Harold L. Moses, M.D. - US grants

In the past project period it was demonstrated that TGFbeta1 rapidly suppresses expression of the protooncogene c-myc at the level of transcriptional initiation in keratinocytes, and that expression of c-myc is necessary for proliferation of these cells. We have identified the TGFbeta control element (TCE) in the human c-myc promoter and further shown that the product of the retinoblastoma tumor susceptibility gene (pRB) is probably necessary for TGFbeta suppression of c-myc, at least in certain cell types. Preliminary data indicate that the tumor suppressor, p53, can also regulate c-myc transcription. Studies on carcinoma cell lines have shown that abnormalities in the rb gene correlate with the loss of the growth inhibitory response to TGFbeta1. Also in the past project period, studies with MMTV-TGFbeta1 transgenic mouse lines have shown a phenotype of inhibition of end-bud growth and branching morphogenesis. Additional studies have shown that focal administration or local overproduction of TGFbeta1 can cause increased connective tissue formation, including angiogenesis, and immunosuppression. Studies with MMTV-TGFalpha transgenic lines in another laboratory have demonstrated that misregulation of TGFalpha in breast epithelial cells leads to hyperplasia and an increased incidence of spontaneous and DMBA-induced tumorigenesis. Based on this and other data, it is hypothesized that in early steps of mammary carcinogenesis, over-expression of TGFalpha promotes, and over-expression of TGFbetas retards or suppresses, the carcinogenic process. With mutations in the carcinoma cells that result in loss of TGFbeta growth inhibition, endogenous TGFbetas then accelerate tumor progression through paracrine effects on stroma, including angiogenesis, and local immunosuppression. It is further hypothesized that the specific alterations that can cause loss of the growth inhibitory response to the TGFbetas are rb deletions, p53 mutations, and/or c-myc misregulation. These hypotheses will be tested through the following specific aims: (1) Continued studies on determining the role of pRB and p53 in the TGFbeta pathway for suppression of c-myc transcription and growth inhibition; characterizing a newly isolated cDNA clone encoding a protein that specifically binds the TCE; and characterizing the effect of p53 on c-myc transcription. (2) Transfection studies to explore the effect of c-myc, pRB and p53 on TGFbeta1 growth inhibition and the effect of TGFbeta1 over-expression in selected carcinoma cell lines. (3) Transgenic mouse studies on development and susceptibility to tumor formation resulting from misregulation of TGFbeta1S223,225, including crossbreeding of homozygous MMTV-TGFalpha and MMTV-TGFbeta1S223,225 lines to determine effect of transgene coexpression on mammary tumor formation; examining the effect of expression of TGFalpha and TGFbeta1S223,225 under the control of the whey acidic protein (WAP) promoter; crossbreeding MMTV-myc and MMTV- TGFbeta1S223,225 or WAP-myc and WAP-TGFbeta1S223,225 transgenic mice to determine effects on breast tumor formation; and experiments with cytokeratin and metallothionein promoters to determine the effects of misregulating TGFbeta1S223,255 on different tissues.

The products of protooncogenes are now widely recognized to play pivotal roles in growth stimulatory pathways and in differentiation. The products of the tumor suppressor genes are now known to exert negative effects on cell proliferation and evidence exists for involvement of these gene products in the pathway for diffusible negative factors. There is overwhelming evidence that changes in the dominant oncogenes and recessive tumor suppressor genes have great importance in the genesis of common human cancers. Major recent advances in growth control include (i) cloning of two new tumor suppressor genes, the Wilm's tumor gene and the type I neurofibromatosis gene; (ii) the characterization of the kit ligand, a novel hematopoietic growth factor, (iii) the description of a new family of hematopoietic receptors and their oncogenic activation; (iv) the description of ras-related gene products, including one encoded by a tumor suppressor gene, that suppress stimulatory signals; (v) the identification tyrosine kinase substrates that function as second messengers; (vi) examples of linkage between cell surface receptors with no previously known signal transduction capability and cytoplasmic tyrosine kinases; (vii) the demonstration that nuclear protooncogene products directly regulate gene transcription and that other known transcriptional regulators can contribute to neoplastic transformation; (viii) definition of roles for cell cycle kinases in growth regulation; (ix) implication of the retinoblastoma tumor susceptibility gene product in the pathway for transcriptional suppression of the protooncogene, c-myc; and (x) demonstration that p53 functions to suppress cell proliferation and that mutations and deletions of p53 gene are common occurrences in human and experimental malignancies. The concurrent meetings on "Positive Growth Control" and "Negative Growth Control" will address these and other major discoveries and approach important cur-rent problems including the interplays between positive and negative growth regulatory pathways. There will be three joint plenary sessions involving participants of both meetings and covering topics of common interest including the cell cycle, ras-related proteins and regulation of transcription. There will be an additional eight plenary sessions in each of the two meetings covering a wide range of topics pertinent to growth regulation and cancer development and progression. Poster sessions will cover topics similar to those addressed in plenary sessions. Meetings on the important and rapidly moving fields of positive and negative growth control are appropriate and timely. Having concurrent meetings on these topics should provide significant synergy and beneficial interactions that otherwise could not be achieved.

