Proinflammatory cytokines enhance estrogen-dependent expression of the multidrug transporter gene ABCG2 through estrogen receptor and NF{kappa}B cooperativity at adjacent response elements

Abstract

Constitutive activation of NFκB in estrogen receptor (ER)-positive breast cancer is associated with tumor recurrence and development of anti-estrogen resistance. Furthermore, a gene expression signature containing common targets for ER and NFκB has been identified and found to be associated with the more aggressive luminal B intrinsic subtype of ER-positive breast tumors. Here, we describe a novel mechanism by which ER and NFκB cooperate to up-regulate expression of one important gene from this signature, ABCG2, which encodes a transporter protein associated with the development of drug-resistant breast cancer. We and others have confirmed that this gene is regulated primarily by estrogen in an ER- and estrogen response element (ERE)-dependent manner. We found that whereas proinflammatory cytokines have little effect on this gene in the absence of 17β-estradiol, they can potentiate ER activity in an NFκB-dependent manner. ER allows the NFκB family member p65 to access a latent NFκB response element located near the ERE in the gene promoter. NFκB recruitment to the gene is, in turn, required to stabilize ER occupancy at the functional ERE. The result of this cooperative binding of ER and p65 at adjacent response elements leads to a major increase in both ABCG2 mRNA and protein expression. These findings indicate that estrogen and inflammatory factors can modify each other's activity through modulation of transcription factor accessibility and/or occupancy at adjacent response elements. This novel transcriptional mechanism could have important implications in breast cancer, where both inflammation and estrogen can promote cancer progression.

FIGURE 1.

TNFα potentiates the up-regulation of ABCG2 mRNA and protein by E₂. A, MCF-7 cells were treated with E₂ (10 nm), TNFα (10 ng/ml), or both for up to 4 h. RNA was isolated, and ABCG2 mRNA levels were examined by qPCR using the ΔΔC_t method. 36B4 was used as an internal control. *, p < 0.05 compared with E₂ alone at the same time point. B, MCF-7 cells were treated for 2 h with or without TNFα (10 ng/ml) in the presence of increasing doses of E₂. *, p < 0.05 compared with E₂ alone at the same dose. C, MCF-7 cells were treated for 2 h with or without E₂ (10 nm) in the presence of increasing doses of TNFα. #, p < 0.05 compared with TNFα alone at the same dose. D, MCF-7 cells were treated with E₂ (E; 10 nm), TNFα (T; 10 ng/ml), or both for 6 h. ABCG2 and β-actin protein levels were examined by Western blotting as described under “Experimental Procedures.” Treatment with vehicle (V) served as a control.

FIGURE 2.

TNFα potentiates ABCG2 expression by enhancing ER occupancy at an essential ERE. A and B, ABCG2 mRNA levels were examined by qPCR in MCF-7 cells pretreated for 2 h with or without the ER antagonist ICI 182,780 (ICI; 1 μm) or a small molecule inhibitor of ERα binding to ERE, 8-[(benzylthio)methyl]theophylline (TPBM; 20 μm), followed by treatment with E₂ (10 nm), TNFα (10 ng/ml), or both for an additional 2 h. *, p < 0.05 compared with E₂ alone. DMSO, dimethyl sulfoxide. C, MCF-7 cells were transfected with 1 μg of an ABCG2-luciferase reporter plasmid in which the ERE located at −180 was intact (−243), mutated (mutERE), or deleted (−115), along with 200 ng of the Renilla luciferase control plasmid pGL4.70. Dual-Luciferase assays were carried out following treatment with E₂, TNFα, or both for 4 h as described under “Experimental Procedures.” *, p < 0.05 compared with E₂ alone. D, MCF-7 cells were treated with E₂, TNFα, or both for up to 60 min. ChIP assay was performed, and ER recruitment to the ABCG2 regulatory region containing the ERE was examined by qPCR. *, p < 0.05 compared with E₂ alone. ER occupancy in response to different treatments is expressed as the percentage of maximum ER occupancy, which was observed at 45 min of E₂ + TNFα treatment. E, ChIP assay was performed with an antibody specific for ERα or a nonspecific IgG antibody as a negative control. qPCR was performed for the ABCG2 regulatory region containing the ERE (Enhancer) or for a far upstream region (Upstream) as a negative control. *, p < 0.05 compared with E₂ alone; nd, non-detectable. F, nuclear extracts were prepared from MCF-7 cells treated with E₂ and TNFα for 45 min and incubated with a DNA probe from the ABCG2 gene in which the ERE was either intact (ERE-WT) or mutated (ERE-mut). DAPA was performed as described under “Experimental Procedures,” and ERα binding to the probes was examined by Western blotting.

