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Molecular Cancer Research 5, 71-85, January 1, 2007. doi: 10.1158/1541-7786.MCR-06-0227
© 2007 American Association for Cancer Research
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Signaling and Regulation

Estrogen-Related Receptor {alpha}1 Transcriptional Activities Are Regulated in Part via the ErbB2/HER2 Signaling Pathway

Eric A. Ariazi1, Richard J. Kraus1, Michael L. Farrell1, V. Craig Jordan2 and Janet E. Mertz1

1 McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin and 2 Fox Chase Cancer Center, Philadelphia, Pennsylvania

Requests for reprints: Janet E. Mertz, McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, 1400 University Avenue, Madison, WI 53706. Phone: 608-262-2383; Fax: 608-262-2824. E-mail: mertz{at}oncology.wisc.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
We previously showed that (a) estrogen-related receptor {alpha}1 (ERR{alpha}1) down-modulates estrogen receptor (ER)–stimulated transcription in low ErbB2–expressing MCF-7 mammary carcinoma cells, and (b) ERR{alpha} and ErbB2 mRNA levels positively correlate in clinical breast tumors. We show here that ERR{alpha}1 represses ER{alpha}-mediated activation in MCF-7 cells because it failed to recruit the coactivator glucocorticoid receptor interacting protein 1 (GRIP1) when bound to an estrogen response element. In contrast, ERR{alpha}1 activated estrogen response element– and ERR response element–mediated transcription in ER{alpha}-positive, high ErbB2–expressing BT-474 mammary carcinoma cells, activation that was enhanced by overexpression of GRIP1. Likewise, regulation of the endogenous genes pS2, progesterone receptor, and ErbB2 by ERR{alpha}1 reflected the cell type–specific differences observed with our reporter plasmids. Importantly, overexpression of activated ErbB2 in MCF-7 cells led to transcriptional activation, rather than repression, by ERR{alpha}1. Two-dimensional PAGE of radiophosphate-labeled ERR{alpha}1 indicated that it was hyperphosphorylated in BT-474 relative to MCF-7 cells; incubation of these cells with anti-ErbB2 antibody led to reduction in the extent of ERR{alpha}1 phosphorylation. Additionally, mitogen-activated protein kinases (MAPK) and Akts, components of the ErbB2 pathway, phosphorylated ERR{alpha}1 in vitro. ERR{alpha}1-activated transcription in BT-474 cells was inhibited by disruption of ErbB2/epidermal growth factor receptor signaling with trastuzumab or gefitinib or inactivation of downstream components of this signaling, MAPK kinase/MAPK, and phosphatidylinositol-3-OH kinase/Akt, with U0126 or LY294002, respectively. Thus, ERR{alpha}1 activities are regulated, in part, via ErbB2 signaling, with ERR{alpha}1 likely positively feedback-regulating ErbB2 expression. Taken together, we conclude that ERR{alpha}1 phosphorylation status shows potential as a biomarker of clinical course and antihormonal- and ErbB2-based treatment options, with ERR{alpha}1 serving as a novel target for drug development. (Mol Cancer Res 2007;5(1):71–86)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The steroid nuclear receptor estrogen receptor {alpha} (ER{alpha}), officially termed NR3A1 (1), is pivotally involved in the etiology of breast cancer. ER{alpha} mediates the effects of estrogens on transcription and is expressed at high levels in approximately three fourths of human breast tumors. It thereby serves as a critical biomarker of clinical course and target for therapy (reviewed in ref. 2). The orphan nuclear receptors estrogen-related receptor {alpha} (ERR{alpha}; NR3B1), ERRß (NR3B2), and ERR{gamma} (NR3B3; ref. 1) exhibit a high degree of sequence similarity with ER{alpha} (reviewed in ref. 3). They do not bind naturally occurring estrogens, but share other biochemical activities with ERs, including binding to estrogen response elements (ERE; refs. 4-8). ERRs also bind to extended nuclear receptor half-site sequences resembling 5'-TNAAGGTCA-3', referred to as ERR response elements (ERRE; refs. 5-7, 9, 10).

ERR{alpha} mRNA levels are similar or greater than ER{alpha} mRNA levels in approximately one fourth of unselected human breast cancers, with the highest levels occurring in tumors lacking functional ER{alpha} (11). Additionally, ERR{alpha} mRNA levels correlate in breast cancers with those of ErbB2 (also called HER2/neu; ref. 11), a marker of tumor aggressiveness. Suzuki et al. (12) reported that ~55% of human breast cancers are positive for ERR{alpha} by immunohistochemistry, with ERR{alpha}-positive status being associated with greatly increased risk of recurrence and adverse clinical outcome. Thus, ERR{alpha} shows potential as a prognosticator and target for breast cancer therapy, with ERR{alpha} possibly playing an important role by substituting for ER{alpha} activities, especially in ErbB2-positive, ER{alpha}-negative, and tamoxifen-resistant tumors.

Whereas ER{alpha} usually regulates gene expression in a ligand-inducible manner, ERR{alpha}1, the 423-amino-acid major isoform encoded by the ESRRA gene (National Center for Biotechnology Information accession NP_004442.3; refs. 4, 5), can constitutively activate transcription in the absence of ligand. ERR{alpha}1 interacts with peroxisome proliferator-activated receptor coactivator-1{alpha} (13, 14) and the p160 family of coactivators, including glucocorticoid receptor interacting protein 1 (GRIP1/SRC2; ref. 15), via a carboxyl-terminal coactivator-binding inverted LxLxxL motif (16). Bulky amino acid side chains almost completely fill the ERR{alpha}1 putative ligand-binding pocket (14, 17), with residue Phe329 recapitulating interactions analogous to ones provided by ligands, thereby promoting binding of coactivators (17).

ERR{alpha}1 has been shown to bind the promoter of the estrogen-inducible pS2 gene (also called TFF1; ref. 18) and to modulate transcription of the estrogen precursor metabolizing genes aromatase (CYP19; ref. 19) and DHEA sulfotransferase (SULT2A1; ref. 20). It also modulates expression of the estrogen-responsive genes lactoferrin (4), osteopontin (21), and even ERR{alpha} itself (also called ESRRA; refs. 22, 23). The effect of ERR{alpha}1 binding to a transcriptional response element can be either negative or positive depending on the specific cell type (8) and promoter context (24). For example, ERR{alpha}1 down-modulates E2-induced transcription in ER{alpha}-positive human mammary carcinoma MCF-7 cells by an active mechanism (8), yet activates gene transcription in ER{alpha}-negative human mammary carcinoma SK-BR-3 cells (19) and a variety of other cell lines, including human cervical carcinoma HeLa cells (8), human endometrial RL95-2 cells (4), human embryonic kidney 293 cells (21), and rat ROS 17/2.8 osteosarcoma cells (21).

The factors determining whether ERR{alpha}1 activates or down-modulates transcription have yet to be fully identified. Epidermal growth factor receptor (EGFR) and ErbB2, members of the ErbB family of transmembrane receptor tyrosine kinases, signal in part through the MAPK and phosphatidylinositol-3-OH kinase (PI3K)/Akt signaling pathways (25). Stimulation of these pathways can lead to activation of unliganded ER{alpha} (26), with overexpression of EGFR and ErbB2 implicated in the failure of antiestrogen therapy in both model systems (27-29) and clinical breast cancers (30-32). Thus, by analogy with ER{alpha}, we hypothesized that signaling via ErbB2 leads to phosphorylation of ERR{alpha}1, thereby modulating its activities. Findings in support of this hypothesis include the following: (a) ERR{alpha}1 can exist as a phosphoprotein (9); (b) human breast tumors that express high lev els of ErbB2 mRNA also frequently express high levels of ERR{alpha} mRNA (11); and (c) SK-BR-3 cells, in which ERR{alpha}1 functions as a constitutive activator (19), contain 2 orders of magnitude more ErbB2 mRNA than MCF-7 cells (33) in which it functions as a down-modulator of transcription (8).

