|
|
||||
|
|
|
|
|
 |
 |
 |
 |
 |
 |
 |
 |
|
|
The John Hopkins University School of Medicine
Overall Abstract Prostate cancer has become one of the most frequently diagnosed cancers in men in the United States and a major cause of cancer morbidity and mortality. The transcendent objective of the Johns Hopkins Prostate Cancer SPORE is to reduce prostate cancer incidence and mortality via the focused pursuit of translational research in prostate cancer. This competitive renewal proposal, which has been revised in response to reviewer critiques, contains five Translational Research Projects, four Core Resources, two Career Development Projects, and two Developmental Research Projects: Research Project #1 exploits the enzyme activities of prostate-specific antigen (PSA) and human glandular kallikrein-2 (hGK2) for the selective activation of prostate-specific pro-drugs, Research Project #2 targets the use of inhibitors of DNA methylation along with inhibitors of histone deacetylation to restore “silenced” gene expression in prostate cancer cells, Research Project #3 builds on an already successful translational research program, the development of cytolytic adenoviruses as prostate cancer gene therapy, by attempting to improve both the selectivity and lethality of replication-restricted cytolytic adenoviruses for prostate cancer cells in such a way as to increase prostate cancer treatment efficacy, Research Project #4 pursues the development of sulforaphane as a prostate cancer prevention agent, and Research Project #5 explores the contribution of genetic determinants of oxidative cell and genome damage and repair to prostate cancer risk in population studies. Each of the Research Projects features translation of innovative scientific concepts to human clinical trials or to population studies. The Research Projects are supported by Core Resources for Administration (Core #1), for a Tissue Archive (Core #2), for Biomarker Development (Core #3), and for Biostatistics (Core #4). PROJECT 1 Prostate cancer remains uniformly fatal once it reaches the hormone-refractory state because current therapies are unable to completely eliminate androgen independent prostate cancer cells. Emerging clinical data suggests that current chemotherapies may prolong survival in a subset of men but these agents have significant dose limiting toxicities. In this proposal, we outline a strategy to target thapsigargin (TO), a potent inhibitor of a ubiquitous intracellular protein, the SERCA pump, whose function is mandatory for survival of all cell types. TG can induce apoptosis at nanomolar concentrations in -a variety of cell types, including prostate cancers, in a proliferation independent manner TO’s cytotoxicity, however, is not cell-type specific and, therefore, TG could not be administered without significant systemic toxicity. To target TO specifically to sites of prostate cancer, a strategy is proposed that takes advantage of the fact that both normal and malignant prostate cells express in high levels the unique serine proteases Prostate-Specific Antigen (PSA) and Human Glandular Kallikrein 2 (hK2) which have the ability to hydrolyze specific peptide sequences. Since neither the blood nor normal tissue other than the prostate express high levels of enzymatically active PSA or hK2, the underlying hypothesis of this proposal is that the proteolytic activity of prostate-specific enzymatic activities (i.e PSA and hK2) can be used to activate prodrugs specifically to cytotoxic metabolites at sites of metastatic prostate cancer while sparing normal host tissue. To target TO’s cytotoxicity, prodrugs will be made consisting of a cytotoxic TG analog coupled to peptides that are recognized as substrates by either PSA or hK2. These inactive peptide-prodrugs can only be effectively hydrolyzed to liberate the cell permeant cytotoxic TO analogs by enzymatically active PSA or hK2. Thus, the specific aims of this proposal are: (1) to evaluate a series of TO peptide prodrugs in vitro in order to select the lead prodrugs for further drug development; (2) to define the lead TO prodrug based on its in vivo antitumor efficacy using human prostate cancer xenografts; (3) to determine whether coupling the lead prodrug identified in specific aim two to a polyethylene glycol (PEG) macromolecular carrier enhances in vivo antitumor efficacy; (4) to identify an hK2 specific peptide substrate and use this substrate to develop an hK2-activated TG prodrug according to strategy outlined in specific aims 1-3. The long-term goals of this project are to complete the necessary preclinical studies required to identify lead PSA and hK2 activated TO prodrugs that can be tested in clinical trials within the Johns Hopkins Oncology Center. PROJECT 2 Inactivation of critical regulatory genes is associated with the development and progression of human neoplasia. Loss of expression is associated with the acquisition of promoter region methylation and changes in histone acetylation leading to a repressive chromatin state. Attempts to relieve this transcriptional repression have lead to clinical trials combining DNA methyltransferase and histone deacetylase inhibitors at Johns