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Published as doi: 10.1096/fj.07-9322com.
 (The FASEB Journal. 2008;22:671-681.)
© 2008 FASEB
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(The FASEB Journal. 2008;22:671-681.)
© 2008 FASEB

Unliganded estrogen receptor {alpha} inhibits breast cancer cell growth through interaction with a cyclin-dependent kinase inhibitor (p21WAF1)

Marie Maynadier*,{dagger},1, Jean-Marie Ramirez*,{dagger},1, Anne-Marie Cathiard*,{dagger}, Nadine Platet*,{dagger}, Delphine Gras*,{dagger}, Michel Gleizes{dagger}, M. Saeed Sheikh{ddagger}, Philippe Nirde*,{dagger} and Marcel Garcia*,{dagger},2

* INSERM, Unité 826, Montpellier, France;

{dagger} University of Montpellier 1, Montpellier, France; and

{ddagger} Department of Pharmacology, State University of New York, Upstate Medical University, Syracuse, New York, USA

2Correspondence: INSERM Unité 826, CRLC, 208 rue des Apothicaires, 34298 Montpellier Cedex 1, France. E-mail: marcel.garcia{at}valdorel.fnclcc.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogens are mitogenic in human breast cancer cells, but the presence of estrogen receptor {alpha} (ER{alpha}) is associated with a favorable prognosis in primary tumors and the molecular basis for this paradoxical relationship remains unknown. Here we show that ER{alpha} and ER{alpha} mutants devoid of ligand and DNA-binding domains inhibit cell growth in three-dimensional matrix as well as tumor formation in nude mice. Using in vitro and intracellular approaches, we have found that ER{alpha}, via its amino acids 184–283, interacts with cyclin-dependent kinase inhibitor p21WAF1. Both proteins exhibit mutual interactions in the absence of estrogens or in the presence of pure antiestrogen ICI182,780, whereas estradiol treatment disrupts their interactions. Cross-linking experiments reveal that these proteins are present in a larger complex of ~200 kDa that also contains cdk2 and cyclin E. We further demonstrate that the unliganded full-length ER{alpha} or the variant having the p21WAF1 interaction region significantly increases p21WAF1 expression, whereas ER{alpha} silencing reduces p21WAF1 levels and silencing of p21WAF1 is sufficient to prevent ER{alpha}-induced growth inhibition. Taken together, our results point to an antiproliferative function of the unliganded ER{alpha} through its physical interactions with p21WAF1 that may also explain the favorable prognosis of ER{alpha}-positive breast cancers.—Maynadier, M., Ramirez, J.-M., Cathiard, A.-M., Platet, N., Gras, D., Gleizes, M., Sheikh, M. S., Nirde, P., Garcia, M. Unliganded estrogen receptor alpha inhibits breast cancer cell growth through interaction with a cyclin-dependent kinase inhibitor (p21WAF1).


Key Words: cell cycle • steroid • proliferation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ESTROGENS ARE CRITICAL REPRODUCTIVE hormones that have multiple effects on various organ systems including the nervous, skeletal, and cardiovascular systems (1 , 2) . Estrogens mediate their effects via estrogen receptors {alpha} and β (ER{alpha} and ERβ) that function as transcription factors. Both ER{alpha} and ERβ are capable of modulating the expression of a variety of genes via their ability to bind directly to the DNA sequences known as estrogen-responsive elements present within the regulatory regions of their target genes (3) . Estrogen-activated ERs can also alter transcription through protein-protein interactions with other transcription factors such as c-Jun (4 5 6) , nuclear factor-{kappa}B (7) , or Sp1 (8) . Indeed, a large body of evidence indicates that growth factors and cytokines participate in ligand-independent activation of ER{alpha} through phosphorylation of the N-terminal region (9) . It is believed, however, that the unliganded ERs present in the higher order multiprotein complexes are mostly inactive (3) .

The mechanisms by which estrogen receptors modulate breast cancer cell growth and progression remain to be fully elucidated. Direct estrogen stimulation of cell growth was clearly demonstrated in breast cancer cell lines (10) and confirmed in vivo by the efficiency of antiestrogen therapy. However, despite estrogens’ mitogenic effect, a substantial amount of evidence suggests that the presence of ER{alpha} in cells could provide protection against tumor spreading and metastasis. Indeed, ER{alpha} expression in breast cancer is associated with a favorable prognosis (11 12 13 14 15) , and ER{alpha}-positive breast cancer cell lines are less invasive and, unlike ER{alpha}-negative cells, do not metastasize (16) . These lines of evidence led us to postulate that, in the absence of estrogen activation, ER{alpha} may serve in a protective role against tumor progression. Previously, we and others have shown that ER{alpha} can decrease the in vitro invasiveness of breast cancer cells (17 , 18) . In this study, we sought to investigate the potential role of unliganded ER{alpha} in breast cancer cell growth. Our results, for the first time, indicate that unliganded ER{alpha} is involved in the inhibition of breast cancer cell growth as well as tumor formation in nude mice. Our results further demonstrate that these inhibitory effects are associated with mutual interactions between ER{alpha} and p21WAF1 and an increase in p21WAF1 expression. These findings, therefore, provide new perspectives on the function of unliganded sex steroid receptors in the homeostasis of target tissues and in the growth of hormone-dependent tumors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids
Expression plasmids for full-length (HEGO) or mutated forms of human ER{alpha} (HE15 and ER11) were constructed in pSG vectors as described previously (19) . The full-length ER{alpha} sequence was then subcloned into pUHC13.3-CMV (pUHC-ER{alpha}), a vector with a tetracycline-repressed promoter (20) . The EcoRI fragment of the HE15 plasmid, which codes for amino acids 1–282 of ER{alpha}, was subcloned into pBK-CMV (Stratagene, La Jolla CA), resulting in the ER15 plasmid. The deletion mutant ER108 (amino acids 1–216) containing, at the 3' end, the Simian virus 40 large T antigen nuclear localization signal (NLS) was described previously (17) . For glutathione S-transferase (GST) pull-down experiments, different fragments of ER{alpha} were amplified by polymerase chain reaction (PCR) and cloned into pGEX-2TK for generation of GST fusion proteins. For double-hybrid assays, p21WAF1 was amplified by PCR and inserted in the BamH1 site after the DNA-binding domain of Gal4 in the pBind vector (Promega, Madison, WI, USA). ER{alpha} was inserted before the activation domain of VP16 in the pAct vector (Promega). The pcDNA3-p21, GST-p21, and GST-p21N plasmids have been described previously (21 22 23) .

