Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention Tumor Immunology: New Perspectives
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Cancer Research 67, 3955-3962, April 15, 2007. doi: 10.1158/0008-5472.CAN-06-3505
© 2007 American Association for Cancer Research
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Endocrinology

Estrogen Receptor ß2 Negatively Regulates the Transactivation of Estrogen Receptor {alpha} in Human Breast Cancer Cells

Chunyan Zhao1, Jason Matthews1,2, Michel Tujague1, Jinghong Wan3, Anders Ström1, Gudrun Toresson1, Eric W-F. Lam4, Guojun Cheng1, Jan-Åke Gustafsson1 and Karin Dahlman-Wright1

1 Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge, Sweden; 2 Department of Pharmacology, University of Toronto, Toronto, Canada; 3 Department of Proteomics, School of Biotechnology, AlbaNova University Center, KTH-Royal Institute of Technology, Stockholm, Sweden; and 4 Cancer Research UK Labs and Section of Cancer Cell Biology, Department of Oncology, Imperial College London, London, United Kingdom

Requests for reprints: Chunyan Zhao, Department of Biosciences and Nutrition, Novum, Karolinska Institutet, S-141 57 Huddinge, Sweden. Phone: 46-8-6089273; Fax: 46-8-7745538; E-mail: chunyan.zhao{at}cnt.ki.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogens, by binding to and activating two estrogen receptors (ER{alpha} and ERß), are critically involved in the development of the mammary gland and breast cancer. An isoform of ERß, ERß2 (also called ERßcx), with an altered COOH-terminal region, is coexpressed with ER{alpha} in many human breast cancers. In this study, we generated a stable cell line from MCF7 breast cancer cells expressing an inducible version of ERß2, along with endogenous ER{alpha}, and examined the effects of ERß2 on the ER{alpha} protein levels and function. We showed that ERß2 inhibited ER{alpha}-mediated transactivation via estrogen response element and activator protein-1 sites of reporter constructs as well as the endogenous genes pS2 and MMP-1. Chromatin immunoprecipitation assays revealed that ERß2 expression caused a significant reduction in the recruitment of ER{alpha} to both the pS2 and MMP-1 promoters. Furthermore, ERß2 expression induced proteasome-dependent degradation of ER{alpha}. The inhibitory effects of ERß2 on ER{alpha} activity were further confirmed in HEK293 cells that lack functional endogenous ERs. We also showed that ERß2 can interact with ER{alpha} both in vitro and in mammalian cells, which is compatible with a model where ERß2/ER{alpha} heterodimers are targeted to the proteasome. Finally, in human breast cancer samples, we observed that expression of ERß2 significantly correlated with ER{alpha}-negative phenotype. Our data suggest that ERß2 could influence ER{alpha}-mediated effects relevant for breast cancer development, including hormone responsiveness. [Cancer Res 2007;67(8):3955–62]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Estrogens bind to and activate two estrogen receptors (ER{alpha} and ERß) and exert their effects through a complex array of signaling pathways that mediate genomic and non-genomic events (1, 2). The ERs are members of the nuclear receptor superfamily of ligand-regulated transcription factors (3). ERs regulate gene expression through distinct DNA response elements. The classic mechanism of estrogen signaling is through an estrogen response element (ERE). The molecular details of this process are well characterized. ER dimerizes and interacts with EREs in target gene promoters, followed by recruitment of a variety of coregulators to alter chromatin structure and facilitate recruitment of the RNA polymerase II transcriptional machinery (2, 4). Estrogen signaling also occurs through alternative mechanisms where liganded ERs are tethered to DNA via association with other transcription factor complexes, including Fos/Jun (activator protein-1 [AP-1]– responsive elements; ref. 5) or SP-1 (GC-rich SP-1 motifs; ref. 6). The mechanistic details of activation through these pathways are less clear. In addition to these ligand-induced transcriptional activities of ER, ligand-independent pathways to activate ERs have been described. Growth factor signaling or stimulation of other signaling pathways leads to activation of kinases that can phosphorylate and thereby activate ERs or associated coregulators in the absence of ligand (7). Furthermore, estrogen may elicit effects through non-genomic mechanisms where estrogen binds to the ER localized outside of the cell nucleus, in turn activating signal transduction pathways in the cytoplasm (8).

The role of ERs in breast cancer has been intensely investigated. ERß is found in both ductal, lobular epithelial and stromal cells of the rodent mammary gland (9). ER{alpha}, on the other hand, is only found in the ductal and lobular epithelial cells but not in stroma (10). It is generally believed that breast tumors, at least initially, are dependent on the stimulatory effects of estrogens. However, many breast tumors eventually progress to an estrogen-independent growth phenotype. Tamoxifen and similar antiestrogens are currently the first-line therapy for treatment of hormone-dependent breast cancer (11). Various ER transcripts have been found in breast carcinomas (10), and data exist supporting protein expression for several of these isoforms (12). Normal and cancer tissues display a variety of profiles regarding ER{alpha}, ERß, and ER splice variants at both mRNA and protein levels (13, 14). This heterogeneity in ER isoform profiles could influence estrogen signaling relevant for breast cancer risk, hormone responsiveness, and survival.

An isoform of ERß, ERß2 (also called ERßcx), encodes a protein of 495-amino-acid residues, with a molecular weight of 55.5 kDa. It uses an alternative exon 8, which encodes for an additional 26 amino acids due to alternative splicing. ERß2 has undetectable affinity for E2 and cannot activate transcription of ERE-driven reporters. When ERß2 is cotransfected with ER{alpha}, it inhibits ligand-induced ER{alpha} transcriptional activity on an ERE reporter gene (15). This intriguing property suggests that ERß2 has an important function in neutralizing the effect of functional ER{alpha}. Expression of ERß2 could also explain tamoxifen resistance in some ER{alpha}-positive breast cancer patients. Indeed, one study reported that expression of ERß2 correlated with a poor response to antiestrogen (13). It has been suggested that expression of ERß2 could have a prognostic value in breast and prostate cancers (13, 16).

