CRBP1调控WIF1/Wnt/β-catenin通路抑制肝癌肿瘤干细胞特性的分子机制

Cancer stem cells are a sub-group of cells, which are considered to be responsible for cancer resistance of chemotherapy and radiation, relapse, and metastasis. Targeting cancer stem cells has a great impact on the strategy of liver cancer treatment. Our previous results showed that over-expression of CRBP1 (cellular retinol binding protein 1) positively associated with overall survival in liver cancer patients. Furthermore, CRBP1 could dramatically inhibit sphere formation capability and tumorigenesis ability of hepatoma cells, and also down-regulate the expression of genes linked to the stemness of liver cancer. Moreover, CRBP1 could up-regulate the expression of WIF1 and inhibit Wnt/β-catenin pathway. Taken together, we hypothesize that CRBP1 can inhibit the stemness of liver cancer stem cells via up-regulating the expression of WIF1, then inhibiting Wnt/β-catenin pathway, which leads to suppress the malignant progression of liver cancer. To evaluate our hypothesis, the following studies will be performed: (1) to determine the roles of CRBP1 inhibiting the stemness of liver cancer stem cells at cellular levels; (2) to explore the mechanism of CRBP1 inhibiting the stemness of liver cancer stem cells via regulating WIF1/Wnt/β-catenin pathway at molecular levels; (3) to confirm the function and mechanism of CRBP1 inhibiting the stemness of liver cancer stem cells in animal models. Our results will demonstrate a novel molecular mechanism by which CRBP1 inhibits the stemness of liver cancer stem cells, which might have major implications in the field of clinical treatment by targeting cancer stem cells, drug development and prognosis to liver cancer patient.

肿瘤干细胞是导致肿瘤患者放化疗抵抗、复发和转移的主要原因之一，靶向肿瘤干细胞对肝癌的治疗具有重要意义。前期研究发现胞内视黄醇结合蛋白CRBP1与肝癌病人生存期呈正相关，显著抑制肝癌细胞的成球和成瘤能力，降低肝癌干性相关基因的表达。进一步研究发现，CRBP1能够上调WIF1，抑制Wnt/β-catenin通路。据此，本项目提出“CRBP1上调WIF1阻断Wnt/β-catenin通路抑制肝癌肿瘤干细胞特性，进而阻止肝癌恶性进展”的假设。为验证这一假设，拟进行以下研究：细胞水平明确CRBP1对肝癌肿瘤干细胞特性的调控作用；分子水平探讨CRBP1调控WIF1/Wnt/β-catenin通路抑制肝癌肿瘤干细胞特性的机制；动物水平验证CRBP1抑制肝癌肿瘤干细胞特性的作用及机制。本项目的开展将阐明CRBP1抑制肝癌肿瘤干细胞特性的作用机制，为靶向肿瘤干细胞的肝癌治疗、药物开发和预后评价提供理论依据。

背景：胞内视黄醇结合蛋白1（Cellular retinol binding protein 1，CRBP-1），是视黄醇的胞内伴侣分子，介导视黄醇的胞内转运和代谢，在多种肿瘤中低表达,本项目旨在探讨CRBP-1对肝癌的调控作用及分子机制。.方法：利用免疫组化检测肝癌组织及癌旁组织中CRBP-1的表达水平，构建过表达CRBP-1的肝癌稳转细胞株，检测过表达CRBP-1对肝癌细胞体外增殖及体内成瘤能力的影响。利用ELISA检测胞内视黄酸的含量变化，利用双荧光素酶报告系统和ChIP实验探讨CRBP-1通过WIF1调控Wnt/β-catenin信号通路的分子机制。.结果：与正常肝组织相比，CRBP-1在肝癌组织中低表达，与肝癌病人的临床指标和生存期密切相关。过表达CRBP-1能够显著抑制体外肝癌细胞的增殖和体内肝癌细胞异体移植瘤的生长。此外，过表达CRBP-1能够显著抑制肝癌细胞的成球能力和肿瘤干性相关基因的表达。进一步机制研究发现，过表达CRBP-1能够增加胞内视黄酸的含量，激活转录因子RARs/RXRs，上调底物蛋白WIF1的表达，进而抑制Wnt/β-catenin信号通路，从而抑制肝癌细胞的干性。.结论：本项目研究结果表明CRBP-1是调控肝癌发生发展的重要因子，可以作为肝癌病人预后的标签蛋白和治疗靶点，对肝癌的临床诊断和治疗具有重要意义。