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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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References

  1. Prat A, Parker JS, Karginova O, Fan C, Livasy C, Herschkowitz JI, He X, Perou CM (2010) Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res 12:R68

    Article  PubMed  Google Scholar 

  2. Bertucci F, Finetti P, Cervera N, Charafe-Jauffret E, Buttarelli M, Jacquemier J, Chaffanet M, Maraninchi D, Viens P, Birnbaum D (2009) How different are luminal A and basal breast cancers? Int J Cancer 124:1338–1348

    Article  PubMed  CAS  Google Scholar 

  3. Kalluri R, Weinberg RA (2009) The basics of epithelial-mesenchymal transition. J Clin Invest 119:1420–1428

    Article  PubMed  CAS  Google Scholar 

  4. Micalizzi DS, Farabaugh SM, Ford HL (2010) Epithelial-mesenchymal transition in cancer: parallels between normal development and tumor progression. J Mammary Gland Biol Neoplasia 15:117–134

    Article  PubMed  Google Scholar 

  5. Zeisberg M, Neilson EG (2009) Biomarkers for epithelial-mesenchymal transitions. J Clin Invest 119:1429–1437

    Article  PubMed  CAS  Google Scholar 

  6. Nadella KS, Jones GN, Trimboli A, Stratakis CA, Leone G, Kirschner LS (2008) Targeted deletion of Prkar1a reveals a role for protein kinase A in mesenchymal-to-epithelial transition. Cancer Res 68:2671–2677

    Article  PubMed  CAS  Google Scholar 

  7. Polyak K, Weinberg RA (2009) Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer 9:265–273

    Article  PubMed  CAS  Google Scholar 

  8. Thiery JP, Acloque H, Huang RY, Nieto MA (2009) Epithelial-mesenchymal transitions in development and disease. Cell 139:871–890

    Article  PubMed  CAS  Google Scholar 

  9. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, Weinberg RA (2008) The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133:704–715

    Article  PubMed  CAS  Google Scholar 

  10. Singh A, Settleman J (2010) EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 29:4741–4751

    Article  PubMed  CAS  Google Scholar 

  11. Blick T, Hugo H, Widodo E, Waltham M, Pinto C, Mani SA, Weinberg RA, Neve RM, Lenburg ME, Thompson EW (2010) Epithelial mesenchymal transition traits in human breast cancer cell lines parallel the CD44(hi/)CD24 (lo/-) stem cell phenotype in human breast cancer. J Mammary Gland Biol Neoplasia 15:235–252

    Article  PubMed  Google Scholar 

  12. Creighton CJ, Chang JC, Rosen JM (2010) Epithelial-mesenchymal transition (EMT) in tumor-initiating cells and its clinical implications in breast cancer. J Mammary Gland Biol Neoplasia 15:253–260

    Article  PubMed  Google Scholar 

  13. Ye Y, Xiao Y, Wang W, Yearsley K, Gao JX, Shetuni B, Barsky SH (2010) ERα signaling through slug regulates E-cadherin and EMT. Oncogene 29:1451–1462

    Article  PubMed  CAS  Google Scholar 

  14. Fujita N, Jaye DL, Kajita M, Geigerman C, Moreno CS, Wade PA (2003) MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer. Cell 113:207–219

    Article  PubMed  CAS  Google Scholar 

  15. Eeckhoute J, Keeton EK, Lupien M, Krum SA, Carroll JS, Brown M (2007) Positive cross-regulatory loop ties GATA-3 to estrogen receptor alpha expression in breast cancer. Cancer Res 67:6477–6483

    Article  PubMed  CAS  Google Scholar 

  16. Dydensborg AB, Rose AA, Wilson BJ, Grote D, Paquet M, Giguere V, Siegel PM, Bouchard M (2009) GATA3 inhibits breast cancer growth and pulmonary breast cancer metastasis. Oncogene 28:2634–2642

    Article  PubMed  CAS  Google Scholar 

  17. Yan W, Cao QJ, Arenas RB, Bentley B, Shao R (2010) GATA3 inhibits breast cancer metastasis through the reversal of epithelial-mesenchymal transition. J Biol Chem 285:14042–14051

    Article  PubMed  CAS  Google Scholar 

  18. Song Y, Washington MK, Crawford HC (2010) Loss of FOXA1/2 is essential for the epithelial-to-mesenchymal transition in pancreatic cancer. Cancer Res 70:2115–2125

