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Originally published In Press as doi:10.1074/jbc.M510809200 on February 3, 2006

J. Biol. Chem., Vol. 281, Issue 14, 9607-9615, April 7, 2006
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Fulvestrant (ICI 182,780)-dependent Interacting Proteins Mediate Immobilization and Degradation of Estrogen Receptor-{alpha}*

Xinghua Long{ddagger} and Kenneth P. Nephew{ddagger}§1

From the {ddagger}Medical Sciences, Indiana University School of Medicine, Bloomington, Indiana 47405 and §Department of Cellular and Integrative Physiology, Indiana University School of Medicine and Indiana University Cancer Center, Indianapolis, Indiana 46202

Received for publication, October 4, 2005 , and in revised form, February 2, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The antiestrogen fulvestrant (ICI 182,780) causes immobilization of estrogen receptor-{alpha} (ER{alpha}) in the nuclear matrix accompanied by rapid degradation by the ubiquitin-proteasome pathway. In this study we tested the hypothesis that fulvestrant induces specific nuclear matrix protein-ER{alpha} interactions that mediate receptor immobilization and turnover. A glutathione S-transferase (GST)-ER{alpha}-activating function-2 (AF2) fusion protein was used to isolate and purify receptor-interacting proteins in cell lysates prepared from human MCF-7 breast cancer cells. After SDS-PAGE and gel excision, mass spectrometry was used to identify two major ER{alpha}-interacting proteins, cytokeratins 8 and 18 (CK8·CK18). We determined, using ER{alpha}-activating function-2 mutants, that helix 12 (H12) of ER{alpha}, but not its F domain, is essential for fulvestrant-induced ER{alpha}-CK8 and CK18 interactions. To investigate the in vivo role of H12 in fulvestrant-induced ER{alpha} immobilization/degradation, transient transfection assays were performed using wild type ER{alpha},ER{alpha} with a mutated H12, and ER{alpha} with a deleted F domain. Of those, only the ER{alpha} H12 mutant was resistant to fulvestrant-induced immobilization to the nuclear matrix and protein degradation. Fulvestrant treatment caused ER{alpha} degradation in CK8·CK18-positive human breast cancer cells, and CK8 and CK18 depletion by small interference RNAs partially blocked fulvestrant-induced receptor degradation. Furthermore, fulvestrant-induced ER{alpha} degradation was not observed in CK8 or CK18-negative cancer cells, suggesting that these two intermediate filament proteins are necessary for fulvestrant-induced receptor turnover. Using an ER{alpha}-green fluorescent protein construct in fluorescence microscopy revealed that fulvestrant-induced cytoplasmic localization of newly synthesized receptor is mediated by its interaction with CK8 and CK18. In summary, this study provides the first direct evidence linking ER{alpha} immobilization and degradation to the nuclear matrix. We suggest that fulvestrant induces ER{alpha} to interact with CK8 and CK18, drawing the receptor into close proximity to nuclear matrix-associated proteasomes that facilitate ER{alpha} turnover.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogen receptor-{alpha} (ER{alpha}),2 a member of the nuclear receptor family, is a ligand-dependent transcription factor that mediates physiological responses to its cognate ligand, 17beta-estradiol (E2), in estrogen target tissues such as the breast, uterus, and bone (1). Because ER{alpha} is a short-lived protein (half-life of 4-5 h), its cellular levels are strictly regulated (2). Although ER{alpha} turnover is a continuous process (2), dynamic fluctuations in receptor levels, mediated primarily by the ubiquitin-proteasome pathway (3-6), occur in response to changing cellular conditions (7-9). In addition, differing ligands have been demonstrated to exert differential effects on steady-state levels of ER{alpha} (10, 11). For example, E2 and the "pure" ER{alpha} antagonists (i.e. ICI 164,384, ICI 182,780, RU 58,668, and ZK-703) (12, 13) induce receptor turnover, whereas the "partial" agonist/antagonist 4-hydroxytamoxifen (4-OHT) stabilizes ER{alpha} (14, 15). E2-mediated ER{alpha} degradation is dependent on transcription, coactivator recruitment, and new protein synthesis, whereas ICI-induced degradation of ER{alpha} is independent of these processes (16-18). Thus, although both E2 and pure antiestrogens induce ER{alpha} degradation, their mechanisms of action differ markedly.

In addition to altering ER{alpha} stability and turnover, different ligands have been shown to have profoundly distinct effects on receptor mobility and cellular localization. For example, ER{alpha} was found localized exclusively in the nucleus after E2 and 4-OHT treatment, whereas ICI caused both nuclear and cytoplasmic receptor localization (13, 19). Stenoien et al. (20), using fluorescence recovery after photobleaching, demonstrated that E2, 4-OHT, and ICI treatment resulted in reduced nuclear mobility of ER{alpha} tagged with cyan fluorescent protein (20). In that study complete fluorescence recovery was not observed after ICI treatment due to immobilization of ER{alpha} to the nuclear matrix (20). Additional studies have further shown a rapid immobilization of the ER{alpha}-ICI complex within the nuclear matrix, with sequestration in a salt-insoluble, nuclear compartment (21, 22), although the precise nature of the receptor-nuclear matrix interaction remains unknown.

Fulvestrant (faslodex, ICI 182,780) belongs to a new class of antihormonal therapy for advanced breast cancer called selective estrogen receptor down-regulators (SERDs) (23, 24). SERDs act as potent antagonists by inducing rapid receptor turnover and display no agonist activity in estrogen target tissues. SERDs differ markedly from the class of molecules called selective estrogen receptor modulators (SERMs), such as 4-OHT, that function as either agonists or antagonists, depending upon the target tissue (24). The pure antagonistic property of fulvestrant is due to a steroidal structure containing a long bulky side chain (25), which induces a distinct conformational change in the ligand binding domain of ER{alpha} (26), specifically in the position of helix 12 (H12), to prevent receptor dimerization and binding to DNA (27). Because specific mutations in H12 can reverse the pure antiestrogenic properties of fulvestrant (28, 29), H12 may contribute to fulvestrant-induced ER{alpha} degradation.

