RhoA的磷酸化在调控弥漫型胃癌细胞增殖中的分子机制研究

Recently, RhoA mutation has been identified as the driven gene in diffuse gastric cancer, especially is RhoA Y42 mutation. However, the mechanism remains not clear. Our results show that C-Met can phosphorylate RhoA in Y42, which significantly enhance the ubiquitination of RhoA. In this study, we will construct stable cell lines and animal model with RhoA Y42F or RhoA WT. Then we will confirm that ubiquitination of RhoA can mediated gastric tumorigenesis,which is relying on the phosphorylation of RhoA by C-Met. Using the C-Met inhibitor/RhoA inhibitor in cell and animal experiments, we try to treat gastric cancer by targeting RhoA mutation. In addition, we continue to detect Met, RhoA Y42 expression in vivo and in vitro.The current study will clarify the molecular mechanism of diffuse gastric cancer tumorigenesis for the first time and also provide new ideas for target therapy in gastric cancer.

近年来的研究表明，RhoA突变是弥漫型胃癌的主要驱动基因之一，其中Y42突变最常见。但其作用机制尚不明确。我们的初步结果显示：C-Met则能通过Y42位磷酸化RhoA来调控RhoA的泛素化。本课题将进一步合成RhoA Y42磷酸化抗体，构建表达RhoA磷酸化位点突变体RhoA Y42F及RhoA WT的稳定的弥漫型胃癌细胞株，证实RhoA对于肿瘤增殖的调控依赖C-Met在Y42位的磷酸化及RhoA的泛素化；同时建立裸鼠皮下移植瘤模型及腹腔转移模型，验证C-Met抑制剂／RhoA抑制剂靶向RhoA突变治疗胃癌的可行性。检测临床标本中Met、RhoAY42等蛋白表达与肿瘤转移及预后的关系。本研究首次阐明了弥漫型胃癌驱动基因RhoA调控的分子机制，将为胃癌的靶向治疗提供全新的思路。

胃癌细胞中c-Met可以结合RhoA,体内外实验均证明c-Met可以在Y42磷酸化RhoA;c-MET/HGF信号通路磷酸化RhoA第42位氨基酸后，RhoA蛋白变得不稳定，进而引起RhoA进入泛素-溶酶体途径降解,RhoA蛋白的降解减少，进一步抑制细胞内RhoA总体活性，从而抑制胃癌的发生及发展。我们进一步发现，Y42突变后，RhoA降解减少，胃癌细胞发生增殖，侵袭迁移的速度明显加快。c-met抑制剂ARQ197能明显抑制转染RhoA-Y42C的胃癌细胞增殖。磷酸化的RhoA和磷酸化的C-Met结合在一起，能更好的预测胃癌的预后。我们同时发现RhoA-Y42C是耐药突变，下调RhoA下游信号通路后，能提高胃癌细胞对化疗效果敏感性。本研究揭示了RhoA突变导致胃癌预后差的机制，有助于识别C-Met靶向治疗的获益人群。