8549

LGR5

BACKGROUND: Accumulating evidence supports the hypothesis that cancer stem cells (CSCs) are essential for cancer initiation, metastasis and drug resistance. However, the functional association of gastric CSC markers with stemness and epithelial-mesenchymal transition (EMT) signature genes is unclear. METHODS: qPCR was performed to measure the expression profiles of stemness and EMT signature genes and their association with putative CSC markers in gastric cancer tissues, cancer cell lines and sphere cells. Western blot analysis was used to confirm the results of the transcript analysis. Cell proliferation, cell migration, drug resistance and sphere cell growth assays were conducted to measure the expansion and invasion abilities of the cells. tumor xenograft experiments were performed in NOD/SCID mice to test cell stemness in vivo. Flow cytometry and immunofluorescence staining were used to analyze cell subpopulations. RESULTS: The expression of LGR5 was strikingly up-regulated in sphere cells but not in cancer tissues or parental adherent cells. The up-regulation of LGR5 was also positively associated with stemness regulators (NANOG, OCT4, SOX2, and AICDA) and EMT inducers (PRRX1, TWIST1, and BMI1). In addition, sphere cells exhibited up-regulated vimentin and down-regulated E-cadherin expression. Using gene-specific primers, we found that the NANOG expression primarily originates from the retrogene NANOGP8. Western blot analysis showed that the expression of both LGR5 and NANOG is significantly higher in sphere cells. LGR5 over-expression significantly enhanced sphere cell growth, cell proliferation, cell migration and drug resistance in MGC803 cells. tumor xenografts in nude mice showed that sphere cells are at least 10 times more efficient at tumor initiation than adherent cells. Flow cytometry analysis showed that ~20% of sphere cells are LGR5+/CD54+, but only ~3% of adherent cells are Lgr5+/CD54+. Immunofluorescence staining supports the above results. CONCLUSION: The LGR5-expressing fraction of CD54+ cells represents gastric cancer CSCs, in which LGR5 is closely associated with stemness and EMT core genes, and NANOG expression is mainly contributed by the retrogene NANOGP8. Sphere cells are the best starting materials for the characterization of CSCs.

BACKGROUND: tumor recurrence, the chief reason for poor prognosis of glioma, is largely attributed to glioma stem cells (GSCs) and epithelial-mesenchymal transition (EMT). However, the mechanisms among them remain unknown. Here, we determined whether leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), known as a stem cell marker for colon cancer and gastric cancer, can serve as a novel GSC marker involved in EMT and a therapeutic target in glioma. METHODS: Stemness properties were examined in FACS-isolated LGR5(+)/LGR5(-) cells. Reported stem cell markers, EMT and the Wnt/beta-catenin pathway were examined in stable LGR5 knockdown or overexpressed GSCs by Western Blot. The treatment experiment was performed in an intracranial orthotopic xenograft model by knockdown of LGR5 or by using the Wnt/beta-catenin pathway inhibitor Wnt-C59. LGR5 expression was determined in 268 glioma specimens by immunohistochemistry. RESULTS: LGR5(+) cells possessed stronger stemness properties compared to LGR5(-) cells. The expression of SOX2, Nanog, CD133, CD44, CD24 and EpCAM was modulated by LGR5. Both LGR5 knockdown and Wnt-C59 reduced tumor invasion and migration and blocked EMT by inhibiting the Wnt/beta-catenin pathway in vitro and suppressed the intracranial orthotopic xenograft growth and prolonged the survival of xenograft mice in vivo. Moreover, LGR5 was positively correlated with Ki67, N-cadherin and WHO grade and negatively correlated with IDH1. Glioma patients with high expression of LGR5 showed significantly poorer prognosis. CONCLUSIONS: LGR5 is a new functional GSC marker and prognostic indicator that can promote EMT by activating the Wnt/beta-catenin pathway and would thus be a novel therapeutic target for glioma.