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Department of Surgery (F.J.F., A.D.K.H., E.W.M., N.J.O., L.S.Y.), St. Vincent’s University Hospital, and Conway Institute of Biomolecular and Biomedical Research (A.D.K.H., L.S.Y.), University College Dublin, Dublin 4, Ireland
Address all correspondence and requests for reprints to: L. S. Young, Department of Surgery, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland. E-mail: leonie.young{at}ucd.ie.
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Abstract |
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and ER-ß function as transcription factors, and both interact with nuclear regulatory proteins to enhance or inhibit transcription. We hypothesized that coregulators are expressed in breast cancer and may be differentially recruited by ERs in the presence of estrogen and tamoxifen. ER-ß was found to be expressed more frequently in node-negative patients (P < 0.05). Expression of steroid receptor coactivator-1 (SRC-1) was associated with nodal positivity (P < 0.05) and resistance to endocrine treatment (P < 0.001). The spatial coexpression of ER-, ER-ß, and the coregulatory proteins was established using immunofluorescence. In both cell lines (MCF-7 and T47D) and in primary breast cancer cell cultures, ß-estradiol up-regulated ER-ß and coregulator protein expression and increased ER-/ER-ß interaction with the estrogen response element (ERE). 4- Hydroxy-tamoxifen (4-OHT) increased ER- and silencing mediator for retinoid and thyroid receptors (SMRT) expression and increased ER-ERE binding. SRC-1 and SMRT were identified at the ER-ERE complex, and interactions between ER isoforms and coregulatory proteins were determined using immunoprecipitation. Both ER- and ER-ß preferentially bound SRC-1 in the presence of ß-estradiol. Conversely, in cells treated with 4-OHT, ER- and ER-ß bound SMRT. Differential recruitment of SRC-1 and SMRT by ER- and ER-ß in the presence of ß-estradiol and 4-OHT may be central to the response of the tumor to endocrine treatment. |
Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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ER is encoded for by two genes, ER- and ER-ß, both of which are members of the nuclear receptor family of ligand-activated transcriptional factors. In response to a ligand, ER dimerizes and binds to DNA response elements on the promoter/enhancer regions of target genes. Although the predominant gene expressed is ER-, expression of ER-ß mRNA has been reported in 40% of breast cancers (3). Differential signaling between ER- and ER-ß has been demonstrated with estrogen and tamoxifen at the AP-1 response element (4). The ratio of ER- to ER-ß may therefore play a distinct role in gene regulation and consequently be important in determining the response to SERMs such as tamoxifen.
The extent of gene regulation is influenced not only by the ligand but also by the presence of specific coregulatory proteins, present at rate-limiting levels, which modulate transcription. Recently, a large number of nuclear receptor interacting proteins have been isolated using various screening strategies. These include steroid receptor coactivator-1 (SRC-1) which has intrinsic histone acetyl-transferase activity enhancing transcription by enabling access of transcription factors and RNA polymerase II core machinery to target DNA (5). In contrast to SRC-1, other proteins were discovered that interact with several unliganded steroid receptors, including the ER. These include nuclear receptor corepressor (NCoR) and its homolog, silencing mediator for retinoid and thyroid hormone receptor (SMRT). Both of these proteins contain transferable repression domains, and several lines of evidence suggest that they function as repressors by recruitment of a complex containing Sin3 histone deacetylase and other regulatory proteins (6, 7). ER is thought to recruit corepressor proteins NCoR and SMRT in the presence of antagonists such as tamoxifen, and this recruitment is thought to be essential for full antagonist activity (8). Therefore, tamoxifen may exert its effect not by a simple passive mechanism driven principally by competitive receptor antagonism but through the active recruitment of coactivator and corepressor proteins to produce a mixed transcriptional phenotype.