Cell culture, transfection, and cell outgrowth assay
MDA-MB-231 and MCF-7 human breast cancer cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) and in F-12/DMEM, respectively, supplemented with 10% fetal bovine serum (FBS). Steroids were withdrawn from cells by 6-day culture in phenol red-free DMEM supplemented with 10% FBS that had been previously stripped with dextran-coated charcoal.

MDA-MB-231 cells were stably transfected with ER15, ER108, and ER11 mutants. pUHC-ER{alpha} was transfected into a subclone of MDA-MB-231 cells that express the trans-acting protein required for tetracycline promoter repression (18) . Stable transfectants were isolated by selection with neomycin (ER15, ER108, and ER11) or hygromycin (pUHC-ER{alpha}). For clone ER15, two neomycin-resistant mass cultures were also selected. Expression of full-length ER{alpha} and mutants was detected by immunofluorescence using the 1D5 antibody directed against the amino-terminal A/B region of human ER{alpha} (DakoCytomation A/S, Copenhagen, Denmark) as described previously (17) . Experiments were performed using transfected cells at passages 4–12, in which the expression of ER{alpha} or the ER mutants was verified.

Outgrowth assays were performed as described previously (24) . Briefly, 20,000 subconfluent cells were resuspended in Matrigel (0.2 ml, 6.3 mg/ml) or in collagen I gel, (0.5 ml, 0.5 mg/ml) (BD, Franklin Lakes, NJ, USA) in 24-well plates. Then, cells were quantified using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay (200 µg/ml MTT for 6 h). We confirmed this assay to be linear for 105 to 5 x 106 embedded cells and gave comparable results as were determined by DNA content estimation using the diaminobenzoic acid fluorescence assay (24) .

Cell cycle analysis
Cell cycle analyses were performed on cells cultured for 4 and 7 days after transfection with small interfering RNA (siRNA) p21WAF1. Cells were harvested with trypsin (5 mg/ml), fixed in 70% cold ethanol for 2 min, washed twice with PBS, and then stained with a solution of 100 µg/ml propidium iodide (Sigma-Aldrich Chimie, Lyon, France) (500 µl for 106 cells) at 4°C overnight. Analysis of DNA content was performed in a Beckman and Coulter apparatus (EPICS XL System II, version 3.0) with a minimum of 15,000 events collected, and these data were analyzed with 2.9 WinMDI software (Scripps Research Institute, La Jolla, CA, USA).

Immunoprecipitation, Western blots, and cross-linking experiments
Cell proteins were extracted by three freeze-thaw cycles in buffer containing 50 mM Hepes, 150 mM NaCl, 0.1% Nonidet P-40, 10% glycerol, 2.5 mM EGTA, and protease inhibitors (Complete; Roche Diagnostics, Indianapolis, IN, USA) and centrifuged at 10,000 g for 15 min. The proteins were quantified by the Bradford method, and a constant amount was immunoprecipitated or analyzed by Western blotting. For immunoprecipitation, proteins were incubated overnight at 4°C with 2 µg of the relevant antibody or control immunoglobulins and then for 2 h at 4°C with prewashed protein G-Sepharose. Immunoprecipitates were recovered by centrifugation, washed four times, and resolved by 10% SDS-PAGE. For Western blot analysis, proteins were resolved by SDS-PAGE and transferred to nitrocellulose. Membranes were probed with antibodies anti-ER{alpha} (1D5 or HC20), p21WAF1 (polyclonal C19; Santa Cruz Biotechology, Inc., Santa Cruz, CA, USA; or monoclonal Ab-1; Oncogene Research Products, San Diego, CA, USA), Cdk2 (M2; Santa Cruz Biotechnology, Inc.), Cdk4 (polyclonal Ab-3; Neomarkers, Fremont, CA, USA), or cyclin E (HE12; Santa Cruz Biotechnology, Inc.). After washing, membranes were incubated with peroxidase-conjugated secondary antibodies (dilution 1:5000; Amersham, Piscataway, NJ, USA) and analyzed with the ECL detection system (Amersham). The apparent molecular weights were determined with prestained standard proteins (Ozyme, St. Quentin-en-Yvelines, France). For protein cross-linking, MCF7 cells were harvested with trypsin, washed twice in PBS, and incubated for 30 min in 0.15 M NaCl and 20 mM Hepes, pH 8, containing the cross-linker, 6 mM disuccinimidyl suberate or vehicle alone.

GST pull-down and in vitro kinase assays
GST fusion proteins were produced in BL21 Escherichia coli and purified by immobilization on glutathione agarose beads as described by the manufacturer (Amersham). GST fusion protein suspension beads (50 µl) were incubated overnight at 4°C with [35S]methionine-labeled proteins generated with the in vitro transcription-coupled translation system (Promega) or with total cellular proteins metabolically labeled with [35S]methionine. After three washes, samples were boiled in Laemmli buffer and analyzed by SDS-PAGE. Signals were amplified by fluorography (Amplify; Amersham), and gels were exposed at –80°C. Quantification of 35S-labeled proteins was performed with a Fuji PhosphorImager.

