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Cancer Research 67, 1352, February 1, 2007. doi: 10.1158/0008-5472.CAN-06-1020
© 2007 American Association for Cancer Research
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Endocrinology

Long-term Treatment with Tamoxifen Facilitates Translocation of Estrogen Receptor {alpha} out of the Nucleus and Enhances its Interaction with EGFR in MCF-7 Breast Cancer Cells

Ping Fan, Jiping Wang, Richard J. Santen and Wei Yue

Department of Internal Medicine, University of Virginia Health Sciences System, Charlottesville, Virginia

Requests for reprints: Wei Yue, Department of Internal Medicine, University of Virginia, P.O. Box 801416, Charlottesville, VA 22908. Phone: 434-924-0399; Fax: 434-924-1284; E-mail: wy9c{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The therapeutic benefit of tamoxifen in patients with hormone-dependent breast cancer is limited by acquired resistance to this drug. To investigate the biological alterations responsible for tamoxifen resistance, an in vitro model was established. After 6-month continuous exposure to tamoxifen (10–7 mol/L), growth of MCF-7 breast cancer cells was no longer inhibited by this antiestrogen. Although there was no significant increase in the basal levels of activated mitogen-activated protein kinase (MAPK), tamoxifen-resistant (TAM-R) cells exhibited enhanced sensitivity to epidermal growth factor (EGF) and estradiol stimulated activation of MAPK. Tamoxifen elicited rapid phosphorylation of MAPK, in contrast to its antagonistic activity in control cells. Blockade of the EGF receptor (EGFR)/MAPK pathway caused more dramatic inhibition of growth of TAM-R cells than the control cells. An increased amount of estrogen receptor {alpha} (ER{alpha}) was coimmunoprecipitated with EGFR from TAM-R cells although the total levels of these receptors were not increased. Notably, ER{alpha} seemed to redistribute to extranuclear sites in TAM-R cells. Increased ER{alpha} immunoreactivity in the cytoplasm and plasma membrane of TAM-R cells was shown by fluorescent microscopy and by Western analysis of isolated cellular fractions. In TAM-R cells, an increased amount of c-Src was coprecipitated with EGFR or ER{alpha}. Blockade of c-Src activity resulted in redistribution of ER{alpha} back to the nucleus and in reduction of its interaction with EGFR. Prolonged blockade of c-Src activity restored sensitivity of TAM-R cells to tamoxifen. Our results suggest that enhanced nongenomic function of ER{alpha} via cooperation with the EGFR pathway is one of the mechanisms responsible for acquired tamoxifen resistance. [Cancer Res 2007;67(3):1352–60]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The antiestrogen tamoxifen has been used since the 1970s for the treatment of breast cancer (1). Although ~50% of patients benefit from this therapy, almost all responsive tumors eventually relapse due to the development of tamoxifen resistance (2). Acquired resistance to tamoxifen is a serious therapeutic problem, and major efforts are now being made to understand the underlying mechanisms responsible (3).

Tamoxifen resistance can be subdivided into de novo and acquired resistance, and the precise mechanisms responsible likely differ. Augmentation of growth factor receptor function seems to play a major role in de novo resistance. An inverse relationship between expression of estrogen receptor {alpha} (ER{alpha}) and epidermal growth factor (EGF) receptor (EGFR) or c-erbB2 was observed in primary breast cancer samples and was associated with decreased sensitivity to endocrine therapy and a poorer prognosis (4, 5). Engineered overexpression of EGFR and c-erbB2 in hormone-dependent breast cancer cells promotes hormone-independent growth (6, 7). A predominant role of growth factors as mitogens may explain the de novo resistance to endocrine therapy.

The mechanisms for acquired tamoxifen resistance are more complex. Usually, TAM-R tumors do not lose ER{alpha}, and the receptor is apparently functioning because a portion of patients with relapsing disease are responsive to secondary endocrine therapies (8, 9). A body of clinical and experimental studies suggests that modification of ER functionality by growth factor pathways might be an important mechanism for acquired tamoxifen resistance (10, 11). Cross talk between ER and EGFR family through phosphorylation of ER{alpha} enhances its transcriptional activity (12, 13). Increased recruitment of coactivators, such as AIB1 (SRC-3; ref. 14), and conformational change (15) after receptor phosphorylation also contribute to enhanced transcription activity of ER{alpha} in tamoxifen-resistant (TAM-R) breast cancer cells.

ERs classically act as ligand-dependent transcription factors in the nucleus; however, recent data have identified a role for rapid extranuclear signaling. The very rapid effects of estrogen on calcium influx, generation of cyclic AMP, and stimulation of kinases including mitogen-activated protein (MAP) kinase (MAPK) represent key examples of the extranuclear events. Compelling evidence indicates that activation of MAPK via extranuclear actions of ER{alpha} requires participation of EGFR in certain cell types (16). The potential link between ER{alpha} and EGFR is G-protein–dependent activation of metalloproteinases that cleave free heparin-binding EGF (HB-EGF) from surrounding protein complexes (16). Extranuclear ER{alpha} has been shown to be biologically important in cell proliferation (17) and survival of breast cancer cells (18).

