title: Maintenance of the stemness in CD44(+) HCT-15 and HCT-116 human colon cancer cells requires miR-203 suppression.
abstract: The purpose of this study was to isolate cancer stem cells (CSCs, also called tumor-initiating cells, TICs) from established human colorectal carcinoma (CRC) cell lines, characterize them extensively and dissect the mechanism for their stemness. Freshly isolated CD44(+) and CD44(-) cells from the HCT-15 human colon cancer cell line were subjected to various analyses. Interestingly, CD44(+) cells exhibited higher soft agar colony-forming ability and in vivo tumorigenicity than CD44(-) cells. In addition, the significant upregulation of the protein Snail and the downregulation of miR-203, a stemness inhibitor, in CD44(+) cells suggested that this population possessed higher invasion/metastasis and differentiation potential than CD44(-) cells. By manipulating the expression of CD44 in HCT-15 and HCT-116 cells, we found that the levels of several EMT activators and miR-203 were positively and negatively correlated with those of CD44, respectively. Further analyses revealed that miR-203 levels were repressed by Snail, which was shown to bind to specific E-box(es) present in the miR-203 promoter. In agreement, silencing miR-203 expression in wild-type HCT-116 human colon cancer cells also resulted in an increase of their stemness. Finally, we discovered that c-Src kinase activity was required for the downregulation of miR-203 in HCT-15 cells, which was stimulated by the interaction between hyaluronan (HA) and CD44. Taken together, CD44 is a critical molecule for modulating stemness in CSCs. More importantly, we show for the first time that the downregulation of miR-203 by HA/CD44 signaling is the main reason for stemness-maintenance in colon cancer cells.
authors.affiliation: Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taiwan.
authors.forename: Sy-Yeuan
authors.initials: SY
authors.lastname: Ju
authors.affiliation: Institute of Pharmacology, National Yang-Ming University, Taiwan; Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan. Electronic address: shchiou@vghtpe.gov.tw.
authors.forename: Shih-Hwa
authors.initials: SH
authors.lastname: Chiou
authors.affiliation: Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taiwan. Electronic address: yeusu@ym.edu.tw.
authors.forename: Yeu
authors.initials: Y
authors.lastname: Su
mesh_terms: D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology; D015536:Down-Regulation; D024721:E-Box Elements; D045325:HCT116 Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000494:pharmacology; D051379:Mice; D008819:Mice, Nude; D035683:MicroRNAs / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015398:Signal Transduction / Q000187:drug effects; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000378:metabolism; D014183:Transplantation, Heterologous; D015854:Up-Regulation; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D018960:Hyaluronan Receptors; C546703:MIRN203 microRNA, human; D035683:MicroRNAs; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D006820:Hyaluronic Acid; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human
keywords: CRC; CSCs; ChIP; EMT; FACS; FCS; HA; PBS; cancer stem cells; chromatin immunoprecipitation; colorectal carcinoma; epithelial–mesenchymal transition; fetal calf serum; fluorescence activated cell sorting; hyaluronan; miRNAs; microRNAs; phosphate buffered saline.; shRNA; short hairpin RNA