In this study, we established stable transfectants of ER{alpha}-positive MCF7 breast cancer cells with tetracycline-regulated ERß2 expression to investigate the influence of ERß2 on ER{alpha} signaling. Collectively, our results indicate that proteasome-dependent degradation of ER{alpha} induced by ERß2 in breast cancer cells may represent a possible molecular mechanism for the antagonistic effect of ERß2 on ER{alpha}-mediated functions. The inhibitory effects of ERß2 on ER{alpha} activity were further confirmed in HEK293 cells that lack functional endogenous ERs. Finally, we show that expression of ERß2 correlated with ER{alpha}-negative phenotype in human breast cancer samples.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Modified MCF-7 human breast cancer cells and HEK293 cells were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 5% FCS and 1% penicillin/streptomycin (Invitrogen) at 37°C in a humidified atmosphere of 5% CO2 in air. For experiments to evaluate the effects of 4,4',4''-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol (PPT), kindly provided by KaroBio AB (Stockholm, Sweden), DMEM without phenol red, and FCS treated with dextran-coated charcoal (DCC-FCS) were used.

Generation of stable MCF7 tet-off ERß2 and HEK293 tet-on ERß2 clones. MCF-7 cells stably transfected with tetracycline-regulated ERß2 expression plasmid were generated in two steps. The cells were first transfected with pTet-tTAK (Life Technologies, Gaithersburg, MD) modified to support puromycin resistance using Lipofectin according to the manufacturer's instructions (Life Technologies). Selection was done with 0.5 µg/mL puromycin (Sigma, St. Louis, MO) in the presence of 1 µg/mL tetracycline (Sigma). A clone showing high levels of induction upon tetracycline withdrawal and low basal activity was selected using the pUHC13-3 control plasmid (Life Technologies). ERß2 cDNA was fused to the flag tag and cloned into pBI-EGFP (Clontech, Palo Alto, CA). This construct was transfected into the highly inducible clone and isolated in step one, together with a neomycin resistance plasmid, and selection was done with 1,000 µg/mL G418 (Calbiochem, La Jolla, CA). For generating stable HEK293 tet-on ERß2 clones, the pBI-EGFP-ERß2 plasmid was transfected into HEK293 tet-on cells, which were obtained from BD Biosciences Clontech (Palo Alto, CA).

Transient transfection and luciferase assays. Transient transfection was done essentially as described previously (17). Briefly, cells were seeded in six-well plates and grown in phenol red–free DMEM supplemented with 5% DCC-FCS for 24 h before transfection. The cells were cotransfected with the reporter plasmid (ERE-TK-Luc or coll517-Luc containing 517 bp of the human collagenase gene promoter including a single AP-1 binding site) and/or ER{alpha} expression plasmid and pRL-TK control plasmid, which contains a Renilla luciferase gene, for normalizing transfection efficiency. Cells were transfected using LipofectAMINE 2000 (Invitrogen/Life Technologies, Carlsbad, CA). After 5 h of transfection, tetracycline was removed, or doxycycline was added 12 h before initiation of treatment with PPT to induce ERß2 expression. Transfected cells were then treated with 10 nmol/L PPT or vehicle for 24 h before harvest and luciferase assay (Biothema, Dalarö, Sweden).

RNA isolation and real-time PCR. Cells were grown for 48 h in phenol red–free DMEM supplemented with 5% DCC-FCS serum. To express ERß2, tetracycline was removed, or doxycycline was added 12 h before addition of 10 nmol/L PPT or vehicle. Real-time PCR was done as described previously (18). Taqman Universal Master Mix (PE Applied Biosystems, Foster City, CA) was used for amplifying MMP-1 gene; for pS2, QPCR Master Mix for Cybergreen (Medprobe, Minneapolis, MN) was used. The PCR primer pairs are as follows: pS2 mRNA, were 5'-CATCGACGTCCCTCCAGAAGAG-3' and 5'-CTCTGGGACTAATCACCGTGCTG-3'; MMP-1 mRNA, 5'-TTGAAGCTGCTTACGAATTTGC-3' and 5'-GTCCCTGAACAGCCCAGTACTT-3'. The probe sequence for MMP-1 was 5'-CAGAGATGAAGTCCGGTTTTTCAAAGGGAA-3'. All target gene transcripts were normalized to the ß-glucuronidase mRNA (PE Applied Biosystems) content and to the time 0 sample. For measurement of expression levels of ERß2 in breast tumor samples, real-time PCR was done using primers specific for ERß2 as described previously (19).

Chromatin immunoprecipitation. MCF7 tet-off ERß2 cells were seeded in 150-mm dishes and grown for 48 h in phenol red–free DMEM supplemented with 5% DCC-FCS serum. For expression of ERß2, tetracycline was removed 12 h before initiation of treatment with ligands. Cells were then treated with 10 nmol/L PPT for the indicated times. Soluble, sonicated chromatin was prepared as previously described (20). Chromatin fractions were immunoprecipitated with 0.5 to 1 µg of the indicated antibodies, and the immune complexes were recovered using protein A/G-Sepharose (50% slurry; Pharmacia, Piscataway, NJ) and processed as described (20). The antibodies used were as follows: ER{alpha}, H-184 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse antihuman IgG (Santa Cruz Biotechnology), and anti-FLAG (M5; Sigma). The immunoprecipitated DNA was amplified by real-time PCR using Platinum SYBR green quantitative PCR supermix uracil DNA glycosylase (Invitrogen). The primer pairs used are as follows: pS2 promoter, 5'-CCGGCCATCTCTCACTATGAA-3' and 5'-CCTCCCGCCAGGGTAAATAC-3'; MMP-1 promoter, 5'-TTGCAACACCAAGTGATTCCA-3' and 5'-CCCAGCCTCTTGCTACTCCA-3'.

Western blotting. Cells were seeded in 100-mm dishes and grown for 48 h in phenol red–free DMEM supplemented with 5% DCC-FCS serum. For expression of ERß2, tetracycline was removed, or doxycycline was added 12 h before initiation of treatment with ligands. Cells were then treated with 10 nmol/L PPT or vehicle for the indicated times, and nuclear extracts were prepared as described in ref. (21). To examine the effect of proteasome inhibition, we pretreated the cells for 2 h with 10 µmol/L MG132 (Sigma) before the removal of tetracycline or addition of doxycycline. After 12 h, cells were harvested, and nuclear extracts were prepared. ER{alpha} was detected using H-184 rabbit polyclonal antibody (Santa Cruz Biotechnology) at 1:10,000 dilution and ECL anti-rabbit IgG, horseradish peroxidase–linked (Amersham Biosciences, Arlington Heights, IL) at 1:100,000 dilution (20). The actin antibody (Sigma) was used at a 1:50,000 dilution. The Image J software (Research Services Branch, National Institute of Mental Health, Bethesda, MD) was used for densitometry of the autoradiographs.

