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Mayo Clinic Rochester
Project 1 Several etiologic factors have been suggested for prostate cancer, including both genetic and environmental factors. Established risk factors include age, race, and family history. It is becoming more apparent that common polymorphisms that result in quantitative or qualitative functional differences in protein products play an important role in modifying disease risk. For prostate cancer, the most compelling hypothesis for increased cancer risk supports a hormonal involvement, which could include enzymes involved in the androgen metabolic pathway and gonadotrophins. Additionally, there is evidence suggesting that estrogen metabolites, as genotoxins, also increase risk for prostate cancer. In this study, we propose to systematically test the hypothesis that common genetic polymorphisms for enzymes that catalyze the synthesis and bioactivation of androgens and estrogen (CYP11A1, CYP17, CYP 19, HSD3B2, HSD17B1, HSD17B3, AKR1C3, SRD5A2), either individually or in combination, are risk factors for prostate cancer. In addition, we hypothesize that common genetic polymorphisms that catalyze the synthesis of catecholestrogens (CYP1A1 and CYP1B1), as well as enzymes that metabolically inactivate estrogens, catecholestrogens, or genotoxins generated from catecholestrogens (COMT, SULT1A1, the GSTs and NQO1), either individually or in combination, might also represent predisposing genetic risk factors for prostate cancer. This hypothesis will be tested in a case-control study that uses two sets of previously identified cases - those with a strong family history of prostate cancer (n=449 from 181 families) and those with a reported negative prostate cancer family history (n=500) - and a population-based control group (n=510). The controls have been extensively screened for prostate cancer by digital rectal examination, serum PSA measurement, transrectal sonographic imaging and when indicated, biopsy. At the conclusion of this project, we will have provided important insight into the potential role of a group of genes important in androgen and estrogen metabolism and generated hypotheses about how these interact in the etiology of prostate cancer. Additionally, by making pairwise comparisons among these three groups of subjects, we will be able to discern whether these common polymorphisms are more strongly associated with familial or sporadic prostate cancer, similarly associated with both or with neither. The overarching goal of this project is to identify genetic susceptibility factors of prostate cancer in order to improve our understanding of the etiology of this disease and potentially identify men at increased risk of developing the disease in whom prevention strategies might be targeted.
Project 2 Over the past five years we have investigated the biochemical and molecular aspects of hK2 and its proform (phK2) and have developed highly sensitive and specific analytic assays for these biomarkers. Our studies have shown that these proteins are localized predominately in the prostate, are hormonally regulated and have potential proteolytic role in the activation of PSA from proPSA. Preliminary studies also have shown that the ratio of serum concentration of hK2 to free-PSA is useful for identifying those patients with borderline elevated PSA concentrations who are most likely to have prostate cancer. Specific Aim 1 will employ the proven molecular, biochemical and analytic techniques used to characterize and measure hK2 to further characterize and develop assays for three new prostate cancer markers: inhibitor - 6 complex (hK2-PI6), hK2 variant (hK2-v) and PSA variant (PSA-v). Vectors will be developed to produce recombinant forms of these proteins and specific monoclonal antibodies will be produced. These antibodies will be used for purification of endogenous proteins, immunohistochemistry studies and development of sensitive and specific immunoassays. Specific Aim 2 involves a collaboration with clinical colleagues in Urology, Oncology and the Cancer Center. Blood, tissue and clinical follow-up information will be collected for three large cohorts of patients. One cohort will include 3000 men undergoing transrectal needle biopsy of the prostate. Another cohort will include 300 men with rising PSA concentrations and negative needle biopsy undergoing saturation biopsy of the prostate under anesthesia. A third cohort will include 240 men with advanced hormone refractory prostate cancer. Each of these cohorts will have PSA, free PSA, hK2 and phK2 measured in their blood to determine the utility of these markers for improving cancer detection and predicting outcomes. In addition, cancer prediction algorithms will be validated with blood from 600 Asian men from Korea undergoing prostate biopsy. Aliquots of blood from the cohorts also will be held frozen to evaluate the utility of hK2-PI6, hK2-v and PSA-v once assays have been developed. The major goal of these studies will be the development of the most practical and accurate assays to improve the detection, diagnosis and management of prostate cancer.
