consumption is 130 Âľg/day (National Academy of Sciences Recommended Dietaty Allowances, 1998)

Epidemiology,

Prostate cancer (PCa) is a complex disease, with a multifactorial etiology involving both genetic and environmental factors. Although there is an association between dietary constituents and the likelihood of developing PCa, a direct causal relationship has not yet been proven. Many antioxidants combine with target tissue and protect the body against harmful affects of free oxygen radicals. Ideal chemopreventive agents should be nontoxic, efficacious, readily available and inexpensive. Epidemiological studies have identified a number of micronutrients, including selenium, as effective PCa prevention agents. If chemoprevention can delay the clinical course of PCa by 5-10 years, there would be a substantial decrease in burden of the disease.

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Venkateswaran: Selenium and Prostate cancer There are more than 100 reported studies and more than 2 dozen animal models on the use of selenium. Two thirds of these studies have shown a clear chemopreventive / antitumorigenic effect of selenium in several organs including the mammary gland, liver, skin, pancreas, esophagus, colon and prostate (Combs and Gray, 1998; Ip, 1998). Half of these reports demonstrate that there is more than a 50% reduction in tumor growth with selenium supplementation along with a selenium-replete diet (Combs, 2001). Sodium selenite is more effective in preventing chemically induced tumors, although tissue levels of selenium are higher with selenomethionine (Ip and Hayes, 1989). These reports have heightened interest in additional human selenium chemoprevention studies and have intensified the search for mechanism involved in suppressing tumorigenesis. This review will examine the evidence linking selenium to the prevention of PCa in addition to providing a perspective on the putative in vitro and in vivo mechanisms of chemoprevention.

lesser extent in the kidney and muscle. Small amounts exist in plasma and other organs (Drasch et al, 2000; Patrick, 2004). Selenium is highly absorbable with no homeostatic control mechanism for its absorption. Absorption from food is efficient and the average dietary intake is between 20-300 µg/day. Selenium deficiency is correlated with reduced serum selenium concentrations of 85-90 µg/L (Levander and Morris, 1984; Yang et al, 1984; Patrick, 2004).

C. Metabolism of selenium The metabolism of selenium is dynamic (Ganther, 1999, 2001). A wide array of metabolic products is generated. Selenoprotein can be produced in the body from various selenium sources. Selenomethionine competes with methionine for absorption in the gut and is integrated and stored in body proteins that contain methionine (Ganther, 1971; Hsieh and Ganther, 1975; Bjornstedt et al, 1992; Veres et al, 1994). It may also get converted to selenocysteine and degraded to hydrogen selenide. Selenite is also metabolized to hydrogen selenide complexed with glutathione. Hydrogen selenide is a key metabolite that acts as a precursor for selenoprotein synthesis and is the excreted form of selenium (Behne and Kyriakopoulos, 2001). Hydrogen selenide is methylated and later excreted in the urine and breath resulting in the characteristic “halitosis” or “garlic breath” (the odor of dimethylselenide excreted through the lungs) associated with selenium toxicity (Behne and Kyriakopoulos, 2001). Methylation of selenium produces less toxic selenium compounds. The monomethylated forms of selenium metabolites have powerful effects on carcinogenesis, while lacking some of the toxic effects produced by other forms such as the inorganic selenite (Ganther, 1971; Ip, 1998). Se-methylselenocysteine, a stable methylated selenium compounds thus serves as a reservoir that provides a steady stream of monomethylated selenium so that a critical level is maintained and growth of cells are inhibited (Ganther, 1999).

II. Selenium Biology A. Forms and dosage of supplemental selenium Selenium is an essential constituent of extracellular and cellular metalloenzymes, glutathione peroxidase, thyroidal and extrathyroidal deiodinase, thioredoxin reductase, and other selenoproteins (Burk, 1986; Lane et al, 1989; Combs, 1999). Selenium is active in a variety of selenoproteins, playing a preventive role in cancer development. The antitumorigenic effect of selenium has always been associated with supranutritional levels of this constituent. In experimental animals, the suggested levels have been > 1mg / kg diet or 0.7 mg / liter drinking water. These doses are 10 times greater than those required to prevent clinical signs of deficiency. Selenoproteins are expressed maximally at dietary levels (0.5 mg/kg in animals). It is unlikely that the anticarcinogenic effects of supranutritional levels of selenium are related to these proteins. Deprivation of selenium has been thought to contribute to carcinogenesis by limiting the expression of one or more selenoproteins that alter the redox system of cells (Combs, 1999). Selenium occurs naturally as selenomethionine, Semethyl-selenomethionine, selenocysteine and selenocystine (Combs and Combs, 1984). The majority of animal model have employed the oxidized inorganic salt. Data from these models cannot be confidently extrapolated to the organo-selenium compounds which are metabolized differently. The beneficial effect of selenium is observed when administered in its naturally occurring form as seleno methionine. Higher animals have no efficient mechanism for methionine synthesis and are unable to synthesize selenomethionine (Combs, 1999). Accordingly, only seleno-cysteine and not seleno-methionine is detected in rats supplemented with selenium as selenite.

D. Selenium toxicity The limit for selenium based on projected lifetime exposure is 5µg/kg body weight/day (US Environmental Protection Agency). The low adverse effect level LOAEL, in humans, has been calculated at 1540±653 µg/day. The no adverse effect level NOAEL, has been calculated at 819±126 µg/day (Whanger et al, 1996). There are reports suggesting that selenium toxicity seen in China requires a daily intake of 2 mg/day (Drasch et al, 2000). There has been no evidence of selenium toxicity observed in the Nutritional Prevention of Cancer Trial at doses of 400 µg of selenium daily. There was no change in hair, nail or skin or garlic breath associated with selenosis. One other selenium trial wherein prostate cancer patients (ascertained by biopsy) were randomized to 1,600 or 3,200 µg/day of selenized yeast for 12 months did not report any selenium-related toxicity, although some side effects were observed (Whanger et al, 1996; Reid et al, 2004; Marshall, 2001).

B. Selenium bioavailability Dietary selenium intake depends on soil selenium levels and the origin and types of food that are consumed. A major portion of selenium is stored in the liver, and to a

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Cancer Therapy Vol 4, page 75 oxidative stress. Alterations in intracellular redox state with modification of cellular antioxidants and antioxidant enzymes may inhibit the therapeutic effectiveness of selenium in PCa therapy (Zhong and Oberley, 2001). Selenite was shown to alter intracellular redox status towards an oxidative state by decreasing the ratio of GSHGSSG. Altering the redox environment of prostate cancer cells with selenite was associated with increased apoptotic potential, sensitizing cells to radiation-induced killing (Husbeck et al, 2005). In a similar study higher manganese superoxide dismutase was seen to play an important role in eliminating superoxide radicals produced as a result of selenite metabolism and contribute to tumor-selective killing by selenite in prostate cancer (Husbeck et al, 2006).

III. Selenium and prostate cancer A. Models in defining chemoprevention agents

efficacy

of

Animal models are central in testing the efficacy of chemopreventive agents. The most feasible, realistic, and potentially effective target for PCa chemoprevention is the progression from PIN to overt cancer. To date more than 100 publications have demonstrated the chemopreventive potential of selenium. These encompass murine studies including spontaneous and induced tumors. Lady transgenics (12T-10) are particularly attractive in this setting, as these animals develop progressive PCa from low-grade intraepithelial neoplasia similar to of PCa, that mimics the variable phenotype of human PCa. The in vivo effects of selenium have been studied in patients with PCa and BPH. Mean plasma selenium levels have been shown to be lower in PCa patients relative to controls and men with BPH. These findings may reflect chronically lower selenium levels in PCa patients or possibly that disease status caused depletion of serum levels (Criqui et al, 1991; Hardell et al, 1995). In placebocontrolled trials with low soil selenium content, the incidence of PCa was significantly lower in the group that received selenium supplementation (Clark et al, 1996, 1998; Fleet, 1997). In addition, Yoshizawa et al, (1998) found that increased selenium levels from individuals who later developed PCa, were associated with a reduced risk of advanced disease. In an established orthotopic prostate tumor model, male nude mice were fed a selenium replete diet supplemented with different forms of selenium (sodium selenate, selenomethionine, methylselenocysteine and selenized yeast) at different concentrations. Results revealed that inorganic selenium (sodium selenate significantly retarded the growth of primary prostatic tumors (Corcoran et al, 2004).

2. Altered carcinogen metabolism Studies on carcinogen metabolism have indicated that supranutritional selenium supplementation can affect carcinogen metabolism, by reducing the initiation of carcinogenesis. Another group has found that dietary levels of selenium (2mg/kg as selenite) reduced the formation of DNA adducts (Chen et al, 1982).

3. Enhanced immune monitoring Certain immune functions can be affected by nutritional intake of selenium. Selenium deprivation impairs the development of both B- and T-cell dependent immune responses in animals (Marsh et al, 1986). Certain cytotoxic activities of natural killer cells and polymorphonuclear cells can be impaired by selenium deprivation. Supranutritional selenium intake can enhance immune surveillance of cancerous cells. Selenium has the capability of stimulating cytotoxic activities of NK cells, lymphocytes and lymphokine-activated killer cells (Ip and White, 1987; Lane et al, 1989; Ryan-Harshman and Aldoori, 2005).

4. Molecular basis

B. Mechanism of anti-carcinogenic action of selenium

i. Cell growth Epidemiological and clinical data suggest that selenium may prevent PCa, but the biological effect of selenium on normal or malignant prostate cells are not known. Recently, it has been shown that selenium induces retardation of DNA synthesis in primary prostate cells (Morris et al, 2005). Both sodium selenite and selenomethionine (0-500 ÂľM) inhibited the growth of prostate cancer cells (LNCaP, PC3, DU145) in a dose dependent manner compared to prostate stromal, epithelial or smooth muscle cells (Menter et al, 2000). The strongest effects were observed on the androgen dependent LNCaP cells; these were more sensitive to growth suppression with selenite than with selenomethionine. Selenium in the form of MSA has been shown to significantly down-regulate the expression of prostatespecific antigen transcript and protein in prostate cancer cells. Selenium suppressed the binding of AR to the androgen responsive element site. It has been implicated that that selenium intervention aimed at complementing the amplitude of androgen signaling could be used in controlling the morbidity of the disease (Dong et al, 2004). In a related study methylseleninic acid specifically and

The inhibitory effects of selenium documented prior to 1985 were in animals that received supplemental dietary selenium. Studies on the inhibitory effect of selenium in vitro were first carried out in the human PCa cell line DU145. Data revealed that concentrations of between 10-12 M and 10-8M may have a slight stimulatory effect on growth of DU145 cells but effect gradually declines to 90% of control at 10-7 M. Beyond this there is a marked inhibition, reaching an ID50 between 1x10-6M and 2x106 M, 4% at 10-5 M, and complete cell death occurs at 10-3M (Webber et al, 1985).

1. Antioxidant protection - oxidative stress Mutagenic oxidative stress is thought to be a major factor in carcinogenesis, as DNA bases are susceptible to electrophilic attack by reactive oxygen species (ROS), and if not corrected resulted in the expression of a malignant phenotype (Combs, 1999). The hypothesis is that antioxidants scavenge free radicals and thus act as anticarcinogens. Anticancer effects of sodium selenite, are mediated via a redox mechanism involving induction of 75

Venkateswaran: Selenium and Prostate cancer rapidly inhibited PSA expression through two mechanisms: inducing PSA protein degradation and suppressing androgen-stimulated PSA transcription. Both these studies implicate important mechanistic implications for prostate specific cancer chemoprevention of selenium (Cho et al, 2004)

iv. Gene expression Studies of the genetic events associated with selenium-induced growth arrest in human prostate cancer have been carried out by gene array (Schlicht et al, 2004; El Bayoumy and Sinha, 2005; Zhao et al, 2004). Methylseleninic acid induced cells were subjected to gene profiling. One of the studies revealed a set of genes (2500 out of 12000) that may have been targets of MSA in impeding cell cycle progression (Nelson et al, 1996; Dong et al, 2002, 2003; Zhao et al, 2004). Each of these has been related to signaling pathways that might mediate the outcome of cell cycle blockade by selenium. One other study showed that MSA modulated expression of many androgen-regulated genes in human prostate cancer cells in vitro (Zhao et al, 2004; Dong et al, 2005). A small set of well-characterized androgen-regulated genes, including those with androgen response regulatory elements show expression changes that are reciprocal to those induced by androgens. However, these have not been implicated as direct targets of androgen signaling pathways. It has been clearly shown that selenium affects a multitude of targets, resulting in amplification of the response. The diversity of this response also makes it difficult for transformed cells to escape the inhibitory effect of selenium (Nelson et al, 1996; Dong et al, 2002, 2003).

ii. Cell cycle Different chemical forms of selenium have varying effects on the cell cycle (Sinha et al, 1996; Sinha and Medina, 1997; Redman et al, 1998; Menter et al, 2000; Dong et al, 2003). The effects are also cell type dependent. Selenomethionine treated normal prostate cells did not exhibit the same proportion of sub-G0-G1 subpopulations as did the prostate carcinoma cells. Androgen dependent LNCaP cells exhibited a higher sub-G0-G1 cell fraction than PC3 or DU145. We have demonstrated that selenomethionine induced cell cycle arrest with accumulation in S-phase in the androgen dependent LNCaP cells but had no effect on androgen independent PC3 cells. Transfection of a functional androgen receptor into PC-3 cells restored selenium sensitivity, demonstrating that the effect was partly receptor mediated (Venkateswaran et al, 2002). Selenite treatment has been demonstrated to result in high levels of superoxide production and a sequential increase in levels of total and phosphorylated p53, and p21 (Zhao et al, 2006). These results are suggestive of the fact that the action of selenite is through the production of superoxide to activate p53, thereby inducing mitochondrial translocation of p53.

5. Nuclear Factor kappa Beta (NF!B) The transcription factor NF!B is a key antiapoptotic factor in mammalian cells (Duffey et al, 1999; Wang et al, 1999; Huang et al, 2001; Gasparian et al, 2002a, b). This has been shown to be suppressed by selenium. NF!B complex is a homo-or heterodimer composed of proteins from the NF!B/Rel family. In non-stimulated cells NF!B resides in the cytoplasm in a complex with the inhibitor protein, together they are named I!B. Selenium inhibition of NF!B activation during early stages of tumorigenesis is a possible mechanism of PCa prevention (Berges et al, 1995). NF!B has been shown to play a role in protecting the cells against diverse apoptotic stimuli including chemo-and radiotherapeutic treatments through the activation of antiapoptotic gene program in cells (Barkett and Gilmore, 1999). There is also evidence that NF!B activation induces cyclin D1, a cyclin that is expressed early in the cell cycle and is crucial to the commitment to DNA synthesis.

iii. Cell death/Apoptosis Methylseleninic acid (MSA) induced apoptosis is accompanied by activation of multiple caspases, mitochondrial release of cytochrome C, PARP cleavage and DNA fragmentation (Jiang et al, 2001, 2002; Hu et al, 2005). These effects are seen only in detached cells, indicating that MSA-induced cell detachment is a prerequisite for caspase activation and the execution of apoptosis. Selenite induced apoptosis is associated with the phosphorylation of c-jun and p38 (Jiang et al, 2001, 2002). Selenite treatment has been shown to cause significant increase in p53 phosphorylation and was an important step that occurred several hours before caspase activation and PARP cleavage (Jiang et al, 2004). Yamaguchi et al, (2005) have recently demonstrated that selenium-based dietary compounds may help to overcome resistance to TRAIL-mediated apoptosis in PCa cells by activating both the extrinsic and intrinsic pathways. Treatment of prostate cancer cells with MSA have provided strong evidence to support an important role of endoplasmic reticulum (ER) stress response in mediating the anticancer effect of selenium (Wu et al, 2005). MSA has been shown to decrease Akt phosphorylation at Thr308 and Ser473, suggesting that selenium-mediated dephosphorylation of Akt was likely to be an additional mechanism in regulating the status of phospho-Akt (Wu et al, 2006).

6. Human selenium binding protein (hsp56) Human selenium binding protein hsp56, is the human homologue of a rodent protein implicated in chemoresistance. This is highly expressed in the androgensensitive LNCaP cells but not in the androgen-insensitive PC3 cells (Yang and Sytkowski, 1998). The expression of this protein is reversibly down-regulated by androgens in vitro. Recently, we have reported that treatment of LNCaP cells with selenomethionine caused G1 arrest with 80% reduction in the S phase, with no effect on PC3. Selenium sensitivity was restored by the presence of a functional androgen receptor. Together, this suggests that selenium modulation of prostate cancer cell growth is mediated by the androgen receptor as well as by hsp56.

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Cancer Therapy Vol 4, page 77 Behne D and Kyriakopoulos A (2001) Mammalian seleniumcontaining proteins. Annu Rev Nutr 21, 453-473. Berges RR, Vukanovic J, Epstein JI, CarMichel M, Cisek L, Johnson DE, Veltri RW, Walsh PC, Isaacs JT (1995) Implication of cell kinetic changes during the progression of human prostatic cancer. Clin Cancer Res 1, 473-480. Bjornstedt M, Kumar S and Holmgren A (1992) Selenodiglutathione is a highly efficient oxidant of reduced thioredoxin and a substrate for mammalian thioredoxin reductase. J Biol Chem 267, 8030-8034. Burk RF (1986) Selenium and cancer: meaning of serum selenium levels. Nutr 116, 1584-1586. Chen J, Goetchius MP, Combs GF, Jr and Campbell TC (1982) Effects of dietary selenium and vitamin E on covalent binding of aflatoxin to chick liver cell macromolecules. J Nutr 112, 350-355. Cho SD, Jiang C, Malewicz B, Dong Y, Young CYF, Kang K-S, Lee Y-S, Ip Cand Lu J (2004) Methyl selenium metabolites decrease prostate-specific antigen expression by inducing protein degradation and suppressing androgen-stimulated transcription. Mol Cancer Ther 3, 605-611. Clark LC, Combs GF Jr, Turnbull BW, Slate EH, Chalker DK, Chow J, Davis LS, Glover RA, Graham GF, Gross EG, Krongrad A, Lesher JL Jr, Park HK, Sanders BB Jr, Smith CL, Taylor JR (1996) A randomized controlled trial. Nutritional Prevention of Cancer Study Group. JAMA 276, 1957-1963. Clark LC, Dalkin B, Krongrad A, Combs GF Jr, Turnbull BW, Slate EH, Witherington R, Herlong JH, Janosko E, Carpenter D, Borosso C, Falk S, Rounder J (1998) Decreased incidence of prostate cancer with selenium supplementation: results of a double-blind cancer prevention trial. Br J Urol 81, 730734. Combs GF Jr (1999) Chemopreventive mechanisms of selenium. Klin 94 Suppl 3, 18-24. Combs GF Jr (2001) Impact of selenium and cancer-prevention findings on the nutrition-health paradigm. Cancer 40, 6-11. Combs GF Jr (2004) Status of selenium in prostate cancer prevention. Br J Cancer 91, 195-199. Combs GF Jr and Combs SB (1984) The nutritional biochemistry of selenium. Annu Rev Nutr 4, 257-280. Combs GF Jr and Gray WP (1998) Chemopreventive agents: selenium. Pharmacol Ther 79, 179-192. Corcoran NM, Najdovska M and Costello AJ (2004) Inorganic selenium retards progression of experimental hormone refractory prostate cancer. J Urol 171, 907-910. Criqui MH, Bangdiwala S, Goodman DS, Blaner WS, Morris JS, Kritchevsky S, Lippel K, Mebane I, Tyroler HA (1991) Associations with cancer mortality in a population-based prospective case-control study. Ann Epidemiol 1, 385-393. Dong Y, Ganther HE, Stewart C and Ip C (2002) Identification of molecular targets associated with selenium-induced growth inhibition in human breast cells using cDNA microarrays. Cancer Res 62, 708-714. Dong Y, Zhang H, Hawthorn L, Ganther HE and Ip C (2003) Delineation of the molecular basis for selenium-induced growth arrest in human prostate cancer cells by oligonucleotide array. J Cancer Res 63, 52-59. Dong Y, Lee SO, Zhang H, Marshall JR, Gao, AC and Ip C (2004) Prostate specific antigen expression is down-regulated by selenium through disruption of androgen receptor signaling. Cancer Res 64, 19-22. Dong Y, Zhang H, Gao AC, Marshall JR and Ip C (2005) Androgen receptor signaling intensity is a key factor in determining the sensitivity of prostate cancer cells to selenium inhibition of growth and cancer-specific biomarkers. Mol Cancer Ther 4, 1047-1055.

C. Synergy with other Antioxidants and chemotherapeutic agents Little information is available on the potential synergy between selenium and other antioxidants. We have also demonstrated the synergistic effect of vitamin E and selenomethionine in combination with a 95% reduction in the growth of LNCaP cells in vitro (Venkateswaran et al, 2004a). A highly significant decrease in the incidence of PCa was observed in Lady transgenic animals treated with a combination of antioxidants (Venkateswaran et al, 2004b). MSA has been shown to enhance apoptosis induced by chemotherapeutic agents (adriamycin and taxol) in prostate cancer, suggesting its use to enhance the effect of anti-cancer agents (Vadgama et al, 2000; Zu and Ip, 2003; Hu et al, 2005). The National Cancer Institute and Southwest Oncology Group have initiated a large scale controlled, randomized trial with PCa prevention as the primary end point. This trial named the SELECT (Selenium and Vitamin E Cancer Prevention Trial) is a phase III, doubleblind, placebo-controlled, 12 year trial designed to assess the effect of selenium and vitamin E, individually and in combination, on the incidence of PCa as determined by routine clinical management (Pak et al, 2002; Klein et al, 2003a, b; Combs, 2004; Meuillet et al, 2004).

IV. Conclusion Robust epidemiological and laboratory evidence suggests that selenium in various forms suppresses growth of cancer cells. Selenium has multiple roles in anticarcinogenesis. Redox-regulated anticancer effects are likely one mechanisms of cancer chemoprevention. Hence, the alteration of intracellular redox state by modifying cellular antioxidants and antioxidants enzymes may regulate the therapeutic effectiveness of selenium in PCa. Chemical transformation of selenium is an important biochemical step in cancer prevention. Selenium acts at an early stage in the progression of carcinogenesis. Selenomethionine not synthesized by humans could have beneficial physiological effects, not shared by other selenium compounds. Hence, future studies should focus on identifying such effects. SELECT, the large-scale, population-based randomized controlled trial will directly test the effect of agents like selenium alone and in combination with vitamin E on the incidence of PCa. Androgens play a critical role in prostate carcinogensis. However, a significant proportion of PCa become androgen unresponsive and refractory to hormonal therapy. Combination studies using antiandrogens and selenium could unravel a multitude of aspects relating to the biochemistry and/or possible functions of selenium both as a chemoprevention and chemotherapeutic agent.

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Cancer Therapy Vol 4, page 81 Cancer Therapy Vol 4, 81-98, 2006

Cancer immunotherapy Review Article

Carole L. Berger*, Joshua Shofner, Juan Gabriel Vasquez, Kavita Mariwalla and Richard L. Edelson Yale University, School of Medicine, Department of Dermatology and Comprehensive Cancer Center

__________________________________________________________________________________ *Correspondence: Carole L. Berger, Ph. D. Yale University, School of Medicine, Department of Dermatology, 333 Cedar Street, New Haven CT 06520; Tel: (203) 737-4024; Fax (203) 785-7637; E-mail: carole.berger@yale.edu Key words: Cancer immunotherapy, Cutaneous T cell lymphoma, Dendritic cells, CD4 and CD8 T cells, immunity, T regulatory cells, Heat shock proteins, immune response, CTCL cells, malignancy, Treg cells, Cytokine therapy, Monoclonal antibody therapy Abbreviations: 8-methoxypsoralen, (8-MOP); antibody-dependent cellular cytotoxicity, (ADCC); antigen presenting cells, (APC); cutaneous T cell lymphoma, (CTCL); cytotoxic T lymphocyte antigen-4, (CTLA-4); dendritic cell, (DC); epidermal growth factor receptor, (EGFR); extracorporeal photopheresis, (ECP); glucocorticoid-induced TNF receptor, (GITR); granulocyte-monocyte colony stimulating factor, (GMCSF); heat shock protein-peptide complexes, (HSPPC); heat shock proteins, (HSP); interleukin 10, (IL10); Keyhole Limpet Hemocyanin, (KLH); Langerhans cell, (LC); lipopolysaccharide, (LPS); monocyte conditioned media, (MCM); natural killer T cells, (NKT); nuclear factor-!B, (NF-!B); pattern recognition receptors, (PRR); psoralen and ultraviolet light therapy, (PUVA); receptor activator of NF-!B, (RANK); T cell receptor, (TCR); TNF-related activation induced cytokine, (TRANCE); Toll-like receptors, (TLR); T-regulatory cells, (Treg); Transimmunization, (TI); ultraviolet A, (UVA); vascular epidermal growth factor, (VEGF); X-linked syndrome, (IPEX) Received: 27 October 2005; Accepted: 11 November 2005; electronically published: March 2006

Abstract This article reviews current advances in the immunotherapy of cancer with a particular focus on our experience with cutaneous T cell lymphoma. We have developed an in vitro model of the growth of this malignancy that has enabled us to decipher the clinical phenotype of the disease and identified new targets for therapeutic intervention. Our studies in this disease can provide a road map for translational immunotherapy, whereby, the clinical features of the disease provide leads that can be followed in the laboratory and the investigational results can be used to interpret the clinical findings. This bidirectional partnership between the clinic and the bench can be exploited to develop a better understanding of cancer and may yield more targeted and less toxic forms of immunotherapy.

advances in basic science can inform the search for relevant treatments. We will then selectively review other forms of immunotherapy that show particular promise in the treatment of CTCL and a variety of other cancers.

I. Introduction Over the past few years the long sought goal of effective, non-toxic cancer immunotherapy has become tantalizingly within reach as our understanding of the immune system and the cells that mediate immunity and tolerance have been clarified by molecular and biologic advances. We are progressing towards the crafting of therapies to improve the treatment of a variety of malignancies and techniques that will aid in diagnosis and monitoring. This review will highlight promising new forms of immunotherapy and demonstrate how a better understanding of the functioning of the immune system has translated into novel therapies for cancer. As a case in point, we will first focus on our studies in cutaneous T cell lymphoma (CTCL) which have elucidated the immunobiology of the disease and identified potential therapeutic targets (Berger et al, 2002a, 2005). Our studies in this disease serve to illustrate the paradigm that

A. Cutaneous T cell lymphoma CTCL is a clonal malignant expansion of antigenexperienced CD4 T cells that localize in the epidermis in direct association with an immature member of the dendritic cell (DC) series, the Langerhans cell (LC, Figure 1). The clonality of the malignant T cells has been confirmed at the phenotypic and molecular level by the demonstration that the CTCL cells all carry an identical T cell receptor (TCR) and by karyotypic and polymerase chain analysis which revealed clonal abnormalities unique for individual patients (Berger et al, 1988; Charley et al, 1990; Volkenandt et al, 1994). These phenotypic and genetic profiles can be used for disease diagnosis and 81

Berger et al: Cancer immunotherapy monitoring of progression. While the clinical observation of the association of CTCL cells with epidermal LC has been known for many years and serves as the diagnostic histopathologic marker of the disease (Rowden et al, 1979), the greater significance of this association of CD4 T cells and antigen presenting cells (APC) for the life cycle of the malignancy was not appreciated although in retrospect it provides obvious clues that should have been pursued. Other intriguing bits of evidence that can be reinterpreted in light of our current observations on the nature of CTCL were made in many laboratories. The expression of a memory phenotype by the malignant T cells indicated that they had already encountered antigen in the periphery (Picker et al, 1990) and CLA, a skin homing molecule, marked the epidermal affinity of the neoplasm (Fuhlbrigge et al, 1997). Studies have identified the presence of apoptotic cells in LC associated with CTCL cells in the epidermis (L端ftl et al, 2002) and increased expression of a negative co-stimulator cytotoxic T lymphocyte antigen-4 (CTLA-4) was identified in CTCL cells infiltrating the epidermis (Kamarashev et al, 1998). In addition, the cytokines interleukin 10 (IL10) and transforming growth factor-beta (TGF-") are produced by CTCL cells (Berger et al, 2002a) and in the case of IL10 cited as a hallmark of the TH2 nature of the malignant T cells (Rook and Heald 1995). The TH1 and 2 paradigm divides CD4 T cells based on their cytokine secretion profile (Kalinski and Moser, 2005) and is discussed in Section D. Furthermore, a long held hypothesis postulated that chronic antigen stimulation played a role in the ontogeny of CTCL (Tan et al, 1974). The significance of this supposition in accordance with our current results has attained heightened importance (see Sections E & F). In fact, the role of chronic inflammation in a variety of malignancies and autoimmune disorders has only recently been appreciated (van Eden, et al, 2003; Zeh III and Lotze

2005) and as we will discuss may be fundamental for our understanding of how these diseases arise and progress (see Section E). The significance of the relationship of CTCL cells and immature DC was not evident until basic science breakthroughs led to methods for the cultivation and identification of DC (Sallusto and Lanzavecchia 1994). Once it was possible to culture DC, the factors that promoted the growth of CTCL cells could be established in an in vitro system (Berger et al, 2002a). Using this approach, we have identified the immunosuppressive nature of the malignancy and determined how the CTCL cells subvert normal immune responses and perpetuate their own survival and growth (Berger et al, 2005). The culture system also permits access to proliferating CTCL cells and immature DC so therapeutic interventions can be tested in vitro for efficacy and toxicity prior to clinical trial evaluation. We will first review the current knowledge of the major cell types that play a role in the immunobiology of CTCL and then link this information to our current understanding of the nature of the disease and the potential for exploitation of these cell types in immunotherapy.

B. Dendritic cells Dendritic cells have assumed a central role in orchestrating both immunity and tolerance and thereby controlling the nature of the immune response. Many dendritic cell subsets have been described based on phenotypic or functional characteristics (Banchereau et al, 2000), but the two main types of DC that are commonly recognized are the myeloid DC which derives from monocytes in the periphery and the lymphoid or plasmacytoid DC which is also present in the blood and is responsible for type I IFN-# and " production during viral infection (Banchereau et al, 2000). Due to their convenient

Figure 1. The pautrier microabscess. CTCL lymphocytes (blue) surround a central immature member of the dendritic cell (DC) series the Langerhans cell in the epidermis. The DC acquire antigen from apoptotic cell death and display self peptides in class II MHC molecules to the T cell receptor of the CD4 + CTCL cells inducing their assumption of a T regulatory phenotype and function (orange).

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Cancer Therapy Vol 4, page 83 access, the majority of immune therapy protocols utilize myeloid DC generated from monocyte precursors. The maturation status of the DC has been demonstrated to have a profound impact on the type of immune response induced (Figure 2). Immature DC are aggressively phagocytic and pinocytic and serve as sentinels surveying the environment for foreign invaders (Banchereau et al, 2000; Chow and Mellman, 2005). Mature DC up-regulate molecules that present antigens to immune effectors (class I and II major histocompatibility antigens) and display accessory molecules that enhance immunity (co-stimulatory molecules, B7-1 and 2, CD40, CD83 and migratory chemokine receptors that provide access to lymph nodes such as CCR7, Banchereau et al, 2000). While immature DC are scavengers that indiscriminately acquire antigen, mature DC are required for effective induction of immunity. However, current studies indicate that even fully mature DC can induce Tregulatory cells (Treg) that promote tolerance and suppress immunity (Yamazaki et al, 2003) and in addition in some cases semi-mature DC may be able to present sufficient class I peptides to stimulate a CD8 T cell response

(Kleindienst et al, 2005). Therefore, although conventional wisdom currently supports mature DC as the cell type of choice for immunotherapy much remains to be learned about the most effective immune vaccine construction for induction of anti-cancer responses. Mature DC may be developed with a variety of substances including: TNF-#; CPG oligodeoxynucleotides; lipopolysaccharide (LPS); monocyte conditioned media and components derived from microbial pathogens (Banchereau et al, 2000). Intriguingly physical disruption (see Section I) as well as CD40-ligand binding to the DC CD40 molecule have been shown to be the most effective means of achieving maturation (Delamarre et al, 2003). DC loaded with peptides, proteins, tumor lysates, apoptotic bodies, transfected with cytokines or fused with tumor cells have been used with varying success as tumor vaccines (Banchereau and Palucka, 2005). Although mature DC effectively stimulate CD8 T cell responses in vitro, clinical efficacy using these cells has been difficult to achieve with partial responses of generally short duration reported

Figure 2. Induction of Immunity or immunoregulation. Immature DC acquire exogenous antigen and display peptides in class I or II MHC molecules. High levels of apoptotic cell death load DC class II molecules with self peptides that can stimulate a CD4 T regulatory response that inhibits normal immunity leading to tolerance and immunosuppression. Cross-priming of peptides into the class I pathway that can be displayed in the full context of co-stimulatory molecules by mature DC induces a CD8 T cell response. CD4 T cell help promotes development of CD8 T cells into competent effector cells that can target class I displayed peptides on tumor cells.

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Berger et al: Cancer immunotherapy (Slovin, 2003). Therefore, it is clear that optimal construction of DC vaccines will require further investigation. DC that present foreign peptides in the context of high levels of co-stimulatory molecules to na誰ve and memory T cells promote both CD4 and CD8 T cell adaptive immunity (see Section D). These immune effectors mediate protection from infection and control of tumor progression and metastasis. DC that present selfpeptide can induce Treg cells and tolerance and immunosuppression (Steinman et al, 2000) and play a critical role in prevention of autoimmune disease (Von Herrah and Harrison, 2003) and promotion of transplant tolerance (Sakaguchi and Wood, 2003). In addition, DC are mediators of innate immunity through secretion of the cytokines IL12 and type I interferons (M端nz et al, 2005). DC have a bidirectional interaction with natural killer T cells (NKT) and $% T cells promoting their activation and destruction of specific target cells, while the innate effectors feedback to trigger DC maturation thereby, furthering both adaptive and innate immunity (Mocikat et al, 2003). The appropriate source of immunogen to load DC for vaccines protocols also remains to be determined. Many protocols have used peptide-pulsed DC matured in vitro and found promising results in animal models of cancer (Steinman and Dhodapkar, 2001). However, this approach is limited by the requirement to tailor the peptide to the HLA-type expressed by the patient and the limited number of known tumor epitopes (Pardoll, 2000; Trombetta and Mellman, 2005). Moreover, cell surface expression of the peptide-MHC complex may be rapidly lost and can only induce an immune response that is limited to the T cell clones specific for the selected epitope (Trombetta and Mellman 2005). Since tumor cells frequently lose expression of antigens, even successful expansion of tumor-reactive T cell clones will be unable to lyse cells that have lost the target antigen. One approach, long advocated by our group (see Section I), has been the use of whole tumor cells killed or rendered apoptotic for DC loading. Immature DC can ingest the apoptotic or killed tumor cells and through a mechanism known as crosspriming express peptides derived from tumor proteins in MHC class I molecules (Chow and Mellman, 2005). Cross-priming of exogenous proteins into the class I pathway distinguishes DC from other APC that can present only endogenous peptides in class I molecules (Trombetta and Mellman 2005). This pathway provides crucial access for ingested peptides derived from proteins of engulfed killed tumor cells to class I MHC molecules where they may be presented and can stimulate a CD8 anti-tumor response (Albert et al, 1998). The limitations that preclude effective DC immunotherapy are manifold and include a lack of understanding of the optimum protocol for maturation, loading and antigen type for induction of successful adaptive anti-tumor immunity, as well as the means for overcoming the rather alarming counter measures that can be deployed by malignancies (see Section H). The goal of successful DC immunotherapy is induction of the major mediators of anti-tumor immunity CD4 and CD8 T cells.

C. The role of CD4 and CD8 T cells in immunity The effector cells of adaptive immunity are the two major T cell subsets defined by their expression of either CD4 or CD8 accessory molecules. CD4 T cells are divided based on cytokine profiles into two major subtypes the TH1 and TH2 cells, where TH1 cells are helper cells that promote cell mediate immunity, secrete IFN-$ and TNF# and carry a TCR that recognizes antigens displayed in class II MHC molecules on an APC. TH2 cells secrete IL 4, 5, 10 and 13 are responsible for humoral immunity and may limit immune responses through IL10 production (Kalinski and Moser, 2005). CD4 T cells comprise the helper T cell subset and promote B cell immunoglobulin production (Kalinski and Moser, 2005) and under appropriate circumstances license CD8 T cell effector functions (Bevan, 2004). CTCL cells are mature CD4 T cells that have been shown to function as helper T cells (Berger et al, 1979; Broder et al, 1997) an observation that appears to conflict with their recently described T-regulatory (Treg) nature (Berger et al, 2005). However, other studies have demonstrated that this dichotomy may be consistent with results obtained with cloned normal Treg cells (Kitani et al, 2000). CD8 T cells are stimulated by peptides displayed in class I MHC molecules on an APC and can mediate tumor cytolysis through production of cytokines such as TNF-# and the secretory granules perforin and granzymes (Raja, 2003). Tumor immunotherapy has been focused in large degree towards induction of a CD8 T cell anti-tumor response that can effectively destroy malignant cells. Although a number of approaches including transfer of ex vivo expanded cytotoxic T cells as well as a spectrum of DC therapies have been designed to optimize the host antitumor response, the overall success of these strategies has been disappointing (Banchereau and Palucka, 2005). Recently, the importance of expanding helper CD4 T cells for the development of effective CD8 T cells has garnered more attention and may improve the efficacy of the induced CD8 T cell response. The emergence of an additional subset of CD4 T cells, the immunosuppressive Treg cells, may limit anti-cancer immunity and the effects of these cells may have to be circumvented before effective immunotherapy can be achieved.

