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UNIVERSITY OF CALIFORNIA, SAN FRANCISCO To view the USCF Prostate SPORE activities, please visit http://cc.ucsf.edu/prostate_spore/index.html. Project 1 Project 1 is aimed at the ligand specific activity of the Androgen Receptor (AR), and the associated alterations in the regulatory influences that occur in prostate cancer. Prostate cancer is a very heterogeneous disease, which likely reflects the multiple mechanisms of its ability to evade hormonal control. AR is a member of the nuclear receptor family of proteins. Lessons from other nuclear receptors suggest that there are multiple layers of transcriptional regulation which are context dependent. These layers include ligand specificity and binding, and the presence of other transcription and coregulator proteins, that are recruited to specific genomic sites. As with the other nuclear receptors of this family, AR is able to positively and negatively regulate transcription, depending on the context of the DNA and the presence of specific factors. The upstream regulatory sequence of the promoter specifies which proteins are able to bind, but it is the composition of the regulatory complexes, that are recruited to that site, that defines the specificity of response. These variables enable AR to integrate multiple signals, and allow for multiple mechanisms by which prostate cancer can obtain growth independence over time. Therefore, we hypothesize that alterations in AR structure and ligand binding and/or the ability of AR to interact with cofactors is at the basis of progression of prostate cancer into hormone-independent disease. We also propose that the understanding of these alterations in transcriptional regulation may allow the identification of specific targets for therapy. To understand and identify target therapeutics at these mechanisms we propose three specific aims:
Aim 1: How does Androgen Receptor (AR) alter its ligand specificity during progression of hormone-refractory disease?
Project 2 Prostate cancer is the most common malignancy and second leading cause of cancer mortality among males in the Western world. Widespread screening for serum PSA has resulted in significant stage migration so that prostate tumors are increasingly identified at early stage and with low to intermediate risk of progressing to metastatic disease - for which there is no cure. One goal of this project is to identify genetic markers that will allow clinicians to predict tumor behavior and stratify patients at intermediate risk of progressing into groups that should go on standard or experimental therapies. Specifically, we will apply array CGH tissue microarray to discover and validate genetic markers that are present in >20% of tumors that identify patients at a relative risk of progressing > 1.5 with 95% confidence. To achieve this, we will screen 50 tumors from patients treated by radical prostatectomy alone that progressed biochemically within 2 years and 50 that did not progress within 4 years for recurrent copy-number abnormalities using megabase resolution array-CGH. Candidate markers will then be evaluated in 800 prostate tumors using tissue microarrays. A second goal of this project is to identify the genes in these regions of genomic abnormality that contribute to prostate cancer progression. To accomplish this, regions of recurrent copy-number abnormality strongly associated with tumor progression will be further narrowed by high-resolution array CGH. Comparative genomic DNA sequence analysis will then be employed to identify candidate tumor suppressor genes and oncogenes implicated in tumor progression. Candidate genes will be analyzed for expression level in 800 tumors using transfection, antisense treatment and/or antibody ablation.
Project 3 Adenovirus vectors are currently being developed as potential therapeutic agents for many types of cancer, including prostate cancer. Promising results have been observed in Phase I and II clinical trials in which they are delivered to tumors by direct injection. However, their clinical utility for prostate cancer therapy depends on developing systems for delivering virus effectively to tumor cells, especially those that have spread from the primary site. Currently, no good animal model exits to facilitate this development process. This is because human adenoviruses do not replicate in rodents, or other laboratory animals. Preclinical development has so far depended on models in which human tumor cells are grown in immune-incompetent mice, and tumors are infected with the human virus vector. This model is a poor predictor of efficacy, because it does not include possible effects of the immune system that almost certainly contribute to the clinical outcome. They also do not predict possible side effects of the virus on normal tissue, since mouse tissue does not support replication. Here we propose to develop a mouse model system for therapeutic adenoviruses that should help address many of the issues needed to optimize clinical testing of these agents. Mouse adenoviruses, MAV-1, is similar in many ways to human adenoviruses, using the same coxsackie and adenovirus receptor CAR-1 to enter cells and infect them. We will build a series of MAV-1 vectors and mutants that will allow us to optimize delivery of virus to primary and metastatic prostate tumors. We will test the possibility that the combination of oncolytic therapy and radiation treatment is more efficacious than either treatment alone. These results will be used to design clinical trials with human oncolytic viruses as single agents or in combination with existing radiation treatment protocols.