In this study the mechanism of fulvestrant-induced ER{alpha} degradation by the ubiquitin-proteasome pathway was investigated. We show that this SERD induces specific ER{alpha} cytokeratins CK8·CK18 interactions, the major intermediate filament proteins found in the nuclear matrix and cytoplasm of ER{alpha}-positive breast cancer cell lines (30). We further demonstrate that H12 is essential for these cytokeratin interactions and, subsequently, receptor immobilization within the nuclear matrix. Furthermore, we show that fulvestrant-mediated receptor degradation and cytoplasmic localization correlate directly with CK8 and CK18 levels in breast cancer cells. Because proteasomes have been shown to be associated primarily with intermediate filaments (31-33), we suggest that fulvestrant induces specific receptor-cytokeratin interactions in the nuclear matrix, bringing ER{alpha} into close proximity to proteasomes for subsequent degradation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The following antibodies and reagents were used in this study: anti-ER{alpha} (HC20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or monoclonal anti-human ER{alpha} (Chemicon International, Inc., Temecula, CA); monoclonal anti-human cytokeratin 8 (RCK102; BD Biosciences) and monoclonal anti-human cytokeratin 18 (RCK106; BD Biosciences); monoclonal anti-cytokeratin peptide 8 (Sigma); mouse anti-glyceraldehyde phosphate dehydrogenase (GAPDH) (Chemicon International); glutathione-Sepharose 4 Fast Flow beads (Amersham Biosciences); SuperSignal West Pico chemiluminescent substrate (Pierce); protease inhibitor mixture set III (Calbiochem-Novabiochem); Lipofectamine Plus reagent, Geneticin, and cell culture reagents (Invitrogen); FuGENE (Roche Applied Science); 4-OHT and MG132 (Sigma); ICI 182,780 (Tocris Cookson Ltd., Ellisville, MO); RNase-free DNase I and BL21 (DE3)pLysS competent cells (Promega, Madison, WI).

Plasmid Construction—Wild type ER{alpha} pSG5-ER{alpha}(HEGO) was kindly provided by Dr. Pierre Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France) and GFP-ER{alpha} (26) by Dr. Michael Mancini (Baylor College of Medicine, Houston, TX). The ER{alpha} helix 12 mutant pRST-7-hER3X (D538N/E542Q/D545N) was kindly provided by Donald McDonnell (Duke University, Durham, NC). pGEX-6P-1-AF2, pGEX-6P-1-AF2{Delta}F, pGEX-6P-1-AF2{Delta}F{Delta}H12, and pGEX-6P-1-ER{alpha}3X-AF2 were constructed by inserting the PCR DNA fragment of interest into pGEX-6P-1 (BamHI and XhoI site). pcDNA3-ER{alpha}{Delta}F, pcDNA3-ER{alpha}3X{Delta}F, and pcDNA3-ER{alpha}{Delta}F{Delta}H12 were generated by inserting the specific PCR DNA fragment into pcDNA3MycHisA (BamHI and XhoI site). pcDNA3-CK8 was generated by inserting the CK8 PCR DNA fragment into pcDNA3MycHisA (BamHI and XhoI site). pcDNA3-CK18 was generated by inserting CK18 PCR DNA fragment into pcDNA3MycHisA (EcoRI and XhoI site). Cloning results were confirmed by subjecting all constructs to DNA sequencing.

Cell Lines—The human cervical carcinoma HeLa cell line and the breast cancer cell lines MCF-7 and its daughter, C4-12 (ER{alpha}-negative, CK8- and 18-positive (34)), are routinely maintained in our laboratory, as described previously (9, 35). MDA-MB-231 and T47D breast cancer cells were purchased from ATCC (Manassas, VA). MDA-MB-231 cells were maintained in Dulbecco's modified Eagle's medium with 50 units/ml penicillin, 50 µg/ml streptomycin, 10 mM Hepes, 6 ng/ml insulin, and 10% fetal bovine serum. T47D cells were maintained in RPMI 1640 medium 2 mM L-glutamine, 1.0 mM sodium pyruvate, 50 units/ml penicillin, 50 µg/ml streptomycin, 10 mM Hepes, 0.2 units/ml insulin, and 10% fetal bovine serum. Before experiments involving transient transfection and hormone treatment, cells were cultured in hormone-free medium (phenol red-free minimum Eagle's medium (MEM) with 5% charcoal-stripped fetal bovine serum) for 3 days.

Stable Transfection of ER{alpha}—C4-12 or HeLa cells were transfected with pcDNA-ER{alpha} (C4-12/ER{alpha} and HeLa/ER{alpha}, respectively) using Lipofectamine Plus Reagent and exposed to antibiotic (G418; 0.5 mg/ml) for 3 weeks. Expression of ER{alpha} in G418-resistant colonies was verified by immunoblotting with anti-ER{alpha}.

Transient Transfection Assay—T47D and HeLa cells were cultured in hormone-free medium for 3 days and transfected with equal amounts of total plasmid DNA (adjusted by the corresponding empty vectors) using Lipofectamine Plus reagent or FuGENE according to the manufacturer's guidelines. Five hours later, the DNA/Lipofectamine mixture was removed, and cells were cultured in hormone-free medium. Unless stated otherwise, 24 h after transfection, cells were treated with the specified drug.

RNA Interference (siRNA)—siRNA transfection reagent, control siRNA, CK8 siRNA, and CK18 siRNA were purchased from Santa Cruz Biotechnology. The CK8 and CK18 siRNAs (singly or both) were transfected into MCF-7 cells according to the manufacturer's protocol; 72 h after transfections, cells were treated with 100 nM ICI 182,780. Whole cell lysates were prepared in 1x SDS sample buffer. Protein levels were examined by Western blotting using specific antibodies.