D. T regulatory cells T regulatory cells have been recently identified as CD4+, CD25+ T cells that inhibit immune responses, prevent autoimmune disease (Von Herrah and Harrison, 2003) and maintain transplant tolerance (Sakaguchi and Wood, 2003). At least two types of Treg cells are recognized, those that arise in the thymus and circulating inducible Treg precursors that comprise 5-10% of the peripheral blood CD4 T cell compartment (Von Herrah and Harrison, 2003). Cell surface markers used to characterize the Treg population include the IL2 receptor (CD25), the inhibitory member of the co-stimulatory family cytotoxic T lymphocyte antigen-4 (CTLA-4), glucocorticoid-induced TNF receptor (GITR) and the

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Cancer Therapy Vol 4, page 85 forkhead transcription factor, Foxp3. Disruption in the Foxp3 gene product, in humans, has been correlated with the disease immune dysregulation polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). IPEX in males is associated with lymphocyte activation autoimmune disorders and fatality due to overproduction of inflammatory cytokines. A similar disease in the scrufy mouse has been related to a mutation in the Foxp3 gene (Khattri et al, 2003). In the mouse, Foxp3 expression is confined to Treg cells and has been proposed as a specific marker restricted to Treg cells (Coffer and Burgering, 2004). Treg cells are anergic and do not proliferate in response to TCR stimulation (Burg et al, 1978; Dalloul et al, 1992). However, Treg cells can be driven to proliferate in vitro in the presence of mature antigen-loaded DC in the absence of added cytokines (Yamazaki et al, 2003). Mature DC present antigen to CD4 T cells and can drive their conversion into Treg and thereby play a role in the regulation of autoimmunity and effector T cell expansions. The mechanism of Treg cell suppression remains controversial and appears to vary based on the culture conditions and the model of immunosuppression monitored (Wang and Wang, 2005). Secretion of the inhibitory cytokines IL10 and TGF-" as well as direct cell contact possibly mediated through CTLA-4 engagement of B7-1 and 2 on the APC have been described in several models. In addition, since Treg express high levels of membrane CD25 depletion of IL2 which is a required growth factor for both antigen responsive CD4 T cells and Treg cells may also serve to inhibit immune responses (Antony and Restifo, 2005). The association of Treg cells with cancers including melanoma (Vignier et al, 2004), lung, ovarian (Woo et al, 2002), pancreatic and breast cancer (Liyanage et al, 2002) has suggested that Treg may play a role in suppressing anti-tumor immune response and perpetuating the malignancy. This observation may explain the limited efficacy of many anti-tumor immunotherapies where induction of an immune response is short-lived and abrogated by the tumor milieu. Therefore, improved immunotherapy will require a means for the depletion or inactivation of the Treg response.

(Lafferty and Cunningham, 1970). Signal 1 (antigen stimulation) in the absence of signal 2 (co-stimulation) is proposed to lead to T cell anergy and non-responsiveness. It was then left to Janeway to explain how APC discriminate between self and foreign molecules (Janeway Jr, 1989). Janeway predicted that the APC would express pattern recognition receptors (PRR) that could detect conserved common molecules displayed by pathogens but not found in the human body. The identification of Tolllike receptors (TLR) on APC that can bind molecules derived from bacteria, viruses and parasites and the demonstration that signaling through Toll receptors leads to DC stimulation and induction of immunity has lent support for this theory (Janeway Jr and Medzhitov, 2002). An alternative point of view was suggested by Matzinger, who reasoned that rather than discriminating between self and non-self antigens the immune system senses danger due to not just microorganisms but any form of stress or damage (Matzinger, 1994). Evidence has accumulated indicating that damaged or dying cells are able to activate DC to promote immunity and that self molecules that represent danger signals include heat shock proteins (HSP), uric acid and nucleotides (Pulendran, 2004). This hypothesis has been of particular relevance to our studies of CTCL since we have demonstrated that CTCL cells display a cell membrane HSP (Berger et al, 1997) that is highly homologous to the ER chaperone BiP (binding protein) or GRP78 (glucose regulated protein). Whether HSP up-regulation in tumor cells is a negative sign that correlates with a poor clinical outcome (Mintz et al, 2003) or whether HSP enhance anti-tumor immunity (Feng et al, 2001) is a yet another enigma that waits to be unraveled and may have significant ramifications for tumor immunotherapy.

F. Heat shock proteins HSP are essential for many of the house-keeping functions of the cell including regulation of protein folding, the removal of misfolded proteins, the stress response and protection from caspase-mediated apoptotic cell death (Nicchitta 2003). Due to the essential nature of their role in the cell, HSP are highly conserved throughout evolution. HSP are up-regulated by the cell under stress conditions including fever, starvation and exposure to proinflammatory cytokines such as TNF and IFN-$, oxidative stress and infection (van Eden et al, 2005). The HSP are divided into a number of families based on molecular size. At least three members of the HSP families, HSP60, 70 and 90 appear to play a major role in inflammation and immunity. Since we have found that the HSP70 family member GRP78 or BiP is abundantly expressed on the cell membrane of CTCL cells (Berger et al, 1997), we propose that BiP may serve as a source of autoantigen that drives CTCL Treg conversion when it is presented in class II MHC molecules on DC to the CTCL cell TCR. BiP is an endoplasmic reticulum chaperone that is responsible for the folding and transport of polypeptide chains (Hendershot, 2004). The expression of BiP on the cell membrane has been confirmed in other cancers and may relate to a chronic stress response and has been associated with a poor prognosis (Triantafilou et al, 2001;

E. Danger signals and the immune response A major paradox in immunology is the ability of the immune system to discern danger while ignoring self tissues thereby, permitting destruction of pathogens without harm to normal tissues. A number of theories have been offered that partially explain this dichotomy including the clonal selection theory which proposes that lymphocytes in the thymus early in the organismsâ&#x20AC;&#x2122; life die rather than proliferate when they encounter self antigens (Burnet 1957) eliminating potentially autoreactive clones. This explanation of central tolerance, however, did not address how the adult develops tolerance to exogenous antigens. An explanation for this part of the puzzle was provided by the two signal paradigm for T cell activation, whereby, the T cells need to see MHC displayed antigen and co-stimulation to initiate an immune response 85

Berger et al: Cancer immunotherapy Mintz et al, 2003). BiP up-regulation appears to play a role in protection of malignant cells from apoptosis, thereby, potentiating tumor cell growth and survival (Reddy et al, 2003). Studies in autoimmune disease have indicated that HSP are a major source of autoantigen(s) that trigger HSPreactive T cells with an immunoregulatory phenotype which can suppress immune responses in inflammatory conditions such as rheumatoid arthritis, type 1 diabetes and potentially atherosclerosis and allergy (van Eden et al, 2005). The role of HSP in autoimmune disease may relate to their highly conserved homology to bacterial HSP. Both HSP60 and 70 are immunodominant proteins that induce specific cellular and humoral immune responses after infection with bacteria, protozoa, fungi and parasites (van Eden et al, 2005). It appears that HSP-responsive cells escape central thymic tolerance and enter the periphery where they may be controlled by HSP-reactive Treg cells (van Eden et al, 2005). HSP also serve as immunogens presented by class I MHC molecules and are targets for cytotoxic T cells (Huang et al, 2000; Feng et al, 2001). HSP70 specific cytotoxic T cells have been generated without the assistance of helper T cell responses (Huang et al, 2000). HSP are also efficiently presented by MHC class II molecules (Anderton et al, 1995) and HSP70 related peptides comprise a substantial proportion of the peptides eluted from class II MHC molecules (Newcomb and Cresswell, 1993). When APC were stressed by heat shock, the responding CD4 T cells displayed a Treg cytokine profile secreting IL10. HSP have been shown to be protective in a variety of experimental disease models independent of the source of the disease inducing antigen (van Eden et al, 2005). Based on this evidence it has been proposed that HSP are involved in the control of inflammatory disease through the induction of Treg cells. Therefore, the inflammation that accompanies malignant transformation may provide an ideal milieu for the upregulation of HSP and the induction of regulatory T cells which could suppress anti-tumor immunity. These current concepts in immunology have had direct impact on our understanding of CTCL and enabled us to decipher many of the clinical clues that were provided by the malignancy. The ability to culture CTCL cells derives directly from an understanding of their relationship to Langerhans cells in the pautrier microabcess (Figure 1) and was dependent on the description of methods for the cultivation of DC.

(Berger et al, 2002a). In this system, co-cultivated CTCL cells and DC proliferate for up to three months while both cell types die within one week when cultured individually (Berger et al, 2002a). These studies also demonstrated that proliferating CTCL cells secrete IL10 and TGF-" two immunosuppressive cytokines that maintain DC immaturity and also inhibit immune responses. The ability to grow CTCL cells has enabled us to obtain sufficient malignant T cells for further evaluation that interpreted in the light of the bourgeoning information about Treg cells led to an explanation for the immunosuppressive nature of CTCL. Since cultured CTCL cells proliferate, they were rapidly rendered apoptotic by antibodies that bound to the TCR or the associated CD3 complex, in the presence of DC bearing co-stimulatory molecules (Berger et al, 2005). When we challenged freshly isolated cultured CTCL cells with DC pulsed with apoptotic malignant T cells, we found that the CTCL cells up-regulated the phenotype and function of Treg cells. Treg CTCL cells express CTLA-4, an inhibitory member of the co-stimulatory family, enhanced levels of membrane CD25 and down-regulate the CD4 T cell TCR indicating engagement by DC class II presented peptides. Treg CTCL cells also up-regulate cytoplasmic FoxP3, a specific marker for Treg cells. Anticlass II antibodies and an inhibitor of the class II pathway were shown to prevent adoption of a Treg profile in CTCL cells confirming the requirement for class II MHC molecules in the induction of a Treg conversion in CTCL. Treg CTCL cells upon stimulation by apoptotic cell loaded DC were found to secrete the immunosuppressive cytokines IL10 and TGF-". Addition of Treg CTCL cells to normal T cells responding to recall antigen or allostimulation resulted in suppression of the normal T cell proliferative or cytokine response (Berger et al, 2005). Further investigation revealed that the mechanism of suppression was not related to the release of immunosuppressive cytokines nor was it mediated by cell contact, indicating that Treg CTCL cells do not employ conventional means for inhibition of normal immune responses. We have recently demonstrated that the suppression of normal immunity mediated by Treg CTCL cells can be partially reversed through the addition of a neutralizing antibody to CTLA-4 as well as with the administration of high doses of IL2 (unpublished results). In addition, sera collected from patients with varying stages of CTCL contained elevated levels of soluble CTLA-4 which correlated with the severity of the disease (unpublished results). Therefore, one means through which CTCL cells suppress immunity may be through secretion of soluble CTLA-4 which could act through interference with effector T cellsâ&#x20AC;&#x2122; access to B7-1 and 2 costimulatory molecules. In addition, depletion of growth factors such as IL2 may inhibit normal T cell antigendriven proliferative responses. These two scientific observations provide obvious avenues for therapeutic exploitation (Section J), through the use of anti-CTLA-4, recombinant IL2 or IL2-binding toxins. In addition, current studies are in progress to determine if there is a role for heat shock proteins as autoantigens in CTCL. These studies may identify opportunities for selective

G. In vitro growth of CTCL CTCL cells isolated from the peripheral blood of patients are anergic and respond poorly to mitogen, antigen and alloantigen stimulation (Burg et al, 1978; Dalloul et al, 1992). Investigation of the immunobiology of the malignancy has been severely hampered by the inability to routinely culture the malignant T cells in vitro and the absence of suitable animal models. We have found that CTCL cells proliferate in the presence of autologous DC cultured with the supportive T cell cytokines IL2 and IL7 and for the DC granulocytemonocyte colony stimulating factor (GMCSF) and IL4 86

Cancer Therapy Vol 4, page 87 targeting of Treg cells to improve the efficacy of immunotherapy (Section J). Although, the interactions of CTCL cells with DC suggest that immunotherapy using DC loaded with apoptotic cells might be precluded in this disease, our clinical experience argues that this approach is not just feasible but efficacious. We have found that a standard therapy, extracorporeal photopheresis (ECP, 65) and a simple modification Transimmunization (TI, Berger et al, 2001) both operate through the induction of differentiating DC derived from monocyte precursors and simultaneous DC loading with apoptotic malignant T cells.

helix where after photoactivation it forms cross-links between pyrimidine bases (Berger et al, 1985). The crosslinks prevent replication and are poorly repaired leading to gradual apoptotic cellular death over a 6 day period. We have demonstrated that the combination of the physical perturbation initiated by the passage of the leukapheresis through the large plastic plate, permitting adherence and release of monocytes, when combined with the apoptotic death of the malignant T cells has profound consequences for the reinfusate returned to the patient (Berger et al, 2001). The monocytes become activated and begin to transition into aggressively phagocytic immature DC while the malignant T cells are rendered apoptotic and are ingested by the DC. Therefore, some semi-mature DC loaded with apoptotic T cells are returned to the patient along with a large number of transitioning DC and apoptotic CTCL cells that may interact inefficiently in vivo. We have developed a modification of ECP which we term Transimmunization (TI, Figure 3) to indicate the transfer of immunogenic peptides from apoptotic cells to DC that can effectively present them to the immune system displayed with the full complement of costimulatory and accessory molecules (Berger et al, 2002b).

H. ECP and TI ECP is a widely used form of immunotherapy that is FDA approved for the treatment of cutaneous T cell lymphoma (Zic, 2003). ECP derived from a combination of therapeutic leukapheresis, a cyto-reductive treatment (Edelson et al, 1974) and psoralen and ultraviolet light therapy (PUVA) used in the treatment of psoriasis and early stage CTCL limited to the epidermis (Gilchrest et al, 1976). In the ECP therapy, a photactivatable drug 8methoxypsoralen (8-MOP) is added to a therapeutic leukapheresis which is passed through an ultraviolet A (UVA) exposure field. The drug intercalates in the DNA

Figure 3. Transimmunization. In the transimmunization (TI) procedure, a leukapheresis is collected in a centrifuge bowl and a photactivatable drug 8-methoxypsoralen (8-MOP) is added and the mixture passed through an ultraviolet A (UVA) exposure field. The 8-MOP/UVA treatment renders nucleated cells apoptotic while adherence and release from the plastic plate induces monocyte activation into the DC pathway. In the TI modification of ECP, the apoptotic CTCL cells and the immature DC are co-cultivated overnight permitting engulfment of the apoptotic cells by the avidly phagocytic DC. The next day the DC loaded with apoptotic CTCL cell derived material are returned to the patient where cross-priming of tumor-associated peptides into the class I pathway can generate CD8 T cells that target CTCL cells in vivo.

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Berger et al: Cancer immunotherapy The TI approach incorporates an overnight incubation step into the standard ECP procedure. During the overnight culture, the monocytes secrete cytokines that comprise the constituents of monocyte conditioned media (MCM) known to potentiate DC maturation (Berger et al, 2002b). In addition, the phagocytosis of apoptotic blebs further drives DC differentiation towards semi-mature DC that increase their expression of CD83, class II MHC, costimulatory molecule CD86 and lose expression of monocyte markers CD14 and CD36 (Berger et al, 2001). These maturing DC are better stimulators in mixed leukocyte cultures than leukapheresis leukocytes confirming their enrichment into APC with high levels of class II MHC molecules. The TI therapy has been used clinically in a phase I trial that has confirmed its excellent safety profile and has shown preliminary signs of efficacy with partial responses in 55% of CTCL patients that had previously failed all forms of therapy (Girardi et al, 2002). Since the TI procedure allows access to all the components of the immune response the addition of exogenous agents such as drugs, antibodies or cytokines is facilitated and can potentially be used to modify the reinfusate and tailor it to the patient. In addition, the level of apoptotic cells reinfused can be controlled and since high levels of apoptosis appear to correlate with induction of a Treg response, it may be possible to extend the therapy to other disorders due to a failure of immunoregulation. Other types of malignancies may also be treated by TI since the nature of the apoptotic cells added to the overnight culture can be simply modified through the use of other mediators of programmed cell death (such as irradiation of isolated

solid tumor cell suspensions) and co-incubation with TI generated transitioning DC overnight. Dissection of the basic science aspects of CTCL has allowed us to identify targets for immunotherapy and also explained why many approaches are limited in efficacy. Strategies to overcome these limitations readily suggest themselves and include anti-CTLA-4 antibodies, toxin conjugated-IL2, inhibition of heat shock proteins and maximizing DC differentiation and antigen presentation. Some of these approaches have already been tested clinically (Table 1) and will be reviewed in the following sections. DC that can effectively present them to the immune system displayed with the full complement of costimulatory and accessory molecules (Berger et al, 2002b). The TI approach incorporates an overnight incubation step into the standard ECP procedure. During the overnight culture, the monocytes secrete cytokines that comprise the constituents of monocyte conditioned media (MCM) known to potentiate DC maturation (Berger et al, 2002b). In addition, the phagocytosis of apoptotic blebs further drives DC differentiation towards semi-mature DC that increase their expression of CD83, class II MHC, costimulatory molecule CD86 and lose expression of monocyte markers CD14 and CD36 (Berger et al, 2001). These maturing DC are better stimulators in mixed leukocyte cultures than leukapheresis leukocytes confirming their enrichment into APC with high levels of class II MHC molecules. The TI therapy has been used clinically in a phase I trial that has confirmed its excellent safety profile and has shown preliminary signs of efficacy with partial responses in 55% of CTCL patients that had previously failed all forms of therapy (Girardi et al, 2002).

Table 1. Overview of the treatment modality, therapy and disease category targeted by the immunotherapy Modality Photochemical Psoralen and ultraviolet light resulting in apoptotic cell loaded dendritic cells Vaccine Dendritic Cell Vaccines

Therapy

Disease Targeted

Photopheresis and Transimmunization

T cell mediated diseases: Cutaneous T Cell Lymphoma, Graft-versus Host Disease, autoimmune disease, transplant rejection

Pulsed with peptides, tumor lysates, hybrid formation, ingestion of apoptotic cells, nucleic acids and viral transfection

Numerous cancers including: renal cell carcinoma, melanoma, colon cancer, lung cancer, neuroblastoma, and prostate cancer.

Alemtuzumab Anti-CD40 Bevacizumab, Cetuximab Gemtuzumab Ibritumaomab, Tositumomab Rituximab, Tastuzumab

B-Cell CLL Non-Hodgkin’s Lymphoma, solid tumors Colon Cancer AML Non-Hodgkin’s Lymphoma Non-Hodgkin’s Lymphoma and Breast Cancer

Antibodies to TGF-", IL10 IL2 ONTAK Antibodies to: CTLA-4, GITR, TRANCE, RANK and chemokine receptors HSPPC-96

Glioblastoma, Non-small Cell Lung Cancer Melanoma, CTCL CTCL Variety of cancers

Monoclonal Antibodies

Cellular Therapy Cytokine Therapy

T Regulatory Cell Control

Heat Shock Proteins

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Variety of cancers

Cancer Therapy Vol 4, page 89 Since the TI procedure allows access to all the components of the immune response the addition of exogenous agents such as drugs, antibodies or cytokines is facilitated and can potentially be used to modify the reinfusate and tailor it to the patient. In addition, the level of apoptotic cells reinfused can be controlled and since high levels of apoptosis appear to correlate with induction of a Treg response, it may be possible to extend the therapy to other disorders due to a failure of immunoregulation. Other types of malignancies may also be treated by TI since the nature of the apoptotic cells added to the overnight culture can be simply modified through the use of other mediators of programmed cell death (such as irradiation of isolated solid tumor cell suspensions) and co-incubation with TI generated transitioning DC overnight. Dissection of the basic science aspects of CTCL has allowed us to identify targets for immunotherapy and also explained why many approaches are limited in efficacy. Strategies to overcome these limitations readily suggest themselves and include anti-CTLA-4 antibodies, toxin conjugated-IL2, inhibition of heat shock proteins and maximizing DC differentiation and antigen presentation. Some of these approaches have already been tested clinically (Table 1) and will be reviewed in the following sections.

co-administration of anti-CTLA-4 with peptide vaccines and these studies also provide similar encouraging results in both objective tumor regression and clinical response (Korman et al, 2005). While the overall regression rates remains somewhat low, these trials do provide proof-ofprinciple that substantiates the proposition that depletion of the T-regulatory population can result in an enhanced tumor vaccine effect and that Treg cells control autoimmunity. As with many existing immunotherapeutics, there remains a profound need for further research into applying these principles to human subjects, with focus on proper dosing schedules and the use of anti-CTLA-4 as a possible therapeutic adjuvant to other cancer vaccines. Institution of anti-CTLA 4 therapy in CTCL may reverse the immunosuppressive consequences of the malignancy and allow induction of more effective anti-tumor immune responses. Aside from anti-CTLA-4 therapy, there are a number of other options that exist for selective targeting of T regulatory cells to enhance the effectiveness of cancer immunotherapy. GITR, a member of the tumor necrosis factor-nerve growth factor receptor family, has been described on the surface of the CD4+ CD25+ T cells and is thought to play a role in regulating suppression. Stimulation of this receptor via an activating antibody has been shown to reverse the induction of suppression and removal of GITR+ cells resulted in organ-specific autoimmunity in murine models (McHugh et al, 2002; Shimizu et al, 2002). Other options include cell signaling molecules such as TNF-related activation induced cytokine (TRANCE) and receptor activator of NF-!B (RANK), which have been shown to be involved in activating signaling pathways of CD4+CD25+ T cells (Green et al, 2002). Depletion of these molecules resulted in a rapid onset of diabetes in murine models. Another alternative for Treg inactivation lies in the close association of T regulatory cells and a number of solid tumors. It has been demonstrated that high levels of CD4+CD25+ T cells are present in lung, ovarian, breast and pancreatic tumor samples (Liyanage et al, 2002; Curiel, et al, 2004) and in ovarian cancer, there appears to be an inverse relationship between the number of T regulatory cells and survival (Curiel, et al, 2004). Researchers have demonstrated that the chemokine CCL22 and the chemokine receptor CCR4 are vital to the migration of T regulatory cells to the tumor site and inhibition of this chemokine resulted in decreased migration of Treg cells (Lee et al, 2005). Therapies that selectively deplete Treg in tumor sites while maintaining the Treg population that controls autoimmunity would enable eradication of immunosuppression while preserving inactivation of autoreactive T cell clones.

I. Targeting Treg cells The expression of CTLA-4 on the surface and in the cytoplasm of Treg cells has suggested that it might provide a logical target for depletion of these immunosuppressive cells that may limit cancer immunotherapy. The first clinical trial focusing on anti-CTLA-4 therapy was conducted with nine patients who had previously been immunized against their ovarian cancer or melanoma. Each of these nine patients received a single IV injection of CTLA-4 monoclonal antibody. In these patients, just over half (55%) experienced some degree of tumor cell death as determined histopathologically or via stabilization of biochemical tumor markers (Hodi et al, 2003). In this pilot study, the only notable side effect was the appearance of a transient rash on nearly all patients, which was easily controlled with antihistamine therapy. In a later trial by Phan and colleagues in 2003, 14 patients with metastatic melanoma were given a peptide pulsed melanoma vaccine along with a standardized dose of anti-CTLA-4 antibody. All patients enrolled in the trial developed T cell reactivity against the immunizing peptides given in conjunction with the anti-CTLA-4. Notably, three patients of the fourteen (21%) experienced objective cancer regression, with two patients having a complete tumor response at one year and one patient having a partial response, while two others experienced mixed responses (regression of certain metastases with growth of others). A significant percentage (43%) of patients experienced grade III/IV autoimmune disease, including three with dermatitis, two with colitis and one with hypophysitis and hepatitis. Each of these patients was treated with supportive care and/or steroid therapy and all patients experiencing autoimmunity recovered from the acute toxicity without later relapse (Phan et al, 2003). A number of other small studies have been conducted looking at various dosing regimens and

J. Cytokine therapy Cytokines also play a major role in the development and functional capacity of T regulatory cells. As previously described, T regulatory cells produce a number of soluble, inhibitory cytokines, such as IL-10 and TGF-". Selective inhibition of these cytokines has been shown to reverse generalized immunosuppression in a number of murine models and in humans (Khoo et al, 1997; 89

Berger et al: Cancer immunotherapy Nakamura et al, 2004). In the realm of cancer immunotherapy, T-cell-specific blockade of TGF-" allows the generation of an immune response capable of eliminating tumors in murine model systems (Gorelik and Flavell, 2001). Phase I clinical trials looking at the efficacy of anti-TGF-" therapy in the setting of glioblastoma and non-small-cell lung cancer, are in progress (Lahn et al, 2005). Blockade of IL-10 has demonstrated enhanced tumor destruction in murine models and may also offer some clinical utility (Piccirillo and Shevach, 2000). Perhaps the best studied cytokine for tumor immunotherapy is IL-2. First investigated in the 1980â&#x20AC;&#x2122;s, IL-2 has been found to enhance the potency of immunotherapy due to its role as activator/expander of tumor-specific T cells. Early clinical success with IL-2 used as monotherapy demonstrated durable regression in 20% and complete response in 9% of renal cell carcinoma patients. Furthermore, in the case of metastatic melanoma, a regression rate of 17% with complete response of 7% was noted (Gaffen and Liu, 2004). These early successes have translated into a continued role for IL-2 today as a vaccine adjuvant in metastatic melanoma and renal cell carcinoma. IL-2 has also been used with good clinical results in CTCL patients (Marolleau et al, 1995). Current studies are underway to determine whether IL-2 can be used synergistically with IL-12 to enhance the anti-tumor effects of a given vaccine (Rook et al, 2003). In vitro studies have shown that a synergism between these two cytokines can augment the immune response in CTCL patients (Zaki et al, 2002). Recent results indicate that although IL-2 appears to be necessary for T cell activation and growth, it also supports the growth and differentiation of T regulatory cells and this may limit the beneficial aspects of this cytokine. However, our preliminary studies suggest that despite the requirement for IL2 to support Treg cell growth, the addition of recombinant IL2 to cocultures of Treg CTCL cells and antigen-stimulated normal T cells reverses immunosuppression of IFN-$ production (unpublished results). Therefore, a role for IL2 in restoration of anti-tumor immunity may be feasible. Similarly, a number of newer studies have looked at the use of alternative cytokines, such as IL-15 and cytokine combinations with vaccines in hopes of more specifically activating the tumor infiltrating lymphocytes without expanding the T regulatory lymphocyte population (Antony and Restifo, 2005). Members of the naturally occurring T regulatory cell subpopulation are CD25+. Given the relative specificity of CD25 to T regulatory cells, antibody dependent cell death via CD25 may serve as a means to eliminate T regulatory cells. Recently, the FDA approved ONTAK, a recombinant cytotoxic protein composed of diphtheria toxin conjugated to the IL-2 binding domain, for use in CTCL patients. Due to its specificity for the IL2 receptor, this antibody should ideally be able to deplete the CD4+CD25+ cell population in vivo in hopes of enhancing the efficacy of adjunct immunotherapy. One caveat, however, is that CD25 is also expressed on newly activated CD4+CD25- T cells as well as effector CD8+ T cells (Annacker et al, 2001). Thus, destruction of the

naturally occurring T regs via a CD25 specific mechanism may potentially destroy some of the effector T cells capable of mounting a clinically salient immune response. Appropriate timing of anti-CD25 therapy appears crucial, as CD25+ depletion before vaccination was more effective than CD25+ depletion after vaccination (Sutmuller et al, 2001). There continues to be great difficulty in distinguishing an actual T regulatory cell from an effector T cell, especially during the midst of an immune response, therefore, the dosing and time course of therapy may be vital to success. An alternative approach to selectively removing the CD25+ population prior to introduction of a tumor vaccine is total lymphocyte ablation in vivo, followed by introduction of the appropriate immunotherapeutic. In a key study conducted at the National Cancer Institute, 35 melanoma patients had their tumors harvested ex vivo for selection of appropriate tumor-infiltrating lymphocytes. Prior to re-infusion of the expanded tumor-specific lymphocyte population, cyclophosphamide and fludarabine were administered to deplete the lymphocyte population in vivo. Marked expansion of tumor-specific T cells was observed and 51% of the melanoma patients achieved objective responses (Dudley et al, 2002; Rosenberg and Dudley, 2004).

K. Monoclonal antibody therapy A prevalent modality in the quest for effective immunotherapeutics is the use of monoclonal antibodies (mAB) as adjuvant to immunotherapy. First discovered in the late 1970â&#x20AC;&#x2122;s (Kohler and Milstein, 1975), monoclonal antibodies have become standard therapy for certain malignancies given their enhancement of the anti-tumor response, relatively safe toxicity profiles and high selectivity. Overall, these antibodies can be divided into a number of specific classes, based on the mechanism by which they exert their effect. The initial classes of antibodies exerted their anti-tumor effects via antibodydependent cellular cytotoxicity (ADCC) or complementdependent cytotoxicity (CDC, Maloney et al, 1994). However, newer classes of antibodies are being studied that are directed against a variety of cellular targets, including growth factors, such as vascular epidermal growth factor (VEGF), epidermal growth factor receptor (EGFR), a number of specific cell signaling molecules and receptors on the cells of the immune system in hopes of enhancing the cellular immune response against cancer (Gutheil et al, 2000; Ciardiello and Tortora, 2001; Gordon et al, 2001). The first anti-tumor antibody approved by the FDA was rituximab in 1997, which was quickly followed by tastuzumab in 1998. These were approved for the treatment of refractory non-Hodgkin lymphoma and HER2/Neu+ breast cancers respectively. Since that time, six additional monoclonal antibodies have been approved for clinical use. These antibodies include anti-CD52 alemtuzumab for refractory B-cell chronic lymphocytic leukemia, anti-CD33 gemtuzumab ozogamycin conjugated to calicheamicin for refractory acute myeloid leukemia, anti-CD20 radioisotope conjugates ibritumaomab and tositumomab for refractory non-Hodgkin lymphoma, to 90

Cancer Therapy Vol 4, page 91 target VEGF bevacizumab for metastatic colon cancer in combination with chemotherapy and anti-EGFR cetuximab for metastatic colon cancer (Lin et al, 2005). As previously described, these antibodies all work either through stimulation of ADCC and CDC or inhibition of specific growth factors/growth factor receptors essential for tumor proliferation. The results of clinical trials using these drugs have recently been reviewed (Harris, 2004) and ongoing clinical trials for other indications are continuing. This limited selection of monoclonal antibodies has enjoyed huge success in their respective oncologic fields and there are now over 400 clinical trials currently underway to determine the efficacy of a huge variety of other anti-cancer antibodies (Gura, 2002). As described earlier, the newest class of monoclonal antibodies is being used in an attempt to augment immunomodulation in conjuction with other immunotherapies, of which only one has entered clinical trials. A primary target is suppression of the aforementioned T regulatory cell, but a number of other potentially beneficial targets exist as well. Anti-4-1BB (CD137) is a surface glycoprotein in the TNF receptor family and it is expressed by activated T and NK cells (Murillo et al, 2003). Treatment of tumor-bearing mice with anti-4-1BB caused tumor regression and addition of anti-4-1BB mAbs help potentiate the effectiveness of adoptive immunotherapy, likely through preventing programmed cell death in lymphocytes (Melero et al, 1997; Guinn et al, 1999; May Jr, et al, 2002). CD40 is a TNF receptor family member expressed on B cells, DC and macrophages and is essential in mediating both cellular and humoral immune responses (Grewal and Flavell 1998). Treatment of B cell malignant mice with anti-CD40 has lead to complete cure in some cases (French et al, 1999). Very early clinical trial data has been published showing a 37.5% disease stabilization rate and 6% partial response rate when anti-CD40 was used for high grade NHL or solid tumors (Vonderheide et al, 2001). In the case of CTCL, use of CD40 ligand has been used to restore IL-12 and TNF-# production in the peripheral mononuclear cells of cancer patients (French et al, 2005), further confirming the possible therapeutic potential involved in the manipulation of CD40 in a number of malignancies. Other directions that antibody research is headed include the use of immuno-modulators, such as IL2, along with monoclonal antibody administration, in hopes of boosting immune effector function, as well as the development of novel immunoconjugates in order to improve efficacy. Each of these new avenues is undertaken with the goal of combining the best aspects of various parts of immunotherapy in order to create the most efficacious treatment.

L. Heat immunotherapy

shock

proteins

an immune response was first demonstrated in 1986 by Srivastava and colleagues, who showed that mice immunized with gp96 produced tumor-specific immunity against the tumors that were used to isolate the HSP, but not against other tumors (Srivastava et al, 1986). This early work in the field was done using the HSP gp96, but it was further validated with later results in which a number of other HSP, including HSP70, HSP90, calreticulin, HSP110 and GRP170 (Udono and Srivastava, 1993; Tamura et al, 1997; Basu and Srivastava, 1999; Wang et al, 2001), were used. In each of these studies, it was shown that the HSP isolated from cancer cells elicited immunity while those HSP isolated from normal tissues did not. This initial work led to subsequent discoveries of the immunostimulatory effects that HSP have on nearly all cells of the immune system, including T cells, B cells, macrophages and dendritic cells (Quintana and Cohen, 2005). Aside from the immunogenicity of HSP, they provide an exciting target for cancer vaccination because of their ability to function as carriers of self and non-self peptides. Given their role as intracellular chaperone, HSP bind a wide variety of peptides in vivo, albeit for a very short time (Reits et al, 2003). These short-lived HSPpeptide complexes are useful immunologic targets because they provide an up-to-date reflection of the internal environment of the cell. In the case of a malignant cell, a number of the HSP-peptide complexes presented on the surface of the cell are unique tumor-associated antigens and thus can be attacked by the immune system of a vaccinated patient. This is supported by initial research which showed that the anti-tumor response generated by HSP-associated tumor cells was derived from peptides bound to the HSP and not directed against the HSP themselves (Li and Srivastava, 1993; Udono and Srivastava, 1993). When alone, neither the HSP nor peptides were immunogenic; only the HSP-peptide complex was able to elicit the desired CD8+ cytotoxic T cell response (Blachere et al, 1997). Thus, the specific immunogenicity of each HSP-peptide preparation is due to the inherent antigenic variety found in the malignancy of interest (Menoret et al, 1999). These discoveries have opened up a new door in anticancer therapy, whereby cancer vaccines can be created using tumor-derived heat shock protein-peptide complexes (HSPPC) as effective immunologic targets. HSP could be loaded in vitro with synthetic peptides and injected back into the patient to achieve a desired immune response. In order to achieve immunity by HSP vaccination, both APC and CD8+ T cells are required, as depletion of either cell type diminishes the protective effects of the vaccine (Udono et al, 1994). Interestingly, it has also been shown that HSP can mediate efficient crosspresentation into the Class I pathway after being endocytosed by APC, via the recently identified CD91 receptor, making them powerful adjuvants and rendering them more effective at generating the desired cytotoxic T cell response (Udono et al, 1994; Jung et al, 2002). In addition to the direct effects on antigen presentation, the interactions of HSP with APC leads to secretion of proinflammatory cytokines such as TNF-#, interleukin-1", IL-12 and GM-CSF; maturation, migration of dendritic

as

A newer approach to producing an effective cancer vaccine is through the use of highly immunogenic heat shock proteins as tumor-associated antigenic adjuvants. This dichomatous role of HSP as both immunostimulant and immunosuppressor has been the subject of much critical debate in recent years. The ability of HSPs to elicit 91

Berger et al: Cancer immunotherapy cells; and translocation of nuclear factor-kappaB (NF-!B, Basu et al, 2000; Singh-Jasuja et al, 2000; Somerson et al, 2001). Given the homology of HSP and their generalized immunogenicity, this allows for the immunization of the host against a wide variety of host-specific tumorassociated antigens. This was initially supported in a number of murine models, which demonstrated that HSP immunization slowed the progression of primary tumors and reduced the overall metastatic burden as well (Tamura et al, 1997). Given its success in murine models, the vaccine was ready for study in human trials. The initial autologous HSP vaccine to be tested in a clinical trial was HSPPC-96 (Oncophage), a gp96 HSP-peptide complex, made from resected tumor tissue and given back to the patient via intradermal or subcutaneous injection. Since 1995, there have been ten phase I or II clinical trials looking at the safety profile of this vaccine as well as the optimal route of administration and dosing schedule (Lewis, 2004). Throughout these studies, the most common side effects noted were transient and included injection site inflammation and low-grade fever. More encouraging was that HSPPC-96 treatment resulted in a better quality of life during treatment when compared to those patients undergoing chemotherapy or high-dose cytokine immunotherapy (Cohen et al, 2002). In addition, no clinical signs of autoimmunity have been documented in the more than 500 patients treated to date. The encouraging safety profile of the HSPPC-96 vaccine has led to a number of phase II and III clinical trials to determine whether or not the vaccine can have a beneficial effect on cancer patients. In the initial pilot trial, 6 of 12 patients with miscellaneous, advanced cancers had a CD8+ response against autologous tumor following HSPPC-96 vaccination (Janetzki et al, 2000). Another trial was conducted in patients with renal cell carcinoma. In 16 patients with stage 4 renal cell carcinoma given a 25!g HSPPC-96 vaccination dose, 3 patients had observable partial responses while on the vaccine and three additional patients showed disease stabilization at one year (Srivastava and Amato, 2001). A phase II study involving melanoma patients showed an increase in blood IFN-$ levels following vaccination and 11 of 23 of these patients showed an increase in melanoma-specific T cells (Belli et al, 2002). The frequency of these measurable immune responses has appeared to correlate well with favorable clinical responses. In the melanoma trial mentioned previously, the majority of the clinical responders were also those that had an immune response to the tumor cells while those without clinical responses also frequently lacked demonstrable immunity to the malignant melanocytes (Srivastava and Amato, 2001; Belli et al, 2002). In this study, five patients of twenty eight with measurable disease post-vaccination showed either objective response or disease stabilization. In a phase II trial looking at patients with colorectal carcinoma, patients clinically free of disease post-surgery had better overall survival if they developed an immune response following HSPPC-96 vaccination than if they did not (Lewis, 2004). Currently, there is sufficient evidence to support the idea that cancer vaccination with HSPPC can produce

measurable anti-tumor responses in cancer patients. Why these measurable immune responses are not always translated into an observable clinical response may relate to the role of HSP as autoantigens that generate a Treg response (van Eden et al, 2005). If high levels of HSP are present in apoptotic cells engulfed by DC, peptides derived from the HSP may be presented in class II MHC molecules and induce Treg cell stimulation. As in other forms of immunotherapy, the immune system may both potentiate and limit the success of the treatment and as previously discussed a combination approach using antiTreg modalities to supplement immunotherapy may allow us to circumvent the difficulties while preserving the benefits. As a whole all of these approaches argue for careful scientific investigation and attention to clinical signs and signals so that the optimal form of immunotherapy can be developed, tested and employed. Since we have better tools and an improved understanding of the immune system, we should be able to pursue therapies that can be rapidly translated from the laboratory to the patient in a safe, efficacious fashion.

