c-MET信号通路与肝癌耐药机制的研究

Hepatocellular carcinoma (HCC) ranks the second place of cancer-related deaths in China. In the last project supported by National Natural Scientific Foundation of China, our team has revealed that sorafenib-resistant HCC cells expressed higher levels of c-MET and phosphoralted c-MET. The results of tissue arrays showed that tissues of human HCC that had been treated with sorafenib had significantly higher positive rates and expression levels of c-MET compared with untreated HCC tissues. Hypoxia, which is closely related to drug resistance in HCC, induced the activation of c-MET in HCC cells. These preliminary results indicate that the c-MET signal pathway may participate in the sorafenib resistance in HCC.However, there are several key questions on the regulatory mechanisms and cross-talks among these pathways remain unclear. The current project aims to explore further the mechanisms accounting for the resistance to sorafenib based on our recent discovery on this area in HCC, our recent preliminary data of studies on c-MET, hypoxia-inducible signaling pathway and already established techniques and methods in therapeutic strategies in HCC. The project will investigate how sorafenib affect the expression and activation of c-MET, the signaling pathways involved in the activation of c-MET by hypoxia, the cross-talks among sorafenib-targetted molecular pathways, c-MET pathway and hypoxia-inducible pathway, and whether blocking c-MET pathway could overcome the resistance of HCC to sorafenib. The answers to these questions will better our understanding of the molecular mechniams involved in the sorafenib resistance in HCC, and help seeking appropriate molecular targets and therapeutic strategies.

肝癌是我国高发恶性肿瘤，在癌症致死原因中居第二位。在国家自然基金资助的项目研究中，课题组发现索拉菲尼耐药肝癌细胞高表达c-MET和磷酸化c-MET，组织芯片发现索拉菲尼治疗后的肝癌组织表达c-MET的水平和阳性率明显上升；另外与肿瘤耐药机制密切相关的缺氧状态显著提高了肝癌细胞c-MET的活化。这些初期研究结果提示c-MET介导的信号通路在肝癌耐药机制起着重要作用。本项目中，课题组利用在肝癌索拉非尼耐药机制和治疗研究方面建立的技术平台和方法，进一步研究索拉非尼影响c-MET表达和活化的分子机制、缺氧影响c-MET活化的分子通路、索拉非尼作用通路、缺氧信号和c-MET信号通路间的相互调节作用以及信号网络的关键分子靶点。对于这些问题的研究将有助于阐明肝癌细胞索拉非尼耐药机制，寻找影响肝癌对索拉菲尼耐药机制的关键分子靶点，为研发新的分子靶向药物提供有力的证据。

肝细胞癌是我国最常见的恶性肿瘤之一。索拉菲尼的上市结束了肝癌治疗无有效药物的历史。然而，索拉菲尼仅能延长晚期肝癌患者2-3个月的生存期，肝癌对索拉菲尼的耐药严重影响治疗效果。因此，迫切研究其耐药机制并寻找新的治疗策略。本课题组针对这一问题进行了如下研究：1）索拉菲尼耐药肝癌细胞高表达c-MET，特异性c-Met抑制剂增强了索拉非尼抑制耐药性HCC细胞生长的效能。Akt抑制剂可以抑制索拉菲尼耐药肝癌细胞的生长，但却同时激活了c-Met信号通路，导致其疗效短暂和治疗失败。双重抑制Akt和c-Met信号通路，可以显著提高索拉菲尼抑制肝癌的效果。这些研究结果为进一步开发Akt和c-Met双重抑制作为索拉菲尼治疗失败后的二线药物的临床效用提供了有力证据。2）Na+/K+-ATPase是一种钠钾离子跨膜转运需要的ATP酶，我们发现索拉菲尼耐药细胞株中Na+/K+-ATPase磷酸化水平增强。正钒酸钠可以抑制Na+/K+-ATPase磷酸化，降低缺氧微环境中肿瘤细胞的HIF-1α合成及核转位，进而逆转肝癌细胞对索拉菲尼的耐药。这项研究提示，正钒酸钠是一种有开发潜力的抗肝癌药物。3）通过对肝癌细胞miRNA微阵列筛选发现索拉菲尼耐药细胞株高表达miR-21。用索拉菲尼处理肝癌细胞后，发现miR-21的表达升高，而PTEN的表达下降。这项研究提示miR-21参与肝癌细胞对索拉菲尼的耐受作用，其主要机理是通过PTEN/Akt通路抑制细胞自噬，miR-21是潜在的克服索拉菲尼耐药和治疗肝癌的靶点。4）通过对亲本和耐药肝癌细胞株采用miRNA芯片检测分析，筛选出耐药细胞株表达最高的六种miRNAs。我们构建了靶向这六种miRNA的人工长链非编码RNA表达载体（Ad5-AlncRNA）。转染Ad5-AlncRNA阻断了miRNA，抑制PTEN的下调和AKT的活化，并增强索拉非尼在体外和动物模型中对肝癌耐药细胞的抑制作用。