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BAYLOR COLLEGE OF MEDICINE
Project 1 Prostate cancer is the most common cancer in American men. The incidence has been increasing dramatically and the age-specific mortality rate is also increasing, though more slowly. The growing disparity between incidence and mortality may reflect the successful early detection of potentially lethal cancers. But, the prevalence of histologic cancers with low malignant potential is so high (~40% in men >50 years old.) that some cancers now being detected may be of little clinical importance. Current techniques for characterizing malignant potential clinically, before the prostate is removed, are inadequate. The biopsy specimen is not sufficiently representative of the cancer in the patient. A biopsy usually underestimates the grade, and staging studies usually underestimate the extent, so the tendency is to treat every detected cancer as potentially lethal. There are few well-established objective markers, other than grade, able to predict prognosis - either the rate of local growth or the probability of metastasis - with sufficient accuracy for appropriate management. The purpose of this project is to develop better methods to assess the threat posed by a prostate cancer. Our first aim is to develop better strategies for needle biopsies so that representative samples of the cancer can be obtained which accurately reflect biological potential before treatment is begun. This aim has been largely completed, and based on our data, additional studies are being performed at our institution as well as other academic centers. Our second aim is to identify new markers of progression and metastatic propensity and to develop a standard, efficient protocol to validate promising markers with respect to establishing prognostic features and outcome. Our assessment and development of candidate markers involve a systematic approach and are based in part on a paradigm that considers that growth and metastatic potential may become uncoupled at early stages of progression. Standard statistical approaches may be inefficient in detecting complex relationships among markers and metastatic potential. These approaches may be complemented by computer-intensive modeling techniques. We have previously reported a novel prognostic indicator, apoptotic index (A.I.), and will continue to develop this marker for its clinical utility. We will also expand our immunohistochemical studies that indicate clustered p53 positive staining combined with positive Ki-67 staining has significant prognostic potential beyond that of either marker alone. The clustered p53 staining pattern has indeed proved to have prognostic value in studies conducted at our institution as well as others. In an effort to further determine the biological and clinical significance of p53 mutation in prostate cancer metastasis, in our third aim we have embarked on a series of studies designed to identify genes that are under the transcriptional regulation of p53 in prostate cancer cells. We have identified two such genes that are being studied with regard to their functional significance as well as their clinical utility as novel biomarkers for prostate cancer metastasis. In a fourth specific aim we are pursuing specific genes that have been identified as being related to metastasis using cell lines derived from primary tumors and their associated metastases together with differential display-PCR.
Project 2 Our understanding of the basic mechanisms underlying the initiation, establishment, and metastasis of prostate cancer is extremely limited. This is due in part to the complex, heterogeneous, and multifocal nature of prostate cancer. Substantial evidence is accumulating that cancer is a genetic disease of the cell cycle. Two classes of mutations impinge directly on the basic cell cycle machinery that controls cell proliferation: 1) those that activate positive growth pathways, such as overexpression of G1 cyclins, and 2) those that inactivate negative growth control pathways. Negative growth control is mediated by tumor suppressor proteins such as Rb and p53 as well as through the action of two newly defined protein families exemplified by p21CIP1 and p16MTS1. These are inhibitors of cyclin-dependent kinases (Cyclin-Kinase Inhibitors, CKIs) which drive cell proliferation. We have recently found that p21 family members are expressed primarily in differentiated cells during development (including prostate epithelium), suggesting that they function to mediate cell cycle arrest during terminal differentiation in particular cell lineages. These proteins, in conjunction with other negative regulators, serve to block cell proliferation in adult tissues, and their activities must be overcome in order to achieve the transformed state. In addition, there is evidence that inappropriate activation of positive components of CDK pathways can also participate in transformation. In the proposed study, we seek to understand how alterations in positive and negative growth regulators contribute to prostate cancer. One line of studies will examine to what extent the levels of cyclin dependent kinase inhibitor and mitotic cyclins are altered in prostate cancer and metastatic disease. Immunohistochemical approaches coupled with pathological analysis of specimens will be used to address this question using antibodies we have generated. Preliminary data indicate reduced levels of the CDK inhibitors p21 and p27 in prostate cancer, but there is little evidence of correlation with tumor grade at this point. Recently, we have discovered human Cdc37 as a component of a signaling pathway required for activation of CDK4. CDK4 is the catalytic subunit of D-type cyclin-kinase complexes and functions at the G1/S transition. We have found that Cdc37 is induced in human prostate cancer. To address the relevance of this to prostate cancer development, we will generate mice expressing Cdc37 in the prostate epithelium. Phenotypic consequences will be examined through pathological and immunohistochemical examination of transgenic prostatic tissue.
