title: Prolonged mammosphere culture of MCF-7 cells induces an EMT and repression of the estrogen receptor by microRNAs.
abstract: Mammosphere culture has been used widely for the enrichment of mammary epithelial stem cells and breast cancer stem cells (CSCs). Epithelial-to-mesenchymal transition (EMT) also induces stem cell features in normal and transformed mammary cells. We examined whether mammosphere culture conditions per se induced EMT in the epithelial MCF-7 breast cancer cell line. MCF-7 cells were cultured as mammospheres for 5 weeks, with dispersal and reseeding at the end of each week. This mammosphere culture induced a complete EMT by 3 weeks. Return of the cells to standard adherent culture conditions in serum-supplemented media generated a cell population (called MCF-7(M) cells), which displays a stable mesenchymal and CSC-like CD(44+)/CD(24-/low) phenotype. EMT was accompanied by a stable, marked increase in EMT-associated transcription factors and mesenchymal markers, and a decrease in epithelial markers and estrogen receptor α (ERα). MCF-7(M) cells showed increased motility, proliferation and chemoresistance in vitro, and produced larger tumors in immunodeficient mice with or without estrogen supplementation. MicroRNA analysis showed suppression of miR-200c, miR-203, and miR-205; and increases in miR-222 and miR-221. Antisense hairpin RNA inhibitor targeting miR-221 resulted in re-expression of ERα in MCF-7(M) cells. This study provides the first example of mammosphere culture conditions inducing EMT and of EMT regulating microRNAs that target ERα.
authors.affiliation: Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030-3505, USA.
authors.forename: I K
authors.initials: IK
authors.lastname: Guttilla
authors.forename: K N
authors.initials: KN
authors.lastname: Phoenix
authors.forename: X
authors.initials: X
authors.lastname: Hong
authors.forename: J S
authors.initials: JS
authors.lastname: Tirnauer
authors.forename: K P
authors.initials: KP
authors.lastname: Claffey
authors.forename: B A
authors.initials: BA
authors.lastname: White
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D019008:Drug Resistance, Neoplasm; D058750:Epithelial-Mesenchymal Transition; D047628:Estrogen Receptor alpha / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000378:metabolism; D010641:Phenotype; D018874:Spheroids, Cellular / Q000502:physiology*; D047368:Tumor Burden / Q000235:genetics
chemical_list: D014408:Biomarkers, Tumor; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D035683:MicroRNAs