2008 Van Andel Research Institute Scientific Report by Van Andel Institute

Cover photo: Prostate tumor PC3 cell treated with PI-3K inhibitor. Morphological architecture of a prostate tumor PC3 cell treated with the phosphoinositol-3 kinase inhibitor LY294002 for 72 hours. PC3 cells were immunostained with phallodin (red) and antibody toward vinculin (green). Nuclei were stained with Hoechst (blue). In this cell, inhibition of PI-3K produced the formation of numerous filopodia and microspikes. This is the result of cellular stress, which will eventually lead to the death of the cell. Photo by Laura Lamb of the Miranti lab.

VARI | 2008

Van Andel Research Institute Scientific Report 2008

Van Andel Research Institute |

Scientific Report

Title page illustration: The glucocorticoid receptor. The figure represents the crystal structure of the glucocorticoid receptor (GR) bound to deacylcortivazol, which is a highly potent ligand against childhood leukemia. The GR protein chain is shown as ribbons, with helix 1 in blue and the coactivator helix in red. The deacylcortivazol molecule is shown within the GR structure in green, with the GR ligand-binding pocket shown by the yellow mesh. Structure by the Xu lab.

VARI | 2008

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 Program

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

iii

Van Andel Research Institute |

Scientific Report

Daniel Nathans Memorial Award

Harald zur Hausen, M.D., and Douglas R. Lowy, M.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

2007 | 2008 Seminars

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

VARI | 2008

Directorâ&#x20AC;&#x2122;s Introduction

Van Andel Research Institute |

Scientific Report

George F. Vande Woude Director’s Introduction A few short years ago, the Van Andel Research Institute was an idea that many said wouldn’t work—an independent research institute located in west Michigan with no tie to a major university. Today it is a thriving organization with an excellent reputation, one that is poised to more than double its size and its contributions to science and human health. Perhaps this is most evident in our ability to compete for external grants; success in the tight competition for grant funding is an important measure of our research quality. The National Institutes of Health (NIH) is a major source of research funding in our disciplines, so I am particularly pleased with the awards our VARI scientists received in the past year. Steve Treizenberg has received a three-year R01 award from the National Institutes of Health (NIH) for his project, “Chromatin and Coactivators in HSV-1 Gene Regulation”. Bart Williams also received an R01 award, for five years, for a project titled “Mouse Models to Characterize the Role of Lrp6 in Metabolic Syndrome”. Finally, Eric Xu received a four-year R01 for his project titled “Structural and Functional Studies of the Nuclear Receptor PPARg”, and it is important to note that Eric now has three active R01 grants. Our congratulations go out to Steve, Bart, Eric, and their labs for the rigorous work that went into making their applications successful. The Department of Defense also funds cancer research on a competitive application basis. Early in 2007, VARI had three awards out of 87 projects recommended for funding by the Breast Cancer Research Program, and this was from more than 1,200 proposals that were reviewed. The projects awarded were Kate Eisenmann’s “A Role for Formin-Mediated Cytoskeletal Regulation in the Mesenchymal-Amoeboid Transition in Breast Cancer Development” (Alberts lab); Carrie Graveel’s “Met Signaling Promotes Mammary Stem Cell Proliferation” (Vande Woude lab); and Jim Resau’s “Intravital Imaging of Developing Breast Cancer Lesion of Defined Genomic Profile in a Mouse” (Resau lab). This was clearly an excellent performance. Showing that this was not an aberration, later in 2007, another three awards were made from the DOD Prostate and Ovarian Cancer program. The successful proposals were from Kate Eisenmann (again!), for “Diaphanous-related Formins in Ovarian Cancer Metastasis” (Alberts lab); Laura Lamb, for “Survival Signaling in Prostate Cancer: Role of Androgen Receptor and Integrins in Regulating Survival” (Miranti lab); and Cindy Miranti for “Mechanisms of KAI1/CD82-induced Prostate Cancer Metastasis” (Miranti lab).

VARI | 2008

We have also been successful in competing for funding from nonfederal sources. Funding was received from the state of Michigan to support the Good Manufacturing Practices Facility project under the direction of Rick Hay. Craig Webb received an award for “Establishment of an Innovative Clinical Research Alliance” from the Michigan Strategic Economic Investment & Commercialization Board. Bin Teh has received awards from the National Foundation for Cancer Research and from the VHL Family Alliance Fund for Cancer Research; Art Alberts received project funding from the J.P. McCarthy Fund; and Jennifer Bromberg-White received a fellowship from the Knight’s Templar Foundation. Congratulations to all for a spectacular showing of top-quality proposals! On another note, congratulations go out to Brian Haab, who was promoted to Senior Scientific Investigator in August 2007. Brian’s Laboratory of Cancer Immunodiagnostics is working on developing new techniques and new diagnostic markers for pancreatic cancer, one of the cancers most difficult to treat successfully. Brian has also been elected to a three-year term on the Board of Directors of the U.S. Human Proteome Organization, which supports and promotes the use of proteomics and provides information about the proteomes of various species. We are pleased to announce the formation of VARI International, headed by Bin Tean Teh. VARI International was formed to organize and formalize the Institute’s international opportunities. Currently, two laboratories with foreign host institutes are in operation: NCCS–VARI Translational Research Laboratory (headed by Bin Tean Teh) at the National Cancer Centre of Singapore, and NMU–VARI Antibody Technology Laboratory (headed by Brian Cao) at Nanjing Medical University. NCCS–VARI is focusing on cancers that are prevalent in Asian countries and on translational cancer research. Since its establishment at the end of 2006, NCCS–VARI has expanded to include five clinical fellows, three postdoctoral fellows, four research technicians, and one bioinformatics scientist. We have competed successfully for several research fellowships from local funding agencies, two scientific papers have been published, and a regional mini-symposium has been organized. NMU–VARI is developing a variety of murine and human monoclonal antibodies and antibody fragments for potential clinical diagnostic and therapeutic applications. Since the establishment of NMU-VARI in 2005, six Ph.D. students and four master’s degree students have been trained, three manuscripts have been published, and four grant applications have been submitted. Of those grant application submissions, two have been funded (one from U.S. funding, the other from China). Cooperative/collaborative arrangements at sites in Australia, Sweden, and France are currently being explored. Establishing such laboratories and determining research projects will take into consideration their ability to synergize and complement VARI’s mission. The Program of Translational Medicine under the direction of Craig Webb has established the essential infrastructure and partnerships that allow VARI to collaborate with other institutions for cutting-edge biomarker-driven clinical research. The Center for Molecular Medicine, in partnership with Spectrum Health Hospitals, was established to perform molecular-based diagnostic testing. A community research network of institutions (ClinXus) has also been formed that provides access to biomarker technologies (molecular and imaging), physician expertise, and patient populations for investigators interested in clinical research. The Program of Translational Medicine has also led to the development of a specific personalized medicine protocol in which genomic technologies are used with the XenoBase bioinformatics tools to identify optimal drug combinations that target the genotype of tumors from late-stage cancer patients. An expanded trial of 200 patients will open for enrollment in 2008.

Van Andel Research Institute |

Scientific Report

In June 2007, VARI was awarded full accreditation by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). This distinction recognizes our institutional commitment to responsible and ethical animal care beyond the standards required by law. Our success in receiving this accreditation was made possible only through the concerted efforts of many people, and this achievement is one of which we can all be proud. Finally in the fall, we presented the Daniel Nathans Memorial Award to Harald zur Hausen and Douglas R. Lowy. Dr. zur Hausen’s lab identified infection by papillomavirus as the main cause of cervical cancer, and Dr. Lowy’s studies helped lead to a new way to prepare vaccines that prevent infection by the virus. The importance of this work in terms of improving human health worldwide is obvious, and we are pleased to have these distinguished researchers join the list of Nathans Award recipients. In conclusion, 2007 has been a wonderful year for VAI. With the dedication and ceaseless efforts of our scientists and strong support from our community, we have built a home on “the hill” that is recognized nationwide for its excellence in research. We continue to exceed even our own expectations and we are eagerly looking forward to the years to come.

VARI | 2008

Laboratory Reports

Van Andel Research Institute |

Scientific Report

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

In 1993, Dr. Art Alberts received his Ph.D. in Physiology and Pharmacology at the University of California, San Diego School of Medicine, where he studied with Jim Feramisco. Dr. Alberts trained as a postdoctoral fellow from 1994 to 1997 with Richard Treisman at the Imperial Cancer Research Fund in London, England, where Dr. Treisman is the current Director. From 1997 through 1999, Dr. Alberts was an Assistant Research Biochemist in the laboratory of Frank McCormick at the University of California, San Francisco. In January 2000, Dr. Alberts joined VARI as a Scientific Investigator; he was promoted in 2006 to Senior Scientific Investigator. Also in 2006, he established and became the Director of the Flow Cytometry core facility.

Staff

Students

Visiting Scientists

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

Aaron DeWard, B.S. Christopher Gorter Albert Rodriguez Katja Strunk

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

VARI | 2008

Research Interests The Laboratory of Cell Structure and Signal Integration is devoted to understanding how defects in cellular architecture affect the progression to malignancy and support the tumorigenic platform. The driving hypothesis is that the cytoskeleton does not only structurally support cell morphology, division, and migration, but with its dynamic nature, it organizes intracellular signaling networks in order to effectively interpret proliferative and migratory responses to extracellular cues. On a molecular basis, we are interested in how cells build and control the cytoskeletal assembly machines and how these molecular machines work in concert within the cell. Through combined molecular, cellular, and genetic approaches, the ultimate goal of the lab is identifying defective nodes in the networks governing cytoskeletal remodeling in order to improve diagnosis and devising molecular tools to correct the defective circuits. Our focus is the role of Rho GTPases in signal transduction networks that control cell proliferation and motility. These highly conserved molecular switches act within growth factor responses by alternating between GTP- and GDP-bound forms. Upon GTP binding, Rho proteins undergo a conformational change that allows them to bind to and modulate the activity of effectors that remodel cell shape, drive motility and division, or alter gene expression patterns. One set of GTPase effector proteins acts as machines that assemble components of the cytoskeleton. The mammalian Diaphanous-related formin (mDia) family of actin-nucleating proteins initiate and control the elongation of new actin filaments. The three conserved mDia proteins (mDia1–3), along with insect Diaphanous protein and their budding yeast counterpart Bni1p, are canonical members of the formin family. With our discovery of one of the first formin proteins, mDia2, we have taken a leading role in their characterization. To study the role of mDia1 in vivo, the murine Drf1 gene was knocked out by conventional gene-targeting methods. Both Drf1+/– and Drf1–/– mice become progressively lympho- and myelodysplastic. Drf1-targeted mice are prone to developing tumors; cancers observed thus far include various leukemias, monocytosis, and plasmocytomas. Overall, mice lacking one or both Drf1 alleles phenocopy human myelodysplastic syndrome. Numerous defects in cytoskeletal remodeling have been observed in immune cells, including impaired T cell adhesion, migration, and the appearance of supernumerary centrosomes, which are indicative of failed cell division. These results were published in the Journal of Biological Chemistry and in Cancer Research. In the first paper with lead author Kate Eisenmann, entitled “T cell responses in mammalian Diaphanous-related formin mDia1 knock-out mice”, we demonstrated a role for mDia1 in normal immune cell function. Disruption of mDia1 leads to fewer T cells in secondary lymphoid organs in Drf1-null animals. T cell adhesion, migration, and proliferation upon activation were all impaired in T cells derived from Drf1-targeted mice. These results pointed to a crucial role for mDia1 in the dynamic regulation of the actin cytoskeleton in activated T cells. The second paper, with lead author Jun Peng, “Myeloproliferative defects following targeting of the Drf1 gene encoding the mammalian Diaphanous-related formin mDia1”, showed that mDia1 also plays an essential role in myelopoiesis. As animals age, they develop myeloproliferative defects in both the bone marrow and peripheral blood. These observations point to a crucial role of mDia1 in maintaining myeloid homeostasis, potentially by functioning as a tumor suppressor or susceptibility gene.

Van Andel Research Institute |

Scientific Report

Overall, the mDia1 knock-out phenotype resembles human chronic myeloproliferative syndrome (MPS) and myelodysplastic syndrome (MDS). Both MPS and MDS have been characterized as preleukemic states, with variable lymphopenia, excess or dysfunctional erythrocytes, chronic myelomonocytic leukemia, ineffective hematopoiesis, and, in some cases, advancing myelofibrosis. Instances of neutrophilic dermatoses (Sweet syndrome) can also accompany MDS and MPS. MDS is a frequent hematologic disorder that typically affects older patients and is thought to be a stem cell disorder. Dysplastic features of the nucleus or cytoplasm, as observed in the mDia1 knock-out mice, and altered cellularity of the bone marrow are also characteristic of MDS. The effect of Drf1 gene targeting and the resulting mDia1 knock-out suggests that the DRF1 gene for human mDia1 is affected in MPS, MDS, or other preleukemic pathologies. Ongoing studies are focused on examining if defects in the human gene encoding mDia1 might be defective in MDS patients.

From left: Matheson, Rodriguez, West, Strunk, DeWard, Guthrey, Leali, Kitchen, Eisenmann, Alberts

Recent Publications Uma, Kamasani, James B. DuHadaway, Arthur S. Alberts, and George C. Prendergast. In press. mDia function is critical for the cell suicide program triggered by farnesyl transferase inhibition. Cancer Biology & Therapy. Sarmiento, Corina, Weigang Wang, Athanassios Dovas, Hideki Yamaguchi, Mazen Sidani, Mirvat El-Sibai, Vera DesMarais, Holly A. Holman, Susan Kitchen, Jonathan M. Backer, Art Alberts, and John Condeelis. 2008. WASP family members and formin proteins coordinate regulation of cell protrusions in carcinoma cells. Journal of Cell Biology 180(6): 1245–1260. Wang, P., M.R. Bowl, S. Bender, J. Peng, L. Farber, J. Chen, A. Ali, Z. Zhang, A.S. Alberts, R.V. Thakker, A. Shilatifard, B.O. Williams, and B.T. Teh. 2008. Parafibromin, a component of the human PAF complex, regulates growth factors and is required for embryonic development and survival in adult mice. Molecular and Cellular Biology 28(9): 2930–2940. Dent, Erik W., Adam V. Kwiatkowski, Leslie M. Mebane, Ulrike Philippar, Melanie Barzik, Douglas A. Rubinson, Stephanie Gupton, J. Edward Van Veen, Craig Furman, Jiangyang Zhang, Arthur S. Alberts, Susumu Mori, and Frank B. Gertler. 2007. Filopodia are required for cortical neurite initiation. Nature Cell Biology 9(12): 1347–1359. Eisenmann, Kathryn M., Richard A. West, Dagmar Hildebrand, Susan M. Kitchen, Jun Peng, Robert Sigler, Jinyi Zhang, Katherine A. Siminovitch, and Arthur S. Alberts. 2007. T cell responses in mammalian Diaphanous-related formin mDia1 knock-out mice. Journal of Biological Chemistry 282(34): 25152–25158. Gupton, Stephanie L., Katherine Eisenmann, Arthur S. Alberts, and Clare M. Waterman-Storer. 2007. mDia2 regulates actin and focal adhesion dynamics and organization in the lamella for efficient epithelial cell migration. Journal of Cell Science 120(19): 3475–3487. Peng, Jun, Susan M. Kitchen, Richard A. West, Robert Sigler, Kathryn M. Eisenmann, and Arthur S. Alberts. 2007. Myeloproliferative defects following targeting of the Drf1 gene encoding the mammalian Diaphanous-related formin mDia1. Cancer Research 67(16): 7565–7571. 8

VARI | 2008

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 was promoted to Senior Scientific Investigator in July 2006.

Staff

Students

Quliang Gu, Ph.D. Ping Zhao, M.S. Tessa Grabinski, B.S.

Guipeng Ding Jenna Manby Rui Sun Ning Xu Aixia Zhang

Van Andel Research Institute |

Scientific Report

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 clinical diagnostic applications. Further, antibodies are making rapid inroads into clinical treatment of a variety of diseases, driven by technological evolution from chimeric and humanized to fully human antibodies. Functioning as an antibody production core facility at VARI, our lab’s primary responsibility is to develop state-of-the-art services and technology platforms for monoclonal antibody (mAb) production and characterization. Our technologies and services include antigen preparation and animal immunization; peptide design and coupling to protein carriers; immunization with living or fixed cells; conventional antigen/adjuvant preparation; and immunizing a wide range of antibody-producing models (including mice, rats, rabbits, and transgenic or knock-out mice). Our work also includes the generation of hybridomas from spleen cells of immunized mice and rats; hybridoma expansion and subcloning; cryopreservation of hybridomas; mAb isotyping; ELISA screening of hybridoma supernatants; mAb characterization by immunoprecipitation, immunohistochemistry, immunofluorescence staining, Western blot, FACS, and in vitro bioassays; conjugation of mAbs to enzymes, biotin/streptavidin, or fluorescent reporters; and development of detection kits such as sandwich ELISA. We contract our services to biotechnology companies, producing and purifying mAbs for their research and for diagnostic kit development. Over the last year, this core has finished 14 antibody development projects for researchers and industrial users in Michigan and nationwide. Michigan’s 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 the CTA in March 2005. The goals of MATC are to provide state-ofthe-art antibody technologies and services to research scientists; to generate, characterize, produce, and purify a wide variety of 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. We also carry out research and collaboration projects, which use both murine mAbs and human antibody fragments generated in our lab, aimed at developing cancer diagnostic and therapeutic applications.