To test the validity of this hypothesis, we examined the effects of ErbB2 signaling on the transcriptional activity and phosphorylated state of ERR{alpha}1 in MCF-7 versus BT-474 cells, another mammary carcinoma cell line with very high ErbB2 levels (33). We found that overexpression of ERR{alpha}1 led to increased accumulation of pS2, progesterone receptor (PgR), and ErbB2 mRNAs in BT-474 cells and decreased accumulation of pS2 and ErbB2 mRNAs in MCF-7 cells. ERR{alpha}1 was hyperphosphorylated in BT-474 cells compared with MCF-7 cells and could be phosphorylated by MAPKs and Akts in vitro. Strikingly, ERR{alpha}1 transcriptional activity was stimulated by overexpression of activated ErbB2 in MCF-7 cells and inhibited in BT-474 cells treated with (a) the ErbB2 inhibitor trastuzumab (Herceptin; ref. 34), (b) the EGFR tyrosine kinase inhibitor gefitinib (Iressa, ZD1839; ref. 35), (c) the MAPK kinase (MEK) inhibitor U0126 (36), or (d) the PI3K inhibitor LY294002 (37). Thus, we conclude that ErbB2 signaling can modulate ERR{alpha}1 activities.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Effects of E2 on Expression of ERR{alpha} in MCF-7 and BT-474 Cells
ERR{alpha}1 can either repress or activate ERE-regulated expression (8), a target sequence to which it binds exclusively as a homodimer (38). Given our finding that ERR{alpha} mRNA levels positively correlate with those of ErbB2 and ErbB3 (11), we speculated that posttranslational modifications mediated by the ErbB2-directed pathway might contribute to regulation of ERR{alpha}1 activities. We studied here two ER{alpha}-positive breast cancer cell lines, MCF-7 and BT474, known to express low and high levels of ErbB2, respectively (39), to examine the effects of ErbB2 on ERR{alpha}1 activities.

First, we measured endogenous ERR{alpha}1 and ER{alpha} protein levels and the effects of estrogen on these levels in these two cell lines. Lysates were prepared from MCF-7 and BT-474 cells cultured in estrogen-free medium and treated for 24 h with 100 pmol/L 17ß-estradiol (E2), its vehicle ethanol as a control, 1 nmol/L E2, or 1 nmol/L E2 plus 1 µmol/L of the complete antiestrogen fulvestrant. The proteins in the lysates were separated by SDS-PAGE, blotted to a filter, and probed with antibodies specific for ER{alpha}, ERR{alpha}, and ß-actin as an internal control (Fig. 1 ). As expected, ER{alpha} levels were similar in the two ER{alpha}-positive cell lines (Fig. 1, lane 1 versus lane 5), down-regulated following treatment with E2 (Fig. 1, lanes 2 and 3 versus lane 1 and lanes 6 and 7 versus lane 5), and further down-regulated in the presence of fulvestrant (Fig. 1, lane 4 versus lane 3 and lane 8 versus lane 7), a drug known to promote proteasome-mediated degradation of ER{alpha} (40). On the other hand, ERR{alpha}1 levels were not significantly affected by the presence of either of these ligands of ER{alpha}, except at the higher concentration of E2 in BT-474 cells (Fig. 1). Furthermore, the ER{alpha}/ERR{alpha} protein ratios were not significantly different between the MCF-7 and BT-474 cells under either the estrogen-free (3.3 versus 4.0, respectively) or 100 pmol/L E2 (1.5 versus 1.3, respectively) growth conditions. Thus, we can assume in the experiments presented below that differential effects on transcription were due to changes in the activities of ERR{alpha}1, not in its levels within the cells.


Figure 1
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  		FIGURE 1. Immunoblot analysis showing endogenous expression of ER{alpha} and ERR{alpha}1 in MCF-7 and BT474 cells. Cells were cultured for 24 h in estrogen-free medium supplemented with ethanol, 100 pmol/L E2, 1 nmol/L E2, or 1 nmol/L E2 plus 1 µmol/L fulvestrant. Endogenous ER{alpha}, ERR{alpha}1, and ß-actin were detected using primary antibodies specific for these proteins followed by IR fluorescent dye–conjugated secondary antibodies. ER{alpha} and ERR{alpha}1 protein levels normalized to ß-actin are shown as units relative to the ER{alpha} level in the control-treated MCF-7 cells (lane 1).

 
ERR{alpha}1 Represses ERE-Regulated Transcription in MCF-7 Cells but Activates Transcription in BT-474 Cells
We initially studied ERR{alpha}1-regulated transcription using minimal synthetic reporter genes containing (a) a palindromic ERE or an ERRE, (b) a TATA box, and (c) an initiator element rather than complex natural promoters. We did so to ensure observed effects were not due to indirect influences of other factors binding to other regulatory elements such as AP1- or Sp1-binding sites. Cells were cotransfected with an ERE(5x)-regulated or ERRE(5x)-regulated dual-luciferase reporter gene set, with the Renilla luciferase plasmid serving as an internal control for the firefly luciferase plasmid (Fig. 2A ). Concurrently, cells were cotransfected in parallel with a TATA-regulated dual-luciferase reporter set (Fig. 2A), which served as an external control for experimental conditions, the physiologic state of the cells, and non–specific effects on the basal transcriptional machinery. The effect and specificity of ERR{alpha}1 was evaluated by cotransfecting the cells with a plasmid expressing wild-type ERR{alpha}1, mutant ERR{alpha}1L413A/L418A, a variant defective in the carboxyl-terminal inverted LxLxxL motif that serves as a coactivator docking site (8), or their parental empty vector. The cells were also cotransfected with a plasmid that expressed GRIP1, a member of the p160 family of coactivators, or its empty parental plasmid. Afterward, the cells were cultured for 40 h in estrogen-free medium supplemented with the indicated compounds, harvested, and assayed for firefly and Renilla luciferase activity.


Figure 2
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  		FIGURE 2. Differential transcriptional activity of ERR{alpha}1 in low ErbB2–expressing MCF-7 cells versus high ErbB2–expressing BT-474 cells. A. Reporter gene sets used in this study. B to E. MCF-7 and BT-474 cells were cotransfected with the ERE(5x) or ERRE(5x)-regulated dual luciferase reporter gene sets along with ERR{alpha}1, ERR{alpha}1L413A/L418A, GRIP1, or empty parental plasmids as indicated. As an external normalization control, cells were also cotransfected in parallel for each condition indicated with the TATA-regulated dual-luciferase reporter set in place of the ERE(5x) or ERRE(5x) reporter sets. Cells were incubated for 40 h in estrogen-free medium supplemented with ethanol (EtOH), 100 pmol/L E2, or 100 pmol/L E2 plus 100 nmol/L fulvestrant as indicated. Columns, mean of samples processed in triplicate; bars, SE. Data are presented relative to the luciferase activity present in the cells assayed in lane 1 of each figure. Hatched columns, cells cotransfected with the GRIP1 expression plasmid; solid columns, cells cotransfected with its empty parental plasmid.

 
As expected, treatment of the ER{alpha}-positive MCF-7 cells with 100 pmol/L E2 induced ERE-regulated transcription ~19-fold (Fig. 2B, lane 7 versus lane 1). Introduction of exogenous wild-type ERR{alpha}1 or mutant ERR{alpha}1L413A/L418A led to a 68% and 71% reduction, respectively, in E2-stimulated transcription in these cells (Fig. 2B, lanes 9 and 11 versus lane 7). The finding that addition of ERR{alpha}1L413A/L418A did not lead to complete loss of the E2-stimulated activity indicates that some of the promoter sites probably remained occupied by ER{alpha} in these high ER{alpha}–expressing cells. Overexpression of the coactivator GRIP1 enhanced E2-stimulated transcription an additional 1.8-fold (Fig. 2B, lane 8 versus lane 7), most likely by stimulating the activity of ER{alpha} bound to the EREs. This finding indicates that GRIP1 was limiting in these cells. Nevertheless, overexpression of GRIP1 failed to overcome the down-modulation by ERR{alpha}1 of the ER{alpha}-stimulated transcription (Fig. 2B, lane 10 versus lane 8). Confirming and extending prior findings (8), we conclude that ERR{alpha}1 acted as a repressor in MCF-7 cells, down-modulating ER{alpha}-stimulated transcription. Importantly, whereas overexpression of the coactivator GRIP1 enhanced ER{alpha} activity, it failed to convert ERR{alpha}1 to an activator.

