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FASEB J. Oct 2011; 25(10): 3695–3707.
PMCID: PMC3177579

Resveratrol, through NF-Y/p53/Sin3/HDAC1 complex phosphorylation, inhibits estrogen receptor α gene expression via p38MAPK/CK2 signaling in human breast cancer cells

Abstract

Agents to counteract acquired resistance to hormonal therapy for breast cancer would substantially enhance the long-term benefits of hormonal therapy. In the present study, we demonstrate how resveratrol (Res) inhibits human breast cancer cell proliferation, including MCF-7 tamoxifen-resistant cells (IC50 values for viability were in the 30–45 μM range). We show that Res, through p38MAPK phosphorylation, causes induction of p53, which recruits at the estrogen receptor α (ERα) proximal promoter, leading to an inhibition of ERα expression in terms of mRNA and protein content. These events appear specifically p53 dependent, since they are drastically abrogated with p53-targeting siRNA. Coimmunoprecipitation assay showed specific interaction between p53, the Sin3A corepressor, and histone deacetylase 1 (HDAC1), which was phosphorylated. The enhancement of the tripartite complex p53/Sin3A/HDAC1, together with NF-Y on Res treatment, was confirmed by chromatin immunoprecipitation analyses, with a concomitant release of Sp1 and RNA polymerase II, thereby inhibiting the cell transcriptional machinery. The persistence of such effects in MCF-7 tamoxifen-resistant cells at a higher extent than parental MCF-7 cells addresses how Res may be considered a useful pharmacological tool to be exploited in the adjuvant settings for treatment of breast cancer developing hormonal resistance.—De Amicis, F., Giordano, F., Vivacqua, A., Pellegrino, M., Panno, M. L., Tramontano, D., Fuqua, S. A. W., Andò, S. Resveratrol, through NF-Y/p53/Sin3/HDAC1 complex phosphorylation, inhibits estrogen receptor α gene expression via p38MAPK/CK2 signaling in human breast cancer cells.

Keywords: tamoxifen resistance, ERα, promoter

Estrogen deprivation and targeting of estrogen receptor α (ERα) action using tamoxifen (T) is the typical strategy for treatment of hormone-dependent breast cancer, although aromatase inhibitors are now quickly replacing T in the clinic. However, tumor regrowth can occur as a consequence of the development of acquired hormonal resistance.

Comprehensive studies in breast cancer model systems support the concept that cancer cells adapt dynamically in response to various antihormonal therapies and up-regulate growth factor pathways required for growth (1, 2). It has been demonstrated that HER2 overexpression in ERα+ MCF-7 human breast cancer cells renders them T resistant (2); also, markedly increased levels of epidermal growth factor receptor (EGFR) plus HER2 protein have been associated with acquired resistance in MCF-7 cells (3). These reports have been substantiated in clinical samples where enhanced mitogen-activated protein kinase (MAPK) activity has been associated with reduced quality and duration of response to T, and shortened disease-free survival in ERα+ breast cancer patients (4).

However, in most cases, ERα remains essential to the problem of resistance due to its intimate crosstalk with growth factor signaling pathways (5). Recent studies report that in breast cancer cells, EGFR/HER2/MAPK, in addition to directly driving cell growth, can also target and phosphorylate key serine residues within the AF-1 domain of ERα in breast cancer cells (6, 7). This, in turn, can maintain EGFR signaling efficiency, possibly through regulating the production and autocrine release of an EGFR ligand (3), thus modulating basal T-resistant cell growth. Furthermore, ERα appears to be functional in T resistance as treatment with the pure antiestrogen fulvestrant (Faslodex), which acts by promoting ERα degradation and down-regulating its expression, can inhibit T-resistant growth both in the clinic and in cell culture models (8).

These studies on the bidirectional molecular crosstalk between ERα and growth factor receptor pathways in endocrine resistance (4) suggest the use of growth factor pathway inhibitors, rather than chemotherapy, for treating resistant disease (3). Therefore, a relatively nontoxic agent that would act on multiple targets but exert minimal toxicity would be a welcome component to our therapeutic portfolio for breast cancer. In recent years, many naturally occurring compounds commonly present in the diet have gained considerable attention as antitumor agents (9). In this regard, resveratrol (Res; 3,5,40-trihydroxy-trans-stilbene), a phytoalexin found in grapes, has been shown to be antiproliferative and protective against various types of cancers (10). This is partly attributable to its antioxidant activities and its inhibition of cyclooxygenase 1 and 2 (11). An accumulation of data has shown that the anticancer properties of Res are related to its ability to down-regulate NFκB activation (12), to cause cell cycle arrest in the G1 phase (13), or to trigger apoptosis in a variety of cancer cell lines (14). Recent data show induction of apoptosis by Res through MAPK-mediated p53 activation (15). Also, Res binds specifically to several peptides such as breast cancer-associated antigen, breast cancer resistance protein, death-associated transcription factor, and ERs (α and β) and can exert either estrogenic or antiestrogenic effects depending on the concentration (16). It may also be important for breast cancer prevention because Res can inhibit breast cancer cell growth in ERα+ and ERα cells (17).