•

Epitope mapping and characterization of a Met-binding peptide using phage-display peptide libraries. This project is to screen for a specific Met-binding peptide from a random-peptide phage-display library that could be used as an in vivo imaging agent (and possibly as a therapeutic carrier) when labeled with radioisotopes or conjugated with chemotherapeutics. A subtractive bio-panning approach on intact cells was used. A Met-binding peptide was obtained that recognizes the Met extracellular domain under native conditions and internalizes upon binding to the Met receptor. In vivo imaging showed that the radiolabeled peptide in a mouse xenograft model had tumor-associated activity. We are modifying this peptide to increase its binding affinity, and we are screening new Met-binding peptides having higher affinity for future clinical applications.

•

Development of highly specific anti-Met mouse mAbs with potential application for clinical immunohistochemical diagnosis. In collaboration with Beatrice Knudsen’s lab at the Fred Hutchinson Cancer Research Center, we have developed a monoclonal antibody, designated MET4, with the goal of accurately and reproducibly measuring MET in formalin-fixed paraffin-embedded (FFPE) tissues. MET4 was selected as the best probe from a pool of MET-avid monoclonal antibodies, based on its specific staining pattern in FFPE preparations of normal human prostate tissues. The reliability of MET4 immunohistochemistry was assessed by comparing MET4-IHC in FFPE cell pellets with immunoblotting analysis, which demonstrated a high avidity of MET4 for formalin-treated MET. These properties encourage further development of MET4 as a multipurpose molecular diagnostic reagent to help guide selection of individual patients being considered for treatment with METantagonistic drugs.

VARI | 2008

•

haracterization of anti-EGFR and anti-Met human Fab fragments and conjugation with chemotherapeutics C to generate reagents for preclinical studies. In collaboration with the Ministry of Health’s Key Laboratory of Antibody Technology in Nanjing Medical University, we screened several Fab fragments (from a naïve human Fab phage library constructed in our lab in late 2004) that specifically recognize Met and EGFR. By modifying and improving bio-panning strategies, we have selected Fab fragments that recognize the Met and EGFR extracellular domains in native confirmation with reasonable affinity. These fragments have internalization properties making them attractive as conjugate reagents for immuno-chemotherapy or immuno-radiation therapy against cancer. We have conjugated anti-EGFR human Fab to paclitaxel as an immuno-chemotherapy reagent and investigated its in vitro anti-tumor efficacy using cell proliferation and apoptosis assays. We will further explore its in vivo anti-tumor efficacy in xenograft or orthotopic animal models, and we will label this Fab fragment with radioisotopes to evaluate its potential as an immuno-radiation reagent for in vivo imaging diagnosis and immuno-radiation therapy.

From left: Zhao, Sun, Nelson, Ding, Grabinski, Cao

Recent Publications Wang, Xin, Jin Zhu, Ping Zhao, Yongjun Jiao, Ning Xu, Tessa Grabinski, Chao Liu, Cindy K. Miranti, Tao Fu, and Brian B. Cao. 2007. In vitro efficacy of immuno-chemotherapy with anti-EGFR human Fab-Taxol conjugate on A431 epidermoid carcinoma cells. Cancer Biology & Therapy 6(6): 980–987. Zhao, Ping, Tessa Grabinski, Chongfeng Gao, R. Scot Skinner, Troy Giambernardi, Yanli Su, Eric Hudson, James Resau, Milton Gross, George F. Vande Woude, Rick Hay, and Brian Cao. 2007. Identification of a Met-binding peptide from a phage display library. Clinical Cancer Research 13(20): 6049–6055.

Van Andel Research Institute |

Scientific Report

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

Student

Paula Davidson, M.S. Caryn Lehner, M.S.

Matthew McElliott

VARI | 2008

Research Interests Through recent advancements in technology, mass spectrometry–based proteomics is now an important and widespread tool in basic and clinical research. In 2005, VARI purchased a Waters Q-Tof mass spectrometry system that remains at the cutting edge of many research applications. This equipment allows us to provide routine mass spectrometry services and to develop new services such as protein profiling for biomarker discovery and protein phosphorylation analysis. Protein identification analysis and protein molecular weight determination are routine services performed on sub-microgram amounts of material to address a wide variety of biological questions. Protein identification via mass spectrometry is mainly used to identify novel protein-protein interactions and can be performed on proteins in SDS-PAGE gels or proteins in solutions. Molecular weight determination of protein solutions is typically employed to confirm the expression and purification of recombinant proteins to be used as reagents in x-ray crystallographic experiments or drug screening/cell-based assays. Our research emphasis is on 1) developing liquid chromatography–mass spectrometry (LC-MS) protein profiling analysis for systems biology research and biomarker discovery and 2) improving methods for identifying and quantifying phosphorylation of proteins.

LC-MS protein profiling Liquid chromatography–mass spectrometry is used at most major research institutions to analyze complex protein mixtures for systems biology research and biomarker discovery. Our lab collaborates with Waters Corporation, a major manufacturer of mass spectrometry and HPLC equipment, to evaluate and improve existing methods while applying LC-MS to the research efforts at VARI and to those of external clients. Our LC-MS system employs a novel data acquisition method unique to Waters mass spectrometers, termed LC-MSE, whereby quantitative and qualitative data are collected in a single analysis. Protein samples are first digested into peptides using trypsin and then analyzed by reverse-phase nanoscale LC-MS. Recording peptide mass, HPLC retention time, and intensity as measured in the mass spectrometer, we digitize the data to allow comparisons across samples. Quantitation is based on the measurement and subsequent comparison of the chromatographic peak area for each peptide across samples. Qualitative protein identification data is collected in a multiplexed, non-intensity-biased fashion concurrent with quantitative data. One current pilot project is a time-course analysis of protein secretion (secretome) from mouse 3T3-L1 preadipocytes as they differentiate in response to treatment with dexamethasone-insulin or with the PPARg antagonist rosiglitasone; a second is the study of the secretome of a cell line model of hypoxia. In addition to mechanismof-action studies, our goal is to use LC-MS to discover candidate biomarkers of disease. Current research efforts focus on sample processing techniques to reproducibly fractionate highly complex samples such as blood plasma, tissue, and urine to allow quantitative analysis. Replicate LC-MS analysis of carefully chosen samples and multivariate data analysis will allow us to differentiate between normal biological variation and disease.

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 targets for drug 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.

Van Andel Research Institute |

Scientific Report

As with most mass spectrometryâ&#x20AC;&#x201C;based methods, mapping phosphorylation sites on proteins begins by enzymatically digesting protein into peptides using trypsin, Lys-C, Staph V8, or chymotrypsin. Peptides are separated by nanoscale reverse-phase HPLC and analyzed by on-line electrospray ionization on a quadrupole time-of-flight (Q-Tof) mass spectrometer. Samples are analyzed using the MSE data acquisition mentioned above. MSE toggles the collision energy in the mass spectrometer between high and low every second throughout the analytic run. Low-collision-energy data acquisition allows peptide mass to be recorded at high sensitivity with high mass accuracy to implicate phosphorylation based on mass alone. The peptide intensity measured in the mass spectrometer is also recorded and used for relative quantitation in time course studies. During high-collision-energy acquisition, all peptides are fragmented to identify the protein(s) from which the peptides were liberated by enzyme digestion and to locate specific phosphorylated amino acids. MSE differs from other mass spectrometry approaches because fragmentation occurs for all peptides, not just for the most abundant peptides. We are currently using this method on several in vitro phosphorylation projects, but our goal is to extend these analyses to in vivo systems to identify novel kinase or phosphatase substrates.

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: Cavey, Lehner, Davidson

VARI | 2008

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

Nick 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

Chih-Shia Lee, M.S. Naomi Asantewa-Sechereh Michelle Dawes Lisa Orcasitas Jennifer Wilcox

Van Andel Research Institute |

Scientific Report

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 of carcinomas, and we hypothesize that signaling through multiple MKK pathways is also essential for sarcomas. One objective of our research is to define the role of MKK signaling in the growth and vascularization of human sarcomas and to determine whether 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 fascinating discoveries that bring us closer to achieving that objective. Yan Ding and Philippe Depeille, postdoctoral fellows in the lab, with the assistance of Elissa Boguslawski, our xenograft technician, had earlier shown that MKKs are active in soft-tissue sarcomas (including Kaposi sarcoma, fibrosarcoma, malignant fibrous histiocytoma, and leiomyosarcoma) and that LeTx can inhibit the in vitro tumorigenic potential of these cells. We believed that the anti-tumoral properties of LeTx primarily stemmed from its ability to substantially decrease the release of many growth factorsâ&#x20AC;&#x201D;notably the pro-angiogenic vascular endothelial growth factor (VEGF)â&#x20AC;&#x201D;from tumor cells, leading to a reduction in tumor growth and vascularization. However, our work this year has changed the way we envision this. As an alternative approach to test the requirement for MKK signaling in fibrosarcoma vascularization in vivo, we established a collaboration with Rick Hay (Laboratory of Noninvasive Imaging and Radiation Biology) to monitor tumor perfusion in xenografts using ultrasound imaging in conjunction with injecting contrast ultrasound microbubbles. We found that inhibition of MKK signaling by LeTx caused a rapid and dramatic decrease in tumor perfusion (Figure 1). Follow-up histologic analysis in collaboration with James Resau (Laboratory of Analytical, Cellular, and Molecular Microscopy) showed this decrease in tumor perfusion was caused by increased extravasation, i.e., tumor blood vessels became leaky (Figure 2). This was unexpected, since published studies have shown that withdrawal of VEGF leads to a regression of neovascularization over the course of weeks, not hours. Our failure to observe similar changes in normal endothelium indicates that the survival requirements for normal and tumor endothelium are distinct. Taken together, our results indicate that while MKK activity is required for tumor cell proliferation, it also plays an important role in tumor vascular function. Further studies are required to delineate the events leading to loss of vascular function, as well as the relative contributions of tumor, stromal, and endothelial cells in this response. Figure 1

Figure 1. Ultrasound analysis of the effects of acute MKK inhibition on tumor blood flow. HT-1080 fibrosarcoma xenograft tumors (approximately 100 mm3 in diameter) were treated with 1 standard dose of either LeTx or inactive LeTx by i.v. injection. Tumor perfusion was evaluated by ultrasound imaging enhanced with contrast microbubbles either immediately prior to treatment or 24 h after treatment. The contrast signals, displayed in the images as green spots, are proportional to the number of microbubbles within the region of interest, which in turn reflects the included volume of flowing blood.

VARI | 2008

Currently, Jenn Bromberg-White, a postdoctoral fellow, is following up these studies with an investigation into the roles these same pathways play in other neovascular diseases such as acute macular degeneration. Chih-Shia Lee, a graduate student, is performing a detailed study of the individual contributions of MKK pathways to melanoma survival, and Jaclyn Lynem, our laboratory technician, is investigating the molecular basis of LF inactivation of MKK. Finally, in our longstanding collaboration with Arthur Frankel, Director of the Scott & White Cancer Research Institute in Texas, we are moving forward with preclinical testing of the therapeutic potential of LeTx in the treatment of malignant melanoma. Figure 2

Figure 2. The effect of acute MKK inhibition on xenograft morphology. Mice bearing HT-1080 xenograft tumors were injected i.v. with inactive LeTx (A) or LeTx (B, C). Twenty-four hours later, tumor (A, B) and kidney (C) tissues were formalin-fixed, paraffin-embedded, sectioned, and stained using hemotoxylin and eosin. Images were obtained at 20X; bars represent 50 Îźm.

From left: Ding, Duesbery, Holman, Boguslawski, Lynem, Lee, Bromberg-White

Van Andel Research Institute |

Scientific Report

Recent Publications Bromberg-White, J.L., and N.S. Duesbery. In press. Biological and biochemical characterization of anthrax lethal factor, a proteolytic inhibitor of MEK signaling pathways. Methods in Enzymology. Kuo, S.R., M.C. Willingham, S.H. Bour, E.A. Andreas, S.K. Park, C. Jackson, N.S. Duesbery, S.H. Leppla, W.J. Tang, and A.E. Frankel. In press. Anthrax toxin-induced shock in rats is associated with pulmonary edema and hemorrhage. Microbial Pathogenesis. Alfano, Randall W., Stephen H. Leppla, Shihui Liu, Thomas H. Bugge, Nicholas S. Duesbery, and Arthur E. Frankel. 2008. Potent inhibition of tumor angiogenesis by the matrix metalloproteinase–activated anthrax lethal toxin: implication for broad anti-tumor efficacy. Cell Cycle 7(6): 745–749. Ding, Yan, Elissa A. Boguslawski, Bree D. Berghuis, John J. Young, Zhongfa Zhang, Kim Hardy, Kyle Furge, Eric Kort, Arthur E. Frankel, Rick V. Hay, James H. Resau, and Nicholas S. Duesbery. 2008. Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma. Molecular Cancer Therapeutics 7(3): 648–658. Huang, Dan, Yan Ding, Wang-Mei Luo, Stephanie Bender, Chao-Nan Qian, Eric Kort, Zhong-Fa Zhang, Kristin VandenBeldt, Nicholas S. Duesbery, James H. Resau, and Bin Tean Teh. 2008. Inhibition of MAPK kinase signaling pathways suppressed renal cell carcinoma growth and angiogenesis in vivo. Cancer Research 68(1): 81–88. Rouleau, Cecile, Krishna Menon, Paula Boutin, Cheryl Guyre, Hitoshi Yoshida, Shiro Kataoka, Michael Perricone, Srinivas Shankara, Arthur E. Frankel, Nicholas S. Duesbery, George F. Vande Woude, Hans-Peter Biemann, and Beverly A. Teicher. 2008. The systemic administration of lethal toxin achieves a growth delay of human melanoma and neuroblastoma xenografts: assessment of receptor contribution. International Journal of Oncology 32(4): 739–748. Depeille, Philippe, John J. Young, Elissa A. Boguslawski, Bree D. Berghuis, Eric J. Kort, James H. Resau, Arthur E. Frankel, and Nicholas S. Duesbery. 2007. Anthrax lethal toxin inhibits growth of and vascular endothelial growth factor release from endothelial cells expressing the human herpes virus 8 viral G protein–coupled receptor. Clinical Cancer Research 13(19): 5926–5934. Young, John J., Jennifer L. Bromberg-White, Cassandra R. Zylstra, Joseph T. Church, Elissa Boguslawski, James H. Resau, Bart O. Williams, and Nicholas S. Duesbery. 2007. LRP5 and LRP6 are not required for protective antigen–mediated internalization or lethality of anthrax lethal toxin. PLoS Pathogens 3(3): e27.

VARI | 2008

MET expression in breast cancer cells

This image was from a breast cancer project funded by DOD IDEA grant with J. Resau as PI. MET is a protein found overexpressed in many cancers. The image shows MET and Her2neu overlaid onto a Nomarskiâ&#x20AC;&#x201C;DIC (differential interference contrast) background of the tissue structure (gray). The holes are where adipose tissue was removed or cleared in histology processing. Her2neu was localized with a DAKO polyclonal antibody (green) and MET was localized with a monoclonal antibody (red). Yellow results from the combination of both green and red costaining or colocalization. This was selected as an Image of Distinction in the Nikon Small World 2007 Competition. Photo by James Resau.

Van Andel Research Institute |

Scientific Report

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

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–FCRDC) in Maryland. In 1983, she joined the Johnson & Johnson Biotechnology Center in San Diego, California. In 1988, she returned to NCI–FCRDC, 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

Animal Caretaker Staff

Lisa DeCamp, B.S. Dawna Dylewski, B.S. Audra Guikema, B.S., L.V.T. Tristan Kempston, B.S. Angie Rogers, B.S. Elissa Boguslawski, RALAT

Sylvia Marinelli, Team leader Crystal Brady Jarred Grams Samuel Johnson Rishard Moody Janelle Post Tina Schumaker Bobbie Vitt

VARI | 2008

Research Interests The goal of the vivarium and the transgenics program 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. 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 to right, standing: Dylewski, Guikema, Grams, Schumaker, Rogers, Eagleson, Brady, Marinelli, Vitt, Post, Jason, Boguslawski, DeCamp From left to right, kneeling: Kempston, Moody, Johnson

Van Andel Research Institute |

Scientific Report

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

Students

Karl Dykema, B.A.

