2007 Van Andel Research Institute Scientific Report by Van Andel Institute

VARI | 2007

Phone 616.234.5000 Fax 616.234.5001 www.vai.org

Scientific Report

333 Bostwick Avenue, N.E., Grand Rapids, Michigan 49503

Van Andel Research Institute Scientific Report 2007

Van Andel Research Institute Scientific Report 2007 Cover photo: The glass sculpture â&#x20AC;&#x153;Lifeâ&#x20AC;?, by Dale Chihuly, in the Van Andel Institute lobby. Photo by David Nadziejka.

Van Andel Research Institute 333 Bostwick Avenue, N.E., Grand Rapids, Michigan 49503 Phone 616.234.5000 Fax 616.234.5001

www.vai.org

VARI | 2007

Van Andel Research Institute Scientific Report 2007

Van Andel Research Institute |

Scientific Report

VARI | 2007

Directorâ&#x20AC;&#x2122;s Introduction

George F. Vande Woude, Ph.D.

Table of Contents

Laboratory Reports

Arthur S. Alberts, Ph.D. Cell Structure and Signal Integration

Brian Cao, M.D. Antibody Technology

Gregory S. Cavey, B.S. Mass Spectrometry and Proteomics

Nicholas S. Duesbery, Ph.D. Cancer and Developmental Cell Biology

Bryn Eagleson, B.S., RLATG Vivarium and Transgenics

Kyle A. Furge, Ph.D. Computational Biology

Brian B. Haab, Ph.D. Cancer Immunodiagnostics

Rick Hay, Ph.D., M.D., F.A.H.A. Noninvasive Imaging and Radiation Biology Office of Translational Programs

Jeffrey P. MacKeigan, Ph.D. Systems Biology

Cindy K. Miranti, Ph.D. Integrin Signaling and Tumorigenesis

James H. Resau, Ph.D. Division of Quantitative Sciences Analytical, Cellular, and Molecular Microscopy Microarray Technology Molecular Epidemiology

Pamela J. Swiatek, Ph.D., M.B.A. Germline Modification and Cytogenetics

Bin T. Teh, M.D., Ph.D. Cancer Genetics

Steven J. Triezenberg, Ph.D. Transcriptional Regulation

George F. Vande Woude, Ph.D. Molecular Oncology

Craig P. Webb, Ph.D. Program for Translational Medicine Tumor Metastasis and Angiogenesis

Michael Weinreich, Ph.D. Chromosome Replication

Bart O. Williams, Ph.D. Cell Signaling and Carcinogenesis

H. Eric Xu, Ph.D. Structural Sciences

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2006 Van Andel Research Institute Symposium

Winning the War against Cancer: From Genomics to Bedside and Back

Daniel Nathans Memorial Award

Tony Hunter, Ph.D., and Tony Pawson, Ph.D.

Postdoctoral Fellowship Program

List of Fellows

Student Programs

Grand Rapids Area Pre-College Engineering Program Summer Student Internship Program

Han-Mo Koo Memorial Seminar Series

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2006 | 2007 Seminars

Van Andel Research Institute Organization Boards Office of the Director VAI Administrative Organization

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VARI | 2007

Directorâ&#x20AC;&#x2122;s Introduction

Van Andel Research Institute |

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George F. Vande Woude Director’s Introduction

Now in our seventh year at a site in downtown Grand Rapids recently dubbed Medical Mile, the Van Andel Institute is embarking on a new phase in its growth as a leading center for biomedical research. An event for which we have been patiently waiting took place on April 12, 2007, when groundbreaking ceremonies officially marked the beginning of construction for the Phase II expansion of the Van Andel Institute. Undeterred by April snow showers, Dave Van Andel got things rolling by maneuvering a GPS-directed John Deere bulldozer to break ground, to the cheers of employees and honored guests who watched from indoors via live video. For the next two years, we will see our Phase II laboratory emerge, fulfilling the plans to increase our research capacity to accommodate 400 new scientists. You can check out the construction at http://www.vai.org/About/Facilities/PhaseII.aspx. In addition to our own project, we are witnessing all around us phenomenal growth in the Medical Mile community. Already established south of us is the St. Mary’s Lacks Cancer Center. To our east and north, construction is underway for Spectrum Health’s new Lemmen-Holton Cancer Pavilion and the Helen DeVos Children’s Hospital. Just adjacent to our campus to the west and north, the Secchia Center that is being built will be headquarters to the College of Human of Medicine (CHM) of Michigan State University (MSU). Second-year CHM students will begin study in Grand Rapids in 2008, while a first-year class of 100 students is scheduled to begin in 2010, leading to a full enrollment of about 400 students. It is hard to match all this excitement, but we have many accomplishments to our credit, and no doubt this has been a key factor stimulating Medical Mile and the growth of the biomedical enterprise. We are all very proud of what is happening.

Personnel It is my pleasure to report that Jim Resau has been promoted to the rank of Distinguished Scientific Investigator. Jim has provided the impetus in developing VARI’s imaging core, and his efforts have led to novel imaging approaches and to many successful collaborations of benefit to our Institute. Jim has also contributed a strong interest and much help with the Van Andel Education Institute (VAEI) educational programs, including the graduate school, the Grand Rapids Area Pre-College Engineering Program, and other student programs of VAEI. He serves as VARI’s Deputy Director for Special Programs and Director of the Quantitative Sciences Division. Congratulations also to Eric Xu on his promotion to Distinguished Scientific Investigator. Eric has made significant scientific contributions to defining the structures of nuclear receptor proteins, including the peroxisome proliferator–activated receptors (PPARs) and the “orphan” nuclear receptors for which the ligand and function are unknown. The importance and excellence of his work is reflected in his success with NIH grants.

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In addition, four of VARI’s original investigators have been promoted to the rank of Senior Scientific Investigator: Art Alberts, Brian Cao, Nick Duesbery, and Bart Williams. Art’s studies on Diaphanous-related formins and the DAD peptide have developed new insights into the assembly of cell structures and the possibility of new approaches to cancer therapy. He has recently played a key role in establishing VARI’s flow cytometry facility. Brian Cao was recognized for the development of VARI’s state-of-the-art antibody technology lab. He has produced novel antibodies for several VARI research programs, developed and improved his lab’s capabilities to meet research needs, and further serves as director of the Michigan Antibody Technology Core of the Core Technology Alliance. Nick Duesbery’s work with anthrax lethal toxin has shown that the lethal factor component of the toxin is a metalloprotease that cleaves MAPK kinases. His lab’s work has increased our understanding of how anthrax toxin works and has also shown that the two-component moiety called “lethal toxin” inhibits the growth of some tumors. In addition to directing his lab, Nick also serves as VARI’s Deputy Director for Research Operations. Bart Williams has pursued the regulation and function of Wnt signaling as it affects various key cellular processes. The breadth of Wnt’s effects has led him from an initial interest in Wnt’s effects in tumorigenesis to the recognition of the role of Wnt in bone development and disease. Bart has also been a major contributor to the development of VARI’s mouse models and to the inception of the VAI graduate school. We congratulate each of these researchers, and we look forward to their continued valuable contributions toward the Institute’s goals. We are pleased to announce the recruitment in 2006 of two exceptional principal investigators (PIs). Jeff MacKeigan, Ph.D., was recruited from Novartis and has established the Laboratory of Systems Biology. Jeff is interested in phosphatases and kinases, how they are regulated, and what signaling pathways they affect. He also brings platform screening technology to our program and has stimulated collaborations with our PIs to use RNAi screens as a genetic tool to understand gene function. Steve Triezenberg, Ph.D., was recruited from MSU and he wears two hats. In addition to Steve’s studies of herpes virus transcription in his newly established Laboratory of Transcriptional Regulation, he is also founding Dean of our new graduate program, established by VAEI. To Steve’s great credit, VAEI’s graduate school has an inaugural class of students that will arrive to begin studies in August 2007. The Ph.D. program, like most of the research at VARI, will focus on the molecular, cellular, and genetic biology of human disease with a pronounced emphasis on translational research. The graduate school will foster the effective transition of students into professional scientists through a unique curriculum employing problem-based learning methods and through workshops to develop the cognate skills of grant and manuscript preparation, financial management, small-group leadership, and career planning.

Programs On June 1, 2006, the Program for Translational Medicine was established under Craig Webb’s direction. This program will push forward our emphasis on moving our research findings into clinical practice and will help to develop “personalized medicine” founded on molecular-based, individual diagnosis and treatment. Craig’s staff will be developing strategies for data collection, integration, and analysis using the XB-BioIntegration Suite (formerly Xenobase), and Craig will work closely with the Office of Translational Programs, directed by Rick Hay, to help achieve VARI’s translational aims.

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The Institute’s entire animal care and use program was evaluated in March 2007 by the Association of Assessment and Accreditation of Laboratory Animal Care, as part of our application for accreditation. AAALAC standards go beyond governmental regulations, and meeting their standards symbolizes quality, promotes scientific validity, demonstrates accountability, and shows commitment to humane animal care. The preliminary results of the review were very favorable, and we anticipate receiving approval and our formal accreditation in a timely manner. Our thanks to Pam Swiatek and Bryn Eagleson and their staffs, as well as to all the others involved in preparing for this evaluation; they did a great job in getting us ready.

Grants In 2006, Eric Xu received his second R01 from NIH for a five-year study of “Structure and Function of Steroid Hormone Receptors”. Also, Brian Haab received his second R21 grant for “Defining Secreted Glycan Alterations in Pancreatic Cancer”. Rick Hay received a state appropriation from the MEDC Michigan Strategic Fund for “Creation of a Good Manufacturing Practices (GMP) Facility”. The project support runs from October 2006 through December 2007. Steve Triezenberg’s graduate student, Sebla Kutluay, received VARI’s first predoctoral grant, for two years from the American Heart Association. Sponsored funding by commercial firms for specific research areas was received by the laboratory of Bin Teh and by my own lab. Other funding was received by various labs from the Breast Cancer Research Foundation, American Cancer Society, and as subgrants through collaborations with other research organizations. 4

Collaborations In late 2006, we established a collaboration for medical education and research between MSU and VARI. The new CHM medical school will establish an innovative molecular medicine curriculum with research in areas including cancer and neurobiology and an emphasis on translational research. The medical school faculty will have laboratory space in our Phase II building upon its completion in 2009, and the school intends to be fully operational in 2010. We anticipate that unique and fruitful collaborations will result from the proximity of the MSU and VARI scientists, and we foresee benefits accruing not only to both institutions, but more importantly to the patients afflicted by the diseases we study. Also in 2006, our joint effort with Spectrum Health has created the Center for Molecular Medicine, which offers molecular-based diagnostics to physicians. Further, a multi-member alliance under the name “ClinXus” offers a venue for novel biomarker-based clinical trials and for future biomarker drug development collaborations with pharmaceutical and biotech firms. In February 2007, we signed a groundbreaking agreement with the National Cancer Center, Singapore (NCCS) to establish a joint translational research program in Singapore. The program will be directed by Bin Teh and will focus on the biological basis for different drug responses in Asian versus non-Asian patients having specific cancers. When we opened our doors in 2000, our commitment to basic sciences and translation was considered new and innovative. Lately, as I travel to other world-class academic institutions, it is clear that everyone has the same burning desire to turn discovery into application. This means that our success will be contingent not only on having the right scientific expertise, but also upon the growth of an ideal medical environment and a very supportive community. I know Grand Rapids has the “right stuff” and is poised to become a leading biomedical center in the next decade. It is an honor to be a part of such an exciting endeavor.

VARI | 2007

Laboratory Reports

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Arthur S. Alberts, Ph.D. Laboratory of Cell Structure and Signal Integration

In 1993, Dr. Alberts received his Ph.D. in physiology and pharmacology at the University of California, San Diego, where he studied with James Feramisco. From 1994 to 1997, he served as a postdoctoral fellow in Richard Treismanâ&#x20AC;&#x2122;s laboratory at the Imperial Cancer Research Fund in London, England. From 1997 through 1999, he was an Assistant Research Biochemist in the laboratory of Frank McCormick at the Cancer Research Institute, University of California, San Francisco. Dr. Alberts joined VARI as a Scientific Investigator in January 2000 and was promoted to Senior Scientific Investigator in 2006.

Staff Laboratory Staff Jun Peng, M.D. Kathryn Eisenmann, Ph.D. Holly Holman, Ph.D. Richard A. West, M.S. Susan Kitchen, B.S.

Students Students Aaron DeWard, B.S. Dagmar Hildebrand, B.S.

Visiting Scientists

Stephen Matheson, Ph.D. Brad Wallar, Ph.D.

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Research Interests Research in the Laboratory of Cell Structure and Signal Integration focuses on the molecular machinery responsible for the reorganization of the cell’s architecture during division and directed migration. Of particular interest is how defects in the machinery drive the progression to malignancy. The goal is to identify key control steps that are altered in disease states and exploit that knowledge to improve diagnostic and prognostic capabilities. We have been targeting key points in the cytoskeletal control system to devise novel targets for molecular therapy. The cytoskeleton comprises microfilaments, microtubules, and intermediate filaments. Each of these structures is a polymer whose assembly from individual monomer subunits is controlled by accessory proteins. While the term “cytoskeleton” implies a static or rigid structure within cells, the various filamentous structures are actually highly dynamic. Microfilaments, for example, are made of polymerized actin; these filaments rapidly polymerize, bundle, bend, depolymerize, or are severed so as to assume different shapes within the cell to fulfill a given function. In some cases, individual strands are woven into networks and contract against each other so that cells can attach to extracellular substrates and crawl along them. For example, actin/microfilament remodeling is crucial in the immune cells’ role to search for and destroy invading pathogens. Cancer cells use such remodeling to migrate from primary tumors (often located at an innocuous site) to a secondary site. At the secondary site, tumor cells grow and damage adjacent tissue, often leading to the eventual death of the patient. This process is called metastasis, and to date there are few, if any, effective anti-cancer therapies that block it. Thus, there is an important need to identify mechanisms that can be effectively targeted to block the spread of tumor cells throughout the body. The Rho family of small GTPases controls critical steps in cytoskeletal remodeling. The GTPases are triggered by signals dictated by activated growth or adhesion receptors and, in turn, bind to “effectors” that govern the machinery assembling the cytoskeleton. Some of these effector proteins directly participate in cytoskeletal remodeling. One fundamentally important set of GTPase effectors is the mammalian Diaphanous-related (mDia) formins. Formins nucleate, processively elongate, and (in some cases) bundle filamentous actin (F-actin) through conserved formin homology-2 (FH2) domains. mDia proteins participate in many cytoskeletal remodeling events including cytokinesis, vesicle trafficking, and filopodia assembly while acting as effectors for Rho small GTPases. Rho proteins govern mDia proteins by regulating an intramolecular autoregulatory mechanism. GTPases binding to the mDia amino-terminal GTPase-binding domain (GBD) sterically hinder the adjacent Dia-inhibitory domain (DID) interaction with the carboxyl-terminal Dia-autoregulatory (DAD) domain (Fig. 1). The release of DAD allows the adjacent FH2 domain to then nucleate and elongate nonbranched actin filaments.

Figure 1.

Figure 1. mDia proteins are autoregulated nucleators of actin. Autoinhibition of mDia is mediated by interaction between the DID and DAD domains. Activated GTP-bound Rho proteins bind to the GBD where they interfere with DAD binding to DID. Then the free FH2 domains, which also function as dimerization interfaces, can nucleate actin monomers and processively elongate actin filaments. Tagged fusion proteins (CFP-Rho GTPase and YFP-mDia) are used in fluorescence resonance energy transfer (FRET) to monitor the sites of protein-protein interactions. Excitation of CFP by a specific wavelength of light results in emitted light at a wavelength that excites YFP, but YFP excitation occurs only if the proteins are close enough to approximate direct binding. This approach is used to generate the data shown in Fig. 2.

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The cytoskeleton not only provides the impetus for cell movement, but it also allows the internal architecture to be organized into different compartments having specific functions in the cellular responses to growth factors. Rho GTPases and the dynamic assembly and disassembly of actin filaments have been shown to have crucial roles in both the internalization and trafficking of growth factor receptors. While all three mammalian Diaphanous-related formins (mDia1, mDia2, and mDia3) have been localized on endosomes, their roles in actin nucleation, filament elongation, and/or bundling remains poorly understood in the context of intracellular trafficking. In a recent publication in Experimental Cell Research, we reported the functional relationship between RhoB, a GTPase known to associate with both early and late endosomes, and the formin mDia2. We were able to show that 1) RhoB and mDia2 interact on endosomes, as seen in Fig. 2 using the FRET approach; 2) GTPase activity—the ability to hydrolyze GTP to GDP—is required for the ability of RhoB to govern endosome dynamics; and 3) the actin dynamics controlled by RhoB and mDia2 is necessary for vesicle trafficking. These studies further suggested that Rho GTPases significantly influence the activity of mDia family formins in driving cellular membrane remodeling through the regulation of actin dynamics. In another recent study, in the journal Current Biology, we reported how Diaphanous-interacting protein (DIP) binds to and regulates the activity of the formin mDia2 and its ability to assemble filopodia. Filopodia are small finger-like projections comprising several bundled nonbranched actin filaments emanating from the leading edge of migrating cells and essentially acting as sensors for directed cell movement. We investigated an interaction occurring between a conserved leucine-rich region (LRR) in DIP and the mDia FH2 domain. While DIP has been shown to interact with and stimulate N-WASp-dependent branched filament assembly via Arp2/3, it interfered with mDia2-dependent filament assembly and bundling. 8 Figure 2.

Figure 2. RhoB and mDia2 interact on a subset of vesicles bearing internalized EGF. CFP-RhoB and YFP-mDia2 interact on vesicles bearing internalized Texas Red–labeled epidermal growth factor. Cells expressing the two FRET probes (4 h after injection) were incubated with fluorescent EGF for 5 min prior to fixation. RhoB-mDia2 FRET occurs on a subset of vesicles (FRET is false-colored green, with Texas Red–EGF shown in red).

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Surprisingly, DIP had no effect on the highly related mDia1. Consistent with a role for mDia2 as a Cdc42 effector, DIP both blocked the formation of filopodia and induced non-apoptotic membrane blebbing, a physiological process involved in both cytokinesis and amoeboid cell movement. DIP-induced blebbing occurred independently of Arp2/3 activity. Figure 3 shows the result of microinjection of DIP LRR into a mouse embryo fibroblast in which a critical subunit of Arp2/3 has been knocked down by siRNA. The experiment reveals a pivotal role for DIP in the control of nonbranched versus branched actin filament assembly mediated, respectively, by Diaphanous-related formins and by activators of Arp2/3. The ability of DIP to trigger blebbing also suggests a role for mDia2 in the assembly of actin filaments at the cell cortex necessary for the maintenance of plasma membrane integrity. Future experiments will address how DIP regulates mDia2 in directed cell movement and during cell division. Figure 3.

Figure 3. DIP LRR–induced plasma membrane blebbing does not require Arp2/3 activity. This mouse embryo fibroblast, which expresses siRNA directed against Arp3, was injected with 0.1 μM recombinant DIP LRR protein along with Texas Red dextran as a marker.

External Collaborators Harry Higgs, Dartmouth Medical School, Hanover, New Hampshire

Recent Publications

From left: Peng, Holman, DeWard, Eisenmann, Kitchen, Alberts, Hildebrand

Eisenmann, Kathryn M., Elizabeth S. Harris, Susan M. Kitchen, Holly A. Holman, Henry N. Higgs, and Arthur S. Alberts. 2007. Dia-interacting protein modulates formin-mediated actin assembly at the cell cortex. Current Biology 17(7): 579–591. Wallar, Bradley J., Aaron D. DeWard, James H. Resau, and Arthur S. Alberts. 2007. RhoB and the mammalian Diaphanousrelated formin mDia2 in endosome trafficking. Experimental Cell Research 313(3): 560–571.

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Brian Cao, M.D. Laboratory of Antibody Technology

Dr. Cao obtained his M.D. from Peking University Medical Center, People’s Republic of China, in 1986. On receiving a CDC fellowship award, he was a visiting scientist at the National Center for Infectious Diseases, Centers for Disease Control and Prevention in Atlanta (1991–1994). He next served as a postdoctoral fellow at Harvard (1994–1995) and at Yale (1995–1996). From 1996 to 1999, Dr. Cao was a Scientist Associate in charge of the Monoclonal Antibody Production Laboratory at the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute, Frederick Cancer Research and Development Center, Maryland. Dr. Cao joined VARI as a Special Program Investigator in June 1999, and he was promoted to Senior Scientific Investigator in July 2006.

Staff Laboratory Staff Ping Zhao, M.S. Tessa Grabinski, B.S.

Students Students Xin Wang Ning Xu Aixia Zhang Jin Zhu

Visiting Scientists

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Research Interests Antibodies are primary tools of biomedical science. In basic research, the characterization and analysis of almost any molecule involves the production of specific monoclonal or polyclonal antibodies that react with it. Antibodies are also widely used in diagnostic applications for clinical medicine. ELISA and radioimmunoassay systems are antibody-based. Analysis of cells and tissues in pathology laboratories includes the use of antibodies on tissue sections and in flow cytometry analyses. Further, antibodies are making rapid inroads into medical therapeutics, driven by technological evolution from chimeric and humanized to fully human antibodies. The therapeutic antibody market has the potential to reach $30 billion by 2010. Our Antibody Technology laboratory has developed several technologies over the last few years: 1) state-of-the-art monoclonal antibody (mAb) production and characterization, followed by scaled-up production and purification; 2) antibody-binding-site epitope mapping using a phage-display peptide library; 3) a human-antibody-fragment phage-display library and screening of specific fragments from the library; and 4) characterization of these human antibody fragments and conjugation with chemotherapeutics to generate immuno-chemotherapeutic reagents for preclinical studies. In collaboration with Nanjing Medical University, China, we constructed our own human naĂŻve Fab fragment phage-display library, with a diversity of 2 Ă&#x2014; 109, in late 2004. In 2005, we screened out several Fab fragments from the library that specifically recognize HGF/SF, Met, and EGFR. By modifying and improving biopanning strategies, we have selected Fab fragments that recognize the Met and EGFR extracellular domains in native conformation with reasonable affinity and, importantly, with the internalization property that makes these Fabs attractive as conjugate reagents for immuno-chemotherapy or immuno-radiation therapy against cancer. In the past year, we have conjugated anti-EGFR human Fab to paclitaxel (Taxol) as an immuno-chemotherapy agent and investigated its in vitro anti-tumor efficacy on A431 epidermoid carcinoma cells using cell proliferation inhibition and apoptosis assays. The Fab-Taxol conjugate inhibited A431 cell proliferation at low concentrations and in a dose-responsive manner; more than 70% inhibition was observed at 52 pM. Furthermore, almost 100% of the cells underwent apoptosis after treatment with Fab-Taxol at 26 pM for 48 hours. The in vitro anti-tumor efficacy is four- to fivefold more potent than Taxol alone. We are modifying the Taxol conjugation conditions and working with other drug conjugations to investigate their in vivo anti-tumor efficacy in xenograft and orthotopic animal models.

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Functioning as an antibody production core facility, this lab has extensive capabilities. Our technologies and services include antigen preparation and animal immunization; peptide design and coupling to protein carriers; DNA immunization (gene-gun technology); immunization with living or fixed cells; conventional antigen/adjuvant preparation; immunizing a wide range of antibody-producing models (including mice, rats, rabbits, human cells, and transgenic or knock-out mice); and in vitro immunization. Our work also includes the generation of hybridomas from spleen cells of immunized mice, rats, and rabbits; hybridoma expansion and subcloning; cryopreservation of hybridomas secreting mAbs; isotyping of mAbs; ELISA screening of hybridoma supernatants; mAb characterization by immunoprecipitation, Western blot, immunohistochemistry, immunofluorescence staining, FACS, and in vitro bioassays; generation of bi-specific mAbs by secondary fusion; conjugation of mAbs to enzymes, biotin/streptavidin, or fluorescent reporters; and development of detection methods/kits such as sandwich ELISA. We also contract services to biotechnology companies, producing and purifying mAbs for their research and for diagnostic kit development. The Michigan Core Technology Alliance (CTA), funded by the state government, was created in 2001. The Antibody Technology Core at VARI and the Hybridoma Core at the University of Michigan in Ann Arbor joined together to form the Michigan Antibody Technology Core (MATC) and became the seventh core of CTA in March 2005. Our goals are to provide state-of-the-art antibody technologies and services to research scientists; to generate, characterize, produce, and purify a wide variety of monoclonal antibodies; to make human antibody fragments and humanize murine mAbs for clinical diagnostic/therapeutic applications; and to advance biomedical research and development. The Antibody Technology Lab at VARI serves as the core’s hub, and Dr. Brian Cao is the director of MATC. 12

From left: Gu, Zhang, Xu, Zhao, Nelson, Grabinski, Cao

Recent Publications Wang, X., J. Zhu, P. Zhao, Y. Jiao, N. Xu, T. Grabinski, C. Liu, C.K. Miranti, T. Fu, and B. Cao. In press. In vitro efficacy of immunochemotherapy with anti-EGFR human Fab-Taxol conjugate on A431 epidermoid carcinoma cells. Cancer Biology & Therapy. Zhang, Y.-W., B. Staal, Y. Su, P. Swiatek, P. Zhao, B. Cao, J. Resau, R. Sigler, R. Bronson, and G.F. Vande Woude. 2007. Evidence that MIG-6 is a tumor-suppressor gene. Oncogene 26(2): 269–276. Tsarfaty, Galia, Gideon Y. Stein, Sharon Moshitch-Moshkovitz, Dafna W. Kaufman, Brian Cao, James H. Resau, George F. Vande Woude, and Ilan Tsarfaty. 2006. HGF/SF increases tumor blood volume: a novel tool for the in vivo functional molecular imaging of Met. Neoplasia 8(5): 344–352.