Recent data suggest that the nuclear/cytoplasmic distribution of ER{alpha} can be altered. Kumar et al. (19) reported that a truncated MTA1 protein was highly expressed in aggressive breast cancer. This protein sequesters ER in the cytoplasm and promotes extracellular signal-regulated kinase (ERK) MAPK signaling. The same group also found that overexpression of c-erbB2 caused nuclear to cytoplasmic translocation of ER{alpha} (20). These data suggest that cooperation of ER{alpha} with growth factor pathways outside the nucleus might contribute to breast cancer progression and resistance to endocrine therapy.

In the present studies, we hypothesize that augmented cooperation of ER{alpha} and EGFR contributes to the development of acquired tamoxifen resistance. We show that prolonged exposure of hormone-sensitive MCF-7 breast cancer cells to tamoxifen causes cytoplasmic translocation of ER{alpha}, which facilitates the interaction of ER{alpha} with EGFRs. Enhanced interaction between ER{alpha} and EGFR is associated with an increased ability of EGF, estradiol (E2), and tamoxifen to stimulate MAPK. Taken together, our result suggest that up-regulation of the extranuclear functions of ER through cooperation with EGFR pathway represents one of the mechanisms of tamoxifen resistance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. Tamoxifen was purchased from Sigma (St. Louis, MO); E2 was from Steraloid, Inc. (Whilton, NH); EGF was from Collaborative Biomedical Products (Bedford, MA); U0126 was from Promega Corporation (Madison, WI); and EGFR tyrosine kinase inhibitor AG1478 and c-Src kinase inhibitor PP2 were from Calbiochem (San Diego, CA). ICI 182,780 (ICI) was a gift from AstraZeneca (Wilmington, DA). Sources of antibodies are as follows: total MAPK (Zymed Laboratories, Inc., South San Francisco, CA), phosphorylated MAPK antibody, Ser473–phosphorylated Akt, and total Akt (Cell Signaling Technology, Beverly, MA); ER{alpha} antibodies (monoclonal D-12; polyclonal HC-20 and H-184), c-Src antibodies (monoclonal and polyclonal), FAK (c-20), 5'-nucleotidase (H-300), and nuclear transport factor 2 (5E8; Santa Cruz Biotechnology); EGFR sheep polyclonal antibody (06-129; Upstate Biotechnology, Lake Placid, NY); Src homology and collagen (Shc) polyclonal antibody (BD Transduction Laboratories, San Diego, CA); MNAR polyclonal antibody (Bethyl Laboratories, Montgomery, TX); and GW182 (Abcam, Inc., Cambridge, MA). Recombinant protein G agarose was from Invitrogen Life Technologies (Carlsbad, CA). Secondary antibodies conjugated with horseradish peroxidase (HRP) were from Amersham Pharmacia Biotech (Piscataway, NJ). Cell culture medium, improved MEM (IMEM), was from Biosource International, Inc. (Camarillo, CA). Fetal bovine serum (FBS), glutamine, and trypsin were from Invitrogen Life Technologies (Carlsbad, CA). Alexa Fluor 568 phalloidin and Alexa Fluor 488 goat anti-rabbit IgG were bought from Molecular Probes (Eugene, OR). All chemicals were obtained from Sigma.

Cell culture. Wild-type MCF-7 cells (kindly provided by Dr. R. Bruggemeier, Ohio State University, Columbus, OH) were grown in IMEM containing 5% FBS. TAM-R cells derived from MCF-7 cells were continuously cultured in the medium containing 10–7 mol/L tamoxifen. The medium for matched control cells contained 0.1% ethanol.

Growth assay. Cells were plated in six-well plates at a density of 60,000 per well in their culture medium. Two days later, the cells were treated as described in figure legends for 5 days with medium change on day 3. The final concentration of vehicle (ethanol or DMSO) was 0.1%. At the end of treatment, cells were rinsed twice with saline. Nuclei were prepared by sequential addition of 1 mL HEPES-MgCl2 solution (0.01 mol/L HEPES and 1.5 mmol/L MgCl2) and 0.1 mL ZAP solution [0.13 mol/L ethylhexadecyldimethylammonium bromide in 3% glacial acetic acid (v/v)], and were counted using a Coulter counter.

Tamoxifen, a partial agonist, exerts slight stimulatory effect on proliferation of wild-type MCF-7 cells in phenol red–free medium containing charcoal-stripped FBS (DCC-FBS); on the other hand, in the medium with phenol red and 5% FBS, tamoxifen is inhibitory. Therefore, the experiments examining the inhibitory effect of tamoxifen was carried out in phenol red containing IMEM with 5% FBS. When testing agonistic action of tamoxifen, cells were treated with tamoxifen or E2 for 5 days in phenol red–free medium containing 1% DCC-FBS.

