泛素连接酶Cbl-b通过STAT3/IL-6促进胃癌发展的机制研究

China has a highest incidence rate of gastric cancer. Dure to the lack of effectively clinical treatment, the median survival time of patients is less than one year. The latest research showed that the formation of "tumor-promoting inflammatory microenvironment" was the key to promote gastric cancer development, therefore, investigation towards molecular mechanisms of "tumor-promoting inflammatory microenvironment" is crucial. The binding of transmembrane receptor gp130 and cytokines can activate two important signal transduction pathways-JAK/STAT3 and SHP2/Ras/ERK, the absence of the latter can lead to JAK/STAT3 hyperactivating, which is the major cause in formation of "tumor-promoting inflammatory microenvironment", suggesting that JAK/STAT3 pathway was negatively regulated by SHP2/Ras/ERK pathway. Our previous studies demonstrated that Cbl-b inhibited the activation of SHP2/Ras/ERK signaling pathway; meanwhile, Cbl/b was overexpressed in gastric cancerous tissues and associated with TNM stage, suggesting that Cbl-b is likely to play a important role in regulation of two signal pathways-JAK/STAT3 and SHP2/RAS/ERK and the development of gastric cancer. Futher, we demonstrated for the first time that Cbl-b in MGC803 cells could promote cell colony formation and tumor pathogenesis in nude mice; at the same time, the activation of STAT3 and the secretion of IL-6 in MGC803 cells depend on the expression of Cbl-b. In this study, we aim at futher defining that Cbl-b is a key factor in the progression of gastric cancer, Cbl-b promotes gastric cancer development through inhibiting SHP2/Ras/ERK pathway and activating JAK/STAT3 pathway. Our research results will provide a new target for the treatment of gastric cancer, help to reduce mortality, and prolong the survival period of gastric cancer patients.

我国胃癌高发，且缺少有效治疗手段。最新研究表明，促癌炎性微环境的形成是促进胃癌发展的关键，因此，研究介导其形成的分子机制至关重要。胃癌发展中JAK/STAT3及SHP2/Ras/ERK两条通路非常重要，Jak/STAT3活化使促癌炎性微环境形成促进胃癌发展，而SHP2/Ras/ERK通路可负向调节其的活化，抑制胃癌发展。我们既往研究证实，Cbl-b抑制SHP2/Ras/ERK通路激活；同时在胃癌组织中高表达并与TNM分期相关，提示Cbl-b可能参与STAT3活化及胃癌发展。进一步，我们首次证实，Cbl-b高表达的胃癌细胞裸鼠体内成瘤性增强，STAT3的活化及IL-6的分泌显著增加，因此Cbl-b可能通过STAT3促进胃癌发展。本研究旨在明确Cbl-b是胃癌发展的关键因子，明确Cbl-b通过抑制SHP2/Ras/ERK、活化JAK/STAT3通路促进胃癌发展，研究结果将为胃癌治疗提供新的靶点

近年来，促癌炎性微环境的形成被认为是促进肿瘤发展的关键，而在胃癌中IL6/STAT3轴的活化是胃癌炎性微环境形成的关键。因此，是何种因素介导了胃癌中IL6/STAT3轴的高度活化，从而启动了胃癌的发生发展是急需研究的。我们的研究证实E3泛素连接酶Cbl-b是胃癌发展的抑癌基因，胃癌患者普遍存在cbl-b的低表达。在细胞水平上我们证实，敲除Cbl-b后胃癌细胞增殖速度明显增快，迁移能力增强,细胞发生了上皮间质转化，并发生干细胞表型的转化。在裸鼠肺转移模型中我们证实，敲除Cbl-b后胃癌细胞经尾静脉注射后在肺部成瘤能力更强，成瘤更大，并侵袭至多个层面。另外我们深入探讨了cbl-b在胃癌进展中的作用机制。我们证实敲除Cbl-b后的胃癌细胞上清中IL-6明显升高、STAT3轴活化明显，这是细胞增殖加快、迁移增强、抗凋亡能力及干细胞特性增加的原因。随后,我们发现cbl-b与TRAF6可以相互结合，cbl-b通过泛素蛋白酶体通路降解TRAF6,敲除cbl-b后TRAF6的泛素化降解以及下游的NFkB通路活化减少。在临床血浆及组织标本层面上，我们发现cbl-b低表达、IL-6和TRAF6的高表达在胃癌患者中提示预后不良。除了探索cbl-b在抑制胃癌进展中的作用及机制，本研究也探讨了cbl-b在化疗耐药中的作用。我们构建了阿霉素继发耐药的胃癌细胞系SGC7901/ADR和乳腺癌细胞系MCF-7/ADR,耐药细胞Cbl-b低表达,发生EMT，并伴有MET活化；在耐药细胞系过表达Cbl-b可增强凋亡、抑制侵袭转移，并逆转EMT；Cbl-b可通过降解EGFR，抑制p-AKT、p-ERK和p-FAK，抑制核转录因子ZEB1和Snail等抑制EMT逆转胃癌细胞和乳腺癌细胞对阿霉素的耐药。此外，建立了胃癌免疫预后模型：应用Cox模型发现胃癌患者肿瘤局部组织中肿瘤细胞表达的PD-L1，免疫细胞表达的PD-L1、PD-1和浸润的CD8+T细胞具有独立预后意义。将四个免疫因子集合在一起建立的预后预测模型，可以将AJCC分期相同的患者进一步区分为高/中/低危人群，为精准治疗提供决策的理论依据。还筛选了胃癌预后相关MicroRNAs，并进一步研究了对胃癌预后有影响的miR-1224-5p是通过抑制FAK的活化、进而抑制NFkB及STAT3通路的活化来抑制胃癌细胞的侵袭转移，进而发挥其抑癌作用机制。