Glutathione S-transferase pull-down assay. Glutathione S-transferase (GST) fusions of ER{alpha}-(309–595) and His-tagged ERß2 LBD (R254 to Q495) were generated by cloning the appropriate DNA sequences into the pGEX2-TK vector (Amersham Pharmacia Biotech) and the pET15b vector (Novagen, Madison, WI), respectively. GST and GST-ER{alpha} proteins were purified on glutathione-Sepharose beads (Sigma) according to standard methods and incubated with partially purified His-tagged ERß2 LBD, prepared as described previously (22), in pull-down buffer [50 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, 1 mmol/L MgCl2, 10% glycerol, and 0.5% NP40] and 1.5% serum bovine albumin. Incubation and rotation were carried out for 2 h at 4°C. After extensive washing with pull-down buffer, the bound proteins were analyzed by SDS-PAGE followed by Western blotting using mouse monoclonal anti-His antibody (Clontech).

Coimmunoprecipitation. Total cell extracts from MCF7 tet-off ERß2 cells were prepared by direct lysis of cells with buffer containing 20 mmol/L HEPES (pH 7.5), 180 mmol/L NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 10% glycerol, 0.5 mmol/L DTT, and 1 mmol/L phenylmethylsulfonyl fluoride. Protein concentrations were measured using Bio-Rad Protein Assay reagent. Cell lysates were incubated with anti-FLAG (M5) antibody at 4°C with rotation for 2 h. Thereafter, prewashed protein G-agarose beads (Amersham Biosciences) were added, and the incubation continued for another 2 h at 4°C followed by four washes with lysis buffer. Subsequently, the immune complex was boiled in electrophoresis sample buffer and analyzed on SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane and visualized using anti-FLAG M5 monoclonal antibody and ER{alpha}, H-184 antibody, respectively.

Human breast tumor samples. Primary breast tumor tissues from 40 patients with invasive ductal carcinoma and undergoing breast cancer surgery were provided by the Charing Cross Hospital, London. All of the samples were frozen in liquid nitrogen immediately after resection and stored at –80°C until use. The studies were approved by the ethical committee of the Karolinska Institute.

Immunohistochemistry. Expression of ER{alpha} and ERß2 in breast tumor samples was measured by immunohistochemistry as previously described (13, 23). The primary antibodies used were ER{alpha} (1D5, 1:30) from DAKO (High Wycombe, United Kingdom) and a specific ERß2 antibody (1:400) produced by us (13). For negative controls, the primary antibody was replaced with PBS alone or with primary antibody after absorption with the corresponding antigen. Sections were incubated in biotinylated goat anti-mouse immunoglobulin (1:200 dilution; Vector Laboratories, Inc., Burlingame, CA) for 2 h at room temperature followed by incubation in avidin-biotin-horseradish peroxidase (Vector Laboratories) for 1 h.

Statistics. Student's t test, Mann-Whitney U test, or Fisher's exact probability test was used to determine significance of differences between groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of ERß2 represses PPT-stimulated ERE and AP-1 activity. To create a system for studying the function of ERß2, we generated stable transfectants of MCF7 breast cancer cells with a tetracycline-regulated vector for expression of ERß2. Native estrogen-responsive MCF7 cells express predominantly ER{alpha} and only very low levels of endogenous ERß2. A Western blot of ERß2 protein with a Flag tag in response to tetracycline withdrawal is shown in Fig. 1A . No detectable Flag-ERß2 protein was expressed in the presence of tetracycline, whereas high levels of Flag-ERß2 protein were observed when the cells were cultured in the absence of tetracycline.


Figure 1
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  		Figure 1. ERß2 expression inhibits ER{alpha}-mediated transactivation. A, MCF7 tet-off FLAG-ERß2 human breast carcinoma cells were cultured in the presence of tetracycline (+ tet) or absence of tetracycline (– tet) for 12 h. ERß2 was expressed only in the absence of tetracycline as measured by anti-flag-tag Western blot. B, transfection of MCF7 tet-off FLAG-ERß2 cells with an ERE-luciferase reporter or a collagenase luciferase reporter shows that the induction of ERß2 by tetracycline withdrawal (+ERß2, black columns) reduces both basal and PPT-induced ERE and AP-1 activity. Columns, mean; bars, SD. Representative results of three independent experiments. *, P < 0.05, significant difference between samples grown in the presence of tetracycline (–ERß2, white columns) or absence of tetracycline (+ERß2, black columns).

 
To investigate the effects of ERß2 on ER{alpha}-mediated transactivation through ERE- and AP-1–response elements, we transfected the MCF7 tet-off FLAG-ERß2 cells with an ERE- or an AP-1-luciferase reporter construct. Cells were grown in the presence or absence of tetracycline, and reporter gene activity after treatment with vehicle or PPT, an agonist selective for ER{alpha}, was determined. The data shown in Fig. 1B support previous studies (15) and show that ERß2 reduced basal as well as PPT-induced ERE activity. In addition, our results show that ERß2 inhibited both the basal and PPT-induced AP-1 activity.

Expression of ERß2 reduces mRNA levels of the endogenous estrogen-regulated genes and inhibits recruitment of ER{alpha} to estrogen-responsive promoters. MCF7 tet-off FLAG-ERß2 cells were cultured in the presence or absence of tetracycline and treated with PPT to determine whether ERß2 inhibits the endogenous expression of estrogen-responsive genes regulated by ER{alpha}. Determination of endogenous expression levels for the ERE-controlled gene pS2 and the AP-1–dependent gene MMP-1 was done by quantitative real-time PCR analysis. As shown in Fig. 2A , PPT stimulates pS2 and MMP-1 mRNA expression after 6 and 12 h of treatment, respectively. Expression of ERß2 suppressed PPT induction of pS2 and MMP-1. These data extend our findings that ERß2 antagonizes ER{alpha}-mediated transactivation from reporter genes to endogenous genes.


