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Positive Cross-Talk Between Estrogen Receptor and NF-Κb in Breast Cancer

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Positive Cross-Talk Between Estrogen Receptor and NF-Κb in Breast Cancer Published OnlineFirst November 17, 2009; DOI: 10.1158/0008-5472.CAN-09-2608 Endocrinology Positive Cross-Talk between Estrogen Receptor and NF-κB in Breast Cancer Jonna Frasor,1 Aisha Weaver,1 Madhumita Pradhan,1 Yang Dai,2 Lance D. Miller,3 Chin-Yo Lin,4 and Adina Stanculescu1 Departments of 1Physiology and Biophysics and 2Bioengineering, University of Illinois at Chicago, Chicago, Illinois; 3Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, North Carolina; and 4Department of Microbiology and Molecular Biology, Brigham Young University, Provo, Utah Abstract aggressive with earlier metastatic recurrence (1–3). Gene expres- Estrogen receptors (ER) and nuclear factor-κB(NF-κB) are sion profiling has further delineated the two types of ER-positive known to play important roles in breast cancer, but these fac- tumors, referred to as intrinsic subtypes luminal A and luminal B, tors are generally thought to repress each other's activity. with the luminal A subtype associated with good patient outcome However, we have recently found that ER and NF-κB can also and the B subtype with a poor survival rate (4, 5). Interestingly, activation of the proinflammatory transcription factor nuclear act together in a positive manner to synergistically increase κ κ gene transcription. To examine the extent of cross-talk be- factor- B(NF-B) may play a role in this dichotomy between ER-positive tumors. Constitutive activation of NF-κBinbreast tween ER and NF-κB, a microarray study was conducted in tumors is associated with more aggressive ER-positive tumors which MCF-7 breast cancer cells were treated with 17β- (6, 7), the development of resistance to endocrine therapy (8, 9), estradiol (E ), tumor necrosis factor α (TNFα), or both. Follow- 2 and progression to estrogen-independent growth (10–12). up studies with an ER antagonist and NF-κB inhibitors show Two ER subtypes have been identified, ERα and ERβ, which me- that cross-talk between E and TNFα is mediated by these 2 diate the biological functions of estrogen primarily through their two factors. We find that although transrepression between ability to function as ligand-activated transcription factors. Both ER and NF-κB does occur, positive cross-talk is more promi- ERs can stimulate gene transcription by directly binding to DNA nent with three gene-specific patterns of regulation: (a) TNFα at estrogen response elements or through tethering to other tran- enhances E action on ∼30% of E -upregulated genes; (b)E 2 2 2 scription factors (13, 14). ERs can also negatively regulate or re- enhances TNFα activity on ∼15% of TNFα-upregulated genes; press transcription in either a direct or indirect manner through and (c)E +TNFα causes a more than additive upregulation 2 interaction with other transcription factors (15, 16). In particular, of ∼60 genes. Consistent with their prosurvival roles, ER and the ability of ERs to repress the transcriptional activity of NF-κB NF-κB and their target gene, BIRC3, are involved in protecting has been well studied. The NF-κB pathway is stimulated by a va- breast cancer cells against apoptosis. Furthermore, genes pos- riety of factors, including proinflammatory cytokines. Following cy- itively regulated by E + TNFα are clinically relevant because 2 tokine binding to its receptor, activation of the IκB kinase (IKK) they are enriched in luminal B breast tumors and their expres- complex occurs, leading to phosphorylation and subsequent degra- sion profiles can distinguish a cohort of patients with poor dation of the inhibitory protein, IκB. This allows release of NF-κB outcome following endocrine treatment. Taken together, family members, p65 and p50, which are sequestered in the cyto- our findings suggest that positive cross-talk between ER and plasm by IκB. Once liberated, p65 and p50 can translocate to the NF-κB is more extensive than anticipated and that these fac- nucleus, bind to DNA at cognate NF-κB response elements, and tors may act together to promote survival of breast cancer cells regulate target gene transcription. NF-κB activation can be re- and progression to a more aggressive phenotype. [Cancer Res pressed by ER through several different mechanisms, including 2009;69(23):8918–25] prevention of NF-κB binding to DNA (17, 18), recruitment of cor- epressors into a complex with NF-κB (19), competition for coacti- Introduction vators (20, 21), or prevention of NF-κB nuclear translocation (22). The estrogen receptor (ER) is expressed in ∼75% of breast tu- The basis for these different mechanisms has not been fully eluci- mors and is a major prognostic and therapeutic determinant. dated but may be related to different cellular backgrounds or to Women with ER-positive tumors have a better prognosis and will gene-specific mechanisms of