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VARI | 2007

Van Andel Research Institute

Scientific Report 2007


Van Andel Research Institute Scientific Report 2007

Cover photo: The glass sculpture “Life”, by Dale Chihuly,

in the Van Andel Institute lobby.

Photo by David Nadziejka.

Van Andel Research Institute

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

Phone 616.234.5000 Fax 616.234.5001 www.vai.org


VARI | 2007

Van Andel Research Institute Scientific Report 2007


Van Andel Research Institute | Scientific Report

ii

Published June 2007.

Copyright 2007 by the Van Andel Institute; all rights reserved.

Van Andel Institute, 333 Bostwick Avenue, N.E.,

Grand Rapids, Michigan 49503, U.S.A.


VARI | 2007

Table of Contents

Director’s Introduction 1

George F. Vande Woude, Ph.D.

Laboratory Reports 5

Arthur S. Alberts, Ph.D.

Cell Structure and Signal Integration 6

Brian Cao, M.D.

Antibody Technology 10

Gregory S. Cavey, B.S.

Mass Spectrometry and Proteomics 13

Nicholas S. Duesbery, Ph.D.

Cancer and Developmental Cell Biology 18

Bryn Eagleson, B.S., RLATG

Vivarium and Transgenics 21

Kyle A. Furge, Ph.D.

Computational Biology 23

Brian B. Haab, Ph.D.

Cancer Immunodiagnostics 26

Rick Hay, Ph.D., M.D., F.A.H.A.

Noninvasive Imaging and Radiation Biology

Office of Translational Programs 31

Jeffrey P. MacKeigan, Ph.D.

Systems Biology 35

Cindy K. Miranti, Ph.D.

Integrin Signaling and Tumorigenesis 39

James H. Resau, Ph.D.

Division of Quantitative Sciences

Analytical, Cellular, and Molecular Microscopy

Microarray Technology

Molecular Epidemiology 46

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

Germline Modification and Cytogenetics 51

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

Cancer Genetics 55

Steven J. Triezenberg, Ph.D.

Transcriptional Regulation 62

George F. Vande Woude, Ph.D.

Molecular Oncology 66

Craig P. Webb, Ph.D.

Program for Translational Medicine

Tumor Metastasis and Angiogenesis 70

Michael Weinreich, Ph.D.

Chromosome Replication 74

Bart O. Williams, Ph.D.

Cell Signaling and Carcinogenesis 78

H. Eric Xu, Ph.D.

Structural Sciences 84

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Van Andel Research Institute | Scientific Report

2006 Van Andel Research Institute Symposium 88

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

Daniel Nathans Memorial Award 94

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

Postdoctoral Fellowship Program 96

List of Fellows

Student Programs 98

Grand Rapids Area Pre-College Engineering Program

Summer Student Internship Program

Han-Mo Koo Memorial Seminar Series 102

2006 | 2007 Seminars

iv

Van Andel Research Institute Organization 107

Boards

Office of the Director

VAI Administrative Organization


VARI | 2007

Director’s Introduction

1


Van Andel Research Institute | Scientific Report

George F. Vande Woude

Director’s Introduction

2

Now in our seventh year at a site in downtown Grand Rapids recently dubbed Medical Mile, the Van Andel Institute is embarking

on a new phase in its growth as a leading center for biomedical research. An event for which we have been patiently

waiting took place on April 12, 2007, when groundbreaking ceremonies officially marked the beginning of construction for

the Phase II expansion of the Van Andel Institute. Undeterred by April snow showers, Dave Van Andel got things rolling

by maneuvering a GPS-directed John Deere bulldozer to break ground, to the cheers of employees and honored

guests who watched from indoors via live video. For the next two years, we will see our Phase II laboratory emerge,

fulfilling the plans to increase our research capacity to accommodate 400 new scientists. You can check out the construction at

http://www.vai.org/About/Facilities/PhaseII.aspx.

In addition to our own project, we are witnessing all around us phenomenal growth in the Medical Mile community. Already

established south of us is the St. Mary’s Lacks Cancer Center. To our east and north, construction is underway for Spectrum

Health’s new Lemmen-Holton Cancer Pavilion and the Helen DeVos Children’s Hospital. Just adjacent to our campus to the west

and north, the Secchia Center that is being built will be headquarters to the College of Human of Medicine (CHM) of Michigan

State University (MSU). Second-year CHM students will begin study in Grand Rapids in 2008, while a first-year class of 100

students is scheduled to begin in 2010, leading to a full enrollment of about 400 students.

It is hard to match all this excitement, but we have many accomplishments to our credit, and no doubt this has been a key factor

stimulating Medical Mile and the growth of the biomedical enterprise. We are all very proud of what is happening.

Personnel

It is my pleasure to report that Jim Resau has been promoted to the rank of Distinguished Scientific Investigator. Jim has provided

the impetus in developing VARI’s imaging core, and his efforts have led to novel imaging approaches and to many successful

collaborations of benefit to our Institute. Jim has also contributed a strong interest and much help with the Van Andel Education

Institute (VAEI) educational programs, including the graduate school, the Grand Rapids Area Pre-College Engineering Program,

and other student programs of VAEI. He serves as VARI’s Deputy Director for Special Programs and Director of the Quantitative

Sciences Division.

Congratulations also to Eric Xu on his promotion to Distinguished Scientific Investigator. Eric has made significant scientific

contributions to defining the structures of nuclear receptor proteins, including the peroxisome proliferator–activated receptors

(PPARs) and the “orphan” nuclear receptors for which the ligand and function are unknown. The importance and excellence of

his work is reflected in his success with NIH grants.


VARI | 2007

In addition, four of VARI’s original investigators have been promoted to the rank of Senior Scientific Investigator: Art Alberts, Brian

Cao, Nick Duesbery, and Bart Williams.

Art’s studies on Diaphanous-related formins and the DAD peptide have developed new insights into the assembly of cell structures

and the possibility of new approaches to cancer therapy. He has recently played a key role in establishing VARI’s flow

cytometry facility.

Brian Cao was recognized for the development of VARI’s state-of-the-art antibody technology lab. He has produced novel

antibodies for several VARI research programs, developed and improved his lab’s capabilities to meet research needs, and

further serves as director of the Michigan Antibody Technology Core of the Core Technology Alliance.

Nick Duesbery’s work with anthrax lethal toxin has shown that the lethal factor component of the toxin is a metalloprotease that

cleaves MAPK kinases. His lab’s work has increased our understanding of how anthrax toxin works and has also shown that the

two-component moiety called “lethal toxin” inhibits the growth of some tumors. In addition to directing his lab, Nick also serves

as VARI’s Deputy Director for Research Operations.

Bart Williams has pursued the regulation and function of Wnt signaling as it affects various key cellular processes. The breadth

of Wnt’s effects has led him from an initial interest in Wnt’s effects in tumorigenesis to the recognition of the role of Wnt in bone

development and disease. Bart has also been a major contributor to the development of VARI’s mouse models and to the

inception of the VAI graduate school.

3

We congratulate each of these researchers, and we look forward to their continued valuable contributions toward the Institute’s

goals.

We are pleased to announce the recruitment in 2006 of two exceptional principal investigators (PIs). Jeff MacKeigan, Ph.D., was

recruited from Novartis and has established the Laboratory of Systems Biology. Jeff is interested in phosphatases and kinases,

how they are regulated, and what signaling pathways they affect. He also brings platform screening technology to our program

and has stimulated collaborations with our PIs to use RNAi screens as a genetic tool to understand gene function.

Steve Triezenberg, Ph.D., was recruited from MSU and he wears two hats. In addition to Steve’s studies of herpes virus transcription

in his newly established Laboratory of Transcriptional Regulation, he is also founding Dean of our new graduate program,

established by VAEI. To Steve’s great credit, VAEI’s graduate school has an inaugural class of students that will arrive to begin

studies in August 2007. The Ph.D. program, like most of the research at VARI, will focus on the molecular, cellular, and genetic

biology of human disease with a pronounced emphasis on translational research. The graduate school will foster the effective

transition of students into professional scientists through a unique curriculum employing problem-based learning methods and

through workshops to develop the cognate skills of grant and manuscript preparation, financial management, small-group leadership,

and career planning.

Programs

On June 1, 2006, the Program for Translational Medicine was established under Craig Webb’s direction. This program will push

forward our emphasis on moving our research findings into clinical practice and will help to develop “personalized medicine”

founded on molecular-based, individual diagnosis and treatment. Craig’s staff will be developing strategies for data collection,

integration, and analysis using the XB-BioIntegration Suite (formerly Xenobase), and Craig will work closely with the Office of

Translational Programs, directed by Rick Hay, to help achieve VARI’s translational aims.


Van Andel Research Institute | Scientific Report

The Institute’s entire animal care and use program was evaluated in March 2007 by the Association of Assessment and

Accreditation of Laboratory Animal Care, as part of our application for accreditation. AAALAC standards go beyond

governmental regulations, and meeting their standards symbolizes quality, promotes scientific validity, demonstrates

accountability, and shows commitment to humane animal care. The preliminary results of the review were very favorable, and

we anticipate receiving approval and our formal accreditation in a timely manner. Our thanks to Pam Swiatek and Bryn Eagleson

and their staffs, as well as to all the others involved in preparing for this evaluation; they did a great job in getting us ready.

Grants

In 2006, Eric Xu received his second R01 from NIH for a five-year study of “Structure and Function of Steroid Hormone Receptors”.

Also, Brian Haab received his second R21 grant for “Defining Secreted Glycan Alterations in Pancreatic Cancer”.

Rick Hay received a state appropriation from the MEDC Michigan Strategic Fund for “Creation of a Good Manufacturing Practices

(GMP) Facility”. The project support runs from October 2006 through December 2007.

Steve Triezenberg’s graduate student, Sebla Kutluay, received VARI’s first predoctoral grant, for two years from the American

Heart Association. Sponsored funding by commercial firms for specific research areas was received by the laboratory of Bin Teh

and by my own lab. Other funding was received by various labs from the Breast Cancer Research Foundation, American Cancer

Society, and as subgrants through collaborations with other research organizations.

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Collaborations

In late 2006, we established a collaboration for medical education and research between MSU and VARI. The new CHM medical

school will establish an innovative molecular medicine curriculum with research in areas including cancer and neurobiology and

an emphasis on translational research. The medical school faculty will have laboratory space in our Phase II building upon its

completion in 2009, and the school intends to be fully operational in 2010. We anticipate that unique and fruitful collaborations

will result from the proximity of the MSU and VARI scientists, and we foresee benefits accruing not only to both institutions, but

more importantly to the patients afflicted by the diseases we study.

Also in 2006, our joint effort with Spectrum Health has created the Center for Molecular Medicine, which offers molecular-based

diagnostics to physicians. Further, a multi-member alliance under the name “ClinXus” offers a venue for novel biomarker-based

clinical trials and for future biomarker drug development collaborations with pharmaceutical and biotech firms.

In February 2007, we signed a groundbreaking agreement with the National Cancer Center, Singapore (NCCS) to establish a joint

translational research program in Singapore. The program will be directed by Bin Teh and will focus on the biological basis for

different drug responses in Asian versus non-Asian patients having specific cancers.

When we opened our doors in 2000, our commitment to basic sciences and translation was considered new and innovative.

Lately, as I travel to other world-class academic institutions, it is clear that everyone has the same burning desire to turn discovery

into application. This means that our success will be contingent not only on having the right scientific expertise, but also upon the

growth of an ideal medical environment and a very supportive community. I know Grand Rapids has the “right stuff” and is poised

to become a leading biomedical center in the next decade. It is an honor to be a part of such an exciting endeavor.


VARI | 2007

Laboratory Reports

5


Van Andel Research Institute | Scientific Report

Arthur S. Alberts, Ph.D.

Laboratory of Cell Structure and Signal Integration

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In 1993, Dr. Alberts received his Ph.D. in physiology and pharmacology at the University of California,

San Diego, where he studied with James Feramisco. From 1994 to 1997, he served as a postdoctoral

fellow in Richard Treisman’s laboratory at the Imperial Cancer Research Fund in London, England. From

1997 through 1999, he was an Assistant Research Biochemist in the laboratory of Frank McCormick

at the Cancer Research Institute, University of California, San Francisco. Dr. Alberts joined VARI as a

Scientific Investigator in January 2000 and was promoted to Senior Scientific Investigator in 2006.

Staff Students Visiting Scientists

Laboratory 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.

Aaron DeWard, B.S.

Dagmar Hildebrand, B.S.

Stephen Matheson, Ph.D.

Brad Wallar, Ph.D.


VARI | 2007

Research Interests

Research in the Laboratory of Cell Structure and Signal Integration focuses on the molecular machinery responsible for the

reorganization of the cell’s architecture during division and directed migration. Of particular interest is how defects in the

machinery drive the progression to malignancy. The goal is to identify key control steps that are altered in disease states and

exploit that knowledge to improve diagnostic and prognostic capabilities. We have been targeting key points in the cytoskeletal

control system to devise novel targets for molecular therapy.

The cytoskeleton comprises microfilaments, microtubules, and intermediate filaments. Each of these structures is a polymer

whose assembly from individual monomer subunits is controlled by accessory proteins. While the term “cytoskeleton” implies a

static or rigid structure within cells, the various filamentous structures are actually highly dynamic. Microfilaments, for example,

are made of polymerized actin; these filaments rapidly polymerize, bundle, bend, depolymerize, or are severed so as to assume

different shapes within the cell to fulfill a given function. In some cases, individual strands are woven into networks and contract

against each other so that cells can attach to extracellular substrates and crawl along them. For example, actin/microfilament

remodeling is crucial in the immune cells’ role to search for and destroy invading pathogens. Cancer cells use such remodeling

to migrate from primary tumors (often located at an innocuous site) to a secondary site. At the secondary site, tumor cells grow

and damage adjacent tissue, often leading to the eventual death of the patient. This process is called metastasis, and to date

there are few, if any, effective anti-cancer therapies that block it. Thus, there is an important need to identify mechanisms that can

be effectively targeted to block the spread of tumor cells throughout the body.

The Rho family of small GTPases controls critical steps in cytoskeletal remodeling. The GTPases are triggered by signals dictated

by activated growth or adhesion receptors and, in turn, bind to “effectors” that govern the machinery assembling the cytoskeleton.

Some of these effector proteins directly participate in cytoskeletal remodeling. One fundamentally important set of GTPase

effectors is the mammalian Diaphanous-related (mDia) formins.

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Formins nucleate, processively elongate, and (in some cases) bundle filamentous actin (F-actin) through conserved formin

homology-2 (FH2) domains. mDia proteins participate in many cytoskeletal remodeling events including cytokinesis, vesicle trafficking,

and filopodia assembly while acting as effectors for Rho small GTPases. Rho proteins govern mDia proteins by regulating

an intramolecular autoregulatory mechanism. GTPases binding to the mDia amino-terminal GTPase-binding domain (GBD)

sterically hinder the adjacent Dia-inhibitory domain (DID) interaction with the carboxyl-terminal Dia-autoregulatory (DAD) domain

(Fig. 1). The release of DAD allows the adjacent FH2 domain to then nucleate and elongate nonbranched actin filaments.

Figure 1.

Figure 1. mDia proteins are autoregulated nucleators

of actin. Autoinhibition of mDia is mediated by interaction

between the DID and DAD domains. Activated GTP-bound

Rho proteins bind to the GBD where they interfere with DAD

binding to DID. Then the free FH2 domains, which also function

as dimerization interfaces, can nucleate actin monomers and

processively elongate actin filaments. Tagged fusion proteins

(CFP-Rho GTPase and YFP-mDia) are used in fluorescence

resonance energy transfer (FRET) to monitor the sites of

protein-protein interactions. Excitation of CFP by a specific

wavelength of light results in emitted light at a wavelength that

excites YFP, but YFP excitation occurs only if the proteins are

close enough to approximate direct binding. This approach is

used to generate the data shown in Fig. 2.


Van Andel Research Institute | Scientific Report

The cytoskeleton not only provides the impetus for cell movement, but it also allows the internal architecture to be organized into

different compartments having specific functions in the cellular responses to growth factors. Rho GTPases and the dynamic

assembly and disassembly of actin filaments have been shown to have crucial roles in both the internalization and trafficking of

growth factor receptors. While all three mammalian Diaphanous-related formins (mDia1, mDia2, and mDia3) have been localized

on endosomes, their roles in actin nucleation, filament elongation, and/or bundling remains poorly understood in the context of

intracellular trafficking.

In a recent publication in Experimental Cell Research, we reported the functional relationship between RhoB, a GTPase known

to associate with both early and late endosomes, and the formin mDia2. We were able to show that 1) RhoB and mDia2 interact

on endosomes, as seen in Fig. 2 using the FRET approach; 2) GTPase activity—the ability to hydrolyze GTP to GDP—is required

for the ability of RhoB to govern endosome dynamics; and 3) the actin dynamics controlled by RhoB and mDia2 is necessary for

vesicle trafficking. These studies further suggested that Rho GTPases significantly influence the activity of mDia family formins in

driving cellular membrane remodeling through the regulation of actin dynamics.

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In another recent study, in the journal Current Biology, we reported how Diaphanous-interacting protein (DIP) binds to and

regulates the activity of the formin mDia2 and its ability to assemble filopodia. Filopodia are small finger-like projections

comprising several bundled nonbranched actin filaments emanating from the leading edge of migrating cells and essentially

acting as sensors for directed cell movement. We investigated an interaction occurring between a conserved leucine-rich

region (LRR) in DIP and the mDia FH2 domain. While DIP has been shown to interact with and stimulate N-WASp-dependent

branched filament assembly via Arp2/3, it interfered with mDia2-dependent filament assembly and bundling.

Figure 2.

Figure 2. RhoB and mDia2 interact on a subset of vesicles bearing internalized EGF. CFP-RhoB and YFP-mDia2 interact on vesicles

bearing internalized Texas Red–labeled epidermal growth factor. Cells expressing the two FRET probes (4 h after injection) were

incubated with fluorescent EGF for 5 min prior to fixation. RhoB-mDia2 FRET occurs on a subset of vesicles (FRET is false-colored

green, with Texas Red–EGF shown in red).


VARI | 2007

Surprisingly, DIP had no effect on the highly related mDia1. Consistent with a role for mDia2 as a Cdc42 effector, DIP both blocked

the formation of filopodia and induced non-apoptotic membrane blebbing, a physiological process involved in both cytokinesis

and amoeboid cell movement. DIP-induced blebbing occurred independently of Arp2/3 activity. Figure 3 shows the result of

microinjection of DIP LRR into a mouse embryo fibroblast in which a critical subunit of Arp2/3 has been knocked down by siRNA.

The experiment reveals a pivotal role for DIP in the control of nonbranched versus branched actin filament assembly mediated,

respectively, by Diaphanous-related formins and by activators of Arp2/3. The ability of DIP to trigger blebbing also suggests a

role for mDia2 in the assembly of actin filaments at the cell cortex necessary for the maintenance of plasma membrane integrity.

Future experiments will address how DIP regulates mDia2 in directed cell movement and during cell division.

Figure 3.

Figure 3. DIP LRR–induced plasma membrane blebbing

does not require Arp2/3 activity. This mouse embryo

fibroblast, which expresses siRNA directed against

Arp3, was injected with 0.1 μM recombinant DIP LRR

protein along with Texas Red dextran as a marker.

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External Collaborators

Harry Higgs, Dartmouth Medical School,

Hanover, New Hampshire

Recent Publications

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

Eisenmann, Kathryn M., Elizabeth S. Harris, Susan M. Kitchen, Holly A. Holman, Henry N. Higgs, and Arthur S. Alberts. 2007.

Dia-interacting protein modulates formin-mediated actin assembly at the cell cortex. Current Biology 17(7): 579–591.

Wallar, Bradley J., Aaron D. DeWard, James H. Resau, and Arthur S. Alberts. 2007. RhoB and the mammalian Diaphanousrelated

formin mDia2 in endosome trafficking. Experimental Cell Research 313(3): 560–571.


Van Andel Research Institute | Scientific Report

Brian Cao, M.D.

Laboratory of Antibody Technology

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Dr. Cao obtained his M.D. from Peking University Medical Center, People’s Republic of China, in 1986.

On receiving a CDC fellowship award, he was a visiting scientist at the National Center for Infectious

Diseases, Centers for Disease Control and Prevention in Atlanta (1991–1994). He next served as a

postdoctoral fellow at Harvard (1994–1995) and at Yale (1995–1996). From 1996 to 1999, Dr. Cao was

a Scientist Associate in charge of the Monoclonal Antibody Production Laboratory at the Advanced

BioScience Laboratories–Basic Research Program at the National Cancer Institute, Frederick Cancer

Research and Development Center, Maryland. Dr. Cao joined VARI as a Special Program Investigator in

June 1999, and he was promoted to Senior Scientific Investigator in July 2006.

Staff

Laboratory Staff

Ping Zhao, M.S.

Tessa Grabinski, B.S.

Students

Students

Xin Wang

Ning Xu

Aixia Zhang

Jin Zhu

Visiting Scientists


VARI | 2007

Research Interests

Antibodies are primary tools of biomedical science. In basic research, the characterization and analysis of almost any molecule

involves the production of specific monoclonal or polyclonal antibodies that react with it. Antibodies are also widely used in

diagnostic applications for clinical medicine. ELISA and radioimmunoassay systems are antibody-based. Analysis of cells

and tissues in pathology laboratories includes the use of antibodies on tissue sections and in flow cytometry analyses. Further,

antibodies are making rapid inroads into medical therapeutics, driven by technological evolution from chimeric and humanized to

fully human antibodies. The therapeutic antibody market has the potential to reach $30 billion by 2010.

Our Antibody Technology laboratory has developed several technologies over the last few years: 1) state-of-the-art monoclonal

antibody (mAb) production and characterization, followed by scaled-up production and purification; 2) antibody-binding-site

epitope mapping using a phage-display peptide library; 3) a human-antibody-fragment phage-display library and screening

of specific fragments from the library; and 4) characterization of these human antibody fragments and conjugation with

chemotherapeutics to generate immuno-chemotherapeutic reagents for preclinical studies.

In collaboration with Nanjing Medical University, China, we constructed our own human naïve Fab fragment phage-display library,

with a diversity of 2 × 10 9 , in late 2004. In 2005, we screened out several Fab fragments from the library that specifically recognize

HGF/SF, Met, and EGFR. By modifying and improving biopanning strategies, we have selected Fab fragments that recognize

the Met and EGFR extracellular domains in native conformation with reasonable affinity and, importantly, with the internalization

property that makes these Fabs attractive as conjugate reagents for immuno-chemotherapy or immuno-radiation therapy against

cancer. In the past year, we have conjugated anti-EGFR human Fab to paclitaxel (Taxol) as an immuno-chemotherapy agent and

investigated its in vitro anti-tumor efficacy on A431 epidermoid carcinoma cells using cell proliferation inhibition and apoptosis

assays. The Fab-Taxol conjugate inhibited A431 cell proliferation at low concentrations and in a dose-responsive manner; more

than 70% inhibition was observed at 52 pM. Furthermore, almost 100% of the cells underwent apoptosis after treatment with

Fab-Taxol at 26 pM for 48 hours. The in vitro anti-tumor efficacy is four- to fivefold more potent than Taxol alone. We are modifying

the Taxol conjugation conditions and working with other drug conjugations to investigate their in vivo anti-tumor efficacy in

xenograft and orthotopic animal models.

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Van Andel Research Institute | Scientific Report

Functioning as an antibody production core facility, this lab has extensive capabilities. Our technologies and services

include antigen preparation and animal immunization; peptide design and coupling to protein carriers; DNA immunization

(gene-gun technology); immunization with living or fixed cells; conventional antigen/adjuvant preparation; immunizing a wide

range of antibody-producing models (including mice, rats, rabbits, human cells, and transgenic or knock-out mice); and in vitro

immunization. Our work also includes the generation of hybridomas from spleen cells of immunized mice, rats, and

rabbits; hybridoma expansion and subcloning; cryopreservation of hybridomas secreting mAbs; isotyping of mAbs; ELISA

screening of hybridoma supernatants; mAb characterization by immunoprecipitation, Western blot, immunohistochemistry,

immunofluorescence staining, FACS, and in vitro bioassays; generation of bi-specific mAbs by secondary fusion; conjugation

of mAbs to enzymes, biotin/streptavidin, or fluorescent reporters; and development of detection methods/kits such as sandwich

ELISA. We also contract services to biotechnology companies, producing and purifying mAbs for their research and for

diagnostic kit development.

