Targeting AR and Akt for the Treatment of Prostate Cancer

描述（由申请人提供）：雄激素受体（AR）在前列腺癌（PCa）的发生和进展中起重要作用。大多数PCa标本表达高水平的AR，这是必要的，足以将雄激素依赖性（AR+）PCa转化为临床上更具侵略性的雄激素非依赖性（AR-）PCa。尽管AR活性的抑制是AR+ PCa治疗的主要手段，但是对于一致致死的AR-PCa没有有效的疗法。研究表明AR和Akt相互激活，并且AR和Akt一起抑制促凋亡信号传导并增强AR+向AR-PCa的转化。靶向AR和Akt信号传导代表了治疗AR+ PCa和预防AR-PCa出现的有力治疗方法。我们最近证明了Withaferin-A（WA），一种天然化合物，通过抑制磷酸化Akt特异性靶向AR-PCa细胞;这种抑制导致细胞培养物和动物模型中前列腺凋亡反应-4（Par-4）依赖性凋亡的激活。类似地，AR+ PCa细胞中AR的抑制促进WA诱导的Par-4活化和凋亡。Akt或AR的过表达抑制WA介导的Par-4活化，并阻断AR+ PCa中的凋亡，表明AR和Akt都负调节PCa细胞中Par-4的促凋亡功能。我们假设WA联合激素消融是控制PCa进展的有效策略。为了解决这个假设，我们将：目标1。表征AR信号传导和Par-4之间的分子联系，并确定WA对AR+ PCa细胞的影响（通过报告构建体研究AR与Par-4启动子的结合，使用AR抗体的ChIP测定，以及促凋亡蛋白和抗凋亡蛋白的Western分析）;目的2.检查Par-4和FOXO 3a之间的关系（由Akt调节），以及WA如何调节AR+ PCa中的这种相互作用（研究FOXO 3a通过报告基因构建体与Par-4启动子的结合，使用FOXO 3a抗体的ChIP测定，迁移率变化测定，促凋亡和抗凋亡蛋白的Western分析，以及FOXO 3a过表达后的TUNEL和膜联蛋白-V测定）;以及Aim 3。在前列腺特异性PTEN敲除小鼠中确定WA对PCa发展的化疗效果（PS PTEN-KO;对PCa进行体内WA功效研究、组织病理学、促存活和促凋亡蛋白的免疫组织化学、增殖指数、凋亡和血清WA水平）。我们预计这些研究将提供分子深入了解WA对AR+ PCa的作用机制，并最终导致治疗PCa的新的治疗策略。

DESCRIPTION (provided by applicant): The androgen receptor (AR) plays an important role in the development and progression of prostate cancer (PCa). Most PCa specimens express high levels of AR, which is necessary and sufficient to convert androgen-dependent (AR+) PCa to the clinically more aggressive androgen-independent (AR-) PCa. Although inhibition of AR activity is a mainstay of AR+ PCa treatment, there are no effective therapies for AR- PCa, which is uniformly lethal. Studies reveal AR and Akt mutually activate one another, and that together AR and Akt inhibit pro-apoptotic signaling and enhance the conversion of AR+ to AR- PCa. Targeting both AR and Akt signaling represents a powerful therapeutic approach for treating AR+ PCa and preventing the emergence of AR- PCa. We recently demonstrated Withaferin-A (WA), a natural compound, specifically targets AR- PCa cells by inhibiting phosphorylated Akt; this inhibition leads to the activation of prostate apoptosis response-4 (Par-4)-dependent apoptosis in cell culture and in animal models. Similarly, inhibition of AR in AR+ PCa cells facilitates WA-induced Par-4 activation and apoptosis. Over-expression of either Akt or AR inhibits WA-mediated Par-4 activation, and blocks apoptosis in AR+ PCa, suggesting both AR and Akt negatively regulate the pro-apoptotic functions of Par-4 in PCa cells. We hypothesize that WA in combination with hormone ablation is an effective strategy to control the progression of PCa. To address this hypothesis, we will: Aim 1. Characterize the molecular link between AR signaling and Par-4, and determine the effects of WA on AR+ PCa cells (investigating AR binding to the Par-4 promoter via reporter constructs, ChIP assays using AR antibodies, and Western analysis of pro- and anti-apoptosis proteins); Aim 2. Examine the relationship between Par-4 and FOXO 3a, which is regulated by Akt, and how WA modulates this interaction in AR+ PCa (studying FOXO3a binding to the Par-4 promoter via reporter constructs, ChIP assays using FOXO3a antibodies, mobility shift assays, Western analysis of pro- and -anti-apoptosis proteins, and TUNEL and annexin-V assays following FOXO3a over-expression); and Aim 3. Determine the chemotherapeutic effects of WA on the development of PCa in prostate specific PTEN knockout mice (PS PTEN-KO; performing in vivo WA efficacy studies on PCa, histopathology, immunohistochemistry of pro-survival and pro-apoptotic proteins, proliferation index, apoptosis, and serum WA levels). We anticipate these studies will provide molecular insight into the mechanism of WA action against AR+ PCa, and will ultimately lead to novel therapeutic strategies for the treatment of PCa.