Expression and regulation of steroid nuclear regulatory proteins may therefore be central to the response of breast cancer cells to estrogen and tamoxifen. In this paper we elucidate the relevance of the nuclear coregulatory proteins SRC-1 and SMRT in human breast cancer. Here we report that SRC-1 is significantly associated with axillary node positivity and recurrence and that SRC-1 and SMRT can be colocalized with ER- and ER-ß within the nucleus of breast cancer epithelial cells. Estrogen and tamoxifen were found to differentially regulate the expression and the recruitment of coregulatory proteins SRC-1 and SMRT to the ER-estrogen response element (ER-ERE) complex in primary breast cell cultures and in breast cancer cell lines.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Patients were selected on the basis of those who received tamoxifen only as adjuvant therapy after surgery. All patients had stage I–II breast cancer at presentation and were assessed by abdominal ultrasound, chest x-ray, and bone scintigraphy before surgery. Sixty-two percent (n = 32) of the group received radiotherapy after surgery and none received adjuvant chemotherapy after their initial surgery. All patients received tamoxifen 20 mg/d for 5 yr, which was discontinued only in those patients who suffered a relapse while on endocrine therapy.
Immunohistochemistry
Five-micron-thick tissue sections were cut from paraffin-embedded breast tumor tissue blocks and mounted on Superfrost Plus slides (BDH, Poole, UK). Sections were dewaxed, rehydrated, and washed in PBS. Endogenous peroxidase was blocked using 3% hydrogen peroxidase in PBS for 10 min. Antigen retrieval was performed by immersing sections in 0.6 M citrate buffer and microwaving on high power for 7 min. Antigens were detected using the Vectastain Elite kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. Briefly, sections were blocked in serum for 90 min. Sections were incubated with the following primary antibodies: rabbit antihuman ER- (1 µg/ml), goat antihuman SRC-1 (1 µg/ml), goat antihuman SMRT (1 µg/ml) (Santa Cruz, CA), and mouse antihuman ER-ß (1 in 20 in PBS) (Serotec, Oxford, UK) for 60 min at room temperature. Sections were subsequently incubated with corresponding biotin-labeled secondary antibody (1 in 2000) for 30 min, followed by peroxidase-labeled avidin-biotin complex. Sections were developed in 3,3-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. Negative controls were performed using matched IgG controls (Dako, Glostrup, Denmark). Sections were examined under a light microscope. Immunostained slides were scored for ER-, ER-ß, SRC-1, and SMRT using the Allred scoring system (9). Each entire slide was evaluated using light microscopy. First, a proportion score was assigned, which represented the estimated portion of positively stained tumor cells (none = 0; <1% = 1; >1% and <10% = 2; >10% and <33% = 3; >33% and <66% = 4; >66% = 5). Next, an intensity score was assigned that represented the average intensity of the positive tumor cells (none = 0; weak = 1; intermediate = 2; strong = 3). The proportion and intensity scores were then added to obtain a total score, which ranged from 0–8. A total score greater than two was taken to indicate positivity. Independent observers, without knowledge of prognostic factors, scored slides.
Immunofluorescent microscopy
Breast cancer sections were prepared as above and incubated in goat serum (ER-) or sheep serum (ER-ß) for 60 min. Rabbit antihuman ER- (10 µg/ml in 10% human serum) or mouse antihuman ER-ß (1:2 dilution with PBS in 10% human serum) was placed on each slide for 90 min. The sections were rinsed in PBS and incubated with the corresponding secondary fluorochrome-conjugated antibody (1 in 100) (Sigma-Aldrich, Steinheim, Germany) for 60 min. The slides were rinsed in PBS, blocked in rabbit serum for 90 min, and washed with PBS. Each slide was incubated with either goat antihuman SRC-1 or goat antihuman SMRT (both 10 µg/ml in 10% human serum) for 90 min, followed by a wash in PBS. The slides were incubated with the corresponding fluorochrome-conjugated antibody (1 in 100) for 60 min. Sections were rinsed in PBS and mounted using fluorescent mounting media (Dako). Sections were examined under a fluorescent microscope. Negative controls were performed using matched IgG.