Project 3 The search for genetic and environmental factors responsible for the increasing incidence of prostate cancer in our society has been hampered by a dearth of molecular and genetic markers associated with either initiation or progression of prostate neoplasia. Recent studies, however, have shown that at least two prostate tumor suppressor (PTS) genes are likely to be present on the short arm of chromosome 8 (8p) near the MSR (8p22) and ANK (8p11-21) loci, respectively, and that these chromosomal regions undergo loss of heterozygosity (LOH) in at least 50% of clinical prostate cancers. The long-term goal of this project is to identify and characterize PTS genes on 8p, as well as to study their respective role(s) in the development of prostate neoplasia. We will focus initially on 8p22, since presumptive LOH hotspots in this region have recently been identified. Many of the resources developed for the 8p22 studies will also be useful for subsequently mapping the 8p11-21 region. We propose to further localize PTS genes on 8p by mapping LOH breakpoints in this region from an additional 150 prostate tumors. As an important part of this effort, we will clone and map the genomic sequences on 8p that are deleted most frequently in prostate cancers using YAC, cosmid, and other large-insert libraries. As a prelude to this proposal, we have already isolated and mapped a series of overlapping yeast artificial chromosome (YAC) clones covering the entire 8p22 region, and we have screened a chromosome-8-specific cosmid library with the goal of identifying and mapping approximately 20 cosmid markers spanning 8p22 at approximately 500 kb intervals. Our ability to rapidly develop a highly successful, large-scale effort in this area has been greatly facilitated by direct access to a large number of well-characterized human prostate cancers through the Baylor College of Medicine SPORE and by access to the core facilities of the Baylor College of Medicine Human Genome Center. We are requesting support for five years to: (1) continue our construction of 8p22 and 8p11-21 chromosomal maps including identification of YAC and cosmid markers spaced at approximately 500 kb intervals throughout both regions; (2) Prepare DNA, touch preps, and cytospins from approximately 150 prostate cancer and non-tumor specimens for mapping LOH hotspots on 8p by the polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH); (3) Prepare large-insert contigs which completely overlap LOH hotspots identified at 8p22 and 8p11-21; (4) Identify candidate PTS genes which map within these LOH hotspots by cDNA screening, exon trapping, and biological assays; and (5) Prepare YAC and cosmid contigs from mouse chromosomal regions syntenic with human 8p22 and 8p11-21. As part of specific aim 3, we will develop a transfection-based functional assay for identification and confirmation of PTS genes using large-insert contigs from LOH hotspot regions. After stable transfection into metastatic rat and mouse prostate carcinoma cell lines, the inserts carrying functional PTS genes will be identified on the basis of their ability to suppress metastasis or growth rate after transplantation into recipient mice. The sensitivity and specificity of this assay will be evaluated initially using a series of YACs and cosmids from 17p, some of which carry a functional p53 gene.
