Positive cross-talk between estrogen receptor and NF-kappaB in breast cancer

Abstract

Estrogen receptors (ER) and nuclear factor-kappaB (NF-kappaB) are known to play important roles in breast cancer, but these factors are generally thought to repress each other's activity. However, we have recently found that ER and NF-kappaB can also act together in a positive manner to synergistically increase gene transcription. To examine the extent of cross-talk between ER and NF-kappaB, a microarray study was conducted in which MCF-7 breast cancer cells were treated with 17beta-estradiol (E(2)), tumor necrosis factor alpha (TNFalpha), or both. Follow-up studies with an ER antagonist and NF-kappaB inhibitors show that cross-talk between E(2) and TNFalpha is mediated by these two factors. We find that although transrepression between ER and NF-kappaB does occur, positive cross-talk is more prominent with three gene-specific patterns of regulation: (a) TNFalpha enhances E(2) action on approximately 30% of E(2)-upregulated genes; (b) E(2) enhances TNFalpha activity on approximately 15% of TNFalpha-upregulated genes; and (c) E(2) + TNFalpha causes a more than additive upregulation of approximately 60 genes. Consistent with their prosurvival roles, ER and NF-kappaB and their target gene, BIRC3, are involved in protecting breast cancer cells against apoptosis. Furthermore, genes positively regulated by E(2) + TNFalpha are clinically relevant because they are enriched in luminal B breast tumors and their expression profiles can distinguish a cohort of patients with poor outcome following endocrine treatment. Taken together, our findings suggest that positive cross-talk between ER and NF-kappaB is more extensive than anticipated and that these factors may act together to promote survival of breast cancer cells and progression to a more aggressive phenotype.

Figure 1. Characterization of Gene Expression Profile in Response to E2 and TNFα Treatment in Breast Cancer Cells

RNA was extracted from MCF-7 cells following treatment with 10 nM E2, 10 ng/ml TNFα, or both for 2 hr and used to carry out microarray studies (Affymetrix, HGU133A). Genes that were significantly regulated by at least one of the treatments were identified and hierarchical clustering was performed. Black bars indicate clusters of genes that are: A) down-regulated by E2, B) up-regulated by E2, C) up-regulated by TNFα, and D) up-regulated by E2+TNFα.

Figure 2. TNFα Modulates Gene Regulation by E2 in a Gene-Specific Manner

(A) The effect of TNFα on E2 up-regulated genes in MCF-7 cells, as identified by microarray, was considered “enhanced” if the fold change by E2+TNFα was >1.5x the fold change by E2 alone or “reversed” if the fold change by E2+TNFα was <0.67x the fold change by E2 alone. (B) The effect of TNFα on three E2-up-regulated genes pS2, c-Fos, and IGFBP4, in MCF-7 cells treated with 10 nM E2, 10 ng/ml TNFα, or both for up to 4 hr was confirmed by QPCR. Fold change was calculated using the ΔΔCt method with 36B4 as an internal control. (*, P<0.05 compared to treatment with E2 alone at the same time point). (C) MCF-7 cells were pretreated with 1 μM of an inhibitor of the IKK complex or the ER antagonist ICI 182,780 (ICI) for 2 hr prior to treatment with E2, TNFα or both for an additional 2 hr. pS2, c-Fos and IGFBP4 mRNA levels were assessed by QPCR. The data are normalized to fold-change obtained with E2 treatment alone. (*, P<0.05 compared to E2 alone; ns, not significant).

