转录共活化因子YAP调控JAK/STAT3通路促进胃癌发生发展的作用和机制研究

The trancriptional co-activator YAP, downstream effector of the Hippo pathway, is a master regulator of organ size through its interaction with various trancription factors. Pathologically, deregulated YAP promotes tumorigenesis by ehnhancing pro-survival signals and inhibiting cellular apoptosis. It has been proved that the activation of YAP is sufficient to induce the secretion of Upd cytokines (IL-6 family) and multiple EGFR ligands, leading to the activation of JAK/STAT3 pathway in intestinal stem cells. Herein, we propose a hypothesis that overexpression of YAP might activate the JAK/STAT3 pathway and play a vital role in maitaining the malignant behavior of gastric cancer cells, either through induction the secretion of STAT3 upstream cytokines, or by directly interaction with STAT3 via its specific domain. In this study, the expression and activation of YAP and STAT3 will be investigated in clinical gastric cancer samples, followed by the correlation analysis between the YAP/STAT3 expression pattern and the clinicopathological parameters. Functionally, the effects of YAP on the STAT3 signaling and cellular behavior will be analyzed by overexpression of YAP or knocking-down endogenous YAP in gastric cancer cell lines.Furthermore, we will test the secretion of cytokines and the interaction between YAP and STAT3, in order to explore the underlying mechanisms of YAP modulating the JAK/STAT3 pathway. In addition, we will construct an inducible YAP knockdown gastric cancer mouse model to confirm our in vitro findings. In summary, we are aiming to reveal the important roles of crosstalk between the Hippo/YAP and the JAK/STAT3 pathways in gastric tumorigenesis, and provide new strategies for the treatment of gastric cancers.

转录共活化因子YAP通过与多种转录因子相互作用，调节细胞生长调控信号，其失调将导致肿瘤的形成。YAP可诱导STAT3上游细胞因子的表达，激活STAT3通路从而促进实验动物肠道干细胞的增殖。这种干细胞的调控模式在肿瘤的发生发展过程中亦具有重要作用。我们推测，胃癌细胞中过度表达的YAP可通过诱导相关细胞因子的表达，或与STAT3结合形成复合物，这两种方式激活STAT3通路。本研究将检测临床病例标本中YAP与STAT3的表达与活化，分析二者及与临床病理特征之间的相关性；构建高表达/沉默YAP细胞模型，研究YAP对胃癌细胞恶性生物学行为的影响；进一步检测细胞因子的表达及蛋白相互作用，研究YAP调节STAT3通路的内在机制；此外，我们还将构建可诱导的YAP沉默胃癌动物模型，在动物体内验证相关机制。本研究将揭示Hippo/YAP与JAK/STAT3通路串话在胃癌发生发展中的影响，为胃癌防治提供新策略。

转录共活化因子YAP通过与多种转录因子相互作用，调节细胞生长调控信号，其失调将导致肿瘤的形成。YAP可诱导STAT3上游细胞因子的表达，激活STAT3通路从而促进实验动物肠道干细胞的增殖。这种干细胞的调控模式在肿瘤的发生发展过程中亦具有重要作用。我们推测，胃癌细胞中过度表达的YAP可通过诱导相关细胞因子的表达，或与STAT3结合形成复合物，这两种方式激活STAT3通路。本研究采用免疫组织化学法检测了临床病例标本中，即正常组织、胃癌前病变组织及进展期胃癌组织中，YAP与STAT3、pSTAT3的表达和定位，以Kruskal-Wallis H检验分析pSTAT3、STAT3及YAP在不同病理组织中的表达，从肠化到胃癌的发展过程中，三者表达逐渐增高，且与病理分级之间存在显著相关性；采用偏相关分析对所有病变组织进行综合观察，发现 pSTAT3与YAP在表达上呈明显线性正相关(r = 0.268, p < 0.01)， YAP与STAT3在表达上也呈 明显线性正相关(r = 0.241, p < 0.01)。.我们发现，胃癌细胞株AGS (p53 wt )、KATOIII (p53 null)两株细胞中STAT3及YAP相对正常胃上皮细胞株GES处于高表达状态，其下游肿瘤增殖、侵袭、转移等相关靶点蛋白表达均有相应升高。 通过siRNA干扰技术分别敲低这两株细胞中的YAP及STAT3的表达，并在mRNA及蛋白水平进行敲除效率验证，我们观察到AGS细胞的增殖能力明显降低，敲减YAP后能够将细胞周期阻滞在G1期，STAT3和YAP siRNA后可增加胃癌细胞株AGS的凋亡。进一步的研究表明，细胞增殖相关蛋白、周期相关蛋白及凋亡相关靶蛋白的表达均有显著性改变。通过细胞因子诱导YAP的高表达，在IL-6的刺激下，p-STAT3(Y705)和YAP都增高了，但是采用STAT3 siRNA将STAT3敲除之后，IL-6对YAP的升高作用并没有消除。因此IL-6可能通过其它通路来增加YAP表达的。过表达YAP能缓解STAT3敲除后对细胞活力的损害作用，但是过表达STAT3却对敲除YAP引起的细胞活力下降没有逆转作用。因此推测，YAP可能对STAT3本身或者其下游通路有促进或者补偿作用，共同参与胃癌的发生和发展。