title: LGR5 Is a Gastric Cancer Stem Cell Marker Associated with Stemness and the EMT Signature Genes NANOG, NANOGP8, PRRX1, TWIST1, and BMI1.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Molecular Biology Lab of Gastric Cancer, School of Life Sciences, Hebei University, Baoding, Hebei Province, China.
authors.forename: Bei
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Medical Oncology, Affiliated Hospital of Hebei University, Baoding, Hebei Province, China.
authors.forename: Queting
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Molecular Biology Lab of Gastric Cancer, School of Life Sciences, Hebei University, Baoding, Hebei Province, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Cao
authors.affiliation: Molecular Biology Lab of Gastric Cancer, School of Life Sciences, Hebei University, Baoding, Hebei Province, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Ma
authors.affiliation: Molecular Biology Lab of Gastric Cancer, School of Life Sciences, Hebei University, Baoding, Hebei Province, China.
authors.forename: Chenxing
authors.initials: C
authors.lastname: Yin
authors.affiliation: Department of Medical Oncology, Affiliated Hospital of Hebei University, Baoding, Hebei Province, China.
authors.forename: Youchao
authors.initials: Y
authors.lastname: Jia
authors.affiliation: Department of Medical Oncology, Affiliated Hospital of Hebei University, Baoding, Hebei Province, China.
authors.forename: Aimin
authors.initials: A
authors.lastname: Zang
authors.affiliation: Molecular Biology Lab of Gastric Cancer, School of Life Sciences, Hebei University, Baoding, Hebei Province, China.
authors.forename: Wufang
authors.initials: W
authors.lastname: Fan
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D015820:Cadherins / Q000235:genetics; D063646:Carcinogenesis / Q000235:genetics; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D005260:Female; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D048054:Mitogen-Activated Protein Kinase 7 / Q000235:genetics; D000071317:Nanog Homeobox Protein / Q000235:genetics; D014411:Neoplastic Stem Cells / Q000473:pathology*; D009944:Organoplatinum Compounds / Q000494:pharmacology; D000077150:Oxaliplatin; D043562:Receptors, G-Protein-Coupled / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D051879:Twist-Related Protein 1 / Q000235:genetics; D015854:Up-Regulation; D014746:Vimentin / Q000235:genetics
chemical_list: D014408:Biomarkers, Tumor; D015820:Cadherins; D018398:Homeodomain Proteins; C116884:LGR5 protein, human; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D009944:Organoplatinum Compounds; C082157:PRRX1 protein, human; D043562:Receptors, G-Protein-Coupled; D051879:Twist-Related Protein 1; D014746:Vimentin; D000077150:Oxaliplatin; C481581:MAPK7 protein, human; D048054:Mitogen-Activated Protein Kinase 7