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Estrogen receptor (ER) and NF-κB are transcription factors with profound effects on breast cancer cell proliferation and survival. While many studies demonstrate that ER and NF-κB can repress each other, we previously identified a gene signature that is synergistically upregulated by these two factors in more aggressive luminal B breast tumors. Herein, we examine a novel mechanism of cross talk between ER and NF-κB that results in the upregulation of the antiapoptotic gene BIRC3 (also known as cIAP2). We demonstrate that NF-κB, acting through two response elements, is required for ER recruitment to an adjacent estrogen response element (ERE) in the BIRC3 promoter. This effect is accompanied by a major increase in NF-κB-dependent histone acetylation around the ERE. Interestingly, CBP, a histone acetyltransferase previously implicated in repressive interactions between ER and NF-κB, plays a permissive role by promoting histone acetylation and ER recruitment, as well as enhanced expression of BIRC3. These findings suggest a new gene regulatory mechanism by which inflammation and NF-κB activation can influence ER recruitment to inherently inactive ER binding sites. This fine-tuning mechanism may explain how two factors that generally repress each other's activity may work together on certain genes to promote breast cancer cell survival and tumor progression.
The estrogen receptor (ER) is expressed in approximately 75% of breast cancers, and women with such tumors are generally treated with endocrine therapies, such as tamoxifen or aromatase inhibitors. However, not all ER-positive tumors respond to these therapies. Through gene expression profiling studies, ER-positive tumors have been delineated into two intrinsic subtypes, luminal A and luminal B (48, 49). Women with the luminal A subtype of breast tumors respond well to therapy and have a good prognosis, whereas the outcome is poor for women with the luminal B subtype of tumors, nearly as poor as that seen in the case of ER-negative tumors. Our lab recently identified a gene signature synergistically upregulated by cross talk between ER and NF-κB that is highly associated with luminal B but not luminal A-type tumors (16). This signature is enriched for cell survival genes, including the cellular inhibitor of apoptosis gene, cIAP2, which is also known as BIRC3. We have previously shown that BIRC3 is upregulated by estradiol (E2) and the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) in a number of ER-positive but not ER-negative cell lines. Using chemical inhibitors and a small interfering RNA (siRNA) approach, our lab has further demonstrated that BIRC3 plays an important role in promoting estrogen-dependent breast cancer cell survival and protecting against TNF-α-induced cell death (51). Understanding the mechanism by which BIRC3 is upregulated by cross talk between ER and NF-κB is therefore of clinical relevance.
The ER subtypes, ERα and ERβ, are ligand-dependent transcription factors that interact with DNA and control transcription of ER target genes in response to estradiol (E2). In breast cancer cells, thousands of ER target genes and binding sites have been identified through genome-wide approaches (6, 15, 19, 31, 35). In the classical mechanism of ER action, binding to DNA occurs at palindromic estrogen response elements (EREs). However, many ER binding sites do not contain recognizable EREs (5, 35, 38); hence, in addition to direct binding to DNA sequences, ER tethering to other transcription factors appears to play a significant role in mediating estrogen action (21, 27, 33, 39, 47). The NF-κB pathway is activated by a variety of extracellular stimuli, such as proinflammatory cytokines and growth factors. In the canonical pathway, activation of the upstream IκB kinase (IKK) kinase complex leads to downstream phosphorylation and subsequent proteasomal degradation of the IκB inhibitory proteins. This liberates NF-κB family members p65 and p50, which then translocate to the nucleus, where they bind to their response elements (NF-κB-REs) and transactivate gene expression. Like ER, NF-κB can interact with many other transcription factors to regulate target gene expression.
ER and NF-κB family members have been shown to influence each other's transcriptional activity. Much work has been done to delineate the multiple mechanisms by which ER can repress NF-κB action to exert an anti-inflammatory effect (7, 13, 28, 41). Similarly, there are several pieces of evidence indicating that NF-κB can repress ER expression and transcriptional activity (2, 10, 14, 23, 36). However, there are relatively few examples of ER and NF-κB working together to increase transcription (1, 30, 45, 53). Recently, we found that these factors can work cooperatively on a number of genes, including PTGES, which codes for prostaglandin E synthase, and ABCG2, which codes for a drug efflux pump (17, 43). For these genes, we demonstrated that NF-κB may potentiate ER action by stabilizing ER occupancy on DNA at functional EREs. For the PTGES gene, cross talk occurs at the ERE itself, whereas for the ABCG2 gene, a weak NF-κB-RE located downstream of the ERE is required. This suggests that many gene-specific mechanisms of positive cross talk can occur between ER and NF-κB and that the specificity may be based on the nature and arrangement of the regulatory elements in the gene.
