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Molecular Cancer Research 2:523-531 (2004)
© 2004 American Association for Cancer Research

Signaling and Regulation

ERß Sensitizes Breast Cancer Cells to Retinoic Acid: Evidence of Transcriptional Crosstalk1

Caroline Rousseau, Jessica N. Nichol, Filippa Pettersson, Marie-Claude Couture and Wilson H. Miller, Jr.

Departments of Oncology and Medicine, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital and McGill University, Montreal, Quebec, Canada

Requests for reprints: Wilson H. Miller Jr., Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, McGill University, 3755 Cote Sainte Catherine Road, Montreal, Quebec, Canada H3T 1E2. Phone: 514-340-8222, ext. 4365; Fax: 514-340-7576. E-mail: wmiller{at}ldi.jgh.mcgill.ca


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The ability of retinoids to inhibit breast cancer cell growth correlates with estrogen receptor (ER) {alpha} status, as shown by the antiproliferative effects of retinoids in ER{alpha}-positive breast cancer cells and their use as chemopreventive agents in premenopausal women. The discovery of ERß, also present in breast cancer cells, has added a new level of complexity to this malignancy. To determine the retinoid response in ERß-expressing breast cancer cells, we used retroviral transduction of ERß in ER-negative MDA-MB-231 cells. Western blot and immunofluorescence confirmed expression and nuclear localization of ERß, whereas functionality was shown using an estrogen response element–containing reporter. A significant retinoic acid (RA)–mediated growth inhibition was observed in the transduced ERß-positive cells as shown by proliferation assays. Addition of estradiol, tamoxifen, or ICI 182,780 had no effect on cell growth and did not alter RA sensitivity. We observed that retinoids altered ERß-mediated transcriptional activity from an estrogen response element, which was confirmed by decreased expression of the pS2 gene, and from an activator protein response element. Conversely, the expression of ERß altered RA receptor (RAR) ß expression, resulting in greater induction of RARß gene expression on RA treatment, without altered expression of RAR{alpha}. Our data provide evidence of transcriptional crosstalk between ERß and RAR in ERß-positive breast cancer cells that are growth inhibited by RA.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Estrogens are potent mitogens in the mammary gland that are required for normal development but are also involved in the progression of mammary carcinoma. The action of estrogen is mediated by binding to the estrogen receptor (ER), a ligand-activated transcriptional factor. Although it was initially believed that the action of estrogen was mediated by a single ER (ER{alpha}), it was subsequently determined that a second ER (ERß) exists (1, 2). The two receptors are highly homologous in the DNA binding domain (region C) and ligand binding domain (region E). However, they greatly differ in the NH2-terminal A/B domain and hinge region (2). The tissue distribution of ER{alpha} and ERß is equally divergent, with ER{alpha} being most highly expressed in the pituitary, vagina, uterus, and breast and ERß in the ovary, prostate, and lung. The presence of ERß in the breast, albeit in lower concentration than ER{alpha}, has led to deliberation regarding its role in mammary development and tumorigenesis (3). Sixty percent to 70% of breast epithelial cells express ERß at all stages of breast development, whereas ER{alpha} expression varies according to the developmental stage of the mammary gland (4). Studies with ER{alpha} knockout mice have shown that ERß does not mediate E2-dependent growth and development of the mammary gland (5). Because many breast tumors express ER{alpha} alone or in combination with ERß (6), there is interest in determining the role of ERß in breast cancer. Some groups have found that ERß correlates with low biological aggressiveness of breast cancer and can even inhibit proliferation and invasion of breast cancer cells (4, 7). In contrast, others have indicated that the ratio of ER{alpha} to ERß changes during breast cancer progression, with increased expression of ERß in relapsed patients exhibiting tamoxifen resistance (8).

The clinical approaches to controlling hormonally responsive breast cancer have primarily focused on ER{alpha} and its target genes. In patients with hormonally responsive breast cancer, current treatment involves blocking the action of ER{alpha} using anti-estrogen therapies. However, hormonal treatment is limited by the development of resistance to tamoxifen and alternative therapies targeting other signaling pathways need therefore be explored.

Retinoids are derivatives of vitamin A that induce differentiation in the treatment of acute promyelocytic leukemia and can cause growth inhibition in a variety of other cell types, including breast cancer cells (9-12). Several natural and synthetic retinoids can inhibit the development of mammary tumors and cause regression of established tumors in rats (13-15). Furthermore, clinical evidence supports the benefit of retinoids for breast cancer prevention in premenopausal women (16, 17).