Cell culture stimulations
After ethical approval, breast tumor specimens were obtained from 30 patients undergoing surgery for removal of a histologically confirmed breast tumor. The tissue was washed in PBS, minced, and placed in 20 ml of Hank’s balanced salt solution without calcium or magnesium (Life Technologies, Inc., Paisley, UK) supplemented with 1 µM EDTA and 1 µM dithiothreitol.
The sample was rotated gently for 40 min followed by centrifugation of the resultant cell suspension at 1700 rpm for 10 min. Cells were then cultured in RPMI containing 5 µg/ml insulin, 10 µg/ml transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM ß-estradiol, 10 mM HEPES, 2 mM glutamine, 10% fetal calf serum (wt/vol), and 5% Ultroser (Gibco, Biosciences, Dublin, Ireland, UK). For breast cancer cell lines, MCF-7 and T47-D breast cancer cell lines (European Collection of Cell Cultures, Wiltshire, UK) were maintained in RPMI media (Life Technologies) supplemented with 5% fetal calf serum, 200 µg/ml penicillin-streptomycin, and 5 µg/ml fungizone (Life Technologies). Cells were incubated in a humidified atmosphere of 5% CO2 at 37 C.
Experiments were carried out when cells reached 90% confluence. Cells were serum and steroid depleted for 24 h before stimulation. Cells were then incubated in the presence and absence of ß-estradiol and 4-hydroxytamoxifen (4-OHT) (10–8 M) for 24 h and harvested. Total protein was extracted using lysis buffer (1% Ipegal, 0.5% deoxycholic acid, 0.1% SDS, and 1x PBS) with Pefabloc (5 µg/ml). Nuclear and cytoplasmic protein was extracted using a NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL) protein extraction kit according to the manufacturer’s instructions. Cell lysates were prepared in an appropriate volume of lysis buffer with protease inhibitors (0.5% Pefabloc) and normalized for protein content.
Flow cytometery
To confirm that the primary breast tumor cells were of epithelial origin, cells were stained with ethidium bromide acradine orange and examined under UV microscopy. On examination 99% of cells had an epithelial morphology and there was 98% cell viability. Flow cytometric analysis using a phycoerythrin-labeled pan-leukocyte marker (CD45 RA and RO) (Becton Dickinson, Oxford, UK) confirmed that less than 1% of the primary breast culture cells were of hemopoietic origin.
Western blotting
Proteins (30–120 µg) were resolved on a polyacrylamide gel (12% for ER- and ER-ß, 7% for SRC-1 and SMRT) at 110 V for 120 min and were transferred to a nitrocellulose membrane (250 mA for 60 min). Membranes were incubated for 60 min in blocking buffer (5% nonfat dry milk and 0.1% Tween in PBS) at room temperature and subsequently with primary antibody, rabbit antihuman ER- (2 µg/ml), rabbit antihuman ER-ß (Zymed Laboratories, Inc., South San Francisco, CA) (2 µg/ml), goat antihuman SRC-1 (2 µg/ml), or SMRT (2 µg/ml) in blocking buffer overnight at 4 C. The membranes were washed before incubation with the corresponding horseradish peroxidase secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (1 in 2000) in blocking buffer for 60 min at room temperature. The membranes were washed and developed with either chemiluminescence (Santa Cruz) or intensified luminescence (Pierce).