Preparation of Whole Cell Extracts—Whole cell extracts were prepared by suspending cells in SDS lysis buffer (62 mM Tris, pH 6.8, 2% SDS, 10% glycerol, and protease inhibitor mixture III). After 15 min of incubation on ice, extracts were sonicated, insoluble materials were removed by centrifugation (15 min at 12,000 x g), and supernatant protein concentrations were determined using a Bio-Rad protein assay kit.

Preparation of Nuclear Extracts and Nuclear Matrix—Nuclear extract was prepared using a nuclear extraction kit (Active Motif, Carlsbad, CA), according to the manufacturer's protocol. Nuclear matrix was prepared following the procedure described by Coutts et al. (30). Briefly, cell nuclei were extracted with nuclear matrix buffer (100 mM NaCl, 300 mM sucrose, 10 mM Tris-HCl, pH 7.4, 2 mM MgCl2, 1% (v/v) thiodiglycol) containing 1 mM phenylmethylsulfonyl fluoride and 0.5% (v/v) Triton X-100. Nuclei were resuspended in digestion buffer (50 mM NaCl, 300 mM sucrose, 10 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 1% (v/v) thiodiglycol, 0.5% (v/v) Triton X-100), digested with DNase I (168 units/ml) for 20 min at room temperature, and then sequentially extracted using 0.25 M ammonium sulfate and 2 M NaCl. Nuclear matrix was resuspended in 1x SDS sample buffer and sonicated.

Western Blot and Quantitation—Whole cell lysates were prepared in 1x SDS sample buffer by sonication, and total protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. ER{alpha} levels were determined by Western blot using a LI-COR (Lincoln, NE) imaging system. The membrane was incubated with primary antibody followed by incubation with infrared dye IR800-labeled goat anti-mouse IgG or IR700-labeled goat anti-rabbit IgG (LI-COR) secondary antibodies and quantitated with LI-COR Odyssey software. For immunoblotting by enhanced chemiluminescence (ECL), primary antibody was detected by horseradish peroxidase-conjugated second antibody and visualized using an enhanced SuperSignal West Pico chemiluminescent substrate.

GST Pull-down Assay—GST pull-down assays were performed as we have described previously (35, 36). To fuse ER{alpha}-AF2 with GST, an ER{alpha}AF2 PCR fragment (amino acids 297-595) was cloned into the BamHI and XhoI sites of the plasmid pGEX-6P-1 and subjected to DNA sequencing to confirm the correct reading frame. The GST-tagged AF2 was then expressed in BL21 cells and purified as described (36, 37). Briefly, overnight cultures of BL21 cells containing the plasmid pGEX-6P-1-GST-ER{alpha}-AF2 were diluted (1:20), cultured in fresh medium for 2 h, and treated with 0.1 mM isopropyl beta-D-thiogalactoside for 3 h. Induced bacteria were then collected by centrifugation and lysed in NETN buffer containing 0.5% Nonidet P-40, 1 mM EDTA, 20 mM Tris, pH 8.0, 100 mM NaCl, and protease inhibitors. GST-ER{alpha}-AF2 was purified on glutathione-Sepharose 4 Fast Flow beads (Amersham Biosciences). MCF-7 cell lysates were prepared by sonicating cells in cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, pH 7.5). Whole cell lysates were then incubated with the glutathione-bound GST-ER{alpha}-AF2 in binding buffer (60 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 7.5, 0.05% Nonidet P-40, 1 mM dithiothreitol, 6 mM MgCl2, and 8% glycerol) in the absence or presence of corresponding ligands or vehicle for 3 h at 4 °C. After washing with binding buffer, ER{alpha}-AF2-bound proteins were eluted, separated by 10% SDS-polyacrylamide, and visualized by Coomassie Blue. Specific proteins were cut from the gel, eluted, and analyzed by MALDI and liquid chromatography mass spectrometry by the Indiana University Protein Analysis Research Center (Indianapolis, IN).


Figure 1
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  		FIGURE 1.
A-C, purification and identification ICI-dependent ER{alpha}-interacting proteins using the GST pull-down assay. Cell lysates were prepared from MCF-7 cells and incubated with immobilized GST-ER{alpha}-AF2 in the presence or absence of ligand (1 µM E2, 4-OHT, and ICI; NH, no hormone). After SDS-PAGE and gel excision, proteins were identified by mass spectrometry. Results were confirmed by Western blotting using specific antibodies. A, Coomassie Blue-stained SDS-polyacrylamide gel of proteins associated with GST-ER{alpha}-AF2. B and C, Western blot confirmation of mass spectrometry using antibodies for CK8 (B) or CK18 (C). *, sample was also washed with high salt buffer (1 M NaCl). D, co-immunoprecipitation of the ER{alpha}-CK8·CK18 interaction in vivo. ER{alpha} was precipitated from MCF-7 cell lysates using an anti-ER{alpha} antibody. The presence of CK8·CK18 in the pull-down complex was examined by immunoblotting using antibodies for CK8 or CK18. To assess the amount of precipitated ER{alpha} in the complex, the same membrane was then re-probed with ER{alpha} antibody. Normal rabbit IgG was used on the negative control. Representative results of two independent experiments, each performed in duplicate, are shown.

 
Co-immunoprecipitation—MCF-7 cell whole cell lysates were prepared in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% Triton X-100, 1 mM Na3VO4, protease inhibitor). Whole cell extract was incubated with protein G-agarose for 30 min at 4 °C. After centrifugation at 12,000 x g for 15 s, the precleared supernatants were incubated with 5 µl of anti-ER{alpha} antibody or IgG at 4 °C for 3 h followed by incubation with 30 µl of protein G-agarose beads for 30 min. The beads were then pelleted by brief centrifugation, washed 3 times with Tris-buffered saline (TBS) and once with TBS containing 0.4 M NaCl, and resuspended in 30 µl of SDS-PAGE loading buffer for SDS-PAGE and Western blotting.

