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 element. 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,

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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 microarray 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 previously 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. Further- alyzed as previously described using 36B4 as an internal control (30). more, this subset of genes shows a unique expression pattern in Primer sequences are available on request. breast tumors of women with poor response to tamoxifen and Cell survival and apoptosis assays. Cell viability was assessed in MCF-7 reduced disease-free and overall survival. cells seeded in 96-well plates using the CellTiter 96 AQueous One Solution (Promega) according to the manufacturer's instructions. Annexin V-FITC labeling was done following the manufacturer's instruction (BD Biosciences), Materials and Methods and the percentage of positively stained cells was assessed by flow cytometry. Statistical analysis. Quantitative PCR, survival, and apoptosis data were β Materials. 17 -Estradiol (E2) was obtained from Sigma. The cytokines analyzed by one-way or two-way ANOVA followed by post hoc Tukey or α β TNF , interleukin (IL)-1 , and IL-6 were obtained from R&D Systems. IKK Bonferroni test, as appropriate. Significance for all statistical tests was α β inhibitor VII, which inhibits both IKK and IKK , was obtained from Cal- set at P < 0.05. Data shown are the mean ± SEM from three to six indepen- biochem. ICI 182,780 was obtained from Tocris. Adenoviral vectors for dent determinations. green fluorescent protein (GFP) and a dominant-negative form of IκBα (IκBα-DN) were very kindly given to us by Dr. Ruxana Sadikot (University of Illinois at Chicago, Chicago, IL). The inhibitor of apoptosis (IAP) antagonist (SMAC mimetic) was very generously provided by Dr. Xiadong Wang Results (University of Texas Southwestern Medical Center, Dallas, TX). To examine the genome-wide cross-talk between ER and NF-κB, Cell culture. MCF-7 cells were obtained from Dr. Benita Katzenellenbo- a microarray study was carried out using RNA from ER-positive gen (University of Illinois at Urbana-Champaign, Urbana, IL) and cultured MCF-7 breast cancer cells that were treated with E2, TNFα, or both in MEM containing 5% calf serum as previously described (24). ZR75-1 and for 2 hours. A total of 395 genes were identified as significantly T47D cells were obtained from Dr. Debra Tonetti (University of Illinois at upregulated or downregulated by at least one of the three treat- Chicago, Chicago, IL) and cultured in RPMI containing 10% fetal bovine ≥ serum. All three cell lines express ERα but not ERβ5; hence, references ments (Supplementary Table S1; fold change 2.0; P < 0.05). Hier- α archical clustering analysis revealed several distinct patterns of to ER in these cell lines indicate ER . Before E2 treatments, cells were cultured in phenol red–free media containing 5% charcoal-dextran–stripped regulation (Fig. 1). As previously shown, a large number of genes serum for at least 3 d. Expression of GFP or IκBα-DN was carried out using are downregulated by E2 treatment (cluster A; ref. 30). Interest- adenoviral vectors as previously described (24, 27, 28). ingly, three distinct subsets of upregulated genes were also iden- Gene expression profiling and analysis. Total RNA was harvested for tified: genes that are upregulated by E2 (cluster B), genes that are cRNA labeling, and hybridization to Affymetrix HGU133A GeneChips was upregulated by TNFα (cluster C), and genes that are upregulated carried out as described previously (27, 29, 30), with three replicates for α by E2 +TNF (cluster D). These three subsets of upregulated each treatment. Arrays were scanned and analyzed using the GeneChip Op- genes were used to examine cross-talk between E2 and TNFα erating Software (Affymetrix). CEL files were processed and normalized us- in more detail. ing “gcrma” function in the R/Bioconductor package (31). Normalized

5 J. Frasor, unpublished observation. 6 http://david.abcc.ncifcrf.gov/ www.aacrjournals.org 8919 Cancer Res 2009; 69: (23). December 1, 2009