The Michigan Core Technology Alliance (CTA), funded by the state government, was created in 2001. The Antibody Technology

Core at VARI and the Hybridoma Core at the University of Michigan in Ann Arbor joined together to form the Michigan Antibody

Technology Core (MATC) and became the seventh core of CTA in March 2005. Our goals are to provide state-of-the-art antibody

technologies and services to research scientists; to generate, characterize, produce, and purify a wide variety of monoclonal

antibodies; to make human antibody fragments and humanize murine mAbs for clinical diagnostic/therapeutic applications; and

to advance biomedical research and development. The Antibody Technology Lab at VARI serves as the core’s hub, and Dr. Brian

Cao is the director of MATC.

12

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

Recent Publications

Wang, X., J. Zhu, P. Zhao, Y. Jiao, N. Xu, T. Grabinski, C. Liu, C.K. Miranti, T. Fu, and B. Cao. In press. In vitro efficacy of immunochemotherapy

with anti-EGFR human Fab-Taxol conjugate on A431 epidermoid carcinoma cells. Cancer Biology & Therapy.

Zhang, Y.-W., B. Staal, Y. Su, P. Swiatek, P. Zhao, B. Cao, J. Resau, R. Sigler, R. Bronson, and G.F. Vande Woude. 2007. Evidence

that MIG-6 is a tumor-suppressor gene. Oncogene 26(2): 269–276.

Tsarfaty, Galia, Gideon Y. Stein, Sharon Moshitch-Moshkovitz, Dafna W. Kaufman, Brian Cao, James H. Resau, George F. Vande

Woude, and Ilan Tsarfaty. 2006. HGF/SF increases tumor blood volume: a novel tool for the in vivo functional molecular imaging

of Met. Neoplasia 8(5): 344–352.


VARI | 2007

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.

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Staff

Laboratory Staff

Paula Davidson, M.S.

Joan Krilich, B.S.

Students

Visiting Scientists


Van Andel Research Institute | Scientific Report

Research Interests

The Mass Spectrometry and Proteomics laboratory provides protein identification analysis and protein molecular weight

determination as core services. Nanogram amounts of protein in SDS-PAGE gels or in solution are digested into peptides and

analyzed by HPLC with on-line electrospray mass spectrometry. Peptides are fragmented in the mass spectrometer to generate

amino acid sequence data that is used to identify proteins by searching protein and DNA databases. Submicrogram amounts

of intact proteins are analyzed by nanoscale liquid chromatography–mass spectrometry (LC-MS) to determine their average

molecular weight; this work is performed using a variety of HPLC columns to optimize recovery and provide reliable results.

These core services are provided to both VARI investigators and external clients. Research in the lab focuses on improving

existing services and developing new methods based on the needs of VARI investigators. Our three main areas of interest are

intact-protein molecular weight determination, phosphopeptide analysis, and protein expression profiling using LC-MS.

Protein LC-MS

14

We use protein LC-MS to confirm correct expression and purification of recombinant proteins from bacteria. The average

molecular weight of a protein is experimentally determined and compared with the calculated weight from the expected amino

acid sequence. Proteins of 50 kDa and larger are analyzed with mass accuracy often better than 0.01%, or ±1 Da per 10 kDa.

Unlike with conventional SDS-PAGE, protein truncation and modifications such as oxidation or acetylation can be accurately

characterized using protein LC-MS. This information is essential when protein reagents are used for labor-intensive and costly

protocols such as x-ray crystallography, antibody production, or drug screening. We have a dedicated LC-MS instrument with

optimized HPLC separation and comprehensive data processing for analyzing complex mixtures of proteins. For proteins that

degrade during purification, we can alter the use of protease inhibitors or minimize degradation through site-directed mutagenesis

of susceptible amino acids. We are also exploring the use of this equipment for biomarker discovery of intact proteins. The goal

is to provide relative quantitation of proteins in disease cell culture models, tumor tissue, and cancer patient body fluids.

Protein phosphorylation analysis

Mapping post-translational modifications of proteins such as phosphorylation is an important yet difficult undertaking in cancer

research. Phosphorylation regulates many protein pathways that could serve as potential drug targets in cancer therapy. In recent

years, mass spectrometry has emerged as a primary tool in determining site-specific phosphorylation and relative quantitation.

Phosphorylation analysis is complicated by many factors, but principally by the low-stoichiometry modifications that may regulate

pathways: we are sometimes dealing with 0.01% or less of phosphorylated protein among a large excess of a nonphosphorylated

counterpart. Our lab collaborates with investigators to map protein phosphorylation using techniques including multiple

enzyme digestion, titanium dioxide phosphopeptide enrichment, and phosphorylation-specific mass spectrometry detection.

Although trypsin is often the enzyme of choice for digesting proteins into peptides for identification, additional enzymes such as

Lys-C, Staph V8, chymotrypsin, thermolysin, or elastase may also be employed. Multiple enzyme digests and titanium dioxide

enrichment are used in combination with precursor ion scanning for –79 m/z on a Waters Q-Tof Premier mass spectrometer.


VARI | 2007

We have developed a robust negative-ion-mode method using nanoscale HPLC that provides specific detection of phosphopeptides

below 20 fmol in the presence of 2 pmol of nonphosphorylated protein. Once detected in the negative mode, phosphopeptides

are sequenced in a subsequent LC-MS analysis in the positive ion mode using accurate mass parent ion selection, a narrow

retention time window, and collision energy ramping. This approach has provided a reliable and sensitive means of analyzing

phosphoproteins in our laboratory. Our current focus is on applying this label-free method to studies requiring relative quantitation

of phosphorylation events.

Protein expression/biomarker discovery

As mass spectrometry instruments and protein separation methods develop, proteomics techniques allow researchers to identify

and quantitate protein samples of increasing complexity. The ultimate goal is to catalog all proteins expressed in a given

cell or tissue as a means of evaluating dynamic physiological events and understanding how all proteins interact to affect a

biological outcome. Traditionally this goal has been approached using 2D gel electrophoresis, image analysis of stained proteins,

and identification of proteins from gels using mass spectrometry. Because of the labor-intensive nature of 2D gels and the

underrepresentation of some protein classes (such as membrane proteins), proteomics has been moving toward solution-based

separations and direct mass spectrometry analysis. Our laboratory recently purchased and installed a Waters Corporation Protein

Expression System for non-gel-based, label-free protein expression analysis. This system represents a paradigm shift in the

field of proteomics, because it provides both quantitative and qualitative data on complex mixtures of proteins in a single LC-MS

analysis. Proteins are enzymatically digested using trypsin and, without any chemical or isotopic labeling, the resulting peptides

are analyzed by LC-MS. The combination of molecular mass and LC retention time establishes a signature for each peptide and

allows comparison across samples. The mass spectrometer signal intensity of each peptide is used for quantitation. Qualitative

protein identification data is obtained by fragmenting all peptides eluting into the mass spectrometer, a feature unique to the

Waters instrument. VARI is one of an elite group of institutions that have this powerful new technology. This system will be used to

map protein pathways under a systems biology approach and to discover potential biomarkers for early detection and diagnosis

in cancer and other diseases.

15

External Collaborators

Gary Gibson, Henry Ford Hospital, Detroit, Michigan

Michael Hollingsworth, Eppley Cancer Center, University of Nebraska, Omaha

Waters Corporation

Core Technology Alliance (CTA)

This laboratory participates in the CTA as a

member of the Michigan Proteomics Consortium.

From left: Davidson, Cavey, Krilich


Van Andel Research Institute | Scientific Report

16

Cells prepared by Miles Qian and Daisuke Matsuda

of the Teh laboratory.

Image by Kristin VendenBeldt of the Resau laboratory.


VARI | 2007

Murine lymph node/vascular tissue.

17

Murine lymph node/vascular tissues stained by immunohistochemisty and photographed using the CRI Nuance camera. Green, pericyte cell marker;

red, CD34 blood vessel marker; blue, nuclear/DNA marker.


Van Andel Research Institute | Scientific Report

Nicholas S. Duesbery, Ph.D.

Laboratory of Cancer and Developmental Cell Biology

18

Dr. Duesbery received a B.Sc. (Hon.) in biology (1987) from Queen’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 Laboratories–Basic Research Program at the National Cancer

Institute, Frederick Cancer Research and Development Center, Maryland. Dr. Duesbery was promoted

to Senior Scientific Investigator and appointed Deputy Director for Research Operations in 2006.

Staff

Students

Jennifer Bromberg-White, Ph.D.

Philippe Depeille, Ph.D.

Yan Ding, Ph.D.

John Young, M.S.

Jaclyn Lynem, B.S.

Elissa Boguslawski

Laura Holman

Students

Visiting Scientists

Chih-Shia Lee, M.S.

Naomi Asantewa-Sechereh

Lisa Orcasitas


VARI | 2007

Research Interests

Many malignant sarcomas such as fibrosarcomas are refractory to available treatments. However, sarcomas possess unique

vascular properties which indicate they may be more responsive to therapeutic agents that target endothelial function.

Mitogen-activated protein kinase kinases (MKKs) have been shown to play an essential role in the growth and vascularization of

carcinomas, and we hypothesize that signaling through multiple MKK pathways is also essential for sarcomas. The objective of

our research is to define the role of MKK signaling in the growth and vascularization of human sarcomas and to determine whether

inhibition of multiple MKKs by agents such as anthrax lethal toxin (LeTx), a proteolytic inhibitor of MKKs, can form the basis of a

novel and innovative approach to the treatment of human sarcoma.

In the past year we have made substantial progress in achieving this objective. Yan Ding, a postdoctoral fellow in the lab, and

Lisa Orcasitas have shown that MKKs are active in fibrosarcoma and that LeTx can inhibit the in vitro tumorigenic potential

of cells derived from human fibrosarcoma. The anti-tumoral properties of LeTx probably stem from its ability to substantially

decrease the release of many growth factors, notably the pro-angiogenic vascular endothelial growth factor (VEGF). In vivo, LeTx

caused a substantial decrease in both tumor volume and mean vascular density of fibrosarcoma xenografts. These changes also

correlated with a decreased level of pro-angiogenic factors, including VEGF. Dr. Ding also found that the ability of LeTx to

decrease the release of VEGF was not limited to fibrosarcoma, but was observed in cell lines derived from various sarcomas

including malignant fibrous histiocytoma and leiomyosarcoma. These results are consistent with the hypothesis that MKK

signaling is required for the growth and vascularization of fibrosarcoma both in vitro and in vivo, and this probably is also true of

other types of soft-tissue sarcomas.

Similarly, using an endothelial model of Kaposi sarcoma, Philippe Depeille, another postdoctoral fellow, and Elissa Boguslawski

showed that in vitro, LeTx 1) decreases proliferation, 2) inhibits tumorigenesis, and 3) dramatically reduces the secretion

of angioproliferative cytokines such as VEGF. Furthermore, in vivo, systemic treatment with LeTx inhibits tumor growth and

vascularization. These findings support the importance of MKK pathways in the release of angioproliferative cytokines that

promote tumor growth and vascularization. Our data suggest that inhibition of MKK signaling may be an effective therapeutic

strategy for the treatment of Kaposi sarcoma.

19

In collaboration with Bart Williams’ lab, John Young, our senior technician, and Jennifer Bromberg-White, a postdoctoral fellow,

investigated the mechanism of anthrax toxin entry into cells. Together they showed that mice or cells lacking LRP6, or a related

protein called LRP5, are still susceptible to anthrax toxin. The discovery that anthrax toxin can enter cells without the help of LRP6

presents a significant challenge to the published models of anthrax toxin function. These findings will help focus the efforts of

scientists working on new ways to treat anthrax.

In collaboration with Arthur Frankel, director of the Scott & White Cancer Research Institute in Texas, we have also tested the

therapeutic potential of LeTx in the treatment of malignant melanoma. Progress to date indicates that melanoma is particularly

sensitive to MKK inhibition. This is likely due in part to the fact that more than 80% of melanoma tumors harbor somatic mutations

that cause constitutive activation of the MKK1 and MKK2 signaling pathways, though indirect evidence suggests that other MKK

pathways also play a role in melanoma progression. Chih-Shia Lee is performing a detailed study of the individual contributions

of MKK pathways to melanoma survival. Jaclyn Lynem and Naomi Asantewa-Sechereh are investigating the molecular basis

of LF inactivation of MKK. We are currently performing preclinical studies to evaluate the potential of LeTx as a therapeutic for

malignant melanoma.


Van Andel Research Institute | Scientific Report

20

From left: Asantewa-Sechereh, Orcasitas, Lynem, Boguslawski, Lee,

Bromberg-White, Duesbery, Holman, Young, Depeille

Recent Publications

Young, J.J., J.L. Bromberg-White, C.R. Zylstra, J. Church, E. Boguslawski, J. Resau, B.O. Williams, and N. Duesbery. In press.

LRP5 and LRP6 are not required for protective antigen-mediated internalization or lethality of anthrax lethal toxin. PLoS Pathogen.

Depeille, P.E., Y. Ding, J.L. Bromberg-White, and N.S. Duesbery. 2007. MKK signaling and vascularization. Oncogene 26(9):

1290–1296.

Abi-Habib, Ralph J., Ravibhushan Singh, Stephen H. Leppla, John J. Greene, Yan Ding, Bree Berghuis, Nicholas S. Duesbery,

and Arthur E. Frankel. 2006. Systemic anthrax lethal toxin therapy produces regressions of subcutaneous human melanoma

tumors in athymic nude mice. Clinical Cancer Research 12(24): 7437–7443.

Bodart, Jean-François L., and Nicholas S. Duesbery. 2006. Xenopus tropicalis oocytes: more than just a beautiful genome.

In Xenopus Protocols: Cell Biology and Signal Transduction, X. Johné Liu, ed. Methods in Molecular Biology series, Vol. 322.

Totowa, N.J.: Humana Press, pp. 43–53.


VARI | 2007

Bryn Eagleson, B.S., RLATG

Vivarium and Laboratory of Transgenics

Bryn Eagleson began her career in laboratory animal services in 1981 with Litton Bionetics at the

National Cancer Institute’s Frederick Cancer Research and Development Center (NCI–Frederick) in

Maryland. In 1983, she joined the Johnson & Johnson Biotechnology Center in San Diego, California.

In 1988, she returned to the NCI–Frederick, where she continued to develop her skills in transgenic

technology and managed the transgenic mouse colony. In 1999, she joined VARI as the Vivarium Director

and Transgenics Special Program Manager.

21

Technical Staff

Lisa DeCamp, B.S.

Laboratory Staff

Dawna Dylewski, B.S.

Audra Guikema, B.S., L.V.T.

Kellie Jilbert, B.S., A.S.

Jamie Bondsfield, A.S.

Elissa Boguslawski, RALAT

Students

Animal Caretaker Staff

Sylvia Marinelli, Team leader

Angie Rogers, B.S.

Crystal Brady

Jarred Grams

Janelle Post

Tina Schumaker

Michael Shearer

Bobbie Vitt

Visiting Scientists


Van Andel Research Institute | Scientific Report

Research Interests

The goal of the vivarium and the transgenics laboratory is to develop, provide, and support high-quality mouse modeling services

for the Van Andel Research Institute investigators, Michigan Technology Tri-Corridor collaborators, and the greater research

community. We use two Topaz Technologies software products, Granite and Scion, for integrated management of the vivarium

finances, the mouse breeding colony, and the Institutional Animal Care and Use Committee (IACUC) protocols and records.

Imaging equipment, such as the PIXImus mouse densitometer and the ACUSON Sequoia 512 ultrasound machine, is available

for noninvasive imaging of mice. VetScan blood chemistry and hematology analyzers are now available for blood analysis.

Also provided by the vivarium technical staff are an extensive xenograft model development and analysis service, rederivation,

surgery, dissection, necropsy, breeding, and health-status monitoring.

Transgenics

22

Fertilized eggs contain two pronuclei, one that is derived from the egg and contains the maternal genetic material and one derived

from the sperm that contains the paternal genetic material. As development proceeds, these two pronuclei fuse, the genetic

material mixes, and the cell proceeds to divide and develop into an embryo. Transgenic mice are produced by injecting small

quantities of foreign DNA (the transgene) into a pronucleus of a one-cell fertilized egg. DNA microinjected into a pronucleus

randomly integrates into the mouse genome and will theoretically be present in every cell of the resulting organism. Expression

of the transgene is controlled by elements called promoters that are genetically engineered into the transgenic DNA. Depending

on the selection of the promoter, the transgene can be expressed in every cell of the mouse or in specific cell populations such as

neurons, skin cells, or blood cells. Temporal expression of the transgene during development can also be controlled by genetic

engineering. These transgenic mice are excellent models for studying the expression and function of the transgene in vivo.

From left: Bondsfield, Eagleson, Jason, Guikema, Shearer, Marinelli, Vitt, Post, Jilbert, Dylewski, Brady,

Schumaker, Rogers, Boguslawski, Grams, DeCamp


VARI | 2007

Kyle A. Furge, Ph.D.

Laboratory of Computational Biology

Dr. Furge received his Ph.D. in biochemistry from the Vanderbilt University School of Medicine in

2000. Prior to obtaining his degree, he worked as a software engineer at YSI, Inc., where he wrote

operating systems for embedded computer devices. Dr. Furge did his postdoctoral work in the

laboratory of George Vande Woude. He became a Bioinformatics Scientist at VARI in June of 2001 and

a Scientific Investigator in May of 2005.

23

Staff

Laboratory Staff

Karl Dykema, B.A.


Van Andel Research Institute | Scientific Report

Research Interests

As high-throughput technologies such as DNA sequencing, gene and protein expression profiling, DNA copy number analysis,

and single nucleotide polymorphism genotyping become more available to researchers, extracting the most significant

biological information from the large amount of data produced by these technologies becomes increasingly difficult.

Computational disciplines such as bioinformatics and computational biology have emerged to develop methods that assist

in the storage, distribution, integration, and analysis of these large data sets. The Computational Biology laboratory at VARI

currently focuses on using mathematical and computer science approaches to analyze and integrate complex data sets in order

to develop a better understanding of how cancer cells differ from normal cells at the molecular level. In addition, members of the

lab provide assistance in data analysis and other computational projects on a collaborative and/or fee-for-service basis.

In the past year the laboratory has taken part in many projects to further the research efforts at VARI. We have worked closely

with the Laboratory of Mass Spectrometry and Proteomics in developing computational infrastructure to support new protein

profiling instrumentation and analysis. We have contributed to several gene expression microarray analysis projects ranging

from mechanisms of oncogene transformation to the identification of genes that are associated with drug sensitivity. We also

work closely with the Laboratory of Cancer Genetics in the development of gene expression–based models for diagnosis and

prognosis of renal cell carcinoma. Moreover, we and other groups have demonstrated that several types of biological information,

in addition to relative transcript abundance, can be derived from high-density gene expression profiling data. Taking advantage

of this additional information can lead to the rapid development of plausible computational models of disease development and

progression.

24

Changes in DNA copy number result in dramatic changes in gene expression within the abnormal region and are detectable

through examination of the population of mRNAs generated from the genes that map to each chromosome. Additionally,

activation of certain oncogenes or inactivation of certain tumor suppressor genes can produce context-independent gene

signatures that can be detected in a gene expression profile. For example, genes that are up-regulated by overexpression of

RAS in breast epithelial cells also tend to be overexpressed in other samples containing activated RAS signaling, such as lung

tumors that contain activating RAS mutations. We have invested a reasonable portion of the past several years developing and

evaluating computational methods to predict deregulated signal transduction pathways and chromosomal abnormalities using

gene expression data. We have worked closely with the Laboratory of Cancer Genetics on computational models to describe the

development and progression of renal cell carcinoma. An example of the successful application of this analytic approach is in

the examination of gene expression profiling data derived from papillary renal cell carcinoma (RCC).


VARI | 2007

Computational analysis of gene expression data derived from papillary RCC revealed that a transcriptional signature indicative of

MYC pathway activation was present in high-grade papillary RCC, but not other high-grade RCCs. Predictions of chromosomal

gains and losses were also generated from the gene expression data, and it was demonstrated that the presence of the MYC

signature was coincident with a predicted amplification of chromosome 8q. Because the c-MYC gene maps to chromosome

8q, a computational model was developed such that amplification of chromosome 8q occurs in the high-grade papillary tumors,

which leads to c-MYC overexpression and activation of the MYC pathway. The importance of MYC activation was confirmed

by both pharmacological and siRNA inhibition of active MYC signaling in a cell line model of high-grade papillary RCC. These

results highlight the effectiveness of using gene expression profiling data to build integrative computational models of tumor

development and progression.

25

From left: Dykema, Furge

Recent Publications

Furge, Kyle A., Jindong Chen, Julie Koeman, Pamela Swiatek, Karl Dykema, Kseniji Lucin, Richard Kahnoski, Ximing J. Yang, and

Bin Tean Teh. 2007. Detection of DNA copy number changes and oncogenic signaling abnormalities from gene expression data

reveals MYC activation in high-grade papillary renal cell carcinoma. Cancer Research 67(7): 3171–3176.

Furge, K.A., M.H. Tan, K. Dykema, E. Kort, W. Stadler, X. Yao, M. Zhou, and B.T. Teh. 2007. Identification of deregulated oncogenic

pathways in renal cell carcinoma: an integrated oncogenomic approach based on gene expression profiling. Oncogene 26(9):

1346–1350.

Furge, Kyle A., Eric J. Kort, Ximing J. Yang, Walter M. Stadler, Hyung Kim, and Bin Tean Teh. 2006. Gene expression profiling in

kidney cancer: combining differential expression and chromosomal and pathway analyses. Clinical Genitourinary Cancer 5(3):

227–231.

Yang, Ximing J., Jun Sugimura, Kristian T. Schafernak, Maria S. Tretiakova, Misop Han, Nicholas J. Vogelzang, Kyle Furge, and

Bin Tean Teh. 2006. Classification of renal neoplasms based on molecular signatures. Journal of Urology 175(6): 2302–2306.


Van Andel Research Institute | Scientific Report

Brian B. Haab, Ph.D.

Laboratory of Cancer Immunodiagnostics

26

Dr. Haab obtained his Ph.D. in chemistry from the University of California at Berkeley in 1998. He then

served as a postdoctoral fellow in the laboratory of Patrick Brown in the Department of Biochemistry at

Stanford University. Dr. Haab joined VARI as a Special Program Investigator in May 2000 and became a

Scientific Investigator in 2004.

Staff

Laboratory Staff

Songming Chen, Ph.D.

Michael Shafer, Ph.D.

Yi-Mi Wu, Ph.D.

Derek Bergsma, B.S.

Sara Forrester, B.S.

Thomas LaRoche, B.S.

Tingting Yue, B.S.

Alex Turner

Students

Krysta Collins

Jennifer Lunger

Devin Mistry

Visiting Scientist

Rasmus Lundquist


VARI | 2007

Research Interests

Members of the Haab laboratory identify protein and carbohydrate abnormalities in the blood of cancer patients and investigate

the significance and potential clinical usefulness of those abnormalities. We develop novel experimental methods to facilitate this

work, and we collaborate with both clinicians and basic scientists to pursue research on pancreatic and prostate cancers.

Low-volume, high-throughput antibody and protein arrays

We have developed the ability to probe multiple proteins or carbohydrate structures using low sample volumes, which provides

a powerful tool for identifying and measuring protein and carbohydrate abnormalities in cancer. Antibody and protein arrays

immobilized on the surface of a microscope slide are the key to such a capability. A biological sample such as blood serum can

be incubated on an array to investigate interactions between the immobilized molecules and the proteins or antibodies in the

sample. Those interactions can be probed to obtain information such as protein abundance, glycosylation level, or protein-protein

interaction level.

The routine use of these tools was made possible by the development of a practical method for processing multiple arrays on

a microscope slide (Fig. 1). A stamp imprints a wax pattern onto the surface of a slide, creating hydrophobic partitions that

segregate various samples. Distinct stamp designs can be used to form differing sizes and numbers of partitions. A design

that imprints 48 arrays on one slide requires only 6 μl of sample per array, with each array composed of 144 distinct spots of

immobilized molecules. Such a design enables the efficient processing of many samples or testing of many conditions in parallel,

as demonstrated in the projects described below. The device for creating these slides is commercially available from The Gel

Company, San Francisco.

