TARGETING FLIP FOR PROSTATE CANCER PREVENTION

描述（由申请人提供）： 转移性雄激素非依赖性前列腺癌（AIPCA）是男性前列腺癌相关死亡的主要原因，对这些患者没有有效的治疗。因此，预防或延迟AIPCA的发展将改善前列腺癌患者的生活质量。基于我们实验室的初步数据，我们假设FLIP的上调导致雄激素非依赖性疾病的发展;因此FLIP转录活性的下调足以防止AIPCA的发展。具体目标1：在TRAMP模型中确定2-ME抑制AIPCA发展的功效。将12周龄的TRAMP小鼠阉割以产生雄激素非依赖性肿瘤。从12周龄开始，将在饮用水中提供递增剂量的2-ME，持续6周。雄激素非依赖性肿瘤的抑制将通过以下方式进行监测：（i）每两周进行一次连续的非侵入性磁共振成像（MRI）;（ii）实验结束时对前列腺进行组织学评价;（iii）确定无肿瘤生存期。将在这些动物的前列腺肿瘤/组织中分析FLIP信号传导组分、调节FLIP诱导的因子（从目的2和3中鉴定）、增殖和凋亡的表达。将测量2-ME及其代谢物的血清水平，以与其预防AIPCA发展的功效相关联。具体目标2：确定（a）雄激素激活FLIP表达和（B）2-ME下调该激活的机制。已经提出了实验来鉴定和表征参与雄激素诱导的FLIP启动子活性的转录激活及其响应于2-ME的调节的转录因子，所述实验使用生物化学和分子方法，例如使用FLIP缺失构建体的瞬时表达测定、共转染、凝胶迁移率变动测定、DNA酶I足迹法和ChIP测定。具体目标3：确定FLIP在2-ME诱导的细胞凋亡中的确切作用。使用缺乏或过表达所鉴定的转录因子的雄激素应答性和非依赖性细胞的稳定细胞系设计了实验，以确定2-ME是否通过（i）降低所鉴定的转录因子的水平和活性，同时降低FLIP的水平;（ii）防止FLIP募集到DISC中;和（iii）转录调节是否足以通过FLIP的激活发展AI表型。具体目标4：确定FLIP的激活是否可预测临床疾病结局。我们将使用免疫组织化学方法评估FLIP在激素难治性和激素缺乏的人前列腺肿瘤中的表达率。从拟议的实验中获得的数据将评估2-ME在预防AIPCA中的潜力，提供AIPCA中FLIP信号通路的机制见解，并确定AIPCA的新标记物。虽然雄激素消融术暂时抑制了前列腺癌的生长，但雄激素非依赖性疾病的最终复发是男性前列腺癌相关死亡的主要原因。这些雄激素非依赖性肿瘤对现有的化疗剂具有抗性。因此，开发靶向参与复发性雄激素非依赖性前列腺肿瘤发展的关键信号通路的新型药物是一个高度优先事项。本申请中提出的靶向Fas相关死亡结构域样白细胞介素-1转化酶样抑制蛋白（FLIP）信号传导以预防雄激素非依赖性前列腺癌发展的临床前研究将对预防人类雄激素非依赖性前列腺癌具有巨大的转化潜力。尽管雄激素消融术暂时抑制了前列腺癌的生长，但雄激素非依赖性疾病的最终复发是男性前列腺癌相关死亡的主要原因。这些雄激素非依赖性肿瘤对现有的化疗剂具有抗性。因此，开发靶向参与复发性雄激素非依赖性前列腺肿瘤发展的关键信号通路的新型药物是一个高度优先事项。本申请中提出的靶向Fas相关死亡结构域样白细胞介素-1转化酶样抑制蛋白（FLIP）信号传导以预防雄激素非依赖性前列腺癌发展的临床前研究将对预防人类雄激素非依赖性前列腺癌具有巨大的转化潜力。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE 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.