The overall goal of this Program Project is to identify the mechanisms that alter normal growth control to bring about neoplastic transformation. Cell proliferation is normally regulated by coordinated mechanisms that stimulate or inhibit growth and modulate differentiation and development. The overall focus of this program is the investigation of the basic control mechanisms through which growth inhibitory proteins and inducers of differentiation modulate cell proliferation and development. Alterations in these controls may lead to cancer. The program is composed of 3 projects that involve the use of biochemical, cell and molecular technologies and transgenic mouse models. Project 2 will use transgenic mice to investigate the role of TGFBeta in the regulation of mammary development, and will test the hypothesis that loss of TGFBeta inhibition and over production of TGFalpha promote development of mammary carcinoma. The mechanism by which TGFbeta inhibits proliferation will also be studied. Project 3 will use molecular biological techniques to determine the function of the three c-Myc proteins in the regulation of growth and differentiation. The capacity of Myc proteins to enhance transcription will be examined, and transgenic mice used to determine effects of the Myc proteins on the induction and suppression of breast tumors. Project 4 will investigate the role of DVR-6 in the growth and differentiation of specific cells and tissues in the mouse embryo. Misexpression of DVR-6 in transgenic mice and generation of DVR-6 null mutants will be used as complementary approaches to elucidate the function of DVR-6 in development. Thy synthesis, secretion and post-transnational modification of DVR-6 by a variety of cells will also be studied. The 3 research projects will be supported by two cores. This program brings together six investigators in 3 projects that integrate the study of cell proliferation with differentiation and development. The proposed studies focus on cell and animal models to characterize the process that alter normal growth control and lead to cancer.

OVERALL DESCRIPTION (Applicant's Description) The Vanderbilt Cancer Center is a comprehensive, multidisciplinary clinical and laboratory research center. The Vanderbilt Cancer Center integrates the cancer-related expertise and resources of the School of Medicine, Vanderbilt University Hospital, and the Vanderbilt Clinic within the Vanderbilt Univer- sity Medical Center; the fully integrated Veterans Administration (VA) Medical Center and the Departments of Chemistry and Molecular Biology in the School of Arts and Sciences. All facilities are located in close proximity on the same campus, a situation which promotes interactions, sharing of resources and collaborations. Established in 1993, the Vanderbilt Cancer Center functions as an organizational unit with a supradepartmental status. The Vanderbilt Cancer Center's specific authorities and responsibilities are: (1) To coordinate and integrate the cancer and cancer-related activities of Vanderbilt University. (2) To conduct, support and enhance cancer research and to integrate cancer-related research throughout the University. (3) To integrate, develop, and conduct cancer education programs. (4) To coordinate and integrate the care of cancer patients at Vanderbilt University Medical Center and the Veterans Administration Medical Center. The research objectives are accomplished through eight research programs and nine shared resources: Programs: (1) Signal Transduction, (2) Regulation of Cell Proliferation, (3) Gastrointestinal Cancer, (4) Cancer Etiology, (5) Cancer Genetics, (6) Breast Cancer, (7) Cancer Pharmacology, (8) Clinical Investigations; Shared Resources: (1) Biostatistics/Data Management, (2) Bioanalytical, (3) Cell Imaging, (4) DNA Sequencing, (5) Genetics, (6) Peptide Sequencing and Amino Acid Analysis, (7) Transgenic Mouse/ES Cell, (8) Human Tissue Acquisition and Pathology, and (9) Viral Vector. Integration of all cancer-related activities under the authority of the Vanderbilt Cancer Center provides an effective administrative structure to promote novel interactive approaches to cancer research, and partial support for this research infrastructure is requested in this application for a Cancer Center Support Grant.

This is the first competing continuation application for the Vanderbilt Cancer Center (VCC) CCSG. The VCC is a matrix center within Vanderbilt University Medical Center (VUMC), and the VCC integrates the cancer-related expertise and resources of the School of Medicine, Vanderbilt University Hospital, and The Vanderbilt Clinic within VUMC; the fully integrated Veterans Administration Medical Center (VAMC); and the Departments of Chemistry and Molecular Biology in the School of Arts and Sciences. All facilities are located in close proximity on the same campus, a situation that promotes interactions, sharing of resources, and collaborations. Established in 1993, the VCC functions as an organizational unit with a supradepartmental status. The VCC s specific authorities and responsibilities are: 1) to coordinate and integrate the cancer and cancer-related activities of Vanderbilt University; 2) to conduct, support and enhance cancer research and to integrate cancer-related activities throughout the University; 3) to integrate, develop and conduct cancer education programs; and 4) to coordinate and integrate the care of cancer patients at VUMC and VAMC. The research objectives are accomplished through seven research programs: Signal Transduction and Cell Proliferation, Cancer Genetics and Genomics, Host Tumor Interactions, Gastrointestinal Cancer, Breast Cancer, Cancer Prevention, and Experimental Therapeutics. Because of new directions and opportunities, the research programs have been reorganized and refined. Significantly, a Cancer Prevention Research Program has been developed with the anticipation of obtaining comprehensive status. Following the reorganization of the research programs, the entire membership of the VCC was evaluated by a stringent set of criteria, and members were assigned to research programs by the individual program leaders or their membership terminated. Eleven shared resources are proposed representing reorganization of clinical research-related shared resources, establishment of two new ones and termination of one. Since the submission of our initial CCSG application in 1994, the VCC has participated in the recruitment of 29 faculty members to enhance programs or shared resources. Start-up funding for 13 of these faculty was provided by the VCC; the remaining 16 were supported initially by institutional or departmental funds with the VCC identifying or helping to identify the need and playing a major role in the recruitment process.

