Journal of Cell Science Epigenetic modulation of the miR-200 family is associated with transition to a breast cancer stem-cell- like state Yat-Yuen Lim 1,2,3 , Josephine A. Wright 1,2 , Joanne L. Attema 1,2 , Philip A. Gregory 1,2 , Andrew G. Bert 1 , Eric Smith 4 , Daniel Thomas 1 , Angel F. Lopez 1,2 , Paul A. Drew 5 , Yeesim Khew-Goodall 1,6 and Gregory J. Goodall 1,2,6, * 1 Division of Human Immunology, Centre for Cancer Biology, SA Pathology, Adelaide, SA 5000, Australia 2 Discipline of Medicine, The University of Adelaide, Adelaide, SA 5005, Australia 3 Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia 4 Discipline of Surgery, The University of Adelaide, Adelaide, SA 5005, Australia 5 School of Nursing and Midwifery, Flinders University, Bedford Park, SA 5042, Australia 6 School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, SA 5005, Australia *Author for correspondence (greg.goodall@health.sa.gov.au) Accepted 27 February 2013 Journal of Cell Science 126, 2256–2266 ß 2013. Published by The Company of Biologists Ltd doi: 10.1242/jcs.122275 Summary The miR-200 family is a key regulator of the epithelial–mesenchymal transition, however, its role in controlling the transition between cancer stem- cell-like and non-stem-cell-like phenotypes is not well understood. We utilized immortalized human mammary epithelial (HMLE) cells to investigate the regulation of the miR-200 family during their conversion to a stem-like phenotype. HMLE cells were found to be capable of spontaneous conversion from a non-stem to a stem-like phenotype and this conversion was accompanied by the loss of miR-200 expression. Stem- like cell fractions isolated from metastatic breast cancers also displayed loss of miR-200 indicating similar molecular changes may occur during breast cancer progression. The phenotypic change observed in HMLE cells was directly controlled by miR-200 because restoration of its expression decreased stem-like properties while promoting a transition to an epithelial phenotype. Investigation of the mechanisms controlling miR-200 expression revealed both DNA methylation and histone modifications were significantly altered in the stem-like and non-stem phenotypes. In particular, in the stem-like phenotype, the miR-200b-200a-429 cluster was silenced primarily through polycomb group-mediated histone modifications whereas the miR-200c-141 cluster was repressed by DNA methylation. These results indicate that the miR-200 family plays a crucial role in the transition between stem-like and non-stem phenotypes and that distinct epigenetic-based mechanisms regulate each miR-200 gene in this process. Therapy targeted against miR-200 family members and epigenetic modifications might therefore be applicable to breast cancer. Key words: Epithelial–mesenchymal transition, Breast cancer stem cells, miR-200, DNA methylation, Histone modifications, Gene regulation Introduction Epithelial-derived tumours contain a heterogeneous population of cells which can be characterized by differences in histopathology and functional properties including proliferative and apoptotic responses to therapies and capacity for anchorage-independent growth (Hanahan and Weinberg, 2011). Recent evidence supports the existence of a cellular hierarchy within epithelial tumours. At the apex of this hierarchy is a tumour-initiating cell (T-IC) or cancer stem cell (CSC) population that can self-renew and differentiate to progeny cells, thus resulting in the observed cellular and functional heterogeneity within epithelial tumours (Polyak and Hahn, 2006; Reya et al., 2001). CSCs have been prospectively isolated from a variety of solid tumours, including breast (Al-Hajj et al., 2003; Ginestier et al., 2007), brain (Singh et al., 2004), colorectal (Dalerba et al., 2007; Ricci-Vitiani et al., 2007), head and neck (Prince et al., 2007), pancreatic (Li et al., 2007), prostate (Patrawala et al., 2007; Patrawala et al., 2006), melanoma (Schatton et al., 2008), and bladder (Chan et al., 2009). The characterization of these rare tumorigenic cells has the potential to provide important prognostic and therapeutic value for epithelial cancers. Breast cancer stem-like cells (bCSCs) enriched in the CD44 hi / CD24 2/low subpopulation are proposed to be largely responsible for cancer progression and metastasis (Al-Hajj, 2007; Al-Hajj et al., 2003). They possess stem-like properties including the ability to self-renew and differentiate into CD44 2/low /CD24 hi progeny, show resistance to standard therapies and increase in numbers after short courses of fractionated irradiation (Phillips et al., 2006). At the molecular level, bCSC gene signatures are associated with decreased overall patient survival, poor metastasis-free survival and tumour recurrence (Liu et al., 2007). Furthermore, bCSCs become more pronounced in tumour tissue following endocrine therapy or chemotherapy, consistent with their selective post-treatment survival (Creighton et al., 2009). Therefore, an improved understanding of bCSCs potential to drive breast cancer progression could inform therapeutic targeting of breast cancer. The epithelial–mesenchymal transition (EMT) is a crucial embryonic developmental process that is characterized by the losses of E-cadherin, cell polarity, cell–cell and cell–matrix contact as well as gain in motility and fibroblast-like morphology 2256 Research Article