Figure 2
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  		Figure 2. Expression of ERß2 inhibits PPT induction of pS2 and MMP-1 mRNA levels and recruitment of ER{alpha} to their promoters. A, real-time PCR results of the effect of ERß2 on expression levels of pS2 and MMP-1. MCF7 tet-off FLAG-ERß2 human breast carcinoma cells were cultured in the presence of tetracycline (–ERß2, white columns) or in the absence of tetracycline (+ERß2, black columns) and treated with 10 nmol/L PPT for the indicated amount of time. Total RNA was isolated, reverse transcribed, and amplified with primers recognizing the mRNA form of pS2 and MMP-1. B, chromatin immunoprecipitation analysis of recruitment of ER{alpha} and ERß2 to the pS2 and MMP-1 promoters. MCF7 tet-off FLAG-ERß2 human breast carcinoma cells were cultured in the presence or absence of tetracycline and treated with 10 nmol/L PPT for 0, 1, and 2 h. Chromatin immunoprecipitation assays were done as described in Materials and Methods with antibodies recognizing ER{alpha} and FLAG (for FLAG-ERß2) in parallel with control IgG. Quantitative real-time PCR of 1 µL of purified immunoprecipitated DNA was done using SYBR Green and primer pairs that amplify the pS2 and MMP-1 promoter regions. Relative promoter enrichment compared with IgG at time 0 in the presence of tetracycline. Columns, mean; bars, SD. Representative results of three independent experiments. *, P < 0.05, significant difference between time-matched samples grown in the presence or absence of tetracycline.

 
The effect of ERß2 expression on the binding of ER{alpha} to the pS2 and MMP-1 promoters was examined by chromatin immunoprecipitation. Cells were treated with PPT for 0, 1, and 2 h, after which chromatin was cross-linked, and protein-DNA complexes were immunoprecipitated with antibodies recognizing normal rabbit IgG, ER{alpha}, or FLAG ERß2. Figure 2B shows that ER{alpha} was recruited to the pS2 and MMP-1 promoter regions. PPT induced significant recruitment of ER{alpha} to the pS2 promoter after 1 and 2 h of treatment, whereas significant recruitment of ER{alpha} to the MMP-1 promoter was observed after 2 h of treatment. In agreement with previous reports, ER{alpha} bound to the pS2 promoter and, to a lesser extent, to the MMP-1 promoter in the absence of ligand (24). Ligand-independent binding of ERß2 to either promoter region was not observed under our assay conditions. The PPT-dependent recruitment of ERß2 to the pS2 and MMP1 promoters was observed in the absence of tetracycline and not in its presence (Fig. 2B). The expression of ERß2 significantly reduced the recruitment of ER{alpha} to both the pS2 and MMP-1 (apparent after 2 h of treatment) promoters, suggesting a plausible mechanism for the ERß2 antagonism of ER{alpha} activity.

ERß2 down-regulates ER{alpha} protein via the proteasome degradation pathway. The effects of ERß2 expression on ER{alpha} protein levels were investigated in MCF7 tet-off FLAG-ERß2 cells grown in the presence or absence of tetracycline. Cells were treated with vehicle or PPT for 0, 2, and 6 h. As shown in Fig. 3A , ERß2 expression caused a decrease in immunoreactive ER{alpha} protein in cells treated with vehicle and PPT, whereas PPT treatment slightly increased ER{alpha} protein levels. These changes in ER{alpha} protein levels were not associated with changes in ER{alpha} mRNA levels (data not shown), suggesting that ERß2 expression may affect ER{alpha} protein stability.


Figure 3
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  		Figure 3. Western blot analysis of the influence of ERß2 on ER{alpha} protein levels. A, MCF7 tet-off FLAG-ERß2 human breast carcinoma cells were cultured in the presence of tetracycline (–ERß2) or in the absence of tetracycline (+ERß2) and treated with either vehicle or PPT for 0, 2, and 6 h. Western blots were done as described in Materials and Methods. B, MCF7 tet-off FLAG-ERß2 human breast carcinoma cells were cultured in the presence of tetracycline or in the absence of tetracycline and treated with either vehicle or proteasome inhibitor (10 µmol/L MG132). Equal amounts of protein extract were resolved by SDS-PAGE and transferred to Hybond-P nylon membrane, and the membranes were probed for ER{alpha}. Data from one experiment that was repeated three independent times.

 
Regulated proteolysis by the proteasome accounts for turnover of most short- and long-lived proteins, including at least some nuclear receptors. To examine if ERß2 down-regulates ER{alpha} through a proteasome-dependent pathway, we tested whether the proteasome inhibitor MG132 (25) would block ERß2-induced down-regulation of ER{alpha}. MCF7 tet-off FLAG-ERß2 cells grown in the presence or absence of tetracycline were pretreated with vehicle or MG132. Nuclear fractions were prepared and analyzed by Western blotting. As expected, expression of ERß2 in cells not treated with proteasome inhibitor reduced the level of ER{alpha} protein compared with cells that did not express ERß2 (Fig. 3B, lane 1 versus 2). Importantly, MG132 blocked the ERß2-induced down-regulation of the ER{alpha} protein levels (lane 3 versus 4). These results suggest that ERß2-induced down-regulation of ER{alpha} protein levels proceeds through the proteasome.

Inhibitory effects of ERß2 on ER{alpha} activity are not restricted to MCF7 cells. The inhibitory effects of ERß2 on ER{alpha} activity were also examined in HEK293 cells that lack functional endogenous ERs (26, 27). Cell clones stably expressing ERß2 were established in HEK293 tet-on cells. The induction of ERß2 protein by doxycycline treatment was verified by Western blotting (Fig. 4A ). To investigate the effects of ERß2 expression on ER{alpha}-mediated transactivation and ER{alpha} protein levels, cells were cotransfected with an expression plasmid for ER{alpha}, an ERE-luciferase reporter construct, and a pRL-TK control plasmid for monitoring the transfection efficiency. Confirming the results seen with the MCF7 cells, ERß2 inhibited both the basal and PPT-induced ERE activity in HEK293 cells (Fig. 4B). ERß2 also suppressed PPT induction of the endogenous pS2 mRNA (data not shown). Furthermore, the level of ER{alpha} protein was reduced when ERß2 was expressed (Fig. 4C, lane 1 versus 2); MG132 blocked the ERß2-induced down-regulation of the ER{alpha} protein levels (lane 3 versus 4). Thus, our results confirmed that ERß2 induced proteasome-mediated degradation of ER{alpha} in HEK293 cells.