M. Dendritic cell immunotherapy Since the publication of the first peptide-pulsed DC cancer vaccine clinical trial in 1995 by Mukherji et al. hundreds of clinical trials have been completed. This great interest in vaccines grew from the widespread success of vaccines in preventing widespread viral diseases, ease of administration and minimal side effects. As previously described, cancer vaccines are designed to treat growing tumors by inducing tumor-specific effector T cells that reduce the tumor mass and via induction of tumor-specific memory T cells to control tumor relapse (Banchereau and Palucka, 2005). Thus, when properly prompted, the immune system's innate, antigen non-specific, immunity and the adaptive, antigen specific, immunity synergize to eradicate pathogens as well as cancers. The induction, coordination and regulation of the adaptive immune systems is ultimately controlled by DC (Steinman, 1991; Banchereau et al, 2000). Although, multiple vaccination models are under investigation such as peptide vaccines, viral vector vaccines and tumor cell vaccines, dendritic cell vaccines have the best response rate (Rosenberg et al, 2004). The completed trials utilizing dendritic cell vaccines have been numerous and varied success observed (Slovin, 2003). By 2003 there were 98 published peer-reviewed articles describing over 1000 patients vaccinated for over two dozen cancer types (Slovin, 2003): adenocarcinoma, bladder, breast, lung, colorectal, CML, duodenal, esophageal, GI carcinoma, glioblastoma, astrocytoma, glucogonoma, gynecologic carcinoma, head and neck, hepatocellular, multiple myeloma, lymphoma, neuroblastoma, ovarian, pancreatic, parathyroid, prostate, sarcoma stomach, thyroid, wilms, neuroendocrine. Nevertheless, the ideal vaccine has yet to be worked out, for example, the source of DC, method for tumor antigen presentation to DC, effectiveness of artificially maturing DC, site of vaccination, number of administrations, vaccine preservation, number of DC necessary and timing 92

Cancer Therapy Vol 4, page 93 of vaccinations remains to be determined. The most effective antigen for use in loading DC is not clear, whether peptide, whole tumor, nucleic acids (Caruso et al, 2005) or viral. The adverse effects associated with DC vaccinations were minor, and mostly self-limited; the most common side effects were fever, injection site reactions and adenopathy. Autoimmunity was uncommon and only a handful of patients experienced conversion to positive ANA, RF, anti-dsDNA and antithyroid titers. Fortunately the autoimmunity was generally not clinically significant. Some conclusions about the best methodology have been reached based on previous research. Vaccine treatments, when successful, are most effective in patients with predominately lymphatic or cutaneously restricted disease (Timmerman et al, 2002; Rosenberg et al, 2004). One explanation for this observation is that in contrast to solid tumors, lymphoid tumors and possibly cutaneous tumors allow direct circulatory access with the best results seen when the vaccines are given subcutaneously or intradermally, rather than intravenously (Oâ&#x20AC;&#x2122;Neill et al, 2004). In almost all cases the DC were collected from the patientâ&#x20AC;&#x2122;s peripheral blood. Some investigators are exploring the possibility of using bone-marrow derived DC in vaccines, since early animal models have been promising ( Mayordomo et al, 1997). Neverthless, DC from whole blood are easily accessible in large numbers and can be subsequently cultured to an even greater number (Dillman et al, 2004). Adjuvants have also been employed to boost the reaction towards vaccines. One very common adjuvant is Keyhole Limpet Hemocyanin (KLH, HĂśltl, 2005). KLH is an inducer of strong CD4+ T cell helper responses as a highly immunogenic neo-antigen. When given in conjunction with tumor antigen, it amplifies the immune response via the production of cytokines in the lymph node microenvironement causing an enhanced CD8+ response. There are currently 22 active clinical trials involving the use of dendritic cells to treat cancers (http://cancernet.nci.nih.gov/clinicaltrials). The types of cancer under investigation include AML, Brain, Breast, Colon, Renal, Lung and Melanoma. All trials are in the first or second phases of testing with 32% in Phase I, 36% in Phase II and 32% in phase I/II of testing. The vaccine composition is rather varied employing autologous dendritic cells paired with a range of potential antigenic substances. Common substances employed in the vaccine design include whole-tumor lysates, tumor antigens, recombinant transfection viruses, RNA and tumor celldendritic cell fusion-type combinations. Adjuvants are commonly added to the vaccines in an attempt to amplify antigenicity and to cause the maturation of monocytes into immature dendritic cells. IL-4 and GM-CSF are often used to dedifferentiate monocytes into immature dendritic cells, that are very potent APCs. The uptake of tumor antigen causes the shift of immature dendritic cells to antigen presenting cells mature dendritic cells. DC vaccines are frequently pulsed with an individual peptide creating CTL that recognize a single tumor epitope. However, tumors by nature are thought to accumulate gene mutations and the antigenic genes are no exception. Thus it is possible that over time the antigens change their structure or may not be

expressed at all (Trefzer et al, 2005). Thus, limiting vaccines to one antigen alone may not be as efficacious as a whole tumor lysate that could provide innumerable antigens for the induction of multiple CTL clones. Future vaccines will need to overcome the immune systems regulatory balances. The immune systems has naturally evolved robust suppressor systems to prevent detrimental antigen-specific responses to self and environmental antigens thereby averting excessive damage to host tissue (Smits et al, 2005). Knowledge regarding the biology of these regulatory mechanisms will become crucial for the successful application of cancer immunotherapy.

II. Concluding comments Several themes suggest themselves as the leitmotiv of this review of cancer immunotherapy. First, a bidirectional knowledge flow between clinical observations and research investigation are crucial for successful immunotherapeutic protocols. The clinical clues form the springboard from which a well planned laboratory study can be launched to dissect and resolve previously mysterious physical signs and symptoms. Once a model has been built in the laboratory, it can be used to identify therapeutic regimens that may be the most beneficial and the least toxic. This paradigm has been exemplified by our current studies in CTCL where our understanding of the immunobiology of the malignancy has elucidated previous cryptic physical manifestations and led us to identify new therapeutic approaches that can be translated into clinical trials. Our in vitro model system of the disease will allow us to screen new therapeutics before they enter the clinic saving time, expense and potential patient harm. In the future, further advances in our understanding of the functioning of the immune system will have immense significance for clinical medicine allowing us to develop new and improved therapies and approaches for disease management. Finally, it is becoming clear that the immune system is a finely balanced interconnected web and that pulling one string may have unforeseen consequences for the entire fabric. It is therefore crucial that we continue to test and learn from our mis-steps so that our future interventions can become ever more sure footed.

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Back row from left to right: Joshua Shofner and Juan Gabriel Vasquez Front row from left to right: Carole L. Berger and Richard Edelson

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Cancer Therapy Vol 4, page 99 Cancer Therapy Vol 4, 99-124, 2006

Role of platelet derived endothelial cell growth factor / thymidine phosphorylase in health and disease Review Article

Michiel de Bruin§, Olaf H. Temmink, Klaas Hoekman, Herbert M. Pinedo, Godefridus J. Peters* VU University Medical Center, Department of Medical Oncology, De Boelelaan 1117, 1081 HV Amsterdam

__________________________________________________________________________________ *Correspondence: Godefridus J. Peters, VU University Medical Center, Department of Medical Oncology, De Boelelaan 1117, 1081 HV Amsterdam; e-mail: gj.peters@vumc.nl Key words: PD-ECGF/TP, disease, MNGIE, angiogenic and tumor promoting effect, apoptosis, L-deoxyribose, fluoropyrimidine sensitivity, Oral fluoropyrimidines Abbreviations: ! activated sequence, (GAS); 2-deoxyribose-1-phosphate, (dR-1-P); 5-chloro-2,4-dihydroxypyridine, (CDHP); 5’deoxyfluorouridine, (5’DFUR); 5-benzylacyclouridine, (BAU); 5-fluorouracil, (5-FU); bovine adrenal capillary endothelial, (BACE); chick chorioallantoic membrane, (CAM-assay); deoxyribose, (dR); dihydropyrimidine dehydrogenase, (DPD); endothelial cell growth factor, (ECGF); embryonic stem, (ES); focal adhesion kinase, (FAK); glyceraldehyde-3-phosphate, (G3P); heme oxygenase-I (HO-I); human umbilical vascular endothelial cells, (HUVEC); inhibitor of alkaline phosphatase, (API); interferon stimulated response element, (ISRE); interferon-", (IFN-"); interferon-!, (IFN-!); interferon, (IFN); interleukin-8, (IL-8); intravenous, (i.v.); L-deoxyribose, (L-dR); matrix metalloproteinase 1, (MMP1); microvessel density, (MVD); Mitochondrial neurogastrointestinal encephalo myopathy, (MNGIE); molecular target of rapamycin, (mTOR); N-acetyl cysteine (NAC); orotate phosphoribosyltransferase, (OPRT); oxonate, (OXO); Platelet derived endothelial cell growth factor, (PD-ECGF); pyrimidine phosphorylase, (PyNPase); reactive oxygen species, (ROS); rheumatoid arthritis, (RA); thymidine kinase 2, (TK2); thymidine phosphorylase inhibitor, (TPI); thymidine phosphorylase, (TP); thymidine, (TdR); thymidine-monophosphate, (TMP); thymidylate synthase, (TS); Trifluorothymidine, (TFT); tumor necrosis factor-", (TNF-"); uridine phosphorylase, (UP); vascular endothelial growth factor, (VEGF) § Current address: Michiel de Bruin, The Netherlands Cancer Institute, Department of Molecular Biology, Plesmanlaan 121, 1066 CX Amsterdam Received: 28 September 2005; Accepted: 08 November 2005; electronically published: March 2006

Summary Platelet-derived endothelial cell growth-factor (PDECGF) is similar to the pyrimidine enzyme thymidine phosphorylase (TP) and hence plays a dual role in cell biology. A high expression is related to malignant angiogenesis and invasion, and is therefore associated with a poor prognosis. It has been postulated that the angiogenic effect of PDECGF/TP is related to the enzymatic activity of TP, which catalyzes the breakdown of thymidine to thymine and deoxyribose-1-phosphate (dR-1-P). The latter, in its parent form or in its sugar form, deoxyribose, may play a role in angiogenesis. It may interfere in cellular energy metabolism or be substrate in a chemical reaction generating reactive oxygen species. L-deoxyribose and a specific TP inhibitor, TPI, can reverse these effects, supporting the role of the enzymatic reaction and that of the sugar. The essential role of TP in cellular metabolism was demonstrated by the finding that a deficiency was associated with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), which is an autosomal, recessive disorder involving mitochondrial DNA alterations. This syndrome was not associated with abnormal vascularization, indicating that TP/PDECGF only plays a role in malignant angiogenesis. Besides the role in angiogenesis, TP also plays an important role in thymidine homeostasis and thus in the synthesis of TMP a precursor in DNA synthesis. A high TP may deplete thymidine and its nucleotides. TP has a also an important pharmacological action. It has a moderate or even negligible role in the activation of the antimetabolite 5-fluorouracil (5FU), but its phosphorolytic activity is essential for the activation of 5’-deoxyfluorouridine (5DFUR) to 5FU. 5DFUR is an intermediate in the activation of the oral 5FU prodrug Capecitabine (Xeloda). Since the expression of TP/PDECGF is high in solid tumors and its stroma, this may be responsible for selective activation of 5DFUR in tumor cells. Since TP is easily inducible by external signals, e.g. radiation, cytokine exposure (tumor necrosis factor, interleukin, interferon), or cytotoxics (e.g.

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De Bruin et al: Role of PD-ECGF/TP in health and disease paclitaxel), combinations of these agents with Capecitabine are currently being explored in the clinic. The mechanism of induction is not completely clear but may involve activation of the translation at the promoter site. TP expression is also regulated by the transcription factor NF-kB, hence targets against this nuclear factor may also affect TP expression and function. These agents may not only affect 5FDUR activation, but also that of the investigational drug Trifluorothymidine (TFT). This drug is a good substrate for TP leading to its inactivation; in order to improve its bioavailability TFT is combined with TPI in the formulation TAS-102, which may have a dual action; TPI may inhibit angiogenesis but is essential for prevention of TFT breakdown. TFT will subsequently be activated. The various complex interactions of TP/PDECGF give it an essential role in cellular functioning and hence it is an ideal target in cancer chemotherapy.

in which angiogenesis and macrophage infiltration play a role in the pathology, one of these is rheumatoid arthritis (RA) (Asai et al, 1993). PD-ECGF was discovered in the late 1980s in platelets and presented as a classical growth factor with angiogenic properties (Miyazono et al, 1987). It was later demonstrated that PD-ECGF is identical to the enzyme thymidine phosphorylase, known to play a role in the pyrimidine metabolism. PD-ECGF will be designated throughout this review as PD-ECGF/TP. This enzyme catalyzes the phosphorolytic cleavage of thymidine (TdR) to thymine and dR-1-P (Figure 1). PD-ECGF/TP participates in many pathological and non-pathological processes (Table 1).

I. Introduction Platelet derived endothelial cell growth factor (PDECGF) plays a dual role in tumor biology. It promotes angiogenesis and plays a role in the metabolism of different fluoropyrimidines. Numerous immunohistochemical and TP-enzyme activity studies have shown that PD-ECGF/TP is upregulated in a broad range of solid tumors compared to normal healthy tissue (Brown and Bicknell, 1998; Ackland and Peters, 1999). It has a pro-angiogenic activity, stimulates the development of metastases (Maeda et al, 1996) and in many studies it was shown to be an independent prognostic factor for poor outcome of the disease (Takebayashi et al, 1996a). Furthermore, PDECGF has been found to be upregulated in other diseases

Figure 1. The phosphorolytic cleavage of TdR, catalyzed by PD-ECGF/TP. TdR can be phosphorylated by thymidine kinase (TK) to TMP, a precursor for DNA synthesis.

Table 1. List of processes in which PD-ECGF/TP is involved, ranging from physiological processes such as TdR homeostasis to patho-physiological processes such as tumor angiogenesis

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Cancer Therapy Vol 4, page 101 To date several hypotheses have been formulated concerning the mechanism of the pro-angiogenic effect, all involve the generation of dR-1-P and dR from the phosphorolytic breakdown of TdR. This makes that there are two approaches possible regarding this enzyme, one is the inhibition of PDECGF/TP, thereby decreasing its angiogenic effect, the other is to utilize PD-ECGF/TP as an activator of specific fluoropyrimidine pro-drugs in order to obtain accumulation of the cytotoxic species of the drug in the tumor (Focher and Spadari, 2001; Marchetti et al, 2001). A higher accumulation of these species in the tumor can be achieved by stimulation of PD-ECGF/TP-activity. This activity can be increased by both cytotoxic compounds such as taxanes (Sawada et al, 1998) and biological agents such as TNF-" and IFNs (Eda et al, 1993).

a survival promoting effect on cortical neurons in culture (Asai et al, 1992b; Ueki et al, 1993). This neurotrophic protein also had a large sequence similarity to PD-ECGF and was thought to be the same protein (Asai et al, 1992b). All three enzymes are considered to be identical (EC 2.4.2.4). PD-ECGF and TP are used interchangeably throughout the literature; however, the use of gliostatin is mainly confined to rheumatoid arthritis and neurologic research.

B. PD-ECGF/TP expression in health and disease The protein is expressed in normal tissues and cells, including macrophages, Kupffer cells, endothelial cells, ovary, salivary gland, brain (Fox et al, 1995; Yoshimura et al, 1990) and placenta (Jackson et al, 1994). In the placenta there is an other form of PD-ECGF/TP protein which contains 5 additional amino acids in the N-terminus and is processed at Thr-6 instead of Ala-11, as is known for the PD-ECGF/TP derived from platelets (Usuki et al, 1990). Besides this differently processed PD-ECGF/TP there appears to be a 27 kD splice variant in the placenta (Jackson et al, 1994). In numerous histochemical studies increased PD-ECGF/TP expression compared to normal tissue, was found in breast- (Moghaddam et al, 1995), bladder- (O'Brien et al, 1995, 1996), gastric- (Yoshimura et al, 1990; Takebayashi et al, 1996b), colorectal(Yoshimura et al, 1990; Takebayashi et al, 1996b), lungcancer (Giatromanolaki et al, 1998b; O'Byrne et al, 2000) and in several other tumors. Increased PD-ECGF/TP expression was confirmed, by measuring TP activity in normal and corresponding tumor tissue by Miwa et al (Miwa et al, 1998). Previous studies focusing on PDECGF/TP as a pyrimidine enzyme had demonstrated a higher expression in colon tumor tissue compared to normal tissue (Peters et al, 1991). High TP has been shown to be a prognostic factor for poor survival in gastric and colorectal cancer (Takebayashi et al, 1996a, b; Matsumura et al, 1998; van Triest et al, 2000), but in esophageal carcinoma there are conflicting reports (Ikeguchi et al, 1999; Koide et al, 1999). PD-ECGF/TP is an independent prognostic factor in gastric carcinoma, where it has been shown that increased PD-ECGF/TP also correlated with increased hepatic metastases, possibly as a reflection of increased vascularization (Maeda et al, 1996). In the study from Takebayashi et al it was shown in colorectal cancer, that only Dukesâ&#x20AC;&#x2122; stage and TPexpression were independent prognostic factors for poor disease outcome. PD-ECGF/TP can be correlated to a poor disease outcome without being correlated to microvessel density (MVD), (Suzuki et al, 2001). In Table 2 adapted from Morita et al (Morita et al, 2001) the results of 58 studies are summarized, in which PD-ECGF/TP was measured (immunohistochemically, PCR or activity). In 31 of these (high) PD-ECGF/TP expression correlated with angiogenesis, in 11 it didnâ&#x20AC;&#x2122;t and in 16 it was not evaluated. Furthermore, in 20 of the studies PD-ECGF/TP correlated with poor prognosis, 13 did not and 25 were not evaluated for this parameter. The lack of a clear association of PD-ECGF/TP with angiogenesis might be due to other activated angiogenic pathways or activation

A. PD-ECGF/TP PD-ECGF is an angiogenic factor discovered in 1987 (Miyazono et al, 1987) and was presented as a classical growth factor. It was reported to be mitogenic in experiments using (3H)-thymidine incorporation as growth parameter (Miyazono et al, 1987). PD-ECGF was cloned and sequenced; its subsequent expression in transformed NIH 3T3 cells resulted in more vascularized tumors compared to the control (Ishikawa et al, 1989). PD-ECGF is located on chromosome 22 and is around 1.8-kb, consisting of 10 exons in a 4.3-kb region (Hagiwara et al, 1991; Stenman et al, 1992). Lysates from PD-ECGF transfected cells were able to induce (3H)-thymidine incorporation in endothelial cells, while conditioned medium from these cells did not have this effect (Ishikawa et al, 1989). This reflected the lack of a hydrophobic leader sequence, necessary for secretion. In this same study it was shown that partially purified PD-ECGF could induce endothelial cell migration and had a strong angiogenic response on the vascular system of the chick chorioallantoic membrane (CAM-assay) (Ishikawa et al, 1989). Sequence analysis of the gene revealed a stretch of 120 amino acids to be identical to thymidine phosphorylase (TP), an enzyme catalyzing the reversible phosphorolysis of thymidine to thymine and 2deoxyribose-1-phosphate (dR-1-P) (Furukawa et al, 1992). TP was known to be active in platelets as part of pyrimidine metabolism (Shaw et al, 1988). Subsequently this enzymatic activity was identified for PD-ECGF (Moghaddam and Bicknell, 1992; Usuki et al, 1992; Sumizawa et al, 1993). The enzymatic activity seems indispensable for the angiogenic effect, since a competitive inhibitor of TP blocked the angiogenic effect (Haraguchi et al, 1994). Site-directed mutagenesis rendered an inactive PD-ECGF/TP protein, which no longer possessed angiogenic activity (Miyadera et al, 1995;Moghaddam et al, 1995). Since (1) the enzymatic activity is indispensable for the angiogenic effect, (2) there is no hydrophobic leader sequence and (3) no receptor for PD-ECGF/TP has been discovered, PD-ECGF/TP is not a classical growth factor. PD-ECGF/TP is also known as gliostatin, which was isolated as a protein possessing a growth inhibitory effect on glial cells but not neuronal cells (Asai et al, 1992a). Gliostatin is neurotrophic and has 101

De Bruin et al: Role of PD-ECGF/TP in health and disease Table 2. Several studies have been categorized according to the outcome of PD-ECGF/TP expression and angiogenesis and prognosis. Overall, there was a positive correlation between PD-ECGF/TP expression and occurrence of angiogenesis (29 / 55 studies) and poor prognosis (19 / 55 studies). Reproduced from Morita et al, 2001 with kind permission from American Chemical Society.

Disease Uterine cervical cancer

PD-ECGF/TP correlation with: Angiogenesis Prognosis Yes / No / ND Yes / No / ND 3/2/0 3/0/2

References

Endometrial cancer

2/1/1

0/3/1

Ovarian cancer Non small cell lung cancer Esophageal cancer

1/0/2 2/0/3

1/0/2 1/1/3

3/0/2

3/2/0

Gastric cancer

4/0/1

3/0/2

Colon cancer

5/0/0

2/0/3

Breast cancer

3/3/2

1/5/2

Pancreatic cancer Bladder cancer

2/0/0 0/1/5

2/0/0 1/2/3

Renal cell carcinoma Head and neck cancer

1/1/0 2/1/0

2/0/0 1/0/2

Prostate Glioma Hepatocellular carcinoma Oral squamous cell carcinoma Overall # studies

1/1/0 1/0/0 0/1/0

0/0/2 0/0/1 0/0/1

Tokumo et al, 1998; Fujimoto et al, 1999; Hata et al, 1999b; Kodama et al, 1999; Ueda et al, 1999 Fujiwaki et al, 1998, 1999b; Sakamoto et al, 1999; Sivridis et al, 2002a Reynolds et al, 1994; Hata et al, 1998; Hata et al, 1999a Koukourakis et al, 1997; Giatromanolaki et al, 1998b; Volm et al, 1998; Volm et al, 1999; Yamashita et al, 1999 Igarashi et al, 1998; Takebayashi et al, 1999; Ikeguchi et al, 1999; Koide et al, 1999; Yamagata et al, 1999 Maeda et al, 1996; Takebayashi et al, 1996b; Takahashi et al, 1998; Yoshikawa et al, 1999; Saito et al, 1999 Takahashi et al, 1996; Takebayashi et al, 1996a; Saeki et al, 1997; Matsumura et al, 1998; Shomori et al, 1999 Toi et al, 1995a, 1997, 1999; Fox et al, 1996, 1997; Engels et al, 1997; Leek et al, 1998; Mimori et al, 1999 Fujimoto et al, 1998; Takao et al, 1998 O'Brien et al, 1996; Kubota et al, 1997; Mizutani et al, 1997; Sawase et al, 1998; Tanaka et al, 1999; Arima et al, 2000 Imazano et al, 1997; Suzuki et al, 2001 Giatromanolaki et al, 1998a; Fukuiwa et al, 1999; Koukourakis et al, 2000 Sugamoto et al, 1999; Sivridis et al, 2002b Nakayama et al, 1998 Yamamoto et al, 1998

1/0/0

0/0/1

Alcalde et al, 1997

31 / 11 / 16

20 / 13 / 25

of different pathways, either resulting in or resulting from diffeent pathological features of the tumor. An inverse relation between PD-ECGF/TP and VEGF has been described for cervical cancers, and there was no correlation between MVD and PD-ECGF/TP (Tokumo et al, 1998). It was described by Oâ&#x20AC;&#x2122;Brien et al that PDECGF/TP expression in invasive bladder tumors was 260 fold higher than in normal mucosa, and 33-fold higher than in superficial tumors (O'Brien et al, 1995). The reverse was true for VEGF, which was lower in invasive compared to superficial tumors (O'Brien et al, 1995). In a study in pancreatic cancer it was found that both VEGF and PD-ECGF/TP were correlated to MVD; VEGF and MVD were not predictive for overall survival, but elevated PD-ECGF/TP was correlated with poor a survival (Fujimoto et al, 1998). Van Triest et al also found coexpression of VEGF and PD-ECGF/TP in colorectal cancer and correlation with MVD. High PD-ECGF/TP also proved to be an independent prognostic factor (van Triest et al, 2000). Co-expression of PD-ECGF/TP and VEGF was found in breast cancer (Toi et al, 1995b). The co-expression of PD-ECGF/TP and VEGF is not uniform and varies per tumor type and study. VEGF is generally correlated with MVD and can be considered as the major pro-angiogenic factor. However, PD-ECGF/TP only seems to play an additional role in certain circumstances and tumor types.

Tumors are heterogeneous tissues consisting of unknown variable contributions of tumor, stromal and infiltrating cells all may express PD-ECGF/TP (Takahashi et al, 1996; Giatromanolaki et al, 1998b; Matsumura et al, 1998; van Triest et al, 2000). It has been shown that TP can be increased in malignant cells rather than in the tumor stroma (Maeda et al, 1996). In some tumors PDECGF/TP seems to be present both in malignant and stromal cells (Takebayashi et al, 1996a). Koukourakis et al reported TP expression of cancer cells, stromal cells (stroma associated fibroblasts) and infiltrating cells like macrophages (Koukourakis et al, 1998). In this study it was shown that PD-ECGF/TP expression in various cells might have different effects and different associations withprognosis. PD-ECGF/TP over-expression in cancer cells was related with poor prognosis, while PD-ECGF/TP expression of the stroma was related to a better survival in a subset of the patients. It was hypothesized that PDECGF/TP in the stroma was a marker of infiltrating macrophages, cells that are evidently involved in tumor biology. Infiltrating macrophages have been identified as producers of pro- and anti-angiogenic factors promoting or inhibiting tumor growth, however, overall they appear to be promoting tumor growth, contributing to the pathology (Bingle et al, 2002). Co-localization of macrophages and PD-ECGF/TP, alone or with other angiogenic factors, e.g. VEGF, interleukin 8 (IL-8) has been studied extensively

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Cancer Therapy Vol 4, page 103 by immunohistochemsitry. In most of these studies coexpression of PD-ECGF/TP and macrophages was observed in melanoma (Torisu-Itakura et al, 2000), glioblastoma multiforme (Hirano et al, 2001), breast (Toi et al, 1999; Ueno et al, 2000), cervical (Fujimoto et al, 2002), colon (Takahashi et al, 1996; Zhang et al, 2004), and prostate cancer (Sivridis et al, 2002). The expression of PD-ECGF/TP is often both cytoplasmic and nuclear (Fox et al, 1996; Yang et al, 2000). In a study on gallbladder adenocarcinomas only nuclear PD-ECGF/TP staining correlated with increased angiogenic activity (Giatromanolaki et al, 2002). In summary, PD-ECGF/TP has been found to have higher expression in tumor tissue compared to normal tissues in a variety of human malignancies, and its expression is not only found in cancer cells but also in the stromal macrophages, lymphocytes and fibroblasts. Overall a high level of PD-ECGF/TP expression is correlated with a higher MVD, more metastases and it appears to be a poor prognostic factor. Two other examples of these particular diseases are osteoarthritis and inflammatory bowel disease (Giatromanolaki et al 2003).

C. Other ECGF/TP

diseases

expressing

adenocarcinoma patients the serum level was higher than those of healthy controls (Katayanagi et al, 2003). This seemed to reflect that the tumor-tissue levels showed an elevated PD-ECGF/TP expression compared to the healthy tissue (Katayanagi et al, 2003). In hepatocellular carcinoma patients serum PD-ECGF/TP was increased in late stage compared to early stage disease, reflecting immunohistochemical stainings showing that PDECGF/TP was increased in tumor compared to normal tissue (Jin-no et al, 1998). In the sera and synovial fluids of patients suffering from rheumatoid arthritis PD-ECGF/TP could be detected at high levels, (Asai et al, 1993). In addition, there was a significant positive correlation between PD-ECGF/TP levels in synovial fluid and in serum (Asai et al, 1993). Other findings suggested that serum PD-ECGF/TP levels could be used as indicator for synovitis and the efficacy of surgical treatment (Muro et al, 2001). The elevated PDECGF/TP levels presumably arise through induction of PD-ECGF/TP in synoviocytes, resulting from aberrant production of cytokines like TNF" and IL1 (Waguri et al, 1997). In conclusion, in both cancer and RA tissues elevated levels of PD-ECGF/TP have been found. This indicates active angiogenesis, known to play an important role in the pathology of both diseases (Folkman, 1995; Koch, 2000). The origin of PD-ECGF/TP in the circulation remains ground for speculation. There are several possibilities such as shedding from tumor cells and synoviocytes or active excretion from the producing cells. Although PD-ECGF/TP lacks a hydrophobic leader (Ishikawa et al, 1989), other pathways might be in place to excrete PD-ECGF/TP. These pathways might be similar to those described for acidic and basic fibroblast growth factor (Powers et al, 2000). The serine residues of PDECGF/TP can be covalently linked to phosphate groups of nucleotides, leading to a nucleotidylated protein, a posttranslational process possibly playing a role in the excretion process (Usuki et al, 1991). There are at least two tumor cell lines, A341 and MKN74 and cytokine treated fibroblast-like-synoviocytes that appear to actively excrete PD-ECGF/TP in the medium (Matsukawa et al, 1996; Waguri et al, 1997). Another less explored possibility to explain the increased PD-ECGF/TP in sera and plasma of cancer and RA patients might be the release of platelet content. It is known that activated platelets deposit the content of their "-granules on (activated) endothelium, thereby releasing several angiogenic and coagulation factors. This can lead to a rise of angiogenic factors at the site of deposition and subsequently in the circulation of patients with active angiogenesis (as in cancer and rheuma patients) (Pinedo et al, 1998). One of the proteins present in " granules is PD-ECGF/TP. It can be speculated that the level of PD-ECGF/TP in peripheral blood mononuclear cells and platelets can be further elevated through PD-ECGF/TP inducing cytokines.

PD-

Increased PD-ECGF/TP mRNA and immunoreactivity were found in lesional psoriasis compared to non-lesional skin (Creamer et al, 1997). In another study it was shown that the thymidine phosphorylase activity was twenty-fold higher in psoriatic lesions than in normal skin (Hammerberg et al, 1991). There is one study, which examined the activity and distribution of PD-ECGF/TP in the nasal mucosa of people with nasal allergy, and observed that the mucosal TP-activity of patients was higher than that of the normal controls. Strong staining of eosinophils was observed, indicating that the enhanced activity might be due to an increased number of PD-ECGF/TP expressing infiltrating cells (Nishimoto et al, 2000). Elevated levels of (circulating) PD-ECGF/TP were found in rheumatoid arthritis patients (Asai et al, 1993; Giatromanolaki et al 2003). The diseases in which an elevation of PDECGF/TP has been described thus far are immune system related and have features of chronic inflammation.

D. Circulating PD-ECGF/TP Pauly et al identified and compared TP activity in the plasma of healthy subjects and cancer patients (Pauly et al, 1977) and found that TP activity was higher in the plasma of cancer patients. This finding was confirmed in the ascites and plasma of tumor bearing animals in which TP activity was elevated compared to healthy animals (Pauly et al, 1978). Although this discovery was disputed by Woodman (Woodman, 1979), it has been found again in later studies (Poon et al, 2001; Shimada et al, 2002; Brostjan et al, 2003). In patients with uterine cervical cancer it was shown that serum PD-ECGF/TP levels had a positive correlation with clinical stage and tumor size (Fujimoto et al, 2000). The prognosis of the patients with high serum PD-ECGF/TP levels was extremely poor (Fujimoto et al, 2000). In a group of gastric

E. MNGIE Mitochondrial neurogastrointestinal encephalo myopathy (MNGIE) is an autosomal, recessive disorder involving mitochondrial DNA alterations (Bardosi et al, 103

De Bruin et al: Role of PD-ECGF/TP in health and disease 1987; Hirano et al, 1994). The onset of the disease is between the first and fifth decades and is characterized by ptosis, progressive external opthalmoparesis, gastrointestinal dysmotility, cachexia, peripheral neuropathy, and leukoencephalopahty. Further analysis of muscle biopsies showed mitochondrial abnormalities such as ragged-red fibers and focal cytochrome c oxidase deficiency sometimes in association with multiple respiratory chain enzyme defects. The mitochondrial DNA in the skeletal muscle is partially depleted, has multiple deletions or both (Nishino et al, 2001). The gene responsible for the disease proved to be PD-ECGF/TP (Nishino et al, 1999). Potential gene mutations in PDECGF/TP exons were screened in 35 patients from 21 distinct families, which resulted in the finding of 16 mutations such as missense, splice site, microdeletions and single nucleotide insertions (Hirano et al, 1998; Nishino et al, 1999). As a result, these mutations in PD-ECGF/TP are associated with a severe reduction of the enzyme activity (Nishino et al, 1999). Heterozygotic activity is between the normal and the impaired activity, which might be a reflection of the heterodimer structure of the protein (Spinazzola et al, 2002). The impairment of the TPenzyme activity leads to increased thymidine plasma levels, up to 60-fold and increased deoxyuridine levels which is also a substrate for PD-ECGF/TP (Marti et al, 2003). Mitochondrial dNTP pools are more dependent on the thymidine salvage pathway than de novo synthesis, and constitutively expressed, mitochondrial thymidine kinase 2 (TK2), converting thymidine to thymidine-monophosphate (TMP), is a result of this dependency. The conversion to TMP, TDP and TTP is the only metabolic pathway for TdR in MNGIE patients. It is hypothesized that due to the high levels of thymidine, the mitochondrial dNTP pools are altered and lead to mtDNA depletions and multiple deletions in these patients (Spinazzola et al, 2002). The impact of PD-ECGF/TP deficiency on the mitochondrial dNTP pool and mtDNA is more severe than on the nuclear pools because: 1) TK2 is constitutively expressed and cytosolic TK1 is not, 2) mitochondria have physically separate nucleotide pools and 3) mtDNA constantly replicates, even in post-mitotic cells. One important fact is that in MNGIE patients so far no vascular abnormalities have been identified, suggesting that absence of TP-activity does not interfere with normal angiogenesis (Nishino et al, 1999).

which would prevent its incorporation into DNA. When PD-ECGF/TP was added to endothelial cells, it increased the uptake of (3H)-thymidine when added as a pulse. An explanation for this might be that PD-ECGF/TP depletes TdR in the medium thereby indirectly decreasing the TdR concentration in the cells; a subsequent pulse would then replenish the intracellular pool of TdR with (3H)thymidine. Increasing concentrations of PD-ECGF/TP led to a bell shaped curve, TdR depletion is replenished to a maximum after PD-ECGF/TP breakdown. In the declining part of the curve the amount of PD-ECGF/TP starts to breakdown the (3H)-thymidine pulse leaving less for replenishment and apparent incorporation (Usuki et al, 1992). When PD-ECGF/TP and (3H)-thymidine were added simultaneously, there was a decrease of (3H)thymidine incorporation, due to direct breakdown of (3H)thymidine by PD-ECGF/TP (Usuki et al, 1992). This phenomenon of increasing (3H)-thymidine apparent incorporation, could be found in both dividing human umbilical cord endothelial cells (HUVECs) and quiescent, non dividing bovine adrenal capillary endothelial (BACE) cells, (Moghaddam and Bicknell, 1992). PD-ECGF/TP might have mediated TdR uptake as a result of the proteinâ&#x20AC;&#x2122;s intrinsic thymidine phosphorylase activity. It appears therefore that the original identification of PDECGF/TP as a mitogenic activator of endothelial cell growth has been based on an erroneous assay since (3H)thymidine incorporation into DNA does not always reflect cellular proliferation. There is however one paper which debates these findings, stating that PD-ECGF/TP reduces the amount of TdR in the surroundings of endothelial cells, thereby reducing the inhibitory effect of TdR on EC growth, thus giving rise to cell growth and to the bell shaped curve (Finnis et al, 1993). In our experiments using human umbilical vascular endothelial cells (HUVEC) in growth assays we found no growth promoting activity of PD-ECGF/TP, using the MTT assay (Figure 2). Parallel to the experiments showing possible mitogenic effects of PDECGF/TP other experiments were undertaken to further clarify the angiogenic effect of PD-ECGF/TP. It was shown that PD-ECGF/TP had a chemotactic effect on endothelial cells in vitro, had angiogenic capacity in the CAM assay and induced more vascularized tumors formed by NIH3T3 cells in nude mice, compared to its mocktransfected counterparts (Ishikawa et al, 1989). Another approach taken to show the angiogenic effect of PDECGF/TP was the use of (gelatine) sponge assays, whereby the sponges with potential angiogenic compounds are implanted in a subcutaneous pouch in mice or rats. After removal the hemoglobin content extracted from the sponge is a measure of the vascularization. It was shown that PD-ECGF/TP indeed increased the hemoglobin content in this assay (Haraguchi et al, 1994; Moghaddam et al, 1995)which could be inhibited by a competitive inhibitor, once again indicating the importance of the enzymatic reaction (Moghaddam et al, 1995). Therefore, despite the fact that the original assay of (3H)-thymidine incorporation might not have been interpreted correctly, there is still ample evidence for angiogenic properties of PD-ECGF/TP.

II. Experimental proof of angiogenesis and possible mechanisms A. Basic observations and transfected models After the original discovery of PD-ECGF, the initial identification of the angiogenic effect was based upon the incorporation of (3H)-thymidine into endothelial cells (Miyazono et al, 1987). Subsequently it was discovered that PD-ECGF was identical to the well known enzyme from pyrimidine metabolism, TP (Furukawa et al, 1992). Due to this fact the (3H)-thymidine incorporation studies need to be viewed in a different light, since the PDECGF/TP converts TdR into thymine (Usuki et al, 1992),

104

Cancer Therapy Vol 4, page 105 Figure 2. Human umbilical vascular endothelial cells (HUVECs), were cultured in the presence of crude endothelial cell growth factor (ECGF), isolated from bovine brain, which was set at 100 %. Addition of PD-ECGF/TP with or with TdR did not result in additional growth (De Bruin et al, unpublished data).

Many cell lines have been transfected with PDECGF/TP, either to study the angiogenic effect or to study its role in fluoropyrimidine sensitivity. The first example comes from Ishikawa et al, (1989) PD-ECGF/TP provided NIH3T3 transfected cells with a growth advantage, resulting in more vascularized tumors. Interestingly, we observed that wild-type NIH3T3 cells, which have no PDECGF/TP did not form tumors in our experiments. However, the ras- and trk- transfected variants had high PD-ECGF/TP and formed well vascularized tumors (Peters et al, 1993). MCF7, breast cancer cells transfected with PD-ECGF/TP, also resulted in tumors with enhanced tumor growth in vivo (Moghaddam et al, 1995). PD-ECGF/TP transfected human epidermoid cells grew more rapidly in xenograft experiments than the parental line which was associated with an elevated MVD. This effect was not observed when cells were transfected with mutant PD-ECGF/TP rendering enzymatic inactive proteins (Nishimoto et al, 1997). Later this model has been used to study the impact of PD-ECGF/TP on apoptosis (Matsushita et al, 1999) and suppression of PD-ECGF/TP mediated growth and angiogenesis by L-deoxyribose (LdR) (Kitazono et al, 1998; Uchimiya et al, 2002). RT112 bladder cells transfected with PD-ECGF/TP, showed an increased invasion capacity in an in vitro bladder invasion assay (Jones et al, 2002). Further testing of these cells in xenograft transplants showed that the RT112-TP cells grew significantly faster than the mock transfected RT112EV (Jones et al, 2002). In order to study the impact on fluoropyrimidine sensitivity, Marchetti et al transfected a rat colon carcinoma cell line, PROb with PD-ECGF/TP, which increased its sensitivity to several fluoropyrimidines. However the effect on tumor growth in vivo was relatively low and appeared to be confined to the initial stages of tumor development; staining for the endothelial cell marker factor VIII was always higher in transfected cells compared to control cells (Marchetti et al, 2001). Ciccolini et al also reported that LST174 PD-

ECGF/TP transfected cells did not grow faster than the wild-type cells, furthermore there was no increase in endothelial markers (Ciccolini et al, 2001). This lack of growth advantage in PD-ECGF/TP transfected mice and human cells has been reported in previous studies from the same group (Evrard et al, 1999b; Ciccolini et al, 2000). In conclusion, in several of the transfected model systems PD-ECGF/TP does increase tumor growth and sometimes MVD and the malignant phenotype. However studies have been published in which the transfer of the PD-ECGF/TP gene did not have an impact on tumor growth. Possibly other angiogenic factors or conditions, not influenced by transfection of PD-ECGF/TP determined the angiogenic potential and tumor growth in these model systems.