Live Cell Microscopy and Drug Treatment—Live fluorescence microscopy was performed by growing cells on 6-well plates and transfection with GFP-ER{alpha} using Lipofectamine or FuGENE and maintained in minimum Eagle's medium with 5% dextran-coated charcoal-stripped fetal bovine serum at 37 °C. Cells were treated with E2 (10 nM), ICI (100 nM), 4-OHT (100 nM), or ICI and cycloheximide (25 µg/ml). Images were taken using a Zeiss Axiovert 40 Inverted Microscope and Axio-Vision software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Fulvestrant Induces ER{alpha}-Intermediate Filament Protein Interactions—Previously it was shown that treatment of breast cancer cells with the pure antagonist ICI resulted in ER{alpha} immobilization and resistance to biochemical extraction within the nuclear matrix (21). For this study we hypothesized that fulvestrant-dependent ER{alpha}-interacting proteins in the nuclear matrix were responsible for this phenomena. To identify putative fulvestrant-dependent ER{alpha} interacting proteins, cell lysates from human breast cancer MCF-7 cells were incubated with immobilized GST-ER{alpha}-activating function-2 (AF2) in the presence of ICI. Interacting proteins were eluted from the beads, separated by SDS-PAGE, and stained with Coomassie Blue. Fulvestrant-specific interacting protein bands (Fig. 1A) were excised from the gel and subjected to mass spectrometry (MALDI and liquid chromatography mass spectrometry) analysis, resulting in two of the proteins being identified as cytokeratins 8 and 18 (CK8 and CK18). To validate those findings, Western blot analysis using CK8- or CK18-specific antibodies, was performed to permit conclusive identification of these putative ER{alpha} binding partners (Fig. 1, B and C). No interaction between ER{alpha} and CK8 or CK18 was observed in the presence of either E2 or 4-OHT (Fig. 1). These ER{alpha}-CK8·CK18 associations were also stable in the presence of high salt (Fig. 1, last lane), consistent with other reports that ER{alpha} is insoluble after immobilization by ICI or RU 58668 (21, 38). To further demonstrate an ER{alpha}-CK8·CK18 interaction in vivo, co-immunoprecipitation was performed using MCF-7 whole cell lysates and an ER{alpha}-specific antibody in the absence or presence of fulvestrant. As shown in Fig. 1D, CK8 and CK18 were seen in the ER{alpha} complex only in the presence of ICI, suggesting that fulvestrant induces an endogenous interaction between ER{alpha} and CK8·CK18.

Expression of CK8·CK18 in ER{alpha}-positive and -negative Cancer Cell Lines—It has been previously shown that both CK8 and CK18 are nuclear matrix-intermediate filament proteins present in ER{alpha}-positive cells (30). To investigate whether a correlation exists between expression of ER{alpha} and/or CK8·CK18, whole cell lysates were prepared from human breast (MCF-7, T47D, MDA-MB-231) and cervical cancer (HeLa) cell lines. Levels of CK8·CK18 and ER{alpha} were determined by Western blot analysis. Differential CK expression was observed between the ER{alpha}-positive and -negative cell lines (Fig. 2A). Furthermore, CK8 and CK18 protein levels were markedly higher in MCF-7 and T47D (ER{alpha}-positive) cells as compared with the ER{alpha}-negative MDA-MB-231 and HeLa cells.

Effect of Fulvestrant on the Association of ER{alpha} with the Nuclear Matrix and Receptor Degradation—Distinct ligands can specifically affect ER{alpha} extractability from the nucleus of breast cancer cells (38). To further characterize the association between ER{alpha} and the nuclear matrix in the presence of antiestrogens, MCF-7 and T47D cells (ER{alpha}-, CK8-, and CK18-positive) were treated with ICI or 4-OHT followed by isolation of nuclear matrix fractions. Nuclear matrix prepared from MDA-MB-231 (ER{alpha}-negative; CK8- and CK18-positive, Fig. 2A) was used as a control. In the nuclear matrix of ER{alpha}-positive cells, CK8 and CK18 were highly abundant (Fig. 2C, upper panel, Coomassie Blue; middle panel, Western blot). In the presence of ICI, the majority of ER{alpha} protein was unextractable and remained tightly associated with the nuclear matrix (Fig. 2C); in contrast, in the presence of 4-OHT, ER{alpha} was loosely associated with the nuclear matrix, readily extractable, and thus, more abundant in the nuclear extract (Fig. 2C, bottom panel). These observations are consistent with the result that fulvestrant induces a salt-resistant ER{alpha}-CK8 and -CK18 interaction (Fig. 1) and that ER{alpha} extractability varies in the presence of different ligands (38).


Figure 2
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  		FIGURE 2.
A, expression of ER{alpha}, CK8, and CK18 in cancer cell lines. Whole cell lysates were prepared in 1x SDS sample buffer from the indicated cancer cell lines, subjected to SDS-PAGE electrophoresis, and transferred to membranes. Western blot analysis was performed using specific antibodies for ER{alpha}, CK8, and CK18. B, fulvestrant induces ER{alpha} immobilization to the nuclear matrix and receptor degradation. MCF7 cells were treated with fulvestrant (ICI 182,780; 10 nM) for the indicated times, and nuclear extract (NE) or nuclear matrix (NM) was prepared as described under "Experimental Procedures." Proteins were separated by SDS-PAGE and analyzed by Western blotting using ER{alpha}-, CK8-, and CK18-specific antibodies. C, association of ER{alpha} with the nuclear matrix in the presence of antiestrogen. Cells were treated with ICI 182,780 or 4-OHT (10 nM for 30 min). Nuclear extract and nuclear matrix was prepared from MCF-7, T47D, and MDA-MB-231 cells, as described under "Experimental Procedures." Proteins were separated by SDS-PAGE and visualized either by Coomassie Blue staining or Western blot analysis using specific antibodies. Upper panel, nuclear matrix proteins stained with Coomassie Blue. Middle panel, CK8·CK18, ER{alpha} levels in NM. Bottom panel, ER{alpha} levels in nuclear extract. Representative results of two independent experiments, each performed in duplicate, are shown.