Cdk2 kinase assays were performed using Cdk2 immunoprecipitates and the histone H1 substrate as described previously (25) . The 32P-labeled histone H1 was quantitated by SDS-PAGE and the PhosphorImager.

Two-hybrid assay
After transient transfection, the interaction of the two fusion proteins resulted in induction of the cotransfected Gal4-responsive firefly luciferase reporter construct (pG5-luc). Luciferase activity was determined and normalized on the basis of the transfection efficiencies by cotransfecting a β-galactosidase expression vector. The expression of the fusion proteins and their expected molecular masses were verified by Western blot analysis followed by incubation with ER{alpha} and p21WAF1 antibodies (data not shown).

Tumor growth in athymic mice
BALB/c nude female mice (Charles River Laboratories, Les Oncins, France) were maintained in sterile conditions and single-cell suspensions of the different stably transfected MDA-MB-231 cell lines were subcutaneously injected under the second mammary fat pad of the mice. Mice were examined every 2 days for tumor appearance, and tumor sizes were estimated by calculating the product of the measurements of two perpendicular diameters. Mice were housed and cared for in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication 85-23)and monitored routinely for evidence of disease.

siRNA transfections
Transfection of a pool of two validated Stealth RNAi for human p21WAF1 and ER{alpha} was performed using the Lipofectamine RNAiMAX reagent according to the manufacturer’s instruction (Invitrogen, Cergy Pontoise, France). ER{alpha} siRNA1 was located in the C domain (5'-GCTACTGTGCAGTGTGCAATGACTA-3', sense) and siRNA2 in the E domain (5'-GCTTAATTCTGGAGTGTACACATTT-3', sense). The p21WAF1 siRNA1 target sequence was 5'-GGQCCUGUCQCUGUCUUGUQCCCUU-3' (sense), and the siRNA2 target sequence was 5'-GAACUUCGACUUUGUCACCGAGACA-3' (sense). siRNA corresponding to firefly luciferase (5'-AACGTACGCGGAATACTTCGA-3', sense) was used as a negative control of transfection efficiency. Cells were plated on six-well plates after transfection with siRNA for p21WAF1 or for ER{alpha} and lysed after 96 or 168 h of incubation, respectively, for protein extraction and Western blot analysis. For growth experiments, siRNA transfected cells were spread on 96-well plates in triplicate and quantified using a MTT assay.

Statistical analysis
Densitometries of Western blotting or immunoprecipitation studies were analyzed with the PC-Bas 2.0 software (Fuji, Stanford, CT, USA). Statistical analysis was performed using the Student’s t test. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of ER mutants with inactive hormone and DNA-binding domains inhibits cell growth and tumor formation in mice
To investigate the effect of unliganded ER{alpha} on tumor cell growth, we stably transfected ER{alpha}-negative MDA-MB-231 cells with the expression vectors carrying full-length ER{alpha} or the deletion variants lacking 1) the C domain [ER11; amino acids (aa) 185–251 deleted], 2) the D/E/F domains (ER15; aa 1–282), or 3) the second zinc finger within the C domain as well as the entire D/E/F domains (ER108; aa 1–216) but with intact NLS (Fig. 1 A).


Figure 1
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  		Figure 1. Characterization of MDA-MB-231 stable transfectants expressing full-length inducible ER{alpha} and ER11, ER15, or ER108 deletion mutants. A) Schematic representation of the four ER{alpha} proteins. The activating functions AF1 and AF2, the DNA (DBD) and hormone (HBD) -binding domains, and the NLSs are indicated. B, C) Characterization of stably transfected MDA-MB-231 cells expressing ER{alpha}, ER15, or ER108. B) Immunofluorescence assays revealed nuclear staining of representative clones with strong (a) or moderate (b) ER{alpha} expression without tetracycline (pUHC-ER{alpha} clone 33). Moderate expression of ER15 in one mass culture (c) and in a single clone expressing high levels of ER108 (d). No staining was observed in control clones or mass cultures transfected with the empty vectors (data not shown). C) Comparison of ER{alpha} expression in extracts from ER{alpha}-positive MCF7 cells and extracts from MDA-MB-231 cells transfected with pBK vector alone (C1, negative control), ER{alpha} (clone 33±tetracycline), ER15, ER108, pSG vector alone (C2, negative control), or ER11.

We also established cells expressing full-length ER{alpha} under inducible conditions using a tetracycline-repressed promoter (pUHC-ER{alpha}) and isolated four independent clones showing moderate to high inducible expression of ER{alpha}. Fig. 1B shows the typical nuclear staining of cells expressing either elevated (a) or moderate (b) levels of full-length ER{alpha}, moderate levels of ER15 (c), and high levels of ER108 (d). All control clones transfected with vectors without ER{alpha} inserts displayed no staining (not shown). The correct molecular masses of the various forms of the exogenous receptors were confirmed by Western blot analysis and, as shown in Fig. 1C , the full-length pUHC-ER{alpha} and the variants including ER11, ER15, and ER108 displayed the expected molecular masses of 62, 55, 28, and 25 kDa, respectively. Densitometric scanning was performed to ascertain the expression of exogenous receptors relative to that of endogenous ER{alpha} in MCF7 breast cancer cells, and the results indicated that pUHC-ER{alpha} (clone 33 –tet), ER15, ER108, and ER11 displayed expression levels of 88 ± 15, 20 ± 6, 194 ± 16, and 25 ± 5%, respectively.