α α TNF modulates gene regulation by E2. To examine how the On the remaining 60% of the E2-upregulated genes, TNF seems presence of TNFα influences E2 action, we compared the effects of to have no effect (Fig. 2A), as shown for cluster B (Fig. 1) and as α E2 and E2 + TNF on the subset of genes that are upregulated by E2 demonstrated by the upregulation of IGFBP4, which is not different α α (n = 46). Unexpectedly, we found that TNF enhances E2 activity between E2 and E2 + TNF at either the 2- or 3-hour time point on ∼30% of these genes (Fig. 2A), which partially accounts for the (Fig. 2B). However, a slight reduction in E2 activity with the addi- genes in cluster D (Fig. 1). The ability of TNFα to enhance E2 action of TNFα was seen after 4 hours. ICI 182,780 completely α tivity is exemplified by pS2, which is robustly upregulated by E2, blocked regulation of IGFBP4 by E2 or E2 +TNF ,whereasthe not affected by TNFα alone, and upregulated to a greater extent IKK inhibitor had no effect (Fig. 2C). Taken together, these findings by the combination of E2 + TNFα when compared with E2 alone indicate that the proinflammatory cytokine, TNFα, acting through α κ (Fig. 2B). The ability of TNF to enhance E2 regulation of pS2 is the NF- B pathway, can influence E2 activity, either positively or mediated by the NF-κB pathway because an inhibitor of the IKK negatively, in a gene-dependent manner. Furthermore, it seems complex prevents TNFα action (Fig. 2C). that at the early time points chosen for study, enhancement of α α ∼ In addition, we found that TNF reverses or represses E2 action E2 activity by TNF is 3× more common than repression. α on <10% of E2-upregulated genes (Fig. 2A), as shown by c-Fos, E2 modulates gene regulation by TNF . We next examined which is upregulated to a greater extent by E2 alone compared how the presence of E2 influences TNFα activity on genes upregu- with E2 + TNFα (Fig. 2B). As is the case for enhanced pS2 regula- lated by TNFα (n = 39). We found that E2 represses or reverses α α ∼ tion, the repression of c-Fos by E2 + TNF is also mediated by the TNF activity on 41% of these genes (Fig. 3A). This finding is ex- NF-κB pathway because the IKK inhibitor prevented repression of pected because the ability of ER to repress NF-κB is well documen- this gene by TNFα (Fig. 2C). In addition, the effect of E2 + TNFα on ted. Regulation of the NF-κB target gene, ICAM1, shows the ability α both pS2 and c-Fos is completely blocked by the ER antagonist, ICI of E2 to repress gene regulation by TNF (Fig. 3B) and that this 182,780, indicating that the different effects of TNFα on E2 action effect is mediated by ER, as evidenced by the reversal of repression require ER activity (Fig. 2C). by ICI 182,780 (Fig. 3C). In addition, we found that E2 has no effect

Figure 2. TNFα modulates gene regulation by E2 in a gene-specific manner. A, the effect of TNFα on E2-upregulated genes in MCF-7 cells, as identified by microarray, was considered enhanced if the fold change by E2 + TNFα was >1.5× the fold change by E2 alone or reversed if the fold change by E2 + TNFα was <0.67× the fold change by E2 alone. B, the effect of TNFα on three E2-upregulated genes, pS2, c-Fos, and IGFBP4, in MCF-7 cells treated with 10 nmol/L E2, 10 ng/mL TNFα,or both for up to 4 h was confirmed by quantitative PCR. Fold change was calculated using the ΔΔCt method with 36B4 as an internal control. *, P < 0.05, compared with treatment with E2 alone at the same time point. C, MCF-7 cells were pretreated with 1 μmol/L of an inhibitor of the IKK complex or the ER antagonist ICI 182,780 (ICI) for 2 h before treatment with E2, TNFα, or both for an additional 2 h. pS2, c-Fos, and IGFBP4 mRNA levels were assessed by quantitative PCR. The data are normalized to fold change obtained with E2 treatment alone. *, P < 0.05, compared with E2 alone; ns, not significant.

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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 upregulated by TNFα in MCF-7 cells was considered enhanced if the fold change by E2 + TNFα was >1.5× the fold change by TNFα alone or reversed if the fold change by E2 + TNFα was <0.67× the fold change by TNFα alone. B, the effect of E2 on the mRNA levels of the TNFα-upregulated genes ICAM1, TNFα, and BIRC3 was examined by quantitative PCR in MCF-7 cells treated with 10 nmol/L E2, 10 ng/mL TNFα, or both for up to 2 h. *, P < 0.05, compared with treatment with TNFα alone at the same time point. C, MCF-7 cells were pretreated with 1 μmol/L of the ER antagonist ICI 182,780 for 2 h before treatment with E2, TNFα, or both for an additional 2 h. ICAM1, TNFα, and BIRC3 mRNA levels were assessed by quantitative PCR. *, P < 0.05, compared with TNFα alone; ns, not significant.