To examine ERR{alpha}1 activity via EREs in the absence of ER{alpha}-stimulated transcription, the cotransfected cells were cultured in estrogen-free medium supplemented with (a) only the drug vehicle, ethanol (Fig. 2B, lanes 1-6), or (b) the complete antiestrogen fulvestrant along with 100 pmol/L E2 (Fig. 2B, lanes 13-18). Under either of these conditions, overexpression of wild-type ERR{alpha}1 led to a barely significant increase in transcription (Fig. 2B, lanes 3, 4, 15, 16 versus lanes 1, 2, 13, 14, respectively). Thus, ERR{alpha}1 exhibited a very low level of activator activity in MCF-7 cells when ER{alpha} is absent.

We likewise examined the ability of ERR{alpha}1 to modulate ERE-regulated transcription in BT-474 cells (Fig. 2D). In the absence of ER{alpha}-stimulated transcription, overexpression of wild-type ERR{alpha}1 led to an ~5-fold increase in ERE-regulated transcription (Fig. 2D, lane 3 versus lane 1 and lane 15 versus lane 13). Overexpression of GRIP1 led to an additional 2-fold enhancement in ERR{alpha}1-stimulated transcription (Fig. 2D, lane 4 versus lane 3 and lane 16 versus lane 15) as well as a 2-fold enhancement in ER{alpha}-stimulated transcription (Fig. 2D, lane 8 versus lane 7). Thus, GRIP1 was limiting in the BT-474 cells. The ERR{alpha}1L413A/L418A mutant variant failed to enhance expression (Fig. 2D, lanes 5, 6, 17, and 18 versus lanes 1, 2, 13, and 14, respectively). Overexpression of wild-type ERR{alpha}1 did not significantly alter the level of ERE-regulated transcriptional activity when the BT-474 cells were incubated in the presence of 100 pmol/L E2 (Fig. 2D, lanes 9 and 10 versus lanes 7 and 8). This finding was likely due to ERR{alpha}1 simply substituting for ER{alpha} as another activator of transcription when it displaced ER{alpha} for binding the EREs. By contrast, the ERR{alpha}1L413A/L418A mutant led instead to a 50% to 60% reduction in E2-stimulated transcription (Fig. 2D, lanes 11 and 12 versus lanes 7 and 8). This reduction in expression was similar to the one observed in E2-stimulated ERE-regulated transcription by wild-type ERR{alpha}1 in MCF-7 cells. Hence, a mutant defective in docking coactivators mimicked in BT-474 cells the repressor activity of wild-type ERR{alpha}1 seen in MCF-7 cells. Thus, in contrast to the results observed in MCF-7 cells, ERR{alpha}1 activated ERE-regulated transcription in BT-474 cells, likely doing so in part via GRIP1 interaction with its carboxyl-terminal coactivator binding motif.

ERR{alpha}1 also regulates transcription via ERREs, including the sequence 5'-TCAAGGTCA-3'. In sharp contrast to the effects observed on ERE-regulated expression, ERRE-regulated expression in MCF-7 cells was only slightly affected by either incubation with E2 (Fig. 2C, lanes 7 and 8 versus lanes 1 and 2) or overexpression of wild-type ERR{alpha}1 (Fig. 2C). Hence, neither ER{alpha} nor ERR{alpha}1 significantly affect ERRE-regulated transcription in MCF-7 cells.

ERRE-regulated expression was also unaffected by incubation with 100 pmol/L E2 in BT-474 cells (Fig. 2E, lanes 7 and 8 versus lanes 1 and 2). However, independent of E2, ERR{alpha}1 activated ERRE-regulated transcription ~2.7- to 3-fold and 5.5- to 6-fold in the absence and presence of GRIP1, respectively (Fig. 2E, lanes 3 and 4 versus lanes 1 and 2; lanes 9 and 10 versus lanes 7 and 8; lanes 15 and 16 versus lanes 13 and 14). Again, the ERR{alpha}1L413A/L418A mutant failed to induce transcription (Fig. 2E, lanes 5 and 6 versus lanes 1 and 2; lanes 11 and 12 versus lanes 7 and 8; lanes 17 and 18 versus lanes 13 and 14), indicating dependence of ERR{alpha}1 activation via ERREs on coactivator docking. Thus, ERR{alpha}1 activated both ERE- and ERRE-regulated transcription in BT-474 cells.

Vanacker et al. (7) previously reported that ER{alpha} can efficiently bind to and activate transcription via an ERRE. We observed here that E2 very weakly induced ERRE-regulated transcription. To address this discrepancy, we examined the responsiveness to E2 of the ERE- and ERRE-regulated reporter gene sets studied above in parallel with a previously described ERR{alpha}1-responsive reporter, pSFREx3-Luc, which contains three copies of the same core extended ERE half-site driving an SV40 minimal early promoter (Fig. 2A; ref. 7). As expected, the ERE(5x)-regulated reporter efficiently responded to E2 in a concentration-dependent manner, with E2 maximally inducing ERE-regulated activity 14-fold in MCF-7 (Fig. 3A ) and 12-fold in BT-474 cells (Fig. 3B). However, supraphysiologic concentrations of E2 up to 1 µmol/L maximally induced the ERRE(5x)-regulated reporter set by 2.1-fold and the SFREx3-regulated reporter set by 1.5-fold in MCF-7 cells (Fig. 3A). Similarly, the maximum level of ERRE(5x)- and SFREx3-regulated reporter activity was 1.6-fold in BT-474 cells (Fig. 3B). Hence, ERREs exhibited only minimal responses to E2-stimulated ER{alpha}, effects that could have been indirect.


Figure 3
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  		FIGURE 3. ER{alpha} activity on a palindromic ERE sequence compared with an extended half-site ERRE sequence. MCF-7 (A) and BT-474 (B) cells were cotransfected as described in Fig. 2 with the indicated dual-luciferase reporter gene sets and cultured in estrogen-free medium supplemented with ethanol. Concentrations of E2 range from 1 pmol/L to 1 µmol/L, or 100 pmol/L E2 plus 100 nmol/L fulvestrant as indicated. Cells were harvested 40 h later and assayed for luciferase activity, with normalization to both the internal and external reporter genes. Points, means of samples processed in triplicate; bars, SE. C. EMSAs showing ERR{alpha}1, but not ER{alpha}, binds the ERRE as well as the ERE with high affinity. Competition EMSAs were done with whole-cell extracts of COS cells transfected with plasmids expressing ER{alpha} or ERR{alpha}1 serving as protein source, a radiolabeled double-stranded oligonucleotide containing an ERE (5'-taagcttAGGTCAcagTGACCTaagctta-3') serving as probe, and unlabeled double-stranded oligonucleotides corresponding to the sequences indicated below the gel serving as competitors. Capitalized letters within boxes, ERE and ERRE extended half-site sequences. White letters on black, mutations.

 
To understand the reason for the lack of responsiveness of the ERRE/SFRE–regulated promoters to E2, we also examined whether ER{alpha} could bind to this ERRE sequence. Competition electrophoretic mobility shift assays were done using a radiolabeled, double-stranded ERE-containing oligodeoxynucleotide as probe; whole-cell extracts obtained from COS cells containing overexpressed ER{alpha} or ERR{alpha}1 as protein source; and unlabeled, double-stranded oligodeoxynucleotide containing an ERE, mutant ERE, or ERRE sequence as competitor (Fig. 3C). As expected, ER{alpha} efficiently bound the ERE (Fig. 3C, lanes 5-7 versus lane 4), but not the mutant ERE (Fig. 3C, lanes 8-10). Contrary to a prior report (7), ER{alpha} also failed to significantly bind the ERRE (Fig. 3C, lanes 11-13 versus lane 4). On the other hand, ERR{alpha}1 efficiently bound both the ERE (Fig. 3C, lanes 16-18, versus lane 15) and the ERRE (Fig. 3C, lanes 22-24), but not the mutant ERE (Fig. 3C, lanes 19-21). Thus, we conclude that ER{alpha} does not significantly bind to the extended half-site ERRE consensus sequence 5'-TCAAGGTCA-3', whereas ERR{alpha}1 can efficiently bind both the consensus sequence and at least some EREs.

