Global identification of genes regulated by estrogen signaling and demethylation in MCF-7 breast cancer cells

https://doi.org/10.1016/j.bbrc.2012.08.007Get rights and content

Abstract

Estrogen signaling and epigenetic modifications, in particular DNA methylation, are involved in regulation of gene expression in breast cancers. Here we investigated a potential regulatory cross-talk between these two pathways by identifying their common target genes and exploring underlying molecular mechanisms in human MCF-7 breast cancer cells. Gene expression profiling revealed that the expression of approximately 140 genes was influenced by both 17β-estradiol (E2) and a demethylating agent 5-aza-2′-deoxycytidine (DAC). Gene ontology (GO) analysis suggests that these genes are involved in intracellular signaling cascades, regulation of cell proliferation and apoptosis. Based on previously reported association with breast cancer, estrogen signaling and/or DNA methylation, CpG island prediction and GO analysis, we selected six genes (BTG3, FHL2, PMAIP1, BTG2, CDKN1A and TGFB2) for further analysis. Tamoxifen reverses the effect of E2 on the expression of all selected genes, suggesting that they are direct targets of estrogen receptor. Furthermore, DAC treatment reactivates the expression of all selected genes in a dose-dependent manner. Promoter CpG island methylation status analysis revealed that only the promoters of BTG3 and FHL2 genes are methylated, with DAC inducing demethylation, suggesting DNA methylation directs repression of these genes in MCF-7 cells. In a further analysis of the potential interplay between estrogen signaling and DNA methylation, E2 treatment showed no effect on the methylation status of these promoters. Additionally, we show that the ERα recruitment occurs at the FHL2 promoter in an E2- and DAC-independent fashion. In conclusion, we identified a set of genes regulated by both estrogen signaling and DNA methylation. However, our data does not support a direct molecular interplay of mediators of estrogen and epigenetic signaling at promoters of regulated genes.

Highlights

► Estrogen signaling and demethylation can both control gene expression in breast cancers. ► Cross-talk between these mechanisms is investigated in human MCF-7 breast cancer cells. ► 137 genes are influenced by both 17β-estradiol and demethylating agent 5-aza-2′-deoxycytidine. ► A set of genes is identified as targets of both estrogen signaling and demethylation. ► There is no direct molecular interplay of mediators of estrogen and epigenetic signaling.

Introduction

It is well established that aberrations in both estrogen signaling and epigenetic modifications can lead to alterations of development and cell growth, causing various malignancies, including breast cancer [1], [2]. 17β-Estradiol (E2), the predominant endogenous estrogen and ER ligand in humans, regulates proliferation and development of breast epithelial cells that can ultimately lead to development of breast cancer [3]. Many studies have explored the mechanisms by which E2 regulates gene expression in breast cancer cells [4]. More recently the importance of epigenetic modifications for regulation of gene expression has been highlighted in breast cancer [5]. In this study we focus on the potential interplay of the epigenetic mark – DNA methylation and estrogen signaling in breast cancer cells.

Estrogens exert their biological functions via estrogen signaling pathways, primarily mediated by estrogen receptors (ERs), ERα and ERβ. ERs belong to the nuclear receptor superfamily of ligand-regulated transcription factors [6]. ERs, when ligand-activated, form dimers that bind to estrogen responsive elements (EREs) and bring coactivators or corepressors to the ER complex, to regulate gene expression. Estrogen also modulates gene expression through a process referred to as transcription factor cross-talk, in which ERs interact with other transcription factors. By non-genomic mechanisms, estrogens bind to ERs localized in the membrane compartment, which leads to activation of signal transduction pathways in the cytoplasm [7]. In addition to these ligand-induced transcriptional activities of ERs, ligand-independent pathways to activate ERs have been described. Growth factor signaling or stimulation of other signaling pathways leads to activation of kinases that can activate ERs by phosphorylation in the absence of ligand [8].

Breast cancer is the most common cancer in women [9]. E2 production and ERα activity are used as targets in hormonal therapy of breast cancer. ERα-expressing (ER+) cancers can be treated with aromatase inhibitors to block estrogen production, or with anti-estrogens, such as tamoxifen, which block ER activity. Tamoxifen is a partial ERα antagonist, competing with E2 for ERα binding and reversing its effect on gene expression [10]. Tamoxifen treatment is suitable for cancers in both pre- and postmenopausal women and has good effects particularly in patients with decreased levels of estrogen. However, 30% of women treated with tamoxifen for 5 years will have recurrent disease within 15 years [11]. As many women eventually develop resistance to current therapies, novel therapeutic alternatives are needed to improve the disease outcome for ER+ breast cancer patients. Regulation of epigenetic modifications might open up new avenues for breast cancer treatment.