Retinoids mediate their effects by binding to a group of nuclear receptors [retinoic acid receptors (RAR) and retinoid X receptors (RXR)] belonging to the superfamily of nuclear receptors that includes ER. These receptors are transcription factors that heterodimerize to bind to RA response elements (RARE) present in the promoter regions of target genes (18). Interestingly, the response to retinoids in breast cancer cell lines seems to correlate with the expression of ER{alpha}, suggesting a possible crosstalk between the RA and the ER pathways (19, 20). We have reported previously that the expression of ER{alpha} in ER{alpha}-negative human breast cancer cell modulates RAR signaling. We found that stable expression of ER{alpha} in the ER-negative human breast cancer cell line MDA-MB-231 led to increased activity of an RARE reporter construct and sensitized the cells to growth inhibition by RA (20, 21). Because breast tumors can express both isoforms of the ER, there exists the potential for ER signaling to be mediated by ER{alpha}, ERß, or both.

To evaluate the effects of ERß on retinoid-mediated growth inhibition, we engineered human ERß (hERß) stably transduced cells from the ER-negative breast cancer cell line, MDA-MB-231, using retroviral technology. We observed several similarities between the stable ERß-expressing cell line and the ER{alpha}-expressing breast cancer cells with regard to the growth and transcriptional response to retinoids. In addition, we noted that retinoids also inhibited the transcriptional activity from estrogen response element (ERE)–driven promoters. Our results provide evidence of transcriptional crosstalk between ERß and RAR and support the use of retinoids to target subpopulations of breast cancer cells expressing functional ERß.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Stable Expression of ERß
To determine if ERß can alter retinoid-mediated growth inhibition and transcription, we generated stable expression of hERß in the ER-negative parental MDA-MB-231 human breast cancer cell line (Fig. 1A). The ERß cDNA was inserted upstream of an internal ribosomal entry site and the enhanced green fluorescence protein (eGFP) gene in the HC2 retroviral vector, thereby allowing us to use flow cytometric analysis to monitor transduction efficiency. MDA-MB-231 cells were transduced with either the empty HC2 retroviral vector containing only eGFP (Ctrl) or the retroviral vector expressing both ERß and eGFP (ERß). To achieve the highest transduction efficiency, cell sorting was done based on green fluorescence and a shift in fluorescence intensity was evident for both the retrovirus control and the ERß-transduced cell lines (Fig. 1B). In all experiments described, both the parental cell line (MDA-MB-231) and the empty retroviral transduced cell line (Ctrl) were used as ER-negative controls. However, to avoid superfluous data, only the empty retroviral transduced cell line (Ctrl) will be shown herein. Transduced polyclonal stable cell lines were tested for their expression of ERß by Western blots done on whole cell extracts. Protein expression of ERß at ~55 kDa was evident in the transduced cell line (ERß) and, as expected, was absent in the cell line expressing only the empty retroviral vector (Ctrl; Fig. 1C). Localization of ERß to the nucleus was further confirmed using immunofluorescence (Fig. 1D).



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  		FIGURE 1. Retroviral-mediated expression of hERß in MDA-MB-231 ER{alpha}-negative breast cancer cells. A. The cDNA of wild-type hERß was cloned into the bicistronic murine stem cell virus-based retroviral vector. This vector contains a packaging signal ({Psi}), multiple cloning site (MCS), internal ribosomal entry site (IRES), and eGFP flanked by two long terminal repeats (LTR). B. Flow cytometric analysis of transduced MDA-MB-231 cells subsequent to cell sorting. Solid lines, eGFP expression in the empty vector transduced cells (Ctrl) and ERß-transduced cells (ERß); dashed lines, untransduced MDA-MB-231 cells. C. ERß protein expression of the transduced cells was analyzed by immunoblots using cell extracts (50 µg) and the QED anti-ERß antibody. ß-actin, loading control. D. Immunostaining of transduced cells using an ERß antibody (right panels) and visualized by fluorescence microscopy. 4',6-Diamidino-2-phenylindole staining (DAPI) was included to visualize the nucleus (left panels).