Jeff Klomp, B.S. Theresa Gipson Craig Johnson

VARI | 2008

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 contributed to several gene expression microarray analysis projects ranging from mechanisms of oncogene transformation to the identification of genes associated with drug sensitivity. For example, in recent work led by the Laboratory of Molecular Oncology, we combined cytogenetic, phenotypic, and gene expression profiling data to help elucidate the role of chromosomal abnormalities during tumor cell progression. We also worked closely with the Laboratory of Cancer Genetics in the development of gene expressionâ&#x20AC;&#x201C;based models for the 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 by examining 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 having 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).

Van Andel Research Institute |

Scientific Report

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: Furge, Johnson, Klomp, Dykema

Recent Publications Camparo, P., V. Vasiliu, V. Molinié, J. Couturier, K. Dykema, D. Petillo, K.A. Furge, E.M. Comperat, M. Laé, R. Bouvier, L. Boccon-Gibbod, Y. Denoux, S. Ferlicot, E. Forest, G. Fromont, et al. In press. Renal translocation carcinomas: clinicopathological, immunohistochemical, and gene expression profiling analysis of 31 cases with a review of the literature. American Journal of Surgical Pathology. Ding, Yan, Elissa A. Boguslawski, Bree D. Berghuis, John J. Young, Zhongfa Zhang, Kim Hardy, Kyle Furge, Eric Kort, Arthur E. Frankel, Rick V. Hay, James H. Resau, and Nicholas S. Duesbery. 2008. Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma. Molecular Cancer Therapeutics 7(3): 648–658. Gao, ChongFeng, Kyle Furge, Julie Koeman, Karl Dykema, Yanli Su, Mary Lou Cutler, Adam Werts, Pete Haak, and George F. Vande Woude. 2007. Chromosome instability, chromosome transcriptome, and clonal evolution of tumor cell populations. Proceedings of the National Academy of Sciences U.S.A. 104(21): 8995–9000.

VARI | 2008

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, became a Scientific Investigator in 2004, and was promoted to Senior Scientific Investigator in 2007.

Staff

Students

Visiting Scientists

Songming Chen, Ph.D. Yi-Mi Wu, Ph.D. Derek Bergsma, B.S. Sara Forrester, B.S. Andrew Porter, B.S. Tingting Yue, B.S. Alex Turner

Krysta Collins Carrie Fiebig Adam Granger Lee Heeringa Kevin Maupin Randi VanOcker

David Nowack, Ph.D.

Van Andel Research Institute |

Scientific Report

Research Interests C-reactive protein C-reactive protein (CRP) is a crucial component of the body’s innate immune system. CRP is involved in the recognition and removal of pathogens and dying cells and in the signaling that controls inflammation. While CRP is crucial to the maintenance of health, recent research has demonstrated a possible involvement of CRP in the development of diseases associated with inflammation. A more complete understanding of CRP functions in normal and disease-associated inflammation could have valuable therapeutic implications. We have used a novel method developed in our laboratory, called Antibody Array Interaction Mapping (AAIM), to uncover possible additional roles for CRP in inflammation and disease. A protein’s function is determined in part by its interactions with other proteins, and identifying and measuring changes in those interactions are keys to understanding protein functions. Current methods of detecting protein-protein interactions—such as immunoprecipitation, mass spectrometry, yeast two-hybrid assay, and protein arrays—are not suitable for measuring changes over multiple samples and may require purified proteins instead of native, biological samples. AAIM complements these methods by allowing quantitative, high-throughput comparisons of protein-protein interaction levels in biological samples. We produce multiple, identical arrays containing antibodies targeting a variety of proteins that might interact with each other. A native, nondenatured biological sample such as serum is incubated on each array, and proteins in the sample are captured by the antibodies according to their specificities. After unbound proteins are washed away, each array is probed with a detection antibody that corresponds to one of the capture antibodies, and the detection antibodies localize on the array wherever their targets are found. The pattern of binding of the detection antibodies can reveal potential protein-protein interactions. Using this tool, we have discovered several novel protein-protein interactions in human serum, including previously unknown interactions between CRP and other inflammation-related proteins. The finding of a subset of CRP circulating in complex with inflammatory mediators suggests previously unrecognized functions or sites of action for CRP. An intriguing aspect of this bound form of CRP is that it appears to be conformationally different than the freely circulating form. The bound CRP is structurally altered in a way that produces potent biological effects distinct from those of normal CRP. We have shown a biological context for the bound form of CRP; now we are seeking to determine how the functions of this bound CRP differ from those of free CRP and how abnormal levels of bound CRP might be involved in inflammation-related pathologies. We also are characterizing the components of circulating multiprotein complexes involving CRP and characterizing the details of those interactions. AAIM has been a valuable tool for the discovery and ongoing study of these multiprotein complexes, especially using monoclonal antibodies with defined specificities for various regions and forms of CRP. Other proteomics methods, performed in the collaboration with the Mass Spectrometry and Proteomics lab at VARI, facilitate this work.

Glycosylation in pancreatic cancer The development of biomarkers for the accurate and early diagnosis of pancreatic cancer has been challenging. Many of the candidate biomarkers are either elevated in other conditions or only in later-stage disease, leading to unacceptably low specificity and sensitivity. A common molecular feature of pancreatic cancer is alteration of the carbohydrate structures (glycans) that are attached to certain proteins. Glycan alterations can appear at a higher rate than changes in protein abundance, and certain glycan structures may be unique to particular disease states, even at early stages of cancer development. Thus, the detection of particular glycans on specific proteins may form the basis of improved pancreatic cancer biomarkers.

VARI | 2008

The key to developing improved markers is the ability to reproducibly measure specific glycans on specific proteins. Many of the carbohydrate structures on proteins in normal and cancer tissues have been characterized using mass spectrometry and enzymatic methods. Those methods are valuable for defining structures, but they do not have the precision or throughput necessary to look at changes in levels between samples, which is necessary to assess biomarker potential. A new method developed in our laboratory provides the means to obtain more detailed information on glycan variation. We use lectins— proteins that bind specific glycan structures—and glycan-binding antibodies to probe the levels of particular glycans on the proteins captured by antibody arrays. This method provides the important feature of allowing comparison between samples of the levels of particular glycans on specific proteins so that we can assess their diagnostic potential. A product based on this technology is now available from GenTel Biosciences (Madison, WI). The class of proteins called mucins shows particularly high levels of glycan alteration in pancreatic cancer. Mucins are longchain, heavily glycosylated proteins on epithelial cell surfaces that have roles in cell protection, interaction with the extracellular space, and regulation of extracellular signaling. Altered carbohydrates on mucins can affect critical processes in cancer such as cell migration or extracellular signaling to the immune system. We have extensively characterized the glycan variations on mucins secreted into the blood of pancreatic cancer patients. In some cases, the levels of certain mucin glycans are altered in cancer patients more often than the levels of the core proteins (Figure 1a). As a result, detection of the glycans performed better as a biomarker than detection of the core proteins (Figure 1b). The efficient analysis of many samples and glycan structures was made possible by the ability to run dozens of samples on a single microscope slide. A device based on that technology, which partitions microscope slides for efficient sample processing, is available from The Gel Company (San Francisco, CA). Our work shows the promise of this approach and points to key directions for further developing biomarkers of pancreatic cancer. Our research now focuses on the goals of identifying the protein carriers of cancer-associated glycans, of identifying the most important cancer-associated glycans and the reagents to detect them, and of applying these discoveries to pancreatic cancer diagnostics (Figure 1c). Figure 1 In addition, we are seeking to better understand the origins of glycan alterations and the functional contribution of these molecules to pancreatic cancer development and progression.

Figure 1. Pancreatic cancer biomarker development. a) Comparison of glycan versus protein detection. The level of the MUC5ac core protein in serum samples from cancer patients and healthy subjects, determined using monoclonal antibody (mAb) sandwich assays, is indicated along the vertical axis. The level of glycan CA 19-9 on MUC5ac, determined using a mAb to capture MUC5ac and another antibody to detect CA 19-9 on the captured protein, is indicated along the horizontal axis. b) Receiver-operator characteristic curve analysis comparing the biomarker performance of core protein versus glycan detection. Each curve gives the sensitivity (rate of true positive detection) and the specificity (rate of true negative detection) for discriminating cancer subjects from control subjects at various thresholds of discrimination. “AUC” is area-under-the-curve, indicating the total discriminating ability of each marker. c) Cluster analysis. The glycan measurements along the vertical axis were taken in the samples indicated along the horizontal axis; the color of each square is the level of each measurement (see the color bar). The rows and columns were ordered (clustered) by similarity, showing consistently increased levels in the cancer patients.

Van Andel Research Institute |

Scientific Report

External Collaborators Philip Andrews, Irwin Goldstein, Gilbert Omenn, and Diane Simeone, University of Michigan, Ann Arbor Randall Brand, University of Pittsburgh, Pennsylvania William Catalona, Northwestern University, Evanston, Illinois Terry Du Clos, University of New Mexico, Albuquerque Ziding Feng and Samir Hanash, Fred Hutchinson Cancer Research Center, Seattle, Washington Michael A. Hollingsworth, University of Nebraska, Omaha Raju Kucherlapati, Harvard Medical School, Boston, Massachusetts Anna Lokshin, University of Pittsburgh, Pennsylvania Alan Partin, Johns Hopkins University, Baltimore, Maryland Lawrence A. Potempa, Immtech Pharmaceuticals, Vernon Hills, Illinois Robert Vessella, University of Washington, Seattle

From left: Yue, Wu, VanOcker, Bergsma, Nelson, Porter, Haab

Recent Publications Chen, Songming, Tom LaRoche, Darren Hamelinck, Derek Bergsma, Dean Brenner, Diane Simeone, Randall E. Brand, and Brian B. Haab. 2007. Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays. Nature Methods 4(5): 437–444. Forrester, Sara, Kenneth E. Hung, Rork Kuick, Raju Kucherlapati, and Brian B. Haab. 2007. Low-volume, high-throughput sandwich immunoassays for profiling plasma proteins in mice: identification of early-stage systemic inflammation in a mouse model of intestinal cancer. Molecular Oncology 1(2): 216–225. Forrester, Sara, Ji Qiu, Leslie Mangold, Alan Partin, David Misek, Brett Phinney, Douglas Whitten, Philip Andrews, Eleftherios Diamandis, Gilbert S. Omenn, Samir Hanash, and Brian B. Haab. 2007. An experimental strategy for quantitative analysis of the humoral immune response to prostate cancer antigens using natural protein microarrays. Proteomics – Clinical Applications 1(5): 494–505. Li, Zheng, Shireesh Srivastava, Xuerui Yang, Sheenu Mittal, Paul Norton, James Resau, Brian Haab, and Christina Chan. 2007. A hierarchical approach employing metabolic and gene expression profiles to identify the pathways that confer cytotoxicity in HepG2 cells. BMC Systems Biology 1: 15 pp.

VARI | 2008

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

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 Natalie Kent, B.S. Jose Toro, B.S.

Students Alaa Abughoush Sara Kunz Jennifer Vogal

Visiting Scientist Physician-Scientist In Training Visiting Scientists

Students

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

Matthew Steensma, M.D.

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

Van Andel Research Institute |

Scientific Report

Research Interests The Laboratory of Noninvasive Imaging & Radiation Biology is devoted both to noninvasive imaging (i.e., depicting anatomic structures and physiology in living organisms without surgery) and to radiation biology (evaluating the consequences of external and internal radiation exposure in living organisms). The lab’s work follows three common themes:

•

Developing and using laboratory models that address medical imaging and radiation exposure problems

•

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

•

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

We depend heavily upon sophisticated instruments and equipment, including nuclear imaging cameras; planar and tomographic (3-D) 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. Our laboratory operates state-of-the-art noninvasive instruments for imaging mice, including a Vevo 770 high-resolution micro-ultrasound imaging system (VisualSonics) and a nanoSPECT/CT imaging unit (BioScan). We are pursuing two major collaborative projects in the area of radiation biology:

•

igel Crompton of Cornerstone University co-directs an effort to predict the sensitivity of a patient’s normal N tissues to irradiation being administered for treatment of cancer. This project is made possible through collaboration with the radiation oncology service at Saint Mary’s Health Care and with the West Michigan Center for Family Health, both in Grand Rapids. For this project, a sample of the patient’s blood is drawn before radiation therapy. That blood sample is then irradiated under precise conditions of exposure, treated with fluorescent molecules that detect certain blood cells (lymphocytes), and analyzed by fluorescence-activated cell sorting (FACS) for evidence of lymphocyte death. We have also been investigating the effects of patient age, gender, and administered radiation dose on the lymphocyte response, and we are now working to determine the molecular basis for patient-to-patient variability. The midpoint results of our five-year clinical trial are being presented this year at the annual meeting of the American Society of Clinical Oncology.

•

I n collaboration with Drs. Weiwen Deng, Aly Mageed, and Anthony Senagore of DeVos Children’s Hospital/ Spectrum Health, we are exploring 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.

VARI | 2008

Our major project in nuclear medicine is to develop and bring into clinical use 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. We are currently translating MARP-based imaging into mice with orthotopic xenografts (see below), as well as undertaking studies in additional animal species in order to gain governmental approval for the first MARP testing in humans. Finally, to support our internal and external collaborators, we operate a multimodality noninvasive imaging program for evaluating the growth, molecular expression, and response to therapy of aggressive human tumor xenografts grown subcutaneously or 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 studying tumors of the brain, adrenals, soft connective tissue, and bone. From studies using this imaging program, one paper (Ding et al. 2008) has been published; two manuscripts have been submitted for publication; and three more are being prepared.

External Collaborators Our lab depends critically on intramural and extramural collaborations to address our research themes. Current extramural collaborators include scientists and physicians at the Department of Veterans Affairs Healthcare System and the University of Michigan Medical Center, both in Ann Arbor; Cornerstone University, West Michigan Heart, P.C., DeVos Children’s Hospital/ Spectrum Health, St. Mary’s Health Care, and West Michigan Center for Family Health, all in Grand Rapids; the University of Illinois in Champaign-Urbana; and VisualSonics, Inc., in Toronto.

Recent Publications Gross, M.D., and R.V. Hay. In press. Molecular imaging of adrenal disease. Molecular Endocrinology. Ding, Yan, Elissa A. Boguslawski, Bree D. Berghuis, John J. Young, Zhongfa Zhang, Kim Hardy, Kyle Furge, Eric Kort, Arthur E. Frankel, Rick V. Hay, James H. Resau, and Nicholas S. Duesbery. 2008. Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma. Molecular Cancer Therapeutics 7(3): 648–658. Zhao, Ping, Tessa Grabinski, Chongfeng Gao, R. Scot Skinner, Troy Giambernardi, Yanli Su, Eric Hudson, James Resau, Milton Gross, George F. Vande Woude, Rick Hay, and Brian Cao. 2007. Identification of a Met-binding peptide from a phage display library. Clinical Cancer Research 13(20): 6049–6055. 31

Van Andel Research Institute |

Scientific Report

Office of Translational Programs The Office of Translational Programs (OTP) is the administrative home 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. These programs include development of translational infrastructure, research project coordination, medical-scientific education oversight, and community outreach. OTP accomplishments during our past year include the following:

•

erving as the administrative home for a new cGMP facility. With funding from the state of Michigan and the S 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 current Good Manufacturing Practices (cGMP) facility that will package pharmaceuticals for early-phase clinical trials commissioned by academic and commercial investigators, primarily in Michigan and the Midwest. Construction of GR-APP at 140 Front Street is complete, and operations will begin in mid 2008.

•

Overseeing VARI’s participation in activities of the Michigan Cancer Consortium (MCC). As an active member of the MCC, VARI is committed to participating in statewide and regional community-based programs to reduce the burden of cancer in Michigan.

•

Coordinating research rotations for physicians-in-training. In collaboration with the Grand Rapids Medical Education and Research Center (MERC), OTP schedules each first-year general surgery resident to spend one month working in a 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 also be arranged for physiciansin-training. For example, Matt Steensma, M.D., is pursuing a one-year laboratory research fellowship with funding through the Orthopaedic Research and Education Foundation under joint mentorship by Dr. Hay and by Dr. David Rispler at MERC.