DNA methylation is a key epigenetic mechanism for the silencing of many genes, including those involved in cell cycle regulation, DNA repair and apoptosis. Methylation of promoter regions is one mechanism for gene inactivation during cancer development, as demonstrated for several tumor suppressor genes [12]. DNA methylation is a chemical modification of DNA – the addition of a methyl group to the 5 position of the cytosine pyrimidine ring. It can be inherited through cell division, as well as added or removed during lifetime, without changing the original DNA sequence. DNA methylation occurs almost exclusively at cytosines located within CpG dinucleotides. Clusters of CpG dinucleotides, known as CpG islands, are associated with the promoter regions of many genes.

Epigenetic abnormalities are associated with cancer, genetic disorders, autoimmune diseases and aging [13]. Unlike genetic damage, epigenetic changes can sometimes be reversed. This creates a potential for the development of therapeutic strategies based on the regulation of the epigenetic status of cells. Several inhibitors of enzymes controlling epigenetic modifications, specifically DNA methyltransferases and histone deacetylases have shown promising anti-tumorigenic effects for some malignancies [14]. 5-aza-2′-deoxycytidine (DAC), a demethylating agent used in this study, is a cytosine analogue and prevents methylation of CpG islands by incorporating into DNA. DAC is used in treatment of myelodysplastic syndromes and it is undergoing investigation for use in treatment of tumors, together with other drugs targeting DNA methylation [15].

Both estrogen signaling and DNA methylation are shown to be involved in cell cycle control, by regulating proliferative and antiproliferative genes [16], [17]. Imbalance in these signaling pathways could lead to malignant transformation. Hence, the number of studies investigating the cross-talk between these two pathways is increasing. Although there is evidence for overlapping gene regulation by estrogen signaling and DNA methylation [18], [19], [20], [21], [22], [23], their common target genes on a genome-wide scale have not yet been reported.

In this study, we for the first time report the comparison of global gene expression profiles regulated by estrogen signaling and demethylation in MCF-7 cells. Furthermore, for a subset of genes identified as regulated by both estrogen signaling and demethylation, we explore potential mechanisms of cross-talk.

Section snippets

Cell culture and RNA extraction

MCF-7 cells, which express endogenous ERα, but not ERβ, were seeded in plates and cultured in phenol red-free DMEM supplemented with 2.5% dextran-coated charcoal-treated heat-inactivated FBS (Hyclone), either with or without 1 μM DAC (Sigma Aldrich), for 72 h. Culture medium with or without treatment was changed every 24 h. The DAC-treated cells were subsequently treated with vehicle (ethanol), and the DAC non-treated cells were subsequently treated with either vehicle or 10 nM E2 for 6 h. Each

Identification of genes regulated by estrogen signaling and demethylation

To identify genes regulated by estrogen signaling and demethylation, we compared the effects of E2 and DAC on global gene expression profiles in MCF-7 cells. 802 genes were identified as up-regulated by E2, while 851 genes were identified as down-regulated by E2. 1017 genes were identified as up-regulated by DAC, suggesting that demethylation is involved in their regulation. To identify possible common targets, we have compared the DAC up-regulated genes with E2-regulated genes. 88 annotated

Discussion

Although there is evidence for interplay between estrogen signaling and DNA methylation, their global overlapping effects on gene regulation have not yet been reported. The aim of this study was to identify the common target genes for estrogen signaling and DNA methylation in human MCF-7 breast cancer cells using a genome-wide gene expression profiling approach. We demonstrate that estrogen signaling and demethylation have the potential to regulate a set of common target genes in MCF-7 cells.

Acknowledgments

The study was supported by Cancerfonden, Karolinska Institute’s faculty funds for partial financing of doctoral students (KID), Karolinska Institute’s Center for Biosciences and the EU integrated project CRESCENDO. We are grateful to the Bioinformatic and Expression Analysis core facility at the Karolinska Institute (BEA, www.bea.ki.se) for performing the microarray assay.

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