Figure 4
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  		Figure 4. Inhibitory effects of ERß2 on ER{alpha} activity in HEK293 cells. A, HEK293 tet-on FLAG-ERß2 cells were cultured in the absence of doxycycline (– Dox) or presence of doxycycline (+ Dox) for 12 h. ERß2 was expressed only in the presence of doxycycline as measured by anti-flag-tag Western blot. B, transfection of HEK293 tet-on FLAG-ERß2 cells with an expression plasmid for ER{alpha} and an ERE-luciferase reporter shows that induction of ERß2 by addition of doxycycline (+ERß2, black columns) reduces both basal and PPT-induced ERE activity. Columns, mean; bars, SD. Representative results of three independent experiments. *, P < 0.05, significant difference between samples grown in the absence of doxycycline (–ERß2, white columns) or presence of doxycycline (+ERß2, black columns). C, HEK293 tet-on FLAG-ERß2 cells were transiently transfected with an expression plasmid for ER{alpha}. The cells were then treated with either vehicle or proteasome inhibitor (10 µmol/L MG132) both in the absence of doxycycline or in the presence of doxycycline. Equal amounts of protein extract were resolved by SDS-PAGE and transferred to Hybond-P nylon membrane, and the membranes were probed for ER{alpha}. Data from one experiment that was repeated three independent times.

 
ERß2 interacts with ER{alpha} in vitro and in mammalian cells. To test whether ERß2 interacts with ER{alpha}, we did GST pull-down assays using His-ERß2 LBD and GST-ER{alpha} LBD fusion proteins. Western blot analysis showed that ERß2 specifically associated with GST-ER{alpha}, but not with GST alone (Fig. 5A ), indicating a direct interaction between both ER subtypes.


Figure 5
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  		Figure 5. ERß2 interacts with ER{alpha} in vitro and in mammalian cells. A, equal amounts of GST-ER{alpha} or GST alone were prepared and incubated with purified His-tagged ERß2 LBD. ER{alpha}-associated proteins were analyzed by Western blotting using a His antibody. Aliquots of purified His-tagged ERß2 LBD protein (3% of input) were also loaded directly onto the gels and analyzed by Western blotting (Input, lane 3). B, MCF7 tet-off FLAG-ERß2 human breast carcinoma cells grown in the presence or absence of tetracycline were lysed. Cell lysates were immunoprecipitated with the M5 monoclonal antibody to FLAG. Immunocomplexes were separated by SDS-PAGE, and proteins were transferred to polyvinylidene difluoride membranes. Blots were probed with an antibody to FLAG or ER{alpha}. In parallel, endogenous ER{alpha} was detected in nonprecipitated lysates (0.5% of the volume used in the immunoprecipitation assays) from the cells in both presence and absence of tetracycline (top). *, position of the heavy-chain IgG cross-reacting with the secondary antibody. Data from one experiment that was repeated three times.

 
To show interactions between ERß2 and ER{alpha} in mammalian cells, we did coimmunoprecipitation assays using the ER{alpha}-expressing MCF7 cell line containing an inducible tet-off FLAG-ERß2 (Fig. 5B). Cell lysates were immunoprecipitated with a mouse monoclonal antibody to the FLAG-tag and probed with a rabbit antibody to ER{alpha}. The anti-FLAG antibody did not precipitate proteins from lysates of cells grown in the presence of tetracycline, but it did precipitate FLAG-ERß2 protein from lysates of cells grown in the absence of tetracycline. Endogenous ER{alpha} was found in FLAG immunoprecipitates from the lysates of cells grown in the absence of tetracycline, but not in the presence of tetracycline. Endogenous ER{alpha} was detected in nonprecipitated lysates from the cells both in the presence or absence of tetracycline. Incubation of cell lysates with beads alone or control IgG failed to immunoprecipitate either FLAG-reactive or ER{alpha} proteins (data not shown). These results indicate that ERß2 associated with endogenous ER{alpha} in mammalian cells.

ER{alpha}-positive breast tumors express lower levels of ERß2. To determine whether our findings that ERß2 expression down-regulates ER{alpha} protein described above for the MCF7 human breast cancer cell line can be extended to breast cancer patient samples, we analyzed samples obtained from breast cancer surgery. A total of 37 individual human invasive ductal carcinoma samples were immunohistochemically analyzed for ER{alpha} protein expression (Fig. 6A and B ). Of these, 18 samples were classified as ER{alpha} positive according to standard criteria (>10% of total cells were positive); the remaining 19 samples were considered as ER{alpha} negative. We then examined expression of ERß2 in these breast tumor samples by a quantitative real-time PCR assay. The level of ERß2 mRNA expression was found to be significantly lower in the ER{alpha}-positive group (48.4 ± 29.4) than in the ER{alpha}-negative group (116.2 ± 67.4; Mann-Whitney U test, P < 0.001). Next, 30 of these tumor samples were further stained with an ERß2 antibody (Fig. 6C and D). Of these, 18 samples were evaluated as positive for ERß2 using this assay, with 60% of cells positive as cutoff value (13).


Figure 6
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  		Figure 6. Representative immunohistochemical staining of ER{alpha} and ERß2 in human breast cancer samples. In a sample in which ER{alpha} staining is positive (A), there is negative nuclear staining with the ERß2 antibody (C). In a sample in which ER{alpha} staining is negative (B), there is positive nuclear staining with the ERß2 antibody (D). The cutoff of 10% and 60% staining was used to define sections positive for ER{alpha} and ERß2, respectively.

