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Targeting AR and Akt for the Treatment of Prostate Cancer

靶向 AR 和 Akt 治疗前列腺癌

基本信息

批准号：
8926871
负责人：
Chendil Damodaran
金额：
$ 30.19万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-15 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8926871
关键词：
Ablation Address Androgen Antagonists Androgen Receptor Androgens Animal Model Antibodies Apoptosis Apoptotic Area Binding Bioavailable Biological Assay Cell Culture Techniques Cell Death Cells Development Diet Disease Progression Dominant-Negative Mutation Dose Electrophoretic Mobility Shift Assay Health Histopathology Hormonal Hormones Immunohistochemistry In Situ Nick-End Labeling In Vitro Induction of Apoptosis Knockout Mice LNCaP Lead Link Malignant Neoplasms Malignant neoplasm of prostate Mediating Modeling Molecular Mus PC3 cell line PTEN gene Pattern Play Prevention Prostate Prostatic Neoplasms Proteins Radiation therapy Receptor Cell Receptor Signaling Refractory Regulation Reporter Research Role S-Phase Fraction Sampling Serum Signal Transduction Small Interfering RNA Specimen Therapeutic Traditional Medicine Tumor Tissue Xenograft procedure androgen independent prostate cancer annexin A5 base cancer therapy chromatin immunoprecipitation effective therapy in vivo in vivo Model inhibitor/antagonist insight killings novel therapeutics prevent pro-apoptotic protein promoter prostate cancer cell receptor binding research study response transgenic adenocarcinoma of mouse prostate treatment strategy tumor tumor growth

项目摘要

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.

描述（申请人提供）：雄激素受体（AR）在前列腺癌（PCa）的发生和进展中发挥重要作用。大多数 PCa 标本表达高水平的 AR，这对于将雄激素依赖性 (AR+) PCa 转化为临床上更具侵袭性的雄激素非依赖性 (AR-) PCa 是必要且充分的。尽管抑制 AR 活性是 AR+ PCa 治疗的主要手段，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，并且 AR 和 Akt 共同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 出现的强大治疗方法。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 Akt 特异性靶向 AR-PCa 细胞；在细胞培养物和动物模型中，这种抑制作用会导致前列腺细胞凋亡反应 4 (Par-4) 依赖性细胞凋亡的激活。同样，在 AR+ PCa 细胞中抑制 AR 可促进 WA 诱导的 Par-4 激活和细胞凋亡。 Akt 或 AR 的过表达会抑制 WA 介导的 Par-4 激活，并阻止 AR+ PCa 中的细胞凋亡，表明 AR 和 Akt 均负向调节 PCa 细胞中 Par-4 的促凋亡功能。我们假设 WA 与激素消融相结合是控制 PCa 进展的有效策略。为了解决这一假设，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 抗体进行 ChIP 分析以及促凋亡蛋白和抗凋亡蛋白的 Western 分析）；目标 2. 检查 Par-4 和 FOXO 3a 之间的关系（受 Akt 调节），以及 WA 如何在 AR+ PCa 中调节这种相互作用（通过报告构建体研究 FOXO3a 与 Par-4 启动子的结合、使用 FOXO3a 抗体进行 ChIP 测定、迁移率变化测定、促凋亡蛋白和抗凋亡蛋白的 Western 分析，以及 FOXO3a 后的 TUNEL 和膜联蛋白-V 测定过度表达）；目标 3. 确定 WA 对前列腺特异性 PTEN 敲除小鼠中 PCa 发育的化疗作用（PS PTEN-KO；对 PCa 进行体内 WA 功效研究、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 对抗 AR+ PCa 的作用机制提供分子见解，并最终带来治疗 PCa 的新治疗策略。