27

Figure 1A. Figure 1B. Figure 1C.

Figure 1. High-throughput sample processing using a novel slide partitioning method. A) Wax is imprinted onto a microscope slide to

form borders around multiple arrays. Wax is melted by the hotplate under the bath, and a slide is inserted upside-down into the holder.

Bringing the lever forward raises a stamp out of the wax bath to touch the slide, imprinting the design onto the slide. Two stamps are

shown in front of the machine. B) Loading samples onto a slide containing 48 arrays. The arrays are spaced by 4.5 mm, which is

compatible with the 9 mm spacing of standard multichannel pipettes. C) Samples loaded onto slides containing 12 (top), 48 (middle),

and 192 (bottom) arrays (96 samples loaded).


Van Andel Research Institute | Scientific Report

Glycans in pancreatic cancer

One of the major interests of the lab is characterizing and studying the changes in carbohydrate structures (glycans) on particular

proteins from pancreatic cancer patients. A novel technique developed in our laboratory enables the measurement of specific

glycans on multiple proteins in biological samples (Fig. 2A, B). We use lectins—proteins that bind specific glycan structures—as

well as glycan-binding antibodies to probe the levels of particular glycans on the proteins captured on the antibody arrays.

Several types of lectins, each with its own carbohydrate binding specificity, can be used to identify the carbohydrate structures

associated with each protein. We can analyze many different patient samples or cell culture conditions, looking at associations

between glycan levels and disease states or at the effects of certain perturbations on glycan structures. This method is in

development for commercial use by GenTel Biosciences (Madison, WI).

Mucins are long-chain, heavily glycosylated proteins on epithelial cell surfaces that have roles in cell protection, interaction with

the extracellular space, and regulation of extracellular signaling. Screening studies in collaboration with Randall Brand and Diane

Simeone have revealed a variety of glycan alterations on mucin molecules from pancreatic cancer patients (a representative

example is shown in Fig. 2C). Altered carbohydrates on mucins can affect critical processes in cancer such as cell migration

or extracellular signaling to the immune system. We are characterizing the glycan structural variation on mucins secreted from

cancer cells and other cells, and we are using cell culture systems to study the origins and effects of those variations. We are

pursuing hypotheses about the effects of extracellular stress from an inflammatory tumor environment on mucin carbohydrate

structures and the resulting interactions of those structures with inflammatory proteins and host cells.

Figure 2A.

28

Figure 2B.

Figure 2C.

Figure 2. Complementary antibody array formats for protein and glycan detection. A) Sandwich assay with fluorescence detection

to measure protein abundance. B) Antibody-lectin assay. The biotinylated lectin binds to glycans on the proteins captured by the

immobilized antibodies. The antibodies are first chemically derivatized to prevent lectin binding to the glycans of the immobilized

capture antibodies. C) Detecting protein and glycan variation in cancer and control sera. Sandwich detection of the MUC1 and CEA

proteins showed similar levels in serum samples from a cancer patient and a control subject (left images). The anti-CA19-9 antibody,

which targets a glycan structure, detected a significant glycan increase on MUC1 and CEA in the cancer serum (right images).


VARI | 2007

Cancer biomarkers

Improved methods of detecting and diagnosing cancer could significantly improve outcomes for many patients. We are seeking

to identify and validate protein biomarkers that could form the basis of clinical cancer diagnostics. The antibody-based assays

that we are using are valuable for this work because they are very reproducible, inexpensive, and high-throughput. In addition,

the use of miniaturized arrays of antibodies allows us to efficiently test many antibodies and samples and to rapidly develop new

assays. We are applying these capabilities in novel approaches to biomarker discovery and validation.

Mouse models of cancer may provide a good resource for biomarker discovery because the genetic and experimental variation

between samples can be closely controlled, thus making the identification of abnormal protein levels easier than with human

clinical specimens. Mass spectrometry studies performed by other members of an NCI-sponsored consortium have identified

candidate biomarkers in mouse models of ovarian and pancreatic carcinomas. Using newly generated antibodies that target

those proteins, we are developing assays to determine the levels of these candidate biomarkers in the mouse models and to

assess their diagnostic value for human cancer. Low-volume methods are crucial for these studies because only a small sample

is available from each mouse. These studies could establish a new paradigm for biomarker discovery and validation.

Longitudinal biomarkers

An NCI-sponsored project in our laboratory focuses on the hypothesis that the diagnostic performance of particular biomarkers

can be improved by using measurements collected on multiple occasions (longitudinal measurements) rather than at just a

single point in time. By looking at changes over time, it may be possible to more accurately distinguish abnormal levels in a

given individual, since that person’s normal level could be used as a reference point. In a collaboration with Robert Vessella

and William Catalona, we are investigating this question for the detection of prostate cancer recurrence. By using various

formats of antibody arrays, we can explore different data types and multiple proteins, which we hope will establish the extent of

diagnostic improvement using longitudinal information. Another collaborator, Ziding Feng, is developing the statistical methods

for analyzing the data, which may have value for other applications of this approach.

29

Tumor-reactive antibodies

We and others have investigated measurements of tumor-reactive antibodies as biomarkers. Certain tumor proteins elicit

an antibody-based immune response in a high percentage of cancer patients. In collaboration with Samir Hanash, Gilbert

Omenn, and others, we have further developed the experimental methods for identifying tumor-reactive antibodies using protein

arrays. We are applying this method to the detection of prostate cancer and prostate cancer recurrence. The changes in the

tumor-reactive antibodies are being assessed using the longitudinal approach described above, which may improve the

diagnostic performance of those biomarkers and give insight into the role of immune response in determining the likelihood of

cancer recurrence.

Pancreatic cancer biomarkers

Other biomarker studies in our lab are focused on pancreatic cancer in collaboration with Anna Lokshin, Michael Hollingsworth,

and others in the Early Detection Research Network (EDRN), which is an NCI-sponsored consortium dedicated to discovering

and validating cancer biomarkers. We use the glycan and protein detection technologies described above to identify and study

biomarkers for the early detection or more accurate diagnosis of pancreatic cancer. We have shown that, in certain cases,

the measurement of a glycan on a protein is more accurate for detecting cancer than the measurement of the protein alone in

traditional antibody assays. We are now seeking to define which protein and glycan alterations have the highest diagnostic and

prognostic significance.


Van Andel Research Institute | Scientific Report

External Collaborators

Philip Andrews, University of Michigan, Ann Arbor

Randall Brand, Evanston Northwestern Healthcare, Evanston, Illinois

William Catalona, Northwestern University, Evanston, Illinois

Ziding Feng, Fred Hutchinson Cancer Research Center, Seattle, Washington

Irwin Goldstein, University of Michigan, Ann Arbor

Samir Hanash, Fred Hutchinson Cancer Research Center, Seattle, Washington

Michael A. Hollingsworth, University of Nebraska, Omaha

Anna Lokshin, University of Pittsburgh, Pennsylvania

Gilbert Omenn, University of Michigan, Ann Arbor

Alan Partin, Johns Hopkins University, Baltimore, Maryland

Diane Simeone, University of Michigan, Ann Arbor

Robert Vessella, University of Washington, Seattle

Recent Publications

From left: Forrester, Porter, Nelson, Haab, Bergsma,

Collins, Lundquist, Chen, Yue, Wu, Turner

30

Chen, S., and B.B. Haab. In press. Antibody microarrays for protein and glycan detection. In Clinical Proteomics, Wiley-VCH.

Chen, S., T. LaRoche, D. Hamelinck, D. Bergsma, D. Brenner, D. Simeone, R.E. Brand, and B.B. Haab. In press. Multiplexed

analysis of glycan variation on native proteins captured by antibody microarrays. Nature Methods.

Forrester, S., J. Qiu, L. Mangold, A.W. Partin, D. Misek, B. Phinney, D. Whitten, P. Andrews, E. Diamandis, G.S. Omenn, S. Hanash,

and B.B. Haab. In press. An experimental strategy for quantitative analysis of the humoral immune response to prostate cancer

antigens using natural protein microarrays. Proteomics.

Omenn, Gilbert S., Raji Menon, Marcin Adamski, Thomas Blackwell, Brian B. Haab, and Weimin Gao, and David J. States. 2007.

The human plasma proteome. In Proteomics of Human Body Fluids: Principles, Methods, and Applications, V. Thongboonkerd,

ed. Totowa, N.J.: Humana Press.

Shafer, Michael W., Leslie Mangold, Alam W. Partin, and Brian B. Haab. 2007. Antibody array profiling reveals serum TSP-1 as

a marker to distinguish benign from malignant prostatic disease. The Prostate 67: 255–267.

Haab, B.B. 2006. Applications of antibody array platforms. Current Opinion in Biotechnology 17(4): 415–421.

Haab, B.B. 2006. Using array-based competitive and noncompetitive immunoassays. In American Association of Cancer

Research Annual Meeting Education Book, Phildelphia: American Association of Cancer Research.

Haab, Brian B., Amanda G. Paulovich, N. Leigh Anderson, Adam M. Clark, Gregory J. Downing, Henning Hermjakob, Joshua

LaBaer, and Mathias Uhlen. 2006. A reagent resource to identify proteins and peptides of interest for the cancer community: a

workshop report. Molecular & Cellular Proteomics 5(10): 1996–2007.

Hung, Kenneth E., Alvin T. Kho, David Sarracino, Larissa Georgeon Richard, Bryan Krastins, Sara Forrester, Brian B. Haab, Isaac

S. Kohane, and Raju Kucherlapati. 2006. Mass spectrometry–based study of the plasma proteome in a mouse intestinal tumor

model. Journal of Proteome Research 5(8): 1866–1878.


VARI | 2007

Rick Hay, Ph.D., M.D., F.A.H.A.

Laboratory of Noninvasive Imaging and Radiation Biology

Dr. Hay earned a Ph.D. in pathology (1977) and an M.D. (1978) at the University of Chicago and the

Pritzker School of Medicine. He became a resident in anatomic pathology and then a postdoctoral

research fellow in the University of Chicago Hospitals and Clinics. Following a postdoctoral fellowship at

the Biocenter/University of Basel (Switzerland), he returned to the University of Chicago as an Assistant

Professor in the Department of Pathology and Associate Director of the Section of Autopsy Pathology

from 1984 to 1992. He moved to the University of Michigan Medical Center in 1992 as a clinical fellow

in the Division of Nuclear Medicine and became Chief Fellow in 1993. From 1994 to 1997 he was a staff

physician, and from 1995 to 1997 the Medical Director in the Department of Nuclear Medicine at St. John

Hospital and Medical Center in Detroit. He joined VARI in 2001 as a Senior Scientific Investigator. In

2002 he was named Assistant to the Director for Clinical Programs, and in 2003 was appointed Deputy

Director for Clinical Programs.

31

Staff

Laboratory Staff

Visiting Scientist

Students

Visiting Scientists

Physician-in-training

Troy Giambernardi, Ph.D.

Kim Hardy, M.A., RT(R), RDMS

Yue Guo, B.S.

Joel Strehl, B.S.

Catherine Walker, B.S.

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

Matthew Steensma, M.D.

Laboratory Staff

Students

Students

Consultants

Visiting Scientists

Elianna Bootzin

Natalie Kent

Sara Kunz

Jose Toro

Rebecca Trierweiler

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

In July 2005, the Laboratory of Noninvasive Imaging and Radiation Biology originated as an outgrowth and expansion of activities

of the Laboratory of Molecular Oncology. This lab is devoted to both noninvasive imaging (i.e., the generation and analysis of

images or depictions of structure and selected functions in living organisms without surgically or mechanically penetrating a body

cavity) and radiation biology (which involves analysis of the consequences of external and internal radiation exposure in living

organisms).

The lab’s work follows three common themes:

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

• Advancement of technology in imaging and radiation biology, including novel agents, probes, and reporters;

new strategies for tackling research problems; and new instrumentation.

• Pursuit of two-way translation between the laboratory and the clinical setting, i.e., using examples of human

disease to design and improve laboratory model systems for study, as well as moving new discoveries from

the laboratory benchtop to the patient’s bedside.

32

We depend heavily upon access to sophisticated instruments and equipment, including nuclear imaging cameras; planar and

tomographic X-ray units; clinical and research ultrasonography units; fluorescence detection systems; and cell and organism

irradiation capability. Because of the equipment- and expertise-intensive nature of our projects, we could not succeed without

the help of our valued collaborators. During this past year we have acquired two new state-of-the-art noninvasive imaging

instruments: a Vevo 770 high-resolution micro-ultrasound imaging system (VisualSonics) and a nanoSPECT/CT imaging unit

(BioScan), and we have continued to pursue research projects in radiation biology, nuclear medicine, and multimodality imaging.

One established research project continues work begun by Nigel Crompton at the Paul Scherrer Institute in Switzerland, now

performed in collaboration with the radiation oncology service at Saint Mary’s Health Care. This project seeks to predict the

sensitivity of a patient’s normal tissues to irradiation that is being administered for treatment of a serious condition such as

cancer. For this project a sample of the patient’s blood is drawn before radiation therapy. The blood sample is then irradiated

(outside the patient) under precise conditions of exposure, treated with fluorescent molecules that detect certain types of blood

cells (lymphocytes), and then analyzed by fluorescence-activated cell sorting (FACS) for evidence of lymphocyte death. In

Switzerland, Dr. Crompton established a close correlation between lymphocyte death and a patient’s normal tissue tolerance to

irradiation. We are now determining whether western Michigan patients respond similarly, as well as investigating the effects of

patient age, gender, and administered radiation dose on the apoptotic response.


VARI | 2007

A new radiation biology project this year, in collaboration with Weiwen Deng and Aly Mageed of DeVos Children’s Hospital,

investigates a new approach for treating graft-versus-host disease in mice undergoing bone marrow transplantation, with planned

extension to human patients in the near future.

Our major established project in nuclear medicine continues work initiated by Dr. Hay and colleagues while he was a member of

the Laboratory of Molecular Oncology. Since 2001 we have been evaluating radioactive antibodies and smaller molecules that

attach to the Met receptor tyrosine kinase, collectively designated Met-avid radiopharmaceuticals (MARPs). Met plays a key

role in causing cancers to become more aggressive, so that they spread to nearby tissues (invasion) and/or travel through the

bloodstream or lymph channels to distant organs (metastasis). We previously showed that both large and small MARPs are useful

for nuclear imaging of Met-expressing human tumors (xenografts) grown under the skin of immunodeficient mice. During the past

year, in collaboration with our colleagues at VARI and with our outside collaborators at DVAHS, ApoLife, and MSU, we have been

evaluating new ways of complexing radioactive atoms with MARPs for improved ease of use and future clinical applications.

In 2006 we began a multimodality noninvasive imaging program for evaluating the growth, Met expression, and response to

therapy of aggressive human tumor xenografts grown orthotopically in immunodeficient mice. Employing a combination of

high-resolution ultrasound with and without contrast agents, planar and tomographic nuclear imaging, and CT imaging, we are

now acquiring data for tumors of the brain, pancreas, adrenals, and bone.

External Collaborators

33

Our lab depends critically on intramural and extramural collaborations to address our research themes. Our extramural

collaborators include scientists and physicians at the Department of Veterans Affairs Healthcare System in Ann Arbor; the

University of Michigan in Ann Arbor; Michigan State University in East Lansing; ApoLife, Inc., in Detroit; Henry Ford

Hospital in Detroit; West Michigan Heart, P.C., in Grand Rapids; DeVos Children’s Hospital in Grand Rapids; St. Mary’s

Health Care in Grand Rapids; Fred Hutchinson Cancer Research Center in Seattle; the Gerald P. Murphy Foundation in

West Lafayette, Indiana; the National Cancer Institute in Bethesda, Maryland; the University of Illinois in Champaign-Urbana;

and VisualSonics, Inc., in Toronto.

Recent Publications

Meng, L.J., N.H. Clinthorne, S. Skinner, R.V. Hay, and M. Gross. 2006. Design and feasibility study of a single photon emission

microscope system for small animal I-125 imaging. IEEE Transactions on Nuclear Science 53(3): 1168–1178.

Hay, R.V., and M.D. Gross. 2006. Scintigraphic imaging of the adrenals and neuroectodermal tumors. In Nuclear Medicine, 2nd

edition, R.E. Henkin, D. Bova, G.L. Dillehay, S.M. Keresh, J.R. Halama, R.H. Wagner, and A.M. Zimmer, eds. Philadelphia: Mosby

Elsevier, pp. 820–844.


Van Andel Research Institute | Scientific Report

Office of Translational Programs

Since July 2005 the Office of Translational Programs (OTP) has been the administrative home base for activities overseen by the

Deputy Director for Clinical Programs. The role of OTP is to promote and facilitate collaborative programs involving the Van Andel

Research Institute and other institutions in the realm of translational medicine.

OTP accomplishments during our second year of formal operation include the following.

• Serving as the administrative home for the GMP facility. With funding from the state of Michigan and the federal Health

Resources and Services Administration, VARI and Grand Valley State University have partnered to build and operate

Grand River Aseptic Pharmaceutical Packaging (GR-APP), a Good Manufacturing Practices facility that will package

pharmaceuticals for early-phase clinical trials commissioned by academic and commercial investigators, primarily in

Michigan and the Midwest. As of this writing, construction of GR-APP is nearing completion, and we expect operations

to begin by autumn of 2007.

• Serving as the administrative home for the West Michigan Chapter of the Michigan Cancer Consortium (MCC).

As an active member of the MCC, VARI is committed to participating in statewide programs to reduce the burden of

cancer in Michigan. In 2005, we and other regional MCC members launched an initiative to develop community-based

programs more relevant to western Michigan. Our first project, designated “C-Works!”, will provide cancer screening

and follow-up services to uninsured working women in Kent County.

34

• Organizing and hosting meetings. In October 2006, OTP hosted the Great Lakes Regional Meeting of the American

Cancer Society at VARI (Troy Giambernardi, Conference Chair) and assisted with preparations for the fall meeting

of the Central Chapter-Society of Nuclear Medicine in Traverse City (Rick Hay, Conference Co-Chair). In November

2006, OTP assisted with local arrangements for the annual meeting of the Michigan Cancer Consortium at DeVos Hall.

• Promoting new interinstitutional collaborations and providing resources for funding proposals. OTP provides a broad

range of administrative assistance, logistical support, grant preparation expertise, meeting venues, and seed funding

for new interinstitutional collaborations seeking extramural funding from state, federal, or private sources. During this

past year we helped secure state funding for ClinXus, a west Michigan–based consortium for conducting innovative

clinical trials, and we are awaiting the outcomes of recent collaborative proposals submitted to NIH and to two private

foundations.

• Coordinating research rotations for physicians-in-training. In collaboration with the Grand Rapids Medical Education

and Research Consortium (MERC), we schedule each first-year general surgery resident to spend one month working

in a designated research laboratory at VARI. This program has been well received by both residents and VARI

investigators. Custom-tailored rotations of variable duration at VARI can be arranged for other physicians-in-training.

Staff

Rick Hay, Ph.D., M.D., F.A.H.A.

Troy Carrigan

Jean Chastain


VARI | 2007

Jeffrey P. MacKeigan, Ph.D.

Laboratory of Systems Biology

Dr. MacKeigan received his Ph.D. in microbiology and immunology at the University of North Carolina

Lineberger Comprehensive Cancer Center in 2002. He then served as a postdoctoral fellow in the

laboratory of John Blenis in the Department of Cell Biology at Harvard Medical School. In 2004, he joined

Novartis Institutes for Biomedical Research in Cambridge, Massachusetts, as an investigator and project

leader in the Molecular and Developmental Pathways expertise platform. Dr. MacKeigan joined VARI in

June 2006 as a Scientific Investigator.

35

Staff

Students

Laboratory Staff Students Visiting Scientists

Brendan Looyenga, Ph.D.

Christina Ludema, B.S.

Natalie Wolters, B.S.

Katie Sian, B.S.

Geoff Kraker


Van Andel Research Institute | Scientific Report

Research Interests

The primary focus of the Systems Biology laboratory is identifying and understanding the genes and signaling pathways that

when mutated contribute to the pathophysiology of cancer. We take advantage of RNA interference (RNAi) and novel proteomic

approaches to identify the enzymes that control cell growth, cell proliferation, and cell survival. For example, after screening the

human genome for more than 600 kinases and 200 phosphatases—called the “kinome” and “phosphatome”, respectively—that

act with chemotherapeutic agents in controlling apoptosis, we identified 73 kinases and 72 phosphatases whose roles in cell

survival were previously unrecognized. We are asking several questions. How are these novel survival enzymes regulated at the

molecular level? What signaling pathway(s) do they regulate? Does changing the number of enzyme molecules present inhibit

waves of compensatory changes at the cellular level (system-level changes)? What are the system-level changes after reduction

or loss of each gene?

Identification of kinases that regulate cell survival

36

We have performed RNAi screens in the presence of apoptosis-inducing chemotherapeutic agents (Taxol, cisplatin, and etoposide)

and identified a group of kinases whose loss of function sensitizes cells to undergo cell death, the most interesting of these being

PINK1 (PTEN-induced kinase 1). PINK1 was originally shown to be up-regulated by the tumor suppressor PTEN. Although

PINK1 does not fall into a particular kinase subfamily, it has a known role in maintaining mitochondrial membrane potential. Other

work has recently shown inherited mutations at chromosomal location 1p36 in familial Parkinson disease, and the two mutated

genes that map to this region are PINK1 (PARK6) and DJ-1 (PARK7). Both genes are responsible for early-onset autosomal

recessive parkinsonism. We had previously noted that DJ-1 is overexpressed in non–small cell lung carcinoma and that

its down-regulation enhances apoptosis. Further, Parkinson disease–causing mutations in LRRK2 (PARK8), which are dominantly

inherited gain-of-function mutations, sensitize neurons to cell death, and a significant fraction of the LRRK2 population is

associated with the mitochondria. We are currently investigating whether the molecular mechanisms of PINK1 and LRRK2 in

cancer and in Parkinson disease are linked.


VARI | 2007

Identification of phosphatases that regulate chemoresistance

Our research has shown that a large percentage of phosphatases and their regulatory subunits contribute to cell survival. This

is a previously unrecognized general role for phosphatases as negative regulators of apoptosis, and it is important because

phosphatases may no longer be simply viewed as enzymes that oppose the action of kinases. This research also identified

a number of phosphatases whose loss of function results in chemoresistance, implicating these proteins as potential tumor

suppressors. In our RNAi study, 5% of all phosphatases were shown to act in this way; an example is MK-STYX. Down-regulation

of MK-STYX resulted in dramatic cellular resistance to cisplatin-, Taxol- or etoposide-induced cell death, which is consistent

with up-regulated survival signals in these cells (Fig. 1). Also, MK-STYX is located at 7q11.23, a chromosome region mutated

in colon cancer. MK-STYX is similar to MKP-1, which inactivates MAPKs; MK-STYX, however, is predicted to be a catalytically

inactive phosphatase. Our observations suggest that MK-STYX acts against cell survival by sequestering pro-survival signaling

components in a way analogous to the “substrate-trapping” effects of catalytically inactive phosphatases.

Figure 1A.

Figure 1B.

37

Figure 1. Identification of MK-STYX as a potential tumor suppressor

phosphatase. Cells were transfected with control siRNA or

MK-STYX siRNA for 48 h and then were treated for an additional

24 h with solvent control (–) or 50 μM cisplatin (+). Cell viability was

visualized by A) crystal violet stain and B) cleavage of full-length

PARP measured by western blot analysis.


Van Andel Research Institute | Scientific Report

Graded MAPK signaling and switch-like c-Fos induction

We also take a systems biology approach to understanding two key molecular pathways, Ras/MAPK and PI3K/mTOR.

In the Ras/MAPK pathway, growth factors activate the small G protein Ras, which recruits Raf to the plasma membrane where

it is activated and phosphorylates MEK1/2, which in turn phosphorylates ERK1/2-MAPKs. Activated ERK1/2 phosphorylates

additional kinases (such as RSK) and specific transcription factors (such as c-Fos and Elk-1) that are important in cellular

proliferation, differentiation, and survival.