However, the mechanism by which ER and NF-κB work together to increase expression of BIRC3 to promote cell survival is not known. In the present study, we show that ER can potentiate TNF-α-dependent BIRC3 expression by binding to an ERE directly upstream of two NF-κB-REs. The ability of ER to access its binding site, however, is NF-κB dependent and is accompanied by CBP-mediated changes in histone acetylation around the ERE. This transcriptional mechanism represents a novel interaction between two potent transcription factors that are known to be important regulators of breast cancer growth and progression. An important implication of our findings is that inflammatory factors can alter where ER binds in the genome and that ER can, in turn, enhance the effect of inflammatory factors on the regulation of their target genes.
17β-Estradiol (E2) and 4-hydroxytamoxifen (TAM) were obtained from Sigma. Cytokines were obtained from R&D Systems. ICI 182,780 (ICI) was purchased from Tocris. The small molecule theophylline, 8-[(benzylthio) methyl]-(7CI, 8CI) (TPBM) was a generous gift from David Shapiro (University of Illinois at Urbana-Champaign [UIUC]). Adenoviral vectors for green fluorescent protein (GFP) and IκBα-DN were kindly provided to us by Michael O'Donnell (University of Illinois at Chicago [UIC]) and Ruxana Sadikot (UIC), respectively, and used as previously described (17, 43). Antibodies for chromatin immunoprecipitation (ChIP) assays, ERα (sc-543), p65 (sc-372), and CBP (sc-369) were obtained from Santa Cruz Biotechnology; anti-histone H3 (ab1791-100) was obtained from Abcam, and anti-histone H4 (17-10047), anti-acetyl-H3 (06-599), and anti-acetyl-H4 (06-866) were obtained from Millipore.
MCF-7 cells were kindly provided by Benita Katzenellenbogen (UIUC). These cells express ERα but not ERβ (8). Cells were cultured in minimal essential medium (MEM) containing 5% calf serum with antibiotics. Prior to E2 treatment, cells were cultured in phenol-red free media containing 5% charcoal-dextran stripped calf serum for at least 3 days.
RNA was isolated and analyzed by reverse transcription-quantitative PCR (RT-qPCR) using primers specific for BIRC3 and for 36B4, which served as an internal control, as previously described (16, 51). Fragments of the BIRC3 promoter (from −527 to +55 and −247 to +55) subcloned into the PGL2-basic reporter plasmid (24) were kindly provided to us by T. H. Lee (Yonsei University, South Korea). Mutations to the ERE and NF-κB-REs were made using the QuikChange Lightning site-directed mutagenesis kit (Stratagene). Wild-type and mutated sequences are given in Table S1 in the supplemental material. MCF-7 cells were transiently transfected with reporter constructs along with the Renilla luciferase construct pGL4.70 (Promega), using Lipofectamine 2000 (Invitrogen) in antibiotic-free Opti-MEM (Invitrogen). Dual-luciferase assays (Promega) were carried out after 4 h of treatment with E2 and/or TNF-α.
ChIP assays were carried out as previously described (43). For initial ChIP assays, chromatin was sonicated three times for 10 s to generate fragments of ~500 bp in size. However, for ChIP studies using qPCR primers tiled along the BIRC3 promoter, sonication was increased to 10 s 30 times to generate fragments ~200 bp in size. Inputs were serially diluted to generate standard curves and DNA enrichment was calculated as percent input for each sample. The percents input for individual experiments were converted to fold change relative to an untreated control. Average fold changes from a minimum of 3 independent experiments were then plotted. Primer sequences for ChIP qPCR are listed in Table S1 in the supplemental material.
siRNA targeting p65, ER, or CBP or a nonspecific control (siNeg) was purchased from Ambion and transfected using the Dharmafect 1 transfection reagent as previously described (51). Treatments for RNA measurements or ChIP assays were carried out 48 h after siRNA transfection.
qPCR and reporter data were analyzed by two-way analysis of variance (ANOVA) followed by a post hoc Bonferroni test. Significance for all statistical tests was set at P < 0.05. The data shown are the mean ± standard error of the mean (SEM) from at least three independent determinations.