Hopkins. These trials need improved methods to assess whether such treatments inhibit their molecular targets and whether relief of the transcriptional repression leads to restoration of gene expression. This translational research proposal seeks to determine the feasibility of gene re-expression as a therapeutic strategy in prostate cancer (PCA) in vitro, in animal models, and in PCA patients . Identification of specific genes important to induce a clinical benefit after therapeutic re-expression will be key to monitor therapy and to determine response outcome. Six aims are outlined. Aim 1 will utilize clinical trials combining inhibitors of methyltransferase (DNA MeTI) and histone deacetylase (HDI) to determine safety and tolerability, obtain early evidence of bioactivity and to gather tissue and plasma samples for analysis in subsequent aims. Aims 2 and 3 will optimize schedule and dose of DNA MeTI and HDI in vitro on identified and newly identified (through cDNA microarrays) targets of transcriptional repression in PCA cell lines. Specific Aims 4 and 5 optimizes methods for studying gene-re-expression in animal models and uses these methods to determine optimal in vivo inhibition of DNA MeTI and HDI. Gene function after restoration will be examined to examine clinical significance of this strategy. Aim 6 returns to the clinical samples in order to optimize and validate the assays developed in vitro and in vivo. Such molecular target assessment will define the limits and feasibility of gene re-expression therapy as a novel strategy for PCA patients. PROJECT 3 We propose that the highly aggressive and unrelentless phenotype of advanced prostate cancer might be overcome in part by utilizing prostate-specific, conditionally replicating adenoviruses. Such vectors are engineered by placing the primary replication regulatory genes (El region) under the control of a prostate specific promoter. In the past, we demonstrated that such a replicating virus is able to preferentially kill prostate cancer cells in vitro, in vivo and even in clinical trials. However, because the PSA promoter and enhancer require active androgen receptor for optimal expression, our vectors were initially restricted to patients with intact androgen production. Recent evidence in our lab has demonstrated that the E1A gene specifically inhibits androgen receptor function, resulting in an unanticipated attenuation of our initial constructs. We propose that fusing the E1A gene to portions of the androgen receptor will overcome this negative feedback loop and simultaneously widen the target population to include prostate cancer patients with androgen dependent and androgen independent disease. In addition, we have observed that combining radiation therapy with the oncolytic viral therapy results in a synergistic augmentation of cell kill, consistent with an adenoviral-mediated radio-sensitization. Such radio- sensitization likely involves the specific inhibition of certain DNA repair complexes by the adenoviral El and E4 proteins. We propose that since such repair complexes require phosphorylation by highly specific protein kinases, additional sensitization might be achieved by inhibiting those protein kinases (DNA-PK ATM and ATR). Thus, we propose that our current prostate specific oncolytic vectors can be improved by the use of tightly regulated prostate specific promoters, by engineering a chimeric fusion protein of E1A with the androgen receptor, by combining the oncolytic vectors with radiation therapy, by sensitization of the target cells to radiation by specific siRNA gene silencing of protein kinases important in DNA repair, and by introducing these incremental vector improvements in an neoadjuvant setting with 3D conformal external beam radiation therapy in high risk patients. PROJECT 4 Studies of the molecular pathogenesis of prostate cancer (PCA) have provided new insights into how PCAs arise and new clues as to how life-threatening PCA might be prevented: somatic inactivation of GSTP 1, the gene encoding the -class glutathione S-transferase, appears to play a critical early role in prostate cancer (PCA) development. Loss of GSTP 1 “caretaker” function renders prostate cells vulnerable to genome damage upon exposure to certain electrophilic and oxidant carcinogens; relentless genome damaging stresses over many years, with accumulating somatic genome abnormalities, may be what ultimately leads to life-threatening PCA. High dietary intake of fruits and vegetables, particularly cruciferous vegetables, may help overcome this vulnerability and reduce risks for life-threatening PCA. Consumption of certain cruciferous vegetables results in the release of isothiocyanate compounds, such as sulforaphane, that trigger the induction of carcinogen-detoxification and anti-oxidant enzymes, including glutathione S-transferases, UDP-glucuronosyl transferases, quinone reductases, etc. By augmenting cellular defenses against carcinogens, carcinogen-detoxification enzyme inducers might compensate for the inadequate carcinogen defenses in prostate cells that accompany somatic GSTP 1 inactivation, providing relief from genome damaging stresses and attenuating