One project in the lab involved the question of whether the evolutionarily conserved MAPK pathway exhibits a switch-like or a

graded response in mammalian cells. Ultrasensitive switch-like responses control cell-fate decisions in many biological settings,

and the regulation of kinase activity is one way in which such behavior can be initiated. Signaling molecules switch between

two discontinuous, stable states with no intermediate; this is referred to as a bistable response (Fig. 2, top panel). Given the

irreversible, all-or-none nature of many cell behaviors, including cell cycle control and apoptosis, significant effort has been

focused on identifying the cellular mechanisms underlying bistability. Our research and that of others has provided solid evidence

for graded MAPK signaling in mammalian cells (Fig. 2, lower panel); that is, as agonist concentration increases, single-cell kinase

activity increases proportionally. Yet we have also found that the proliferative response to growth factor stimulation is switch-like,

demonstrating that the ultrasensitive step in the MAPK pathway occurs at the level of MAPK nuclear concentration and switch-like

c-Fos induction. Although c-Fos induction and cell cycle entry in mammalian cells is switch-like, graded MAPK activation could

have an important role in cell survival, since many MAPK targets regulating cell survival are in the cytoplasm.

38

Figure 2.

Figure 2. Total cell population MAPK measurements.

Single cells exhibiting a bistable (all-or-none) response or graded

response (linear).

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


VARI | 2007

Cindy K. Miranti, Ph.D.

Laboratory of Integrin Signaling and Tumorigenesis

Dr. Miranti received her M.S. in microbiology from Colorado State University in 1982 and her Ph.D. in

biochemistry from Harvard Medical School in 1995. She was a postdoctoral fellow in the laboratory

of Joan Brugge at ARIAD Pharmaceuticals, Cambridge, Massachusetts, from 1995 to 1997 and in the

Department of Cell Biology at Harvard Medical School from 1997 to 2000. Dr. Miranti joined VARI as a

Scientific Investigator in January 2000. She is also an Adjunct Assistant Professor in the Department of

Physiology at Michigan State University.

39

Staff

Laboratory Staff

Mathew Edick, Ph.D.

Suganthi Sridhar, Ph.D.

Kristin Saari, M.S.

Lia Tesfay, M.S.

Laura Lamb, B.S.

Veronique Schulz, B.S.

Susan Spotts, B.S.

Students

Students

Eric Graf

Gary Rajah

Visiting Scientists


Van Andel Research Institute | Scientific Report

Research Interests

Our laboratory is interested in the mechanisms by which integrin receptors, interacting with the extracellular matrix (ECM), regulate

cell processes involved in the development and progression of cancer. Using tissue culture models, biochemistry, molecular

genetics, and mouse models, we are defining the cellular and molecular events involved in integrin-dependent adhesion and

downstream signaling that are important for prostate tumorigenesis and metastasis.

Integrins are transmembrane proteins that serve as receptors for ECM proteins. By interacting with the ECM, integrins stimulate

intracellular signaling transduction pathways to regulate cell shape, proliferation, migration, survival, gene expression, and

differentiation. Integrins do not act autonomously, but “crosstalk” with receptor tyrosine kinases (RTKs) to regulate many of these

cellular processes. Studies in our lab indicate that integrin-mediated adhesion to ECM proteins activates epidermal growth factor

receptors EGFR/ErbB2 and the HGF/SF receptor c-Met. Integrin-mediated activation of these RTKs is ligand-independent and

required for the activation of a subset of intracellular signaling molecules in response to cell adhesion.

The prostate gland and cancer

40

Tumors that develop from cells of epithelial origin, i.e., carcinomas, represent the largest tumor burden in the United States.

Prostate cancer is the most frequently diagnosed cancer in American men and the second leading cause of cancer death in

men. Patients who at the time of diagnosis have androgen-dependent and organ-confined prostate cancer are relatively easy

to cure through radical prostatectomy or localized radiotherapy, but patients with aggressive and metastatic disease have fewer

options. Androgen ablation can significantly reduce the tumor burden in the latter patients, but the potential for relapse and the

development of androgen-independent cancer is high. Currently there are no effective treatments for patients who reach this

stage of disease.

In the human prostate gland, α3β1 and α6β4 integrins on epithelial cells bind to the ECM protein laminin 5 in the basement

membrane. In tumor cells, however, the α3 and β4 integrin subunits disappear—as does laminin 5—and the tumor cells express

primarily α6β1 and adhere to a basement membrane containing laminin 10. There is also an increase in expression of the RTKs

EGFR and c-Met in the tumor cells. Two fundamental questions are whether the changes in integrin and matrix interactions that

occur in tumor cells are required for or help to drive the survival of tumor cells, and whether crosstalk with RTKs is important.


VARI | 2007

Integrins and RTKs in prostate epithelial cell survival

How integrin engagement of various ECMs regulates survival pathways in normal and tumor cells is poorly understood.

We recently initiated studies to determine how adhesion to matrix regulates cell survival in normal epithelial cells. We

have shown that integrin-induced activation of EGFR in normal primary prostate epithelial cells is required for survival on their

endogenous matrix, laminin 5. The ability of EGFR to support integrin-mediated cell survival on laminin 5 is mediated through

α3β1 integrin and requires signaling downstream to Erk. Surprisingly, we found that the death induced by inhibition of EGFR

in normal primary prostate cells is not mediated through or dependent on classical caspase-mediated apoptosis. The presence

of an autophagic survival pathway (Fig. 1), regulated by adhesion to matrix, prevents the induction of caspases when EGFR is

inhibited. Suppression of autophagy is sufficient to induce caspase activation and apoptosis in laminin 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 the maintenance of an autophagic survival pathway (Fig. 2).

Figure 1.

Figure 2.

Normal

Autophagic

41

Figure 1. Induction of autophagy in primary prostate epithelial cells

as shown by punctate staining of the autophagic LC3 protein using

fluorescence microscopy.

Figure 2. Laminin-mediated survival pathways in primary prostate

epithelial cells.

Interestingly, both of these pathways are absent in at least one metastatic prostate cancer cell line, PC3. Accordingly,

integrin-mediated survival of PC3 cells does not depend on EGFR or Erk, but is instead dependent on PI-3K. The PI-3K pathway is

inhibitory to autophagy. We are currently testing additional prostate tumor cells lines to determine if this switch in matrix-mediated

survival pathways is found in all prostate cancers.

Our next step is to determine how integrins regulate survival through autophagy. Since loss of autophagy results in activation

of caspases and classical apoptosis, we have been searching for signaling pathways whose inhibition also results in caspase

activation. We have tentatively identified two important molecules, the RTK c-Met and the anti-apoptotic protein Bcl-XL. Inhibition

of either molecule leads to caspase-induced cell death, indicating that they may be involved in regulating integrin-mediated

autophagy. Future studies in our lab will be aimed at deciphering this pathway.


Van Andel Research Institute | Scientific Report

The androgen receptor in integrin-mediated survival

All primary and metastatic prostate cancers express the intracellular steroid receptor for androgen, AR. In the normal gland, the

AR-expressing cells do not interact with the ECM in the basement membrane; however, all AR-expressing tumor cells do adhere

to the ECM in the basement membrane. In normal cells, AR expression suppresses growth and promotes differentiation, but in

tumor cells AR expression promotes cell growth and is required for cell survival. The mechanisms that lead to the switch from

growth inhibition and differentiation to growth promotion and survival are unknown. Our hypothesis is that adhesion to the ECM

by the tumor cells is responsible for driving the switch in AR function.

When prostate tumor cells are placed in culture, they lose expression of AR. The reason for this is not clear, but it may have to

do with loss of the appropriate ECM-containing basement membrane. When we introduce AR into prostate tumor cells, it actually

suppresses their growth and induces cell death. However, if we place the AR-expressing tumor cells on laminin (the ECM found

in tumors), these cells no longer die. The mechanisms responsible for this change in survival are unknown. Preliminary studies

indicate that there are changes in integrin expression upon expression of AR that may enhance specific signaling pathways

when those integrins bind to matrix. We are currently determining which cell survival pathways are activated by AR upon integrin

engagement.

CD82 and integrin signaling in prostate cancer metastasis

42

Death from prostate cancer is due to the development of metastatic disease, which is difficult to control and occurs by mechanisms

that are not understood. One approach we are taking is to characterize genes that are specifically associated with metastatic

prostate cancer. CD82/KAI1 is a metastasis suppressor gene whose expression is specifically lost in metastatic cancer but not

in primary tumors. Interestingly, CD82/KAI1 is known to associate with both integrins and RTKs. Our goal has been to determine

how loss of CD82/KAI1 expression promotes metastasis.

We have found that reexpression of CD82/KAI1 in metastatic tumor cells suppresses laminin-specific migration and invasion

via suppression of both integrin- and ligand-induced activation of the RTK c-Met. Interestingly, c-Met is often overexpressed

in metastatic prostate cancer. Thus, CD82/KAI1 normally acts to regulate signaling through c-Met such that upon CD82 loss in

tumor cells, signaling through c-Met is increased, leading to increased invasion. We are currently determining the mechanism

by which CD82/KAI1 down-regulates c-Met signaling. Our studies indicate that c-Met and CD82 do not directly interact, and

CD82 may act to suppress c-Met signaling indirectly by dispersing c-Met aggregates present on metastatic tumor cells. We

have developed mutants of CD82 in order to determine which part of the CD82 molecule is required for suppression of

c-Met activity. Also, we have determined that reexpression of CD82 in tumor cells induces a physical association between CD82

and a related family member, CD9. We are determining whether this association is important for suppressing c-Met activity.

We have also initiated mouse studies to demonstrate the importance of CD82 in regulating metastasis in vivo. Using orthotopic

injection of wild-type or CD82-expressing metastatic prostate tumor cells directly into the prostate, we found that CD82 also

suppresses metastasis in vivo. We are continuing these studies to determine if CD82’s ability to specifically affect c-Met is

responsible for metastasis suppression. In addition, we are generating mice in which CD82 expression is specifically lost in

the epithelial cells of the prostate gland. This approach will allow us to determine if CD82 is important for the normal biology of

prostate epithelial cells in vivo. Furthermore, we will be able to determine if loss of CD82 in the mouse prostate gland will lead to

an increased ability to produce metastatic prostate cancer.

.


VARI | 2007

External Collaborators

Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington

Senthil Muthuswamy, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

Ilan Tsarfaty, Tel Aviv University, Israel

Valera Vasioukin, Fred Hutchinson Cancer Research Center, Seattle, Washington

Xin Zhang, University of Tennessee, Memphis

43

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

Recent Publications

Edick, M.J., Tesfay, L., Lamb, L.E., Knudsen, B.S., and Miranti, C.K. In press. Inhibition of integrin-mediated crosstalk with

EGFR/Erk or Src signaling pathways in autophagic prostate epithelial cells induces caspase-independent death. Molecular

Biology of the Cell.

Sridhar, S.C., and C.K. Miranti. In press. Tumor metastasis suppressor KAI1/CD82 is a tetraspanin. In Contemporary Cancer

Research: Metastasis, C. Rinker-Schaeffer, M. Sokoloff, and D. Yamada, eds.

Wang, X. , J. Zhu, P. Zhao, Y. Jiao, N. Xu, T. Grabinski, C. Liu, C.K. Miranti, T. Fu, and B. Cao. In press. In vitro efficacy of immunochemotherapy

with anti-EGFR human Fab-Taxol conjugate on A431 epidermoid carcinoma cells. Cancer Biology & Therapy.

Knudsen, Beatrice S., and Cindy K. Miranti. 2006. Impact of cell adhesion changes on proliferation and survival during prostate

cancer development and progression. Journal of Cellular Biochemistry 99(2): 345–361.


Van Andel Research Institute | Scientific Report

44

Image courtesy of Qian Xie, Yue Guo, Rick Hay, and

George Vande Woude, Van Andel Research Institute;

Helayne Sherman, West Michigan Heart, P.C.; and Ai Lockard, VisualSonics, Inc.


VARI | 2007

Evaluating the blood supply of cancer with ultrasound.

45

Human glioblastoma (brain cancer) cells were grown as a tumor beneath the skin of a laboratory mouse. Small bubbles about the size of individual blood

cells were then injected into the mouse’s bloodstream. During the next few minutes the tumor was imaged by ultrasound, using a device similar to sonar.

Echoes from the bubbles, shown in green, depict complex branching patterns of tiny blood vessels growing around and within the tumor to supply it with

nutrients and oxygen. We are using the ultrasound technique to monitor how new types of anticancer medicine change the abundance and branching

of tumor blood vessels in mice, with the hope of applying this technology in the near future to patients with cancer.


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

46

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 Laboratories–Basic Research Program

at the National Cancer Institute, Frederick Cancer Research and Development Center, Maryland, from

1992 to 1999. He joined VARI as a Special Program Senior Scientific Investigator in June 1999 and

in 2003 was promoted to Deputy Director. In 2004, Dr. Resau assumed as well the direction of the

Laboratory of Microarray Technology to consolidate the imaging and quantification of clinical samples

in a CLIA-type research laboratory program. In 2005, Dr. Resau was made the Division Director of the

quantitative laboratories (pathology-histology, microarray, proteomics, epidemiology, and bioinformatics),

and in 2006 he was promoted to Distinguished Scientific Investigator.

Staff

Laboratory Staff

Eric Kort, M.D.

Brendan Looyenga, Ph.D.

Bree Berghuis, B.S., HTL

(ASCP), QIHC

Eric Hudson, B.S.

Paul Norton, B.S.

Ken Olinger, B.S.

David Satterthwaite, B.S.

Kristin VandenBeldt, B.S.

JC Goolsby

Students

Pete Haak, B.S.

Alicia Coleman

Kate Jackson

Wei Luo, B.A.

Nick Miltgen

Kara Myslivec

Sara Ramirez

Jourdan Stuart

Mohan Thapa, M.S.

Huong Tran

Grant Van Eerden

Visiting Scientist

Yair Andegeko


VARI | 2007

Research Interests

The Division of Quantitative Sciences includes the laboratories of Analytical, Cellular, and Molecular Microscopy (ACMM), the

Laboratory of Microarray Technology (LMT), the Laboratory of Computational Biology, the Laboratory of Molecular Epidemiology,

and the Laboratory of Mass Spectrometry and Proteomics. The Division’s laboratories use objective measures to define pathophysiologic

events and processes. For example, the LMT measures the expression of genes relative to a control or a standard.

When pathology and tissue organization is combined with expression, one can better determine not only what the change is but

also possible causation, treatment targets, and effects of treatment. The Molecular Epidemiology laboratory builds objective data

and pathology correlations to infer causation and prognosis.

The ACMM laboratory has programs in pathology, histology, and imaging to describe and visualize changes in cell, tissue, or

organ structure. Our imaging instruments allow us to visualize cells and their components with striking clarity, allowing researchers

to determine where in a cell specific molecules are located. We also use a laser for microdissection of cells from a sample. The

laboratory provides paraffin-block (SPIN program) and frozen-section (TAS program) staining of tissues. An archive of pathology

tissues in the paraffin blocks (Van Andel Tissue Repository; VATR) is being accumulated with the cooperation of local hospitals,

and the data on the samples is being converted to computerized files. The lab also carries out research that will improve our

ability to quantify images, so that we will be able to not only state that a particular protein is present in an image, but also answer

the questions of how much is there and with what other molecules is it co-localized? We are able to image using either fluorescent

(e.g., FITC, GFP) or chromatic agents (e.g., DAB, H&E) and separate the components using our confocal, Nuance, or Maestro

instruments.

47

The Laboratory of Microarray Technology provides gene expression analysis using cDNA microarrays. High-throughput robotics

are used to maintain and process cDNA clone sets for the human, mouse, rat, and canine genomes. The clones are used to

produce both cDNA and spotted oligonucleotide microarrays that are evaluated using strict quality control and quality assurance

criteria developed using the Clinical Laboratory Improvement Amendments (CLIA) as a model. These criteria allow the

laboratory to function in a manner consistent with fully accredited clinical laboratories. In 2006 we produced and used 790

cDNA microarrays, and we also produced 112 custom protein microarrays. In addition, the laboratory has expanded its services

to include Agilent and Operon commercial oligonucleotide microarrays. The use of these products will remove much of the

internal quality control and quality assurance burden, and they will also facilitate the requirement to perform array comparative

genomic hybridization, chromatin immunoprecipitation (chip-on-chip), and splice variant analysis.

Hauenstein Parkinson’s Center

Throughout 2006 we have continued our collaboration with the Hauenstein Parkinson’s Center to collect patient blood samples

and controls from 114 individuals. Mutations in the parkin gene in a series of families with more than one generation affected by

Parkinson disease are being investigated by DNA sequence analysis and will be correlated to gene expression data obtained

from microarray analysis.


Van Andel Research Institute | Scientific Report

Blood spot arrays

State laws in the U.S.A. mandate that blood be drawn from all newborn infants to screen for a variety of health-threatening

conditions. The assays consume only a small portion of the blood samples, which are collected on filter paper (“Guthrie”) cards.

Many states archive the leftover cards, often in unrefrigerated storage. Pete Haak and Eric Kort have successfully isolated

mRNA from archived unfrozen neonatal blood spots obtained as long as nine years ago. Using both quantitative RT-PCR and

multiplex gene expression analysis with cDNA arrays, we can detect RNA from hundreds to thousands of genes in these samples.

Furthermore, we have shown through use of freshly spotted blood cards that the genes detected approximate those found in

whole blood and purified buffy coat. These preliminary experiments demonstrate the feasibility of detecting and identifying RNA

amplified from unfrozen stored neonatal blood spots. The application of high-throughput assays to the analysis of these widely

available samples may be a valuable resource for the study of perinatal markers and determinants of subsequent disease

development. The coming year will see this technology applied to the study of cerebral palsy and neuroblastoma.

Mouse models of Parkinson disease

48

As part of the VAI initiative into Parkinson disease, we have begun to generate novel rodent models of dopaminergic cell loss in

the brain in collaboration with Bart Williams. One of the key tools for these studies is the transgenic dopamine-transporter/cre

(DAT-cre) mouse line, which specifically expresses the cre recombinase in dopaminergic neurons of the brain. In combination

with other transgenic and knock-out mouse lines, the DAT-cre mice will allow us to address the response of such neurons to toxic

stimuli in the context of specific gene deletions and additions. Several of the ongoing and future projects based on the DAT-cre

mouse model are briefly described below.

• Imaging and isolation of primary dopaminergic neurons from mouse brain. We have performed a genetic cross

between the DAT-cre strain and ROSA26 reporter strain to generate mice that specifically express the LacZ reporter

gene in dopaminergic neurons. The DAT-cre/ROSA26 mice will permit us to visualize and quantify live dopaminergic

neurons in vivo. With these mice we will assess the effect of cytotoxic agents (e.g., MMTP, rotenone, or

6-hydroxydopamine) on the number of dopaminergic cells, and more importantly, assess the ability of mice to recover

from these insults. These studies will provide insight into the regenerative capacity of the brain when dopaminergic

neurons are lost or injured. The DAT-cre/ROSA26 mice will also provide a source of highly pure dopaminergic neurons

for in vitro studies. Dopaminergic neurons from these mice will be isolated from brain tissue treated with DDAOgalactoside

and will be identified from the cellular population by fluorescence-activated cell sorting in the VAI flow

cytometry core facility.

• Dopaminergic cell regeneration as a function of age. The relationship between age and the likelihood of developing

Parkinson disease is well established, though the causal nature of this relationship is unclear. One hypothesis is that the

capacity of the brain to regenerate damaged neurons decreases with age, consistent with a gradual loss of brain stem

cells that give rise to new dopaminergic neurons. To test this hypothesis in a mammalian system, we are planning a

genetic cross between DAT-cre and pu TK mice, the latter specifically expressing herpes simplex virus thymidine

kinase (hsvTK) in cells that contain cre recombinase. Cells expressing hsvTK are sensitive to the antiviral compound

ganciglovir (G418) and undergo programmed cell death after systemic treatment. Using the DAT-cre/pu TK model,

we will eliminate dopaminergic neurons at various ages (3, 6, 9, and 12 months) and assess the regenerative potential

of these mice using behavioral and histological parameters. These studies will indicate both the absolute and relative

capacities of the mammalian brain to regenerate dopaminergic neurons as a function of age, thereby providing

information about the value of therapies intended to stimulate the endogenous regenerative capacity of the brain in

Parkinson disease patients.


VARI | 2007

• Effect of hypoxia-inducible factor signaling on dopaminergic cell survival. Dopaminergic neurons are exquisitely

sensitive to oxidative stress, which is defined by an increase in toxic reactive oxygen species. Reactive oxygen

species lead to cell death by direct mechanisms, such as damage to important cellular biomolecules, and indirect

ones, such as the induction of cell death pathways. The latter effect may be offset by cell survival pathways, which

increase thethreshold signal intensity required to induce cell death. Because both chemically induced and idiopathic

Parkinson disease are characterized by increased oxidative stress in dopaminergic neurons, therapies that increase

cell survival pathways in these neurons may be broadly applicable as a treatment to decrease cell death in patients.

The PI-3-kinase (PI3K)/Akt pathway is a highly conserved cell survival pathway operating in virtually all mammalian cell types.

This pathway is tightly regulated by the phosphatase PTEN, which directly opposes the kinase activity of PI3K. We have crossed

DAT-cre mice to mice with a conditionally inactivated allele for PTEN (PTEN flox/flox ). Expression of the cre recombinase in these

mice leads to a genetic deletion of PTEN, thereby increasing Akt activity. DAT-cre/PTEN flox/flox mice and their wild-type littermates

will be treated with the neurotoxin MPTP, which induces high levels of oxidative stress in dopaminergic neurons. We will compare

the mice using behavioral and histological parameters to determine whether increased Akt activity leads to greater cell survival

after an oxidative stress insult.

Educational highlights

This year we had one student from GRAPCEP, two students from the MSU-CVM program, and a guest student from Bath University

in the United Kingdom. Our GRAPCEP mentorship program continues to be funded by Pfizer for a seventh year. Dr. Resau is

a member of the graduate school committee that established the VAEI Graduate School, which will increase our research and

educational opportunities.

49

From left, back row: Goolsby, Satterthwaite, Norton, Resau, Haak, Hudson;

front row: Kort, Luo, VandenBeldt, Berghuis, Jason, Ramirez, Looyenga


Van Andel Research Institute | Scientific Report

Recent Publications

Baldus, S.E., E.J. Kort, P. Schirmacher, H.P. Dienes, and J.H. Resau. In press. Quantification of MET and hepatocyte growth

factor/scatter factor expression in colorectal adenomas, carcinomas, and non-neoplastic epithelia by quantitative laser scanning

microscopy. International Journal of Oncology.

Kort, E.J., M.R. Moore, E.A. Hudson, B. Leeser, G.M. Yeruhalmi, R. Leibowitz-Amit, G. Tsarfaty, I. Tsarfaty, S. Moshkovitz, and J.H.

Resau. In press. Use of organ explant and cell culture. In Mechanisms of Carcinogenesis, Hans Kaiser, ed. Dordrecht, The

Netherlands: Kluwer Academic.

Lindemann, K.K., J. Resau, J. Nährig, E. Kort, B. Leeser, K. Anneke, A. Welk, J. Schäfer, G.F. Vande Woude, E. Lengyel, and N.

Harbeck. In press. Differential expression of c-Met, its ligand HGF/SF, and HER2/neu in DCIS and adjacent normal breast tissue.

Histopathology.

Whitwam, T., M.W. VanBrocklin, M.E. Russo, P.T. Haak, D. Bilgili, J.H. Resau, H.-M. Koo, and S.L. Holmen. In press. Differential

oncogenic potential of activated RAS isoforms in melanocytes. Oncogene.

Young, J.J., J.L. Bromberg-White, C.R. Zylstra, J. Church, E. Boguslawski, J. Resau, B.O. Williams, and N. Duesbery. In press. LRP5

and LRP6 are not required for protective antigen–mediated internalization or lethality of anthrax lethal toxin. PLoS Pathogen.

50

Bruxvoort, Katia J., Holli M. Charbonneau, Troy A. Giambernardi, James C. Goolsby, Chao-Nan Qian, Cassandra R. Zylstra,

Daniel R. Robinson, Pradip Roy-Burman, Aubie K. Shaw, Bree D. Buckner-Berghuis, Robert E. Sigler, James H. Resau, Ruth

Sullivan, Wade Bushman, and Bart O. Williams. 2007. Inactivation of Apc in the mouse prostate causes prostate carcinoma.

