Metastatic androgen independent prostate cancer (AIPCA) is the major cause of prostate cancer related deaths in men with no effective treatment for these patients. Therefore preventing or delaying development of AIPCA will improve the quality of life for prostate cancer patients. Based on preliminary data from our laboratory we hypothesize that upregulation of FLIP leads to the development of androgen independent disease;therefore down regulation of FLIP transcriptional activity is sufficient to prevent the development of AIPCA .
Specific aim 1 : Establish the efficacy of 2-ME in inhibiting the development of AIPCA in the TRAMP model. 12-week- old TRAMP mice will be castrated to develop androgen independent tumors. Escalating doses of 2-ME will be provided in drinking water for 6 weeks starting at 12 weeks of age. Suppression of androgen independent tumors will be monitored by (i) sequential non-invasive magnetic resonance imaging (MRI) every two weeks;(ii) histological evaluation of the prostate at the termination of the experiment and (iii) determination of tumor- free survival. Expression of FLIP signaling components, factors that regulate FLIP induction (identified from aims 2 and 3), proliferation and apoptosis will be analyzed in prostate tumor/tissue from these animals. Serum levels of 2-ME and its metabolites will be measured to correlate with its efficacy in preventing the development of AIPCA.
Specific aim 2 : Determine the mechanism through which (a) androgens activate expression of FLIP and (b) 2-ME down regulates this activation. Experiments have been proposed to identify and characterize transcription factors involved in androgen-induced transcriptional activation of FLIP promoter activity and its modulation in response to 2-ME using biochemical and molecular approaches such as transient expression assays using FLIP deletion constructs, co-transfections, gel mobility shift assays, DNase I foot printing and ChIP assays.
Specific aim 3 : Determine the precise role of FLIP in 2-ME induced apoptosis. Experiments have been designed using stable cell lines of androgen-responsive and -independent cells lacking or overexpressing the identified transcription factor to establish whether 2-ME induces apoptosis by (i) reducing the levels and activity of the identified transcription factor with concomitant reduction in the levels of FLIP;(ii) preventing the recruitment of FLIP into DISC;and (iii) whether transcriptional regulation is sufficient for the development of AI phenotype through the activation of FLIP.
Specific aim 4 : Determine whether activation of FLIP predicts clinical disease outcome. We will assess the prevalence of FLIP expression in human prostate tumors that are hormone refractory and hormone naove using immunohistochemistry. Data obtained from the proposed experiments will evaluate the potential of 2-ME in the prevention of AIPCA, provide mechanistic insight into FLIP signaling pathway in AIPCA and identify novel markers for AIPCA. Although androgen ablation inhibits prostate cancer growth temporarily, eventual recurrence of androgen independent disease is the major cause of prostate cancer related deaths in men. These androgen independent tumors are resistant to existing chemotherapeutic agents. Therefore development of novel agents targeting critical signaling pathways involved in the development of recurring androgen independent prostate tumors is a high priority. Preclinical studies proposed in this application targeting Fas-associated death domain like interleukin-1 converting enzyme like inhibitory protein (FLIP) signaling to prevent the development of androgen independent prostate cancer will have tremendous translational potential for prevention of androgen independent prostate cancer in humans.
Although androgen ablation inhibits prostate cancer growth temporarily, eventual recurrence of androgen independent disease is the major cause of prostate cancer related deaths in men. These androgen independent tumors are resistant to existing chemotherapeutic agents. Therefore development of novel agents targeting critical signaling pathways involved in the development of recurring androgen independent prostate tumors is a high priority. Preclinical studies proposed in this application targeting Fas-associated death domain like interleukin-1 converting enzyme like inhibitory protein (FLIP) signaling to prevent the development of androgen independent prostate cancer will have tremendous translational potential for prevention of androgen independent prostate cancer in humans.
