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Cancer Research 68, 8805, November 1, 2008. doi: 10.1158/0008-5472.CAN-08-1594
© 2008 American Association for Cancer Research
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Endocrinology

Estrogen-Related Receptor {alpha} Is Critical for the Growth of Estrogen Receptor–Negative Breast Cancer

Rebecca A. Stein1, Ching-yi Chang1, Dmitri A. Kazmin1, James Way3, Thies Schroeder2, Melanie Wergin2, Mark W. Dewhirst2 and Donald P. McDonnell1

1 Department of Pharmacology and Cancer Biology and 2 Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina and 3 GlaxoSmithKline, Research Triangle Park, North Carolina

Requests for reprints: Donald P. McDonnell, Department of Pharmacology and Cancer Biology, Duke University Medical Center, Box 3813, Durham, NC 27710. Phone: 919-684-6035; Fax: 919-681-7139; E-mail: donald.mcdonnell{at}duke.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Expression of estrogen-related receptor {alpha} (ERR{alpha}) has recently been shown to carry negative prognostic significance in breast and ovarian cancers. The specific role of this orphan nuclear receptor in tumor growth and progression, however, is yet to be fully understood. The significant homology between estrogen receptor {alpha} (ER{alpha}) and ERR{alpha} initially suggested that these receptors may have similar transcriptional targets. Using the well-characterized ER{alpha}-positive MCF-7 breast cancer cell line, we sought to gain a genome-wide picture of ER{alpha}-ERR{alpha} cross-talk using an unbiased microarray approach. In addition to generating a host of novel ERR{alpha} target genes, this study yielded the surprising result that most ERR{alpha}-regulated genes are unrelated to estrogen signaling. The relatively small number of genes regulated by both ER{alpha} and ERR{alpha} led us to expand our study to the more aggressive and less clinically treatable ER{alpha}-negative class of breast cancers. In this setting, we found that ERR{alpha} expression is required for the basal level of expression of many known and novel ERR{alpha} target genes. Introduction of a small interfering RNA directed to ERR{alpha} into the highly aggressive breast carcinoma MDA-MB-231 cell line dramatically reduced the migratory potential of these cells. Although stable knockdown of ERR{alpha} expression in MDA-MB-231 cells had no effect on in vitro cell proliferation, a significant reduction of tumor growth rate was observed when these cells were implanted as xenografts. Our results confirm a role for ERR{alpha} in breast cancer growth and highlight it as a potential therapeutic target for estrogen receptor–negative breast cancer. [Cancer Res 2008;68(21):8805–12]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Estrogen-related receptor {alpha} (ERR{alpha}; NR3B) is an orphan member of the nuclear receptor (NR) superfamily that shares considerable structural homology to the classical estrogen receptor {alpha} (ER{alpha}) and ERβ. Although its primary function seems to be the activation of fatty acid oxidation and mitochondrial biogenesis in tissues that have high-energy demands (e.g., cardiac and skeletal muscle), its expression has also been associated with a negative outcome in breast and ovarian cancers (1–3). This latter finding is somewhat paradoxical, given that cancers are generally thought to rely predominantly on glycolytic metabolism (4). It is unclear therefore, considering the known biology of ERR{alpha}, how this receptor is involved in tumor pathophysiology.

One proposed mechanism regarding the role of ERR{alpha} in breast cancer biology stems from the observation that ERR{alpha} and ER{alpha} share a high degree of structural similarity. It has been hypothesized in breast cancer that ERR{alpha} may function as a modulator of estrogen signaling. Indeed, cross-talk between the ER{alpha} and ERR{alpha} signaling pathways has been observed at several levels (5–8). It was initially suggested that ERR{alpha} might function as an alternate receptor for estrogens. However, it now seems that neither physiologic nor synthetic ER{alpha} ligands are likely to interact significantly with ERR{alpha}. Indeed, crystallographic analysis of apo-ERR{alpha} indicates that, even without ligand, this orphan receptor adopts an active conformation, recruiting cofactors and initiating transcription (9).

Despite the inability of ERR{alpha} to respond directly to estrogens and antiestrogens, this receptor may affect ER{alpha} signaling at the level of target gene transcription. Notably, ER{alpha} and ERR{alpha} are most highly homologous within their DNA-binding domains. Not surprisingly, therefore, direct DNA-binding studies and reporter gene assays have indicated that many classical palindromic estrogen response elements (ERE) can function as estrogen-related receptor response elements (ERRE; ref. 10). The majority of the ERREs mapped in genes involved in metabolism, however, do not interact in a significant manner with ER{alpha}. That EREs may represent a subset of ERREs supports a model in which ERR{alpha} exhibits activities both related and unrelated to ER{alpha} signaling in breast cancer. Definition of the ERR{alpha} transcriptome in breast cancer will likely help to determine the extent to which its role in this context involves cross-talk with the ER{alpha} signaling pathway and identify the mechanisms by which this receptor affects tumor pathology.