Differential Regulation of Endogenous Cellular pS2, PgR, and ErbB2 Genes by ERR{alpha}1 in MCF-7 versus BT-474 Cells
Does ERR{alpha}1 also differentially regulate expression of endogenous cellular genes in a cell type–dependent manner? To begin to answer this question, we examined the promoter regions of cellular genes implicated in breast cancer for potential ERREs by searching a eukaryotic promoter database3 (41) and Genbank.4 The binding affinities of ERR{alpha}1 for these putative sites relative to a consensus ERRE were determined by semiquantitative competition EMSAs done with the consensus ERRE-containing double-stranded oligonucleotide serving as the radiolabeled probe DNA. ERR{alpha}1 bound these sites with a variety of affinities (Table 1 ). Interestingly, ERR{alpha}1 bound the PgR site 1 with a higher relative binding affinity (RBA, 1.85) than the reference ERRE although they contain the same ERRE extended half-site sequence. Thus, the precise context of an ERRE can modulate ERR{alpha}1 binding affinity for it. ERR{alpha}1 also bound quite well to the ErbB2 (RBA, 1.08), PgR site 2 (RBA, 0.90), and pS2 site 2 (RBA, 0.50) sequences.


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  		Table 1. ERR{alpha}1 Relative Binding of Affinities (RBAs) for Sequences in Promoters of Human Genes Implicated in Breast Cancer

 
To examine the effects of ERR{alpha}1 on expression of the endogenous cellular genes pS2, PgR, and ErbB2 in MCF-7 and BT-474 cells, cells were cotransfected in parallel with plasmids encoding enhanced green fluorescent protein (EGFP) and either wild-type ERR{alpha}1 or its empty parental vector. After incubation for 48 h in estrogen-free medium to avoid effects due to ER{alpha}, EGFP-positive cells were isolated by fluorescence-activated cell sorting. RNA was purified from these EGFP-positive cells and assayed by quantitative real-time PCR for amounts of pS2, PgR, ErbB2, and ERR{alpha} mRNA relative to cellular 18S rRNA as an internal control (Fig. 4 ). Consistent with this protocol having worked successfully, ERR{alpha} mRNA levels were found to be 50- to 67-fold higher in the cells (isolated by fluorescence-activated cell sorting) transfected with the ERR{alpha}1 expression plasmid compared with the ones transfected with the empty parental plasmid (data not shown). Whereas overexpression of ERR{alpha}1 in MCF-7 cells led to a modest decrease or no change in expression of these three genes (Fig. 4A-C, lane 2 versus lane 1), it led to a 2- to 11-fold increase in their expression in BT-474 cells (Fig. 4A-C, lane 4 versus lane 3). Furthermore, with the basal level of ErbB2 mRNA already 5-fold higher in BT-474 cells than in MCF-7 cells (Fig. 4C, lane 3 versus lane 1), the resulting differential expression of ErbB2 increased to a highly significant 30-fold when ERR{alpha}1 was overexpressed (Fig. 4C, lane 4 versus lane 2). Thus, we conclude that ERR{alpha}1 differentially regulates endogenous target genes as well as synthetic reporter ones in a cell type–dependent manner.


Figure 4
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  		FIGURE 4. Effects of ERR{alpha}1 overexpression in MCF-7 (black columns) and BT-474 cells (hatched columns) on expression of the endogenous cellular genes encoding pS2 (A), PgR (B), and ErbB2 (C) mRNA. Cells were cotransfected with pEGFP and the ERR{alpha}1 expression plasmid or its empty parent plasmid, pcDNA3.1 and incubated under estrogen-free conditions. Twenty-four hours later, EGFP-positive cells were isolated by fluorescence-activated cell sorting. The pS2, PgR, and ErbB2 RNAs in cells were analyzed by quantitative real-time PCR, with normalization to the 18S rRNA present in the same RNA samples. Data are shown relative to empty parental plasmid transfected MCF-7 cells (lane 1). Columns, mean of samples processed in quadruplicate; bars, SE.

 
Extent of Phosphorylation of ERR{alpha}1 Correlates with its Ability to Activate Transcription
To begin to determine the mechanism(s) of ERR{alpha}1 cell type–dependent activity, we examined ERR{alpha}1 phosphorylation status. MCF-7 and BT-474 cells were incubated with [32P]Pi; ERR{alpha}1 was immunoprecipitated from protein extracts prepared from these cells; and its phosphorylated isoforms were resolved by two-dimensional PAGE. Although BT-474 cells were found to contain predominantly one highly phosphorylated isoform of ERR{alpha}1, MCF-7 cells contained several differentially phosphorylated isoforms of ERR{alpha}1 (Fig. 5A ). Given the 32P label was roughly equally distributed among three isoforms of ERR{alpha}1 in the MCF-7 cells, the percentage of ERR{alpha}1 by moles in the most highly phosphorylated isoform in these cells was at most 15%. Thus, BT-474 cells contained a much larger percentage of their ERR{alpha}1 in a highly phosphorylated isoform than did the MCF-7 cells.


Figure 5
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  		FIGURE 5. Phosphorylation of ERR{alpha}1 in situ and in vitro. A. Autoradiograms of two-dimensional gels showing the in situ phosphorylated states of ERR{alpha}1 in MCF-7 cells, BT-474 cells, and BT-474 cells incubated with a murine mAb to ErbB2 (HER2). MCF-7 and BT-474 cells were transfected in parallel with the wild-type ERR{alpha}1 expression plasmid. Twenty-seven hours later, the cells were metabolically labeled by incubation for 4 h in phosphate-free medium supplemented with [{gamma}32P]ATP. Afterward, whole-cell extracts were prepared. ERR{alpha}1 was immunoprecipitated with an anti-GST-hERR{alpha}1 polyclonal antiserum and resolved by two-dimensional PAGE. The mAb 4D5–treated cells incorporated less 32P because they were growth inhibited; a second, longer exposure of this same gel is shown directly below the original one. B. In vitro phosphorylation of ERR{alpha}1 using activated MAPKs and Akts. Equal amounts of 6xHis-tagged ERR{alpha}1 were incubated in parallel with activated MAPK1, MAPK2, Akt1, and Akt2 along with [{gamma}32P]ATP. The products were resolved by 4% to 12% gradient SDS-PAGE and visualized by autoradiography. Myelin basic protein (MBP) was included in the reactions as an internal positive control. C. Localization of sites of phosphorylation of ERR{alpha}1 in vitro by activated MAPK2. Equimolar amounts of the indicated GST-ERR{alpha}1 fusion proteins were incubated with activated MAPK2 and [{gamma}32P]ATP. PHAS-I and GST-ß-globin (molecular weight 41 kDa) were incubated likewise in parallel as positive and negative controls, respectively. The products were resolved by 12% SDS-PAGE and visualized with a PhosphorImager.

 
The monoclonal antibody (mAb) 4D5 is the murine precursor of the humanized antibody trastuzumab. They share the same epitope-reacting regions, disrupting the ErbB2 signaling pathway without affecting the overall amount of ERR{alpha}1 per cell (Fig. 7). Incubation of BT-474 cells with antibody 4D5 led to a significant reduction in the extent of ERR{alpha}1 phosphorylation, with the appearance of several phosphorylated isoforms of ERR{alpha}1 in a pattern somewhat similar to the one observed with the MCF-7 cells (Fig. 5A). The lower amount of phospo-labeled ERR{alpha}1 observed in the 4D5-treated cells was due to this treatment being inhibitory to cell growth (data not shown). Thus, we confirmed prior reports that ERR{alpha}1 is a phosphoprotein (9, 38). We also conclude that ERR{alpha}1 was phosphorylated in vivo at several sites, with the extent of phosphorylation being cell type dependent and reduced by disruption of the ErbB2 signaling pathway. Importantly, the extent of phosphorylation correlated with the transcriptional activity of ERR{alpha}1: The partially phosphorylated isoforms present in MCF-7 cells likely functioned as repressors, whereas the highly phosphorylated isoform(s) present in BT-474 cells probably functioned as activators.