Project 4 A major goal of cancer research has been to identify antigens on the surface of tumor cells which are qualitatively or quantitatively different from antigens on normal cells. Such antigens can be targeted by monoclonal antibodies which can form the basis of therapeutic drugs or be used in diagnostic and prognostic tests. For this work, we propose to utilize a novel approach to develop antibody based molecules for prostate cancer therapy and diagnosis which overcomes the limitations of conventional hybridoma technology. Antibodies will be generated from human antibody diversity libraries using phage display technology. This will yield antibodies which are entirely human in sequence. Using recently developed methodology, libraries will be directly selected on prostate tumors leading to enrichment for antibodies which not only bind prostate cell surface receptors, but which bind in a manner that triggers receptor mediated endocytosis. Such antibodies are likely to be especially useful therapeutically, either by modulating receptor function leading to tumor growth inhibition or apotosis, or by delivering toxic molecules to tumor cells. A novel high throughput assay will be used to identify those monoclonal antibodies which bind and are internalized into prostate tumor cells but are not bound and internalized into normal cells. Antibody specificity for prostate cancer will be confirmed using immunocytochemistry and rodent and human tissue arrays in collaboration with the SPORE Tissue Core and Animal Technology Core. For antibodies with the appropriate pattern of reactivity, additional characterization will include: 1) identification of antigen bound using either immunoprecipitation and protein sequencing or cDNA library screening; and 2) in vitro and in vivo studies of antibody efficacy on tumor growth. After 5 years, we anticipate the identification of multiple lead candidate antibodies which can be developed for prostate cancer therapy or diagnosis. In addition, the tumor specific antigens recognized by the antibodies may prove useful as vaccine candidates or as targets for small molecule drug discovery programs.
Project 5 Genetic studies on cancer susceptibility in humans have shown that there is enormous variation between individuals in the probability of developing cancer in several target organs, including the prostate. The identification of the genes that confer susceptibility or resistance to prostate cancer would have important clinical implications that may lead to novel methods for the prediction of individual risk of tumor progression, as well as strategies for prevention or therapy. The control of prostate cancer development by genetic and environmental factors is extremely complex, and studies to date using human subjects have not led to the identification of the major molecular determinants of risk. Animal models offer significant advantages for the detection of gene-environment interactions because of our availability to control both the host genetic background and the degree of exposure to causative agents. The recent development of animal models for prostate cancer greatly facilitates the study of the complex genetics of prostate cancer susceptibility. These models also provide appropriate systems for the testing of new approaches to therapeutic intervention. This proposal will focus on identification of genetic modifiers of prostate cancer risk by exploiting strain-dependent variation in susceptibility to prostate cancer in TRAMP mice (Transgenic Adenocarcinoma of Mouse Prostate). Previous work has shown that expression of the SV40largeT and small t antigens driven by a probasin promoter induces cancer of the prostate in C57BL/6 mice. The latency of the disease is considerably shorter when the transgene is bred on to a FVB/n genetic background, indicating that genetic factors clearly affect tumor progression. We will exploit these strain-dependent differences to map the loci that control prostate cancer susceptibility using both backcross and intercross approaches. These studies will be greatly facilitated by previous mapping studies of skin tumor modifier loci using the same strain combinations. The relationship between tumor modifier loci and tumor suppressor genes or oncogenes that show somatic LOH or amplification during tumor development will be investigated using microsatellite based allelotypes of tumor DNA samples isolated from both intraspecific (C57/Bl X FVB/n F1) or interspecific (Mus musculus X Mus spretus F1) mouse tumors. We will utilize both genomic microarrays of mouse BACs to detect copy number changes in tumor genomes, together with cDNA microarrays carrying up to 25,000 mouse cDNAs to detect changes in gene expression patterns at different stages of mouse prostate cancer progression. These investigations will be carried out in parallel with collaborators analyzing somatic genetic alterations in human prostate cancers (project 2). In future studies, any candidate genes or loci that emerge from the mouse models will be tested in human prostate cancer patients and appropriate controls to determine whether they affect the probability of human prostate cancer development, progression or survival.
