Abstract 5318: The breast epithelial stem cell line D492 overexpressing miR200c-141 is resistant to mesenchymal transition.

Abstract MicroRNAs (miRs) are highly conserved, small RNA molecules that regulate key biological processes. miRs can act as tumor suppressors or oncogenes and their expression is often deregulated in cancers. The miR200 family is found in two clusters at chromosome 1 (miR200ba-429) and 12 (miR200c-141). Downregulation of the miR200 family has been linked to epithelial to mesenchymal transition (EMT) and recent studies show that members of this family are regulators of epithelial integrity in many tissues. We have recently shown that D492, a breast epithelial cell line with stem cell properties which generates branching structures when cultured in reconstituted basement membrane (rBM), undergoes EMT in coculture with endothelial cells. This is evidenced by formation of a stable spindle-like phenotype, suppression of keratin expression and cadherin switch from E- to N-cadherin (Sigurdsson et al. PLoS ONE 2011). In this study we have mapped the miRNA expressional changes in D492 compared to its mesenchymal-derivative D492M. Among the most profound changes were suppression of the miR200 family. Treatment of D492M with the demethylating agent 5-aza-2′-deoxycytidine reversed the EMT phenotype and reestablished E-cadherin expression. Bisulfite sequencing showed that the promoter region of miR200c-141 was methylated in D492M but not in D492. No methylation was seen in the promoter regions of E-cadherin or miR200ba-429 in either cell line. Overexpression of miR200c-141 in D492M reversed the EMT phenotype resulting in loss of N-cadherin and Snail expression and gain of E-cadherin and EpCAM expression. Interestingly, overexpression of miR200c-141 in D492M failed to reverse keratin expression back to an epithelial phenotype. When cultured in rBM D492M-200c-141 cells form irregular branching and showed no EMT phenotype. Interestingly, overexpression of miR200c-141 in D492M and D492 inhibits endothelial induced mesenchymal transition in rBM. This indicates that silencing of the miR-200c-141 with promoter methylation is associated with endothelial induced-EMT. In conclusion, our data suggests that overexpression of miR-200c-141 in D492 and D492M inhibits and reverses EMT, respectively, and this support the view that miR200c-141 may be a potent tumor suppressor and important regulator of epithelial integrity in the human breast. Citation Format: Bylgja Hilmarsdottir, Valgardur Sigurdsson, Jon T. Bergthorsson, Franzdottir R. Sigridur, Magnusson K. Magnus, T Gudjonsson. The breast epithelial stem cell line D492 overexpressing miR200c-141 is resistant to mesenchymal transition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5318. doi:10.1158/1538-7445.AM2013-5318