Dissecting the role of UBE2J1 in prostate cancer

项目总结 前列腺癌(PCA)是美国男性中诊断最多的癌症，许多患者患有前列腺癌 原发性前列腺癌最终会发展为转移性疾病。转移性疾病通常对电流具有抵抗力 “标准护理”治疗，雄激素受体(AR)靶向治疗，如苯扎鲁胺。抵抗力 AR靶向治疗在很大程度上限制了晚期PCa患者的临床结果。因此，有一个 揭示抗药性的分子机制和识别新的脆弱性方面尚未得到满足的需求 克服它。通过全面的体内文库筛选，我们已经鉴定出泛素结合酶 E2J1(UBE2J1)，在8-15%的前列腺癌患者中丢失，作为反应的首要候选修饰物之一 AR靶向治疗。通过CRISPR介导的基因敲除，我证明了UBE2J1在 LNCaP/AR PCA细胞对AR靶向治疗具有抵抗力。此外，UBE2J1的枯竭也使 对雄激素剥夺疗法(ADT)的抵抗。从机制上讲，我观察到显著增加了 AR和AR靶基因在UBE2J1缺失细胞中的表达，提示UBE2J1缺失可能减缓 下调泛素介导的AR降解。这一假说进一步得到了对 当UBE2J1缺失时，AR调节的增强子位点上的AR结合显著增加。令人惊讶的是，恢复 用两个创新的AR PROTAC在UBE2J1缺失细胞中的AR降解挽救了对ADT的敏感性。 此外，由于UBE2J1缺失促进ER相关降解途径(ERAD)，它可能挽救细胞死亡 由内质网应激诱导的微环境引起，包括治疗。我观察到了一个意义重大的 增加关键的内质网应激调节剂GRP78。总的来说，我的初步数据表明UBE2J1的丢失 可能通过受损的AR降解和促进ERAD和 挽救AR靶向治疗引起的肿瘤细胞凋亡。我提出了两个具体的目标来检验这一点 UBE2J1缺失导致AR抗性的假说及分子机制 心理治疗。对于第一个目标，我将在体外全面阐明UBE2J1耗竭的后果 并在体内用其他PCa细胞系和SCID小鼠建立异种移植模型。对于第二个目标，我将揭示 UBE2J1导致AR靶向治疗耐药的机制。我将探索UBE2J1，作为E2 泛素结合酶，靶向AR的降解，并调节AR信号。另一方面，我会 检测有UBE2J1缺失的细胞中的ERAD和ER应激，并确定挽救细胞死亡是否是 AR靶向治疗的耐药机制。这项研究的成功完成将为我们带来新的 对AR靶向治疗耐药机制的洞察，促进我们对AR靶向治疗的理解 AR降解和ERAD与治疗耐药的关系。它还可能导致识别出 克服耐药性的新治疗方法，从而改善患者的临床结局 拥有先进的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.