EMSAs
Nuclear proteins were prepared as described above. For EMSAs, 2 µg of nuclear extract was incubated for 20 min in the presence of 20 mM HEPES (pH 7.9), 5 mM MgCl2, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 8% Ficoll, 600 mM KCl, 500 ng/µL poly-deoxyinosinic-dexycytidylic acid, 50 mM dithiothreitol, and -[32P]dCTP-labeled double-stranded olignucleotide for ERE, 5'-ATGCGTAAGGTCAGAGTCACCTTGC-3', with the consensus sequence for ERE underlined. For supershift experiments, antibodies against ER-, ER-ß, SRC-1, and SMRT were added after the initial 20-min incubation, then incubated for an additional 20 min. The samples were electrophoresed through a 5.5% nondenaturing polyacrylamide gel in 0.5x Tris-borate-EDTA buffer. For competition studies, the reaction was performed as described with 50x molar excess of unlabeled probe. Positive controls were recombinant ER- and ER-ß (Panvera, Carlsbad, CA), and negative controls were nuclear extracts from the ER-negative SKBR3 breast cancer cells.
To determine the relative expression of proteins at the ERE, EMSA gels were transferred to a nitrocellulose membrane (250 mA for 90 min) and were subsequently immunoblotted with antibodies directed against ER-, ER-ß, SRC-1, and SMRT.
Immunoprecipitation
Complex formation between coregulators and ER- and ER-ß was examined by using breast tumor cell lysates. Whole-cell lysates were prepared as described above. One hundred micrograms of the lysate were immunoprecipitated with 2 µg of either anti-SRC-1 or anti-SMRT (Santa Cruz) for 60 min at 4 C. The precipitates were collected for 1 h on protein A/G-agarose (Santa Cruz). After washing with RIPA buffer, precipitates were resuspended in Laemmli SDS sample buffer and resolved on 12% SDS-PAGE. After transfer to nitrocellulose, the proteins were probed with either anti-ER- (Santa Cruz) (2 µg/ml) or anti-ER-ß (Zymed) (2 µg/ml), followed by the corresponding peroxidase-conjugated secondary antibody (1 in 2000 for ER-, 1 in 5000 for ER-ß). Labeled bands were detected using chemiluminescence (Santa Cruz) or intensified luminescence (Pierce).
Clinicopathological parameters
Variables analyzed included tumor size, tumor grade, presence of lymphovascular invasion, and axillary node status. A recurrence was defined as any local (chest wall) or systemic recurrence during the follow-up period.
Statistical analysis
Statistical analysis was carried out using the 
2 test for categorical variables. Two-sided P values of <0.05 were considered to be statistically significant.
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Results |
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ER-, ER-ß, SRC-1, and SMRT were localized within paraffin-embedded human breast cancer tissue using immunohistochemistry (n = 52). Strong positive staining for ER- was detected in the nuclei of invasive ductal and invasive lobular breast tumor epithelial cells, whereas expression within the cytosol was found to be negligible (Fig. 1A
). Using antibodies directed against the wild-type ER-ß, recognizing the ER-ß1 isoform of the protein, consistent, strong ER-ß staining was detected in the nuclei and to a lesser extent in the cytosol of tumor epithelial cells (Fig. 1B).
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, C and D). Specific staining was absent when the primary antibody was substituted with matched IgG.
Qualitative expression of ER-, ER-ß, and the coregulators SRC-1 and SMRT were correlated with clinicopathological data (Table 1). ER-ß expression in breast tumor tissue was found to be significantly associated with a positive response to endocrine treatment (P < 0.05). Conversely, expression of the coactivator protein SRC-1 was associated with poor outcome. SRC-1 was found to be expressed more frequently in node-positive compared with node-negative patients (P < 0.05) in a cohort with a median follow-up of 56 months. Furthermore, SRC-1 correlated with insensitivity to endocrine therapy as 92% of patients who suffered a recurrence while on endocrine treatment expressed SRC-1, whereas only 10% of those that did not have recurrent disease were SRC-1 positive (P < 0.001).
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Given evidence that SRC-1 and SMRT interact directly with steroid receptors to modulate steroid-dependent signaling, the coregulatory proteins would be expected to be expressed in the same cell as ER. Immunofluorescence staining was undertaken to confirm subcellular localization of SRC-1 and SMRT and to identify coexpression with ER- and ER-ß. SRC1 and SMRT were found to be expressed in the nucleus of the cell. Both SRC-1 and SMRT were found to be colocalized with ER- and ER-ß within a subset of breast tumor epithelial cells (Fig. 2).