Cancer Research 67(6): 2490–2496.

Qian, Chao-Nan, James H. Resau, and Bin Tean Teh. 2007. Prospects for vasculature reorganization in sentinel lymph nodes.

Cell Cycle 6(5): 514–517.

Wallar, Bradley J., Aaron D. DeWard, James H. Resau, and Arthur S. Alberts. 2007. RhoB and the mammalian Diaphanousrelated

formin mDia2 in endosome trafficking. Experimental Cell Research 313(3): 560–571.

Zhang, Y.-W., B. Staal, Y. Su, P. Swiatek, P. Zhao, B. Cao, J. Resau, R. Sigler, R. Bronson, and G.F. Vande Woude. 2007. Evidence

that MIG-6 is a tumor-suppressor gene. Oncogene 26(2): 269–276.

Moshitch-Moshkovitz, Sharon, Galia Tsarfaty, Dafna W. Kaufman, Gideon Y. Stein, Keren Shichrur, Eddy Solomon, Robert H. Sigler,

James H. Resau, George F. Vande Woude, and Ilan Tsarfaty. 2006. In vivo direct molecular imaging of early tumorigenesis and

malignant progression induced by transgenic expression of GFP-Met. Neoplasia 8(5): 353–363.

Qian, Chao-Nan, Bree Berghuis, Galia Tsarfaty, MaryBeth Bruch, Eric J. Kort, Jon Ditlev, Ilan Tsarfaty, Eric Hudson, David G.

Jackson, David Petillo, Jindong Chen, James H. Resau, and Bin Tean Teh. 2006. Preparing the “soil”: the primary tumor induces

vasculature reorganization in the sentinel lymph node before the arrival of metastatic cancer cells. Cancer Research 66(21):

10365–10376.

Tsarfaty, Galia, Gideon Y. Stein, Sharon Moshitch-Moshkovitz, Dafna W. Kaufman, Brian Cao, James H. Resau, George F. Vande

Woude, and Ilan Tsarfaty. 2006. HGF/SF increases tumor blood volume: a novel tool for the in vivo functional molecular imaging

of Met. Neoplasia 8(5): 344–352.

Yao, Xin, Chao-Nan Qian, Zhong-Fa Zhang, Min-Han Tan, Eric J. Kort, James H. Resau, and Bin Tean Teh. 2006. Two distinct

types of blood vessels in clear cell renal cell carcinoma have contrasting prognostic implications. Clinical Cancer Research

13(1): 161–169.

.


VARI | 2007

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

Laboratory of Germline Modification and Cytogenetics

Dr. Swiatek received her M.S. (1984) and Ph.D. (1988) degrees in pathology from Indiana University.

From 1988 to 1990, she was a postdoctoral fellow at the Tampa Bay Research Institute. From 1990

to 1994, she was a postdoctoral fellow at the Roche Institute of Molecular Biology in the laboratory of

Tom Gridley. From 1994 to 2000, Dr. Swiatek was a research scientist and Director of the Transgenic

Core Facility at the Wadsworth Center in Albany, N.Y., and an Assistant Professor in the Department of

Biomedical Sciences at the State University of New York at Albany. She joined VARI as a Special

Program Investigator in August 2000. She has been the chair of the Institutional Animal Care and Use

Committee since 2002 and is an Adjunct Assistant Professor in the College of Veterinary Medicine at

Michigan State University. Dr. Swiatek received her M.B.A. in 2005 from Krannert School of Management

at Purdue University. She was promoted to Senior Scientific Investigator in 2006.

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Staff

Laboratory Staff

Students

Visiting Scientists

Sok Kean Khoo, Ph.D., Associate Laboratory Director

Laura Ayotte, B.S.

Julie Koeman, B.S., CLSp(CG)

Kellie Sisson, B.S.

Kaye Johnson, B.A.

Diana Lewis


Van Andel Research Institute | Scientific Report

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.

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In addition to traditional gene-targeting technologies, the germline modification lab can produce mouse models in which the

gene of interest is inactivated in a target organ or cell line instead of in the entire animal. These models, known as conditional

knock-outs, are particularly useful in studying genes that, if missing, cause the mouse to die as an embryo. The lab also has

the ability to produce mutant embryos that have a wild-type placenta using tetraploid embryo technology; this is useful when

the gene-targeted mutation prevents implantation of the mouse embryo in the uterus. We also assist in the development of

embryonic stem (ES) or fibroblast cell lines from mutant embryos, which allows for in vitro studies of the gene mutation.

Our gene-targeting service encompasses three major procedures: DNA electroporation, clone expansion and cryopreservation,

and microinjection. Gene targeting is initiated by mutating the genomic DNA of interest and inserting it into ES cells via

electroporation. The mutated gene integrates into the genome and, by a process called homologous recombination, replaces one

of the two wild-type copies of the gene in the ES cells. Clones are identified, isolated, and cryopreserved, and genomic DNA is

extracted from each clone and delivered to the client for analysis. Correctly targeted ES cell clones are thawed, established into

tissue culture, and cryopreserved in liquid nitrogen. Gene-targeting mutations are introduced by microinjection of the pluripotent

ES cell clones into 3.5-day-old mouse embryos (blastocysts). These embryos, containing a mixture of wild-type and mutant ES

cells, develop into mice called chimeras. The offspring of chimeras that inherit the mutated gene are heterozygotes possessing

one copy of the mutated gene. The heterozygous mice are bred together to produce “knock-out mice” that completely lack the

normal gene and have two copies of the mutant gene.

Embryo/sperm cryopreservation

We provide cryopreservation services for archiving and reconstituting valuable mouse strains. These cost-effective procedures

decrease the need to continuously breed valuable mouse models, and they provide added insurance against the loss of custom

mouse lines due to disease outbreak or a catastrophic event. Mouse embryos at various stages of development, as well as

mouse sperm, can be cryopreserved and stored in liquid nitrogen; they can be thawed and used, respectively, by implantation

into the oviducts of recipient mice or by in vitro fertilization of oocytes.


VARI | 2007

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 recipient’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 recipient’s genome from

each generation are identified, and these mice are bred by the client. Using speed congenics, 99.9% of congenicity can be

achieved in five generations (1 to 1.5 years).

53

Michigan Animal Model Consortium

The VARI Germline Modification and Cytogenetics lab directs the Michigan Animal Model Consortium (MAMC), one of the ten

Core Technology Alliance (CTA) collaborative core facilities located at the University of Michigan, Michigan State University,

Wayne State University, Western Michigan University, Kalamazoo Valley Community College, Grand Valley State University, and

VARI. The other facilities offer research services in proteomics, bioinformatics, structural biology, genomics, biological imaging,

bioscience commercialization, high-throughput compound screening, good manufacturing practices, and antibody technology.

The MAMC labs were developed with funding from the Michigan Economic

Development Corporation and provide efficient mouse modeling services

to researchers studying human diseases. MAMC’s long-term goal is to

offer a comprehensive set of cutting-edge services that, through continuous

enhancements and development, will define our organization as a single

point-of-service site for animal models research. Centralized provision

of services maximizes research productivity and decreases time to

discovery and is in high demand by academia and pharmaceutical and

biotechnology companies, which are increasingly looking to outsource to

service centers. Through its well-organized service structure and staff

of experts, MAMC supports the growth of the life science industry in

Michigan, which is congruent with the CTA goals.

From left, front row: Koeman, Sisson, Swiatek, Ayotte

back row: Johnson, Lewis, Khoo


Van Andel Research Institute | Scientific Report

MAMC service offerings

Animal model development

• Mouse transgenics. Transgenic technology is used to produce genetically engineered mice expressing foreign genes

and serving as models for human disease research. Microinjection delivers the foreign DNA into the pronucleus of a

one-cell fertilized egg. This service is provided using various strains of laboratory mice, with production of three

transgenic founder mice guaranteed from each procedure.

• Gene targeting. By transfecting mouse embryonic stem cells with inactivating, homologous DNAs, target gene

expression can be shut down. Genetically engineered mice are produced by microinjecting mutant stem cells into

mouse embryos and breeding the progeny to mutant homozygosity. This service is provided using 129 or C57BL/6

embryonic stem cells.

• Xenotransplantation. Human cancer cells are injected into immunodeficient mice to produce human-derived

tumors. Protocols are designed to test anti-tumor treatment regimens that can lead to prognostic, diagnostic, or

therapeutic procedures for humans.

Animal model analysis

• Cytogenetics. Mouse and rat chromosomal abnormalities and genetic loci are visually observed using Giemsa stain,

SKY, or FISH techniques.

• Necropsy. Mice are dissected postmortem and tissues are fixed for histological analysis, with necropsy reports

generated using voice-recognition software.

54

• Histology. Histological sections are prepared from mouse tissues using microtomes and cryostats; they are processed

and stained using automated instruments and then are microscopically analyzed.

• Veterinary pathology. A board-certified veterinary pathologist holding the D.V.M. and Ph.D. degrees provides

expert microscopic analysis and project consultation.

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

Animal model maintenance and preservation

• Mouse rederivation. All mouse strains entering the specific pathogen–free breeding facility are rederived to specific

pathogen–free mouse status using embryo transfer techniques.

• Animal technical services. Veterinary services such as injections, measurements, mating set-up, and tail biopsies are

performed by the animal technician staff.

• Contract breeding. Wild-type mouse strains and genetically engineered animal models are maintained for research

purposes by breeding the strains in a specific pathogen–free environment.

• Embryo/sperm cryopreservation. Genetically engineered mice are preserved for archival purposes, disease control,

genetic stability, and economic efficiency using germplasm cryopreservation techniques.

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

Recent Publications

Furge, Kyle A., Jindong Chen, Julie Koeman, Pamela Swiatek, Karl Dykema, Kseniji Lucin, Richard Kahnoski, Ximing J. Yang, and

Bin Tean Teh. 2007. Detection of DNA copy number changes and oncogenic signaling abnormalities from gene expression data

reveals MYC activation in high-grade papillary renal cell carcinoma. Cancer Research 67(7): 3171–3176.

Zhang, Y.-W., B. Staal, Y. Su, P. Swiatek, P. Zhao, B. Cao, J. Resau, R. Sigler, R. Bronson, and G.F. Vande Woude. 2007. Evidence

that MIG-6 is a tumor-suppressor gene. Oncogene 26(2): 269–276.

Mukhopadhyay, Rita, Ye-Shih Ho, Pamela J. Swiatek, Barry P. Rosen, and Hiranmoy Bhattacharjee. 2006. Targeted disruption of

the mouse Asnal gene results in embryonic lethality. FEBS Letters 580(16): 3889–3894.


VARI | 2007

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

Laboratory of Cancer Genetics

Dr. Teh obtained his M.D. from the University of Queensland, Australia, in 1992, and his Ph.D. from

the Karolinska Institute, Sweden, in 1997. Before joining the Van Andel Research Institute (VARI), he

was an Associate Professor of medical genetics at the Karolinska Institute. Dr. Teh joined VARI as a

Senior Scientific Investigator in January 2000. His research mainly focuses on kidney cancer, and he is

currently on the Medical Advisory Board of the Kidney Cancer Association. Dr. Teh was promoted to

Distinguished Scientific Investigator in 2005.

55

Staff

Laboratory Staff

Chao-Nan (Miles) Qian, M.D., Ph.D.

Peng-Fei Wang, M.D., Ph.D.

Xin Yao, M.D., Ph.D.

Eric Kort, M.D.

Daisuke Matsuda, M.D.

Jindong Chen, Ph.D.

Leslie Farber, Ph.D.

Kunihiko Futami, Ph.D.

Dan Huang, Ph.D.

Sok Kean Khoo, Ph.D.

Students

Visiting Scientists

Yan Li, Ph.D.

Douglas Luccio-Camelo, Ph.D.

David Petillo, Ph.D.

Zhongfa (Jacob) Zhang, Ph.D.

Stephanie Bender, M.S.

Wangmei Luo, M.S.

Mark Betten, B.S.

Aaron Massie, B.S.

Michael Westphal, B.S.

Sabrina Noyes, B.S.


Van Andel Research Institute | Scientific Report

Research Interests

Kidney cancer, or renal cell carcinoma (RCC), is the tenth most common cancer in the United States (35,000 new cases and

more than 13,000 deaths a year). Its incidence has been increasing, a phenomenon that cannot be accounted for by the wider

use of imaging procedures. We have established a comprehensive and integrated kidney research program, and our major

research goals are 1) to identify the molecular signatures of different subtypes of kidney tumors, both hereditary and sporadic,

and to understand how genes function and interact in giving rise to the tumors and their progression; 2) to identify and develop

diagnostic and prognostic biomarkers for kidney cancer; 3) to identify and study novel and established molecular drug targets

and their sensitivity and resistance; and 4) to develop animal models for drug testing and preclinical bioimaging.

Our program to date has established a worldwide network of collaborators; a tissue bank containing fresh-frozen tumor pairs (over

1,000 cases) and serum; and a gene expression profiling database of 500 tumors, with long-term clinical follow-up information for

half of them. Our program includes positional cloning of hereditary RCC syndromes and functional studies of their related genes,

microarray and bioinformatic analysis, generation of RCC mouse models, and more recently, molecular therapeutic studies.

Hereditary RCC syndromes

We are currently focusing on the cloning of the gene responsible for familial clear cell renal cell carcinoma, which is a separate

entity from von Hippel-Lindau (VHL) and from familial RCC with a chromosome-3 translocation. These efforts involve the use of

high-density, single-nucleotide-polymorphism (SNP) microarrays and correlation with our existing gene expression profiles.

56


VARI | 2007

Microarray gene expression profiling and bioinformatics

High-density SNP genotyping has been performed on some of the specimens registered in our RCC expression database.

We are currently focusing on analysis and data mining. Clinically, we continue to subclassify the tumors by correlation with

clinicopathological information. One example is the study of the unclassified group of tumors for which the histological diagnosis

is “unknown”. We have also identified a specific set of genes that can distinguish chromophobe (malignant) from oncocytoma

(benign), two types that share a high degree of similarity in their expression profiles. Our database has proven to be very useful

in RCC research, since we can obtain differential expression of any gene in seconds; this has led to numerous collaborations. We

are currently combining SNP and expression data to identify novel RCC-related genes.

Mouse models of kidney cancer and molecular therapeutic studies

We have generated several kidney-specific conditional knock-outs including APC, PTEN, and VHL. The first two knock-outs

give rise to renal cysts and tumors, whereas VHL remains neoplasia-free; double knock-outs are also being studied. We have

successfully generated nine xenograft RCC models via subcapsular injection that have characteristic clinical features and

outcomes. Tumors and serum have been harvested for a baseline data set. We are currently performing in vitro and in vivo

studies on several new drugs for kidney cancer.

Molecular and cellular studies

We use numerous well-characterized kidney cancer cell lines to study the functions of novel kidney cancer–related genes by

overexpressing or down-regulating the genes. In addition, we perform cell cycle, proliferation, and migration assays to assess

the cellular effects of these genes. These studies are usually coupled with in vivo studies.

57

External Collaborators

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

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


Van Andel Research Institute | Scientific Report

Recent Publications

Al-sarraf, N., S. Mahmood, J.N. Reif, J. Hinrichsen, B.T. Teh, E. McGovern, P. De Meyts, K.J. O’Byrne, and S.G. Gray. In press.

DOK4/IRS-5 expression is altered in clear cell renal cell carcinoma. International Journal of Cancer.

Daly, A.F., J.-F. Vanbellinghen, S.K. Khoo, M.-L. Jaffrain-Rea, L.A. Naves, M.A. Guitelman, A. Murat, P. Emy, A.-P.

Gimenez-Roqueplo, G. Tamburrano, G. Raverot, A. Barlier, W. De Herder, A. Penfornis, E. Ciccarelli, et al. In press. Aryl hydrocarbon

receptor interacting protein gene mutations in familial isolated pituitary adenomas: analysis in 73 families. Journal of Clinical

Endocrinology and Metabolism.

Evans, Andrew J., Ryan C. Russell, Olga Roche, T. Nadine Burry, Jason E. Fish, Vinca W.K. Chow, William Y. Kim, Arthy Saravanan,

Mindy A. Maynard, Michelle L. Gervais, Roxana I. Sufan, Andrew M. Roberts, Leigh A. Wilson, Mark Betten, Cindy Vandewalle, et

al. 2007. VHL promotes E2 box–dependent E-cadherin transcription by HIF-mediated regulation of SIP1 and Snail. Molecular

and Cellular Biology 27(1): 157–169.

Furge, Kyle A., Jindong Chen, Julie Koeman, Pamela Swiatek, Karl Dykema, Kseniji Lucin, Richard Kahnoski, Ximing J. Yang, and

Bin Tean Teh. 2007. Detection of DNA copy number changes and oncogenic signaling abnormalities from gene expression data

reveals MYC activation in high-grade papillary renal cell carcinoma. Cancer Research 67(7): 3171–3176.

58

Furge, K.A., M.H. Tan, K. Dykema, E. Kort, W. Stadler, X. Yao, M. Zhou, and B.T. Teh. 2007. Identification of deregulated oncogenic

pathways in renal cell carcinoma: an integrated oncogenomic approach based on gene expression profiling. Oncogene 26(9):

1346–1350.

Gad, S., S.H. Lefèvre, S.K. Khoo, S. Giraud, A. Vieillefond, V. Vasiliu, S. Ferlicot, V. Molinié, Y. Denoux, N. Thiounn, Y. Chrétien,

A. Méjean, M. Zerbib, G. Benoît, J.M. Hervé, G. Allègre, B. Bressac-de Paillerets, B.T. Teh, and S. Richard. 2007. Mutations in

BHD and TP53 genes, but not in HNF1β gene, in a large series of sporadic chromophobe renal cell carcinoma. British Journal

of Cancer 96(2): 336–340.

Greenman, Christopher, Philip Stephens, Raffaella Smith, Gillian L. Dalgliesh, Christopher Hunter, Graham Bignell, Helen Davies,

Jon Teague, Adam Butler, Claire Stevens, Sarah Edkins, Sarah O’Meara, Imre Vastrik, Esther E. Schmidt, Tim Avis, et al. 2007.

Patterns of somatic mutation in human cancer genomes. Nature 446(7132): 153–158.

Lin, Fan, Ping L. Zhang, Ximing J. Yang, Jianhui Shi, Tom Blasick, Won K. Han, Hanlin L. Wang, Steven S. Shen, Bin T. Teh, and

Joseph V. Bonventre. 2007. Human kidney injury molecule-1 (hKIM-1): a useful immunohistochemical marker for diagnosing

renal cell carcinoma and ovarian clear cell carcinoma. American Journal of Surgical Pathology 31(3): 371–381.

Qian, Chao-Nan, James H. Resau, and Bin Tean Teh. 2007. Prospects for vasculature reorganization in sentinel lymph nodes.

Cell Cycle 6(5): 514–517.

Wang, Kim L., David M. Weinrach, Chunyan Luan, Misop Han, Fan Lin, Bin Teh, and Ximing J. Yang. 2007. Renal papillary

adenoma—a putative precursor of papillary renal cell carcinoma. Human Pathology 38(2): 239–246.

Yang, X.J., M. Takahashi, K.T. Schafernak, M.S. Tretiakova, J. Sugimura, N.J. Vogelzang, and B.T. Teh. 2007. Does “granular cell”

renal cell carcinoma exist? Molecular and histopathological reclassification. Histopathology 50(5): 678–680.

Adley, Brian P., Anita Gupta, Fan Lin, Chunyan Luan, Bin T. Teh, and Ximing J. Yang. 2006. Expression of kidney-specific

cadherin in chromophobe renal cell carcinoma and renal oncocytoma. American Journal of Clinical Pathology 126(1): 79–85.


VARI | 2007

Adley, Brian P., Veronica Papavero, Jun Sugimura, B.T. Teh, and Ximing J. Yang. 2006. Diagnostic value of cytokeratin 7 and

parvalbumin in differentiating chromophobe renal cell carcinoma from renal oncocytoma. Analytical and Quantitative Cytology

and Histology 28(4): 228–236.

Furge, Kyle A., Eric J. Kort, Ximing J. Yang, Walter M. Stadler, Hyung Kim, and Bin Tean Teh. 2006. Gene expression profiling in

kidney cancer: combining differential expression and chromosomal and pathway analyses. Clinical Genitourinary Cancer 5(3):

227–231.

Pimenta, Flávio J., Letícia F.G. Silveira, Gabriela C. Taveres, Andreza C. Silva, Paolla F. Perdigão, Wagner H. Castro, Marcus V.

Gomez, Bin T. Teh, Luiz De Marco, and Ricardo S. Gomez. 2006. HRPT2 gene alterations in ossifying fibroma of the jaws. Oral

Oncology 42(7): 735–739.

Qian, Chao-Nan, Bree Berghuis, Galia Tsarfaty, MaryBeth Bruch, Eric J. Kort, Jon Ditlev, Ilan Tsarfaty, Eric Hudson, David G.

Jackson, David Petillo, Jindong Chen, James H. Resau, and Bin Tean Teh. 2006. Preparing the “soil”: the primary tumor induces

vasculature reorganization in the sentinel lymph node before the arrival of metastatic cancer cells. Cancer Research 66(21):

10365–10376.

Tretiakova, M., M. Turkyilmaz, T. Grushko, M. Kocherginsky, C. Rubin, B. Teh, and X.J. Yang. 2006. Topoisomerase IIα expression

in Wilms’ tumour: gene alterations and immunoexpression. Journal of Clinical Pathology 59(12): 1272–1277.

Yang, Ximing J., Jun Sugimura, Kristian T. Schafernak, Maria S. Tretiakova, Misop Han, Nicholas J. Vogelzang, Kyle Furge, and

Bin Tean Teh. 2006. Classification of renal neoplasms based on molecular signatures. Journal of Urology 175(6): 2302–2306.

59

Yao, Xin, Chao-Nan Qian, Zhong-Fa Zhang, Min-Han Tan, Eric J. Kort, James H. Resau, and Bin Tean Teh. 2006. Two distinct

types of blood vessels in clear cell renal cell carcinoma have contrasting prognostic implications. Clinical Cancer Research

13(1): 161–169.

Zhang, Chun, Dong Kong, Min-Han Tan, Donald L. Pappas, Jr., Peng-Fei Wang, Jindong Chen, Leslie Farber, Nian Zhang, Han-

Mo Koo, Michael Weinreich, Bart O. Williams, and Bin Tean Teh. 2006. Parafibromin inhibits cancer cell growth and causes G1

phase arrest. Biochemical and Biophysical Research Communications 350(1): 17–24.

Zynger, Debra L., Nikolay D. Dimov, Chunyan Luan, Bin Tean Teh, and Ximing J. Yang. 2006. Glypican 3: a novel marker in

testicular germ cell tumors. American Journal of Surgical Pathology 30(12): 1570–1575.


Van Andel Research Institute | Scientific Report

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Photo by Lia Tesfay of the Miranti lab

and Jim Resau of the Resau lab.


VARI | 2007

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

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


Van Andel Research Institute | Scientific Report

Steven J. Triezenberg, Ph.D.

Laboratory of Transcriptional Regulation

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Dr. Triezenberg received his bachelor’s degree in biology and education at Calvin College in Grand

Rapids, Michigan. His Ph.D. training in cell and molecular biology at the University of Michigan was

followed by postdoctoral research in the laboratory of Steven L. McKnight at the Carnegie Institution of

Washington. Dr. Triezenberg was a faculty member of the Department of Biochemistry and Molecular

Biology at Michigan State University for more than 18 years, where he also served as Associate Director

of the Graduate Program in Cell and Molecular Biology. Dr. Triezenberg was recruited to VAI to serve as

the founding Dean of the Van Andel Institute Graduate School and as a Scientific Investigator in the Van

Andel Research Institute, arriving in May 2006.

Laboratory Staff

Staff

Martha Roemer, M.S.

Student

Sebla Kutluay, B.S.


VARI | 2007

Research Interests

The genetic information encoded in DNA must first be transcribed in the form of RNA before it can be translated into the proteins

that do most of the work in a cell. Some genes must be expressed more or less constantly throughout the life of any eukaryotic

cell. Others must be turned on (or turned off) in particular cells either at specific times or in response to a specific signal or event.

Thus, regulation of gene expression is a key determinant of cell function. Our laboratory explores the mechanisms that regulate

the first step in that flow, the process termed transcription.

