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

项目摘要 在美国，前列腺癌（PCa）被预测为癌症相关的第二大原因。 2020年美国男性的死亡率在初步诊断为PCa后，进行了根治性前列腺切除术、放射治疗和 雄激素剥夺疗法（ADT）用于治疗原发性肿瘤。当癌症复发时，阉割- 通过抗雄激素药物，如恩杂鲁胺（XTANDI®）， 醋酸阿比特龙（ZYTIGA®）或阿帕鲁胺（ERLEADA™）。虽然这些药物最初非常有效， 患者通过尚不完全了解的机制迅速产生耐药性。因此 迫切需要确定耐药机制以改善CRPC的治疗结果。蛋白 体内平衡（蛋白质平衡）缺陷和致癌激活在肿瘤发生过程中发挥重要作用; 然而，抗雄激素抗性前列腺癌中涉及蛋白质稳态调节仍然是初步了解的。我们 已经报道了泛素介导的蛋白水解途径和蛋白酶体活性在 恩杂鲁胺和阿比特龙耐药PCa细胞。因此，雄激素受体（AR）及其变体形式AR-1是雄激素受体（AR）的一个重要组成部分。 V7蛋白通过分子伴侣-泛素-蛋白酶体- 系统改造分子伴侣（Hsp 70）/E3泛素连接酶（Stub 1）机制调节全长AR和 AR变体蛋白质稳态。通过小分子抑制Hsp 70促进Hsp 70、AR-V7和Stub 1接近 其显著破坏AR/AR-V7基因程序并抑制前列腺肿瘤生长。这项建议 开创了一个新的范式，探索驱动下一代抗雄激素的潜在机制 CRPC中的耐药性。该计划的最终目标是剖析分子伴侣泛素的作用， 蛋白酶体系统在抗雄激素抵抗中的作用，并开发新的药物途径， 用抗AR剂靶向新辅助治疗CRPC患者的个性化治疗。我们将提供一本小说 下一代抗雄激素抵抗的机制通过蛋白质稳态损伤，并揭示， 抗雄激素耐药CRPC中的Hsp 70/Stub 1机制可能触发致癌蛋白的积累， 作为AR变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 通过生物化学测定询问Hsp 70/Stub 1/AR-V7三元复合物，并揭示 Hsp 70活性在诱导AR-V7降解中的抑制机制通过Stub 1的邻近。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，我们将提供理由， 通过调节Hsp 70/Stub 1纠正蛋白质稳态失衡，作为潜在的治疗策略， 克服CRPC患者对AR靶向治疗的耐药性， 培养物（CRC）和患者来源的异种移植物（PDX）模型来剖析体内机制。这项建议 将填补CRPC患者克服下一代抗雄激素耐药的差距，并指出 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.