In the present study, we report that Res produces a G1/S-phase cell cycle arrest and can antagonize resistance to T in ERα+ breast cancer cells. Concomitant to increased expression of p53, Res treatment causes a down-regulation of ERα protein, mRNA, and gene promoter activity. We demonstrate that these effects are crucially mediated by p53 via its recruitment to the ERα proximal promoter.

MATERIALS AND METHODS

Materials

Res, 4-hydroxytamoxifen (OHT), aprotinin, leupeptin, phenylmethylsulfonyl fluoride (PMFS), and sodium orthovanadate were from Sigma (Milan, Italy). PD 169316 and TBB were from Calbiochem (Darmstadt, Germany). Antibodies used in this study were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell culture

MCF-7, ZR-75.1, and MDA-MB-321 cells were cultured as described previously (18). T-resistant MCF7-TR1 and MCF7-TR2 cells were generated in the laboratory of S.A.W.F. as described previously (19). Hormone stimulation was performed, after 48 h of serum starvation, in 1% dextran charcoal-stripped fetal bovine serum to reduce steroid concentration (18).

MTT assay

Cells were treated as indicated, and antiproliferative effects were assessed by the MTT assay as described previously (20).

Plasmids

Plasmids used were XETL (21), wtp53 (22), p53 truncated (mp53 7–11; ref. 23), p53 mutant (R175H; ref. 24), deletion fragments of the ERα gene promoter (25), p53–1 gene promoter (26), and human p21 promoter (a kind gift from Dr. T. Sakai, Kyoto Prefectural University of Medicine, Kyoto, Japan). The Renilla luciferase expression vector pRL-TK (Promega, Milan, Italy) was used as a transfection standard.

FACS analysis

Cells were treated as indicated, and analysis was performed as described previously (26).

Reverse transcription and real-time PCR

Cells were treated as indicated and processed as described previously (19). The primers were 5′-AGAGGGCATGGTGGAGATCTT-3′ (ERα forward), 5′-CAAACTCCTCTCCCTGCAGATT-3′ (ERα reverse); 5′-GTGGAAGGAAATTTGCGTGT-3′ (p53 forward), 5′-CCAGTGTGATGATGGTGAGG-3′ (p53 reverse); and 5′-GGCGTCCCCCAACTTCTTA-3′ (18S forward), 5′-GGGCATCACAGACCTGTTATT-3′ (18S reverse).

Immunoprecipitation and Western blotting

Cells were treated as indicated, lysed in IP buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1 mM EDTA; and 0.5% Nonidet P-40) containing phosphatase and protease inhibitors, then processed as described previously (18). GAPDH was used as loading control.

Transfections and luciferase assays

Transfections were done as described previously (18), using Fugene 6 reagent (Roche Diagnostics, Milan, Italy). Luciferase activity was measured with the Dual Luciferase kit (Promega).

Lipid-mediated transfection of siRNA duplexes

RNA oligonucleotides directed against p53, Sin3A, and NF-Y were purchased from Invitrogen (Carlsbad, CA, USA). Cells were transfected using Lipofectamine 2000 reagent (Invitrogen, Paisley, UK) according to the manufacturer's instructions and then treated as indicated.

Chromatin immunoprecipitation (ChIP) assays

Cells were treated for 24 h before harvesting for the assay, performed as described previously (18). ERα promoter primers used for PCR were 5′-GTCGTTCATTTCATTTCAA-3′ (forward) and 5′-TGGAAACATTACGTATACTC-3′ (reverse), containing the region from −165 to −65 bp, and 5′-GTGGCCATTGTTGACCTACAG-3′ (forward) and 5′-CTGTAGGTCAACAATGGCCAC-3′ (reverse), upstream from this region.

Site-directed mutagenesis

Mutagenesis was performed on fragment A of the ERα promoter using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA, USA) following the manufacturer's instructions. The sequence for the sense primer was 5′-AGTGAGTGTTTAAGCGTTTGTCAGGGCAAGGCA-3′. The plasmids were then sequenced to confirm the mutation of the desired site.

Electrophoretic mobility shift assay (EMSA)

EMSA was performed as described previously (25), with a few modifications. Cells were treated for 6 h before harvesting for the assay. The sequence of ERα-CCAAT oligonucleotide used as probe or the unlabeled competitor was 5′-GTGATGTTTAAGCCAATGTCAG-3′, mutated 5′- GTGATGTTTAAGCGTTTGTCAG-3′. To test specific binding, nuclear extracts were preincubated with mouse monoclonal NF-YA antibody or normal rabbit IgG. The reactions were separated on 6% polyacrylamide gel in 0.25× Tris borate–EDTA for 3 h at 150 V.