Figure 3. E2 Modulates Gene Regulation by TNFα in a Gene-Specific and Time-Dependent Manner

(A) The effect of E2 on the subset of genes up-regulated by TNFα in MCF-7 cells was considered “enhanced” if the fold change by E2+TNFα was >1.5x the fold change by TNFα alone or “reversed” if the fold change by E2+TNFα was <0.67x the fold change by TNFα alone. (B) The effect of E2 on the mRNA levels of the TNFα-up-regulated genes ICAM1, TNFα and BIRC3 was examined by QPCR in MCF-7 cells treated with 10 nM E2, 10 ng/ml TNFα, or both for up to 2 hr. (*, P<0.05 compared to treatment with TNFα alone at the same time point.) (C) MCF-7 cells were pretreated with 1 μM of the ER antagonist ICI 182,780 (ICI) for 2 hr prior to treatment with E2, TNFα or both for an additional 2 hr. ICAM1, TNFα, and BIRC3 mRNA levels were assessed by QPCR. (*, P<0.05 compared to TNFα alone; ns, not significant.)

Figure 4. Common E2 and TNFα Target Gene Regulation in MCF-7 Cells

(A) MCF-7 cells were treated with 10 nM E2, 10 ng/ml TNFα, or both for up to 4 hr and PHLDA1 and IL17RB mRNA were assessed by QPCR. (*, P<0.05 compared to treatment with E2 alone at the same time point; #, P<0.05 compared to treatment with TNFα alone at the same time point). (B) PHLDA1 mRNA was examined by QPCR in MCF-7 cells treated with 10 nM E2 in the absence or presence of 10 ng/ml TNFα, IL-1β, or IL-6 for 2 hr (*, P<0.05 compared to E2 alone; #, P<0.05 compared to TNFα alone; ^, P<0.05 compared to IL-1β alone) or in the ERα positive breast cancer cell lines ZR75-1 or T47D treated with E2, TNFα or both for 8 hr (*, P<0.05 compared to all other groups in that cell line). (C) PHLDA1 mRNA was measured in MCF-7 cells treated with E2 and/or TNFα for 2 hr following a 2 hr pretreatment with 1 μM of the ER antagonist ICI 182,780 or 24 hr following infection with adenoviral vectors expressing GFP (control) or a dominant negative form of IκBα (IκBα-DN), which prevents activation of NFκB, and treatment with E2, TNFα, or both for 2 hr (*, P<0.05 compared to all other groups).

Figure 5. ER and NFκB-dependent Breast Cancer Cell Survival

MCF-7 cells were treated with 10 nM E2, 50 ng/ml TNFα, or both. (A) Cell viability was assessed after 30 hr by MTS assay or (B) apoptosis was measured after 48 hr by Annexin V staining (*, P<0.05 compared to TNFα). (C) MCF-7 cells were treated with E2, TNFα or both in the presence or absence of the ER antagonist ICI 182,780 (1 μM), an inhibitor of the IKK complex (1 μM), or an inhibitor of IAP proteins (10 nM) and cell viability was assessed. (*, P<0.05 compared to TNFα).

Figure 6. Genes Regulated by E2+TNFα are Enriched in ER+ and Luminal B Breast Tumors and are Associated with Response to Tamoxifen Therapy

(A) A set of 80 genes up-regulated by E2+TNFα was assessed in a compendium of breast tumor gene expression profiles. Tumors in which these genes were statistically over- (red) or under-expressed (green) were identified using Genomica software. The ER status, intrinsic subtype, and grade of each tumor are indicated in brown. Enrichment of the 80-gene list in ER+ and Luminal B tumors was observed (red) while reduced expression of the 80-gene set was seen in ER-, basal, and grade 3 tumors (green). Numbers in parenthesis indicate the negative log of the P-value for the enrichment of the 80 gene-set within each tumor group. (B) Microarray gene expression data from the tumors of 81 patients treated with tamoxifen following surgery were used to perform hierarchical clustering of patients using the expression profiles of the 80 genes up-regulated by E2+TNFα. Four distinct patient clusters were identified and differentially colored. (C) Kaplan-Meier survival analysis was carried out to track disease-free, distant metastases-free, and disease-specific survival over time for each cluster of patients. Colors of the survival curves correspond with the coloring of the clusters. P-values from likelihood-ratio tests are shown to indicate differences between the survival curves from each of the patient clusters for each type of outcome examined.