RNF43通过泛素化降解E-cadherin促进肺腺癌上皮间质转化的分子机制研究

The aggressiveness of lung adenocarcinoma is ascribed to be a poor prognosticator for overall survival in patients with lung adenocarcinoma. To the date, the mechanism underlying the metastasis of lung adenocarcinoma remains unclear. Epithelial-mesenchymal transition (EMT) is characterized as a prometastatic factor in malignant diseases. Previous study reported that c-Src-phosphorylated E-cadherin was ubiquitinated and degraded by Hakai to facilitate EMT. However, we found that Hakai was surprisingly undetectable in lung adenocarcinomas. Furthermore, it was preliminarily proven that RNF43 expressing in a high level in lung adenocarcinomas ubiquitinated and degraded E-cadherin followed by c-Src-mediated phosphorylation to facilitated EMT phenotype. Accordingly, we hypothesize that a brand-new E3 ubiquitin ligase, RNF43, compensates Hakai loss to decrease E-cadherin in order to facilitate EMT through the interaction with some other membrane protein. We plan to conduct the research on clarify the expression levels of human E3 ubiquitin ligase using protein and gene chip. Then, E3 ubiquitin ligase exclusive for E-cadherin, RNF43, is further confirmed by siRNA library screening. It is likely to disclose a novel molecular mechanisem by which E-cadherin is phosphorylated and ubiquitinated to facilitate EMT and to further investigate the biological role of EMT in lung adenocarcinoma. It is expected to provide a theoretical screen for a novelty anti-EMT target and to shed a ligh on the therapy of lung adenocarcinoma.

高侵袭性严重影响肺腺癌的预后，其分子机制不明。有研究报道c-Src磷酸化修饰的E-cadherin可被Hakai泛素化降解，介导上皮间质转化（EMT），促进细胞侵袭转移。课题组前期研究发现在肺腺癌中Hakai表达缺失，全新E3泛素化酶RNF43可泛素化降解被c-Src磷酸化的E-cadherin，介导EMT，而RNF43与E-cadherin并无作用结构域。我们推测：肺腺癌中RNF43与其他膜蛋白结合，激活其RING结构域，泛素化降解被c-Src磷酸化的E-cadherin。本课题将应用蛋白及基因芯片在肺腺癌及癌旁组织中探索人类E3泛素化酶及相关分子的表达，结合siRNA library筛查，明确RNF43泛素化降解E-cadherin介导肺腺癌EMT的分子机制，揭示c-Src对E-cadherin的磷酸化与其泛素化的关系，探索EMT对肺腺癌生物学行为的影响，提供肺腺癌EMT新干预靶点。

本课题在计划时间内完成了大部分研究内容，基本达到了我们的研究目标，获得了比较好的临床应用前景的转化方向。我们首先明确了在肺腺癌当中磷酸化Caspase-8是非常重要的EMT关键分子。在肺腺癌当中，c-Src能够有效特异性磷酸化Caspase-8，而c-Src被磷酸化Caspase-8过分活化，进而对E-cadherin进行磷酸化修饰，导致RNF43对磷酸化E-cadherin进行泛素化修饰，导致讲解，启动EMT过程。c-Src对Caspase-8的磷酸化修饰导致肺腺癌细胞对paclitaxel化疗敏感性下降，只能通过坏死性凋亡对肺腺癌细胞进行杀伤，该作用效果较差，dasatinib能够逆转c-Src对Caspase-8的磷酸化，联合应用c-FLIP shRNA能够极大的放大paclitaxel的化疗作用，我们课题组在原有脂质体紫杉醇基础上，在脂质体内包裹c-FLIP shRNA，发挥很好的作用。此外，我们课题组在肺腺癌细胞中筛选出了全新长编码RNA——lncCRLA，lncCRLA为肺腺癌EMT过程中转录表达的产物，能够与RIPK1相互作用从而限制paclitaxel介导的坏死性凋亡作用，lncCRLA能够分布于肺腺癌外泌体，被肿瘤细胞释放入血，lncCRLA能够对肺腺癌化疗疗效产生指向性作用，同时，我们课题组借助单细胞测序和空间转录组技术，在单细胞和空间层面分析了EMT机制以及肺腺癌细胞的异质性，lncCRLA在肺腺癌初始细胞中高表达，可能对肺腺癌发生具有指向性作用，我们课题组对lncCRLA的临床应用抱有非常大的希望。此外，我们对RNF43在肺腺癌当中的表达也进行了探索，该分子在肺腺癌当中的表达率接近95%，因此我们课题组下一步将针对该分子开展ADC药物或者Car-T治疗的探索。