Dissecting the role of UBE2J1 in prostate cancer

剖析 UBE2J1 在前列腺癌中的作用

• 批准号: 10386745
• 负责人: Carla Sofia Rodriguez-Tirado
• 金额: $ 3.8万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10386745
• 关键词: American; Androgen Receptor; Apoptosis; Cause of Death; Cell Death; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Disease; Endoplasmic Reticulum Degradation Pathway; Enhancers; GRP78 gene; Genes; Genomics; Goals; Growth; Homeostasis; Impairment; In Vitro; Knock-out; LNCaP; Lead; Libraries; Malignant neoplasm of prostate; Mediating; Molecular; Outcome; Patient-Focused Outcomes; Patients; Receptor Signaling; Recurrence; Resistance; Role; SCID Mice; Site; Ubiquitin; Ubiquitin-Conjugating Enzymes; Work; Xenograft Model; advanced prostate cancer; androgen deprivation therapy; cancer diagnosis; castration resistant prostate cancer; endoplasmic reticulum stress; enzalutamide; experimental study; improved; in vivo; innovation; insight; knock-down; men; neoplastic cell; new therapeutic target; novel; novel therapeutic intervention; novel therapeutics; prostate cancer cell; prostate cancer cell line; protein degradation; receptor; receptor binding; resistance mechanism; response; restoration; small hairpin RNA; standard of care; targeted treatment; therapy resistant; tumor microenvironment

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.

项目摘要 前列腺癌（PCA）是美国男性和许多患者中诊断得最多的癌症 原发性PCA最终将发展转移性疾病。转移性疾病通常对电流有抵抗力 “护理标准”治疗，雄激素受体（AR）靶向疗法，例如恩扎拉胺。抵抗 AR靶向疗法在很大程度上限制了晚期PCA患者的临床结果。因此，有一个 未满足的需要揭示抗性的分子机制，并确定了新的脆弱性 克服它。通过全面的体内库屏幕，我们已经确定了泛素结合酶 E2 J1（UBE2J1），在8-15％的PCA患者中丢失，是最高的候选响应改性剂之一 进行AR靶向疗法。通过CRISPR介导的淘汰赛，我证明了UBE2J1的部署 LNCAP/AR PCA细胞对AR靶向疗法的耐药性。此外，UBE2J1部署也承认 对雄激素剥夺疗法（ADT）的抗性。从机械上讲，我观察到显着增加 ube2j1耗竭细胞中AR和AR靶基因的表达，表明UBE2J1的丧失可能会减慢 下泛素介导的AR降解。观察到 在UBE2J1耗竭时，AR调节的增强子位点的AR结合显着增加。令人惊讶的是，恢复 UBE2J1中的AR降解删除了具有两个创新的AR Protac的细胞，对ADT的敏感性做出了反应。 此外，随着UBE2J1-alss促进ER相关的降解途径（ERAD），它可能会挽救细胞死亡 由ER应力引起的微环境（包括治疗）引起的。我观察到了重要的 ER应力调节器的增加GRP78。总的来说，我的初步数据表明ube2j1的损失 通过AR降解和促进ERAD和 营救由AR靶向治疗引起的肿瘤细胞凋亡。我提出了两个特定的目的来研究这一点 假设并阐明了UBE2J1-als的功能和分子机制，赋予了对AR的抗性 治疗。为了第一个目标，我将全面阐明Ube2J1部署的后果 和体内，使用其他PCA细胞系和SCID鼠标Xenographic模型。为了第二个目标，我将揭示 UBE2J1引起AR靶向治疗耐药性的机制。我将探讨ube2j1是否为E2 泛素结合酶，靶向AR降解并调节AR信号传导。另一方面，我会 检查使用UBE2J1部署的细胞中的ERAD和ER应激 抵抗AR靶向治疗的机制。这项研究的成功完成将有助于小说 洞悉对AR靶向疗法的抵抗机制，促进我们对 AR降解与抗治疗耐药性之间的联系。它也可能导致识别 新的治疗方法以克服抗药性，从而改善了患者的临床结果 使用高级PCA。