 
Growth and Transcriptional Response to ERß Ligands
Several groups have shown previously that reintroducing ER{alpha} into an ER{alpha}-negative cell line alters the proliferative response to the ER{alpha} agonist, estradiol (22, 23). In the ERß stably transduced cell line (ERß), treatment with estradiol, tamoxifen, or ICI 182,780 resulted in no significant change in proliferation (Fig. 2A). This agrees with a previous study in which the growth rate of ERß-positive breast cancer cells was unaffected by estradiol or ICI 182,780 (4). To validate the functionality of the expressed ERß, we investigated the transcriptional activity from a reporter construct containing three tandem EREs in the presence of known ERß ligands. We observed an induction of transcription in response to estradiol and complete inhibition of transcription from this reporter in the presence of tamoxifen and ICI 182,780 in the ERß-positive cells. As expected, these ligands had no effect in the cell line expressing only the retroviral vector (Ctrl). Increased baseline transcription in the ERß cells may be attributed to residual estrogen in medium or to ligand-independent receptor activity. These data confirm the functionality of ERß in the stably transduced cell line.



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  		FIGURE 2. Effect of ERß ligands on the growth and transcriptional properties of ERß-transduced cells. A. After 6 days of treatment with 10–7 mol/L estradiol (E2), 10–7 mol/L tamoxifen (OHT), or 10–7 mol/L ICI 182,780 (ICI), cell number was assessed using sulforhodamine B staining as described in Materials and Methods. Columns, average of two separate experiments in quadruplicate. B. ERß-transduced cells exhibit ligand-dependent transcriptional activity from an ERE3-tk-CAT reporter. Columns, average of two independent experiments in triplicate, normalized with ßGAL.

 
Growth Response of the ERß Stable Transfectant to RA
We compared the growth inhibitory effect of RA in ER{alpha}-positive (ER{alpha}) and ERß-positive cells (ERß). The ER{alpha} stable cells are an ER{alpha}-positive subclone (S30) of MDA-MB-231 and were not derived from retroviral transduction (22). However, both cell lines were derived from the identical parental cell line, MDA-MB-231. After 6 days of treatment with RA, we observed significant growth inhibition only in the cells expressing ER{alpha} or ERß (Fig. 3A). To determine if the growth inhibitory action of RA is altered by ERß ligands, we compared the effect of RA in the presence of the ERß agonist (E2) and antagonist (ICI 182,780). As seen in Fig. 3B, 6 days of treatment with RA alone resulted in ~50% growth reduction for ERß-expressing breast cancer cells, and this remained unchanged regardless of the presence of ER agonistic or antagonistic ligand.



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  		FIGURE 3. ERß-transduced cells are growth inhibited by RA. A. Viable MDA-MB-231 cells stably expressing either the empty retroviral vector (Ctrl), ER{alpha}, or ERß were assessed using sulforhodamine B staining as described in Materials and Methods after 6 days of 10–5 mol/L RA treatment and compared with untreated cells. B. RA-mediated growth inhibition in ERß-transduced cells is unaltered by estradiol (10–7 mol/L, E2) or ICI 182,870 (10–7 mol/L, ICI) after 6 days. Columns, average of at least three different experiments in triplicate. *, P < 0.05, statistically significant from the parental cell line (A) or treatment with vehicle (B), calculated using Dunnett's test.

 


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  		FIGURE 6. Retinoid receptor expression and regulation by RA in ER-positive cells. A. RAR{alpha} Northern blot analysis of total cellular RNA (20 µg) isolated after 24 hours of 10–5 mol/L RA (or vehicle) treatment. ß-actin, loading control. B. Basal RARß2 expression is suppressed but inducible by RA in ER-expressing cells. Reverse transcription-PCR analysis of RARß2 in cells treated with RA (10–6 mol/L) or vehicle for 24 hours using ß-actin to control for variability in cDNA.