Immunoblotting. Cells grown in 60-mm dishes were washed with PBS, incubated on ice for 5 min with 0.5 mL lysis buffer [20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L sodium orthovanadate, 2.5 mmol/L sodium PPi, 1% Triton X-100, 1 mmol/L ß-glycerophosphate, 1 µg/mL leupeptin and aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF)], pulse sonicated, and centrifuged at 14,000 rpm for 10 min at 4°C. Cell lysates were stored at –80°C until analysis. Total protein content of the lysate was determined by a standard Bradford assay using the reagent from Bio-Rad Laboratories (Hercules, CA). Fifty micrograms of total protein were separated on 10% SDS polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was probed with primary antibodies dissolved in TBS containing 5% bovine serum albumin followed by incubation with secondary antibody conjugated with HRP (1:2,000) and reaction with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology Inc., Rockford, IL). Protein bands were visualized by exposing the membrane to X-ray film and quantified using a Molecular Dynamics (Piscataway, NJ) scanner and ImageQuant program.

Immunoprecipitation. Cells grown in 100-mm dishes were washed with cold PBS and extracted with 1 mL lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 5 mmol/L EDTA, 25 mmol/L NaF, 2 mmol/L NaVO4, 5% glycerol, 1% Triton X-100, 10 µg/mL leupeptin, aprotinin, and pepstatin). Samples were incubated on ice for 30 min, sonicated, and centrifuged at 14,000 rpm for 10 min at 4°C. Supernatants containing 1 mg total protein were incubated with antibody against the target protein at 4°C for 4 h before addition of 40 µL Protein G beads, and incubation was continued at 4°C overnight. The protein G beads with immunocomplex were centrifuged at 14,000 rpm for 20 s. The supernatant was carefully removed. The beads were washed twice with 1 mL buffer II (20 mmol/L MOPS, 2 mmol/L EGTA, 5 mmol/L EDTA, 25 mmol/L NaF, 40 mmol/L ß-glycerophosphate, 10 mmol/L sodium PPi, 2 mmol/L NaVO4, 0.5% Triton X-100, 1 mmol/L PMSF, 10 µg/mL leupeptin, aprotinin, and pepstatin) and then boiled in 50 µL of 2x Laemmli buffer. The samples were subjected to electrophoresis in 7.5% to 10% SDS polyacrylamide gel and immunoblotting as described above.

Immunofluorescent microscopy. Cells grown on sterile glass coverslips in six-well plate were fixed in 4% paraformaldehyde in PBS at room temperature for 20 min. Cells were permeabilized in cold acetone at –20°C for 2 to 4 min. After background blocking with 5% normal goat serum in PBS for 1 h, the cells were incubated with the anti-ER{alpha} antibody (H-184) overnight at 4°C followed by incubation with Alexa Fluor 488–labeled secondary antibody at room temperature for 1 h. Alexa Fluor 546–labeled phalloidin were used for filamentous actin counterstaining. The cells incubated with secondary antibody only served as the control for antibody specificity. Confocal images were captured under Zeiss LSM 510 confocal Microscope using LSM Image Browser software (Zeiss, Thornwood, NY).

Apoptosis ELISA assay. Apoptosis was measured using Cell Death Detection ELISA kit (Roche Diagnostics, Indianapolis, IN) following the manufacturer's instruction. Briefly, cells were plated into 12-well plates at the density of 8 x 104 per well. Two days later, the cells were treated with testing compounds for desired period of time. The cell lysate was prepared by incubation of the cell monolayer with 0.5 mL lysis buffer at room temperature for 30 min followed by centrifugation at 14,000 rpm for 10 min at 4°C. A parallel set of plates with identical treatment was prepared for cell counting. The result was expressed as absorbance at 405 nm normalized by cell number.

Knockdown of EGFR with specific small interfering RNA. A smart pool of double-stranded small interfering RNA (siRNA) against EGFR and nonspecific siRNA were obtained from Dharmacon Tech (Lafayette, CO) and used according to the manufacturer's instructions. TAM-R cells cultured in 100-mm dishes were shifted to DMEM without antibiotics at least 1 day before transfection. A 4-mL mixture of siRNA in Opti-MEM I (100 nmol/L) and LipofectAMINE 2000 (Invitrogen Life Technologies) was incubated with cells for 4 to 6 h and then replaced by culture medium with 5% FBS. The cells were harvested 48 h after transfection.

Subcellular protein fractionation. Plasma membrane protein was extracted using Mem-PER Eukaryotic Membrane Protein Extraction Reagent kit, and nuclear and cytoplasmic proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents kit following the manufacturer's instruction. The kits were purchased from Pierce Biotechnology.