B. Mechanistic background of the angiogenic and tumor promoting effect of PDECGF/TP Most research on PD-ECGF/TP was performed to show its presence with immunohistochemical stainings; but there are very few studies on the mechanistic background. The enzymatic activity seems indispensable for the angiogenic effect of PD-ECGF/TP. The lack of a hydrophobic leader sequence and a receptor, makes that it is not a classical angiogenic factor. Since enzymatic activity is of importance for the angiogenic activity of PDECGF/TP, the focus of research has been on the products produced in the enzymatic reaction: thymine, dR-1-P and deoxyribose (dR), the dephosphorylated product of dR-1-P (Brown and Bicknell, 1998) and to a lesser extent the decrease of TdR. Sengupta et al observed that dR and dR1-P promoted endothelial tube formation and the regeneration of an endothelial monolayer in the wound assay in vitro without stimulating mitogenesis. In vivo, TP and dR increased vascularization in the sponge assay, in which the clearance of a radiolabel (133Xe) was a measure 105

De Bruin et al: Role of PD-ECGF/TP in health and disease for the extent of vascularization of the sponge (Sengupta et al, 2003). It was shown that PD-ECGF/TP had chemotactic activity in vitro (Ishikawa et al, 1989). dR was able to induce migration of bovine aortic endothelial (BAE) cells similar to PD-ECGF/TP. The results were repeated in the CAM assay, in which dR and PDECGF/TP could induce development of the vascular system. Hotchkiss et al, (2003a) found that the production of dR-1-P and dR explained how PD-ECGF/TP, expressed by either tumor cells or monocytes mediates endothelial cell migration. Tumor cells, transfected with PDECGF/TP or monocyte-like cell lines THP1 and U937 naturally expressing PD-ECGF/TP were used. Purified PD-ECGF/TP can mediate a chemotactic effect in the endothelial migration assay, for which TdR is required. This confirmed the necessity of enzymatic activity, since the effect was abrogated in the absence of substrate. The importance of enzymatic activity was further strengthened by inhibition of migration in the presence of an inhibitor of PD-ECGF/TP. In contrast to Sengupta et al, (2003), Hotchkiss et al found no effect of PD-ECGF/TP in a wound assay (Hotchkiss et al, 2003a), in their view confirming the need of a gradient of dR. It was shown that dR was 10 times more potent in inducing migration than dR-1-P, while the combined effect of PD-ECGF/TP plus TdR was comparable to dR alone. The hypothesis that dR1-P needs to be converted to dR before inducing maximal chemotactic effects, was tested and confirmed by the addition of an inhibitor of alkaline phosphatase (API), which attenuated not only the migration induced by dR-1P but also by PD-ECGF/TP. The inhibition of PDECGF/TP induced migration by API, while blocking the dephosphorylation of dR-1-P confirmed that this conversion to dR was part of the mechanism. Altogether, the data using cell lines, naturally expressing high PDECGF/TP or by transfection, underlined that migration was dependent on dR produced by PD-ECGF/TP. These cell lines released more dR than dR-1-P. dR-1-P is formed intracellularly and is not able to diffuse to the extracellular space. Its conversion to dR is likely due to an intracellular phosphatase resulting in a chemotactic dR concentration gradient after subsequent extracellular release (Hotchkiss et al, 2003a). These authors provided the first substantial evidence that dR-1-P is converted to dR. The exact mechanism by which dR might exert chemotactic effects on the endothelial cells is largely unknown. It has been proposed that a chemotactic gradient is detected at opposite sides of the cell, followed by migration through pseudopodal processes. However these actions are usually mediated through specific receptors, which have so far not been identified for dR on endothelial cells (Brown and Bicknell, 1998). Corneal endothelial cells indeed migrate in the direction of simple sugars which could be utilized as an energy source(Vogel et al, 1993). Hotchkiss et al, (2003b) identified the formation of focal adhesions and the phosphorylation of focal adhesion kinase (FAK) in HUVECs after both PD-ECGF/TP or dR stimulation. FAK is a non-receptor protein kinase that is recruited to focal adhesions by integrin engagement with the extracellular matrix and plays a central role in

mediating cell attachment and migration. It was observed that VEGF, PD-ECGF/TP and dR similarly stimulated the formation of these focal adhesions and phosphorylation of FAK in HUVECs. There was however a difference in the involvement of specific integrins. PD-ECGF/TP migration was blocked by antibodies against "5#1 and "V#3, whereas VEGF induced migration seemed to involve only "V#3 (Hotchkiss et al, 2003b). Seeliger et al describe that dR activates p70/S6 kinase, this activation could be blocked with rapamycin, an inhibitor of the molecular target of rapamycin (mTOR), thereby explaining the blockage of the pro-angiogenic effect of dR in an endothelial migration assay and in the rat aortic ring assay (Seeliger et al, 2004). Malik and Parsons investigated the interaction of integrin receptors with ECM proteins resulting in the activation of p70/S6 kinase, it was shown that for this activation there is at least a partial requirement for FAK (Malik and Parsons, 1996). These two observations together with the finding of Hotchkiss et al (Hotchkiss et al, 2003b) all implicate that dR induced signaling is through FAK and p70/S6 kinase. Furthermore, p70/S6 kinase has been shown to signal survival and inhibits the pro-apoptotic molecule BAD (Harada et al, 2001). At high non-physiological concentrations the reducing sugar dR can induce apoptosis in peripheral blood mononuclear cells (Barbieri et al, 1994) and fibroblasts (Kletsas et al, 1998). This cytotoxic effect was observed in cycling and in G0 arrested cells and could be reversed by the antioxidant N-acetyl cysteine (NAC) indicating a role for oxidative stress induced by dR (Kletsas et al, 1998). One process, which might be responsible for the induction of cell death, involves the high reducing capability of dR and dR-1-P. Reducing sugars are involved in non-enzymatic glycosylation of proteins: the so called Maillard reaction (Monnier, 1990). The Maillard reaction leads to the formation of specific protein adducts, known as advanced glycosylation end products (AGEs). The reaction is initiated by nonenzymatic condensation of a reducing sugar with an amine, occurring preferentially on lysine and arginine groups or N-terminal groups. The reactivity of the sugars is a function of the anomerization rate of the sugar, meaning the larger the percentage of a given sugar in the open chain form, the more reactive (Bunn and Higgins, 1981). The reaction rate is inversely proportional to the number of carbon atoms in the sugar, being lowest for hexoses and highest for trioses, like glyceraldehyde. Furthermore phosphorylated sugars are much more reactive than their non-phosphorylated counterparts (Monnier, 1990) After the condensation Schiff base adducts and Amadori products are formed. These unstable intermediates react via a series of non-enzymatic reactions to form AGEs. During these reactions, specifically in the transition metal-catalyzed autooxidation, free radicals are produced (Monnier, 1990) (Bierhaus et al, 1998). AGE formation plays a role in aging processes and diabetes related pathologies; this role appears to have two aspects: alterations of proteins due to glycosylation and induction of reactive oxygen species (ROS) (Baynes, 2001). Brown et al, (2000) postulated that the Maillard reaction could be the biological activity which might

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Cancer Therapy Vol 4, page 107 underlie the angiogenic effect of PD-ECGF/TP. They also suggested that the angiogenic effect of PD-ECGF/TP, which was increased in a PD-ECGF/TP overexpressing bladder tumor cell line RT112, might be related to an increase of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF) and matrix metalloproteinase 1 (MMP1) production after addition of TdR. A specific thymidine phosphorylase inhibitor (TPI) (Fukushima et al, 2000) and thymine inhibited this effect, both through abrogation of the enzymatic reaction (thymine by shifting the balance of the reaction). PD-ECGF/TP mediated TdR breakdown was associated with induction of ROS, since the expression of the oxidative stress marker heme oxygenase-I (HO-I) increased in transfected cells after incubation with TdR. This induction could be inhibited by N-acetyl cysteine (NAC), substantiating the evidence for ROS formation (Brown et al, 2000). It was hypothesized that dR, dR-1-P and dR-5-P, which potentially can be formed from dR-1-P through the action of phosphoribomutase (E.C. 5.4.2.7) react in the Maillard reaction (Monnier, 1990), thereby inducing ROS, leading to the upregulation of the described angiogenic factors (Brown et al, 2000). This is a novel finding that PDECGF/TP indirectly induces angiogenesis, without a direct interaction between products or substrates of the enzymatic reaction and endothelial cells. Nakajima et al found similar results showing that PD-ECGF/TP transfected KB epidermoid tumor cells, excreted IL-8 and VEGF in higher quantities (Nakajima et al, 2004). In human malignant melanoma it was found that there was an apparent co-expression of PD-ECGF/TP and HO-I in infiltrating macrophages (Torisu-Itakura et al, 2000), supporting the ROS hypothesis. These hypotheses on the link between TP-activity and angiogenesis are based on the formation of dR-1-P and further metabolism, in most cases to dR. Although all

papers concerning the angiogenic effect of PD-ECGF/TP mention the formation of dR from dR-1-P as a fact, evidence offering proof for this is scarce, and needs more exploration. Little is known of the cellular metabolism of dR-1-P. It can either be dephosphorylated to dR and leave the cell, or isomerized by phosphoribomutase to dR-5-P which can enter glycolytic metabolism. Anand and Anand question the fate of dR-1-P, mentioning the lack of knowledge about the metabolic pathways responsible for dR-1-P breakdown and conversion(Anand and Anand, 2000). Previous studies did not actually evaluate dR-1-P accumulation and disappearance after TdR exposure. De Bruin et al, (2003) studied the generation of dR-1-P and its accumulation after incubation with TdR in an intact colon cancer cell line and its PD-ECGF/TP transfected variant. Thymine production was measured in the medium providing an accurate estimate of the minimal amount of dR-1-P produced inside the cells. Subsequent analysis of cellular dR-1-P content showed that less than 1% of the estimated dR-1-P was detected, indicating that there was a very rapid disappearance of dR-1-P. This was confirmed in cellular extracts when dR-1-P was added directly and incubated and the majority of dR-1-P disappeared within 10 minutes (De Bruin et al, 2003a). In order to obtain insight to where the dR-1-P is disappearing deoxyribose or dR-5-P were added to influence the dephosphorylation or conversion into dR-5-P, respectively. Deoxyribose did not influence the disappearance whereas dR-5-P resulted in an initial increase followed by a decrease (Figure 3) (De Bruin et al, 2004). This disappearance could be decrease by dR-5-P and not by deoxyribose indicating that dR-1-P is converted to dR-5-P and is then on route to be metabolised in the glycolysis or pentose phosphate pathway.

Figure 3. The effect of 5000 pmol dR or dR-5-P on the flux of 500 pmol dR-1-P in Colo320TP1 cell lysates. To all incubations 10 ÂľM TPI was added to prevent formation of TdR. dR-1-P rapidly disappears in time, however dR-5P is able to slow down this process. After an initial rise, indicating that isomerization by phosphopentomutase is an equilibrium reaction, dR-1-P also disappears here.

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De Bruin et al: Role of PD-ECGF/TP in health and disease This indicates that there must be a rapid metabolism of dR-1-P since dR-1-P is unable to cross the cell membrane. There are at least two possibilities, it is either dephosphorylated to dR which subsequently is able to cross the membrane into the medium, or dR-1-P is isomerized to dR-5-P by phosphopentomutase (E.C. 5.4.2.7) and then split into glyceraldehyde-3-phosphate (G3P) plus acetaldehyde by deoxyriboaldolase (E.C. 4.1.2.4) (Sgarrella et al, 1997; Carta et al, 2001) (Figure 4). G3P can then enter the glycolytic pathway to yield ATP. This pathway has been described for bacteria, e.g. Bacillus cereus (Sgarrella et al, 1992) but has not yet been fully confirmed in eukaryotic cells although the enzymatic activity needed for this pathway was identified in amniotic WISH cells (Carta et al, 2001). dR-1-P may enter the glycolytic pathway thus providing energy to the cell. PDECGF/TP therefore could be able to contribute to the maintenance of adequate levels of ATP in the cell by releasing the dR-1-P moiety from thymidine. The angiogenic effects of PD-ECGF/TP and the metabolite dR1-P may have to be (re)viewed in the light of these possible pathways, and the speed with which dR-1-P fluxes through these cells and potentially affects oxidative

and energy status. If dR-1-P enters the glycolytic pathway, it will not leave the cell as the chemotactic dR. The question that remains is can the deoxyribose moiety be used for energy generation and result in a pro-angiogenic action? Furthermore, this question can be reevaluated for each cell type that is known to overexpress PD-ECGF/TP, e.g. macrophages, stromal and tumor cells. It is possible that the overload of rerouting into the glycolysis leads to down-stream metabolite accumulation, making dR-1-P and G3P available for Maillard reactions. Thus, several pathways could be potentially responsible for the disappearance of dR-1-P: 1) dephosphorylation and excretion from the cell, 2) routing into the glycolytic pathway, in the form of dR-5-P and G3P and 3) formation of AGE plus induction of reactive oxygen species (ROS) (Figure 4). In conclusion, the pro-angiogenic effect of PDECGF/TP has been suggested to be associated with the formation of dR. The product produced in the enzymatic reaction of PD-ECGF/TP however is dR-1-P, and its dephosphorylation and the rate at which this occurs is largely ignored within the field.

Figure 4. Possible pathways that can be followed by dR-1-P when it is converted or broken down. All the potential products could play a role in the pro-angiogenic capacity of PD-ECGF/TP. Routing into the glycolysis or pentose phosphate pathway contributes to the cellular energy demand, while all products might be involved in generation of reactive oxygen species through the formation of advanced glycosyslation endproducts (AGEs) thereby indirectly upregulating secondary pro-angiogenic factors. Finally dR can diffuse out of the cell and have a direct interaction and influence on endothelial cells.

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Cancer Therapy Vol 4, page 109 seems to be independent of the enzymatic activity (Mori et al, 2002). The KB/CV and its PD-ECGF/TP transfected counterpart KB/TP epidermoid cancer cells have been used extensively to study the role of PD-ECGF/TP in tumor biology (Matsushita et al, 1999). This model system in combination with TPI (Fukushima et al, 2000) has shown that PD-ECGF/TP is involved in increased tumor growth, increased angiogenesis, and decreased apoptosis (Matsushita et al, 1999). Furthermore, it was shown that TPI suppressed the development of liver metastases of KB/TP cells injected in the spleen (Takao et al, 2000). The properties of KB/TP cells, the potential angiogenic mechanisms through the metabolites of the TP reaction and the finding that dR protected against hypoxia induced apoptosis have prompted the investigation of the effect of L-dR on KB/TP cells. L-dR could counteract the effect of PD-ECGF/TP transfection in the KB cells and induced a decrease of tumor growth in xenografted mice and an increased apoptotic index in vivo. L-dR suppressed both dR induced endothelial cell chemotaxis and tube formation in vitro (Uchimiya et al, 2002). L-dR was also able to eliminate the inhibitory effect of dR on hypoxia induced apoptosis (Kitazono et al, 1998). In follow-up research using the KB/TP model system it was shown that L-dR was able to reduce the enhanced metastatic capacity of the transfected cells, lowering the number of metastatic nodules. The vascular area of the metastatic nodules was lower after treatment with L-dR and PD-ECGF/TP mediated resistance to apoptosis was also counteracted by L-dR (Nakajima et al, 2004). In vitro the invasive activity of KB/TP cells was only higher under hypoxic conditions. This might be indicative for an energetic advantage if dR1-P is shuttled into the glycolytic pathway. Similar to the cells described by Brown et al (Brown et al, 2000), the KB/TP cells had increased levels of IL-8 and VEGF in their conditioned medium. L-dR was able to reduce the excretion of these two angiogenic factors (Nakajima et al, 2004). L-dR seems to counteract the effects of PDECGF/TP overexpression, possibly, by interfering with the effects induced by dR, blockage of chemotaxis or preventing dR to enter the glycolysis. The authors suggest that L-dR might be a useful anti-metastatic agent for tumors overexpressing PD-ECGF/TP, thereby also interfering with the pro-angiogenic effect of PDECGF/TP.

C. The Influence of PD-ECGF/TP on apoptosis and the effect of L-deoxyribose on angiogenesis and apoptosis Takebayashi et al, (1996a) found PD-ECGF/TP to be an independent poor prognostic factor. Similarly Moghaddam et al, (1995) reported an increase in growth of breast carcinomas expressing PD-ECGF/TP, but without an increase of MVD. These results were the reason for Matsuura et al, (1999) to investigate whether PD-ECGF/TP might possess other functions than inducing angiogenesis, which might contribute to an adverse disease outcome. They examined the role of PD-ECGF/TP in apoptotic cell death, tumor cell proliferation and regulation of p53 in colorectal carcinomas. In vivo PDECGF/TP expression increased MVD and reduced apoptotic cell death, regardless of p53 expression and tumor stage (Matsuura et al, 1999). Possibly apoptosis is reduced in well vascularized tumors. Similar results were reported in gastric and esophageal cancers, where PDECGF/TP was associated with MVD increase and decrease in apoptosis (Osaki et al, 2000; Ikeguchi et al, 2001a,b; Okamoto et al, 2001). However, there was no correlation between the apoptotic index and PD-ECGF/TP expression in cervical cancer (Fujiwaki et al, 1999a). A relation between PD-ECGF/TP and hypoxiainduced apoptosis was found in the human epidermoid carcinoma KB cells. A PD-ECGF/TP transfected and nontransfected variant of the KB cells were used. Both had similar growth rates under normoxic conditions but there was growth advantage for the KB/TP cells under hypoxic conditions (Kitazono et al, 1998). This advantage disappeared when the highly specific thymidine phosphorylase inhibitor (TPI) (Fukushima et al, 2000) was added. The cells transfected with PD-ECGF/TP were resistant to hypoxia induced apoptosis. The abrogation of this protective effect through inhibition of the enzymatic activity with TPI and the fact that metabolites of the TP reaction, thymine and deoxyribose (dR), could mimic the protective effect in mock-transfected KB/CV cells, indicates that enzymatically active PD-ECGF/TP confers this resistance (Kitazono et al, 1998). Besides hypoxia induced apoptosis, PD-ECGF/TP transfected Jurkat cells were protected against cisplatin induced apoptosis, however here the enzymatically inactive mutant PDECGF/TP conferred the same effect (Ikeda et al, 2003). A potential molecular mechanism for the inhibition of apoptosis by dR, described by Ikeda et al, (2002) was the suppression of pro-apoptotic events and proteins, like down-regulation of cytochrome C release, decreasing the activity of caspases 3, 8 and 9 and prevention of the downregulation of the anti-apoptotic proteins Bcl-2 and Bcl-XL after hypoxic stimulation. Furthermore it was shown that dR substantially suppressed the induction of hypoxia inducible factor 1-" (HIF-1-"), which might be another part of the anti-apoptotic effect dR. It appeared that dR enhanced the ubiquination of HIF-1-", which regulates the levels of this protein, which is downregulated under hypoxic conditions (Ikeda et al, 2002). In contradiction with this finding is the observation that protection conferred by PD-ECGF/TP to FAS induced apoptosis

III. Role of PD-ECGF/TP fluoropyrimidine sensitivity

in

Besides its angiogenic action, the enzymatic activity of PD-ECGF/TP plays a role in fluoropyrimidine sensitivity, being able to activate 5-fluorouracil (5-FU) and 5â&#x20AC;&#x2122;-deoxyfluorouridine (5â&#x20AC;&#x2122;DFUR, doxifluridine, furtulon) (Ackland and Peters, 1999). The antimetabolite 5FU was introduced in 1957 by Heidelberger et al (Heidelberger et al, 1957) and is still used for the treatment of a wide range of solid tumors alone, but usually in combination with other chemotherapeutic agents. Solid tumors treated with 5FU include breast, colorectal, head and neck and cervical carcinomas (Pinedo and Peters, 1988). It is the most widely prescribed agent 109

De Bruin et al: Role of PD-ECGF/TP in health and disease for colorectal cancer. The major targets of 5FU are 1) thymidylate synthase (TS), which is inhibited by metabolized 5FU, and 2) DNA and 3) RNA incorporation. With some exceptions, high expression of PDECGF/TP was correlated with an unfavorable outcome. However, in several studies it was found that increased PD-ECGF/TP expression was related with a better outcome of treatment. Adjuvant treatment of PDECGF/TP expressing, node positive breast carcinomas with cyclophosphamide, methotrexate and 5FU (CMF) had a significant survival benefit compared to PDECGF/TP negative tumors (Fox et al, 1997;Gasparini et al, 1999;Yang et al, 2002). A possible explanation might be that the effect of methotrexate is enhanced due to PDECGF/TP mediated TdR breakdown. Patterson et al, (1995, 1998) indeed described that high TP can moderate thymidine dependent rescue of TS inhibited cells. This of course depends on the intracellular TdR concentration.

Depletion of TdR increases dependency on the salvage of TdR, which in turn is lowered by high PD-ECGF/TP. Metzger et al, (1998) and Salonga et al, (2000) found that patients expressing high PD-ECGF/TP mRNA had a worse prognosis. This effect might be attributed to the (potential) angiogenic effect of PD-ECGF/TP. PD-ECGF/TP has a broad substrate specificity. The structures of several fluoropyrimidines, natural deoxynucleosides, and the thymidine phosphorylase inhibitor are shown in Figure 5. The potential actions of PD-ECGF/TP in the metabolism of various fluoropyrimidines are depicted in Figure 6. TP activates 5â&#x20AC;&#x2122;DFUR to 5FU by cleaving off the 5-deoxyribose moiety, while TP can theoretically activate 5FU by addition of dR1-P to 5-fluoro-2â&#x20AC;&#x2122;-deoxyuridine; a precursor of FdUMP, providing enough dR-1-P is present. FdUMP inhibits TS, responsible for de novo thymidylate synthesis.

Figure 5. Structures of the natural deoxyynucleoside thymidine, the base thymine, deoxyribose-1-phosphate moiety, thymidine phosphorylase inhibitor and fluoropyrimidines.

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Figure 6. Scheme showing the possible metabolic pathways for 5FU via PD-ECGF/TP, UP and orotate phosphoribosyltransferase (OPRT) and its different targets: TS inhibition via FdUMP and incorporation of FdUTP and FUTP into DNA and RNA, respectively. 5’DFUR is an intermediate in the conversion of Capecitabine, to 5FU. Finally the metabolic fate of TFT is shown, which can be degraded by PD-ECGF/TP or activated by TK, resulting in TS inhibition and DNA incorporation. The bold arrows give the main route of activation; others are possible but dependent on the availability of sometimes rare co-substrates.

PD-ECGF/TP may play a more important role when an additional source of dR-1-P is provided as co-substrate for the activation reaction (Peters et al, 1987). In previous studies (Patterson et al, 1995; Kato et al, 1997; Evrard et al, 1999a, b; Marchetti et al, 2001) the effect of TP on 5FU and 5'DFUR was demonstrated. For instance PDECGF/TP transfected MCF7 cells had an increased sensitivity of 165-fold to 5’DFUR (Patterson et al, 1995), PC9 transfected cells 153-fold (Kato et al, 1997), and PROb transfected cells 10-fold (Marchetti et al, 2001). In most of these studies, there was also increased sensitivity to 5FU but always lower than that for 5'DFUR. Other studies report that after transfection with PD-ECGF/TP the sensitivity increase for 5FU was higher than that of 5'DFUR (Evrard et al, 1999a, b), which is possibly due to an increased availability of dR-1-P in these cells, necessary for activation of 5FU by PD-ECGF/TP. Increase in dR-1-P availability in cells greatly enhances 5FU sensitivity mediated by TP (Peters et al, 1987; Schwartz et al, 1994; Ciccolini et al, 2001). This different role of PDECGF/TP in 5FU and 5'DFUR cytotoxicity is due to the fact that 5’DFUR is a prodrug of 5FU and needs an extra activation step. Activation of 5’DFUR can only occur through its conversion to 5FU, but three different pathways can mediate that of 5FU. Other activation pathways such as uridine phosphorylase (UP) and particularly orotate phosphoribosyltransferase (OPRT) (Peters et al, 1989) seem to be much more important. Thereafter the drugs might exert a similar mechanism of action.

causing some myelosuppression and gastrointestinal toxicity. Oral 5FU was abandoned decades ago because of irregular absorption, caused, among other things, by the variable levels of dihydropyrimidine dehydrogenase (DPD) the enzyme responsible for 5FU breakdown. 5FU is usually administered to the patient via bolus injection or continuous intravenous (i.v.) administration, the latter being a labor intensive approach, often requiring hospitalization of the patient, and resulting in considerable risk of venous thrombosis or infection around the catheters (Malet-Martino and Martino, 2002; Hoff et al, 2001). For these reasons new oral fluoropyrimidine formulations have been developed, to replace continuous infusions. These have improved safety and retain at least equal efficacy compared with continuous infusions, improving the quality of life by reducing the stress of hospitalization since it can be taken at home and providing the patient with a certain freedom (Venturini, 2002). Liu and colleagues showed in 1997 that patients preferred oral chemotherapy but regardless of the route of administration, patients were unwilling to accept a lower response rate and/or a shorter duration of response. However, when a similar efficacy is expected, it was specifically shown that patients preferred oral fluoropyrimidines compared to the Mayo scheme of i.v. administered 5FU/LV (Borner et al, 2002). Over the years, attempts have been made to produce more effective fluoropyrimidines suitable for oral administration. Ftorafur (Ft; 1-(2-tetrahydrofuryl)-5fluorouracil, Tegafur or Futraful) is a second-generation oral fluoropyrimidine, used in several combinations to improve its bioavailability. UFT consists of Ft combined with uracil in a 1:4 molar ratio, uracil being a natural substrate for DPD which prevents 5FU’s breakdown by competition. This was designed to produce a constant

A. Oral fluoropyrimidines: UFT, S1 and capecitabine 5FU is a relatively active anticancer agent, but has some disadvantages. It is a relatively non-toxic drug, 111

De Bruin et al: Role of PD-ECGF/TP in health and disease reserve of 5FU and to minimize the production of inactive products (Fujii et al, 1979; Hoff, 2000; Kohne and Peters, 2000; Malet-Martino and Martino, 2002). S1, its successor follows a different strategy (Shirasaka et al, 1996). This combination exists of Ft and two other compounds, 5chloro-2,4-dihydroxypyridine (CDHP) and potassium oxonate (OXO) (molar ratio Ft : CDHP : OXO; 1 : 0.4 : 1). CDHP and OXO have no antitumor activity and act as modulators of 5FU in the metabolism. CDHP functions as an inhibitor of DPD, thereby increasing the period of high 5FU in the circulation. CDHP is 200-fold more potent than uracil in inhibiting DPD (Tatsumi et al, 1987). OXO is added to limit the gastrointestinal toxicity of Ft. This toxic effect is the result of phosphoribosylation of 5FU to 5FUMP by OPRT. OXO accumulates specifically in normal gastrointestinal tissues compared to tumors (Shirasaka et al, 1993; Yoshisue et al, 2000), preventing activation of 5FU in normal mucosa but not in the tumor. A third rationally designed prodrug of 5FU is capecitabine (Xeloda). Capecitabine was developed to circumvent the toxicity of 5â&#x20AC;&#x2122;DFUR, which was converted to 5FU by PD-ECGF/TP. Capecitabine is an oral fluoropyrimidine carbamate that is converted to 5FU in three steps. The first step is catalyzed by carboxyl esterase located almost exclusively in the liver, the second step by cytidine deaminase expressed in the liver and various types of tumors and the last by PD-ECGF/TP (Figure 7) which is higher in tumors than in normal tissues thus ensuring an enhanced efficacy (Miwa et al, 1998). The design of capecitabine potentially had two advantages: enhanced activation at the tumor site and a decrease of drug concentration in the healthy tissue thereby decreasing systemic toxicity. Xenograft models were used to demonstrate the antitumor effect of capecitabine (Ishikawa et al, 1998a). Furthermore, it was shown that in tumor bearing mice the tumor concentrations of 5FU were considerably (114-209 fold) higher than in plasma (Ishikawa et al, 1998b). These results have been confirmed in a clinical trial, where it was shown that 5FU was on average 3.2 times higher in tumor-tissue than in adjacent non-tumor tissue and that 5FU concentration in tumor tissue was 21 times higher than in plasma (Schuller et al, 2000). In two phase III trials capecitabine proved to be at least equivalent to the standard 5FU/leucovorin treatment (Van Cutsem et al, 2000, 2001; Hoff et al, 2001). The dose limiting toxicities however were similar to those of i.v. administered fluoropyrimidines. Most common were the hand foot syndrome and diarrhea. So, since capecitabine is at least as effective as the 5FU/LV standard, and it provides considerable benefits to the well being of the patient, it has become one of the most widely prescribed oral anti-cancer drugs.

1972; Warrell, Jr. et al, 1979). Although TFT had antitumor effects, the development was discontinued due to side effects and rapid degradation of TFT to trifluorothymine by PD-ECGF/TP. The mechanism of action is through inhibition of TS (Santi and Sakai, 1971;Eckstein et al, 1994) and via incorporation into the DNA (Fujiwara et al, 1970; Emura et al, 2004b; Temmink et al, 2005). TFT has shown efficacy in 5FU resistant tumor cell lines, bypassing the resistance mechanisms of these cells (Fukushima et al, 2000; Murakami et al, 2000). The inactivation of TFT can be prevented by combining it with a specific thymidine phosphorylase inhibitor (TPI, 5chloro-6-(1-(2-iminopyrrolidinyl) methyl)-2,4(1H,3H)pyrimidinedione hydrochloride) which increased the bioavailability (Fukushima et al, 2000; Takao et al, 2000), allowing further development of TFT in this combination as an oral fluoropyrimidine. This combination of TFT and TPI in the molar ratio 1: 0.5, called TAS-102, can be administered orally, and is currently tested in phase I trials (Thomas et al, 2002). Orally administered TAS-102 prevents systemic degradation of TFT resulting in increased plasma levels compared to TFT alone (Fukushima et al, 2000). Another advantage of this combination is that the inhibition of PD-ECGF/TP can also decrease the angiogenic potential of PD-ECGF/TP. There was no effect of TPI on TFT sensitivity (De Bruin et al, 2003b) on a cellular level, not even in highly TP overexpressing cells, which was unexpected because it has been demonstrated that TFT is a good substrate for TP (Fukushima et al, 2000). Possibly activation of TFT by thymidine kinase (TK) is very efficient, preventing inactivation by TP. Emura et al (Emura et al, 2004a) found that the in vivo response of several tumor types was independent of TP, but, the ratio of TK / TP significantly correlated with tumor growth inhibition. This suggested that the balance of the relationship of activation and degradation, TP/TK affected the antitumor effect of TAS102.

C. Diverse phosphorylase

roles

of

thymidine

Compared with 5â&#x20AC;&#x2122;DFUR, the role of PD-ECGF/TP differs for 5FU because a role as activator is dependent on the availability of the co-substrate dR-1-P. For the 5FU prodrug Ft, present in the combinations UFT and S1 there is no direct role for PD-ECGF/TP. Although it has been postulated that Ft might be activated by PD-ECGF/TP (Sugata et al, 1986), recent studies show that the activation of Ft is mediated by cytochrome P450 enzymes (Komatsu et al, 2000) which have a considerable but variable expression in colon cancer cell lines (Yu et al, 2001). In addition, Ft cytotoxicity was not affected by TPI or increased in cells with high TP (De Bruin et al, 2003b). The oral prodrug capecitabine has been specifically designed to utilize the commonly found overexpression of PD-ECGF/TP at the tumor site, ensuring specific accumulation of 5FU in tumor tissue. An indirect effect on the sensitivity of the different fluoropyrimidines could arise from PD-ECGF/TP through a decrease of the TdR pools in the tumor environment, limiting thymidine salvage.

B. Trifluorothymidine plus thymidine phosphorylase inhibitor; TAS-102 Trifluorothymidine (TFT) was synthesized in the early 1960s by Heidelberger et al (Heidelberger and Anderson, 1964; Heidelberger et al, 1964). TFT has previously been used in antiviral therapy and has been evaluated for cancer therapy as a single agent or in combination (Ansfield and Ramirez, 1971; Dexter et al, 112

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Figure 7. Activation scheme of Xeloda, the first step is catalyzed by carboxyl esterase (CE), the second by cytidine deaminase (CDA), and ultimately 5â&#x20AC;&#x2122;deoxy-5- fluorouridine is activated by PD-ECGF/TP.

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De Bruin et al: Role of PD-ECGF/TP in health and disease Summarized, PD-ECGF/TP can have a dual role in the tumor. Its angiogenic activity promotes tumor growth and progression providing a target for intervention via inhibition of PD-ECGF/TP, e.g. by TPI. The other role is the utilization of the overexpression of PD-ECGF/TP in order to activate fluoropyrimidine prodrugs (Focher and Spadari, 2001; Marchetti et al, 2001).

forms an activated transcription complex. IFN ! results in STAT 1 phosphorylation and homodimerization (Borden, 1998). The activated STAT transcription factors can then bind to their consensus sequences in the DNA to activate transcription. Furthermore, various chemotherapeutic agents including taxanes, cyclophosphamide and mitomycin C (Sawada et al, 1998), and X-ray radiation (Sawada et al, 1999) can upregulate PD-ECGF/TP. The latter two may induce PD-ECGF/TP indirectly via upregulation of TNF-" or IFN-! (Blanquicett et al, 2002). Fukushima et al investigated PD-ECGF/TP upregulation due to IFN-" and paclitaxel in vitro and in vivo. Cell lines with high PDECGF/TP mostly had high STAT1 levels and PDECGF/TP and could no longer be induced by IFN-", whereas low PD-ECGF/TP expressing cell lines in which expression could be induced, had low inducible STAT1. Furthermore in clinically resected tumors PD-ECGF/TP and STAT1 were measured simultaneously and almost all tumors had high expression of both PD-ECGF/TP and STAT1 (Fukushima et al, 2002). There is also a role for NF$B. Although, no direct involvement of NF$B has been shown in relation to PDECGF/TP expression, Zhu and Schwartz, (2003) recently suggested that NF$B and TNFRII may be involved in the regulation of PD-ECGF/TP gene expression. We found that monocytic cells exposed to a NF$B inhibitor, sulfasalazine, had an altered pathway and a marked downregulation of TNFRII, confirming the finding of Zhu et al in a different system. Microenvironmental conditions, such as hypoxia and low pH can also upregulate PD-ECGF/TP expression possibly explaining the preferential presence around necrotic areas (Griffiths et al, 1997; Griffiths and Stratford, 1998). Others have shown increased PDECGF/TP expression at the infiltrating tumor edge (Maeda et al, 1996). Since it has been shown, in numerous transfection studies, that PD-ECGF/TP enhanced the efficacy of at least 5’DFUR (and thus of capecitabine), PD-ECGF/TP is used as a target to modulate fluoropyrimidine sensitivity (Morita et al, 2001). Xenograft models of human breast and colon cancers exposed to paclitaxel, docetaxel, mitomycin C or cyclophosphamide showed increased PDECGF/TP concentrations. In combination therapy, taxol, docetaxel and X-ray radiation with capecitabine or 5’DFUR were more effective than either alone (Sawada et al, 1998, 1999). Similar results were found by Endo et al, (1999) who found that cyclophosphamide preferentially upregulated pyrimidine phosphorylase activity (PyNPase) in the human tumor, whereas no change was detected in several other tissues of the tumor bearing and treated mouse. This upregulation of pyrimidine phosphorylase (PyNPase) resulted in a synergistic effect between cyclophosphamide and 5’DFUR or capecitabine, without enhancement of toxicity (Endo et al, 1999). Attempts to enhance the effects of 5’DFUR and capecitabine by specifically upregulating PD-ECGF/TP is a straightforward approach. However the results of the attempts to influence 5FU are less clear. 5FU has been combined with IFNs in order to increase its efficacy,

IV. Pyrimidine phosphorylases; Thymidine phosphorylase and Uridine phosphorylase Of note, besides PD-ECGF/TP there is a closely related pyrimidine phosphorylase active in human tumors: uridine phosphorylase (UP, EC 2.4.2.3) (Pizzorno et al, 2002). Since both TP and UP have broad and sometimes overlapping substrate specificity depending on the tissue in which they are measured, it can be difficult to distinguish between them. Two specific inhibitors TPI for PD-ECGF/TP and 5-benzylacyclouridine (BAU) (Niedzwicki et al, 1982) for UP, can be used as tools when the phosphorylase activity in cells or tissues is measured. Furthermore there are also interspecies differences, which might limit the use of certain model systems. El-Kouni et al, (1993) described that specificity of TP and UP for substrates varied between two different organs and cancers from mouse and humans. Using UP knockout embryonic stem (ES) cells, Cao et al found a 10-fold increase in IC50 for 5FU compared to normal ES cells. Furthermore there was a 16-fold increase in the IC50s for 5’DFUR (Cao et al, 2002). This is indicative for a role for UP in (oral) fluoropyrimidines and is in contrast to the finding that transfection of MCF7 cells with the UP gene did not influence the effect of 5FU or 5'DFUR (Cuq et al, 2001).