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Gregory S. Cavey, B.S. Laboratory of Mass Spectrometry and Proteomics

Mr. Cavey received his B.S. degree from Michigan State University in 1990. Prior to joining VARI he was employed at Pharmacia in Kalamazoo, Michigan, for nearly 15 years. As a member of a biotechnology development unit, he was group leader for a protein characterization core laboratory. More recently as a research scientist, he was principal in the establishment and application of a state-of-the-art proteomics laboratory for drug discovery. Mr. Cavey joined VARI as a Special Program Investigator in July 2002.

Staff Laboratory Staff Paula Davidson, M.S. Joan Krilich, B.S.

Students

Visiting Scientists

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Research Interests The Mass Spectrometry and Proteomics laboratory provides protein identification analysis and protein molecular weight determination as core services. Nanogram amounts of protein in SDS-PAGE gels or in solution are digested into peptides and analyzed by HPLC with on-line electrospray mass spectrometry. Peptides are fragmented in the mass spectrometer to generate amino acid sequence data that is used to identify proteins by searching protein and DNA databases. Submicrogram amounts of intact proteins are analyzed by nanoscale liquid chromatography–mass spectrometry (LC-MS) to determine their average molecular weight; this work is performed using a variety of HPLC columns to optimize recovery and provide reliable results. These core services are provided to both VARI investigators and external clients. Research in the lab focuses on improving existing services and developing new methods based on the needs of VARI investigators. Our three main areas of interest are intact-protein molecular weight determination, phosphopeptide analysis, and protein expression profiling using LC-MS.

Protein LC-MS

We use protein LC-MS to confirm correct expression and purification of recombinant proteins from bacteria. The average molecular weight of a protein is experimentally determined and compared with the calculated weight from the expected amino acid sequence. Proteins of 50 kDa and larger are analyzed with mass accuracy often better than 0.01%, or ±1 Da per 10 kDa. Unlike with conventional SDS-PAGE, protein truncation and modifications such as oxidation or acetylation can be accurately characterized using protein LC-MS. This information is essential when protein reagents are used for labor-intensive and costly protocols such as x-ray crystallography, antibody production, or drug screening. We have a dedicated LC-MS instrument with optimized HPLC separation and comprehensive data processing for analyzing complex mixtures of proteins. For proteins that degrade during purification, we can alter the use of protease inhibitors or minimize degradation through site-directed mutagenesis of susceptible amino acids. We are also exploring the use of this equipment for biomarker discovery of intact proteins. The goal is to provide relative quantitation of proteins in disease cell culture models, tumor tissue, and cancer patient body fluids.

Protein phosphorylation analysis Mapping post-translational modifications of proteins such as phosphorylation is an important yet difficult undertaking in cancer research. Phosphorylation regulates many protein pathways that could serve as potential drug targets in cancer therapy. In recent years, mass spectrometry has emerged as a primary tool in determining site-specific phosphorylation and relative quantitation. Phosphorylation analysis is complicated by many factors, but principally by the low-stoichiometry modifications that may regulate pathways: we are sometimes dealing with 0.01% or less of phosphorylated protein among a large excess of a nonphosphorylated counterpart. Our lab collaborates with investigators to map protein phosphorylation using techniques including multiple enzyme digestion, titanium dioxide phosphopeptide enrichment, and phosphorylation-specific mass spectrometry detection. Although trypsin is often the enzyme of choice for digesting proteins into peptides for identification, additional enzymes such as Lys-C, Staph V8, chymotrypsin, thermolysin, or elastase may also be employed. Multiple enzyme digests and titanium dioxide enrichment are used in combination with precursor ion scanning for –79 m/z on a Waters Q-Tof Premier mass spectrometer.

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We have developed a robust negative-ion-mode method using nanoscale HPLC that provides specific detection of phosphopeptides below 20 fmol in the presence of 2 pmol of nonphosphorylated protein. Once detected in the negative mode, phosphopeptides are sequenced in a subsequent LC-MS analysis in the positive ion mode using accurate mass parent ion selection, a narrow retention time window, and collision energy ramping. This approach has provided a reliable and sensitive means of analyzing phosphoproteins in our laboratory. Our current focus is on applying this label-free method to studies requiring relative quantitation of phosphorylation events.

Protein expression/biomarker discovery As mass spectrometry instruments and protein separation methods develop, proteomics techniques allow researchers to identify and quantitate protein samples of increasing complexity. The ultimate goal is to catalog all proteins expressed in a given cell or tissue as a means of evaluating dynamic physiological events and understanding how all proteins interact to affect a biological outcome. Traditionally this goal has been approached using 2D gel electrophoresis, image analysis of stained proteins, and identification of proteins from gels using mass spectrometry. Because of the labor-intensive nature of 2D gels and the underrepresentation of some protein classes (such as membrane proteins), proteomics has been moving toward solution-based separations and direct mass spectrometry analysis. Our laboratory recently purchased and installed a Waters Corporation Protein Expression System for non-gel-based, label-free protein expression analysis. This system represents a paradigm shift in the field of proteomics, because it provides both quantitative and qualitative data on complex mixtures of proteins in a single LC-MS analysis. Proteins are enzymatically digested using trypsin and, without any chemical or isotopic labeling, the resulting peptides are analyzed by LC-MS. The combination of molecular mass and LC retention time establishes a signature for each peptide and allows comparison across samples. The mass spectrometer signal intensity of each peptide is used for quantitation. Qualitative protein identification data is obtained by fragmenting all peptides eluting into the mass spectrometer, a feature unique to the Waters instrument. VARI is one of an elite group of institutions that have this powerful new technology. This system will be used to map protein pathways under a systems biology approach and to discover potential biomarkers for early detection and diagnosis in cancer and other diseases.

External Collaborators Gary Gibson, Henry Ford Hospital, Detroit, Michigan Michael Hollingsworth, Eppley Cancer Center, University of Nebraska, Omaha Waters Corporation

Core Technology Alliance (CTA) This laboratory participates in the CTA as a member of the Michigan Proteomics Consortium.

From left: Davidson, Cavey, Krilich

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Cells prepared by Miles Qian and Daisuke Matsuda of the Teh laboratory. Image by Kristin VendenBeldt of the Resau laboratory.

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Murine lymph node/vascular tissue.

Murine lymph node/vascular tissues stained by immunohistochemisty and photographed using the CRI Nuance camera. Green, pericyte cell marker; red, CD34 blood vessel marker; blue, nuclear/DNA marker.

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Nicholas S. Duesbery, Ph.D. Laboratory of Cancer and Developmental Cell Biology

Dr. Duesbery received a B.Sc. (Hon.) in biology (1987) from Queenâ&#x20AC;&#x2122;s University, Canada, and both his M.Sc. (1990) and Ph.D. (1996) degrees in zoology from the University of Toronto, Canada, under the supervision of Yoshio Masui. Before his appointment as a Scientific Investigator at VARI in April 1999, he was a postdoctoral fellow in the laboratory of George Vande Woude in the Molecular Oncology Section of the Advanced BioScience Laboratoriesâ&#x20AC;&#x201C;Basic Research Program at the National Cancer Institute, Frederick Cancer Research and Development Center, Maryland. Dr. Duesbery was promoted to Senior Scientific Investigator and appointed Deputy Director for Research Operations in 2006.

Staff

Students

Jennifer Bromberg-White, Ph.D. Philippe Depeille, Ph.D. Yan Ding, Ph.D. John Young, M.S. Jaclyn Lynem, B.S. Elissa Boguslawski Laura Holman

Students

Visiting Scientists

Chih-Shia Lee, M.S. Naomi Asantewa-Sechereh Lisa Orcasitas

VARI | 2007

Research Interests Many malignant sarcomas such as fibrosarcomas are refractory to available treatments. However, sarcomas possess unique vascular properties which indicate they may be more responsive to therapeutic agents that target endothelial function. Mitogen-activated protein kinase kinases (MKKs) have been shown to play an essential role in the growth and vascularization of carcinomas, and we hypothesize that signaling through multiple MKK pathways is also essential for sarcomas. The objective of our research is to define the role of MKK signaling in the growth and vascularization of human sarcomas and to determine whether inhibition of multiple MKKs by agents such as anthrax lethal toxin (LeTx), a proteolytic inhibitor of MKKs, can form the basis of a novel and innovative approach to the treatment of human sarcoma. In the past year we have made substantial progress in achieving this objective. Yan Ding, a postdoctoral fellow in the lab, and Lisa Orcasitas have shown that MKKs are active in fibrosarcoma and that LeTx can inhibit the in vitro tumorigenic potential of cells derived from human fibrosarcoma. The anti-tumoral properties of LeTx probably stem from its ability to substantially decrease the release of many growth factors, notably the pro-angiogenic vascular endothelial growth factor (VEGF). In vivo, LeTx caused a substantial decrease in both tumor volume and mean vascular density of fibrosarcoma xenografts. These changes also correlated with a decreased level of pro-angiogenic factors, including VEGF. Dr. Ding also found that the ability of LeTx to decrease the release of VEGF was not limited to fibrosarcoma, but was observed in cell lines derived from various sarcomas including malignant fibrous histiocytoma and leiomyosarcoma. These results are consistent with the hypothesis that MKK signaling is required for the growth and vascularization of fibrosarcoma both in vitro and in vivo, and this probably is also true of other types of soft-tissue sarcomas. Similarly, using an endothelial model of Kaposi sarcoma, Philippe Depeille, another postdoctoral fellow, and Elissa Boguslawski showed that in vitro, LeTx 1) decreases proliferation, 2) inhibits tumorigenesis, and 3) dramatically reduces the secretion of angioproliferative cytokines such as VEGF. Furthermore, in vivo, systemic treatment with LeTx inhibits tumor growth and vascularization. These findings support the importance of MKK pathways in the release of angioproliferative cytokines that promote tumor growth and vascularization. Our data suggest that inhibition of MKK signaling may be an effective therapeutic strategy for the treatment of Kaposi sarcoma. In collaboration with Bart Williamsâ&#x20AC;&#x2122; lab, John Young, our senior technician, and Jennifer Bromberg-White, a postdoctoral fellow, investigated the mechanism of anthrax toxin entry into cells. Together they showed that mice or cells lacking LRP6, or a related protein called LRP5, are still susceptible to anthrax toxin. The discovery that anthrax toxin can enter cells without the help of LRP6 presents a significant challenge to the published models of anthrax toxin function. These findings will help focus the efforts of scientists working on new ways to treat anthrax. In collaboration with Arthur Frankel, director of the Scott & White Cancer Research Institute in Texas, we have also tested the therapeutic potential of LeTx in the treatment of malignant melanoma. Progress to date indicates that melanoma is particularly sensitive to MKK inhibition. This is likely due in part to the fact that more than 80% of melanoma tumors harbor somatic mutations that cause constitutive activation of the MKK1 and MKK2 signaling pathways, though indirect evidence suggests that other MKK pathways also play a role in melanoma progression. Chih-Shia Lee is performing a detailed study of the individual contributions of MKK pathways to melanoma survival. Jaclyn Lynem and Naomi Asantewa-Sechereh are investigating the molecular basis of LF inactivation of MKK. We are currently performing preclinical studies to evaluate the potential of LeTx as a therapeutic for malignant melanoma.

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From left: Asantewa-Sechereh, Orcasitas, Lynem, Boguslawski, Lee, Bromberg-White, Duesbery, Holman, Young, Depeille

Recent Publications Young, J.J., J.L. Bromberg-White, C.R. Zylstra, J. Church, E. Boguslawski, J. Resau, B.O. Williams, and N. Duesbery. In press. LRP5 and LRP6 are not required for protective antigen-mediated internalization or lethality of anthrax lethal toxin. PLoS Pathogen. Depeille, P.E., Y. Ding, J.L. Bromberg-White, and N.S. Duesbery. 2007. MKK signaling and vascularization. Oncogene 26(9): 1290–1296. Abi-Habib, Ralph J., Ravibhushan Singh, Stephen H. Leppla, John J. Greene, Yan Ding, Bree Berghuis, Nicholas S. Duesbery, and Arthur E. Frankel. 2006. Systemic anthrax lethal toxin therapy produces regressions of subcutaneous human melanoma tumors in athymic nude mice. Clinical Cancer Research 12(24): 7437–7443. Bodart, Jean-François L., and Nicholas S. Duesbery. 2006. Xenopus tropicalis oocytes: more than just a beautiful genome. In Xenopus Protocols: Cell Biology and Signal Transduction, X. Johné Liu, ed. Methods in Molecular Biology series, Vol. 322. Totowa, N.J.: Humana Press, pp. 43–53.

VARI | 2007

Bryn Eagleson, B.S., RLATG Vivarium and Laboratory of Transgenics

Bryn Eagleson began her career in laboratory animal services in 1981 with Litton Bionetics at the National Cancer Institute’s Frederick Cancer Research and Development Center (NCI–Frederick) in Maryland. In 1983, she joined the Johnson & Johnson Biotechnology Center in San Diego, California. In 1988, she returned to the NCI–Frederick, where she continued to develop her skills in transgenic technology and managed the transgenic mouse colony. In 1999, she joined VARI as the Vivarium Director and Transgenics Special Program Manager.

Technical Staff Lisa DeCamp, B.S. Laboratory Staff Dawna Dylewski, B.S. Audra Guikema, B.S., L.V.T. Kellie Jilbert, B.S., A.S. Jamie Bondsfield, A.S. Elissa Boguslawski, RALAT

Animal Caretaker Staff Sylvia Marinelli, Team leader Students Visiting Scientists Angie Rogers, B.S. Crystal Brady Jarred Grams Janelle Post Tina Schumaker Michael Shearer Bobbie Vitt

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Research Interests The goal of the vivarium and the transgenics laboratory is to develop, provide, and support high-quality mouse modeling services for the Van Andel Research Institute investigators, Michigan Technology Tri-Corridor collaborators, and the greater research community. We use two Topaz Technologies software products, Granite and Scion, for integrated management of the vivarium finances, the mouse breeding colony, and the Institutional Animal Care and Use Committee (IACUC) protocols and records. Imaging equipment, such as the PIXImus mouse densitometer and the ACUSON Sequoia 512 ultrasound machine, is available for noninvasive imaging of mice. VetScan blood chemistry and hematology analyzers are now available for blood analysis. Also provided by the vivarium technical staff are an extensive xenograft model development and analysis service, rederivation, surgery, dissection, necropsy, breeding, and health-status monitoring.

Transgenics

Fertilized eggs contain two pronuclei, one that is derived from the egg and contains the maternal genetic material and one derived from the sperm that contains the paternal genetic material. As development proceeds, these two pronuclei fuse, the genetic material mixes, and the cell proceeds to divide and develop into an embryo. Transgenic mice are produced by injecting small quantities of foreign DNA (the transgene) into a pronucleus of a one-cell fertilized egg. DNA microinjected into a pronucleus randomly integrates into the mouse genome and will theoretically be present in every cell of the resulting organism. Expression of the transgene is controlled by elements called promoters that are genetically engineered into the transgenic DNA. Depending on the selection of the promoter, the transgene can be expressed in every cell of the mouse or in specific cell populations such as neurons, skin cells, or blood cells. Temporal expression of the transgene during development can also be controlled by genetic engineering. These transgenic mice are excellent models for studying the expression and function of the transgene in vivo.

From left: Bondsfield, Eagleson, Jason, Guikema, Shearer, Marinelli, Vitt, Post, Jilbert, Dylewski, Brady, Schumaker, Rogers, Boguslawski, Grams, DeCamp

VARI | 2007

Kyle A. Furge, Ph.D. Laboratory of Computational Biology

Dr. Furge received his Ph.D. in biochemistry from the Vanderbilt University School of Medicine in 2000. Prior to obtaining his degree, he worked as a software engineer at YSI, Inc., where he wrote operating systems for embedded computer devices. Dr. Furge did his postdoctoral work in the laboratory of George Vande Woude. He became a Bioinformatics Scientist at VARI in June of 2001 and a Scientific Investigator in May of 2005.

Staff Laboratory Staff Karl Dykema, B.A.

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Research Interests As high-throughput technologies such as DNA sequencing, gene and protein expression profiling, DNA copy number analysis, and single nucleotide polymorphism genotyping become more available to researchers, extracting the most significant biological information from the large amount of data produced by these technologies becomes increasingly difficult. Computational disciplines such as bioinformatics and computational biology have emerged to develop methods that assist in the storage, distribution, integration, and analysis of these large data sets. The Computational Biology laboratory at VARI currently focuses on using mathematical and computer science approaches to analyze and integrate complex data sets in order to develop a better understanding of how cancer cells differ from normal cells at the molecular level. In addition, members of the lab provide assistance in data analysis and other computational projects on a collaborative and/or fee-for-service basis. In the past year the laboratory has taken part in many projects to further the research efforts at VARI. We have worked closely with the Laboratory of Mass Spectrometry and Proteomics in developing computational infrastructure to support new protein profiling instrumentation and analysis. We have contributed to several gene expression microarray analysis projects ranging from mechanisms of oncogene transformation to the identification of genes that are associated with drug sensitivity. We also work closely with the Laboratory of Cancer Genetics in the development of gene expressionâ&#x20AC;&#x201C;based models for diagnosis and prognosis of renal cell carcinoma. Moreover, we and other groups have demonstrated that several types of biological information, in addition to relative transcript abundance, can be derived from high-density gene expression profiling data. Taking advantage of this additional information can lead to the rapid development of plausible computational models of disease development and progression.

Changes in DNA copy number result in dramatic changes in gene expression within the abnormal region and are detectable through examination of the population of mRNAs generated from the genes that map to each chromosome. Additionally, activation of certain oncogenes or inactivation of certain tumor suppressor genes can produce context-independent gene signatures that can be detected in a gene expression profile. For example, genes that are up-regulated by overexpression of RAS in breast epithelial cells also tend to be overexpressed in other samples containing activated RAS signaling, such as lung tumors that contain activating RAS mutations. We have invested a reasonable portion of the past several years developing and evaluating computational methods to predict deregulated signal transduction pathways and chromosomal abnormalities using gene expression data. We have worked closely with the Laboratory of Cancer Genetics on computational models to describe the development and progression of renal cell carcinoma. An example of the successful application of this analytic approach is in the examination of gene expression profiling data derived from papillary renal cell carcinoma (RCC).

VARI | 2007

Computational analysis of gene expression data derived from papillary RCC revealed that a transcriptional signature indicative of MYC pathway activation was present in high-grade papillary RCC, but not other high-grade RCCs. Predictions of chromosomal gains and losses were also generated from the gene expression data, and it was demonstrated that the presence of the MYC signature was coincident with a predicted amplification of chromosome 8q. Because the c-MYC gene maps to chromosome 8q, a computational model was developed such that amplification of chromosome 8q occurs in the high-grade papillary tumors, which leads to c-MYC overexpression and activation of the MYC pathway. The importance of MYC activation was confirmed by both pharmacological and siRNA inhibition of active MYC signaling in a cell line model of high-grade papillary RCC. These results highlight the effectiveness of using gene expression profiling data to build integrative computational models of tumor development and progression.

From left: Dykema, Furge

Recent Publications Furge, Kyle A., Jindong Chen, Julie Koeman, Pamela Swiatek, Karl Dykema, Kseniji Lucin, Richard Kahnoski, Ximing J. Yang, and Bin Tean Teh. 2007. Detection of DNA copy number changes and oncogenic signaling abnormalities from gene expression data reveals MYC activation in high-grade papillary renal cell carcinoma. Cancer Research 67(7): 3171–3176. Furge, K.A., M.H. Tan, K. Dykema, E. Kort, W. Stadler, X. Yao, M. Zhou, and B.T. Teh. 2007. Identification of deregulated oncogenic pathways in renal cell carcinoma: an integrated oncogenomic approach based on gene expression profiling. Oncogene 26(9): 1346–1350. Furge, Kyle A., Eric J. Kort, Ximing J. Yang, Walter M. Stadler, Hyung Kim, and Bin Tean Teh. 2006. Gene expression profiling in kidney cancer: combining differential expression and chromosomal and pathway analyses. Clinical Genitourinary Cancer 5(3): 227–231. Yang, Ximing J., Jun Sugimura, Kristian T. Schafernak, Maria S. Tretiakova, Misop Han, Nicholas J. Vogelzang, Kyle Furge, and Bin Tean Teh. 2006. Classification of renal neoplasms based on molecular signatures. Journal of Urology 175(6): 2302–2306.

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Brian B. Haab, Ph.D. Laboratory of Cancer Immunodiagnostics

Dr. Haab obtained his Ph.D. in chemistry from the University of California at Berkeley in 1998. He then served as a postdoctoral fellow in the laboratory of Patrick Brown in the Department of Biochemistry at Stanford University. Dr. Haab joined VARI as a Special Program Investigator in May 2000 and became a Scientific Investigator in 2004.

Staff

Students

Visiting Scientist

Songming Chen, Ph.D. Michael Shafer, Ph.D. Yi-Mi Wu, Ph.D. Derek Bergsma, B.S. Sara Forrester, B.S. Thomas LaRoche, B.S. Tingting Yue, B.S. Alex Turner

Krysta Collins Jennifer Lunger Devin Mistry

Rasmus Lundquist

Laboratory Staff

VARI | 2007

Research Interests Members of the Haab laboratory identify protein and carbohydrate abnormalities in the blood of cancer patients and investigate the significance and potential clinical usefulness of those abnormalities. We develop novel experimental methods to facilitate this work, and we collaborate with both clinicians and basic scientists to pursue research on pancreatic and prostate cancers.

Low-volume, high-throughput antibody and protein arrays We have developed the ability to probe multiple proteins or carbohydrate structures using low sample volumes, which provides a powerful tool for identifying and measuring protein and carbohydrate abnormalities in cancer. Antibody and protein arrays immobilized on the surface of a microscope slide are the key to such a capability. A biological sample such as blood serum can be incubated on an array to investigate interactions between the immobilized molecules and the proteins or antibodies in the sample. Those interactions can be probed to obtain information such as protein abundance, glycosylation level, or protein-protein interaction level. The routine use of these tools was made possible by the development of a practical method for processing multiple arrays on a microscope slide (Fig. 1). A stamp imprints a wax pattern onto the surface of a slide, creating hydrophobic partitions that segregate various samples. Distinct stamp designs can be used to form differing sizes and numbers of partitions. A design that imprints 48 arrays on one slide requires only 6 Îźl of sample per array, with each array composed of 144 distinct spots of immobilized molecules. Such a design enables the efficient processing of many samples or testing of many conditions in parallel, as demonstrated in the projects described below. The device for creating these slides is commercially available from The Gel Company, San Francisco.

Figure 1A.

Figure 1B.

Figure 1C.

Figure 1. High-throughput sample processing using a novel slide partitioning method. A) Wax is imprinted onto a microscope slide to form borders around multiple arrays. Wax is melted by the hotplate under the bath, and a slide is inserted upside-down into the holder. Bringing the lever forward raises a stamp out of the wax bath to touch the slide, imprinting the design onto the slide. Two stamps are shown in front of the machine. B) Loading samples onto a slide containing 48 arrays. The arrays are spaced by 4.5 mm, which is compatible with the 9 mm spacing of standard multichannel pipettes. C) Samples loaded onto slides containing 12 (top), 48 (middle), and 192 (bottom) arrays (96 samples loaded).