Statistical analysis. All reported values are the means ± SE. Statistical comparisons were determined with two-tailed Student's t tests. Results were considered statistically significant if the P value was <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Long-term tamoxifen treatment alters the responsiveness of MCF-7 cells to tamoxifen and E2. As shown in earlier studies, medium supplemented with FBS contains sufficient estrogen and growth factors to support growth of MCF-7 cells (21). Acute exposure of wild-type MCF-7 cells to tamoxifen (10–7 mol/L) for 5 days in this medium causes 30% to 35% reduction in cell number. Exposure of MCF-7 cells to 0.1% ethanol for up to 12 months did not change the inhibition rate of tamoxifen (Fig. 1A ). In contrast, the inhibitory effect of tamoxifen was diminished in the cells continuously exposed to tamoxifen over a period of 12 months (Fig. 1B). As expected, E2 (10–10 mol/L) did not significantly stimulate MCF-7 cells grown in medium containing phenol red and 5% FBS. Lack of stimulation reflected the fact that estrogen in the serum and phenol red in the medium provided maximal stimulation of growth, which masked the stimulatory effect of estrogen (22). Exposure to ethanol over a 12-month period did not alter these responses (Fig. 1C). Treatment with tamoxifen for up to 6 months did not alter the pattern of response to E2. In contrast, the rate of E2-induced growth stimulation was gradually increased in the cells preexposed to tamoxifen for longer periods of time (i.e., for 7–12 months; Fig. 1D). No growth stimulation by tamoxifen was observed in long-term tamoxifen-treated cells under estrogen-deprived conditions (data not shown). These results indicate that long-term tamoxifen treatment enhanced the sensitivity of MCF-7 cells to the mitogenic effect of E2 (Fig. 1D), while inducing tamoxifen resistance (Fig. 1B). The cells cultured in tamoxifen-containing medium for longer than 6 months were named TAM-R cells.


Figure 1
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  		Figure 1. Altered acute responses of MCF-7 cells to tamoxifen and E2 after long-term tamoxifen exposure. MCF-7 cells cultured for 0 to 12 mo with 10–7 mol/L tamoxifen or ethanol were plated in six-well plates at a density of 60,000 per well in IMEM containing 5% FBS. The cells were then treated with tamoxifen (TAM; 10–7 mol/L; A and B) or E2 (10–10 mol/L; C and D) for 5 d, and cell number was counted. For tamoxifen-preexposed cells, tamoxifen was continuously present in the medium before the 5-d treatment with fresh medium containing either tamoxifen or E2.

 
Basal phosphorylation of p44/p42 MAPK increases transiently during development of tamoxifen resistance. Activation of growth factor receptors and their downstream kinase cascades is an important mechanism involved in acquired resistance to tamoxifen (23, 24). The MAPK and the phosphatidylinositol 3-kinase pathways are the major cascades in mediation of mitogenic signals in hormone-dependent breast cancer. To monitor the basal levels of activation of these two signaling pathways during tamoxifen treatment, MCF-7 cells continuously cultured with tamoxifen (10–7 mol/L) in IMEM with phenol red and 5% FBS were collected at various time points, and the levels of phosphorylated MAPK and Akt were determined by Western analyses. The levels of phosphorylated MAPK increased as early as 5 days after treatment with tamoxifen and remained high for 2 months (Fig. 2A ). There was no significant difference in the levels of phosphorylated MAPK between tamoxifen-treated and ethanol-treated cells at 3 months (Fig. 2A) and thereafter (data not shown). The levels of phosphorylated Akt during tamoxifen treatment remained unchanged (Fig. 2A).


Figure 2
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  		Figure 2. Basal and stimulator-induced phosphorylation of MAPK and Akt. A, effect of tamoxifen treatment on activation of MAPK and Akt. Cell lysates were prepared from MCF-7 cells cultured in the presence of tamoxifen (10–7 mol/L) or ethanol (EtOH) for various periods as indicated. Phosphorylation of p44/p42 (p-p44/p42) MAPK and Akt was examined by Western blot analysis using specific antibodies against phosphorylated proteins. To examine stimulator-induced phosphorylation of MAPK, cells were cultured for 24 h (96 h for treatment with E2 or tamoxifen) in phenol red–free medium containing 1% DCC-FBS and then incubated with EGF at indicated concentrations for 15 min (B), or with E2 (10–10 mol/L; C) or tamoxifen (10–7 mol/L; D) for various periods before preparation of cell lysates and Western blot analysis. Numbers under phosphorylated MAPK bands, fold changes of phosphorylated MAPK normalized by total MAPK. Representative of three repeatable experiments.

 
Enhanced responses to EGF and E2 stimulation of p44/42 MAPK in TAM-R cells. Although the basal levels of phosphorylated MAPK returned to the control levels after 3-month treatment with tamoxifen, TAM-R cells were more sensitive to the stimulatory effect of EGF. As shown in Fig. 2B, in TAM-R cells a significant increase in the level of phosphorylated MAPK (1.9-fold) was induced by EGF at the concentration of 0.1 ng/mL, whereas the control cells did not respond to this concentration of EGF at all. The responses increased with the concentration of EGF. At each concentration, TAM-R cells displayed higher levels of phosphorylated MAPK than the control cells.

The rapid response to E2-stimulated activation of MAPK was also increased in TAM-R cells. Compared with the control cells, TAM-R cells showed earlier and more drastic increase in the level of phosphorylated MAPK after E2 treatment (Fig. 2C). In TAM-R cells, tamoxifen (10–7 mol/L) induced phosphorylation of MAPK at 3 to 15 min (Fig. 2D). Peak stimulation of MAPK (>5-fold) occurred at 3 to 5 min, which then decreased. In contrast, tamoxifen showed slight inhibition or slight stimulation (data not shown) on MAPK activation in matched control cells (Fig. 2D) depending on the degree of deprivation of growth factors and estrogen in the medium. The duration of tamoxifen-induced MAPK phosphorylation was similar to that induced by E2. These results indicate that the nongenomic effect of estrogen increased in TAM-R cells. In addition, tamoxifen acts as an agonist to elicit MAPK activation in TAM-R cells.