Project 6 Project 6 will develop and evaluate a succession of new immunological therapeutics for patients with prostate cancer based on CTLA-4 blockade. CTLA-4, a molecule which inhibits T cell response to antigens, is expressed on T cells and binds the B7 molecule found on antigen-presenting cells. In vivo blockade of CTLA-4 with a murine monoclonal anti-CTLA-4 antibody results in enhanced antitumor active immune responses in several murine tumor models. CTLA-4 blockade has significant anti-tumor effects in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model when used with a GM-CSF transfected cell vaccine, where the vaccine alone has no effect. The immune response elicited is at least in part directed against normal prostate antigen(s). The first Phase I Trial of anti-CTLA-4 antibody as a single agent was carried out at UCSF in prostate cancer patients, and was completed in the fall of 2000. No toxicity was noted. It is likely that CTLA-4 blockade will have increased clinical efficacy when combined with a therapeutic strategy resulting in antigen presentation or dendritic cell activation. Several therapeutic approaches targeted at increased antigen presentation have been developed and evaluated in clinical trials conducted at UCSF, including the systemic administration of GM-CSF, and the use of antigen-pulsed dendritic cells. Project 6 will evaluate the efficacy of CTLA-4 blockade in combination with systemically administered GM-CSF, first in a preclinical model utilizing the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model, and then in a phase I/II clinical trial. The cellular requirements for rejection of transplantable TRAMP tumors,as well as for induction of auto-immune prostatitis in mice will be evaluated. An understanding of these requirements may help future therapeutic efforts. A combination therapy clinical trial (anti-CTLA-4 antibody plus systemic GM-CSF) will be undertaken in prostate cancer patients with minimal residual disease after prostatectomy or radiation therapy. Subsequent CTLA blockade-based combination trials will be undertaken, utilizing GM-CSF transfected allogeneic prostate cancer cells or antigen-pulsed dendritic cells. This project will seek to identify new antigenic targets for subsequent therapeutic interventions. In the preclinical model,the efficacy of CTLA-4 blockade and immunization with prostate-related proteins (prostate stem cell antigen, 2 prostate-associated serine esterases, and novel antigens identified by T cell and antibody-based expression cloning) in the induction of autoimmune prostatitis and in rejection of TRAMP tumors in mice will be evaluated. Clinically, pre-immune and immune sera from patients treated on the proposed clinical trials will be used to undertake western blotting against prostate cancer cell line lysates. Candidate antigens will be identified by expression cloning. The utility of novel antigens in the induction of autoimmune prostatitis or rejection of TRAMP tumors will be evaluated in an animal model. Particularly promising antigens could then be considered for use in vaccination or pulsed dendritic cell strategies. 4 antibody plus systemic GM-CSF) will be undertaken in prostate cancer patients with minimal residual disease after prostatectomy or radiation therapy. Subsequent CTLA blockade-based combination trials will be undertaken, utilizing GM-CSF transfected allogeneic prostate cancer cells or antigen-pulsed dendritic cells. This project will seek to identify new antigenic targets for subsequent therapeutic interventions. In the preclinical model,the efficacy of CTLA-4 blockade and immunization with prostate-related proteins (prostate stem cell antigen, 2 prostate-associated serine esterases, and novel antigens identified by T cell and antibody-based expression cloning) in the induction of autoimmune prostatitis and in rejection of TRAMP tumors in mice will be evaluated. Clinically, pre-immune and immune sera from patients treated on the proposed clinical trials will be used to undertake western blotting against prostate cancer cell line lysates. Candidate antigens will be identified by expression cloning. The utility of novel antigens in the induction of autoimmune prostatitis or rejection of TRAMP tumors will be evaluated in an animal model. Particularly promising antigens could then be considered for use in vaccination or pulsed dendritic cell strategies.
