EphA2调控Hippo信号通路促进胃癌侵袭转移的机制研究

Epithelial-mesenchymal transition (EMT) is a critical event in early invasion and metastasis of cancer. EphA2 and Hippo signaling pathway play an important role in cancer invasion and metastasis. Complex cross-talk is exist between Hippo signaling and Wnt/β-catenin signaling. However, the relationship between EphA2 and Hippo signaling, and whether EphA2 participate in the cross-talk of Hippo and Wnt/β-catenin signaling are still obscure. Our previous studies showed that EphA2 could promote EMT through Hippo signaling pathway. The project plans to investigate the effect of EphA2 expression and activation on Hippo signaling pathway. Next, we analyse the molecular mechanisms that whether EphA2 regulating Hippo signaling pathway through competitive binding 14-3-3 with transcript co-activator YAP/TAZ. Moreover, The EphA2 regulates the cross-talk between Hippo and Wnt signaling is also assessed. Finally, we try to identify the process that EphA2 promote gastric cancer invasion and metastasis by inducing EMT through inhibiting Hippo signaling pathway. In this project, the possibility of EphA2 as an upstream membrane receptor of Hippo signaling pathway was studied for the first time. The mechanism study of this project not only clarify the mechanim of EphA2 promoting gastric cancer invasion and metastasis by regulating Hippo signaling, but also provide a novel strategy for gastric cancer target therapy.

上皮-间质转化 (EMT) 是肿瘤侵袭转移的关键事件。EphA2 和Hippo通路在调控EMT 和肿瘤侵袭转移中均发挥重要作用，Hippo与Wnt通路之间存在复杂的相互作用。但EphA2 和Hippo通路是否关联，以及EphA2是否介入Hippo-Wnt的相互作用不清楚。我们前期研究提示，EphA2 可能通过Hippo通路调控胃癌细胞EMT和侵袭转移。为此，项目拟研究EphA2对胃癌细胞Hippo 通路活性的影响；探索EphA2与YAP/TAZ 竞争性结合14-3-3 蛋白，抑制Hippo 通路活性并介入Hippo-Wnt通路相互作用的分子机制；分析EphA2 是否通过抑制Hippo 通路，诱导EMT，促进胃癌细胞侵袭转移。项目首次探讨EphA2作为Hippo通路上游因子的可能性，不仅可阐明EphA2调控Hippo通路促进胃癌侵袭转移的机制，而且有望为胃癌的靶向治疗提供新策略。

胃癌侵袭、转移以及复发是影响进展期患者生存期的主要因素。本课题组致力于研究EphA2介导的胃癌侵袭转移。本项目通过研究发现EphA2-YAP信号通路是在胃癌侵袭转移以及耐药过程中发挥重要作用。首先，通过慢病毒/质粒转染胃癌细胞株，获得稳定传代的EphA2过表达/沉默表达的胃癌细胞株。通过一系列生物功能学实验发现，EphA2表达促进胃癌细胞、增殖以及转移，同时通过细胞蛋白质组学测序发现，EphA2在胃癌细胞中与YAP结合，同时，co-IP实验证实了这一现象。YAP作为Hippo信号通路的核心组件，一系列由Hippo上游信号分子或非Hippo通路信号分子调控的YAP的入核，被认为是Hippo通路介导细胞增殖侵袭的重要事件，是近年来研究的热点。通过研究EphA2过表达/沉默表达的胃癌细胞株发现，EphA2通过直接磷酸化激活YAP，从而抑制YAP降解，促进YAP入核，从而发挥生物学功能。其次，通过耐药实验发现EphA2过表达促进胃癌细胞耐受5-FU。同时，发现5-FU能阻断YAP表达以及入核从而抑制肿瘤细胞生长，Verteporfin能协同5-FU而抑制YAP生物学功能。我们在以免疫缺陷小鼠的动物模型中，进一步证实了EphA2-YAP这一信号通路在介导胃癌细胞增殖侵袭转移的作用。结合临床，我们课题组收集了2016-2017年于湘雅医院行胃癌根治手术患者病理标本以及临床病理学资料，通过免疫组化实验和临床病理学资料分析发现，EphA2表达与YAP和pYAP表达正相关，且EphA2、YAP表达与胃癌患者早期复发相关。本研究表明，在胃癌细胞中，EphA2-YAP信号通路促进细胞增殖侵袭、转移以及耐药形成，同时，抑制EphA2-YAP通路能有效降低胃癌细胞耐受5-FU，可作为胃癌患者，特别是耐5-FU胃癌患者的治疗胃癌提供新的分子靶点。