Dissecting the Role of Proteostasis in Anti-Androgen Resistant Prostate Cancer

项目摘要 在美国，前列腺癌(PCA)被预测为与癌症相关的第二大原因 2020年美国男性死亡人数。在初步诊断为前列腺癌后，根治性前列腺切除术，放射治疗和 雄激素剥夺疗法(ADT)用于治疗原发肿瘤。当癌症复发时，去势- 耐药前列腺癌(CRPC)可通过抗雄激素药物治疗，如苯扎鲁胺(XTANDI®)、 阿比特龙(ZYTIGA®)或阿帕鲁胺(ERLEADA™)。虽然这些药物最初非常有效， 患者很快就会通过尚未完全了解的机制产生耐药性。因此，在那里 迫切需要确定耐药机制以改善CRPC的治疗结果。蛋白 动态平衡(蛋白平衡)缺陷和致癌激活在肿瘤发生中起重要作用； 然而，参与抗雄激素耐药的PCa的蛋白平衡调节仍然是一个初步的了解。我们 已报道泛素介导的蛋白分解途径和蛋白酶体活性受到抑制。 对苯扎鲁胺和阿比特龙耐药的PCa细胞。因此，雄激素受体(AR)及其变异体AR- V7蛋白通过伴侣-泛素-蛋白酶体稳定并积累在这些耐药细胞中。 系统改造。伴侣(Hsp70)/E3泛素连接酶(Stub1)机制调节全长AR和 AR变异型蛋白平衡。小分子抑制HSP70促进HSP70、AR-V7和Stub1的亲和性 这显著扰乱了AR/AR-V7基因程序，抑制了前列腺癌的生长。这项建议 启动了一种新的范式来探索驱动下一代抗雄激素的潜在机制 CRPC中的抗性。这个项目的最终目标是剖析伴侣-泛素-的作用。 蛋白酶体系统在抗雄激素耐药中的作用，并开发新的药物途径，提供联合 抗AR药物靶向新辅助治疗对慢性前列腺癌患者的个体化治疗。我们将提供一部小说 下一代抗雄激素耐药的蛋白抑制机制及其机制研究 抗雄激素耐药CRPC中的HSP70/Stub1机制可能触发致癌蛋白的积聚 作为AR变异体和糖皮质激素受体(GR)，由于不能清除蛋白质。我们将在功能上 通过生化分析询问Hsp70/Stub1/AR-V7三元复合体，并发现 抑制热休克蛋白70活性诱导AR-V7降解的机制我们 将通过泛素化结合位点揭示AR和AR-V7泛素化的潜在机制 鉴定和大规模泛素残基测序。重要的是，我们将提供以下理由 通过调节Hsp70/Stub1纠正蛋白平衡失衡作为一种潜在的治疗策略 克服慢性前列腺癌患者对AR靶向治疗的耐药性并发展条件重编程细胞 培养(CRC)和患者来源的异种移植(PDX)模型，以剖析体内的机制。这项建议 将填补CRPC患者在克服下一代抗雄激素耐药方面的空白，并为 通过改进目前的AR靶向治疗来进行未来的研究。

PROJECT ABSTRACT In the United States, prostate cancer (PCa) is predicted to be the second leading cause of cancer related death in men in the United States in 2020. After initial diagnosis of PCa, radical prostatectomy, radiation and androgen deprivation therapy (ADT) are used to treat the primary tumors. When cancer recurs, castrate- resistant prostate cancer (CRPC) is treated by anti-androgen drugs, such as enzalutamide (XTANDI®), abiraterone acetate (ZYTIGA®) or apalutamide (ERLEADA™). Although these drugs are highly effective initially, patients quickly develop resistance through mechanisms that are not completely understood. Therefore, there is an urgent need to identify resistant mechanisms to improve the treatment outcome of CRPC. Protein homeostasis (proteostasis) deficiency and oncogenic activation plays important roles during tumorigenesis; however, proteostasis modulation involved in anti-androgen resistant PCa is still rudimentarily understood. We have reported that ubiquitin mediated proteolysis pathway and proteasome activity are suppressed in enzalutamide and abiraterone resistant PCa cells. As a result, androgen receptor (AR) and its variant form AR- V7 protein are stabilized and accumulated in these resistant cells through chaperone-ubiquitin-proteasome- system alteration. The chaperone (Hsp70)/E3 ubiquitin ligase (Stub1) machinery regulates full length AR and AR variant proteostasis. Hsp70 inhibition by small molecules promotes Hsp70, AR-V7 and Stub1 proximity which significantly disrupts AR/AR-V7 gene programs and suppresses prostate tumor growth. This proposal initiates a new paradigm to explore the underlying mechanisms driving next generation anti-androgen resistance in CRPC. The ultimate goal of this program is to dissect the roles of chaperone-ubiquitin- proteasome-system in anti-androgen resistance and develop new pharmaceutical approaches to provide co- targeting neoadjuvant with anti-AR agents to personalized CRPC patients’ treatment. We will provide a novel mechanism of next generation anti-androgen resistance via proteostasis impairment and uncover that Hsp70/Stub1 machinery in anti-androgen resistant CRPC may trigger accumulation of oncogenic proteins such as AR variants and glucocorticoid receptor (GR) due to inability of protein clearance. We will functionally interrogate the Hsp70/Stub1/AR-V7 ternary complex through biochemical assays, and uncover the mechanisms of inhibition of Hsp70 activity in inducing AR-V7 degradation through the proximity of Stub1. We will unveil the underlying mechanisms of AR and AR-V7 ubiquitination through the ubiquitination binding sites identification and large-scale ubiquitin remnants sequencing. Importantly, we will provide the rationale to correct proteostasis imbalance through modulation of Hsp70/Stub1 as a potential therapeutic strategy to overcome resistance to AR-targeted therapies in CRPC patients and develop conditional reprogramed cell cultures (CRCs) and patient derived xenograft (PDX) models to dissect the mechanisms in vivo. This proposal will fill the gap in overcoming next generation anti-androgen resistance in CRPC patients and point the way to future research by improving current AR-targeted therapies.