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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.

LGR5 Is a Gastric Cancer Stem Cell Marker Associated with Stemness and the EMT Signature Genes NANOG, NANOGP8, PRRX1, TWIST1, and BMI1