Over the past 20 years, my laboratory has used infection by herpes simplex virus as an experimental context for exploring the

mechanisms of transcriptional activation. In the past 10 years, we have also asked similar questions in a very different biological

context, the acclimation of plants to cold temperature.

Transcriptional activation during herpes simplex virus infection

Herpes simplex virus type 1 (HSV-1) causes the common cold sore or fever blister. The initial lytic or productive infection by

HSV-1 results in obvious symptoms in the skin and mucosa, typically in or around the mouth. After the initial infection resolves,

HSV-1 finds its way into nerve cells, where the virus can “hide” in a latent mode for long times—essentially for the lifetime of the

host organism. Occasionally, some trigger event (such as emotional stress, damage to the nerve from a sunburn, or a root canal

operation) will cause the virus to reactivate, producing new viruses in the nerve cell and sending those viruses back to the skin

to cause a recurrence of the cold sore.

The DNA genome of HSV-1 encodes approximately 80 different proteins. However, the virus does not have its own machinery for

expressing those genes; instead, it must divert the gene expression machinery of the host cell. That process is triggered by a

viral regulatory protein designated VP16, whose function it is to stimulate transcription of the first viral genes to be expressed in

the infected cell (the immediate-early, or IE, genes).

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VP16 recruitment of host cell transcription machinery

The prevailing model for the mechanism of transcriptional activation is that a portion of an activating protein (such as VP16) called

the activation domain (AD) can bind to the host cell RNA polymerase II or to its accessory proteins. In this manner, VP16 recruits

or tethers accessory proteins to the genes that are to be activated. Over the years, several accessory proteins (also known as

general transcription factors) have been implicated as potential targets for VP16. Of those, the evidence seems to point most

directly at TFIID, a multi-protein complex that includes the TATA-binding protein (TBP). TBP itself can bind rather efficiently to

the VP16 activation domain, and mutations in VP16 that disrupt transcriptional activation also disrupt the interaction with TBP.

We have pursued the structure of the VP16-TBP interaction by methods including X-ray crystallography and nuclear magnetic

resonance. We have also tested the hypothesis that VP16 can influence the orientation of TBP on the TATA-box DNA of a target

gene promoter. This hypothesis, proposed by other laboratories, is based on the fact that both TBP and the TATA sequence to

which it binds are quite symmetric, and yet TBP can effectively support transcription in only a single orientation. We developed

a new quantitative method for assessing TBP orientation and using this method have now demonstrated that TBP binds in a

well-oriented manner even in the absence of VP16. Moreover, on a TATA site engineered to be completely symmetric, to which

TBP binds in both orientations, the VP16 activation domain has no significant influence. This work resolves a long-standing issue

regarding TBP orientation and eliminates one hypothesis for the mechanism of transcriptional activation.


Van Andel Research Institute | Scientific Report

Chromatin-modifying coactivators in herpes virus infections

Eukaryotic DNA is typically packaged as chromatin, in which the DNA is wrapped around “spools” of histone proteins, and

these spools are then further arranged into higher-order structures. This packaging creates an impediment to transcription,

during which RNA polymerase must separate the two strands of DNA. The impediment can be overcome with the help of

chromatin-modifying coactivator proteins, some of which alter the histone proteins by post-translational modifications

(e.g., acetylation or methylation) and others of which can slide or remove the histone proteins to permit access by RNA

polymerase to the DNA.

Experiments using the VP16 activation domain in artificial contexts (for example, in yeast genetic assays) have indicated that

VP16 can recruit various coactivator proteins to target genes. However, the HSV-1 viral DNA is not packaged with histones in

the infectious virion, and prior evidence suggested the viral DNA remained largely chromatin-free during infection. Therefore, we

wondered whether VP16 would recruit these coactivators to viral IE genes, and if so whether those coactivators would be acting

on histone proteins (which didn’t seem to be present) or on some other target. Our results have clearly indicated that VP16 can

recruit certain coactivators to IE genes during lytic infection. We have also shown that at least some histone proteins do associate

with viral DNA, although perhaps not to the same extent as with cellular DNA. We are currently exploring further which histones

associate with viral DNA, how quickly they are put in place, the mechanisms used to put them in place, and what VP16 and other

regulatory proteins might do to counteract the repressive effects of chromatin, which could be considered a molecular defense

mechanism.

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Can a curry spice block herpes infections?

Curcumin, the bright yellow component of the curry spice turmeric, affects eukaryotic cells in several ways. Another laboratory

has reported that curcumin could block the histone acetyltransferase activity of two coactivator proteins, p300 and CBP. Because

we had shown that VP16 can recruit p300 and CBP to viral IE gene promoters, we tested whether curcumin, as an inhibitor of p300

or CBP activity, would block viral IE gene expression and thus block HSV infection. Indeed, curcumin has dramatic effects on IE

gene expression and substantial effects on virus infection (Fig. 1). We are now trying to determine whether that effect is indeed

channeled through the p300 and CBP proteins or whether it arises from another of the biological activities of curcumin.

Figure 1.

- curcumin + curcumin

Figure 1. HSV-1 infection of Vero cell monolayers.

HSV-1 infection results in plaques or holes in a monolayer of

cultured human cells (left). In the presence of curcumin (right),

plaques are generally smaller and the cells within the plaques

are not as completely obliterated. Photo by M. Roemer.


VARI | 2007

Gene activation during cold acclimation in plants

Although plants and their cells obviously have very different forms and functions than animals and their cells, the mechanisms used

for expressing genetic information are quite similar. About ten years ago, we applied our emerging interest in chromatin-modifying

coactivators to an interesting question in plant biology. Some plants, including the prominent experimental organism Arabidopsis,

can sense low (but nonfreezing) temperature in a way that provides protection from subsequent freezing temperatures (Fig. 2).

This process is known as cold acclimation. Michael Thomashow, an MSU plant scientist, has explored the genes expressed

during this process, and we collaborated with his laboratory to explore the mechanisms involved. We have characterized one

particular histone acetyltransferase, termed GCN5, and two of its accessory proteins, ADA2a and ADA2b. Mutations in the genes

encoding these coactivator proteins result in diminished expression of cold-regulated genes. Moreover, histones located at these

cold-regulated genes become more highly acetylated during initial stages of cold acclimation. We are now working to determine

whether GCN5 and the ADA2 proteins are partially or fully responsible for this cold-induced acetylation. We are also collaborating

with groups in Greece and Pennsylvania to explore the distinct biological activities of the two ADA2 proteins. This work may help

us understand whether the mechanisms by which plants express their genes can be effectively modulated so as to protect crop

plants from loss in yield or viability due to environmental stresses such as low temperature.

Figure 2.

Non-acclimated

Acclimated

Figure 2. Acclimation of Arabidopsis seedlings.

Arabidopsis seedlings were grown on agar plates for three weeks

at 20 °C. The plants in the right panel were chilled at 4 °C for two

days. All plants were then subjected to subfreezing temperatures

(–5 °C) for one day and then were returned to warm temperatures

to recover. The acclimated plants remain healthy and green; the

nonacclimated plants lose much of their color and die.

Photo by K. Pavangadkar.

65

External Collaborators

From left: Triezenberg, Roemer, Kutluay

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


Van Andel Research Institute | Scientific Report

George F. Vande Woude, Ph.D.

Laboratory of Molecular Oncology

66

Dr. Vande Woude received his M.S. (1962) and Ph.D. (1964) from Rutgers University. From 1964–1972,

he served first as a postdoctoral research associate, then as a research virologist for the U.S. Department

of Agriculture at Plum Island Animal Disease Center. In 1972, he joined the National Cancer Institute as

Head of the Human Tumor Studies and Virus Tumor Biochemistry sections and, in 1980, was appointed

Chief of the Laboratory of Molecular Oncology. In 1983, he became Director of the Advanced Bioscience

Laboratories–Basic Research Program at the National Cancer Institute’s Frederick Cancer Research and

Development Center, a position he held until 1998. From 1995, Dr. Vande Woude first served as Special

Advisor to the Director, and then as Director for the Division of Basic Sciences at the National Cancer

Institute. In 1999, he was recruited to become the first Director of the Van Andel Research Institute.

Staff

Laboratory Staff

Student

Guest Researchers

Yu-Wen Zhang, M.D., Ph.D.

Chongfeng Gao, Ph.D.

Carrie Graveel, Ph.D.

Qian Xie, Ph.D.

Dafna Kaufman, M.Sc.

Matt VanBrocklin, M.S.

Jack DeGroot, B.S.

Betsy Haak, B.S.

Liang Kang, B.S.

Rachel Kuznar, B.S.

Benjamin Staal, B.S.

Ryan Thompson, B.S.

Yanli Su, A.M.A.T.

Angelique Berens

David Wenkert, M.D.

Yuehai Shen, Ph.D.

Edwin Chen, B.S.


VARI | 2007

Research Interests

A mouse model of mutationally activated Met

Signaling through Met and its ligand, HGF/SF, has been implicated in most types of human cancer. Compelling genetic evidence

for the role of Met stems from the discovery that activating gain-of-function mutations are found in human kidney cancers and

in other cancer types (http://www.vai.org/met/). To study how Met-activating mutations are involved in tumor development, we

generated mice bearing mutations in the endogenous Met locus representative of both the inherited and the sporadic mutations

found in human cancers. On a C57BL/6 background, the different mutant Met lines developed unique tumor profiles, including

carcinomas, sarcomas, and lymphomas. We have found that the differences in tumor types and latency may be due to signaling

differences triggered by the specific mutation in a tissue- or stem cell–specific pattern. Cytogenetic analysis of all tumor types

shows frequent trisomy of the Met locus. Moreover, it is the mutant met allele that is amplified and likely to be required for tumor

progression. When mutant Met was transferred to the FVB/N mouse background, these animals developed aggressive mammary

tumors. Therefore, understanding the signaling specificity of these mutations is essential for developing successful cancer

therapeutics. Our mutant mice provide a valuable model for testing Met inhibitors and for understanding the molecular events

crucial for Met-mediated tumorigenesis.

A novel mouse model for preclinical studies

67

We have generated a severe combined immune deficiency (SCID) mouse strain carrying a human HGF/SF transgene. This mouse

provides a species-compatible ligand for propagating human tumor cells expressing human Met. The growth of Met-expressing

human tumor xenografts can be significantly enhanced in this transgenic mouse relative to growth in nontransgenic hosts. This

immunocompromised strain is vital for examining the role of Met in human tumor malignancy. We are developing metastasis

models and generating orthotopic xenografts of human tumor cells. This model is being used for preclinical testing of drugs or

compounds targeting the HGF/SF-Met complex and downstream signaling pathways.

Understanding the “multiple personalities” of cancer cells

Several years ago, we asked whether tumor cells can switch between proliferative and invasive phenotypes. We discovered

that tumor cells can indeed switch, and they can do so rapidly; they may also express both proliferative and invasive features.

We have established in vitro methods for selecting highly proliferative or highly invasive tumor cell populations that may mimic

the in vivo process of clonal selection during tumor progression. We have determined that chromosomal instability correlates

with the proliferative and invasive phenotypes. Using spectral karyotyping (SKY) and M-Fish, we observe significant changes in

chromosome content with each phenotype, and the changes show remarkable concordance with changes in gene expression.

Regional gene expression changes appear to favor the expression of specific genes appropriate for the invasive or proliferative

phenotype. Moreover, the ratio of chromosomal changes closely parallels the ratio of gene expression in the chromosome. These

results show that chromosome instability and the resulting heterogeneous chromosome composition provide the diversity in gene

expression to allow tumor cell clonal evolution.


Van Andel Research Institute | Scientific Report

Examining how geldanamycin inhibits tumor cell invasion

Our lab has been studying the mechanism by which geldanamycin (GA) inhibits urokinase activation of plasmin from plasminogen

(uPA). Previously, we have shown that a subset of GA-related drug derivatives inhibits HGF/SF-induced activation of plasmin in

canine MDCK cells. We found that such inhibition also occurs in several human glioblastoma tumor cell lines. Curiously, these

GA drugs inhibit HGF/SF-induced uPA activation and block MDCK cell scattering and glioblastoma tumor cell invasion in vitro at

concentrations below that required to exhibit a measurable effect on Met degradation through HSP90. This inhibition is observed

only with HGF/SF-mediated activation and only when the magnitude of HGF/SF-uPA induction is 1.5 times basal uPA-plasmin

activity.

Inhibition of MAPK in melanoma

68

Extracellular signals activate mitogen-activated protein kinase (MAPK) cascades, potentiating biological activities such as cell

proliferation, differentiation, and survival. Constitutive activation of MAPK signaling pathways is implicated in the development

and progression of many human cancers, including melanoma. Mutually exclusive activating mutations in NRas or BRAF are

found in about 85% of all melanomas, resulting in constitutive activation of the MAPK pathway (Ras-BRaf-MEK-Erk-Rsk). We

have previously demonstrated that inhibition of this pathway with small-molecule MEK inhibitors selectively induces apoptosis in

human melanoma cells but not in normal melanocytes both in vitro and in vivo. These results support the concept that the MAPK

pathway represents a tumor-specific survival signaling pathway in melanoma cells and that targeting members of this pathway

may be an effective therapeutic strategy. Understanding the mechanisms by which constitutive MAPK promotes survival and

defining the minimal vital MAPK pathway components required for the development and progression of melanoma may have

direct translational implications. Preliminary data suggest that MAPK activation actively suppresses several pro-apoptotic Bcl-2

family members. We are currently using the specific small-molecule MEK inhibitor PD184352 together with molecular biological

approaches to selectively modulate the expression and function of these molecules in order to validate and develop them as

novel therapeutic targets for treating melanoma and other MAPK-associated cancers.

External Collaborators

Francesco DeMayo, Baylor College of Medicine, Houston, Texas

Ermanno Gherardi, MRC Center, Cambridge, England

Nadia Harbeck, Technische Universität, Munich, Germany

Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington

Ernest Lengyel, University of Chicago, Illinois

Patricia LoRusso, Karmanos Cancer Institute, Detroit, Michigan

Benedetta Peruzzi, National Cancer Institute, Bethesda, Maryland

Alnawaz Rehemtulla, Brian Ross, and Richard Simon, University of Michigan, Ann Arbor

Ilan Tsarfaty, Tel Aviv University, Israel

Robert Wondergem, East Tennessee State University, Johnson City


VARI | 2007

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

Recent Publications

Lindemann, K.K., J. Resau, J. Nährig, E. Kort, B. Leeser, K. Anneke, A. Welk, J. Schäfer, G.F. Vande Woude, E. Lengyel, and N.

Harbeck. In press. Differential expression of c-Met, its ligand HGF/SF, and HER2/neu in DCIS and adjacent normal breast tissue.

Histopathology.

69

Zhang, Y.W., and G.F. Vande Woude. In press. Mig-6, signal transduction, stress response, and cancer. Cell Cycle.

Sawada, Kenjiro, A. Reza Radjabi, Nariyoshi Shinomiya, Emily Kistner, Hilary Kenny, Amy R. Becker, Muge A. Turkyilmaz, Ravi

Salgia, S. Diane Yamada, George F. Vande Woude, Maria S. Tretiakova, and Ernst Lengyel. 2007. c-Met overexpression is

a prognostic factor in ovarian cancer and an effective target for inhibition of peritoneal dissemination and invasion. Cancer

Research 67(4): 1670–1679.

Zhang, Y.-W., B. Staal, Y. Su, P. Swiatek, P. Zhao, B. Cao, J. Resau, R. Sigler, R. Bronson, and G.F. Vande Woude. 2007. Evidence

that MIG-6 is a tumor-suppressor gene. Oncogene 26(2): 269–276.

Gherardi, Ermanno, Sara Sandin, Maxim V. Petoukhov, John Finch, Mark E. Youles, Lars-Göran Öfverstedt, Ricardo N. Miguel,

Tom L. Blundell, George F. Vande Woude, Ulf Skoglund, and Dmitri I. Svergun. 2006. Structural basis of hepatocyte growth

factor/scatter factor and MET signalling. Proceedings of the National Academy of Sciences U.S.A. 103(11): 4046–4051.

Lee, Jae-Ho, Chong Feng Gao, Chong Chou Lee, Myung Deok Kim, and George F. Vande Woude. 2006. An alternatively spliced

form of Met receptor is tumorigenic. Experimental and Molecular Medicine 38(5): 565–573.

Moshitch-Moshkovitz, Sharon, Galia Tsarfaty, Dafna W. Kaufman, Gideon Y. Stein, Keren Shichrur, Eddy Solomon, Robert H. Sigler,

James H. Resau, George F. Vande Woude, and Ilan Tsarfaty. 2006. In vivo direct molecular imaging of early tumorigenesis and

malignant progression induced by transgenic expression of GFP-Met. Neoplasia 8(5): 353–363.

Tsarfaty, Galia, Gideon Y. Stein, Sharon Moshitch-Moshkovitz, Dafna W. Kaufman, Brian Cao, James H. Resau, George F. Vande

Woude, and Ilan Tsarfaty. 2006. HGF/SF increases tumor blood volume: a novel tool for the in vivo functional molecular imaging

of Met. Neoplasia 8(5): 344–352.


Van Andel Research Institute | Scientific Report

Craig P. Webb, Ph.D.

Program for Translational Medicine

Laboratory of Tumor Metastasis and Angiogenesis

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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 Laboratories–Basic Research Program at the National Cancer

Institute, Frederick Cancer Research and Development Center, Maryland (1995–1999). Dr. Webb joined

VARI as a Scientific Investigator in October 1999.

Staff

Laboratory Staff

Students

Visiting Scentists

Visiting Scientists

Student

David Cherba, Ph.D.

Jessica Hessler, PhD.

Jeremy Miller, Ph.D.

David Monsma, Ph.D.

Emily Eugster, M.S.

Sujata Srikanth, M.Phil.

Dawna Dylewski, B.S.

Brian Hillary, B.A.

Marcy Ross, B.S.

Stephanie Scott, B.S.

Katherine Koehler

Philip Grimley, M.D.

David Reinhold, Ph.D.

Guenther Tusch, Ph.D.

Molly Dobb


VARI | 2007

Research Interests

The Program for Translational Medicine was launched on June 1, 2006. While maintaining a research effort focused on enhancing

our understanding of the molecular basis of tumor metastasis, the program is also developing community capabilities around

translational research and the future clinical applications of molecular-based medicine. These efforts are very much focused on

the practical implementation of biomarkers for improving diagnostic and therapeutic strategies against chronic human diseases,

including cancer. The program has recently launched a proof-of-concept personalized medicine initiative to identify novel

treatment options for patients with late-stage cancer. The research portion of this community protocol includes the enhancement

of computer-based predictive models, by overlaying knowledge of molecular networks and drug-target interactions to identify

potential combination targeting strategies for late-stage disease. These predictions are evaluated for efficacy in xenograft models

for each patient’s tumor. Our informatics system, XB-BioIntegration Suite (XB-BIS), has also been enhanced to permit reporting of

molecular drug information back to the medical oncologists, who may use the information for treatment decisions. In the coming

years, we plan on expanding our molecular profiling efforts to identify drugable targets within the cancer stem cell components of

several malignancies and to improve our predictive modeling and reporting capabilities, with the hope of identifying the optimal

combinational strategies for treating cancers using FDA-approved agents and/or drugs in the drug discovery pipeline.

Our research typically begins with the analysis of human specimens. We aim to identify molecular correlates of important clinical

phenotypes, such as the propensity to metastasize and drug resistance. The standardized collection of human specimens,

along with information about the patient’s medical history, diagnosis, treatment, and response to therapy, represents a crucial

component of our research. Identifying the molecular correlates of a given phenotype, whether nucleic acid or protein, often

represents the first step in our translational pipeline. We have developed the essential workflow and integrated informatics that

are required to manage and interpret complex data sets of longitudinal clinical/preclinical and molecular data across different

experimental platforms. XB-BIS is now interfaced with the electronic medical records system of Spectrum Health, through the

co-development of an IRB data exchange portal maintained by the Spectrum Health Research Department. This permits the

transfer of de-identified medical record information from consenting patients into XB-BIS so that it can be combined with molecular

data generated from the processing of the patient’s tissue, blood, or urine. In 2006, XB-BIS was commercialized and has been

licensed by XB-TransMed Solutions (http://www.xbtransmed.com), who now provide professional support services related to the

sales and support of the tool, while our laboratory maintains focus on XB-BIS research and development.

71

In the research lab, and increasingly within the Center for Molecular Medicine, we use various molecular technologies to generate

the molecular data pertaining to a clinical or preclinical sample. XB-BIS permits the analysis of these data in conjunction with

the clinical/preclinical information, and coupled with systems biology tools such as GeneGo’s MetaCore TM product or Ingenuity’s

IPA suite, we identify potential diagnostic signatures that can predict clinically meaningful phenotypes. For example, using

Affymetrix gene expression analysis, we have identified tumor profiles associated with metastatic outcome in colorectal cancer

and with patient survival in mesothelioma. These signatures are now being validated within the CLIA/CAP-accredited Center for

Molecular Medicine, a joint venture between VAI and Spectrum Health.

Potential therapeutic intervention strategies are also identified and validated in the laboratory using a variety of approaches

including RNA interference and/or existing therapeutic agents in the appropriate model systems. At this time, our focal diseases

are pancreatic cancer and multiple myeloma. We have begun to identify potential new targets in these tumors and are using

both inducible shRNA systems for gene knock-down and targeted nanoparticles to validate possible intervention strategies in

mouse xenograft models developed and characterized within our laboratory. While our research is focused on discovering new

diagnostic and therapeutic strategies for metastatic and refractory disease, the translational infrastructure we have developed

can be applied to a broad spectrum of other diseases. The optimal therapeutic target is no longer the disease based on organ

site, but rather the molecular networks driving the clinical phenotype within the disease and the individual.


Van Andel Research Institute | Scientific Report

Community initiatives

As our research discoveries move closer to clinical application, we continue working to increase the readiness of the community

to offer advances in molecular medicine. To translate our discoveries into human benefit, we must work in highly coordinated,

multidisciplinary partnerships with community institutions. The synergistic goals are to benefit human health and promote Grand

Rapids as a leader in translational medicine. The combination of powerful informatics, regulated diagnostics, and clinical trial

coordination have aligned our collective community strengths with industrial demand and the FDA’s critical path intitiative.

The following initiatives were successfully launched in 2006.

• Innovative Clinical Research Alliance. Under the name “ClinXus”, this is a multi-institutional alliance that offers new

biomarker-driven clinical trials to patients and physicians, and it will provide a single destination for pharmaceutical and

biotech companies looking to carry out biomarker-drug co-development. In 2006, ClinXus obtained a $1.5 million state

grant to accelerate the development of this community alliance. Partner institutions include VARI, Spectrum Health, Saint

Mary’s Health Care, Jasper Clinic, Grand Valley Medical Specialists, and Grand Valley State University. Other members

will join in 2007 as we continue to expand our collective capabilites for innovative clinical research. More information can

be found at http://www.clinxus.com.

72

• The Center for Molecular Medicine. The CMM is a joint venture with Spectrum Health that is bringing cutting-edge,

molecular-based diagnostic tests to physicians and their patients. It can offer a broad range of molecular services

and has recently been certified by the College of American Pathology to run molecular diagnostic tests, including the

Roche AmpliChip cytochrome p450 test indicating the correct dose for many prescription drugs. More information can

be found at http://www.cmmdx.org.