In MCF-7 breast cancer cells, BIRC3 expression is upregulated by TNF-α in a dose-dependent manner, and this upregulation is further enhanced by the combination of TNF-α and E2 (Fig. 1A). E2 in combination with interleukin-1β (IL-1β), but not IL-6, can also enhance BIRC3 expression over that seen with the respective cytokine alone (Fig. 1B). In the absence of cytokines, E2 has no effect on BIRC3 expression across a wide range of doses (Fig. 1C). However, in the presence of cytokines, such as TNF-α, a typical E2 dose-dependent increase in BIRC3 expression is observed. The effect of E2 is prevented by a number of ER antagonists, including TAM (Fig. 1D) and ICI 182,780 (16), indicating that ER is required for E2 activity. These data demonstrate that although E2 cannot regulate BIRC3 expression on its own, it is capable of potentiating cytokine action on this gene.
Previous studies have shown that cytokines regulate BIRC3 expression in an NF-κB-dependent manner through the first (NF1) and third (NF3) of three NF-κB-REs located in the promoter of the gene (24). We find that inhibition of the NF-κB pathway with IκBα-DN (Fig. 2A) or mutation of both NF-κB sites (Fig. 2B) not only prevents TNF-α from regulating expression of BIRC3, but also completely blocks E2 from potentiating TNF-α action, suggesting that both E2 and TNF-α require NF-κB activity to mediate their effects on the BIRC3 gene. We confirmed that IκBα-DN potently inhibits nuclear translocation of NF-κB family members, p65 and p50, in MCF-7 cells (see Fig. S1 in the supplemental material). The NF-κB family member p65 has been shown to bind to the promoter region containing NF1 and NF3 in response to cytokines (24). p65 recruitment to this region was assessed in breast cancer cells by ChIP assays, which demonstrated that the combination of E2 and TNF-α had little effect on p65 occupancy compared to that seen with TNF-α alone (Fig. 2C). It is therefore unlikely that E2 potentiates TNF-α action by influencing p65 recruitment to the promoter, which is in contrast to what we have previously shown for the ABCG2 gene (43).
Further analysis of the BIRC3 promoter sequence revealed a putative ERE located 156 bp upstream of NF1 (Fig. 3A). Deletion (−247) or mutation of this ERE (mERE) completely prevented E2 from potentiating TNF-α action but had little effect on TNF-α alone (Fig. 3B). This finding suggests that although the ERE does not appear to be functional in response to E2 alone, it possesses a latent functionality that becomes apparent in response to both E2 and TNF-α—or, in other words, TNF-α allows this ERE to function. Although the sequence of the ERE is near consensus (GGGCA TAT TGACC), we mutated it to a full consensus site (GGTCA CCG TGACC) according to MatInspector (44) because the single-nucleotide difference is at an essential position in the sequence. A BIRC3 promoter construct containing the consensus ERE also failed to respond to E2, indicating that the lack of functionality of the ERE is not because of a weak binding sequence (Fig. 3C). In addition, TPBM, a small molecule that prevents ER binding to DNA (37), prevented potentiation of BIRC3 expression by E2 plus TNF-α over TNF-α alone, suggesting that ER binding to the BIRC3 ERE is essential for enhanced regulation of the gene (Fig. 3D).
To examine recruitment of ER to the BIRC3 promoter, ChIP assays were carried out. In agreement with the lack of an E2 effect on BIRC3 expression, E2 had no effect on ER recruitment to the BIRC3 promoter region containing the ERE (Fig. 4A; see Fig. S2 in the supplemental material). However, the combination of E2 and TNF-α led to a rapid and robust recruitment of ER, which was sustained over 60 min and completely prevented by blockade of the NF-κB pathway with IκBα-DN (Fig. 4A and B). These results indicate that E2 alone cannot bring about ER recruitment to the BIRC3 gene and that activation of NF-κB is necessary to allow ER to be recruited by the combination of E2 and TNF-α. This mechanism appears to be gene specific since TNF-α can actually enhance (17, 43), repress, or have no effect on (see Fig. S3 in the supplemental material) ER recruitment to other ER target genes.