V. Regulation of PD-ECGF/TP expression and (Bio)modulation of fluoropyrimidines The PD-ECGF/TP promoter does not contain ‘TATA’ or ‘CCAAT’ boxes, sequences recognized by RNA polymerase II, prevalent in most eukaryotic genes (Hagiwara et al, 1991). The exact mechanism of regulation of PD-ECGF/TP gene expression is yet unknown, however the promoter contains six to nine Sp1 transcription factor binding sites (i.e. GC-box) postulated to contribute to both basal and inducible expression (Hagiwara et al, 1991; Zhu et al, 2002). PD-ECGF/TP can be upregulated by cytokines such as tumor necrosis factor-" (TNF-"), IL-1, interferon-! (IFN-!) (Eda et al, 1993) and IFN-" (Schwartz et al, 1992; Tevaearai et al, 1992; Makower and Wadler, 1999; Morita and Tokue, 1999). Other identified transcription binding sites in the PD-ECGF/TP promoter are an interferon stimulated response element (ISRE) (Schwartz et al, 1998) and a ! activated sequence (GAS) (Goto et al, 2001). The ISRE and GAS are sequences through which interferon (IFN)-mediated signaling acts. IFN-", # and ! act via the signal transducer and activator of transcription (STAT) family of transcription factors. IFN-" and # phosphorylate STAT1 and 3, which with a third cytoplasmic protein 114

Cancer Therapy Vol 4, page 115 resulting in abrogation of 5FU associated TS increase, augmentation of 5FU plasma levels and increased 5FU induced DNA damage (Schwartz et al, 1992;Makower and Wadler, 1999;van der Wilt et al, 1997;Horowitz et al, 1995). Although initially the response rates appeared to be higher when 5FU was combined with IFN-", randomized trials showed that the survival was equivalent, with in some instances an increased toxicity when IFN was added (Kemeny and Younes, 1992; Atzpodien et al, 1994; Greco et al, 1996; Labianca et al, 1996; Makower and Wadler, 1999).

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VI. Conclusion There are many data indicating a role for PDECGF/TP in (tumor) angiogenesis. Numerous immunohistological studies showed correlation between MVD and PD-ECGF/TP expression. Furthermore PDECGF/TP is often an independent marker for poor prognosis. The investigations focusing on the mechanism of the angiogenic effect of PD-ECGF/TP are far less in number. The main focus of research has been the enzymatic reaction catalyzed by PD-ECGF/TP and more specifically the products from the reaction. dR appears to play a key role in the pro-angiogenic and anti-apoptotic effects. However, the route and mechanism of this metabolite remain partly unknown. So far Induction of endothelial cell migration, enhanced metastasis and migration of cancer cells and induction of other angiogenic factors appears to play a role. The fact that LdR can counteract most effects of dR and PD-ECGF/TP is reasonable proof that dR is responsible for the effects. Although PD-ECGF/TP cleaves TdR to thymine and dR1-P, not much is known of the elimination of dR-1-P or generation of dR from dR-1-P. So further research upstream of dR is warranted. Besides this apparent role in tumor progression PDECGF/TP is also a key player in fluoropyrimidine sensitivity. It proved to be able to generate 5FU from 5â&#x20AC;&#x2122;DFUR, a feature used as strategy in the development of capecitabine. The application of capecitabine has proven that specific activation by PD-ECGF/TP is feasible and effective. Furthermore, it is capable of breaking down TFT into an inactive form. In the drug combination TAS-102 (TFT plus TPI), TFT is protected from breakdown by the inhibitor. A combination with a PD-ECGF/TP inhibitor is very appealing. TPI has been shown to inhibit some of the pro-angiogenic and anti-apoptotic features of PDECGF/TP. Other articles describe L-dR as good candidate for combination with fluoropyrimidines, still being able to take advantage of the high PD-ECGF/TP levels in tumors but inhibiting the pro-tumor effects down-stream of the PD-ECGF/TP reaction. PD-ECGF/TP is an enzyme with two faces in tumor development and treatment. It promotes tumor growth in various manners, but due to its up-regulation, it can also be utilized as a target for drug modulation.

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Cancer Therapy Vol 4, page 125 Cancer Therapy Vol 4, 125-134, 2006

The protective effect of vitamin C on Azathioprine induced seminiferous tubular structural changes and cytogenetic toxicity in albino rats Research Article

Fardous S. Karawya1 and Abeer F. El-Nahas2,* 1 2

Department of Histology, Faculty of Medicine and Department of Genetics, Faculty of Veterinary Medicine, Alexandria University, Egypt

__________________________________________________________________________________ *Correspondence: Abeer F. El-Nahas. Faculty of Veterinary Medicine, Edfina, Behera, P. O. Post 22758, Egypt; Fax +2 045 2960450 e-mail: abeerelnahas@hotmail.com Key words: Azathioprine, testis, genotoxicity, vitamin C Abbreviations: Azathioprine, (AZA); haematoxylin and eosin, (H&E); reactive oxygen species, (ROS) Received: 30 August 2005; Revised; 4 October 2005 Accepted: 15 November 2005; electronically published: March 2006

Summary Azathioprine (AZA) is widely used as an anticancer and immunosuppressive drug. Wide range of adverse effects including reproductive toxicity, mutagenesis and carcinogenesis has been demonstrated with its administration. This study was planned to assess the effect of short-term large dose of AZA (150 mg/kg b.wt by gavage as a single dose) and Long-term small dose (15 mg/kg b.wt by gavage for two months) on testicular gametogenic activity and bone marrow chromosomes of adult male albino rats. Also to evaluate the possible protective effect of vitamin C gavaged 14 days after short term AZA treatment or co-administered with long term AZA treatment. Evident toxicity of both treatments of AZA on testicular tissue through significant reduction in testicular weight, and severe damage of germinal epithelium of seminiferous tubules. Moreover, both treatments have genotoxic effect through significant reduction of mitotic index, increased number of micronucleus, aberrant cells and structural chromosomal aberration (fragment, deletion and ring chromosome). Short-term large dose of AZA associated also with increased number of polyploid cells. Vitamin C provided protection to testicular tissue in both treatments indicated by increased testicular weight and restoration of germinal epithelium. Vitamin C provided partial protection to genetic material appeared in decreasing number of aberrant cells through decreasing number of polyploid cells caused by short-term large dose AZA treatment. This protection is not extending to altered mitotic index, micronuclus or structural chromosomal aberrations in both treatments or to the number of aberrant cells caused by long term AZA treatment. We conclude that vitamin C provide protection to testicular tissue and genomic stability by reducing number of polyploid cells. antibody producing cells of the immune system, blood cells, hair cells, gonadal cells and malignant cells (Diasio and Lobuglio, 1991). Inspite of its serious side effects, they can be of great value in treatment, they can prolong life, preserve function, reduce symptoms, and sometimes may serve to put the disease into remission (Oka and Yoshimura, 1996). AZA is immunomodulatory drug often used to treat inflammatory bowel disease, autoimmune diseases, prevent rejection of transplanted organs and also used as anticancer drug (Schein and Winokur, 1975; Tage-Jensen et al, 1987; Dejaco et al, 2001; McMullaen et al, 2001; Langer et al, 2003; Marcen et al, 2003). It is inhibitor of purine metabolism leading to DNA damage. Upon its

I. Introduction Cytotoxic/immunosuppresive drugs are agents used to treat and cure many forms of malignancies. Because of its potent immunosuppressive properties, it has received considerable attention for the control of several clinical settings where the goal of therapy is to suppress an unwanted immune response (Paul and Bruce, 1991). The major current indications for drugs include the control of organ rejection after transplantation, prevention of Rh hemolytic disease of the new born and non-neoplastic disorders associated with altered immune reactivity (Annward et al, 1990; Paul and Bruce, 1991; Diasio and Lobuglio, 1991; Chaki, 1999). These drugs work by targeting and damaging cells that grow at rapid rate as 125

Karawya and El-Nahas: The protective effect of vitamin C on Azathioprine in albino rats administration, it rapidly converted into several compounds, including the active 6-mercaptopurine (Diasio and Lobuglio, 1991; William et al, 1998). AZA can affect rapidly growing cells including bone marrow and gastrointestinal cells, resulting in leukopenia, thrombocytopenia, increased susceptibility to infections and hepatotoxicity (Arber et al, 1991; Rosenkranz and Klopman, 1991; Olshan et al, 1994; Johnson et al, 1995; Rojapakse et al, 2000; Lowry et al, 2001; Kersten et al, 2002; Norgad et al, 2004). Treatment of certain types of cancer with cyclical combined chemotherapy with or without radiotherapy has dramatically increased the rate of long-term remission. The increased survival of patients has focused attention on the chronic effects of these cytotoxic agents on the function of normal tissues, which is not manifested until the damage is extensive (Annward et al, 1990). Unfortunately, the destructive effects of these treatments on spermatogenesis are well documented. Iatrogenic infertility and sterility are serious side effects of cytotoxic chemotherapy in young patients with relatively normal life expectation (Heikens et al, 1996; Gerres et al, 1998; Soriano et al. 2000; Silva et al, 2002; Tal et al, 1985; Rueffer et al, 2001; Das et al, 2002). AZA severely affect spermatogenesis in rat, it significantly lowered sperm count in couda epididymis and caused dose dependent damage of the seminiferous tubules (Iwasaki et al, 1996). Furthermore, patients received a combination of Cyclosporine- Azathioprine and Prednisone has low testesterone level and impairment of hypothalamic pituitary gonadal axis (Ramirez et al, 1991). The most serious complication among patients undergoing immunosuppressive therapy is the risk of developing cancer. Many of these drugs used have mutagenic properties and also contribute to increased cancer risk (Schein and Winokur, 1975; Mitrou et al, 1979; Baker et al, 1987). AZA is mutagenic, genotoxic and several types of tumors are associated with prolonged treatment with it (Clark, 1975; van Went, 1979; Nagafuchi and Miyazaki, 1991; Langer et al, 2003; Marcen et al, 2003). Cytotoxic drugs disturb oxidant-antioxidant balance and the oxidative damage to sperm, testis and genetic material is thought to be responsible for serious effects on male fertility and genomic stability (Ahotupa and Huhtaniemi, 1992; Michael et al, 1999; Blasiak et al, 2002). The potential role of dietary antioxidants as tocopherol, ascorbic acid, !-carotin, etc to reduce the activity of free radical-induced reactions has drawn increasing attention (McCall and Balz, 1999; Oliveira and Fortes, 2003). The purpose of this study was to assess the effect of short and long term AZA treatment on seminiferous tubules and bone marrow chromosomes and the possible protective effect of vitamin C in adult male albino rats.

B. Animals 80 adult male albino rats (150-200 gm) body weight were used. The animals were kept in standard housing conditions and freely supplied with food and water for one week before the experiment.

C. Experimental design The animals were divided into two major groups i. Short term experiment which include: Group 1: Received AZA (150 mg/kg body weight) gavaged as a single dose. The animals sacrificed after 14 days post treatment. The same dose was previously used to evaluate hepatotoxicity and carcinogenecity of AZA in rat (IARC, 1981; Arber et al, 1991). The used dose is larger than that of human as the smaller the animal, the larger the dose/kg b.wt (Paget and Barnes, 1964) Group 2: Received AZA as in group 1 in conjunction with vitamin C (100 mg/kg body weight) gavaged and maintained for 14 days after AZA treatment. ii. Long term experiment Group1: Received AZA (15mg/kg body weight) gavaged daily for two months. The used dose is about 1/10 of the acute dose used in this study. Group 2: Received AZA as in group1 simultaneously with vitamin C (100 mg/kg b.wt for two months Two control groups 10 animals each were used for each experiment: Control 1: Received distilled water orally Control 2: Received vitamin C (100mg/kg b.wt) dissolved in distilled water. At the end of each experiment the animals were sacrificed by decapitation after ether anesthesia and were subjected to the following studies:

D. Histological examination Both testes were removed and weighted then fixed quickly in Bouinâ&#x20AC;&#x2122;s fluid and processed for light microscopic examination using haematoxylin and eosin (H & E) (Drury and Wallington 1980).

E. Cytogenetic analysis Bone marrow from one femur was obtained to perform analysis of chromosomal aberrations and from other femur to analyze micronuclei.

1. Chromosomal aberrations The animals were sacrificed 1-2 hrs after injection of 4 mg/kg b.wt colchicine. Bone marrow preparation was made according to Giri et al, 1986. The cells spread into clean slides. The slide were air-dried stained with Gur Giemsa and 50 well spread metaphases per animal were selected for analysis of chromosomal aberrations. The mitotic indices were calculated from 1000 cells per animals.

2. Micronucleus technique Micronucleus preparations were prepared according to Schmid et al, 1976. 1000 polychromatic erythrocytes were demonstrated for each animal. The micronuclei represent condensed or chromosome fragments that remain after the nucleus expelled.

II. Materials and methods A. Drug A commercial available formulation of AZA tablets 50 mg was used.

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Cancer Therapy Vol 4, page 127 treated with 150 mg/kg b.wt, there were loss of both the basic tubular morphology and most of germinal epithelium. Relatively, few spermatogonial stem cells were observed, the cells displayed hyperchromatic nuclei, and vaculated cytoplasm (Figures 1 B, C, D). Testicular tissues of some animals showed irregular and severely regressed tubules with predominant vacuolated sertoli cells and widening of interstitial spaces due to presence of odema (Figures 1 E, F). Administration of vitamin C 14 days post AZA treatment caused a remarkable sparing of germ cell line. All types of germinal cells were present within the epithelium of many tubules. Tubular architecture was preserved and germinal epithelium showed a normal maturation progression (Figures 1 G, H). Testicular tissue of the rats received AZA (15 mg/kg b.wt) revealed moderate affection of many seminiferous tubules. Large number of germ cells were detached from sertoli cells and sloughed in the lumen of the tubules leading to obstruction and enlargement of the testis

F. Statistical analysis Evaluation of mean frequencies between treated and control groups by Student’s t-test. Results were considered statistically significant at P<0.05.

III. Results A. Histological examination Both short and long-term AZA treatments significantly lowered testicular weight compared with the control (Table 1). Administration of vitamin C for 14 days after single large dose significantly increased testis weight compared with AZA treated groups. While coadministration of vitamin C with AZA for two months significantly restore testicular weight to normal (Table 1). Microscopical examination of testicular tissues from the control and vitamin C treated animals showed normal cytoarchitecture and maturation of germinal epithelium (Figures 1A). This in sharp contrast to the testis of AZA treated animals (Figures 1B, Figures 2A). In these animals

Table 1. The effect of vitamin C administration on testicular weight in rat treated with Azathioprine as a single large dose or small doses for two months. Parameters Control Vit C Aza Aza + Vit C

Acute 1.4±0.2a 1.3±0.2a 0.3±0.1c 0.6±0.1b

Chronic 1.6±0.1a 1.5±0.1a 1.0±0.2b 1.2±0.1b

Each value represent mean ± SD of 10 animals Values with different letters at the same column were significantly differed. P < 0.05.

Figure 1. Photomicrograph of rat testis. (A) The control, normal spermatogenesis was found. (B, C, D, E, F) Rat received AZA (150 mg/kg b.wt), where (B) show complete disorganization and atrophy of seminiferous tubules with widening of its lumen and absence of sperm (x100). (C, D) Few spermatogenic cells with deeply stained nuclei and vaculated cytoplasm and Sertoli cells (x400). (E, F) Interstitial odema and severly regressed irregular tubules lined mainly with few spermatogonial cells and Sertoli cells (x100, 400). (G, H) Increased number of normal tubules with active spermatogenesis due to vitamin C administration with AZA (x100, 400).

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Karawya and El-Nahas: The protective effect of vitamin C on Azathioprine in albino rats which was detected in some animals (Figures 2 A, B, C, D). Some tubules lined only with sertoli cells (Figures 2 E, F). Odema and widening of interstitial spaces also noted in this group. The testicular gametogenic disorders induced by AZA in this group were reversed with evident improvement of spermatogenesis by vitamin C coadministration (Figures 2 G, H).

(Table 2, 3. Figures 3 A, B, C). However, short-term large dose of AZA caused a significant increase in polyploid cells (Table 3. Figures 3 D). Administration of vitamin C for 14 days after short term AZA treatment (150 mg/kg b.wt) or its simultaneous treatment for two months with AZA (15 mg/kg bawd) has no protective effect on altered number of micronucleus, number of dividing cells, or structural chromosomal aberrations (Table 2, 3). The protective effect of vitamin C appeared in decreasing number of aberrant cells through decreasing number of polyploid cells caused by short term AZA treatment (Table 3). Meanwhile, co-administration of vitamin C for two months with AZA did not reduce number of aberrant cells.

B. Cytogenetic analysis Short and long term treatment with AZA caused a significant reduction in the number of dividing cells (mitotic index) and a significant increased number of MN, aberrant cells and structural chromosomal aberrations which include fragment, deletions and ring chromosome

Figure 2. Photomicrograph of rat testis received AZA (15 mg/kg b.wt) for two months. (A) Many degenerated seminiferous tubules with loss of architecture and sloughing of degenerated germ cells in the lumen (x100). (B, C, D) Variable degrees of abnormal spermatogenesis with slaughing of spermatid and spermatocytes in the lumen of the tubules (x400). (E, F) Some tubules lined only with vacuolated Sertoli cells (x100, 400). (G, H) Reversed and evident improvement of spermatogenesis in most seminiferous tubules due to vitamin C administration with AZA (x100, 400).

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Cancer Therapy Vol 4, page 129 Table 2. Number of aberrant cells, MN and MI in mice treated with Azathioprine and/or vitamin C Parameters Short run exp. Control Vit C Aza Aza + Vit C Long run exp. Control Vit C Aza Aza + Vit C

MN*

MI**

1.2±0.1b 1.3±0.8b 4.9±1.7a 4.5±0.6a

10.8±2.1a 11.5±0.9a 5.1±0.5b 4.4±0.3b

1.8±0.8b 2.2±0.8b 7.4±2.3a 5.0 ±1.2a

10.1±2.0a 10.9±1.5a 7.5±2.1b 9.1±2.8b

Each value represent mean ± SD of 10 animals, 50 cells scored per animal * Micronucleus incidence in 1000 cells. ** No. of dividing cells in 1000 cells. Values with different letters at the same column were significantly differed. P < 0.05.

Table 3. Number of aberrant cells and different types of chromosomal aberrations in mice treated with Azathioprine and/or vitamin C. Group Short run exp. Control Vit C Aza Aza + Vit C Long run exp. Control Vit C Aza Aza + Vit C

No. of Aberrant cells

Fragment

Deletion

Ring

Polyploidy chromosme

2.1±0.8c 1.9±0.6c 14.4±1.1a 8.4±3.6b

1.6±0.8b 1.1±0.2b 7.4±1.8a 6.0 ±0.5a

0.8±0.5b 1.2±0.2b 4.8±0.8a 4.1±0.4a

0.4±0.2b 0.9±0.1b 2.2±0.8a 2.3±0.4a

0.1±0.7b 0.5±0.2b 6.8±3.0a 1.2±0.4b

2.4±1.1b 2.2±0.8b 15.6±2.3a 14.2±3.5a

1.4±1.1b 1.3±1.2b 11.0±3.5a 8.2±0.6a

1.0±0.7b 1.2±0.6b 5.6±4.1a 4.4±1.5a

0.4±0.2b 0.6±0.8b 2.4±1.6a 2.4±2.1a

0.3±0.1a 0.5±0.2a 0.9±0.4a 0.6±0.3a

Each value represent mean ± SD of 10 animals, 50 cells scored per animal. Values with different letters at the same column were significantly differed. P < 0.05.

Figure 3. Metaphase spreads from bone marrow of rat treated with Azathioprine (A-C) show structural chromosomal aberrations, the arrow indicate fragment (A), deletion (B), ring chromosome (C), and polyploidy (D) as a numerical change. Original magnification x 100.

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Karawya and El-Nahas: The protective effect of vitamin C on Azathioprine in albino rats testicular testesterone level in testesterone suppressed rat, reduce the number of spermatogonia and spermatocytes to 60% of normal suggesting the role of testesterone in the maintenances of these cell population. Also absence of testesterone lead to loss of spermatid adhesion, preventing their further maturation. Aumuller et al, 1992; lee et al, 1999, proved the basis of testesterone dependency of spermatid/ Sertoli cell cytoskeleton. Normally junctional area termed the ectoplasmic specialization develops between Sertoli cells and round spermatids, disrupted in the absence of testesterone and caused sloughing of spermatids into the lumen. The most popular mechanism of AZA induced cellular damage was lipid peroxidation. AZA with other cytotoxic drugs are associated with the induction of oxidative stress by generation of free radicals and reactive oxygen species (ROS), which interfere with testicular gametogenic activities. Our results showed that vitamin C provide significant protection of testicular tissue and spermatogenesis when administered in both AZA treatments. This is in agreement of Das et al, 2002, who proved testicular protection against cyclophosphamide toxicity by vitamin C administration, this suggesting the role of vitamins in prevention of cytotoxic drug-induced testicular damage. The sperm are extremely sensitive to free radicals damage due to active generation of free radicals, lack of defensive enzymes and high concentration of polyunsaturated fatty acids. Without proper membrane fluidity, enzymes are activated which can lead to impaired motility, abnormal structure, loss of viability and death of sperms (Baker et al, 1996; Hsu et al, 1998). These factors make the health of sperms critically dependent upon antioxidant. Michael et al, (1999), demonstrated that free radicals or oxidative damage to sperm is thought to be responsible for many cases of idiopathic oligospermia with high levels of free radicals found in semen of infertile men. Fraga et al, 1991; Chen et al, 2001, observed that when dietary vitamin C was reduced the seminal ascorbic acid decreased and the number of sperm with damaged DNA increased. These results indicated that dietary vitamin C plays a critical role in protecting against sperm damage. In our study both small and large doses of AZA increased number of micronucleus, structural and numerical chromosomal aberrations (with large dose only). van Went, 1979, observed a dose-dependent increase in the number of the cells with micronucleus in rat and mice caused by AZA treatment and increased number of structural chromosomal aberrations in lymphocyte cultures of children on long-term AZA therapy. Both treatments with AZA cause a significant reduction in the number of dividing cells. Nagafuchi and Miyazaki, 1991, observed a dose dependent increase in DNA single strand breaks with concomitant cytotoxicity associated with AZA treatment. The role of vitamin C in reducing genotoxicity induced by many agents has been proved (Gajecka et al, 1999; Nefic, 2001; Siddique et al, 2005). Vitamin C reduced the clastogenic effect induced by anticancer drugs dexorubicine and idarubicin (Antunes and Takahashi,

IV. Discussion Cytotoxic drugs that are widely used as immunosuppressive and anti-inflammatory agents in patients with neoplastic conditions are of long-range concern due to the problem of cumulative organ toxicity that is not manifested until damage is extensive. These considerations have arisen because of their wide spread use in recent years (Oka and Yoshimura, 1996; Bunn and Kelly, 1998). In view of the marked cytotoxicity of most anticancer drugs, it exerts adverse effects in young patients (Whitehead et al, 1981). This study has provided an insight into some fertility problems and genotoxicity associated with AZA treatment. Testicular weights and microscopic examination of testicular tissue showed that short and long term administration of AZA have diverse effects on male fertility. Single large dose of AZA (150 mg/kg b.wt) showed germinal aplasia and the seminiferous tubules were extremely atrophied, most of the cells within the tubules were sertoli cells and occasionally germ cell with pyknotic nuclei and vaculated cytoplasm were seen. There is absence of many stages of spermatogenesis compared with the control group. The reduction in the testis weight is indirectly indicative of the effect on spermatogenesis. Dhabhar et al, 1993, proved that Sertoli cells which secrete inhibin are resistant to these cytotoxic agents. Ramirez et al, 1991; Iwasaki et al, 1996, proved that AZA induced impairment of spermatogenesis by direct inhibition of germinal epithelium or indirect by influencing the axis between hypothalamus-pituitary and gonads. These effects were proved morphologically by testicular atrophy and azoospermia. Furthermore, Annward et al, 1990, found that rapidly dividing cells are more sensitive to cytotoxic drugs than quiescent cells, hence sterility and ovarian dysfunction appear to be less common in females treated with these drugs than in males indicating that the ovary with its lower germ cells proliferative rate may be partially protected from cytotoxic drugs (Annward et al, 1990). Furthermore, Mclachlan et al, 1996, observed that acute withdrawal of testesterone by the use of leydig cell cytotoxin produce destructive pattern of spermatogenic cells degeneration with sharp increase in the number of pyknotic nuclei and vaculated cytoplasm which was observed in our study. Using similar criteria, Dekretser et al, 1972, reported that isolated germinal epithelium damage is rare and that leydig cell function is nearly always impaired as well, and other reports described gynecomastia in pubertal boys treated with cytotoxic drugs that was manifestation of leydig cell dysfunction (Sherin et al, 1978). These mean that low testestorone level due to damage of leydig cells responsible for the morphological changes observed in the testis. On the other hand testicular tissue of animals treated with AZA (15 mg/kg b.wt) orally daily for two months revealed moderate degenerative changes in most seminiferous tubules. Many germ cells were detached from Sertoli cells and sloughed in the lumen of seminiferous tubules, the slaughed cells contained desqumated spermatid and spermatocytes. In the present study, adhesion of round spermatids approved to be lost resulting in their sloughing into the lumen. Richburg and Boekelheide, 1996, proved that chronic reduction of 130

Cancer Therapy Vol 4, page 131 damage in Wistar rat bone marrow cells. Mutat Res 419, 137-43. Arber N, Zajicek G, Nordenberg J and Zkelsidi Y (1991) Azathioprine treatment increases hepatocyte turnover. Gastroenetrology 101, 1083-1086. Au muller G, Schulze C and Viebahn C (1992) Intermediate filaments in Sertoli cells. Microsco Res Tech 20, 50-72. Baker HWG, Brindle J and Levine DS (1996) Protective effect of antioxidant on the impairment of sperm motility by activated polymorphonuclear leucocytes. Fetil Steril 65, 411-419. Baker GL, Kahl LE, Zee BC, Stolzer BL, Agarwal AK, Medsger TA Jr (1987) Malignancy following treatment of rheumatoid arthritis with cyclophosphamide. Long-term case-control follow-up study. Am J Med 83, 1-9. Blasiak J, Gloc E, Wozniak K, Mlynarski W, Stolarska M, Skorski T and Majsterek I (2002) Genotoxicity of idarubicin and its modulation by vitamins C and E and amifostine. Chem Biol Interact 140, 1-18. Bunn PA and Kelly K (1998) New chemotherapeutic agents prolong survival and improve quality of life in non-small cell lung cancer: a review of the literature and future directions. Clin Cancer Res 4, 1087-100. Cabrera G (2000) Effect of five dietary antimutagens on the genotoxicity of six mutagens in the microscreen prophageinduction assay. Environ Mol Mutagen 36, 206-20. Chaki SP, Srinivas M, Chaube SK (1999) Effect of cyclosporine on human sperm motility in vitro. Arch Androl 43, 215-220 Chen JS, Sensini C, Barelli M, Manca D and Menesinichen MG (2001) Ascorbic acid induced spectrin reorganization in bull epididymal spermatozoa. Cell Biol Toxicol 16, 77-82. Clark JM (1975) The mutagenicity of azathioprine in mice, Drosophila melanogaster and Neurospora crassa. Mutat Res 28, 87-99. Das UB, Mallich M, Debnath JM and Ghosh D (2002) Protective effect of ascorbic acid on cyclophosphamide-induced testicular gametogenic and androgenic disorders in male rats. Asian J Androl 4, 201-207. Dekretser DM, Burger HG and Forlune D (1972) Hormonal, Histological and chromosomal studies in adult male with testicular disorders. J Clin Endocrinol Metab 35, 392-401. Dhabhar BN, Malhotra H, Joseph R, Garde S, Bhasin S Sheth A and Advani SH (1993) Gonadal function in prepubertal boys following treatment for Hodgkinâ&#x20AC;&#x2122;s disease. Am J Pediat Hematol Oncol 15, 306-310. Dejaco C, Mittermaier C, Reinisch W, Gasche C, Waldhoer T, Strohmer H, Moser G (2001) Azathioprine treatment and male fertility in inflammatory bowel disease. Gastroenterology 121, 1048-53. Diasio RB and Lobuglio AF (1991) Immunomodulators: Immunosuppresive agents and Immunstimulants. In: Goodman GA, Rall TW Nies AS, Taylor P, editors. The pharmacological basis of theraputics. New York: Pergamon press pp, 1291-1308. Drury RAB, Wallington EA (1980) Carletonâ&#x20AC;&#x2122;s Histology Technique 5th ed Oxford, Newyork, Torento. Oxford University Press pp. 140-142. Ferguson LR, Whiteside G, Holdaway KM and Baguley BC (1996) Application of fluorescence in situ hybridisation to study the relationship between cytotoxicity, chromosome aberrations, and changes in chromosome number after treatment with the topoisomerase II inhibitor amsacrine. Environ Mol Mutagen 27, 255-262. Frago C (1991) Ascorbic acid protects against oxidative DNA damage in human sperm. Proc Nat Acad Sci USA 88, 11003-11006. Gajecka M, Kujawski LM, Gawecki J and Szyfter K (1999) The protective effect of vitamins C and E against B (a)P-induced

1998; Tavares et al, 1998; Pillanse et al, 2002; Blasiak et al, 2002). However, in our results vitamin C did not exert any protective effect on AZA induced structural chromosomal aberrations. Pillanse et al, 1990, found that ascorbic acid provide protection from cyclophosphamide induced teratogenic effect in mouse embryo, this protection is not associated with prevention of DNA strand breaks. Furthermore, vitamin C did not provide protection from genotoxic effect of toxophene, dichorovos and nitrosomorpholine compounds (Cabrera 2000; Robichova et al, 2004). Large dose of AZA associated with increased polyploidy cells. Motwani et al, 2000, suggested that cell with compremized G1 checkpoint in response to microtubule inhibitors enter S phase with 4n DNA, endoreduplicate and become polyploid cells. We suggested that large dose of AZA may act as microtubules inhibitors. Ferguson et al, 1996, observed increased number of polyploidy cells associated with high dose of amsacrine (antileukemic drug). Meanwhile, the use of small and large dose of this drug led to chromosomal fragments. Treatment of the rats with vitamin C 14 days following AZA treatment (150 mg/kg. b.wt.) caused a significant reduction in polyploidy cells. Motwani et al, 2000, showed that endoreduplication and polyploidation can prevented by inhibition of cycline-dependent kinase, resulted in the arrest of cells in pseudo G1 state and dramatic decrease in cells containing >4n DNA. Thomas et al, 2005, proved that vitamin C delay the accumulation and activation of cell cycle control kinases. Defects in cell cycle checkpoints can lead to chromosome abnormality, aneuploidy, and genomic instability, all of which can contribute to tumorigenesis (Lannutti et al, 2005; Vries et al, 2005). The antitumour effect of vitamin C has been proved (Roomi et al, 2005; Thomas et al, 2005). They proved that vitamin C transiently arrest cancer cell cycle progression in S phase and G (2)/M boundary by delaying the accumulation and activation of cell division control kinases/cycline complex. We suggesting that vitamin C provide genomic stability by preventing polyploidy In conclusion effort should be made to identify more careful design of non-toxic chemotherapy regimes. Vitamin C provided significant protection to the spermatogenic cells and provided genomic stability but did not protect the cells from structural chromosomal aberration. It may be used with other antioxidants for full protection of genetic material.

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Abeer F. El-Nahas

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Cancer Therapy Vol 4, page 135 Cancer Therapy Vol 4, 135-142, 2006

The usefulness of oral TS-1 treatment for potentially curable gastric cancer patients with intraperitoneal free cancer cells Research Article

Yutaka Yonemura1,*, Yoshio Endou2, Etsurou Bando1, Taiichi Kawamura1, Gorou Tsukiyama1, Shouji Takahashi1, Naoko Sakamoto5, Kiyoshi Tone6, Kimihide Kusafuka6, Ichirou Itoh6, Masashi Kimura7, Masakazu Fukushima3, Takuma Sasaki4, Narikazu Boku8 1

Peritoneal Dissemination Program, Shizuoka Cancer Center, Shizuoka, Japan Department of Experimental Therapeutics, Cancer Research Institute, Kanazawa University, Kanazawa, Japan 3 Cancer Research Laboratory and Institute for Pathogenic Biochemistry in Medicine, Taiho Pharmaceutical Co., Ltd, Japan 4 Deaprtment of Pharmacology, Aichi-Gakuin University, Aichi, Japan 5 Department of Surgery, Self Defence Force Fuji Hospital, Shizuoka, Japan 6 Department of Pathology, Shizuoka Cancer Center, Shizuoka, Japan 7 Department of Pharmacology, Shizuoka Cancer Center, Shizuoka, Japan 8 Department of Medical Oncology, Shizuoka Cancer Center, Shizuoka, Japan 2

__________________________________________________________________________________ *Correspondence: Dr. Yutaka Yonemura, Peritoneal Dissemination Program, 1007 Shimo-nagakubo, Nagaizumi-machi, Suntou-gun, Shizuoka, 411-8777, Japan; Phone: 81-55-989-5235; Fax: 81-55-989-5634; E-mail: y.yonemura@scchr.jp Key words: Gastric cancer, peritoneal dissemination, TS-1, S-1, lavage cytology Abbreviations: 5-chloro-2, 4-dihydroxypyridine, (CDHP); 5-fluorouracil, (5-FU); area under the curve, (AUC); orotate phosphoribosytransferase, (ORPT); oxonate, (Oxo); platinum, (Pt) Received: 16 January 2006; Revised: 24 January 2006 Accepted: 30 January 2006; electronically published: March 2006

Summary Prognosis of patients with positive peritoneal wash cytology (Cy1) but no macroscopic peritoneal dissemination (P0) is very poor. We assessed the effects of TS-1, a novel oral derivatives of 5-fluorouracil, as a postoperative chemotherapy for these patients. Positive cytology by peritoneal washing was found in 101 potentially curable gastric cancer patients with peritoneal free cancer cells (P0/Cy1 status). After radical gastrectomy, 35 patients were treated with oral TS-1 (80mg/m2) for 28 consecutive days and 14 day rest, and the schedule was repeated every 6 weeks (TS-1 group). The other 66 patients did not receive any chemotherapy (Control group). A total of 220 courses were administered with a median of 7 courses and the range from 2 to 25 courses. The patients of TS-1 group survived significantly longer than that of Control group (P<0.0001). Two year survival rates of Control group and TS-1 group were 9% and 53%, respectively. Recurrence was not found in 15 (43%) for TS-1 group and 3 (5%) for Control group. Peritoneal recurrences of TS-1 treatment and Control group were found in 11 (31%) and 34 (52%) patients (P<0.05). Cox proportional hazard model showed TS-1 treatment as an independent prognostic factor, and the relative risk by TS-1 treatment was 0.17-fold lower than that of Control group. Major adverse reactions included myelosuppression and gastrointestinal toxicities, but they were generally mild and no treatment-related deaths. TS-1 treatment is safe and effective as a postoperative chemothrapy for patients with P0/Cy1 status.

died of peritoneal dissemination even after curative resection (Chu et al, 1989). More than 30% of potentially curable gastric cancer patients show positive lavaged

I. Introduction Prognosis of patients with advanced gastric cancer is still poor, because many patients with T3 and T4 tumor 135

Yonemura et al: Oral TS-1 treatment for gastric cancer patients with i.p. free cancer cells university hospital. Just after laparotomy, peritoneal cavity was inspected and palpated and the findings of the status of T-grade, N-grade, peritoneal dissemination and liver metastasis were recorded. Then, 200 ml of physiological saline was injected into the peritoneal cavity, and peritoneal washings were collected from the left subdiaphragmatic space and Douglasâ&#x20AC;&#x2122; pouch and heparin was used as an anticoagulant. The fluid was centrifuged 5 min at 1500 rpm, and the cytospins were obtained with Auto Smea CF-12D (Sakura Seiki Co., Ltd., Tokyo Japan). Five slides per on person were prepared and two and one were stained with Papanicolau method and Alcian Blue staining. The other two were fixed 10 minutes in cold absolute acetone for immunocytochemistry. The monoclonal antibodies used in the study were antihuman carcinoembryonic antigen (TAKARA Bio INC., Tokyo, Japan) and anti-human epithelial antigen (DAKO, Copenhagen, Denmark). Immunocytochemical staining of cytospins was performed by means of a sensitive streptoavidin, biotin immunohistoperoxidase method (LSAB KIT, DAKO). The enzyme activity was developed using 3-amino 9-ethyl carbazole as chromogen substrate. The immunocytochemical findings were evaluated independently by two investigators with no knowledge of the cytopathologic diagnosis. Peritoneal washings were considered as positive when at least one of the five slides showed cells compatible with cancer cells (Benevolo et al, 1998). Among 1039 potentially curable patients, 114 patients showed positive cytology. The incidences of positive cytology in T1, T2, T3, and T4 tumors were 0.2 % (1/411), 8.3% (34/409), 38.9% (70/180) and 23.1% (9/35), respectively. The 114 patients underwent subtotal or total gastrectomy with D1+! or D2 dissection. Thirty-five patients (TS-1 group) were received oral TS-1 treatment after gastrectomy and the 66 patients were not treated with any chemotherapy (Control group). The other 13 patients were treated with systemic chemotherapy except TS-1 therapy. All the patients were received informed consent about the regimens, which are considered to be effective for gastric cancer from the medical oncologists. The medical oncologists explained the results of clinical trials of the regimens, schedule, effective rates and the precise adverse effects after chemotherapy. The selection of the regimen was made by the patients. Eligibility for TS-1 treatment required adequate organ functions: hemoglobin >=9.0 gr/dl, WBC >=4000-12000; platelets>= 100000/ml, AST and ALT <=100 IU/ml, serum alkaline phosphatase within twice the normal upper limit, bilirubin <=1.5mg/dl; creatinine within upper normal limit; and informed consent from the patients. Among 13 patients who were treated with systemic chemotherapy except TS-1 therapy, .MTX/5-FU therapy was done in 6 patients, and the other patients selected the CPT11/MMC in two, TS-1/CDDP in two, CPT-11/CDDP in two, and 5FU/Taxol in one.

cytology by peritoneal washing (Band et al, 1999), and almost all the patients died of peritoneal recurrence (Band et al, 1999). Accordingly, the patients are grouped as P0/Cy1 in the Japanese rules of gastric cancer (Japanese Research Society for Gastric Cancer, 2004). P0 means no macroscopic peritoneal dissemination and Cy1 is the status showing positive cytology by the peritoneal washing which is recommended to be done just after laparotomy (Japanese Research Society for Gastric Cancer, 2004). Because the prognosis of the patients with P0/Cy1 status is very poor as that of patients with established peritoneal dissemination, these patients are classified as staged 4 (Japanese Research Society for Gastric Cancer, 1995). However, no standard treatment for the patients with P0/Cy1 status is proposed in the guidelines (Japanese Research Society for Gastric Cancer, 2004). Intravenous 5FU infusion (Cullinan et al, 1985) or in combination with other anticancer drugs, FAM (MacDonald et al, 1980), FAMTX (Wils et al, 1991) have been used for chemotherapy of advanced gastric cancer. However, there has been no report to study the efficacy of systemic chemotherapy on patients with P0Cy1 status (MacDonald et al, 1980; Cullinan et al, 1985; Wils et al, 1991). TS-1 is a new oral fluorinated pyrimidine agent, which contains tegaful, 5-chloro-2, 4-dihydroxypyridine (CDHP) and potassium oxonate (Oxo) in a molar ratio of 1:0.4:1 (Shirasaka et al, 1996). Dihydropyridine dehydrogenase DPD, which are found in a high concentration in the liver, rapidly degrade 5-fluorouracil (5-FU). CDHP is a specific inhibitor of DPD and the inhibition of the 5-FU by CDHP is very important for the efficacy of 5-FU. In the experimental model, high and constant 5-FU concentrations were maintained by continuous infusion of 5FU in combination with CDHP (Tatsumi et al, 1987). However, in the model, diarrhea due to 5FU is a severe dose-limiting factor. Oxo is an inhibitor of orotate phosphoribosytransferase (ORPT) and acts as a protector against 5-FU-induced GI toxicity without loss of antitumor activity (Shirasaka et al, 1996). Accordingly, TS-1 might be more effective in the treatment of cancer patients than continuous infusion of 5-FU from the point of anti-tumor potency and toxicity. Because prolonged exposure is desirable from the view of anti-tumor mechanisms of 5-FU, oral administration of TS-1 is certainly the most appealing rout of administration, as compared with intravenous infusion of 5-FU (Van Groeningen et al, 2000). Hirata et al reported that plasma concentrations of 5-FU for 4-week consecutive administration of TS-1 maintained the concentrations enough to kill cancer cells during the treatment period (Hirata et al, 1999). In the present study, the effect of oral TS-1 was studied on the survival of patients diagnosed as P0/Cy1.