    Article  PubMed  CAS  Google Scholar 

  19. Creighton CJ, Li X, Landis M, Dixon JM, Neumeister VM, Sjolund A, Rimm DL, Wong H, Rodriguez A, Herschkowitz JI, Fan C, Zhang X, He X, Pavlick A, Gutierrez MC, Renshaw L, Larionov AA, Faratian D, Hilsenbeck SG, Perou CM, Lewis MT, Rosen JM, Chang JC (2009) Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc Natl Acad Sci USA 106:13820–13825

    Article  PubMed  CAS  Google Scholar 

  20. Brabletz S, Brabletz T (2010) The ZEB/miR-200 feedback loop–a motor of cellular plasticity in development and cancer? EMBO Rep 11:670–677

    Article  PubMed  CAS  Google Scholar 

  21. Dykxhoorn DM (2010) MicroRNAs and metastasis: little RNAs go a long way. Cancer Res 70:6401–6406

    Article  PubMed  CAS  Google Scholar 

  22. Greene RM, Pisano MM (2010) Palate morphogenesis: current understanding and future directions. Birth Defects Res C 90:133–154

    Article  CAS  Google Scholar 

  23. de Herreros AG, Peiro S, Nassour M, Savagner P (2010) Snail family regulation and epithelial mesenchymal transitions in breast cancer progression. J Mammary Gland Biol Neoplasia 15:135–147

    Article  PubMed  Google Scholar 

  24. O’Day E, Lal A (2010) MicroRNAs and their target gene networks in breast cancer. Breast Cancer Res 12:201

    Article  PubMed  Google Scholar 

  25. Charafe-Jauffret E, Ginestier C, Iovino F, Wicinski J, Cervera N, Finetti P, Hur MH, Diebel ME, Monville F, Dutcher J, Brown M, Viens P, Xerri L, Bertucci F, Stassi G, Dontu G, Birnbaum D, Wicha MS (2009) Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer Res 69:1302–1313

    Article  PubMed  CAS  Google Scholar 

  26. Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS (2003) In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17:1253–1270

    Article  PubMed  CAS  Google Scholar 

  27. Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP (2007) Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3:12

    Article  PubMed  Google Scholar 

  28. Phoenix KN, Vumbaca F, Fox MM, Evans R, Claffey KP (2010) Dietary energy availability affects primary and metastatic breast cancer and metformin efficacy. Breast Cancer Res Treat 123:333–344

    Article  PubMed  CAS  Google Scholar 

  29. Greene SB, Herschkowitz JI, Rosen JM (2010) The ups and downs of miR-205: identifying the roles of miR-205 in mammary gland development and breast cancer. RNA Biol 7:300–304

    Article  PubMed  CAS  Google Scholar 

  30. Gjerdrum C, Tiron C, Hoiby T, Stefansson I, Haugen H, Sandal T, Collett K, Li S, McCormack E, Gjertsen BT, Micklem DR, Akslen LA, Glackin C, Lorens JB (2010) Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival. Proc Natl Acad Sci USA 107:1124–1129

    Article  PubMed  CAS  Google Scholar 

  31. Sullivan NJ, Sasser AK, Axel AE, Vesuna F, Raman V, Ramirez N, Oberyszyn TM, Hall BM (2009) Interleukin-6 induces an epithelial-mesenchymal transition phenotype in human breast cancer cells. Oncogene 28:2940–2947

    Article  PubMed  CAS  Google Scholar 

  32. Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, Clark L, Bayani N, Coppe JP, Tong F, Speed T, Spellman PT, De Vries S, Lapuk A, Wang NJ, Kuo WL, Stilwell JL, Pinkel D, Albertson DG, Waldman FM, McCormick F, Dickson RB, Johnson MD, Lippman M, Ethier S, Gazdar A, Gray JW (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10:515–527

    Article  PubMed  CAS  Google Scholar 

  33. Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, Pilotti S, Pierotti MA, Daidone MG (2005) Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 65:5506–5511

    Article  PubMed  CAS  Google Scholar 

  34. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100:3983–3988

    Article  PubMed  CAS  Google Scholar 

  35. Shimono Y, Zabala M, Cho RW, Lobo N, Dalerba P, Qian D, Diehn M, Liu H, Panula SP, Chiao E, Dirbas FM, Somlo G, Pera RA, Lao K, Clarke MF (2009) Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells. Cell 138:592–603

    Article  PubMed  CAS  Google Scholar 

  36. Zhao JJ, Lin J, Yang H, Kong W, He L, Ma X, Coppola D, Cheng JQ (2008) MicroRNA-221/222 negatively regulates estrogen receptor alpha and is associated with tamoxifen resistance in breast cancer. J Biol Chem 283:31079–31086