FIGURE 3.

NFκB activity is required for potentiation of E₂-regulated ABCG2 expression and ER occupancy by TNFα. A and B, ABCG2 mRNA levels were determined by qPCR in MCF-7 cells treated for 2 h with 10 ng/ml IL-1β or IL-6 with or without 10 nm E₂ or following infection with adenoviral constructs for either GFP (control) or IκBα-DN for 24 h prior to treatment with E₂ and/or TNFα for 2 h. *, p < 0.05 compared with E₂ or IL-1β alone. C and D, ChIP assays for ER were carried out 24 h after infection of cells with GFP or IκBα-DN adenoviral constructs or 2 h after pretreatment with an IKK inhibitor (1 μm), followed by treatment with E₂, TNFα, or both for 45 min. *, p < 0.05 compared with E₂ alone in the same group.

FIGURE 4.

TNFα potentiation of ABCG2 expression requires a latent NFκBRE located upstream of the ERE. A, the diagram of the ABCG2 promoter shows the known ERE at −180 and two potential NFκBREs (N1 and N2) identified by the bioinformatics software program MatInspector (56). B, MCF-7 cells were transfected with 1 μg of an ABCG2-luciferase reporter plasmid in which N1, N2, or both were mutated (mut) along with 200 ng of the Renilla luciferase control plasmid pGL4.70. Dual-Luciferase assays were carried out following treatment with E₂, TNFα, or both for 4 h. *, p < 0.05 compared with E₂ alone for the same reporter.

FIGURE 5.

NFκB family member p65 is recruited to the N1 site of the ABCG2 promoter in an ER-dependent manner. A, MCF-7 cells were pretreated for 2 h with or without the ER antagonist ICI 182,780 (ICI; 1 μm), followed by treatment with E₂, TNFα, or both for 45 min. ChIP assay was carried out, and p65 recruitment to the ABCG2 promoter was examined by qPCR. *, p < 0.05 compared with TNFα. B, nuclear extracts were prepared from MCF-7 cells treated with E₂ and TNFα for 45 min and incubated with a DNA probe from the ABCG2 gene in which N1 was either intact (N1-WT) or mutated (N1-mut). DAPA was performed as described under “Experimental Procedures,” and p65 binding to the probes was examined by Western blotting. C, MCF-7 cells were treated with E₂ (E), TNFα (T), or both for 5 or 15 min, and whole-cell (WCE) and nuclear (NE) extracts were prepared. Phosphorylated and total IκBα and β-actin protein levels were examined in whole-cell extracts, and phosphorylated and total p65 and histone H3 protein levels were examined in nuclear extracts by Western blotting.

FIGURE 6.

Conversion of the NFκBRE of ABCG2 to a consensus p65 sequence renders the promoter responsive to TNFα. A, the sequence of the N1 site of ABCG2 is compared with a consensus p65-binding motif (20). B, reporter activity was measured by a Dual-Luciferase assay in MCF-7 cells transfected with 1 μg of ABCG2 reporter plasmid in which the N1 site was intact (−243) or mutated to a consensus p65-binding site (N1-Cons) and with 200 ng of Renilla luciferase control plasmid pGL4.70. Transfection was carried out, and luciferase activity was measured after 4 h of treatment with E₂, TNFα, or both. *, p < 0.05, TNFα compared with none (control); ns, not significant.

FIGURE 7.

Proposed model for TNFα potentiation of E₂-regulated ABCG2 expression involves cooperativity between ER and p65 at adjacent response elements. E₂ is capable of up-regulating ABCG2 expression through ER binding to an ERE in the ABCG2 promoter, whereas in the absence of E₂, TNFα has no effect on ABCG2 transcription. However, in the presence of both E₂ and TNFα, we found that ER is required to allow recruitment of the NFκB subunit p65 to a latent NFκBRE that displays no activity in the absence of E₂ but is required for TNFα potentiation of E₂ activity. Once recruited, p65 is capable of stabilizing ER occupancy on the gene. This reciprocity between ER and NFκB at adjacent response elements ultimately leads to enhanced regulation of ABCG2.