Mek1介导的新型信号通路在幽门螺旋杆菌诱导的胃癌中的机制研究

Despite the identification of Helicobacter pylori's role in gastric cancer development and classification of the bacterium as a class I carcinogen in 1994, gastric carcinoma remains the third leading cause of cancer-related mortality especially in East Asian countries, China, Japan and Korea. H. pylori CagA toxin induces cell elongation which is characteristics of epithelial-mesenchymal transition (EMT) and pivotal pathogenesis for inflammation and gastric cancer development including metastasis. Although the molecular mechanism is not fully understood, H. pylori is known to modulate MAPK/Erk pathway (also known as the Ras-Raf-Mek-Erk pathway) for cell elongation. We demonstrated that the sequential activation of AR/EGF, the EGF receptor, A/B-Rafs and Mek1 in the MAPK pathway is required for the mechanism underlying H. pylori-induced cell elongation. However, surprisingly, Erk1/2 signal molecules are not required for the cell elongation. It strongly suggests that H. pylori exploits the Mek1-mediated signal pathway for inducing cell elongation. In this study, we will elucidate the novel Mek1-mediated signal pathway and its roles in H. pylori-induced cell elongation/EMT and furthermore gastric cancer development including metastasis. Firstly, we will identify which domain and which phosphorylation site of Mek1 molecule are responsible for the cell elongation phenomena. Secondly, we will construct knock-in Mek1 mutant in gastric cancer cell lines using CRISPER Cas9 technique and we will identify new binding molecules to the domain or phosphorylation site of Mek1, which sheds light on the novel Mek1-mediated signal pathway in details. Thirdly, we will perform orthotopic xenograft in mouse with these knock-in gastric cancer mutant cell lines to identify the roles of Mek1-mediated signal pathway in gastric cancer development including metastasis. Thus, this study should facilitate the development of therapeutic strategies against gastric disease development including gastric cancer.

胃癌是世界范围内造成癌症相关死亡的第三大原因，而幽门螺旋杆菌感染是胃癌发生的最重要危险因素之一。该细菌的致癌蛋白CagA可激活宿主MAPK/Erk通路而诱导上皮间质转化相关的细胞伸长表型，然而其机制尚未完全阐明。我们首先发现MAPK/Erk通路中的双调蛋白、EGF、EGFR1、A-/B-Raf和Mek1序列激活为细胞伸长表型所必需，然而Erk并不参与。这提示Mek1介导着其他未知信号通路，且该通路在幽门螺旋杆菌介导的细胞延长表型中具有关键作用。在本项目的研究中，我们将首先筛选出介导该表型的Mek1位点，而后使用基因编辑技术构建相应的Mek1位点突变细胞系。利用该细胞系和野生型细胞系进行免疫沉淀、免疫共沉淀和质谱分析，确认该位点磷酸化状态及其下游信号分子；而后通过小鼠模型进行原位异体转移实验，最终阐明Mek1介导的新型信号通路及其在幽门螺旋杆菌诱导的胃癌中的机制，为胃癌预防和治疗提供新思路。

胃癌是世界范围内造成癌症相关死亡的第三大原因，而幽门螺杆菌感染是胃癌发生的最重要危险因素之一。该细菌的致癌蛋白CagA可激活宿主MAPK/Erk通路而诱导上皮间质转化相关的细胞伸长表型，然而其机制尚未完全阐明。本项目研究发现MAPK/Erk通路中的双调蛋白、EGF、EGFR1、A-/B-Raf和Mek1序列激活为细胞伸长表型所必需，然而Erk并不参与。这提示Mek1存在新的信号位点并介导其他未知信号通路，在幽门螺旋杆菌介导的细胞伸长表型（HICE）中具有关键作用。本项目通过siRNA沉默，Mek1突变体质粒转染，CRISPR/Cas9基因编辑细胞系以及动物模型等技术，进一步发现Mek1 T55的磷酸化在HICE中起关键作用，并可介导不同胃上皮细胞系发生迁移现象，与间充质上皮细胞转化（EMT）相关。该信号通路的激活并非依赖于已知的Ser218和Ser222磷酸化位点。通过免疫共沉淀技术，我们发现Mek1在行蛋白印迹的高温加热后，T55磷酸化未能检出，这可能与其空间构象改变有关，进一步通过Mek1 T55磷酸化特性行抗体及免疫荧光证实Mek1 T55在幽门螺杆菌感染后可被磷酸化，且Mek1 E51，Q58在协同T55激活下游通路中起重要作用。进一步我们在实验小鼠中构建了三种不同的Mek1突变E51/T55/Q58A，可能由于胚胎致死，我们未能成功构建Mek1敲除小鼠，我们将利用这些基因工程小鼠进一步研究Mek1 T55介导的信号通路在幽门螺杆菌诱导的胃癌中的作用机制。 通过本项目研究我们确立了Mek1分子上存在的新信号位点，并证实其在HICE和EMT中其关键作用。接下来项目将进一步完善动物实验，完整阐明Mek1介导的新型信号通路。本项目研究有助于我们完善对MAPK通路的认识，对于阐明幽门螺杆菌如何通过Mek1介导胃癌的发生和转移具有重要意义，可为相关药物开发提供潜在靶点。