Project 4 The therapeutic options for prostate cancer are currently limited to prostatectomy or radiation for cure of localized disease and hormone therapy for palliation of advanced cancers. Standard chemotherapy is largely ineffective in this disease. Novel therapeutic approaches are therefore needed. The retinoids (natural and synthetic derivatives of vitamin A), have emerged in the last decade as the premier class of cancer chemopreventive agents, and have been studied extensively in preclinical and clinical investigations. In recent studies, combination of retinoids with alpha-interferon produced impressive responses in patients with advanced epithelial cancers, demonstrating that retinoids may have therapeutic as well as chemopreventive applications. Finally, preclinical data suggest that vitamin D and its derivatives may have anticancer properties, and epidemiological studies have implicated low levels of vitamin D in the progression from latent to clinical prostate cancer. In preliminary work, our group identified differences in the concentration of endogenous retinoids between the normal human prostate, benign prostatic hyperplasia (BPH) and prostate cancer. In addition, we showed that treatment with fenretinide (4-HPR, a synthetic retinoid) reduced the incidence and the volume of ras+myc-induced prostate cancers in mice, and reduced the number of bone metastases in a metastatic variant of this model. Interestingly, fenretinide treatment caused a several-fold increase in the tissue retinoic aid concentration in both the murine cancers and in human prostates. Finally, we identified the presence of vitamin D receptors in normal and cancerous human prostate tissue and showed that both vitamin D and a nonhypercalcemic analog of vitamin D (EB 1089) inhibited the growth of the human prostate cancer cell line LNCaP. Furthermore, the combination of vitamin D and 9-cis retinoic acid acted synergistically to inhibit growth in this cell line. Our project is focused on developing a biological therapy for prostate cancer and has preclinical and clinical components. We will continue to study endogenous retinoid metabolism in normal and cancerous human prostates, analyze vitamin D/retinoid combinations in vitro, and use animal models to study new retinoids as well as retinoid/interferon and retinoid/vitamin D combinations in vivo. Molecular pathways following the administration of retinoids and of retinoid/vitamin D combinations will be tested in human prostate cancer cell lines and human prostate stromal cell lines using a subtractive hybridization technique. Clinical phase I/II studies will be conducted to assess the effects of retinoids and of retinoid combinations in different stages of prostate cancer. The clinical studies will be tailored to incorporate information from the preclinical animal testing concerning the optimal drug combination and the optimal timing of therapy. Related Ongoing Clinical Trials: Dov Kadmon, PI - (Selenium and Vitamin E Chemo-prevention Trial (SELECT) for Prostate Cancer September 2000 for estimated accrual start-up)
Project 5 Our general programmatic strategy for gene therapy is to develop approaches that impact on primary prostate cancer as well as its metastases using cytotoxic/cytokine in situ gene therapy approaches. Specifically, our strategy involves: 1) the initiation of cytotoxic activities and the promotion of immunocyte infiltration into primary prostate cancer using replication defective adenovirus-mediated transduction of specific genes, such as Herpes Simplex Virus-thymidine kinase (HSV-tk) plus ganciclovir (GCV), to the maturation and further specification of an antitumor immune response using immunomodulatory and co-stimulatory molecules such as IL-12 and B7 also delivered via adenoviral vectors, and 2) the identification and characterization of immunogenic prostate cancer cell specific antigens and their subsequent use to replace and/or augment in situ gene therapies as described in 1 and 2. During the course of these studies, we developed useful preclinical model systems based on cell lines derived from both primary and metastatic tumor generated by the mouse prostate reconstitution model. Our initial studies demonstrated that HSV-tk+GCV gene therapy not only suppressed the growth of local orthotopic tumors through necrotic and apoptotic activities, but also significantly reduced the number and size of pre-established metastatic lung foci. Encouraged by these efficacy studies, we then addressed the local toxicity of HSV-tk+GCV therapy and systemic distribution of the HSV-tk vector following transduction into the mouse prostate gland. These studies indicated that the local inflammatory activities produced by the administration of this vector were minimal and that vector spread outside of the prostate was very limited and well within the limits that we perceived as being safe for humans. With these preclinical data we initiated the first in situ gene therapy Phase I trial in patients whose disease had recurred following initial radiation therapy. In addition, we have initiated neoadjuvant gene therapy using the HSV-tk+GCV protocol based on our preclinical studies demonstrating antimetastatic activity. Although these studies are still in progress, they have produced important information regarding safety, potential efficacy and biological activities of this gene therapy protocol in prostate cancer patients. In specific aim 1 of this proposal, we will continue these therapeutic trials as well as to test