Figure 7
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  		FIGURE 7. Effects of modulation of the ErbB2 signaling pathway on ERR{alpha}1-mediated activation of transcription. A and B. BT-474 cells were cotransfected as described in Fig. 2 with the ERE(5x)-regulated dual-luciferase reporter set along with the indicated expression plasmids and, likewise, in parallel with the TATA-regulated reporter set. They were incubated for 40 h in estrogen-free medium supplemented with (A) 20 µg/mL nonspecific mouse IgG, 20 µg/mL trastuzumab, or 1 µmol/L gefitinib as indicated; or (B) DMSO as the drug vehicle control, 20 µmol/L U0126, or 20 µmol/L LY294002 as indicated. Columns, mean of samples processed in triplicate relative to the levels present in the cells in lanes 1; bars, SE. Hatched columns, cells cotransfected with the GRIP1 expression plasmid; solid columns, cells cotransfected with its empty parental plasmid. C. Immunoblot analysis of overexpressed ERR{alpha}1 in BT-474 cells treated as in A and B. The membranes were probed with antibodies that react specifically with ERR{alpha}1 and ß-actin as a control. Reactive proteins were visualized by enhanced chemiluminescence and autoradiography. Lanes 1 to 3, samples from A; lanes 4 and 5, samples from B done on a different day. D. Immunoblot analysis of the phosphorylation status of Akt and MAPK in BT-474 cells treated as in A and B. The membranes were probed with antibodies that react specifically with phosphorylated Akt (p-Akt), total Akt, phosphorylated p42/44 MAPK (p-MAPK), and total p42/44 MAPK.

 
Activated MAPKs and Akts Phosphorylate ERR{alpha}1 In vitro
We next tested whether ERR{alpha}1 can serve in vitro as a substrate of MAPKs and Akts, downstream kinases in the ErbB2 signaling pathway. We incubated equal amounts of Escherichia coli–produced, carboxyl-terminal 6xHis-tagged ERR{alpha}1 (ERR{alpha}1-His) with activated MAPK1, MAPK2, Akt1, or Akt2 in the presence of [{gamma}-32P]ATP and resolved the resulting phosphorylated products by 4% to 12% gradient SDS-PAGE. Myelin basic protein was included in each reaction as an internal control. Each of these four kinases phosphorylated ERR{alpha}1 in vitro (Fig. 5B). Interestingly, differences were observed in the mobilities of the phosphorylated proteins, consistent with multiple sites on ERR{alpha}1 being phosphorylated by the MAPKs (Fig. 5B, lanes 1 and 2) and fewer sites being phosphorylated by the Akts (Fig. 5B, lanes 3 and 4).

To begin to localize sites of phosphorylation by 1-423 MAPK2, full-length glutathione S-transferase (GST)-ERR{alpha}11-423 and truncated variants of it were synthesized in and purified from E. coli. Equimolar amounts of each protein were incubated with activated p42 MAPK and [{gamma}32P]ATP and resolved by 12% SDS-PAGE (Fig. 5C). Phosphorylated heat- and acid-stable protein regulated by insulin (PHAS-I) and GST-ß-globin1-123 were assayed in parallel as positive and negative controls, respectively. As expected, activated MAPK efficiently phosphorylated PHAS-1, but not GST-ß-globin1-123 (Fig. 5C, lane 1 versus lane 5, respectively). MAPK phosphorylated each of the GST-ERR{alpha}1 fusion proteins, with significantly more label incorporated into GST-ERR{alpha}11-423 than into GST-ERR{alpha}11-376 and GST-ERR{alpha}11-173 (Fig. 5C, lane 4 versus lanes 3 and 2). Thus, ERR{alpha}1 can serve as a substrate of MAPK1/2 and Akt1/2, with multiple phosphorylation sites likely present within the protein, including at least one within the carboxyl-terminal domain.

Overexpression of ErbB2 in MCF-7 Cells Converts ERR{alpha}1 to an Activator
Given that ERR{alpha}1 transcriptional activity correlated with the cell ErbB2 status (Figs. 2 and 4), we desired to test more directly whether altering cellular ErbB2 signaling would affect ERR{alpha}1 transcriptional activity. One approach we used was to cotransfect MCF-7 cells in parallel with (a) the ERE (5x)-regulated and TATA-regulated dual luciferase reporter sets; (b) the expression plasmid encoding wild-type ERR{alpha}1; and (c) pErbB2Act, an expression plasmid encoding an activated (oncogenic) form of rat ErbB2 (rat neu; ref. 42) or its empty vector as a control. The cells were cultured in estrogen-free medium in the absence or presence of 1 µmol/L fulvestrant to prevent complications from ER{alpha}. As observed above (Fig. 2B), overexpression of ERR{alpha}1 alone led to minimal activation of ERE-regulated transcription (Fig. 6A , lane 2 versus lane 1 and lane 6 versus lane 5). Addition of ErbB2Act without exogenous ERR{alpha}1 stimulated ERE-regulated transcription ~2-fold under both estrogen-free conditions and in the presence of fulvestrant (Fig. 6A, lane 3 versus lane 1 and lane 7 versus lane 5, respectively). Hence, this activation by ErbB2Act was probably mediated via endogenous ERR{alpha}1, not ER{alpha}. Strikingly, overexpression of ERR{alpha}1 together with ErbB2Act stimulated ERE-regulated transcription almost 4-fold (Fig. 6A, lane 4 versus lane 1 and lane 8 versus lane 5). This effect of ErbB2Act occurred without a change in the level of ERR{alpha}1 (Fig. 6B, lanes 2, 4, 6, and 8). Thus, we conclude that ERR{alpha}1 transcriptional activity can be altered by ErbB2 signaling.


Figure 6
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  		FIGURE 6. Overexpression of ErbB2Act (activated rat neu oncogene) leads to activation of ERR{alpha}1 in MCF-7 cells. A. MCF-7 cells were cotransfected with the ERE(5x)-regulated dual-luciferase sets described in Fig. 2, plasmids expressing ERR{alpha}1 or their empty expression plasmid, and ErbB2Act or its empty plasmid as indicated. Cells were harvested 48 h later. Columns, mean of samples processed in triplicate relative to the level present in the cells in lane 1; bars, SE. Solid columns, cells incubated in charcoal-stripped serum (CS-FBS); cross-hatched columns, cells incubated in charcoal-stripped serum supplemented with E–6 mol/L fulvestrant (FUL). B. Immunoblot analysis of ERR{alpha}1 present in MCF-7 cells treated as in A. The membrane was probed with antibodies specific to ERR{alpha}1 and ß-actin followed by horseradish peroxidase–conjugated secondary antibodies and visualized by enhanced chemiluminescence and autoradiography.

 
Inhibition of ErbB2 Signaling Abrogates Transcriptional Activation by ERR{alpha}1
We next investigated whether blocking specific components within the ErbB2 signaling pathway led to inhibition of transcriptional activation by ERR{alpha}1. BT-474 cells were cotransfected with the reporter gene sets and expression plasmids as described in Fig. 2D (lanes 1-4). They were subsequently incubated for 40 h in estrogen-free medium supplemented with 20 µg/mL nonspecific murine IgG as a control, the humanized anti-ErbB2 mAb trastuzumab at 20 µg/mL, or the small-molecule EGFR inhibitor gefitinib at 1 µmol/L (Fig. 7A ). Gefitinib blocks transphosphorylation of ErbB2 by EGFR, thereby indirectly inhibiting ErbB2 (35). As expected, overexpression of ERR{alpha}1 in the IgG-treated cells led to a 4- to 5-fold activation of ERE(5x)-regulated transcription (Fig. 7A, lanes 3 and 4 versus lanes 1 and 2). Incubation with trastuzumab led to an ~85% reduction in ERE-regulated transcription by ERR{alpha}1 (Fig. 7A, lanes 7 and 8 versus lanes 3 and 4) to a level even below that observed in the presence of only endogenous ERR{alpha}1 in the absence of the drug (Fig. 7A, lanes 1 and 2). This large reduction was probably due to treatment with trastuzumab altering as well the transcriptional activity of the endogenous ERR{alpha}1 (Fig. 7A, lanes 5 and 6 versus lanes 1 and 2). Incubation with gefitinib led to an even greater ~90% reduction in ERE-regulated transcription by ERR{alpha}1 (Fig. 7A, lanes 11 and 12 versus lanes 3 and 4). Overexpression of GRIP1 largely failed to reverse the effect of the drug treatments (Fig. 7A, even-numbered lanes). Immunoblot analysis with an ERR{alpha}1-specific antiserum showed that incubation with the drugs had not affected accumulation of ERR{alpha}1 in the cells (Fig. 7C, lanes 1-3). Immunoblot analysis with antisera specific to the phosphorylated versus unphosphorylated forms of MAPK and Akt confirmed that these drug treatments had, indeed, inhibited activation of the MEK/MAPK and PI3K/Akt signaling pathways in these cells (Fig. 7D, lanes 1-3). Thus, we conclude that disruption of the ErbB2 signaling pathway with either trastuzumab or gefitinib prevented ERR{alpha}1 from functioning as an activator of ERE-regulated transcription in BT-474 cells, likely doing so in part by inhibiting addition of specific posttranslational phosphorylations of ERR{alpha}1 necessary for it to exist in its activator form. Moreover, blocking the ErbB2-directed cascade of signaling events rendered ERR{alpha}1 unresponsive to GRIP1-mediated coactivation.