TAM-R cells are dependent on EGFR/MAPK signaling pathway for growth. Consistent with enhanced sensitivity to EGF-stimulated MAPK activation, TAM-R cells became more sensitive to the growth inhibition by small-molecule inhibitors that block kinase activity of EGFR or MAP/ERK kinase (MEK). The dose-response curve of TAM-R cells to AG1478 was shifted to the left compared with that of control cells (Fig. 3A ). The estimated IC50 of AG1478 in TAM-R cells was ~3-fold lower than that in the control cells. AG1478-induced growth inhibition of TAM-R cells was at least partially due to enhanced response of these cells to the apoptotic effect of AG 1478. A significantly higher portion of TAM-R cells underwent apoptosis than the control cells when exposed to the same concentration of AG1478 (Fig. 3B). Similarly, the specific MEK inhibitor, U0126, caused more growth inhibition in TAM-R cells than in the control (Fig. 3C). These results suggest that MCF-7 cells become more dependent on the EGFR/MAPK pathway for growth after long-term exposure to tamoxifen.


Figure 3
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  		Figure 3. Enhanced sensitivity to growth inhibition of AG1478 and U0126 in TAM-R cells. A, growth response to AG1478. TAM-R and control cells were plated in six-well plates at a density of 60,000 per well in IMEM containing 5% FBS. The cells were then treated with different concentrations of EGFR inhibitor AG1478 for 5 d, and cell number was counted. Points, average of four independent experiments; bars, SE. B, apoptotic response to AG1478. Cells were plated at the density of 8 x 104 per well into 12-well plates. A parallel set of plates were used for cell counting. Two days later, the cells were treated with AG1478 (5 µmol/L) for 48 h before ELISA for apoptosis. C, growth response to the MEK inhibitor U0126 was evaluated under the same conditions as described in (A). **, P < 0.01; *, P < 0.05, compared with the control cells.

 
Enhanced interaction between EGFR and ER{alpha} in TAM-R cells. The MAPK signaling pathway can be activated by the members of the EGFR family as well as ER{alpha} via a nongenomic mechanism. Increases in EGFR and/or c-erbB2 expression would be expected to be responsible for enhanced sensitivity of these cells to EGF stimulation because overexpression of EGFR and/or c-erbB2 has been reported in de novo or acquired tamoxifen resistance of breast cancer cells (4). To our surprise, expression levels of EGFR, ER{alpha} (Fig. 4A ), and c-erbB2 (data not shown) did not change during the first 3 months of adaptation nor after 6 to 12 months (data not shown). Interestingly, in the cells treated with tamoxifen for 4 months or longer, a significantly increased amount of ER{alpha} was coimmunoprecipited with EGFR although there was no change in the levels of these receptors compared with the control cells that had been treated with ethanol (Fig. 4A). Knockdown of EGFR with specific siRNA in TAM-R cells reduced the amount of ER{alpha} that interacted with EGFR as well as EGF-stimulated MAPK phosphorylation (Fig. 4B).


Figure 4
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  		Figure 4. Interaction between ER{alpha} and EGFR was increased in TAM-R cells. A, cell lysates were prepared from three different passages (#1–#3) of TAM-R cells and matched controls that had been exposed to tamoxifen or ethanol for >4 mo. Interaction between EGFR and ER{alpha} was examined by immunoprecipitation (IP) with antibody against EGFR followed by immunoblotting (IB) for ER{alpha}. Numbers under ER{alpha} bands, fold changes. B, TAM-R cells grown in 100-mm dishes were transfected with EGFR siRNA or nonspecific siRNA. Cell lysates were prepared 48 h after transfection for immunoprecipitation and immunoblot. C, TAM-R and control cells were cultured in serum-free medium for 24 h and then incubated with EGFR inhibitor AG1478 (5 µmol/L, 1 h) or pure antiestrogen ICI (10–8 mol/L, 1 and 4 h) before preparation of cell lysates for immunoprecipitation. The amount of total input protein for immunoprecipitation is 1 mg.

 
We then examined whether the interaction between EGFR and ER{alpha} could be blocked by a specific EGFR kinase inhibitor or by a pure antiestrogen. Preincubation of TAM-R cells with the selective inhibitor of EGFR AG1478 (5 µmol/L) for 1 h did not significantly reduce the amount of ER{alpha} bound to EGFR (Fig. 4C), suggesting that kinase activity of EGFR is not critical for its binding to ER{alpha}. In control and TAM-R cells, treatment with the antiestrogen ICI (10–8 mol/L) increased the amount of ER{alpha} coprecipitated with EGFR at 30 min (data not shown) and 1 h and slightly decreased at 4 h of treatment (Fig. 4C). These results provided supportive evidence that the cytoplasmic level of ER{alpha} is an important factor determining its interaction with EGFR because it is known that ICI compounds cause cytoplasmic translocation of ER{alpha} and accelerate its degradation (25). Immunofluorescent microscopy showed increased amount of ER{alpha} in the cytoplasm of both the control and TAM-R cells treated with ICI (data not shown).