Statistical analysis

Data represent means ± sd of 3 experiments. Data were analyzed by Student's t test using GraphPad Prism 4 software (GraphPad, San Diego, CA, USA). Values of P < 0.05 were considered statistically significant.

RESULTS

Res treatment decreases breast cancer cell proliferation

Res, depending on the concentration, can induce cell proliferation (concentration of 5 μM), cell growth arrest (15 μM or higher), or apoptosis in cultured cells (16, 17), and studies on cancer chemopreventive effects of Res in various stages of carcinogenesis (11, 14) reveal that in each case, Res was used at high concentration/dose (10–60 μM). Based on these considerations to examine the effects of Res on breast cancer cell proliferation, we initially tested several cell lines, including ERα+ breast cancer cells (hormone-dependent parental MCF-7 cells and two different clones of acquired T-resistant cells, namely MCF-7-TR1 and MCF-7-TR2) and ER MDA-MB-231 cells. Proliferating cells were exposed to OHT (100 nM or 1 μM) and/or different concentrations of Res (20, 50, and 100 μM), and then analyzed in MTT growth assays. As expected, after 4 d of 1 μM OHT, the treatment significantly reduced the numbers of MCF-7 cells (Fig. 1A); in contrast, OHT treatment (100 nM or 1 μM) did not affect MCF-7-TR cell numbers (Fig. 1B, C).

Figure 1.
Res inhibits breast cancer cell proliferation. MTT assay. Serum-starved MCF-7 (A), MCF-7 TR2 (B), MCF-7 TR1 (C), or MDA-MB-231 cells (D) were exposed to vehicle (−), 10 μM ICI, or different concentrations of Res and/or OHT in medium containing ...

Res (20 μM) inhibited basal cell proliferation of MCF-7 cells (21%) and both clones of MCF-7 TR (25.5–25%). With increasing doses of Res (50 and 100 μM), MCF-7 cell numbers were reduced by 46 to 67%, respectively (IC50 45 μM); MCF-7-TR growth was also reduced significantly in a dose-response fashion. It is worth noting that MCF-7 TR1 cells appear to require lower concentrations of Res to inhibit cell growth (IC50 29 μM). We next tested different doses of Res in combination with OHT (100 nM and 1 μM) in MCF-7 and MCF-7 TR cells. Res cotreatment greatly counteracted the resistance to T in both MCF-7 TR clones.

It is documented that Res has some structural similarities to diethylstilbene, a synthetic estrogen. Recent studies show that Res competes with 125I-labeled estradiol for binding to the human ER (16); also, Res can modulate cell signaling in metastatic breast cancer cells through the ER (27). To test whether Res effects could be mediated by binding to ERα, we pretreated the cells with ICI 182,780, a potent and specific antagonist with excellent growth-inhibitory effects in several cell and animal models of human breast cancer, which induces ERα degradation through ubiquitin-mediated mechanism (8). ICI as well as OHT was effective at reducing the growth (Fig. 1A, C). The combination of 1 μM ICI 182,780 and 50 μM Res did not alter the inhibitory effects of Res on cell proliferation (Fig. 1A–C). Also, ER MDA-MB-231 cells exhibited a reduction in cell numbers after Res treatment in a dose response fashion (IC50 42 μM; Fig. 1D), suggesting that multiple mechanisms, also not involving ERα, could be responsible for the effects of Res in breast cancer cells.

Res induces cell cycle arrest in breast cancer cells through increased expression of p21 and p53

To confirm these results independently and to determine the phase of the cell cycle at which Res exerts its growth-inhibitory effects, we performed FACS analysis. Exponentially growing MCF-7 and MCF-TR1 cells were treated with different concentrations of Res (20, 50, and 100 μM) and/or 1 μM ICI for 24 h and analyzed (Fig. 2A). Different Res concentrations increased the population of G0/G1 phase (percentage increase of different concentrations tested vs. untreated control: MCF-7 16.0, 19.0, and 28.8%; MCF-7 TR1 21.3, 38.8, and 44.0%), with a concomitant decrease in the percentage of cells in S phase (percentage decrease of concentrations tested vs. untreated control: MCF-7 36.0, 37.0, and 57.5%; MCF-7 TR1 51.4, 75, and 76%), suggesting a G1 arrest. However, 50 μM Res action on cell cycle progression was unaffected by the combination with ICI. These results suggest that inhibition of cell cycle progression could be one of the events associated with the selective antiproliferative efficacy of Res in MCF-7 and T-resistant breast cancer cells.

Figure 2.
Res induces G1 arrest in breast cancer cells through increased expression of p21. A) FACS analysis. Serum-starved cells were exposed to vehicle (−), 10 μM ICI, or different concentrations of Res and/or OHT in medium containing 1% dextran ...