We next conducted a series of experiments to examine whether TNF-α influenced ER recruitment through chromatin remodeling around the ERE of the BIRC3 gene. Formaldehyde-assisted isolation of regulatory elements (FAIRE), micrococcal nuclease (MNase) sensitivity, and restriction enzyme accessibility assays all failed to detect any changes in chromatin architecture at the ERE, but chromatin remodeling was observed at the NF-κB-REs (see Fig. S4 in the supplemental material) (data not shown). However, TNF-α induced a robust enrichment of acetylated histones 3 and 4 (AcH3 and AcH4) around the ERE (Fig. 5A; see Fig. S5 and S6 in the supplemental material). A similar increase in acetylation throughout this region, as well as increased AcH4 nearer the transcription start site, was observed in response to E2 plus TNF-α (see Fig. S6). The effect of TNF-α on acetylation around the ERE was blocked by siRNA targeting p65 (Fig. 5B and C), indicating that TNF-α acting through NF-κB leads to an increase in histone acetylation at this site. This finding suggests that TNF-α-stimulated histone acetylation may be a potential mechanism by which NF-κB influences ER recruitment to the BIRC3 promoter.
To determine which histone acetyltransferases (HATs) may be involved in histone acetylation around the ERE and subsequent ER recruitment, siRNAs for a number of coactivators and HATs known to interact with ER and p65 were used (see Fig. S7 in the supplemental material). As expected, knockdown of ER and p65 reduced regulation of BIRC3. Of the HATs, we found that CBP significantly reduced BIRC3 expression in response to E2 plus TNF-α treatment (Fig. 6A). CBP knockdown, as well as ER knockdown, significantly reduced ER recruitment to the BIRC3 promoter in the presence of E2 plus TNF-α (Fig. 6B). Furthermore, knockdown of CBP significantly reduced AcH3 and AcH4 enrichment around the ERE (Fig. 6C). The presence of CBP at the BIRC3 promoter was observed by ChIP assay but was not regulated by E2 and/or TNF-α treatment (Fig. 6D). Taken together, these data suggest that CBP is required for regulation of BIRC3, and this may be due to its ability to acetylate histones around the ERE and promote ER access to the gene, thereby leading to potentiation of BIRC3 gene expression.
In this study, we find that BIRC3, an important antiapoptotic factor in breast cancer cells (51), is upregulated by TNF-α and that this regulation is greatly potentiated by E2, despite the fact that E2 cannot regulate the gene on its own. This occurs through a novel mechanism of cross talk between ER and NF-κB not previously observed. We find that E2 potentiates TNF-α action through a near-consensus ERE that is located upstream of two functional NF-κB-REs. The endogenous ERE appears to be inaccessible to ER in the presence of E2 alone, but NF-κB activation leads to a high degree of histone acetylation around the ERE. This is mediated by CBP, which allows ER to access its binding site and work synergistically with NF-κB to potently and rapidly increase transcription of the BIRC3 gene.
At first we expected that the lack of functionality of the ERE could be the result of its sequence. It has been demonstrated that the sequence of the ERE can affect the binding affinity of ER for its response element and in turn influences transcription of the target gene (18, 32). Furthermore, we have shown that cooperativity between ER and NF-κB occurs between a nonconsensus NF-κB-RE and an ERE for another gene whose expression is increased by E2 and TNF-α (43). However, the nature of the BIRC3 ERE is nearly identical to a consensus sequence, and conversion to a perfect consensus sequence does not significantly increase ER activity, suggesting another factor may be preventing ER from interacting with this binding site. This is in accordance with the finding that far more high-confidence ER binding sites have been identified computationally than are actually detected by genome-wide ChIP-on-chip studies for ER recruitment with E2 alone (6, 52).