 
RA Alters ERß-Mediated Gene Expression
The expression of ER{alpha}-regulated genes can be altered by RA (24, 25). To determine if similar transcriptional effects would be observed in ERß-positive cells, we studied the expression of pS2, a known ER-responsive gene containing an ERE in its promoter region. Expression of ERß increases basal expression of pS2 in absence of ligand as compared with the ER-negative Ctrl. Although pS2 is induced by stable transduction of ERß alone, treatment with estradiol increases the expression of this gene. In ERß-positive cells, induction of pS2 by estradiol is inhibited by 24 hours treatment with RA (Fig. 4A). Densitometry analysis was done on two independent experiments to determine the pS2/glyceraldehyde-3-phosphate dehydrogenase ratio. E2 treatment enhanced pS2 expression 2-fold, whereas treatment with RA alone or with E2 decreased pS2 expression by 50% of the untreated cells. In addition, on a synthetic promoter containing three tandem EREs, RA inhibits transcription, and this inhibition is maintained even in the presence of estradiol (Fig. 4B). These data indicate that ER-mediated transcription is inhibited by RA and that an ER agonist cannot rescue the transcriptional inhibitory effect of RA.



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  		FIGURE 4. Analysis of gene regulation in ERß-transduced breast cancer cells. A. Northern blot analysis of pS2 expression in empty retroviral vector (Ctrl) or ERß-transduced cells (ERß). Cells were treated with 10–5 mol/L RA, 10–7 mol/L estradiol (E2), or a combination of both ligands for 24 hours. Total cellular RNA (20 µg) was loaded in each lane as well as the corresponding glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B. RA inhibits transcription from an ERE. Cells were cotransfected with ERE3-tk-CAT and CMV-ßGAL and treated with the indicated ligands for 48 hours.

 
RA Decreases Induced Activator Protein Activity in ERß-Positive Breast Cancer Cells
The growth inhibitory effect of retinoids in breast cancer cells has often been attributed to its inhibitory action on activator protein (AP-1)–mediated transcription (26, 27). In contrast, ER{alpha} can increase AP-1 activity in response to estradiol. Using a reporter construct from the collagenase promoter containing an AP-1 response element [Coll(–73)Luc], we studied the effect of ERß ligands and RA on AP-1 activity using treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) to confirm the functionality of the reporter. Unexpectedly, we observed an increase in AP-1 activity on E2 treatment in the ERß stable transfectants, whereas both 4-hydroxytamoxifen (OHT) and ICI 182,780 treatment did not alter AP-1 transcription (Fig. 5). These results were unanticipated because, unlike ER{alpha}, E2 has been characterized previously as an inhibitor of ERß-mediated AP-1 activity and OHT as an activator (28). The Ctrl cell line was unaffected by ER ligands or RA (data not shown). When compared with vehicle-treated cells, treatment with RA alone did not significantly alter AP-1-mediated transcription. However, when ERß-expressing cells are cotreated with RA and E2, the AP-1 stimulatory action of estradiol, as well as that of TPA, is blocked by RA.



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  		FIGURE 5. Analysis of AP-1-mediated transcriptional activity in ERß-transduced breast cancer cells. ERß-transduced cells were transfected with 1 µg Coll(–73)Luc and 1 µg CMV-ßGAL expression vector and treated with vehicle, 10–7 mol/L estradiol (E2), 10–7 mol/L tamoxifen (OHT), 10–7 mol/L ICI 182,780 (ICI), 100 ng/mL 12-O-tetradecanoylphorbol-13-acetate (TPA), or a combination of each ligand with 10–6 mol/L RA for 24 hours. Columns, mean transcriptional activity of at least two independent experiments in triplicate; bars, SEM.

 
ERß Alters the Expression of RARß2
To determine if there was reciprocal crosstalk between ERß and RAR, we also observed the effect of ERß on RAR-mediated transcription. Some groups have shown that an increased level of RAR{alpha} correlates with RA-mediated growth inhibition (29, 30), whereas others attribute the inhibition of growth by RA to induction of RARß2 (31). To determine the level of these nuclear receptors in our stably transfected cell lines, we assessed the expression of RAR{alpha} and RARß2 by Northern blot and semiquantitative reverse transcription-PCR, respectively (Fig. 6). We noted no significant change in expression level of RAR{alpha} in the Ctrl cell line, the ER{alpha}-positive or ERß-positive stable transfectants, before or after RA treatment (Fig. 6A). Because the induction of RARß2 has been associated with response to retinoids in certain types of cancer, we also assessed the expression of this gene in our system by semiquantitative reverse transcription-PCR. It is well documented that RARß2 is present at much lower levels than RAR{alpha} in breast cancer cells, rendering it difficult to perform a simple Northern or Western blot. In contrast to RAR{alpha}, we observed significant differences in the level of expression of RARß2. Expression of ß-actin, the linearity of which was verified by titration, shows that the samples contained equivalent amounts of RNA. In the ER{alpha} or ERß stable transfectants, expression of RARß2 was suppressed in the absence of RA and, unlike the ER-negative cells, RARß2 expression levels were strongly induced in response to RA (Fig. 6B).

