Dissecting the role of UBE2J1 in prostate cancer

项目摘要 前列腺癌（PCa）是美国男性中诊断最多的癌症， 原发性PCa最终将发展为转移性疾病。转移性疾病通常对电流有抵抗力， “标准护理”治疗、雄激素受体（AR）靶向疗法，如恩杂鲁胺。抗 AR靶向治疗在很大程度上限制了晚期PCa患者的临床结局。因此，有一个 揭示耐药性的分子机制和确定新的脆弱性， 通过全面的体内文库筛选，我们鉴定了泛素结合酶， E2 J1（UBE 2 J1），在8-15%的PCa患者中丢失，是最佳候选反应调节剂之一 AR靶向治疗。通过CRISPR介导的基因敲除，我证明了UBE 2 J1的缺失与CRISPR介导的基因敲除有关。 LNCaP/AR PCa细胞赋予对AR靶向疗法的抗性。此外，UBE 2 J1缺失还赋予 雄激素去甲肾上腺素治疗（ADT）。从机制上讲，我观察到 UBE 2 J1缺失的细胞中AR和AR靶基因的表达，表明UBE 2 J1的缺失可能会减慢 下调泛素介导的AR降解。这一假设得到了一项观察的进一步支持。 在UBE 2 J1耗尽后，AR调节增强子位点中的AR结合显著增加。引人注目的是， 用两种创新的AR PROTAC在UBE 2 J1缺失细胞中的AR降解拯救了对ADT的敏感性。 此外，由于UBE 2 J1-缺失促进ER相关降解途径（ERAD），因此它可能挽救细胞死亡 由ER应激诱导微环境引起，包括治疗处理。我观察到一个显着的 增加关键ER应激调节因子GRP 78。总的来说，我的初步数据表明UBE 2 J1的丢失 可能通过受损的AR降解和促进ERAD赋予AR靶向治疗抗性， 挽救AR靶向治疗引起的肿瘤细胞凋亡。我提出两个具体目标来研究这个问题 UBE 2 J1缺失导致AR抗性的功能和分子机制 疗法对于第一个目标，我将全面阐明UBE 2 J1缺失的后果， 以及使用其他PCa细胞系和SCID小鼠异种移植模型进行体内研究。对于第二个目标，我将揭示 UBE 2 J1导致AR靶向治疗耐药的机制。我将探讨UBE 2 J1作为E2是否 泛素缀合酶，靶向AR降解并调节AR信号传导。另一方面，我会 检查UBE 2 J1缺失的细胞中的ERAD和ER应激，并确定挽救细胞死亡是否是UBE 2 J1缺失的原因。 对AR靶向治疗的抗性机制。这项研究的成功完成将有助于新的 深入了解AR靶向治疗的耐药机制，推进我们对 AR降解和ERAD与治疗抗性之间的联系。它也可能导致识别 克服耐药性的新治疗方法，从而改善患者的临床结局 先进的PCa

PROJECT SUMMARY Prostate cancer (PCa) is the most diagnosed cancer among American men and many patients with primary PCa will eventually develop metastatic disease. Metastatic disease is often resistant to the current “standard-of-care” treatment, androgen receptor (AR) targeted therapies, such as enzalutamide. Resistance to AR targeted therapies largely limits the clinical outcome of patients with advanced PCa. Therefore, there is an unmet need for revealing the molecular mechanisms of resistance and identifying novel vulnerabilities to overcome it. Through a comprehensive in vivo library screen, we have identified Ubiquitin Conjugating Enzyme E2 J1 (UBE2J1), which is lost in 8-15% of patients with PCa, as one of the top candidate modifiers of response to AR targeted therapy. Through CRISPR-mediated knock-out, I demonstrated that UBE2J1 depletion in LNCaP/AR PCa cells confers resistance to AR targeted therapies. Furthermore, UBE2J1 depletion also confers resistance to Androgen Deprivation Therapy (ADT). Mechanistically, I have observed significantly increased expression of AR and AR target genes in cells with UBE2J1 depletion, suggesting the loss of UBE2J1 may slow down the ubiquitin mediated degradation of AR. This hypothesis is further supported by the observation of a significant increase of AR binding in AR regulated enhancer sites upon UBE2J1 depletion. Strikingly, restoring AR degradation in UBE2J1 deleted cells with two innovative AR PROTACs rescues the sensitivity to ADT. Furthermore, as UBE2J1-loss promotes ER associated degradation pathway (ERAD), it might rescue cell death caused by a ER stress inducing microenvironment including therapy treatment. I have observed a significant increase of key ER-stress regulator, GRP78. Collectively, my preliminary data suggests the loss of UBE2J1 may confer AR targeted therapy resistance through impaired AR degradation and promoting ERAD and rescuing tumor cell apoptosis caused by AR targeted therapy. I propose two specific aims to examine this hypothesis and elucidate the function and molecular mechanism of UBE2J1-loss conferred resistance to AR therapy. For the first aim, I will comprehensively elucidate the consequence of UBE2J1 depletion both in vitro and in vivo, using other PCa cell lines and SCID mouse xenograft models. For the second aim, I will reveal the mechanism by which UBE2J1 causes AR-targeted therapy resistance. I will explore whether UBE2J1, as an E2 ubiquitin conjugating enzyme, targets AR for degradation and regulates AR signaling. On the other hand, I will examine ERAD and ER stress in cells with UBE2J1 depletion and determine whether rescuing cell death is the mechanism of resistance to AR targeted therapy. The successful completion of this study will contribute novel insights into the mechanism of resistance to AR targeted therapies, advancing our understanding of the connection between AR degradation and ERAD to therapy resistance. It may also lead to the identification of new therapeutic approaches to overcome resistance, consequently improving the clinical outcome of patients with advanced PCa.