 
In the ERß2 positive tumors, 14 of 18 were evaluated as ER{alpha} negative, whereas 10 of 12 were ER{alpha} positive in the ERß2-negative group. Thus, consistent with the results above, ERß2 protein staining was associated with absence of ER{alpha} protein staining (P < 0.05, Fisher's exact probability test).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using a stable cell line derived from MCF7 breast cancer cells expressing ERß2 in an inducible fashion, we show that ERß2 interacts with ER{alpha} both in vitro and in mammalian cells, and that ERß2 induces proteasome-dependent degradation of ER{alpha}. We propose that the ERß2-induced proteasome-dependent degradation of ER{alpha} is caused by the formation of ERß2/ER{alpha} heterodimers. We suggest that ERß2-mediated degradation of ER{alpha} is at least one mechanism whereby expression of ERß2 inhibits recruitment of ER{alpha} to the estrogen-responsive promoters, leading to suppression of ER{alpha}-regulated genes. We thus present a molecular mechanism by which ERß2 could antagonize ER{alpha} activity in breast cancer cells.

Although the possible mechanisms have remained unclear, a few studies have shown that ERß2 inhibits the ER{alpha}-mediated transactivation through the classic ERE-pathway in reporter systems (15, 28). Our work confirms these observations (Fig. 1B). We extend these studies and show that ERß2 inhibits the expression of the endogenous pS2 gene that contains an ERE site within its promoter (29). Furthermore, we studied AP-1 sites. The MMP-1 gene is one of several hormone-responsive genes in breast cancer cells regulated by ER{alpha}/AP-1 (30), and this gene was used as a model to investigate the effect of ERß2 on ER{alpha} transactivation through nonclassic AP-1–mediated pathway. Our results show that ERß2 expression inhibits PPT-induced MMP-1 mRNA and reporter gene activity in cells transfected with AP-1-luciferase reporter constructs. The mechanism of the inhibitory effect of ERß2 on ER{alpha} activity was further investigated by chromatin immunoprecipitation assay. Treatment with PPT induced a dramatic increase in the occupancy of the pS2 and MMP-1 gene promoters by ER{alpha}. However, the recruitment of ERß2 to either promoter was much weaker than of ER{alpha} even when ERß2 was overexpressed. This is presumably due to the much lower DNA binding ability of ERß2 than ER{alpha} (28). We observed that the expression of ERß2 significantly reduced the recruitment of ER{alpha} to both the pS2 and MMP-1 promoters. This suggests that ERß2-mediated reduction of ER{alpha}-mediated transcriptional activity is related to the reduced recruitment of ER{alpha} to the estrogen-responsive regions of these promoters. Consistent with this, our laboratory has previously shown that wild-type ERß modulates ER{alpha} activity by altering the recruitment of ER{alpha}, c-Fos, and c-Jun to estrogen-responsive promoters (20).

The ubiquitin proteasomal degradation multicomplex accounts for turnover of most short-lived proteins, including nuclear receptors (31, 32). Previous studies have shown that estradiol-mediated ER{alpha} degradation occurs through the 26 S proteasome pathway (33, 34). Our results show that ERß2 decreases ER{alpha} protein levels in MCF7 cells, and that an inhibitor of proteasomal degradation (MG132) blocks ERß2-induced down-regulation of ER{alpha}. This is consistent with the ERß2-inducing proteasomal degradation of ER{alpha}. In our study, treatment up to 6 h with the ER{alpha} agonist PPT did not cause a decrease in ER{alpha} protein levels. The discrepancy between our findings and a report showing down-regulation of ER{alpha} following a 24-h treatment with estradiol (25) could be explained by differences in the duration of ligand treatment. The ERß2 and ER{alpha} are coimmunoprecipitated in MCF7 cells (Fig. 5), suggesting a possible mechanism of ERß2-induced proteasomal degradation of ER{alpha} that involves initial interaction of ERß2 with ER{alpha}. Indeed, a mechanism in which protein-protein interactions activate the ubiquitin-proteasome pathway for degradation of one or both interacting proteins has been suggested by Wormke et al. (35).

We have shown a direct protein-protein interaction between ERß2 and ER{alpha} in vitro and in mammalian cells. Similarly, previous studies showed that transient coexpression of wild-type ERß and ER{alpha} leads to formation of heterodimers, binding to a synthetic ERE in vitro (36, 37). The major dimer interface between ER{alpha} and ERß has been mapped to a conserved region of the hormone binding domain corresponding to helix 10. Indeed, the amino acid sequence of helix 10 is also conserved between wild-type ERß and ERß2. The last 61 amino acids of ERß, which are replaced by a unique 26-amino-acid sequence in ERß2, encode part of helix 11 and helix 12, but leaving helix 10 unchanged. It is therefore not surprising that ERß2 forms heterodimers with ER{alpha}. A recent study indicated that coexpression of ERß and ER{alpha} can uniquely regulate gene expression (38). During the process of breast cancer progression, ER{alpha} and ERß2 coexist, and the ratio of ER{alpha} to ERß2 changes (39), suggesting that ERß2 may be of biological importance during breast cancer development.

The molecular mechanisms behind the inhibitory effect of ERß2 on ER{alpha}-mediated transactivation may involve a number of different pathways. For example, after the heterodimerization between ERß2 and ER{alpha}, the ERß2/ER{alpha} complex may dissociate from the estrogen-responsive promoters, resulting in repression of ER{alpha} target gene expression. In addition, heterodimerization may hinder the recruitment of coactivators to the receptors (e.g., due to steric hindrance or because heterodimerization induces receptor conformations that are nonpermissive for transactivation). However, it is unlikely that ERß2 and ER{alpha} act as a heterodimer on an ER{alpha}-responsive promoter because our findings show that ERß2 was much less efficiently recruited to such promoters. Furthermore, it has previously been reported that heterodimerization between NRs sometimes inhibits receptor activity. For example, heterodimerization between ERR{gamma} and ERR{alpha} was found to inhibit the transcriptional activities of both receptors (40). Results of the present study suggest another possible mechanism where ERß2 induces proteasome-dependent degradation of ER{alpha}, resulting in limiting levels of this protein, thus leading to suppression of ER{alpha} transcriptional activity. This model is supported by other studies investigating nuclear receptor crosstalk with other signaling systems (35, 41). For instance, decreased ER{alpha} levels may contribute to the decreased expression of some E2-responsive genes in breast cancer cells cotreated with E2 plus TCDD.