Since G1/S cell cycle arrest has been shown to be mediated by cyclin-dependent kinase inhibitors, such as p21 (28), we assessed its expression as a candidate mediator of the antiproliferative response. MCF-7 and MCF-7-TR1 cells were incubated with 20 μM Res for 24 h prior to Western blot analysis (Fig. 2B). We found that p21 protein levels were significantly enhanced by Res, while p27 levels were reduced. Studies have demonstrated that Rb is phosphorylated in a cell-cycle-dependent manner and that progression of cells through G1/S transition requires inactivation of Rb by phosphorylation (29). Specifically, Rb is hypophosphorylated in G0/G1 and becomes hyperphosphorylated at or near the G1/S boundary. Res (20 μM) treatment caused a marked decrease in expression of the hyperphosphorylated form of pRb (Fig. 2B), which was unaffected by cotreatment with ICI. Treatment with higher concentrations of Res (100 μM) produced similar results (data not shown); therefore, we performed the next experiments using low doses of Res (20 μM).

The p53 tumor suppressor gene is considered to be a “master cellular regulator,” which on stress stimuli becomes activated and promotes cell cycle arrest and apoptosis (30). To test whether Res-induced cell cycle arrest could be associated with modulation of p53 expression, cells were treated for different times with Res and assayed by Western blot analysis (Fig. 3A). Res induced higher p53 levels in all the ERα+ breast cancer cells examined, with maximal levels seen at 12–24 h after treatment. To analyze the molecular basis for Res-induced regulation of p53 further, we also examined the effects of Res treatment on p53 mRNA levels in MCF-7 and MCF-7-TR1 cells, and we found increase in the steady-state level of p53 mRNA (Fig. 3B).

Figure 3.
Res treatment induces p53 and down-regulates ERα expression in a time-dependent manner. A) Immunoblot analysis. Cells were treated with 20 μM Res at different times as indicated. B) RT-PCR assay. mRNA expression of p53 in MCF-7 cells and ...

It is reported that part of the activation of the p53 protein involves p38MAPK, which triggers antiproliferative response (26). Therefore, to investigate whether the intracellular signaling pathway might be involved in mediating or contributing to Res effects, we evaluated the expression levels of the phospho-activated isoforms of the p38MAPK at different times of Res treatment. As shown in Fig. 3C, significant activation of p38 was observed after 10 min exposure to Res and remained highly activated through the course of experiment (36 h). The relevance of activated p38 in triggering Res effects on p53 expression was challenged by using two p38 inhibitors, PD169316 and SB203580, known to block the kinase activities of p38 isoforms (31). In agreement with previous reports (31), in the presence of PD169316, the Res induced p38 phosphorylation was still present, while p53 up-regulation was prevented (Fig. 3D). Similar results were obtained using SB203580 (data not shown).

Induction of p53 by Res treatment is associated with down-regulation of ERα expression and transcriptional activity

Most studies have focused on the transactivation function of p53; however, p53 can also repress transcription from various promoters, and among the genes down-regulated by p53 are those that have also been shown to be estrogen responsive (32). Increased expression of ERα, which drives breast cancer cell proliferation, is an early event in breast carcinogenesis, and elevated levels of ERα in benign breast epithelium is itself a risk factor for progression to invasive breast disease (5). Also, ERα may play a permissive role in the progression of breast cancer toward a drug-resistant phenotype (5, 6) due to its crosstalk with membrane growth factor receptors.

We thus hypothesized that Res's effects in ERα+ breast cancer cells could involve the regulation of ERα itself. As shown in Fig. 3A, ERα protein expression levels were decreased after 24–36 h of treatment, while p53 levels were increased substantially. Moreover, preventing up-regulation of p53 by PD169316 abrogated Res down-regulatory effects on ERα expression (Fig. 3D). T treatment is known to cause an increase in the steady-state levels of ERα and Res cotreatment abrogated ERα stabilization, in both MCF-7 and MCF-7 TR1 cells, with a concomitant induction of p53 (Fig. 3E).

To investigate functional effects of down-regulation of ERα, we then analyzed the effects of Res treatment on ERα genomic activity. To this aim, a luciferase reporter plasmid containing a consensus estrogen-responsive element (XETL) was transiently transfected into MCF-7 cells. Res treatment caused a significant decrease in E2 luciferase activity (Fig. 4A). We also evaluated protein levels of known estrogen-regulated genes, IRS1 and cyclin D1, and found that Res treatment substantially decreased basal (Fig. 4B) and E2-induced levels (Fig. 4C) of cyclin D1 and IRS1. Elevated IRS1 levels have been correlated with resistance in a number of different systems, perhaps due to its crosstalk with ERα (33); thus, Res could be a potential new therapeutic for affecting several mechanisms of clinical resistance.

Figure 4.
Res treatment decreases E2-induced signal and ERα mRNA. A) ERE luciferase reporter assay. XETL was transiently transfected into MCF-7 cells treated with vehicle (−) or 10 nM E2 and/or different concentrations of Res. After 18 h, cells ...