specific combination therapies involving adenoviral vector delivered HSV-tk+GCV together with adenoviral vector delivered IL-12 using our preclinical animal model systems. In further preclinical studies we have demonstrated efficacy and acceptable toxicity levels of adenoviral vector delivered IL-12 in orthotopic mouse prostate cancer model systems. In these studies a single dose of recombinant adenoviral vector transducing p35 and p40 generated high levels of in vitro activities without direct cytotoxic response. Using an optimal dose of this vector, we were able to generate specific antitumor activities, including reduction of tumor volume, and to enhance survival. In pre-established lung metastasis assays this gene therapy protocol was highly effective in generating systemic activities as suppressive development of distant metastases. We have further shown that IL-12+B7 results in enhanced therapeutic activity. Specifically, IL-12+B7 therapy resulted in increased infiltration of CD8 positive T cells within the tumor and resulted in increased survival relative to IL-12 gene therapy alone. Further studies using adenoviral vector transduced IL-12 and IL-12+B7 cell based vaccines also demonstrated that this approach was capable of protecting a significant percentage of animals from later tumor challenges using mouse prostate cancer model systems. In specific aim 2 we will continue to elucidate the therapeutic activities and toxicities of IL-12 and IL-12+B7 in situ gene therapies as well as cell-based vaccine approaches. We have further submitted an IND to initiate adenoviral vector delivered IL-12 in patients who have had recurrence after radiation therapy in order to initially test the toxicity of this approach in a Phase I clinical trial. We have continued our initial studies combining irradiation therapy plus HSV-tk+GCV gene therapy and the results have demonstrated highly synergistic therapeutic effects. These pre-clinical studies resulted in the initiation of clinical trials that are now ongoing and involve combination HSV-tk+GCV plus antiandrogen/irradiation therapy approaches. In specific aim 3, we use our preclinical mouse model systems to test various combination strategies involving irradiation therapy and both replication-defective and replication-competent adenovirus-mediated HSV-tk+GCV and IL-12 gene therapy approaches. As stated in specific aim 4 of this proposal, we will generate novel replication defective and replication competent adenoviral vector systems using novel promoters such as the caveolin-1 promoter.
Project 6 The long-term objective of these studies is to develop, characterize, and refine novel strategies to manipulate prostate-specific gene expression in order to facilitate the establishment of transgenic mouse models and gene therapies for translational prostate cancer research. The rationale for these studies is that direct genetic manipulation of the prostate translates into transgenic models that heredetably exhibit reproducible pathologies characteristic of human disease. These can be used simultaneously by investigators around the world to study the molecular basis of initiation, transformation and progression of prostate cancer. In addition, these studies translate into the design of prostate-specific gene-based therapies that can be tested rapidly in the animal models. To this end, the principal investigator and his collaborators will continue development of a novel system based on the regulatory elements of the rat probasin (PB) gene shown during the term of the initial SPORE award to target developmentally and hormonally regulated heterologous gene expression specifically to the prostate in transgenic mice. Lines of transgenic mice have already been established carrying either a PB-ras construct that reproducibly develop prostate hyperplasia or a PB-Tag construct that reproducibly develop prostate cancer. Having demonstrated that the murine prostate can be transformed as a consequence of targeted oncogene expression, it will be a primary objective of this project to thoroughly characterize the initiation, development and progression of prostate disease in these mice at the histologic, pathologic and molecular levels and establish PB-myc mice to complement these models. Since the role of p53 in the development of prostate cancer metastases has been demonstrated using p53-deficient mice, a prostate-specific gene knock-out system based on the cre-lox recombinase strategy will be developed to facilitate further characterization of the loss of wild-type tumor suppressor genes and other genes identified through genetic studies to correlate with prostatic disease. This approach will also facilitate investigation into the role of the retinoblastoma (Rb) protein in the progression of prostate cancer since Rb-deficient mice are not sufficiently viable for these studies. In order to further facilitate translational research, the prostate-specific expression system will be used to generate a novel prostate-specific p53-based gene therapy strategy designed to exploit the p53-dependent apoptotic pathway for the treatment of prostate cancer. The efficacy of this strategy will be tested in the transgenic models. Therefore, the strategies designed to manipulate prostate-specific gene expression developed in this project will be used both to identify biological factors involved in the progression of prostate cancer and to development and test innovative medical strategies for the prevention and treatment of prostate cancer.