B. Drugs The patients were assigned on the basis of body surface area to receive one of the following doses twice a daily, after breakfast and dinner.: body surface area < 1.25 m2, 40 mg; < 1.5 m2, 50 mg; >=1.5 m2, 60 mg. TS-1 (Taiho Pharmacoceutical Co., Ltd., Tokyo, Japan) was administered at the respective dose for 28 days, followed by a 2-week rest. This schedule was repeated every 6 weeks until the occurrence of recurrence, unacceptable toxicities or patientsâ&#x20AC;&#x2122; refusal. Compliance was assessed by patient interviews with each investigator, with a schedule calendar with regular monitoring.

II. Materials and methods A. Patients During the last 5 years from September, 26, 2000 to December, 23, 2005, 1039 patients with gastric cancer underwent curative resection at Shizuoka Cancer Center and Kanazawa

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Cancer Therapy Vol 4, page 137 and 8 weeks after operation in 22 (63%), 9 (26%) and 4 (11%) patients. A total of 220 courses were administered to the 35 patients in TS-1 Group with a median of 7 courses and the range from 2 to 25 courses. All the 35 patients received TS-1 as outpatients, but three patients were treated as inpatients due to interstitial pneumonia, and severe general malaise. Two patients required dose reduction from 120mg/day to 100mg/day, and from 100mg/day to 80mg/day due to adverse reactions. Survival curves of the two groups were shown in Figure 1. The patients of TS-1 group survived significantly longer than that of Control group (P<0.0001). Two year survival rates of Control group and TS-1 group were 9% and 53%, respectively. The mean days between operation and recurrence were 372 (±63) days for TS-1 group, but was 220 (±221) days for Control group (P<0.05). Recurrence was not found in 15 (43%) for TS-1 group and 3 (5%) for Control group. Recurrence sites after TS-1 treatment were 11, 5, 2 and 1 in the peritoneum, lymph nodes, bone and liver, respectively. In Control group, 34, 10, 8 and 5 patients had recurrences in peritoneum, lymph nodes, liver and bone.

C. Statistical analyses Survival was calculated by the method of Kaplan-Meier, and Cox proportional hazard model was used for the multivariate analyses using SPSS software (SPSS® Version 10.1, Chicago, Illinois, USA). Results are presented as the mean plus or minus the standard deviation of the mean. The chi-squared test and Student’s-t test were used to analyze data. Differences associated with a P value of 0.05 or less were considered to be statistically significant.

III. Results Clinicopathologic factors of the two groups were shown in Table 1. There was no statistical difference in macroscopic type, wall invasion, lymph node metastasis and histologic type between Control and TS-1 group (Table 1). Operation methods of both groups were listed in Table 2. D2 dissection was done in 55 (83%) and 25 (71%) of Control and TS-1 group, respectively. Combined resection of spleen was performed in 7 (11%), and 6 (17%) of Control and TS-1 group. TS-1 was administered during day 25 and day 56 after operation. TS-1 administration was started from 4, 6

Table 1. Patients' characteristics

Macroscopic type Type 0 Type 1 Type 2 Type 3 Type 4 Wall invasion T1 T2 T3 T4 Lymph nodes N0 N1 N2 N3 Histologic type differentiated poorly differentiated Age

Control group

TS-1 gropu

1 2 20 34 9

0 1 6 16 12

1 24 33 8

0 5 27 3

8 19 21 18

7 10 15 3

26 40 62.9 ± 12.4

9 26 61.7 ±10.4

Control group

TS-1 group

11 55

10 25

35 31

19 16

7

6

Table 2. Operation methods

Lymph node dissection D1+! D2 Operations Subtotal gastrectomy total gastrectomy Combined resection splenectomy

137

Yonemura et al: Oral TS-1 treatment for gastric cancer patients with i.p. free cancer cells Group

No

1 y.s.r

2 y.s.r

MST

TS-1 group

35

88%

53%

21.1 m

Control group

66

44%

9%

9.1 m

Figure 1. Survival curves of patients with peritoneal free cancer cells. TS-1 group: patients treated with 80 mg/m2 of oral TS-1 after radical gastrectomy. Control group: Patients did not receive any chemotherapy after radical gastrectomy.

MST of the 13 patients was 5.0 months and the one year survival rate was 60%, but no two year survivor. There was no statistical survival difference between Control group and the group treated with other regimen except TS-1. Table 3 shows the results of generalized Wilcoxon test and multivariate analyses using Cox proportional hazard model. The status of lymph node metastasis (pN0/N1 vs. pN3/N4), T grade (T2 vs. T3 vs. T4) andhistologic type (differentiated type vs. poorly differentiated type) were not independent prognostic factors. In contrast, TS-1 treatment was an independent prognostic factor, and the relative risk for death by TS-1 treatment was 0.17-fold lower than that of Control group. The adverse effects during treatment are listed in Table 4. Major adverse reactions included myelosuppression and gastrointestinal toxicities. However, they were generally mild and no treatment-related deaths. Two patients developed grade 3 leukopenia, one in the

first, and two in the second courses of the treatment. Three patients developed severe malaise and required hospitalization.

IV. Discussion Prognosis of patients with P0/Cy1 status is very poor even after radical gastrectomy, due to the high incidence of peritoneal recurrence. These results show the importance to control peritoneal recurrence in the patients with P0/Cy1 status. Cox proportional hazard model showed the TS-1 treatment alone as an independent prognostic factor, but T and N grade did not emerged as the independent prognosticators. The reason may be speculated that the occult peritoneal dissemination already exists, because almost all patients died of peritoneal dissemination.

Table 4. Adverse effects during TS-1 treatment Grade (No of patients) 2 3 0 2

4 0

Incidence of Grade 3-4 6%

Toxicity Hematological

1 5

Leukopenia Anemia Thrombocytopenia

3 1

0 0

0 0

0 0

0% 0% 0%

Stomatitis

3

0

0

0

0%

Diarrhea Nausea, vomiting Malaise

4 6 3

0 0 0

0 0 3

0 0 0

0% 0% 9%

Non-hematological

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Cancer Therapy Vol 4, page 139 Table 3. Results of generalized WIIcoxon test and multivariate analysis

Prognostic parameter Lypmh node metatstasis

Wilcoxon test X2 P

X2

Cox hazard model P Relative risk

95% CI

N0-N1 vs N2-N3 Histologic type

0.014

0.904

0.133

0.715

1.58

0.74

3.28

Differentiated vs. Poorly differentiated T-grade

1.646

0.919

0.316

0.25

0.98

0.51

1.23

T2 vs. T3 T3 vs. T4 Treatment

0.103 2.444

0.748 0.118

0.154 0.156

0.695 0.216

0.83 1.82

0.42 0.7

1.71 4.68

28.799

0.0001>

22.475

0.001>

0.17

1.04

3.34

TS-1 vs. No treatment

In contrast, lymph node metastasis or local recurrence might be prevented by extended gastrectomy. Accordingly, peritoneal recurrence must be more important prognostic factor than T or N grade in patients with P0/Cy1 status. TS-1 may inhibit the growth of peritoneal dissemination. Many good clinical responses in patients with advanced gastric cancer by TS-1 therapy have been reported (Sakata et al, 1998; Abe et al, 2003). However, the outcome was markedly poorer in the patients with established peritoneal dissemination than in those with lymph node or liver metastasis (Abe et al, 2003). One of the reasons why the peritoneal dissemination resists against systemic administration of anticancer drugs is the poor penetration of drugs into the peritoneal cavity. Accordingly, only a small amount of drugs reaches the peritoneal cavity after intravenous administration, because drug distribution is limited due to the peritoneal blood barrier (Sugarbaker et al, 1993; Jacquet and Sugarbaker, 1996). In contrast, IP chemotherapy offers potential therapeutic advantages over systemic chemotherapy by generating high IP concentrations of drugs (Los et al, 1989; Markman, 1991). Los et al, reported that the area under the curve (AUC) for bound and free platinum (Pt) in the peritoneal cavity after IP treatment of cisplatinum is 6 times higher than the AUCs in the peritoneal cavity after IV treatment in rats (Los et al, 1989; Markman, 1991). However, the problem of IP chemotherapy is the rapid clearance of drugs from the peritoneal cavity, and the intermittent administration of drugs does not allow the exposure of anti-cancer drugs to the peritoneal cancer tissue for a long period. In addition, even distribution of anti-cancer drugs in the peritoneal cavity can not be obtained due to adhesion after gastrectomy. So far, there was no report to confirm the efficacy of anti-cancer drugs for the prevention of peritoneal recurrence in the patients with peritoneal free cancer cells. Yamagata et al reported the effects of TS-1 on patients with peritoneal dissemination from gastric cancer, but the number of patients is only 7 (Yamagata et al, 2004). The present study clearly showed the effect of TS-1 on the survival improvement of patients with P0/Cy1 status.

In the experimental peritoneal dissemination models, 5-FU concentrations in ascites after oral administration of TS-1 were significant high levels of around 300-500 ng/ml at 1-6 hours (Mori et al, 2003; Yonemura et al, 2005). In contrast, 5-FU levels in ascites after oral administration of 5-FU was almost nil (Mori et al, 2003; Yamagata et al, 2004). TS-1 contains CDHP, which exhibits a very high activity in inhibiting DPD. DPD in a high concentration in the peritoneal mononuclear cells, degrades 5-FU, but CDHP concentration in ascites showed high level from 30 min. after oral administration of TS-1 (Yonemura et al, 2005). As a result, CDHP inhibit the rapid degradation of 5-FU by DPD in the ascites. Accordingly, high and constant 5-FU concentrations were maintained in the ascites (Mori et al, 2003; Yonemura et al, 2005). Furthermore, Mori et al reported in 2003 that 5-FU concentrations in the experimental peritoneal dissemination were significantly higher than in the levels of ascites 1 hour after oral TS-1 administration, and maintained at significant high levels even after 8 hours (Mori et al, 2003). Hirata et al reported the plasma concentrations of 5FU after TS-1 administration for advanced gastric cancer patients ranged between 3 ng/ml to 128ng/ml, and the levels maintained during the treatment period of 28 days. The 5-FU concentrations in the ascites after 120mg/day of oral TS-1 ranged from 30 to 80 ng/ml, and the levels maintained during the treated period (Iiduka et al, 2002). Furthermore, CDHP levels in ascites of human gastric cancer patients also maintained at enough levels to inhibit DPD activities (Iiduka et al, 2002). The high concentrations and long exposure duration of 5-FU in the peritoneal cavity after TS-1 may be effective against peritoneal free cancer cells. Furthermore, high concentrations of CDHP may also prevent 5-FU degradation in the peritoneal cavity. In addition, the regimen is safe, because of the absence of any grade 4 toxicities. However, TS-1 related toxicities developed during 1st and 2nd coursed, because patients did not fully recover from operation. TS-1 treatment could be started from 6 weeks after operation in nine (26%) of 35 patients. However, tumor growth may occur during the postoperative weeks before TS-1

139

Yonemura et al: Oral TS-1 treatment for gastric cancer patients with i.p. free cancer cells Japanese Research Society for Gastric Cancer (1995) The General Rules for Gastric Cancer Study (1st English edn). Kanehara Shuppan: Tokyo. Japanese Research Society for Gastric Cancer (2004) Guidelines for treatment on Carcinoma of the Stomach, (2nd Edition), Japan: Kanehara Shuppan: Tokyo. Los G, Mutsaers PHA, van der Vijgh WJ, Baldew GS, Graaf PW, McVie JG (1989) Direct diffusion of cisdiamminedichloroplatinum (II) in intraoeritoneal rat tumors after intraperitoenal chemotherapy: A comparison with systemic chemotherapy. Cancer Res 49, 3380-3384. MacDonald JS, Philip SS, Woolley PV, Smythe T, Ueno W, Hoth D, Smith F, Boiron M, Gisselbrecht C, Brunet R, Lagarde C (1980) 5-Fluorouracil, doxorubicin and mitomycin (FAM) combination chemotherapy for advanced gastric cancer. Ann Intern Med 93, 533-536. Markman M (1991) Intraperitoneal chemotherapy. Semin Oncol 18, 248-254. Mori T, Fujiwara Y, Yano M, Tamura S, Yasuda T, Takiguchi S (2003) Experimental study to evaluate the usefulness of S-1 in a model of peritoneal dissemination of gastric cancer. Gastric Cancer 6, 13-18. Sakata Y, Ohtsu A, Horikoshi K, Mitachi Y, Taguchi T (1998) Late phase II study of novel oral fluoropyrimidine anticancer drugs S-1 (1M tegaful-0.4M gimestat-1M otastat potassium) in advanced gastric cancer patients. Eur J Cancer 34, 17151720 Shirasaka T, Nakano K, Takechi H, Satake H, Uchida J, Fujioka A, Saito H, Okabe H, Oyama K, Takeda N, Fukushima M (1996) Antitumor activity of 1M tegaful,-0.4M 5-chrolo-2,4dyhydroxypiridine-1M potassium oxonate (S-1) against human colon carcinoma orthotopically implanted into nude rats. Cancer Res 56, 2602-2606. Sugarbaker PH, Stuart OA, Vidal-Jove J, Pessagno AM, DeBruijn EA (1993) Studies of the peritoneal-plasma barrier after systemic mitomycin C administration. Cancer Treat Res 4, 188-194. Tatsumi K, Fukushima M, Shirasaka T, Fujii S (1987) Inhibitory effect of pyrimidine, barbituric acid and pyrimidine derivatives on 5-fluorouracil degradation in rat liver extracts. Jpn J Cancer Res 78,748-755. Van Groeningen CJ, Peters GJ, Schornagel JH, Gall S, Noodhuis P, de Vries MJ, Turner SL, Swart MS, Pinedo HM, Hanauske AR, Giaccine G (2000) Phase I clinical and pharmacologic study of oral S-1 in patients with advanced gastric solid tumor. J Clin Oncol 18, 2772-2779. Wils JA, Klein HO, Wagener DJ, Bleiberg H, Reis H, Korsten F, Conroy T, Fickers M, Leyvraz S, Buyse M (1991) Sequential high-dose methotrexate and fluorouracil combined with doxorubicin--a step ahead in the treatment of advanced gastric cancer: a trial of the European Organization for Research and Treatment of Cancer Gastrointestinal Tract Cooperative Group. J Clin Oncol 9, 827-31. Wu CC, Chen JT, Chang MC, Ho WL, Chen CY, Yeh DH, Liu TJ, Pâ&#x20AC;&#x2122;eng FK (1997) Optimal surgical strategy for potentially curable serosa-involved gastric carcinoma with intraperitoneal free cancer cells. J Am Coll Surg 184, 611617. Yamagata S, Nakata B, Hirakawa K (2004) Dihydropyrimidine dehydrogenase inhibitory fluoropyrimidine S-1 may be effective against peritoneal dissemination in gastric cancer. Oncol Rep 12, 973-978. Yonemura Y, Bandou E, Kinoshita K, Kawamura T, Takahashi S, Endou Y, Sasaki T (2003) Effective therapy for peritoneal

administration. Earlier start of TS-1 administration should be recommended, but the recovery after operation is not enough in some patients. We are now trying to treat the patients by early postoperative intraperitoneal chemotherapy before TS-1 therapy (Yu et al, 2001; Yonemura et al, 2003). Prognosis of patients with P0/Cy1 status is similar to the prognosis of those who underwent palliative resection (Wu et al, 1997). Accordingly, Wu proposed that gastrectomy without additional lymphadenectomy is recommended for the patients with P0/Cy1 status. As shown in the present paper, TS-1 is effective to improve the survival and to control peritoneal recurrence for patients with P0/Cy1 status. From these studies, the dissection of lymph node metastases may be important to improve the survival in patients with P0/Cy1 status. TS-1 treatment is safe and effective as a postoperative chemotherapy for patients with P0/Cy1 status, and radical gastrectomy plus postoperative TS-1 therapy are recommended for the patients.

References Abe S, Kojima M, Tamura H, Kurihara H, Kitago M, Kobayashi T, Nakamura T, Ogihara T (2003) A clinical result of TS-1 in advanced and recurrent gastric cancer in our hospital. Gan To Kagaku Ryoho 30963-970. Band E, Yonemura Y, Takeshita Y, Taniguchi K, Yasui T, Yoshimitsu Y, Fushida S, Fujimura T, Nishimura G, Miwa K (1999) Intraoperativve lavage for cytological examination in 1, 297 patients with gastric carcinoma. Amer J Surg 178, 256-262. Benevolo M, Mottolese M, Coamelli M, Tedesco M, Giannarelli D, Vasselli S, Carlini M, garofalo A, Natali PG (1998) Diagnostic and prognostic value of peritoneal immunocytology in gastric cancer. J Clin Oncol 16, 34063411. Chu ZD, Lang NP, Thompson C, Osteen PK, Westbrook KC (1989) Peritoneal carcinomatosis in nongynecological malignancy. A prospective study of prognostic factors. Cancer 63, 364-367. Cullinan SA, Moertel CG, Fleming TR, Rubin JR, Krook JE, Everson LK, Windschitl HE, Twito DI, Marschke RF, Foley JF, et al (1985) A comparison of three chemotherapeutic regimens in the treatment of advanced pancreatic and gastric carcinoma. Fluorouracil vs. fluorouracil and doxorubicin vs fluorouracil, doxorubicin, and mitomycin. JAMA 253, 20612067. Hirata K, Horikoshi N, Aiba K, Okazaki M, Denno R, Sasaki K, Nakano Y, Ishizuka H, Yamada Y, Uno S, Taguchi T, Shirasaka T (1999) Pharmacokinetic study of S-1, a novel oral fluorouracil antitumor drug. Clin Cancer Res 5, 20002005. Iiduka R, Takahashi S, Kakihara N, Matsumura H, Takenaka A (2002) Concentration of FT and CDHP and 5-FU in the ascites fluid of patient with peritoneal dissemination after new anti-cancer drug TS-1 oral administration. Jpn J Cancer Chemother 29, 1251-1253. Jacquet P, Sugarbaker PH (1996) Peritoneal-plasma barrier. In P.H. Sugarbaker (ed). Peritoneal Carcinomatosis: Principles of Management, Kluwar Academic Pubisher, Boston, pp5363.

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Cancer Therapy Vol 4, page 141 dissemination in gastric cancer. Surg Oncol Clin N Am 12, 635-648. Yonemura Y, Endou Y, Tochiori S, Banou E, Kawamura T, Shimada T, Miyamoto K, Tanaka M, Sasaki T (2005) effects of chemotherapy on experimental peritoneal dissemination of gastric cancer. Jpn J Cancer Chemother 32,1635-1639.

Yu W, Whang I, Chung HY, Averbach A, Sugarbaker PH (2001) Indications for early postoperative intraperitoneal chemotherapy of advanced gastric cancer: results of a prospective randomized trial. World J Surg 25 985-90.

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Cancer Therapy Vol 4, page 143 Cancer Therapy Vol 4, 143-152, 2006

Molecular basis for androgen independency in prostate cancer Review Article

JesĂşs Gil MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Campus, W12 0NN London, UK

__________________________________________________________________________________ *Correspondence: JesĂşs Gil, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Campus, W12 0NN London, UK; Phone: +44 (0) 20 8383 8263; Fax: +44 (0) 20 8383 8306; e-mail: jesus.gil@csc.mrc.ac.uk Key words: Prostate cancer, androgen independency, androgen receptor, c-myc. Abbreviations: androgen receptor, (AR); androgen responsive elements, (ARE); androgen-independent prostate cancer, (AIPC); benign prostatic hyperplasia, (BPH); dihydrotestosterone, (DHT); gonadotropin releasing hormone (GnRH) luteininizing hormone, (LH); prostate cancer (PCa); prostate-specific antigen, (PSA); prostatic intraepithelial neoplasia, (PIN) Received: 11 January 2006; Revised: 22 February 2006 Accepted: 23 March 2006; electronically published: April 2006

Summary The growth of normal prostate is dependent on androgen stimulation. In a similar fashion, arising prostate tumors are androgen-dependent and consequently androgen ablation is the therapy of choice for prostate cancer reappearing after initial treatment or invading secondary organs. When tumors become resistant to this therapy, these are more aggressive and often present androgen-independent characteristics. Therefore the conversion from androgen-dependent to androgen-independent tumors constitutes an important event in prostate cancer progression from the therapeutic point of view and because of its incidence on mortality. Extensive efforts have been used to investigate the molecular basis of androgen-independency as they could predict the therapeutic outcome of particular tumors and eventually serve for applying tailored pharmacogenetics to prostate cancer. In this review we will try to summarize our knowledge of the genetic events governing the conversion to androgen independency in prostate tumorigenesis.

the later stages of this disease (Feldman BJ and Feldman D, 2001).

I. Introduction More than 10 % of American men older than 60 are affected by prostate cancer (PCa). Similarly PCa is highly prevalent in western countries, being the second most diagnosed malignancy after skin cancer and more importantly, the second most common cause of cancerrelated male mortality (Greenlee et al, 2000). In addition, PCa incidence its directly associated with aging, meaning that with increased life expectancies, the number of PCa cases is predicted to raise over the coming years (Nelson et al, 2003). Prostate cancer is a relatively slowly progressing and indolent disease and when it remains localised it can be controlled first with surgery and later by anti-androgen therapies. However tumors reappearing after anti-androgen treatment are usually irresponsive to the existing treatments, more aggressive and prone to metastasize. Thus, androgen-independent prostate cancer (AIPC) constitutes a real life threat that accounts for the gross part of PCa mortality. Therefore, understanding the genetic basis for the progression to AIPC could contribute to the establishment of rationale alternative therapies for

In adult men, the prostate gland is a small acornshaped tissue with ductal-acinar histology that lacks lobular organization. It is located below the bladder, surrounding the urethra and secretes proteins that are incorporated to the seminal fluid. However, it lacks a discernible role in fertility and its loss has no impact on viability. Therefore, the practical interest in studying the biology and development of the prostate is linked entirely to its pathological relevance. Morphologically the human prostate can be divided in 3 distinct regions termed peripheral, transitional and central zones. This morphological distinction is important from the pathological perspective as prostate cancer (PCa) originates mainly from the peripheral zone, while the transition zone give rises to benign prostate hyperplasias (BPH) that are not evolutionary related to prostate cancer. At the cellular level, the human prostate is composed of ducts and acini embedded in a stromal matrix of

143

Gil: Androgen-independent prostate cancer fibroblastic and myofibroblastic cells. In the glandular epithelium there are two predominant epithelial cell types, namely secretory luminal cells and basal epithelial cells (Bonkhoff and Remberger, 1996). Basal cells are set in the exterior part of the prostate ducts and they secrete components of the basement membrane. The luminal cells are located in the inner layer of the ducts and are involved in secreting components that are incorporated to the prostatic fluid. Interestingly, luminal cells express androgen receptor (AR), the prostate specific antigen (PSA) and are dependent on androgen for their proliferation, while basal cells are androgen-independent and do not express these markers. In addition, there is also a small proportion of a third cell type of non-epithelial origin in the prostate, neuro-endocrine cells, which precise role is not completely understood. Interestingly it is believed that a subset of the basal cells could present a stem cell-like potential giving rise to and being able to replenish the luminal cells. These human prostatic epithelial stem cells have been identified as !2"1hi/CD133+, have a high proliferative potential in vitro and can reconstitute prostatic-like acini in nude mice (Richardson et al, 2004). The stem cell properties are underscored by the observation that hormone replacement in castrated animals results in a quick regeneration of the whole prostate gland (Sugimura et al, 1986). In addition, luminal cells are post-mitotic while basal cells are not. As in any other organ, there exists a significant interaction and crosstalk between the different cell types (epithelial and stromal) and this interaction impacts on the overall growth and development of the prostate (Liu et al, 1997). The different cell types of the prostate can be identified by the expression of diverse cellular markers. For example, luminal cells are characterised mainly by expressing cytokeratin 8 and 18. On the other hand, basal cells express cytokeratin 5 and 14 in conjunction with other relevant markers such as CD44 (Liu et al, 1997). A more detailed analysis of cytokeratin expression in the prostate have identified different cell subpopulations expressing mixed combinations of these markers, what has been interpreted as suggestive of intermediate precursors originated during the differentiation of prostate cells (Xue et al, 1998). Further work in this direction will probably be interesting for understanding both normal prostate development and the formation and evolution of PCa lesions

Histological analysis of prostate lesions have identified a type of pre-malignant neoplastic lesions, referred as prostatic intraepithelial neoplasia (PIN) as the precursor of human prostate tumors. PIN lesions typically appear in the prostate peripheral zone and are multifocal in nature similarly to that observed with PCa (Abate-Shen and Shen, 2000). PIN has been classified in 4 grades according to their severity. Importantly, prostate ducts affected by PIN present disruption of the basal layer while still retaining an intact basement membrane, therefore impeding the stromal invasion by neoplastic cells. In contrast, invasive prostate carcinomas can invade the stromal tissue, as the basal lamina is lost from the prostate ducts. A reliable indicator of PCa development is the presence of high levels of PSA in serum. Elevated levels of PSA in serum are observed in invasive carcinomas but not in PIN (Mazzucchelli et al, 2000). The available clinical tests for measuring the levels of PSA in serum have allowed for an early detection of the disease. Therefore, intervention at earlier stages of PCa is being implemented and in the long term may contribute to improved survival. Several systems are used to classify the severity of PCa. The procedure most commonly used is known as Gleason score and was devised by Dr. Donald F. Gleason more than 30 years ago. It is based on the histological classification of haematoxylin/eosin stained prostate sections between five grades. They vary from 2 to 10 as the grade of the two more abundant patterns observed in every case is added up to calculate the Gleason score. Different studies have validated the accuracy of the Gleason score system by correlating it with patient survival or progression to metastatic state (Gleason, 1992).

A. Treatments for prostate cancer Primary prostate cancer which remains localised to the prostate glands can be controlled by either prostatectomy or radiation therapy. The later usually removes or destroys local tumors. However, different strategies have to be applied for dealing with PCa after it invades other organs or for treating PCa tumors that reappear after the initial intervention. In these circumstances deprivation of androgens is the therapy of choice (Eisenberger et al, 1998). However it has to be considered that the effectiveness of androgen ablation in the management of advanced prostate cancer is limited. After certain time (the average being 2 years), PCa evolves to an androgen-independent disease with a poor prognosis and unfortunately with no curative therapies available (Feldman BJ and Feldman D, 2001). During the final stages, tumors progressively invade seminal vesicles and finally metastasize in other organs, primarily to the bone, resulting in osteoblastic tumors (Logothetis and Lin, 2005). Usually a lost of androgen dependency is observed at these final steps of the disease. This is partially caused by the selective pressure exerted by androgen ablation therapy.

III. Prostate cancer, progression and treatment There are several abnormal pathologies of the prostate each one presenting different origins, progression and consequences. Hyperplasia on the transition zone of the prostate and a subsequent obstruction of the urethra is extremely frequent in aging men (Isaacs and Coffey, 1989). This condition is known as benign prostatic hyperplasia (BPH) and itself constitutes the second most common reason for surgery in men over 65 (Oesterling, 1995). However as we noted before, this fairly common malignancy does not constitute a precursor phase of PCa, which arises from a different region of the prostate.

IV. Androgen receptor signalling Growth factors enhance proliferation and promote survival being involved in maintaining the normal 144

Cancer Therapy Vol 4, page 145 homeostasis of tissues and organs. This is particularly true under physiological and pathological circumstances for the prostate as it is dependent on androgens for its normal growth and development. Also prostate cancer remains highly dependent on androgens during its initial stages. The main circulating androgen on males is testosterone that is produced in the testis and the adrenal glands (Wilson et al, 1983). Once inside prostate cells, the enzyme 5!-reductase converts it to the metabolically more active dihydrotestosterone (DHT) (Bruchovsky and Wilson, 1999). DHT act as the main physiological ligand of androgen receptor (AR) and it is much more active than testosterone, having higher affinity for the AR. The AR is a classical nuclear receptor, consisting of a ligand-binding domain, an amino terminal activating domain and two Znfingers that constitute the DNA-binding domain (Gao et al, 2005). Similar to other nuclear receptors, upon binding of ligand, the AR suffers a conformational change that facilitate its binding to different transcriptional coactivators that link it with the general transcriptional machinery (Hart, 2002). Many transcriptional targets of the AR have been identified, amongst them PSA being one prototypic target with a critical relevance for prostate cancer diagnosis (Kim and Coetzee, 2004). After its activation by androgens, AR can bind to DNA target sequences located on or near androgen-dependent genes, termed androgen responsive elements (AREs). Such elements are present in known androgen responsive genes such as the ones encoding for PSA or AR (Berger and Watson, 1989). Using different genomic approaches a broad set of genes that are responsive to androgens have been identified (Nelson et al, 2002). Overall, through the modulation of multiple targets, androgens and AR mediate key processes involved in the normal development and function of the prostate (Bonkhoff and Remberger, 1996; Isaacs et al, 1992). During development, the pattern of expression of the AR dictates the androgen-responsiveness of the prostate and impact on its differentiation. In the mature prostate androgen does not only regulate the division and proliferation of prostate cells but also governs a cell death programme. In general, androgen also regulates several aspects of prostate cellular metabolism.

or maximal androgen blockade is a more thorough approach that combines an anti-androgen together with super-agonists of GnRH (Labrie et al, 1993). In this way, not only the androgen production in the testis is blocked, but also anti-androgens can avoid the activation of the androgen receptor by the residual levels of androgen that continues to be produced in the adrenal gland. It is important to remark that any of these strategies do not achieve a complete depletion of androgen, but result in androgen deprivation. Because of that, Scher and Sawyers proposed that the term â&#x20AC;&#x2DC;castration-resistantâ&#x20AC;&#x2122; is more adequate to define these tumors (Scher and Sawyers, 2005). Overall, the fallout of these therapies is that they cannot be considered a cure of prostate cancer, but they just temporally avoid its progression for a limited time before the appearance of resistant tumors resulting in the inevitable progression of this disease. The current standard therapy for patients who have progressed on androgen deprivation is docetaxel and prednisone (Debes and Tindall, 2004). Although effective in prolonging life, this therapy is not curative and new approaches to treat AIPC are under development and consequently others should be investigated.

VI. Conversion independency

to

androgen

The transition of the prostate cancer cell to an androgen independent phenotype is a complex process that involves selection and outgrowth of pre-existing clones of androgen-independent cells as well as selection for genetic events that help the cancer cells survive and grow in androgen-independent conditions. These two mechanisms share a common initial point: prostate cancers are heterogeneous tumours comprised of different subpopulations of cells that therefore would respond differently to anti-androgens. This tumour heterogeneity may thus reflect a combination of their multifocal origin, ability to adapt to the environment and the genetic instability of the tumor (Abate-Shen and Shen, 2000). Similarly to what happens with our current understanding of the genetic mechanism involved in the transition from cancer to a metastatic stage (Bernards and Weinberg, 2002), there are different theories for explaining the conversion of androgen-dependent PCa to androgen-independent prostate cancer (AIPC) (Feldman BJ and Feldman D, 2001). The initial PCa tumors already contain a heterogeneous mix of cells with different genetic backgrounds and the selective pressure exerted by antiandrogen therapy favours the outgrowth of a population over other. One possibility is that cells resistant to androgen ablation emerge amongst androgen-resistant prostate stem cells. Although this remains a hypothesis, the recent identification of prospective prostate cancer stem cells suggests that such a scenario could be plausible (Collins et al, 2005). In any case, the anti-androgen treatment adds up to the global pressure of the tumor to outgrowth and survive and can contribute to select for cells able to proliferate independently of androgens. Thus, either these mutations happen as an early event that is selected during the evolution process, or perhaps the

V. Anti-androgen therapy The observation that castration induces regression of PCa, in a similar way as it induces involution of the prostate gland in animal models was the first suggestion that PCa relies on androgens for its maintenance and progression. Therefore, it became obvious that targeting this growth-addiction offered a therapeutic opportunity to tackle PCa progression (Huggins, 1967). Anti-androgen intervention can be implemented in different ways; one is orchiectomy (surgical removal of the testicles), but the most usual approaches involve pharmacological control. The first of these chemical approaches consist in the use of super-agonists of the Gonadotropin releasing hormone (GnRH) (Labrie et al, 1986). They downregulate the GnRH receptors, leading to a suppressor of luteinizing hormone (LH) release, that finally causes an inhibition of testosterone secretion in the testis. Total androgen ablation 145

Gil: Androgen-independent prostate cancer selection is triggered at a later stage because the mutations already conferred some advantage for tumor growth, even under androgen-dependent conditions (Abate-Shen and Shen, 2000). Therefore, one suggestion is that intermittent anti-androgen therapy could be successful in suppressing tumor growth without exerting such a strong pressure for the appearance of AIPC that would delay the progression to the more malignant stages of PCa (Abate-Shen and Shen, 2000). The fact that anti-androgen therapy exerts such a pressure for the selection of additional lesions during PCa is underscored for the diversity of mutations that can be observed upon different anti-androgen treatments, as we will describe more extensively below.

androgen independent (Taplin and Balk, 2004). A comparison with the primary tumors from where these AIPC tumors originated shows that the AR amplification happened after treatment, what suggest that the pressure exerted by anti-androgen therapy probably allows for a selection of pre-existing clones in which the AR gene had been amplified (Visakorpi et al, 1995a). An interesting observation is that tumors with the AR gene amplified present a better outcome that the ones without AR amplifications (Koivisto et al, 1997). Although we termed the tumors that present amplification of the AR gene as androgen independent, probably is more accurate name them as hypersensitive to low concentrations of androgen, as they still depend of the minimum amounts of androgen present in the organism to proliferate. This is consistent with the proposed nomenclature of â&#x20AC;&#x2DC;castration-resistantâ&#x20AC;&#x2122; tumors proposed by Scher and Sawyers (Scher and Sawyers, 2005). Therefore, it can be suggested that total androgen ablation may be an advantage respect to monotherapy as it will reduce even further the effective levels of circulating androgens in blood (Palmberg et al, 2000). Interestingly, an upregulation of the AR mRNA levels was the only consistent change observed in a set of isogenic prostate cancer xenograft models after progression to androgen independency (Chen et al, 2004). The conclusion inferred from this study is that even a subtle upregulation of AR mRNA levels causes a state of increased sensitivity to low levels of circulating androgens. Similar results could be predicted if increased expression of AR co-activators is achieved either because of their gene amplification or because their transcriptional upregulation.

VII. Molecular pathways involved in AIPC An overview of the signal transduction pathways triggered by androgen signaling suggests that alterations occurring at different levels could potentially result in the maintenance of androgen-independent growth. The different genetic events that are involved in AIPC progression (summarized in Table 1) can be classified taking into consideration their different ways of action (Feldman BJ and Feldman D, 2001).

A. Mechanisms dependent on AR and androgens. 1. Amplification of AR There are several mechanisms for explaining how proliferation is sustained in a way dependent on the androgen receptor (AR) upon anti-androgen treatment or castration. Amplifications of the AR gene are observed in more than 30 % of the tumors that later will become

Table 1. Mechanisms of progression to androgen-independent prostate cancer (AIPC) Mechanism Dependent on AR and androgens

Example Amplification of AR gene

Increased AR mRNA levels Increased androgen (DHT) levels Dependent on AR but independent on androgens

Independent on AR and androgens

AR activated by flutamide

Details More than 30% tumors

5!-reductase polymorphism AR T877A mutation

AR activated by glucocorticoids Activation of AR by growth factor receptors Activation of AR by Her2/neu Activation of PI3K/AKT pathway

AR L701H, T877A mutations

Activation of antiapoptotic pathways Activation of downstream routes by c-myc

i.e. Bcl2 expression

146

IGF-1, KGF, EGF PTEN deletions in PCa

Amplifications of c-myc in more than 70% of AIPC tumors

References Visakorpi et al, 1995a; Taplin and Balk, 2004 Chen et al, 2004 Labrie et al, 1996 Horoszewicz et al, 1980 Zhao et al, 2000 Culig et al, 1994 Craft et al, 1999 Li et al, 1997 Vivanco and Sawyers, 2002 Gleave et al, 1999 Bernard et al, 2003

Cancer Therapy Vol 4, page 147 Some of these mutations in the AR gene have also been found to make the AR responsive to different ligands. For example a double mutation in the AR gene (T877A together with L701H) results in the AR being susceptible to activation by glucocorticoids (Zhao et al, 2000). Finally, AR mutations being selected during antiandrogen treatment can also be mimicked in animal models of AIPC conversion, further underscoring their importance.

2. Production of increased levels of androgen. There are some hints that under anti-androgen therapy despite a sharp decrease of overall androgen in blood, the availability of DHT does not decrease to the similar extent (Labrie et al, 1986). One of the possible explanations is a compensatory regulatory loop that accounts for an increased conversion of testosterone to DHT. Interestingly polymorphisms in the 5!-reductase gene had been reported and some of these polymorphisms have been linked to a hyperactive enzyme being produced. These polymorphisms are observed more frequently in men of African origin that present an increased risk of PCa (Makridakis et al, 1997). No association of these mutations with AIPC conversion has been described, but the predisposition of individuals carrying these polymorphisms cannot be ignored.

