c-Met新转录变异体在胃癌中的作用机制及临床意义研究

c-Met is an important target in treatment of gastric cancer, which has been proved in several clinical trials. However, how to choose suitable patients for targeted therapy is still needed to be resolved. It was reported that transcript variants are close related to the tumorigenesis and progression, as well as drug resistance of targeted therapy. In the preliminary study, we cloned a novel c-Met transcript variant through bioinformatics and validated it in transcriptional level. The novel c-Met transcript variant only contain the N terminal of wild c-Met and shortage of C terminal of wild c-Met, which indicates the transcript variant might have different role compared with wild c-Met. It indicates that c-Met transcript variant may play “false positive” in patient selection for targeting c-Met treatment. This study will validate the novel c-Met transcript variant protein expression in gastric cell lines and tissues. Receptor-ligand interaction, biological behavior and signaling transduction were used to illustrate the role and molecular mechanism between novel c-Met transcript variant and wild c-Met. We will further investigate the role of c-Met transcript variant in patient selection for targeting therapy of c-Met in gastric cancer. This study is on target to reveal the functional mechanism of novel c-Met transcript variant in gastric cancer. It will also provide important foundations of theory and application for individual therapy of targeting c-Met in gastric cancer.

c-Met是治疗胃癌的一个重要靶点，然而现有证据表明抗c-Met治疗仅在部分胃癌患者中有效，如何选择潜在有效患者进行靶向c-Met治疗是目前亟需解决的科学问题。研究显示多个靶点基因的转录变异体与患者选择及耐药密切相关。在前期研究工作中，申请者通过生物信息学成功克隆了一个新的c-Met转录变异体，并在转录水平验证了该变异体的存在。该变异体缺少C端的酪氨酸激酶结构域，提示其在胃癌中的作用与野生c-Met不同，在靶向c-Met治疗筛选患者中可能起“假阳性”作用。本研究拟在蛋白质水平验证该转录变异体在胃癌中的表达，通过受体-配体相互结合、生物学行为、信号传导等阐述c-Met新转录变异体与野生型c-Met的功能关系及其作用机制，并进一步明确其在靶向c-Met治疗晚期胃癌中的患者选择作用。该项目在阐明c-Met转录变异体作用机制的基础上，将为临床靶向c-Met精准治疗胃癌患者提供新的理论和应用基础。

本研究是以c-Met为靶点探讨其临床应用和机制，通过本课题的研究，具体的工作内容和主要结果如下：.1）完成 c-Met 转录变异体的克隆、重组质粒构建工作；体内外 c-Met 转录变异体对胃癌细胞凋亡、增殖、迁移、侵袭等生物学行为的影响。在晚期胃癌的标本中验证了c-Met新转录变异体的表达；进一步探索了c-Met转录变异体的生物学机制，发现c-Met转录变异体没有与全长c-Met的作用；在对晚期胃癌和靶向c-Met治疗晚期胃癌的病例中分析发现c-Met转录变异体可能是假阳性的可能机制；.2）本研究基于c-Met靶点探讨了c-Met在化疗前后的表达变化情况，共计纳入了122例晚期胃癌患者，通过IHC和CISH方法检测晚期胃癌治疗前后标本中c-Met的变化。研究发现c-Met高表达患者预后较差；39%的患者c-Met阳性在治疗后变为阴性；35%的患者阴性在治疗后变为阳性(P=0.007)；c-Met和HER2有部分患者为共表达。提示治疗前活检对于精准靶点治疗有重要作用。本研究已投稿BMC Cancer；.3）本课题探讨了自噬在c-Met抑制剂耐药中的作用。发现Met磷酸化（pMet）能明显抑制c-Met表达阳性细胞系MKN-45和SNU-5，且四种c-Met抑制剂均可诱导p62表达下调，LC3 II/I比例增加；使用自噬抑制剂Bafilomycin A1与Hydroxychloroquine处理细胞后，可以阻断c-Met抑制剂介导的自噬，且证实c-Met抑制剂诱导自噬是通过引起自噬体形成而不是阻滞自噬降解途径。进一步的机制探索研究中发现自噬产生时伴随mTOR和ULK1磷酸化水平显著下降，说明mTOR失活与ULK1激活参与了自噬发生，且这种现象可被c-Met配体HGF及mTOR激动剂MHY逆转，进一步说明c-Met抑制剂通过c-Met/mTOR/ULK1级联反应诱导自噬。基于上述研究结果，我们深入探索了联合自噬抑制剂和c-Met抑制剂的抑瘤作用，体外细胞中发现自噬抑制剂联合c-Met抑制剂可明显抑制细胞的增殖，且细胞凋亡比例增加，体外动物移植瘤模型中进一步证实自噬抑制剂联合c-Met抑制剂具有协同抑瘤作用。该部分已被Cell death and disease接收发表。.通过本课题的研究，为深入理解c-Met及其靶向c-Met治疗晚期胃癌提供了研究基础。