DESCRIPTION: (Adapted from the investigator's abstract) There is compelling evidence derived from studies demonstrating inactivating mechanisms in human tumors and genetically modified mice indicating that the TGF-b signal transduction pathway is tumor suppressive. There is also compelling evidence that TGF-b can enhance the progression of mammary tumors. Carcinoma cells frequently lose the growth inhibitory response to TGF-b accompanied by an increased production of TGF-b. The locally high levels of TGF-b can then have effects on host cells (immunosuppressive and angiogenesis) or tumor cells (increased plasticity and motility) that would favor progression to a more malignant phenotype. Recent advances in the understanding of TGF-b signal transduction have provided some insights into how these diverse effects of TGF- b may occur. Current evidence indicates that the SMAD pathway is necessary for growth inhibition while the Rho proteins and the stress-activated kinase (JNK, p38) pathways are likely operative both in transcriptional regulation and cell shape changes. Based on this background, the applicant has formulated the following interrelated hypotheses: 1. Endogenous TGF-b's acting through the TGF-b receptors function to suppress both mammary epithelial proliferation and tumor formation. 2. Partial loss of type II TGF-b receptor (TbRII)) function in mammary epithelial cells will lead to loss of TGF-b growth inhibition and increased mammary tumor formation and synergize with oncogene activation to give highly aggressive tumors due in part to continued TGF-b autocrine stimulation of fibroblastoid morphology and increased mobility changes likely mediated through Rho proteins and stress activated kinase pathways. 3. Complete loss of TbRII in mammary epithelial cells will lead to increased mammary tumors but these tumors will be less aggressive due in part to absence of autocrine TGF-b action on tumor cells. The following specific aims are proposed to test these hypotheses: 1) Characterize spontaneous mammary tumor formation in mice expressing both MMTV-DNIIR (dominant negative type II receptor construct) and MMTV-TGF or MMTV-c-neu transgenes providing a model of partial loss of TbRII function. 2) Characterize the developmental phenotype of the mammary gland and mammary tumor formation in association with conditional knock-out of TbRII in mammary epithelial cells by cross breeding floxed TbRII mice (Tgfbr2floxE2) with MMTV-Cre mice. This will provide a model of complete loss of TbRII. 3) Determine the effect of decreased functional TbRII levels and different TGF-b signaling pathways on growth inhibition and conversion to a fibroblastoid morphology in NMUMG cells and cultures of carcinoma cells from aims 1 and 2.

It is estimated to affect between one million and one and a quarter million people worldwide and over 350,000 persons in North America. Disease susceptibility appears to be influenced by genetic determinants, location of residence through adolescence, and possibly age of exposure to certain infectious agents. Recent work in the MS laboratory at Vanderbilt University has found an association between CNS infection with Chlamydia pneumoniae and MS. The objectives of this application are to examine the role antibiotic therapy has on C. pneumoniae infection in the cerebrospinal fluid (CSF) of MS patients. We hypothesize that appropriate antibiotic therapy will markedly reduce or eradicate C. pneumoniae infection in the CSF of MS patients and that the clinical course of their disease will improve following treatment. A randomized clinical trial to examine the efficacy of two different antibiotic regimens versus placebo will be done over a six-month period of time in C. pneumoniae positive MS patients. Efficacy will be determined by eradication of C. pneumoniae in a culture system and a 90% reduction of DNA products using a quantitative polymerase chain reaction (PCR) assay. After an effective anti-chlamydial regimen has been found, a second randomized clinical trial will examine clinical outcome measures in a cohort of secondary progressive multiple sclerosis (SPMS) patients. Half of these patients will receive antibiotics and interferon-?1b and the other half interferon-?1b alone. These patients will be followed for two years and the primary outcome measure will be measures of sustained progression or disability. These studies will determine both the optimal antibiotic treatment of CNS C. pneumoniae infection in MS patients and if antibiotic therapy favorable alters the natural history of MS.

It is well known that minority groups suffer disproportionately from cancers with disparities existing in both the mortality and incidence rates. Other well-recognized problems are the paucity of minority scientists and physicians working in cancer research and the disproportionate number of minorities being entered into clinical trials. Meharry Medical College (MMC) has a distinguished history of training a large percentage of the African American physicians currently active in the United States. While MMC has long been active in cancer research, the promise of these activities has not yet reached fruition. Vanderbilt- Ingram Cancer Center (VICC) is a research intensive NCI-designated cancer center with very strong basic, translational, clinical, prevention and population-based research programs. MMC and VICC are located approximately two miles apart within Nashville. There has been a long history of faculty interactions between the two institutions, and the more recent establishment of the Meharry-Vanderbilt Alliance has provided the infrastructure for the rapid development of a MSI/CC partnership with support from the CCSG supplement. The overall objectives of this U54 application are to strengthen and expand the existing partnership for cancer research between MMC and the VICC to achieve three ends: (1) to expand the existing partnership for cancer research between MMC and the VICC to achieve three ends: (1) to increase and stabilize the competitive cancer research capability of MMC; (2) to create, stable, long term collaborative relationships between MMC and VICC in conducting activities focused on the disproportionate incidence, mortality and morbidity in minority populations in the mid-south. The programs we have designed to achieve these ends are guided by modest cores for administration at each institution and for planning and evaluation. Research activities for the project are contained in a development core that consists of components in basic science, cancer prevention, outcomes and outreach, and clinical investigations. We propose funding of six pilot projects and four shared resources. Two basic scientists, one medical oncologist, one surgical oncologists and two epidemiologists, will be recruited to join the MMC faculty with joint appointments at Vanderbilt under the auspices of this grant. We propose career development of MMC faculty through formal training programs and training of a minority medical oncologist, minority research nurses and data managers, minority Ph.D. candidates, and MMC medical students in basic and clinical cancer research.