 
To monitor ER{alpha} immobilization and degradation, nuclear extract and nuclear matrix were prepared from MCF-7 cells treated with fulvestrant for 0-4 h. As shown in Fig. 2B, rapid (<30 min) immobilization of ER{alpha} from the nuclear extract to the nuclear matrix was observed followed by receptor degradation 1 h after the onset of ICI treatment. In addition, CK8 and CK18 were both localized in the insoluble nuclear matrix (Fig. 2B). Taken together, these observations demonstrate that after treatment with fulvestrant, ER{alpha} is rapidly sequestered in a salt-insoluble nuclear compartment before being degraded.

Helix 12 Is Required for Fulvestrant-dependent Interaction of ER{alpha} with CK8 and CK18 and Antiestrogen-induced Immobilization of ER{alpha} to the Nuclear Matrix and Receptor Degradation—Previous studies have suggested a role of two domains of ER{alpha} in ICI-induced receptor immobilization and degradation; that is, H12 and the F domain. Furthermore, Katzenellenbogen and coworkers (29) showed that mutations in H12 conferred resistance to ICI-induced degradation. Furthermore, to examine whether these two domains are required for fulvestrant-dependent interactions with CKs, several ER{alpha} AF2 mutant GST fusion proteins were constructed; AF2{Delta}F, with the F domain of AF2 deleted, AF2{Delta}F{Delta}H12, completely lacking both F domain and helix 12, AF2-3X, with 3 mutated amino acids in H12 (D538N/E542Q/D545N), AF23X{Delta}F, containing H12 mutations and lacking the F domain (Fig. 3A). In the presence of fulvestrant, the F domain deletion constructs remained capable of interacting with both CK8 and CK18, demonstrating that the F domain is not required for the ER{alpha}-CK interaction (Fig. 3B). However, removal of H12 or point mutations introduced into this region completely abolished fulvestrant-induced receptor-CK8·CK18 interactions (Fig. 3B). Interestingly, no interaction between ERbeta and either CK8 or CK18 was observed after ICI treatment (Fig. 3B, last lane, ERbetaAF2). In MCF-7 cells (39, 40) and rat efferent ductules (40), ERbeta appears to be resistant to fulvestrant-induced degradation, and our results further indicate that the lack of CK interactions may play a role in the inability of fulvestrant to degrade this ER isoform.

Having demonstrated that H12 is required for fulvestrant-induced interaction of ER{alpha} with CK8 and CK18, it was of interest to test whether H12 and the F domain are required for ER{alpha} immobilization. Plasmids containing wild type ER{alpha} (wtER{alpha}), ER{alpha}{Delta}F, or ER{alpha}3X were transfected into the MDA-MB-231 breast cancer cell line (ER{alpha}-negative; CK8- and CK18-positive, Fig. 2A). Transfected MDA-MB-231 cells were treated with ICI or E2 for 30 min (this short treatment duration causes ER{alpha} immobilization but not degradation). Whole cell lysates and nuclear extracts were prepared, and ER{alpha} protein levels were determined by Western blot analysis. After E2 treatment, both wtER{alpha} and ER{alpha}{Delta}F were extractable by nuclear extraction buffer (Fig. 3C); however, after treatment with ICI, neither construct was extractable (Fig. 3C). No effect of E2 or ICI on the extractability of the mutant ER{alpha}3X was observed (Fig. 3C). Taken together, these results indicate that H12 is essential for fulvestrant-induced immobilization of ER{alpha} to the nuclear matrix.

It was recently demonstrated that mutations in H12 could influence tamoxifen-mediated ER{alpha} stability (41). To examine whether H12 contributes to fulvestrant-mediated receptor degradation, T47D breast cancer cells (CK8- and CK18-positive, Fig. 2A) were transiently transfected with full-length ER{alpha}3X (point-mutated helix 12) or wtER{alpha}. Receptor levels were assessed by Western blot analysis after treatment with ICI for 1 h. As shown in Fig. 3D, degradation of wtER{alpha}, but not ER{alpha}3X, was observed after ICI treatment, suggesting that an intact H12 is required for fulvestrant-induced ER{alpha} degradation.

Because the F domain of ER{alpha} contains a PEST sequence (residues 555-567), a proposed signal for rapid intracellular breakdown of proteins (42), it was of interest to investigate whether this domain may be involved in fulvestrant-induced ER{alpha} degradation. T47D cells were transected with plasmids expressing ER{alpha}{Delta}F, ER{alpha}3X{Delta}F, or ER{alpha}{Delta}F{Delta}H12 and treated with ICI for 1 h. The relative stability of each mutant ER{alpha} was then assessed using Western blot analysis using a monoclonal antibody against the N-terminal region of ER{alpha}, which recognizes receptors with C-terminal deletions. As shown in Fig. 4, a decrease in the level of ER{alpha}{Delta}F protein was observed after ICI treatment; in contrast, both ER{alpha}3X{Delta}F and ER{alpha}{Delta}F{Delta}H12 were resistant to fulvestrant-induced degradation. Moreover, ER{alpha}3X{Delta}F levels actually increased after treatment with the antiestrogen, likely due to blockage of basal turnover of the mutant receptor (Fig. 4). In support of this possibility, treatment with the proteasome inhibitor MG132, an inhibitor of basal ER{alpha} protein turnover (43) increased levels of ER{alpha} protein (Fig. 4, A and B). Collectively, these results indicate that the F domain is not required for fulvestrant-induced ER{alpha} degradation, in contrast to H12. Our observations also support those of Pakdel et al. (43), who reported that the F domain is dispensable for E2-induced degradation of ER{alpha} (43).


