IntroductionThe 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.ResultsmiR-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.ConclusionsOur 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.
Therapies targeting estrogen receptor α (ERα) including selective estrogen receptor modulators (SERMs), e.g., tamoxifen; selective estrogen receptor downregulators (SERDs), i.e., 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 by acquired resistance. 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 review focuses on miRNAs implicated as disruptors of antiestrogen therapies, their bona fide gene targets, and associated pathways promoting endocrine resistance.
Endocrine-resistance develops in ~ 40% of breast cancer patients after tamoxifen (TAM) therapy. Although microRNAs are dysregulated in breast cancer, their contribution to endocrine-resistance is not yet 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. We also demonstrated that 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 miR-29b-1/a has tumor suppressor activity in TAM-resistant cells and does not appear to play a role in mediating TAM resistance.
Aberrant microRNA expression contributes to breast cancer progression and endocrine resistance. We reported that although tamoxifen stimulated miR-29b-1/a transcription in tamoxifen (TAM)-resistant breast cancer cells, ectopic expression of miR-29b-1/a did not drive TAM-resistance in MCF-7 breast cancer cells. However, miR-29b-1/a overexpression significantly repressed TAM-resistant LCC9 cell proliferation, suggesting that miR-29b-1/a is not mediating TAM resistance but acts as a tumor suppressor in TAM-resistant cells. The target genes mediating this tumor suppressor activity were unknown. Here, 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 cellspecific 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,luciferase reporter assays, and western blot also verified the ATP synthase subunit genes ATP5G1 and ATPIF1 as bone fide miR29b-1/a targets. Our results suggest that miR-29 repression of TAMresistant breast cancer cell proliferation is mediated in part through repression of genes important in mitochondrial bioenergetics. microRNAs (miRNA, miRs) are 22 nt non-coding RNAs that recognize and bind complementary seed sequences in the 3′-UTR region of a target messenger RNA (mRNA) 1, 2 . This results in translational repression and/or transcriptional degradation of the target gene. By targeting several mRNAs, miRNAs regulate several cellular and biological processes including cell cycle, cell proliferation and cell differentiation, apoptosis, cellular respiration and glycolysis (reviewed in refs 3-6). Aberrant miRNA expression mediates disease initiation and progression in breast and other cancers 7 . Seventy percent of breast tumors express estrogen receptor alpha (ERα) implying eligibility for endocrine therapies including selective estrogen receptor modulators (SERMs), e.g., Tamoxifen (TAM), and aromatase inhibitors (AI), e.g., letrozole. The efficacy of endocrine therapy is limited by relapse in ~40% of patients 8,9 . Endocrine resistance is mediated by multiple mechanisms depending on the type of therapy used (reviewed in refs 10-14). Alterations in miRNA expression are also implicated in endocrine-resistance (reviewed in refs 15 and 16).Previously, we reported miR-29b-1 and miR-29a were downregulated by TAM in TAM-sensitive (TAM-S) MCF-7 BC cells and upregulated in TAM-resistant (TAM-R) LCC2, LCC9, and LY2 BC cells 17 . There are four miR-29 family members in the human genome: miR-29b-2 and miR-29c (chromosome 1q32.2) and miR-29b-1 and miR-29a (chromosome 7q32.3). miR-29b-1 and miR-29a are separated by ~652 bp on the same pri-miRNA transcript [1...
Anacardic acid (AnAc), a potential dietary agent for preventing and treating breast cancer, inhibited the proliferation of estrogen receptor α (ERα) positive MCF-7 and MDA-MB-231 triple negative breast cancer cells. To characterize potential regulators of AnAc action, MCF-7 and MDA-MB-231 cells were treated for 6 h with purified AnAc 24:1n5 congener followed by next generation transcriptomic sequencing (RNA-seq) and network analysis. We reported that AnAc-differentially regulated miRNA transcriptomes in each cell line and now identify AnAc-regulated changes in mRNA and lncRNA transcript expression. In MCF-7 cells, 80 AnAc-responsive genes were identified, including lncRNA MIR22HG. More AnAc-responsive genes (886) were identified in MDA-MB-231 cells. Only six genes were commonly altered by AnAc in both cell lines: SCD, INSIG1, and TGM2 were decreased and PDK4, GPR176, and ZBT20 were increased. Modeling of AnAc-induced gene changes suggests that AnAc inhibits monounsaturated fatty acid biosynthesis in both cell lines and increases endoplasmic reticulum stress in MDA-MB-231 cells. Since modeling of downregulated genes implicated NFκB in MCF-7, we confirmed that AnAc inhibited TNFα-induced NFκB reporter activity in MCF-7 cells. These data identify new targets and pathways that may account for AnAc’s anti-proliferative and pro-apoptotic activity.
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.
Acquired tamoxifen (TAM) resistance is a significant clinical problem in treating patients with estrogen receptor α (ERα) + breast cancer. We reported that ERα increases nuclear respiratory factor-1 (NRF-1), which regulates nuclear-encoded mitochondrial gene transcription, in MCF-7 breast cancer cells and NRF-1 knockdown stimulates apoptosis. Whether NRF-1 and target gene expression is altered in endocrine resistant breast cancer cells is unknown. We measured NRF-1and metabolic features in a cell model of progressive TAM-resistance. NRF-1 and its target mitochondrial transcription factor A (TFAM) were higher in TAM-resistant LCC2 and LCC9 cells than TAM-sensitive MCF-7 cells. Using extracellular flux assays we observed that LCC1, LCC2, and LCC9 cells showed similar oxygen consumption rate (OCR), but lower mitochondrial reserve capacity which was correlated with lower Succinate Dehydrogenase Complex, Subunit B in LCC1 and LCC2 cells. Complex III activity was lower in LCC9 than MCF-7 cells. LCC1, LCC2, and LCC9 cells had higher basal extracellular acidification (ECAR), indicating higher aerobic glycolysis, relative to MCF-7 cells. Mitochondrial bioenergetic responses to estradiol and 4-hydroxytamoxifen were reduced in the endocrine-resistant cells compared to MCF-7 cells. These results suggest the acquisition of altered metabolic phenotypes in response to long term antiestrogen treatment may increase vulnerability to metabolic stress.
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