Whereas a physiologic role for ERR{alpha} as a regulator of estrogen signaling remains to be described, its role as a modulator of metabolic processes in muscle, heart, and liver is well characterized. In these settings, ERR{alpha} enhances β oxidation of fatty acids and regulates flux through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) by transcriptional induction of rate-limiting enzymes in each process (11–14). Although certain tumors rely heavily on glycolysis, the capacity to generate ATP through OXPHOS is not always completely lost. For example, cancer cells may retain the ability to use a variety of oxidative substrates, including fatty acids, glutamine, and ketone bodies (15, 16). Recent studies have shown that the TCA cycle and OXPHOS pathway are used in tumors not only to generate ATP but also to supply the biosynthetic precursors required for sustained proliferation (17). Considering the complexity of tumor metabolism, the extent to which ERR{alpha} function in breast cancer is related to either its metabolic functions or its interaction with the ER{alpha} signaling pathway remains to be determined.

In view of this potential duality in ERR{alpha} function, we sought to define the ER{alpha}-related and ER{alpha}-unrelated transcriptional effects of ERR{alpha} activity in breast cancer cells. The significant number of genes affected by ERR{alpha} activity, but not by estrogen treatment, led to the hypothesis that an ER{alpha}-independent activity of ERR{alpha} may partially account for the negative correlation between ERR{alpha} expression and patient outcomes. We used a xenograft model of ER{alpha}-negative breast cancer to test the effect of ER{alpha}-independent ERR{alpha} activity on breast cancer pathology and found that ERR{alpha} knockdown significantly inhibited tumor growth. These studies provide compelling evidence that ERR{alpha} is involved in breast cancer growth and justify further investigation into the use of ERR{alpha} as a therapeutic target.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell culture. MDA-MB-231 cells were cultured in DMEM, AU565 and BT474 cells in RPMI, and MCF-7 cells in DMEM/F12 (Invitrogen). Stable MDA-MB-231 luciferase cells and derived cells were grown in DMEM supplemented with antibiotics.

Generation and transduction of adenoviruses. The generation of PGC-1{alpha} adenoviruses has been described (18). Cells were infected at a multiplicity of infection (MOI) of 20 to 100 (2 h at 20°C). For microarray experiments, MCF-7 cells were cultured in estrogen-free media (48 h) before adenoviral infection (MOI, 50), treated 12 h later, and harvested after an additional 12 h. In experiments with small interfering RNA (siRNA) and PGC-1{alpha} infections, cells were infected with siRNA adenovirus 48 h before an infection with PGC-1{alpha} (11). Immunoblot analysis verified that ERR{alpha} knockdown did not alter ER{alpha} expression (Supplementary Fig. S1).

RNA preparations and analysis. Total RNA was isolated using Qiagen or Bio-Rad RNA purification columns and DNase treated (Bio-Rad). An iScript cDNA synthesis kit (Bio-Rad) was used (1 µg RNA). Real-time PCR was performed [0.25 µL cDNA, 0.4 µmol/L specific primers (Supplementary Table S1), and Bio-Rad iQ SYBRGreen supermix], and results were quantified using the 2–{Delta}{Delta}Ct method (19).

Microarray data analysis. Probe synthesis, hybridization on Affymetrix HG133A GeneChips (Affymetrix), and scanning were performed at the Expression Analysis Institute. Probe level analysis was performed in dChip (20). Normalization and statistical cutoffs are described in detail in Supplementary Materials and Methods.

Immunoblotting. Protein extracts were separated by 8% SDS-PAGE and transferred onto nitrocellulose membrane. ERR{alpha} protein was detected using a mouse anti-ERR{alpha} monoclonal antibody (18), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected using goat anti-GAPDH (V-18; Santa Cruz Biotechnology, Inc.).

Retroviral constructs and gene silencing. ERR{alpha} siRNA oligonucleotides (11) and control siRNA oligonucleotides (18) were ligated into PSR-GFP/Neo (OligoEngine). Stable cell lines were constructed and sorted using standard techniques, as described in Supplementary Materials and Materials. To achieve ERR{alpha} re-expression, an siRNA-resistant ERR{alpha} (ERRsiR) was generated by introducing silent mutations into the siRNA-targeted region.