Project Summary - Abstract Emerging data indicate that TGF-ss signaling in both epithelial cells and fibroblasts, in addition to controlling cell proliferation, also regulates the microenvironment by modulating the expression of growth factors, chemokines and cytokines. Loss of TGF-ss signaling in both mammary epithelial cells and stromal fibroblasts can result in increased production of chemokines that recruit bone marrow-derived cells (BMDCs), which then influence the initiation and progression of carcinomas. By conditional knockout of the type II TGFss receptor gene (Tgfbr2) in a subset of tissue fibroblasts using FSP1-Cre (Tgfbr2FSPKO), we have shown that loss of TGF-ss signaling enhances the initiation and progression of carcinomas in adjacent epithelia. Because of the short life span of the homozygous Tgfbr2FSPKO animals, we have employed recombination of Tgfbr2FSPKO and control mammary fibroblasts with mouse mammary carcinoma cells that are transplanted beneath the renal capsule of nude mice. The results of these studies show that Tgfbr2FSPKO fibroblasts enhance carcinoma growth, invasion and metastasis. Mechanisms identified so far include increased secretion of HGF, MSP and TGF-¿ by Tgfbr2FSPKO fibroblasts relative to control fibroblasts with increased activating phosphorylation of cognate receptors on adjacent carcinoma cells. Inhibition of c-Met attenuates the effects of the Tgfbr2FSPKO fibroblasts. Further, the Tgfbr2FSPKO fibroblasts secrete increased levels of several chemokines with associated increased infiltration of BMDCs that are known to promote carcinoma progression. Based on these observations and other data, we hypothesize that TGF-ss signaling in a subset of mammary gland fibroblasts is tumor suppressive for adjacent epithelia by regulating expression of growth factors, cytokines and chemokines. As a corollary, altered TGF-ss signaling in these stromal cells enhances mammary carcinoma initiation and progression by regulating secretion of both selected growth factors (i.e., HGF, MSP, TGF-¿) that act in a paracrine manner on the epithelial cells and chemokines that recruit BMDCs to the tumor site. The BMDCs in turn express high levels of TGF-ss and MMPs that further promote tumor progression. These hypotheses can only be tested in animal models, and we will examine them through the following specific aims. Specific aim 1. Determine the effect of TGF-ss signaling in different subsets of mammary fibroblasts on mammary gland development and oncogene-induced mammary carcinomas. Specific aim 2. Determine mechanisms and effects of recruitment of BMDC caused by Tgfbr2 knockout in different subsets of fibroblasts on mammary carcinoma initiation and progression. Specific aim 3. Determine whether altered mammary fibroblast TGF-ss signaling contributes to progression of human breast cancer.

The objective of the Transgenic Mouse/ES Cell Shared Resource to investigators production is assist in the and use of transgenic and gene knockout mice. Through an interdisciplinary approach, the Resource has been able to provide a broader array of services than are available elsewhere to a large base of investigators. The Shared Resource focuses on quality control, the increased potential for collegial interactions, and immediate access to individuals with deep knowledge of transgenic mice. Experienced personnel develop new procedures and applications, such as those that facilitate the use of site-specific recombinases to perform tissue-specific gene knockouts. As the technology for genome manipulation continues to expand, the Transgenic Mice/ES Cell Shared Resource is likely to grow with it. Specifically, the Shared Resource offers pronuclear DNA microinjection; ES cell microinjection into blastocysts; assisted reproduction; gene targeting; and conditional knockouts utilizing the Cre or FIp(e) site-specific recombinase system; embryo cryopreservation and long-term storage; and sperm cryopreservation and storage. Dr. Cathleen Pettepher is the Director of the Transgenic Mouse/ES Cell Shared Resource and offers consultation to investigators, reviews and approves requests for gene targeting experiments and DNA or ES cell microinjections, and tracks the efficiency and quality of the services offered. She has a highly-trained staff that helps her serve VICC researchers.

DESCRIPTION (provided by applicant): The importance of stromal-epithelial interactions in mammary gland development and tumorigenesis is well established. These interactions likely involve autocrine and paracrine action of multiple growth factors, including members of the TGF-beta family, which are expressed in both stroma and epithelium. We have previously demonstrated that expression of a dominant-negative type II TGF-beta receptor construct (DNIIR) in mammary stroma using a metallothionein (MT) promoter results in increased lateral branching. Current evidence indicates that DNIIR causes only partial blockage of TGF-beta signaling. To accomplish complete knock-out of the type II TGF-beta receptor gene (Tgfbr2) in mammary stromal cells we have crossed FSP1-Cre mice with the Tgfbr2(floxE2/floxE2) mice. FSP1 (fibroblast-specific protein-I) is expressed in stromal fibroblasts. As shown under Preliminary Studies, these mice have a phenotype different from that observed with partial abrogation of TGF-beta signaling in stromal cells in the MT-DNIIR mice. With complete knock-out of Tgfbr2 in stromal cells there was loss of mammary fat cells with a markedly hypercellular stroma and decreased ductal branching with hypoplastic epithelial cells. Animals with FSP1-Cre and heterozygous floxed Tgfbr2 [Tgfbr2(floxE2/wt)] making them haploinsufficient for Tgfbr2 in stromal cells have normal mammary gland development, but defective regression following lactation. Thus, we have a unique model for studying the role of TGF-beta signaling in stromal cells on mammary gland development and tumorigenesis. Based on this information, we have formulated the following hypotheses: (1) TGF-beta signaling in stroma is essential for normal development of the mammary gland and plays a role in the invasive and metastatic potential of carcinoma cells. (2) Stromal haploinsufficiency of Tgfbr2 leads to incomplete regression following lactation and alters the invasion and metastatic capabilities of mammary tumors induced by coexpressed oncogenes. The Specific Aims are proposed to test these hypotheses are: 1. Characterization of Cre expression pattern, recombination and phenotype of FSP1-Cre:Tgfbr2(floxE2/floxE2) mice. 2. Characterization of Cre expression pattern, recombination and phenotype of mice haploinsufficient for Tgfbr2 in stroma, FSP1-Cre:Tgfbr2(floxE2/wt) mice. 3. Characterization of Tgfbr2 null stromal cells and determination of their effect on invasion and metastasis of carcinoma cells.

human therapy evaluation; multiple sclerosis; Chlamydiaceae; antibiotics; cerebrospinal fluid; central nervous system; patient oriented research; clinical research; human subject;