    Article  PubMed  CAS  Google Scholar 

  37. Adams BD, Cowee DM, White BA (2009) The role of miR-206 in the epidermal growth factor (EGF) induced repression of estrogen receptor-alpha (ERα) signaling and a luminal phenotype in MCF-7 breast cancer cells. Mol Endocrinol 23:1215–1230

    Article  PubMed  CAS  Google Scholar 

  38. Adams BD, Furneaux H, White BA (2007) The micro-ribonucleic acid (miRNA) miR-206 targets the human estrogen receptor-alpha (ERα) and represses ERα messenger RNA and protein expression in breast cancer cell lines. Mol Endocrinol 21:1132–1147

    Article  PubMed  CAS  Google Scholar 

  39. Spizzo R, Nicoloso MS, Lupini L, Lu Y, Fogarty J, Rossi S, Zagatti B, Fabbri M, Veronese A, Liu X, Davuluri R, Croce CM, Mills G, Negrini M, Calin GA (2010) miR-145 participates with TP53 in a death-promoting regulatory loop and targets estrogen receptor-alpha in human breast cancer cells. Cell Death Differ 17:246–254

    Article  PubMed  CAS  Google Scholar 

  40. Charafe-Jauffret E, Ginestier C, Birnbaum D (2009) Breast cancer stem cells: tools and models to rely on. BMC Cancer 9:202

    Article  PubMed  Google Scholar 

  41. Taube JH, Herschkowitz JI, Komurov K, Zhou AY, Gupta S, Yang J, Hartwell K, Onder TT, Gupta PB, Evans KW, Hollier BG, Ram PT, Lander ES, Rosen JM, Weinberg RA, Mani SA (2010) Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes. Proc Natl Acad Sci USA 107:15449–15454

    Article  PubMed  CAS  Google Scholar 

  42. Fuxe J, Vincent T, de Herreros AG (2010) Transcriptional crosstalk between TGFβ and stem cell pathways in tumor cell invasion: role of EMT promoting Smad complexes. Cell Cycle 9:2363–2374

    Google Scholar 

  43. Iliopoulos D, Hirsch HA, Struhl K (2009) An epigenetic switch involving NF-kappaB, Lin28, Let-7 microRNA, and IL6 links inflammation to cell transformation. Cell 139:693–706

    Article  PubMed  CAS  Google Scholar 

  44. Gao SP, Mark KG, Leslie K, Pao W, Motoi N, Gerald WL, Travis WD, Bornmann W, Veach D, Clarkson B, Bromberg JF (2007) Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas. J Clin Invest 117:3846–3856

    Article  PubMed  CAS  Google Scholar 

  45. Sansone P, Storci G, Tavolari S, Guarnieri T, Giovannini C, Taffurelli M, Ceccarelli C, Santini D, Paterini P, Marcu KB, Chieco P, Bonafe M (2007) IL-6 triggers malignant features in mammospheres from human ductal breast carcinoma and normal mammary gland. J Clin Invest 117:3988–4002

    Article  PubMed  CAS  Google Scholar 

  46. Lo HW, Hsu SC, Xia W, Cao X, Shih JY, Wei Y, Abbruzzese JL, Hortobagyi GN, Hung MC (2007) Epidermal growth factor receptor cooperates with signal transducer and activator of transcription 3 to induce epithelial-mesenchymal transition in cancer cells via up-regulation of TWIST gene expression. Cancer Res 67:9066–9076

    Article  PubMed  CAS  Google Scholar 

  47. Wu X, Chen H, Parker B, Rubin E, Zhu T, Lee JS, Argani P, Sukumar S (2006) HOXB7, a homeodomain protein, is overexpressed in breast cancer and confers epithelial-mesenchymal transition. Cancer Res 66:9527–9534

    Article  PubMed  CAS  Google Scholar 

  48. Zhau HE, Odero-Marah V, Lue HW, Nomura T, Wang R, Chu G, Liu ZR, Zhou BP, Huang WC, Chung LW (2008) Epithelial to mesenchymal transition (EMT) in human prostate cancer: lessons learned from ARCaP model. Clin Exp Metastasis 25:601–610

    Article  PubMed  CAS  Google Scholar 

  49. Rao X, Di Leva G, Li M, Fang F, Devlin C, Hartman-Frey C, Burow ME, Ivan M, Croce CM, Nephew KP (2010) MicroRNA-221/222 confers breast cancer fulvestrant resistance by regulating multiple signaling pathways. Oncogene 30:1082–1097

    Article  PubMed  Google Scholar 

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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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  3. X. Hong
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  4. J. S. Tirnauer
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  5. K. P. Claffey
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  6. B. A. White
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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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  • DOI: https://doi.org/10.1007/s10549-011-1534-y

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