The importance of leucine-rich repeat containing g-protein coupled receptor 5 (LGRS5) in proliferation of hepatocellular carcinoma cell

Every year, approximately 700 thousands of newly diagnosed liver cancer cases are reported and approximately 85% to 90 of all these cases are hepatocellular carcinoma (HCC). HCC is the third most common cause of cancer related death and the most well-known risk factors for HCC are chronic viral hepatitis, chronic alcohol consumption, obesity, and chemicals such as aflatoxin. Presence of non-mutational or mutational Wnt/beta-catenin signaling deregulation in the one-third of all HCC indicates the importance of this pathway in hepatocarcinogenesis. For example, FZD-7, which is a Wnt receptor, is overexpressed in 90% of all HCC cases. Furthermore abnormal cytoplasmic and nuclear beta-catenin accumulation was found in 20 to 40% of HCC cases. However the lack of correlation between increased nuclear ß- catenin level and expression of its target proteins give us the idea that signaling pathways other than Wnt/beta-catenin might regulate expression of the target genes and/or activation of Wnt/beta-catenin signaling network might control other target genes which have not been described yet. In our previous studies we observed that Lithium mediated Wnt/beta-catenin signalization caused a 10-fold increased in the expression of LGR5 in HuH-7 cells (HCC cell line). LGR5, which is defined as one of the target genes of Wnt/beta-catenin pathway in the intestine, is reported to be a stem cell marker in hair follicle and especially it is used for describing colon cancer stem cells. In this thesis, expression of LGR5 in HCC cell lines and role of its expression in proliferation were investigated. Firstly, we showed that, among 9 HCC cell lines (SNU398, SNU-475, SK-Hep1, HepG2, PLC, Mahlavu, Hep3B, SNU449, HuH7), while LGR5 expressed significantly in poorly invasive and motile epithelial like HCC cell lines cell lines, comprising hepatic cancer stem cell population, it has no or low expression level in the others. Secondly, when without any induction that triggers Wnt / beta-catenin signaling pathway, LGR5 was overexpressed in HuH-7 cell lines by transient transfection with pCMV6-AC-GFP-LGR5 plasmid in the presence of mock cell controls, no significant effect was observed on proliferation. Since results obtained from mock controls comprising empty plasmid vector, and parental controls were considerably different from each other, it is worth repeating the experiments. In addition to LGR5 signalization and effects of this signalization on cell biology, we also investigated role of the triple complex of LGR5/Wnt3a/R-Spondin1 on cell biology and on Wnt/beta-catenin signaling pathway in the LGR5-overexpressed cells. While R-Spondin1 had no influence on transcriptional activity of Tcf/Lef, Wnt3a had little effect on Tcf/Lef activity when used alone. However, R-Spondin1 strongly synergized with Tcf/Lef activity induced by Wnt3a. In HuH7 cells, which were transiently transfected with the pCMV6-AC-GFP-LGR5 plasmid, overexpression of LGR5 did not affect either Wnt3a or Wnt3a/R-Spondin1 mediated signalization. This result implicated that Wnt3a and Wnt3a/R-Spondin1 mediated signalization was independent from LGR5-induced signalization. Wnt3a and/or R-Spondin treatment did not alter cell proliferation of LGR5-transiently transfected HuH7 cells compared to mock-transfected control cells. Overall, although LGR5 has no significant role in proliferation, its expression in HpCSC including poorly motile and invasive cell lines let us to think that it may involve in other biological behaviors of cells and this should be investigated in the future.