DESCRIPTION (provided by applicant): Support is requested to partially underwrite the costs of a conference entitled "Overcoming Colorectal Cancer Disparities" to be held November 2nd to 4th 2005 at the conference center on Peabody campus of Vanderbilt University. The general purpose of this conference is to assemble the top researchers in this field along with promising young investigators, trainees and more senior faculty, in order to exchange and disseminate information on current research aimed at addressing colorectal cancer disparities in the United States. Attention will be focused on senior faculty members who may be willing to adapt their current research to include questions which may help address and overcome cancer disparities in their areas of research expertise. One workshop entitled "Career Development" will provide practical advice for career development, specifically on the topic of creating and sustaining a research program. This workshop will be designed to cover the needs of researchers involved in basic, clinical and/or control research and include information pertinent for trainees and Jr. faculty from underserved populations. The conference will be publicized in scientific journals, on the Internet, and announcements will be mailed to members of neuroscience societies, as well as to individuals known to be active in the field. Electronic publicity will be accomplished by Meharry and Vanderbilt-linked conference web site with details of the program and on-line registration, and by listing it on established web sites providing meeting announcements. A concerted effort will be made to attract junior investigators and those considered underrepresented in science fields, such as women and ethnic minorities. A specific number of travel awards will be provided to aid in this effort.

Strong ties have developed between cancer researchers at Meharry Medical College (MMC) and the[unreadable] Vanderbilt-lngram Comprehensive Cancer Center (VICC) over the past five years of their formal partnership.[unreadable] The bedrock of this pioneering collaboration has been the U54 from the NCI. Our partnership is dedicated to[unreadable] enhancing the strengths and eliminating the weaknesses of our two institutions as we move toward our[unreadable] shared goal of eliminating cancer disparities in the US. The U54 award has moved our collaborative[unreadable] research program more forcefully toward a relationship that is based on reciprocity and stronger mutual[unreadable] benefit. Until recently, MMC viewed itself primarily as a teaching institution and, therefore, did not have a[unreadable] critical mass of researchers in cancer. MMC, with pivotal help from U54 funds, is growing its capacity but[unreadable] has a thriving concentration of cancer researchers in cancer biology, cancer epidemiology and clinical[unreadable] cancer research. The U54 has provided the major impetus for a bold restructuring of the basic science[unreadable] departments at MMC that will assure release time for MMC investigators. The Division of Cancer Biology,[unreadable] headed by Dr. Adunyah, Co-Pi of the U54 at MMC will consist of faculty with 80% release time for research.[unreadable] In cooperation with the U54 Pis, mentoring committees consisting of faculty from MMC and VICC will assist[unreadable] faculty in developing competitive research programs. This restructure was due, in part, to recommendations[unreadable] made by our Program Steering Committee at their 2003 and 2004 meetings. Funds provided by this U54 will[unreadable] solidify our collaboration. During this second phase of our partnership, we concentrate on amassing[unreadable] sufficient infrastructure for cancer research that will strengthen reciprocity between MMC and VICC. In[unreadable] addition to funding research studies, funds will be used to recruit investigators and strengthen infrastructure[unreadable] in basic, epidemiologic and clinical cancer research at MMC. A critical mass of researchers in all these[unreadable] areas will strengthen the quality of MMC/VICC collaborative efforts. A sustained and comprehensive Cancer[unreadable] Partnership between MMC and VICC is of immense benefit to the two participating institutions as well as the[unreadable] mid-South region of the US. Our major collaborative successes including the Southern Community Cohort[unreadable] Study of urban and rural African Americans and Caucasians in the mid-South testify to our potential for[unreadable] meeting our goal of eliminating cancer disparities between African Americans and Caucasians.[unreadable]

ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): It is now widely accepted that the TGF-beta signaling pathways can both suppress tumor formation and promote tumor progression and that both effects are mediated largely through tumor cell autonomous TGF-beta signaling. Data generated during this grant period with conditional knockout of TGF-beta signaling in epithelia support the hypothesis that epithelial cell autonomous TGF-beta signaling is tumor suppressive and demonstrate that metastases can not only occur but are enhanced with knockout of the type II TGF-beta receptor (Tgfbr2) in mammary carcinoma cells. However, systemic inhibition of TGF-beta signaling markedly suppressed pulmonary metastases in the MMTV-c-neu/DNIIR mice. This indicates that the effect of systemic inhibitors of TGF-beta in suppressing metastasis is largely on host cells. These results have modified our hypotheses concerning the mechanism of TGF-beta promotion of tumor progression. We now propose that tumor cell autonomous signaling by TGF-beta can suppress rather than enhancing metastases, and that TGF-beta signaling in host cells is a significant component of both the tumor suppressive and the tumor promotion effects of TGF-beta in vivo. We will test these hypotheses through the following specific aims by determining the changes in the carcinoma cells and their microenvironment associated with knockout of the type II TGF-beta receptor gene, Tgfbr2, in tumor cells that lead to increased metastases. Specific Aim 1. Determine the effects of systemic inhibition of TGF-beta signaling on mammary tumor formation and metastases from MMTV-c-neu and MMTV-PyVmT-induced mammary tumors in the context Tgfbr2 knockout in mammary epithelial cells effected by both MMTV-Cre and WAP-Cre. Specific Aim 2. Determine the influence of timing of loss of TGF-beta signaling during mammary tumor formation and progression on metastatic spread using inducible MMTV-Cre. Specific Aim 3. Determine mechanisms for enhanced metastatic capability with loss of tumor cell TGF-beta signaling by examining both tumor cells and their microenvironment.

