Constitutive expression of AhR and BRCA-1 promoter CpG hypermethylation as biomarkers of ERaα-negative breast tumorigenesis

Abstract

Background: Only 5-10 % of breast cancer cases is linked to germline mutations in the BRCA-1 gene and occurs early in life. Conversely, sporadic breast tumors, which represent 90-95 % of breast malignancies, have lower BRCA-1 expression, but not mutated BRCA-1 gene, and tend to occur later in life in combination with other genetic alterations and/or environmental exposures. The latter may include environmental and dietary factors that activate the aromatic hydrocarbon receptor (AhR). Therefore, understanding if changes in expression and/or activation of the AhR are associated with somatic inactivation of the BRCA-1 gene may provide clues for breast cancer therapy. Methods: We evaluated Brca-1 CpG promoter methylation and expression in mammary tumors induced in Sprague-Dawley rats with the AhR agonist and mammary carcinogen 7,12-dimethyl-benzo(a)anthracene (DMBA). Also, we tested in human estrogen receptor (ER)aα-negative sporadic UACC-3199 and ERaα-positive MCF-7 breast cancer cells carrying respectively, hyper- and hypomethylated BRCA-1 gene, if the treatment with the AhR antagonist aα-naphthoflavone (aαNF) modulated BRCA-1 and ERaα expression. Finally, we examined the association between expression of AhR and BRCA-1 promoter CpG methylation in human triple-negative (TNBC), luminal-A (LUM-A), LUM-B, and epidermal growth factor receptor-2 (HER-2)-positive breast tumor samples. Results: Mammary tumors induced with DMBA had reduced BRCA-1 and ERaα expression; higher Brca-1 promoter CpG methylation; increased expression of Ahr and its downstream target Cyp1b1; and higher proliferation markers Ccnd1 (cyclin D1) and Cdk4. In human UACC-3199 cells, low BRCA-1 was paralleled by constitutive high AhR expression; the treatment with aαNF rescued BRCA-1 and ERaα, while enhancing preferential expression of CYP1A1 compared to CYP1B1. Conversely, in MCF-7 cells, aαNF antagonized estradiol-dependent activation of BRCA-1 without effects on expression of ERaα. TNBC exhibited increased basal AhR and BRCA-1 promoter CpG methylation compared to LUM-A, LUM-B, and HER-2-positive breast tumors. Conclusions: Constitutive AhR expression coupled to BRCA-1 promoter CpG hypermethylation may be predictive markers of ERaα-negative breast tumor development. Regimens based on selected AhR modulators (SAhRMs) may be useful for therapy against ERaα-negative tumors, and possibly, TNBC with increased AhR and hypermethylated BRCA-1 gene.