Next, we assessed the effect of unliganded full-length ER{alpha} on cell growth using the four pUHC-ER{alpha} clones. Our results indicated (Table 1 ) that in the absence of estradiol (E2), cell growth in the monolayer cultures was significantly reduced by 28.9% on induction of ER{alpha} after tetracycline removal for 4 days. It should be noted that the growth-suppressive effect of ER{alpha} is probably superior to 28.9% because significant ER{alpha} expression persisted in the tetracycline-treated controls (+tet vs. –tet; stained nuclei increased from 5.4 to 25.8%). In contrast, the control clones exhibited only 4% growth inhibition after tetracycline withdrawal. To demonstrate the E2-independence of this growth suppressive effect, we tested the outgrowth of cells expressing ER15, a variant without the hormone-binding domain, or ER108, a mutant lacking the second zinc finger whose removal is sufficient to totally prevent DNA binding (26) and dimerization capacities (27) . The growth of these cells was compared with that of controls in two different three-dimensional matrices, including Matrigel, a reconstituted basement membrane, and collagen I gel (Fig. 2 ). In Matrigel, expression of ER15 or ER108 significantly decreased growth (mean 29.5%) compared with controls (C1 and C2) (Fig. 2A ). The stellate morphology of MDA-MB-231 cells (C1) with sprouting protrusions was strongly inhibited by ER15 and ER108 expression (Fig. 2A , right panels). ER15 or ER108 cells displayed rounded morphological features with rare protrusions into Matrigel. A similar morphological effect and growth inhibition (mean 32%) was also observed in collagen matrix with the ER108 mutant (Fig. 2B ). In contrast, the growth of the ER11 clone was not significantly modified compared with control cells, indicating that deletion of the C region, including the first zinc finger, prevented the ER{alpha}-mediated growth inhibition (Fig. 2B ).


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  		Table 1. Growth inhibition induced by tetracycline-modulated ERa expression


Figure 2
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  		Figure 2. Inhibition of cell outgrowth in Matrigel and collagen matrices by ER{alpha} mutants deleted in hormone and/or DNA binding domains in MDA-MB-231 cells. For outgrowth assays, MDA-MB-231 cells transfected with ER108 ({triangleup}), ER15 ({circ}) or ER11 ({diamondsuit}) or with the corresponding control vectors, C1 (•) or C2 ({blacktriangleup}) were embedded in Matrigel matrix (A) or collagen gels (B). Cells were quantified after different times in culture using an MTT assay, as described in Materials and Methods. Values represent mean ± SD of triplicate cultures. Right panels in A: typical phase-contrast optical microphotographs of cells in Matrigel after 4 days in culture (x200). Right panels in B: p-nitrotetrazolium violet cell staining after 7 days in collagen I culture (x200). One representative experiment of three is shown.

We also explored whether the unliganded ER{alpha} or ER{alpha} mutants could prevent tumor formation by assessing the tumorigenicity of different clones after subcutaneous injection in nude mice. Two pairs of clones, expressing either high or moderate levels of ER{alpha}, were injected in untreated mice and compared with controls (Fig. 3 A). High expression of ER{alpha} (~80% of MCF7 levels) fully inhibited tumor formation. Lower ER{alpha} expression (~20% of MCF7 levels) decreased tumor growth by 97% and tumor incidence by 50% at 41 days postinjection (Fig. 3A ). Moderate expression of ER15 mass cultures (Fig. 3B ) also significantly reduced tumor growth by 86%, whereas tumor incidence was not significantly reduced. Finally, the expression of ER15 or ER108 in individual clones also decreased tumor growth by 68 and 62%, respectively (Fig. 3C, D ). The higher inhibition of tumor growth obtained with full-length ER{alpha}, relative to that of the ER{alpha} mutants, can be explained by the additive effect due to the binding of endogenous estrogens, previously shown to prevent tumor growth of ER{alpha}-expressing MDA-MB-231 cells (28) . Together these data indicate that the expression of full-length ER{alpha} or of the truncated ER{alpha} variants, unable to bind estrogens and DNA, reduces cell proliferation in culture and tumor growth in nude mice.


Figure 3
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  		Figure 3. Tumor growth of MDA-MB-231 cells expressing full-length or mutants of ER{alpha} in athymic mice. Nude mice (5 to 6 weeks old) were injected subcutaneously at two distinct sites with an equal mixture of two stable MDA-MB-231 transfectants. A) Clones with moderate ({circ}), or stronger pUHC-ER{alpha} expression ({blacktriangleup}) or control clones (•). B) ER15 clones ({circ}) pBK control clones (•). C) ER15 mass cultures ({circ}) or pBK control cultures (•). D) ER108 clones ({circ}) or pSG control clones (•). The total number of injected cells/site was 3.3 x 106 (A, C), 2.5 x 106 (B) or 2 x 106 (D). Mean ± SD of tumor area. Tumor incidence (number of tumors/number of injections) for each group is indicated in brackets.

Physical interaction between p21WAF1 and the first zinc finger region of ER{alpha}
Our preceding findings prompted us to investigate whether ER{alpha} mediates these effects by interacting with other proteins. To identify which proteins are implicated, we used, as bait, the first zinc finger domain (aa 178–215; GST-ER{alpha} 178–215) of ER{alpha} because ER108, which contains this region, inhibited cell growth, whereas ER11, which lacks the two zinc finger domains, did not (Fig. 2B ). GST pull-down assays were performed with [35S]methionine-labeled protein extracts from human MCF7 and MDA-MB-231 breast cancer cells. Among a small number of proteins specifically retained by GST-ER{alpha} 178–215 (and not by GST alone) a 21-kDa protein was noted (Fig. 4 A). We wondered whether the 21-kDa protein was the cyclin-dependent kinase inhibitor p21WAF1 (29 , 30) , because previous findings had indicated that p21WAF1 could be displaced from Cdk2-cyclinE complexes after ER{alpha} activation by E2 (31) . Next, we tested the interaction of in vitro translated human p21WAF1 with a series of GST fusion proteins containing different ER{alpha} deletion mutants to confirm whether p21WAF1 interacts with ER{alpha}. Figure 4B, C shows that p21WAF1 did not interact with GST alone, GST-ER{alpha} mutant 2–184 containing the A/B ER{alpha} domain, and GST-ER{alpha} mutants 283–330 and 313–595, corresponding to the D/E/F domains. However, three GST-ER{alpha} mutants containing sequences within the 184–283 region interacted with p21WAF1 albeit with different affinities. The differences in affinity were confirmed by several independent experiments. The fact that mutants containing the adjacent sequences 178–215 and 251–312 both interacted with p21WAF1 suggested the presence of two independent binding sites within the 184–283 amino acid region with the 178–215 amino acid region displaying higher affinity (Fig. 4B, C ). In reciprocal experiments, we found that the N-terminal 1–90 amino acids were involved in interactions between p21WAF1 and ER{alpha} (Fig. 4D ). Because the indicated N-terminal region of p21WAF1 contains a Cdk-binding site, we used GST-Cdk2 to further investigate the potential effect of ER{alpha} on p21WAF1 and Cdk2 interactions. We found that the presence of ER{alpha} did not prevent p21WAF1-Cdk2 interactions (data not shown).