To test whether inhibition of ErbB2 signaling modulates ERR{alpha}1 activity by affecting the activities of downstream components in this pathway, we likewise examined the effects on ERR{alpha}1 activity of incubation of BT-474 cells with U0126 and LY294002, direct inhibitors of MEK and PI3K, respectively (see Fig. 8 ). In the cells treated with only DMSO, the solvent for these drugs, overexpression of ERR{alpha}1 led, as expected, to an ~5-fold activation of ERE-regulated transcription (Fig. 7B, lane 3 versus lane 1). Incubation with 20 µmol/L U0126 led to an ~50% reduction in ERR{alpha}1-induced transcription regardless of whether GRIP1 was also overexpressed (Fig. 7B, lanes 7 and 8 versus lanes 3 and 4). The effect of incubation with 20 µmol/L LY294002 was even greater, inhibiting ERR{alpha}1-mediated activation of ERE-regulated transcription by 75% to 85% (Fig. 7B, lanes 11 and 12 versus lanes 3 and 4). Again, immunoblot analysis showed that the drug-treated cells still accumulated ERR{alpha}1 (Fig. 7B, lanes 4 and 5), with U0126 having led to inhibition of MAPK phosphorylation without affecting Akt status (Fig. 7D, lane 4) and LY294002 having led to inhibition of Akt phosphorylation without affecting MAPK status (Fig. 7D, lane 5). Therefore, the MEK/MAPK and PI3K/Akt signaling pathways contribute to the ability of ERR{alpha}1 to activate ERE-regulated transcription in BT-474 cells.


Figure 8
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  		FIGURE 8. Model for modulation of transcription by ERR{alpha}1 via the ErbB2 signaling pathway. Only a few of the numerous players in the ErbB2 signaling pathways are indicated, along with the steps in these pathways blocked by the drugs used in this study. See text for details.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
We showed here that ERR{alpha}1 down-modulated E2-induced ERE-regulated transcription in low ErbB2–expressing MCF-7 cells, doing so even when the coactivator GRIP1 was overexpressed (Fig. 2B). This inability of GRIP1 to overcome repression by ERR{alpha}1 was not due to lack of functionality because GRIP1 efficiently enhanced ERE-regulated expression mediated by an amino-terminal deleted variant of ERR{alpha}1 in these cells.5 Thus, the failure of wild-type ERR{alpha}1 to respond to GRIP1 must lie with its intrinsic properties in this cell line. On the other hand, wild-type ERR{alpha}1 functioned instead as a ligand-independent activator of ERE-regulated transcription in BT-474 cells, activity that was further stimulated by overexpression of GRIP1 (Fig. 2D). Thus, ERR{alpha}1 transcriptional activity and ability to recruit GRIP1 is cell type dependent.

ERR{alpha}1 also bound to (Fig. 3C, lanes 14-24) and activated transcription via ERREs (Fig. 2E) in BT-474 cells. Putative ERREs were identified in the promoter regions of multiple cellular genes implicated in breast cancer (Table 1), three of which, pS2, PgR, and ErbB2, were shown here to be up-regulated in response to ERR{alpha}1 in BT-474 cells, but not in MCF-7 cells (Fig. 4). This is the first report showing that ERR{alpha}1 affects ErbB2 expression, with ErbB2 mRNA levels activated almost 30-fold above the level observed in MCF-7 cell. Given that ErbB2 is an indicator of aggressive tumor growth, its regulation by ERR{alpha}1 provides another potential link for ERR{alpha}1 playing a role in the development of some breast cancers.

We also showed here that ERR{alpha}1 can exist in multiple phosphorylated isoforms in vitro (Fig. 5B and C) and in situ (Fig. 5A). The extent of this phosphorylation was significantly greater, on average, in BT-474 cells than in MCF-7 cells and reduced by treatment of BT-474 cells with the murine version of trastuzumab (Fig. 5A). Furthermore, ERR{alpha}1 could serve directly as a substrate of activated MAPKs (Fig. 5B, lanes 1 and 2, and C) and Akts (Fig. 5B, lanes 3 and 4) in vitro. Importantly, ERR{alpha}1 was shown to be a target of ErbB2 signaling: (a) Overexpression of an activated ErbB2 oncogene converted ERR{alpha}1 from a repressor to an activator of ERE-regulated transcription in MCF-7 cells (Fig. 6); and (b) disruption of ErbB2 signaling in BT-474 cells with trastuzumab, gefitinib, U0126, or LY294002 converted ERR{alpha}1 from an activator to a repressor of ERE-regulated transcription (Fig. 7). Therefore, we conclude that ERR{alpha}1 transcriptional activity is regulated, in part, by the ErbB2 signaling pathway affecting the precise state of phosphorylation of ERR{alpha}1 (Fig. 8).

Cross-talk between ER{alpha} and ERR{alpha}1
ER{alpha} and ERR{alpha}1 can both bind EREs, competing for binding to them (refs. 6-8; Fig. 3C). Vanacker et al. (7) reported that ER{alpha} can also bind to an ERRE, activating transcription 6- to 10-fold through this sequence in an E2-stimulated manner. In contrast, we observed only minimal (i.e., 1.5- to 2-fold) activation of two different ERRE/SFRE–regulated reporters in MCF-7 and BT-474 cells following addition of E2, conditions that led to 12- to 14-fold activation of our minimal ERE-regulated reporter (Fig. 3A and B). This finding was expected because ER{alpha} was unable to significantly bind a consensus ERRE (Fig. 3C, lanes 1-13). Differences between our experiments and the previous report include the following: (a) use of an ER{alpha}-negative rat osteosarcoma cell line transfected with an ER{alpha} expression plasmid for the transcription assays instead of breast carcinoma cell lines that endogenously express high levels of ER{alpha}; and (b) use of ER{alpha}-programmed reticulocyte lysates for the protein source for the EMSAs instead of whole-cell lysates of COS cells transfected with an ER{alpha} expression plasmid. Thus, the ER{alpha} protein levels in our reporter gene assays and EMSAs were probably significantly lower than they were in the previously reported experiments. We conclude that ER{alpha} probably does not significantly interact with an ERRE when present at physiologic concentrations; however, it remains possible that ER{alpha} exhibits a low affinity for some ERREs when its concentration is nonphysiologically high. Because ERR{alpha}1 exhibits a strong preference over ER{alpha} for binding to ERREs, there probably exists specific ERR{alpha}1-regulated genes that could serve as biomarkers of ERR{alpha}1 activities.

Coregulators of ERR{alpha}1
GRIP1 has been shown to recognize the nuclear receptor box within the COOH terminus of ERR{alpha}1, enhancing transcriptional activity (16). Why, then, did GRIP1 fail to significantly enhance ERR{alpha}1 transcriptional activity in MCF-7 cells (Fig. 2B and C)? Barry et al. (10) have reported that the exact sequence of the nine-nucleotide extended half-site sequence and the state of phosphorylation of ERR{alpha}1 (38) affect whether ERR{alpha}1 preferentially binds to an ERRE as a monomer or homodimer; ERR{alpha}1 acts as a repressor when bound as a monomer because it cannot recruit coactivators such as PGC-1{alpha}. However, the transcriptional activity of ERR{alpha}1 and its ability to recruit the coactivator GRIP1 to an ERE was shown here to be dependent on cell type (Fig. 2B versus D) and ErbB2 status (Fig. 6). Since ERR{alpha}1 binds to an ERE only as a homodimer (Fig. 3C; ref. 38), an alternative mechanism(s) must also exist by which ERR{alpha}1 transcriptional activity can be regulated. Based on the data presented here, we hypothesize that the recruitment of coactivators such as GRIP1 is determined, at least in part, by the phosphorylation status of specific amino acid residues within ERR{alpha}1 (Fig. 8).