Project 3 Prostate adenocarcinoma accounts for nearly one-third of all invasive cancers not involving the skin in American men. Prostate cancer has a marked degree of clinical variability compared to other cancers and even with accurate stage determination and appropriate therapy, a finite number of men will suffer tumor progression. Thus, one of the highest priorities in prostate cancer research is to identify new laboratory tests, especially genetic markers, which can more accurately predict the rate of progression for a given prostate tumor and how it might respond to appropriate therapy. Comparative genomic hybridization (CGH) studies have shown that 8q24 is commonly overrepresented and/or amplified in prostate cancer. Using fluorescence in situ hybridization (FISH) we have recently shown that 8q24 overrepresentation is a marker of clinical progression in stage pT2N0M0, pT3N0M0, and pT2-3N1-3M0 prostate cancer. It should be noted that we define overrepresentation as a significant increase in 8q24 copy number relative to centromere 8 copy number; thus overrepresentation includes overt amplification. 8q24 contains c-myc. Thus c-myc could be the target of the overrepresentation. However, it is becoming clear that the actual (or only) target of many overrepresented regions is not necessarily the first gene identified in that region. Very few studies of 8q24 overrepresentation mapping have been reported. In Specific Aim 1, we propose to map the extent of the minimally overrepresented region at 8q24 using FISH, CGH, and genomic CGH (gCGH) technology. In Specific Aim 2, we will isolate the genes from the minimal region(s) of overrepresentation using various approaches including database searches and cDNA selection. The expression level of these genes will be evaluated in tumors with overrepresentation to determine which genes are candidates for mutational and functional studies. In Specific Aim 3, we will extend and validate the clinical-translational relevance of 8q24 overrepresentation. This project will identify the actual target of overrepresentation at 8q24. It may be that we will identify c?myc as the common target of the 8q24 overrepresentation event. If so, then we will have identified a known target for gene therapy and will have validated a means to stratify patients for this therapy. Importantly, it is possible that other genes are a common target in 8q24, or at least that they are commonly co-overrepresented with c-myc. Such genes will define new targets for gene and/or immunotherapy in prostate cancer and will potentially provide new prognostic and predictive markers.
Project 4 Because the immune system has the capacity to recognize and in many cases destroy tumor cells, significant efforts are being devoted to the development of immune-based therapies for cancer. Both cytotoxic T lymphocytes (CTL) and helper T lymphocytes (HTL) have been shown to react with antigens expressed by tumor cells and as a result, establish protective and therapeutic effects. Since CTL and HTL recognize antigens in the form of peptide complexes with major histocompatibility complex (MHC) surface molecules, it is necessary to identify the chemical nature of tumor-derived peptides that can elicit T-cell responses capable of inhibiting tumor-cell growth. The overall objective of the proposed study is to identify peptides derived from sequences of several known prostatic-associated antigens (PAA) that will be capable of stimulating CTL and HTL against prostate tumor cells. We have selected several PAA which are preferentially expressed on cells of prostatic epithelial origin including transformed cells. The amino acid sequences of these PAA have been screened for the presence of peptides containing MHC binding motifs. Those peptides that display a high degree of probability of binding to MHC molecules will be synthesized and tested for their capacity to elicit in vitro T-cell responses to naturally processed PAA as final proof that they indeed represent T-cell epitopes. The ultimate goal of our work is to utilize these tumor-reactive T-cell epitopes to develop immunotherapeutic approaches to treat prostatic cancers. To accomplish this goal, we propose the following specific aims:
The completion of these aims should facilitate the development of novel broadly applicable T-cell based immune therapies such as epitope-based vaccines and adoptive T-cell therapy for the treatment of early and advanced prostate cancer.