Core 1 The Administrative Core will be the operational backbone of the SPORE. It will coordinate all SPORE meetings, retreats, & research conferences, generate final progress reports for submission to the NCI, oversee the budget of each Project and Core, set up subcontracts, and arrange a yearly retreat for SPORE investigators and a yearly meeting of the External Advisory Board. Supplemental research funds from other sources will also be managed by this Core and distributed to SPORE Projects, Pilot Projects, and Cores as instructed by the Steering Committee. It will send out and post notices about the availability of developmental funds and manage these applications as they are submitted. The Administrative Core will administrate or maintain minutes of all Steering Committee meetings. The Core will provide some individual support or problem- solving capability for every Project and Core at some point during the year. The Core will consist of the Co-PI of the SPORE, the Steering Committee, the Administrator of the SPORE, and an Administrative Assistant. The Administrator will be a member of the Steering Committee. The group will advise and work with each Project leader in making final decisions about new initiatives, recommendations for hiring new faculty with the support of SPORE funds, and the redistribution of resources if that should be considered necessary. The salary of the SPORE Principal Investigator will be paid partially out of this Core, and all travel to/from the yearly SPORE investigator meetings in Rockville/Bethesda will be covered by this budget, as well.
Core 2 The primary aim of the Tissue Core is the collection and preparation of human prostate tissue urine and blood for distribution to SPORE investigators. Tissue is collected from primary tumors from prostatectomy and TRUS core samples as well as FNA or excisional biopsy material of metastatic disease. All efforts are made to optimize tissue sample acquisition for protein and nucleic acid preservation. Tissues are banked fresh, frozen or formalin fixed and paraffin embedded. In addition to prospective tissue samples, archival paraffin blocks of prostate tissue are also collected. A detailed histologic review is performed on all cases, and on each frozen or paraffin block distributed to investigators. The tissue is evaluated for morphologic quality, histologic features, tumor grade, percent of tumor present, and other pertinent histologic features. The Core works with SPORE investigators to prepare tissue in a suitable manner for research. Laser microdissection facilities, and a prostate cancer tissue microarray library for in situ and immunohistochemistry studies are also available.
Core 3 There are two aspects to the informatics challenges in this application: data warehousing and data analysis. The data warehousing will be based on a scalable RDBMS such as Oracle 8, with extensive access provided through the Web via standard browsers. The critical requirements include extensive read and write access to primary data tables, support for complex custom-designed queries, and implementation of server-side custom applications for visualization and quantitative statistical analysis of the data. The data will include quantitation of biological materials by array-CGH, expression microarrays, and tissue arrays and will require linkage to databases containing tissue tracking information and patient outcomes. The data analysis problems are also complex and will require novel methods. In cases where the number of measurements per biological sample vastly exceeds the number of independent samples, standard statistical analyses can have difficulty detecting truly significant correlations. We will develop and deploy methods for high-dimensional data visualization as well as for detecting subtle correlations both between genetic loci and biological outcomes and among genetic loci or functional pathways. The goal is an integrated data processing and analytical system capable of supporting the research projects' basic needs as well as providing quantitatively supportable hypotheses about the basis of prostate cancer progression.
Core 4 Prostate cancer survivors and advocates formed a core group in September 1998 after meeting with Deborah Collyar, leader of UCSF's Breast Cancer Advocacy Core. Members have become an integral part of the Prostate Cancer Program at UCSF. They are actively involved in many aspects of the program, including:
The primary goal of this Core is to support the research efforts of the SPORE. Specifically, the advocates will facilitate patient access to clinical research trials; participate with other members of the SPORE to study clinically relevant questions, solutions of which have either a near- or long-term positive impact on those who have or are at risk of developing prostate cancer; improve accrual to clinical trials; and help to develop additional resources to support the research objectives of the SPORE.