Long-term tamoxifen treatment results in nucleus to cytoplasm redistribution of ER{alpha} in MCF-7 cells. Because the levels of ER{alpha} and EGFR were not increased in TAM-R cells, enhanced interaction between EGFR and ER{alpha} might result from redistribution of ER{alpha}. To test this hypothesis, immunofluorescent staining of ER{alpha} was carried out in TAM-R and control cells. As shown in Fig. 5A , the nuclei of the control cells were intensively stained green, indicating that ER{alpha} was predominantly located in the nucleus. Compared with ethanol-treated cells, TAM-R cells were larger with more spread cytoplasm. Green fluorescence was no longer restricted to the nucleus. Bright green spots were scattered throughout the cytoplasm and on the cell borders. In the experiment where the primary anti-ER{alpha} antibody was omitted, there was no green staining at all, indicating that the green fluorescence shown in Fig. 5A was specific staining of ER{alpha} (see Supplementary Fig. S1).


Figure 5
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  		Figure 5. Altered subcellular distribution of ER{alpha} in TAM-R cells. A, cells cultured on coverslips for 2 d were fixed and incubated with anti-ER{alpha} antibody overnight at 4°C. After a thorough wash with PBS, the cells were incubated with Alexa Fluor 488–labeled secondary antibody (green) for 1 h. Actin was stained with Alexa Fluor 546 labeled phalloidin (red). Confocal images were captured under Zeiss LSM 510 confocal microscope (x40 objective). B, protein levels of ER{alpha} in subcellular fractions. Nuclei, cytoplasm, and plasma membrane were isolated as described in Materials and Methods. An equal amount of proteins from different fractions was subjected to SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by Western blot for ER{alpha}. 5'-Nucleotidase, GW182, and nuclear transport factor 2 (NTF2) were used as markers for plasma membrane, cytosol, and nucleus, respectively. Numbers under the ER{alpha} bands, fold changes of ER{alpha} normalized by the marker of each fraction.

 
To further confirm that long-term tamoxifen treatment increased the pool of cytoplasmic ER{alpha}, fractions of the plasma membrane, nucleus, and cytoplasm were prepared from TAM-R and control cells. ER{alpha} level in each fraction was monitored by Western blot. 5'-Nucleotidase, GW182, and nuclear transport factor 2 were used as markers of plasma membrane, cytoplasm, and nucleus, respectively. In the control cells, ER{alpha} was recovered from both cytoplasmic and nuclear fractions with higher levels in the nucleus. In TAM-R cells, ER{alpha} was detectable in the membrane fraction. The level of ER{alpha} in cytoplasm was increased, whereas the level of nuclear ER{alpha} was reduced compared with the control cells (Fig. 5B). These data, together with the immunofluorescent results, indicate that long-term tamoxifen treatment caused redistribution of ER{alpha} to the cytoplasm and plasma membrane and that this facilitated interaction between EGFR and ER{alpha}.

Kinase active c-Src is involved in the interaction between EGFR and ER{alpha}. A number of proteins have been reported to serve as adaptor proteins participating in the nongenomic actions of ER{alpha}. Preference for certain adaptors varies in different types of cells. To determine whether enhanced interaction between EGFR and ER{alpha} required an adaptor protein, several candidate proteins were examined by immunoblots following immunoprecipitation with antibodies against EGFR or ER{alpha}. We found that all adaptor proteins examined, including c-Src, Shc, MNAR, and FAK, were coimmunoprecipited by anti-EGFR or anti-ER{alpha} antibodies in both TAM-R and control cells. Among those adaptor proteins, only c-Src was found to be significantly increased to complex with EFGR and ER{alpha} in TAM-R cells (Fig. 6A ). Inhibition of c-Src kinase with PP2 reduced the interaction of ER{alpha} with EGFR in both TAM-R cells and control cells (Fig. 6B). The effect was more dramatic in TAM-R cells. PP2 treatment promoted translocation of ER{alpha} from the cytoplasm back to the nucleus. As shown in Fig. 6C, green fluorescence (ER{alpha} staining) was concentrated in the nuclei of TAM-R cells treated with PP2 for 48 h. Similarly, the levels of ER{alpha} in the membrane and cytoplasmic fractions were reduced and the level of nuclear ER{alpha} was increased by PP2 in TAM-R cells (Fig. 5B). More importantly, longer treatment of TAM-R cells with PP2 completely restored their sensitivity to tamoxifen (Fig. 6D). These results suggest that c-Src plays a critical role in retaining ER{alpha} in the cytoplasm and facilitating the formation of EGFR-ER{alpha} complexes during development of tamoxifen resistance.