Although the majority of human breast cancers express ERß2, and the level of expression of ERß2 often exceeds that of wild-type ERß in these cancers (42, 43), the clinical significance of ERß2 still remains to be defined. Clinical studies indicate that expression of ERß2 in breast cancer correlates with a poor response to antiestrogen (13). In this regard, it is of interest that ERß2 reduces ER{alpha} protein levels because the presence of ER{alpha} and progesterone receptors (PR) is predictive for response to endocrine therapy and improved disease-free survival (44). Approximately 50% to 60% of women with ER{alpha}-positive breast cancer benefit from endocrine therapy. In contrast, only a small minority of ER{alpha}/PR–negative patients respond to endocrine therapy (45). In this study, we show that high levels of ERß2 were expressed in ER{alpha}-negative breast tumors, implying that the presence of ERß2 in breast cancer might lead to tamoxifen resistance. Our findings are concordant with the observations that a decrease of ERß2 is associated with the development of ER{alpha}-expressing breast cancer (46).

In summary, we have shown that ERß2 binds directly to ER{alpha} and regulates ER{alpha} protein levels and transcriptional activity in a negative manner. Further studies are required to understand the distinct role of ERß2 in estrogen-dependent cell proliferation and development of hormone-dependent tumors.


   Acknowledgments
 
Grant support: Swedish Cancer Fund, KaroBio AB, and Susan Komen Foundation (G. Cheng).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Potential conflict of interest: J-Å. Gustafsson is cofounder, shareholder, deputy board member, and consultant of KaroBio AB.

