YAP在胰腺癌发生发展中功能及TRAF6对YAP调控作用的研究

Pancreatic cancer is the most lethal disease and the mortality and morbidity is almost equal. The failure of early diagnosis and resection is the important reason for the desperate results of pancreatic cancer; therefore the research focusing on the mechanism of tumorigenicity has become the hot spot for pancreatic cancer. The former research has found that Hippo signaling pathway is important in turmorigenicity, while YAP is the key protein in this cell signaling pathway. First we have found that the YAP is over-expressed in the pancreatic cancer cell lines and tissues; and the over-expression of YAP is associated with the poor prognosis of the patients. Then we have found that TRAF6 is over-expressed in pancreatic cancer tissues and TRAF6 is associated with YAP in vitro, while TRAF6 is the key factor in many cell signaling pathways. Therefore we speculate that TRAF6 could regulate the Hippo-YAP pathway also. In this project we will use the cell lines, animal models and clinical samples to further study the mechanism of YAP in pancreatic cancer tumorigenicity by plasmid transfection, siRNA interference technologies, and we will discuss the regulating function and mechanism of TRAF6 on the Hippo-YAP pathway. The results of this project will further reveal the mechanism of pancreatic cancer tumorigenicity and enrich the knowledge on Hippo-YAP pathway and supply new idea and target for the therapy of pancreatic cancer.

胰腺癌恶性程度极高，其发病率与死亡率几乎相等，针对其发生发展机制的研究是目前胰腺癌研究领域的热点。既往研究提示Hippo信号通路在肿瘤发生发展中具有重要作用，而YAP是Hippo信号通路重要成员。我们前期工作发现在胰腺癌组织标本中高表达YAP，且高表达YAP与患者预后呈负相关；同时我们发现胰腺癌组织中高表达TRAF6，而TRAF6是多种信号通路的调控因子，前期研究提示TRFA6蛋白可以与YAP蛋白相结合，因而我们推测TRAF6蛋白可能对Hippo-YAP信号通路进行调控。因此在本申请项目中，我们将利用细胞系、动物模型、临床样本等，通过质粒转染、siRNA干扰等技术进一步明确YAP在胰腺癌发生发展中的功能，并探讨TRAF6对Hippo-YAP信号通路的调控作用及机制。项目研究获得结果将进一步揭示胰腺癌发生发展的分子机制，并丰富对Hippo信号通路的认识，为胰腺癌治疗提供新思路及治疗靶点。

胰腺癌恶性程度极高，其发病率与死亡率几乎相等，针对其发生发展机制的研究是目前胰腺癌研究领域的热点。既往研究提示Hippo信号通路在肿瘤发生发展中具有重要作用，而YAP是Hippo信号通路重要成员。我们前期工作发现在胰腺癌组织标本中高表达YAP，且高表达YAP与患者预后呈负相关；同时我们发现胰腺癌组织中高表达TRAF6，而TRAF6是多种信号通路的调控因子，我们推测TRAF6蛋白可能对Hippo-YAP信号通路进行调控。在本项目研究中我们主要研究内容为TRAF6在胰腺癌侵袭迁移中作用，以及TRAF6对YAP信号通路的调控作用机制。结果提示TRAF6在胰腺癌细胞系及组织标本中高表达，且过表达TRAF6能够促进胰腺癌细胞的增殖及迁移；而下调TRAF6表达能够抑制胰腺癌细胞的增殖及迁移。进一步我们研究了TRAF6对YAP信号通路的调控作用。结果提示下调TRAF6表达可以显著抑制胰腺癌细胞YAP表达，同时能够抑制YAP信号通路下游靶基因Cyr61、CyclinE及CTGF的表达。而TRAF6促进胰腺癌细胞系的迁移及克隆形成是通过YAP完成。在TRAF6过表达胰腺癌细胞系内下调YAP表达可以去除TRAF6过表达促进胰腺癌迁移作用。此外沉默YAP表达可以损害过表达TRAF6促进胰腺癌非锚定生长能力。为进一步研究TRAF6对YAP信号通路作用位点，我们研究了TRAF6与YAP上游分子MST间关系。通过免疫荧光实验发现，TRAF6与MST蛋白在胰腺癌细胞内具有共同定位；而GST pull down实验发现，胰腺癌细胞内GST-TRAF6能够与内源性的MST形成复合物；此外免疫共沉淀实验同样证实内源性TRAF6能够与MST相互作用。文献报道TRAF6具有泛素化酶作用，我们进一步研究证实TRAF6能够促进MST蛋白的讲解。通过GEPIA数据库搜索，结果提示在胰腺癌标本MST表达与胰腺癌患者预后呈负相关，而MST表达与TRAF6表达呈负相关。综合上述结果，我们提出TRAF6通过促进MST蛋白降解，进而激活YAP信号通路促进胰腺癌细胞的增殖及迁移。本研究结果丰富了对肿瘤重要信号通路Hippo-YAP的认识，同时能够为胰腺癌的治疗提供新的治疗靶点。