In addition to ERR{alpha}1, GRIP1 itself is also a phosphoprotein target of EGFR signaling via MAPK whose phosphorylation is required for full activity (43). Thus, the effects of activated ErbB2 and the drug inhibitors of EGFR/ErbB2 signaling on transcription (Figs. 6 and 7) could have been due to changes in the phosphorylation status of GRIP1 and ERR{alpha}1.

PGC-1{alpha} can also function as a strong coactivator of ERR{alpha}1 (13). However, it is not present in the mammary cell lines studied here (data not shown). SRC-1 and SRC-3/AIB1 have also been shown to stimulate ERR{alpha}1 activity in transient transfection assays in some mammalian cell lines, albeit only modestly (15, 16). Thus, GRIP1 is likely the major, physiologically relevant coactivator of ERR{alpha}1 in mammary cells. Hence, we hypothesize that ERR{alpha}1/GRIP1 complexes likely substitute for ER{alpha}/AIB1 complexes as the major activators driving ERE-regulated expression in some breast cancers, especially ER{alpha}-negative ones. In these cases, drugs that specifically disrupt these complexes may serve as a novel therapy.

Ligand-Independent Regulation of ERR{alpha}1 Activities via ErbB2 Signaling
Based on the results presented here, we hypothesize the following model for regulation of ERR{alpha}1 activities (Fig. 8). In cells expressing ErbB2 at low levels (e.g., MCF-7), ERR{alpha}1 exists, on average, in a minimally phosphorylated state in which it binds EREs as a homodimer, yet fails to respond to GRIP1-dependent coactivation. Thus, it inhibits transcription. In cells expressing ErbB2 at high levels (e.g., BT-474), ErbB2, as either a homodimer or heterodimer with other ErbB family members, signals additional or alternative phosphorylations of ERR{alpha}1, at least in part, through MEK/MAPK and PI3K/Akt signaling pathways. This highly phosphorylated form of ERR{alpha}1 binds to both EREs and ERREs as a homodimer, activating transcription via interactions with cellular coactivators such as GRIP1. Thus, changes in specific sites of phosphorylation of ERR{alpha}1 induced via the ErbB2 signaling pathway convert ERR{alpha}1 between repressor and activator of transcription.

The precise mechanism(s) that regulates the interaction between GRIP1 and ERR{alpha}1 is still unclear. Barry et al. (38) have proposed that ERR{alpha}1 can switch between monomer and homodimer, with only the homodimer form binding coactivators. Another possibility is that the repressor domain(s) of ERR{alpha}1 interacts with cellular corepressors, blocking binding of coactivators. Castet et al. (44) recently reported that the corepressor RIP140 inhibits ERR{alpha}-mediated trans-activation of ERE-dependent expression. Other data consistent with a corepressor(s) regulating the activity of ERR{alpha}1 include (a) identification of a repressor domain within the NH2-terminal region of ERR{alpha}1 (45) and (b) overexpression of ERR{alpha}1E97G,A98S,A101V, a variant of ERR{alpha}1 that fails to bind DNA due to mutations in its DNA-binding domain P-box, derepressing ERE-regulated transcription in MCF-7 cells, presumably by sequestering a corepressor (8). A third possibility is that changes in phosphorylation lead to changes in other posttranslational modifications in ERR{alpha}1, thereby affecting the coregulators with which it interacts. Consistent with this latter hypothesis is the recent finding that ERR{alpha}1 is also sumoylated; mutation of one of these sites of sumoylation also affects ERR{alpha}1 transcriptional activity.5 These three mechanisms are not mutually exclusive.

Other kinase/phosphatase signaling pathways probably also target ERR{alpha}1, leading to changes in ERR{alpha}1 activities via alterations in its specific sites of phosphorylation. For example, Barry and Giguère (38) recently reported that protein kinase C{delta}, an enzyme whose activity is stimulated by epidermal growth factor or phorbol 12-myristate 13-acetate, can phosphorylate ERR{alpha}1 within its DNA-binding domain, thereby enhancing both the binding of ERR{alpha}1 homodimers to ERREs and transcription. They further showed that stimulation of ERR{alpha}1 phosphorylation by incubation of their MCF-7 cells with phorbol 12-myristate 13-acetate can lead to an ~2-fold activation of transcription of the pS2 gene via an ERRE present within its promoter. Likely, numerous cellular kinases and phosphatases activated through signaling pathways can affect specific sites of phosphorylation that exist within the A/B,5 C (38), and E/F (Fig. 5C) domains of ERR{alpha}1. Depending on which of these multiple specific sites becomes phosphorylated, ERR{alpha}1 functions as a repressor or activator to modulate expression of numerous ERE- and ERRE-regulated cellular genes.

Role of ERR{alpha} in Breast Cancer
The ErbB family of tyrosine kinase receptors signals diverse pathways that play roles in the development of aggressive breast cancers and their resistance to antihormonal therapy. Hence, factors whose activities are both estrogen-independent and sensitive to disruptors of ErbB2 signaling likely contribute to some tamoxifen-resistant and ER-negative breast cancers. ERR{alpha}1 meets the following criteria: (a) the activator form of ERR{alpha}1 can functionally substitute for ER{alpha} in ErbB2-overexpressing cells (Figs. 2D and 6), and (b) blockade of ErbB2 signaling or its downstream effectors, e.g., MEK/MAPK or PI3K/Akt, leads to conversion of ERR{alpha}1 from an activator to a repressor, eliminating the ability of ERR{alpha}1 to substitute for ER{alpha} (Fig. 7). Thus, ER-positive breast tumors expressing high levels of ErbB2 along with the activator form of ERR{alpha} will likely not respond well to hormonal-blockade therapies; rather, they may respond well instead to ErbB2-based therapies such as trastuzumab. Given that ERR{alpha}1 likely down-modulates the activity of ER{alpha} in some ER{alpha}-positive tumors while it functionally substitutes for ER{alpha} in other tumors leading to estrogen-independent activation of key genes involved in breast cancer, ERR{alpha}1 and its phosphorylation status should be evaluated as biomarkers of prognosis and determinants of specific therapeutic treatments. Moreover, ERR{alpha} may have use, in itself, as a target for a new class of drugs, possibly for use in combination with some current therapies.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Lines
MCF-7/WS8 mammary carcinoma cells were used in all studies in which MCF-7 cells are indicated; they were clonally derived from MCF-7 cells by selection for sensitivity to growth stimulation by E2 (46, 47). BT-474 cells were obtained from the American Type Culture Collection (Manassas, VA). Both cell lines were maintained in estrogenized medium (i.e., phenol red-containing RPMI 1640, 10% whole fetal bovine serum, 6 ng/mL insulin, 2 mmol/L glutamine, 100 µmol/L nonessential amino acids, and 100 units of penicillin and streptomycin per milliliter). Two days before reseeding of cells for an experiment, the medium was changed to phenol red- and estrogen-free medium containing charcoal-stripped fetal bovine serum (48). The monkey kidney COS-M6 cell line was cultured as previously described (8). Cells were maintained at 37°C in a humidified 5% CO2 atmosphere.

Cellular Treatment Agents
E2 (Sigma-Aldrich, St. Louis, MO) and the complete antiestrogen fulvestrant (ICI 182,780, Faslodex; a generous gift from AstraZeneca, Macclesfield, United Kingdom) were dissolved in ethanol. Control, nonspecific murine IgG (reagent grade, Sigma-Aldrich) was dissolved in PBS. Trastuzumab (Herceptin; purchased from the Lurie Cancer Center pharmacy) was dissolved in bacteriostatic water. A hybridoma cell line that secretes the mAb 4D5, a murine precursor of trastuzumab directed against the ectodomain of ErbB2 (HER2), was obtained from the American Type Culture Collection; IgG was purified from the ascites fluid of a mouse inoculated with this cell line. Gefitinib (Iressa, ZD1839; a generous gift from AstraZeneca) was initially dissolved in DMSO, followed by further dilution in ethanol. U0126 (Promega, Madison, WI) and LY294002 (Promega) were dissolved in DMSO. All test agents were added to the medium at a 1:1,000 (v/v) dilution.