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and ER-ß in primary breast tumor cell cultures and in breast tumor cell lines
Complete and incomplete consensus binding sites for ER have been located within the proximal promoter region of both ER- and ER-ß. The ability of estrogen and 4-OHT to regulate the protein expression of ER- and ER-ß in breast cancer cell lines and primary breast cancer cells was determined. Clinicopathological data of primary breast cancer cultures are shown in Table 2. ER- was expressed predominantly in the nuclear fraction in primary breast tumor cells and in MCF-7 cells. 4-OHT induced an up-regulation in ER- expression in the primary breast cultures and in the cell line, whereas ß-estradiol induced no appreciable change in ER- expression (Fig. 3A). ER-ß was also found to be expressed predominantly in the nuclear portion of the primary breast cancer cells and in the ER-ß-positive breast cancer cell line T47-D. ER-ß expression was up-regulated in the presence of ß-estradiol, whereas 4-OHT induced a marked decrease (Fig. 3B).
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Using bioinformatic techniques (MatInspector, Genomatrix Software), putative incomplete EREs were identified in the promoter region of SRC-1 and SMRT (Fig. 4). Because estrogen has been demonstrated to bind incomplete estrogen half-sites, the ability of estrogen and 4-OHT to regulate the protein expression of SRC-1 and SMRT was examined (10). Both SRC-1 and SMRT were found to be expressed in the nuclear but not in the cytoplasmic fraction of MCF-7 cells. An increase in SRC-1 protein expression was detected in the presence of ß-estradiol but not 4-OHT (Fig. 4A). Conversely, SMRT protein expression was found to be up-regulated in the presence of both ß-estradiol and 4-OHT (Fig. 4B).
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- and ER-ß-ERE binding and recruitment of SRC-1 and SMRT
To determine the ability of ER- and ER-ß to bind to the ERE in the presence of ß-estradiol and 4-OHT and, once bound, to identify SRC-1 and SMRT in the transcription factor-DNA complex, gel shift assays were performed. Using published oligonucleotide sequences, the ability of nuclear extracts from nontreated primary breast cancer cell cultures (Fig. 5A) and MCF-7 breast cancer cells (data not shown) to bind to the DNA-binding motif was compared with cells treated with ß-estradiol and 4-OHT. ER-ERE binding was induced in the presence of ß-estradiol and to a lesser extent 4-OHT in comparison with control. A supershift induced by preincubation of the nuclear extracts with anti-ER- and -ER-ß antibodies established that both ER isoforms were present in the protein-DNA complex. Increased ER-ERE binding was observed in the presence of 4-OHT and ß-estradiol compared with control. The presence of ER and the coregulatory proteins in the transcription factor-DNA complex was investigated by preincubation with antibodies directed against SRC-1 and SMRT. ER- and ER-ß were both found to be present at the ERE, both under control conditions and in the presence of ß-estradiol and 4-OHT. SRC-1 was found to complex at the ERE in untreated cells and in the presence of ß-estradiol, whereas SMRT was found to be present with 4-OHT.
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, ER-ß, SRC-1, and SMRT (Fig. 5B). Bands detected using specific antibodies were found to migrate to the same height as those detected using the radiolabeled ERE. Under basal conditions in breast cancer cells, a greater SRC-1 expression was seen at the DNA complex in comparison with SMRT. In the presence of ß-estradiol, SRC-1 expression was induced at the ER-ERE complex, whereas there was no expression of SRC-1 at the protein-DNA complex detected in the presence of 4-OHT. In contrast, SMRT expression was increased at the ER-ERE in the presence of 4-OHT in comparison with control cells and cells treated with ß-estradiol.
