Mechanisms of hormonal regulation of CAD gene expression and inhibition by Aryl hydrocarbon receptor agonist in human breast cancer cells

The CAD gene is trifunctional and expresses carbamoylphosphate synthetase/aspartate carbamyltransferase/dihydroorotase, which are required for pyrimidine biosynthesis. CAD gene activities are induced in MCF-7 human breast cancer cells, and treatment of MCF-7 or ZR-75 cells with 17b-estradiol (E2) resulted in a 3-5 fold increase in CAD mRNA levels in both cell lines. E2 induced reporter gene activity in MCF-7 and ZR-75 cells transfected with a construct containing the growth-responsive -90/+115 (pCAD1) region of the CAD gene promoter, which contains three upstream GC-rich and two downstream E-box motifs. Deletion and mutation analysis of the CAD gene promoter demonstrated that only the GC boxes that bind Sp1 protein were required for E2-responsiveness. Results of gel shift and chromatin immunoprecipitation (CHIP) assays show that both Sp1 and estrogen receptor a (ERa) interact with the GC-rich region of the CAD gene promoter. Moreover, hormone-induced transactivation of pCAD1 was inhibited by cotransfection with dominant-negative Sp1 expression plasmid and small inhibitory RNA for Sp1. These results demonstrate that, in common with many other genes involved in E2-induced cell proliferation, the CAD gene is also regulated by a nonclassical ERa/Sp1-mediated pathway. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands suppress several E2-induced responses in the rodent uterus and mammary tumors and in human breast cancer cells. TCDD inhibited hormone-induced activation of CAD mRNA levels and reporter gene activity in MCF-7 and ZR-75 cells transfected with E2-responsive pCAD promoter constructs. E2-mediated transactivation of pCAD constructs with a mutant inhibitory dioxin responsive element DRE (iDRE) were also inhibited by TCDD suggesting that inhibitory AhR-ERa/Sp1 crosstalk was iDRE-independent. It was not possible to determine whether the levels of ERa in cells cotreated with E2 plus TCDD were limiting since the proteasome inhibitor MG132 itself directly decreased CAD mRNA levels. Using fluorescence resonance energy transfer (FRET), it was shown that both E2 and TCDD enhanced AhR-ERa interactions. E2 also induced interactions between ERa and Sp1. However cotreatment with TCDD abrogated this effect. Results of this study demonstrate a unique model of AhR-ERa crosstalk where the liganded AhR inhibits ERa-Sp1 interactions and also recruits ERa to Ahresponsive gene promoters such as CYP1A1.