Proliferation and migration assays. For proliferation assays, 5,000 cells were plated per well of a 96-well plate. Cell number was assayed using FluoReporter Blue Fluorometric dsDNA Quantitation kit (Molecular Probes). For migration assays, serum-starved cells migrated for 4 h prior to being stained and counted. See Supplementary Materials and Methods for details.

In vivo tumor formation. All animal handling and procedures were approved (Duke University Medical Center). Cells (2 x 106) in 50% Matrigel, (BD Matrigel Matrix, BD Biosciences) in PBS were injected into the axillary mammary fat pad of 6-wk-old to 8-wk-old female athymic nu/nu mice (10 mice per group; ref. 21). Primary tumor volume was quantified using calipers (volume = width2 x length / 2), and tumors were removed either at a maximum of 1,500 mm3 or upon ulceration. Tumors were excised and frozen in liquid N2.

Statistical analyses. Proliferation, migration, and RT-qPCR data are represented as mean ± SE for biological replicates, unless otherwise noted. Significance was evaluated by Mann-Whitney U test for all biological data (GraphPad) and Student's t test, as indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
The majority of ERR{alpha} target genes in breast cancer are unrelated to ER{alpha} signaling. The relationship between the ER{alpha} and ERR{alpha} transcriptomes was evaluated in MCF-7 breast cancer cells using a classical microarray approach. Because a small molecule ligand has not been identified for ERR{alpha}, its transcriptional activity in these studies was induced using its known coactivator PGC-1{alpha} (peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha}) as a protein ligand (22). Although complementary approaches, such as knocking down of ERR{alpha}, were subsequently used, PGC-1{alpha} was used as a coactivator to highlight ERR{alpha} target genes by providing a large dynamic range of regulation amenable to microarray technology. PGC-1{alpha} enhances the transcriptional activity of several NRs and non-NR transcription factors, resulting in the regulation of a variety of metabolic processes, including lipid metabolism, mitochondrial biogenesis, and oxidative metabolism (14, 23). To isolate the ERR{alpha}–PGC-1{alpha} complex in these studies, we used a customized PGC-1{alpha} that selectively binds to and activates the ERRs (ERRspPGC1). Non–NR-dependent activities of PGC-1{alpha} were controlled for using a variant PGC-1{alpha} in which the leucines within the NR-interacting domain had been mutated to alanines (L2L3M). We previously reported the construction, validation, and use of ERRspPGC1 (termed PGC-1{alpha} 2 x 9) and L2L3M in the context of hepatic gene regulation and function of ERR{alpha} (18).

For the microarray study, MCF-7 cells were grown in estrogen-free conditions for 48 hours before adenoviral infection with wild-type (WT) PGC-1{alpha}, ERRspPGC1, L3L3M, or β-galactosidase. When maximal expression of the PGC-1{alpha} construct was achieved (12 hours), cells were treated with either vehicle or estradiol (10 nmol/L) for an additional 12 hours. This enabled the identification of genes regulated by ERR{alpha} or ER{alpha} in breast cancer cells and the analysis of pathway overlap. We confirmed the equivalent expression of each PGC-1{alpha} variant and that ERRspPGC1 activates known ERR{alpha} target genes (e.g., medium chain acyl dehydrogenase) similarly to WT PGC-1{alpha} but does not activate ER{alpha} targets [e.g., progesterone receptor (PR); Fig. 1C and Supplementary Fig. S2]. We also verified that canonical ER{alpha} target genes (e.g., PR) were activated by estradiol treatment under our experimental conditions (Supplementary Fig. S2).