Figure 4
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  		Figure 4. Interaction of ER{alpha} with the cyclin-dependent kinase inhibitor, p21WAF1. A) Interaction of labeled breast cancer cell extracts with the GST-ER{alpha} (178–215) fusion protein. Cellular extracts from MCF7 and MDA-MB-231 breast cancer cells were metabolically labeled with [35S]methionine for 18 h, extracted and incubated with either GST alone or GST-ER{alpha} (178–215), and then immobilized on GST Sepharose beads. The retained proteins were analyzed by SDS-PAGE and fluorography. A 21-kDa protein specifically retained by GST-ER{alpha} (178–215) is indicated by the arrow. B–D) Binding of p21WAF1 to ER{alpha} and ER mutants. B) [35S]Methionine-labeled p21WAF1 was synthesized using a transcription-translation kit and incubated with GST alone or the indicated GST-ER mutants. The [35S]methionine-labeled proteins retained on glutathione agarose beads were analyzed on sodium dodecyl sulfate gels, as described in Materials and Methods. C) Schematic representation of ER{alpha} and data from B. D) [35S]Methionine-labeled ER{alpha} was incubated with GST alone, GST-p21WAF1 (full-length p21WAF1), or GST-p21N (1–90 N-terminal amino acids of p21WAF1), and the retained receptor was then analyzed.

Estrogen inhibits interaction between ER{alpha} and p21WAF1 in breast cancer cells
We also investigated the interactions between ER{alpha} and p21WAF1 in MCF7 cells by using mammalian cell two-hybrid screening. In this assay, the luciferase activity due to transactivation is increased when VP16-ER{alpha} and GAL4-p21WAF1 bind with each other. Figure 5 A, B shows that luciferase activity is increased in response to pure antiestrogen ICI182,780 or when the concentration of VP16-ER{alpha} is increased further, confirming their interactions and indicating that pure antiestrogen favors their binding. In contrast, E2, when used at different concentrations, inhibited binding between ER and p21WAF1 (Fig. 5A, B ). In MDA-MB-231 cells, ER{alpha}-p21WAF1 complexes were similarly formed in the presence of 10 nM ICI182,780 and were dissociated by 100 nM E2 treatment (data not shown).


Figure 5
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  		Figure 5. Formation of ER{alpha}-p21WAF1 complexes in MCF7 breast cancer cells and their dissociation in the presence of estradiol. A) MCF7 cells deprived of steroid for 5 days were cotransfected with the expression vectors for the two hybrid proteins Gal4-p21WAF1 and VP16-ER{alpha} and with a luciferase expression vector under the control of the Gal4-promoter. The interaction of p21WAF1 with ER{alpha} was quantified by luciferase activity after 30 h of treatment with either the indicated concentrations of E2 ({diamond}) or the pure antiestrogen ICI182,780 ({blacksquare}) or a combination of ICI182,780 + 1 µM E2 ({blacktriangleup}) as described in Materials and Methods. B) The interaction of p21WAF1 with ER{alpha} was determined as in (A) after transfection of increasing concentrations of VP16-ER{alpha}. The steroid-deprived cells were maintained without hormones (100%) or treated with either 10 nM E2 ({diamond}) or 10 nM ICI182,780 ({blacksquare}) for 30 h. The negative control (VP16-ER{alpha}=0) was VP16 alone. The values in A and B are means ± SD and were confirmed in at least two experiments.

To demonstrate interactions between endogenous ER{alpha} and p21WAF1, MCF7 cells were used in the cross-linking experiments to covalently link these proteins. ER{alpha}-p21WAF1 complexes were fixed by adding the cross-linker in living cells and were characterized by immunoprecipitation, gel electrophoresis, and Western blotting (Fig. 6 A). After immunoprecipitation a 200-kDa complex was detected in Western blots using anti-ER{alpha}, p21WAF1, or Cdk2 antibodies. In addition, experiments performed on total cell extracts after cross-linking also confirmed the presence of this complex, which was detected using anti-ER{alpha}, p21WAF1, and Cdk2 antibodies and also an anti-cyclin E antibody (Fig. 6B ). Moreover, we found that in these extracts the 200-kDa complex was not detected using an anti-Cdk4 antibody, suggesting that ER{alpha}-p21WAF1 complexes appear to preferably interact with Cdk2 (data not shown).