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

VARI | 2008

mTOR staining of transitional cell carcinoma

A pseudofluorescent image captured with the CRi Nuance imaging system of phospho-mTOR immunohistochemical staining of a human transitional cell carcinoma. The sample shows high expression of phospho-mTOR in the tumor cells (red) among normal interstitial tissue. The cellular nuclei are stained blue. The tissue was prepared by Chao-Nan Qian of the Teh laboratory and imaged by Kristin VandenBeldt of the Resau laboratory.

Van Andel Research Institute |

Scientific Report

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

Students

Brendan Looyenga, Ph.D. Christina Ludema, B.S.

Katie Sian, B.S. Natalie Wolters, B.S. Joe Church Halley Crissman Alyse DeHaan Sara Herman Geoff Kraker Matthew McElliott

VARI | 2008

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, proliferation, and 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 several essential kinases and phosphatases whose roles in cell survival were previously unrecognized. We are asking several questions. How are these 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?

Novel modulators of chemotherapeutic sensitization Kinases and phosphatases play an integral role in balancing the survival and apoptotic signals within a cell. In an attempt to define proteins with a major role in these processes, we tested an RNAi library against all known kinases and phosphatases in the human genome and assayed various phenotypes, including sensitization to apoptosis and chemoresistance. A group of apoptosis sensitizers was identified whose siRNA knock-out conferred a marked increase in cell survival as well as a striking chemoresistant phenotype (Figure 1). One of these proteins, MK-STYX, resembles the dual-specificity phosphatases implicated in MAP kinase signaling, but it is catalytically inactive due to a cysteine-to-serine mutation at its active site. When MK-STYX is knocked down via RNAi, the cells display a profound decrease in apoptosis; MK-STYX-overexpressing cells, on the other hand, are sensitized to apoptotic signals. We propose that MK-STYX could function as a dead phosphatase, sequestering potential phosphoproteins that promote survival. Through further experiments, we plan to characterize MK-STYX and elucidate its mechanism of apoptotic sensitization; these studies may identify a survival signal that would constitute a novel target for chemotherapy.

Figure 1

Figure 1. Human kinase and phosphatase siRNA library screen. HeLa cells were transfected with siRNAs directed against all known and putative human phosphatases and kinases. Cells were incubated for 72 h to allow target knockdown, and apoptosis was measured by a DNA-fragmentation ELISA. The graph shows relative apoptosis for 600 kinase and 200 phosphatase siRNA targets.

Van Andel Research Institute |

Scientific Report

Monitoring cellular signaling Phosphatidylinositol-3-kinase (PI3K) phosphorylates the 3´ ring position of phosphatidylinositol to generate lipid products important for signal transduction, membrane trafficking, and other cellular processes. The identification of PI3Ks as key players in cellular functions ranging from vesicular trafficking to cell survival merits further study to identify factors acting immediately upand downstream of these lipid kinases, as well as characterizing the phosphatase regulating these molecular pathways. Roles for PI3K isoforms in amino acid sensing and in signaling through the mTOR pathway, as well as in autophagy, have also recently emerged. Note that these functions of PI3Ks might not merely rely on their lipid kinase activity, since they are large enzymes that could also serve as platforms for the assembly of protein complexes. Understanding is needed of the mechanisms of PI3K signaling involved in these various cellular functions.

Parkinson disease–associated genes in cancer Renal cell carcinoma (RCC) is an aggressive cancer that is highly metastatic and refractory to all forms of systemic cancer therapy. Using bioinformatic analysis and over 150 RCC tumor samples, we have identified Parkinson disease–associated (PD) kinases as a novel molecular constituent of the renal tubule epithelium whose expression is specifically down-regulated during the progression of papillary RCC (Figure 2). These PD kinases are highly expressed in the brain and kidney and have been previously linked to familial Parkinson disease. Activating mutations in these genes sensitize cells to oxidative stress and lead to increased death of neural cells, implying that these genes may function as a sensor of oxidative stress in the renal epithelium and induce cell death pathways in response to toxic levels of reactive oxygen species. Selective loss of each gene in more aggressive and metastatic RCC tumors suggests that this protein may also be a tumor suppressor. The goal of this project is to define the relationship between oxidative stress management and malignant tumor progression in the kidney, with a particular emphasis on the role of kinase signaling. This project is a collaboration with VARI’s Kyle Furge and Bin Teh.

Figure 2

Figure 2. PD genes are located within a conserved RCC amplicon on chromosome 12. Expression profiles from more than 150 normal and RCC tissue samples were obtained by microarray analysis with the Affymetrix HGU-133 Plus 2.0 chip.

VARI | 2008

Colorectal cancer Chemoresistance is a therapeutic problem that severely limits successful treatment of most human cancers. This is particularly true of colorectal cancer, in which the development of resistance is common: most anti-cancer regimens are ineffective, with the five-year survival rates for late-stage colorectal cancer being only 8%. How colorectal cancer resistance develops is largely unknown, and the response to therapy varies based on individual patient tumors. With this in mind, how can we prevent cancer emergence or progression at the level of individual tumors? Recent studies have shown that a large percentage of colorectal tumors have mutations in a key gene, for class I PI3K. While mutations play an important causative role in colorectal cancer, it is currently unclear how these mutations can be exploited as drug targets and whether we can develop targeted cancer agents based on the gene. We have ongoing projects to determine the molecular pathways (and genes) that can be used to prevent progression of precancerous lesions to colorectal cancer. Further, we are defining each pathway activation in each patientâ&#x20AC;&#x2122;s tumor and comparing the pathways with a novel chemopreventive agent against PI3K/mTOR.

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. One project in the lab involves the question of whether the evolutionarily conserved pathways exhibit 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. 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.

From left: Wolters, Nelson, MacKeigan, Looyenga, DeHaan, Church, Crissman, Sian, McElliott

Recent Publications Elis, W., E. Triantafellow, N.M. Wolters, K.R. Sian, G. Caponigro, J. Borawski, L.A. Gaither, L.O. Murphy, P. Finan, and J.P. MacKeigan. 2008. Downregulation of class II PI3Ka expression below a critical threshold induces apoptotic cell death. Molecular Cancer Research 6(4): 614â&#x20AC;&#x201C;623. Wolters, N.M., and J.P. MacKeigan. 2008. From sequence to function: using RNAi to elucidate mechanisms of human disease. Cell Death and Differentiation 15(5): 809â&#x20AC;&#x201C;819. 37

Van Andel Research Institute |

Scientific Report

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 and an Assistant Professor in the Van Andel Education Institute.

Staff

Students

Kristin Saari, M.S. Lia Tesfay, M.S. Veronique Schulz, B.S.

Jelani Zarif, M.S. Laura Lamb, B.S. Susan Spotts, B.S. Erica Bechtel Eric Graf Fraser Holleywood Gary Rajah

VARI | 2008

Research Interests Our laboratory is interested in understanding the mechanisms by which integrin receptors, interacting with the extracellular matrix, 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 extracellular matrix (ECM) proteins. By interacting with the ECM, integrins stimulate intracellular signaling transduction pathways that 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 in 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 present at the time of diagnosis with androgen-dependent and organ-confined prostate cancer are relatively easy to cure through radical prostatectomy or localized radiotherapy. However, patients with aggressive and metastatic disease have fewer options. Androgen ablation can significantly reduce the tumor burden in these 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, a3b1 and a6b4 integrins on epithelial cells bind to the ECM protein laminin 5 in the basement membrane. In tumor cells, however, the a3 and b4 integrin subunits disappear—as does laminin 5—and the tumor cells express primarily a6b1 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 in our lab 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 for cell survival.

Integrins and RTKs in prostate epithelial cell survival How integrin engagement of different ECMs regulates survival pathways in normal and tumor cells is poorly understood. We have previously demonstrated that integrin-induced activation of EGFR in normal primary prostate epithelial cells is required for survival of these cells on their endogenous matrix, laminin 5. The ability of EGFR to support integrin-mediated cell survival on laminin 5 is mediated through a3b1 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 caspasemediated apoptosis. The presence of an autophagic survival pathway, 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 5–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 maintenance of an autophagic survival pathway. We have begun studies to determine how integrins regulate cell survival through autophagy. When we block expression of the RTK c-Met in primary prostate epithelial cells adherent to laminin 5, they also die. In this case death is due to classical caspase-mediated apoptosis. Since autophagy must be inhibited in these cells to induce apoptosis, these results suggest that c-Met may regulate autophagy. Future studies in our lab are aimed at deciphering this pathway and determining how this pathway is altered during tumor progression. 39

Van Andel Research Institute |

Scientific Report

Role of 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 change 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 change in AR function by initiating crosstalk between AR and integrins. 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. We have determined that AR expression results in increased expression of a6b1 integrin, the receptor for laminin. Thus, AR-expressing tumor cells are likely to survive better when they remain adherent to the laminin-rich ECM that is present in the prostate gland. Survival under these conditions appears to depend on the ability of AR to enhance expression of the laminin receptor, a6b1 integrin; we are currently determining how AR regulates the expression of this integrin.

Role of 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. The mechanisms involved in progression to metastatic disease 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 by studying the role of CD82/KAI1 in integrin and RTK crosstalk. 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. So far our investigations indicate that c-Met and CD82 do not directly interact, and CD82 may act to suppress c-Met signaling indirectly by dispersing the c-Met aggregates on metastatic tumor cells into monomers, thus blocking signaling. We are developing mutants of CD82 to determine which part of the CD82 molecule is required for suppression of c-Met activity. In addition, we have determined that reexpression of CD82 in tumor cells induces a physical association between CD82 and a related family member, CD9. Loss of CD9 prevents CD82 from suppressing c-Met. We are currently determining whether CD82/CD9 association with integrins is required to suppress c-Met. We have also initiated several 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. The ability of some prostate cancer cells to metastasize depends on activation of c-Met. Using mice that are able to specifically activate c-Met, we have been able to demonstrate that these tumor cells will only metastasize when c-Met is active. Under these conditions, reexpression of CD82 completely suppresses metastasis. In addition, we have generated mice in which CD82 expression is specifically lost in the epithelial cells of the prostate gland. These mice will be crossed to mice that develop only primary tumors to determine if the loss of CD82 is sufficient to induce prostate cancer metastasis.

VARI | 2008

External Collaborators Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Valeri Vasioukin, Fred Hutchinson Cancer Research Center, Seattle, Washington Andries Zijlstra, Vanderbilt University, Nashville, Tennessee

From left, standing: Bechtel, Rajah, Graf, Zarif, Holleywood, Spotts: seated: Schulz, Miranti, Guthrey, Tesfay, Saari, Lamb

Recent Publications Edick, Mathew J., Lia Tesfay, Laura E. Lamb, Beatrice S. Knudsen, and Cindy K. Miranti. 2007. Inhibition of integrin-mediated crosstalk with epidermal growth factor receptor/Erk or Src signaling pathways in autophagic prostate epithelial cells induces caspase-independent death. Molecular Biology of the Cell 18(7): 2481–2490. Tolbert, W. David, Jennifer Daugherty, Chongfeng Gao, Qian Xie, Cindy Miranti, Ermanno Gherardi, George Vande Woude, and H. Eric Xu. 2007. A mechanistic basis for converting a receptor tyrosine kinase agonist to an antagonist. Proceedings of the National Academy of Sciences U.S.A. 104(37): 14592–14597. Wang, Xin, Jin Zhu, Ping Zhao, Yongjun Jiao, Ning Xu, Tessa Grabinski, Chao Liu, Cindy K. Miranti, Tao Fu, and Brian B. Cao. 2007. In vitro efficacy of immuno-chemotherapy with anti-EGFR human Fab-Taxol conjugate on A431 epidermoid carcinoma cells. Cancer Biology & Therapy 6(6): 980–987. 41

Van Andel Research Institute |

Scientific Report

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 CLIAtype 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

Students

Visiting Scientist

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

Pete Haak, B.S. Heather Born Danielle Burgenske Janell Carruthers Halley Crissman Sara Herman Wei Luo Bryan Mendez Tarrick Mussa Sara Ramirez Aleesa Schlientz Huong Tran Yarong Yang

Yair Andegeko

VARI | 2008

Research Interests The Division of Quantitative Sciences includes the laboratories of Analytical, Cellular, and Molecular Microscopy (ACMM), the Laboratory of Microarray Technology, the Laboratory of Computational Biology, the Laboratory of Molecular Epidemiology, and the Laboratory of Mass Spectrometry and Proteomics. The Division’s laboratories use objective measures to define pathophysiologic events and processes. 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, and enable researchers to determine where in a cell specific molecules are located. An archive of pathology tissues in paraffin blocks (Van Andel Tissue Repository) is being accumulated with the cooperation of local hospitals, and the data on the samples is being converted to computerized files in collaboration with Tom Barney from VAI-IT. The lab also carries out research that will improve our ability to quantify images. 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 Agilent commercially prepared arrays as well as “home-brewed” cDNA microarrays. In 2007 we produced and used 305 cDNA microarrays and 150 custom protein microarrays. We also used 107 Agilent arrays to genomically characterize a variety of tissues and samples, including archived human blood samples from newborns.

Hauenstein Parkinson’s Center Throughout 2007 we continued our collaboration with the Hauenstein Parkinson’s Center, collecting blood samples and controls from 154 individuals. Mutations in the parkin gene in a set of families with more than one generation affected by Parkinson disease are being studied by DNA sequence analysis and will be correlated to gene expression data from microarray analysis.

Identification of novel Parkinson-modifying genes with siRNA screening Small interfering RNA (siRNA) technology allows the specific knockdown of any mRNA/protein pair. Combined with information from the human genome, this technology has given rise to libraries of siRNAs targeted to every known or predicted gene in the genome. Under the direction of VARI’s Jeff MacKeigan, postdoctoral fellow Brendon Looyenga has begun to use a subset of the siRNA library developed by Qiagen to individually target several classes of enzymes having pharmaceutical potential. We are searching for genes involved in Parkinson disease that may be drug targets for rationally designed therapies. We are attempting to identify molecules that attenuate oxidative stress–induced toxicity in dopaminergic neurons; our initial focus is on phosphatases and kinases. To date we have screened all of the phosphatases in the human genome and have identified several potential candidates that regulate neuronal cell death in response to 6-hydroxydopamine, a toxic compound used to induce oxidative stress in Parkinson research. We are validating these initial screening studies and are planning the assays required to screen all kinases in the human genome as well. We hope to extend these studies to include nuclear hormone receptors and G protein–coupled receptors.

Mouse models of Parkinson disease James Resau and Brendan Looyenga are generating novel rodent models of dopaminergic cell loss in the brain in collaboration with VARI’s Bart Williams. A key tool 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. The DAT-cre mice will allow us to address the response of such neurons to specific gene deletions and additions; projects based on the DAT-cre mouse model include the following.

Van Andel Research Institute |

Scientific Report

â&#x20AC;˘

I maging and isolation of primary dopaminergic neurons from mouse brain. Brendan Looyenga has 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.

â&#x20AC;˘

opaminergic cell regeneration as a function of age. The relationship between age and the likelihood of D 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 will cross DAT-cre and puDTK mice, the latter specifically expressing herpes simplex virus thymidine kinase (hsvTK) in cells that contain cre recombinase. Cells expressing hsvTK are sensitive to ganciglovir (G418) and undergo programmed cell death after systemic treatment. Using the DAT-cre/ puDTK model, we will eliminate dopaminergic neurons at various ages and assess the regenerative potential of these mice. These studies will provide information about the value of therapies intended to stimulate the endogenous regenerative capacity of the brain in Parkinson disease patients.

â&#x20AC;˘

ffect of hypoxia-inducible factor signaling on dopaminergic cell survival. Dopaminergic neurons are exquisitely E sensitive to oxidative stress (reactive oxygen species), which can 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 mechanism may be offset by cell survival pathways, which increase the threshold signal intensity required to induce cell death. Because Parkinson disease is characterized by increased oxidative stress in dopaminergic neurons, therapies that increase cell survival pathways in these neurons may be broadly applicable to decrease cell death in patients.

Other highlights Our GRAPCEP mentorship program continues for an eighth year and is now funded by Schering Plough. This year we had three students from GRAPCEP. Dr. Resau is a member of the graduate school committee that established the VAEI Graduate School, which will increase our research and educational opportunities. Also in 2007, Jim Resau had an image selected as one of the Nikon Small World top 100 images (see p.19), and Bree Berghuis, working with Carrie Graveel, had an image of cMet staining selected for the June 2008 Ventana Calendar.