Project 7 The growth and differentiation of prostatic epithelial cells is initiated and mediated through androgen regulation of prostatic stromal cells. Upon stimulation by androgen, signals from stromal cells direct ductal morphogenesis and cytodifferentiation of epithelial cells to form glandular acini. Little is known about the molecular mechanisms of androgen action in prostatic stromal cells. Prostate cancer is an androgen-dependent disease. Alterations in the axis of stromal-epithelial interactions are likely in prostatic carcinoma. It is our hypothesis that prostate stroma-specific molecular pathways are fundamental in the endocrine and paracrine regulation of prostatic epithelial cell proliferation and differentiation, and that such pathways are central to androgen regulated prostate cancer initiation and progression. To address this hypothesis and identify key molecular mechanisms, the specific aims of this proposal are centered on the role of prostate stroma gene expression in several aspects of prostate cancer. Specifically, we want to study the role of stromal and androgen-dependent genes, such as COUP-TFs, in the initiation and progression of prostate tumors and to define the promoters of these genes for future gene targeting specifically expressed in prostate stroma cells. We will use transgenic mice, gene specific knockout (homologous recombination) and in vitro mouse prostate reconstitution system (MPR) to achieve these goals. We expect results obtained from these studies will help us understanding the role of stroma in prostate carcinogenesis. In addition, we hope to identify a genetic probe for prostate cancer and eventually devise a method for genetic therapy. Further specific aims have been initiated that more directly address the mechanisms of androgen action in prostate cancer cells. These studies seek to understand the potential biological and clinical significance of the SRC steroid coactivator gene family in regard to the development of prostate cancer and, potentially, prostate cancer metastasis.
Core 1 The purpose of the Administrative Core is to support the translational research objectives of the SPORE by providing the management and coordination of the daily activities of the program; providing administrative direction and structure for the investigators; and by providing scientific direction of the SPORE, through the External Advisory Board, Internal and External Advisory Committees, the Career Development Program and the Developmental Research Program, under the direction of the Principal Investigator, Timothy C. Thompson, Ph.D.
Core 2 Over the past three years, the Baylor SPORE in Prostate Cancer Pathology Core has systematically developed the personnel, procedures and infrastructure required to procure, catalog and store large numbers of clinical specimens, both serum and prostate tissue. Through rigorously controlled procedures for specimen collection and processing, followed subsequently by quality control analysis of those specimens, the Baylor Pathology Core has demonstrated its reliability in developing and maintaining this important resource. The Pathology Core investigators, in conjunction with the SPORE Scientific Directors, have also developed systems to regulate specimen disbursement to the large number of SPORE investigators utilizing this resource, ensuring that the appropriate numbers and types of specimens are available while developing priorities to guard the most valuable specimens for the highest priority projects. The Core has also provided routine processing of samples for SPORE investigators, including whole-mount processing and cancer mapping, frozen section and paraffin histology, image analysis, flow cytometry, electron microscopy, immunohistochemistry, and in situ hybridization. Building on these strong foundations, the Pathology Core will continue to procure and maintain the serum and prostate tissue bank, while working to improve its performance and expand its role in the SPORE research program. The Core will coordinate with research project investigators to aid in the collection of new types of specimens, including human plasma, buffy coat DNA/RNA, and lymphocyte cell lines from individuals from families with familial prostate cancer, as well as to help coordinate the development of a mouse prostate cancer tissue bank using tissue from SPORE-supported animal model systems, e.g., the MPR model system and the TRAMP transgenic mouse system. The Core is poised to embark on a major new effort to harvest metastatic prostate cancer specimens, both hormone naive and hormone refractory, through a warm autopsy program as well as through collaborative associations with other institutions. The Core will continue to integrate its functions more closely with the SPORE Prostate Information System (SPIS), streamlining the processes of procurement and disbursement of samples, providing widespread on-line access to the Pathology Core inventory of specimens with matched corresponding clinical information, and finally setting the stage for Internet accessibility of the Pathology Core inventory to other institutions interested in using those specimens for prostate cancer research. Finally, the Pathology Core is developing the capacity to produce and process tissue microarrays of highly selected prostate cancer specimens and to perform laser capture microscopy.