Project 5 The goal of this project is to exploit the highly potent cytotoxic properties of a novel class of genes, called Fusogenic Membrane Glycoproteins (FMG), for the gene therapy of prostate cancer. Many viruses kill their target cells by causing cell fusion through binding of the viral envelope protein on an infected cell with its cellular receptor on neighboring cells. The result is the formation of large, multi-nucleated syncytia which eventually become non-viable and die. We have used gene transfer of the cDNAs of three different types of FMG to prostate tumor cells. The cytotoxicities of these FMG were consistently greatly superior to that of conventional suicide genes and the local bystander killing effects were at least one log greater than those of the HSVtk/Ganciclovir system. FMG tested so far kill target cells via non-apoptotic mechanisms with the concomitant induction of immune stimulatory signals such as heat shock proteins. We now hypothesize that these properties of FMG-mediated tumor cell killing can be exploited, and enhanced, to generate more effective gene therapies for prostate cancer. We will characterize in detail the mechanisms by which FMG gene transfer leads to cell death to understand what regulates the efficiency of syncytial killing and how to improve it for therapeutic purposes. We will investigate how the mechanisms of syncytial killing can be enhanced in vivo to stimulate potent immune responses against tumor metastases. This will be done by constructing vectors in which additional immune stimulatory genes, such as GM-CSF, are co-expressed with FMG. In addition, we will take full advantage of collaborations within the SPORE group to investigate whether co-expression of an FMG with the sodium iodide symporter (NIS) gene can augment the cytotoxicity of FMG alone by and allowing increased tumor cell killing in combination with radioiodine treatment. We will also generate FMG-induced prostate tumor cell-dendritic cell hybrids for anti-tumor vaccination, in close collaboration with the expertise of Dr. Esteban Celis as a co-member of the SPORE group. We propose to make a series of viral vectors to transfer the cDNAs of different FMG into prostate tumor cells to identify the most effective FMG for the gene therapy of prostate cancer. Finally, we will construct retroviral and adenoviral vectors which incorporate tight transcriptional regulatory elements of the PSA promoter to allow targeting of FMG expression to prostate cells to increase the safety of these potent genes for progression to clinical trials. We propose using a GALV adenoviral vector system for a Phase I/II clinical trial in years 4 and 5 of the funding period.
Project 6: Prostate cancer is the second leading cause of cancer death of American men. Metastatic prostate cancer is considered essentially incurable. In marked contrast, thyroid cancers can be effectively treated and, at times, cured even when widespread metastasis is present, because of the ability of the cells to concentrate iodine, making therapy with radioactive iodine possible and effective. The studies described in this proposal are aimed at transferring the gene for the thyroidal sodium-iodide symporter (NIS), the structure that is responsible for iodide trapping by thyroid cells, into prostate cancer cells. In addition, the potential role of this transfer as a means of gene therapy for metastatic prostate cancer is examined. The studies involve targeting expression of the NIS gene using prostate specific promoters in order to achieve prostate specific gene expression. Our preliminary studies have demonstrated the feasibility of this gene transfer in vitro and in vivo, in that high level and prostate specific iodide uptake has been established in LNCaP cell tumors in mice and those tumors have been successfully treated with 131I. The experiments outlined in this proposal will further examine the feasibility and efficacy of NIS gene transfer in vivo using mouse models, mechanisms of maximizing NIS protein expression and activity, and the cell killing effect of 131I in these models in vitro and in vivo. Further, our studies will examine the immune response within immunocompetent host mice following radioiodine killing of NIS transfected murine prostate tumors and the influence of that response upon the appearance and progression of native prostate cancer in transgenic mice (TRAMP), which naturally develop prostate cancer. Finally the proposal describes a phase I/II clinical trial of adenoviral mediated NIS gene transfer in patients with recurrent prostate cancer. Our studies, will serve to examine the potential of NIS gene transfer to prostate cancer as a method of therapy of metastatic disease and are the first so described. In addition, successful demonstration of radioiodine effect in our prostate cancer model will serve to stimulate interest in NIS as a therapeutic gene for other cancer types.
Core 1 The Administrative Core will function so that maximum potential for translational objectives can be achieved among the Projects and the Cores. It will have the following responsibilities:
Core 2 The Prostate Cancer Tissue Procurement Core of this Specialized Program of Research Excellence (SPORE) provides a coordinated, centralized, dedicated program for procurement and processing of biospecimens obtained from prostate cancer patients. Human biospecimens are one of the most valuable and unique resources available for translational research at the Mayo Clinic, Rochester, and the GOAL of the Tissue Procurement Core is to procure prostate tissues from every prostate cancer patient undergoing radical prostatectomy at the Mayo Clinic. The Tissue Procurement Core will coordinate acquisition of both normal and neoplastic prostate tissues for translational research, and a portion of normal and prostate cancer tissue from each patient will be obtained fresh and stored frozen to provide investigators with DNA and RNA, and the remainder of the tissue will be available in paraffin blocks stored at the Mayo Clinic Tissue Registry. The Tissue Procurement Core will also serve as a resource of expertise, collaborative effort, and service for pathology, immunohistochemistry, in situ hybridization, laser capture microdissection, reverse-transcriptase polymerase chain reaction (RT-PCR), tissue microarrays, and digital image analysis (DIA). The Core will interface and be electronically integrated with the Prostate Cancer Patient Registry and the Biostatistics Core to provide investigators with clinically annotated biospecimens. The collection, banking, and use of biospecimens will be performed with appropriate patient consent and institutional approval. The Tissue Procurement Core will interact and collaborate with other Prostate SPOREs to promote resource sharing, and integrate scientific projects of mutual interest.