2. Activation of AR independently of ligand It has been suggested that alterations in proteins that act in conjunction with AR in transcriptional control could be involved in the progression to AIPC (Adachi et al, 2000). However, the existing evidence comes only from cell culture data. Perhaps one of the best indications that this could be meaningful comes from the fact that mutations on the AR affecting to residues involved in their interaction with co-activators are also involved in AIPC. Based on work performed in other tumor types (specially in lymphoma) it has been proposed that the complex tumor suppressor and oncogene networks altered during cancer progression also influence the responses to cancer therapies (Schmitt et al, 2002). Thus, it can be envisioned that mutations that happen in tumors and result positively selected because they can favour tumor progression may impact in AIPC. In this sense the AR pathway is inter-connected with other signaling transduction pathways and therefore alterations in those pathways would result in a modification of AR function that could provoke AIPC. For example, other growth survival pathways driven by extracellular receptors have been found to crosstalk and thus sustain AR-signaling. In that sense different evidence has been provided for the ability to cause AIPC by growth factors such as epidermal growth factor (EGF), insulinlike growth factor 1 (IGF-1) and keratinocyte growth factor (KGF) among others (Culig et al, 1994). Probably IGF-1 is the best studied example, but interestingly it is known that all of them, IGF-1, KGF and EGF act upstream of AR signaling, as the androgen independent effects they exert can be blocked by anti-androgens such as casodex. Conversely, this would downplay the effect that they can have during AIPC in conditions of complete anti-androgen therapy. A clearer connection has been identified between the pathways triggered by receptor tyrosine kinase and AR signaling. Similar results have also been obtained for studies performed in other endocrine-dependent tumors (such as breast or ovarian cancers). The prototypic case of a growth factor receptor involved in hormoneindependency is Her2/neu (Craft et al, 1999). Her2/neu is over-expressed or amplified in a subset of both breasts and ovarian tumours. Those studies have provided the foundation for developing agents interfering with Her2/neu signaling and their use in cancer control (Allen, 2002). Notably, these drugs have already been used successfully against non-small-cell lung cancer and certain metastatic breast tumours and are being applied now in the clinic.

B. Mechanisms dependent on AR but independent on androgens 1. AR sensitive to activation by alternative ligands As we have noted, AR expression is conserved in many of the AIPC cases (Buchanan et al, 2001). In a significant subset of these cases (the exact percentage varying from report to report), gain of function mutations in the ligandbinding domain of the AR are observed. However, it seems clear that these mutations are selected as a consequence of anti-androgen treatment. These AR gain of function mutations were first described in LNCaP cells (Veldscholte et al, 1994). LNCaP cells were derived from a metastasis to the lymph node of a patient with prostate cancer that had been treated with the anti-androgen flutamide (Horoszewicz et al, 1980). Their AR presents a mutation resulting in the substitution of threonine for alanine at position 877 (T877A). As a result the AR mutant present in LNCaP cells becomes susceptible to be activated by ligands different of DHT. Notably flutamide, that it is used as an antiandrogen or androgen antagonist during therapy, behaves as an activator of the T877A AR (Horoszewicz et al, 1980). In this way, flutamide fuels the progression of PCa once this mutation on the AR is selected. Interestingly, the same AR mutation is observed in a number of different cases of AIPC. Also there is a subset of patients in clinic that present a decrease of the PSA levels after flutamide withdrawal, what would be consistent with flutamide upregulating the expression of PSA, an AR-dependent gene (Horoszewicz et al, 1980). Interestingly however, LNCaP cells which growth is stimulated by flutamide by virtue of the T877A mutations, still remain sensitive to casodex, other anti-androgen (Horoszewicz et al, 1980). This has been explained in structural studies examining the binding site of both drugs in the AR molecule (Horoszewicz et al, 1980). The T877A mutation also makes the AR sensitive to other ligands such as estrogens and progesterone. In addition to the T877A mutation a whole set of other AR mutations have been identified and catalogued (http://www.androgendb.mcgill.ca).

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Gil: Androgen-independent prostate cancer Interestingly, studies performed on AIPC cell lines derived from xenografts from castrated mice did consistently show an upregulation of Her2/neu expression, what suggest that in AIPC operates a similar mechanism to that operating in breast and ovarian cancer (Craft et al, 1999). Forced over-expression of Her2/neu recapitulates those observations, thus suggesting that is sufficient for conversion to AIPC. Her2/neu growth promotion under AIPC conditions is also dependent on AR signaling, but differently to what it has been shown for IGF-1 and EGF, casodex cannot inhibit this process, therefore suggesting that this pathway is independent on the AR-ligand binding domain (Craft et al, 1999; Yeh et al, 1999). These results hint that, similarly to what already is in practise in the clinics for breast tumors, inhibition of Her2/neu signaling (through the use of antagonist antibodies, such as herceptin) could help to treat PCa. Also, perhaps this therapy should be used in combination with chemotherapeutic agents such as it is used in metastatic breast cancer (Slamon et al, 2001). Preliminary work performed in prostate cancer xenografts and cells suggested that indeed this could be the case (Agus et al, 1999). However, there is no current clinical data to support the use of drugs targeting Her2/neu for prostate cancer (Gross et al, 2004). The crosstalk between the Her2/neu pathway and AR signaling ultimately results in the phosphorylation of AR what impacts into its inappropriate activation. As a result, it can be hypothesized that the components lying in the interphase between both pathways could drive AIPC conversion when its regulation is lost. The pathway has begun to be unveiled and involves at least MAPK signaling (Yeh et al, 1999). Additionally the phosphatidylinositol-3-OH kinase (PI3K) pathway is also activated by Her2/neu (Yakes et al, 2002) and it is also implicated in cross-talking with the AR (Lin et al, 2001). In fact, activation of the PI3K/AKT pathway can drive AIPC in a manner that is independent on both AR and androgens, as we will discuss below.

regulators, increased cell cycle progression, decreased transcription of pro-apoptotic genes through inhibition of forkhead transcription factors, altered metabolism, or changes in mRNA translation that ultimately impact on cell death. Mutations that activate this signaling route, include amplifications of components of the PI3K pathway and the inactivation of the negative regulator lipid phosphatase PTEN, are common in human malignancies and amongst them PCa (Vivanco and Sawyers, 2002). The PI3K pathway is engaged and activated by multiple extracellular receptors. PI3K is a lipid kinase that phosphorylates phosphatidyl inositols. This phosphorylation process is antagonized by PTEN that is a lipid phosphatase that removes the 3-phosphate from 3phosphorylated inositol lipids. The phosphatidyl inositol tri-phosphate lipids act as second messengers and activate AKT. AKT is a protein kinase that once activated controls multiple targets involved in cell cycle progression or apoptosis survival amongst others. Among the substrates that AKT phosphorylates are the CDK inhibitor p27KIP1, the apoptotic proteins BAD, AR, FOXO and many others (Datta et al, 1999; Reed, 2002). Experiments analysing androgen-independent xenografts have shown increased levels of AKT activity (Graff et al, 2000). Interestingly, the ability of AKT to phosphorylate AR could be linked to this androgen independency (Lin et al, 2001). In addition, as the PI3K/AKT pathway is a critical regulator of apoptosis, it may be thought that part of the effect exerted by this gene in AIPC could be through the regulation of apoptosis by modulating independent parallel pathways. Androgen, as we mentioned before, drives prostate cells into active cell cycle progression. Conversely androgen withdrawal results in a combination of a tight cell cycle arrest and apoptosis. Consequently, it can be thought that if alternative survival pathways are activated, the anti-proliferative effects caused by androgen withdrawal can be suppressed either partially or totally. Particularly, overexpression of apoptosis modulators may be an obvious target to improve cell survival. One of the first genes suggested to play such a role was Bcl2, that under normal circumstances has its expression restricted to basal cells that do not dependent on androgens. Conversely Bcl2 is not expressed in luminal cells that are androgen-dependent (McDonnell et al, 1992). Overexpression of Bcl2 is observed in the initial stages previous to PCa such as in PIN. Interestingly, aberrant expression of Bcl2 has been linked to hormoneindependent prostate carcinomas (Gleave et al, 1999) and specifically Bcl2 is expressed during AIPC conversion while the parental tumors were showed not to express initially Bcl2. Presumably other modulators of the apoptotic network could have similar effects in driving AIPC-progression.

C. Mechanisms independent on AR and androgens 1. Activation of parallel survival pathways An alternative mechanism for progression to AIPC is based on the induction of a positive growth signal independent on the AR that can overcome the growth inhibition imposed by anti-androgen therapies, thus establishing what has been defined as a bypass pathway (Feldman BJ and Feldman D, 2001). A role for the PI3K/AKT pathway in PCa had been already suggested through the analysis of mutations happening in cancer. Particularly, the tumor suppressor PTEN, was first identified because it was found altered in prostate amongst other types of tumors (Li et al, 1997). The PI3K pathway integrates receptor tyrosine kinase signaling with the apoptotic network. One key mediator of PI3K signaling is the protein kinase AKT, that phosphorylates multiple downstream effectors causing deep changes in cellular physiology (Cantley, 2002). How AKT promotes survival is not completely understood, but it may involve direct phosphorylation of apoptotic

2. Activation of downstream pathways Previous work has suggested the involvement of cmyc in prostate cancer and particularly in the progression to AIPC (Nupponen et al, 1998). In this case, the mechanism by which c-myc drives the progression of AIPC relies on the activation of pathways located

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Cancer Therapy Vol 4, page 149 downstream of the AR. Different studies had narrowed down a common amplicon detected during the conversion to AIPC to a short region spanning the chromosome 8q and containing the c-myc gene (Visakorpi et al, 1995b; Nupponen et al, 1998). Fluorescence in situ hybridization have shown amplification of the c-myc gene in more than 70% of AIPCs (Nupponen et al, 1998) and a significant increase of c-myc amplification is observed after anti-androgen treatment (Kaltz-Wittmer et al, 2000). However, the mechanism used by c-myc to drive AIPC progression was not clear. Recently we examined the effect of c-myc in AIPC progression by using LNCaP cells treated with the antiandrogen casodex (Bernard et al, 2003). In that work, we showed that overexpression of c-myc was sufficient to induce androgen-independent growth in casodex treated cells. Our data suggested that c-myc did not act through an increase of AR activity because c-myc did not alter the levels of AR-dependent genes such as PSA or PSMA. Functional data further confirm these results as neuroendocrine differentiation induced by AR inhibition (Ahlgren et al, 2000; Ismail et al, 2002; Wright et al, 2003) was not overcome by c-myc expression. In addition,

AR silencing in c-mycâ&#x20AC;&#x201C;expressing cells did not prevent cell growth. More interestingly, what our results suggest is that c-myc is indeed a downstream target of AR, as the cmyc protein level is regulated by AR activity and c-myc is required for androgen-dependent growth (a schema depicting c-myc mode of action is presented in Figure 1). The precise mechanism of c-myc regulation by AR is not known, but our data suggest that it can happen at a posttranscriptional level. We have also demonstrated that c-myc expression could immortalize human primary prostate epithelial cells by overriding the p16INK4a/Rb pathway and inducing hTERT expression (Gil et al, 2005). The fact that c-myc can be activated by AR activity (Quarmby et al, 1987) supports a role for c-myc in the early stages of prostate cancer. Accordingly, it was demonstrated that directed cmyc expression in the prostate induces development of a prostatic intraepithelial neoplasia in the mouse (Zhang et al, 2000). Therefore, during PCa c-myc act probably as a pleiotropic oncogene that becomes activated or overexpressed and gives growth advantage to early tumors, but later on the progress of the tumor is also able to confer androgen independency.

Figure 1. Role of c-myc in AIPC conversion. (A) In normal prostate cells that do not present c-myc amplifications (upper cartoon), the expression of c-myc mRNA is regulated by androgens and once expressed, c-myc contributes to sustain cell growth. After androgen deprivation or when cells are treated with anti-androgens (lower cartoon), cells stop growing, c-myc and PSA level are downregulated and cells overcome neuroendocrine differentiation. (B) In prostate cells presenting c-myc amplification or overexpression (upper cartoon) androgen withdrawal or treatment with anti-androgens results in neuroendocrine differentiation and a decline in PSA levels, but cell growth is maintained thanks to the high expression of c-myc that controls multiple growth promoting targets (lower cartoon).

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During AIPC, c-myc is needed for AR-induced proliferation because it controls the activity of major growth related proteins. Consequently, without AR activity, but with constitutive expression of c-myc, the cells grow regardless of AR signaling, even if AR expression is maintained. In addition c-myc is an essential gene which deletion impairs the growth of both androgendependent and -independent cell lines. Therefore, in addition to the classical hypotheses of increased AR signaling or establishment of a bypass pathway (Feldman BJ and Feldman D, 2001), c-myc may induce androgenindependent growth through the activation of a downstream pathway. Indeed, we have shown that c-myc is regulated by AR and is required for AR-dependent as well as -independent growth, suggesting that c-myc may be involved in the development of AIPC, including that resulting from an increase of AR signaling.

IX. Conclusions The hypothesis that mutations which drive cancer progression could also have a profound impact on the responses to therapies have already been proved for other types of oncogenic lesions (Schmitt et al, 2000). In this context, PCa, together with other hormone dependent cancers such as breast cancer and ovarian cancer constitute unique types as their growth rely heavily on hormones and the hormone dependency is also targeted during the anticancer therapy. Because of that, a better knowledge of the molecular mechanisms that can permit growth under anti-androgen conditions could result in the adoption of novel therapeutic approaches, either targeting genes identified through this process, or using our improved knowledge of the molecular events involved in AIPC to implement combined therapies. Also the opposite road can be devised, if any of the novel therapeutic approaches (either conventional or targeted) that are being developed for other tumor types are successful in treating PCa, the molecular pathways underlying the mechanism of action of the drugs can be investigated in the context of PCa. Overall, a better molecular knowledge will contribute to better treatments for avoiding this mortal stage of PCa, which incidence is predicted to scale in the coming years due to increased aging.

Acknowledgements JesĂşs Gil is supported by the Medical Research Council.

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JesĂşs Gil

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Cancer Therapy Vol 4, page 153 Cancer Therapy Vol 4, 153-162, 2006

Nutritional patterns and lung cancer risk in Uruguayan men Research Article

Eduardo De Stefani1, Alvaro L. Ronco2, Paolo Boffetta3, Hugo Deneo-Pellegrini1, Pelayo Correa4, Giselle Acosta1, Luis Piñeyro Gutiérrez5 and María Mendilaharsu1 1

Grupo de Epidemiología, Departamento de Anatomía Patológica, Hospital de Clínicas, Facultad de Medicina, Montevideo, Uruguay 2 División de Epidemiología, Instituto de Radiología, Hospital Pereira Rossell, Montevideo, Uruguay 3 International Agency for Research on Cancer, Lyon, France 4 Department of Pathology, Louisiana State University Health Sciences, New Orleans, Louisiana, USA 5 Instituto de Neumología, Facultad de Medicina, Montevideo, Uruguay

__________________________________________________________________________________ *Correspondence: Dr. Eduardo De Stefani, Avenida Brasil 3080 dep. 402, Montevideo, Uruguay; Tel.: (598) 2 708 23 14; Fax: (598) 2 402 08 10; E-Mail: estefani@adinet.com.uy Key words: lung cancer, factor analysis, principal components, dietary patterns Abbreviations: food-frequency questionnaire (FFQ); heterocyclic amines, (HCA); odds ratios, (ORs)

Supported by a grant N° ECE/98/17 from International Agency for Research on Cancer, Lyon, France All authors contributed to the statistical analysis, interpretation of the data, and preparation of the manuscripts. Received: 26 January 2006; Revised: 20 February 2006 Accepted: 10 April 2006; electronically published: April 2006

Summary Lung cancer is the more frequent malignancy among Uruguayan men. The age-adjusted incidence rate is 76.5 per 100,000 inhabitants of Montevideo. In comparisons between American registries, the rate of Uruguay is only second, following the rate observed among Black men in United States. Doubtless the main etiologic factor of lung cancer in Uruguay is tobacco smoking. Uruguayan men display a high prevalence rate of smoking and this population is also characterized by an elevated use of black tobacco cigarettes and by the use of hand-rolled cigarettes. Both types of cigarettes have been considered as particularly risky. Also occupation and dietary factors have been considered as risk factors for this malignancy. Previous studies on diet and lung cancer suggested that vegetables, fruits, carotenoids and fat were associated with lung cancer risk. A case-control study which included 846 newly diagnosed and microscopically confirmed male cases with lung cancer and 846 male hospitalized patients with non-neoplastic diseases was conducted in Montevideo, Uruguay. Both series of participants were directly interviewed by two trained social workers in the four major public hospitals in Montevideo. Cases and controls were frequency matched on age and residence. The patients were analyzed by factor analysis (principal components) with an eigenvalue of 1.25 as a limit for retained principal components. The analysis allowed to retain four factors which were labelled as “healthy”, “western”, “fatty” and “cheese and rice”. The “cheese and rice” and “healthy” patterns were protective (OR for the higher score of the “cheese and rice” factor 0.55, 95 % CI 0.37-0.80), whereas the “western” and the “fatty” patterns were directly associated with a significant increase in risk of lung cancer (OR for the “fatty” pattern 2.33, 95 % CI 1.65-3.28). We concluded that factor analysis is a valuable statistical method which allows to reduce complex sets of data in a smaller number of factors. These, in turn, are possibly more explicative than the traditional logistic analysis of individual foods or food groups. Aside from the recognized and well-known role of tobacco smoke in the etiology of lung cancer, the role of diet should be taken into account as an important non-tobacco risk factor for this malignancy.

Uruguayan men, with an age-adjusted incidence rate of 76.5 per 100,000 inhabitants of Montevideo (Parkin et al, 2002). In fact, in comparisons between American

I. Introduction Lung cancer is the most frequent malignancy among

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De Stefani et al: Nutritional patterns and lung cancer risk in Uruguayan men (response rate 97.2 %). The cases were distributed by cell type as follows: squamous cell (308 patients, 36.4 %), small cell (105, 12.4 %), adenocarcinoma (212, 25.1 %), large cell (37, 4.4 %) and unclassified carcinoma (184, 21.7 %) using the World Health Organization (WHO) classification for tumors of the lung and pleura (Travis et al, 1999). The relatively large number of unclassified cancers was, in part, to the fact that 87 % of these tumors were of peripheral location and most of them (64 %) were microscopically diagnosed by cytology.

registries, the rate of Uruguay is only second, following the rate observed among Black men in United States (Parkin et al, 2002). Undoubtly the main etiologic factor of lung cancer in Uruguay is tobacco smoking (Carámbula et al, 1995). Uruguayan men display a high prevalence rate of smoking and this population is also characterized by an elevated use of black tobacco cigarettes and by the use of hand-rolled cigarettes (Carámbula et al, 1995; Engeland et al, 1996; De Stefani et al, 1997a). Both types of cigarettes have been considered as particularly risky (Engeland et al, 1996; De Stefani et al, 1997a). Also occupation and dietary factors have been considered as risk factors for this malignancy (Hueper, 1966; Ziegler et al, 1996; World Cancer Research Fund, 1997). Traditionally, research on the role of diet and lung cancer risk have been conducted, both in prospective and case-control studies, by individual analysis of foods and/or nutrients (Ziegler et al, 1996; World Cancer Research Fund, 1997). Rather recently, factor analysis, that is a statistical method for simplifying and reducing complete sets of data into a rather small number of factors, has begun to be employed in the field of dietary epidemiology (Randall et al, 1992; Slattery et al, 1998; De Stefani et al, 1999; McCann et al, 2001; Palli et al, 2001; Terry et al, 2001a, b; Handa and Kreiger, 2002; Markaki et al, 2003; Masaki et al, 2003; Sieri et al, 2004; De Stefani et al, 2005; Fung et al, 2005). Initially used in the field of psychology (Pearson, 1901; Spearman, 1904), this method has been employed in the field of social sciences, economy and other sciences (Harman, 1976; Kim and Mueller, 1978; Kline, 2002). The role of dietary patterns in the etiology of lung cancer was studied in two previous reports (Tsai et al, 2003; Balder et al, 2005). The first study (Tsai et al, 2003) used cluster analysis and reported that a “healthy” pattern had a protective effect against lung cancer. However, after controlling for smoking this inverse association was no longer significant. The second study used factor analysis and reported a protective effect for a so-called “salad” pattern whereas the “pork, processed meat and potatoes” pattern was directly associated with lung cancer risk (Balder et al, 2005). We considered that a research on foods and lung cancer risk, using exploratory factor analysis, could be worthwhile in order to replicate previous studies in the field (Tsai et al, 2003; Balder et al, 2005). This is the objective of the present research.

B. Selection of controls In the same time period and in the same hospitals, all male patients hospitalized for conditions not related with tobacco smoking, alcohol drinking and without recent changes in their diets were considered as eligible for the study. One thousand and six hundred (1600) patients were identified through the log book of admissions. Forty two patients refused the interview, leaving a final number of 1158 potential controls (response rate 96.5 %). Among them 846 controls were frequency matched to cases on age (in ten-years intervals) and residence (Montevideo, other counties) and retained in the current analysis. Controls presented the following diseases: eye disorders (178 patients, 21.0 %), abdominal hernia (170, 20.1 %), fractures (130, 15.4 %), injuries (77, 9.1 %), skin diseases (67, 7.9 %), acute appendicitis (66, 7.8 %), varicose veins (42, 5.0 %), hydatid cyst (40, 4.7 %), urinary stones (25, 3.0 %), blood disorders (21, 2.5 %), prostate hypertrophy (18, 2.1 %) and osteoarticular diseases (12, 1.4 %).

C. Questionnaire Cases and controls were interviewed shortly after admittance in the hospitals. All the interviews were performed face-to-face by two trained social workers. No proxy interviews were accepted. The questionnaire administered to the participants included the following sections: (1) sociodemographics, (2) a complete occupational history based in job titles and its duration, (3) family history of lung cancer or other cancers in first degree relatives, (4) self-reported height and weight five years before the date of the interview, (5) a complete history on tobacco smoking, including age at start, age of quit, average number of cigarettes smoked per day, type of tobacco used (based in brands), type of cigarette, filter use, degree of inhalation (no inhalation, mouth and chest), (6) a complete history of alcohol drinking including age at start, age of quit, number of glasses drunked per week or day, type of alcoholic beverage, (7) a maté drinking section including age at start, age at quit, liters or fractions of liter ingested per day (or week), temperature of the beverage and (8) a food-frequency questionnaire (FFQ) on 64 food items. This FFQ is considered as representative of the usual diet of Uruguayans and allowed the estimation of total energy intake. Furthermore, the FFQ was not validated but it was tested for reproducibility with good results (see Appendix I). Queries about food intake concerned to consumption five years before the date of the interview, both for cases and controls. We have already reported results on selected nutritional aspects and lung cancer which are partially based on this population (Deneo-Pellegrini et al, 1996; De Stefani et al, 1999).

II. Methods In the time period 1995-2001, a case-control study on environmental risk factors and lung cancer was conducted in Montevideo, Uruguay.

D. Foods included in the study The following food and food groups were included in the factor analysis: red meat (beef, lamb), white meat (poultry, fish), processed meat (bacon, sausage, mortadella, salami, saucisson, hot dog, ham, salted meat), cheese, butter, whole milk, total eggs (boiled eggs, fried eggs), desserts (milk with sugar, rice pudding, custard, marmalade, ice cream, cake), white rice, fresh vegetables (carrot, tomato, lettuce, onion), cooked vegetables (garlic, swiss chard, spinach, potato, sweet potato, winter squash, beetroot, zucchini, cabbage, cauliflower, red pepper), citrus

A. Selection of cases All newly diagnosed and microscopically confirmed cases of lung carcinoma were considered eligible for the present study. Thus, 870 patients with lung carcinoma were identified in the four major hospitals in Montevideo, Uruguay. Twenty one patients presented extensive disease with brain metastases and four patients refused the interview. These patients were excluded from the study, leaving a final number of 846 participants

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Cancer Therapy Vol 4, page 155 fruits (orange, tangerine), other fruits (apple, pear, grape, peach, plum, banana, fig, fruit cocktail), coffee with milk, maté, and total alcohol (beer, wine, hard liquor).

relatives, body mass index (continuous), total energy intake (continuous), and the following tobacco variables: smoking status, years since quit among former smokers (categorical), cigarettes per day among current smokers (categorical), age at start smoking (continuous). Since scores are conditional on each other, the four scores were included simultaneous in the models (Balder et al, 2005). Scores was introduced in this basic model as categorical variables. The categories were defined in quartiles, following the distribution of the controls. Departure from the multiplicative model was determined by assessing the likelihood ratio test statistic. An ! level of 0.05 was used as the indicator of statistical significance and, accordingly, 95 % CIs were reported. All p-values were derived from two-sided statistical tests. Each factor was entered as continuous into the model in order to estimate the p-value for trend. Comparisons between cell types of lung cancer for food patterns were estimated by polytomous (multinomial) regression (Rothman and Greenland, 1998). All the calculations were performed with the STATA software programme (STATA, 1999).

E. Statistical analysis Factor analysis (principal components) was conducted to derive food patterns based on the 16 foods and food groups. This analysis was conducted using the principal components factor procedure in STATA (1999). The factors were rotated by the varimax function to achieve a simpler structure with greater interpretability. In determining the number of factors to retain we used eigenvalues greater than 1.25 and the Scree test. Positive loadings indicates that the foods are directly correlated with the factor, and negative loadings indicate that the foods are inversely correlated with the factor. Labeling of the food patterns was entirely subjective and was based on our interpretation of the loadings observed in the correlation matrix. Scores for the retained factors were obtained by the score command in STATA (1999). Correlations between selected variables and scores were estimated by the procedures of Pearson and Spearman (STATA, 1999). Odds ratios of lung cancer for estimated scores were estimated by multiple unconditional logistic regression (Breslow and Day, 1980). The basic model included the following terms: age (continuous), residence, urban/rural status, education (categorical), family history of lung cancer among first degree

III. Results Distribution of cases and controls by sociodemographic variables and selected risk factors are shown in Table 1. As a result of the matched design, cases

Table 1. Distribution of cases and controls by sociodemographics and selected risk factors Cases Variable Age (in years)

Residence Urban/rural status Education (years)

Income (US dollars)

Family history of lung cancer Body mass index

Total energy intake

Tobacco smoking

Category 30-39 40-49 50-59 60-69 70-79 80-89 Montevideo Other counties Urban Rural 0-2 3-5 6+ ! 144 145+ Unknown No Yes ! 22.8 22.9-24.8 24.9-27.0 27.1+ ! 1722 1723-2071 2072-2466 2467+ Never smokers Former 20+yrs Former 10-19 yrs Former 1-9 yrs Current 1-9 cig/day Current 10-19 c/day Current 20-29 c/day Current 30+ cig/day

Nº 14 93 186 334 200 19 410 436 645 201 224 333 289 326 326 194 765 81 282 216 165 183 171 181 211 283 19 35 51 154 19 84 183 301 846

Nºpatients

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% 1.6 11.0 22.0 39.5 23.6 2.3 48.5 51.5 76.4 23.8 26.5 39.4 34.1 38.5 38.5 22.9 90.4 9.6 33.3 25.5 19.5 21.6 20.2 21.4 24.9 33.5 2.3 4.1 6.0 18.2 2.2 9.9 21.6 35.6 100.0

Controls Nº % 14 1.6 93 11.0 186 22.0 334 39.5 200 23.6 19 2.3 410 48.5 436 51.5 664 78.5 182 21.5 204 24.1 311 36.8 331 39.1 322 38.1 318 37.6 206 24.3 811 95.9 35 4.1 218 25.8 205 24.2 214 25.3 209 24.7 211 24.9 212 25.1 212 25.1 211 24.9 146 17.3 86 10.2 88 10.4 124 14.7 84 9.9 133 15.7 96 11.3 89 10.5 846 100.0

De Stefani et al: Nutritional patterns and lung cancer risk in Uruguayan men and controls were similar concerning age and residence. Also the distribution of controls and cases were rather similar concerning urban/rural status and monthly income. Cases were slightly less educated than controls and were significantly leaner when compared with controls. On the other hand total energy intake was significantly higher among cases than controls and family history of lung cancer among first-degree relatives displayed an increased risk of the malignancy (OR 2.4, 95 % CI 1.6-3.7). Finally, tobacco smoking, as expected, was strongly and directly associated with lung cancer risk (OR for current heavy smokers 26.0, 95 % CI 13.3-59.9, p-value for linear trend <0.0001). Four major food patterns were retained (Table 2). Factor or pattern 1 was labeled “cheese and rice” since it reflected the correlated intake of some dairy foods, white rice and processed meat, explaining 14.2 % of the variance. Factor 2 was labeled as “Western” since it presented high loadings of red meat and alcohol. This

pattern explained 9.5 % of the variance. Pattern 3 was labeled as “fatty” and was rich in whole milk and coffee with milk. This factor explained the 8.5 % of the variance. Factor 4 was labeled as “healthy” and presented high correlations of white meat, fresh vegetables, cooked vegetables, citrus fruits and non-citrus fruits and explained 8.0 % of the variance. Correlations between patterns and selected variables for controls are shown in Table 3. The “cheese and rice” pattern was directly associated with total energy inatake and with lactose. The “western” factor was composed by patients who were less educated. Also this pattern was significantly correlated with smoking intensity, total energy intake, saturated fat, monounsaturated fat, polyunsaturated fat and with cholesterol. The “western” pattern was negatively correlated with total carbohydrates, calcium and lactose. The “fatty” pattern displayed elder patients and there was a significant

Table 2. Factor-loading matrix among controls1 Foods Red meat White meat Processed meat Cheese Butter Whole milk Eggs Desserts White rice Fresh vegetables Cooked vegetables Citrus fruits Other fruits Coffee w/milk Maté Alcohol Variance (%) Nº of zeros Nºof high loadings

Factor 1 (Cheese & Rice) 0.04 0.30 0.47 0.67 0.68 0.01 0.14 0.47 0.46 -0.04 0.15 -0.11 0.13 0.00 0.07 -0.14 14.2 5 5

Factor 2 (Western) 0.67 -0.27 0.40 -0.07 0.05 -0.02 0.38 0.08 0.10 0.10 0.24 0.18 -0.12 0.01 0.55 0.45 9.5 5 4

Factor 3 (Fatty) 0.05 -0.08 0.09 -0.04 0.01 0.77 0.08 0.27 -0.09 0.01 0.17 0.08 0.03 0.78 -0.24 -0.01 8.5 10 2

Factor 4 (Healthy) 0.03 0.46 -0.02 0.14 -0.04 0.09 0.08 0.22 -0.01 0.66 0.43 0.57 0.72 -0.03 0.04 -0.11 8.0 8 5

Table 3. Energy-adjusted Pearson correlation coefficients between food patterns and selected variables. Variable Age Education Body mass index Smoking Total energy Saturated fat MUFA PUFA Cholesterol Carbohydrates Vitamin C Vitamin A "-carotene Vitamin E Folate Calcium Lactose

Cheese & rice -0.05 0.06 0.01 -0.05 0.46 -0.04 -0.11 0.04 -0.03 0.06 -0.12 0.00 -0.01 0.05 -0.08 0.11 0.40

Western -0.21 -0.06 -0.07 0.23 0.51 0.30 0.43 0.27 0.40 -0.43 0.07 -0.00 0.01 -0.10 0.06 -0.30 -0.20

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Fatty 0.13 -0.06 -0.04 -0.00 0.48 0.13 -0.10 -0.10 0.01 -0.05 0.05 0.03 -0.01 0.03 0.19 0.57 0.28

Healthy 0.09 0.07 0.06 -0.03 0.28 -0.16 -0.18 -0.09 0.00 0.16 0.64 0.25 0.23 0.66 0.59 0.14 -0.06

Cancer Therapy Vol 4, page 157 positive correlation with total energy intake (r=0.48) and with saturated fat, folate, lactose and calcium. The “healthy” pattern showed a strong inverse association with saturated and monounsaturated fat. This pattern showed a positive correlation with total energy intake, vitamin C,

vitamin A, "-carotene, vitamin E and folate. Odds ratios of lung cancer (all histologies) for food patterns are shown in Table 4. The “cheese and rice”

Table 4. Odds ratios of lung cancer (all histologies) for food patterns1 Food Pattern Cheese and rice

Western

Fatty foods

Healthy

Cases/Controls 210/211 261/212 225/212 150/211 p-value for trend 98/211 170/212 240/212 338/211 p-value for trend 165/211 159/212 191/212 331/211 p-value for trend 260/211 203/211 198/213 185/211 p-value for trend

OR1 1.0 1.28 1.09 0.69 1.0 1.70 2.58 3.83 1.0 1.02 1.30 2.20 1.0 0.79 0.69 0.64

95%CI 0.97-1.69 0.83-1.45 0.51-0.93 0.008 1.23-2.34 1.89-3.53 2.81-5.23 <0.0001 0.76-1.39 0.96-1.74 1.66-2.92 <0.0001 0.60-1.04 0.52-0.91 0.48-0.85 0.001

OR2 1.0 1.17 0.93 0.55 1.0 1.38 1.85 2.05 1.0 1.00 1.31 2.33 1.0 0.77 0.75 0.74

95%CI 0.86-1.61 0.67-1.30 0.37-0.80 0.001 0.96-1.99 1.28-2.67 1.37-3.06 <0.0001 0.71-1.41 0.94-1.84 1.65-3.28 <0.0001 0.56-1.05 0.55-1.04 0.53-1.04 0.06

1

Age-adjusted. Adjusted for age, residence, urban/rural status, education, family history of lung cancer among first-degree relative, body mass index, smoking status, years since cessation, number of cigarettes smoked per day among current smokers, age at start smoking and total energy intake. 2

Table 5. Odds ratios of lung cancer for food patterns by smoking status1 Pattern Cheese and rice Low score 2 3 High score Heterogeneity 0.10 Western Low score 2 3 High score Heterogeneity 0.52 Fatty Low score 2 3 High score Heterogeneity 0.51 Healthy Low score 2 3 High score Heterogeneity 0.06

Former smokers Cases/Controls OR 95%CI 70/116 1.0 reference 63/117 0.83 0.50-1.36 78/108 0.96 0.57-1.59 48/103 0.41 0.22-0.76 p-value for trend 0.02 Cases/Controls OR 95%CI 40/144 1.0 reference 58/115 1.49 0.87-2.54 76/105 1.98 1.14-3.44 85/80 2.19 1.17-4.08 p-value for trend 0.009 Cases/Controls OR 95%CI 43/97 1.0 reference 45/106 0.95 0.53-1.69 59/114 1.01 0.58-1.75 112/127 1.36 0.79-2.34 p-value for trend 0.18 Cases/Controls OR 95%CI 62/83 1.0 reference 62/110 0.58 0.34-0.99 65/119 0.57 0.33-0.97 70/132 0.48 0.28-0.83 p-value for trend 0.02

1

Current smokers Cases/Controls OR 95%CI 140/95 1.0 reference 198/95 1.40 0.92-2.13 147/104 0.86 0.55-1.35 102/108 0.59 0.35-0.98 0.008 Cases/Controls OR 95%CI 58/67 1.0 reference 112/97 1.20 0.71-2.04 164/107 1.60 0.96-2.67 253/131 1.83 1.05-3.17 0.01 Cases/Controls OR 95%CI 122/114 1.0 reference 114/106 1.00 0.65-1.54 132/98 1.46 0.94-2.26 219/84 3.36 2.12-5.31 <0.0001 Cases/Controls OR 95%CI 198/128 1.0 reference 141/101 0.87 0.58-1.29 133/94 0.86 0.57-1.31 115/79 0.91 0.58-1.42 0.59

Adjusted for age, residence, urban/rural status, education, family history of lung cancer among first-degree relatives, body mass index, cigarettes per day, years since quit and total energy intake.

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De Stefani et al: Nutritional patterns and lung cancer risk in Uruguayan men pattern was inversely associated with lung cancer risk. (OR 0.55, 95 % CI 0.37-0.80, p-value for trend=0.001). The “western” pattern was strongly and directly associated with lung cancer risk. The OR of the higher category was associated with a strong increase in risk (OR 2.05, 95 % CI 1.37-3.06, p-value for trend<0.0001). Also the “fatty” factor was directly associated with lung cancer risk (OR 2.33, 95 % CI 1.65-3.28, p-value for trend<0.0001). The “healthy“ pattern was inversely associated with lung cancer risk (OR for the high score 0.74, 95 % CI 0.531.04, p-value for trend=0.06). Odds ratios of lung cancer for food patterns by smoking status are shown in Table 5. The “cheese and rice” pattern displayed a significant reduction in risk among former smokers and current smokers (OR for former smokers 0.41, 95 % CI 0.22-0.76, p-value for trend=0.02). Also the “western” pattern was positively associated with lung cancer risk in both strata of smokers, although the effect was more impressive among former smokers (OR 2.19, 1.17-4.08, p-value for trend=0.009). The “fatty” pattern was much more directly associated among current smokers (OR 3.36, 95 % CI 2.12-5.31, pvalue for trend<0.0001). Finally, the “healthy” pattern showed a strong reduction in risk among former smokers, but not among current users of cigarettes (OR for former smokers 0.48, 95 % CI 0.28-0.83, p-value for trend=0.02).

The p-value for heterogeneity was close to significance (pvalue=0.06). The effect of the four patterns in different cell types is shown in Table 6. The “cheese and rice” pattern displayed an inverse association with squamous cell carcinoma (p-value for trend=0.001), whereas small cell carcinoma and adenocarcinoma of the lung were not associated with this factor. Large cell carcinoma and other lung cancers were moderately protective (p-value for trend=0.03). On the other hand, the “western” pattern displayed a significant increase in risk for squamous cell carcinoma and for adenocarcinoma of the lung. The “fatty” pattern was positively associated with all histologies. The highest risky effect was observed among squamous cell and adenocarcinoma of the lung. The “healthy” pattern was significantly protective for large cell carcinoma and othe types of lung cancer, whereas the remaining cell types were not associated with risk.

