c-Met通过激活NRF2抑制细胞铁死亡（ferroptosis）促进肝癌瑞戈非尼耐药的机制研究

Due to lower early diagnosis rate and drug resistance ,hepatocellular carcinoma(HCC) has poor prognosis. The only US Food and Drug Administration (FDA)-approved second-line systemic therapy for hepatocellular carcinoma is regorafenib; however, clinically, the problem of regorafenib resistance gradually stands out. And the mechanism of the resistance to regorafenib is not clear. Ferroptosis is a new form of regulated cell death that is a process driven by accumulated iron-dependent lipid ROS. Recent studies showed that ferroptosis is intimately related to the occurrence and development of malignant tumor and the sensitivity of chemotherapeutic drugs, and the inhibition of ferroptosis could sensitize sorafenib in liver cancer. Our previous studies showed that the c-Met pathway was activated in sorafenib-resistant HCC cells and inhibition of c-Met could reverse sorafenib resistance. These results provide strong evidence for further investigation on the clinical utility of dual inhibition of Akt and c-Met as a second-line therapy following acquired resistance to sorafenib in HCC. what's more, c-Met is associated with some important regulators of ferroptotic cell death,such as lipid peroxidation and ROS,glutathione metabolism,and NRF2 pathway, etcetera. Our preliminary data suggested that regorafenib could induce ferroptosis with activation of c-Met and NRF2 in HCC. According to the above, we present the hypothesis that c-Met promotes regorafenib resistance through inhibiting ferroptosis via activating NRF2 in hepatocellular carcinoma. To verify this hypothesis, we will perform experiments using the methods of lentivirus, immunohistochemistry, Western blot, qPCR, nude mice transplanted tumor model, Lipid peroxidation assay , Glutathione assay, etc. To investigate the influences and mechanisms of c-Met on the chemosensitivity of regorafenib in HCC through inhibition of ferroptosis. The project will provide new therapeutic targets and interventions for HCC from the perspective of ferroptosis.

由于早诊困难与耐药问题，肝细胞癌预后较差。2017年瑞戈非尼获美国FDA批准用于既往接受索拉非尼治疗期间病情进展的不可切除性肝细胞癌患者的二线治疗,其耐药问题在临床上逐渐凸显，但机制不清。铁死亡是一种新型的细胞死亡方式，实验显示其与肿瘤的发生发展及耐药密切相关。我们前期发现索拉非尼耐药肝癌细胞中c-Met通路异常活化，而用c-Met抑制剂可逆转耐药。此外，研究表明c-Met与铁死亡的重要调控因子如脂质过氧化与ROS、谷胱甘肽代谢及NRF2通路等有相关性，近期发现瑞戈非尼可诱导肝癌细胞铁死亡且伴随c-Met及NRF2异常活化。基于此，我们提出c-Met通过激活NRF2抑制细胞铁死亡促进肝癌瑞戈非尼耐药的假设，拟采用细胞及动物肿瘤模型为平台，应用慢病毒感染、组化、铁死亡水平、WB、qPCR等检测手段，探讨c-Met靶向铁死亡对肝癌瑞戈非尼敏感性的影响及机制，为改善肝癌瑞戈非尼疗效提供新思路。

肝癌是发病率和死亡率最高的恶性肿瘤之一，传统化疗药物疗效低，分子靶向药物是肝癌研究的主要方向，但因耐药性的出现导致疗效远不够理想。2017年瑞戈非尼获美国FDA批准，用于既往接受索拉非尼治疗期间，病情进展的不可切除性肝细胞癌患者的二线治疗,其耐药问题在临床上也逐渐凸显，但机制不清。我们课题组在之前的研究中发现索拉非尼耐药肝癌细胞中c-Met通路异常活化，而用c-Met抑制剂可逆转耐药；近期发现瑞戈非尼可诱导肝癌细胞铁死亡且伴随c-Met及NRF2异常活化，基于以上因素，本课题组对相关问题进行了研究，首先通过浓度递增法诱导获得了瑞戈非尼肝癌HepG2耐药细胞株，然后成功建立3D球型细胞培养模型，3D球体模型耐药和非耐药细胞株均可稳定生长。检测发现c-Met蛋白在2D细胞培养和3D瑞戈非尼耐药细胞株模型中高表达，进一步应用c-Met抑制剂capmatinib可以逆转瑞戈非尼耐药，并且和热疗（43℃）具有协同作用。在此基础之上我们进行了联合药物治疗，证实c-Met抑制剂卡博替尼（Cabozantinib）和新型CDK4/6抑制剂SHR6390联合用药对肝癌细胞系的协同抑制作用。联合用药不仅能够协同抑制细胞的增殖，同时通过细胞的运动和侵袭实验证实，其协同抑制肿瘤细胞的转移和侵袭。此外SHR6390可以增强卡博替尼对c-Met通路的抑制作用，使其蛋白表达明显下调。最后通过建立裸鼠肝癌皮下移植瘤模型证实，联合治疗组与单药治疗组相比，显著抑制皮下瘤生长，表明其在体内具有协同抗肝癌作用。为了寻找新的肝癌治疗靶点和预后标记物，课题组进行了人类基因序列分析，得出五个潜在的预后标志物(RP11502I4.3,SPINK5, CHRM3, SLC5A12, and RP11-467L13.7)，同时发现巨噬细胞与患者肿瘤突变风险显著相关，为后续的研究提供了方向和依据。