Core 3 The Medical Informatics Core of the SPORE serves investigators and other cores by providing three primary services: (1) sample size calculations and statistical power analyses, (2) SPORE Prostate Information System design and support, and (3) statistical analysis and data modeling assistance. The overall goal of these services is to improve the effectiveness and efficiency of SPORE researchers and their projects. Sample size calculations are an integral component of all project initiatives and specimen requests. Before embarking on a new project, a researcher needs to justify that he/she will have a reasonable chance of detecting a significant result if it actually exists. Power calculations help to ensure that enough observations can be obtained within the scope of the project. Power also adds strength to the conclusion that an effect, which was not observed, actually does not exist by possibly suggesting that the chances were good that, had the effect actually been present, it would have been found. Conversely, because resources are finite, power calculations prevent investigators from collecting too many observations and thereby wasting precious specimens, time, and money. Through these sample size calculations, the Medical Informatics Core is very active in the management of specimen resources. All resource requests must be approved by the director of the Medical Informatics Core, who sits on the Resource Allocation Committee, as to the appropriateness of their sample sizes. The next phase of development in the SPORE Prostate Information System, described below, will feature an electronic specimen management and request function so that power calculations can be obtained by the investigator without shuffling paper forms. The SPORE Prostate Information System (SPIS) is the common database system for all SPORE investigators. SPIS is a centralized database management system which contains over 500 data elements and features an X-Windows interface that is identical whether the end user is running an IBM or compatible personal computer, a Macintosh, or a Unix x-terminal. Furthermore, the SPIS is accessible anywhere on Internet in an on-line, real-time mode. The Medical Informatics Core manages development of SPIS by translating investigator needs into system design and modification requests. The Medical Informatics Core also performs administration and support of the SPIS. Statistical analysis and support is another feature of the Medical Informatics Core. Many investigators make use of such assistance for data analysis and interpretation. Support is provided in different forms, at the researchers request. Assistance in statistical test selection may be all that is needed for a particular investigator. Other investigators require complete experimental design work along with statistical testing and interpretation. The Core is constantly expanding in analytical techniques. In addition to traditional statistical analyses, computational methods such as Markov models, neural networks, and decision tree construction are performed routinely. The SPORE Prostate Information System, where much of the SPORE data resides, is directly accessed by the statistical package (SAS) used by the Core. This allows extremely efficient statistical analysis as the data are centrally located, and no data exchange needs to occur. Both the Core Principal Manager and Database Manager are well experienced in SAS.
Core 4 The ability to manipulate the murine genome by the introduction of foreign genes or the modification of endogenous genes has offered researchers a potent tool to establish animal models for human diseases. The overall goal of the Transgenic Core is to allow SPORE investigators to manipulate the murine genome to create animal models for the investigation of the development of prostate cancer. Once animal models are established, SPORE investigators can use these animals to develop and evaluate the potential of therapeutic agents for the cure and prevention of prostate cancer. During the last three years the Transgenic Core has produced transgenics by the microinjection of DNA into the pronucleus of the one-cell mouse embryo. First, the Transgenic Core produced mice to evaluate the potential of specific promoter elements in targeting gene expression to the prostate. Once the investigators identified the DNA elements that would target gene expression to the prostate, the Transgenic Core generated transgenic animals with oncogenes under the control of these prostate specific elements. In doing so, the Transgenic Core has produced transgenic mice, that develop prostate cancer. The Transgenic Core will continue to produce transgenic mice by DNA microinjection in order to allow investigators to evaluate the usefulness of potential models for prostate cancer. In addition, the Transgenic Core will expand to allow investigators to take advantage of Embryonic Stem Cell technology to investigate the role of specific regulatory proteins in the development of prostate cancer. The Transgenic Core will also continue to serve as a resource to preserve the animal models already established by SPORE investigators.
Core 5 The microarray core facility is a new program within the SPORE that has been funded by a special supplement. This supplement has allowed the purchase of a Genepix 4000 Microarray Scanner and a Genomic Solutions Hybridization Station. Since the arrival of this equipment in early February, we have begun optimizing RNA labeling, hybridization conditions and scanning protocol using 768 spot test arrays generously provided by the Baylor Microarray Core Facility. We have achieved reasonable hybridizations, but in the immediate future we plan to carry out further optimizations and determine the reproducibility and sensitivity of this technique. This will allow us to begin immediate analysis when the University of Michigan SPORE supplies large-scale microarrays. In the interim we can perform microarray analysis for SPORE investigators using large-scale microarrays from the Baylor Microarray Core provided by individual investigators.
SPORE WEB SITE INFORMATION SPORE Investigators
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