DESCRIPTION (provided by applicant): This U01 proposal is designed to be part of the network for the study of tumor microenvironment (TMEN) and seeks to improve our understanding of the critical steps involved the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) to a level that will permit the rational development of effective stromal specific therapeutic agents. Pancreatic cancer is the fourth leading cause of cancer death in the US. In 2004, 31,270 deaths were recorded. This disease is devastating and currently specific therapies are lacking and it leads to rapid death of all patients, coupled with pain and despair. The economic toll of pancreatic cancer is estimated at about $3.7 billion dollars to the US healthcare system. The central focus of this project is to elucidate the molecular mechanisms by which fibroblasts/mesenchymal cells, myeloid/immune cells (MIC) and extracellular matrix (ECM) may contribute to the origin and progression of PDAC. While, invasive PDAC is significantly associated with a marked desmoplastic reaction (one of highest of all human tumors), significant recruitment of fibroblasts/myofibroblasts and ensuing fibrosis, the functional contribution of stromal fibroblasts in PDAC pathogenesis is not known. This application tests the central hypothesis that "stromal fibroblasts are rate limiting determinants of PDAC progression". The new mouse models described in this project will provide a basic knowledge regarding the role of stromal fibroblasts, immune cells and ECM biochemistry in PDAC and explore potential PDAC microenvironment specific therapeutic opportunities. The proposal brings together the laboratories of Drs. Kalluri, Moses and Weaver, with complementary expertise to the study PDAC microenvironment. The proposal is divided into three specific aims. Specific Aim 1: Determine the functional contribution of stromal fibroblasts in the pathogenesis of PDAC. Specific aim 2. Determine the role of chemokines and host myeloid/immune cells (MICs) in the pathogenesis of PDAC. Specific Aim 3. Determine whether increased tissue tension drives pancreatic adenocarcinoma aggression.

Strong ties have developed between cancer researchers at Meharry Medical College (MMC), Tennessee State University (TSU) and Vanderbilt-lngram Cancer Center (VICC) resulting in this formal Cancer Partnership. Our Partnership is dedicated to enhancing the strengths and eliminating the weaknesses of our three institutions as we move toward our shared goal of eliminating cancer disparities in the US. The U54 award is the bedrock of this collaboration and has moved our collaborative research more forcefully toward a relationship that is based on reciprocity and stronger mutual benefit. The overall objectives of this competing renewal application are: (1) to increase and stabilize the competitive cancer research capability of MMC and TSU;(2) to create stable, long-term collaborative relationships between MMC, TSU and the VICC in cancer research, research training, career development and cancer outreach;and (3) to improve the effectiveness of VICC research, training, career development, cancer education and cancer outreach activities specifically designed to benefit minority populations in the region served by VICC. Funds provided by this U54 will solidify our collaboration. During this phase of our Cancer Partnership, we continue to concentrate on amassing sufficient infrastructure for cancer research that will strengthen reciprocity between MMC, TSU and VICC. In addition to funding two full projects and two pilot studies involving collaborators from all three institutions, funds will be used to recruit investigators to TSU and VICC and to strengthen infrastructure in basic, epidemiologic and clinical cancer research through the support of core facilities providing expertise in biostatistics, clinical trial accruals, histopathology and cancer outreach. All activities involve coordinated, well-planned interactions between MMC, TSU and VICC and will be monitored by an Internal Advisory Committee composed of members from each institution and a Program Steering Committee composed of external, nationally-recognized cancer investigators and representation from the National Cancer Institute. A sustained and comprehensive Cancer Partnership is of immense benefit to the three participating institutions as well as the mid-South region of the US. The institution's complementary strengths will help the Cancer Partnership establish effective cancer research and increase training opportunities at two minority-serving institutions enhance community- and population-based science research targeting minority cancer-related disparities and increase training of minority scientists at an NCI-funded Comprehensive Cancer Center.

This application is for a cooperative agreement to continue the Cancer Partnership as a triad with participation from Vanderbilt-lngram Cancer Center (VICC), Meharry Medical College (MMC) and Tennessee State University (TSU). Program administrators of the National Cancer Institute (NCI) of the National Institutes of Health (NIH) will be part of the Program Steering Committee for this Program and will be consulted and kept apprised of progress. As such. Dr. Nelson Aguila, Diversity Training Branch in the Center to Reduce Cancer Health Disparities at the NCI, has provided the requisite approval for the submission of this triad application. A copy of this approval letter is included in Section 14 Letters of Support. The Partnership was developed in 1999 and continues with the goal of strengthening cancer research capabilities of MMC and TSU as well as providing exposure and resources for faculty at VICC focusing on cancer disparities research, mentoring junior faculty and training the next generation of minority students and postdoctoral fellows. The administrative leadership teams of the three institutions are jointly responsible for the overall management, coordination and support of the components in the Partnership. We have a longstanding and close working relationship with the administrative teams of our Partner organizations, resulting in numerous supportive interactions. The internal activities of the Partnership will be accomplished by the respective Administrative Cores in close collaboration with the Internal Advisory Committee and Program Steering Committees of the Partnership. Figure 1 represents the organizational structure of the parties involved in the Partnership. The figure also depicts direct lines of communication between the Administrative Cores based at the VICC, MMC and TSU and the other participating components.