Core 5 The main function of this core will be to provide the scientific oversight as well as the infrastructure required for the conduct of clinical research in prostate cancer at the UCSF Cancer Center. The major goals of this program will be to 1) provide scientific oversight and prioritize prostate cancer clinical research directions, 2) provide a clinical trials unit which, working closely with the tissue and informatics cores, will be responsible for clinical trial development and conduct, regulatory compliance, contract negotiation, data entry, data quality assurance, audits and report generation, 3) maximize the potential for translational projects by providing clinical input for all SPORE projects, from project inception to completion, 4) develop a high risk (prostate cancer susceptibility) research clinic, targeted at men at high risk for developing prostate cancer, 5) develop a research clinic for prostate cancer patients at high risk for recurrence after conventional local therapy, and 6) develop a health related quality of life (HRQOL) research program. The Clinical Research Core is not meant to provide day to day data management or support for any studies except those funded by the SPORE, but it will serve to integrate information on all prostate cancer clinical trials in an effort to both increase accrual, and to provide the clinical context within which investigator-initiated trials of novel agents developed by SPORE projects can be developed.
Core 6 The Animal Technology Core of the Prostate Cancer SPORE group is designed to serve investigators through consultation, training and carrying out procedures required for the proper and efficient use of transgenic models of prostate cancer and other in vivo models of prostatic research. The Animal Technology Core is staffed with researchers with decades of experience in the area of prostate and animal models of prostate cancer headed by Dr. Gerald Cunha. The goals of the Animal Technology Core are as follows: (1) Provide consultation services on mouse prostate and transgenic models of prostate carcinogenesis. (2) Evaluate existing transgenic models of prostate cancer and recommend utilization of specific models most appropriate to the goals of the research project. (3) Develop new transgenic models of prostate cancer as needed. (4) Provide training and/or participate in the harvesting of individual lobes of the mouse prostate at all stages of development (neonatal to adult) and all stages of pathogenesis (normal to invasive cancer). (5) Perform prostatic ductal microdissection. (6) Perform injection of viral suspensions into mouse prostate, perform direct intra-ductal injection of viral suspensions and perform injection of viral suspensions into the internal iliac artery, which supplies the prostate. (7) Perform subcutaneous and subrenal capsule grafting into host animals of prostatic carcinoma cell lines, transplantable mouse prostatic tumors, or human prostatic cancer xenografts. (8) Perform neonatal rat prostate organ culture assay for testing monoclonal antibodies and anti-sense oligonucleotides for their ability to inhibit prostatic growth and differentiation. (9) Develop and characterize rabbit monoclonal antibodies selectively reactive for human prostatic carcinoma cells or tumor stroma. Almost all of the proposed full projects as well as the pilot projects will utilize the technical resources of the Animal Technology Core. As new projects are integrated into the Prostate Cancer SPORE group, it is likely that they will also benefit from the experience, expertise and service of the staff of this core.
Developmental Research Program There are four general goals of the Developmental Research Program. Each goal will be accomplished using a combination of institutional funding and an NCI Prostate SPORE grant. 1) To attract outstanding UCSF scientists and clinical investigators to the field of prostate cancer, and to promote innovative collaborative research between the two groups. 2) To encourage collaborations between UCSF investigators with diverse skills, which will result in new research projects. 3) To assist in the development of the careers of UCSF junior faculty in the basic and clinical sciences. 4) To recruit to UCSF junior faculty in basic and clinical areas of research, which will enhance and expand the current research program. In order to facilitate accomplishing goals 1-3, funding has been allocated to initiate Pilot Research Projects. Five Pilot Research Projects, ~$30,000/each, were funded in 1999 from UCSF Prostate Cancer Center resources. The projects ranged from identifying proteases in human and transgenic animal models of prostate cancer to constructing a mutant telomerase that arrests the growth of human prostate cancer cells. Other projects are focused on identifying genomic abnormalities in prostate cancer, and characterizing the role of prostate cancer stroma in promoting tumor progression. Potential Pilot Research Projects which are likely to be funded should the Prostate SPORE be awarded include the following: 1). Interference with telomeres to treat prostate cancer. 2). Cellular and molecular mechanisms of prostatic apoptosis. 3). The impact of ethnicity on health-related quality of life outcomes following diagnosis and treatment of prostate cancer. Recruitment of three new faculty members specializing in prostate cancer research by the Departments of Medical Oncology, Radiation Oncology, and Urology are now under way (Goal #4). These positions will be funded by institutional resources. |
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