∼ α α α on the regulation of 44% of TNF -upregulated genes (Fig. 3A), as as compared with TNF alone (Fig. 3B). The effect of E2 on TNF shown by cluster C (Fig. 1). However, this subset contains several regulation of BIRC3 is completely prevented by ICI 182,780, indicat- NF-κB target genes that are known to be repressed by ER. For ex- ing an essential role for ER in enhancing NF-κB activity on this ample, our microarray data indicate that E2 treatment has no ef- gene (Fig. 3C). These findings indicate that although ER represses fect on TNFα upregulation of its own gene, but this is in contrast NF-κB activity on many genes, ER can also enhance NF-κB activity to several reports in the literature, which show that E2 can repress in a gene-specific manner. TNFα autoregulation (19, 36, 37). This conflict was resolved by a Common E2 and TNFα target genes. Previous work from our time-course experiment, which revealed that E2 can repress regu- laboratory showed that the PTGES gene is upregulated indepen- lation of TNFα mRNA but only at the earlier time points examined, dently by E2 and TNFα and in a more than additive manner by and that repression by E2 is lost at 2 hours, the time point at which the combination of E2 + TNFα (24). Our microarray data suggest the microarray was carried out (Fig. 3B). Treatment with ICI at the that a similar regulation occurs on a large subset of common E2 2-hour time point had no effect on the autoregulation of TNFα and TNFα target genes (n = 63), which make up the majority of (Fig. 3C). These findings suggest that our microarray data may cluster D (Fig. 1). These genes, as represented by PHLDA1 and α α have underestimated the ability of E2 to repress TNF activity IL17RB (Fig. 4A), are significantly upregulated by E2 + TNF com- and that E2 repression of some TNFα-regulated genes may be time pared with either E2 or TNFα alone across a 4-hour time course. dependent. To examine regulation of PHLDA1 in more detail, additional One particularly unanticipated finding from our study is that E2 studies were carried out using different proinflammatory cytokines can enhance TNFα activity on ∼15% of TNFα-upregulated genes and different ER-positive breast cancer cell lines. The combination (Fig. 3A and some genes in cluster D of Fig. 1). This enhancement is of E2 + TNFα or E2 + IL-1β, but not E2 + IL-6, led to a significantly shown for BIRC3 (c-IAP2), which is an important antiapoptotic greater upregulation of PHLDA1 in MCF-7 cells compared with E2 gene of the IAP family and is known to be upregulated by NF-κB or cytokine alone (Fig. 4B). A significant upregulation of PHLDA1 (38). The upregulation of BIRC3 is greatly enhanced by E2 + TNFα by E2 + TNFα was also observed in two other ER-positive breast www.aacrjournals.org 8921 Cancer Res 2009; 69: (23). December 1, 2009