DESCRIPTION (provided by applicant): Obesity has been associated with increased incidence of malignancies including colon cancer. As the epidemics of obesity and associated diabetes and metabolic syndrome continue to grow, it is clinically relevant to understand the influence that a steatotic (fatty) liver microenvironment has on the growth of tumors; both metastatic and primary. Preliminary studies show an increased burden of metastatic disease in a mouse model of diet-induced non-alcoholic fatty liver disease (NAFLD). Microarray analysis indicates that the matrix metalloproteinases MMP12 and MMP13 are markedly elevated in the steatotic microenvironment, leading to the hypothesis that tumor cells react to an enhanced host tissue MMP response in the microenvironment of hepatite steatosis and steatohepatitis, which in turn plays a role in dictating the biological behavior of the tumor at its site of metastasis. To test this hypothesis, the specific aims of this proposal utilize mouse models of NAFLD as well as experimental liver metastasis models. In vivo techniques of bone marrow and liver transplantation are proposed to examine cellular mechanisms of action of these MMPs. These are coupled with sophisticated multiphoton microscopy techniques that allow visualization of early metastases in intact mouse livers and analysis of interactions of these tumor cells with the liver microenvironment. Finally, this proposal seeks to determine the molecular mechanisms by which MMP12 and/or MMP13 influences metastatic efficiency in the steatotic liver microenvironment. We hypothesize that MMP-mediated proteolysis of cytokines, chemokines, and/or adipokines establishes microenvironmental cues that influence the survival and establishment of metastatic cancer cells in the steatotic liver. Findings in a mouse model will be corroborated with human liver tissue samples to assure relevance to human disease. The long term objective of these studies is to provide information on the molecular events that contribute to the establishment of colon carcinoma metastasis in the fatty liver that will then lead to more refined targeted therapeutic strategies for the treatment of metastatic colorectal cancer. As a greater percentage of the population lives with NAFLDi the impact of this microenvironment on the growth of metastatic tumors must be better understood. The combination of selective inhibition of stromal and epithelial derived MMPs may become part of rational strategies to treat metastatic colorectal cancer. RELEVANCE (See Instructions): 7 The rising incidence of non alcoholic fatty liver disease (NAFLD) associated with obesity is a problem of increasing clinical significance. In addition, obesity is clinically associated with an increased prevalence of certain malignancies including colon cancer and breast cancer. This proposal seeks to probe the molecular mechanisms by which microenvironmental liver steatosis and steatohepatitis impacts metastasis of colorectal cancer cells to the liver.

The main goal of the MMC-VICC-TSU Cancer Partnership (MVTCP) training and education program is to increase the number and quality of minority researchers dedicated to making an impact in cancer research. Our aims are to (1) recruit outstanding minority undergraduate and graduate students and Oncology Fellows to cancer research, (2) provide these trainees with exceptional mentoring and training so that they are equipped to make a difference in the impact of cancer on the population, and (3) motivate them to seek further cancer research training which could lead to development of careers that include cancer research focus. In order to accomplish this goal our partnership will recruit and train 6 Ph.D., 5 MSPH and 5 MPH students in our cancer research programs at MMC and TSU. The Partnership will also support 6 Clinical Oncology Fellows in training in the Vanderbilt Oncology Fellowship Program. Table 1 indicates the proposed number of Ph.D., MSPH/MPH and Clinical Oncology Trainees to be supported by the grant in each year.

Strong ties have developed between cancer researchers at Meharry Medical College (MMC), Tennessee State University (TSU) and Vanderbilt-lngram Cancer Center (VICC) resulting in this formal Cancer Partnership. Our Partnership is dedicated to enhancing the strengths and eliminating the weaknesses of our three institutions as we move toward our shared goal of eliminating cancer disparities in the US. The U54 award is the bedrock of this collaboration and has moved our collaborative research more forcefully toward a relationship that is based on reciprocity and stronger mutual benefit. The overall objectives of this competing renewal application are: (1) to increase and stabilize the competitive cancer research capability of MMC and TSU; (2) to create stable, long-term collaborative relationships between MMC, TSU and the VICC in cancer research, research training, career development and cancer outreach; and (3) to improve the effectiveness of VICC research, training, career development, cancer education and cancer outreach activities specifically designed to benefit minority populations in the region served by VICC. Funds provided by this U54 will solidify our collaboration. During this phase of our Cancer Partnership, we continue to concentrate on amassing sufficient infrastructure for cancer research that will strengthen reciprocity between MMC, TSU and VICC. In addition to funding two full projects and two pilot studies involving collaborators from all three institutions, funds will be used to recruit investigators to TSU and VICC and to strengthen infrastructure in basic, epidemiologic and clinical cancer research through the support of core facilities providing expertise in biostatistics, clinical trial accruals, histopathology and cancer outreach. All activities involve coordinated, well-planned interactions between MMC, TSU and VICC and will be monitored by an Internal Advisory Committee composed of members from each institution and a Program Steering Committee composed of external, nationally-recognized cancer investigators and representation from the National Cancer Institute. A sustained and comprehensive Cancer Partnership is of immense benefit to the three participating institutions as well as the mid-South region of the US. The institution's complementary strengths will help the Cancer Partnership establish effective cancer research and increase training opportunities at two minority-serving institutions enhance community- and population-based science research targeting minority cancer-related disparities and increase training of minority scientists at an NCI-funded Comprehensive Cancer Center.

In the next five years, the primary goal of our recruitment efforts will focus on enhancement of the biostatistical, behavioral and population-based research components of our Partnership. To that end, the first aim of our new faculty recruitment plan will be to recruit one senior biostatistician to MMC, one cancer behavioral research scientist to VICC and one population-based investigator to TSU. We strongly believe that recruitment of investigators with expertise in these identified areas of need will continue to strengthen our Cancer Partnership.