Effect of ERß on the Transcriptional Activity of a Transiently Expressed RARE
To further characterize the effect of ERß on RARß2 expression, we transiently expressed a reporter driven by the RARE of the RARß2 promoter in the stably transduced cell lines and assessed transcriptional activity in the absence and presence of 10–6 mol/L all-trans retinoic acid (Fig. 7). We observed that cells expressing the ERß receptor, as compared with the ER-negative cells, displayed significantly lower basal activity from this promoter in the absence of RA (Fig. 7B). Transient transfection of only the tk-CAT part of the reporter did not differ between the cell lines, indicating that the effect of ER{alpha} or ERß on transcriptional activity was due to the ßRARE and not any part of the thymidine kinase promoter (data not shown). We detected an ~10-fold decrease in basal activity from the ßRE-tk-CAT in the presence of ER{alpha} or ERß (Fig. 7B). On addition of RA, there was a strong induction of transcriptional activity in the cells that had a suppressed baseline and a weaker induction in the parental cells (Fig. 7A). This transcriptional effect was only observed on an RARE, because reporter constructs containing vitamin D response element, thyroid hormone response element, or peroxisome proliferator response element were unaffected by the expression of ER{alpha} or ERß (data not shown). Thus, expression of ER{alpha} or ERß results in a greater induction of RARß2 expression when compared with ER-negative cells.



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  		FIGURE 7. Expression of ER{alpha} or ERß increases the fold induction of transcriptional activity from the ßRARE promoter by suppressing basal activity in the absence of RA. A. Retinoid-induced transcriptional activity in the absence (Ctrl) or presence of ER{alpha} or ERß. Cells were transfected with 1 µg ßRE-tk-CAT and 1 µg CMV-ßGAL expression vector and treated with 10–6 mol/L RA or vehicle for 48 hours. Box, fold induction (Fold Ind.) for each cell line. Columns, mean transcriptional activity of at least three independent experiments in triplicate; bars, SEM. B. Transcriptional activity from the ßRE-tk-CAT in absence of ligand. Columns, chloramphenicol acetyltransferase (CAT) activity relative to the ER-negative cell line (Ctrl). *, P < 0.05, statistically significant from the Ctrl, calculated using Dunnett's test.

 
An Active ERß Is Required to Maintain RARß2 Inducibility
The ligands OHT and ICI 182,780 both inhibit the transcriptional activity of ERß. We tested the activity of the ßRARE reporter in the presence of these antagonists and the agonistic ligand estradiol in the ERß-positive cells (Fig. 8A). As expected, OHT, ICI 182,780, and estradiol had no effect on the Ctrl empty retroviral transfected cell line. In cells stably expressing ERß, OHT and ICI 182,780 completely released inhibition of the RAR-mediated transcription and restored baseline activity to the same level as seen in the ER-negative cell line (Ctrl). Addition of estradiol, which activates ERß via the AF-2 domain, further decreases ßRARE activity in these cells. Because it is known that these ligands can alter the expression levels of ER{alpha}, we verified their effects on the expression of the stably transduced ERß cells after 24 hours of treatment. Although there were no changes in mRNA expression (data not shown), the protein levels were significantly altered in the presence of the various ligands (Fig. 8B). As reported previously with ER{alpha}, estradiol and ICI 182,780 both down-regulate expression of ERß. Although the expression of ERß is similarly down-regulated by both of these ligands, only the antagonist ICI 182,870 increases the transcriptional activity from the ßRARE. These data indicate that inhibition of ERß transcriptional activity allows transcription from the ßRARE. Conversely, activation of ERß transcription with estradiol inhibits ßRARE-mediated transcription.