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Glycans in pancreatic cancer One of the major interests of the lab is characterizing and studying the changes in carbohydrate structures (glycans) on particular proteins from pancreatic cancer patients. A novel technique developed in our laboratory enables the measurement of specific glycans on multiple proteins in biological samples (Fig. 2A, B). We use lectinsâ&#x20AC;&#x201D;proteins that bind specific glycan structuresâ&#x20AC;&#x201D;as well as glycan-binding antibodies to probe the levels of particular glycans on the proteins captured on the antibody arrays. Several types of lectins, each with its own carbohydrate binding specificity, can be used to identify the carbohydrate structures associated with each protein. We can analyze many different patient samples or cell culture conditions, looking at associations between glycan levels and disease states or at the effects of certain perturbations on glycan structures. This method is in development for commercial use by GenTel Biosciences (Madison, WI). Mucins are long-chain, heavily glycosylated proteins on epithelial cell surfaces that have roles in cell protection, interaction with the extracellular space, and regulation of extracellular signaling. Screening studies in collaboration with Randall Brand and Diane Simeone have revealed a variety of glycan alterations on mucin molecules from pancreatic cancer patients (a representative example is shown in Fig. 2C). Altered carbohydrates on mucins can affect critical processes in cancer such as cell migration or extracellular signaling to the immune system. We are characterizing the glycan structural variation on mucins secreted from cancer cells and other cells, and we are using cell culture systems to study the origins and effects of those variations. We are pursuing hypotheses about the effects of extracellular stress from an inflammatory tumor environment on mucin carbohydrate structures and the resulting interactions of those structures with inflammatory proteins and host cells. Figure 2A.

Figure 2B.

Figure 2C.

Figure 2. Complementary antibody array formats for protein and glycan detection. A) Sandwich assay with fluorescence detection to measure protein abundance. B) Antibody-lectin assay. The biotinylated lectin binds to glycans on the proteins captured by the immobilized antibodies. The antibodies are first chemically derivatized to prevent lectin binding to the glycans of the immobilized capture antibodies. C) Detecting protein and glycan variation in cancer and control sera. Sandwich detection of the MUC1 and CEA proteins showed similar levels in serum samples from a cancer patient and a control subject (left images). The anti-CA19-9 antibody, which targets a glycan structure, detected a significant glycan increase on MUC1 and CEA in the cancer serum (right images).

VARI | 2007

Cancer biomarkers Improved methods of detecting and diagnosing cancer could significantly improve outcomes for many patients. We are seeking to identify and validate protein biomarkers that could form the basis of clinical cancer diagnostics. The antibody-based assays that we are using are valuable for this work because they are very reproducible, inexpensive, and high-throughput. In addition, the use of miniaturized arrays of antibodies allows us to efficiently test many antibodies and samples and to rapidly develop new assays. We are applying these capabilities in novel approaches to biomarker discovery and validation. Mouse models of cancer may provide a good resource for biomarker discovery because the genetic and experimental variation between samples can be closely controlled, thus making the identification of abnormal protein levels easier than with human clinical specimens. Mass spectrometry studies performed by other members of an NCI-sponsored consortium have identified candidate biomarkers in mouse models of ovarian and pancreatic carcinomas. Using newly generated antibodies that target those proteins, we are developing assays to determine the levels of these candidate biomarkers in the mouse models and to assess their diagnostic value for human cancer. Low-volume methods are crucial for these studies because only a small sample is available from each mouse. These studies could establish a new paradigm for biomarker discovery and validation.

Longitudinal biomarkers

An NCI-sponsored project in our laboratory focuses on the hypothesis that the diagnostic performance of particular biomarkers can be improved by using measurements collected on multiple occasions (longitudinal measurements) rather than at just a single point in time. By looking at changes over time, it may be possible to more accurately distinguish abnormal levels in a given individual, since that personâ&#x20AC;&#x2122;s normal level could be used as a reference point. In a collaboration with Robert Vessella and William Catalona, we are investigating this question for the detection of prostate cancer recurrence. By using various formats of antibody arrays, we can explore different data types and multiple proteins, which we hope will establish the extent of diagnostic improvement using longitudinal information. Another collaborator, Ziding Feng, is developing the statistical methods for analyzing the data, which may have value for other applications of this approach.

Tumor-reactive antibodies

We and others have investigated measurements of tumor-reactive antibodies as biomarkers. Certain tumor proteins elicit an antibody-based immune response in a high percentage of cancer patients. In collaboration with Samir Hanash, Gilbert Omenn, and others, we have further developed the experimental methods for identifying tumor-reactive antibodies using protein arrays. We are applying this method to the detection of prostate cancer and prostate cancer recurrence. The changes in the tumor-reactive antibodies are being assessed using the longitudinal approach described above, which may improve the diagnostic performance of those biomarkers and give insight into the role of immune response in determining the likelihood of cancer recurrence.

Pancreatic cancer biomarkers

Other biomarker studies in our lab are focused on pancreatic cancer in collaboration with Anna Lokshin, Michael Hollingsworth, and others in the Early Detection Research Network (EDRN), which is an NCI-sponsored consortium dedicated to discovering and validating cancer biomarkers. We use the glycan and protein detection technologies described above to identify and study biomarkers for the early detection or more accurate diagnosis of pancreatic cancer. We have shown that, in certain cases, the measurement of a glycan on a protein is more accurate for detecting cancer than the measurement of the protein alone in traditional antibody assays. We are now seeking to define which protein and glycan alterations have the highest diagnostic and prognostic significance.

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External Collaborators Philip Andrews, University of Michigan, Ann Arbor Randall Brand, Evanston Northwestern Healthcare, Evanston, Illinois William Catalona, Northwestern University, Evanston, Illinois Ziding Feng, Fred Hutchinson Cancer Research Center, Seattle, Washington Irwin Goldstein, University of Michigan, Ann Arbor Samir Hanash, Fred Hutchinson Cancer Research Center, Seattle, Washington Michael A. Hollingsworth, University of Nebraska, Omaha Anna Lokshin, University of Pittsburgh, Pennsylvania Gilbert Omenn, University of Michigan, Ann Arbor Alan Partin, Johns Hopkins University, Baltimore, Maryland Diane Simeone, University of Michigan, Ann Arbor Robert Vessella, University of Washington, Seattle

Recent Publications 30

From left: Forrester, Porter, Nelson, Haab, Bergsma, Collins, Lundquist, Chen, Yue, Wu, Turner

Chen, S., and B.B. Haab. In press. Antibody microarrays for protein and glycan detection. In Clinical Proteomics, Wiley-VCH. Chen, S., T. LaRoche, D. Hamelinck, D. Bergsma, D. Brenner, D. Simeone, R.E. Brand, and B.B. Haab. In press. Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays. Nature Methods. Forrester, S., J. Qiu, L. Mangold, A.W. Partin, D. Misek, B. Phinney, D. Whitten, P. Andrews, E. Diamandis, G.S. Omenn, S. Hanash, and B.B. Haab. In press. An experimental strategy for quantitative analysis of the humoral immune response to prostate cancer antigens using natural protein microarrays. Proteomics. Omenn, Gilbert S., Raji Menon, Marcin Adamski, Thomas Blackwell, Brian B. Haab, and Weimin Gao, and David J. States. 2007. The human plasma proteome. In Proteomics of Human Body Fluids: Principles, Methods, and Applications, V. Thongboonkerd, ed. Totowa, N.J.: Humana Press. Shafer, Michael W., Leslie Mangold, Alam W. Partin, and Brian B. Haab. 2007. Antibody array profiling reveals serum TSP-1 as a marker to distinguish benign from malignant prostatic disease. The Prostate 67: 255–267. Haab, B.B. 2006. Applications of antibody array platforms. Current Opinion in Biotechnology 17(4): 415–421. Haab, B.B. 2006. Using array-based competitive and noncompetitive immunoassays. In American Association of Cancer Research Annual Meeting Education Book, Phildelphia: American Association of Cancer Research. Haab, Brian B., Amanda G. Paulovich, N. Leigh Anderson, Adam M. Clark, Gregory J. Downing, Henning Hermjakob, Joshua LaBaer, and Mathias Uhlen. 2006. A reagent resource to identify proteins and peptides of interest for the cancer community: a workshop report. Molecular & Cellular Proteomics 5(10): 1996–2007. Hung, Kenneth E., Alvin T. Kho, David Sarracino, Larissa Georgeon Richard, Bryan Krastins, Sara Forrester, Brian B. Haab, Isaac S. Kohane, and Raju Kucherlapati. 2006. Mass spectrometry–based study of the plasma proteome in a mouse intestinal tumor model. Journal of Proteome Research 5(8): 1866–1878.

VARI | 2007

Rick Hay, Ph.D., M.D., F.A.H.A. Laboratory of Noninvasive Imaging and Radiation Biology

Dr. Hay earned a Ph.D. in pathology (1977) and an M.D. (1978) at the University of Chicago and the Pritzker School of Medicine. He became a resident in anatomic pathology and then a postdoctoral research fellow in the University of Chicago Hospitals and Clinics. Following a postdoctoral fellowship at the Biocenter/University of Basel (Switzerland), he returned to the University of Chicago as an Assistant Professor in the Department of Pathology and Associate Director of the Section of Autopsy Pathology from 1984 to 1992. He moved to the University of Michigan Medical Center in 1992 as a clinical fellow in the Division of Nuclear Medicine and became Chief Fellow in 1993. From 1994 to 1997 he was a staff physician, and from 1995 to 1997 the Medical Director in the Department of Nuclear Medicine at St. John Hospital and Medical Center in Detroit. He joined VARI in 2001 as a Senior Scientific Investigator. In 2002 he was named Assistant to the Director for Clinical Programs, and in 2003 was appointed Deputy Director for Clinical Programs.

Staff Laboratory Staff Troy Giambernardi, Ph.D. Kim Hardy, M.A., RT(R), RDMS Yue Guo, B.S. Joel Strehl, B.S. Catherine Walker, B.S.

Laboratory Staff

Students

Elianna Bootzin Natalie Kent Sara Kunz Jose Toro Rebecca Trierweiler

Visiting Scientist Physician-in-training Visiting Scientists

Students

Nigel Crompton, Ph.D., D.Sc.

Students

Consultants

Matthew Steensma, M.D.

Visiting Scientists

Helayne Sherman, M.D., Ph.D., F.A.C.C. Milton Gross, M.D., F.A.C.N.P.

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Research Interests In July 2005, the Laboratory of Noninvasive Imaging and Radiation Biology originated as an outgrowth and expansion of activities of the Laboratory of Molecular Oncology. This lab is devoted to both noninvasive imaging (i.e., the generation and analysis of images or depictions of structure and selected functions in living organisms without surgically or mechanically penetrating a body cavity) and radiation biology (which involves analysis of the consequences of external and internal radiation exposure in living organisms). The lab’s work follows three common themes:

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Development and use of laboratory models that address medical imaging and radiation exposure problems.

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Advancement of technology in imaging and radiation biology, including novel agents, probes, and reporters; new strategies for tackling research problems; and new instrumentation.

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Pursuit of two-way translation between the laboratory and the clinical setting, i.e., using examples of human disease to design and improve laboratory model systems for study, as well as moving new discoveries from the laboratory benchtop to the patient’s bedside.

We depend heavily upon access to sophisticated instruments and equipment, including nuclear imaging cameras; planar and tomographic X-ray units; clinical and research ultrasonography units; fluorescence detection systems; and cell and organism irradiation capability. Because of the equipment- and expertise-intensive nature of our projects, we could not succeed without the help of our valued collaborators. During this past year we have acquired two new state-of-the-art noninvasive imaging instruments: a Vevo 770 high-resolution micro-ultrasound imaging system (VisualSonics) and a nanoSPECT/CT imaging unit (BioScan), and we have continued to pursue research projects in radiation biology, nuclear medicine, and multimodality imaging. One established research project continues work begun by Nigel Crompton at the Paul Scherrer Institute in Switzerland, now performed in collaboration with the radiation oncology service at Saint Mary’s Health Care. This project seeks to predict the sensitivity of a patient’s normal tissues to irradiation that is being administered for treatment of a serious condition such as cancer. For this project a sample of the patient’s blood is drawn before radiation therapy. The blood sample is then irradiated (outside the patient) under precise conditions of exposure, treated with fluorescent molecules that detect certain types of blood cells (lymphocytes), and then analyzed by fluorescence-activated cell sorting (FACS) for evidence of lymphocyte death. In Switzerland, Dr. Crompton established a close correlation between lymphocyte death and a patient’s normal tissue tolerance to irradiation. We are now determining whether western Michigan patients respond similarly, as well as investigating the effects of patient age, gender, and administered radiation dose on the apoptotic response.

VARI | 2007

A new radiation biology project this year, in collaboration with Weiwen Deng and Aly Mageed of DeVos Children’s Hospital, investigates a new approach for treating graft-versus-host disease in mice undergoing bone marrow transplantation, with planned extension to human patients in the near future. Our major established project in nuclear medicine continues work initiated by Dr. Hay and colleagues while he was a member of the Laboratory of Molecular Oncology. Since 2001 we have been evaluating radioactive antibodies and smaller molecules that attach to the Met receptor tyrosine kinase, collectively designated Met-avid radiopharmaceuticals (MARPs). Met plays a key role in causing cancers to become more aggressive, so that they spread to nearby tissues (invasion) and/or travel through the bloodstream or lymph channels to distant organs (metastasis). We previously showed that both large and small MARPs are useful for nuclear imaging of Met-expressing human tumors (xenografts) grown under the skin of immunodeficient mice. During the past year, in collaboration with our colleagues at VARI and with our outside collaborators at DVAHS, ApoLife, and MSU, we have been evaluating new ways of complexing radioactive atoms with MARPs for improved ease of use and future clinical applications. In 2006 we began a multimodality noninvasive imaging program for evaluating the growth, Met expression, and response to therapy of aggressive human tumor xenografts grown orthotopically in immunodeficient mice. Employing a combination of high-resolution ultrasound with and without contrast agents, planar and tomographic nuclear imaging, and CT imaging, we are now acquiring data for tumors of the brain, pancreas, adrenals, and bone.

External Collaborators Our lab depends critically on intramural and extramural collaborations to address our research themes. Our extramural collaborators include scientists and physicians at the Department of Veterans Affairs Healthcare System in Ann Arbor; the University of Michigan in Ann Arbor; Michigan State University in East Lansing; ApoLife, Inc., in Detroit; Henry Ford Hospital in Detroit; West Michigan Heart, P.C., in Grand Rapids; DeVos Children’s Hospital in Grand Rapids; St. Mary’s Health Care in Grand Rapids; Fred Hutchinson Cancer Research Center in Seattle; the Gerald P. Murphy Foundation in West Lafayette, Indiana; the National Cancer Institute in Bethesda, Maryland; the University of Illinois in Champaign-Urbana; and VisualSonics, Inc., in Toronto.

Recent Publications Meng, L.J., N.H. Clinthorne, S. Skinner, R.V. Hay, and M. Gross. 2006. Design and feasibility study of a single photon emission microscope system for small animal I-125 imaging. IEEE Transactions on Nuclear Science 53(3): 1168–1178. Hay, R.V., and M.D. Gross. 2006. Scintigraphic imaging of the adrenals and neuroectodermal tumors. In Nuclear Medicine, 2nd edition, R.E. Henkin, D. Bova, G.L. Dillehay, S.M. Keresh, J.R. Halama, R.H. Wagner, and A.M. Zimmer, eds. Philadelphia: Mosby Elsevier, pp. 820–844.

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Office of Translational Programs Since July 2005 the Office of Translational Programs (OTP) has been the administrative home base for activities overseen by the Deputy Director for Clinical Programs. The role of OTP is to promote and facilitate collaborative programs involving the Van Andel Research Institute and other institutions in the realm of translational medicine. OTP accomplishments during our second year of formal operation include the following.

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Serving as the administrative home for the GMP facility. With funding from the state of Michigan and the federal Health Resources and Services Administration, VARI and Grand Valley State University have partnered to build and operate Grand River Aseptic Pharmaceutical Packaging (GR-APP), a Good Manufacturing Practices facility that will package pharmaceuticals for early-phase clinical trials commissioned by academic and commercial investigators, primarily in Michigan and the Midwest. As of this writing, construction of GR-APP is nearing completion, and we expect operations to begin by autumn of 2007.

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Serving as the administrative home for the West Michigan Chapter of the Michigan Cancer Consortium (MCC). As an active member of the MCC, VARI is committed to participating in statewide programs to reduce the burden of cancer in Michigan. In 2005, we and other regional MCC members launched an initiative to develop community-based programs more relevant to western Michigan. Our first project, designated “C-Works!”, will provide cancer screening and follow-up services to uninsured working women in Kent County.

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Organizing and hosting meetings. In October 2006, OTP hosted the Great Lakes Regional Meeting of the American Cancer Society at VARI (Troy Giambernardi, Conference Chair) and assisted with preparations for the fall meeting of the Central Chapter-Society of Nuclear Medicine in Traverse City (Rick Hay, Conference Co-Chair). In November 2006, OTP assisted with local arrangements for the annual meeting of the Michigan Cancer Consortium at DeVos Hall.

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Promoting new interinstitutional collaborations and providing resources for funding proposals. OTP provides a broad range of administrative assistance, logistical support, grant preparation expertise, meeting venues, and seed funding for new interinstitutional collaborations seeking extramural funding from state, federal, or private sources. During this past year we helped secure state funding for ClinXus, a west Michigan–based consortium for conducting innovative clinical trials, and we are awaiting the outcomes of recent collaborative proposals submitted to NIH and to two private foundations.

•

Coordinating research rotations for physicians-in-training. In collaboration with the Grand Rapids Medical Education and Research Consortium (MERC), we schedule each first-year general surgery resident to spend one month working in a designated research laboratory at VARI. This program has been well received by both residents and VARI investigators. Custom-tailored rotations of variable duration at VARI can be arranged for other physicians-in-training.

Staff Rick Hay, Ph.D., M.D., F.A.H.A. Troy Carrigan Jean Chastain

VARI | 2007

Jeffrey P. MacKeigan, Ph.D. Laboratory of Systems Biology

Dr. MacKeigan received his Ph.D. in microbiology and immunology at the University of North Carolina Lineberger Comprehensive Cancer Center in 2002. He then served as a postdoctoral fellow in the laboratory of John Blenis in the Department of Cell Biology at Harvard Medical School. In 2004, he joined Novartis Institutes for Biomedical Research in Cambridge, Massachusetts, as an investigator and project leader in the Molecular and Developmental Pathways expertise platform. Dr. MacKeigan joined VARI in June 2006 as a Scientific Investigator.

Staff

Laboratory Staff Brendan Looyenga, Ph.D. Christina Ludema, B.S. Natalie Wolters, B.S.

Students

Katie Sian, B.S. Geoff Kraker

Visiting Scientists

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Research Interests The primary focus of the Systems Biology laboratory is identifying and understanding the genes and signaling pathways that when mutated contribute to the pathophysiology of cancer. We take advantage of RNA interference (RNAi) and novel proteomic approaches to identify the enzymes that control cell growth, cell proliferation, and cell survival. For example, after screening the human genome for more than 600 kinases and 200 phosphatases—called the “kinome” and “phosphatome”, respectively—that act with chemotherapeutic agents in controlling apoptosis, we identified 73 kinases and 72 phosphatases whose roles in cell survival were previously unrecognized. We are asking several questions. How are these novel survival enzymes regulated at the molecular level? What signaling pathway(s) do they regulate? Does changing the number of enzyme molecules present inhibit waves of compensatory changes at the cellular level (system-level changes)? What are the system-level changes after reduction or loss of each gene?

Identification of kinases that regulate cell survival

We have performed RNAi screens in the presence of apoptosis-inducing chemotherapeutic agents (Taxol, cisplatin, and etoposide) and identified a group of kinases whose loss of function sensitizes cells to undergo cell death, the most interesting of these being PINK1 (PTEN-induced kinase 1). PINK1 was originally shown to be up-regulated by the tumor suppressor PTEN. Although PINK1 does not fall into a particular kinase subfamily, it has a known role in maintaining mitochondrial membrane potential. Other work has recently shown inherited mutations at chromosomal location 1p36 in familial Parkinson disease, and the two mutated genes that map to this region are PINK1 (PARK6) and DJ-1 (PARK7). Both genes are responsible for early-onset autosomal recessive parkinsonism. We had previously noted that DJ-1 is overexpressed in non–small cell lung carcinoma and that its down-regulation enhances apoptosis. Further, Parkinson disease–causing mutations in LRRK2 (PARK8), which are dominantly inherited gain-of-function mutations, sensitize neurons to cell death, and a significant fraction of the LRRK2 population is associated with the mitochondria. We are currently investigating whether the molecular mechanisms of PINK1 and LRRK2 in cancer and in Parkinson disease are linked.

VARI | 2007

Identification of phosphatases that regulate chemoresistance Our research has shown that a large percentage of phosphatases and their regulatory subunits contribute to cell survival. This is a previously unrecognized general role for phosphatases as negative regulators of apoptosis, and it is important because phosphatases may no longer be simply viewed as enzymes that oppose the action of kinases. This research also identified a number of phosphatases whose loss of function results in chemoresistance, implicating these proteins as potential tumor suppressors. In our RNAi study, 5% of all phosphatases were shown to act in this way; an example is MK-STYX. Down-regulation of MK-STYX resulted in dramatic cellular resistance to cisplatin-, Taxol- or etoposide-induced cell death, which is consistent with up-regulated survival signals in these cells (Fig. 1). Also, MK-STYX is located at 7q11.23, a chromosome region mutated in colon cancer. MK-STYX is similar to MKP-1, which inactivates MAPKs; MK-STYX, however, is predicted to be a catalytically inactive phosphatase. Our observations suggest that MK-STYX acts against cell survival by sequestering pro-survival signaling components in a way analogous to the “substrate-trapping” effects of catalytically inactive phosphatases.

Figure 1A.

Figure 1B.

Figure 1. Identification of MK-STYX as a potential tumor suppressor phosphatase. Cells were transfected with control siRNA or MK-STYX siRNA for 48 h and then were treated for an additional 24 h with solvent control (–) or 50 μM cisplatin (+). Cell viability was visualized by A) crystal violet stain and B) cleavage of full-length PARP measured by western blot analysis.

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Graded MAPK signaling and switch-like c-Fos induction We also take a systems biology approach to understanding two key molecular pathways, Ras/MAPK and PI3K/mTOR. In the Ras/MAPK pathway, growth factors activate the small G protein Ras, which recruits Raf to the plasma membrane where it is activated and phosphorylates MEK1/2, which in turn phosphorylates ERK1/2-MAPKs. Activated ERK1/2 phosphorylates additional kinases (such as RSK) and specific transcription factors (such as c-Fos and Elk-1) that are important in cellular proliferation, differentiation, and survival. One project in the lab involved the question of whether the evolutionarily conserved MAPK pathway exhibits a switch-like or a graded response in mammalian cells. Ultrasensitive switch-like responses control cell-fate decisions in many biological settings, and the regulation of kinase activity is one way in which such behavior can be initiated. Signaling molecules switch between two discontinuous, stable states with no intermediate; this is referred to as a bistable response (Fig. 2, top panel). Given the irreversible, all-or-none nature of many cell behaviors, including cell cycle control and apoptosis, significant effort has been focused on identifying the cellular mechanisms underlying bistability. Our research and that of others has provided solid evidence for graded MAPK signaling in mammalian cells (Fig. 2, lower panel); that is, as agonist concentration increases, single-cell kinase activity increases proportionally. Yet we have also found that the proliferative response to growth factor stimulation is switch-like, demonstrating that the ultrasensitive step in the MAPK pathway occurs at the level of MAPK nuclear concentration and switch-like c-Fos induction. Although c-Fos induction and cell cycle entry in mammalian cells is switch-like, graded MAPK activation could have an important role in cell survival, since many MAPK targets regulating cell survival are in the cytoplasm.

Figure 2.

Figure 2. Total cell population MAPK measurements. Single cells exhibiting a bistable (all-or-none) response or graded response (linear).

From left: Wolters, Ludema, Kraker, Looyenga, MacKeigan, Nelson, Sian

VARI | 2007

Cindy K. Miranti, Ph.D. Laboratory of Integrin Signaling and Tumorigenesis

Dr. Miranti received her M.S. in microbiology from Colorado State University in 1982 and her Ph.D. in biochemistry from Harvard Medical School in 1995. She was a postdoctoral fellow in the laboratory of Joan Brugge at ARIAD Pharmaceuticals, Cambridge, Massachusetts, from 1995 to 1997 and in the Department of Cell Biology at Harvard Medical School from 1997 to 2000. Dr. Miranti joined VARI as a Scientific Investigator in January 2000. She is also an Adjunct Assistant Professor in the Department of Physiology at Michigan State University.

Staff

Students

Mathew Edick, Ph.D. Suganthi Sridhar, Ph.D. Kristin Saari, M.S. Lia Tesfay, M.S. Laura Lamb, B.S. Veronique Schulz, B.S. Susan Spotts, B.S.

