Role of MIR-29B-1 and MIR-29A in endocrine-resistant breast cancer.

Therapies targeting estrogen receptor α (ERα) including selective estrogen receptor modulators (SERMs), e.g., tamoxifen (TAM); selective estrogen receptor downregulators (SERDs), e.g., fulvestrant (ICI 182,780); and aromatase inhibitors (AI), e.g., letrozole, are successfully used in treating breast cancer patients whose initial tumor expresses ERα. Unfortunately, the effectiveness of endocrine therapies is limited as ~ 40% of breast cancer patients will eventually acquire resistance to them. The role of miRNAs in the progression of endocrine-resistant breast cancer is of keen interest in developing biomarkers and therapies to counter metastatic disease. This dissertation begins with a review on miRNAs implicated in breast cancer, their bona fide gene targets, and associated pathways promoting endocrine resistance. Although microRNAs are dysregulated in breast cancer, their contribution to endocrine-resistance is not yet fully understood. Previous microarray analysis identified miR-29a and miR-29b-1 as repressed by TAM in MCF-7 endocrine-sensitive breast cancer cells but stimulated by TAM in LY2 endocrine-resistant breast cancer cells. Here we examined the mechanism for the differential regulation of these miRs by TAM in MCF-7 versus TAM-resistant LY2 and LCC9 breast cancer cells and the functional role of these microRNAs in these cells. Knockdown studies revealed that ERα is responsible for TAM regulation of miR-29b-1/a transcription. Transient overexpression of miR-29b-1/a decreased MCF-7, LCC9, and LY2 proliferation and inhibited LY2 cell migration and colony formation but did not sensitize LCC9 or LY2 cells to TAM. Furthermore, TAM reduced DICER1 mRNA and protein in LY2 cells, a known target of miR-29. Supporting this observation, anti-miR-29b-1 or anti-miR-29a inhibited the suppression of DICER by 4-OHT. These results suggest that miR-29b-1/a have tumor suppressor activity in TAM-resistant cells and do not appear to play a role in mediating TAM resistance. The target genes mediating miR-29b-1/a tumor suppressor activity were unknown. Here, using RNA sequencing, we identify miR-29b-1 and miR-29a target transcripts in both MCF-7 and LCC9 cells. We find that miR-29b-1 and miR-29a regulate common and unique transcripts in each cell line. The cell-specific and common downregulated genes were characterized using the MetaCore Gene Ontology (GO) enrichment analysis algorithm. LCC9-sepecific miR-29b-1/a-regulated GO processes include oxidative phosphorylation, ATP metabolism, and apoptosis. Extracellular flux analysis of cells transfected with anti- or pre- miR-29a confirmed that miR-29a inhibits mitochondrial bioenergetics in LCC9 cells. qPCR and luciferase reporter assays also verified the ATP synthase subunit genes ATP5G1 and ATPIF1 as bona fide miR29b-1/a targets. Our results suggest that miR-29 repression of TAM-resistant breast cancer cell proliferation is mediated in part through repression of genes important in mitochondrial bioenergetics. There is a critical need to develop sensitive circulating biomarkers that accurately identify signaling pathways altered in breast cancer patients resistant to endocrine therapies. Serum miRNAs have the potential to serve as biomarkers of the progression of endocrine-resistant breast cancer due to their cancer-specific expression and stability. Exosomal transfer of miRNAs has been implicated in metastasis and endocrine-resistance. This dissertation ends with a review on miRNAs in breast tumors and in serum, including exosomes, from breast cancer patients that are associated with resistance to tamoxifen

Identification and Roles of miR-29b-1-3p and miR29a-3p-Regulated and Non-Regulated lncRNAs in Endocrine-Sensitive and Resistant Breast Cancer Cells

Despite improvements in the treatment of endocrine-resistant metastatic disease using combination therapies in patients with estrogen receptor α (ERα) primary tumors, the mechanisms underlying endocrine resistance remain to be elucidated. Non-coding RNAs (ncRNAs), including microRNAs (miRNA) and long non-coding RNAs (lncRNA), are targets and regulators of cell signaling pathways and their exosomal transport may contribute to metastasis. Previous studies have shown that a low expression of miR-29a-3p and miR-29b-3p is associated with lower overall breast cancer survival before 150 mos. Transient, modest overexpression of miR-29b1-3p or miR-29a-3p inhibited MCF-7 tamoxifen-sensitive and LCC9 tamoxifen-resistant cell proliferation. Here, we identify miR-29b-1/a-regulated and non-regulated differentially expressed lncRNAs in MCF-7 and LCC9 cells using next-generation RNA seq. More lncRNAs were miR-29b-1/a-regulated in LCC9 cells than in MCF-7 cells, including DANCR, GAS5, DSCAM-AS1, SNHG5, and CRND. We examined the roles of miR-29-regulated and differentially expressed lncRNAs in endocrine-resistant breast cancer, including putative and proven targets and expression patterns in survival analysis using the KM Plotter and TCGA databases. This study provides new insights into lncRNAs in endocrine-resistant breast cancer

Genome-wide miRNA response to anacardic acid in breast cancer cells.

MicroRNAs are biomarkers and potential therapeutic targets for breast cancer. Anacardic acid (AnAc) is a dietary phenolic lipid that inhibits both MCF-7 estrogen receptor α (ERα) positive and MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation with IC50s of 13.5 and 35 μM, respectively. To identify potential mediators of AnAc action in breast cancer, we profiled the genome-wide microRNA transcriptome (microRNAome) in these two cell lines altered by the AnAc 24:1n5 congener. Whole genome expression profiling (RNA-seq) and subsequent network analysis in MetaCore Gene Ontology (GO) algorithm was used to characterize the biological pathways altered by AnAc. In MCF-7 cells, 69 AnAc-responsive miRNAs were identified, e.g., increased let-7a and reduced miR-584. Fewer, i.e., 37 AnAc-responsive miRNAs were identified in MDA-MB-231 cells, e.g., decreased miR-23b and increased miR-1257. Only two miRNAs were increased by AnAc in both cell lines: miR-612 and miR-20b; however, opposite miRNA arm preference was noted: miR-20b-3p and miR-20b-5p were upregulated in MCF-7 and MDA-MB-231, respectively. miR-20b-5p target EFNB2 transcript levels were reduced by AnAc in MDA-MB-231 cells. AnAc reduced miR-378g that targets VIM (vimentin) and VIM mRNA transcript expression was increased in AnAc-treated MCF-7 cells, suggesting a reciprocal relationship. The top three enriched GO terms for AnAc-treated MCF-7 cells were B cell receptor signaling pathway and ribosomal large subunit biogenesis and S-adenosylmethionine metabolic process for AnAc-treated MDA-MB-231 cells. The pathways modulated by these AnAc-regulated miRNAs suggest that key nodal molecules, e.g., Cyclin D1, MYC, c-FOS, PPARγ, and SIN3, are targets of AnAc activity

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

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

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

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

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

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

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