Figure 6
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  		Figure 6. Covalent cross-linking of a 200-kDa complex containing ER{alpha}, p21WAF1, Cdk2-cyclin E, and hormonal modulation of the complex. MCF7 cells were grown in the absence of estrogens for 48 h and then incubated for 30 min in the presence (+) of the cross-linker (CL), 6 mM disuccinimidyl suberate or with vehicle alone (–). After protein extraction, a constant amount (150 µg of proteins) was analyzed on SDS-PAGE either (A) after immunoprecipitation with an anti-ER{alpha} antibody (HC20, 1D5) or with control rabbit immunoglobulins (IgG) or (B) directly (total cell extracts). Proteins were transferred to nitrocellulose and detected by Western blotting (WB) using antibodies against ER{alpha} [HC20 or 1D5 in A or 1D5 in B], p21WAF1 [C19 in A or Ab-1 in B], Cdk2 (M2), or cyclin E (HE12). The apparent molecular masses, according to standard proteins, are 62 kDa for ER{alpha}, 35 kDa for Cdk2, and 50 kDa for cyclin E. C, D) Effects of hormonal treatments on the levels of Cdk2, p21WAF1, 200 kDa complex, and Cdk2 activity. MCF7 cells were maintained for 5 days in hormone-deprived medium and then treated with either 10 nM E2 (for 5 or 24 h), with 10 nM ICI182,780 (ICI) for 24 h, or with ethanol vehicle alone (C=control). C) Cdk2 and p21WAF1 were quantified using the respective antibodies in Western blots and the 200-kDa complex using ER{alpha} antibody after cross-linking. D) Cdk2 activity was determined on Cdk2 immunoprecipitates from 500 µg of protein from MCF7 cells on histone H1 as substrate, as described in Materials and Methods. The 200-kDa complex and histone phosphorylation were semiquantified by densitometric scanning. Data are means ± SD from three experiments. *P < 0.05 relative to control (Student’s t test).

We next investigated the effects of hormone treatment on the formation of the 200-kDa complex and on the expression of proteins present in these complexes. MCF7 cells were either left untreated or treated with E2 or ICI182,780 and, as shown in Fig. 6C , p21WAF1 levels were increased ~1.5-fold by antiestrogen treatment and decreased by 2- to 4-fold after E2 treatment for 5 and 24 h. The Cdk2 levels, in contrast, remain constant under all conditions, indicating that the effect was specific for p21WAF1 (Fig. 6C ). ICI182,780 treatment also increased the abundance of 200-kDa cross-linked complexes that were completely dissociated after exposure to E2 for 5 and 24 h (Fig. 6C, D ).

In parallel, Cdk2 kinase activity was also determined in the immunoprecipitates, and Cdk2 kinase activity was increased by ~2- and 10-fold after E2 treatment for 5 and 24 h, respectively. In contrast, a 24-h treatment with the antiestrogen ICI182,780 did not affect Cdk2 activity. It is possible that the formation of ER{alpha}- and p21WAF1-containing complexes in the absence of E2 or in the presence of the antiestrogen may involve Cdk2 inhibition. Conversely, E2 treatment may rapidly dissociate these complexes and thereby activate Cdk2. Based on our results, it is tempting to suggest that mutual interactions between ER and p21WAF1 may in fact inhibit Cdk2.

To gain further evidence that E2 dissociates interactions between ER{alpha} and p21WAF1, MCF-7 cells were treated with ICI182,780 alone for 48 h or with ICI182,780 and then E2 (1 µM) during the last 24 h. Cells were then subjected to sequential protein extractions in low-salt (TE buffer: 10 mM Tris, pH 7.4, and 1 mM EDTA) and high-salt (TE buffer+0.4 M KCl) conditions, and Western blot analyses were performed. Our results indicated that, in the presence of antiestrogen, ~60% of the ER{alpha} was localized in the low-salt extracts containing the majority of p21WAF1, whereas E2 exposure caused ~90% of the ER{alpha} to shift to the high-salt extracts (data not shown).

Overall, our results presented so far indicate that, in the absence of estrogens (or in the presence of the pure antiestrogen, ICI182,780), ER{alpha} and p21WAF1 interact with each other within a 200-kDa complex that also contains Cdk2 and cyclin E and that their mutual interactions are inhibited by E2.

Unliganded ER{alpha} or ER{alpha} mutants containing the p21WAF1-binding domain increase p21WAF1 expression
We also investigated the effect of unliganded ER{alpha} on p21WAF1 expression and, as shown in Fig. 7 A, the pUHC-ER{alpha} clone exhibits ~2-fold higher levels of p21WAF1 than the control cells (mock). In ER108 or ER15 clones, p21WAF1 levels were increased ~2.9- and ~5.9-fold, respectively, compared with the control cells. In contrast, in the ER11 clone that expresses an ER mutant devoid of the p21WAF1-binding region, the p21WAF1 levels were not significantly affected. Next, we sought to investigate the effect of ER{alpha} depletion on p21WAF1 expression, and for this purpose we used MCF-7 cells in which ER{alpha} expression was knocked down with specific siRNAs. Figure 7B shows that RNAi-mediated knockdown of endogenous ER{alpha} significantly inhibits p21WAF1 expression levels. Taken together, these data indicate that the expression of full-length ER{alpha} or ER{alpha} mutants containing the p21WAF1-binding region increases p21WAF1 expression in a ligand-independent manner.


Figure 7
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  		Figure 7. Modulation of p21WAF1 concentration by unliganded ER{alpha} and estradiol. A) Modulation of p21WAF1 by ER{alpha} or ER{alpha} mutants without ligand or/and DNA binding domains. The expression of ER{alpha} and p21WAF1 was determined by Western blotting on similar amounts of total protein extracts from different cell lines. Data are expressed as the percentage of the values obtained in the concomitant experiment with MCF7 cells. The number of experiments is indicated in parenthesis. wt MCF7 = wild-type MCF7 cells; MDA-MB-231 mock = mean of the three mock-transfected cell clones (pUHC, pBK, and pSG vectors alone); pUHC-ER{alpha}, ER15, ER108, and ER11 = stably transfected cell clones already described in the legend to Fig. 1 . B) Inhibition of ER{alpha} expression in MCF7 cells. Western blots of lysates from MCF7 cells transfected with ER{alpha} and control (Ctrl) siRNAs. Cell lysates were collected 7 days after completion of transfection. Membranes were immunoblotted to quantitate ER{alpha} and p21WAF1 expression and reprobed for actin. Molecular masses are shown on the right. Blots were quantified by densitometric analysis. Data (mean±SD) are from two independent experiments. *P < 0.05, **P < 0.01 relative to controls (Student’s t test).