The ability of ER to differentially recruit SRC-1 and SMRT in the presence of ß-estradiol and 4-OHT was confirmed using coimmunoprecipitation (Fig. 5C). Both ER- and ER-ß preferentially interacted with SRC-1 in the presence of ß-estradiol, compared with 4-OHT. 4-OHT diminished the ER-SRC-1 binding seen in the presence of ß-estradiol. Conversely, 4-OHT induced corepressor SMRT-ER interaction. ß-Estradiol failed to induce ER-SMRT interaction compared with basal conditions and had no effect on 4-OHT-induced SMRT-ER interaction.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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and ER-ß have been performed on breast cell lines. Here we report ex vivo studies on human breast cancer tissue and in vitro functional studies on primary breast cancer cells, cultured from purified breast epithelial cancer cells. We present evidence of SRC-1 and SMRT coexpression with the steroid receptors ER- and ER-ß and their differential regulation and recruitment to the ER-ERE complex with ß-estradiol and 4-OHT in human breast cancer.
We found ER- and ER-ß to be widely expressed in the nuclei of breast cancer epithelial cells. The role of ER-ß in mammary tumor progression and its value as a prognostic marker remains controversial. Clinical studies have suggested that ER-ß protein may be associated with well differentiated tumors (18), whereas molecular data propose that ER-ß can signal through AP-1 sites to induce agonist activity of SERMs, including that of tamoxifen (19). In this study ER-ß protein expression was found to be significantly associated with a positive response to endocrine treatment. To determine the functional effect of estrogen and the selective estrogen receptor modulator tamoxifen on the protein expression and DNA interaction of ER- and ER-ß, we developed primary breast cancer cell cultures from breast cancer tissue. The development of primary breast cancer cultures has been previously described; however, work elucidating functional alterations in steroid receptor expression remains limited (20). In this study, there was no alteration in ER- protein expression observed in the presence of ß-estradiol, whereas ER- protein up-regulation occurred in the presence of 4-OHT. These findings are consistent with previous studies examining the regulation of ER- expression (21). Conversely, in primary breast cancer cell cultures, an up-regulation of ER-ß protein was observed with ß-estradiol, whereas treatment with 4-OHT induced a decrease in ER-ß expression. Increased ER-ß mRNA expression in the presence of ß-estradiol has previously been described in the T47D breast tumor cell line (22).
In breast cancer, ER interacts with its response element (ERE) on target genes to regulate transcription. The ability of ER- and ER-ß to interact with ERE in breast cancer cell lines in the absence of ligand and in the presence of estrogen and tamoxifen has been previously demonstrated (23). Here, in primary breast cancer cell cultures, estrogen and to a lesser extent 4-OHT induced ER-ERE interactions compared with untreated cells. There was no difference between the recruitment of ER- in relation to ER-ß noted in the presence of either ligand. Based on these observations, we suggest that the expression of ER-ß as a beneficial prognostic indicator in breast cancer may not be a straightforward ligand-nuclear receptor-activated event but may involve other signaling mechanisms.