Figure 3
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  		FIGURE 3.
A and B, helix 12 of ER{alpha} is essential for receptor interactions with CK8 and CK18. A, schematic diagram of ER{alpha} and the AF2 constructs used for GST pull-down. B, analysis of ER{alpha}-AF2 mutants and their interactions with CK8 or CK18. Cell extracts were prepared from MCF-7 cells and incubated with immobilized ER{alpha}-AF2 GST fusion proteins in the presence or absence of 1 µM ligand (E2, 4-OHT, and ICI; NH, no hormone). Interacting proteins CK8 and CK18 were detected by Western blotting using CK8- or CK18-specific antibodies. Bottom panel, Coomassie Blue-stained SDS-polyacrylamide gels of GST fusion proteins. DBD, DNA binding domain LBD, ligand binding domain. C, helix 12, but not the F domain, is required for fulvestrant-induced ER{alpha} immobilization. MDA-MB-231 cells were transiently transfected with wtER{alpha},ER{alpha}{Delta}F, or ER{alpha}-3X and treated with Me2SO, 100 nM E2, or fulvestrant (ICI) for 30 min. Whole cell lysates and nuclear extracts (NE) were prepared as described under "Experimental Procedures." Upper panel,ER{alpha} levels were measured and analyzed using Western blotting and a LI-COR imaging system, as described under "Experimental Procedures." Lower panel, quantitative analysis of ER{alpha} protein level in NE, normalized by loading control using LICOR Odyssey software. DMSO, dimethyl sulfoxide. D, helix 12 is required for fulvestrant-induced ER{alpha} degradation. T47D cells were transiently transfected with wtER{alpha} and ER{alpha}-3X. Transfected cells were treated with either 100 nM fulvestrant (ICI) or 100 nM 4-OHT for 2 h. Upper panel, whole cell lysates were prepared as described under "Experimental Procedures," subjected to SDS-PAGE, and transferred to membranes. ER{alpha} levels were measured and analyzed using Western blot and a LICOR imaging system, as described under "Experimental Procedures." Lower panel, quantitative analysis of Western blot of ER{alpha} protein from panel A, normalized by GAPDH using LICOR Odyssey software. Representative results of two independent experiments, each performed in duplicate, are shown.

 
Fulvestrant-induced Degradation of ER{alpha} Is Dependent on Cellular Levels of CK8 and CK18—Having established that fulvestrant induces an interaction between ER{alpha}, the nuclear matrix, and CK8 and CK18, it was important to define the role of these intermediate filaments in antiestrogen-mediated receptor degradation. To test receptor stability in the presence or absence of these CKs, we utilized C4-12 cells, an ER{alpha}-negative, CK8·CK18-positive breast cancer cell line derived from MCF-7 (34) and HeLa cells (negative for ER{alpha}, CK8, and CK18). These cell lines were stably transfected with wtER{alpha} and treated with ICI for 1-4 h; ER{alpha} protein levels were then measured by Western blot. After treatment with ICI, marked degradation of ER{alpha} was observed in C4-12 cells (Fig. 5A) but not in HeLa cells (Fig. 5B), indicating that the presence of CK8 and CK18 is essential for receptor turnover by the pure antiestrogen. To investigate the effect of CK8·CK18 overexpression on fulvestrant-induced ER{alpha} degradation, HeLa cells (negative for CK8·CK18 and ER{alpha}) were co-transfected with CK8 and CK18 (singly or both) along with ER{alpha}, and the transfected cells were treated with ICI for 2h. ER{alpha} protein levels were subsequently determined by immunoblot analysis. As shown in Fig. 6A, overexpression of CK8·CK18 restored the ability of fulvestrant to degrade ER{alpha} in HeLa cells. We then examined whether fulvestrant-induced ER{alpha} degradation could be inhibited by CK8·CK18-specific small interference RNAs (siRNA). MCF-7 cells (CK8·CK18-positive) were transfected with CK8 or CK18 siRNAs (singly or both) and treated with ICI for 2 h. CK8·CK18 and ER{alpha} protein levels were measured by Western blotting. As shown in Fig. 6B, CK8·CK18 siRNAs decreased the level of CK8 and CK18, and fulvestrant-induced ER{alpha} degradation was less dramatic in these MCF-7 cells.


Figure 4
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  		FIGURE 4.
The F domain of ER{alpha} is not required for fulvestrant-induced receptor degradation. T47D cells were transiently transfected with ER{alpha}{Delta}F and ER{alpha}3X{Delta}F and ER{alpha}{Delta}F{Delta}H12 (depicted in Fig. 3) and treated with fulvestrant (100 nM ICI 182,780) or MG132 (5 µM) for 1 h. Whole cell lysates were prepared, and ER{alpha} levels were measured and analyzed using Western blotting and a LICOR imaging system, as described under "Experimental Procedures." Western blot image (A) and quantitative analysis (B) were prepared using normalization to GAPDH and LICOR Odyssey software. Representative results of two independent experiments, each performed in duplicate, are shown. DMSO, dimethyl sulfoxide; MG, MG132.

