Prolonged mammosphere culture of MCF-7 cells induces an EMT and repression of the estrogen receptor by microRNAs

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 CD⁴⁴⁺/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.