PO-276 Prognostic significance of LGR5, an intestinal stem cell marker, in colorectal cancers. (running head: LGR5 in colorectal cancers)

Introduction Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) has been suggested as a promising cancer stem cell marker in colorectal cancers (CRCs). However, the clinical significance of LGR5 expression in CRCs remains controversial. Here, we investigated the expression profile of LGR5 and determined the prognostic impact of LGR5 in a large cohort of CRC samples. Material and methods LGR5 expression was determined with fresh-frozen CRC tissues by real time-PCR analysis. Tissue microarrays (TMAs) containing 1133 CRC samples were constructed, on which RNA in situ hybridization (ISH) for LGR5 was performed. The functional effects of LGR5 expression on cancer cell proliferation and migration was assessed by in vitro transfection technique. Results and discussions LGR5 expression was higher in CRCs than in normal mucosa, and was not associated with other cancer stem cell markers. LGR5 positivity was observed in 43% of CRCs and positively correlated with old age, well to moderate differentiation, and nuclear b-catenin expression. Enhanced LGR5 expression remained during the adenoma-carcinoma transition, but substantially declined in the budding cells. Because nuclear β-catenin expression was consistently observed in the budding cells and TGFβ1 treatment or SNAIL overexpression did not result in a decrease in the LGR5 expression in LOVO cells, Wnt or EMT pathway are not likely to be responsible for LGR5 suppression. LGR5 expression showed negative correlations with microsatellite instability and CpG island methylator phenotype, and was not associated with KRAS and BRAF mutations, suggesting an implication of LGR5 in chromosomal instability (CIN) pathway in CRC carcinogenesis. Notably, LGR5 positivity was an independent prognostic marker for better prognosis. LGR5 overexpression led to reduced tumour growth by decreasing ERK phosphorylation along with decreased colony forming and migration abilities in DLD1 cells. Likewise, knockdown of LGR5 expression resulted in a decline in the colony forming and migration capacities in LOVO cells. These data suggest the suppressive role of LGR5 in CRC progression. Conclusion Enhanced LGR5 expression remains persistent during the adenoma to carcinoma progression, and is associated with CIN pathway of colon carcinogenesis. Abrupt LGR5 down-regulation observed in the budding cancer cells at the invasive fronts is less likely due to altered Wnt or EMT signalling pathways. LGR5 was an independent prognostic marker for better clinical outcomes in CRCs.