 


Figure 5
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  		FIGURE 5.
Fulvestrant induces ER{alpha} degradation in CK8-, CK18-positive C4-12 cells but not in CK8-, CK18-negative HeLa cells. C4-12 cells (derived from MCF-7 breast cancer cells) (A) and HeLa cells (B) were stably transfected with wtER{alpha} and treated with fulvestrant (100 nM ICI 182,780) for 1, 2, or 4 h. Whole cell lysates were prepared, subjected to SDS-PAGE, and blotted. ER{alpha} levels were measured and analyzed using Western blotting and LICOR imaging system (described under "Experimental Procedures."). Upper panel, Western blot image. Lower panel, quantitative analysis of Western blot of ER{alpha} protein from panel A, normalized to GAPDH using LICOR Odyssey software. Representative results of two independent experiments, each performed in duplicate, are shown.

 
Cytoplasmic Localization of ER{alpha} Is Associated with CK8 and CK18—A unique but poorly understood property of pure antagonists like the ICI compounds (13, 19) and RU 58668 (44) is the induction of cytoplasmic localization of ER{alpha}. Intermediate filament proteins CK8 and CK18 have been shown to be located in both the nuclear matrix as well as in the cytoplasm (30). To investigate whether fulvestrant-mediated cytoplasmic localization of ER{alpha} is associated with CK8 and CK18, we transfected an ER{alpha}-GFP plasmid into CK8- and CK18-positive or -negative cell lines (MCF-7, T47D, or HeLa cells, respectively; Fig. 2). Transfected cells were then treated with ICI in the presence or absence of the protein synthesis inhibitor cycloheximide or the partial antagonist 4-OHT. In untreated cells and cells treated with 4-OHT, expression of ER{alpha}-GFP was exclusively nuclear (Fig. 7, first and last columns, respectively). After treatment of MCF-7 and T47D cells with ICI, dramatic cytoplasmic localization of ER{alpha} was observed (Fig. 7, second column). This was completely blocked by cycloheximide treatment (Fig. 7, third column), consistent with a previous report demonstrating the requirement of new protein synthesis for fulvestrant-induced cytoplasmic ER{alpha} localization (44). In contrast to observations in MCF7 and T47D cells, in HeLa cells treated with fulvestrant markedly less cytoplasmic localization was observed based on both the percentage of cells displaying ER{alpha}-GFP in the cytoplasm and cytoplasmic ER{alpha}-GFP intensity (Fig. 7, last row).3 After 8 h of ICI treatment, most (>50%)3 MCF-7 and T47D cells showed some degree of cytoplasmic localization; however, <10% of the CK8·CK18-negative HeLa cells displayed cytoplasmic localization, in agreement with a previous report (19). Collectively, these results indicate that the presence of CK8 and CK18 is necessary for fulvestrant-induced cytoplasmic localization of ER{alpha}.


Figure 6
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  		FIGURE 6.
Cytokeratins 8, 18 facilitate fulvestrant-induced ER{alpha} degradation. A, HeLa cells (negative for CK8·CK18 and ER{alpha}) were transiently transfected with CK8·CK18 (singly or both) and ER{alpha} in the absence or presence of fulvestrant (100 nM ICI 182,780). Protein levels were measured and analyzed using Western blotting and LICOR imaging system (described under "Experimental Procedures"). Upper panel, Western blot image. Lower panel, quantitative analysis of Western blots from panel A (normalized to GAPDH using LICOR Odyssey software). DMSO, dimethyl sulfoxide. B, knockdown of endogenous CK8·CK18 inhibits fulvestrant-induced ER{alpha} degradation. MCF-7 cells were transfected with siRNAs for CK8 or CK18 (singly or both) in the absence or presence of fulvestrant (100 nM ICI 182,780). Protein levels were measured and analyzed using Western blotting and LICOR imaging system. Upper panel, Western blot image. Lower panel, quantitative analysis of Western blots from panel B (normalized to GAPDH using LICOR Odyssey software). Representative results of two independent experiments, each performed in duplicate, are shown. CTRL, control.

 


Figure 7
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  		FIGURE 7.
Cytoplasmic localization of ER{alpha} after treatment of breast cancer cells with fulvestrant. MCF7, T47D, and HeLa cells were transiently transfected with pEGFP-C1-hER{alpha} and treated with fulvestrant (100 nM ICI 182,780) or fulvestrant plus 25 µg/ml cycloheximide (CHX) or 100 nM 4-hydroxytamoxifen (4-OHT) or Me2SO (NH, no hormone). Images were taken 8 h after drug treatment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The antiproliferative effects of fulvestrant (ICI 182,780) on breast cancer cells are due to rapid degradation of ER{alpha} protein (12, 21). While the drug acts by immobilizing ER{alpha} to the nuclear matrix followed by rapid receptor turnover, the molecular mechanism has not been fully established. In this study we identified two fulvestrant-dependent ER{alpha}-interacting proteins, CK8 and CK18, members of the nuclear matrix intermediate filament family of structural proteins (30). We show that CK8 and CK18 are involved in fulvestrant-induced ER{alpha} immobilization and degradation, and we further demonstrate that H12 of ER{alpha} is essential for the fulvestrant-dependent interaction with CK8 and CK18. Although ER{alpha} has long been known to associate with the nuclear matrix (45), our findings are the first demonstration of a fulvestrant-dependent interaction between ER{alpha} and intermediate filament proteins in the nuclear matrix. Because proteasomes are closely associated with intermediate filaments (31-33), we suggest that SERD-induced rapid degradation of ER{alpha} is due to specific interactions with CK8 and CK18 by bringing the receptor into close proximity to the 26 S proteasome protein degradation machinery.

