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)是前列腺癌相关死亡的主要原因，这些患者没有有效的治疗方法。因此，预防或延缓前列腺癌的发展将提高前列腺癌患者的生活质量。根据我们实验室的初步数据，我们假设Flio的上调会导致雄激素非依赖性疾病的发展；因此，Flio转录活性的下调足以防止AIPCa的发展。具体目的1：确定2-ME在TRAMP模型中抑制AIPCa发展的有效性。12周大的流浪鼠将被阉割，患上雄激素非依赖性肿瘤。从12周龄开始，饮用水中将增加剂量的2-ME，为期6周。对雄激素非依赖性肿瘤的抑制将通过(I)每两周进行一次连续的非侵入性磁共振成像(MRI)；(Ii)在实验结束时对前列腺进行组织学评估；(Iii)确定无肿瘤存活率。在这些动物的前列腺肿瘤/组织中，将分析FliP信号组件的表达、调节FliP诱导的因子(从AIMS 2和3中确定)、增殖和凋亡。2-ME及其代谢物的血清水平将被检测，以与其预防AIPCA发展的有效性相关。具体目标2：确定(A)雄激素激活FliP表达和(B)2-ME下调这种激活的机制。已有实验建议使用生化和分子方法来鉴定和表征参与雄激素诱导的翻转启动子活性转录激活及其对2-ME的调控的转录因子，例如使用翻转缺失构建的瞬时表达分析、共转染、凝胶迁移率改变分析、DNase I足印和芯片分析。具体目标3：确定FLIP在2-ME诱导的细胞凋亡中的确切作用。实验用稳定的雄激素反应和缺乏或过度表达已识别的转录因子的独立细胞系来确定2-ME是否通过(I)降低已识别的转录因子的水平和活性并伴随着FLIP水平的降低而诱导细胞凋亡；(Ii)防止FLIP被招募到盘中；以及(Iii)转录调控是否足以通过激活FLIP来促进AI表型的发展。具体目标4：确定FLIP的激活是否可以预测临床疾病的结果。我们将使用免疫组织化学方法评估激素不敏感和激素不稳定的前列腺癌中FLIP表达的发生率。这些实验数据将评估2-ME在预防AIPCA中的潜力，为AIPCA中翻转信号通路的机制提供洞察力，并为AIPCA寻找新的标记。虽然雄激素治疗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药物具有耐药性。因此，针对雄激素非依赖性前列腺癌复发过程中涉及的关键信号通路的新型药物的开发是当务之急。本申请中提出的针对Fas相关死亡结构域的临床前研究，如白细胞介素1转换酶样抑制蛋白(FLIP)信号转导，以防止雄激素非依赖性前列腺癌的发展，将在预防人类雄激素非依赖性前列腺癌方面具有巨大的翻译潜力。公共卫生相关性虽然雄激素治疗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药物具有耐药性。因此，针对雄激素非依赖性前列腺癌复发过程中涉及的关键信号通路的新型药物的开发是当务之急。本申请中提出的针对Fas相关死亡结构域的临床前研究，如白细胞介素1转换酶样抑制蛋白(FLIP)信号转导，以防止雄激素非依赖性前列腺癌的发展，将在预防人类雄激素非依赖性前列腺癌方面具有巨大的翻译潜力。

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.