Figure 6
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  		Figure 6. Role of c-Src in formation of EGFR/ER{alpha} complexes. A, TAM-R and control cells were grown in 60-mm dishes to 90% confluence before preparation of cell lysates. Monoclonal antibodies were used to immunoprecipitate EGFR or ER{alpha} with total input protein of 1 mg. Proteins coprecipitated were detected by immunoblot. B, effect of PP2 (5 µmol/L, 48 h) on ER{alpha} binding to EGFR. C, confocal images of ER{alpha} in control and TAM-R cells treated with vehicle or c-Src inhibitor PP2 (5 µmol/L) for 48 h. D, growth responses of wild-type MCF-7 cells, TAM-R cells, and PP2-treated TAM-R cells to acute treatment of tamoxifen. PP2 (5 µmol/L) treatment lasted for 8 mo. Cell numbers were counted as described in Materials and Methods. Columns, percentage reduction compared with the vehicle control of each cell type.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This report shows that long-term exposure to tamoxifen resulted in an increased association of ER{alpha} with the EGFR and in enhanced sensitivity to EGF-stimulated MAPK activation. As a result, TAM-R cells exhibited enhanced sensitivity to the effects of the EGFR inhibitor, AG1478, both with respect to proliferation and apoptosis. The increased binding of ER{alpha} with the EGFR, as shown by immunoprecipitation studies, was associated with a redistribution of ER{alpha} to extranuclear sites. A key finding to explain this redistribution and its biological significance is that a c-Src inhibitor prevented the translocation of ER{alpha} to the cytoplasm and reversed tamoxifen resistance. Taken together, these findings provide new insight into the process of enhanced extranuclear actions of E2, which occur during the process of development of tamoxifen resistance.

It is commonly accepted that activation of growth factor receptor pathways, especially the EGFR family, is responsible for both de novo and acquired tamoxifen resistance. Patients with hormone-dependent breast cancer overexpressing EGFR and c-erbB2 exhibit lower clinical response rates and/or shorter duration of responses to antiestrogen therapy (24, 26). Inhibition of c-erbB2 and MAPK restored the inhibitory effect of tamoxifen on ER{alpha} transcriptional activity and cell growth in breast cancer cells overexpressing c-erbB2 (27). Therefore, up-regulation of transcriptional activity of ER{alpha} by growth factor pathways has been considered a mechanism for development of resistance to endocrine therapy in hormone-dependent breast cancer (12, 14, 28).

Our results suggest that activation of the MAPK signaling via cooperation between ER{alpha} and EGFR pathways is an important mechanism for acquired tamoxifen resistance. The ER{alpha} and EGFR pathways exert their biological functions through their own receptors and via cross talk with each other. Activation of EGFR increases ligand-independent and ligand-dependent transcriptional activity of ER{alpha}. On the other hand, extranuclear ERs use the EGFR pathway to induce rapid activation of MAPK and phosphatidylinositol 3-kinase, which has been shown to be involved in estrogen-stimulated proliferation and protection from apoptosis in breast cancer cells (17, 18). Results from the current studies indicate that the interaction of ER{alpha} with EGFR is augmented when the ER in breast cancer cells is continuously exposed to tamoxifen. Enhanced interaction between ER{alpha} and EGFR increases the sensitivity to E2 and EGF. Tamoxifen, under this circumstance, is no longer inhibitory but stimulatory on MAPK activation. The up-regulated system receives EGF, E2, and tamoxifen as stimulatory factors to signal the MAPK cascade, which becomes a predominant proliferation pathway in TAM-R cells.

It would be expected that enhanced interaction between EGFR and ER{alpha} resulted from overexpression of EGFR, ER{alpha}, or both. To our surprise, neither EGFR nor ER{alpha} was up-regulated in TAM-R cells when determined by Western analysis. One significant change after long-term tamoxifen exposure is translocation of ER{alpha} to the cytoplasm. This phenomenon is due to tamoxifen treatment rather than alteration in ER{alpha} expression because it does not occur in the same cell line under estrogen-deprived condition (LTED). Elevation in the levels of extranuclear ER{alpha} increases the opportunity to associate with factors that facilitates activation of the MAPK pathway via nongenomic mechanism. We did find that tamoxifen treatment dramatically increased ER{alpha} levels in cytosol, as shown by confocal microscopy and confirmed by Western blot analysis of cell fractions. Additional evidence came from the results of ICI treatment. The pure antiestrogens, ICI compounds, are also called ER down-regulators because they cause translocation of ER{alpha} to the cytoplasm, enhance the rate of degradation, and shorten ER{alpha} half-life (29, 30). Dauvois et al. (25) reported that cytoplasmic translocation of ER{alpha} was detectable within 15 min of ICI treatment and continued to increase for 1 h. Consistent with these reports, we observed an increase in the amount of ER{alpha} that interacted with EGFR in both MCF-7 and TAM-R cells after 30 min and 1 h of culture with ICI. The interaction was reduced by 4 h. These results indicate that cytoplasmic translocation of ER{alpha} is required for enhanced interaction between ER{alpha} and EGFR. Long-term tamoxifen treatment induces translocation of ER{alpha} from the nucleus to the cytoplasm.