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  		FIGURE 8. ERß ligands alter transcriptional activity from the ßRE-tk-CAT. A. Baseline transcriptional activity of the ßRE-tk-CAT was evaluated after treating the cells 1 hour post-transfection with vehicle, 10–7 mol/L tamoxifen (OHT), 10–7 mol/L ICI 182,780 (ICI), or 10–7 mol/L estradiol (E2). Columns, chloramphenicol acetyltransferase (CAT) activity relative to the ER-negative cell line (Ctrl). *, P < 0.05, statistically significant differences between the vehicle and the treated samples. B. Immunoblot of whole cell extracts (50 µg) depicting the effect of the ERß ligands mentioned above on ERß protein expression after 24 hours. Cells were also immunoblotted for ß-actin as a loading control.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Breast cancer is a hormone-dependent malignancy, with standard therapy directed at regulating ER-mediated signaling. In premalignant and malignant breast lesions, ER expression is significantly increased (32). The discovery of the ERß in both normal and malignant breast tissues has added a new level of complexity, although the expression of ERß in the breast is less abundant than ER{alpha} and there is controversy regarding its role in breast physiology and tumorigenesis (33).

Retinoids have shown some therapeutic potential for the treatment of breast cancer (16, 17). Although they do not target ER directly, there is considerable evidence correlating the presence of ER{alpha} with RA sensitivity (10, 20, 34-36). Importantly, clinical trials using a retinoid derivative have shown efficacy in preventing contralateral breast cancer in premenopausal women exclusively, further suggesting a role for ER{alpha} in RA-mediated growth inhibition (37). Because ERß can also be detected in breast cancer cells, we wished to determine its prognostic implications in the management of breast cancer with retinoids. For this purpose, we engineered ERß-positive breast cancer cells using retroviral transfection of the ER{alpha}-negative MDA-MB-231 cell line. Although several ERß isoforms have been identified in human breast cancer tissue and cell lines, we used the wild-type hERß of 530 amino acids (6).

Expression of ERß was confirmed by Western blot and immunofluorescence. Functionality of the transduced ERß was shown by transient transfection, using a synthetic ERE-containing reporter construct, and by estradiol-mediated activation of the pS2 gene. Although stable expression of ERß restored ligand-dependent transcription, the growth properties of the stable cell lines were unaffected by the ERß ligands estradiol, tamoxifen, or ICI 182,780. These results agree with those reported previously in which ER ligands did not alter the proliferation of ERß-expressing stable cell lines (4, 38). These data suggest that the estradiol-driven neoplastic process of the breast that has been described for ER{alpha} may not be pertinent to ERß. Although ER ligands did not alter the proliferation of ERß-transduced cells, we observed that retinoids inhibited proliferation of ERß-positive and ER{alpha}-positive breast cancer cells and that cell growth was inhibited regardless of cotreatment with ER ligands.

The mechanism for retinoid-mediated growth inhibition is not well understood. However, there is evidence that retinoids suppress estradiol-mediated proliferation and transcriptional activity and can antagonize the proliferative effects of AP-1 (27, 39). In the ERß-transduced cells, we show that RA can repress ERE-mediated transcription and decreases estradiol-activated endogenous gene expression (pS2). However, repression of ERß activity alone cannot explain the growth inhibitory properties of retinoids because ER ligands do not alter the proliferation of ERß-positive breast cancer cells.

Increased AP-1 activity generally leads to activation of cell proliferation signals (40, 41). Because the growth inhibitory mechanisms of retinoids have, in part, been attributed to the antagonism of this activity, we explored the possibility that ERß cells may have altered AP-1 activity in response to RA. Several groups have shown that, unlike ER{alpha}, anti-estrogens activate ERß-mediated AP-1 activity, whereas E2 is antagonistic (28, 42). In contrast, using a reporter construct from the collagenase promoter containing an AP-1 response element [Coll(–73)Luc], we noted that E2 increased AP-1 activity in these cells. Despite the activation of AP-1 activity, the proliferation rate of ERß-positive cells was unaffected by treatment with E2. Furthermore, the anti-estrogens OHT and ICI 182,780 did not alter AP-1-mediated transcription in our stably transduced ERß cells. These results, which contradict those observed in transient transfections of ERß, are in accordance with those reported in another ERß stably transfected MDA-MB-231 cell line in which anti-estrogens did not activate AP-1 response elements (38). We also report that treatment with RA did not significantly alter basal AP-1-mediated transcription but decreased E2-induced and TPA–induced AP-1 activity. Although RA antagonism of AP-1 activity is not specific for E2 induction of this reporter, we have nevertheless shown that RA can alter ERß-mediated transcription from both ERE-driven and AP-1-driven promoters in breast cancer cells stably expressing ERß.

