Targeting GRP78-dependent AR-V7 protein degradation overcomes castration-resistance in prostate cancer therapy
Targeting GRP78-dependent AR-V7 protein degradation overcomes castration-resistance in prostate cancer therapy
复制标题
靶向 GRP78 依赖性 AR-V7 蛋白降解可克服前列腺癌治疗中的去势抵抗
DOI：
10.7150/thno.41849
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发表时间：
2020-01-01
期刊：
影响因子：
12.4
通讯作者：
Huang, Hongbiao
中科院分区：
文献类型：
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作者：
Liao, Yuning;Liu, Yuan;Huang, Hongbiao
原理：雄激素受体剪接变体7（AR-V7）是去势抵抗性前列腺癌（CRPC）发展的主要原因。然而，AR-V7在前列腺癌治疗中的翻译后修饰水平的调节和功能仍然知之甚少。在这里，我们进行了一个天然产物的文库筛选，以确定负责人前列腺癌细胞系中AR-V7蛋白降解的潜在小分子。方法：利用天然产物库筛选AR-V7抑制剂。Co-IP和生物量谱测定用于鉴定AR-V7相互作用蛋白，而蛋白质印迹、共聚焦显微镜、RNA干扰和基因转染用于验证这些相互作用。采用细胞活力、EDU染色和集落形成测定来检测细胞生长和增殖。流式细胞仪检测细胞周期分布。使用小鼠异种移植模型研究体内抗CRPC作用。结果如下：该筛选确定了吴茱萸次碱，中药吴茱萸的主要成分之一，作为一种新的化学物质，通过K48连接的泛素化选择性地诱导AR-V7蛋白降解。在机械上，这种作用依赖于吴茱萸次碱诱导GRP 78-AR-V7蛋白复合物的形成，其进一步募集E3连接酶SIAH 2以直接促进AR-V7的泛素化。因此，GRP 78依赖性AR-V7蛋白降解的遗传和药理学激活恢复了细胞培养和动物模型中去势抵抗性前列腺癌对抗雄激素治疗的敏感性。结论：这些发现不仅为克服前列腺癌治疗中的去势抵抗提供了新的方法，而且增加了我们对分子伴侣和泛素连接酶在塑造蛋白质稳定性方面的相互作用的理解。
Rationale: Androgen receptor splice variant 7 (AR-V7) is a leading cause of the development of castration-resistant prostate cancer (CRPC). However, the regulation and function of AR-V7 at levels of post-translational modifications in prostate cancer therapy remain poorly understood. Here, we conducted a library screen of natural products to identify potential small molecules responsible for AR-V7 protein degradation in human prostate cancer cell lines. Methods: A natural product library was used to screen the inhibitor of AR-V7. Co-IP and biomass spectrum assays were used to identify the AR-V7-interacting proteins, whereas western blot, confocal microscopy, RNA interfering, and gene transfection were used to validate these interactions. Cell viability, EDU staining, and colony formation assays were employed to detect cell growth and proliferation. Flowcytometry assays were used to detect the distribution of cell cycle. Mouse xenograft models were used to study the anti-CRPC effects in vivo. Results: This screen identified rutaecarpine, one of the major components of the Chinese medicine Evodia rutaecarpa, as a novel chemical that selectively induces AR-V7 protein degradation via K48-linked ubiquitination. Mechanically, this effect relies on rutaecarpine inducing the formation of a GRP78-AR-V7 protein complex, which further recruits the E3 ligase SIAH2 to directly promote the ubiquitination of AR-V7. Consequently, the genetic and pharmacological activation of the GRP78-dependent AR-V7 protein degradation restores the sensitivity of castration-resistant prostate cancer to anti-androgen therapy in cell culture and animal models. Conclusions: These findings not only provide a new approach for overcoming castration-resistance in prostate cancer therapy, but also increase our understanding about the interplay between molecular chaperones and ubiquitin ligase in shaping protein stability.