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Northwestern University
Project 1 Clusterin is a sulfated glycoprotein, which is expressed in almost all cells. Its level in benign prostate is relatively low. Prostate cancer cells express more clusterin. The intensity immunohistochemical staining for clusterin in prostate cancer tissues correlates with the Gleason scores. Our recent results have demonstrated that clusterin is a novel antiapoptotic mediator in prostate cancer. The objective of the present application is to translate these laboratory observations into clinical settings. We propose to establish its utility as a prognostic marker in prostate cancer. Our hypothesis is that the level of clusterin expression is associated with resistance to apoptosis in prostate cancer cells, thus may serve as a prognostic marker. Four specific aims are proposed. Specific Aim 1 will manipulate the level of clusterin expression in human established prostate cancer cells, LNCaP, by our newly developed retrovirus gene transfer technique. We propose to inoculate 50% clusterin over-expressed cells (GFP) and 50% wild type LNCaP cells into orthotopic sites of nude mice. Results will be compared with that from the control tumors in which empty vector-GFP transfected cells (50%) will be mixed with wild type (50%). Specific Aim 2 will be a study to predict tumor development in high grade PIN. Needle biopsy specimens, containing high grade PIN, will be used for immunohistochemical staining for clusterin. In high-grade PIN specimens, we attempt to correlate the level of clusterin and bcl-2 expression with those that later developed prostate cancer. Specific Aim 3 is also a retrospective study in which prostatic tissues obtained from archival specimens will be used. Prostatectomy patients with an undetectable serum PSA will be selected for the study. The level of clusterin and bcl-2 expression in each case will be correlated with the Gleason grade. A rise in serum PSA will be considered as tumor recurrence. Specific Aim 4 will be a prospective study, in which clusterin levels in serum specimens, along with PSA and PAP, will be determined in prostate cancer patients who were diagnosed of having metastatic disease and receiving hormonal therapy. Serum levels of PSA, PAP, or clusterin will be used to correlate with failure to androgen ablation therapy. Results will be analyzed to determine if serum clusterin levels can be used, along with serum PSA and PAP, to predict endocrine treatment failure.
Project 2 The long-term objective of this proposal is to elucidate the role of androgen-response genes in prostate cancer pathogenesis. Androgen controls homeostasis of the prostate through androgen action pathway, a cascade of molecular and cellular events triggered by androgen leading to cell proliferation, differentiation, and/or death. Our preliminary studies indicate that part of androgen action pathway, via calreticulin, is essential for maintaining anchorage-dependence of prostatic epithelial cells. Our preliminary studies showed that calreticulin is abundantly expressed and regulated by androgen in prostatic epithelial cells. Further analysis showed that calreticulin can regulate cell adhesion and intracellular Ca ++ homeostasis in cultured prostate cancer cells. Our recent data indicate that calreticulin overexpression markedly suppressed anchorage-independent growth of prostate cancer cells in soft agar and inhibited metastasis of xenograft tumors in nude mice. Immunohistochemical staining of human prostate cancer specimens indicated that calreticulin expression is frequently down-regulated in prostate cancer cells in vivo and that the down-regulation is more frequent in high Gleason grade tumors. Our research hypothesis is that escape from androgen-dependent anchorage-dependence, via calreticulin down-regulation, is an essential step leading to prostate cancer metastasis . Four specific aims are proposed.
Project 3 For tumors to grow and metastasize, they induce the generation of new blood vessels, a process referred to as angiogenesis. Inhibition of angiogenesis, thus provides an important new therapeutic target. The angiogenesis inhibitor angiostatin is a kringle containing internal fragment of plasminogen, which has been shown to inhibit cancer growth in numerous animal models, including prostate cancer. In prior research by Soff and colleagues, the mechanism by which plasminogen is converted to angiostatin was elucidated, which involves plasmin autoproteolysis within kringle 5 [Gately et al, 1997] [Gately et al, 2000]. The isoform resulting from this process, referred to as Angiostatin4.5 (AS4.5) since it includes kringles 1 through 4 and most of 5, has been shown to be a naturally occurring angiogenesis inhibitor [Gately et al, 2000]. Most recently, we have demonstrated that the human PC-3 prostate carcinoma cell line expresses a high affinity receptor for plasmin on the cell surface, which binds plasmin and facilitates proteolytic conversion of plasmin to AS4.5. Plasmin, when bound to the PC-3 receptor, thus undergoes autoproteolysis, to yield AS4.5. The plasmin receptor protects plasmin catalytic activity from the neutralizing activity of 2-antiplasmin ( 2AP), found in plasma, which may explain how Angiostatin4.5 is generated in vivo, despite the presence of 2AP in plasma. In this project, we shall: (1) Complete the purification, and identification of the plasmin receptor, and determine which cells and under what circumstances the receptor is expressed, (2) Determine the mechanism by which the plasmin receptor promotes plasmin autoproteolysis, (3) Determine if the levels of the receptor correlate with the plasma levels of Angiostatin4.5 in patients with cancer, and (4) determine if the levels of the receptor correlate with the local and metastatic progression in patients with prostate cancer.