Received 9/21/06. Revised 1/14/07. Accepted 1/31/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hall JM, Couse JF, Korach KS. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 2001;276:36869–72.[Free Full Text]
  2. Nilsson S, Gustafsson JA. Estrogen receptor action. Crit Rev Eukaryot Gene Expr 2002;12:237–57.[CrossRef][Medline]
  3. Mangelsdorf DJ, Thummel C, Beato M, et al. The nuclear receptor superfamily: the second decade. Cell 1995;83:835–9.[CrossRef][Medline]
  4. Klinge CM. Estrogen receptor interaction with estrogen response elements. Nucleic Acids Res 2001;29:2905–19.[Abstract/Free Full Text]
  5. Kushner PJ, Agard DA, Greene GL, et al. Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol 2000;74:311–7.[CrossRef][Medline]
  6. Saville B, Wormke M, Wang F, et al. Ligand-, cell-, and estrogen receptor subtype (alpha/beta)-dependent activation at GC-rich (Sp1) promoter elements. J Biol Chem 2000;275:5379–87.[Abstract/Free Full Text]
  7. Kato S, Endoh H, Masuhiro Y, et al. Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 1995;270:1491–4.[Abstract/Free Full Text]
  8. Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin WW, Liao JK. Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 2000;407:538–41.[CrossRef][Medline]
  9. Palmieri C, Cheng GJ, Saji S, et al. Estrogen receptor beta in breast cancer. Endocr Relat Cancer 2002;9:1–13.[Abstract]
  10. Gotteland M, Desauty G, Delarue JC, Liu L, May E. Human estrogen receptor messenger RNA variants in both normal and tumor breast tissues. Mol Cell Endocrinol 1995;112:1–13.[CrossRef][Medline]
  11. Jensen EV, Jordan VC. The estrogen receptor: a model for molecular medicine. Clin Cancer Res 2003;9:1980–9.[Abstract/Free Full Text]
  12. Poola I, Speirs V. Expression of alternatively spliced estrogen receptor alpha mRNAs is increased in breast cancer tissues. J Steroid Biochem Mol Biol 2001;78:459–69.[CrossRef][Medline]
  13. Saji S, Omoto Y, Shimizu C, et al. Expression of estrogen receptor (ER) (beta)cx protein in ER(alpha)-positive breast cancer: specific correlation with progesterone receptor. Cancer Res 2002;62:4849–53.[Abstract/Free Full Text]
  14. Fuqua SA, Schiff R, Parra I, et al. Expression of wild-type estrogen receptor beta and variant isoforms in human breast cancer. Cancer Res 1999;59:5425–8.[Abstract/Free Full Text]
  15. Ogawa S, Inoue S, Watanabe T, et al. Molecular cloning and characterization of human estrogen receptor betacx: a potential inhibitor ofestrogen action in human. Nucleic Acids Res 1998;26:3505–12.[Abstract/Free Full Text]
  16. Fujimura T, Takahashi S, Urano T, et al. Differential expression of estrogen receptor beta (ERbeta) and its C-terminal truncated splice variant ERbetacx as prognostic predictors in human prostatic cancer. Biochem Biophys Res Commun 2001;289:692–9.[CrossRef][Medline]
  17. Zhao C, Xu L, Otsuki M, et al. Identification of a functional variant of estrogen receptor beta in an African population. Carcinogenesis 2004;25:2067–73.[Abstract/Free Full Text]
  18. Kietz S, Thomsen JS, Matthews J, Pettersson K, Strom A, Gustafsson JA. The Ah receptor inhibits estrogen-induced estrogen receptor beta in breast cancer cells. Biochem Biophys Res Commun 2004;320:76–82.[CrossRef][Medline]
  19. Zhao C, Lam EW, Sunters A, et al. Expression of estrogen receptor beta isoforms in normal breast epithelial cells and breast cancer: regulation by methylation. Oncogene 2003;22:7600–6.[CrossRef][Medline]
  20. Matthews J, Wihlen B, Tujague M, Wan J, Strom A, Gustafsson JA. Estrogen receptor (ER) beta modulates ERalpha-mediated transcriptional activation by altering the recruitment of c-Fos and c-Jun to estrogen-responsive promoters. Mol Endocrinol 2006;20:534–43.[Abstract/Free Full Text]
  21. Kinyamu HK, Archer TK. Estrogen receptor-dependent proteasomal degradation of the glucocorticoid receptor is coupled to an increase in mdm2 protein expression. Mol Cell Biol 2003;23:5867–81.[Abstract/Free Full Text]
  22. Zhao C, Toresson G, Xu L, Koehler KF, Gustafsson JA, Dahlman-Wright K. Mouse estrogen receptor beta isoforms exhibit differences in ligand selectivity and coactivator recruitment. Biochemistry 2005;44:7936–44.[CrossRef][Medline]
  23. Cheng G, Li Y, Omoto Y, et al. Differential regulation of estrogen receptor (ER)alpha and ERbeta in primate mammary gland. J Clin Endocrinol Metab 2005;90:435–44.[Abstract/Free Full Text]
  24. Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M. Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 2000;103:843–52.[CrossRef][Medline]
  25. Nawaz Z, Lonard DM, Dennis AP, Smith CL, O'Malley BW. Proteasome-dependent degradation of the human estrogen receptor. Proc Natl Acad Sci U S A 1999;96:1858–62.[Abstract/Free Full Text]
  26. Kahlert S, Nuedling S, van Eickels M, Vetter H, Meyer R, Grohe C. Estrogen receptor alpha rapidly activates the IGF-1 receptor pathway. J Biol Chem 2000;275:18447–53.[Abstract/Free Full Text]
  27. Duong BN, Elliott S, Frigo DE, et al. AKT regulation of estrogen receptor {beta} transcriptional activity in breast cancer. Cancer Res 2006;66:8373–81.[Abstract/Free Full Text]
  28. Peng B, Lu B, Leygue E, Murphy LC. Putative functional characteristics of human estrogen receptor-beta isoforms. J Mol Endocrinol 2003;30:13–29.[Abstract]
  29. Barkhem T, Haldosen LA, Gustafsson JA, Nilsson S. Transcriptional synergism on the pS2 gene promoter between a p160 coactivator and estrogen receptor-alpha depends on the coactivator subtype, the type of estrogen response element, and the promoter context. Mol Endocrinol 2002;16:2571–81.[Abstract/Free Full Text]
  30. DeNardo DG, Kim HT, Hilsenbeck S, Cuba V, Tsimelzon A, Brown PH. Global gene expression analysis of estrogen receptor transcription factor cross talk in breast cancer: identification of estrogen-induced/activator protein-1-dependent genes. Mol Endocrinol 2005;19:362–78.[Abstract/Free Full Text]
  31. Molinari E, Gilman M, Natesan S. Proteasome-mediated degradation of transcriptional activators correlates with activation domain potency in vivo. EMBO J 1999;18:6439–47.[CrossRef][Medline]
  32. Wijayaratne AL, McDonnell DP. The human estrogen receptor-alpha is a ubiquitinated protein whose stability is affected differentially by agonists, antagonists, and selective estrogen receptor modulators. J Biol Chem 2001;276:35684–92.[Abstract/Free Full Text]
  33. El Khissiin A, Leclercq G. Implication of proteasome in estrogen receptor degradation. FEBS Lett 1999;448:160–6.[CrossRef][Medline]
  34. Alarid ET, Bakopoulos N, Solodin N. Proteasome-mediated proteolysis of estrogen receptor: a novel component in autologous down-regulation. Mol Endocrinol 1999;13:1522–34.[Abstract/Free Full Text]
  35. Wormke M, Stoner M, Saville B, et al. The aryl hydrocarbon receptor mediates degradation of estrogen receptor alpha through activation of proteasomes. Mol Cell Biol 2003;23:1843–55.[Abstract/Free Full Text]
  36. Pace P, Taylor J, Suntharalingam S, Coombes RC, Ali S. Human estrogen receptor beta binds DNA in a manner similar to and dimerizes with estrogen receptor alpha. J Biol Chem 1997;272:25832–8.[Abstract/Free Full Text]
  37. Cowley SM, Hoare S, Mosselman S, Parker MG. Estrogen receptors alpha and beta form heterodimers on DNA. J Biol Chem 1997;272:19858–62.[Abstract/Free Full Text]
  38. Monroe DG, Secreto FJ, Subramaniam M, Getz BJ, Khosla S, Spelsberg TC. Estrogen receptor alpha and beta heterodimers exert unique effects on estrogen- and tamoxifen-dependent gene expression in human U2OS osteosarcoma cells. Mol Endocrinol 2005;19:1555–68.[Abstract/Free Full Text]
  39. Speirs V, Carder PJ, Lane S, Dodwell D, Lansdown MR, Hanby AM. Oestrogen receptor beta: what it means for patients with breast cancer. Lancet Oncol 2004;5:174–81.[CrossRef][Medline]
  40. Huppunen J, Aarnisalo P. Dimerization modulates the activity of the orphan nuclear receptor ERRgamma. Biochem Biophys Res Commun 2004;314:964–70.[CrossRef][Medline]
  41. Hoivik D, Willett K, Wilson C, Safe S. Estrogen does not inhibit 2,3,7, 8-tetrachlorodibenzo-p-dioxin-mediated effects in MCF-7 and Hepa 1c1c7 cells. J Biol Chem 1997;272:30270–4.[Abstract/Free Full Text]
  42. Iwao K, Miyoshi Y, Egawa C, Ikeda N, Noguchi S. Quantitative analysis of estrogen receptor-beta mRNA and its variants in human breast cancers. Int J Cancer 2000;88:733–6.[CrossRef][Medline]
  43. Leygue E, Dotzlaw H, Watson PH, Murphy LC. Altered estrogen receptor alpha and beta messenger RNA expression during human breast tumorigenesis. Cancer Res 1998;58:3197–201.[Abstract/Free Full Text]
  44. Katzenellenbogen BS, Frasor J. Therapeutic targeting in the estrogen receptor hormonal pathway. Semin Oncol 2004;31:28–38.[Medline]
  45. Robertson JF, Cannon PM, Nicholson RI, Blamey RW. Oestrogen and progesterone receptors as prognostic variables in hormonally treated breast cancer. Int J Biol Markers 1996;11:29–35.[Medline]
  46. Park BW, Kim KS, Heo MK, et al. The changes of estrogen receptor-beta variants expression in breast carcinogenesis: decrease of estrogen receptor-beta2 expression is the key event in breast cancer development. J Surg Oncol 2006;93:504–10.[CrossRef][Medline]



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