Strong ties have developed between cancer researchers at Meharry Medical College (MMC), Tennessee State University (TSU) and Vanderbilt-lngram Cancer Center (VICC) resulting in this formal Cancer Partnership. Our Partnership is dedicated to enhancing the strengths and eliminating the weaknesses of our three institutions as we move toward our shared goal of eliminating cancer disparities in the US. The U54 award is the bedrock of this collaboration and has moved our collaborative research more forcefully toward a relationship that is based on reciprocity and stronger mutual benefit. The overall objectives of this competing renewal application are: (1) to increase and stabilize the competitive cancer research capability of MMC and TSU; (2) to create stable, long-term collaborative relationships between MMC, TSU and the VICC in cancer research, research training, career development and cancer outreach; and (3) to improve the effectiveness of VICC research, training, career development, cancer education and cancer outreach activities specifically designed to benefit minority populations in the region served by VICC. Funds provided by this U54 will solidify our collaboration. During this phase of our Cancer Partnership, we continue to concentrate on amassing sufficient infrastructure for cancer research that will strengthen reciprocity between MMC, TSU and VICC. In addition to funding two full projects and two pilot studies involving collaborators from all three institutions, funds will be used to recruit investigators to TSU and VICC and to strengthen infrastructure in basic, epidemiologic and clinical cancer research through the support of core facilities providing expertise in biostatistics, clinical trial accruals, histopathology and cancer outreach. All activities involve coordinated, well-planned interactions between MMC, TSU and VICC and will be monitored by an Internal Advisory Committee composed of members from each institution and a Program Steering Committee composed of external, nationally-recognized cancer investigators and representation from the National Cancer Institute. A sustained and comprehensive Cancer Partnership is of immense benefit to the three participating institutions as well as the mid-South region of the US. The institution's complementary strengths will help the Cancer Partnership establish effective cancer research and increase training opportunities at two minority-serving institutions, enhance community- and population-based science research targeting minority cancer-related disparities and increase training of minority scientists at an NCI-funded Comprehensive Cancer Center.

Summary In this competing renewal application we propose to continue the support of our mature Cancer Partnership as a triad with participation from Meharry Medical College (MMC), Vanderbilt-Ingram Cancer Center (VICC), and Tennessee State University (TSU). Our triad represents a balanced focus on population science, basic, and clinical research. The administrative leadership teams of the three institutions are jointly responsible for the overall management, coordination, and support of the components in the Partnership. We have a long-standing and collaborative working relationship with the administrative teams of our Partner organizations, resulting in numerous supportive interactions and enhanced productivity. The outstanding and experienced leadership for the Cancer Partnership is comprised of: (1) Drs. Samuel Adunyah (contact PI) and Phillip Lammers as Co- Principal Investigators for MMC; (2) Drs. Harold Moses (contact PI) and Ann Richmond as Co-Principal Investigators for the VICC; and (3) Drs. Baqar Husaini (contact PI) and Margaret Whalen as Co-Principal Investigators for TSU. The Principal Investigators are strong in their conviction that the current U54 grant has been a catalyst for a monumental shift in research culture at the partnering institutions. They also share the belief that the Partnership is at a critical point in its maturation and have worked together to craft this application such that it fulfills its mission. The Administrative Core provides day-to-day administrative support for the Partnership and coordinates and compiles both documents and data from all participants in the Partnership for requisite submissions to the funding agency, such as proposals, progress reports, and periodic requests for special data from the agency. Importantly, the Core engages in event planning and production activities for the Partnership: 1) Executive Committee, 2) Internal Advisory Committee (IAC), 3) Monthly Seminar Series, 4) Mentoring Advisory Committee, 5) Scientific Review Committee, 6) Annual Retreat, 7) Recruited Faculty Feedback Session, 8) Education Oversight Committee, 9) Community Advisory Board. In summary, the administrative core is committed to providing the necessary services to support smooth working relationships with the Principal Investigators at VICC, MMC and TSU, institutional senior leaders, and other key personnel, as well as with the National Cancer Institute. Administrative core personnel will accomplish this through administrative operations that enable streamlined and effective assistance to the Partnership.

SUMMARY Particular emphasis is devoted in the US to identify the determinants of, and eventually remedies, for the disproportionate share of the cancer burden borne by underrepresented minorities. Incidence rates for many forms of cancer are higher among blacks than whites. The disparity is compounded by lower rates of relative survival for almost all cancers, so that age-adjusted mortality rates are substantially higher among African Americans than whites for about two-thirds of all types of cancers. Our Cancer Partnership provides an exceptional environment to focus the efforts of diverse investigators working across many disciplines to address health disparities in NCI designated cancer centers and to develop research infrastructure, capacity and effectiveness of minority-serving institutions. Collectively, these efforts offer the best opportunity to overcome cancer disparities and optimize research resources and infrastructure. Our Partnership has matured and developed strong, successful collaborative interactions based on excellence, true reciprocity and mutual benefit to achieve this goal. Moreover, this Partnership is geographically placed in a region with the highest cancer incidence in the US, pointing to the enormous potential for improved outcomes through the proposed work. We are dedicated to enhancing the strengths and eliminating the weaknesses of our three institutions as we move toward our shared goal of eliminating cancer disparities. The overall objectives of this competing renewal application are to: (1) increase and stabilize the competitive cancer research capability of Meharry Medical College (MMC) and Tennessee State University (TSU); (2) create stable, long-term collaborative relationships between MMC, TSU and the Vanderbilt Ingram Cancer Center (VICC) in cancer research, research education, career development and cancer outreach; (3) expand access to clinical trials for minority populations served by Nashville General Hospital (NGH)/MMC, and (4) improve the effectiveness of VICC research, career development, education and outreach activities specifically designed to benefit minority populations served by VICC. All activities will be monitored by an Internal Advisory Committee and the Program Steering Committee with representation from the NCI. A sustained and comprehensive Cancer Partnership is of immense benefit to the three participating institutions as well as the mid-South region of the US. The common mission statement for our Cancer Partnership is to conduct rigorous basic, translational and clinical research directed towards the reduction of disparities in the incidence and treatment of cancer through a multidisciplinary collaborative approach to research, education and community engagement. As a triad we will amplify cancer research capabilities in the greater Nashville academic and healthcare community, improve and document outcomes in the diagnosis and treatment of cancer in minority and underserved communities, minimize or eliminate measurable disparities in the incidence of cancer among minority populations, and fortify collaborations among the three partner organizations.