Lgr5-Wnt/β-catenin信号通路对肝癌干细胞特性的调控作用及其分子机制研究

Hepatocellular carcinoma (HCC) is the 5th most common cancer and the second most frequent cause of cancer death in men worldwide. HCC is believed to arise from cancer stem cells (CSCs)/ tumor initiating cells (T-ICs), which are responsible for tumor relapse, metastasis, and chemoresistance. Leucine-rich repeat-containing G protein-coupled receptor 5(LGR5) marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs).Lgr5 palyed important roles in regulating embryogenesis and self-renewal of adult stem cells. Lgr5 also has recently been found to be overexpressed in some types of human cancers.However, the precise function of Lgr5 in HCC and liver CSCs remain largely unknown. In our previous study, we identified OV6 as a new liver CSCs marker, and clarified several important singaling pathways regulating liver CSCs tumorigenicity and chemoresistance. We also found Lgr5 positive HCC cells exihibited stronger self-renewal ability and tumoriginicity . Additionally,we observed that expression of Lgr5 enhaunced Wnt/β-catenin signaling activity in HCC cells.In this project, by using molecular and cellular biology methods (spheroid assay, chemoresistance assay, NOD/SCID mice tumorigenicity experiments, experimental metastasis model), we will test the role of Lgr5 on regulating liver CSCs function（e.g. self-renewal, stemness, tumorigenicity, chemoresistance, invasion and metastasis ability）. We will also clarify the molecular mechanism involved. Success of the project will contribute to further understanding the role of liver CSCs in HCC progression. The importance of Lgr5-Wnt/β-catenin axis also provides important target to develop novel therapeutic agent against liver CSCs.

Lgr5是维持胚胎发育和成体干细胞自我更新的重要分子，但其对肝癌和肝癌干细胞的调控作用及机制仍知之甚少。申请人已建立肝癌干细胞研究平台，对肝癌干细胞标志物、分子调控网络、干预靶点进行了系统研究，论文发表于Gastroenterology、Hepatology等杂志。前期通过组织样本和细胞系，我们发现Lgr5在肝癌干细胞中高表达，Lgr5+肝癌细胞或过表达Lgr5的肝癌细胞具有更强致瘤性和自我更新能力。进一步研究还表明，Lgr5能增强肝癌细胞Wnt/β-catenin通路活性。本项目拟在此基础上，结合原代肝癌标本、人肝癌PDX模型和细胞系，从体外和体内两个水平详细研究Lgr5对肝癌干细胞自我更新、耐药、成瘤、侵袭转移等特性的影响，阐述Lgr5激活Wnt/β-catenin信号通路的具体机制，并探索靶向Lgr5-Wnt/β-catenin通路在抑制肝癌干细胞致瘤性、耐药和侵袭转移中的潜在价值。

肝癌干细胞概念的提出，为深入认识肝癌发生进展机制及寻找肝癌诊治新策略提供了理论基础。研究证实Lgr5是多种组织的干细胞标记物，并且和肿瘤的发生、发展密切相关。本研究结合大鼠DEN诱导肝癌模型、肝癌细胞系、临床肝癌样本，一方面从体内和体外分别证实了Lgr5阳性肝癌细胞的干细胞特征，阐明了Lgr5的干性基因功能；另一方面，解析了Lgr5通过调控Wnt／β-catenin通路增强肝癌细胞的成瘤性，从而调控肝癌的发展的分子机制。本项目的主要研究发现包括：1）明确了Lgr5在肝癌干细胞中的表达升高，Lgr5表达升高与肝癌的不良预后相关；2）在肝癌细胞中过表达Lgr5可增强肝癌细胞的干细胞特性（干性基因表达、耐药性、成球能力及体内成瘤能力），反之，干扰Lgr5可抑制肝癌细胞的干细胞特性；3）分子机制方面，Lgr5通过增强Wnt/β-catenin信号通路活性促进肝癌发展；4）在临床样本中，进一步证实了Lgr5表达与肝癌干细胞亚群数量、β-catenin核定位正相关；5）高剂量维生素C和二甲双胍可选择性杀伤肝癌干细胞（Lgr5阳性）。项目研究期间，共发表标注本基金号的SCI论文7篇，总影响因子超过40分，其中10分以上论文2篇；本课题共培养博士研究生3名，项目申请人2016年获得国家自然科学 基金“优秀青年基金”；2018年获评上海市“青年科技英才”； 申请专利1项。