Abstract
Triple negative breast cancers (TNBC) are the most aggressive and lethal breast cancers (BC). The aryl hydrocarbon receptor (AHR) is often overexpressed in TNBC, and its activation results in the epigenetic silencing of BRCA1, which is a necessary factor for the transcriptional activation of estrogen receptor (ER)α. The dietary isoflavone genistein (GEN) modulates BRCA1 CpG methylation in BC cells. The purpose of this study was to investigate the effect of GEN on BRCA1 epigenetic regulation and AHR activity in vivo and TNBC cells. Mice were administered a control or GEN-enriched (4 and 10 ppm) diet from gestation through post-natal day 50. Mammary tissue was analyzed for changes in BRCA1 regulation and AhR activity. TNBC cells with constitutively hypermethylated BRCA1 (HCC38) and MCF7 cells were used. Protein levels and mRNA expression were measured by Western blot and real-time PCR, respectively. BRCA1 promoter occupancy and CpG methylation were analyzed by chromatin immunoprecipitation and methylation-specific PCR, respectively. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. GEN administered in the diet dose-dependently decreased basal Brca1 methylation and AHR activity in the mammary gland of adult mice. HCC38 cells were found to overexpress constitutively active AHR in parallel with BRCA1 hypermethylation. The treatment of HCC38 cells with GEN upregulated BRCA1 protein levels, which was attributable to decreased CpG methylation and AHR binding at BRCA1 exon 1a. In MCF7 cells, GEN prevented the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent localization of AHR at the BRCA1 gene. These effects were consistent with those elicited by control AHR antagonists galangin (GAL), CH-223191, and α-naphthoflavone. The pre-treatment with GEN sensitized HCC38 cells to the antiproliferative effects of 4-hydroxytamoxifen. We conclude that the dietary compound GEN may be effective for the prevention and reversal of AHR-dependent BRCA1 hypermethylation, and the restoration of ERα-mediated response, thus imparting the sensitivity of TNBC to antiestrogen therapy.
Highlights
Breast cancers (BC) are the most common malignancies and causes of cancer mortality in women worldwide [1]
Western blot measurements confirmed the E2cells, we found by methylation-specific PCR (MSP) that the relative ratio of mBRCA1/umBRCA1 was on average ~10-fold higher dependent upregulation of BRCA1 in MCF7 cells, which served as a positive control for the detection (p = 0.0158) in HCC38 cell cultures (Figure 2B)
Similar stimulatory effects were observed with protein lysates from HCC38 cells treated with were observed with protein lysates from HCC38 cells treated with the aryl hydrocarbon receptor (AHR) antagonists NF (2 μM, the AHR antagonists NF (2 μM, Figure 3B), CH-223191 (10 μM, Figure 3C), and GAL
Summary
Breast cancers (BC) are the most common malignancies and causes of cancer mortality in women worldwide [1]. Triple negative breast cancers (TNBC) account for ~15–20% of BC cases and refer to a heterogenous group of BC that lacks expression of estrogen receptor (ER)α, progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) [2]. Nutrients 2019, 11, 2559 treatment option for TNBC, since these tumors lack molecular targets for therapy. Only ~20–30% of TNBC patients have a pathological complete response to neoadjuvant therapy [3]. Women who inherit BRCA1 mutations have a ~72% lifetime risk of developing BC [5], the majority of which are TNBC [6]. Similar to BRCA1 mutation carriers, the hypermethylation of BRCA1 is associated with a BRCA1-deficient phenotype (i.e., BRCAness) [7]
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