JOURNAL ARTICLE

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

Shaimaa Ahmed, Annie Wang, Trine Celius, Jason Matthews
Toxicological Sciences: An Official Journal of the Society of Toxicology 2014, 138 (1): 89-103
24299737
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.

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