Because transcriptional crosstalk has been reported between ER{alpha} and RAR, we also examined the effect of ERß expression on RA-mediated pathways. The growth inhibitory action of retinoids has often been attributed to increased expression of RAR{alpha} and to RARß2 induction (43-45). We found that the expression of RAR{alpha} was unchanged in response to RA but that the basal expression of RARß2 RNA, as determined by reverse transcription-PCR (Fig. 6B) and RNase protection assay (data not shown), was significantly lower in cells expressing ER{alpha} or ERß than in parental ER-negative cells. Induction of RARß2 expression has been associated with retinoid response in a variety of cancer cell types and provides another example of the crosstalk between ER-mediated and RAR-mediated pathways in human breast cancer cells (44-46). Using ER ligands, we determined that the function of ERß in altering RAR-mediated transcription takes precedence over its expression levels. Although both ICI 182,780 and E2 decrease ERß protein expression, these ligands oppose each other in their action on the RARß promoter. We have shown previously that the NH2-terminal region of ER{alpha}, including the DNA binding domain, was important for mediating transcriptional crosstalk with RAR. Although ERß varies greatly from ER{alpha} in the NH2-terminal region, there are some similarities. For example, it has been reported that both receptors can bind p300 at the NH2 terminal in absence of ligand (47). Therefore, it remains a possibility that ERß or ER{alpha} interaction with RAR transcription pathway may involve squelching for limited known or unknown cofactors. Stable cell lines using ERß deletion mutants or ERß variants will provide greater insight into this transcriptional interaction. In addition, it may be of interest to study the effect of ERßcx expression on retinoid activity in breast cancer cells. This isoform has been detected in human breast cancer, shows preferential dimerization with ER{alpha}, and has a dominant negative effect on ligand-dependent ER{alpha} reporter gene transactivation (48).

In conclusion, we provide evidence of nuclear receptor signaling crosstalk between ERß and RAR in human breast cancer cells. Given the promiscuity of coactivators and corepressors with different nuclear receptors, it is not surprising that crosstalk exists between the different nuclear receptor families. We show that RA can significantly decrease the growth of ERß-positive breast cancer cells in the presence or absence of ER ligands, thereby supporting the use of retinoids for the management of ERß-positive breast cancer.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Cells
MDA-MB-231 (clone 10A) and the ER{alpha}-positive subclone (S30) were obtained courtesy of Dr. V.C. Jordan (Northwestern University Medical School, Chicago, IL). All cell lines were routinely cultured in {alpha}-MEM phenol red–free medium (Life Technologies, Inc., Burlington, Ontario, Canada) supplemented with 5% charcoal-stripped serum. For the culture of S30 cells, 0.5 µg/mL G418 (Life Technologies) was added to the above medium. All cells were maintained in 5% CO2 at 37°C in a humidified atmosphere.

Construction of Stable Cell Lines
The hERß expression vector (1,590 bp) was kindly provided by Dr. S. Mader (Département de Biochimie, Université de Montréal, Montreal, Quebec, Canada). Retroviral vectors were constructed by cloning the above cDNA in the multiple cloning site of the murine stem cell virus retroviral vector (HC2). This technique has been described previously in detail (21). Concentrated virus was used to infect MDA-MB-231 cells. Pooled populations of transduced cells were routinely analyzed by flow cytometry for eGFP expression, thus confirming expression of the bicistronic RNA and the stable expression of ERß.

Western Blots
Whole cell extracts were isolated from confluent 150 mm plates of transduced cells as described previously (21). Whole cell extracts were also isolated from cells treated with 10–7 mol/L tamoxifen (kindly provided by Dr. A.E. Wakeling, Zeneca, Macclesfield, United Kingdom), 10–7 mol/L ICI 182,780 (Sigma Chemical Co., St. Louis, MO), and 10–7 mol/L estradiol (Sigma Chemical) and treated for 24 hours. Protein lysates (50 µg) were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). Membranes were probed using the ERß antibody (QED Biosciences, San Diego, CA) and incubated with secondary antibody at a 4,000-fold dilution prior to analysis by chemiluminescence. Membranes were exposed to anti-ß-actin antibody (Sigma Chemical) to control for loading.