prostatic carcinogenesis. The translational research hypothesis to be addressed in this research project is that dietary carcinogen-detoxification enzyme inducers, such as sulforaphane, might protect against PCA development by increasing defenses against genome damage inflicted by electrophilic and oxidant carcinogens. To accelerate the translation of this hypothesis to human clinical studies, three Specific Aims are presented: (i) a determination of the relationship between dietary isothiocyanate intake and PCA risk in a prospective nested case-control study, (ii) an assessment of the chemopreventive activity of sulforaphane in a rat model of PCA driven by prostate inflammation and the dietary carcinogen 2-amino-i-methyl-6-phenylimidazo (PhIP), and (iii) the introduction of carcinogen-detoxification enzyme inducers into “proof-of-principle” clinical trials as PCA prevention agents. PROJECT 5 Genetic influences on prostate cancer susceptibility are strongly suggested by twin and family studies, although definitive progress with respect to specific gene identification has been slow. In this proposal, we plan to examine common sequence variations in genes affecting levels of oxidative stress in the prostate as potential risk factors for prostate cancer. Evidence supporting an i role for oxidative damage in prostate cancer etiology has been obtained from multiple sources, including epidemiologic studies implicating anti-oxidants as risk lowering agents for prostate cancer, molecular studies demonstrating ubiquitous inactivation of GST B, a key cellular defense mechanism against oxidative damage, in prostate carcinogenesis, and the implication of oxidative damage in the formation of Proliferative Inflammatory Atrophy PJA the recently described precursor lesion for prostate cancer. Because of this potential role in carcinogenesis, we propose that a critical subset of genes to examine in terms of susceptibility to prostate cancer are those involved in the generation and disposition Of reactive oxygen species. Specifically, we will test the hypothesis that sequence variants of such genes are associated with prostate cancer risk. The list of genes to be studied will include genes previously implicated in the biology of ROS (e.g. oxidases, peroxidases, transferases, DNA repair enzymes), with priority given to those with abundant expression in normal, preneoplastic or cancerous prostatic tissue as determined by cDNA microarray analysis. We will expand our existing microarray analyses of prostate tissues to include both PIN and PIA lesions to finalize the set of candidate genes. Mass spectrometry will then be used to evaluate polymorphisms of these candidate genes in highly informative study populations of 400 prostate cancer cases and 400 matched controls that are part of an ongoing case-control study. We anticipate that multiple genes will emerge as having allelic variants that confer increased risk of prostate cancer. The subset of genes with the strongest association with prostate cancer risk will then be validated’ in an independent, well-characterized case-control population sampled from within an ongoing cohort study. Besides clues that the proposed research may provide in terms of identification of new targets for therapy and possible preventive strategies for prostate cancer, this study will define a series of genetic markers that may form a panel to identify men at high risk. Such a panel could be used for risk stratification in clinical trials to identify men who may benefit most from a particular form of chemotherapy or chemoprevention aimed at reducing oxidative damage in the prostate. CORE 1 The Administrative Core will be responsible for managerial oversight of all Johns Hopkins Prostate Cancer SPORE Program activities In addition, the Core will help facilitate interactions between Johns Hopkins Prostate Cancer SPORE Program Investigators and Investigators associated with other Prostate Cancer -- SPORE Programs, and help orchestrate productive responses to new National Cancer Institute initiatives The managerial structure of the Johns Hopkins Prostate Cancer SPORE Program, With its Principal Investigator, Co-Principal Investigator, Executive Committee, Internal Oversight Committee, and External Scientific Advisory Board, has been designed to promote translational research by creating a prostate cancer research culture that transcends academic Departments, medical disciplines, and individual research skills, and by providing with high quality -monitoring, evaluation, and oversight of the SPORE portfolio of - - Research Projects, Core Resources,-the Career Development Program, and the Developmental Research Program. The Administrative Core will provide communications resources, including teleconferencing, travel funds, and administrative staffing for all its managerial activities. CORE 2 The Pathology Core will procure, bank, and distribute prostate tissues to promote translational prostate cancer research by SPORE and other investigators, and, will work closely with each investigator to facilitate the achievement of the specific aims of the individual projects. To carry out these functions, our specific aims are the following: 1) To maintain and enhance a repository of prostate tissues containing neoplastic and non-neoplastic samples from both fresh frozen and paraffin blocks and to distribute these samples to SPORE investigators. 