p21WAF1 elevation is a prerequisite for growth inhibition by unliganded ER{alpha}
To determine the role of p21WAF1 in the growth inhibition induced by unliganded ER{alpha}, we used p21WAF1 siRNAs to knock down its expression (Fig. 8 ). Western blot analysis demonstrated partial (~50%) or near complete inhibition of p21WAF1 expression in ER108 cells 4 or 7 days after transfection with p21WAF1 siRNA compared with control siRNA (Fig. 8A ). Assessment of cell growth in p21WAF1-silenced ER108 cells demonstrated that the reduction in growth inhibition was linked to the degree and duration of p21WAF1 silencing (Fig. 8B, C ). Cell growth in a MDA-MB-231 control clone (C2: vector alone) was not affected by p21WAF1 silencing. Moreover, cell cycle studies indicated that the percentage of cells in the S + G2/M phases was lower in ER108 cells compared with control cells, and this difference was abolished on p21WAF1 silencing (Fig. 8D ). These findings indicate that the increase in p21WAF1 expression is required for the growth inhibition associated with unliganded ER{alpha}.


Figure 8
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  		Figure 8. p21WAF1 silencing reverses growth inhibition mediated by ER{alpha} mutants. A) Western blot analysis of cell lysates from ER108 cells transfected with p21WAF1 and control (Ctrl) siRNAs. Cell lysates were collected 4 and 7 days after completion of transfection. Data are means ± SE of triplicates from one representative experiment of two. B) Growth of control MDA-MB-231 cells (C2) or ER108 cells transfected with 2 nM control (Ctrl, white bars) or p21WAF1 siRNA (gray bars for 1 nM and black bars for 2 nM). Cells were seeded at 103 cells/well and after 4 days cells were quantified using an MTT assay as described in Materials and Methods. Data (mean±SD) are from one representative of two independent experiments, performed in triplicate. C) Growth of control cells (C2, {blacktriangleup}) or ER108 ({blacksquare}) after transfection with 2 nM p21WAF1 or control (Ctrl) siRNAs. Cells were quantified at different times of culture using an MTT assay. Values represent means ± SD of one experiment (representative of four independent experiments) performed in triplicate. D) P21WAF1 silencing favors cell proliferation. ER108 cells were transfected with control (Ctrl) or p21WAF1 siRNAs for 7 days and then prepared for flow cytometry as described in Materials and Methods. Data are mean ± SE of three independent experiments and presented as a percentage of cells in the S + G2/M phases. *P < 0.05, **P < 0.01 relative to respective controls.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
ER{alpha} cell growth inhibition is independent from hormone and DNA binding
Although ER{alpha} mediates mitogenic activity of estrogens in human breast cancer cells, its expression is associated with a favorable prognosis (11 12 13 14 15) and lack of tumorigenicity in ovariectomized nude mice (32) . To understand this discrepancy, we investigated whether ER{alpha} would exhibit a protective role against tumor formation in the absence of estrogens.

Our data demonstrate that the expression of unliganded ER{alpha} or deletion variants unable to bind estrogens or DNA (but containing the p21WAF1-binding domain) inhibits cancer cell growth in three-dimensional cultures on collagen and Matrigel matrices and also prevents tumor growth in nude mice. Several lines of evidence indicate that this growth inhibition is unlikely to occur due to nonspecific squelching of common transcription factors but rather to involve ER interactions with specific proteins such as p21WAF1. Indeed, ER{alpha} mutants unable to bind transcriptional coactivators through the E domain have an antiproliferative effect that is not observed with a mutant lacking the p21WAF1-binding domain. It is noteworthy that a high proportion of the ER{alpha} cellular pool in breast cancers exists in the unliganded form as measured by clinical ligand-binding assays that detect only the free receptor (33) . Thus, the ER{alpha} concentrations used in our study, which are equivalent to levels found in primary breast cancers, may have a physiological protective role. It is, therefore, conceivable that ER{alpha} may have a dual function, 1) the well-known mitogenic role involving the estrogen-activated receptor (10 , 34) and 2) the growth inhibitory function engaging the unliganded receptor as demonstrated in the present study.

In a woman’s life, there are two periods when estrogens are low (before menarche and after menopause), and unbound ER{alpha} concentrations become particularly important. The present data suggest that the unliganded ER{alpha} may contribute to maintaining estrogen target tissues in the latency phase with a low cellular renewal that precedes the proliferation phase induced by hormonal surge. There are epidemiological studies that support our findings about the antiproliferative effect of unliganded ER{alpha}. For instance, the "estrogens window hypothesis," which correlates the length of E2 exposure between menarche and first pregnancy (35) or between menarche and menopause (36) with an increased risk for breast cancer, could also be inversely interpreted as a protective effect of the length of estrogen-deprived periods. On the other hand, the high efficacy of the first-line therapy with aromatase inhibitors for breast cancers in postmenopausal women, which is normally attributed to estrogen withdrawal (37) , could also be involved the growth inhibitory function of the unliganded ER{alpha}. In fact, treatments with aromatase inhibitors have been shown to increase unliganded ER{alpha} levels (37) .