Core 3 The Gene Discovery/Bioinformatics Core (GDBC) of this Specialized Program of Research Excellence (SPORE) provides a program to analyze public and private genomic data related to prostate and to supply leads to the other SPORE projects of interesting genes that would be utilized to accomplish their goals as well as to facilitate discovery of novel prostate genes that would be useful for diagnosis or therapy. With the human genome project close to completion, and sequence information pouring into databases from many other projects (e.g. CGAP) genomic data is becoming one of the most valuable resources for cancer projects. The GDBC aims to analyze and utilize this information in order to be a resource of interesting leads to genes for all the other SPORE projects. More specifically, the GDBC will analyze the Expressed Sequence Tags (ESTs) that derive from high quality cDNA libraries produced from normal and tumor prostate cells. This analysis, in conjunction with data from micro-array gene expression profiling, will provide diagnostic markers for Project x, and candidates for immune therapy and Gene therapy (Project y, z). Analysis of the sequences derived from the human genome project and in conjunction with data from cDNA libraries and expression profiling of micro-arrays can aid the Prognostic Marker Projects. This goal will be achieved by analyzing the information in the Expressed Sequence Tag (EST) database which contains more than two million partial sequences of cDNA clones from cDNA libraries extracted from different human organs or cell types. About 70,000 ESTs are derived from normal prostate, prostate cancer, and prostate cell-lines. By comparing these ESTs with each other using computer algorithms, we can group ESTs in clusters that correspond to distinct genes, estimate the level of expression of genes, and define their expression patterns in different tissues or cell types. Similarly, we will obtain differential expression levels of genes in cancer vs. normal prostate tissue. Such genes can be markers for diagnosis or predictors for the aggressiveness of the disease. We will also select clusters corresponding to genes that express in prostate but not in any essential tissues and can be used as targets for immunotherapy.
CORE 4 The Clinical Follow-up Core provides patients to support the translational and clinical portions of Projects 2, 3, 4, 5, and 6 of the SPORE. This patient follow-up data will be gathered prospectively on patients who undergo prostate biopsy, primary therapy for early stage prostate cancer or therapy for hormone refractory prostate cancer. These therapies consist of radical retropubic prostatectomy, external beam radiotherapy, prostate brachytherapy by permanent interstitial implantation or hormonal therapy. Patients who have failed primary external beam radiotherapy will be identified and recruited for Projects 5 and 6 of the SPORE. Patients will be recruited for participation in the SPORE translational research and clinical trials in this core which will operate jointly in the Departments of Urology, Radiation Oncology, and Medical Oncology. The follow-up test schedule for primary patients is specified from treatment initiation. Database managers in each department will assist in recruiting patients for these studies, schedule tests and follow-up appointments, and gather data. Prospective follow-up data collection in support of the biomarker Projects 2 and 3 and will include serum prostate specific antigen (PSA), physical examination, and disease status. Data storage and analysis will be conducted by the Biostatistics Core.
Core 5 The Biostatistics Core provides statistical collaboration and data management support for each of the SPORE projects, the developmental projects, and the Cores. Each of the projects presented in this application reflects input from members of the Biostatistics Core on study design and analysis plan. The Biostatistics Core will provide statistical support across many different fields, including statistical genetics, epidemiological studies, basic sciences including gene array, and clinical trials. This comprehensive nature of the Biostatistics Core assures each SPORE investigator access to statistical expertise that includes collaborative development of study designs and analysis plans, state of the art data analysis and interpretation, data management resources, and abstract and manuscript preparation. The Biostatistics Core also provides a mechanism for the management and integration of both existing and newly collected data through consistent and compatible data handling. Areas of support include database development, data form development and processing, quality control, data collection and entry, and data archiving. This Core complements and assists the efforts of other Cores such as the clinical follow-up Tissue Procurement Cores with superior data management and experience with tissue registries such as the Radical Prostatectomy database. The Biostatistics Core builds upon the innovative and time-tested procedures and systems developed by one of the largest statistical groups in the country whose members have collaborated on more than 8,000 clinical and basic science research studies since 1966. |
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