Eric Graf Gary Rajah

Laboratory Staff

Students

Visiting Scientists

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Research Interests Our laboratory is interested in the mechanisms by which integrin receptors, interacting with the extracellular matrix (ECM), regulate cell processes involved in the development and progression of cancer. Using tissue culture models, biochemistry, molecular genetics, and mouse models, we are defining the cellular and molecular events involved in integrin-dependent adhesion and downstream signaling that are important for prostate tumorigenesis and metastasis. Integrins are transmembrane proteins that serve as receptors for ECM proteins. By interacting with the ECM, integrins stimulate intracellular signaling transduction pathways to regulate cell shape, proliferation, migration, survival, gene expression, and differentiation. Integrins do not act autonomously, but “crosstalk” with receptor tyrosine kinases (RTKs) to regulate many of these cellular processes. Studies in our lab indicate that integrin-mediated adhesion to ECM proteins activates epidermal growth factor receptors EGFR/ErbB2 and the HGF/SF receptor c-Met. Integrin-mediated activation of these RTKs is ligand-independent and required for the activation of a subset of intracellular signaling molecules in response to cell adhesion.

The prostate gland and cancer

Tumors that develop from cells of epithelial origin, i.e., carcinomas, represent the largest tumor burden in the United States. Prostate cancer is the most frequently diagnosed cancer in American men and the second leading cause of cancer death in men. Patients who at the time of diagnosis have androgen-dependent and organ-confined prostate cancer are relatively easy to cure through radical prostatectomy or localized radiotherapy, but patients with aggressive and metastatic disease have fewer options. Androgen ablation can significantly reduce the tumor burden in the latter patients, but the potential for relapse and the development of androgen-independent cancer is high. Currently there are no effective treatments for patients who reach this stage of disease. In the human prostate gland, α3β1 and α6β4 integrins on epithelial cells bind to the ECM protein laminin 5 in the basement membrane. In tumor cells, however, the α3 and β4 integrin subunits disappear—as does laminin 5—and the tumor cells express primarily α6β1 and adhere to a basement membrane containing laminin 10. There is also an increase in expression of the RTKs EGFR and c-Met in the tumor cells. Two fundamental questions are whether the changes in integrin and matrix interactions that occur in tumor cells are required for or help to drive the survival of tumor cells, and whether crosstalk with RTKs is important.

VARI | 2007

Integrins and RTKs in prostate epithelial cell survival How integrin engagement of various ECMs regulates survival pathways in normal and tumor cells is poorly understood. We recently initiated studies to determine how adhesion to matrix regulates cell survival in normal epithelial cells. We have shown that integrin-induced activation of EGFR in normal primary prostate epithelial cells is required for survival on their endogenous matrix, laminin 5. The ability of EGFR to support integrin-mediated cell survival on laminin 5 is mediated through Îą3Î˛1 integrin and requires signaling downstream to Erk. Surprisingly, we found that the death induced by inhibition of EGFR in normal primary prostate cells is not mediated through or dependent on classical caspase-mediated apoptosis. The presence of an autophagic survival pathway (Fig. 1), regulated by adhesion to matrix, prevents the induction of caspases when EGFR is inhibited. Suppression of autophagy is sufficient to induce caspase activation and apoptosis in laminin 5â&#x20AC;&#x201C;adherent primary prostate epithelial cells. Thus, adhesion of normal cells to matrix regulates survival through at least two mechanisms, crosstalk with EGFR and Erk and the maintenance of an autophagic survival pathway (Fig. 2).

Figure 1.

Figure 2.

Normal

Autophagic

Figure 1. Induction of autophagy in primary prostate epithelial cells as shown by punctate staining of the autophagic LC3 protein using fluorescence microscopy.

Figure 2. Laminin-mediated survival pathways in primary prostate epithelial cells.

Interestingly, both of these pathways are absent in at least one metastatic prostate cancer cell line, PC3. Accordingly, integrin-mediated survival of PC3 cells does not depend on EGFR or Erk, but is instead dependent on PI-3K. The PI-3K pathway is inhibitory to autophagy. We are currently testing additional prostate tumor cells lines to determine if this switch in matrix-mediated survival pathways is found in all prostate cancers. Our next step is to determine how integrins regulate survival through autophagy. Since loss of autophagy results in activation of caspases and classical apoptosis, we have been searching for signaling pathways whose inhibition also results in caspase activation. We have tentatively identified two important molecules, the RTK c-Met and the anti-apoptotic protein Bcl-XL. Inhibition of either molecule leads to caspase-induced cell death, indicating that they may be involved in regulating integrin-mediated autophagy. Future studies in our lab will be aimed at deciphering this pathway.

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The androgen receptor in integrin-mediated survival All primary and metastatic prostate cancers express the intracellular steroid receptor for androgen, AR. In the normal gland, the AR-expressing cells do not interact with the ECM in the basement membrane; however, all AR-expressing tumor cells do adhere to the ECM in the basement membrane. In normal cells, AR expression suppresses growth and promotes differentiation, but in tumor cells AR expression promotes cell growth and is required for cell survival. The mechanisms that lead to the switch from growth inhibition and differentiation to growth promotion and survival are unknown. Our hypothesis is that adhesion to the ECM by the tumor cells is responsible for driving the switch in AR function. When prostate tumor cells are placed in culture, they lose expression of AR. The reason for this is not clear, but it may have to do with loss of the appropriate ECM-containing basement membrane. When we introduce AR into prostate tumor cells, it actually suppresses their growth and induces cell death. However, if we place the AR-expressing tumor cells on laminin (the ECM found in tumors), these cells no longer die. The mechanisms responsible for this change in survival are unknown. Preliminary studies indicate that there are changes in integrin expression upon expression of AR that may enhance specific signaling pathways when those integrins bind to matrix. We are currently determining which cell survival pathways are activated by AR upon integrin engagement.

CD82 and integrin signaling in prostate cancer metastasis

Death from prostate cancer is due to the development of metastatic disease, which is difficult to control and occurs by mechanisms that are not understood. One approach we are taking is to characterize genes that are specifically associated with metastatic prostate cancer. CD82/KAI1 is a metastasis suppressor gene whose expression is specifically lost in metastatic cancer but not in primary tumors. Interestingly, CD82/KAI1 is known to associate with both integrins and RTKs. Our goal has been to determine how loss of CD82/KAI1 expression promotes metastasis. We have found that reexpression of CD82/KAI1 in metastatic tumor cells suppresses laminin-specific migration and invasion via suppression of both integrin- and ligand-induced activation of the RTK c-Met. Interestingly, c-Met is often overexpressed in metastatic prostate cancer. Thus, CD82/KAI1 normally acts to regulate signaling through c-Met such that upon CD82 loss in tumor cells, signaling through c-Met is increased, leading to increased invasion. We are currently determining the mechanism by which CD82/KAI1 down-regulates c-Met signaling. Our studies indicate that c-Met and CD82 do not directly interact, and CD82 may act to suppress c-Met signaling indirectly by dispersing c-Met aggregates present on metastatic tumor cells. We have developed mutants of CD82 in order to determine which part of the CD82 molecule is required for suppression of c-Met activity. Also, we have determined that reexpression of CD82 in tumor cells induces a physical association between CD82 and a related family member, CD9. We are determining whether this association is important for suppressing c-Met activity. We have also initiated mouse studies to demonstrate the importance of CD82 in regulating metastasis in vivo. Using orthotopic injection of wild-type or CD82-expressing metastatic prostate tumor cells directly into the prostate, we found that CD82 also suppresses metastasis in vivo. We are continuing these studies to determine if CD82â&#x20AC;&#x2122;s ability to specifically affect c-Met is responsible for metastasis suppression. In addition, we are generating mice in which CD82 expression is specifically lost in the epithelial cells of the prostate gland. This approach will allow us to determine if CD82 is important for the normal biology of prostate epithelial cells in vivo. Furthermore, we will be able to determine if loss of CD82 in the mouse prostate gland will lead to an increased ability to produce metastatic prostate cancer. .

VARI | 2007

External Collaborators Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Senthil Muthuswamy, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Ilan Tsarfaty, Tel Aviv University, Israel Valera Vasioukin, Fred Hutchinson Cancer Research Center, Seattle, Washington Xin Zhang, University of Tennessee, Memphis

From left: Lamb, Saari, Spotts, Rajah, Tesfay, Graf, Schulz, Miranti

Recent Publications Edick, M.J., Tesfay, L., Lamb, L.E., Knudsen, B.S., and Miranti, C.K. In press. Inhibition of integrin-mediated crosstalk with EGFR/Erk or Src signaling pathways in autophagic prostate epithelial cells induces caspase-independent death. Molecular Biology of the Cell. Sridhar, S.C., and C.K. Miranti. In press. Tumor metastasis suppressor KAI1/CD82 is a tetraspanin. In Contemporary Cancer Research: Metastasis, C. Rinker-Schaeffer, M. Sokoloff, and D. Yamada, eds. Wang, X. , J. Zhu, P. Zhao, Y. Jiao, N. Xu, T. Grabinski, C. Liu, C.K. Miranti, T. Fu, and B. Cao. In press. In vitro efficacy of immunochemotherapy with anti-EGFR human Fab-Taxol conjugate on A431 epidermoid carcinoma cells. Cancer Biology & Therapy. Knudsen, Beatrice S., and Cindy K. Miranti. 2006. Impact of cell adhesion changes on proliferation and survival during prostate cancer development and progression. Journal of Cellular Biochemistry 99(2): 345â&#x20AC;&#x201C;361.

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Image courtesy of Qian Xie, Yue Guo, Rick Hay, and George Vande Woude, Van Andel Research Institute; Helayne Sherman, West Michigan Heart, P.C.; and Ai Lockard, VisualSonics, Inc.

VARI | 2007

Evaluating the blood supply of cancer with ultrasound.

Human glioblastoma (brain cancer) cells were grown as a tumor beneath the skin of a laboratory mouse. Small bubbles about the size of individual blood cells were then injected into the mouseâ&#x20AC;&#x2122;s bloodstream. During the next few minutes the tumor was imaged by ultrasound, using a device similar to sonar. Echoes from the bubbles, shown in green, depict complex branching patterns of tiny blood vessels growing around and within the tumor to supply it with nutrients and oxygen. We are using the ultrasound technique to monitor how new types of anticancer medicine change the abundance and branching of tumor blood vessels in mice, with the hope of applying this technology in the near future to patients with cancer.

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James H. Resau, Ph.D. Division of Quantitative Sciences Laboratory of Analytical, Cellular, and Molecular Microscopy Laboratory of Microarray Technology Laboratory of Molecular Epidemiology

Dr. Resau received his Ph.D. from the University of Maryland School of Medicine in 1985. He has been involved in clinical and basic science imaging and pathology-related research since 1972. Between 1968 and 1994, he was in the U.S. Army (active duty and reserve assignments) and served in Vietnam. From 1985 until 1992, Dr. Resau was a tenured faculty member at the University of Maryland School of Medicine, Department of Pathology. Dr. Resau was the Director of the Analytical, Cellular and Molecular Microscopy Laboratory in the Advanced BioScience Laboratoriesâ&#x20AC;&#x201C;Basic Research Program at the National Cancer Institute, Frederick Cancer Research and Development Center, Maryland, from 1992 to 1999. He joined VARI as a Special Program Senior Scientific Investigator in June 1999 and in 2003 was promoted to Deputy Director. In 2004, Dr. Resau assumed as well the direction of the Laboratory of Microarray Technology to consolidate the imaging and quantification of clinical samples in a CLIA-type research laboratory program. In 2005, Dr. Resau was made the Division Director of the quantitative laboratories (pathology-histology, microarray, proteomics, epidemiology, and bioinformatics), and in 2006 he was promoted to Distinguished Scientific Investigator.

Staff Laboratory Staff

Students

Visiting Scientist

Eric Kort, M.D. Brendan Looyenga, Ph.D. Bree Berghuis, B.S., HTL (ASCP), QIHC Eric Hudson, B.S. Paul Norton, B.S. Ken Olinger, B.S. David Satterthwaite, B.S. Kristin VandenBeldt, B.S. JC Goolsby

Pete Haak, B.S. Alicia Coleman Kate Jackson Wei Luo, B.A. Nick Miltgen Kara Myslivec Sara Ramirez Jourdan Stuart Mohan Thapa, M.S. Huong Tran Grant Van Eerden

Yair Andegeko

VARI | 2007

Research Interests The Division of Quantitative Sciences includes the laboratories of Analytical, Cellular, and Molecular Microscopy (ACMM), the Laboratory of Microarray Technology (LMT), the Laboratory of Computational Biology, the Laboratory of Molecular Epidemiology, and the Laboratory of Mass Spectrometry and Proteomics. The Divisionâ&#x20AC;&#x2122;s laboratories use objective measures to define pathophysiologic events and processes. For example, the LMT measures the expression of genes relative to a control or a standard. When pathology and tissue organization is combined with expression, one can better determine not only what the change is but also possible causation, treatment targets, and effects of treatment. The Molecular Epidemiology laboratory builds objective data and pathology correlations to infer causation and prognosis. The ACMM laboratory has programs in pathology, histology, and imaging to describe and visualize changes in cell, tissue, or organ structure. Our imaging instruments allow us to visualize cells and their components with striking clarity, allowing researchers to determine where in a cell specific molecules are located. We also use a laser for microdissection of cells from a sample. The laboratory provides paraffin-block (SPIN program) and frozen-section (TAS program) staining of tissues. An archive of pathology tissues in the paraffin blocks (Van Andel Tissue Repository; VATR) is being accumulated with the cooperation of local hospitals, and the data on the samples is being converted to computerized files. The lab also carries out research that will improve our ability to quantify images, so that we will be able to not only state that a particular protein is present in an image, but also answer the questions of how much is there and with what other molecules is it co-localized? We are able to image using either fluorescent (e.g., FITC, GFP) or chromatic agents (e.g., DAB, H&E) and separate the components using our confocal, Nuance, or Maestro instruments. The Laboratory of Microarray Technology provides gene expression analysis using cDNA microarrays. High-throughput robotics are used to maintain and process cDNA clone sets for the human, mouse, rat, and canine genomes. The clones are used to produce both cDNA and spotted oligonucleotide microarrays that are evaluated using strict quality control and quality assurance criteria developed using the Clinical Laboratory Improvement Amendments (CLIA) as a model. These criteria allow the laboratory to function in a manner consistent with fully accredited clinical laboratories. In 2006 we produced and used 790 cDNA microarrays, and we also produced 112 custom protein microarrays. In addition, the laboratory has expanded its services to include Agilent and Operon commercial oligonucleotide microarrays. The use of these products will remove much of the internal quality control and quality assurance burden, and they will also facilitate the requirement to perform array comparative genomic hybridization, chromatin immunoprecipitation (chip-on-chip), and splice variant analysis.

Hauenstein Parkinsonâ&#x20AC;&#x2122;s Center Throughout 2006 we have continued our collaboration with the Hauenstein Parkinsonâ&#x20AC;&#x2122;s Center to collect patient blood samples and controls from 114 individuals. Mutations in the parkin gene in a series of families with more than one generation affected by Parkinson disease are being investigated by DNA sequence analysis and will be correlated to gene expression data obtained from microarray analysis.

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Blood spot arrays State laws in the U.S.A. mandate that blood be drawn from all newborn infants to screen for a variety of health-threatening conditions. The assays consume only a small portion of the blood samples, which are collected on filter paper (“Guthrie”) cards. Many states archive the leftover cards, often in unrefrigerated storage. Pete Haak and Eric Kort have successfully isolated mRNA from archived unfrozen neonatal blood spots obtained as long as nine years ago. Using both quantitative RT-PCR and multiplex gene expression analysis with cDNA arrays, we can detect RNA from hundreds to thousands of genes in these samples. Furthermore, we have shown through use of freshly spotted blood cards that the genes detected approximate those found in whole blood and purified buffy coat. These preliminary experiments demonstrate the feasibility of detecting and identifying RNA amplified from unfrozen stored neonatal blood spots. The application of high-throughput assays to the analysis of these widely available samples may be a valuable resource for the study of perinatal markers and determinants of subsequent disease development. The coming year will see this technology applied to the study of cerebral palsy and neuroblastoma.

Mouse models of Parkinson disease

As part of the VAI initiative into Parkinson disease, we have begun to generate novel rodent models of dopaminergic cell loss in the brain in collaboration with Bart Williams. One of the key tools for these studies is the transgenic dopamine-transporter/cre (DAT-cre) mouse line, which specifically expresses the cre recombinase in dopaminergic neurons of the brain. In combination with other transgenic and knock-out mouse lines, the DAT-cre mice will allow us to address the response of such neurons to toxic stimuli in the context of specific gene deletions and additions. Several of the ongoing and future projects based on the DAT-cre mouse model are briefly described below. •

Imaging and isolation of primary dopaminergic neurons from mouse brain. We have performed a genetic cross between the DAT-cre strain and ROSA26 reporter strain to generate mice that specifically express the LacZ reporter gene in dopaminergic neurons. The DAT-cre/ROSA26 mice will permit us to visualize and quantify live dopaminergic neurons in vivo. With these mice we will assess the effect of cytotoxic agents (e.g., MMTP, rotenone, or 6-hydroxydopamine) on the number of dopaminergic cells, and more importantly, assess the ability of mice to recover from these insults. These studies will provide insight into the regenerative capacity of the brain when dopaminergic neurons are lost or injured. The DAT-cre/ROSA26 mice will also provide a source of highly pure dopaminergic neurons for in vitro studies. Dopaminergic neurons from these mice will be isolated from brain tissue treated with DDAOgalactoside and will be identified from the cellular population by fluorescence-activated cell sorting in the VAI flow cytometry core facility.

•

Dopaminergic cell regeneration as a function of age. The relationship between age and the likelihood of developing Parkinson disease is well established, though the causal nature of this relationship is unclear. One hypothesis is that the capacity of the brain to regenerate damaged neurons decreases with age, consistent with a gradual loss of brain stem cells that give rise to new dopaminergic neurons. To test this hypothesis in a mammalian system, we are planning a genetic cross between DAT-cre and pu TK mice, the latter specifically expressing herpes simplex virus thymidine kinase (hsvTK) in cells that contain cre recombinase. Cells expressing hsvTK are sensitive to the antiviral compound ganciglovir (G418) and undergo programmed cell death after systemic treatment. Using the DAT-cre/pu TK model, we will eliminate dopaminergic neurons at various ages (3, 6, 9, and 12 months) and assess the regenerative potential of these mice using behavioral and histological parameters. These studies will indicate both the absolute and relative capacities of the mammalian brain to regenerate dopaminergic neurons as a function of age, thereby providing information about the value of therapies intended to stimulate the endogenous regenerative capacity of the brain in Parkinson disease patients.

VARI | 2007

â&#x20AC;˘

Effect of hypoxia-inducible factor signaling on dopaminergic cell survival. Dopaminergic neurons are exquisitely sensitive to oxidative stress, which is defined by an increase in toxic reactive oxygen species. Reactive oxygen species lead to cell death by direct mechanisms, such as damage to important cellular biomolecules, and indirect ones, such as the induction of cell death pathways. The latter effect may be offset by cell survival pathways, which increase thethreshold signal intensity required to induce cell death. Because both chemically induced and idiopathic Parkinson disease are characterized by increased oxidative stress in dopaminergic neurons, therapies that increase cell survival pathways in these neurons may be broadly applicable as a treatment to decrease cell death in patients.

The PI-3-kinase (PI3K)/Akt pathway is a highly conserved cell survival pathway operating in virtually all mammalian cell types. This pathway is tightly regulated by the phosphatase PTEN, which directly opposes the kinase activity of PI3K. We have crossed DAT-cre mice to mice with a conditionally inactivated allele for PTEN (PTENflox/flox). Expression of the cre recombinase in these mice leads to a genetic deletion of PTEN, thereby increasing Akt activity. DAT-cre/PTENflox/flox mice and their wild-type littermates will be treated with the neurotoxin MPTP, which induces high levels of oxidative stress in dopaminergic neurons. We will compare the mice using behavioral and histological parameters to determine whether increased Akt activity leads to greater cell survival after an oxidative stress insult.

Educational highlights This year we had one student from GRAPCEP, two students from the MSU-CVM program, and a guest student from Bath University in the United Kingdom. Our GRAPCEP mentorship program continues to be funded by Pfizer for a seventh year. Dr. Resau is a member of the graduate school committee that established the VAEI Graduate School, which will increase our research and educational opportunities.

From left, back row: Goolsby, Satterthwaite, Norton, Resau, Haak, Hudson; front row: Kort, Luo, VandenBeldt, Berghuis, Jason, Ramirez, Looyenga

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Scientific Report

Recent Publications Baldus, S.E., E.J. Kort, P. Schirmacher, H.P. Dienes, and J.H. Resau. In press. Quantification of MET and hepatocyte growth factor/scatter factor expression in colorectal adenomas, carcinomas, and non-neoplastic epithelia by quantitative laser scanning microscopy. International Journal of Oncology. Kort, E.J., M.R. Moore, E.A. Hudson, B. Leeser, G.M. Yeruhalmi, R. Leibowitz-Amit, G. Tsarfaty, I. Tsarfaty, S. Moshkovitz, and J.H. Resau. In press. Use of organ explant and cell culture. In Mechanisms of Carcinogenesis, Hans Kaiser, ed. Dordrecht, The Netherlands: Kluwer Academic. Lindemann, K.K., J. Resau, J. Nährig, E. Kort, B. Leeser, K. Anneke, A. Welk, J. Schäfer, G.F. Vande Woude, E. Lengyel, and N. Harbeck. In press. Differential expression of c-Met, its ligand HGF/SF, and HER2/neu in DCIS and adjacent normal breast tissue. Histopathology. Whitwam, T., M.W. VanBrocklin, M.E. Russo, P.T. Haak, D. Bilgili, J.H. Resau, H.-M. Koo, and S.L. Holmen. In press. Differential oncogenic potential of activated RAS isoforms in melanocytes. Oncogene. Young, J.J., J.L. Bromberg-White, C.R. Zylstra, J. Church, E. Boguslawski, J. Resau, B.O. Williams, and N. Duesbery. In press. LRP5 and LRP6 are not required for protective antigen–mediated internalization or lethality of anthrax lethal toxin. PLoS Pathogen.

Bruxvoort, Katia J., Holli M. Charbonneau, Troy A. Giambernardi, James C. Goolsby, Chao-Nan Qian, Cassandra R. Zylstra, Daniel R. Robinson, Pradip Roy-Burman, Aubie K. Shaw, Bree D. Buckner-Berghuis, Robert E. Sigler, James H. Resau, Ruth Sullivan, Wade Bushman, and Bart O. Williams. 2007. Inactivation of Apc in the mouse prostate causes prostate carcinoma. Cancer Research 67(6): 2490–2496. Qian, Chao-Nan, James H. Resau, and Bin Tean Teh. 2007. Prospects for vasculature reorganization in sentinel lymph nodes. Cell Cycle 6(5): 514–517. Wallar, Bradley J., Aaron D. DeWard, James H. Resau, and Arthur S. Alberts. 2007. RhoB and the mammalian Diaphanousrelated formin mDia2 in endosome trafficking. Experimental Cell Research 313(3): 560–571. Zhang, Y.-W., B. Staal, Y. Su, P. Swiatek, P. Zhao, B. Cao, J. Resau, R. Sigler, R. Bronson, and G.F. Vande Woude. 2007. Evidence that MIG-6 is a tumor-suppressor gene. Oncogene 26(2): 269–276. Moshitch-Moshkovitz, Sharon, Galia Tsarfaty, Dafna W. Kaufman, Gideon Y. Stein, Keren Shichrur, Eddy Solomon, Robert H. Sigler, James H. Resau, George F. Vande Woude, and Ilan Tsarfaty. 2006. In vivo direct molecular imaging of early tumorigenesis and malignant progression induced by transgenic expression of GFP-Met. Neoplasia 8(5): 353–363. Qian, Chao-Nan, Bree Berghuis, Galia Tsarfaty, MaryBeth Bruch, Eric J. Kort, Jon Ditlev, Ilan Tsarfaty, Eric Hudson, David G. Jackson, David Petillo, Jindong Chen, James H. Resau, and Bin Tean Teh. 2006. Preparing the “soil”: the primary tumor induces vasculature reorganization in the sentinel lymph node before the arrival of metastatic cancer cells. Cancer Research 66(21): 10365–10376. Tsarfaty, Galia, Gideon Y. Stein, Sharon Moshitch-Moshkovitz, Dafna W. Kaufman, Brian Cao, James H. Resau, George F. Vande Woude, and Ilan Tsarfaty. 2006. HGF/SF increases tumor blood volume: a novel tool for the in vivo functional molecular imaging of Met. Neoplasia 8(5): 344–352. Yao, Xin, Chao-Nan Qian, Zhong-Fa Zhang, Min-Han Tan, Eric J. Kort, James H. Resau, and Bin Tean Teh. 2006. Two distinct types of blood vessels in clear cell renal cell carcinoma have contrasting prognostic implications. Clinical Cancer Research 13(1): 161–169. .