From left: Scott, Dobb, Hessler, Hillary, Koehler, Monsma, Cherba,

Reinhold, Dylewski, Srikanth, Ross, Eugster, Webb


VARI | 2007

External Collaborators

Academic Surgical Associates, Grand Rapids, Michigan

Barbara Ann Karmanos Institute, Detroit, Michigan

Cancer & Hematology Centers of Western Michigan, P.C., Grand Rapids

DeVos Children’s Hospital, Grand Rapids, Michigan

Digestive Disease Institute, Grand Rapids, Michigan

GeneGo, Inc., St. Joseph, Michigan

Grand Valley Medical Specialists, Grand Rapids, Michigan

Grand Valley State University, Grand Rapids, Michigan

Henry Ford Hospital, Detroit, Michigan

Jasper Clinical Research & Development, Inc., Kalamazoo, Michigan

Johns Hopkins University, Baltimore, Maryland

M.D. Anderson Cancer Center, Houston, Texas

MMPC, Grand Rapids, Michigan

New York University, New York City

Oncology Care Associates, St. Joseph, Michigan

Pfizer (Ann Arbor, Michigan; Saint Louis, Missouri; Groton, Connecticut)

ProNAi Therapeutics, Kalamazoo, Michigan

Saint Mary’s Health Care, Grand Rapids, Michigan

Schering-Plough Research Institute, New Jersey

Spectrum Health, Grand Rapids, Michigan

TGEN, Phoenix, Arizona

Uniformed Services University of the Health Sciences, Bethesda, Maryland

University of Michigan, Ann Arbor

University of California, San Francisco

West Michigan Heart, Grand Rapids, Michigan

73

Recent Publications

Kuick, Rork, David E. Misek, David J. Monsma, Craig P. Webb, Hong Wang, Kelli J. Peterson, Michael Pisano, Gilbert S. Omenn,

and Samir M. Hanash. 2007. Discovery of cancer biomarkers through the use of mouse models. Cancer Letters 249(1): 40–48.


Van Andel Research Institute | Scientific Report

Michael Weinreich, Ph.D.

Laboratory of Chromosome Replication

74

Dr. Weinreich received his Ph.D. in biochemistry from the University of Wisconsin–Madison in 1993.

He then was a postdoctoral fellow in the laboratory of Bruce Stillman, director of the Cold Spring

Harbor Laboratory, New York, from 1993 to 2000. Dr. Weinreich joined VARI as a Scientific Investigator

in March 2000.

Staff

Dorine Savreux, Ph.D.

FuJung Chang, M.S.

Amber Crampton, B.Sc.

Carrie Gabrielse, B.S.

Vickie Harkins, B.S.

Students

Ying-Chou Chen, M.S.

Charles Miller, B.S.

David Dornboss, Jr.

Louise Haste

Kate Leese


VARI | 2007

Research Interests

We are studying how cells accurately replicate their DNA, a process that begins at specific DNA sequences termed replication

origins. There are approximately 400 replication origins in budding yeast and as many as 10,000 in human cells. Coordinating

the activation of these origins for DNA synthesis during the cell cycle is a daunting task. We know that origins recruit many

proteins prior to the DNA synthetic period (S-phase) that are required for the assembly and activation of replication forks. These

proteins include Cdt1p, Cdc6p, and the origin recognition complex (ORC), which binds directly to origin DNA. Cdt1p, Cdc6p,

and ORC cooperate to load the MCM DNA helicase at the origin in an ATP-dependent reaction. There are perhaps a score of

additional proteins that assemble at the origin following MCM loading before DNA synthesis can begin. In our lab we are studying

how Cdc6p activity is influenced by chromatin structure and ATP binding.

We previously isolated genetic suppressors of a cdc6-4 temperature-sensitive (ts) mutant that inactivated the SIR2 gene. Sir2p is

a histone H3 and H4 deacetylase, and therefore its loss leads to increased H3 and H4 acetylation within chromatin. Although loss

of SIR2 allowed growth of the cdc6-4 strain at high temperature, we have found that Sir2p inhibits only specific origins. We have

systematically identified multiple SIR2-regulated origins on chromosomes III and VI. Our studies so far indicate that these origins

share a common organization including an inhibitory element through which Sir2p acts. Origins in Saccharomyces cerevisiae

have a modular structure (Fig. 1) that includes an ORC binding site (A and B1 elements) and a loading site for the MCM helicase

(B2 element). We have identified an inhibitory sequence in SIR2-regulated origins, termed the I S element, located downstream

of B2. This element is responsible for Sir2p inhibition at these origins. Recent high-resolution mapping along chromosome

III indicates that the I S element maps squarely within a positioned nucleosome. This nucleosome is directly adjacent to or

overlaps the B2 element and therefore might influence MCM helicase loading. In support of this, we have found that

excluding this nucleosome from the B2 element abolishes the activity of the I S element. Furthermore, the I S element acts in a

distance-dependent manner, which is consistent with an effect through this positioned nucleosome.

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Figure 1.

Figure 1.

S. cerevisiae origins have a modular structure consisting of an

essential A element and important B elements. These elements

direct binding of proteins in the “pre-replicative complex” (pre-RC)

that forms during G1 phase. An inhibitory element (I S ) is present

at some origins and likely interferes with pre-RC assembly.


Van Andel Research Institute | Scientific Report

How might SIR2 inhibit DNA replication? We believe that this occurs through deacetylation of histone H4 K16. Sir2p deacetylates

the histone H3 acetyl-lysine residues K9 and K14 as well as H4 K16. We found that an H4 mutation of K16 to Q that mimics

the acetylated state suppresses the cdc6-4 and mcm2-1 ts mutations; H3 K9Q or K14Q mutations do not suppress these ts

mutations. Taken together, our data suggest that a local nucleosome acetylated on H4 K16 facilitates MCM helicase loading and

that a nucleosome impinging on B2, if it is deacetylated on K16, inhibits MCM loading. We would like to understand the molecular

function of the I S element, which is presumably affecting this histone H4 modification. Based on the frequency of SIR2-regulated

origins on chromosome III and VI, we expect that a significant number of origins (about 80 of 400) will be subject to this type of

regulation.

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The Cdc7p-Dbf4p kinase promotes DNA replication and assists in repair of certain DNA lesions. Cdc7p-Dbf4p is a two-subunit

serine/threonine kinase required for initiating DNA replication, and it acts after assembly of the MCM helicase as diagramed in

Fig 2. Cdc7p is the kinase subunit but it has no activity in the absence of the Dbf4p regulatory subunit. We have analyzed Dbf4p

using a structure-function approach to determine the residues required for its essential role in DNA replication. We found that

about 40% of the Dbf4p N-terminus is dispensable for its essential replication function, but that it encodes a conserved 100-

amino-acid region with similarity to the BRCT motif. We have called this sequence the BRDF motif for BRCT and DBF4 similarity.

The BRCT domain folds into a modular structure and is often found in proteins that participate in the DNA damage response. The

BRCT domain likely binds to phosphoproteins and therefore allows regulated targeting to proteins modified by phosphorylation,

as occurs following activation of the DNA damage checkpoint. Yeast dbf4 mutants altering this motif are sensitive to replication

fork arrest, suggesting that the BRDF domain targets the kinase to stalled replication forks (Fig. 3). In support of this interpretation,

we have performed domain-swapping experiments and identified a heterologous BRCT domain that will function in place of

the Dbf4p BRDF domain. We are testing whether these two domains target Dbf4p to the same or similar substrates. It appears

therefore, that the Dbf4p BRDF motif has a role in maintaining replication fork stability, likely through targeting Cdc7p kinase to

non-origin sites. This is a separable activity to the essential role of Dbf4p in promoting the initiation of replication.

Figure 2.

Figure 2.

DNA synthesis requires Cdc7p-Dbf4p kinase, which is thought to act on the pre-RC to

promote Cdc45p and GINS binding. Assembly of a “pre-initiation complex” facilitates

origin unwinding to give ssDNA.

Figure 3.

A.

Figure 3.

A) Schematic representation showing elements

conserved among all Dbf4p orthologs.

The N-terminal BRDF domain is dispensable

for DNA replication. B) We propose this

BRCT-like domain directs Cdc7p-Dbf4p kinase

to stalled replication forks via recognition of

a phosphorylated protein in the replisome.

B.


VARI | 2007

We are also studying the human Cdc7-Dbf4 protein kinase, called here HsCdc7-Dbf4. The HsCdc7 protein is up-regulated in

about 50% of the NCI 60 tumor cell lines representing the most common forms of cancer in the USA. In contrast, HsCdc7 protein

has very low abundance or is undetectable in normal cells and tissues. It may be that nondividing cells down-regulate HsCdc7

expression. We have further determined by immunohistochemistry that HsCdc7 protein is highly expressed in some primary

human tumors. Since HsCdc7 is an essential kinase required for DNA replication, its increased expression level in some tumors

and tumor cell lines may reflect higher rates of cellular proliferation. Alternatively, since HsCdc7 is involved in other aspects

of chromosome metabolism (e.g., DNA repair) and functions in the S-phase checkpoint, its increased expression may offer an

advantage to tumor cells that have higher rates of chromosome instability.

It was therefore interesting when we discovered several years ago that knockdown of HsCdc7 expression using RNAi results in

an apoptotic response in some cancer cell lines but not in normal cells. We have been examining the molecular differences for

this apoptotic response. Apoptosis occurs in cells lines that are either p53 wild type or phenotypically null for p53. There is good

published evidence that in response to HsCdc7 depletion, wild-type cells undergo a G1 and G2/M arrest that is p53-dependent

and protects against apoptosis. However, in some cancer cell lines, even in the absence of p53 function, HsCdc7 knockdown

does not induce apoptosis, although these cells are otherwise competent to undergo the apoptotic program in response to various

stimuli. Since HsCdc7 is required for DNA replication and apparently plays a role in other aspects of chromosome metabolism,

we think that these findings have significance for inhibiting the growth and/or viability of certain types of tumor cells.

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Recent Publications

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

Gabrielse, Carrie, Charles T. Miller, Kristopher H. McConnell, Aaron DeWard, Catherine A. Fox, and Michael Weinreich. 2006.

A Dbf4p BRCA1 C-terminal-like domain required for the response to replication fork arrest in budding yeast. Genetics 173(2):

541–555.

Zhang, Chun, Dong Kong, Min-Han Tan, Donald L. Pappas, Jr., Peng-Fei Wang, Jindong Chen, Leslie Farber, Nian Zhang,

Han-Mo Koo, Michael Weinreich, Bart O. Williams, and Bin Tean Teh. 2006. Parafibromin inhibits cancer cell growth and causes

G1 phase arrest. Biochemical and Biophysical Research Communications 350(1): 17–24.


Van Andel Research Institute | Scientific Report

Bart O. Williams, Ph.D.

Laboratory of Cell Signaling and Carcinogenesis

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Dr. Williams received his Ph.D. in biology from Massachusetts Institute of Technology in 1996. For three

years, he was a postdoctoral fellow at the National Institutes of Health in the laboratory of Harold

Varmus, former Director of NIH. Dr. Williams joined VARI as a Scientific Investigator in July 1999 and was

promoted to Senior Scientific Investigator in 2006.

Staff

Charlotta Lindvall, M.D., Ph.D.

Dan Robinson, Ph.D.

Cassandra Zylstra, B.S.

Students

Sarah Mange

Amanda Field


VARI | 2007

Research Interests

Our laboratory is interested in understanding how alterations in the Wnt signaling pathway cause human disease. Specifically,

we have focused our efforts on the functions of the Wnt co-receptors, Lrp5 and Lrp6. Wnt signaling is an evolutionarily conserved

process that functions in the differentiation of most tissues within the body. Given its central role in growth and differentiation, it

is not surprising that alterations in the pathway are among the most common events associated with human cancer. In addition,

several other human diseases, including osteoporosis, have been linked to altered regulation of this pathway.

We also work on understanding the role of Wnt signaling in bone formation. Our interest is not only from the perspective of normal

bone development, but also in trying to understand whether aberrant Wnt signaling plays a role in the predisposition of some

common tumor types (for example, prostate, breast, lung, and renal tumors) to metastasize to and grow in bone. The long-term

goal of this work is to provide insights that could be used in developing strategies to lessen the morbidity and mortality associated

with skeletal metastasis.

Wnt signaling in normal bone development

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Mutations in the Wnt receptor, Lrp5, have been causally linked to alterations in human bone development. We have characterized

a mouse strain deficient for Lrp5 and shown that it recapitulates the low-bone-density phenotype seen in human patients deficient

for Lrp5. We have furthered this study by showing that mice carrying mutations in both Lrp5 and the related Lrp6 protein have

even more-severe defects in bone density.

To test whether Lrp5 deficiency causes changes in bone density due to aberrant signaling through β-catenin, we created mice

carrying an osteoblast-specific deletion of β-catenin (OC-cre;β-catenin-flox/flox mice). In collaboration with Tom Clemens of the

University of Alabama at Birmingham, we found that alterations in Wnt/β-catenin signaling in osteoblasts lead to changes in the

expression of RANKL and osteoprotegerin (OPG). Consistent with this, histomorphometric evaluation of bone in the mice with

osteoblast-specific deletions of either Apc or β-catenin revealed significant alterations in osteoclastogenesis.

We are currently addressing how other genetic alterations linked to Wnt/β-catenin signaling affect bone development and osteoblast

function. We have generated mice with a conditional allele of Lrp6 that can be inactivated via cre-mediated recombination, and

we will assess the role of Lrp6 in terminal osteoblast differentiation. We are also generating mice carrying a conditional deletion

of Lrp5 in differentiated osteoblasts, and we will characterize their phenotype. Finally, we are working to determine what other

signaling pathways in osteoblasts may impinge on β-catenin signaling to control osteoblast differentiation and function.

General mechanisms of Wnt signaling

There are many levels of regulating the reception of Wnt signals. The completion of the Human Genome Project has shown

that there are 19 different genes encoding Wnt proteins, 9 encoding Frizzled proteins, and the genes encoding Lrp5 and Lrp6.

In addition, there are several proteins that can inhibit Wnt signaling by binding to components of the receptor complex and

interfering with normal signaling, including the Dickkopfs (Dkks) and the Frizzled-related proteins (FRPs). One of the long-term

goals of our laboratory is to understand how specificity is generated for the different signaling pathways, with a specific focus on

understanding the molecular functions of Lrp5 and Lrp6.


Van Andel Research Institute | Scientific Report

Wnt signaling in prostate development and cancer

Two hallmarks of advanced prostate cancer are the development of skeletal osteoblastic metastasis and the ability of the tumor

cells to become independent of androgen for survival. The association of Wnt signaling with bone growth, plus the fact that

β-catenin can bind to the androgen receptor and make it more susceptible to activation with steroid hormones other than DHT,

make Wnt signaling an attractive candidate for explaining some phenotypes associated with advanced prostate cancer. We

have created mice with a prostate-specific deletion of the Apc gene. These mice develop fully penetrant prostate hyperplasia by

four months of age, and these tumors progress to frank carcinomas by seven months. We have found that these tumors initially

regress under androgen ablation but show signs of androgen-independent growth some months later.

Wnt signaling in mammary development and cancer

We are also addressing the relative roles of Lrp5 and Lrp6 in Wnt1-induced mammary carcinogenesis. A deficiency in Lrp5

dramatically inhibits the development of mammary tumors in this context. A germline deficiency for Lrp5 or Lrp6 results in

delayed mammary development. As Lrp5-deficient mice are viable and fertile, we have focused our initial efforts on these mice.

In collaboration with Caroline Alexander’s laboratory, we have found dramatic reductions in the number of mammary progenitor

cells in these mice. We are continuing to examine the mechanisms underlying this reduction.

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VARI mutant mouse repository

With support from the Van Andel Institute, my laboratory maintains a repository of mutant mouse strains to support the general

development of animal models of human disease. We distribute these strains at a nominal cost to interested laboratories.

External Collaborators

Bone development

Mary Bouxsein, Beth Israel Deaconness Medical Center, Boston, Massachusetts

Thomas Clemens, University of Alabama–Birmingham

Marie Claude Faugere, University of Kentucky, Lexington

David Ornitz and Fanxin Long, Washington University, St. Louis, Missouri

Matthew Warman, Harvard University, Boston, Massachusetts

Prostate cancer

Wade Bushman and Ruth Sullivan, University of Wisconsin–Madison

Mammary development

Caroline Alexander, University of Wisconsin–Madison

Yi Li, Baylor Breast Center, Houston, Texas

Jeffrey Rubin, National Cancer Institute, Bethesda, Maryland

Mechanisms of Wnt signaling

Kathleen Cho, University of Michigan, Ann Arbor

Kang-Yell Choi, Yansei University, Seoul, South Korea

Eric Fearon, University of Michigan, Ann Arbor

Silvio Gutkind, National Institute of Dental and Craniofacial Research, Bethesda, Maryland

Kun-Liang Guan, University of Michigan, Ann Arbor


VARI | 2007

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

Recent Publications

Lindvall, C., W. Bu, B.O. Williams, and Y. Li. In press. Wnt signaling, stem cells, and the cellular origin of breast cancer.

Stem Cell Reviews.

Wu, R., N.D. Handrix, R. Kuick, Y. Zhai, D.R. Schwartz, A. Akyol, S. Hanash, D. Misek, H. Katabuchi, B.O. Williams, E.R. Fearon,

and K.R. Cho. In press. Mouse model of human endometroid adenocarcinoma based on somatic defects in the Wnt β-catenin

and PI3K/Pten signaling pathways. Cancer Cell.

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Young, J.J., J.L. Bromberg-White, C.R. Zylstra, J. Church, E. Boguslawski, J. Resau, B.O. Williams, and N. Duesbery. In

press. LRP5 and LRP6 are not required for protective antigen-mediated internalization or lethality of anthrax lethal toxin.

PLoS Pathogens.

Bruxvoort, Katia J., Holli M. Charbonneau, Troy A. Giambernardi, James C. Goolsby, Chao-Nan Qian, Cassandra R. Zylstra,

Daniel R. Robinson, Pradip Roy-Burman, Aubie K. Shaw, Bree D. Buckner-Berghuis, Robert E. Sigler, James H. Resau, Ruth

Sullivan, Wade Bushman, and Bart O. Williams. 2007. Inactivation of Apc in the mouse prostate causes prostate carcinoma.

Cancer Research 67(6): 2490–2496.

Liu, X., K.M. Bruxvoort, Cassandra R. Zylstra, J. Liu, R. Cichowski, Marie-Claude Faugere, Mary L. Bouxsein, C. Wan, Bart O.

Williams, and Thomas L. Clemens. 2007. Lifelong accumulation of bone in mice lacking Pten in osteoblasts. Proceedings of the

National Academy of Sciences U.S.A. 104(7): 2259–2264.

Inoki, Ken, Hongjiao Ouyang, Tianqing Zhu, Charlotta Lindvall, Yian Wang, Xiaojie Zhang, Qian Yang, Christina Bennett, Yoku

Harada, Kryn Stankunas, Cun-yu Wang, Xi He, Ormond A. MacDougald, Ming You, Bart O. Williams, and Kun-Liang Guan. 2006.

TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell

126(5): 955–968.

Lindvall, Charlotta, Nicole C. Evans, Cassandra R. Zylstra, Yi Li, Caroline M. Alexander, and Bart O. Williams. 2006. The Wnt

signaling receptor LRP5 is required for mammary ductal stem cell activity and Wnt1-induced tumorigenesis. Journal of Biological

Chemistry 281(46): 35081–35087.

Zhang, Chun, Dong Kong, Min-Han Tan, Donald L. Pappas, Jr., Peng-Fei Wang, Jindong Chen, Leslie Farber, Nian Zhang,

Han-Mo Koo, Michael Weinreich, Bart O. Williams, and Bin Tean Teh. 2006. Parafibromin inhibits cancer cell growth and causes

G1 phase arrest. Biochemical and Biophysical Research Communications 350(1): 17–24.


Van Andel Research Institute | Scientific Report

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Photo taken by Veronique Schulz of the Mirant lab.


VARI | 2007

Human melanoma cells.

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


Van Andel Research Institute | Scientific Report

H. Eric Xu, Ph.D.

Laboratory of Structural Sciences

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Dr. Xu went to Duke University and the University of Texas Southwestern Medical Center, where he

earned his Ph.D. in molecular biology and biochemistry. Following a postdoctoral fellowship with Carl

Pabo at MIT, he moved to GlaxoWellcome in 1996 as a research investigator of nuclear receptor drug

discovery. Dr. Xu joined VARI as a Senior Scientific Investigator in July 2002 and was promoted to

Distinguished Scientific Investigator in March 2007.

Staff

Laboratory Staff

Jiyuan Ke, Ph.D.

Schoen Kruse, Ph.D.

Augie Pioszak, Ph.D.

David Tolbert, Ph.D.

Yong Xu, Ph.D.

Students

Chenghai Zhang, Ph.D.

X. Edward Zhou, Ph.D.

Jennifer Daugherty, B.S.

Amanda Kovach, B.S.

Kelly Powell, B.S.

Visiting Scientist

Visiting Scientists

Ross Reynolds, Ph.D.


VARI | 2007

Research Interests

Our laboratory is employing multidisciplinary approaches to study the structures and functions of protein complexes that play key

roles in major signaling pathways, and to use the resulting structural information to develop therapeutic agents for the treatment

of human disease, including cancer and diabetes. Currently we are focusing on three families of proteins: nuclear hormone

receptors, the Met tyrosine kinase receptor, and G protein–coupled receptors, because these proteins, beyond their fundamental

roles in biology, are important drug targets for many human diseases.

Nuclear hormone receptors

The nuclear hormone receptors form a large family comprising ligand-regulated and DNA-binding transcriptional factors. The

family includes receptors for classic steroid hormones such as estrogen, progesterone, androgens, and glucocorticoids, as well

as receptors for peroxisome proliferator activators, vitamin D, vitamin A, and thyroid hormones. One distinguishing fact about

these classic receptors is that they are among the most successful targets in the history of drug discovery: every receptor has

one or more cognate synthetic ligands currently being used as medicines. The nuclear receptors also include a class of “orphan”

receptors for which no ligand has been identified. In the last two years, we have developed the following projects centering on the

structural biology of nuclear receptors.

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Peroxisome proliferator–activated receptors

The peroxisome proliferator–activated receptors (PPARα, δ, and γ) are key regulators of glucose and fatty acid homeostasis and

as such are important therapeutic targets for treating cardiovascular disease, diabetes, and cancer. To understand the molecular

basis of ligand-mediated signaling by PPARs, we have determined crystal structures of each PPAR’s ligand-binding domain

(LBD) bound to diverse ligands including fatty acids, the lipid-lowering fibrate drugs, and a new generation of anti-diabetic drugs,

the glitazones. We have also determined the crystal structures of these receptors bound to coactivators or co-repressors. We

are developing this project into the structures of large PPAR fragment/DNA complexes.

Human glucocorticoid and mineralocorticoid receptors

The human glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are classic steroid hormone receptors that are key

to a wide spectrum of human physiology including immune/inflammatory responses, metabolic homeostasis, and control of blood

pressure. Both are well-established drug targets. GR ligands such as dexamethasone (Dex) and fluticasone propionate (FP) are

used to treat asthma, leukemia, and autoimmune diseases; MR ligands such as spironolactone and eplerenone are used to treat

hypertension and heart failure. However, the clinical use of these ligands is limited by undesirable side effects partly associated

with their receptor cross-reactivity or low potency. Thus, the discovery of highly potent and more-selective ligands for GR and MR

is an important goal of pharmaceutical research.

We have determined a crystal structure of the GR LBD bound to dexamethasone and the MR LBD bound to corticosterone, both

of which are in complex with a coactivator peptide motif. These structures provide a detailed basis for the specificity of hormone

recognition and coactivator assembly by GR and MR. Currently we are studying receptor-ligand interactions by crystallizing GR

and MR with various steroid or nonsteroid ligands. In collaboration with Brad Thompson and Raj Kumar at the University of Texas

Medical Branch at Galveston, we are also extending our studies to the structure of a large GR fragment bound to DNA.


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.

The growth of prostate cancer cells that harbor a mutated AR is then no longer dependent on androgen, making anti-hormone

therapy ineffective. This form of hormone-independent prostate cancer is highly aggressive and is responsible for most deaths

from prostate cancer. The development of effective therapies requires a detailed understanding of the structure and functions

of the central molecule, i.e., the androgen receptor and its interactions with hormones and co-regulators. In this project, we are

aiming to determine the structures of the mutated AR proteins that alter the response to anti-hormone therapy. In collaboration

with Donald MacDonnell at Duke University, we are working on the crystal structure of the full-length AR/DNA complex.