The highly conserved C region (mainly corresponding to exon 2) of the unliganded ER{alpha} appears to be important for its antiproliferative effects. It is possible, therefore, that some naturally occurring ER{alpha} variants generated via differential mRNA splicing may display a similar activity. In fact, ER{alpha} {Delta}3 (second zinc finger deleted) and ER{alpha} {Delta}5 (hormone-binding domain altered) have been detected in breast cancers (38) , and Erenburg et al. (39) have shown that ER{alpha} {Delta}3 has intrinsic antiproliferative activity. Further experiments are required to determine whether ER{alpha} variants or other receptors with high homology to ER{alpha}, such as ERβ or estrogen-related receptors, are growth inhibitors in their unliganded form.

Unliganded ER{alpha} interacts with p21WAF1 and increases p21WAF1 expression
To date, the role of unliganded ER{alpha} only in its interaction with transcription factors has been investigated. The observation that growth inhibition was associated with some ER{alpha} mutants, which cannot bind to estrogens or DNA, favored a mechanism involving protein-protein interactions rather than a transcriptional function of the receptor. First, we identified the first zinc finger of ER{alpha} as the potential interacting domain, and then we identified the interacting proteins using GST pull-down and double hybrid screening. The direct binding of p21WAF1 to ER{alpha} was demonstrated in the absence of hormone or in the presence of a pure antiestrogen. That ER{alpha} bound to a pure antiestrogen such as ICI182,780, behaving as an unliganded receptor, is not surprising as this type of ligand slightly modifies receptor conformation in contrast to partial agonist ligands. The observation that E2 prevents ER{alpha} and p21WAF1 interactions suggests that an estrogen-induced conformational change in the ER{alpha} structure may dissociate it from p21WAF1. Such a conformational change in the ER{alpha} structure has been described for estrogen action, which allows the binding of cofactors as well as the receptor dimerization necessary for the transcriptional activation of specific promoters (3) . In addition, the presence of unliganded ER{alpha} and p21WAF1 in a large 200 kDa complex containing both Cdk2 and cyclin E and their dissociation on estrogen exposure suggest that ER{alpha} might affect the catalytic activity of the Cdk2-cyclin E complex. Indeed, previous studies have shown that complexes between p21WAF1 and Cdks/cyclins can exist in both catalytically active and inactive forms and that their activity may depend on the presence of additional proteins (40 , 41) . The dissociation of ER{alpha} from p21WAF1 in the 200 kDa complex by E2 could provide an explanation for previous results showing that E2 rapidly dissociates p21WAF1 from the Cdk2-cyclin E complex by an unknown mechanism to activate the entry of cells into the cell cycle (31 , 42) . Our data are also consistent with previous findings demonstrating that p21WAF1 was the predominant inhibitor of Cdk2-cyclin E activity in cells treated with antiestrogens (31 , 43 , 44) . Our results also demonstrated that the expression levels of ER{alpha} and p21WAF1 are coupled because an increase in the ER{alpha} levels is associated with a significant increase in p21WAF1 levels in MDA-MB-231 cells and ER{alpha} silencing decreases p21WAF1 expression in MCF7 cells. The increase in the p21WAF1 expression appears 1) to depend on ER{alpha} and p21WAF1 interactions as ER11, a mutant defective in p21WAF1 binding, does not affect the levels of p21WAF1 and 2) does not require ER{alpha} hormone and DNA binding domains because ER15 and ER108 mutants positively influence the p21WAF1 expression level. However, the precise mechanism by which p21WAF1 expression is increased on its interaction with ER{alpha} requires further investigation.

Involvement of p21WAF1 in ER{alpha} antiproliferative activity
Our results implicate p21WAF1 in ER{alpha}-dependent growth inhibition as is evidenced by the fact that the antiproliferative activity and an increase in p21WAF1 levels correlate only when the expressed ER{alpha} mutants contain the p21WAF1 binding site. This notion is further supported by our finding that p21WAF1 silencing prevents ER{alpha}-induced growth inhibition. However, the involvement of other proteins that interact with ER{alpha} is not excluded.

Thus, our findings are in agreement with previous data indicating that ER{alpha} and p21WAF1 are central to the control of proliferation in human breast cancer cells. Deregulation of p21WAF1 expression is sufficient to prevent the growth of ER{alpha}-positive breast cancer cells (23) , and several lines of evidence indicate that p21WAF1 inactivation has a key role in the early phase of E2-induced mitogenesis. Indeed, in the presence of estrogens, activation of Cdk2-cyclin E complexes has been observed through lower targeting of newly synthesized p21WAF1 to Cdk2-cyclin E (25) and through p21WAF1 redistribution to both cyclin D1-Cdk4 (42) and cAMP response element-binding protein binding protein-ER{alpha} complexes (45) . Furthermore, in antiestrogen-pretreated cell lysates, an inhibitory activity toward Cdk2-cyclin E could mainly be attributed to p21WAF1 and is relieved by E2 treatment (43 , 44) . Finally, a positive association has been found in the expression of p21WAF1 and ER{alpha} in in situ ductal carcinomas (46) and other breast cancers (47 , 48) suggesting that they could participate in the control of proliferation.

In conclusion, these findings provide evidence of a new function for unliganded ER{alpha} as a negative regulator of cell growth. This effect involving interactions between ER{alpha} with p21WAF1 and an increase in p21WAF1 expression is likely to be important for the homeostasis of estrogen target tissues and improved understanding of the mechanisms responsible for the favorable prognosis of ER{alpha}-positive breast cancers.


   ACKNOWLEDGMENTS
 
We thank Sandrine Bonnet for animal care and Jean-Yves Cance for artwork. We thank P. Balaguer, D. Chalbos, P. Chambon, C. Jessus, and A. Valette for the generous gift of cell lines and plasmids. This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Association pour la Recherche sur le Cancer, the French Ministère de la Recherche et de l’Enseignement Supérieur (fellowship to N.P.), and the Ligue contre le cancer, Comité de l’Hérault (fellowship to M.M.).


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication July 5, 2007. Accepted for publication September 6, 2007.


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 RESULTS
 DISCUSSION
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