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

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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-7M cells), which displays a stable mesenchymal and CSC-like CD44+/CD24−/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-7M 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-7M cells. This study provides the first example of mammosphere culture conditions inducing EMT and of EMT regulating microRNAs that target ERα.

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Acknowledgments

We wish to thank Dr. Lisa Mehlmann, Department of Cell Biology, UCHC, for her assistance with microscopy. Financial support is from R21 DK073456, NIH/NIDDK (BW); NIH/NCI CA064436 (KPC).

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Authors and Affiliations

  1. Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, 06030-3505, USA

    I. K. Guttilla, K. N. Phoenix, X. Hong, K. P. Claffey & B. A. White

  2. Center for Molecular Medicine, University of Connecticut Health Center, Farmington, CT, 06030, USA

    J. S. Tirnauer

  3. NEAG Comprehensive Cancer Center, University of Connecticut Health Center, Farmington, CT, 06030, USA

    J. S. Tirnauer & K. P. Claffey

  4. Center for Vascular Biology, University of Connecticut Health Center, Farmington, CT, 06030-6501, USA

    K. N. Phoenix, X. Hong & K. P. Claffey

  5. Breast Cancer Research Program, NEAG Comprehensive Cancer Center, University of Connecticut Health Center, Farmington, CT, 06030-6501, USA

    K. P. Claffey

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  1. I. K. Guttilla
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  2. K. N. Phoenix
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Correspondence to K. P. Claffey or B. A. White.

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Guttilla, I.K., Phoenix, K.N., Hong, X. et al. Prolonged mammosphere culture of MCF-7 cells induces an EMT and repression of the estrogen receptor by microRNAs. Breast Cancer Res Treat 132, 75–85 (2012). https://doi.org/10.1007/s10549-011-1534-y

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