We also considered that the endogenous chromatin structure may be preventing ER from interacting with its binding site. The fact that many transcription factor binding sites are occluded during DNA packaging has been proposed as a common mechanism to confer a higher degree of regulation to gene expression in response to various stimuli (11). Furthermore, previous studies have demonstrated that other transcription factors, such as FOXA1, are necessary to facilitate ER recruitment to specific regions of chromatin (26). NF-κB has previously been shown to enhance DNA accessibility around the NF-κB-RE to which it binds. For example, at the granulocyte-macrophage colony-stimulating factor gene (GM-CSF) promoter, NF-κB causes increase in chromatin accessibility via remodeling through recruitment of the ATPase component of the SWI/SNF chromatin remodeling complex, BRG1 (4, 22). More recently, constitutive activation of NF-κB along with AP1 has been associated with increased accessibility of the IL-6 promoter in highly invasive breast cancer cells (40). However, in the case of the BIRC3 gene, NF-κB had little to no effect on the chromatin structure around the ERE, suggesting that NF-κB may be influencing ER recruitment through another mechanism. We did observe a significant enrichment of acetylated histones around the ERE in the presence of TNF-α or E2 plus TNF-α, which suggested that NF-κB interaction with HATs may increase accessibility of the ERE to ER. Previous studies have shown that histone acetylation can contribute to DNA accessibility to transcription factors by loosening histone-DNA contacts (3).
In our study, a number of HATs were identified that are required for E2 plus TNF-α-induced expression of BIRC3, several of which are known to interact independently with ER or NF-κB. CBP was of particular interest to us since it was required not only for enhanced BIRC3 expression, but also for ER recruitment as well as histone acetylation around the ERE. Previous studies have shown that ER recruitment to the CRH promoter is accompanied by an increase in H3 acetylation, as well as CBP recruitment (34). CBP has also been shown to be important for NF-κB action on the BIRC3 gene (25). In MCF-7 breast cancer cells, CBP appears to be present constitutively at the BIRC3 promoter. This has also been shown for other genes where CBP is thought to play a role in the rapid induction of transcription (42, 46). For the c-Fos gene, CBP is constitutively present at a serum response element where it interacts with the transcription factor, Elk-1 (42). Similarly, CBP is constitutively present at the mitogen-activated protein kinase phosphatase 1 gene (MKP-1) promoter, potentially through interactions with Sp3 and CREB transcription factors (46). The BIRC3 promoter contains a large number of putative response elements in close proximity to the ERE, including binding sites for AP-1, SP1, PPAR, C/EBP, and STAT transcription factors. All of these are known to interact with CBP, but determining which factors are responsible for maintaining CBP at the BIRC3 promoter requires further investigation.
Notably, CBP has been described to play a role in both synergy and repression between transcription factors. CBP appears to be required for synergy between NF-κB and STAT1 on the CXCL10 gene (12), while several studies describe a role for CBP, or the related factor p300, in mutual transrepression between ER and NF-κB (20, 41, 50). These studies suggest that transrepression may occur through (i) a competition between ER and NF-κB for the limited amounts of cellular CBP or p300 or (ii) ER causing displacement of CBP from NF-κB target gene promoters (41, 50). One study suggested that a repressive complex involving all three players may occur. However, this was not supported by a mammalian two-hybrid study that demonstrated a synergistic action between ER, NF-κB, and CBP (20). In the case of BIRC3 regulation, we propose that CBP plays a synergistic role between ER and NF-κB, potentially because of the nature and arrangement of the ER and NF-κB binding sites. Loss of CBP is correlated with reduced histone acetylation and ER binding, suggesting that the function of CBP is to assist ER in accessing the ERE, potentially through increased histone acetylation. Alternatively, CBP could function to acetylate nonhistones, such as ER and p65. These posttranslational modifications are associated with increased DNA binding and activity of both transcription factors (9, 29) and therefore require further investigation. A third possibility is that CBP may play a scaffolding role, allowing formation of a more stable complex, consisting of ER, p65, and other HATs, which cause acetylation of histones around the ERE.
In conclusion, we find that ER and NF-κB cooperate with each other via adjacent response elements in the BIRC3 promoter and cause enhanced regulation of the gene by a novel mechanism. Our findings indicate that inflammatory factors can confer functionality upon the ERE of BIRC3 by recruiting HATs and inducing histone acetylation at this site. These epigenetic modifications appear necessary to allow ER recruitment to the site, which permits ER to work together with NF-κB to increase expression of BIRC3. This scenario may be clinically important for those ER-positive breast tumors that have concomitant inflammation. Estrogen and proinflammatory cytokines may thus act together to cause enhanced transcription of prosurvival factors, leading to tumor promotion and more aggressive tumors.
This work was funded by the National Institutes of Health grant CA130932-2 (J.F.).
Published ahead of print 14 November 2011
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