Targeting triple-negative breast cancer cells with the histone deacetylase inhibitor panobinostat

INTRODUCTION: Of the more than one million global cases of breast cancer diagnosed each year, approximately fifteen percent are characterized as triple-negative, lacking the estrogen, progesterone, and Her2/neu receptors. Lack of effective therapies, younger age at onset, and early metastatic spread have contributed to the poor prognoses and outcomes associated with these malignancies. Here, we investigate the ability of the histone deacetylase inhibitor panobinostat (LBH589) to selectively target triple-negative breast cancer (TNBC) cell proliferation and survival in vitro and tumorigenesis in vivo. METHODS: TNBC cell lines MDA-MB-157, MDA-MB-231, MDA-MB-468, and BT-549 were treated with nanomolar (nM) quantities of panobinostat. Relevant histone acetylation was verified by flow cytometry and immunofluorescent imaging. Assays for trypan blue viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation, and DNA fragmentation were used to evaluate overall cellular toxicity. Changes in cell cycle progression were assessed with propidium iodide flow cytometry. Additionally, qPCR arrays were used to probe MDA-MB-231 cells for panobinostat-induced changes in cancer biomarkers and signaling pathways. Orthotopic MDA-MB-231 and BT-549 mouse xenograft models were used to assess the effects of panobinostat on tumorigenesis. Lastly, flow cytometry, ELISA, and immunohistochemical staining were applied to detect changes in cadherin-1, E-cadherin (CDH1) protein expression and the results paired with confocal microscopy in order to examine changes in cell morphology. RESULTS: Panobinostat treatment increased histone acetylation, decreased cell proliferation and survival, and blocked cell cycle progression at G2/M with a concurrent decrease in S phase in all TNBC cell lines. Treatment also resulted in apoptosis induction at 24 hours in all lines except the MDA-MB-468 cell line. MDA-MB-231 and BT-549 tumor formation was significantly inhibited by panobinostat (10 mg/kg/day) in mice. Additionally, panobinostat up-regulated CDH1 protein in vitro and in vivo and induced cell morphology changes in MDA-MB-231 cells consistent with reversal of the mesenchymal phenotype. CONCLUSIONS: This study revealed that panobinostat is overtly toxic to TNBC cells in vitro and decreases tumorigenesis in vivo. Additionally, treatment up-regulated anti-proliferative, tumor suppressor, and epithelial marker genes in MDA-MB-231 cells and initiated a partial reversal of the epithelial-to-mesenchymal transition. Our results demonstrate a potential therapeutic role of panobinostat in targeting aggressive triple-negative breast cancer cell types

Dual regulation by microRNA-200b-3p and microRNA-200b-5p in the inhibition of epithelial-to-mesenchymal transition in triple-negative breast cancer

Epithelial to mesenchymal transition (EMT) involves loss of an epithelial phenotype and activation of a mesenchymal one. Enhanced expression of genes associated with a mesenchymal transition includes ZEB1/2, TWIST, and FOXC1. miRNAs are known regulators of gene expression and altered miRNA expression is known to enhance EMT in breast cancer. Here we demonstrate that the tumor suppressive miRNA family, miR-200, is not expressed in triple negative breast cancer (TNBC) cell lines and that miR-200b-3p over-expression represses EMT, which is evident through decreased migration and increased CDH1 expression. Despite the loss of migratory capacity following re-expression of miR-200b-3p, no subsequent loss of the conventional miR-200 family targets and EMT markers ZEB1/2 was observed. Next generation RNA-sequencing analysis showed that enhanced expression of pri-miR-200b lead to ectopic expression of both miR-200b-3p and miR-200b-5p with multiple isomiRs expressed for each of these miRNAs. Furthermore, miR-200b-5p was expressed in the receptor positive, epithelial breast cancer cell lines but not in the TNBC (mesenchymal) cell lines. In addition, a compensatory mechanism for miR-200b-3p/200b-5p targeting, where both miRNAs target the RHOGDI pathway leading to non-canonical repression of EMT, was demonstrated. Collectively, these data are the first to demonstrate dual targeting by miR-200b-3p and miR-200b-5p and a previously undescribed role for microRNA processing and strand expression in EMT and TNBC, the most aggressive breast cancer subtype