E2 elicits rapid activation of MAPK via extranuclear ER{alpha} in a variety of cell types (31–34). Membrane localization of ER{alpha} usually occurs via protein-protein interactions. Previous studies from our laboratory showed that E2-induced rapid activation of MAPK in MCF-7 cells required the interaction of ER{alpha} with insulin-like growth factor-I (IGF-I) receptor (IGF-IR) via the adapter protein Shc (17, 35). This interaction brought ER{alpha} to the plasma membrane and facilitated ER{alpha}-mediated rapid E2 action (17, 35). Other proteins, such as c-Src, FAK, MNAR (36), and striatin (37), are also reported to form complex with ERs and to be involved in membrane localization of ERs. In TAM-R cells, an increased amount of c-Src was recovered from the complex of EGFR and ER{alpha}. The amount of other adaptor proteins coimmunoprecipited with EGFR or ER{alpha} was unchanged or slightly reduced. These data suggest that one role of kinase active c-Src in TAM-R cells is to retain ER{alpha} in the cytoplasm. The precise molecular mechanism to explain how relocalization of ER{alpha} occurs in TAM-R cells is unclear at the present time. The fact that PP2 treatment reduced the levels of cytoplasmic ER{alpha} implies that c-Src may also be involved in a dynamic process of ER{alpha} translocation. Further studies are needed to investigate in detail the mechanisms mediating this process.

Several lines of evidence indicate the involvement of c-Src in extranuclear signaling of ER{alpha} (38). c-Src activity can be activated by estrogen in breast cancer cells. Mammary epithelial cells derived from c-Src-null mice exhibit delayed MAPK activation in response to exogenous E2 (39). ER{alpha} acts as a G protein coupled receptor (40). The activation of the G proteins increases c-Src activity, which leads to activation and secretion of matrix metalloproteinases (MMP) MMP2 and MMP9. Activated MMPs cleave HB-EGF from cell matrix, and released HB-EGF in turn activates EGFR (16). In addition to ER{alpha}, GPR30 may also play a role in mediating E2-induced rapid activation of c-Src (41). c-Src kinase can increase EGFR activity by phosphorylation of EGFR at tyrosine residues 845 and 1,101. Overexpression of both EGFR and c-Src in murine fibroblasts synergistically increased DNA synthesis and tumor formation in nude mice (42, 43). These results suggest that c-Src not only serves as an adaptor protein in the EGFR/ER{alpha} complex but also acts in concert with these two signal molecules. Synergistic interaction of EGFR, ER{alpha}, and c-Src enhances the responsiveness of MCF-7 breast cancer cells to the mitogenic stimulation of EGF and E2, which allows the cells to survive and proliferate in the presence of tamoxifen.

We noted that TAM-R cells are extremely sensitive to EGF stimulation. This could result from cross talk between growth factor receptors and interactions of EGFR with a non–receptor tyrosine kinase such as c-Src (43). A number of studies suggest that IGF-IR can signal to MAPK through EGFR in different cell types, including breast cancer cells (44, 45). Knowlden et al. (46) recently reported up-regulation of IGF-IR function and transactivation of EGFR by IGF-II in TAM-R breast cancer cells. Interestingly, they found that c-Src was the link between these two pathways. The current study could not rule out the involvement of IGF-IR in tamoxifen resistance. However, the lack of activation of Akt indicates that IGF-IR signaling is not the predominant pathway in support of proliferation in this stage of tamoxifen resistance.

Several endocrine-resistant breast cancer models have been developed. Although up-regulation of growth factor receptor signaling is a common feature, these models display variable activation of kinase cascades. Constitutive MAPK activation was also observed in our long-term estrogen-deprived MCF-7 cells (LTED; ref. 47) and in the models reported by others (48). In contrast to LTED cells, the levels of phosphorylated MAPK in tamoxifen-treated MCF-7 cells increased transiently during the first 2 months and returned to the level of control cells. Another controversial area is the expression of growth factor receptors. For example, EGFR was reported to be 10-fold higher in one antiestrogen-resistant model (49) but has no significant change in the other (50). These differences may reflect the ways that breast cancer cells adapt to different endocrine therapies (estrogen deprivation or drug treatment) and may be related to the biological characteristics of different cell lines. Our model suggests a novel mechanism of tamoxifen resistance. Enhanced interaction of ER{alpha}, EGFR, and c-Src facilitates the activation of the MAPK cascade triggered by EGF or by E2 or tamoxifen via extranuclear function of ER{alpha} (online Supplementary Fig. S2). Cooperation of the ER{alpha} and EGFR pathways allows more efficient usage of the mitogenic factors in the living environment and regrow in the presence of tamoxifen. This model provides experimental evidence for development of new therapeutic strategies to delay tamoxifen resistance or to restore sensitivity to this drug in the cases that resistance has developed.


   Acknowledgments
 
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.

We thank Dr. Zhenguo Zhang for his instruction on immunoprecipitation and immunofluorescent staining, and Jan Redick and Christine Davis for assistance with capturing the confocal microscopic images taken at the Advanced Microscopy Facility of the University of Virginia.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 3/17/06. Revised 10/18/06. Accepted 11/13/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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