Reduced Expression of miR-200 Family Members Contributes to Antiestrogen Resistance in LY2 Human Breast Cancer Cells

<div><p>Introduction</p><p>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.</p><p>Results</p><p>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-2′-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.</p><p>Conclusions</p><p>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.</p></div

ZEB1 mRNA expression in MCF-7, LCC1, LCC2, LCC9 and LY2 cells.

<p>Cells were serum-starved for 48 h and treated with EtOH (vehicle control), 10 nM E₂, or 100 nM 4-OHT for 6 h. ZEB1 expression was quantified by qPCR. Values are the mean ± SEM of 3 experiments and are expressed as fold relative to EtOH-treated MCF-7. *p<0.05 <i>versus</i> EtOH-treated MCF-7.</p

Model of function of miR-200 family members in endocrine resistance in breast cancer cells.

<p>A, In endocrine-sensitive breast cancer cells, <i>e.g</i>., MCF-7, miR-200b and miR-200c are expressed, resulting in low ZEB1 protein expression. The lack of ZEB1 allows expression of E-cadherin. Vimentin is not expressed. B, In endocrine-resistant cells that have undergone EMT, miR-200 family expression is low, resulting in increased ZEB1 protein which inhibits E-cadherin expression. Vimentin, N-cadherin, and Slug expression is increased. Slug directly inhibits miR-200b expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062334#pone.0062334-Liu1" target="_blank">[69]</a>. Grey lettering indicates reduced expression and dashed lines indicate reduced regulation.</p

5-aza-dC and TSA increase miR-200b and miR-200c expression in LY2 cells.

<p>(A–F) LY2 or MCF-7 cells were treated with 2.5 µM 5-aza-dC for 72 h with 100 ng/ml TSA added for the last 16 h of treatment. The expression of miR-200b, miR-200c or ZEB1 mRNA was determined by qPCR. Values are the mean ± SEM of triplicate determinations. G, LY2 cells were treated with 2.5 µM 5-aza-dC (AZA) alone for 72 h or with 100 ng/ml TSA added for the last 24 h of treatment. Whole cell lysates were separated by 10% SDS-PAGE and western blotted for ZEB1 expression. The membrane was stripped and reprobed for α-tubulin. ZEB1/α-tubulin expression was normalized to non-treated control cells. Values are ± SEM of duplicate determinations. *p<0.05 versus untreated.</p

miR-200b and miR-200c inhibit LY2 cell migration in a wound healing assay.

<p>LY2 cells were plated in a 6-well plate (3000 cells/well), transfected with negative control, pre-miR-200b, or pre-miR-200c for 24 h. Cells were wounded by scratching using a p200 pipette tip at time zero (0 h). Cells were washed with medium to remove displaced cells. The wound closure was analyzed at the indicated times using NIH Image J software. Values are the mean ± SEM of four separate measurements. *p<0.01 versus negative control-transfected cells. There were no statistical differences between cells transfected with pre-miR-200b versus pre-miR-200c.</p

Overexpression of miR-200b and miR-200c inhibits expression of mesenchymal markers and increases E-cadherin in LY2 cells.

<p>(A–C) LY2 cells were not transfected (Non-TF), Mock-transfected (RNAiMAX), or transfected with negative control, pre-miR-200b, or pre-miR-200c for 24 h before preparing RNA or WCE for subsequent analysis. A, <i>ZEB1</i>, <i>ZEB2</i>, E-cadherin (<i>CDH1</i>), and vimentin (<i>VIM</i>) expression was quantified by qPCR. Values are the mean ± SEM. *p<0.05 versus negative control for each gene. B–C, Protein expression of Zeb1 as well as EMT markers N-cadherin, vimentin, and Slug, and epithelial marker E-cadherin was analyzed by western blotting normalized to the expression of α-tubulin. Non-transfected values are set to one.</p

Knockdown of miR-200b or miR-200c does not promote resistance of MCF-7 to 4-OHT or fulvestrant.

<p>MCF-7 cells were transfected with a negative control, anti-miR-200b, or anti-miR-200c. 1 d post transfection, cells were treated with 100 nM 4-OHT or 100 nM fulvestrant for 4 d prior to MTT assay. A, CT values for miR-200b and miR-200c in the cells transfected as indicated for 1 or 5 d. Values are the mean ± SEM of 3 determinations. B, MTT values are the mean ± SEM of 3 experiments. *p<0.05 versus EtOH-treated, control-transfected MCF-7 cells; ^#p<0.05 versus EtOH-treated, miR-200b- or miR-200c- transfected cells.</p

Expression of miR-200 family members in MCF-7, LCC1, LCC2, LCC9 and LY2 cells.

<p>Cells were serum-starved for 48 h and then treated with vehicle control EtOH, 10 nM E₂, or 100 nM 4-OHT for 6 h. miR-200 expression was quantified by qPCR. Values are the mean ± SEM of 3–4 experiments and are expressed as fold relative to EtOH-treated MCF-7 expression for each miRNA. *p<0.05 <i>versus</i> MCF-7 EtOH-treated.</p

miRNAs upregulated by AnAc MCF-7 cells.

<p>The genomic location of each miRNA was identified in miRAD <a href="http://bmi.ana.med.uni-muenchen.de/miriad/" target="_blank">http://bmi.ana.med.uni-muenchen.de/miriad/</a> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184471#pone.0184471.ref034" target="_blank">34</a>]. Verified targets are those experimentally validated targets of the indicated miRNA as demonstrated by 3’-UTR luciferase reporter assay in the cited reference. Since many publications do not include whether the 5p or 3p arm of the miRNA was studied, if the sequence of the miRNA was provided, it was searched in miRBase.org to identify which arm was used in the target gene 3’-UTR luciferase reporter assay.</p

miRNAs upregulated by AnAc in MDA-MB-231 cells.

<p>The genomic location of each miRNA was identified in miRAD <a href="http://bmi.ana.med.uni-muenchen.de/miriad/" target="_blank">http://bmi.ana.med.uni-muenchen.de/miriad/</a> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184471#pone.0184471.ref034" target="_blank">34</a>]. Verified targets are those experimentally validated targets of the indicated miRNA as demonstrated by 3’-UTR luciferase reporter assay. Since many publications do not include whether the 5p or 3p arm of the miRNA was studied, if the sequence of the miRNA was provided, it was searched in miRBase.org to identify which arm was used in the target gene 3’-UTR luciferase reporter assay.</p