α– cancer cell lines, ZR75-1 and T47D (Fig. 4B). The ER is required type, also significantly overexpress the E2 + TNF positive cross- for regulation of PHLDA1 by E2 + TNFα because the ER antagonist, talk genes. Luminal B tumors have previously been shown to be ICI 182,780, completely blocked regulation (Fig. 4C). The fact that more aggressive ER-positive tumors associated with a worse pa- both TNFα and IL-1β, but not IL-6, can regulate this gene suggests tient outcome, whereas luminal A tumors are also ER-positive an important role for the NF-κB pathway in the regulation of but associated with a better patient outcome (5). These findings PHLDA1. Expression of IκBα−DN, which cannot be phosphorylated suggest that the combination of E2 and proinflammatory cytokines, or degraded and thereby blocks NF-κB activity, confirms that the acting through ER and NF-κB, may coordinately regulate specific NF-κB pathway is also required for the enhanced upregulation of genes associated with more aggressive ER-positive tumors. In con- PHLDA1 by E2 + TNFα (Fig. 4C). These findings indicate that the trast, grade 3 and basal subtype tumors underexpress the positive scope of positive cross-talk between ER and NF-κB in breast cancer cross-talk gene set. This most likely is related to the fact that the may be broader than expected, rather than being limited to one par- majority of basal and grade 3 tumors are ER negative. ticular cytokine in a single cell line. The association of the 80 positive cross-talk genes with key clin- ER and NF-κB target genes in breast cancer cell survival and ical parameters suggests that they may be involved in response to human breast tumors. In total, 80 genes were identified as being endocrine treatments and patient survival. To investigate whether upregulated to a greater extent by E2 + TNFα than by E2 or TNFα the expression of the 80-gene signature is prognostic of patient alone based on three different patterns of regulation: (a) TNFα en- outcome, we examined the expression of these genes in a cohort α hanced E2 activity, (b)E2 enhanced TNF activity, and (c) upregu- of women with ER-positive breast tumors (n = 81) who had been lation by E2 + TNFα. The top three biological functions associated treated with tamoxifen and followed over time. Hierarchical clus- with these 80 genes were transcriptional regulation, metabolism, tering of patients using expression levels of the 80 genes grouped and apoptosis/cell death, as identified using functional annotation them into four clusters with distinct expression profiles (Fig. 6B). clustering of enriched GO terms. The overall enrichment score for each cluster ranged from 2.63 to 1.49, but only the transcriptional regulation cluster reached significance (Benjamini Hochberg cor- rected P = 0.01). The identification of apoptosis-related function for these genes is consistent with the prosurvival roles for both ER and NF-κB. To investigate this further, we took advantage of the fact that treatment of some breast cancer cell lines, including the MCF-7 cells used in these studies, with higher doses of TNFα can induce apoptosis through prolonged activation of the c-jun – NH2-terminal kinase pathway (39 41). Treatment of MCF-7 cells with TNFα for 30 to 48 hours caused a >50% reduction in cell viability and a 4-fold increase in the number of apoptotic cells (Fig. 5A and B). The effect of TNFα on cell viability and apoptosis is completely reversed by the combination of E2 + TNFα, indicating that E2 prevents TNFα-induced apoptosis. We find that the ability α of E2 to prevent TNF induced cell death is blocked by ICI 182,780, as well as by an inhibitor of the IKK complex (Fig. 5C), indicating that both ER and NF-κB are required for E2 to promote cell survival in the presence of TNFα. To examine whether BIRC3, an antiapoptotic factor that is highly upregulated by the combination of E2 + TNFα (Fig. 3B), may be an important effector of ER and NF- κB-mediated cell survival, we used an IAP antagonist that blocks the activity of BIRC3 (42, 43). We found that the IAP antagonist completely prevented an increase in cell survival by E2 + TNFα, which suggests that the ER-negative and NF-κB-dependent upregulation of BIRC3 plays an essential role in breast cancer cell survival (Fig. 5C). To further examine the significance of the 80-gene signature re- α sulting from positive cross-talk by E2 + TNF in breast tumor biology, we used a recently described breast tumor compendium Figure 4. Common E2 and TNFα target gene regulation in MCF-7 cells. A, α that contains gene expression profiles for ∼1,200 well-annotated MCF-7 cells were treated with 10 nmol/L E2, 10 ng/mL TNF , or both for up to 4 h, and PHLDA1 and IL17RB mRNA were assessed by quantitative PCR. breast tumors (34). We first identified tumors in the compendium *, P < 0.05, compared with trezatment with E2 alone at the same time point; that significantly overexpress or underexpress genes from the pos- #, P < 0.05, compared with treatment with TNFα alone at the same time point. itive cross-talk gene set by 1.8-fold or more (Fig. 6A). We next ex- B, PHLDA1 mRNA was examined by quantitative PCR in MCF-7 cells treated with 10 nmol/L E2 in the absence or presence of 10 ng/mL TNFα, IL-1β, or IL-6 amined whether this gene expression profile was enriched in for 2 h (*, P < 0.05, compared with E2 alone; #, P < 0.05, compared with TNFα tumors with certain attributes or characteristics, including ER sta- alone; ^, P < 0.05, compared with IL-1β alone) or in the ERα-positive breast cancer cell lines ZR75-1 or T47D treated with E2, TNFα, or both for 8 h tus, intrinsic subtype [luminal A, luminal B, Her2, basal, or normal (*, P < 0.05, compared with all other groups in that cell line). C, PHLDA1 mRNA (4)], or grade. As expected, we find that the positive cross-talk gene was measured in MCF-7 cells treated with E2 and/or TNFα for 2 h following a set is significantly overexpressed in ER-positive tumors and under- 2-h pretreatment with 1 μmol/L of the ER antagonist ICI 182,780 or 24 h following infection with adenoviral vectors expressing GFP (control) or IκBα-DN, which expressed in ER-negative tumors (Fig. 6A). In addition, tumors of prevents activation of NF-κB, and treatment with E2, TNFα, or both for 2 h. the luminal B intrinsic subtype, but not those of the luminal A sub- *, P < 0.05, compared with all other groups.