Endocrine Disruptor Regulation of MicroRNA Expression in Breast Carcinoma Cells

Several environmental agents termed "endocrine disrupting compounds" or EDCs have been reported to bind and activate the estrogen receptor-α (ER). The EDCs DDT and BPA are ubiquitously present in the environment, and DDT and BPA levels in human blood and adipose tissue are detectable in most if not all women and men. ER-mediated biological responses can be regulated at numerous levels, including expression of coding RNAs (mRNAs) and more recently non-coding RNAs (ncRNAs). Of the ncRNAs, microRNAs have emerged as a target of estrogen signaling. Given the important implications of EDC-regulated ER function, we sought to define the effects of BPA and DDT on microRNA regulation and expression levels in estrogen-responsive human breast cancer cells.To investigate the cellular effects of DDT and BPA, we used the human MCF-7 breast cancer cell line, which is ER (+) and hormone sensitive. Our results show that DDT and BPA potentiate ER transcriptional activity, resulting in an increased expression of receptor target genes, including progesterone receptor, bcl-2, and trefoil factor 1. Interestingly, a differential increase in expression of Jun and Fas by BPA but not DDT or estrogen was observed. In addition to ER responsive mRNAs, we investigated the ability of DDT and BPA to alter the miRNA profiles in MCF-7 cells. While the EDCs and estrogen similarly altered the expression of multiple microRNAs in MCF-7 cells, including miR-21, differential patterns of microRNA expression were induced by DDT and BPA compared to estrogen.We have shown, for the first time, that BPA and DDT, two well known EDCs, alter the expression profiles of microRNA in MCF-7 breast cancer cells. A better understanding of the molecular mechanisms of these compounds could provide important insight into the role of EDCs in human disease, including breast cancer

ERE-luciferase assay in MCF-7 cells.

<p>Cells were incubated overnight in media containing 5% charcoal-stripped FBS and were then transfected with an ERE-luciferase plasmid. After 4 hrs, drugs were added to the cells as indicated and luciferase levels were measured 18 hrs. later.</p

SuperArray Analysis of Genes Altered by E₂, BPA and DDT Treatment.

<p>Gene expression profile of select genes from a SuperArray analysis showing significant changes in expression with E₂, BPA or DDT treatment. Numbers indicate significant –fold changes in gene expression.</p

Quantitative PCR confirmation of select genes altered by estrogen, BPA, and DDT in MCF-7 cells.

<p>MCF-7 cells were treated with drug as indicated and total RNA was isolated, reverse-transcribed into cDNA, and subjected to real-time RT-PCR analysis for quantitation. Results are shown for (A) SERPINB5 (maspin) and (B) Bcl2 gene expression. Treatment of MCF-7 cells was as follows: Ethanol (vehicle), 1 nM E₂, 10 µM BPA, and 10 µM DDT. Results are expressed as the mean fold induction ± S.E.M. (***, <i>p</i><0.001; **, <i>p</i> = 0.01 – 0.001; *, <i>p</i> = 0.05 - 0.01).</p

MicroRNA microarray of MCF-7 breast cancer cells treated with E₂, BPA and DDT.

<p>Venn diagram showing the relative expression profiles of microRNA's from MCF-7 cells treated with estrogen vs BPA vs DDT.</p

The effect of BPA and DDT on endogenous miR-21 target genes in MCF-7 cells.

<p>MCF-7 cells were treated for 18 hrs. with 0, 1, 10 and 25 µM BPA or DDT followed by RT-PCR of PDCD4 and maspin.</p

MicroRNA microarray heatmaps of MCF-7 cells treated with vehicle, estrogen, BPA, or DDT.

<p>(A) Estrogen versus control (B) BPA versus control and (C) DDT versus control. Arrowheads represent expression of miR-21.</p

Effects of E₂, BPA and DDT on miR-21 gene expression in MCF-7 and MCF-7F cells.

<p>MCF-7 cells (A) and MCF-7F cells (B) were treated with either vehicle, 1 nM E₂, 10 µM BPA, or 10 µM DDT. Total microRNA was extracted followed by RT-PCR. * = p<0.05, ** = p<0.001.</p