Res down-regulates ERα mRNA via a region between −245 and +212 bp of its promoter

To investigate further the molecular basis for regulation of ERα expression by Res, we also examined its effects on ERα mRNA levels in MCF-7 and resistant cells, treated for 24 h with increasing concentrations. Res significantly down-regulated ERα, as assessed by real-time RT-PCR (Fig. 4D). OHT alone, as expected, increased ERα mRNA, and Res reversed this effect, suggesting that Res could modulate the expression of coactivators and corepressors, which in turn can influence T action (34).

To determine whether ERα was a direct Res gene target, we next treated MCF-7-TR1 cells with the translational inhibitor cycloheximide (Chx) in the absence or presence of Res. Chx blocked down-regulation of ERα mRNA produced by Res (Fig. 4E), indicating that the latter does not have a direct effect, which, in contrast, could be due to other regulatory proteins.

Since Res down-regulated ERα mRNA, we evaluated whether Res treatment could act at the level of gene transcription through regulatory regions of the ERα promoter. Therefore, we first examined the ERα promoter region from −4100 to +212 bp via a bioinformatics approach, using the U.S. National Center for Biotechnology Information (NCBI) Genome database (http://www.ncbi.nlm.nih.gov). The region examined in this study contains multiple regulatory elements, including binding sites for AP-1, NF-κB, Oct-1, Sp1, CCAAT binding proteins, CREB-2, USF1, and 1/2 PRE (18, 25).

Five overlapping ERα promoter deletion constructs, −245 to +212 bp (fragment A), −735 to +212 bp (fragment B), −1000 to +212 bp (fragment C), −2769 to +212 bp (fragment D), and −4100 to +212 bp (fragment E), all relative to the first transcriptional ATG start site (depicted in Fig. 5A) and previously described (25), were transfected transiently into MCF-7 and MCF-7-TR1 cells, and the data are shown as relative promoter activity in luciferase units. We found that Res treatment significantly and reproducibly reduced the activity of all the fragments tested in MCF-7 and in MCF-7 TR1 cells (Fig. 5B), indicating that the basal promoter region from −245 to +212 bp, which contains important regulatory elements necessary for ERα transcription, was implicated in Res-mediated down-regulation of ERα activity. As control of specific action of Res, we evaluated the activity of p21 promoter or p53-1 gene promoter (from −1800 to +12 bp) after Res treatment (Fig. 5B′).

Figure 5.
Res treatment down-regulates ERα promoter activity, and this effect is mediated by p53. A) Schematic representation of deletion fragments of the ERα gene promoter. Fragment coordinates are expressed relative to the primary transcription ...

Down-regulatory effects of Res on ERα expression are dependent on p53

We next examined whether p53 might mediate ERα down-regulation by Res. We performed p53 siRNA-knockdown experiments during Res treatment of MCF-7 and MCF-7-TR1 cells. Addition of a p53-targeting siRNA resulted in a remarkable decrease in p53 protein levels as well as a reduction of its downstream target p21 (Fig. 5C). p53 knockdown greatly reversed the down-regulation of ERα protein levels by Res. These results are consistent with those shown in Fig. 3D. Similar results were obtained at mRNA levels (Fig. 5D).

To determine whether p53 directly controls the ERα promoter, we cotransfected the fragment E reporter and wtp53 expression vector and saw a significant decrease in ERα promoter activity, which was decreased further by the addition of Res (Fig. 5E). Expression of a p53 truncated mutant plasmid, containing exons 7–11 (mp53 7–11), lacking regions known to be involved in protein–protein interaction (35), had no effect. In contrast, p53 mutant with a missense mutation (R175H), which has been detected in cancer, and defective for sequence-specific DNA binding to a SV40 site, could enhance ERα promoter activity. Therefore, these results confirm that wtp53 is important for repression of ERα promoter activity.

p53 associates with histone deacetylase 1 (HDAC1) and mSin3A on the ERα promoter after Res treatment

Since the ERα promoter lacks a consensus p53 binding site, the mechanism of p53 regulation of ERα transcription could involve physical interactions with transcriptional cofactors, including CARM, CBP, and c-Jun, as previously demonstrated by Fuchs-Young and colleagues (36); the recruitment of HDAC; or possibly other chromatin-modifying factors and subsequent interference with the functions of transcriptional activators (37). It has been shown that the interaction of p53 with the corepressor protein Sin3A couples it to HDAC1, and that Sin3A is required for wtp53 to repress transcription (35). To address the possibility that p53 utilizes HDACs and Sin3A to repress ERα gene transcription, we performed coimmunoprecipitation assays from MCF-7 and MCF-7-TR1 cells. Res enhanced interactions between p53, Sin3A, and HDAC1 (Fig. 6A). We did not detect interactions between p53 and the corepressors NCoR and SMRT with Res treatment (data not shown).

Figure 6.
p53 associates with HDAC-1 and Sin3A on the ERα promoter after Res treatment. A) Coimmunoprecipitation assay. Cells were treated for 24 h with vehicle (−) or 20 μM Res and/or PD and or TBB. Cell lysates were immunoprecipitated ...