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Figure 5. ER- and NF-κB-dependent breast cancer cell survival. MCF-7 cells were treated with 10 nmol/L E2, 50 ng/mL TNFα, or both. Cell viability was assessed after 30 h by MTS assay (A), or apoptosis was measured after 48 h by Annexin V staining (B). *, P < 0.05, compared with 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 μmol/L), an inhibitor of the IKK complex (1 μmol/L), or an inhibitor of IAP proteins (10 nmol/L), and cell viability was assessed. *, P < 0.05, compared with TNFα.

Survival analysis of the four patient clusters indicated distinct out- and their downstream target genes may alter the genetic program- comes for disease recurrence (disease-free survival; Fig. 6C), metas- ming of ER-positive tumors and subsequently affect response to tases (distant metastasis–free survival; Fig. 6C), and death from endocrine therapy and patient survival. breast cancer (disease-specific survival; Fig. 6C) following tamoxifen treatment. Expression profiles of the 80 genes clearly dis- tinguished patients with good outcomes (Fig. 6B and C, green Discussion cluster and curves) from those with poor outcomes (Fig. 6B and The major mechanism of cross-talk between ER and NF-κB that C, red cluster and curves). Survival differences between the four pa- has been described is transrepression, whereby ER represses NF- tient clusters were statistically significant for disease-free (P = κB activity and NF-κB represses ER activity (44, 45). Our microar- 0.0128) and distant metastasis–free (P = 0.0138) survival and nearly ray results confirm that transrepression does occur between these significant for disease-specific survival (P = 0.0782). These findings two factors in breast cancer cells and that ER repression of NF-κB suggest that the interactions between the ER and NF-κB pathways activity is more common than NF-κB repression of ER activity.

Figure 6. Genes regulated by E2 + TNFα are enriched in ER-positive and luminal B breast tumors and are associated with response to tamoxifen therapy. A, a set of 80 genes upregulated by E2 + TNFα was assessed in a compendium of breast tumor gene expression profiles. Tumors in which these genes were statistically overexpressed (red) or underexpressed (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-positive and luminal B tumors was observed (red), whereas reduced expression of the 80-gene set was seen in ER-negative, basal, and grade 3 tumors (green). Numbers in parentheses 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 upregulated 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 metastasis–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. www.aacrjournals.org 8923 Cancer Res 2009; 69: (23). December 1, 2009