cross-talk. likely receive endocrine therapy, such as tamoxifen or an aroma- In contrast, very few reports have indicated that positive tran- tase inhibitor. However, not all ER-positive tumors respond to en- scriptional cross-talk can occur between ER and NF-κB (23–26). In docrine therapy and, frequently, de novo or acquired resistance each case, the mechanisms for positive cross-talk seem to involve a occurs. These ER-positive tumors, which tend to retain ER expres- complex formation containing the ER and NF-κB family members sion but without typical response to tamoxifen, are generally more at either an estrogen response element or a NF-κB response ele- ment. Previously, we have found that activation of ER and NF-κB in breast cancer cells, via treatment with estradiol (E2)andthe proinflammatory cytokine tumor necrosis factor α (TNFα), leads Note: Supplementary data for this article are available at Cancer Research Online to enhanced transcription of the prostaglandin E synthase (http://cancerres.aacrjournals.org/). 2 Requests for reprints: Jonna Frasor, Department of Physiology and Biophysics, (PTGES) gene (24). However, the extent to which this positive University of Illinois at Chicago, 835 South Wolcott Avenue, MC 901, Chicago, IL cross-talk between ER and NF-κB occurs in breast cancer cells is 60612. Phone: 312-355-2583; Fax: 312-996-1414; E-mail: [email protected]. ©2009 American Association for Cancer Research. not known. This lack of information prompted us to examine the doi:10.1158/0008-5472.CAN-09-2608 genome-wide transcriptional cross-talk between ER and NF-κB, Cancer Res 2009; 69: (23). December 1, 2009 8918 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst November 17, 2009; DOI: 10.1158/0008-5472.CAN-09-2608 ER and NF-κB Cross-Talk data were further analyzed using GeneSpring software and significantly regulated genes were identified by the following criteria: ≥2.0 fold change α α and P < 0.05 for at least one treatment condition (E2, TNF ,orE2 + TNF ) compared with untreated control, as previously described (30, 32). After identification of regulated genes, modulation of gene regulation was considered “enhanced” if the fold change by E2 + TNFα was greater than α 1.5× the fold change seen with either E2 or TNF alone. Similarly, gene regulation was considered “reversed” if the fold change by E2 +TNFα was less than 0.67× the fold change seen with E2 or TNFα alone. All micro- array data are publicly available through GEO (accession no. GSE11467). Gene Ontology (GO) term enrichment was carried out using the Functional Annotation Clustering tool in DAVID,6 as previously described (33). Enrich- ment of gene expression patterns in human breast tumors was determined using data from a breast tumor compendium and Genomica software as pre- viously described (34). Survival analysis was carried out using gene expression profiles from the Uppsala patient population, as previously described with some modifications (27, 35). A total of 81 ER-positive tumors from patients Figure 1. Characterization of gene expression profile in response to E and who subsequently underwent endocrine therapy with tamoxifen only were 2 α– TNFα treatment in breast cancer cells. RNA was extracted from MCF-7 cells used to assess the role of E2 + TNF regulated genes in patient outcome. α following treatment with 10 nmol/L E2, 10 ng/mL TNF , or both for 2 h and used Expression profiles of E2 +TNFα–regulated genes were used to cluster to carry out microarray studies (Affymetrix, HGU133A). Genes that were patients using average linkage hierarchical clustering. Kaplan-Meier esti- significantly regulated by at least one of the treatments were identified and hierarchical clustering was done. Black bars, clusters of genes that are mates were used to compute survival curves for disease-free survival, distant – downregulated by E2 (A), upregulated by E2 (B), upregulated by TNFα (C), metastasis free survival, and disease-specific survival, and the significance of and upregulated by E2 + TNFα (D). the hazard ratios between the four major patient clusters was determined by the P of the likelihood ratio test, as was described previously (35). RNA isolation and quantitative PCR. RNA isolation was carried out and interestingly, we found that positive cross-talk is predominant using Trizol according to the manufacturer's instructions (Invitrogen). To- compared with repression. We identified a large subset of genes tal RNA (0.5 μg) was reverse transcribed using Moloney murine leukemia that are synergistically upregulated by the combination of E2 and virus reverse transcriptase (Invitrogen). The resulting product was diluted TNFα in an ER- and NF-κB-dependent manner. This subset of to 100 μL with double-distilled water and 2 μL were used for each subse- genes is highly enriched in luminal B tumors and may contribute quent quantitative PCR reaction. Quantitative PCR was carried out and an- to ER- and NF-κB-dependent breast cancer cell survival.
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