Phosphorylation is a post-translational modification that enhances enzymatic activity (38), and on Res, HDAC1 was phosphorylated (Fig. 6A). An important effector of p38MAPK is CK2, a key activator of HDAC1 (39); therefore we investigated the possibility that Res regulated ERα by directly modulating HDAC1 phosphorylation via CK2, possibly enhancing interaction with p-p38 MAPK (40). Immunoprecipitation assay revealed that Res enhanced the physical interaction between CK2 and p-p38 (Fig. 6B), which allows for HDAC1 phosphorylation, since pretreatment with a p38 MAPK inhibitor (PD169316) or a CK2 inhibitor (TBB) substantially reduced this effect (Fig. 6A).

Thus, these results support the hypothesis that a complex between p53, HDAC1, and Sin3A exists in vivo.

The requirement for Sin3A in Res-mediated down-regulation of ERα was tested by knocking down its expression with siRNA. MCF-7 cells were transfected with NS RNA or Sin3A siRNA and then treated with Res or left untreated. Western blot analysis revealed that Res increased Sin3A expression, and in the presence of a specific Sin3A siRNA, ERα repression produced by Res was prevented (Fig. 6C).

To assess the contribution of the p53, HDAC1, and Sin3A complex to repression of the ERα gene, we performed ChIP assays (Fig. 6D). MCF-7 and MCF-7-TR1 cells were treated for 24 h with Res to allow for elevated expression of endogenous p53. Protein-DNA complexes immunoprecipitated with antibodies directed against p53, HDAC1, Sin3A, Sp1, or RNAPol II. Since our results showed that Res mediates down-regulation of ERα via a region from −245 to +212 bp of its promoter, and previous data indicated that Sp1 binding was important for transcriptional regulation of ERα (25), the PCR primers used encompass these sites. Enhanced recruitment of p53 to the ERα promoter was seen in both cell types with Res treatment; as a control, we did not see recruitment to an unrelated ERα promoter region located upstream of the −245- to +212- bp region not containing GC-rich boxes (data not shown).

On the basis of our results demonstrating a complex between p53 and HDAC1, we next studied the recruitment of specific HDACs to the ERα promoter. HDAC1 was the most strongly recruited following Res treatment of cells. However, we also detected HDAC3 in a ligand-independent manner (data not shown). Among the different corepressors previously described (18) that can interact with p53 (30, 37), we found Sin3A to be recruited to the ERα promoter after treatment in MCF 7 and MCF-7 TR1 cells, while SMRT and NCoR were not detected (data not shown). Concomitant with the increased recruitment of p53, RNAPol II and Sp1 were released.

Res produces p53 interaction with NF-Y transcription factor at the ERα promoter in vivo

The promoter region examined in this study lacks a canonical p53-binding sequence but contains, at position −90 bp adjacent to a Sp1 site, a CCAAT box recognized by the transcription factor NF-Y, which is a known effector of cellular signaling mediated by p38MAPK (41). NF-Y has been shown to be required for p53-mediated inhibition of several genes and associates with p53 in vitro and in vivo (42). Therefore, we hypothesized that Res could efficiently inhibit ERα gene transcription by NF-Y/p53 complexes at the CCAAT box.

Mutation of 3 bp of CCAAT box/NF-Y site of the A fragment ERα promoter results in loss of Res-responsiveness, addressing how this nuclear transcription factor could be the effector of Res signaling (Fig. 7A).

Figure 7.
Res induces NF-Y binding to CCAAT region in ERα promoter. A) Site-directed mutagenesis of the CCAAT box present in fragment A. Constructs were transiently transfected in MCF-7 cells treated with vehicle (−) or 20 μM Res. After ...

To test DNA-protein interactions in the promoter region, we performed EMSA, using oligonucleotides from the ERα promoter sequence from −112 to −91 bearing the CCAAT box (Fig. 7B). One strong DNA-protein complex was detected on Res (Fig. 7B, lane 2) whose appearance was competed effectively by a 100-fold molar excess of unlabeled probe (Fig. 7B, lane 3), demonstrating the specificity of the DNA-binding complex. This inhibition was no longer observed using a mutated CCAAT oligonucleotide as competitor (Fig. 7B, lane 4). We also tested whether NF-YA is involved in the formation of the protein-DNA complexes detected in the EMSA, using the mouse monoclonal anti-NF-YA, which almost completely disrupted the DNA-protein complex band (Fig. 7B, lane 5). Normal rabbit IgG addition did not affect protein–DNA complex formation (Fig. 7B, lane 6). Moreover, using ChIP (Fig. 6D) and ChIP-re-ChIP assay (Fig. 7C) with primers amplifying a region encompassing the Sp1 and CCAAT sites, we found that together p53 and NF-Y bind this ERα promoter region in vivo after Res treatment. These data indicate that NF-Y binds to the ERα promoter at CCAAT sites and may mediate p53 binding at ERα promoter. This results in enhancement of the tripartite complex p53/Sin3A/HDAC1 with Res treatment and a concomitant release of Sp1 and RNAPol II, thereby inhibiting ERα transcription. Finally, to demonstrate the crucial role of NF-Y in the observed ERα down-regulation by Res, we performed NF-Y siRNA-knockdown experiments in MCF-7 cells treated with Res or left untreated. Western blot analysis revealed that a specific NFy siRNA prevented the ERα protein down-regulation produced by Res (Fig. 7D).