VARI | 2007

Pamela J. Swiatek, Ph.D., M.B.A. Laboratory of Germline Modification and Cytogenetics

Dr. Swiatek received her M.S. (1984) and Ph.D. (1988) degrees in pathology from Indiana University. From 1988 to 1990, she was a postdoctoral fellow at the Tampa Bay Research Institute. From 1990 to 1994, she was a postdoctoral fellow at the Roche Institute of Molecular Biology in the laboratory of Tom Gridley. From 1994 to 2000, Dr. Swiatek was a research scientist and Director of the Transgenic Core Facility at the Wadsworth Center in Albany, N.Y., and an Assistant Professor in the Department of Biomedical Sciences at the State University of New York at Albany. She joined VARI as a Special Program Investigator in August 2000. She has been the chair of the Institutional Animal Care and Use Committee since 2002 and is an Adjunct Assistant Professor in the College of Veterinary Medicine at Michigan State University. Dr. Swiatek received her M.B.A. in 2005 from Krannert School of Management at Purdue University. She was promoted to Senior Scientific Investigator in 2006.

Staff Laboratory Staff

Students

Sok Kean Khoo, Ph.D., Associate Laboratory Director Laura Ayotte, B.S. Julie Koeman, B.S., CLSp(CG) Kellie Sisson, B.S. Kaye Johnson, B.A. Diana Lewis

Visiting Scientists

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Research Interests The Germline Modification and Cytogenetics lab is a full-service lab that functions at the levels of service, research, and teaching to develop, analyze, and maintain mouse models of human disease. Our lab applies a business philosophy to core service offerings for both the VARI community and external entities. Our mission is to support mouse model and cytogenetics research with scientific innovation, customer satisfaction, and service excellence.

Gene targeting Mouse models are produced using gene-targeting technology, a well-established, powerful method for inserting specific genetic changes into the mouse genome. The resulting mice can be used to study the effects of these changes in the complex biological environment of a living organism. The genetic changes can include the introduction of a gene into a specific site in the genome (gene “knock-in”) or the inactivation of a gene already in the genome (gene “knock-out”). Since these mutations are introduced into the reproductive cells known as the germline, they can be used to study the developmental aspects of gene function associated with inherited genetic diseases.

In addition to traditional gene-targeting technologies, the germline modification lab can produce mouse models in which the gene of interest is inactivated in a target organ or cell line instead of in the entire animal. These models, known as conditional knock-outs, are particularly useful in studying genes that, if missing, cause the mouse to die as an embryo. The lab also has the ability to produce mutant embryos that have a wild-type placenta using tetraploid embryo technology; this is useful when the gene-targeted mutation prevents implantation of the mouse embryo in the uterus. We also assist in the development of embryonic stem (ES) or fibroblast cell lines from mutant embryos, which allows for in vitro studies of the gene mutation. Our gene-targeting service encompasses three major procedures: DNA electroporation, clone expansion and cryopreservation, and microinjection. Gene targeting is initiated by mutating the genomic DNA of interest and inserting it into ES cells via electroporation. The mutated gene integrates into the genome and, by a process called homologous recombination, replaces one of the two wild-type copies of the gene in the ES cells. Clones are identified, isolated, and cryopreserved, and genomic DNA is extracted from each clone and delivered to the client for analysis. Correctly targeted ES cell clones are thawed, established into tissue culture, and cryopreserved in liquid nitrogen. Gene-targeting mutations are introduced by microinjection of the pluripotent ES cell clones into 3.5-day-old mouse embryos (blastocysts). These embryos, containing a mixture of wild-type and mutant ES cells, develop into mice called chimeras. The offspring of chimeras that inherit the mutated gene are heterozygotes possessing one copy of the mutated gene. The heterozygous mice are bred together to produce “knock-out mice” that completely lack the normal gene and have two copies of the mutant gene.

Embryo/sperm cryopreservation We provide cryopreservation services for archiving and reconstituting valuable mouse strains. These cost-effective procedures decrease the need to continuously breed valuable mouse models, and they provide added insurance against the loss of custom mouse lines due to disease outbreak or a catastrophic event. Mouse embryos at various stages of development, as well as mouse sperm, can be cryopreserved and stored in liquid nitrogen; they can be thawed and used, respectively, by implantation into the oviducts of recipient mice or by in vitro fertilization of oocytes.

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Cytogenetics Our lab also directs the VARI cytogenetics core, which uses advanced molecular techniques to identify structural and numerical chromosomal aberrations in mouse, rat, and human cells. Tumor, fibroblast, blood, or ES cells can be grown in tissue culture, growth-arrested, fixed, and spread onto glass slides. Karyotyping of chromosomes using Leishman- or Giemsa-stained (G-banded) chromosomes is our basic service; spectral karyotyping (SKY) analysis of metaphase chromosome spreads in 24 colors can aid in detecting subtle and complex chromosomal rearrangements. Fluorescence in situ hybridization (FISH) analysis, using indirectly or directly labeled bacterial artificial chromosome (BAC) or plasmid probes, can also be performed on metaphase spreads or on interphase nuclei derived from tissue touch preps or nondividing cells. Sequential staining of identical metaphase spreads using FISH and SKY can help identify the integration site of a randomly integrated transgene. Recently, FISH has been widely used to validate microarray data by confirming amplification/gain or deletion/loss of chromosomal regions of interest.

Speed congenics Congenic mouse strain development traditionally involves a series of backcrosses, transferring a targeted mutation or genetic region of interest from a mixed genetic donor background to a defined genetic recipient background (usually an inbred strain). This process requires about ten generations (2.5 to 3 years) to attain 99.9% of the recipientâ&#x20AC;&#x2122;s genome. Since congenic mice have a more defined genetic background, phenotypic characteristics are less variable and the effects of modifier genes can be more pronounced. Speed congenics, also called marker-assisted breeding, uses DNA markers in a progressive breeding selection to accelerate the congenic process. For high-throughput genotyping, we use the state-of-the-art Sentrix BeadChip technology from Illumina, which contains 1,449 mouse single nucleotide polymorphisms (SNPs). These SNPs are strain-specific and cover the 10 most commonly used inbred mouse strains for optimal marker selection. The client provides the genomic DNA of male mice from the second, third, and fourth backcross generations for genotyping. The males having the highest percentage of the recipientâ&#x20AC;&#x2122;s genome from each generation are identified, and these mice are bred by the client. Using speed congenics, 99.9% of congenicity can be achieved in five generations (1 to 1.5 years).

Michigan Animal Model Consortium The VARI Germline Modification and Cytogenetics lab directs the Michigan Animal Model Consortium (MAMC), one of the ten Core Technology Alliance (CTA) collaborative core facilities located at the University of Michigan, Michigan State University, Wayne State University, Western Michigan University, Kalamazoo Valley Community College, Grand Valley State University, and VARI. The other facilities offer research services in proteomics, bioinformatics, structural biology, genomics, biological imaging, bioscience commercialization, high-throughput compound screening, good manufacturing practices, and antibody technology. The MAMC labs were developed with funding from the Michigan Economic Development Corporation and provide efficient mouse modeling services to researchers studying human diseases. MAMCâ&#x20AC;&#x2122;s long-term goal is to offer a comprehensive set of cutting-edge services that, through continuous enhancements and development, will define our organization as a single point-of-service site for animal models research. Centralized provision of services maximizes research productivity and decreases time to discovery and is in high demand by academia and pharmaceutical and biotechnology companies, which are increasingly looking to outsource to service centers. Through its well-organized service structure and staff of experts, MAMC supports the growth of the life science industry in Michigan, which is congruent with the CTA goals.

From left, front row: Koeman, Sisson, Swiatek, Ayotte back row: Johnson, Lewis, Khoo

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Scientific Report

MAMC service offerings Animal model development •

Mouse transgenics. Transgenic technology is used to produce genetically engineered mice expressing foreign genes and serving as models for human disease research. Microinjection delivers the foreign DNA into the pronucleus of a one-cell fertilized egg. This service is provided using various strains of laboratory mice, with production of three transgenic founder mice guaranteed from each procedure.

•

Gene targeting. By transfecting mouse embryonic stem cells with inactivating, homologous DNAs, target gene expression can be shut down. Genetically engineered mice are produced by microinjecting mutant stem cells into mouse embryos and breeding the progeny to mutant homozygosity. This service is provided using 129 or C57BL/6 embryonic stem cells.

• Xenotransplantation. Human cancer cells are injected into immunodeficient mice to produce human-derived tumors. Protocols are designed to test anti-tumor treatment regimens that can lead to prognostic, diagnostic, or therapeutic procedures for humans.

Animal model analysis • Cytogenetics. Mouse and rat chromosomal abnormalities and genetic loci are visually observed using Giemsa stain, SKY, or FISH techniques. • Necropsy. Mice are dissected postmortem and tissues are fixed for histological analysis, with necropsy reports generated using voice-recognition software. • Histology. Histological sections are prepared from mouse tissues using microtomes and cryostats; they are processed and stained using automated instruments and then are microscopically analyzed. 54

• Veterinary pathology. A board-certified veterinary pathologist holding the D.V.M. and Ph.D. degrees provides expert microscopic analysis and project consultation.

• DNA isolation. DNA is isolated from mouse tail biopsies using the AutogenPrep 960 instrument.

Animal model maintenance and preservation

• Mouse rederivation. All mouse strains entering the specific pathogen–free breeding facility are rederived to specific pathogen–free mouse status using embryo transfer techniques. • Animal technical services. Veterinary services such as injections, measurements, mating set-up, and tail biopsies are performed by the animal technician staff. • Contract breeding. Wild-type mouse strains and genetically engineered animal models are maintained for research purposes by breeding the strains in a specific pathogen–free environment. • Embryo/sperm cryopreservation. Genetically engineered mice are preserved for archival purposes, disease control, genetic stability, and economic efficiency using germplasm cryopreservation techniques.

• Cancer model repository. Mouse cancer models of research interest are maintained through breeding strategies.

VARI | 2007

Bin T. Teh, M.D., Ph.D. Laboratory of Cancer Genetics

Dr. Teh obtained his M.D. from the University of Queensland, Australia, in 1992, and his Ph.D. from the Karolinska Institute, Sweden, in 1997. Before joining the Van Andel Research Institute (VARI), he was an Associate Professor of medical genetics at the Karolinska Institute. Dr. Teh joined VARI as a Senior Scientific Investigator in January 2000. His research mainly focuses on kidney cancer, and he is currently on the Medical Advisory Board of the Kidney Cancer Association. Dr. Teh was promoted to Distinguished Scientific Investigator in 2005.

Staff Laboratory Staff

Students

Chao-Nan (Miles) Qian, M.D., Ph.D. Peng-Fei Wang, M.D., Ph.D. Xin Yao, M.D., Ph.D. Eric Kort, M.D. Daisuke Matsuda, M.D. Jindong Chen, Ph.D. Leslie Farber, Ph.D. Kunihiko Futami, Ph.D. Dan Huang, Ph.D. Sok Kean Khoo, Ph.D.

Yan Li, Ph.D. Visiting Scientists Douglas Luccio-Camelo, Ph.D. David Petillo, Ph.D. Zhongfa (Jacob) Zhang, Ph.D. Stephanie Bender, M.S. Wangmei Luo, M.S. Mark Betten, B.S. Aaron Massie, B.S. Michael Westphal, B.S. Sabrina Noyes, B.S.

Van Andel Research Institute |

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Research Interests Kidney cancer, or renal cell carcinoma (RCC), is the tenth most common cancer in the United States (35,000 new cases and more than 13,000 deaths a year). Its incidence has been increasing, a phenomenon that cannot be accounted for by the wider use of imaging procedures. We have established a comprehensive and integrated kidney research program, and our major research goals are 1) to identify the molecular signatures of different subtypes of kidney tumors, both hereditary and sporadic, and to understand how genes function and interact in giving rise to the tumors and their progression; 2) to identify and develop diagnostic and prognostic biomarkers for kidney cancer; 3) to identify and study novel and established molecular drug targets and their sensitivity and resistance; and 4) to develop animal models for drug testing and preclinical bioimaging. Our program to date has established a worldwide network of collaborators; a tissue bank containing fresh-frozen tumor pairs (over 1,000 cases) and serum; and a gene expression profiling database of 500 tumors, with long-term clinical follow-up information for half of them. Our program includes positional cloning of hereditary RCC syndromes and functional studies of their related genes, microarray and bioinformatic analysis, generation of RCC mouse models, and more recently, molecular therapeutic studies.

Hereditary RCC syndromes We are currently focusing on the cloning of the gene responsible for familial clear cell renal cell carcinoma, which is a separate entity from von Hippel-Lindau (VHL) and from familial RCC with a chromosome-3 translocation. These efforts involve the use of high-density, single-nucleotide-polymorphism (SNP) microarrays and correlation with our existing gene expression profiles. 56

VARI | 2007

Microarray gene expression profiling and bioinformatics High-density SNP genotyping has been performed on some of the specimens registered in our RCC expression database. We are currently focusing on analysis and data mining. Clinically, we continue to subclassify the tumors by correlation with clinicopathological information. One example is the study of the unclassified group of tumors for which the histological diagnosis is â&#x20AC;&#x153;unknownâ&#x20AC;?. We have also identified a specific set of genes that can distinguish chromophobe (malignant) from oncocytoma (benign), two types that share a high degree of similarity in their expression profiles. Our database has proven to be very useful in RCC research, since we can obtain differential expression of any gene in seconds; this has led to numerous collaborations. We are currently combining SNP and expression data to identify novel RCC-related genes.

Mouse models of kidney cancer and molecular therapeutic studies We have generated several kidney-specific conditional knock-outs including APC, PTEN, and VHL. The first two knock-outs give rise to renal cysts and tumors, whereas VHL remains neoplasia-free; double knock-outs are also being studied. We have successfully generated nine xenograft RCC models via subcapsular injection that have characteristic clinical features and outcomes. Tumors and serum have been harvested for a baseline data set. We are currently performing in vitro and in vivo studies on several new drugs for kidney cancer.

Molecular and cellular studies 57

We use numerous well-characterized kidney cancer cell lines to study the functions of novel kidney cancerâ&#x20AC;&#x201C;related genes by overexpressing or down-regulating the genes. In addition, we perform cell cycle, proliferation, and migration assays to assess the cellular effects of these genes. These studies are usually coupled with in vivo studies.

External Collaborators We have extensive collaborations with researchers and clinicians in the United States and overseas.

From left: Zhang, Qian, Massie, Noyes, Westphal, Farber, Kort, Chen, Petillo, Matsuda, Teh

Van Andel Research Institute |

Scientific Report

Recent Publications Al-sarraf, N., S. Mahmood, J.N. Reif, J. Hinrichsen, B.T. Teh, E. McGovern, P. De Meyts, K.J. O’Byrne, and S.G. Gray. In press. DOK4/IRS-5 expression is altered in clear cell renal cell carcinoma. International Journal of Cancer. Daly, A.F., J.-F. Vanbellinghen, S.K. Khoo, M.-L. Jaffrain-Rea, L.A. Naves, M.A. Guitelman, A. Murat, P. Emy, A.-P. Gimenez-Roqueplo, G. Tamburrano, G. Raverot, A. Barlier, W. De Herder, A. Penfornis, E. Ciccarelli, et al. In press. Aryl hydrocarbon receptor interacting protein gene mutations in familial isolated pituitary adenomas: analysis in 73 families. Journal of Clinical Endocrinology and Metabolism. Evans, Andrew J., Ryan C. Russell, Olga Roche, T. Nadine Burry, Jason E. Fish, Vinca W.K. Chow, William Y. Kim, Arthy Saravanan, Mindy A. Maynard, Michelle L. Gervais, Roxana I. Sufan, Andrew M. Roberts, Leigh A. Wilson, Mark Betten, Cindy Vandewalle, et al. 2007. VHL promotes E2 box–dependent E-cadherin transcription by HIF-mediated regulation of SIP1 and Snail. Molecular and Cellular Biology 27(1): 157–169. Furge, Kyle A., Jindong Chen, Julie Koeman, Pamela Swiatek, Karl Dykema, Kseniji Lucin, Richard Kahnoski, Ximing J. Yang, and Bin Tean Teh. 2007. Detection of DNA copy number changes and oncogenic signaling abnormalities from gene expression data reveals MYC activation in high-grade papillary renal cell carcinoma. Cancer Research 67(7): 3171–3176.

Furge, K.A., M.H. Tan, K. Dykema, E. Kort, W. Stadler, X. Yao, M. Zhou, and B.T. Teh. 2007. Identification of deregulated oncogenic pathways in renal cell carcinoma: an integrated oncogenomic approach based on gene expression profiling. Oncogene 26(9): 1346–1350. Gad, S., S.H. Lefèvre, S.K. Khoo, S. Giraud, A. Vieillefond, V. Vasiliu, S. Ferlicot, V. Molinié, Y. Denoux, N. Thiounn, Y. Chrétien, A. Méjean, M. Zerbib, G. Benoît, J.M. Hervé, G. Allègre, B. Bressac-de Paillerets, B.T. Teh, and S. Richard. 2007. Mutations in BHD and TP53 genes, but not in HNF1β gene, in a large series of sporadic chromophobe renal cell carcinoma. British Journal of Cancer 96(2): 336–340. Greenman, Christopher, Philip Stephens, Raffaella Smith, Gillian L. Dalgliesh, Christopher Hunter, Graham Bignell, Helen Davies, Jon Teague, Adam Butler, Claire Stevens, Sarah Edkins, Sarah O’Meara, Imre Vastrik, Esther E. Schmidt, Tim Avis, et al. 2007. Patterns of somatic mutation in human cancer genomes. Nature 446(7132): 153–158. Lin, Fan, Ping L. Zhang, Ximing J. Yang, Jianhui Shi, Tom Blasick, Won K. Han, Hanlin L. Wang, Steven S. Shen, Bin T. Teh, and Joseph V. Bonventre. 2007. Human kidney injury molecule-1 (hKIM-1): a useful immunohistochemical marker for diagnosing renal cell carcinoma and ovarian clear cell carcinoma. American Journal of Surgical Pathology 31(3): 371–381. Qian, Chao-Nan, James H. Resau, and Bin Tean Teh. 2007. Prospects for vasculature reorganization in sentinel lymph nodes. Cell Cycle 6(5): 514–517. Wang, Kim L., David M. Weinrach, Chunyan Luan, Misop Han, Fan Lin, Bin Teh, and Ximing J. Yang. 2007. Renal papillary adenoma—a putative precursor of papillary renal cell carcinoma. Human Pathology 38(2): 239–246. Yang, X.J., M. Takahashi, K.T. Schafernak, M.S. Tretiakova, J. Sugimura, N.J. Vogelzang, and B.T. Teh. 2007. Does “granular cell” renal cell carcinoma exist? Molecular and histopathological reclassification. Histopathology 50(5): 678–680. Adley, Brian P., Anita Gupta, Fan Lin, Chunyan Luan, Bin T. Teh, and Ximing J. Yang. 2006. Expression of kidney-specific cadherin in chromophobe renal cell carcinoma and renal oncocytoma. American Journal of Clinical Pathology 126(1): 79–85.

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Adley, Brian P., Veronica Papavero, Jun Sugimura, B.T. Teh, and Ximing J. Yang. 2006. Diagnostic value of cytokeratin 7 and parvalbumin in differentiating chromophobe renal cell carcinoma from renal oncocytoma. Analytical and Quantitative Cytology and Histology 28(4): 228–236. Furge, Kyle A., Eric J. Kort, Ximing J. Yang, Walter M. Stadler, Hyung Kim, and Bin Tean Teh. 2006. Gene expression profiling in kidney cancer: combining differential expression and chromosomal and pathway analyses. Clinical Genitourinary Cancer 5(3): 227–231. Pimenta, Flávio J., Letícia F.G. Silveira, Gabriela C. Taveres, Andreza C. Silva, Paolla F. Perdigão, Wagner H. Castro, Marcus V. Gomez, Bin T. Teh, Luiz De Marco, and Ricardo S. Gomez. 2006. HRPT2 gene alterations in ossifying fibroma of the jaws. Oral Oncology 42(7): 735–739. Qian, Chao-Nan, Bree Berghuis, Galia Tsarfaty, MaryBeth Bruch, Eric J. Kort, Jon Ditlev, Ilan Tsarfaty, Eric Hudson, David G. Jackson, David Petillo, Jindong Chen, James H. Resau, and Bin Tean Teh. 2006. Preparing the “soil”: the primary tumor induces vasculature reorganization in the sentinel lymph node before the arrival of metastatic cancer cells. Cancer Research 66(21): 10365–10376. Tretiakova, M., M. Turkyilmaz, T. Grushko, M. Kocherginsky, C. Rubin, B. Teh, and X.J. Yang. 2006. Topoisomerase IIα expression in Wilms’ tumour: gene alterations and immunoexpression. Journal of Clinical Pathology 59(12): 1272–1277. Yang, Ximing J., Jun Sugimura, Kristian T. Schafernak, Maria S. Tretiakova, Misop Han, Nicholas J. Vogelzang, Kyle Furge, and Bin Tean Teh. 2006. Classification of renal neoplasms based on molecular signatures. Journal of Urology 175(6): 2302–2306. Yao, Xin, Chao-Nan Qian, Zhong-Fa Zhang, Min-Han Tan, Eric J. Kort, James H. Resau, and Bin Tean Teh. 2006. Two distinct types of blood vessels in clear cell renal cell carcinoma have contrasting prognostic implications. Clinical Cancer Research 13(1): 161–169. Zhang, Chun, Dong Kong, Min-Han Tan, Donald L. Pappas, Jr., Peng-Fei Wang, Jindong Chen, Leslie Farber, Nian Zhang, HanMo Koo, Michael Weinreich, Bart O. Williams, and Bin Tean Teh. 2006. Parafibromin inhibits cancer cell growth and causes G1 phase arrest. Biochemical and Biophysical Research Communications 350(1): 17–24. Zynger, Debra L., Nikolay D. Dimov, Chunyan Luan, Bin Tean Teh, and Ximing J. Yang. 2006. Glypican 3: a novel marker in testicular germ cell tumors. American Journal of Surgical Pathology 30(12): 1570–1575.

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Photo by Lia Tesfay of the Miranti lab and Jim Resau of the Resau lab.

VARI | 2007

Normal mouse prostate tissue fixed and stained to visualize CD82 protein (brown).

CD82 is a metastasis suppressor protein. Its presence is seen in the groups of epithelial cells that surround the lumens in this normal prostate tissue.

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Steven J. Triezenberg, Ph.D. Laboratory of Transcriptional Regulation

Dr. Triezenberg received his bachelorâ&#x20AC;&#x2122;s degree in biology and education at Calvin College in Grand Rapids, Michigan. His Ph.D. training in cell and molecular biology at the University of Michigan was followed by postdoctoral research in the laboratory of Steven L. McKnight at the Carnegie Institution of Washington. Dr. Triezenberg was a faculty member of the Department of Biochemistry and Molecular Biology at Michigan State University for more than 18 years, where he also served as Associate Director of the Graduate Program in Cell and Molecular Biology. Dr. Triezenberg was recruited to VAI to serve as the founding Dean of the Van Andel Institute Graduate School and as a Scientific Investigator in the Van Andel Research Institute, arriving in May 2006.

Laboratory Staff

Staff

Student

Martha Roemer, M.S.

Sebla Kutluay, B.S.

VARI | 2007

Research Interests The genetic information encoded in DNA must first be transcribed in the form of RNA before it can be translated into the proteins that do most of the work in a cell. Some genes must be expressed more or less constantly throughout the life of any eukaryotic cell. Others must be turned on (or turned off) in particular cells either at specific times or in response to a specific signal or event. Thus, regulation of gene expression is a key determinant of cell function. Our laboratory explores the mechanisms that regulate the first step in that flow, the process termed transcription. Over the past 20 years, my laboratory has used infection by herpes simplex virus as an experimental context for exploring the mechanisms of transcriptional activation. In the past 10 years, we have also asked similar questions in a very different biological context, the acclimation of plants to cold temperature.

Transcriptional activation during herpes simplex virus infection Herpes simplex virus type 1 (HSV-1) causes the common cold sore or fever blister. The initial lytic or productive infection by HSV-1 results in obvious symptoms in the skin and mucosa, typically in or around the mouth. After the initial infection resolves, HSV-1 finds its way into nerve cells, where the virus can â&#x20AC;&#x153;hideâ&#x20AC;? in a latent mode for long timesâ&#x20AC;&#x201D;essentially for the lifetime of the host organism. Occasionally, some trigger event (such as emotional stress, damage to the nerve from a sunburn, or a root canal operation) will cause the virus to reactivate, producing new viruses in the nerve cell and sending those viruses back to the skin to cause a recurrence of the cold sore. The DNA genome of HSV-1 encodes approximately 80 different proteins. However, the virus does not have its own machinery for expressing those genes; instead, it must divert the gene expression machinery of the host cell. That process is triggered by a viral regulatory protein designated VP16, whose function it is to stimulate transcription of the first viral genes to be expressed in the infected cell (the immediate-early, or IE, genes).

