LGR5 Facilitates Liver Cancer Via Wnt/β-catenin Pathway

With rapid morbidity of cancer worldwide, people are more and more apprehensive of significant hazard of tumor imposed on public health. Meanwhile, Weinberg updated traditional concept of cancer by proposing new hallmarks of tumor, which includes self-sufficiency in growth signals, insensitivity to antigrowth signals, resisting cell death, limitless replicative potential, sustained angiogenesis, tissue invasion and metastasis, avoiding immune destruction, tumor promotion inflammation, deregulating cellular energetics and genome instability and mutation.In China liver diseases are so epidemic that incidence of liver tumor keeps high level in a long term. Liver tumor mainly contains primary liver cancer and metastatic liver cancer. According to different cell types, primary liver tumor has been classified to hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined hepatocellular and cholangiocarcinoma (HCC-CC). Approximately seventy-five percent of primary liver cancer belongs to HCC.Cancer stem cells (CSCs), a subpopulation in tumor tissue, possess more proliferative ability and manifest properties of stem cells, which play a crucial role in chemotherapy resistance and neoplasm recurrence. Liver cancer stem cells (LCSCs) or Tumor Initiating Cells (TICs) have been identified in liver tumor recently, which contributes to exploring mechanism and novel strategies for liver tumor. It is still a conundrum to identify and target the featured subgroup cells. Therefore, many molecules are recognized as biomarkers of HCC. Among them, Epcam, CD 133 and OV6 have been widely applied to identify cancer stem cells. However, these molecules are not exclusive enough to mark cancer stem cells particularly in HCC.LGR5, leucine-rich repeat containing G protein-coupled receptor 5, also called GPR49. LGR5, a new targeted gene of Wnt signal pathway, is structurally related to members of the glycoprotein hormone receptor family, which contains 107 amino acids and 7 transmembrane domains. Apart from being a biomarker of stem cells in tissues, it has been demonstrated that LGR5 is over expressed and strongly related to tumorigenesis. In intestinal cancer, LGR5 positive tumor cells has proved to be CSCs. LGR5, as a biomarker of CSCs, subsequently is used extensively in intestinal cancer. Nevertheless, how LGR5 react in liver tumor is still ambiguous. To explore the mechanism of LGR5 in liver cancer, we put forward that high-expressed LGR5 regulates tumorigenesis of CSCs through Wnt pathway.Methods To investigate expression in hepatic progenitor cells and human liver cancer, we constructed 2-AAF/PH model to study LGR5 via immunohistochemistry(IHC) assay. Next, LGR5 expression of RNA and protein were examined in rat hepatomas induced by diethylnitrosamine (DEN). Confirming the relationship of LGR5 and HCC preliminarily we detected the propotion of LGR5 positive cells by flow cytometry, subsequently isolated LGR5 positive and negative cells from HCC cells via magnetic activated cell sorting (MACS) for the propose of phenotype comparison. To testify the influence of LGR5 over-expression or down-regulaion on tumorigenesis, we constructed stale transfected HCC cell lines based on HCC-LM3 and PVTT cell lines, respectively. Co-localizations of LGR5 and other biomarkers of CSCs were confirmed via IHC and immunofluresence (IF). Sphere assay shed light on stem cell potential of LGR5 in liver tumor. RT-PCR also showed LGR5 which played a role of sternness correlated with genes associated with tumorigenesis. NOD/SCID mice were inoculate with cells of LGR5 high-expression, down-regulation, positive and negative to investigate tumorigenicity in vivo. To probe the principles how LGR5 facilitates neoplasm in HCC, we developed functional experiments associated with Wnt/β-catenin signal pathway, including RT-PCR, Western blot, immuno-preticipation (IP) and so forth.Results Contrasting with hepatocytes LGR5 highly expressed in hepatic progenitors, which implied tumorigenesis of LGR5. Using human specimens, we found that LGR5 expressed differentially in HCC patients. Importantly, LGR5 positive HCC cells exerted potential of stem cells in vivo and in vitro. It has been demonstrated that over-expression of LGR5 potentiated sternness of HCC cells and vice versa. Furthermore, LGR5 functiones astargeted gene of Wnt signal pathway through inhibiting APC/Axin/GSK-3β, degradation complex of β-catenin, to stimulate tumorigenesis. Analyzing pathological and clinical data of HCC patients, we imposed the positive correlation between expression of LGR5 and clinical prognosis.Conclusion We imposed that LGR5 is competent to be a biomarker of LCSCs given the evidence of LGR5 positive HCC cells functioned as CSCs. Our study showed that LGR5 being targeted gene of Wnt signal pathway inhibited APC/Axin/GSK-3β, degradation complex of β-catenin, to stimulate tumorigenesis. This study focuses on reporting the exclusive role of LGR5 in regulating LCSCs, and the results suggest that LGR5 may be a functional biomarker for HCC.