IV. Discussion According to our results, principal components analysis retained four patterns which were labeled as follows: “cheese and rice”, “western”, “fatty” and “healthy”. Whereas the “western” and the “fatty” factors

Table 6. Odds ratios of different histologies of lung cancer for food patterns1

Cell type Squamous cell Small cell Adenocarcinoma Other types

Score 2 OR 1.06 1.50 1.06 1.44

95%CI 0.71-1.57 0.82-2.78 0.67-1.68 0.92-2.24

Cell type Squamous cell Small cell Adenocarcinoma Other types

Score 2 OR 1.73 1.09 1.46 1.04

95%CI 1.04-2.88 0.48-2.47 0.83-2.58 0.60-1.80

Cell type Squamous cell Small cell Adenocarcinoma Other types

Score 2 OR 0.93 0.69 1.14 1.14

95%CI 0.59-1.48 0.34-1.40 0.68-1.92 0.70-1.85

Cell type Squamous cell Small cell Adenocarcinoma Other types

Score 2 OR 1.01 0.74 0.82 0.63

95%CI 0.67-1.51 0.40-1.36 0.52-1.28 0.41-0.97

Factor 1 (Cheese and rice) Score 3 Score 4 OR 95%CI OR 0.87 0.57-1.33 0.41 0.78 0.39-1.56 0.80 1.13 0.71-1.81 0.73 0.99 0.61-1.60 0.57 Factor 2 (Western) Score 3 Score 4 OR 95%CI OR 2.31 1.39-3.84 2.35 1.70 0.78-3.73 1.45 1.68 0.94-2.98 2.78 1.60 0.94-2.72 1.49 Factor 3 (Fatty) Score 3 Score 4 OR 95%CI OR 1.21 0.77-1.90 2.26 1.22 0.64-2.33 1.81 1.59 0.96-2.63 2.60 1.33 0.81-2.18 2.17 Factor 4 (Healthy) Score 3 Score 4 OR 95%CI OR 1.07 0.71-1.61 0.82 0.76 0.41-1.40 0.89 0.77 0.49-1.21 0.90 0.54 0.34-0.85 0.54

1

95%CI 0.25-0.69 0.38-1.68 0.42-1.27 0.32-1.02

p-value trend 0.001 0.20 0.32 0.03

95%CI 1.36-4.08 0.62-3.37 1.53-5.05 0.82-2.71

p-value trend 0.003 0.28 <0.0001 0.10

95%CI 1.45-3.53 0.95-3.44 1.58-4.28 1.33-3.55

p-value trend <0.0001 0.02 <0.0001 0.001

95%CI 0.53-1.29 0.47-1.68 0.56-1.44 0.33-0.88

p-value trend 0.43 0.68 0.82 0.003

Adjusted for age, residence, urban/rural status, education, family history of lung cancer among first-degree relative, body mass index, smoking status, years since cessation, number of cigarretes smoked per day among current smokers, age at start smoking and total energy intake. 2 Pattern 1: squamous cell vs adenocarcinoma=0.05/ 3 Pattern 4: squamous cell vs other types=0.04 4 Pattern 4:adenocarcinoma vs other types=0.04

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Cancer Therapy Vol 4, page 159 were positively associated with lung cancer risk, both the “cheese and risk” and “healthy” patterns were somehow protective. The “cheese and rice” pattern showed high loadings for white meat, processed meat, cheese, butter, desserts and rice. Taking into account that six different food groups contributed in an important loading, we considered the possibility of labeling this pattern as a “mixed” pattern. Finally, since both cheese and rice were protective in lung cancer we decided to label this pattern as “cheese and rice” factor. This pattern was not identified in previous studies on cancer and factor analysis. The “cheese and rice” pattern showed high correlations with processed meat, cheese, butter, desserts and rice. All these variables have been traditionally directly associated with risk of lung cancer (Ziegler et al, 1996; World Cancer Research Fund, 1997; Slattery et al, 1998; Terry et al, 2001a). Nevertheless, some studies reported a protective effect of cheese in non-smokers (Brennan et al, 2000). In our study, the “cheese and rice” food pattern has been inversely associated with lung cancer risk. In the Dutch prospective study the “sweet” pattern has some common characteristics (Balder et al, 2005). Further studies are needed in order to clarify the possible mechanisms by which these foods could be protective in lung cancer. On the contrary, the “western” pattern, which has high loadings for red meat and alcohol, replicated similar patterns in other studies which employed factor analysis (Slattery et al, 1998; Terry et al, 2001a). Red meat, processed meat, maté drinking and alcohol drinking have been considered as risk factors for the lung mucosa or bronchial lining (Harris et al, 1996; Ferguson and Harris, 1998; Slattery et al, 1998; Tsai et al, 2003). Our “western” pattern is close to the “pork, processed meat, potatoes” pattern of Balder et al, (2005). Red meat consumption has been considered as risk factors for two possible mechanisms: 1) through its high content of saturated fat and/or 2) through its high amount of HCA’s resulting from the cooking method of this meat (Slattery et al, 1998; Tsai et al, 2003). Since both chemicals have been suggested as carcinogens, its activity together with the carcinogenic chemicals present in tobacco smoke (Sinha et al, 1998), suggest that heavy smokers and high consumers of welldone red meat are, possibly, at high risk of developing lung cancer. To our knowledge, our study on maté drinking and lung cancer is the only report which suggested a possible direct effect of this herbal tea in lung cancer (De Stefani et al, 1997b). For this reason, it is impossible to be sure that the results are not due to residual confounding from tobacco or due to other confounders (IARC, 1980). Concerning alcohol consumption, a recent meta-analysis suggested that alcohol could be a risk factor only on at very large doses (Korte et al, 2002). Therefore, although our findings regarding the “western” pattern are somehow reassuring in replicating previous findings, it is necessary to be cautious since exploratory factor analysis is essentially subjective both in the selection of the variables and in its interpretation (De Stefani et al, 2005; Fung et al, 2005). The “fatty” factor was associated with a significantly increase in risk of lung cancer and this applies to all the cell types. The only variables with high loadings for this

pattern were whole milk and coffee with milk. It is noteworthy that Uruguayan population consumed high amounts of whole milk (rich in fat) and coffee with milk, being pure coffee an unfrequent habit. Therefore, this pattern is rich in foods which contains high amounts of fat, mainly saturated fat. Thus, our findings support the role of saturated fat in lung carcinogenesis, replicating previous findings (Terry et al, 2001a; Balder et al, 2005). The “healthy” factor displayed high loadings of white meat, fresh vegetables, cooked vegetables, citrus fruits and non-citrus fruits. rich in carotenoids, winter squash, potato, and white rice. These foods are considered as protective. The “healthy” pattern was particulary protective among former smokers and among patients with large cell carcinoma. Vegetables which contained "carotene, like carrot and sweet potato, were inversely associated with lung cancer risk. A recent study reported that sweet potato is a rich source of cis-"-Cryptoxanthin, "-carotene, and 9-cis "-Carotene (Benamotz and Fishler, 1998). These stereoisomeric forms of carotenoids deserve more attention in the field of protection of lung cancer (Benamotz and Fishler, 1998). Potatoes have been considered as inversely associated with some cancers (Cambie and Ferguson, 2003). It is possible that cell walls from potatoes adsorb heterocyclic amines (HCA) in the intestinal lumen (Harris et al, 1996; Ferguson and Harris, 1998). It is well-known that HCA could be carcinogens for lung tissue (Sinha et al, 1998; Weisburger, 2002). As it was suggested by Weisburger, (2002), HCA could act as initiators and fats as promoters in lung carcinogenesis. Our “healthy” pattern was different with the patterns identified by Balder et al, (2005), although the “cooked vegetables” pattern presented some charcteristics in common. Whereas the “salad vegetables” of the Netherlands Cohort Study was essentially protective, the “healthy” pattern of Tsai et al. was no longer significant after controlling for tobacco smoking (Tsai et al, 2003; Balder et al, 2005). In traditional studies (both prospective and case-control), the role of vegetables and fruits were inversely associated with lung cancer risk (Ziegler et al, 1996; World Cancer Research Fund, 1997; IARC, 2003). However, the study of Feskanich et al, (2000) reported results close to the null for fruit and vegetables intake. Perhaps residual confounding from smoking could attenuate the inverse association with plant foods. In this sense we adjusted the estimates for fruit and vegetables consumption using a index which included smoking status, number of cigarettes per day among current smokers, years since quit and age at start smoking. The use of factor analysis raises some concerns. The first problem is related with the construction and analysis of the FFQ. In other words, the FFQ could be not adequate for the purposes of the study. We devoted a considerable time in order to constructing our FFQ and we consider that it is representative of the usual diet among Uruguayans. An important decision in factor analysis is the choice of the number of factors to be retained (McCann et al, 2001; Terry et al, 2001a; Handa and Kreiger, 2002). In order to retain the number of factors which explains more of the variance than a single variable, we set an eigenvalue of 1.25. In spite of some controversial findings, the factors

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De Stefani et al: Nutritional patterns and lung cancer risk in Uruguayan men retained by exploratory factor analysis, have biological sense. More precisely, the “western” pattern, the “healthy” pattern and the “fatty” factor are plausible, since all three are in accordance with the Uruguayan diet. Also they could be replicable in other datasets (Harman, 1976; Pearson, 1901; Kline, 2002). The retained factors explained 45 % of the total variance of the model. This percentage is rather good when compared with other studies which employed factor analysis in cancer epidemiology (De Stefani et al, 1999; McCann et al, 2001; Palli et al, 2001; Terry et al, 2001b; Handa and Kreiger, 2002; Masaki et al, 2003; Sieri et al, 2004; De Stefani et al, 2005; Fung et al, 2005). Also the communalities were reasonably high. As other case-control studies, our study has limitations. Aside from the usual problems of selection and classification biases, our study could suffer from recall bias. At difference with prospective studies, case-control studies has this common problem. Although the interviews were conducted blindly by the two social workers, it is impossible to discard some degree of faulty recall. It is true that our participants are mostly unawere of the role of diet in lung cancer or of other diseases, but it is impossible to discard that some patients were more healthy than others. We have excluded proxy interviews, but sometimes proxies are more exact then the participants. Our study has strengths. We excluded other hospitals which could be sources of patients with lung cancer since they not were a source of controls. Although we do not match by hospital, the proportion of cases and controls were rather similar. The statistical power of the study allowed to detect as significant an OR of 1.3. Another strength is the high

response rate both for cases and controls.

V. Conclusions In summary, we conducted a case-control on foods and risk of lung cancer. To do so, we used exploratory factor analysis (principal components) and we retained four factors with an eigenvalue of 1.25. These patterns explained 40.2 % of the total variance. Whereas the “cheese and rice” and “healthy” patterns were significantly protective, the “western” and the “fatty” patterns were directly associated with lung cancer risk. As important variables, white meat, tomatoes, green leafy vegetables, onions, carotenoid vegetables, winter squash, potatoes, citrus fruits and white rice deserve attention in further studies. Also, some foods rich in fat could be also protective, like cheese. On the other hand, red meat, processed meat, maté consumption, alcohol drinking, whole milk and coffee with milk appears to be associated with lung cancer risk. It is our opinion that is too soon to suggest preventive messages. Perhaps, the only preventive message is to quit smoking.

Acknowledgements This research was supported by the International Agency for Research on Cancer.

Appendix I Results of the reproducibility test

Table 1. Means and Pearson correlation coefficients for food groups Means Food groups1 Red meat White meat Processed meat Total meat Dairy foods Eggs Desserts Grains Raw vegetables Cooked vegetables Total vegetables Citrus fruits Other fruits Total fruits All tubers Legumes Total vegetables & fruits Coffee3 Tea3 Alcohol4

First interview 355.4 86.2 212.1 660.2 606.1 122.8 165.7 1035.9 254.4 603.6 858.1 132.6 323.1 455.8 337.4 37.9 1313.8

Correlations2 Second interview 369.0 93.7 258.6 735.7 597.8 130.9 177.8 1072.5 271.6 662.9 934.5 111.9 309.3 421.2 332.5 38.1 1355.7

rho 0.77 0.60 0.55 0.67 0.64 0.45 0.50 0.69 0.64 0.39 0.46 0.46 0.51 0.54 0.66 0.70 0.59

124.7 65.9 80.0

110.2 83.8 81.1

0.33 0.51 0.70

1

Servings/year Energy-adjusted Pearson correlations 3 Mililiters/day 4 Mililiters/ethanol day 2

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Cancer Therapy Vol 4, page 161 Table 2. Means and Pearson correlation coefficients for nutrients Means Nutrient Energy Protein Carbohydrates Total fat Saturated fat MUFA1 PUFA2 Linoleic acid Linolenic acid Cholesterol Vitamin A "-Carotene Total carotenoids Vitamin C Vitamin E Vitamin B6 Vitamin B12 Thiamine Riboflavin Folate Fiber Calcium Iron Sodium

First interview 2015 102.8 263.6 116.2 46.4 45.4 11.9 10.4 1.3 533 11918 5596 10356 123 3.8 1.5 6.5 1.4 1.9 194.3 22.1 646 16.7 954

Correlations Second interview Crude rho 2098 0.78 108.4 0.61 264.0 0.75 123.3 0.63 48.7 0.67 48.4 0.66 12.9 0.55 11.3 0.54 1.3 0.65 655 0.53 13082 0.49 5782 0.50 10641 0.45 132 0.51 4.0 0.50 1.6 0.60 6.8 0.68 1.6 0.67 1.9 0.71 211.3 0.56 22.6 0.66 662 0.56 17.7 0.61 1042 0.57

Energy-adjusted rho 0.43 0.69 0.62 0.69 0.67 0.49 0.48 0.59 0.47 0.50 0.50 0.49 0.46 0.39 0.44 0.71 0.45 0.61 0.49 0.61 0.58 0.46 0.51

1

Monounsaturated fat Polyunsaturated fat

2

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Cancer Therapy Vol 4, page 163 Cancer Therapy Vol 4, 163-170, 2006

Advantages of a unique DNA-based vaccine in comparison to paclitaxel in treatment of an established intracerebral breast cancer in mice Research Article

Terry Lichtor1, Roberta P Glick1, Henry Lin1, Amla Chopra2, InSug O-Sullivan2 and Edward P Cohen2 1 2

Department of Neurological Surgery, Rush University Medical Center and John H Stroger Hospital of Cook County Department of Microbiology and Immunology, University of Illinois at Chicago; Chicago, Illinois

__________________________________________________________________________________ *Correspondence: Terry Lichtor, MD, PhD, Department of Neurosurgery, Rush University Medical Center, 1900 West Polk Street, Chicago, Illinois 60612, Telephone: 312-864-5120, Fax: 312-864-9606, E-Mail: Terry_Lichtor@rush.edu Key words: DNA-Based Vaccine, IL-2, breast cancer, intracerebral cannula Abbreviations: Intracerebrally, (i.c.); intraperitoneal, (i.p.); tumor associated antigens, (TAA)

This work was supported by a grant from the CINN Foundation awarded to Drs Lichtor and Glick, and by NIDCR grant number 1 RO1 DEO13970-01A2 awarded to Dr. Cohen.

Received: 19 April 2006; Accepted: 03 May 2006; electronically published: May 2006

Summary In this study we compared the benefits of treating C3H/He mice with an established intracerebral breast carcinoma by immunization with a unique DNA-based vaccine to chemotherapy with paclitaxel. Prior studies revealed the immunotherapeutic properties of a vaccine prepared by transfer of genomic DNA from breast cancer cells into a highly immunogenic cell line. Here, C3H/He mice with an established intracerebral breast cancer were treated either by injection into the tumor bed through a unique cannula system with the cell based vaccine or with paclitaxel administered intraperitoneally. Both treatment strategies were effective in prolonging survival and stimulating a systemic anti-tumor immune response (p< 0.025). However, unlike mice treated with the vaccine, the animals that received paclitaxel alone displayed significant toxic side effects. No additional therapeutic advantage was detected when these two treatment strategies were combined. The vaccine tended to provide a somewhat better therapeutic and clearly better systemic immunologic effect based on two independent spleen cell assays in comparison to paclitaxel.

from an adenocarcinoma of the breast into a highly immunogenic mouse fibroblast cell line. The cells used as DNA-recipients were modified in advance of DNAtransfer to secrete various immune-augmenting cytokines. The rationale was that genes specifying tumor associated antigens (TAA) would be expressed in a highly immunogenic form by the transfected cells. As the transferred DNA is integrated into the genome of the recipient cells, and replicated as the cells divide, the vaccine could be prepared with DNA derived from microgram amounts of tumor tissue. It is likely that the multiple mutant and/or dysregulated genes in the breast cancer cells specifying an array of unidentified weakly immunogenic tumor associated antigens were expressed in a highly immunogenic form by the transfected cells. Since

I. Introduction We recently reported on the immunotherapeutic properties of a unique DNA-based cell vaccine for treatment of intracerebral (i.c.) breast cancer in C3H/He mice (Lichtor et al, 2005). In particular we showed that C3H/He mice injected with a cell mixture containing a breast carcinoma (SB5b) along with a vaccine prepared by transfection of mouse fibroblasts with DNA from the breast carcinoma (SB5b) survived longer than mice in various control groups. Systemic cellular tumor immunity was generated in the mice injected intracerebrally with the transfected cells, which was mediated predominantly by CD8+ T cells (Lichtor et al, 2005). The vaccine was prepared by transfer of sheared genomic DNA-fragments

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Lichtor et al: Treatment of a brain tumor with a DNA-based vaccine University, Osaka, Japan) (Yamada et al, 1987). The vector specified the human IL-2 gene and the neor gene (confers resistance to the neomycin analog, G418) (Cobere-Garapin et al, 1981). Every third passage, the cells were placed in growth medium containing 300 µg/ml G418. Under these circumstances, the quantity of IL-2 secreted by the cells after three months of continuous culture was equivalent to that of cells from primary cultures. Like unmodified cells, LM fibroblasts transduced with pZipNeoSVIL-2 divided approximately every 24 hours.

the tumor cell population is known to be heterogeneous and includes cells that are resistant to cellular immune mechanisms, the tumor cell population must include a subpopulation of breast cancer cells that are resistant to host immune mechanisms. In an attempt to more closely simulate the clinical situation, mice bearing an established i.c. malignant breast carcinoma were treated with the DNA-based vaccine or with paclitaxel, a chemotherapeutic agent commonly used in breast cancer therapy (Conte et al, 2004; Eralp et al, 2004). Paclitaxel has been shown to be active against gliomas and various brain metastases, although its use in treatment of brain tumors is limited due to low blood-brain barrier permeability (Koziara et al, 2004). There is synergy between radiation therapy and paclitaxel in treatment of mice astrocytoma, yet clinical trials involving patients bearing a supratentorial high-grade glioma undergoing combination therapy of external beam radiation along with paclitaxel have not established any additional benefit of paclitaxel (Langer et al, 2001). This study was designed both to compare and determine the possible benefits of combining paclitaxel with immunotherapy in the treatment of C3H/He mice bearing an established, highly aggressive intracerebral breast cancer. The mice were treated by injection into the tumor bed with the DNA-based vaccine, with paclitaxel administered intraperitoneally or by paclitaxel followed by immunization with the DNA-based vaccine. The results indicated that the survival of mice with an established intracerebral breast cancer was prolonged by treatment with either paclitaxel or the DNA-transfected fibroblasts (p < 0.025), but survival of mice receiving the combined therapy did not exceed that of tumor-bearing mice receiving either form of treatment alone.

C. Modification of cytokine-secreting fibroblasts to express H-2Kb class I-determinants LM cells, of C3H/He mouse origin, express H-2k determinants. Allogeneic class I-determinants are strong immune adjuvants. To further augment their non-specific immunogenic properties, the LM fibroblasts were further modified to express H-2Kb class-I determinants as described previously (Lichtor et al, 2005). Confirmation of the expression of H-2K b-determinants by the fibroblasts was confirmed by quantitative immunofluorescence measurements; more than 99 percent of the transduced fibroblasts stained positively for H-2Kb-determinants. Under similar conditions, nontransduced fibroblasts or fibroblasts stained with FITC-conjugated isotype serum failed to stain. The expression of H-2Kb-determinants was a stable property of the cells, and the intensity of staining for H-2Kb determinants was essentially unchanged after three months of continuous culture. Therefore the LM fibroblasts (LMKb/IL-2) possess both syngeneic and allogeneic determinants when injected into C57Bl/6 mice (H-2 b).

D. Transfection of modified fibroblasts with sheared genomic DNA from a breast carcinoma that arose spontaneously in a C3H/He mouse (SB5b) Genomic DNA was isolated (Qiagen, Chatsworth, CA) from a mammary adenocarcinoma (SB-5b) that arose spontaneously in a C3H/He mouse. The genomic DNA was used to transfect the modified fibroblasts, using the method described by Wigler et al, (1979), as modified. Briefly, high molecular weight DNA was sheared by three passages through a 25-gauge needle. The approximate size of the DNA at the time it was used in the experiments was 25 kb, as determined by agarose gel electrophoresis. Afterward, 100 µg of sheared DNA was mixed with 10 µg pCDNA6/V5-HisA, a plasmid which gives resistance to the antibiotic Blasticidin. The sheared tumor-DNA and plasmid DNA (the DNA : plasmid ratio was 10 : 1 to ensure that cells that were converted to Basticidin-resistance took up DNA from the breast carcinoma cells as well) were then mixed with Lipofectamine 2000, according to the manufacturer’s instructions (Life Technologies, Carlsbad, CA). The DNA/Lipofectamine mixture was added to a population of 1 X 107 actively proliferating modified fibroblasts cells divided into ten dishes containing 1 X 106 cells each. Eighteen hours afterward, the medium was replaced with fresh growth medium. The fibroblasts were maintained for 14 days in growth medium containing 2-5 µg/ml Blasticidin HCl (Invitrogen, Carlsbad, CA). One hundred percent of the cells transfected with tumor-DNA alone maintained in the Basticidin growth medium died within this period. The surviving colonies (at least 2.5 X 104) were pooled and maintained as a cell line for use in the experiments.

II. Materials and Methods A. Cell lines and experimental animals Four to six-week-old pathogen-free C3H/He (H-2 k) mice were obtained from Charles River Breeding Laboratories (Portage, MI). The mice were maintained in the animal care facilities of the University of Illinois, according to National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. They were 6-8 weeks old when used in the experiments. SB-5b cells were derived from an adenocarcinoma of the breast, which formed spontaneously in a C3H/He mouse in our animal colony. The SB-5b cells were grown by in vitro passage. LM cells, a fibroblast cell line of C3H/He mouse origin, were obtained from the American Type Culture Collection (Manassas, VA). All the cells were maintained at 370C in a humidified 7% CO2/air atmosphere in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FBS (Sigma, St Louis, MO) and antibiotics (Life Technologies) (growth medium).

B. Modification of fibroblasts to secrete IL-2 To augment their non-specific immunogenic properties, before transfection, the fibroblasts modified to secrete IL-2 (LMIL-2 cells) were prepared as described previously (Kim et al, 1992). In brief, a gene specifying human IL-2 [the biological properties of human IL-2 in mice are equivalent to that of mouse IL-2 (Kim et al, 1992)] was transduced into LM fibroblasts with the retroviral vector pZipNeoSVIL-2 (obtained originally from T. Taniguchi, Institute for Molecular and Cellular Biology, Osaka

E. Intracerebral injection of C3H/He mice with SB-5b breast cancer cells As a model of intracerebral metastatic breast cancer in patients, C3H/He mice were injected intracerebrally with the breast cancer cells through a small cannula (Griffitt et al, 1999)

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Cancer Therapy Vol 4, page 165 that was modified as follows for injection of the tumor cells and the modified fibroblasts. Small screws (0-80 X 1/16; 1.6 mm in length) were obtained from Plastics One (Roanoke, VA) and a .022 diameter hole was subsequently drilled through the center of the screw. Anesthetized mice and were placed into a stereotactic frame and a small burr hole was placed with a D#60 drill bit (Plastics One, Roanoke, VA) over the right frontal lobe in the region of the coronal suture. The screws bearing a central hole were subsequently secured into the small burr hole using Elmer’s Super Glue Gel. The mice were allowed to recover and on specified days injections were made using a Hamilton syringe containing a 26 gauge needle with a small piece of solder placed 5 mm from the tip of the needle to maintain a uniform depth of injection. The total injection volume was 5-10 µl.

G. ELISPOT IFN-! assays Spleen cells from C3H/He mice injected i.c. with the various cell constructs were analyzed for T cells reactive with SB-5b cells in ELISPOT IFN-! assays. T cells recovered by Histopaque density gradient from the spleens of mice injected i.c. with the transfected cells were co-incubated with SB-5b tumor cells (the ratio of spleen cells : SB-5b cells = 10:1) for 16 hours at 370C in wells precoated with a high-affinity monoclonal antibody for IFN-! according to the manufacturer’s instructions (BD Pharmingen, San Diego, CA). After incubation, the cells were washed before the addition of biotinylated anti-IFN-! detection antibody and horse radish peroxidase labeled streptavidin (Streptavidin-HRP). The spots were counted using computer-assisted image analysis (ImmunoSpot Series 2 analyzer: Cellular Technology Limited, Cleveland, OH).

F. T cell mediated cytotoxicity toward breast cancer cells in mice immunized with the transfected fibroblasts

H. Statistical analysis

A CellTiter 96 aqueous non-radioactive cell proliferation assay kit (Promega, Madison WI) was used to measure T cell mediated cytotoxicity toward the breast cancer cells in mice immunized with the transfected fibroblasts. Effector T cell [recovered from the spleens of immunized mice by Histopaque (Sigma) density gradient (Kim and Cohen, 1994)] and mitomycin C-treated (50 !g/ml for 45 min) SB-5b target cells were cocultured at 370 C for 18 hrs at a 30:1 effector:target cell ratio. Afterward, the non-adherent cells were removed, washed and viable SB-5b cells were added at various E:T ratios for 4 hrs at 370 C in a 7% CO2/air atmosphere. The number of remaining viable cells was measured by methylthiazolyl tetrazolium salt (MTS), which is bioreduced by viable cells into a formazan product that can be detected at 490 nm. Negative control wells were treated with 2% Triton-100 to cause total lysis of the cells. Positive control wells did not receive effector cells. Next 20 !l of MTS and 1 !l of phenazine methosulfate (PMS), an electron coupling reagent, were mixed and added to each well, followed by incubation at 370C for 1-4 hrs in a 7% CO 2/air atmosphere after which the absorbance was read. The percent specific lysis was calculated from the absorbance using the formula as follows:

Student’s t test was used to determine the statistical differences between the survival of mice in various experimental and control groups. A p value less than 0.05 was considered significant.

III. Results A. Treatment of intracerebral breast cancer in C3H/He mice with Paclitaxel Paclitaxel is a potent inhibitor of cell division (Gaitanos et al, 2004; Nettles et al, 2004; Ross et al, 2004). It blocks cells in the G2/M phase of replication through its effect on the formation and function of microtubules in the cell. To determine the effect of paclitaxel on an intracerebral breast cancer, naïve C3H/He mice were injected intracerebrally into the right frontal lobe with the malignant cells (SB5b). One day afterward, the mice received a single intraperitoneal injection of varying amounts of paclitaxel (range = 1.75 to 2.75 mg/kg). The results (Figure 1) indicated that the two higher doses of

Experimental Group " Negative Control #100 Positive Control " Negative Control

Figure 1. Treatment of C3H/He mice with intracerebral SB5b breast carcinoma with paclitaxel. C3H/He mice (6 animals per group) were injected intracerebrally with 1.0 X 104 SB5b cells into the right frontal lobe. The mice received a single intraperitoneal injection of paclitaxel on the following day. Mean survival time (MST) in days: Untreated Control, 23.1 ± 2.3; Paclitaxel 1.75 mg/kg, 21.8 ± 2.3; Paclitaxel 2.25 mg/kg, 25.2 ± 4.4; Paclitaxel 2.75 mg/kg, 25.8 ± 8.0.

!

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Lichtor et al: Treatment of a brain tumor with a DNA-based vaccine paclitaxel (2.25 and 2.75 mg/kg) resulted in a modest but not statistically significant effect in prolonging the survival of mice with an intracerebral breast cancer. In the experiments to follow, mice with intracerebral breast cancer receiving the combined therapy were treated with a single intraperitoneal injection of 2.25 mg/kg of paclitaxel before the first immunization.

assay was used to measure IL-2 secretion by the transduced fibroblasts. The results indicated that antibiotic-resistant cells transduced with pZipNeoSVIL-2 (specifies IL-2) formed 2214 pg IL-2/ml/106 cells/72 hrs. Every third passage, the transduced fibroblasts were passaged in medium containing antibiotic. Under these circumstances, equivalent quantities of IL-2 were present after three months of continuous culture. The generation time of transduced and non-transduced fibroblasts, approximately every 24 hours, was equivalent.

B. The effect of paclitaxel on the white blood cell count in C3H/He mice Paclitaxel is highly toxic. Since the development of an effective immune response is dependent on white cell proliferation following antigen administration, peripheral white blood counts were measured at varying times after an injection of paclitaxel. The results (Figure 2) indicate that four days after injection of 2.25 mg/kg paclitaxel, the white blood count had returned to pre-injection levels consistent with a recovery from the toxic effects of the drug.

D. Expression of H-2Kb, MHC class 1determinants, by LM fibroblasts transduced with the vector, pBR327H-2Kb H-2Kb-determinants are allogeneic in C3H/He mice (H-2 ). Allogeneic MHC-determinants are strong immune adjuvants (deZoeten et al, 2002). To further augment their immunogenic properties, the cytokine-secreting fibroblasts used as DNA recipients were subsequently modified to express H-2Kb-determinants. A plasmid, pBR327H-2Kb, was used for this purpose. The results indicated that more than 99 percent of the transduced fibroblasts stained positively for H-2Kb-determinants. Under similar conditions, nontransduced fibroblasts or fibroblasts stained with FITC-conjugated isotype serum failed to stain. The expression of H-2Kb-determinants was a stable property of the cells, and the intensity of staining for H-2Kb determinants was essentially unchanged after three months of continuous culture. k

C. Cytokine-secretion by LM mouse fibroblasts transduced with a plasmid vector specifying IL-2 To augment their nonspecific immunogenic properties, the fibroblasts used as recipients of DNA from the breast cancer cells were modified before DNA-transfer to secrete IL-2. A gene specifying IL-2 was introduced into the cells by transduction with a plasmid vector. The vector also specified a gene conferring resistance to neomycin, an antibiotic used for selection. An ELISA

Figure 2. Peripheral white cell count following intraperitoneal injection of paclitaxel. C3H/He mice age received a single intraperitoneal injection of paclitaxel (2.25 mg/kg). Blood samples were then taken from 2 mice each day for one week in order to determine the peripheral white blood cell count. The blood samples were obtained infraorbitally and counted using a hemocytometer. The white blood cell count is the number of cells X 106. Error bars represent one standard deviation.

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Cancer Therapy Vol 4, page 167 either therapy alone. Finally it should be noted that those animals treated with paclitaxel exhibited significant lethargy and cachexia that was not observed in either the controls or those animals treated only with DNAtransfected fibroblasts modified to secrete IL-2.

E. Treatment of mice bearing an established intracerebral breast cancer with transfected fibroblasts modified to secrete IL2 and/or paclitaxel The therapeutic properties of intratumoral injections of the DNA-based vaccine were compared with those of paclitaxel in the treatment of mice with an established intracerebral breast cancer. A cannula was placed into the right frontal lobe of C3H/He mice. One day afterward the animals received an injection intracerebrally (i.c.) through the cannula with 1.0 X 104 SB-5b breast carcinoma cells. On the following day those animals treated with paclitaxel received a single intraperitoneal (i.p.) injection of 2.25 mg/kg paclitaxel. On days two and nine following tumor injection, the animals treated with the vaccine received 1.0 X 106 transfected fibroblasts introduced through the cannula into the tumor region. The results (Figure 3) indicated that mice with an established breast cancer injected either i.c. with fibroblasts transfected with tumor DNA and modified to secrete IL-2 or i.p. with pacitaxel survived significantly longer than untreated mice (p < 0.025). In addition mice with an established breast cancer that received a combination of i.p. paclitaxel followed by immunization with the transfected fibroblasts survived significantly longer (p < 0.05) than untreated mice. However there was no difference in survival in the mice treated by a combination of paclitaxel and transfected fibroblasts in comparison to those animals treated with

F. T cell mediated toxicity toward breast cancer in mice immunized with transfected fibroblasts modified to secrete IL-2 and/or paclitaxel To determine if the immunity in mice injected i.c. with the transfected fibroblasts was systemic, spleen cells from mice injected i.c. with the transfected cells were analyzed for cytotoxic effects toward the breast cancer cells. An MTS based cytotoxicity assay was used for this purpose. The analysis was performed two weeks after the i.c. injection of breast cancer cells. The results (Figure 4) indicated that the spleen cell-mediated cytotoxic responses of greatest magnitude were in mice injected i.c. with breast cancer cells and transfected fibroblasts modified to secrete IL-2. Somewhat lesser cytotoxic effects were present in mice with an established i.c. breast cancer treated with paclitaxel with or without transfected fibroblasts modified to secrete IL-2. Thus, systemic immune responses directed toward the breast cancer cells were induced in mice injected i.c. with either IL-2 secreting transfected cells or paclitaxel.

Figure 3. Treatment of an established intracerebral breast cancer with paclitaxel and/or cytokine-secreting allogeneic fibroblasts transfected with a spontaneous breast neoplasm (SB5b). A cannula was inserted into the right frontal lobe of C3H/He mice (ten animals/group). On the following day each animal received through the cannula a single injection of 1.0 X 104 SB5b cells. On the following day those animals treated with paclitaxel received a single intraperitoneal (i.p.) injection of 2.25 mg/kg paclitaxel. On the following day (day two following tumor injection) and one week later (day 9 following tumor injection) those animals treated with the vaccine received 1.0 X 106 syngeneic/allogeneic fibroblasts transfected with DNA from the breast cancer cells and modified to secrete IL-2 introduced through the cannula into the tumor region. Mean survival time (MST) in days: Untreated, 16.6 ± 1.4; Paclitaxel, 18.4 ± 1.5; Vaccine, 19.2 ± 3.0; Paclitaxel + Vaccine; 18.4 ± 2.6. Probability values were as follows: Paclitaxel vs untreated, p < 0.005; Vaccine vs untreated, p < 0.025; Paclitaxel + vaccine vs untreated, p < 0.05.

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Figure 4. MTS assay for determination of cytotoxicity from spleen cells taken from the animals 2 weeks following the intracerebral injection of tumor cells. The target cells used in this study were SB5b breast cancer cells, and the effector (spleen cell) to target cell ratios (E:T) were 50:1 and 100:1. Mononuclear cells from the spleens of the immunized mice obtained through Ficoll-Hypaque centrifugation were used for this assay. The error bars represent one standard deviation. Probability values were as follows: Paclitaxel vs untreated, p= 0.005, 0.011 and 0.025 at E: T ratio 25:1, 50:1 and 100:1 respectively; Vaccine vs untreated, p= 0.011, 0.005 and 0.028 at E: T ratio 25:1, 50:1 and 100:1 respectively; Paclitaxel + vaccine vs untreated, p= 0.020, 0.001 and 0.029 at E: T ratio 25:1, 50:1 and 100:1 respectively; Paclitaxel + vaccine vs Paclitaxel, p= 0.102, 0.7, 0.22 at E: T ratio 25:1, 50:1 and 100:1 respectively.

Elispot-IFN-! assays were used as an additional means of determining if T cells directed toward the breast cancer cells were present in the spleens of mice in the various treatment groups. T cells were recovered by Hypaque density gradient centrifugation from the spleens of mice at two weeks following the i.c. injection of the breast cancer cells. The cells were co-incubated with the breast cancer cells for 16 hours at 370C in wells before the non-adherent cells were transferred to the ELISPOT plates

containing wells precoated with a high-affinity monoclonal antibody for INF-!. After further steps, the number of spots was determined by a computer aided spot counter. The results indicated that the highest number of spots was present in spleen cells from mice with established breast cancer treated with fibroblasts transfected with tumor DNA and modified to secrete IL-2 (Figure 5).

Figure 5. ELISPOT assay detecting INF-! secretion by spleen cells (number of spots/106 cells) in the animals two weeks following injection of tumor cells. Mononuclear cells from the spleens of the immunized mice obtained through Ficoll-Hypaque centrifugation were used in this assay. The assay was performed in the presence (SB5b stimulated) and absence (unstimulated) of SB5b tumor cells. The frequency of tumor-specific effector cells in the spleen before vaccination was 0.002%. Probability values were as follows: Paclitaxel vs untreated, p = 0.030; Vaccine vs untreated, p = 0.048; Paclitaxel + vaccine vs untreated, p = 0.029; Paclitaxel + vaccine vs Paclitaxel, p= 0.064.

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Cancer Therapy Vol 4, page 169 response and therapeutic synergy, it is difficult to find such an interval in this animal model in which the life expectancy is approximately three weeks following i.c. injection of this highly aggressive tumor. It remains possible that other chemotherapeutic agents may have synergistic effects when administered in combination with immunotherapeutic treatments including the DNA-based vaccine used in this study. Finally given that the vaccine and paclitaxel have distinct mechanisms which may not complement each other, it is conceivable that some increase in the therapeutic benefits of these two treatments might exist using a different dosing schedule perhaps for example if the vaccine is given prior to administration of paclitaxel.

IV. Discussion This study has demonstrated that anti-tumor immune responses are generated in C3H/He mice with an established i.c. breast cancer injected i.c. with cytokinesecreting mouse fibroblasts transfected with unfractionated genomic DNA from the breast cancer cells. The immunity was sufficient to prolong survival, although the mice eventually died of the disease. A major advantage of this type of vaccine is that the fibroblasts could be genetically modified in advance of DNA-transfer to augment their immunogenic properties. In this instance, the cells were modified to express allogeneic class I MHC-determinants (allogeneic MHC-determinants are strong immune adjuvants and ensure that the cells will be rejected) and to secrete IL-2. The prolonged survival of mice with i.c. breast cancer treated solely by immunization with the cytokine-secreting cells points toward the potential of this form of therapy in patients with breast cancer metastatic to the brain. Because the tumor cell population is known to be heterogeneous and includes cells that are resistant to cellular immune mechanisms, a subpopulation of malignant breast cancer cells, resistant to host immune mechanisms must have survived. For further control, a combination of therapeutic strategies will be required. In this study we compared the benefits of combining immunotherapy with paclitaxel, a standard chemotherapeutic agent. The dose and dosing schedule of paclitaxel used in this study is identical to that used in similar studies with this animal model (Chopra et al, 2006). Although paclitaxel suppresses the peripheral white cell population for several days, an anti-tumor immune response was found in the spleen cells taken from those animals treated with both paclitaxel and the cellular vaccine. However the best systemic immunologic effect was detected in those animals treated with vaccine alone. A statistically significant prolongation of survival (p< 0.025) was found in mice receiving either form of treatment alone. Combination therapy did not appear to provide synergistic potential. The results of this study are consistent with other reports that paclitaxel is effective in the treatment of metastatic brain tumors (Cortes et al, 2003; Koziara et al, 2004). It is also evident that paclitaxel is toxic since animals treated with paclitaxel were cachectic and lethargic. The toxic side effects largely attributed to the paclitaxel solvent, Cremophor EL, have limited the use of paclitaxel in patients (Cortes et al, 2003). Furthermore the suppression of the peripheral white blood cell count attributed to paclitaxel, although relatively brief, makes paclitaxel along with most chemotherapeutic agents somewhat antagonistic when administered with immunotherapeutic treatment strategies. Nevertheless the combination of systemic chemotherapy along with immunotherapy has been used to treat patients with advanced-stage carcinoma (Yin et al, 2005). It has been proposed that dying tumor cells, particularly those killed by chemotherapy, engage with anti-tumor immune responses (Lake and Robinson, 2005). Although immunization at an appropriate interval following chemotherapy may result in an enhanced tumor immune

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