VP16 recruitment of host cell transcription machinery The prevailing model for the mechanism of transcriptional activation is that a portion of an activating protein (such as VP16) called the activation domain (AD) can bind to the host cell RNA polymerase II or to its accessory proteins. In this manner, VP16 recruits or tethers accessory proteins to the genes that are to be activated. Over the years, several accessory proteins (also known as general transcription factors) have been implicated as potential targets for VP16. Of those, the evidence seems to point most directly at TFIID, a multi-protein complex that includes the TATA-binding protein (TBP). TBP itself can bind rather efficiently to the VP16 activation domain, and mutations in VP16 that disrupt transcriptional activation also disrupt the interaction with TBP. We have pursued the structure of the VP16-TBP interaction by methods including X-ray crystallography and nuclear magnetic resonance. We have also tested the hypothesis that VP16 can influence the orientation of TBP on the TATA-box DNA of a target gene promoter. This hypothesis, proposed by other laboratories, is based on the fact that both TBP and the TATA sequence to which it binds are quite symmetric, and yet TBP can effectively support transcription in only a single orientation. We developed a new quantitative method for assessing TBP orientation and using this method have now demonstrated that TBP binds in a well-oriented manner even in the absence of VP16. Moreover, on a TATA site engineered to be completely symmetric, to which TBP binds in both orientations, the VP16 activation domain has no significant influence. This work resolves a long-standing issue regarding TBP orientation and eliminates one hypothesis for the mechanism of transcriptional activation.

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Chromatin-modifying coactivators in herpes virus infections Eukaryotic DNA is typically packaged as chromatin, in which the DNA is wrapped around “spools” of histone proteins, and these spools are then further arranged into higher-order structures. This packaging creates an impediment to transcription, during which RNA polymerase must separate the two strands of DNA. The impediment can be overcome with the help of chromatin-modifying coactivator proteins, some of which alter the histone proteins by post-translational modifications (e.g., acetylation or methylation) and others of which can slide or remove the histone proteins to permit access by RNA polymerase to the DNA. Experiments using the VP16 activation domain in artificial contexts (for example, in yeast genetic assays) have indicated that VP16 can recruit various coactivator proteins to target genes. However, the HSV-1 viral DNA is not packaged with histones in the infectious virion, and prior evidence suggested the viral DNA remained largely chromatin-free during infection. Therefore, we wondered whether VP16 would recruit these coactivators to viral IE genes, and if so whether those coactivators would be acting on histone proteins (which didn’t seem to be present) or on some other target. Our results have clearly indicated that VP16 can recruit certain coactivators to IE genes during lytic infection. We have also shown that at least some histone proteins do associate with viral DNA, although perhaps not to the same extent as with cellular DNA. We are currently exploring further which histones associate with viral DNA, how quickly they are put in place, the mechanisms used to put them in place, and what VP16 and other regulatory proteins might do to counteract the repressive effects of chromatin, which could be considered a molecular defense mechanism.

Can a curry spice block herpes infections? Curcumin, the bright yellow component of the curry spice turmeric, affects eukaryotic cells in several ways. Another laboratory has reported that curcumin could block the histone acetyltransferase activity of two coactivator proteins, p300 and CBP. Because we had shown that VP16 can recruit p300 and CBP to viral IE gene promoters, we tested whether curcumin, as an inhibitor of p300 or CBP activity, would block viral IE gene expression and thus block HSV infection. Indeed, curcumin has dramatic effects on IE gene expression and substantial effects on virus infection (Fig. 1). We are now trying to determine whether that effect is indeed channeled through the p300 and CBP proteins or whether it arises from another of the biological activities of curcumin.

Figure 1.

- curcumin

+ curcumin

Figure 1. HSV-1 infection of Vero cell monolayers. HSV-1 infection results in plaques or holes in a monolayer of cultured human cells (left). In the presence of curcumin (right), plaques are generally smaller and the cells within the plaques are not as completely obliterated. Photo by M. Roemer.

VARI | 2007

Gene activation during cold acclimation in plants Although plants and their cells obviously have very different forms and functions than animals and their cells, the mechanisms used for expressing genetic information are quite similar. About ten years ago, we applied our emerging interest in chromatin-modifying coactivators to an interesting question in plant biology. Some plants, including the prominent experimental organism Arabidopsis, can sense low (but nonfreezing) temperature in a way that provides protection from subsequent freezing temperatures (Fig. 2). This process is known as cold acclimation. Michael Thomashow, an MSU plant scientist, has explored the genes expressed during this process, and we collaborated with his laboratory to explore the mechanisms involved. We have characterized one particular histone acetyltransferase, termed GCN5, and two of its accessory proteins, ADA2a and ADA2b. Mutations in the genes encoding these coactivator proteins result in diminished expression of cold-regulated genes. Moreover, histones located at these cold-regulated genes become more highly acetylated during initial stages of cold acclimation. We are now working to determine whether GCN5 and the ADA2 proteins are partially or fully responsible for this cold-induced acetylation. We are also collaborating with groups in Greece and Pennsylvania to explore the distinct biological activities of the two ADA2 proteins. This work may help us understand whether the mechanisms by which plants express their genes can be effectively modulated so as to protect crop plants from loss in yield or viability due to environmental stresses such as low temperature. Figure 2.

Non-acclimated

Acclimated

Figure 2. Acclimation of Arabidopsis seedlings. Arabidopsis seedlings were grown on agar plates for three weeks at 20 °C. The plants in the right panel were chilled at 4 °C for two days. All plants were then subjected to subfreezing temperatures (–5 °C) for one day and then were returned to warm temperatures to recover. The acclimated plants remain healthy and green; the nonacclimated plants lose much of their color and die. Photo by K. Pavangadkar.

From left: Triezenberg, Roemer, Kutluay

External Collaborators Kanchan Pavangadkar and Michael F. Thomashow, Michigan State University, East Lansing Amy S. Hark, Muhlenberg College, Allentown, Pennsylvania Kostas Vlachonasios, Aristotle University of Thessaloniki, Greece

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George F. Vande Woude, Ph.D. Laboratory of Molecular Oncology

Dr. Vande Woude received his M.S. (1962) and Ph.D. (1964) from Rutgers University. From 1964–1972, he served first as a postdoctoral research associate, then as a research virologist for the U.S. Department of Agriculture at Plum Island Animal Disease Center. In 1972, he joined the National Cancer Institute as Head of the Human Tumor Studies and Virus Tumor Biochemistry sections and, in 1980, was appointed Chief of the Laboratory of Molecular Oncology. In 1983, he became Director of the Advanced Bioscience Laboratories–Basic Research Program at the National Cancer Institute’s Frederick Cancer Research and Development Center, a position he held until 1998. From 1995, Dr. Vande Woude first served as Special Advisor to the Director, and then as Director for the Division of Basic Sciences at the National Cancer Institute. In 1999, he was recruited to become the first Director of the Van Andel Research Institute.

Staff

Laboratory Staff Yu-Wen Zhang, M.D., Ph.D. Chongfeng Gao, Ph.D. Carrie Graveel, Ph.D. Qian Xie, Ph.D. Dafna Kaufman, M.Sc. Matt VanBrocklin, M.S. Jack DeGroot, B.S.

Betsy Haak, B.S. Liang Kang, B.S. Rachel Kuznar, B.S. Benjamin Staal, B.S. Ryan Thompson, B.S. Yanli Su, A.M.A.T.

Student

Guest Researchers

Angelique Berens

David Wenkert, M.D. Yuehai Shen, Ph.D. Edwin Chen, B.S.

VARI | 2007

Research Interests A mouse model of mutationally activated Met Signaling through Met and its ligand, HGF/SF, has been implicated in most types of human cancer. Compelling genetic evidence for the role of Met stems from the discovery that activating gain-of-function mutations are found in human kidney cancers and in other cancer types (http://www.vai.org/met/). To study how Met-activating mutations are involved in tumor development, we generated mice bearing mutations in the endogenous Met locus representative of both the inherited and the sporadic mutations found in human cancers. On a C57BL/6 background, the different mutant Met lines developed unique tumor profiles, including carcinomas, sarcomas, and lymphomas. We have found that the differences in tumor types and latency may be due to signaling differences triggered by the specific mutation in a tissue- or stem cellâ&#x20AC;&#x201C;specific pattern. Cytogenetic analysis of all tumor types shows frequent trisomy of the Met locus. Moreover, it is the mutant met allele that is amplified and likely to be required for tumor progression. When mutant Met was transferred to the FVB/N mouse background, these animals developed aggressive mammary tumors. Therefore, understanding the signaling specificity of these mutations is essential for developing successful cancer therapeutics. Our mutant mice provide a valuable model for testing Met inhibitors and for understanding the molecular events crucial for Met-mediated tumorigenesis.

A novel mouse model for preclinical studies We have generated a severe combined immune deficiency (SCID) mouse strain carrying a human HGF/SF transgene. This mouse provides a species-compatible ligand for propagating human tumor cells expressing human Met. The growth of Met-expressing human tumor xenografts can be significantly enhanced in this transgenic mouse relative to growth in nontransgenic hosts. This immunocompromised strain is vital for examining the role of Met in human tumor malignancy. We are developing metastasis models and generating orthotopic xenografts of human tumor cells. This model is being used for preclinical testing of drugs or compounds targeting the HGF/SF-Met complex and downstream signaling pathways.

Understanding the â&#x20AC;&#x153;multiple personalitiesâ&#x20AC;? of cancer cells Several years ago, we asked whether tumor cells can switch between proliferative and invasive phenotypes. We discovered that tumor cells can indeed switch, and they can do so rapidly; they may also express both proliferative and invasive features. We have established in vitro methods for selecting highly proliferative or highly invasive tumor cell populations that may mimic the in vivo process of clonal selection during tumor progression. We have determined that chromosomal instability correlates with the proliferative and invasive phenotypes. Using spectral karyotyping (SKY) and M-Fish, we observe significant changes in chromosome content with each phenotype, and the changes show remarkable concordance with changes in gene expression. Regional gene expression changes appear to favor the expression of specific genes appropriate for the invasive or proliferative phenotype. Moreover, the ratio of chromosomal changes closely parallels the ratio of gene expression in the chromosome. These results show that chromosome instability and the resulting heterogeneous chromosome composition provide the diversity in gene expression to allow tumor cell clonal evolution.

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Examining how geldanamycin inhibits tumor cell invasion Our lab has been studying the mechanism by which geldanamycin (GA) inhibits urokinase activation of plasmin from plasminogen (uPA). Previously, we have shown that a subset of GA-related drug derivatives inhibits HGF/SF-induced activation of plasmin in canine MDCK cells. We found that such inhibition also occurs in several human glioblastoma tumor cell lines. Curiously, these GA drugs inhibit HGF/SF-induced uPA activation and block MDCK cell scattering and glioblastoma tumor cell invasion in vitro at concentrations below that required to exhibit a measurable effect on Met degradation through HSP90. This inhibition is observed only with HGF/SF-mediated activation and only when the magnitude of HGF/SF-uPA induction is 1.5 times basal uPA-plasmin activity.

Inhibition of MAPK in melanoma

Extracellular signals activate mitogen-activated protein kinase (MAPK) cascades, potentiating biological activities such as cell proliferation, differentiation, and survival. Constitutive activation of MAPK signaling pathways is implicated in the development and progression of many human cancers, including melanoma. Mutually exclusive activating mutations in NRas or BRAF are found in about 85% of all melanomas, resulting in constitutive activation of the MAPK pathway (Ras-BRaf-MEK-Erk-Rsk). We have previously demonstrated that inhibition of this pathway with small-molecule MEK inhibitors selectively induces apoptosis in human melanoma cells but not in normal melanocytes both in vitro and in vivo. These results support the concept that the MAPK pathway represents a tumor-specific survival signaling pathway in melanoma cells and that targeting members of this pathway may be an effective therapeutic strategy. Understanding the mechanisms by which constitutive MAPK promotes survival and defining the minimal vital MAPK pathway components required for the development and progression of melanoma may have direct translational implications. Preliminary data suggest that MAPK activation actively suppresses several pro-apoptotic Bcl-2 family members. We are currently using the specific small-molecule MEK inhibitor PD184352 together with molecular biological approaches to selectively modulate the expression and function of these molecules in order to validate and develop them as novel therapeutic targets for treating melanoma and other MAPK-associated cancers.

External Collaborators Francesco DeMayo, Baylor College of Medicine, Houston, Texas Ermanno Gherardi, MRC Center, Cambridge, England Nadia Harbeck, Technische Universit채t, Munich, Germany Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Ernest Lengyel, University of Chicago, Illinois Patricia LoRusso, Karmanos Cancer Institute, Detroit, Michigan Benedetta Peruzzi, National Cancer Institute, Bethesda, Maryland Alnawaz Rehemtulla, Brian Ross, and Richard Simon, University of Michigan, Ann Arbor Ilan Tsarfaty, Tel Aviv University, Israel Robert Wondergem, East Tennessee State University, Johnson City

VARI | 2007

From left: Gao, Thompson, Xie, Graveel, Kaufman, Vande Woude, Staal, Bassett, Haak, Nelson, DeGroot, Su, Zhang

Recent Publications Lindemann, K.K., J. Resau, J. Nährig, E. Kort, B. Leeser, K. Anneke, A. Welk, J. Schäfer, G.F. Vande Woude, E. Lengyel, and N. Harbeck. In press. Differential expression of c-Met, its ligand HGF/SF, and HER2/neu in DCIS and adjacent normal breast tissue. Histopathology. Zhang, Y.W., and G.F. Vande Woude. In press. Mig-6, signal transduction, stress response, and cancer. Cell Cycle. Sawada, Kenjiro, A. Reza Radjabi, Nariyoshi Shinomiya, Emily Kistner, Hilary Kenny, Amy R. Becker, Muge A. Turkyilmaz, Ravi Salgia, S. Diane Yamada, George F. Vande Woude, Maria S. Tretiakova, and Ernst Lengyel. 2007. c-Met overexpression is a prognostic factor in ovarian cancer and an effective target for inhibition of peritoneal dissemination and invasion. Cancer Research 67(4): 1670–1679. Zhang, Y.-W., B. Staal, Y. Su, P. Swiatek, P. Zhao, B. Cao, J. Resau, R. Sigler, R. Bronson, and G.F. Vande Woude. 2007. Evidence that MIG-6 is a tumor-suppressor gene. Oncogene 26(2): 269–276. Gherardi, Ermanno, Sara Sandin, Maxim V. Petoukhov, John Finch, Mark E. Youles, Lars-Göran Öfverstedt, Ricardo N. Miguel, Tom L. Blundell, George F. Vande Woude, Ulf Skoglund, and Dmitri I. Svergun. 2006. Structural basis of hepatocyte growth factor/scatter factor and MET signalling. Proceedings of the National Academy of Sciences U.S.A. 103(11): 4046–4051. Lee, Jae-Ho, Chong Feng Gao, Chong Chou Lee, Myung Deok Kim, and George F. Vande Woude. 2006. An alternatively spliced form of Met receptor is tumorigenic. Experimental and Molecular Medicine 38(5): 565–573. Moshitch-Moshkovitz, Sharon, Galia Tsarfaty, Dafna W. Kaufman, Gideon Y. Stein, Keren Shichrur, Eddy Solomon, Robert H. Sigler, James H. Resau, George F. Vande Woude, and Ilan Tsarfaty. 2006. In vivo direct molecular imaging of early tumorigenesis and malignant progression induced by transgenic expression of GFP-Met. Neoplasia 8(5): 353–363. Tsarfaty, Galia, Gideon Y. Stein, Sharon Moshitch-Moshkovitz, Dafna W. Kaufman, Brian Cao, James H. Resau, George F. Vande Woude, and Ilan Tsarfaty. 2006. HGF/SF increases tumor blood volume: a novel tool for the in vivo functional molecular imaging of Met. Neoplasia 8(5): 344–352.

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Craig P. Webb, Ph.D. Program for Translational Medicine Laboratory of Tumor Metastasis and Angiogenesis

Dr. Webb received his Ph.D. in cell biology from the University of East Anglia, England, in 1995. He then served as a postdoctoral fellow in the laboratory of George Vande Woude in the Molecular Oncology Section of the Advanced BioScience Laboratoriesâ&#x20AC;&#x201C;Basic Research Program at the National Cancer Institute, Frederick Cancer Research and Development Center, Maryland (1995â&#x20AC;&#x201C;1999). Dr. Webb joined VARI as a Scientific Investigator in October 1999.

Staff Laboratory Staff David Cherba, Ph.D. Jessica Hessler, PhD. Jeremy Miller, Ph.D. David Monsma, Ph.D. Emily Eugster, M.S. Sujata Srikanth, M.Phil. Dawna Dylewski, B.S.

Students Brian Hillary, B.A. Marcy Ross, B.S. Stephanie Scott, B.S. Katherine Koehler

Visiting Scentists Visiting Scientists Philip Grimley, M.D. David Reinhold, Ph.D. Guenther Tusch, Ph.D.

Student Molly Dobb

VARI | 2007

Research Interests The Program for Translational Medicine was launched on June 1, 2006. While maintaining a research effort focused on enhancing our understanding of the molecular basis of tumor metastasis, the program is also developing community capabilities around translational research and the future clinical applications of molecular-based medicine. These efforts are very much focused on the practical implementation of biomarkers for improving diagnostic and therapeutic strategies against chronic human diseases, including cancer. The program has recently launched a proof-of-concept personalized medicine initiative to identify novel treatment options for patients with late-stage cancer. The research portion of this community protocol includes the enhancement of computer-based predictive models, by overlaying knowledge of molecular networks and drug-target interactions to identify potential combination targeting strategies for late-stage disease. These predictions are evaluated for efficacy in xenograft models for each patient’s tumor. Our informatics system, XB-BioIntegration Suite (XB-BIS), has also been enhanced to permit reporting of molecular drug information back to the medical oncologists, who may use the information for treatment decisions. In the coming years, we plan on expanding our molecular profiling efforts to identify drugable targets within the cancer stem cell components of several malignancies and to improve our predictive modeling and reporting capabilities, with the hope of identifying the optimal combinational strategies for treating cancers using FDA-approved agents and/or drugs in the drug discovery pipeline. Our research typically begins with the analysis of human specimens. We aim to identify molecular correlates of important clinical phenotypes, such as the propensity to metastasize and drug resistance. The standardized collection of human specimens, along with information about the patient’s medical history, diagnosis, treatment, and response to therapy, represents a crucial component of our research. Identifying the molecular correlates of a given phenotype, whether nucleic acid or protein, often represents the first step in our translational pipeline. We have developed the essential workflow and integrated informatics that are required to manage and interpret complex data sets of longitudinal clinical/preclinical and molecular data across different experimental platforms. XB-BIS is now interfaced with the electronic medical records system of Spectrum Health, through the co-development of an IRB data exchange portal maintained by the Spectrum Health Research Department. This permits the transfer of de-identified medical record information from consenting patients into XB-BIS so that it can be combined with molecular data generated from the processing of the patient’s tissue, blood, or urine. In 2006, XB-BIS was commercialized and has been licensed by XB-TransMed Solutions (http://www.xbtransmed.com), who now provide professional support services related to the sales and support of the tool, while our laboratory maintains focus on XB-BIS research and development. In the research lab, and increasingly within the Center for Molecular Medicine, we use various molecular technologies to generate the molecular data pertaining to a clinical or preclinical sample. XB-BIS permits the analysis of these data in conjunction with the clinical/preclinical information, and coupled with systems biology tools such as GeneGo’s MetaCoreTM product or Ingenuity’s IPA suite, we identify potential diagnostic signatures that can predict clinically meaningful phenotypes. For example, using Affymetrix gene expression analysis, we have identified tumor profiles associated with metastatic outcome in colorectal cancer and with patient survival in mesothelioma. These signatures are now being validated within the CLIA/CAP-accredited Center for Molecular Medicine, a joint venture between VAI and Spectrum Health. Potential therapeutic intervention strategies are also identified and validated in the laboratory using a variety of approaches including RNA interference and/or existing therapeutic agents in the appropriate model systems. At this time, our focal diseases are pancreatic cancer and multiple myeloma. We have begun to identify potential new targets in these tumors and are using both inducible shRNA systems for gene knock-down and targeted nanoparticles to validate possible intervention strategies in mouse xenograft models developed and characterized within our laboratory. While our research is focused on discovering new diagnostic and therapeutic strategies for metastatic and refractory disease, the translational infrastructure we have developed can be applied to a broad spectrum of other diseases. The optimal therapeutic target is no longer the disease based on organ site, but rather the molecular networks driving the clinical phenotype within the disease and the individual.

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Community initiatives As our research discoveries move closer to clinical application, we continue working to increase the readiness of the community to offer advances in molecular medicine. To translate our discoveries into human benefit, we must work in highly coordinated, multidisciplinary partnerships with community institutions. The synergistic goals are to benefit human health and promote Grand Rapids as a leader in translational medicine. The combination of powerful informatics, regulated diagnostics, and clinical trial coordination have aligned our collective community strengths with industrial demand and the FDA’s critical path intitiative. The following initiatives were successfully launched in 2006.

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Innovative Clinical Research Alliance. Under the name “ClinXus”, this is a multi-institutional alliance that offers new biomarker-driven clinical trials to patients and physicians, and it will provide a single destination for pharmaceutical and biotech companies looking to carry out biomarker-drug co-development. In 2006, ClinXus obtained a $1.5 million state grant to accelerate the development of this community alliance. Partner institutions include VARI, Spectrum Health, Saint Mary’s Health Care, Jasper Clinic, Grand Valley Medical Specialists, and Grand Valley State University. Other members will join in 2007 as we continue to expand our collective capabilites for innovative clinical research. More information can be found at http://www.clinxus.com.

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The Center for Molecular Medicine. The CMM is a joint venture with Spectrum Health that is bringing cutting-edge, molecular-based diagnostic tests to physicians and their patients. It can offer a broad range of molecular services and has recently been certified by the College of American Pathology to run molecular diagnostic tests, including the Roche AmpliChip cytochrome p450 test indicating the correct dose for many prescription drugs. More information can be found at http://www.cmmdx.org.

From left: Scott, Dobb, Hessler, Hillary, Koehler, Monsma, Cherba, Reinhold, Dylewski, Srikanth, Ross, Eugster, Webb

VARI | 2007

External Collaborators Academic Surgical Associates, Grand Rapids, Michigan Barbara Ann Karmanos Institute, Detroit, Michigan Cancer & Hematology Centers of Western Michigan, P.C., Grand Rapids DeVos Children’s Hospital, Grand Rapids, Michigan Digestive Disease Institute, Grand Rapids, Michigan GeneGo, Inc., St. Joseph, Michigan Grand Valley Medical Specialists, Grand Rapids, Michigan Grand Valley State University, Grand Rapids, Michigan Henry Ford Hospital, Detroit, Michigan Jasper Clinical Research & Development, Inc., Kalamazoo, Michigan Johns Hopkins University, Baltimore, Maryland M.D. Anderson Cancer Center, Houston, Texas MMPC, Grand Rapids, Michigan New York University, New York City Oncology Care Associates, St. Joseph, Michigan Pfizer (Ann Arbor, Michigan; Saint Louis, Missouri; Groton, Connecticut) ProNAi Therapeutics, Kalamazoo, Michigan Saint Mary’s Health Care, Grand Rapids, Michigan Schering-Plough Research Institute, New Jersey Spectrum Health, Grand Rapids, Michigan TGEN, Phoenix, Arizona Uniformed Services University of the Health Sciences, Bethesda, Maryland University of Michigan, Ann Arbor University of California, San Francisco West Michigan Heart, Grand Rapids, Michigan

Recent Publications Kuick, Rork, David E. Misek, David J. Monsma, Craig P. Webb, Hong Wang, Kelli J. Peterson, Michael Pisano, Gilbert S. Omenn, and Samir M. Hanash. 2007. Discovery of cancer biomarkers through the use of mouse models. Cancer Letters 249(1): 40–48.

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Michael Weinreich, Ph.D. Laboratory of Chromosome Replication

Dr. Weinreich received his Ph.D. in biochemistry from the University of Wisconsinâ&#x20AC;&#x201C;Madison in 1993. He then was a postdoctoral fellow in the laboratory of Bruce Stillman, director of the Cold Spring Harbor Laboratory, New York, from 1993 to 2000. Dr. Weinreich joined VARI as a Scientific Investigator in March 2000.

Staff Dorine Savreux, Ph.D. FuJung Chang, M.S. Amber Crampton, B.Sc. Carrie Gabrielse, B.S. Vickie Harkins, B.S.

