Targeting AR and Akt for the Treatment of Prostate Cancer

描述(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标本都表达高水平的AR，这是将雄激素依赖型(AR)PCa转化为临床上更具侵袭性的雄激素非依赖性(AR-)PCa所必需且充分的条件。虽然抑制AR活性是AR-PCa治疗的主要方法，但AR-PCa没有有效的治疗方法，因为AR-PCa都是致命的。研究表明，AR和Akt相互激活，AR和Akt共同抑制促凋亡信号，促进AR向AR-PCA的转化。同时靶向AR和Akt信号通路是治疗AR-PCa和预防AR-PCa发生的有效治疗手段。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来特异性地靶向AR-PCA细胞；这种抑制导致在细胞培养和动物模型中激活依赖于前列腺癌凋亡反应-4(PAR-4)的细胞。同样，AR-Pca细胞中AR的抑制促进了WA诱导的PAR-4的激活和凋亡。Akt或AR过表达均可抑制Wa介导的PAR-4激活，并阻断AR-Pca细胞的凋亡，提示AR和Akt均负向调节Pca细胞中Par-4的促凋亡功能。我们推测，WA联合激素消融是控制PCa进展的有效策略。为了解决这一假设，我们将：目的1.表征AR信号与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、使用AR抗体的芯片分析以及促和抗凋亡蛋白的Western分析来研究AR与PAR-4启动子的结合)；目的2.研究受Akt调控的PAR-4和FOXO-3a之间的关系，以及WA如何调节AR-Pca中的这种相互作用(通过构建报告结构、使用FOXO3a抗体的芯片分析、迁移率改变分析、促和抗凋亡蛋白的Western分析、以及FOXO3a过表达后的TUNEL和Annexin-V检测)；以及目的3.确定WA对前列腺特异性PTEN基因敲除小鼠(PS PTEN-KO；进行体内Wa疗效研究，包括PCA、组织病理学、促生存和促凋亡蛋白的免疫组织化学、增殖指数、细胞凋亡和血清Wa水平)。我们预计这些研究将为WA抗AR-PCa的作用机制提供分子洞察力，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.