Plasmids
Plasmid pcDNA3.1-hERR{alpha}1 (ERR{alpha}1), encoding the full-length, 423-amino-acid major human isoform of ERR{alpha}, and plasmid pcDNA3.1-hERR{alpha}1L413A/L418A (ERR{alpha}1L413A/L418A), encoding a variant of ERR{alpha}1 defective in the carboxyl-terminal coactivator-binding LxLxxL motif, have been previously described (8). Plasmid pcDNA3.1-hERR{alpha}11-376 (ERR{alpha}11-376), generated by PCR-based subcloning, encodes a carboxyl-terminal truncated variant of ERR{alpha}1 lacking the coactivator-binding LxLxxL motif. Plasmid pcDNA3-GRIP1 encodes the coactivator GRIP1 (49). The replication-defective retrovirus pJRneu has been previously described (42); it encodes the activated form of the rat neu oncogene (ErbB2Act). Plasmid pEGFP encodes enhanced green fluorescent protein (TaKaRa; Clontech, Palo Alto, CA).

Plasmids pTA-ffLuc and pTA-srLuc, containing TATA-box basal promoter firefly and Renilla luciferase reporter genes, respectively, were constructed by insertion via HindIII linkers of the nucleotides –31 to +31 region of the herpes simplex virus thymidine kinase promoter into pGL3-Basic and phRG-B (Promega), respectively. Plasmid pERE(5x)TA-ffLuc, containing five tandem copies of the consensus palindromic ERE, was constructed by insertion into the XhoI and BglII sites of pTA-ffLuc of the oligodeoxynucleotides 5'-tcgagagAGGTCActgTGACCTctgagagAGGTCActgTGACCTctctcagAGGTCActgTGACCTctgcgagAGGTCActgTGACCTctgcgagAGGTCActgTGACCTcta-3' and 5'-gatctagAGGTCAcagTGACCTctcgcagAGGTCAcagTGACCTctcgcagAGGTCAcagTGACCTctgagagAGGTCAcagTGACCTctctcagAGGTCAcagTGACCTctc-3'. Plasmid pERRE(5x)TAffLuc, containing five tandem copies of a consensus ERRE, was constructed by insertion into pTA-ffLuc of the oligodeoxynucleotides 5'-tcgagtcaAGGTCAgagctcgtcaAGGTCActgcagctcaAGGTCAgctagcgtcaAGGTCAgcatgcgtcaAGGTCAa-3' and 5'-gatctTGACCTtgacgcatgcTGACCTtgacgctagcTGACCTtgagctgcagTGACCTtgacgagctcTGACCTtgac-3'. EREs and ERREs are underlined, with core ERE half-sites indicated in uppercase letters. The previously described plasmid pSFREx3-Luc (refs. 6, 50; generous gift of Jean-Marc Vanacker) contains three copies of a consensus ERRE/SFRE (5'-TCAAGGTCA-3') upstream of the SV40 minimal early promoter regulating firefly luciferase expression. Plasmid phRL-TK contains the herpes simplex virus thymidine kinase promoter driving expression of a humanized Renilla luciferase (Promega).

Transient Transfection Reporter Gene Assays
Transient transfection assays were done using a dual-luciferase system (Promega) in which pTA-srLuc served as an internal control (Fig. 2A). The basal TATA promoter–regulated set (Fig. 2A) was transfected in parallel with the ERE- or ERRE/SFRE–regulated sets as an external control for the physiologic state of the cells and effects of test agents on basal transcription activity. All data were normalized to both of these controls.

After culture for 2 days in estrogen-free medium, MCF-7 and BT-474 cells were seeded in estrogen-free medium into 24-well plates at densities of 100,000 and 150,000 per well, respectively. The amounts of the indicated DNAs cotransfected per well using TransIT LT1 reagent (Mirus, Madison, WI) were as follows: 150 ng of pcDNA3.1-ERR{alpha}1, pcDNA3.1- hERR{alpha}1L413A/L418A, and pcDNA3.1; 200 ng of pGRIP1 or its empty parental plasmid, pcDNA3.1; 200 ng of pJRneu or its empty parental plasmid, pJR (42); 200 ng of pERE(5x)-TA-ffLuc, pERRE(5x)-TA-ffLuc, pTA-ffLuc, or pSFRE3x-Luc; and 50 ng of pTA-srLuc or phRLTK. Four hours later, the cells were placed in fresh medium containing the indicated treatment agents until harvested.

EMSAs
To assess the binding affinities of ERR{alpha}1 for putative ERREs located within promoter regions of cellular genes, competition EMSAs were done as previously described (5, 8). In brief, COS-M6 cells were transfected with pcDNA3.1-ERR{alpha}1 (4 µg per 100-mm-diameter dish) using TransIT LT1 reagent. Forty-eight hours later, whole-cell extracts were prepared as the source of ERR{alpha}1 protein. Twenty micrograms of ERR{alpha}1-containing protein extract was preincubated on ice for 15 min with the indicated unlabeled competitor oligonucleotides in reaction buffer (8). Afterward, 1 ng of radiolabeled double-stranded oligodeoxynucleotide containing the sequence 5'-agcagtggcgatttgTCAAGGTCAcacagt-3' serving as probe DNA was added, and incubation was continued for 15 min at room temperature before electrophoresis in a nondenaturing 5% polyacrylamide gel at 200 V for 2 h at 4°C. Gels were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Immunoshift assays were done by addition of the rabbit polyclonal antiserum anti-GST-hERR{alpha}117-329 (5). Competition EMSAs were done likewise with whole-cell extracts prepared from COS-M6 cells transfected with pCMV-ER{alpha} or pcDNA3.1-hERR{alpha}1 serving as protein source, radiolabeled double-stranded oligonucleotide containing the sequence 5'-taagcttAGGTCAcagTGACCTaagctta-3' serving as probe DNA, and unlabeled oligodeoxynucleotide containing the indicated sequence serving as competitor.

Cellular RNA Analyses
Cells were seeded in 15-cm plates at ~70% confluency, transfected with 8 µg pEGFP plus 14 µg of pcDNA3.1-hERR{alpha}1 or pcDNA3.1 using TransIT LT1 reagent, and incubated under estrogen-free conditions. Twenty-four hours later, EGFP-positive cells (100,000-500,000 per group) were sorted and collected by fluorescence-activated cell sorting (Beckman Coulter Epics Elite ESP equipped with Elite software; Beckman Coulter, Miami, FL). Total RNA, isolated from these EGFP-positive cells with an RNeasy Micro kit (Qiagen), was converted to first-strand cDNA using SuperScript III with a combination of random hexamers and oligo(dT) as primers (Invitrogen). Quantitative real-time PCR assays were done as previously described (11, 50) with the Taqman Universal or SYBR Green PCR Master Mixes and an ABI 7700 sequence detection system (Applied Biosystems, Foster City, CA). The pS2 forward primer was 5'-GAGGCCCAGACAGAGACGTG-3', pS2 reverse primer was 5'-CCCTGCAGAAGTGTCTAAAATTCA-3' and the pS2 probe was 5'-[FAM]-CTGCTGTTTCGACGACACCGTTCG-[QSY7]-3'. The PgR forward primer was 5'-CGTGCCTATCCTGCCTCTCAA-3' and the PgR reverse primer was 5'-CCGCCGTCGTAACTTTCGT-3'. Abundances were determined by comparison with a standard curve generated by serial dilution of known quantities of the PCR product (11), with internal normalization to 18S rRNA.

Immunoblot Analyses
Proteins were extracted in Cell Lysis Buffer (Cell Signaling Technology, Beverly, MA) supplemented with Protease Inhibitor Cocktail Set III and Phosphatase Inhibitor Cocktail Set II (Calbiochem, San Diego, CA). Protein amounts were quantified with Coomassie Protein Assay Reagent (Pierce Biotechnology, Rockford, IL) with bovine serum albumin as the standard. Proteins (20 µg