Students Ying-Chou Chen, M.S. Charles Miller, B.S. David Dornboss, Jr. Louise Haste Kate Leese

VARI | 2007

Research Interests We are studying how cells accurately replicate their DNA, a process that begins at specific DNA sequences termed replication origins. There are approximately 400 replication origins in budding yeast and as many as 10,000 in human cells. Coordinating the activation of these origins for DNA synthesis during the cell cycle is a daunting task. We know that origins recruit many proteins prior to the DNA synthetic period (S-phase) that are required for the assembly and activation of replication forks. These proteins include Cdt1p, Cdc6p, and the origin recognition complex (ORC), which binds directly to origin DNA. Cdt1p, Cdc6p, and ORC cooperate to load the MCM DNA helicase at the origin in an ATP-dependent reaction. There are perhaps a score of additional proteins that assemble at the origin following MCM loading before DNA synthesis can begin. In our lab we are studying how Cdc6p activity is influenced by chromatin structure and ATP binding. We previously isolated genetic suppressors of a cdc6-4 temperature-sensitive (ts) mutant that inactivated the SIR2 gene. Sir2p is a histone H3 and H4 deacetylase, and therefore its loss leads to increased H3 and H4 acetylation within chromatin. Although loss of SIR2 allowed growth of the cdc6-4 strain at high temperature, we have found that Sir2p inhibits only specific origins. We have systematically identified multiple SIR2-regulated origins on chromosomes III and VI. Our studies so far indicate that these origins share a common organization including an inhibitory element through which Sir2p acts. Origins in Saccharomyces cerevisiae have a modular structure (Fig. 1) that includes an ORC binding site (A and B1 elements) and a loading site for the MCM helicase (B2 element). We have identified an inhibitory sequence in SIR2-regulated origins, termed the IS element, located downstream of B2. This element is responsible for Sir2p inhibition at these origins. Recent high-resolution mapping along chromosome III indicates that the IS element maps squarely within a positioned nucleosome. This nucleosome is directly adjacent to or overlaps the B2 element and therefore might influence MCM helicase loading. In support of this, we have found that excluding this nucleosome from the B2 element abolishes the activity of the IS element. Furthermore, the IS element acts in a distance-dependent manner, which is consistent with an effect through this positioned nucleosome.

Figure 1.

Figure 1. S. cerevisiae origins have a modular structure consisting of an essential A element and important B elements. These elements direct binding of proteins in the â&#x20AC;&#x153;pre-replicative complexâ&#x20AC;? (pre-RC) that forms during G1 phase. An inhibitory element (IS) is present at some origins and likely interferes with pre-RC assembly.

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How might SIR2 inhibit DNA replication? We believe that this occurs through deacetylation of histone H4 K16. Sir2p deacetylates the histone H3 acetyl-lysine residues K9 and K14 as well as H4 K16. We found that an H4 mutation of K16 to Q that mimics the acetylated state suppresses the cdc6-4 and mcm2-1 ts mutations; H3 K9Q or K14Q mutations do not suppress these ts mutations. Taken together, our data suggest that a local nucleosome acetylated on H4 K16 facilitates MCM helicase loading and that a nucleosome impinging on B2, if it is deacetylated on K16, inhibits MCM loading. We would like to understand the molecular function of the IS element, which is presumably affecting this histone H4 modification. Based on the frequency of SIR2-regulated origins on chromosome III and VI, we expect that a significant number of origins (about 80 of 400) will be subject to this type of regulation.

The Cdc7p-Dbf4p kinase promotes DNA replication and assists in repair of certain DNA lesions. Cdc7p-Dbf4p is a two-subunit serine/threonine kinase required for initiating DNA replication, and it acts after assembly of the MCM helicase as diagramed in Fig 2. Cdc7p is the kinase subunit but it has no activity in the absence of the Dbf4p regulatory subunit. We have analyzed Dbf4p using a structure-function approach to determine the residues required for its essential role in DNA replication. We found that about 40% of the Dbf4p N-terminus is dispensable for its essential replication function, but that it encodes a conserved 100amino-acid region with similarity to the BRCT motif. We have called this sequence the BRDF motif for BRCT and DBF4 similarity. The BRCT domain folds into a modular structure and is often found in proteins that participate in the DNA damage response. The BRCT domain likely binds to phosphoproteins and therefore allows regulated targeting to proteins modified by phosphorylation, as occurs following activation of the DNA damage checkpoint. Yeast dbf4 mutants altering this motif are sensitive to replication fork arrest, suggesting that the BRDF domain targets the kinase to stalled replication forks (Fig. 3). In support of this interpretation, we have performed domain-swapping experiments and identified a heterologous BRCT domain that will function in place of the Dbf4p BRDF domain. We are testing whether these two domains target Dbf4p to the same or similar substrates. It appears therefore, that the Dbf4p BRDF motif has a role in maintaining replication fork stability, likely through targeting Cdc7p kinase to non-origin sites. This is a separable activity to the essential role of Dbf4p in promoting the initiation of replication. Figure 2.

Figure 2. DNA synthesis requires Cdc7p-Dbf4p kinase, which is thought to act on the pre-RC to promote Cdc45p and GINS binding. Assembly of a â&#x20AC;&#x153;pre-initiation complexâ&#x20AC;? facilitates origin unwinding to give ssDNA.

Figure 3.

Figure 3. A) Schematic representation showing elements conserved among all Dbf4p orthologs. The N-terminal BRDF domain is dispensable for DNA replication. B) We propose this BRCT-like domain directs Cdc7p-Dbf4p kinase to stalled replication forks via recognition of a phosphorylated protein in the replisome.

VARI | 2007

We are also studying the human Cdc7-Dbf4 protein kinase, called here HsCdc7-Dbf4. The HsCdc7 protein is up-regulated in about 50% of the NCI 60 tumor cell lines representing the most common forms of cancer in the USA. In contrast, HsCdc7 protein has very low abundance or is undetectable in normal cells and tissues. It may be that nondividing cells down-regulate HsCdc7 expression. We have further determined by immunohistochemistry that HsCdc7 protein is highly expressed in some primary human tumors. Since HsCdc7 is an essential kinase required for DNA replication, its increased expression level in some tumors and tumor cell lines may reflect higher rates of cellular proliferation. Alternatively, since HsCdc7 is involved in other aspects of chromosome metabolism (e.g., DNA repair) and functions in the S-phase checkpoint, its increased expression may offer an advantage to tumor cells that have higher rates of chromosome instability. It was therefore interesting when we discovered several years ago that knockdown of HsCdc7 expression using RNAi results in an apoptotic response in some cancer cell lines but not in normal cells. We have been examining the molecular differences for this apoptotic response. Apoptosis occurs in cells lines that are either p53 wild type or phenotypically null for p53. There is good published evidence that in response to HsCdc7 depletion, wild-type cells undergo a G1 and G2/M arrest that is p53-dependent and protects against apoptosis. However, in some cancer cell lines, even in the absence of p53 function, HsCdc7 knockdown does not induce apoptosis, although these cells are otherwise competent to undergo the apoptotic program in response to various stimuli. Since HsCdc7 is required for DNA replication and apparently plays a role in other aspects of chromosome metabolism, we think that these findings have significance for inhibiting the growth and/or viability of certain types of tumor cells.

Recent Publications

From left: Chen, Miller, Savreux, Weinreich, Crampton, Gabrielse, Chang, Haste

Gabrielse, Carrie, Charles T. Miller, Kristopher H. McConnell, Aaron DeWard, Catherine A. Fox, and Michael Weinreich. 2006. A Dbf4p BRCA1 C-terminal-like domain required for the response to replication fork arrest in budding yeast. Genetics 173(2): 541â&#x20AC;&#x201C;555. Zhang, Chun, Dong Kong, Min-Han Tan, Donald L. Pappas, Jr., Peng-Fei Wang, Jindong Chen, Leslie Farber, Nian Zhang, Han-Mo Koo, Michael Weinreich, Bart O. Williams, and Bin Tean Teh. 2006. Parafibromin inhibits cancer cell growth and causes G1 phase arrest. Biochemical and Biophysical Research Communications 350(1): 17â&#x20AC;&#x201C;24.

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Bart O. Williams, Ph.D. Laboratory of Cell Signaling and Carcinogenesis

Dr. Williams received his Ph.D. in biology from Massachusetts Institute of Technology in 1996. For three years, he was a postdoctoral fellow at the National Institutes of Health in the laboratory of Harold Varmus, former Director of NIH. Dr. Williams joined VARI as a Scientific Investigator in July 1999 and was promoted to Senior Scientific Investigator in 2006.

Staff Charlotta Lindvall, M.D., Ph.D. Dan Robinson, Ph.D. Cassandra Zylstra, B.S.

Students Sarah Mange Amanda Field

VARI | 2007

Research Interests Our laboratory is interested in understanding how alterations in the Wnt signaling pathway cause human disease. Specifically, we have focused our efforts on the functions of the Wnt co-receptors, Lrp5 and Lrp6. Wnt signaling is an evolutionarily conserved process that functions in the differentiation of most tissues within the body. Given its central role in growth and differentiation, it is not surprising that alterations in the pathway are among the most common events associated with human cancer. In addition, several other human diseases, including osteoporosis, have been linked to altered regulation of this pathway. We also work on understanding the role of Wnt signaling in bone formation. Our interest is not only from the perspective of normal bone development, but also in trying to understand whether aberrant Wnt signaling plays a role in the predisposition of some common tumor types (for example, prostate, breast, lung, and renal tumors) to metastasize to and grow in bone. The long-term goal of this work is to provide insights that could be used in developing strategies to lessen the morbidity and mortality associated with skeletal metastasis.

Wnt signaling in normal bone development Mutations in the Wnt receptor, Lrp5, have been causally linked to alterations in human bone development. We have characterized a mouse strain deficient for Lrp5 and shown that it recapitulates the low-bone-density phenotype seen in human patients deficient for Lrp5. We have furthered this study by showing that mice carrying mutations in both Lrp5 and the related Lrp6 protein have even more-severe defects in bone density. To test whether Lrp5 deficiency causes changes in bone density due to aberrant signaling through β-catenin, we created mice carrying an osteoblast-specific deletion of β-catenin (OC-cre;β-catenin-flox/flox mice). In collaboration with Tom Clemens of the University of Alabama at Birmingham, we found that alterations in Wnt/β-catenin signaling in osteoblasts lead to changes in the expression of RANKL and osteoprotegerin (OPG). Consistent with this, histomorphometric evaluation of bone in the mice with osteoblast-specific deletions of either Apc or β-catenin revealed significant alterations in osteoclastogenesis. We are currently addressing how other genetic alterations linked to Wnt/β-catenin signaling affect bone development and osteoblast function. We have generated mice with a conditional allele of Lrp6 that can be inactivated via cre-mediated recombination, and we will assess the role of Lrp6 in terminal osteoblast differentiation. We are also generating mice carrying a conditional deletion of Lrp5 in differentiated osteoblasts, and we will characterize their phenotype. Finally, we are working to determine what other signaling pathways in osteoblasts may impinge on β-catenin signaling to control osteoblast differentiation and function.

General mechanisms of Wnt signaling There are many levels of regulating the reception of Wnt signals. The completion of the Human Genome Project has shown that there are 19 different genes encoding Wnt proteins, 9 encoding Frizzled proteins, and the genes encoding Lrp5 and Lrp6. In addition, there are several proteins that can inhibit Wnt signaling by binding to components of the receptor complex and interfering with normal signaling, including the Dickkopfs (Dkks) and the Frizzled-related proteins (FRPs). One of the long-term goals of our laboratory is to understand how specificity is generated for the different signaling pathways, with a specific focus on understanding the molecular functions of Lrp5 and Lrp6.

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Wnt signaling in prostate development and cancer Two hallmarks of advanced prostate cancer are the development of skeletal osteoblastic metastasis and the ability of the tumor cells to become independent of androgen for survival. The association of Wnt signaling with bone growth, plus the fact that β-catenin can bind to the androgen receptor and make it more susceptible to activation with steroid hormones other than DHT, make Wnt signaling an attractive candidate for explaining some phenotypes associated with advanced prostate cancer. We have created mice with a prostate-specific deletion of the Apc gene. These mice develop fully penetrant prostate hyperplasia by four months of age, and these tumors progress to frank carcinomas by seven months. We have found that these tumors initially regress under androgen ablation but show signs of androgen-independent growth some months later.

Wnt signaling in mammary development and cancer We are also addressing the relative roles of Lrp5 and Lrp6 in Wnt1-induced mammary carcinogenesis. A deficiency in Lrp5 dramatically inhibits the development of mammary tumors in this context. A germline deficiency for Lrp5 or Lrp6 results in delayed mammary development. As Lrp5-deficient mice are viable and fertile, we have focused our initial efforts on these mice. In collaboration with Caroline Alexander’s laboratory, we have found dramatic reductions in the number of mammary progenitor cells in these mice. We are continuing to examine the mechanisms underlying this reduction.

VARI mutant mouse repository With support from the Van Andel Institute, my laboratory maintains a repository of mutant mouse strains to support the general development of animal models of human disease. We distribute these strains at a nominal cost to interested laboratories.

External Collaborators Bone development Mary Bouxsein, Beth Israel Deaconness Medical Center, Boston, Massachusetts Thomas Clemens, University of Alabama–Birmingham Marie Claude Faugere, University of Kentucky, Lexington David Ornitz and Fanxin Long, Washington University, St. Louis, Missouri Matthew Warman, Harvard University, Boston, Massachusetts Prostate cancer Wade Bushman and Ruth Sullivan, University of Wisconsin–Madison Mammary development Caroline Alexander, University of Wisconsin–Madison Yi Li, Baylor Breast Center, Houston, Texas Jeffrey Rubin, National Cancer Institute, Bethesda, Maryland Mechanisms of Wnt signaling Kathleen Cho, University of Michigan, Ann Arbor Kang-Yell Choi, Yansei University, Seoul, South Korea Eric Fearon, University of Michigan, Ann Arbor Silvio Gutkind, National Institute of Dental and Craniofacial Research, Bethesda, Maryland Kun-Liang Guan, University of Michigan, Ann Arbor

VARI | 2007

From left, standing: Williams, Robinson; seated: Zylstra, Lindvall

Recent Publications Lindvall, C., W. Bu, B.O. Williams, and Y. Li. In press. Wnt signaling, stem cells, and the cellular origin of breast cancer. Stem Cell Reviews. Wu, R., N.D. Handrix, R. Kuick, Y. Zhai, D.R. Schwartz, A. Akyol, S. Hanash, D. Misek, H. Katabuchi, B.O. Williams, E.R. Fearon, and K.R. Cho. In press. Mouse model of human endometroid adenocarcinoma based on somatic defects in the Wnt β-catenin and PI3K/Pten signaling pathways. Cancer Cell. Young, J.J., J.L. Bromberg-White, C.R. Zylstra, J. Church, E. Boguslawski, J. Resau, B.O. Williams, and N. Duesbery. In press. LRP5 and LRP6 are not required for protective antigen-mediated internalization or lethality of anthrax lethal toxin. PLoS Pathogens. Bruxvoort, Katia J., Holli M. Charbonneau, Troy A. Giambernardi, James C. Goolsby, Chao-Nan Qian, Cassandra R. Zylstra, Daniel R. Robinson, Pradip Roy-Burman, Aubie K. Shaw, Bree D. Buckner-Berghuis, Robert E. Sigler, James H. Resau, Ruth Sullivan, Wade Bushman, and Bart O. Williams. 2007. Inactivation of Apc in the mouse prostate causes prostate carcinoma. Cancer Research 67(6): 2490–2496. Liu, X., K.M. Bruxvoort, Cassandra R. Zylstra, J. Liu, R. Cichowski, Marie-Claude Faugere, Mary L. Bouxsein, C. Wan, Bart O. Williams, and Thomas L. Clemens. 2007. Lifelong accumulation of bone in mice lacking Pten in osteoblasts. Proceedings of the National Academy of Sciences U.S.A. 104(7): 2259–2264. Inoki, Ken, Hongjiao Ouyang, Tianqing Zhu, Charlotta Lindvall, Yian Wang, Xiaojie Zhang, Qian Yang, Christina Bennett, Yoku Harada, Kryn Stankunas, Cun-yu Wang, Xi He, Ormond A. MacDougald, Ming You, Bart O. Williams, and Kun-Liang Guan. 2006. TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell 126(5): 955–968. Lindvall, Charlotta, Nicole C. Evans, Cassandra R. Zylstra, Yi Li, Caroline M. Alexander, and Bart O. Williams. 2006. The Wnt signaling receptor LRP5 is required for mammary ductal stem cell activity and Wnt1-induced tumorigenesis. Journal of Biological Chemistry 281(46): 35081–35087. Zhang, Chun, Dong Kong, Min-Han Tan, Donald L. Pappas, Jr., Peng-Fei Wang, Jindong Chen, Leslie Farber, Nian Zhang, Han-Mo Koo, Michael Weinreich, Bart O. Williams, and Bin Tean Teh. 2006. Parafibromin inhibits cancer cell growth and causes G1 phase arrest. Biochemical and Biophysical Research Communications 350(1): 17–24.

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Photo taken by Veronique Schulz of the Mirant lab.

VARI | 2007

Human melanoma cells.

Human melanoma cells were fixed and stained to show the nuclei (blue), actin stress fibers (red), and focal adhesions (green dots).

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H. Eric Xu, Ph.D. Laboratory of Structural Sciences

Dr. Xu went to Duke University and the University of Texas Southwestern Medical Center, where he earned his Ph.D. in molecular biology and biochemistry. Following a postdoctoral fellowship with Carl Pabo at MIT, he moved to GlaxoWellcome in 1996 as a research investigator of nuclear receptor drug discovery. Dr. Xu joined VARI as a Senior Scientific Investigator in July 2002 and was promoted to Distinguished Scientific Investigator in March 2007.

Staff Laboratory Staff Jiyuan Ke, Ph.D. Schoen Kruse, Ph.D. Augie Pioszak, Ph.D. David Tolbert, Ph.D. Yong Xu, Ph.D.

Students Chenghai Zhang, Ph.D. X. Edward Zhou, Ph.D. Jennifer Daugherty, B.S. Amanda Kovach, B.S. Kelly Powell, B.S.

Visiting Scientist Visiting Scientists Ross Reynolds, Ph.D.

VARI | 2007

Research Interests Our laboratory is employing multidisciplinary approaches to study the structures and functions of protein complexes that play key roles in major signaling pathways, and to use the resulting structural information to develop therapeutic agents for the treatment of human disease, including cancer and diabetes. Currently we are focusing on three families of proteins: nuclear hormone receptors, the Met tyrosine kinase receptor, and G protein–coupled receptors, because these proteins, beyond their fundamental roles in biology, are important drug targets for many human diseases.

Nuclear hormone receptors The nuclear hormone receptors form a large family comprising ligand-regulated and DNA-binding transcriptional factors. The family includes receptors for classic steroid hormones such as estrogen, progesterone, androgens, and glucocorticoids, as well as receptors for peroxisome proliferator activators, vitamin D, vitamin A, and thyroid hormones. One distinguishing fact about these classic receptors is that they are among the most successful targets in the history of drug discovery: every receptor has one or more cognate synthetic ligands currently being used as medicines. The nuclear receptors also include a class of “orphan” receptors for which no ligand has been identified. In the last two years, we have developed the following projects centering on the structural biology of nuclear receptors.

Peroxisome proliferator–activated receptors

The peroxisome proliferator–activated receptors (PPARα, δ, and γ) are key regulators of glucose and fatty acid homeostasis and as such are important therapeutic targets for treating cardiovascular disease, diabetes, and cancer. To understand the molecular basis of ligand-mediated signaling by PPARs, we have determined crystal structures of each PPAR’s ligand-binding domain (LBD) bound to diverse ligands including fatty acids, the lipid-lowering fibrate drugs, and a new generation of anti-diabetic drugs, the glitazones. We have also determined the crystal structures of these receptors bound to coactivators or co-repressors. We are developing this project into the structures of large PPAR fragment/DNA complexes.

Human glucocorticoid and mineralocorticoid receptors

The human glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are classic steroid hormone receptors that are key to a wide spectrum of human physiology including immune/inflammatory responses, metabolic homeostasis, and control of blood pressure. Both are well-established drug targets. GR ligands such as dexamethasone (Dex) and fluticasone propionate (FP) are used to treat asthma, leukemia, and autoimmune diseases; MR ligands such as spironolactone and eplerenone are used to treat hypertension and heart failure. However, the clinical use of these ligands is limited by undesirable side effects partly associated with their receptor cross-reactivity or low potency. Thus, the discovery of highly potent and more-selective ligands for GR and MR is an important goal of pharmaceutical research. We have determined a crystal structure of the GR LBD bound to dexamethasone and the MR LBD bound to corticosterone, both of which are in complex with a coactivator peptide motif. These structures provide a detailed basis for the specificity of hormone recognition and coactivator assembly by GR and MR. Currently we are studying receptor-ligand interactions by crystallizing GR and MR with various steroid or nonsteroid ligands. In collaboration with Brad Thompson and Raj Kumar at the University of Texas Medical Branch at Galveston, we are also extending our studies to the structure of a large GR fragment bound to DNA.

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The human androgen receptor

The androgen receptor (AR) is the central molecule in the development and progression of prostate cancer, and as such it serves as the molecular target of anti-androgen therapy. However, most prostate cancer patients develop resistance to such therapy, mainly due to mutations in this hormone receptor that alter its three-dimensional structure and allow AR to escape repression. The growth of prostate cancer cells that harbor a mutated AR is then no longer dependent on androgen, making anti-hormone therapy ineffective. This form of hormone-independent prostate cancer is highly aggressive and is responsible for most deaths from prostate cancer. The development of effective therapies requires a detailed understanding of the structure and functions of the central molecule, i.e., the androgen receptor and its interactions with hormones and co-regulators. In this project, we are aiming to determine the structures of the mutated AR proteins that alter the response to anti-hormone therapy. In collaboration with Donald MacDonnell at Duke University, we are working on the crystal structure of the full-length AR/DNA complex.

Structural genomics of nuclear receptor ligand-binding domains

The LBDs of nuclear receptors contain key structural elements that mediate ligand-dependent regulation of these receptors, and as such, LBDs have been the focus of intense structural studies. There are only a few orphan nuclear receptors for which the LBD structure remains unsolved. In the past two years, we have focused on structural characterization of two orphan receptors: constitutive androstane receptor (CAR) and steroidogenic factor-1 (SF-1). The CAR structure reveals a compact LBD fold containing a small pocket that is only half the size of the pocket in PXR, a receptor closely related to CAR. The constitutive activity of CAR appears to be mediated by a novel linker helix between the C-terminal AF-2 helix and helix 10. On the other hand, SF-1 is regarded as a ligand-independent receptor, but its LBD structure reveals the presence of a phospholipid ligand in a surprisingly large pocket; its size is more than twice that of the pocket in the mouse LRH-1, a closely related receptor. The bound phospholipid is readily exchanged and modulates SF-1 interactions with coactivators. Mutations designed to reduce the size of the SF-1 pocket or to disrupt hydrogen bonds formed with the phospholipid abolish SF-1/coactivator interactions and reduce SF-1 transcriptional activity. These findings establish that SF-1 is a ligand-dependent receptor and suggest an unexpected link between nuclear receptors and phospholipid signaling pathways.

The Met tyrosine kinase receptor MET is a tyrosine kinase receptor that is activated by hepatocyte growth factor/scatter factor (HGF/SF). Aberrant activation of the Met receptor has been linked to the development and metastasis of many types of solid tumors and has been correlated with poor clinical prognosis. HGF/SF has a modular structure with an N-terminal domain, four kringle domains, and an inactive serine protease domain. The structure of the N-terminal domain with a single kringle domain (NK1) has been determined. Less is known about the structure of the Met extracellular domain. The molecular basis of the MET receptorâ&#x20AC;&#x201C;HGF/SF interaction and the activation of MET signaling by this interaction remains poorly understood. In collaboration with George Vande Woude and Ermanno Gherardi, we are developing this project to solve the crystal structure of the Met receptor/HGF complex.

VARI | 2007

G Protein–coupled receptors G protein–coupled receptors (GPCRs) form the largest family of receptors in the human genome; they are receptors for diverse signals carried by photons, ions, small chemicals, peptides, and hormones. These receptors account for over 40% of drug targets, but the structure of these receptors remains a challenge because they are seven-transmembrane receptors. Currently, there is only one reported GPCR structure, for an inactive form of bovine rhodopsin. Many important questions regarding GPCR ligand binding and activation remain unanswered. From our standpoint, GPCRs are similar to nuclear hormone receptors with respect to regulation by protein-ligand and protein-protein interactions. Due to their importance, we have decided to take on studies of the structural basis of ligand binding in, and activation of, GPCRs.