DISCUSSION

Interest in Res surged when it was reported that it also has anticancer activities (15). Res has been characterized as an antiinflammatory, anticarcinogenic, antitumorigenic, cell cycle inhibitory, cardioprotective phytoestrogen (10, 11). In agreement with such diverse pleiotropic effects, it was also shown to activate ERK (14) and inhibit a plethora of enzymes and other regulatory proteins, such as ribonucleotide reductase (43), F0F1–ATPase/ATP synthase (44), and protein kinase D (45). It also inhibits the transcription of CYP 4501Y1 (46) and cyclooxygenase-2 (21). It is possible that such apparently diverse modes of action are due to the unique ability of Res to influence multiple cellular modules rather than having a single target as in the case of many synthetic drugs.

Here we show that Res effectively inhibits the growth of breast cancer cells, and we demonstrate that it exerts its effects through multiple mechanisms of action, including blockade of cells at the G1/S phase of the cell cycle; activation of p38MAPK/CK2 signaling, modulating HDAC1 phosphorylation; and induction of p53 expression, leading to transcriptional inhibition of ERα.

We show that the effect of Res on T-resistant breast cancer cell proliferation was significant. Previous studies report that one mechanism whereby Res inhibits cell proliferation is related to its action on cell cycle progression (13). Our FACS data indicate a G1 arrest with Res in breast cancer cells. Supporting these data, our findings indicated a significant decrease in the expression of hyperphosphorylated form of pRb, which is a marker of the G1/S boundary and an increase in p21. It is well accepted that p21 is a critical mediator of p53-dependent cell cycle arrest. Res treatment significantly increased p53 in a time-dependent manner, reaching maximal expression after 24 h. Therefore, it is not surprising that p53 expression must be tightly controlled by Res to ensure that p53-induced cell cycle arrest functions properly to prevent molecular events that could lead to cancer cell growth.

Specifically, growth of T-resistant breast cancer cells is dependent on crosstalk between growth factors and nuclear or membrane ERα (5). Mice with elevated ERα expression develop ductal hyperplasia, lobular hyperplasia, and ductal carcinoma in situ, which demonstrates the consequences of unregulated ERα levels at all stages of breast cancer development (47). It has also been proposed that ESR1 is amplified in subsets of breast cancers and in precancerous breast diseases (48). Therefore, it is reasonable that ERα down-regulators are attracting significant clinical interest.

Here, we found that Res decreased ERα genomic activity and expression, at both the mRNA and protein levels, via p-38MAPK/CK2/p53 in MCF-7 and MCF-7 T-resistant cells, and also reversed the effects of T. ChIP assay of ERα promoter indicated a release of Sp1 and RNAPol II and enhancement of the tripartite corepressor complex p53/Sin3A/HDAC1, which could also participate in the displacement of coactivators responsible for agonist effects of T in MCF-7 TR. In particular, it was reported that overexpression of coactivators and down-regulation of corepressors can negate the inhibitory effects of endocrine therapy, especially in the case of SERMs (34). A significant finding in our study is that sustained p38 activation by Res plays a critical role in the modulation of p53 and ERα expression, which is central in the regulation of ER+ breast cancer cell proliferation. A potential mechanism could involve cell surface receptors, such as integrin αVβ3, which contain receptor sites for Res, activating ERK/p53-dependent responses in breast cancer cells (49).

We also defined the molecular mechanisms through which Res interferes with ERα gene transcription. Our functional experiments using 5 deletion constructs of the ERα promoter from −4100 to +212 bp showed that Res down-regulatory effects acted through a region from +212 to −245 bp of the promoter, which contains elements required for basal ERα transcriptional activity and at least two potential binding sites for Sp1, as well as a CCAAT box (25). We discovered that p53 could directly influence ERα promoter activity, since transiently overexpressed wtp53 decreased transcriptional activity of the ERα gene promoter in luciferase assays. ChIP results further confirmed that Res treatment induces the recruitment of p53 to this region despite the lack of a p53 consensus site.

Emerging evidence suggests that p53 may regulate cellular process through a coordinated program that includes both the activation and the repression of cellular genes (30), and among genes down-regulated by p53 there are estrogen-regulated genes (31). It has been reported that coexpression of wtp53 in T47-D cells blocked estrogen-dependent cell growth, and also that the wtp53 physically interacted with ER in vivo and repressed the estrogen-activated transcriptional activity (50). This notion is supported by clinical observations suggesting a strong inverse correlation between the wtp53 and ER proteins (51). Alternatively, wtp53 may suppress other signal transduction pathways (MAPK or the PI3 kinase), which in turn influence ER-dependent signaling (52).