Pure antiestrogens, like fulvestrant, can be converted to full estrogen agonists by specific mutations in H12 (28, 29). H12 makes up most of the C-terminal helix within the ligand binding domain of ER{alpha} (46) and appears to be required for recruiting coactivators and co-repressors, serving as a "molecular switch" that connects ligands with coregulators (47). This helix is required for ICI-induced immobilization, as demonstrated by Stenoien et al. (20) using fluorescence recovery after photobleaching, and mutations in H12 can abrogate E2-mediated degradation (3-6), suggesting that the H12 coactivator binding surface is required for ligand-mediated ER{alpha} down-regulation. Furthermore, antiestrogens have been shown to change ER{alpha} stability by altering the position of H12 (26). To test whether H12 is essential for receptor-CK8 and -CK18 interactions and, thus, the ability of fulvestrant to immobilize and degrade ER{alpha}, we examined the interaction between several GST-ER{alpha}-AF2 mutants and CK8 and CK18. Point mutations or deletion of H12, but not loss of F domain function, abolished CK8 and CK18 interactions, demonstrating that the F domain is not required for fulvestrant-induced ER{alpha} immobilization. Based on these results, we suggest that in the presence of fulvestrant, H12 interacts with CK8·CK18 and immobilizes ER{alpha} within the nuclear matrix for subsequent degradation.

Because the interaction of ER{alpha} with CK8 and CK18 is specific for fulvestrant, it is likely that H12 assumes a different position when bound by ICI, as compared with 4-OHT, resulting in receptor degradation versus stabilization. Indeed, a recent report showing differences in antiestrogen-induced relocation of hydrophobic residues in H12 strongly supports this possibility (26). Of the ER{alpha} antagonists examined, ICI caused the greatest exposure of surface hydrophobicity, whereas 4-OHT caused the least exposure (26). Thus, it seems plausible that ICI-induces a conformational change that allows H12 to interact with CK8 and/or CK18. Nonetheless, it is not clear how an ER{alpha}-CK8·CK18 interaction triggers rapid receptor turnover; however, proteasomes have recently been shown to be closely associated with intermediate filaments and, thus, likely facilitate this process (31-33).

It has previously been shown that pure antiestrogens (ICI 182,780, RU 58668) can disrupt ER{alpha} nucleocytoplasmic shuttling and cause receptor cytoplasmic localization (13), a process that requires new protein synthesis (19). It is also known that both CK8 and CK18 are located in the cytoplasm and the nuclear matrix (30). In the present study, ER{alpha} cytoplasmic localization was observed only in CK8·CK18-positive cells, suggesting that these intermediate filaments play a role in retaining ER{alpha} in the cytoplasm after fulvestrant treatment. In support of this hypothesis, Htun et al. (19) reported that cytoplasmic retention of ER{alpha} varied between breast cancer cell lines, with greater cytoplasmic localization seen in ER{alpha}-positive MCF-7 and T47D cells as compared with ER{alpha}-negative MDA-MB-231 cells. Although an explanation for this observation was not offered (19), our findings that CK8 and CK18 are differentially expressed in these cell lines provides a plausible rationale. Interestingly, whereas other cytokeratins are present in the nuclear matrix (e.g. CK5·CK19), these do not interact with ER{alpha},3 and the basis for the specificity of ER{alpha} for CK8 and CK18 remains unclear.

Although it is well established that the level of ER{alpha} in breast tumors is a valuable predictor of a patient's response to antiestrogen therapies such as tamoxifen and fulvestrant (48), CK8 and CK18, via their correlation with tumor differentiation (49), have also been used in cancer diagnosis. Furthermore, up-regulation of CK8·CK18 expression was associated with good prognosis in breast cancer patients (49, 50), whereas their down-regulation was correlated with a poor clinical outcome (51). We have previously shown that breast cancer cells with a disrupted ubiquitin-like NEDD8 pathway can acquire antiestrogen resistance (8) and that tumors from patients who developed resistance to fulvestrant can retain ER{alpha} expression (52). Taken together, it seems reasonable to suggest that disruption of ER{alpha} degradation may contribute fulvestrant-resistant breast cancer. Because CK8 and CK18 are associated with fulvestrant-mediated ER{alpha} degradation, their decreased levels would likely disrupt fulvestrant-mediated ER{alpha} immobilization and degradation, which are both essential for the antiproliferative activity of this antiestrogen (8). Thus, we speculate that down-regulation of CK8·CK18 may be involved in fulvestrant resistance; furthermore, a H12 mutant ER{alpha} would likely be resistant to fulvestrant-mediated degradation, supporting the observation that H12 mutations can contribute to endocrine-resistant breast cancer (53-56). In conclusion, fulvestrant resistance is clearly multifactorial. We are currently investigating the role of the NEDD8 pathway and the nuclear matrix proteins CK8 and CK18 in antiestrogen-resistant breast cancer.


   FOOTNOTES
 
* This work was supported by The American Cancer Society Research and Alaska Run for Women Grant TBE-104125 and the United States Army Medical Research Acquisition Activity Awards DAMD 17-02-1-0418 and -0419. 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. Back

1 To whom correspondence should be addressed: Medical Sciences, Indiana University School of Medicine, 302 Jordan Hall, 1001 E. 3rd St., Bloomington, IN 47405-4401. Tel.: 812-855-9445; Fax: 812-855-4436; E-mail: knephew{at}indiana.edu.

2 The abbreviations used are: ER{alpha}, estrogen receptor-{alpha}; CK, cytokeratin; E2, 17beta-estradiol; GFP, green fluorescent protein; ICI, ICI 182,780; 4-OHT, 4-hydroxytamoxifen; siRNA, small interference RNA; SERD, selective estrogen receptor down-regulator; GAPDH, glyceraldehyde phosphate dehydrogenase; GST, glutathione S-transferase; AF2, activating function-2; wt, wild type; H12, helix 12. Back

3 X. Long and K. P. Nephew, unpublished results. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Pierre Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France) for providing pSG5-ER{alpha} (HEGO), Dr. Michael A. Mancini (Baylor College of Medicine, Houston, TX) for GFP-ER{alpha}, and Dr. Donald P. McDonnell (Duke University, Durham, NC) for pRST-7-hER3X. We thank Drs. Meiyun Fan and Curtis Balch (Indiana University School of Medicine) for their critical review of this manuscript.



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