From left: Olinger, Luo, Hudson, Jason, Carruthers, Berghuis, Resau, Goolsby, Ramirez, Kort, VandenBeldt, Looyenga

VARI | 2008

Recent Publications Haak, P., J. Busik, E. Kort, M. Tikhonenko, N. Paneth, and J. Resau. In press. Archived unfrozen neonatal blood spots are amenable to quantitative gene expression analysis. Neonatology. Ding, Yan, Elissa A. Boguslawski, Bree D. Berghuis, John J. Young, Zhongfa Zhang, Kim Hardy, Kyle Furge, Eric Kort, Arthur E. Frankel, Rick V. Hay, James H. Resau, and Nicholas S. Duesbery. 2008. Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma. Molecular Cancer Therapeutics 7(3): 648–658. Huang, Dan, Yan Ding, Wang-Mei Luo, Stephanie Bender, Chao-Nan Qian, Eric Kort, Zhong-Fa Zhang, Kristin VandenBeldt, Nicholas S. Duesbery, James H. Resau, and Bin Tean Teh. 2008. Inhibition of MAPK kinase signaling pathways suppressed renal cell carcinoma growth and angiogenesis in vivo. Cancer Research 68(1): 81–88. Baldus, Stephan E., Eric J. Kort, Peter Schirmacher, Hans P. Dienes, and James H. Resau. 2007. 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 31(1): 199–204. Depeille, Philippe, John J. Young, Elissa A. Boguslawski, Bree D. Berghuis, Eric J. Kort, James H. Resau, Arthur E. Frankel, and Nicholas S. Duesbery. 2007. Anthrax lethal toxin inhibits growth of and vascular endothelial growth factor release from endothelial cells expressing the human herpes virus 8 viral G protein–coupled receptor. Clinical Cancer Research 13(19): 5926–5934. Li, Zheng, Shireesh Srivastava, Xuerui Yang, Sheenu Mittal, Paul Norton, James Resau, Brian Haab, and Christina Chan. 2007. A hierarchical approach employing metabolic and gene expression profiles to identify the pathways that confer cytotoxicity in HepG2 cells. BMC Systems Biology 1: 15 pp. Lindemann, K., J. Resau, J. Nährig, E. Kort, B. Leeser, K. Anneke, A. Welk, J. Schäfer, G. F. Vande Woude, E. Lengyel, and N. Harbeck. 2007. Differential expression of c-Met, its ligand HGF/SF and HER2/neu in DCIS and adjacent normal breast tissue. Histopathology 51(1): 54–62. Ott, Mickey, Alan T. Davis, Wayne VanderKolk, James H. Resau, David H. DeHeer, Clifford B. Jones, Chad Stouffer, and Edward W. Kubek. 2007. The protective effect of the blood brain barrier from systemic cytokines in an animal femur fracture model. Journal of Trauma 63(3): 591–595. Whitwam, T., M.W. VanBrocklin, M.E. Russo, P.T. Haak, D. Bilgili, J.H. Resau, H.-M. Koo, and S.L. Holmen. 2007. Differential oncogenic potential of activated RAS isoforms in melanocytes. Oncogene 26(31): 4563–4570. Young, John J., Jennifer L. Bromberg-White, Cassandra R. Zylstra, Joseph T. Church, Elissa Boguslawski, James H. Resau, Bart O. Williams, and Nicholas S. Duesbery. 2007. LRP5 and LRP6 are not required for protective antigen–mediated internalization or lethality of anthrax lethal toxin. PLoS Pathogens 3(3): e27. Zhao, Ping, Tessa Grabinski, Chongfeng Gao, R. Scot Skinner, Troy Giambernardi, Yanli Su, Eric Hudson, James Resau, Milton Gross, George F. Vande Woude, Rick Hay, and Brian Cao. 2007. Identification of a Met-binding peptide from a phage display library. Clinical Cancer Research 13(20): 6049–6055.

Van Andel Research Institute |

Scientific Report

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, and in 2006 she was promoted to Senior Scientific Investigator.

Staff

Student

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

Katie Koelzer

VARI | 2008

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. The germline modification lab can also 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 can produce mutant embryos that have a wild-type placenta using tetraploid embryo technology, which 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, to allow 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.

Van Andel Research Institute |

Scientific Report

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 (about 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. 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; it is in demand by academia, and also by 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: Sisson, Swiatek, Khoo, Koeman, Lewis, Mowry

VARI | 2008

MAMC service offerings

Animal model development

•

ouse transgenics. Transgenic technology is used to produce genetically engineered mice expressing foreign M 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 uses various strains of laboratory mice, with production of three transgenic founder mice guaranteed from each procedure.

•

ene targeting. By transfecting mouse embryonic stem cells with inactivating, homologous DNAs, target G 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

•

ytogenetics. Mouse and rat chromosomal abnormalities and genetic loci are visually observed using Giemsa C stain, SKY, or FISH techniques.

•

ecropsy. Mice are dissected postmortem and tissues are fixed for histological analysis, with necropsy N reports generated using voice-recognition software.

•

istology. Histological sections are prepared from mouse tissues using microtomes and cryostats; they are H processed and stained using automated instruments and then are microscopically analyzed.

•

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

•

ouse rederivation. All mouse strains entering the specific pathogen–free breeding facility are rederived to M specific pathogen–free status using embryo transfer techniques.

•

nimal technical services. Veterinary services such as injections, measurements, mating set-up, and tail A 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.

•

mbryo/sperm cryopreservation. Genetically engineered mice are preserved for archival purposes, disease E control, genetic stability, and economic efficiency using germplasm cryopreservation techniques.

•

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

Van Andel Research Institute |

Scientific Report

Lipid signaling in osteosarcoma

A cell-based RNAi screen of human genes identified regulators of lipid signaling. Shown is an osteosarcoma cell marked by a lipid-binding domain fused to enhanced green fluorescent protein (green) and with the nucleus Hoechst-stained (blue); imaging was by fluorescence microscopy. Photo by Katie Sian of the MacKeigan lab.

VARI | 2008

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, 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 Chao-Nan (Miles) Qian, M.D., Ph.D. Peng-Fei Wang, M.D., Ph.D. Eric Kort, M.D. Daisuke Matsuda, M.D. Jindong Chen, Ph.D. Leslie Farber, Ph.D. Dan Huang, Ph.D. Yan Li, Ph.D. David Petillo, Ph.D. Racheal Zhang, Ph.D.

Zhongfa (Jacob) Zhang, Ph.D. Stephanie Bender, M.S. Wangmei Luo, M.S. Robert Antecki, B.S. Elizabeth Block, B.S. Stephanie Kloostra, B.S. Aaron Massie, B.S. Sabrina Noyes, B.S. Michael Westphal, B.S.

Students

Visiting Scientist

Michael Avallone Lindsay Barnett Kristin Buzzitta Bill Wondergem

Laura Lowe Furge, Ph.D.

Van Andel Research Institute |

Scientific Report

Research Interests Kidney cancer, or renal cell carcinoma (RCC), is the tenth most common cancer in the United States (51,000 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 1,500 cases) and serum; and a gene expression profiling database of 600 tumors, with long-term clinical follow-up information for half of them. Our program includes molecular subclassification using microarray gene expression profiling and bioinformatic analysis, generation of RCC mouse models, and more recently, molecular therapeutic studies.

RCC genomics We have been using high-density single nucleotide polymorphism (SNP) arrays to genotype RCC samples, and by combing this data and the gene expression data (see below), we have identified the candidate chromosomal regions and genes that are involved in different subsets of tumors.

Gene expression profiling and bioinformatics To date, we have studied over 600 RCC specimens. We are currently focusing on analysis and data mining. Clinically, we continue to subclassify the tumors by correlation with clinicopathological information, including rarer forms of RCC such as translation-related papillary RCC, mixed epithelial and stromal tumors, and adult Wilms. We are also in the process of trying to understand the underlying molecular signatures of some of the so-called unclassified group of tumors for which the histological diagnosis is â&#x20AC;&#x153;unknownâ&#x20AC;?. Our database has proven to be very useful in RCC research, since we can obtain differential expression data for any gene in seconds.

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 including urothelial cancer of the renal pelvis, whereas the VHL knock-out remains neoplasiafree; 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.

VARI | 2008

Targeted therapeutic studies We have focused on several targets we identified using gene expression profiling studies. In vitro and in vivo studies are being performed to verify these targets and their role in therapeutics. These include cell-cycle, proliferation, and migration assays to assess the cellular effects of these genes. In vivo studies are performed to understand the involvement of blood vessels in drug response.

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

From left: Teh, Antecki, Li, Furge, Huang, Kort, Noyes, Block, Petillo, Zhang, Matsuda, Chen, Kloostra

Recent Publications Camparo, P., V. Vasiliu, V. Molinié, J. Couturier, K. Dykema, D. Petillo, K.A. Furge, E.M. Comperat, M. Laé, R. Bouvier, L. Boccon-Gibbod, Y. Denoux, S. Ferlicot, E. Forest, G. Fromont, et al. In press. Renal translocation carcinomas: clinicopathological, immunohistochemical, and gene expression profiling analysis of 31 cases with a review of the literature. American Journal of Surgical Pathology. Chuang, Shang-Tian, Kurt T. Patton, Kristian T. Schafernak, Veronica Papavero, Fan Lin, Robert C. Baxter, Bin Tean Teh, and Ximing J. Yang. 2008. Over expression of insulin-like growth factor binding protein 3 in clear cell renal cell carcinoma. Journal of Urology 179(2): 445–449. Huang, Dan, Yan Ding, Wang-Mei Luo, Stephanie Bender, Chao-Nan Qian, Eric Kort, Zhong-Fa Zhang, Kristin VandenBeldt, Nicholas S. Duesbery, James H. Resau, and Bin Tean Teh. 2008. Inhibition of MAPK kinase signaling pathways suppressed renal cell carcinoma growth and angiogenesis in vivo. Cancer Research 68(1): 81–88.

Van Andel Research Institute |

Scientific Report

Morris, M.R., D. Gentle, M. Abdulrahman, N. Clarke, M. Brown, T. Kishida, M. Yao, B.T. Teh, F. Latif, and E.R. Maher. 2008. Functional epigenomics approach to identify methylated candidate tumour suppressor genes in renal cell carcinoma. British Journal of Cancer 98(2): 496–501. Selvarajan, S., L-H. Sii, A. Lee, G. Yip, B-H. Bay, M-H. Tan, B.T. Teh, and P-H. Tan. 2008. Parafibromin expression in breast cancer: a novel marker for prognostication? Journal of Clinical Pathology 61(1): 64–67. Wang, Pengfei, Michael R. Bowl, Stephanie Bender, Jun Peng, Leslie Farber, Jindong Chen, Asif Ali, ZhongFa Zhang, Arthur S. Alberts, Rajesh V. Thakker, Ali Shilatifard, Bart O. Williams, and Bin Tean Teh. 2008. Parafibromin, a component of the human PAF complex, regulates growth factors and is required for embryonic development and survival in adult mice. Molecular and Cellular Biology 28(9): 2930–2940. Yang, Ximing J., Ming Zhou, Ondrej Hes, Steven Shen, Rongshan Li, Jose Lopez, Rajal B. Shah, Yu Yang, Shang-Tian Chuang, Fan Lin, Maria M. Tretiakova, Eric J. Kort, and Bin Tean Teh. 2008. Tubulocystic carcinoma of the kidney: clinicopathological and molecular characterization. American Journal of Surgical Pathology 32(2): 177–187. Al-sarraf, Nael, Johanne Nørvig Reiff, Jane Hinrichsen, Shaukat Mahmood, Bin Tean Teh, Eilish McGovern, Pierre De Meyts, Kenneth J. O’Byrne, and Steven G. Gray. 2007. DOK4/IRS-5 expression is altered in clear cell renal cell carcinoma. International Journal of Cancer 121(5): 992–998. Daly, Adrian F., Jean-François Vanbellinghen, Sok Kean Khoo, Marie-Lise Jaffrain-Rea, Luciana A. Naves, Mirtha A. Guitelman, Arnaud Murat, Philippe Emy, Anne-Paule Gimenez-Roqueplo, Guido Tamburrano, Gérald Raverot, Anne Barlier, Wouter De Herder, Alfred Penfornis, Enrica Ciccarelli, et al. 2007. Aryl hydrocarbon receptor interacting protein gene mutations in familial isolated pituitary adenomas: analysis in 73 families. Journal of Clinical Endocrinology and Metabolism 92(5): 1891–1896. Haven, C.J., M. van Puijenbroek, M.H. Tan, B.T. Teh, G.J. Fleuren, T. van Wezel, and H. Morreau. 2007. Identification of MEN1 and HRPT2 somatic mutations in paraffin-embedded (sporadic) parathyroid carcinomas. Clinical Endocrinology 67(3): 370–376. Yao, Xin, Chao-Nan Qian, Zhong-Fa Zhang, Min-Han Tan, Eric J. Kort, James H. Resau, and Bin Tean Teh. 2007. Two distinct types of blood vessels in clear cell renal cell carcinoma have contrasting prognostic implications. Clinical Cancer Research 13(1): 161–169.

VARI | 2008

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.

Staff

Student

Glen Alberts, B.S. Jennifer Klomp, M.S. Xu Lu, Ph.D.

Sebla Kutluay, B.S. Sarah DeVos 55

Van Andel Research Institute |

Scientific Report

Research Interests The genetic information encoded in DNA must first be copied, 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, transcription, using infection by herpes simplex virus and the acclimation of plants to cold temperature as experimental contexts.

Transcriptional activation in herpes simplex virus infection Herpes simplex virus type 1 (HSV-1) causes the common cold sore or fever blister. The initial lytic infection by HSV-1 results in the obvious symptoms, typically in or around the mouth. After the initial infection, HSV-1 finds its way into nerve cells, where the virus can hide in a latent mode for long timesâ&#x20AC;&#x201D;essentially for the lifetime of the host. Occasionally, some event (such as emotional stress or damage to the nerve from a sunburn or a root canal operation) will cause the latent virus to reactivate, producing new viruses and recurrence of the cold sore. The DNA genome of HSV-1 encodes approximately 80 different proteins. The virus does not have its own machinery for expressing those genes, so it diverts the gene expression machinery of the host cell. That process is triggered by a viral regulatory protein designated VP16, whose function is to stimulate transcription of the first viral genes to be expressed (the immediate-early, or IE, genes). In the prevailing model for the mechanism of transcriptional activation, the activation domain of an activating protein (such as VP16) can bind to the host cell RNA polymerase II or to its accessory proteins. In this manner, VP16 recruits or tethers these accessory proteins to the genes that are to be activated.

Chromatin-modifying coactivators Eukaryotic DNA is typically packaged as chromatin, in which the DNA is wrapped around â&#x20AC;&#x153;spoolsâ&#x20AC;? of histone proteins, and these spools are 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 is overcome with the help of chromatin-modifying coactivator proteins; some chemically alter the histones and others remove the histones so that RNA polymerase can access the DNA. VP16 can recruit various coactivator proteins to target genes, and results from our lab have clearly indicated that VP16 can recruit certain coactivators to IE genes during lytic infection. We have also shown that some histone proteins do associate with viral DNA, although perhaps not to the same extent as with cellular DNA. Yet, the fact that coactivators are present on viral DNA is not sufficient evidence that they play a significant role in transcriptional activation. We have tested whether particular coactivators are necessary for effective expression of HSV-1 IE genes during lytic infection, using siRNA knockdown of certain coactivators or using mutant cell lines having disrupted expression or activity of a coactivator. We were surprised to find that viral genes were expressed efficiently regardless of what we did to diminish the coactivator activity. These results indicate that our initial hypothesis was wrong; the coactivators, although present, are not required for viral gene expression during lytic infection. Another possibility is that the coactivators are required to reactivate the viral genes from the latent or quiescent state, and we will test that hypothesis during the coming year.

VARI | 2008

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 had 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 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; however, subsequent experiments indicate that this effect is not mediated by the p300 and CBP proteins. For example, the effects on viral infection were observed using lower curcumin concentrations than those required to substantially inhibit global histone H3 acetylation. Moreover, we detected no effect of curcumin on the presence of H3 at viral gene promoters or on the acetylation of H3 at those promoters. These results suggest that curcumin affects VP16-mediated recruitment of RNA polymerase II to IE gene promoters by a mechanism independent of p300/CBP histone acetyltransferase activity. We conclude that curcumin does block herpes infections, but we donâ&#x20AC;&#x2122;t yet know the mechanism by which it does so.