The coregulators SRC-1 and SMRT function through interaction with nuclear receptors to activate or repress transcription. Nuclear expression of the coactivator amplified in breast cancer (AIB1) has been observed in both breast cancer tissue and cell lines (24). In this study, we found both SRC-1 and SMRT to be localized primarily within the nucleus of breast epithelial cells using immunohistochemistry and immunofluorescence. As both SRC-1 and SMRT have been shown to interact in vitro with ER- and ER-ß to regulate ER-dependent signaling, it would be expected that the coregulators would be expressed in the same cells. Azorsa et al. (25) have demonstrated colocalization of the coactivator AIB1 and ER in AIB1-amplified cells lines, although in previous work the expression of SRC-1 and ER- were found to be segregated in the rat mammary epithelium (26). In this study, we found the coregulators SRC-1 and SMRT to be coexpressed with both ER- and ER-ß in human breast cancer epithelial cells. This indicates that these regulatory proteins may have a potential impact on the functional expression of ER-target genes in breast cancer. We therefore determined the ability of estrogen and 4-OHT to recruit coregulators to the ER-ERE site. Previous reports suggest that unliganded steroid receptors are occupied by corepressor proteins. Of interest, in this study in breast cancer cells under basal conditions, the coactivator protein SRC-1 and not the corepressor SMRT was present at the ER-ERE. Nuclear steroid receptors, including ER, have been shown to interact with coactivators and corepressors (27). In this study, differential recruitment of SRC-1 and SMRT to the ER-ERE in primary breast cell cultures was observed in the presence of ß-estradiol and 4-OHT. In the presence of ß-estradiol, SRC-1 was recruited to the response element, whereas in the presence of tamoxifen, corepressor ER-ERE binding was induced. To further determine whether clinical associations between ER-ß positivity and response to endocrine treatment could be due to differential interaction between ER- and ER-ß and coregulatory proteins in the presence of estrogen and tamoxifen, we investigated steroid receptor regulatory protein interactions. Previous studies have proposed that a defined hierarchy governs interactions between ER isoforms and the SRC/p160 family of coactivators, suggesting that AIB 1 is the preferred partner for ER- (28). There was no difference, however, between the ER isoforms in the ability to recruit either SRC-1 or SMRT demonstrated here. Both ER- and ER-ß isoforms interacted with SRC-1 in the presence of ß-estradiol and, conversely, SMRT in the presence of 4-OHT. These data and our observations that 4-OHT down-regulates expression of ER-ß protein with a corresponding increase in ER- protein suggests that associations between ER-ß positivity and sensitivity to endocrine treatment may be due in part to reduced levels of ER-ß protein in the presence of tamoxifen.
Recent observations by Sohn et al. (29) have suggested that steroid receptors exist in a state of equilibrium between the active and inactive state and that the ratio of coactivator to corepressor in a given cell type may determine the potency of transcriptional activation. We have shown that in human breast cancer cells SRC-1 is recruited to the ER-ERE complex in the presence of estrogen, whereas SMRT is recruited in the presence of 4-OHT. However, the composition of the complex may be determined not only by the ligand-occupied steroid receptor but also by the levels of coactivator and corepressor present at the DNA-protein complex. Bioinformatic assessment of the promoter region of the coregulators SRC-1 and SMRT revealed putative ERE imperfect half-sites. Mathematical studies by Tyulmenkov et al. (10) have shown a correlation between estrogen transcriptional activity and the homology of the response element, suggesting a possible involvement of ER in the modulation of these coregulatory proteins. We therefore looked for the ability of estrogen and tamoxifen to modulate the expression of these coregulators. In MCF-7 breast cells, SRC-1 and SMRT protein expression was seen exclusively in the nuclear fraction. Differential steroid regulation of coregulator expression in breast cancer cells has previously been examined for the coactivators AIB1 and receptor interacting protein (RIP140) (30, 13).
Here, SRC-1 protein expression was detected in untreated MCF-7 breast cancer cells, and the protein expression was up-regulated in the presence of ß-estradiol but not 4-OHT. Conversely, SMRT was not found under basal conditions, although expression was detected when cells were treated with both ß-estradiol and 4-OHT. These data are in contrast to reports of SRC-1 and SMRT expression in pituitary cells, which demonstrated decreased coregulator mRNA expression subsequent to estrogen treatment (31). Tamoxifen may therefore achieve ER-target gene silencing not only through the recruitment of SMRT to the ER-ERE but also by increasing the protein levels available to interact with the liganded ER. Development of resistance to endocrine treatment may therefore occur through alterations in the delicate balance between coactivators and corepressors such as SRC-1 and SMRT at the steroid receptor response element complex.
