Pterostilbene leads to DNMT3B-mediated DNA methylation and silencing of OCT1-targeted oncogenes in breast cancer cells

Abstract

Transcription factor (TF)-mediated regulation of genes is often disrupted during carcinogenesis. The DNA methylation state of TF-binding sites may dictate transcriptional activity of corresponding genes. Stilbenoid polyphenols, such as pterostilbene (PTS), have been shown to exert anticancer action by remodeling DNA methylation and gene expression. However, the mechanisms behind these effects still remain unclear. Here, the dynamics between oncogenic TF OCT1 binding and de novo DNA methyltransferase DNMT3B binding in PTS-treated MCF10CA1a invasive breast cancer cells has been explored. Using chromatin immunoprecipitation (ChIP) followed by next generation sequencing, we determined 47 gene regulatory regions with decreased OCT1 binding and enriched DNMT3B binding in response to PTS. Most of those genes were found to have oncogenic functions. We selected three candidates, PRKCA, TNNT2, and DANT2, for further mechanistic investigation taking into account PRKCA functional and regulatory connection with numerous cancer-driving processes and pathways, and some of the highest increase in DNMT3B occupancy within TNNT2 and DANT2 enhancers. PTS led to DNMT3B recruitment within PRKCA, TNNT2, and DANT2 at loci that also displayed reduced OCT1 binding. Substantial decrease in OCT1 with increased DNMT3B binding was accompanied by PRKCA promoter and TNNT2 and DANT2 enhancer hypermethylation, and gene silencing. Interestingly, DNA hypermethylation of the genes was not detected in response to PTS in DNMT3B-CRISPR knockout MCF10CA1a breast cancer cells. It indicates DNMT3B-dependent methylation of PRKCA, TNNT2, and DANT2 upon PTS. Our findings provide a better understanding of mechanistic players and their gene targets that possibly contribute to the anticancer action of stilbenoid polyphenols.

Introduction

DNA methylation is one of the components of the epigenome which strongly dictates the availability for transcriptional machinery, including transcription factors (TFs), to bind DNA and instigate transcription [1,2]). Specifically, increased methylation of regulatory regions of tumor suppressor genes (TSGs) or decreased methylation of regulatory regions of oncogenes has been found to result in corresponding changes in gene expression, namely silencing of TSGs or upregulation of oncogenes in many cancer types including breast cancer [3], [4]–5]. Several groups have identified TFs that are sensitive to DNA methylation status at the recognized elements or their proximity. For instance, TFs such as NRF1, CTCF, NF-κB, CREB, and OCT1 have impaired binding to DNA when cytosines are methylated around their respective TF binding sites [6], [7], [8], [9], [10]–11]. Numerous pieces of evidence show that changes in gene expression during carcinogenesis is often related to genes with differentially methylated regions and those regions are enriched for binding sites of methylation-sensitive TFs. Hence, the interplay between DNA methylation and TF binding within regulatory regions of cancer-related genes is of great interest and may at least partially explain the transcriptional dysregulation occurring during carcinogenesis and potential mechanisms of the action of anticancer agents, including dietary bioactive compounds [12].

Bioactive compounds present in our diet have been shown to remodel DNA methylation patterns and impact regulation of DNA methylation machinery in cancer models [3,4,13]. Specifically, stilbenoid polyphenols, resveratrol (RSV) and its dimethylated analogue pterostilbene (PTS), that are present abundantly in grapes and blueberries, respectively, have been shown to elicit bidirectional effects on DNA methylation status, change binding of DNA methyltransferases (DNMTs), and alter TF occupancy within differentially methylated regions [3,4]. A classic example is the capacity of RSV to effectively reverse cancer-specific hypermethylation and silencing of numerous TSGs such as BRCA1, RASSF1A, and PTEN [14], [15]–16]. Additionally, PTS led to an increase in DNA methylation of the Fasn gene promoter, which averted Fasn upregulation upon obesogenic diet in rats [17]. Furthermore, using genome-wide technology, we have previously identified differentially methylated genes in response to RSV and followed up with mechanistic studies to describe epigenetic and transcriptional regulators associated with remodeling of the DNA methylation patterns in response to stilbenoids, RSV and PTS [3,4]. We were the first group to show that treatment of breast cancer cells with stilbenoids results in DNA hypermethylation of regulatory regions of numerous genes with oncogenic and pro-metastatic functions and silences them. Interestingly, we specifically showed that 80% of regions hypermethylated in response to stilbenoids encompass a putative binding site for OCT1 [3].

OCT1 is a ubiquitous transcription factor that controls a wide range of target genes including genes involved in immune response, metabolic regulation, and stem cell function [18]. OCT1 expression levels are increased in certain malignancies such as gastric, breast, lung, and thyroid cancer and have been suggested to have a role in tumor initiation and progression [19]. Moreover, regions across the genome that are implicated in cancer malignancies have also been shown to be enriched with OCT1 binding sites, and elevated OCT1-mediated transcription is associated with poor prognosis in different cancers [20,21]. Analysis of the regulatory region in IL2 gene demonstrated that 90% methylation at a CpG site within OCT1 binding site motif correlates with IL2 transcriptional inactivity in MCF-7 breast cancer cells [10]. The same CpG site was demethylated in stimulated human T cells that express IL2 [10]. The sensitivity of OCT1 to DNA methylation within its binding region has also been shown in other genes such as CDX2, DAPK and HSPA2 [22], [23]–24].

Previously, we have shown that stilbenoid treatment restores DNA methylation at the MAML2 regulatory enhancer region leading to MAML2 silencing, which subsequently inhibits tumorigenic properties of breast cancer cells [3]. These changes corresponded with increased binding of DNMT3B, a de novo DNMT, and decreased binding of OCT1 within the tested MAML2 region. Of note, PTS exerted those effects at lower concentrations (7 µM) compared with RSV (15 µM), which supports the higher bioavailability and metabolic stability of PTS observed by others in in vivo studies [25]. These results have given us the basis for focusing on PTS and suggesting that OCT1-targeted oncogenes may be silenced through a mechanism whereby stilbenoid compounds recruit DNMT3B to regulatory regions of oncogenes to increase methylation and consequently reduce transcriptional activity. To further investigate these mechanistic players and their roles in modulating expression of genes with potential oncogenic functions, we have performed chromatin immunoprecipitation (ChIP) followed by next-generation sequencing to analyze binding events of OCT1 and DNMT3B at a genome-wide scale. We then elucidated a DNMT3B-dependent mechanism of hypermethylation and silencing of OCT1-targeted oncogenes which may, at least partially, contribute to the anticancer effects of stilbenoid polyphenols.