Gene activation during cold acclimation of 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 popular experimental organism Arabidopsis, can sense low but nonfreezing temperature in a way that provides protection from subsequent freezing temperatures. This process is known as cold acclimation. Michael Thomashow, an MSU plant scientist, identified genes that are expressed during this process and a transcription factor that activates these genes in response to low temperature. We have collaborated with the Thomashow 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. However, contrary to our expectations, the GCN5 and ADA2 proteins are not responsible for this cold-induced acetylation. In fact, weâ&#x20AC;&#x2122;ve tested several other Arabidopsis histone acetyltransferases, and none (on their own) seem solely responsible for this acetylation. It seems likely that redundant mechanisms are at work, such that when we disrupt one pathway, another pathway compensates. We are also collaborating with groups in Greece and Pennsylvania to explore the distinct biological activities of the two ADA2 proteins. Although the two proteins have very similar sequences and both are expressed throughout the plant, mutations in the genes encoding these two proteins have very different phenotypes. The ada2b mutants are very short, have smaller cells than normal, and are sterile. In contrast, the ada2a mutants seem quite normal in most attributes (Figure 1). Plants with mutations in both ADA2a and ADA2b are strikingly similar to plants with mutations in GCN5. We suspect that GCN5 can partner with either ADA2a or ADA2b and that these two distinct complexes affect different sets of genes and thus different developmental and stress response pathways. 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 1

Figure 1. Growth of Arabidopsis plants, wild type and mutants.

Van Andel Research Institute |

Scientific Report

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

From left: Lu, Alberts, Kutluay, Triezenberg, Klomp

Recent Publications Kutluay, Sebla B., James Doroghazi, Martha E. Roemer, and Steven J. Triezenberg. 2008. Curcurmin inhibits herpes simplex virus immediate-early gene expression by a mechanism independent of p300/CBP histone acetyltransferase activity. Virology 373(2): 239â&#x20AC;&#x201C;247. Shooltz, Dean D., Glen L. Alberts, and Steven J. Triezenberg. 2008. One-step affinity purification of recombinant TATA binding proteins utilizing a modular protein interaction partner. Protein Expression and Purification 59(2): 297â&#x20AC;&#x201C;301.

VARI | 2008

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 founding Director of the Van Andel Research Institute.

Staff

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

Alysha Kett

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

Van Andel Research Institute |

Scientific Report

Laboratory of Molecular Oncology The Laboratory of Molecular Oncology is focused on understanding the numerous and diverse roles that Met and HGF/SF play in malignant progression and metastasis. Our work involves a wide variety of cancers, animal models, and drug therapies. The combination of studies, coupled with our examination of Met signaling, will lead to a greater understanding of tumor progression and new knowledge for developing and delivering novel targeted therapies.

Clonal selection of proliferative and invasive cells in metastasis Malignant progression leads to metastasis, which is the primary cause of death due to cancer. Metastasis begins with proliferating tumor cells that become invasive and detach from the primary tumor mass, invading the extracellular matrix, entering the bloodstream or lymphatic vessels, and establishing metastases or secondary tumors as proliferating colonies at distant sites. Because phenotypic switching between proliferation and invasion is critical to malignant progression, we use in vitro and in vivo methods to select subclones of glioblastoma tumor cells that are either highly proliferative or highly invasive. The molecular signaling pathways that accompany phenotypic switching change dramatically in response to HGF/SF. We discovered that invasive cells signal through the Ras/MAPK pathway, while the c-myc pathway is highly expressed in proliferative clones. Using gene expression analysis, spectral karyotyping (SKY), and fluorescent in situ hybridization (FISH), we observed that subtle and specific changes in chromosome content ratio are virtually the same as the changes in the chromosome transcriptome ratio, showing that major changes in gene expression are mediated by gains or losses in chromosome content. Importantly, a significant number of the genes whose expression change is greater than twofold are functionally consistent with changes in the proliferative or invasive phenotypes. Our results imply that chromosome instability can provide the diversity of gene expression that allows a tumor to switch between proliferative and invasive phenotypes during tumor progression.

Met Induces mammary tumors in mice and is associated with human basal breast cancers We are also investigating the role that the MET oncogene plays in breast cancer progression and metastasis through a novel mouse model of mutationally activated Met. We discovered that mutationally activated Met induces a high incidence of mammary tumors in mice. These mammary tumors have several unique pathological characteristics and contain high levels of extrachromosomal Met amplification. In addition, all of the tumors lack progesterone receptor expression and only half express ErbB2. These characteristics are similar to those of aggressive forms of human breast cancer and led us to examine how Met is associated with the various human breast cancer subtypes. Recently, gene expression studies have identified several distinct breast cancer subtypes that correlate with clinical outcome. These molecular subtypes include three main groups of estrogen receptor (ER)â&#x20AC;&#x201C;negative tumors (basal, ErbB2, and normallike/unclassified) and at least two types of ER-positive tumors (luminal A and luminal B). A study of Met expression data from existing human breast cancer datasets indicated that Met was significantly expressed in basal-like cancers relative to nonbasal cancers. To further examine Met expression patterns in human breast cancer, we used a human breast cancer tissue microarray containing 139 patient samples (in collaboration with Dr. Matthew Ellis and associates, Washington University). High Met staining was associated with the basal and ErbB2 subtypes and was inversely associated with the luminal subtypes. To confirm this observation, Met expression was compared to ER status and was found to negatively correlate with ER expression. These results show that Met protein levels are increased in the majority of breast cancer cases, but that the protein levels are highest in the more aggressive basal subtypes. Therefore, Met can be a novel therapeutic target for those patients with the most aggressive tumors and, currently, the fewest therapeutic options.

VARI | 2008

Noninvasive imaging of glioblastoma progression in a novel mouse model One major deficiency of the existing glioblastoma tumor cell lines used in mouse orthotopic models is in their lack of invasiveness. We have determined that the invasive phenotype of human glioblastoma cells is greatly enhanced in cells that develop extensive metastatic foci in the lungs, skeletal muscle, and lymph nodes after tail vein injection. Importantly, all individual infiltrative cells express Met, indicating that Met would be an effective target for inhibiting glioblastoma growth and invasion. One of the cell lines, when inoculated orthotopically, displays extensive infiltrative growth into normal mouse brain tissue. The brain tumor growth generates necrosis with pseudopalisades and closely resembles malignant glioblastoma in humans. In this model, osteolysis occurs at the inoculation site and, as a result, the tumor grows both intra- and extracranially. This growth pattern provides a transcranial acoustic window, allowing observation of tumor growth and vascularization with high resolution micro-ultrasound. Such observation allows real time monitoring of orthotopic brain tumor growth, for assessing intracranial tumor vascularity and for evaluating the therapeutic efficacy of antitumor agents. We determined that increases in the opening of the skull are proportional to tumor growth, and therefore ultrasound provides a surrogate measurement of tumor growth. With this cell line, we can measure tumor growth orthotopically in the brain, subcutaneously as tumor xenografts, and as metastatic growth in experimental lung metastases assays. We have shown that an anti-HSP90 drug, the geldanamycin derivative 17-(allylamino)-17-demethoxygeldanamycin (17AAG), inhibits tumor growth in all three model systems.

The role of Mig-6 in Met signaling and tumor suppression Mig-6 is one of several feedback regulators that we have found is rapidly induced by HGF/SF-Met signaling, as well as by other receptor tyrosine kinases such as EGFR. Mig-6 is a scaffolding adaptor protein that upon induction can negatively regulate EGFR and Met signaling. Mig-6 is located on human chromosome 1p36, a locus that is frequently associated with many human cancers. We have discovered that Mig-6 may function as a tumor suppressor, because mutations in the MIG-6 gene have been observed in human lung cancers, and disruption of Mig-6 in mice leads to lung, gallbladder, and bile duct cancers. Mig-6 may also play an important role in stress response and tissue homeostasis, as mice having a Mig-6 deficiency develop degenerative joint diseases that might be triggered by mechanical joint stress. We are currently investigating how Mig-6 regulates EGFR and Met signal transduction and what role Mig-6 may play in the development and progression of cancer and of degenerative joint disease.

External Collaborators Donald Bottaro and Benedetta Peruzzi, National Cancer Institute, Bethesda, Maryland Sandra Cottingham, Spectrum Health Hospitals, Grand Rapids, Michigan Francesco DeMayo, Baylor College of Medicine, Houston, Texas Ermanno Gherardi, MRC Center, Cambridge, England Sherri Davies and Matthew Ellis, Washington University, St. Louis, Missouri Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Ernest Lengyel and Ravi Salgia, University of Chicago, Illinois Patricia LoRusso, Karmanos Cancer Institute, Detroit, Michigan 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

Van Andel Research Institute |

Scientific Report

From left, back row: Graveel, DeGroot, Haak, Vande Woude, Gao, Staal, Kaufman, Essenburg, Zhang front row: Kang, Nelson, Su, Xie, Berens, Thompson

Recent Publications Knudsen, B., and G.F. Vande Woude. In press. Showering c-Met-dependent cancers with drugs. Current Opinion in Genetics and Development. Rouleau, Cecile, Krishna Menon, Paula Boutin, Cheryl Guyre, Hitoshi Yoshida, Shiro Kataoka, Michael Perricone, Srinivas Shankara, Arthur E. Frankel, Nicholas S. Duesbery, George F. Vande Woude, Hans-Peter Biemann, and Beverly A. Teicher. 2008. The systemic administration of lethal toxin achieves a growth delay of human melanoma and neuroblastoma xenografts: assessment of receptor contribution. International Journal of Oncology 32(4): 739–748. Zhao, Ping, Tessa Grabinski, Chongfeng Gao, R. Scot Skinner, Troy Giambernardi, Yanli Su, Eric Hudson, James Resau, Milton Gross, George F. Vande Woude, Rick Hay, and Brian Cao. 2007. Identification of a Met-binding peptide from a phage display library. Clinical Cancer Research 13(20): 6049–6055. Zhang, Yu-Wen, and George F. Vande Woude. 2007. Mig-6, signal transduction, stress response and cancer. Cell Cycle 6(5): 507–513. Tolbert, W. David, Jennifer Daugherty, Chongfeng Gao, Qian Xie, Cindy Miranti, Ermanno Gherardi, George Vande Woude, and H. Eric Xu. 2007. A mechanistic basis for converting a receptor tyrosine kinase agonist to an antagonist. Proceedings of the National Academy of Sciences U.S.A. 104(37): 14592–14597. Lindemann, K., J. Resau, J. Nährig, E. Kort, B. Leeser, K. Anneke, A. Welk, J. Schäfer, G. F. Vande Woude, E. Lengyel, and N. Harbeck. 2007. Differential expression of c-Met, its ligand HGF/SF and HER2/neu in DCIS and adjacent normal breast tissue. Histopathology 51(1): 54–62. Gao, ChongFeng, Kyle Furge, Julie Koeman, Karl Dykema, Yanli Su, Mary Lou Cutler, Adam Werts, Pete Haak, and George F. Vande Woude. 2007. Chromosome instability, chromosome transcriptome, and clonal evolution of tumor cell populations. Proceedings of the National Academy of Sciences U.S.A. 104(21): 8995–9000.

VARI | 2008

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; he now also oversees the Program for Translational Medicine.

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

Visiting Scentists Visiting Scientists Marcy Ross, B.S. Stephanie Scott, B.S. Danielle Welch, B.S. Katherine Koehler

Richard Leach, M.D. David Reinhold, Ph.D.

Students Molly Dobb Hailey Hines Catherine Perrin James Smith, Jr.

Van Andel Research Institute |

Scientific Report

Research Interests Molecular biomarkers are widely expected to revolutionize the current trial-and-error practice of medicine by enabling a more predictive discipline in which therapies target the molecular constitution of individual patients and their disease. This concept is often termed “personalized medicine”. Biomarkers are being widely evaluated for their ability to assess disease risk, detect and monitor disease over time, accurately identify disease stage, approximate prognosis, and predict optimal targeted treatments. The Program for Translational Medicine was launched in 2006 to extend the Institute’s translational research capabilities, with a focus on the development of molecular biomarker strategies with clinical implications. The program’s activities have focused on building the critical translational infrastructure and technologies, the fostering of clinical and industrial partnerships, and the coordination of the multidisciplinary project teams required to implement molecular-based approaches in medicine. The Program of Translational Medicine, with its multidisciplinary partners, strives to create an efficient pipeline between the clinic and the research laboratory for efficient discovery and clinical application of novel biomarker strategies. We also work to increase the readiness of the community to implement advances in molecular medicine, benefiting human health and promoting West Michigan as a leader in biomarker research.

Translational informatics To accelerate the implementation of personalized medicine, the consolidation and real-time analysis of standardized molecular and clinical/preclinical data is critical. Thus, much of our effort over the past several years has focused on the development of an integrated informatics solution known as the XenoBase BioIntegration Suite (XB-BIS; see http://xbtransmed.com). XB-BIS supports essential features of data management, data analysis, knowledge management, and reporting within an integrated framework, enabling the efficient exchange of information between the basic research laboratory and the clinic (Figure 1). XB-BIS has recently been licensed to industrial and academic partners with an interest in biomarker research, drug development, and the development of molecular-based diagnostics.

Figure 1

Figure 1. The XenoBase BioIntegration Suite (XB-BIS). The suite serves as a portal and common interface for consolidating clinical, preclinical, and molecular data, and it incorporates analytical, visualization, and reporting tools, which have historically been used in isolation. XB-BIS allows for the bidirectional flow of real-time data, information, and extracted knowledge between the multiple clinical and laboratory research components of a translational project.

VARI | 2008

Community partnerships Productive partnerships are pivotal to our efforts in biomarker research and personalized medicine. In the Center for Molecular Medicine (CMM, http://www.commex.org), the Van Andel Institute and Spectrum Health Hospitals have created a CLIA-certified/CAP-accredited clinical diagnostics laboratory for biomarker qualification and the development of associated diagnostic assays. CMM offers cutting-edge molecular diagnostic tests, and also performs fee-for-service genomics and proteomics work for commercial firms. Under Dr. Daniel Farkas, a national leader in molecular diagnostics, CMM provides access to molecular technologies of today that will become central to our personalized medicine initiatives in the future. ClinXus (http://www.clinxus.org) was developed to coordinate the West Michigan translational research enterprise. ClinXus was awarded a Michigan 21st Century Jobs Fund grant to support early-stage development and operations, and it was recently recognized for its efforts to develop biomarker strategies in translational studies by membership in the Predictive Safety and Testing Consortium (PSTC) of the Critical Path Institute. The PSTC brings pharmaceutical companies together to share and validate each other’s safety testing methods under advisement of the FDA and the European Medicines Agency. Membership in this prestigious consortium will help ensure that West Michigan remains at the forefront of biomarker research and development and will further the community’s rapidly emerging life sciences and healthcare industry.

Predictive therapeutics protocol Translational research represents the interface where hypotheses advance to studies that ultimately provide definitive information for a clinical decision. A fundamental challenge in clinical cancer research remains how to make best use of current biomarker technologies, advances in computational biology, the expanding pharmacopeia, and a rapidly expanding knowledge of disease networks to deliver targeted treatments to cancer patients with optimal therapeutic index. Our research is focused on developing, testing, and refining biomarker-driven analytical methods to systematically predict combinations of drugs that target the perturbed molecular systems within a tumor. We have also begun to consider means by which such information should be conveyed to the treating physician in support of medical decision-making. We recently completed a feasibility study of 50 late-stage pediatric and adult cancer patients: tumor-derived gene expression profiles were analyzed to identify potential drugs to target perturbed molecular components of each patient’s specific tumor. With patient consent, tumor biopsies are collected, qualified by pathology, and processed within the CMM to generate a standardized gene expression profile for the tumor. These molecular data are uploaded into XB-BIS along with pertinent clinical data, and these are compared with other patient samples. Deregulated patterns of gene expression are identified and analyzed within XB-BIS to identify drugs that have predicted efficacy based upon the genomic data. A report scoring a series of drugs for predicted efficacy is generated within XB-BIS, and this is conveyed to the treating physician in an actionable and electronic format for consideration in treatment planning. The process from patient consent to molecular report must be completed in 10 days, which provides significant workflow challenges. In parallel, a series of tumor grafts is established in immune-compromised mice, and these appear to closely resemble the human disease at the phenotypic and genotypic level. This resource is being used to test biomarker-driven predictive models (and the identified drugs) in a more systematic fashion and to evaluate novel targeted agents in partnership with industrial sponsors. Over the long term, the treatments are captured within XB-BIS together with critical outcome variables, allowing the predictive analytical methods to be refined and optimized. Anecdotal signs of success in a handful of patients have provided the impetus to launch a follow-up study with an expanded population of 220 patients and a more rigorous statistical design. In conjunction with our laboratory efforts to isolate, characterize, and target the putative cancer stem-cell subpopulation of metastatic tumors, biomarker-driven approaches that identify a rational treatment regimen targeting the molecular composition of the patient’s tumor hold promise for the future treatment of metastatic and refractory malignancies.