2) To continue to design and produce tissue microarrays using human prostate tissues, cell lines, and xenografts. 3) To provide histopathologic diagnoses of tissue specimens and to perform immunohistochemistry (IHC), interpretation and analysis of IHC slides. 4) To provide a facility and pathology expertise for laser capture microdissection. 5) To provide a platform for gene expression profiling using cDNA microarrays. 6) To function as a national resource for the distribution of prostate tissue and tissue microarrays. Approximately 1000 radical prostatectomies are performed per year at our institution. Approximately 5% of these are from African Americans and this number is increasing. We currently have 2941 frozen tissue blocks from 723 patients, 1407 of which have been annotated. We have 41 tissue arrays containing over 10,000 tissue cores from prostate specimens from 662 patients, 220 of which have very long term follow-up data. The core facility has developed an open source tissue microarray database and imaging system that can be used for collaborative studies of biomarkers. The database houses all pathology information from the specimens in our collection and has been redesigned to assure the protection of patient confidentiality and privacy. The database is linked to those presently available in the department of Urology and Core #4 which contains detailed clinical follow-up information. We currently have over 8,900 patients who have had radical prostatectomies at our institution since 1982 in the Urology and pathology clinical databases. We will work closely with Core #4 to develop additional tissue microarrays from this population. The continued enhancement of these valuable pathology resources will facilitate development of new prevention and treatment strategies. CORE 3 The Biomarker Development Core (3) will be involved in translational research related to: 1) development of clinically useful assays for biomarkers discovered within this SPORE project, 2) testing the clinical utility of new biomarkers on large series of banked sera 3) discovery of novel biomarkers for prostate cancer using SELDI a technique available within our facility. Drs. Partin and Chan are NCI- EDRN (Early Detection Research Network) investigators and Dr. Partin serves on the Steering Committee for the EDRN. Through this important resource, we have/are archiving over 1000 serum samples per year from men undergoing early detection, staging, treatment and monitoring for prostate cancer. These samples will be available through an EDRN approval process for biomarker testing. In addition, through this resource, we will have access to other important EDR including urine, and plasma (archived by other EDRN: investigators). Finally, Dr. Chan is the director of an Industrial sponsored SELDI facility (Ciphergen Biosystems) and has access to proteomic technology and supplies through this relationship which will allow us to investigate new biomarkers utilizing this new technology. In summary this core facility will have a team with a long track-record in biomarker development testing, a vast collection of biomaterial for translational testing of new biomarkers, and available technology to develop and discover new biomarkers for prostate cancer to support this SPORE application. CORE 4 The Biostatistics Core of the proposed Johns Hopkins SPORE in prostate cancer will consist of experienced biostatisticians and database programmers from the Oncology Center, the Departments of Urology, Epidemiology and Pathology. A long history of collaboration already exists between the director of the core and several principal investigators, including the SPORE P1. The core is designed to:
The Core will have an integral role in the scientific development, execution, analysis of all projects in the SPORE. Core investigators have extensive and complementary experiences in quantitative methods for biomedical applications, including both clinical and basic science studies. They are committed to taking a direct interest in the substantive issues being investigated; to participating in regular project and program meetings, and to providing rigorous and innovative input on all quantitative matters arising in the projects. By contributing to multiple projects, they will also be in a position to promote interdisciplinary interactions among projects. The Johns Hopkins Prostate SPORE Investigators and Addresses John T. Isaacs, Ph.D. Samuel Denmeade, M.D. Michael Carducci, M.D. James Herman Roberto Pili, M.D. Sharyn D. Baker, Pharm.D. Ming Zhao, Ph.D. Ronald Rodriguez, M.D., Ph.D. Theodore DeWeese, M.D. William G. Nelson, M.D., Ph.D. H. Ballentine Carter, M.D. Thomas Kensler, Ph.D. Bruce W. Trock, Ph.D. Angelo DeMarzo, M.D., Ph.D. William B. Isaacs, Ph.D. Kathy J. Helzlsouer, M.D. Elizabeth Platz, M.P.H., Sc.D. Jonathan Epstein, M.D. Jun Luo, Ph.D. Alan W. Partin, M.D., Ph.D. Daniel Chan, Ph.D. Steven Piantadosi, M.D., Ph.D. |
 |
|||
|
|