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）的发生和发展中起着重要作用。大多数PCa标本表达高水平的AR，这是将雄激素依赖型（AR+） PCa转化为临床更具侵袭性的雄激素非依赖型（AR-） PCa的必要和充分条件。尽管抑制AR活性是AR+ PCa治疗的主要方法，但对于AR- PCa尚无有效的治疗方法，这是一致致命的。研究表明，AR和Akt相互激活，AR和Akt共同抑制促凋亡信号，增强AR+向AR- PCa的转化。同时靶向AR和Akt信号是治疗AR+ PCa和防止AR- PCa发生的有效治疗方法。我们最近证明了Withaferin-A （WA）是一种天然化合物，通过抑制磷酸化的Akt特异性靶向AR- PCa细胞；在细胞培养和动物模型中，这种抑制导致前列腺凋亡反应-4 （Par-4）依赖性凋亡的激活。同样，在AR+ PCa细胞中，抑制AR有助于wa诱导的Par-4激活和凋亡。Akt或AR过表达均可抑制wa介导的Par-4激活，阻断AR+ PCa细胞的凋亡，提示AR和Akt均负向调节Par-4在PCa细胞中的促凋亡功能。我们假设WA联合激素消融是控制前列腺癌进展的有效策略。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（通过报告基因构建、AR抗体的ChIP检测和促凋亡蛋白和抗凋亡蛋白的Western分析研究AR与Par-4启动子的结合）；目标2。研究Par-4与Akt调控的FOXO3a之间的关系，以及WA如何在AR+ PCa中调节这种相互作用（通过报告基因构建、FOXO3a抗体的ChIP分析、迁移位移分析、促凋亡蛋白和抗凋亡蛋白的Western分析、FOXO3a过表达后的TUNEL和annexin-V分析来研究FOXO3a与Par-4启动子的结合）；和Aim 3。确定WA对前列腺特异性PTEN敲除小鼠（PS PTEN- ko；进行WA对PCa的体内疗效研究、组织病理学、促生存和促凋亡蛋白的免疫组化、增殖指数、凋亡和血清WA水平）的化疗作用。我们期望这些研究将提供WA作用于AR+ PCa的分子机制，并最终为PCa的治疗提供新的治疗策略。

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.