title: Zinc finger nuclease-mediated knockout of AHR or ARNT in human breast cancer cells abolishes basal and ligand-dependent regulation of CYP1B1 and differentially affects estrogen receptor α transactivation.
abstract: In this study, we used zinc finger nuclease-mediated knockout of the aryl hydrocarbon receptor (AHR) or AHR nuclear translocator (ARNT) in MCF7 and AHR knockout in MDA-MB-231 human breast cancer cells to investigate cross talk among AHR, ARNT, and estrogen receptor α (ERα). Knockout of AHR or ARNT prevented the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent induction of all AHR target genes examined. Knockout of AHR or ARNT also significantly reduced basal cytochrome P4501B1 (CYP1B1) expression levels, which were restored with overexpression of either protein but not with a DNA binding-deficient AHR mutant. Basal and TCDD-, 17β-estradiol (E2)-, or TCDD + E2-dependent recruitment of AHR, ARNT, ERα, NCoA3, and RNA polymerase II to CYP1B1 as well as CYP1B1 mRNA levels were abolished in MCF7-AHR((ko)) and MDA-MB-231 AHR(ko) cells. However, reduced but significant E2-dependent recruitment of ERα, NCoA3, and RNA polymerase II to CYP1B1 and weak increases in CYP1B1 mRNA levels were observed in MCF7 ARNT((ko)) cells. Interestingly, E2-dependent increases in trefoil factor 1, but not growth regulation by estrogen in breast cancer 1 (GREB1) mRNA levels, were dependent on ARNT expression. Moreover, the TCDD-dependent increases in the proteolytic degradation of ERα were prevented by the loss of AHR or ARNT. Our data show that AHR and ARNT play critical roles in the basal, TCDD, and E2-induced regulation of CYP1B1 but also reveal distinct roles for both proteins in ERα transactivation.
authors.affiliation: Department of Pharmacology & Toxicology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Shaimaa
authors.initials: S
authors.lastname: Ahmed
authors.forename: Annie
authors.initials: A
authors.lastname: Wang
authors.forename: Trine
authors.initials: T
authors.lastname: Celius
authors.forename: Jason
authors.initials: J
authors.lastname: Matthews
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000235:genetics*; D001483:Base Sequence; D065633:Cytochrome P-450 CYP1B1; D003851:Deoxyribonucleases; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000235:genetics*; D017353:Gene Deletion; D055785:Gene Knockdown Techniques; D006801:Humans; D008024:Ligands; D061986:MCF-7 Cells; D008969:Molecular Sequence Data; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D055503:Protein Multimerization; D020239:Receptor Cross-Talk; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics*; D015533:Transcriptional Activation / Q000187:drug effects*; D016335:Zinc Fingers
chemical_list: C497396:ARNT protein, human; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D008024:Ligands; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D004958:Estradiol; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1; D003851:Deoxyribonucleases
keywords: 2,3,7,8-tetrachlorodibenzo-p-dioxin; aryl hydrocarbon receptor; aryl hydrocarbon receptor nuclear translocator; cytochrome P4501A1; cytochrome P4501B1; estrogen receptor α; zinc finger nucleases.