Structural genomics of nuclear receptor ligand-binding domains

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The LBDs of nuclear receptors contain key structural elements that mediate ligand-dependent regulation of these receptors,

and as such, LBDs have been the focus of intense structural studies. There are only a few orphan nuclear receptors for which

the LBD structure remains unsolved. In the past two years, we have focused on structural characterization of two orphan

receptors: constitutive androstane receptor (CAR) and steroidogenic factor-1 (SF-1). The CAR structure reveals a compact LBD

fold containing a small pocket that is only half the size of the pocket in PXR, a receptor closely related to CAR. The constitutive

activity of CAR appears to be mediated by a novel linker helix between the C-terminal AF-2 helix and helix 10. On the other

hand, SF-1 is regarded as a ligand-independent receptor, but its LBD structure reveals the presence of a phospholipid ligand in a

surprisingly large pocket; its size is more than twice that of the pocket in the mouse LRH-1, a closely related receptor. The bound

phospholipid is readily exchanged and modulates SF-1 interactions with coactivators. Mutations designed to reduce the size

of the SF-1 pocket or to disrupt hydrogen bonds formed with the phospholipid abolish SF-1/coactivator interactions and reduce

SF-1 transcriptional activity. These findings establish that SF-1 is a ligand-dependent receptor and suggest an unexpected link

between nuclear receptors and phospholipid signaling pathways.

The Met tyrosine kinase receptor

MET is a tyrosine kinase receptor that is activated by hepatocyte growth factor/scatter factor (HGF/SF). Aberrant activation of

the Met receptor has been linked to the development and metastasis of many types of solid tumors and has been correlated

with poor clinical prognosis. HGF/SF has a modular structure with an N-terminal domain, four kringle domains, and an inactive

serine protease domain. The structure of the N-terminal domain with a single kringle domain (NK1) has been determined. Less

is known about the structure of the Met extracellular domain. The molecular basis of the MET receptor–HGF/SF interaction and

the activation of MET signaling by this interaction remains poorly understood. In collaboration with George Vande Woude and

Ermanno Gherardi, we are developing this project to solve the crystal structure of the Met receptor/HGF complex.


VARI | 2007

G Protein–coupled receptors

G protein–coupled receptors (GPCRs) form the largest family of receptors in the human genome; they are receptors for diverse

signals carried by photons, ions, small chemicals, peptides, and hormones. These receptors account for over 40% of drug

targets, but the structure of these receptors remains a challenge because they are seven-transmembrane receptors. Currently,

there is only one reported GPCR structure, for an inactive form of bovine rhodopsin. Many important questions regarding GPCR

ligand binding and activation remain unanswered. From our standpoint, GPCRs are similar to nuclear hormone receptors with

respect to regulation by protein-ligand and protein-protein interactions. Due to their importance, we have decided to take on

studies of the structural basis of ligand binding in, and activation of, GPCRs.

External Collaborators

Doug Engel, University of Michigan, Ann Arbor

Ermanno Gherardi, University of Cambridge, UK

Steve Kliewer, University of Texas Southwestern Medical Center, Dallas

David Mangelsdorf, University of Texas Southwestern Medical Center, Dallas

Donald MacDonnell, Duke University, Durham, North Carolina

Stoney Simmons, National Institutes of Health, Bethesda, Maryland

Scott Thacher, Orphagen Pharmaceuticals, San Diego, California

Brad Thompson and Raj Kumar, University of Texas Medical Branch at Galveston

Ming-Jer Tsai, Baylor College of Medicine, Houston, Texas

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From left, standing: E. Xu, Daugherty, Tolbert, Kovach, Powell, Zhang, Zhou

kneeling: Kruse, Pioszak, Y. Xu, Ke

Recent Publications

Choi, Mihwa, Antonio Moschetta, Angie L. Bookout, Li Peng, Michihisa Umetani, Sam R. Holmstrom, Kelly Suino-Powell, H. Eric

Xu, James A. Richardson, Robert D. Gerard, David J. Mangelsdorf, and Steven A. Kliewer. 2006. Identification of a hormonal

basis for gallbladder filling. Nature Medicine 12(11): 1253–1235.


Van Andel Research Institute | Scientific Report

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2006 Van Andel Research Institute Symposium


VARI | 2007

Winning the War against Cancer:

From Genomics to Bedside and Back

In September 2006, the Van Andel Research Institute honored the lifetime achievements of George F. Vande Woude with a

symposium titled “Winning the War against Cancer: From Genomics to Bedside and Back”. Organized by Nicholas Duesbery,

Tony Hunter, and Bin Teh, the three-day symposium featured noted speakers, including three Nobel laureates; presentation of the

Daniel Nathans Award; and a reception honoring Dr. Vande Woude. More than 250 scientists attended the meeting.

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Symposium photos by Jindong Chen.


Van Andel Research Institute | Scientific Report

Invited Speakers

Jerry Adams

Walter & Eliza Hall Institute

Tim Hunt

Clare Hall Laboratories

Tony Pawson

Mount Sinai Hospital Research Institute

James P. Allison

Memorial Sloan-Kettering Cancer Center

Tony Hunter

Salk Institute for Biological Studies

Bruce Ponder

Cancer Research U.K

Anton Berns

Nederlands Kanker Institute

Arnold Levine

Institute for Advanced Study

Martine Roussel

St. Jude Children’s Research Hospital

J. Michael Bishop

University of California, San Francisco

David M. Livingston

Dana-Farber Cancer Institute

Janet Rowley

University of Chicago

Joan S. Brugge

Harvard Medical School

James L. Maller

University of Colorado School of Medicine

Joseph Schlessinger

Yale University School of Medicine

Suzanne Cory

Walter & Eliza Hall Institute

Paul A. Marks

Memorial Sloan-Kettering Cancer Center

Phillip A. Sharp

Massachusetts Institute of Technology

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Michael Dean

National Cancer Institute–Frederick

Frank McCormick

University of California, San Francisco

Louis Staudt

National Cancer Institute

Edward Harlow

Harvard Medical School

William Muller

McGill University

Craig Thompson

Abramson Family Cancer Research Institute

Stephen Hughes

National Cancer Institute–Frederick

Morag Park

McGill University

George Vande Woude

Van Andel Research Institute

Karen Vousden

Beatson Institute for Cancer Research


VARI | 2007

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Van Andel Research Institute | Scientific Report


VARI | 2007

All of us who have had the privilege of working with George over the years join in appreciation

and thanks for his constant interest, wisdom, insights, and humor.

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Duesbery, N.S., and B.T. Teh. 2007. Cancer: biology and therapeutics—a tribute to George Vande Woude.

Oncogene 26(9): 1258–1259.

Teh, B.T., and N. Duesbery. 2007. A tribute to George F. Vande Woude, a man of character: 2006 Scientific Symposium

“Winning the War against Cancer: From Genomics to Bedside and Back.” Cancer Research 67(6): 2394–2395.


Van Andel Research Institute | Scientific Report

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Daniel Nathans Memorial Award


VARI | 2007

Daniel Nathans Memorial Award

The Daniel Nathans Memorial Award was established in memory of Dr. Daniel Nathans, a distinguished member of our scientific

community and a founding member of VARI’s Board of Scientific Advisors. We established this award to recognize individuals

who emulate Dan and his contributions to biomedical and cancer research. It is our way of thanking and honoring him for his help

and guidance in bringing Jay and Betty Van Andel’s dream to reality. The Daniel Nathans Memorial Award was announced at our

inaugural symposium, “Cancer & Molecular Genetics in the Twenty-First Century”, in September 2000.

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Previous Award Recipients

2000 Richard D. Klausner, M.D.

2001 Francis S. Collins, M.D., Ph.D.

2002 Lawrence H. Einhorn, M.D.

2003 Robert A. Weinberg, Ph.D.

2004 Brian Druker, M.D.

2005 Tony Hunter, Ph.D., and Tony Pawson, Ph.D.

Tony Hunter, Ph.D.

Tony Pawson, Ph.D.


Van Andel Research Institute | Scientific Report

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Postdoctoral Fellowship Program


VARI | 2007

Postdoctoral Fellowship Program

The Van Andel Research Institute provides postdoctoral training opportunities to Ph.D. scientists beginning their research careers.

The fellowships help promising scientists advance their knowledge and research experience while at the same time supporting

the research endeavors of VARI. The fellowships are funded in three ways: 1) by the laboratories to which the fellow is assigned;

2) by the VARI Office of the Director; or 3) by outside agencies. Each fellow is assigned to a scientific investigator who oversees

the progress and direction of research. Fellows who worked in VARI laboratories in 2006 and early 2007 are listed below.

Jennifer Bromberg-White

Dan Huang

Michael Shafer

Penn. State University College of Medicine, Hershey

VARI mentor: Nick Duesbery

Peking Union Medical College, China

VARI mentor: Bin Teh

Michigan State University, East Lansing

VARI mentor: Brian Haab

Philippe Depeille

Schoen Kruse

Suganthi Sridhar

University of Montpellier, France

VARI mentor: Nicholas Duesbery

University of Colorado, Boulder

VARI mentor: Eric Xu

Southern Illinois University, Carbondale

VARI mentor: Cindy Miranti

Yan Ding

Brendan Looyenga

Peng Fei Wang

Peking Union Medical College, China

VARI mentor: Nicholas Duesbery

University of Michigan, Ann Arbor

VARI mentor: James Resau

Fourth Military Medical University, China

VARI mentor: Bin Teh

Mathew Edick

Douglas Luccio-Camelo

Yi-Mi Wu

University of Tennessee, Memphis

VARI mentor: Cindy Miranti

University of Brazil, Rio de Janeiro

VARI mentor: Bin Teh

National Tsin-Hua University, Taiwan

VARI mentor: Brian Haab

97

Kathryn Eisenmann

University of Minnesota, Minneapolis

VARI mentor: Arthur Alberts

Daisuke Matsuda

Kitasato University, Japan

VARI mentor: Bin Teh

Yong Xu

Shanghai Institute of Materia Medica, China

VARI mentor: Eric Xu

Leslie Farber

George Washington University, Washington, D.C.

VARI mentor: Bin Teh

Augen Pioszak

University of Michigan, Ann Arbor

VARI mentor: Eric Xu

Xin Yao

Tianjin Medical University, China

VARI mentor: Bin Teh

Kunihiko Futami

Tokyo University of Fisheries, Japan

VARI mentor: Bin Teh

Daniel Robinson

University of California, Davis

VARI mentor: Bart Williams

Chenghai Zhang

Virus Institute of the CDC, China

VARI mentor: Eric Xu

Quliang Gu

Dorine Savreux

Xiaoyin Zhou

Sun Yat-sen University of Medicine, China

VARI mentor: Brian Cao

Virology University, France

VARI mentor: Michael Weinreich

University of Alabama – Birmingham

VARI mentor: Eric Xu

Carrie Graveel

University of Wisconsin – Madison

VARI mentor: George Vande Woude

Jessica Hessler

University of Michigan, Ann Arbor

VARI mentor: Craig Webb

Holly Holman

University of Glasgow, U.K.

VARI mentor: Arthur Alberts


Van Andel Research Institute | Scientific Report

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Student Programs


VARI | 2007

Grand Rapids Area Pre-College Engineering Program

The Grand Rapids Area Pre-College Engineering Program (GRAPCEP) is administered by Davenport University and jointly

sponsored and funded by Pfizer, Inc., and VARI. The program is designed to provide selected high school students, who have

plans to major in science or genetic engineering in college, the opportunity to work in a research laboratory. In addition to

research methods, the students also learn workplace success skills such as teamwork and leadership. The three 2006 GRAPCEP

students were

Alicia Coleman (Resau/Duesbery)

Creston High School

Megan Spencer (Holmen)

Creston High School

Ware-Van Brunt (Webb)

Creston High School

99

From left: Ware-Van Brunt, Coleman, Spencer


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 2006 to March 2007, VARI hosted 62 students from 23 colleges and universities in formal summer internships under

the Frederik and Lena Meijer Student Internship Program and in other student positions during the year. An asterisk (*) indicates

a Meijer student intern.

100

Andrews University, Berrien Springs, Michigan

Christopher Armstrong* (Xu)

Aquinas College, Grand Rapids, Michigan

Krysta Collins (Haab)

Natalie Kent (Hay)

Sara Kunz (Hay)

Rebecca Trierweiler (Hay)

Calvin College, Grand Rapids, Michigan

David Dornboss, Jr. (Weinreich)

Jonathan Dudley (Vande Woude)

Amanda Field* (Williams)

Alysha Kett* (Vande Woude)

Geoff Kraker (MacKeigan)

Kate Leese (Weinreich)

Sarah Mange (Williams)

Devin Mistry (Haab)

Jose Toro (Hay)

Bill Wondergem (Teh)

Case Western Reserve University, Cleveland

Elianna Bootzin* (Hay)

Central Michigan University, Mount Pleasant

Sarah DeVos* (Teh)

Franciscan University, Steubenville, Ohio

Joan Krilich* (Cavey)

Grand Rapids Community College, Michigan

Wei Luo (Resau)

Grand Valley State University, Allendale, Michigan

Angelique Berens (Vande Woude)

Eric Graf (Miranti)

Nick Miltgen (Resau)

Gary Rajah* (Miranti)

Lisa Orcasitas (Duesbery)

Sara Ramirez (Resau)

Brittany Stropich* (Alberts)

Indiana University, Bloomington

Erin Jefferson* (Webb)

Kalamazoo College, Kalamazoo, Michigan

Adam Granger (Holmen)

Marquette University, Milwaukee, Wisconsin

Michael Avallone (Teh)

Miami University, Oxford, Ohio

Grant Van Eerden (Resau)

Michigan State University, East Lansing

David Achila (Xu/Weinreich)

Ying-Chou Chen, M.S. (Weinreich)

Michelle Dawes (Duesbery)

Aaron DeWard (Alberts)

Pete Haak, B.S. (Resau)

Kate Jackson (Resau)

Andrew Kraus (Vande Woude)

Sebla Kutluay, B.S. (Triezenberg)

Chih-Shia Lee, M.S. (Duesbery)

Charles Miller (Weinreich)

Kara Myslivec (Resau)

Katie Sian, B.S. (MacKeigan)


VARI | 2007

2006 summer intern students

Nanjing Medical University, China

Xin Wang (Cao)

Ning Xu (Cao)

Aixia Zhang (Cao)

Jin Zhu (Cao)

Northern Illinois University, Dekalb

Mohan Thapa (Resau)

Purdue University, West Lafayette, Indiana

Brent Goodman* (Furge)

University of Bath, United Kingdom

Naomi Asantewa-Sechereh (Duesbery)

Louise Haste (Weinreich)

University of Illinois, Champaign-Urbana

Huong Tran (Resau)

University of Mannheim, Germany

Dagmar Hildebrand (Alberts)

Stefan Kutscheidt (Miranti)

University of Michigan, Ann Arbor

Katherine Koelzer* (Swiatek)

Erin Lambers (Duesbery)

Jennifer Lunger* (Haab)

Renee VanderLaan* (Holmen)

University of North Carolina, Chapel Hill

Jourdan Stuart* (Resau)

University of Notre Dame, South Bend, Indiana

Kristin Buzzitta (Teh)

Joe Church* (Duesbery)

Margaret Condit (Teh)

Western Michigan University, Kalamazoo

Mallory Walters (Holmen)

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Van Andel Research Institute | Scientific Report

102

Han-Mo Koo Memorial Seminar Series


VARI | 2007

Han-Mo Koo Memorial Seminar Series

This seminar series is dedicated to the memory of Dr. Han-Mo Koo, who was a VARI Scientific Investigator from 1999 until his

passing in May of 2004.

January 2006

W. Michael Kuehl, National Cancer Institute

“Molecular pathogenesis of multiple myeloma”

Kenneth Bradley, University of California, Los Angeles

“Anthrax lethal toxin”

Valina L. Dawson, Johns Hopkins University

“Life and death signaling by PAR in the brain”

Ted M. Dawson, Johns Hopkins University

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

Andy Futreal, Wellcome Trust Sanger Institute

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

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February

Morag Park, McGill University, Montreal

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

Nicholas J. Vogelzang, Nevada Cancer Institute

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

March

Teresa L. Burgess, Amgen, Inc.

“Fully human monoclonal antibodies to hepatocyte growth factor”

Kenneth L. van Golen, University of Michigan

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

Thomas W. Glover, University of Michigan

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


Van Andel Research Institute | Scientific Report

April

Stephen J. O’Brien, National Cancer Institute

“Genetic architecture of complex diseases: lessons from AIDS”

Richard Treisman, Cancer Research, U.K.

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

Dean Felsher, Stanford University

“Molecular and cellular basis of oncogene addiction”

May

Partho Ghosh, University of California, San Diego

“Met as a target for bacterial intracellular invasion”

Ming-Jer Tsai, Baylor College of Medicine

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

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Laura S. Schmidt, National Cancer Institute–Frederick

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

David Drubin, University of California, Berkeley

“Harnessing actin dynamics for endocytic trafficking”

Thomas Clemens, University of Alabama at Birmingham

“Oxygen sensing and osteogenesis”

June

Robert J. Motzer, Memorial Sloan-Kettering Cancer Center

“Targeted therapy for metastatic renal cell carcinoma”

Tom Blumenthal, University of Colorado

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

July

Rudolf Jaenisch, Whitehead Institute and Massachusetts Institute of Technology

“Nuclear cloning, stem cells, and pluripotency”


VARI | 2007

August

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

“p53, mitochondria, and apoptosis”

Gregory S. Fraley, Hope College

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

September

Stephen A. Krawetz, Wayne State University

“Genome reprogramming and the paternal contribution at fertilization”

Hilary Koprowski, Thomas Jefferson University

“Rabies at the dawn of the 21st century”

October

Y. Eugene Chen, University of Michigan

“Nitro-lipids and PPARs in metabolic syndrome”

Jacques Pouyssegur, Institute of Signaling, University of Nice

“Hypoxia signaling and cancer progression”

105

November

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

“BRCA1 and tumorigenesis in animal models”

Dafna Bar-Sagi, New York University

“RAS signaling: new trails in familiar territory”

December

Kun-Liang Guan, University of Michigan

“Regulation and function of the TSC-mTOR pathway”

January 2007

Moses Lee, Hope College

“Regulation of the topoisomerase IIα gene using polyamides that bind to the inverted CCAAT

box present in the promoter”


Van Andel Research Institute | Scientific Report

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 Science

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

106

Melanie H. Cobb, University of Texas Southwestern Medical Center

“MAP kinase signaling in pancreatic beta cells”


VARI | 2007

Van Andel Research Institute Organization

107


Van Andel Research Institute | Scientific Report

David L. Van Andel,

Chairman and CEO, Van Andel Institute

VARI Board of Trustees

David L. Van Andel, Chairman and CEO

Fritz M. Rottman, Ph.D.

James B. Wyngaarden, M.D.

108

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 Institute’s research programs. The members are

Alan Bernstein, Ph.D.

Joan Brugge, Ph.D.

Webster Cavenee, Ph.D.

Frank McCormick, Ph.D.

Davor Solter, M.D., Ph.D.


VARI | 2007

Office of the Director

George F. Vande Woude, Ph.D.

Director

Deputy Director for Clinical Programs

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

Deputy Director for Special Programs

James H. Resau, Ph.D.

Deputy Director for Research Operations

Nicholas S. Duesbery, Ph.D.

109

Director for Research Administration

Administrator to the Director

Science Editor

Roberta Jones

Michelle Bassett

David E. Nadziejka

Administration Group

From left, standing:

Chastain, Lewis, Koehler, Noyes,

Stougaard, Carrigan, Johnson, Resau;

Seated:

Holman, Jason, Nelson,

Novakowski, Rappley


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.

110

Executive

Steven R. Heacock, Chief Administrative Officer and General Counsel

R. Jack Frick, Chief Financial Officer

Ann Schoen, Executive Assistant

Communications and Development

Joseph P. Gavan, Vice President

Jaime Brookmeyer

Sarah Friedman

Stephanie Hehl

Sarah Lamb

Facilities

Samuel Pinto, Manager

Jason Dawes

Ken De Young

Christen Dingman

Shelly King

Richard Sal

Richard Ulrich

Pete VanConant

Jeff Wilbourn

Finance

Timothy Myers, Controller

Sandi Essenberg

Stephanie Green

Richard Herrick

Keri Jackson

Angela Lawrence

Laura Lohr

Heather Ly

Susan Raymond

Andrew Schmidt

Jamie VanPortfleet

Glassware and Media Services

Richard M. Disbrow, CPM, Manager

Bob Sadowski

Marlene Sal

Grants and Contracts

Carolyn W. Witt, Director

Anita Boven

Nicole Higgins

Sara O’Neal

David Ross

Human Resources

Linda Zarzecki, Director

Margie Hoving

Pamela Murray

Angela Plutschouw

Information Technology

Bryon Campbell, Ph.D., Chief Information Officer

David Drolett, Manager

Bill Baillod

Tom Barney

Phil Bott

Nathan Bumstead

Charles Grabinski

Kenneth Hoekman

Kimberlee Jeffries

Jason Kotecki

Theo Pretorius

Thad Roelofs

Russell Vander Mey

Candy Wilkerson

Investments Office

Kathleen Vogelsang

Ted Heilman

Procurement Services

Richard M. Disbrow, CPM, Manager

Heather Frazee

Chris Kutchinski

Shannon Moore

Amy Poplaski

John Waldon

Public Affairs

John VanFossen

Security

Kevin Denhof, CPP, Chief

Christen Dingman

Sandra Folino

Maria Straatsma

Contract Support

Mary Morgan, Librarian

(Grand Valley State University)

Jim Kidder, Safety Manager

(Michigan State University)


VARI | 2007

111


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

John C. Kennedy

Board of Scientific Advisors

Michael S. Brown, M.D., Chairman

Richard Axel, M.D.

Joseph L. Goldstein, M.D.

Tony Hunter, Ph.D.

Phillip A. Sharp, Ph.D.

112

Van Andel Research Institute

Board of Trustees

David Van Andel, Chairman

Fritz M. Rottman, Ph.D.

James B. Wyngaarden, M.D.

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.

Van Andel Research Institute

Director

George Vande Woude, Ph.D.

Chief Administrative Officer

and General Counsel

Steven R. Heacock

VP Communications

and Development

Joseph P. Gavan

Van Andel Education Institute

Director

Gordon Van Harn, Ph.D.

Chief Financial Officer

R. Jack Frick


VARI | 2007

Van Andel Research Institute

DIRECTOR – George Vande Woude, Ph.D.

Deputy Directors

Clinical Programs Rick Hay, Ph.D., M.D.

Special Programs James Resau, Ph.D.

Research Operations Nick Duesbery, Ph.D.

Director for Research Administration

Roberta Jones

SCIENTIFIC ADVISORY BOARD

Alan Bernstein, Ph.D.

Joan Brugge, Ph.D.

Webster Cavenee, Ph.D.

Frank McCormick, Ph.D.

Davor Solter, Ph.D.

BASIC SCIENCE

SPECIAL PROGRAMS

113

Cancer Cell Biology

Brian Haab, Ph.D.

Cancer Immunodiagnostics

George Vande Woude, Ph.D.

Molecular Oncology

Craig Webb, Ph.D.

Tumor Metastasis & Angiogenesis

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

Animal Models

Nicholas Duesbery, Ph.D.

Cancer & Developmental Cell Biology

Bart Williams, Ph.D.

Cell Signaling & Carcinogenesis

Cancer Genetics

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

Cancer Genetics

Structural Biology

Eric Xu, Ph.D.

Structural Sciences

Systems Biology

Jeffrey MacKeigan, Ph.D.

Systems Biology

Brian Cao, M.D.

Antibody Technology

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

Germline Modification

Bryn Eagleson, A.A.

Transgenics and Vivarium

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

Cytogenetics

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

Sequencing

Art Alberts, Ph.D.

Flow Cytometry

Division of Quantitative Sciences

James Resau, Ph.D.

James Resau, Ph.D.

Analytical, Cellular,

& Molecular MIcroscopy

James Resau, Ph.D.

Microarray Technology

Kyle Furge, Ph.D.

Computational Biology

Greg Cavey, B.S.

Mass Spectrometry and

Proteomics

James Resau, Ph.D.

Molecular Epidemiology

Animal Imaging

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

Noninvasive Imaging

& Radiation Biology

Gene Regulation

Steven Triezenberg, Ph.D.

Transcriptional Regulation

Dean of VAI Graduate School


The Van Andel Institute and/or its affiliated organizations (VARI and VAEI), through its responsible managers, recruits, hires, upgrades,

trains, and promotes in all job titles without regard to race, color, religion, sex, national origin, age, height, weight, marital status,

disability, pregnancy, or veteran status, except when an accommodation is unavailable or it is a bona fide occupational qualification.

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

Phone 616.234.5000 Fax 616.234.5001 www.vai.org

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