SUMO化/去SUMO化修饰介导的p27kip1核输出机制在胆管癌化疗抵抗中作用的研究

Cancer cells appear to acquire intracellular mechanisms to export anti-cancer nuclear proteins and molecular targets of anti-neoplastic agents during the progression of malignant transformation. p27kip1 was one of the most important cyclin-dependent kinase inhibitors (CKI) and targeting gene for chemotherapy, play a significant role in the regulation of cell cycle. Our previous research verified that mislocalization of nuclear p27kip1 into the cytoplasm was found in bile duct carcinoma, leaded to carcinogenesis, and resistance to therapeutic drugs, but the exact mechanism is unknown. Small ubiquitin-like modifier pathway was discovered in recent years, which is new way by posttranslational modification to some kinds of nuclear protein, involved in transcriptional regulation nuclear transport, signal transduction and cell cycle regulation and other functions in cells. So it is possible potential mechanisms that regulation of p27kip1 distribution between nuclear and cytoplasm. According to this hypothesis，our research propose to explore the entire celluar processes of p27kip1 SUMOylation，which requires an ATP-dependent enzymatic cascade consisting of the E1-activating enzyme Aos1/Uba2, the E2-conjugating enzyme Ubc9, and generally one of several known SUMO E3 ligases，in addition p27kip1 post-translational modifications、p27kip1 nucleocytoplasmic transport and dissociation in cytoplasm by using our successfully established cell simulation system for studying SUMO modification or demodification of an individual protein in vitro . To make it clear that the effect of p27kip1 SUMOylation status on p27kip1 pool，which was dynamically distribute from nucleus to cytoplasm. To support the hypothesis that p27kip1 nuclear translocation mediated by SUMOylation and deSUMOylation was the molecule mechanisms for chemotherapy resistance in cholangiocarcinoma cell. With this finding we expect to provide new gene therapy target and experimental basis for the new theory of correcting subcellular mislocalization of p27kip1 and recovering chemotherapy sensitization

肿瘤细胞在恶性转化过程中获得内在化疗抗性的重要方式就是增强肿瘤抑制因子及靶向药物分子的核输出。 p27kip1作为重要的细胞周期负调控因子及抗肿瘤药物关键靶分子，我们前期发现并证实：其异常核外定位及表达与胆管癌化疗抗性密切相关，但机制不明。SUMO和去SUMO化是近年来发现的一种新的蛋白质翻译后修饰方式，广泛参与细胞内转录调节、核-质转运和细胞周期调控等过程，因此有可能在肿瘤的发生及化疗抵抗中发挥重要作用。本课题拟通过已成功构建的“SUMO化”和“去SUMO化”细胞内模拟系统，深入研究p27kip1的SUMO化转录后修饰功能网通过SUMO活化、结合、连接、核质转运和解离过程影响p27kip1池核质动态分布机制，并明确关键调控因子。以期阐明SUMO和去SUMO化介导的p27kip1核转位是胆管癌获得化疗抗性的内在机制。为最终重建p27kip1亚细胞定位的化疗增敏治疗提供新的靶点及实验依据。

背景：肿瘤细胞获得内在化疗抗性的重要方式就是增强肿瘤抑制因子及靶向药物分子的核输出。P27kip1作为细胞周期关卡关键抑制因子及许多化疗药物作用中重要的靶分子，其核功能失活业已证实与众多恶性肿瘤的化疗抵抗密切相关，但机制不明。.内容：通过免疫组织化学、双荧光素酶报告基因系统、免疫荧光显微镜观察SUMO化修饰功能网络对p27kip1的核质内动态分布的影响；通过构建潜在位点突变体，利用免疫沉淀及免疫印迹技术分别验证p27kip1与SUMO化修饰所涉及核质运输过程和E3连接酶复合体（RanBP2/RanGAP1*SUMO1/Ubc9）在p27kip1的SUMO翻译后修饰机制中的关键作用。并通过MTT、流式细胞术、免疫印迹和裸鼠移植瘤模型等方法分别在体外、体内检测上述机制在胆管癌细胞化疗药物敏感性中的作用。.结果：p27kip1是SUMO化的天然底物，p27kip1的 SUMO1作用位点是73位赖氨酸；p27kip1的SUMO1化修饰过程是激活的SUMO蛋白通过与E2酶(Ubc9)蛋白结合，在SUMO连接酶E3(RanBP2等)的帮助下与p27kip1赖氨酸残基形成共价键而完成结合，Ubc9通过核质转运蛋白CRM1及E3连接酶复合体共同介导了p27kip1的SUMO化和核输出；RanBP2通过增强p27kip1的SUMO1在73位赖氨酸修饰作用而促进CRM1介导p27kip1的核输出并促进胆管癌细胞增殖；而敲减Ubc9可显著抑制胆管癌TFK1细胞的体内、外增殖能力；并可显著抑制TFK1细胞体外迁移能力并增加胆管癌化疗敏感性。.结论：Ubc9、RanBP2、CRM1等作为p27kip1的SUMO化和核输出关键蛋白与E3连接酶复合体共同介导了p27kip1的SUMO化和核输出；SUMO化修饰介导的p27kip1核转位在胆管癌中的发生、发展中和化疗抵抗中发挥重要作用。因此，有望成为胆管癌治疗新的靶点。