A number of studies have investigated the mechanisms associated with p53 repression (36). One of the mechanisms reported involves interference with the functions of activators. For instance, p53 represses the α-fetoprotein (AFP) gene by inhibiting the promoter binding of hepatic nuclear factor 3 (HNF-3), a transcription factor that activates AFP transcription. The overlapping of p53 and HNF-3 binding sites in the AFP promoter results in competitive binding and displacement of HNF-3 from the AFP promoter. Transcriptional repression by p53 that is dependent on noncompetitive DNA binding has also been reported, and it can occur in the apparent absence of p53 binding to a classical consensus DNA element. In this model, repression may be achieved through the physical interaction of p53 with transcriptional activators. Consistent with these notions, p53 has been shown to bind Sp1, rendering the protein inactive for Sp1-mediated transcription. Our ChIP experiments showed a marked decrease in Sp1 binding to the ERα promoter concomitant with increased p53 recruitment, which displaced RNAPol II and thus repressed ERα gene expression. These results support previous findings illustrating that overexpression of p53 in Hela cells leads to loss of estradiol and genistein-induced human ERα transactivity (31).

With respect to RNAPol II inhibition mediated by p53, several mechanisms have been documented. These include repression through the recruitment of HDAC and, possibly, other chromatin-modifying factors or corepressors (30, 36, 37). Corepressors function as counterparts to coactivators, revealing that nuclear receptor-mediated transcription is subjected to both positive and negative regulation. The relative expression of coactivators and corepressors within a cell influences the ability to regulate gene expression. Transient transfection assays first demonstrated that both NCoR and SMRT can selectively repress the agonist activity of T and RU486 on ERα and PR, respectively (53). Subsequently, it was demonstrated that injecting antibodies to NCoR or SMRT promoted the agonist activity of T and that T was a relatively potent ERα agonist in fibroblasts derived from NCoR-null mice (54). Specifically, in our study, the inhibitory effects of Res on the ERα promoter transcriptional machinery were associated with increased expression of Sin3A (explaining the Res efficacy in T-resistant cells) and HDAC1 phosphorylation. Our results highlight the dominant role of Sin3A corepressor together with p-p38/CK2 activation mediating phosphorylation of HDAC1. We speculate that HDAC1 phosphorylation could be critical for the formation of p53 and Sin3A-HDAC1 complexes at the ERα promoter that realize the outcome of p53 binding. In this regard, previous studies report that the proline-rich domain of p53 was found to be important for its transrepression activity (35, 40). Indeed this region is required for the interaction between p53 and mSin3A, an association that is believed to form the basis of repression for certain p53 target genes (35, 37). This could explain our results showing that only wtp53 overexpression was able to repress the ERα promoter. Site-directed mutagenesis, EMSA, and ChIP experiments demonstrated that NF-Y at the CCAAT box mediates the Res effects on the ERα promoter; furthermore, siRNA-knockdown experiments highlight the crucial role of NF-Y in mediating Res effects on ERα expression.

Intriguingly, our findings have more emphasis by studies demonstrating that ERα itself can autoregulate its promoter without directly binding to DNA (55). Therefore, it is tempting to speculate that the effects of Res on the ERα promoter could be amplified by the down-regulation of ERα protein. We also propose that the responses observed provoked by Res then attenuate cell growth in T-resistant clones by the feedback inhibition of steady-state ERα mRNA levels. We found that such an effect could be attributed, at least in part, to transcriptional regulation.

Based on the results of this study, we propose a model (Fig. 8) for the inhibitory action of Res on the proliferation of T-resistant breast cancer cells through regulation of ERα gene transcription, which is mediated by up-regulation of p53, which is recruited to the ERα minimal promoter. Concomitant p38/CK2 activation, phosphorylating HDAC1, which interacts with Sin3A, provides the formation of a tripartite complex, p53/HDAC1/Sin3A, interacting with NF-Y, interfering with Sp1 DNA binding. Alterations in chromatin structure may reduce promoter accessibility to the transcriptional machinery, which would explain the decrease in RNAPol II binding.

Figure 8.
Proposed model for Res-induced repression of ERα promoter in ER+ breast cancer cells. See text for details. Ac, acetylation.

In summary, we elucidate the mechanisms by which Res down-regulates ERα content. This appears particularly relevant when ERα signaling and ERα crosstalk with growth factor pathways become crucial to turning on breast cancer cells to a hormone-resistant phenotype.

This work was supported by Programma di Ricerca di Rilevante Interesse Nazionale–Ministro dell'Istruzione, dell'Università e della Ricerca, Ex 60%–2010, and U.S. National Institutes of Health/National Cancer Institute grant R01 CA72038 to S.A.W.F.

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