Project 4 Erectile dysfunction (ED) is a devastating complication of treatment for prostate cancer. Post-radical prostatectomy ED (pRP-ED) is a major quality of life issue with social and financial ramifications for patients. All treatments for pRP-ED are met with significantly lower success rates when compared to other ED groups (i.e., diabetes, etc). This complication occurs despite attempts to preserve cavernous nerve (CN) integrity with bilateral or unilateral nerve-sparing prostatectomies. The exact mechanism involved with post-radical prostatectomy (pRP) ED is not clear other than the obvious CN nerve interruption during dissection and the development of penile fibrosis. It is currently thought that postoperative penile fibrosis despite normal arterial flow is the cause of ED. If we are to impact the incidence of this complication and improve the chances of successful rehabilitation then a better understanding of the mechanism of injury is required. This will require knowledge of the molecular signaling events responsible for pRP-ED. Once the mechanisms are elucidated avenues for intervention and prevention will be expanded. Our goal is to characterize the molecular mechanisms of pRP-ED using our animal model. We postulate that pRP-ED is a result of downregulated NOS-I, TGFb1 and ET-1. We hypothesize that downregulated penile TGFb1 and ET-1 induce a cascade of molecular events that culminate in the loss of penile smooth muscle (SM) cells and their replacement with a less compliant extracellular matrix (ECM) (fibrosis). Four specific aims are proposed: 1) to test the hypothesis that nitric oxide synthase isoforms are altered after CN injury and result in a decreased TGFb1, 2) to test the hypothesis that expression of NOS-I and NOS-II in CN injured penes is downregulated, 3) to test the hypothesis that protein content and expression of ET-1 and ET-B receptor are decreased with CN injury, 4) to test the hypothesis that the CN injury impacts the % of SM, ECM and fibrosis. Results will aid the understanding of how CN injury contributes to ED and elucidate preventative mechanisms.
Project 5 Our laboratory seeks to understand the molecular regulation of metastatic behavior in human prostate cancer, and to therapeutically manipulate that process. Epidemiological evidence suggest that consumption of genistein, a constituent of soy and a putative prostate cancer chemoprevention agent, is associated with a lower incidence of metastatic prostate cancer, as well as significant decreases in prostate cancer mortality. Laboratory investigations by our group have identified genistein-mediated decreases in matrix metalloproteinase (MMP) and cell invasion as potentially causal. We hypothesize that specific effector pathways are necessary, and possible unique, regulators of MMP-2 and cell invasion in human prostate cancer, and that they play a role in the therapeutic modulation of these processes by genistein. Seeking to understand the regulation and therapeutic modulation of metastatic behavior in human prostate cancer, our group has provided key insights. We were the first ot show tha prostate cancer cell motility was inhibited by a chemopreventive agent, thus identifying this as a therapeutic target for future agents. Investigating mechanism, we showed that the TGFβ binding protein, endoglin, acts to suppress motility in human prostate, and that TGFβ-mediated effects upon adhesion and invasion require p38 MAP kinase, which turns out to be a target of genistein action. We have completed a phase I study of genistein, demonstrating for the first time that diet can modulate cell signaling in humans, and have now implemented a phase II study to measure antimetastatic activity, both studies in men with prostate cancer. The specific aims of the project are:
For both aims, genstein’s effect underlying processes will be evaluated, first in model systems, and then in humans.
ADMINISTRATIVE CORE The Administrative Core of the SPORE Program provides effective leadership and program coordination to all participants in the SPORE Program. In addition to managing the finances for all SPORE projects and Cores, the Administrative Core organizes monthly SPORE meetings, quarterly meetings of the Internal Advisory Committee and the yearly meeting of the External Advisory Committee. The Core generates the progress reports to the National Cancer Institute. The Core is responsible for overseeing the Developmental Research Program and the Career Development Program. The Administrative Core is also responsible for sending investigators to interact with other SPORE investigators at the National SPORE meeting in July and the yearly prostate inter-SPORE meeting to be held in Chicago in 2004. All of these responsibilities are accomplished by the SPORE directors, Scientific Program Director, Program Administrator and Grants and Contracts Administrator.
TISSUE PROCUREMENT CORE The Tissue Procurement Core of the SPORE in Prostate Cancer Program is successfully procuring tissue samples, blood samples (serum, plasma, cells and blood spots) and urine samples from those patients who consent to participate in the IRB approved SPORE sample procurement. Formalin-fixed paraffin-embedded tissue is available on each case. As of February, 2004, 553 patients have consented to participate in SPORE sample procurement (475 from Northwestern Memorial Hospital, 51 VA Lakeside and 27 from Evanston Northwestern Healthcare). All of the patient clinical data including demographic information, pathologic data, and laboratory test data are being entered into a dedicated prostate SPORE database designed by the SPORE bioinformatics group. In addition, all tissue data is entered into a separate, but linked tissue database. The Core is in the process of conducting quality control on tissue samples and database information. Services provided by Tissue Procurement Core:
BIOSTATISTICAL/BIOINFORMATICS CORE The Biostatistics/Bioinformatics Core designs and maintains the Prostate SPORE database of clinical and tissue specimen data for all patients who consent to participate in the SPORE study. The Core has expertise in database technology, systems programming and data entry which is essential to managing all aspects of SPORE informatics. The SPORE database is web-based application written in Cold Fusion. There are 165 variables in the Clinical database. There is a separate, but related Tissue database with 30 tissue-specific variables in addition to relevant clinical and demographic information. The Biostatistics/Bioinformatics Core meets weekly with the Tissue Procurement Core to discuss issues regarding data acquisition and analysis. The Biostatistics/Bioinformatics Core provides SPORE investigators with biostatistical input relating to study design and data analyses. Dr. Jovanovic assisted Dr. Bergan with his project on endoglin in human prostate cancer. He has also provided statistical analysis for Dr. Gann related to his use of biomarkers in predicting prostate cancer related outcomes. Drs. Soff and Jovanovic are collaborating to analyze data on angiostatin in human prostate cancer specimens. |
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