Northern Blots
Expression of RAR{alpha} and pS2 were analyzed by Northern blot as described previously (10). Briefly, total RNA (20 µg) was isolated 24 hours post-treatment with indicated ligands and electrophoresed on a 1% formaldehyde/agarose gel and blotted onto nitrocellulose filter. The filters were hybridized to radiolabeled probes of RAR{alpha} (PstI fragment) or pS2, washed, and autoradiographed.

Immunofluorescence
Cells were grown on coverslips until semiconfluent monolayers were obtained and fixed with 4% cold paraformaldehyde in PBS. Coverslips were washed with PBS-0.5% Triton X-100 containing 10% FCS (Life Technologies) for 5 minutes at room temperature. Incubation with 1:50 anti-ERß (N-19, Santa Cruz Biotechnology, Santa Cruz, CA) diluted in PBS-0.1% Triton X-100 was done for 3 hours in a humid chamber. Cells were washed extensively with PBS-0.01% Triton X-100 and staining was done using Alexa Fluor 546–conjugated anti-goat secondary antibody (Molecular Probes, Eugene, OR) diluted 1:1,000 in PBS-0.01% Triton X-100 for 30 minutes. Cells were washed again and 2 µg/mL 4',6-diamidino-2-phenylindole (Molecular Probes) solution was added for 5 minutes to visualize nuclei. Coverslips were mounted onto glass slides using Immuno-Mount (Shandon, Inc., Pittsburgh, PA) and cells were visualized with an Olympus BX51 fluorescence microscope (Olympus, Melville, NY). An oil immersion (100x) objective was selected for the observations.

Cell Proliferation Studies
Cells were seeded in 24-well plates at a density of 2,000 cells per well. In the treated cells, a final concentration of 10–5 mol/L all-trans retinoic acid, 10–7 mol/L E2, 10–7 mol/L OHT, or 10–7 mol/L ICI 182,780 was replenished 1 day after seeding and subsequently on days 3 and 5. Controls for treated cells contained identical concentrations of vehicle alone. After 6 days of treatment, cells were fixed in 10% trichloroacetic acid and subsequently stained with sulforhodamine B (Sigma Chemical). Sulforhodamine B is an aminoxanthene dye that binds to basic amino acid residues and gives an index of culture cell protein that is linear with cell number (49). Bound sulforhodamine B was solubilized in 10 mmol/L unbuffered Tris and absorbance was measured at 570 nm in a microplate reader for quadruplicate samples.

Analysis of RARß Expression
Expression of RARß was assessed by reverse transcription-PCR as described previously (21). Briefly, cDNA was prepared from 1 µg RNA and PCR amplification was done using RARß2 primers. To ensure the validity of the ß-actin PCR as a loading control, the ß-actin samples were titrated and we confirmed that the PCR result was within linear range.

Transient Transfections and Chloramphenicol Acetyltransferase Assays
Cells were plated at 2 x 105 cells per well in six-well plates and allowed to adhere overnight in phenol red–free {alpha}-MEM medium supplemented with 5% charcoal-stripped fetal bovine serum. Transfections were done using FuGENE (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's guidelines. Transfections of the ßRE-tk-CAT and ERE3-tk-CAT have been described previously (21). Briefly, reporter plasmid (1 µg) was transfected with pCMV-ßGAL plasmid (1 µg) as an internal control using a ratio of 2:1 FuGENE (Boehringer Mannheim) to DNA. Cells were treated with the indicated ligands after 5 hours and harvested 48 hours post-transfection. For transfection of an AP-1 response element, the above methodology was followed with the Coll(–73)Luc reporter, except that cells were treated with the indicated ligands for 24 hours. Luciferase assay was done in accordance with the manufacturer's guidelines (Promega, Madison, WI) and measured using a Lumat LB-9507 luminometer (Perkin-Elmer Instruments, Darmstadt, Germany). 12-O-tetradecanoylphorbol-13-acetate (100 ng/mL) was used as a positive control.

Statistical Analysis
Results from representative experiments are shown as means of the number of replicates. Statistical analysis was done using Dunnett's test, wherein statistical significance was noted for P < 0.05.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
1 Canadian Breast Cancer Research Initiative, Canadian Institute for Health Research (W.H. Miller), U.S. Army Medical Research and Materiel Command Breast Cancer Research Program award DAMD17-97-1-7167 (C. Rousseau), and DAMD17-03-1-0472 (F. Pettersson). Back

Received March 8, 2004; revised June 16, 2004; accepted July 26, 2004.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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