鉴定TSC2对mTOR抑制剂雷帕霉素治疗肝癌的影响效果及相关分子机制

The mTOR pathway is the key regulatory pathway in (hepatocellular carcinoma，HCC) and important targets for drug development . The previous study confirmed that all mTOR targeted drugs failed to get better survival in phase II clinical studies in HCC. It is presumed that the main reason is related to the choice of drug indication. TSC2 is a key regulator of mTOR pathway，and it was previously reported that the genetic variations could affect the efficacy of mTOR inhibitors in HCC. We have found that the incidence of mutated TSC2 was significantly higher than that in European, American and Japanese populations, and most of the somatic mutations were functional variants. In addition, TSC2 and TP53 were identified to be co-mutated in the HBV positive HCCs. Accordingly, the project will explore the relationship between TSC2 and rapamycin treatment sensitivity, and establish the role of TS2C gene mutation in HCC, and clarify the effect of mutation on rapamycin sensitivity. Moreover, based on the mouse model with HBV background, TSC2 and TP53 will be co-knocked out by Crispr/Cas9 technology in the liver to construct the tumor model, and then to analyze the evolution of somatic mutations , and to identify mechanisms of the synergistic effects on HCC and the effects on rapamycin sensitivity. So，this study will define the selection criteria of rapamycin treatment in HCC with TSC2 as the bio-marker, and provide the basic theory for the clinical application of mTOR inhibitors in HCC, and further promote the precision medicine of HCC.

mTOR通路是肝细胞肝癌的关键调控通路和药物研发的重要靶点,研究证实mTOR靶向药的肝癌二期临床均告失败。推测其原因与药物适应症的选择有关。TSC2是mTOR关键调控分子，先前研究报道其基因变异会影响mTOR抑制剂在肝癌中的疗效。我们前期发现，相比欧美日人群，我国肝癌人群TSC2体细胞突变发生率显著增高，且多为功能变异，TSC2和TP53在HBV阳性肝癌中存在共突变效应。据此项目组将利用肝癌细胞系探究TSC2与雷帕霉素治疗敏感性的关系，确立TS2C基因突变在肝癌中的作用，明确突变对雷帕霉素敏感性的影响。并以HBV模型小鼠为背景，利用Crispr/Cas9技术共敲除TSC2和TP53，构建肝脏成瘤模型，以此分析突变进化规律，鉴定其协同作用机制以及对雷帕霉素敏感性的影响，阐明TSC2作为判定雷帕霉素适应症的分子标记的标准，为mTOR抑制剂在肝癌中的临床应用提供实验依据，推动肝癌的精准治疗。

肿瘤基因组学研究表明大概10-15%的肝细胞肝癌患者会在TSC1/2基因上检测到体细胞突变。TSC1/2是mTOR信号通路的关键基因，同时也是免疫抑制剂雷帕霉素的作用靶点。因雷帕霉素的抗癌作用，我们首先在24株肝癌细胞系中评估了雷帕霉素对肿瘤的抑制效果；其次，我们通过mRNA测序和外显子测序技术见了TSC1/2基因的表达水平和突变情况；最后，在雷帕霉素不敏感的TSC2高表达的JHH-7细胞株中，进行类TSC2基因的定点突变，全面的分析了TSC2基因突变与雷帕霉素抗癌敏感性之间的关系。通过研究我们初步确认TSC2基因表达水平与雷帕霉素敏感性线性相关，基因表达越低，敏感性越高。此外，TSC2基因突变同样影响雷帕霉素的抗肝癌效果，功能缺失突变对其影响更大。本研究为肝癌的个体化治疗提供了新的理论基础。