External Collaborators Doug Engel, University of Michigan, Ann Arbor Ermanno Gherardi, University of Cambridge, UK Steve Kliewer, University of Texas Southwestern Medical Center, Dallas David Mangelsdorf, University of Texas Southwestern Medical Center, Dallas Donald MacDonnell, Duke University, Durham, North Carolina Stoney Simmons, National Institutes of Health, Bethesda, Maryland Scott Thacher, Orphagen Pharmaceuticals, San Diego, California Brad Thompson and Raj Kumar, University of Texas Medical Branch at Galveston Ming-Jer Tsai, Baylor College of Medicine, Houston, Texas

From left, standing: E. Xu, Daugherty, Tolbert, Kovach, Powell, Zhang, Zhou kneeling: Kruse, Pioszak, Y. Xu, Ke

Recent Publications Choi, Mihwa, Antonio Moschetta, Angie L. Bookout, Li Peng, Michihisa Umetani, Sam R. Holmstrom, Kelly Suino-Powell, H. Eric Xu, James A. Richardson, Robert D. Gerard, David J. Mangelsdorf, and Steven A. Kliewer. 2006. Identification of a hormonal basis for gallbladder filling. Nature Medicine 12(11): 1253–1235.

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2006 Van Andel Research Institute Symposium

VARI | 2007

Winning the War against Cancer: From Genomics to Bedside and Back In September 2006, the Van Andel Research Institute honored the lifetime achievements of George F. Vande Woude with a symposium titled â&#x20AC;&#x153;Winning the War against Cancer: From Genomics to Bedside and Backâ&#x20AC;?. Organized by Nicholas Duesbery, Tony Hunter, and Bin Teh, the three-day symposium featured noted speakers, including three Nobel laureates; presentation of the Daniel Nathans Award; and a reception honoring Dr. Vande Woude. More than 250 scientists attended the meeting.

Symposium photos by Jindong Chen.

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Invited Speakers

Jerry Adams

Tim Hunt

Tony Pawson

Walter & Eliza Hall Institute

Clare Hall Laboratories

Mount Sinai Hospital Research Institute

James P. Allison

Tony Hunter

Bruce Ponder

Memorial Sloan-Kettering Cancer Center

Salk Institute for Biological Studies

Cancer Research U.K

Anton Berns

Arnold Levine

Martine Roussel

Nederlands Kanker Institute

Institute for Advanced Study

St. Jude Children’s Research Hospital

J. Michael Bishop

David M. Livingston

Janet Rowley

University of California, San Francisco

Dana-Farber Cancer Institute

University of Chicago

Joan S. Brugge

James L. Maller

Joseph Schlessinger

Harvard Medical School

University of Colorado School of Medicine

Yale University School of Medicine

Suzanne Cory

Paul A. Marks

Phillip A. Sharp

Walter & Eliza Hall Institute

Memorial Sloan-Kettering Cancer Center

Massachusetts Institute of Technology

Michael Dean

Frank McCormick

Louis Staudt

National Cancer Institute–Frederick

University of California, San Francisco

National Cancer Institute

Edward Harlow

William Muller

Craig Thompson

Harvard Medical School

McGill University

Abramson Family Cancer Research Institute

Stephen Hughes

Morag Park

George Vande Woude

National Cancer Institute–Frederick

McGill University

Van Andel Research Institute

Karen Vousden Beatson Institute for Cancer Research

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VARI | 2007

All of us who have had the privilege of working with George over the years join in appreciation and thanks for his constant interest, wisdom, insights, and humor. 93

Duesbery, N.S., and B.T. Teh. 2007. Cancer: biology and therapeutics—a tribute to George Vande Woude. Oncogene 26(9): 1258–1259. Teh, B.T., and N. Duesbery. 2007. A tribute to George F. Vande Woude, a man of character: 2006 Scientific Symposium “Winning the War against Cancer: From Genomics to Bedside and Back.” Cancer Research 67(6): 2394–2395.

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Daniel Nathans Memorial Award

VARI | 2007

Daniel Nathans Memorial Award

The Daniel Nathans Memorial Award was established in memory of Dr. Daniel Nathans, a distinguished member of our scientific community and a founding member of VARI’s Board of Scientific Advisors. We established this award to recognize individuals who emulate Dan and his contributions to biomedical and cancer research. It is our way of thanking and honoring him for his help and guidance in bringing Jay and Betty Van Andel’s dream to reality. The Daniel Nathans Memorial Award was announced at our inaugural symposium, “Cancer & Molecular Genetics in the Twenty-First Century”, in September 2000. 95

Previous Award Recipients 2000 2001 2002 2003 2004 2005

Richard D. Klausner, M.D. Francis S. Collins, M.D., Ph.D. Lawrence H. Einhorn, M.D. Robert A. Weinberg, Ph.D. Brian Druker, M.D. Tony Hunter, Ph.D., and Tony Pawson, Ph.D.

Tony Hunter, Ph.D.

Tony Pawson, Ph.D.

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Postdoctoral Fellowship Program

VARI | 2007

Postdoctoral Fellowship Program The Van Andel Research Institute provides postdoctoral training opportunities to Ph.D. scientists beginning their research careers. The fellowships help promising scientists advance their knowledge and research experience while at the same time supporting the research endeavors of VARI. The fellowships are funded in three ways: 1) by the laboratories to which the fellow is assigned; 2) by the VARI Office of the Director; or 3) by outside agencies. Each fellow is assigned to a scientific investigator who oversees the progress and direction of research. Fellows who worked in VARI laboratories in 2006 and early 2007 are listed below.

Jennifer Bromberg-White

Dan Huang

Michael Shafer

Penn. State University College of Medicine, Hershey VARI mentor: Nick Duesbery

Peking Union Medical College, China VARI mentor: Bin Teh

Michigan State University, East Lansing VARI mentor: Brian Haab

Philippe Depeille

Schoen Kruse

Suganthi Sridhar

University of Montpellier, France VARI mentor: Nicholas Duesbery

University of Colorado, Boulder VARI mentor: Eric Xu

Southern Illinois University, Carbondale VARI mentor: Cindy Miranti

Yan Ding

Brendan Looyenga

Peng Fei Wang

Peking Union Medical College, China VARI mentor: Nicholas Duesbery

University of Michigan, Ann Arbor VARI mentor: James Resau

Fourth Military Medical University, China VARI mentor: Bin Teh

Mathew Edick

Douglas Luccio-Camelo

Yi-Mi Wu

University of Tennessee, Memphis VARI mentor: Cindy Miranti

University of Brazil, Rio de Janeiro VARI mentor: Bin Teh

National Tsin-Hua University, Taiwan VARI mentor: Brian Haab

Kathryn Eisenmann

Daisuke Matsuda

Yong Xu

University of Minnesota, Minneapolis VARI mentor: Arthur Alberts

Kitasato University, Japan VARI mentor: Bin Teh

Shanghai Institute of Materia Medica, China VARI mentor: Eric Xu

Leslie Farber

Augen Pioszak

Xin Yao

George Washington University, Washington, D.C. VARI mentor: Bin Teh

University of Michigan, Ann Arbor VARI mentor: Eric Xu

Tianjin Medical University, China VARI mentor: Bin Teh

Kunihiko Futami

Daniel Robinson

Chenghai Zhang

Tokyo University of Fisheries, Japan VARI mentor: Bin Teh

University of California, Davis VARI mentor: Bart Williams

Virus Institute of the CDC, China VARI mentor: Eric Xu

Quliang Gu

Dorine Savreux

Xiaoyin Zhou

Sun Yat-sen University of Medicine, China VARI mentor: Brian Cao

Virology University, France VARI mentor: Michael Weinreich

University of Alabama â&#x20AC;&#x201C; Birmingham VARI mentor: Eric Xu

Carrie Graveel University of Wisconsin â&#x20AC;&#x201C; Madison VARI mentor: George Vande Woude

Jessica Hessler University of Michigan, Ann Arbor VARI mentor: Craig Webb

Holly Holman University of Glasgow, U.K. VARI mentor: Arthur Alberts

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Student Programs

VARI | 2007

Grand Rapids Area Pre-College Engineering Program The Grand Rapids Area Pre-College Engineering Program (GRAPCEP) is administered by Davenport University and jointly sponsored and funded by Pfizer, Inc., and VARI. The program is designed to provide selected high school students, who have plans to major in science or genetic engineering in college, the opportunity to work in a research laboratory. In addition to research methods, the students also learn workplace success skills such as teamwork and leadership. The three 2006 GRAPCEP students were

Alicia Coleman

(Resau/Duesbery)

Creston High School

Megan Spencer

(Holmen)

Creston High School

Ware-Van Brunt

(Webb)

Creston High School

From left: Ware-Van Brunt, Coleman, Spencer

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Summer Student Internship Program The VARI student internships were established to provide college students with an opportunity to work with professional researchers in their fields of interest, to use state-of-the-art equipment and technologies, and to learn valuable people and presentation skills. At the completion of the 10-week program, the students summarize their projects in an oral presentation. From January 2006 to March 2007, VARI hosted 62 students from 23 colleges and universities in formal summer internships under the Frederik and Lena Meijer Student Internship Program and in other student positions during the year. An asterisk (*) indicates a Meijer student intern.

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Andrews University, Berrien Springs, Michigan

Grand Valley State University, Allendale, Michigan

Christopher Armstrong* (Xu)

Aquinas College, Grand Rapids, Michigan

Krysta Collins (Haab) Natalie Kent (Hay) Sara Kunz (Hay) Rebecca Trierweiler (Hay)

Calvin College, Grand Rapids, Michigan

David Dornboss, Jr. (Weinreich) Jonathan Dudley (Vande Woude) Amanda Field* (Williams) Alysha Kett* (Vande Woude) Geoff Kraker (MacKeigan) Kate Leese (Weinreich) Sarah Mange (Williams) Devin Mistry (Haab) Jose Toro (Hay) Bill Wondergem (Teh)

Case Western Reserve University, Cleveland

Elianna Bootzin* (Hay)

Central Michigan University, Mount Pleasant

Sarah DeVos* (Teh)

Franciscan University, Steubenville, Ohio

Joan Krilich* (Cavey)

Grand Rapids Community College, Michigan

Wei Luo (Resau)

Angelique Berens (Vande Woude) Eric Graf (Miranti) Nick Miltgen (Resau) Gary Rajah* (Miranti) Lisa Orcasitas (Duesbery) Sara Ramirez (Resau) Brittany Stropich* (Alberts)

Indiana University, Bloomington

Erin Jefferson* (Webb)

Kalamazoo College, Kalamazoo, Michigan

Adam Granger (Holmen)

Marquette University, Milwaukee, Wisconsin

Michael Avallone (Teh)

Miami University, Oxford, Ohio

Grant Van Eerden (Resau)

Michigan State University, East Lansing

David Achila (Xu/Weinreich) Ying-Chou Chen, M.S. (Weinreich) Michelle Dawes (Duesbery) Aaron DeWard (Alberts) Pete Haak, B.S. (Resau) Kate Jackson (Resau) Andrew Kraus (Vande Woude) Sebla Kutluay, B.S. (Triezenberg) Chih-Shia Lee, M.S. (Duesbery) Charles Miller (Weinreich) Kara Myslivec (Resau) Katie Sian, B.S. (MacKeigan)

VARI | 2007

2006 summer intern students

Nanjing Medical University, China

University of Notre Dame, South Bend, Indiana

Kristin Buzzitta (Teh) Joe Church* (Duesbery) Margaret Condit (Teh)

Xin Wang (Cao) Ning Xu (Cao) Aixia Zhang (Cao) Jin Zhu (Cao)

Northern Illinois University, Dekalb

Mohan Thapa (Resau)

Purdue University, West Lafayette, Indiana

Brent Goodman* (Furge)

University of Bath, United Kingdom Naomi Asantewa-Sechereh (Duesbery) Louise Haste (Weinreich)

University of Illinois, Champaign-Urbana

Huong Tran (Resau)

University of Mannheim, Germany Dagmar Hildebrand (Alberts) Stefan Kutscheidt (Miranti)

University of Michigan, Ann Arbor Katherine Koelzer* (Swiatek) Erin Lambers (Duesbery) Jennifer Lunger* (Haab) Renee VanderLaan* (Holmen)

University of North Carolina, Chapel Hill

Jourdan Stuart* (Resau)

Western Michigan University, Kalamazoo

Mallory Walters (Holmen)

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Han-Mo Koo Memorial Seminar Series

VARI | 2007

Han-Mo Koo Memorial Seminar Series This seminar series is dedicated to the memory of Dr. Han-Mo Koo, who was a VARI Scientific Investigator from 1999 until his passing in May of 2004.

January 2006

W. Michael Kuehl, National Cancer Institute

“Molecular pathogenesis of multiple myeloma”

Kenneth Bradley, University of California, Los Angeles

“Anthrax lethal toxin”

Valina L. Dawson, Johns Hopkins University

“Life and death signaling by PAR in the brain”

Ted M. Dawson, Johns Hopkins University

“Genetic clues to the mysteries of Parkinson’s disease” 103

Andy Futreal, Wellcome Trust Sanger Institute

“Surveying somatic mutations in human cancer by targeted re-sequencing”

February

Morag Park, McGill University, Montreal

“The Met receptor tyrosine kinase: from tubes to tumorigenesis”

Nicholas J. Vogelzang, Nevada Cancer Institute

“Treatment options in metastatic renal cell carcinoma: an embarrassment of riches”

March

Teresa L. Burgess, Amgen, Inc.

“Fully human monoclonal antibodies to hepatocyte growth factor”

Kenneth L. van Golen, University of Michigan

“Understanding the roles of Rho and Rac GTPases in prostate cancer bone metastasis”

Thomas W. Glover, University of Michigan

“Mechanisms and significance of chromosome fragile site instability in cancer”

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April

Scientific Report

Stephen J. O’Brien, National Cancer Institute

“Genetic architecture of complex diseases: lessons from AIDS”

Richard Treisman, Cancer Research, U.K.

“Regulation of the SRF transcription factor via cytoskeletal and MAP kinase signaling pathways”

Dean Felsher, Stanford University

“Molecular and cellular basis of oncogene addiction”

May

Partho Ghosh, University of California, San Diego

“Met as a target for bacterial intracellular invasion”

Ming-Jer Tsai, Baylor College of Medicine

“Role of nuclear receptor co-regulator SRC-3/AIB1 in prostate cancer”

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Laura S. Schmidt, National Cancer Institute–Frederick

“Understanding the genetics of kidney cancer through familial renal cancer studies”

David Drubin, University of California, Berkeley

“Harnessing actin dynamics for endocytic trafficking”

Thomas Clemens, University of Alabama at Birmingham

“Oxygen sensing and osteogenesis”

June

Robert J. Motzer, Memorial Sloan-Kettering Cancer Center

“Targeted therapy for metastatic renal cell carcinoma”

Tom Blumenthal, University of Colorado

“Widespread operons in the C. elegans genome: why and how”

July

Rudolf Jaenisch, Whitehead Institute and Massachusetts Institute of Technology

“Nuclear cloning, stem cells, and pluripotency”

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August

Douglas R. Green, St. Jude Children’s Research Hospital

“p53, mitochondria, and apoptosis”

Gregory S. Fraley, Hope College

“Food, fat, and sex: how the brain integrates energetics and reproduction”

September

Stephen A. Krawetz, Wayne State University

“Genome reprogramming and the paternal contribution at fertilization”

Hilary Koprowski, Thomas Jefferson University

“Rabies at the dawn of the 21st century”

October

Y. Eugene Chen, University of Michigan

“Nitro-lipids and PPARs in metabolic syndrome”

Jacques Pouyssegur, Institute of Signaling, University of Nice

“Hypoxia signaling and cancer progression”

November

Chuxia Deng, National Institute of Diabetes and Digestive and Kidney Diseases

“BRCA1 and tumorigenesis in animal models”

Dafna Bar-Sagi, New York University

“RAS signaling: new trails in familiar territory”

December

Kun-Liang Guan, University of Michigan

“Regulation and function of the TSC-mTOR pathway”

January 2007

Moses Lee, Hope College

“Regulation of the topoisomerase IIα gene using polyamides that bind to the inverted CCAAT box present in the promoter”

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February

Scientific Report

Raj Kumar, University of Texas Medical Branch

“Structure and functions of the steroid receptors”

David Kimelman, University of Washington

“Tales of tails: the importance of Bmp signaling in embryogenesis”

Arthur L. Haas, Louisiana State University

“ISG15 and ubiquitin as antagonistic regulators of cell transformation”

March

S. Stoney Simons, Jr., National Institutes of Health

“A systems biology approach to steroid hormone action: towards a quantitative understanding of whole cell responses to steroid hormones”

John D. Shaughnessy, Jr., University of Arkansas for Medical Science

“Using genomics to better understand the biology and clinical course of multiple myeloma”

106

Melanie H. Cobb, University of Texas Southwestern Medical Center

“MAP kinase signaling in pancreatic beta cells”

VARI | 2007

Van Andel Research Institute Organization

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Van Andel Research Institute |

Scientific Report

David L. Van Andel, Chairman and CEO, Van Andel Institute VARI Board of Trustees

David L. Van Andel, Chairman and CEO Fritz M. Rottman, Ph.D. James B. Wyngaarden, M.D.

Board of Scientific Advisors 108

The Board of Scientific Advisors advises the CEO and the Board of Trustees, providing recommendations and suggestions regarding the overall goals and scientific direction of VARI. The members are

Michael S. Brown, M.D., Chairman Richard Axel, M.D. Joseph L. Goldstein, M.D. Tony Hunter, Ph.D. Phillip A. Sharp, Ph.D.

Scientific Advisory Board The Scientific Advisory Board advises the VARI Director, providing recommendations and suggestions specific to the ongoing research, especially in the areas of cancer, genomics, and genetics. It also coordinates and oversees the scientific review process for the Instituteâ&#x20AC;&#x2122;s research programs. The members are

Alan Bernstein, Ph.D. Joan Brugge, Ph.D. Webster Cavenee, Ph.D. Frank McCormick, Ph.D. Davor Solter, M.D., Ph.D.

VARI | 2007

Office of the Director

George F. Vande Woude, Ph.D. Director

Deputy Director for Clinical Programs

Deputy Director for Special Programs

Deputy Director for Research Operations

Rick Hay, Ph.D., M.D.

James H. Resau, Ph.D.

Nicholas S. Duesbery, Ph.D.

Director for Research Administration

Administrator to the Director

Science Editor

Roberta Jones

Michelle Bassett

David E. Nadziejka

Administration Group From left, standing: Chastain, Lewis, Koehler, Noyes, Stougaard, Carrigan, Johnson, Resau; Seated: Holman, Jason, Nelson, Novakowski, Rappley

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Scientific Report

Van Andel Institute Administrative Organization The organizational units listed below provide administrative support to both the Van Andel Research Institute and the Van Andel Education Institute.

Executive

Human Resources

Steven R. Heacock, Chief Administrative Officer and General Counsel R. Jack Frick, Chief Financial Officer Ann Schoen, Executive Assistant

Linda Zarzecki, Director Margie Hoving Pamela Murray Angela Plutschouw

Communications and Development Joseph P. Gavan, Vice President Jaime Brookmeyer Sarah Friedman Stephanie Hehl Sarah Lamb

Facilities

110

Samuel Pinto, Manager Jason Dawes Ken De Young Christen Dingman Shelly King Richard Sal Richard Ulrich Pete VanConant Jeff Wilbourn

Finance Timothy Myers, Controller Sandi Essenberg Stephanie Green Richard Herrick Keri Jackson Angela Lawrence Laura Lohr Heather Ly Susan Raymond Andrew Schmidt Jamie VanPortfleet

Glassware and Media Services Richard M. Disbrow, CPM, Manager Bob Sadowski Marlene Sal

Grants and Contracts Carolyn W. Witt, Director Anita Boven Nicole Higgins Sara Oâ&#x20AC;&#x2122;Neal David Ross

Information Technology Bryon Campbell, Ph.D., Chief Information Officer David Drolett, Manager Bill Baillod Tom Barney Phil Bott Nathan Bumstead Charles Grabinski Kenneth Hoekman Kimberlee Jeffries Jason Kotecki Theo Pretorius Thad Roelofs Russell Vander Mey Candy Wilkerson

Investments Office Kathleen Vogelsang Ted Heilman

Procurement Services Richard M. Disbrow, CPM, Manager Heather Frazee Chris Kutchinski Shannon Moore Amy Poplaski John Waldon

Public Affairs John VanFossen

Security Kevin Denhof, CPP, Chief Christen Dingman Sandra Folino Maria Straatsma

Contract Support Mary Morgan, Librarian (Grand Valley State University) Jim Kidder, Safety Manager (Michigan State University)

VARI | 2007

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Scientific Report

Van Andel Institute

Van Andel Institute Board of Trustees David Van Andel, Chairman Peter C. Cook Ralph W. Hauenstein John C. Kennedy

Board of Scientific Advisors Michael S. Brown, M.D., Chairman Richard Axel, M.D. Joseph L. Goldstein, M.D. Tony Hunter, Ph.D. Phillip A. Sharp, Ph.D.

112

Van Andel Research Institute Board of Trustees

Chief Executive Officer David Van Andel

Van Andel Education Institute Board of Trustees David Van Andel, Chairman Donald W. Maine Gordon Van Harn, Ph.D. Gordon Van Wylen, Sc.D.

David Van Andel, Chairman Fritz M. Rottman, Ph.D. James B. Wyngaarden, M.D.

Van Andel Research Institute Director

Van Andel Education Institute Director

George Vande Woude, Ph.D.

Gordon Van Harn, Ph.D.

Chief Administrative Officer and General Counsel Steven R. Heacock

VP Communications and Development Joseph P. Gavan

Chief Financial Officer R. Jack Frick

VARI | 2007

Van Andel Research Institute

DIRECTOR â&#x20AC;&#x201C; George Vande Woude, Ph.D.

Deputy Directors

SCIENTIFIC ADVISORY BOARD

Clinical Programs Rick Hay, Ph.D., M.D. Special Programs James Resau, Ph.D. Research Operations Nick Duesbery, Ph.D.

Director for Research Administration

Alan Bernstein, Ph.D. Joan Brugge, Ph.D. Webster Cavenee, Ph.D. Frank McCormick, Ph.D. Davor Solter, Ph.D.

Roberta Jones

BASIC SCIENCE

SPECIAL PROGRAMS 113

Division of Quantitative Sciences

Cancer Cell Biology

Animal Models

Brian Haab, Ph.D. Cancer Immunodiagnostics

Nicholas Duesbery, Ph.D. Cancer & Developmental Cell Biology

Brian Cao, M.D. Antibody Technology

George Vande Woude, Ph.D. Molecular Oncology

Bart Williams, Ph.D. Cell Signaling & Carcinogenesis

Pamela Swiatek, Ph.D., M.B.A. Germline Modification

Craig Webb, Ph.D. Tumor Metastasis & Angiogenesis

Cancer Genetics

Bryn Eagleson, A.A. Transgenics and Vivarium

Signal Transduction

Bin Teh, M.D., Ph.D. Cancer Genetics

Pamela Swiatek, Ph.D., M.B.A. Cytogenetics

Art Alberts, Ph.D. Cell Structure & Signal Intergration

Structural Biology

Bin Teh, M.D., Ph.D. Sequencing

Cindy Miranti, Ph.D. Integrin Signaling & Tumorigenesis

Eric Xu, Ph.D. Structural Sciences

Art Alberts, Ph.D. Flow Cytometry

DNA Replication & Repair

Systems Biology

Greg Cavey, B.S. Mass Spectrometry and Proteomics

Michael Weinreich, Ph.D. Chromosome Replication

Jeffrey MacKeigan, Ph.D. Systems Biology

James Resau, Ph.D. Molecular Epidemiology

Animal Imaging

Gene Regulation

Rick Hay, Ph.D., M.D. Noninvasive Imaging & Radiation Biology

Steven Triezenberg, Ph.D. Transcriptional Regulation Dean of VAI Graduate School

James Resau, Ph.D.

James Resau, Ph.D. Analytical, Cellular, & Molecular MIcroscopy James Resau, Ph.D. Microarray Technology Kyle Furge, Ph.D. Computational Biology

The Van Andel Institute and/or its affiliated organizations (VARI and VAEI), through its responsible managers, recruits, hires, upgrades, trains, and promotes in all job titles without regard to race, color, religion, sex, national origin, age, height, weight, marital status, disability, pregnancy, or veteran status, except when an accommodation is unavailable or it is a bona fide occupational qualification.

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VARI | 2007

Phone 616.234.5000 Fax 616.234.5001 www.vai.org

Scientific Report

333 Bostwick Avenue, N.E., Grand Rapids, Michigan 49503

Van Andel Research Institute Scientific Report 2007