TGF-β介导非经典豪猪信号通路促进肝癌细胞对索拉非尼耐药

Sorafenib resistance is a major challenge in the treatment of advanced hepatocellular carcinoma. We previously demonstrated that poorly-differentiated Huh-7 subpopulation featured with aberrant activation of hedgehog signaling (Hh) pathway, whereas blocking the canonical pathway of hedgehog signaling did not significantly improve drug resistance. Therefore, we postulate that TGF-β-mediated non-canonical hedgehog signaling may play a significant role in maintaining sorafenib resistance of hepatoma cells. In the present study we aim to demonstrate that TGF-β/SMAD/Gli-2 signaling pathway initiates downstream events, such as epithelial mesenchymal transition, drug resistance and apoptosis, which are responsible for sorafenib resistance of poorly-differentiated hepatoma cells. To test our hypothesis, different hedgehog signaling activity xenograft models in immunodeficient mice will be established to elucidate the underlying mechanisms of aberrant activation of non-canonical hedgehog pathway by TGF-β involved in sorafenib resistance. We also investigate whether blocking TGF-β/SMAD/GLI-2 signaling by specific modalities affects hedgehog signaling activity and drug sensitivity. The completion of this study will enable to understand the molecular basis of non-canonical hedgehog signaling pathway mediating sorafenib resistance, and may identify new targets to improve the outcome of HCC chemotherapy.

索拉非尼耐药是进展期肝细胞癌的治疗难题。我们前期研究发现耐药的肝癌细胞亚群高表达豪猪信号活性，但抑制经典豪猪信号通路不能改善其对索拉非尼的敏感性，而TGF-β介导的非经典豪猪信号通路在低分化、索拉非尼耐药的肝癌细胞亚群中异常活化，可能是一条重要的参与肝癌细胞索拉非尼耐药的信号通路。我们推测TGF-β通过TGF-β/SMAD/GLI-2途径活化非经典豪猪信号通路可能是低分化肝癌细胞索拉非尼耐药的分子基础。本课题设计将在原有研究基础上，采用前期分选所得高豪猪信号活性、索拉非尼耐药的Huh-7-DN细胞作为工具细胞株，并建立免疫缺陷小鼠HCC移植瘤模型，明确TGF-β活化非经典豪猪信号通路的途径，初步探索非经典豪猪信号通路在索拉非尼耐药中的作用和机制。同时明确抑制TGF-β/SMAD/GLI-2途径对肝癌细胞豪猪信号活性和药物敏感性的影响，以期为临床治疗进展期肝癌索拉非尼耐药寻找新的干预靶点。

背景：原发和继发耐药是进展期肝细胞癌的治疗难题。前期研究发现低分化肝癌细胞亚群豪猪（Hedgehog）信号通路异常活化，呈现上皮细胞间质转化、高转移性和高耐药性特征，但对于低分化肝癌细胞中豪猪信号通路活化的机制尚不明确。因此，本研究着重探讨豪猪通路异常活化促进低分化肝癌细胞耐药的分子机制。主要结果：76.2%（16/21）临床肝癌组织中豪猪信号通路异常活化，38.1%（8/21）癌组织高表达GLI2和TGF-β1/SMAD3蛋白，33.3%（7/21）癌组织高表达耐药相关ABCC1蛋白，但未同时表达SHh蛋白。低分化肝癌细胞中GLI2、TGF-β1/SMAD3和ABCC1蛋白表达量显著高于高分化肝癌细胞。采用iRNA干扰技术抑制SMAD3表达可显著降低GLI2和ABCC1基因表达；予豪猪信号通路抑制剂LDE225和GANT61，或SMAD3抑制剂SIS3联合索拉非尼干预低分化肝癌细胞，均可显著改善低分化肝癌细胞对索拉非尼的敏感性，表明非经典豪猪信号通路TGF-β1/SMAD3/GLI2参与低分化肝癌细胞的耐药调控。生物信息学提示ABCC1启动子区存在GLI2和SMAD3同源结合序列，通过构建ABCC1启动子区、GLI2和SMAD3结合序列突变位点双荧光素酶报告基因系统，发现GLI2和SMAD3均促进ABCC1的转录。免疫共沉淀实验证实SMAD3通过与GLI2形成复合物，促进后者进入细胞核，启动下游靶基因的转录。结论：TGF-β1/SMAD3/GLI2信号轴介导非经典豪猪信号通路活化，启动下游耐药相关ABCC1等靶基因转录，是低分化肝癌细胞对索拉非尼耐药的分子基础。