Van Andel Research Institute |

Scientific Report

From left: Koehler, Eugster, Miller, Webb, Monsma, Ross, Srikanth, Hines, Cherba, Scott, Dylewski

External Collaborators Acad emic Surgical Associates, Advanced Radiology Services, Cancer & Hematology Centers of Western Michigan, P.C., DeVos Childrenâ&#x20AC;&#x2122;s Hospital, Digestive Disease Institute, Grand Valley Medical Specialists, Grand Valley State University, MMPC, Saint Maryâ&#x20AC;&#x2122;s Health Care, Spectrum Health, and West Michigan Heart, all of Grand Rapids, Michigan Barbara Ann Karmanos Institute, and Henry Ford Hospital, Detroit, Michigan GeneGo, Inc., and Oncology Care Associates, St. Joseph, Michigan Jasper Clinical Research & Development, Inc. and ProNAi Therapeutics, Kalamazoo, Michigan Johns Hopkins University, Baltimore, Maryland M.D. Anderson Cancer Center, Houston, Texas Mary Crowley Cancer Center, Dallas, Texas Michigan State University, East Lansing, Michigan New York University, New York City Pfizer (Ann Arbor, Michigan; Saint Louis, Missouri; Groton, Connecticut) Schering-Plough Research Institute, New Jersey TGEN, Phoenix, Arizona University of Michigan, Ann Arbor University of California, San Francisco

Recent Publications Cherba, D., and C.P. Webb. In press. Systems biology of personalized medicine. In Bioinformatics for Systems Biology, second ed., Stephen Krawetz, ed. Humana Press. Littman, B., J. Thompson, and C.P. Webb. In press. Where are we heading/What do we really need? In Biomarkers in Drug Development, Michael Bleavins, Ramin Rahbari, Malle Jurima-Romet, and Claudio Carini, eds. New York: Wiley. Webb, C.P. In press. Personalized medicine: the need for system integration in the design of targeted therapies. In Computational and Systems Biology: Applications and Methods, Richard Mazzarella, ed.

VARI | 2008

Differentiating prostate epithelial cells

Prostate epithelial cells (PECs) were treated with keratinocyte growth factor and dihydrotestosterone to induce differentiation. PECs are positive for integrin beta 1 (green) and laminin 5 (red), while differentiated cells are negative for both. Nuclei were stained with Hoechst (blue). By forcing the cells to differentiate, cells that cannot be isolated for cell culture under normal conditions can be studied, allowing for improved understanding of normal prostate biology. Photo by Laura Lamb of the Miranti lab.

Van Andel Research Institute |

Scientific Report

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. Carrie Gabrielse, B.S.

Students Ying-Chou Chen, M.S. Charles Miller, B.S. Anthony Gaca Christina Gourlay Louise Haste Christina Untersperger

VARI | 2008

Research Interests We are studying how cells accurately replicate their DNA, a process that begins at specific DNA sequences termed replication origins. Various genome-wide approaches have identified from 320 to 420 possible replication origins in budding yeast, the model organism we study. In G1 phase, each origin assembles approximately 40 polypeptides in a temporally defined order, culminating in the initiation of DNA replication at the G1/S phase boundary. The first stage of this process is called pre-replicative complex assembly and requires the origin recognition complex (ORC), Cdc6p, and Cdt1p. ORC directly binds to DNA and then recruits Cdt1p and Cdc6p during early G1 phase. These three proteins cooperate to load the MCM DNA helicase at origins in an ATP-dependent reaction. Cyclin-dependent kinases and the Cdc7p-Dbf4p kinase then catalyze the association of additional proteins with the MCM helicase, ultimately causing the initiation of bi-directional DNA synthesis (Figure 1). In our lab we are studying how Cdc6p-ATP functions to load the MCM helicase within a chromatin context in budding yeast. We also are studying the Cdc7p-Dbf4p kinase in both yeast and human cells. We previously discovered that deletion of SIR2, encoding a histone deacetylase, rescued the temperature sensitivity of several mutants that were defective in pre-RC assembly, including a cdc6-4 mutant. We screened the replication origins on chromosomes III and VI to identify those origins that were inhibited by SIR2 and identified five SIR2-sensitive origins: ARS305, ARS315, ARS317, ARS603, and ARS606. We determined the detailed structure of two origins on chromosome III and found that these origins contain inhibitory elements distal to the ORC binding site. By utilizing data from another group that mapped stably positioned nucleosomes on chromosome III, we found that these inhibitory elements were positioned within stably bound nucleosomes. Furthermore, the positioned nucleosomes were very close to or overlapping the site of pre-RC assembly. Origins that were not inhibited by SIR2 did not have nearby nucleosomes in this region. Since genetically, SIR2 inhibited origins through the inhibitory element, we suggest that the acetylation state of this nucleosome affects pre-RC assembly. We do not know whether Sir2p is acting directly at these inhibitory sites or indirectly by regulating another gene, but we put forth the following model to explain our findings (Figure 2). Because pre-RC assembly occurs on naked DNA and because nucleosomes within the origin inhibit pre-RC assembly, some origins may exist within a nucleosome environment that is not optimal for pre-RC assembly. In those cases, either a particular modification of a nearby nucleosome or a protein that binds to the nucleosome in a SIR2-dependent manner inhibits pre-RC assembly. Since Sir2p was previously thought to only act at very specific heterochromatic regions in the genome, we are interested to discover exactly how Sir2p acts at these euchromatic sites.

Figure 1

Figure 1. The stages of replication initiation. In the first stage, a multi-subunit complex called the prereplicative complex (pre-RC) is assembled at replication origins. This complex consists of ORC, Cdt1p, Cdc6p, and the MCM helicase. In the second stage, Cdc7p-Dbf4p and Cdk kinases activate the MCM helicase, resulting in origin unwinding and the association of DNA polymerases at the origin.

Van Andel Research Institute |

Scientific Report

Figure 2

Figure 2. Model for SIR2mediated inhibition of DNA replication. A) Schematic of the ARS315 element that contains an inhibitory sequence element (IS) downstream of the site for pre-RC assembly. B) The IS element positions a nucleosome close to B2, which is inhibitory for pre-RC assembly. Deletion of SIR2 or mutation of the IS element allows repositioning of the nucleosome to allow pre-RC assembly.

Cdc7p-Dbf4p is a two-subunit protein kinase required for initiating DNA replication after MCM helicase loading. The Cdc7p kinase subunit binds Dbf4p, which activates its kinase activity. Although Cdc7p-Dbf4p is required to promote DNA replication, it also has an undefined role in the repair of certain DNA lesions. In order to define the amino acids required for its roles in DNA replication and repair, we are analyzing Dbf4p using a mutational approach. We found that the Dbf4p N-terminus is dispensable for DNA replication, but it encodes functions that participate in the repair of DNA lesions and the firing of late-replication origins. An N-terminal BRCT-like motif may mediate these activities. It is also possible that Dbf4p maintains replication fork stability by targeting Cdc7p kinase to stalled replication forks. Interestingly, these are separable activities from Dbf4p’s essential role in promoting initiation of DNA replication. In addition, we have identified two regions within Dbf4p—a C-terminal Zn-finger motif and a separate region—that mediate binding to and activation of the Cdc7p kinase. The C-terminal region is also not essential for Dbf4p activity, but loss of this region dramatically lowers Cdc7p kinase activity. We also are studying the human Cdc7-Dbf4 protein kinase. We previously raised monoclonal antibodies that recognize both human subunits and used these to screen human cancer cell lines and primary human tumors for HsCdc7-Dbf4 abundance. Although both subunits are expressed at very low levels in normal cycling cells (and are perhaps absent in post-mitotic cells), they are up-regulated in a substantial number of tumor cell lines. HsCdc7 protein is also highly expressed in some primary breast and colon tumors. By screening expression data from a panel of more than 650 primary human tumors, we also found that CDC7 and DBF4 mRNA expression are coordinately up-regulated in many tumors of diverse origin. Similarly, we screened several primary tumors and tumor cell lines for gene copy changes in CDC7 and DBF4. We found that the DBF4 gene copy number is often elevated in those cells expressing higher levels of Cdc7-Dbf4 kinase. Because HsCdc7 is an essential kinase for DNA replication, its increased expression level in some tumors and tumor cell lines may reflect higher rates of cellular proliferation. However, we find no correlation between doubling time and Cdc7-Dbf4 expression in the NCI60 tumor cell lines. Furthermore, several published studies suggest that increased Cdc7-Dbf4 expression can inhibit the growth of rodent cell lines but has no effect on the growth of human cells. We suggest that since HsCdc7-Dbf4 is likely involved in other aspects of chromosome metabolism (e.g., DNA repair) and functions in the S-phase checkpoint, its increased expression in some tumor cell lines may offer an advantage for handling the chromosome instability that occurs in many human tumors. Our aim is to understand the mechanism(s) that allow increased expression of Cdc7-Dbf4 kinase in tumor cells and to investigate its phenotypic effects.

VARI | 2008

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

External Collaborators Angelika Amon, Massachusetts Institute of Technology, Cambridge Catherine Fox, University of Wisconsinâ&#x20AC;&#x201C;Madison Carol Newlon, University of Medicine and Dentistry of New Jersey, Newark Alain Verreault, University of Montreal, Quebec, Canada

Recent Publications Crampton, Amber, FuJung Chang, Donald L. Pappas, Jr., Ryan L. Frisch, and Michael Weinreich. 2008. An ARS element inhibits DNA replication through a SIR2-dependent mechanism. Molecular Cell 30(2): 156â&#x20AC;&#x201C;166.

Van Andel Research Institute |

Scientific Report

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. Kyle VanKoevering, B.S. Cassandra Zylstra, B.S. 72

Students Tristan Kempston Audrey Sanders Brent Vanderhart, B.S.

VARI | 2008

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 useful 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 further shown 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 b-catenin, we created mice carrying an osteoblast-specific deletion of b-catenin (OC-cre;b-catenin-flox/flox mice). In collaboration with Tom Clemens of the University of Alabama at Birmingham, we found that alterations of Wnt/b-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 b-catenin revealed significant alterations in osteoclastogenesis. We are addressing how other genetic alterations linked to Wnt/b-catenin signaling affect bone development and osteoblast function. We have generated mice with conditional alleles of Lrp6 and Lrp5 that can be inactivated via cre-mediated recombination, and we will assess the roles of these genes at different stages of osteoblast differentiation. Finally, we are working to determine what other signaling pathways may impinge on b-catenin signaling to control osteoblast differentiation and function.

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, and a germline deficiency for Lrp5 or Lrp6 results in delayed mammary development. Because Lrp5-deficient mice are viable and fertile, we have focused our initial efforts on these mice. In collaboration with Caroline Alexanderâ&#x20AC;&#x2122;s laboratory, we have found dramatic reductions in the number of mammary progenitor cells in these mice, and we are examining the mechanisms underlying this reduction. We have also found that Lrp6 plays a key role in mammary development, and we are focusing on the mechanisms underlying this unique role. Finally, we are defining the relative roles of b-catenin and mTOR signaling in the initiation and progression of Wnt1-induced mammary tumors. We are particularly interested in the role(s) of these pathways in regulating the proliferation of normal mammary progenitor cells, as well as of tumor-initiating cells.

Van Andel Research Institute |

Scientific Report

Wnt signaling in metabolic syndrome Several studies have linked mutations in Lrp5 and/or Lrp6 to the development of diabetes, dyslipedemias, and hypertension in humans and mice. We are exploring the roles of these genes in this context by creating mice carrying conditional deletions in hepatocytes or in adipocytes and evaluating their phenotypes.

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 b-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.

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.

VARI mutant mouse repository With support from the Institute, our 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.

From left: Vanderhart, Zylstra, Sanders, Lindvall, Williams, VanKoevering

VARI | 2008

External Collaborators Bone development Mary Bouxsein, Beth Israel Deaconness Medical Center, Boston, Massachusetts Thomas Clemens, University of Alabama–Birmingham 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

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 California, San Diego Malathy Shekhar, Wayne State University, Detroit, Michigan Aaron Zorn, University of Cincinnati

Recent Publications Robinson, D.R., C.R. Zylstra, and B.O. Williams. In press. Wnt signaling and prostate cancer. Current Drug Targets. Shekhar, Malathy P.V., Brigitte Gerard, Robert J. Pauley, Bart O. Williams, and Larry Tait. 2008. Rad6B is a positive regulator of b-catenin stabilization. Cancer Research 68(6): 1741–1750. Wang, Pengfei, Michael R. Bowl, Stephanie Bender, Jun Peng, Leslie Farber, Jindong Chen, Asif Ali, ZhongFa Zhang, Arthur S. Alberts, Rajesh V. Thakker, Ali Shilatifard, Bart O. Williams, and Bin Tean Teh. 2008. Parafibromin, a component of the human PAF complex, regulates growth factors and is required for embryonic development and survival in adult mice. Molecular and Cellular Biology 28(9): 2930–2940. Hinoi, Takao, Aytekin Akyol, Brian K. Theisen, David O. Ferguson, Joel K. Greenson, Bart O. Williams, Kathleen R. Cho, and Eric R. Fearon. 2007. Mouse model of colonic adenoma-carcinoma progression based on somatic Apc inactivation. Cancer Research 67(20): 9721–9730. Lindvall, Charlotta, Wen Bu, Bart O. Williams, and Yi Li. 2007. Wnt signaling, stem cells, and the cellular origin of breast cancer. Stem Cell Reviews 3(2): 157–168. Wu, Rong, Neali Hendrix-Lucas, Rork Kuick, Yali Zhai, Donald R. Schwartz, Aytekin Akyol, Samir Hanash, David E. Misek, Hidetaka Katabuchi, Bart O. Williams, Eric R. Fearon, and Kathleen R. Cho. 2007. Mouse model of human ovarian endometroid adenocarcinoma based on somatic defects in the Wnt b-catenin and PI3K/Pten signaling pathways. Cancer Cell 11(4): 321–333. Young, John J., Jennifer L. Bromberg-White, Cassandra R. Zylstra, Joseph T. Church, Elissa Boguslawski, James H. Resau, Bart O. Williams, and Nicholas S. Duesbery. 2007. LRP5 and LRP6 are not required for protective antigen–mediated internalization or lethality of anthrax lethal toxin. PLoS Pathogens 3(3): e27.

Van Andel Research Institute |

Scientific Report

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 Abhishek Bandyopadhyay, Ph.D. Jiyuan Ke, Ph.D. Schoen Kruse, Ph.D. Raghu Malapaka, Ph.D. Karsten Melcher, Ph.D. Augie Pioszak, Ph.D. David Tolbert, Ph.D. 76

Yong Xu, Ph.D. Chenghai Zhang, Ph.D. X. Edward Zhou, Ph.D. Jennifer Daugherty, B.S. Amanda Kovach, B.S. Naomi Parker, B.S. Kelly Powell, B.S.

Students

Visiting Scientist

Cee Wah Chan Aoife Conneely Xiang Gao Mien Nguyen Rachel Talaski Peipei Zhong

Ross Reynolds, Ph.D.

VARI | 2008

Research Interests Our laboratory uses multidisciplinary approaches to study the structures and functions of protein complexes that play key roles in major signaling pathways. Currently we are focusing on three families of proteins: nuclear hormone receptors, the Met tyrosine kinase receptor, and G protein–coupled receptors, because beyond their fundamental roles in biology, these proteins 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 transcription 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. These classic receptors are among the most successful targets in the history of drug discovery: every receptor has one or more cognate synthetic ligands being used as medicines. The nuclear receptors also include a class of “orphan” receptors for which no ligand has been identified. In the last several years, we have developed the following projects centering on the structural biology of nuclear receptors.

Peroxisome proliferator–activated receptors The peroxisome proliferator–activated receptors (PPARa, d, and g) are key regulators of glucose and fatty acid homeostasis and as such are important therapeutic targets for treating cardiovascular disease, diabetes, and cancer. 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 approaches to 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. 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.

Van Andel Research Institute |

Scientific Report

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. This hormone-independent cancer is highly aggressive and is responsible for most deaths from prostate cancer. In this project, we are aiming to determine the structures of 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.

Orphan nuclear receptors 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; more than twice the size 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 the 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 correlates 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; thus, 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.

G proteinâ&#x20AC;&#x201C;coupled receptors G proteinâ&#x20AC;&#x201C;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 molecules. Currently, there is only one reported GPCR structure, for an inactive form of bovine rhodopsin. 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. Currently, we are focusing on hormone recognition by Class B GPCRs, and we have recently determined the first structure of parathyroid hormone bound to the extracellular domain of its receptor.