To test the hypothesis that development of insensitivity to endocrine treatment may be due in part to alterations in the ratio of coactivator to corepressor, we examined the expression of SRC-1 and SMRT in our cohort of breast cancer patients. The relative expression of coregulatory proteins has previously been associated with tumor progression and the development of resistance to estrogen modulatory therapies (11). However, there is little consensus in the literature as to the nature of these changes. Although coactivators SRC-1 and AIB1 have both been associated with tumor progression at the mRNA level, studies investigating coactivator RNA expression and resistance to estrogen modulators failed to demonstrate a significant correlation (32, 33). Using immunohistochemistry, we have demonstrated a positive correlation between SRC-1 protein expression and nodal positivity and disease recurrence while on endocrine treatment in human breast cancer tissue.
Endocrine therapies including tamoxifen have an integral role in the adjuvant management of breast cancer; however, whereas most ER-positive patients will initially respond, many will eventually relapse. ER- and ER-ß interact with coregulatory proteins to modulate gene transcription. These data indicate that in human breast cancer tamoxifen induces target gene silence by recruitment and regulation of corepressor proteins such as SMRT. The development of resistance may therefore be due in part to the alterations in the relative level of coactivator to corepressor at the ER-ERE. This hypothesis is supported by clinical observations of a significant association between SRC-1 and resistance to endocrine therapy. Elucidation of the role of the coregulatory proteins in the aberrant functionality of the steroid receptors may provide a global target in the treatment of breast cancer.
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Footnotes |
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Abbreviations: AIB1, Amplified in breast cancer coactivator; ER, estrogen receptor; ERE, estrogen response element; 4-OHT, 4-hydroxytamoxifen; SERM, selective ER modulator; SMRT, silencing mediator for retinoid and thyroid receptors; SRC-1, steroid receptor coactivator-1.
Received June 17, 2003.
Accepted September 29, 2003.
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Y.-K. Leung, P. Mak, S. Hassan, and S.-M. Ho Estrogen receptor (ER)-beta isoforms: A key to understanding ER-beta signaling PNAS, August 29, 2006; 103(35): 13162 - 13167. [Abstract] [Full Text] [PDF]
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L. C Murphy and P. H Watson Is oestrogen receptor- {beta} a predictor of endocrine therapy responsiveness in human breast cancer? Endocr. Relat. Cancer, June 1, 2006; 13(2): 327 - 334. [Abstract] [Full Text] [PDF]
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S.-Q. Kuang, L. Liao, S. Wang, D. Medina, B. W. O'Malley, and J. Xu Mice Lacking the Amplified in Breast Cancer 1/Steroid Receptor Coactivator-3 Are Resistant to Chemical Carcinogen-Induced Mammary Tumorigenesis Cancer Res., September 1, 2005; 65(17): 7993 - 8002. [Abstract] [Full Text] [PDF]
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D. G. Monroe, F. J. Secreto, M. Subramaniam, B. J. Getz, S. Khosla, and T. C. Spelsberg Estrogen Receptor {alpha} and {beta} Heterodimers Exert Unique Effects on Estrogen- and Tamoxifen-Dependent Gene Expression in Human U2OS Osteosarcoma Cells Mol. Endocrinol., June 1, 2005; 19(6): 1555 - 1568. [Abstract] [Full Text] [PDF]
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E. Myers, A. D.K. Hill, G. Kelly, E. W. McDermott, N. J. O'Higgins, Y. Buggy, and L. S. Young Associations and Interactions between Ets-1 and Ets-2 and Coregulatory Proteins, SRC-1, AIB1, and NCoR in Breast Cancer Clin. Cancer Res., March 15, 2005; 11(6): 2111 - 2122. [Abstract] [Full Text] [PDF]
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 F J Fleming, E Myers, G Kelly, T B Crotty, E W McDermott, N J O'Higgins, A D K Hill, and L S Young Expression of SRC-1, AIB1, and PEA3 in HER2 mediated endocrine resistant breast cancer; a predictive role for SRC-1 J. Clin. Pathol., October 1, 2004; 57(10): 1069 - 1074. [Abstract] [Full Text] [PDF]
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