Because NF-κB activation is associated with tumor invasiveness, PHLDA1 has been shown to be required for survival of cells follow- metastasis, and drug resistance (46), the fact that ER represses ing serum starvation (48). Furthermore, PHLDA1 has been de- NF-κB activity on numerous genes may represent one mechanism tected in breast tumors and seems to correlate with better by which ER-positive breast tumors are actually less aggressive patient outcome in ER-negative tumors and with worse outcome than ER-negative tumors. This concept has been previously in ER-positive tumors (49). The function and expression of BIRC3, suggested for ER repression of RelB, a member of the NF-κB family, in contrast, have not been well examined in breast tumors. How- in breast cancer (47). However, results from our microarray anal- ever, the IAP family of apoptosis inhibitors does play an antiapop- ysis clearly show that transrepression is only one of many mechan- totic function in different cancer cells and may contribute to isms of cross-talk that occur between these two pleiotropic tumor resistance to therapeutic drugs (43, 50, 51). Our findings transcription factors. show that inhibition of BIRC3 using an IAP antagonist (also known Three different types of positive cross-talk between ER and NF-κB as a SMAC mimetic), which can bind and inhibit the activity of IAP were identified in our study. First, TNFα can enhance E2 activity on proteins, completely prevented cell survival in response to E2 and ∼ α κ 30% of E2-upregulated genes, as shown by pS2. Second, E2 can en- TNF . We propose that whereas both ER and NF- B have individ- hance TNFα activity on ∼15% of TNFα-upregulated genes, such as ual prosurvival functions, they may also act together to regulate the BIRC3. And third, E2 + TNFα acting together cause a more than ad- expression of additional antiapoptotic genes, such as BIRC3, which ditive upregulation of a specific subset of genes, including PHLDA1 can further contribute to the enhanced survival of breast cancer and IL17RB. Although repression between ER and NF-κB has been cells. In addition to BIRC3, other genes may contribute to more ag- well documented, few instances of a positive interaction between ER gressive breast tumors. For example, IL17RB has recently been ob- and NF-κB have been reported (23–26). For example, ER and NF-κB served to have a prognostic function in breast tumors when its act together to upregulate prostaglandin E synthase (24), serotonin expression is compared with the level of another gene, HOXB13 1A receptor (26), prolactin in pituitary cells (23), and cyclin D1 in (52). The ratio of these two genes may also be predictive of tumor mammary cells (25). Thus, our study adds significant new informa- responsiveness to tamoxifen (53). Intriguingly, activation of NF-κB tion to the field by highlighting the extensive degree of positive is associated with antiestrogen resistance (6, 8, 9), but whether NF- cross-talk that occurs between ER and NF-κB in breast cancer cells. κB activation can alter the HOXB13 to IL17RB ratio and how this The concept that ER and NF-κB can work together to positively influences response to tamoxifen require further investigation. upregulate the expression of particular genes could have important In summary, our findings indicate that a spectrum of cross-talk implications in breast cancer disease progression. Recent evidence between ER and NF-κB can be observed in breast cancer cells, and indicates that ER-positive breast tumors with constitutively active that, in particular, these two factors act together to enhance the NF-κB are more aggressive and less responsive to tamoxifen (7). expression of numerous genes. It is possible that the coordinated Our findings suggest that the positive transcriptional cross-talk actions of ER and NF-κB lead to enhanced cell survival, reduced between ER and NF-κB may enhance the expression of genes response to therapeutic agents such as tamoxifen, and the devel- involved in the promotion of more aggressive tumors. This is sup- opment of more aggressive breast tumors. Thus, transcriptional ported by the observation that these genes are enriched in luminal cross-talk between ER and NF-κB represents a potential therapeu- B tumors, a more aggressive subtype of ER-positive breast tumors tic target for breast cancer treatment, and further investigation in- (5), but not in luminal A tumors, which are also ER-positive but have to this possibility is warranted. a better patient outcome. Furthermore, the gene expression profile of tumors from women who failed to respond to tamoxifen and had Disclosure of Potential Conflicts of Interest poor disease-free and overall survival indicates that the expression levels of these genes, including some that are highly expressed in The authors have no potential conflicts of interest to disclose. poor outcome patients, are associated with response to endocrine therapy. Acknowledgments One potential reason why these tumors are more aggressive is that both ER and NF-κB are transcription factors with well- Received 7/15/09; revised 9/14/09; accepted 9/24/09; published OnlineFirst 11/17/09. Grant support: National Cancer Institute grant R01CA130932 (J. Frasor). characterized prosurvival or antiapoptotic activities. Whereas ER The costs of publication of this article were defrayed in part by the payment of page and NF-κB can act individually to upregulate cell survival genes, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. we find that they can also act together to enhance the expression We thank Benita Katzenellenbogen for her support in initiating this project of additional cell survival genes, including PHLDA1 and BIRC3. (National Cancer Institute grant R01CA18119 to B.S. Katzenellenbogen).

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www.aacrjournals.org 8925 Cancer Res 2009; 69: (23). December 1, 2009

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2009 American Association for Cancer Research. Correction Cancer Research Correction: Positive Cross-Talk between Estrogen Receptor and NF-κB in Breast Cancer

In this article (Cancer Res 2009;69:8918–25), which was published in the December 1, 2009 issue of Cancer Research (1), both instances of “ER-negative and NF-κB-dependent” in the last paragraph of the Introduction, and “ER-negative and NF-κB- dependent” in the Fig. 5 legend, should read as “ER- and NF-κB-dependent”. Also, “ER-negative positive” in the first paragraph of the Results should read as “ER- positive”. The online article has been changed to reflect this correction and no longer matches the print. Reference 1. Frasor J, Weaver A, Pradhan M, Dai Y, Miller LD, Lin C-Y, Stanculescu A. Positive cross-talk between estrogen receptor and NF-κB in breast cancer. Cancer Res 2009;69:8918–25.

854 Cancer Res; 70(2) January 15, 2010 Published OnlineFirst November 17, 2009; DOI: 10.1158/0008-5472.CAN-09-2608

Positive Cross-Talk between Estrogen Receptor and NF-κB in Breast Cancer

Jonna Frasor, Aisha Weaver, Madhumita Pradhan, et al.

Cancer Res 2009;69:8918-8925. Published OnlineFirst November 17, 2009.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-09-2608

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