Reduced Expression of miR-200 Family Members Contributes to Antiestrogen Resistance in LY2 Human Breast Cancer Cells Tissa T. Manavalan . , Yun Teng . , Lacey M. Litchfield . , Penn Muluhngwi, Numan Al-Rayyan, Carolyn M. Klinge* Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, Kentucky, United States of America Abstract Introduction: The role of miRNAs in acquired endocrine-resistant breast cancer is not fully understood. One hallmark of tumor progression is epithelial-to-mesenchymal transition (EMT), characterized by a loss of cell adhesion resulting from reduced E-cadherin and increased cell mobility. miR-200 family members regulate EMT by suppressing expression of transcriptional repressors ZEB1/2. Previously we reported that the expression of miR-200a, miR-200b, and miR-200c was lower in LY2 endocrine-resistant, mesenchymal breast cancer cells compared to parental, endocrine sensitive, epithelial MCF-7 breast cancer cells. Here we investigated the regulation of miR-200 family members and their role in endocrine- sensitivity in breast cancer cells. Results: miR-200 family expression was progressively reduced in a breast cancer cell line model of advancing endocrine/ tamoxifen (TAM) resistance. Concomitant with miR-200 decrease, there was an increase in ZEB1 mRNA expression. Overexpression of miR-200b or miR-200c in LY2 cells altered cell morphology to a more epithelial appearance and inhibited cell migration. Further, miR-200b and miR-200c overexpression sensitized LY2 cells to growth inhibition by estrogen receptor (ER) antagonists TAM and fulvestrant. Knockdown of ZEB1 in LY2 cells recapitulated the effect of miR-200b and miR-200c overexpression resulting in inhibition of LY2 cell proliferation by TAM and fulvestrant, but not the aromatase inhibitor exemestane. Demethylating agent 5-aza-29-deoxycytidine (5-aza-dC) in combination with histone deacetylase inhibitor trichostatin A (TSA) increased miR-200b and miR-200c in LY2 cells. Concomitant with the increase in miR-200b and miR-200c, ZEB1 expression was decreased and cells appeared more epithelial in morphology and were sensitized to TAM and fulvestrant inhibition. Likewise, knockdown of ZEB1 increased antiestrogen sensitivity of LY2 cells resulting in inhibition of cell proliferation. Conclusions: Our data indicate that reduced miRNA-200b and miR-200c expression contributes to endocrine resistance in breast cancer cells and that the reduced expression of these miR-200 family members in endocrine-resistant cells can be reversed by 5-aza-dC+TSA. Citation: Manavalan TT, Teng Y, Litchfield LM, Muluhngwi P, Al-Rayyan N, et al. (2013) Reduced Expression of miR-200 Family Members Contributes to Antiestrogen Resistance in LY2 Human Breast Cancer Cells. PLoS ONE 8(4): e62334. doi:10.1371/journal.pone.0062334 Editor: Aamir Ahmad, Wayne State University School of Medicine, United States of America Received January 17, 2013; Accepted March 20, 2013; Published April 23, 2013 Copyright: ß 2013 Manavalan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health R01 CA138410 to CMK. LML was supported by a predoctoral fellowship from National Institute of Environmental Health Sciences T32 ES011564. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: carolyn.klinge@louisville.edu . These authors contributed equally to this work. Introduction EMT (epithelial-to-mesenchymal transition) is a hallmark of metastatic cancer [1]. EMT is induced by activation of signaling pathways, e.g., TGF-b, Wnt, and Notch [2,3]. EMT is character- ized by loss of the epithelial marker E-cadherin resulting from gene methylation or repression by upregulation of transcriptional repressors Zinc finger E-box binding homeobox domain proteins ZEB1 (also known as TCF1 or dEF1) or ZEB2 (also known as SIP1), Snail1/2, and TWIST [4,5,6,7]. As a result of the reduction in E-cadherin and cell-cell interactions, cells acquire a mesenchy- mal phenotype distinguished by the expression of vimentin and N- cadherin [8]. Members of the miR-200 family and miR-221/222 are implicated in EMT and metastasis [9]. Reduced expression of these miRNAs has been reported in metastatic breast cancer [10,11,12]. Other miRNAs with roles in maintaining the epithelial phenotype of cells include miR-203, miR-205, miR-99a, miR-99b, miR-130a and miR-34a/b/c [13,14,15,16,17]. The link between the development of endocrine-resistance and EMT in breast cancer is still not clearly understood. While most patients with estrogen receptor a (ERa)-positive tumors initially benefit from endocrine therapies, e.g., tamoxifen (TAM), and aromatase inhibitors, e.g., exemestane, resistance develops in ,40– PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e62334