VARI | 2008

External Collaborators Doug Engel, University of Michigan, Ann Arbor Ermanno Gherardi, University of Cambridge, United Kingdom 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: Kruse, Chan, Zhou, Pioszak, Bandyopadhyay, Zhang, Zhong, Ke, Malapaka, Y. Xu, Melcher, Tolbert; seated: Guthrey, Parker, Conneely, Kovach, Powell, H.E. Xu

Recent Publications Pioszak, Augen A., and H. Eric Xu. 2008. Molecular recognition of parathyroid hormone by its G protein-coupled receptor. Proceedings of the National Acadamy of Sciences U.S.A. 105(13): 5034–5039. Suino-Powell, Kelly, Yong Xu, Chenghai Zhang, Yong-guang Tao, W. David Tolbert, Stoney S. Simons, Jr., and H. Eric Xu. 2008. Doubling the size of the glucocorticoid receptor ligand binding pocket by deacylcortivazol. Molecular and Cellular Biology 28(6): 1915–1923. Guo, Dongsheng, Joy Sarkar, Kelly Suino-Powell, Yong Xu, Kojiro Matsumoto, Yuzhi Jia, Songtao Yu, Sonal Khare, Kasturi Haldar, M. Sambasiva Rao, Jennifer E. Foreman, Satdarshan P.S. Monga, Jeffrey M. Peters, H. Eric Xu, and Janardan K. Reddy. 2007. Induction of nuclear translocation of constituitive androstane receptor by peroxisome proliferator–activated receptor a synthetic ligands in mouse liver. Journal of Biological Chemistry 282(50): 36766–36776. Tolbert, W. David, Jennifer Daugherty, Chongfeng Gao, Qian Xie, Cindy Miranti, Ermanno Gherardi, George Vande Woude, and H. Eric Xu. 2007. A mechanistic basis for converting a receptor tyrosine kinase agonist to an antagonist. Proceedings of the National Academy of Sciences U.S.A. 104(37): 14592–14597.

Van Andel Research Institute |

Scientific Report

Daniel Nathans Memorial Award

VARI | 2008

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.

Award Recipients 2000 2001 2002 2003 2004 2005 2006

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. Harald zur Hausen, M.D., and Douglas R. Lowy, M.D.

Dr. Harald zur Hausen (left) and Dr. Douglas R. Lowy (right) with Dr. George Vande Woude, who presented the Nathans Awards.

Van Andel Research Institute |

Scientific Report

Postdoctoral Fellowship Program

VARI | 2008

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 2007 and early 2008 are listed below.

Abhishek Bandyopadhyay

Brendon Looyenga

Yi-Mi Wu

University of Cambridge, U.K. VARI mentor: Eric Xu

University of Michigan, Ann Arbor VARI mentor: James Resau

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

Jennifer Bromberg-White

Xu Lu

Yong Xu

Pennsylvania State University College of Medicine, Hershey VARI mentor: Nicholas Duesbery

University of Texas Health Sciences Center, San Antonio VARI mentor: Steven Triezenberg

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

Philippe Depeille

Venkata Malapaka

Chenghai Zhang

University of Montpellier, France VARI mentor: Nicholas Duesbery

Western Michigan University, Kalamazoo VARI mentor: Eric Xu

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

Yan Ding

Daisuke Matsuda

Xiaoyin Zhou

Peking Union Medical College, China VARI mentor: Nicholas Duesbery

Kitasato University, Japan VARI mentor: Bin Teh

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

Kathryn Eisenmann

Augen Pioszak

University of Minnesota, Minneapolis VARI mentor: Arthur Alberts

University of Michigan, Ann Arbor VARI mentor: Eric Xu

Leslie Farber

Daniel Robinson

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

University of California, Davis VARI mentor: Bart Williams

Quliang Gu

Virology University, France VARI mentor: Michael Weinreich

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

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

Dorine Savreux Peng Fei Wang Fourth Military Medical University, Shannxi, China VARI mentor: Bin Teh

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

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

Dan Huang Peking Union Medical College, China VARI mentor: Bin Teh

Schoen Kruse University of Colorado, Boulder VARI mentor: Eric Xu

Yan Li Peking Union Medical College, China VARI mentor: Bin Teh

From left: Eisenmann, Kruse, Bromberg-White, Pioszak, Malapaka, Lu, Zhang, Looyenga, Xu

Van Andel Research Institute |

Scientific Report

Student Programs

VARI | 2008

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 Schering Plough and VARI. The program is designed to provide selected high school students, who have plans to major in science or genetic engineering in college, with 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 four 2007 GRAPCEP students were

Bryan Mendez

(Resau/Duesbery)

Creston High School

Tarrick Mussa

(Resau/Duesbery)

Creston High School

Aleesa Schlientz

(Resau/Duesbery)

Creston High School

Jennifer Vogal

(Hay)

Creston High School

From left: Mussa, Schlientz, Vogal, Mendez

Van Andel Research Institute |

Scientific Report

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 2007 to March 2008, VARI hosted 74 students from 22 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.

Anderson University, Indiana

Kalamazoo College, Kalamazoo, Michigan

James Smith, Jr. (Webb)

Adam Granger (Haab)

Aquinas College, Grand Rapids, Michigan

Marquette University, Milwaukee, Wisconsin

Elizabeth Block (Teh) Krysta Collins* (Haab) Christina Gourley (Weinreich) Sara Kunz* (Hay) Mien Nguyen* (Xu) Audrey Sanders (Williams) Randi VanOcker (Haab)

Calvin College, Grand Rapids, Michigan

Christopher Gorter* (Alberts) Lee Heeringa (Haab) Alysha Kett* (Vande Woude) Geoff Kraker (MacKeigan) Bill Wondergem (Teh)

Central Michigan University, Mount Pleasant

Lindsay Barnett (Teh) Sarah DeVos* (Triezenberg)

Ferris State University, Big Rapids, Michigan

Carrie Fiebig (Haab)

Grand Rapids Community College, Michigan

Wei Luo (Resau) Albert Rodriguez (Alberts)

Michael Avallone (Teh)

McGill University, Montreal, Quebec, Canada

Halley Crissman (Resau/MacKeigan)

Michigan State University, East Lansing

Heather Born (Resau) Ying-Chou Chen, M.S. (Weinreich) Michelle Dawes (Duesbery) Aaron DeWard, B.S. (Alberts) Anthony Gaca* (Weinreich) Pete Haak, B.S. (Resau) Sebla Kutluay, B.S. (Triezenberg) Laura Lamb, B.S. (Miranti) Chih-Shia Lee, M.S. (Duesbery) Charles Miller, B.S. (Weinreich) Katie Sian, B.S. (MacKeigan) Susan Spotts, B.S. (Miranti) Rachel Talaski (Xu) Jelani Zarif, M.S. (Miranti)

Nanjing Medical University, China

Guipeng Ding (Cao) Ning Xu (Cao) Aixia Zhang (Cao)

Grand Valley State University, Allendale, Michigan

Northern Illinois University, DeKalb

Alaa Abughoush (Hay) Erica Bechtel* (Miranti) Janell Carruthers (Resau) Molly Dobb (Webb) Eric Graf (Miranti) Craig Johnson (Furge) Tristan Kempston* (Williams) Kevin Maupin (Haab) Lisa Orcasitas (Duesbery) Gary Rajah (Miranti) Sara Ramirez (Resau)

Yarong Yang (Resau)

Spring Arbor University, Michigan

Jenna Manby* (Cao)

Sun Yat-sen University, Guangzhou, China

Rui Sun (Cao)

VARI | 2008

Kneeling, left to right: Church, Smith, Gaca, Kempston, Gorter, Koelzer, Buzzitta, Devos Standing, left to right: Crissman, Firestone, Manby, Dawes, Hines, Avallone, Born, Kunz, Wilcox, Burgenske, Bechtel, Block, Barnett, VanKoevering, Nguyen, Gao, Talaski, McElliott, Herman

University of Bath, United Kingdom

University of Ulster, Northern Ireland

Naomi Asantewa-Sechereh (Duesbery) Cee Wah Chen (Xu) Aoife Conneely (Xu) Louise Haste (Weinreich) Fraser Holleywood (Miranti) Christina Untersperger (Weinreich)

University of Illinois, Champaign-Urbana

Huong Tran (Resau)

University of Mannheim, Germany

Katja Strunk (Alberts)

University of Michigan, Ann Arbor

Alyse DeHaan* (MacKeigan) Xiang Gao (Xu) Theresa Gipson* (Furge) Sara Herman* (Resau/MacKeigan) Hailey Hines (Webb) Katie Koelzer* (Swiatek) Matthew McElliott* (Cavey/MacKeigan) Catherine Perrin* (Webb) Kyle VanKoevering (Williams) Jennifer Wilcox* (Duesbery)

University of Notre Dame, South Bend, Indiana

Peipei Zhong (Xu)

University of Wisconsin â&#x20AC;&#x201C; Green Bay

Danielle Burgenske (Resau)

Other Van Andel Institute Interns Aquinas College, Grand Rapids, Michigan

Tanja Barunovic (Grants and Contracts)

Davenport University, Grand Rapids, Michigan

Randall Edler (Information Technology) Philip Straatsma (Information Technology)

Ferris State University, Big Rapids, Michigan

Eric Firestone (Facilities)

Grand Valley State University

Andy Schmidt (Finance)

University of Michigan, Ann Arbor

Mitchell Zoerhoff (Communications)

Kristin Buzzitta* (Teh) Joe Church (MacKeigan)

Van Andel Research Institute |

Scientific Report

Han-Mo Koo Memorial Seminar Series

VARI | 2008

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 2007

Moses Lee, Hope College

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

February

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 Sciences

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

Melanie H. Cobb, University of Texas Southwestern Medical Center

“MAP kinase signaling in pancreatic beta cells”

April

Christopher G. Wood, M.D. Anderson Cancer Center

“Redefining the role of surgery for renal cell carcinoma in the era of targeted therapy”

Jules J. Berman, Writer/consultant

“New trends in biomedical informatics”

Van Andel Research Institute |

May

Scientific Report

Shiv Greval, National Cancer Institute

“Heterochromatin: a versatile platform of the genome”

Ermanno Gherardi, MRC Centre

“Structure/function of HGF/SF and MET”

Caroline Alexander, University of Wisconsin–Madison

“Stem cells and cancer”

David A. Cheresh, University of California, San Diego

“Signaling mechanism in angiogenesis and metastasis”

June

Bruce R. Ksander, Schepens Eye Research Institute

“A gene therapy to prevent the growth and spread of uveal melanomas”

August

Peggy Farnham, University of California, Davis

“Using ChIP-chip to characterize mechanisms of transcriptional repression in pluripotent and differentiated mammalian cells”

Daniel D. Von Hoff, Translational Genomics Research Institute

“The oncologists’ 6th vital sign—a context of vulnerability”

Arul M. Chinnaiyan, University of Michigan

“Recurrent gene fusions in prostate cancer: a new class of biomarkers and therapeutic targets”

Eddy Arnold, Rutgers University

“Engineering of high-resolution HIV-1 reverse transcriptase crystals and the concept of strategic flexibility in evading drug resistance”

VARI | 2008

September

Michael Glotzer, University of Chicago

“Dividing the spoils—positioning the plane of cell division”

Andries Zijlstra, Vanderbilt University

“Intravital imaging of tumor cell motility: the role of migration in metastasis”

Harald zur Hausen, German Cancer Research Institute

The Daniel Nathans Memorial Scientific Lecture: “Mechanisms of viral oncogenesis” The Daniel Nathans Memorial Lay Lecture: “Infectious causes of human cancer”

Douglas R. Lowy, National Cancer Institute

The Daniel Nathans Memorial Scientific Lecture: “Preventing cervical cancer by HPV vaccination and other approaches” The Daniel Nathans Memorial Lay Lecture: “Prevention and treatment of cancers caused by infections”

Michael Ohh, University of Toronto

“HIF-centric tumour suppressor model of VHL”

October

Atul Butte, Stanford University

“Exploring genomic medicine using translational bioinformatics”

Phil Hieter, University of British Columbia

“Chromosome instability in yeast and cancer”

Dimiter S. Dimitrov, National Cancer Institute

“Human monoclonal antibodies against viruses and cancer”

Robert L. Nussbaum, University of California, San Francisco

“Molecular genetic approach to Parkinson disease”

Van Andel Research Institute |

November

Scientific Report

Timothy P. Cripe, Children’s Hospital Medical Center, Cincinnati

“Got herpes? Developing oncolytic virus therapy for pediatric cancer”

Gary D. Stoner, Ohio State University

“A food-based approach to the prevention of G.I. tract cancers”

Stephen P. Bell, Massachusetts Institute of Technology

“A G1 checkpoint coordinating origin selection and cell cycle progression”

Diane M. Simeone, University of Michigan

“Pancreatic cancer stem cells”

G. David Roodman, University of Pittsburgh

“Paget’s disease: virus or gene?”

February 2008

David N. Zachs, University of Michigan

“Photoreceptor survival during disease: life hanging in the balance”

Anthony J. Senagore, Spectrum Health

“Economic issues in translational medicine”

March

Robert M. Strieter, University of Virginia

“CXC chemokines in angiogenesis and metastases of cancer”

Graham J. Burton, University of Cambridge

“Trophoblast invasion in human pregnancy: functions, mechanisms, and regulation”

Valeri I. Vasioukhin, Fred Hutchinson Cancer Research Center

“Mechanisms of prostate cancer initiation and progression”

VARI | 2008

Van Andel Research Institute Organization

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 James Fahner, M.D. Fritz M. Rottman, Ph.D.

Board of Scientific Advisors 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.

VARI | 2008

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

Administrative Assistants From left, Lewis, Koehler, Spears, Holman, Chastain, Guthrey, Noyes, Nelson, Rappley, Jason, Koo, Resau Not present: Novakowski

Van Andel Research Institute |

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

Finance

David Van Andel, Chairman and CEO Steven R. Heacock, Chief Administrative Officer and General Counsel R. Jack Frick, Chief Financial Officer Christy Goss, Executive Assistant Ann Schoen, Executive Assistant Laura Lohr

Timothy Myers, Controller Cory Cooper Sandi Essenburg Stephanie Green Richard Herrick Keri Jackson Angela Lawrence Laura Lohr Heather Ly Susan Raymond Jamie VanPortfleet Mitchell Zoerhoff

Business Development Jerry Callahan, Ph.D., Vice President Brent Mulder, Ph.D. Thomas DeKoning Jennifer McGrail Linda Chamberlain, Ph.D., consultant

Communications and Development Joseph P. Gavan, Vice President Jaime Brookmeyer Tim Hawkins Sarah Lamb Sarah Smedes Laurie Ward

Facilities Samuel Pinto, Manager Michelle Bies Jeff Cooling Jason Dawes Ken De Young Shelly King Tracy Lewis Dave Marvin Karen Pittman Richard Sal Richard Ulrich Pete VanConant Jeff Wilbourn

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

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

Human Resources Linda Zarzecki, Director Margie Hoving Stephanie Koelewyn Pamela Murray Angela Plutschouw

VARI | 2008

Information Technology

Procurement Services

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

Richard M. Disbrow, C.P.M., Manager Heather Frazee Chris Kutchinski Shannon Moore Amy Poplaski John Waldon

Investments Office

Sarah Lowen, Librarian (Grand Valley State University) Jim Kidder, Safety Manager (Michigan State University)

Kathleen Vogelsang, Director Benjamin Carlson Ted Heilman

Security Kevin Denhof, CPP, Chief Amy Davis Sean Mooney Maria Straatsma Chris Wilson

Contract Support

Van Andel Research Institute |

Scientific Report

Van Andel Institute

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

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 James Fahner, M.D. Fritz M. Rottman, Ph.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 | 2008

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.

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

Director for Research Administration

Roberta Jones

BASIC SCIENCE

SPECIAL PROGRAMS

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

Craig Webb, Ph.D. Tumor Metastasis & Angiogenesis

Cancer Genetics

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

Signal Transduction Art Alberts, Ph.D. Cell Structure & Signal Intergration Cindy Miranti, Ph.D. Integrin Signaling & Tumorigenesis

DNA Replication & Repair Michael Weinreich, Ph.D. Chromosome Replication

James Resau, Ph.D.

Bryn Eagleson, A.A. Transgenics and Vivarium

James Resau, Ph.D. Analytical, Cellular, & Molecular MIcroscopy

Structural Biology

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

James Resau, Ph.D. Microarray Technology

Eric Xu, Ph.D. Structural Sciences

Art Alberts, Ph.D. Flow Cytometry

Kyle Furge, Ph.D. Computational Biology

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

Systems Biology Jeffrey MacKeigan, Ph.D. Systems Biology

Animal Imaging

Gene Regulation

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

Steven Triezenberg, Ph.D. Transcriptional Regulation

Greg Cavey, B.S. Mass Spectrometry and Proteomics James Resau, Ph.D. Molecular Epidemiology

Dean of VAI Graduate School

Van Andel Research Institute |

Scientific Report

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.

Printed by Spectrum Graphics, Inc.

100