Figure 1
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  		Figure 1. Definition of the overlap between ERR{alpha} and ER{alpha} signaling in MCF-7 cells. A, number of genes regulated by ERR{alpha} (induced or repressed by infection of ERRspPGC1 compared with L2L3M) versus genes regulated by ER{alpha} (induced or repressed by estrogen treatment) and those that belong to both groups (15 genes). 620 ERR{alpha}-regulated and 99 ER{alpha}-regulated genes were identified using a Student's t test with a P value cutoff of P < 0.01 and fold change of >50% up or down. A comparison was then made to determine the subset of genes regulated by either pathway, in which expression levels were significantly different (P < 0.01) between MCF-7 cells expressing ERRspPGC1 (vehicle treated) and MCF-7 cells treated with estradiol (expressing control L2L3M). Genes in which this secondary analysis showed no significant difference between ERR{alpha} and estrogen-induced expression are shown in light gray. B, hierarchical clustering of genes regulated by ER{alpha} and ERR{alpha}, illustrated on a subset of MCF-7 microarray conditions. Cells were infected with adenovirus and treated 12 h later with vehicle or estradiol (10 nmol/L). ERR{alpha}-regulated genes were defined as the set of genes affected by ERRspPGC1 (I, II, III, V) and ER{alpha} targets as genes induced or repressed by addition of estradiol (predominately in classes II, III, IV). C, real-time PCR of representative genes from each class found in the microarray. Four different infections (negative control β-galactosidase and WT PGC-1{alpha} in addition to the two PGC-1{alpha} mutants) were analyzed with vehicle or estradiol treatment. 36B4 was used as internal normalization control, and a representative experiment was shown for each gene. Medium chain acyl dehydrogenase (ACADM) represents genes regulated only by ERR{alpha} activation (class I) and ret proto-oncogene (RET) represents genes regulated only by ER{alpha} (class IV). Note that in addition to the jointly regulated genes in classes II and III [e.g., CXCL12 (stromal derived factor-1) and cyclin E2], class V was found to contain some dually regulated genes (e.g., cyclin G2).

 
Analysis of microarray expression data revealed that the vast majority of genes were similarly regulated by ERRspPGC1 and WT PGC-1{alpha} compared with either control condition (β-galactosidase or PGC-1{alpha} L2L3M; Supplementary Fig. S3). This result agrees with our previous findings (24) and suggests that ERR{alpha} may be a major conduit of PGC-1{alpha} signaling in breast cancer cells. We did find, however, that WT PGC-1{alpha} regulated a subset of genes differently than ERRspPGC1 (particularly in estradiol treated samples), which is likely due to PGC-1{alpha} coactivation of other NRs (e.g., ER{alpha}; ref. 25). Therefore, to determine the degree of cross-talk between ERR{alpha} and ER{alpha}, we identified genes regulated by either ERRspPGC1 (vehicle treated, PGC-1{alpha} L2L3M versus ERRspPGC1 infected) or estradiol (PGC-1{alpha} L2L3M infected, vehicle versus estradiol). Applying identical statistical thresholds for each comparison (Student's t test P < 0.01, fold change >50%), 620 ERR{alpha}-regulated and 99 ER{alpha}-regulated genes were identified (Supplementary Table S2). Initial inspection revealed that only 15 probesets were present in both groups, and we next sought to address the difference in the gene expression patterns between the samples with activated ERR{alpha} versus samples with activated ER{alpha} (ERRspPGC1-infected, vehicle-treated samples versus L2L3M-infected, estradiol-treated samples). This analysis indicated that the effects of ERR{alpha} and ER{alpha} may not be as distinct as was thought based on the small number of genes regulated by both receptors in our initial analysis. Specifically, 34% of the 704 initially identified genes were not differentially regulated by ERR{alpha} versus ER{alpha} (Fig. 1A, light gray region; Supplementary Table S2).

Unsupervised hierarchical clustering of the genes regulated by either ERR{alpha} or ER{alpha} revealed five major expression patterns (Fig. 1B). We validated genes subject to each pattern of regulation by RT-qPCR (Fig. 1C and Supplementary Fig. S4). The majority of genes are induced predominately by ERR{alpha}, with little ER{alpha} contribution (class I). Class II represents genes that are positively regulated by ER{alpha} and ERR{alpha}, whereas class III is composed of genes up-regulated by ER{alpha} and repressed by ERR{alpha}. Although direct competition for DNA binding may explain the activity of ERR{alpha} on class III genes, it may also be due to cofactor competition, a mechanism that is likely to be relevant in cells where both receptors are activated. The reduction of basal levels of gene expression by ERRspPGC1 on some genes in this group raises the possibility that the ERR{alpha} actively represses genes through a mechanism independent of ER{alpha}. Genes in class IV are up-regulated by estradiol with little or no contribution of ERR{alpha}. Finally, the dominant feature of class V genes is repression by ERR{alpha}. Within this class, however, resides a subset of genes repressed by both ERR{alpha} and ER{alpha} (e.g., cyclin G2). Although these results, taken together, suggest some overlap between the ER{alpha} and ERR{alpha} pathways in breast cancer cells, they also show that a large portion of ERR{alpha} activity may be unrelated to ER{alpha} signaling. This finding motivated our continued investigations into the role of ER{alpha}-independent ERR{alpha} activity in breast cancer.

Functional classes of genes regulated by ERR{alpha} in breast cancer cell lines. As a first step in defining the functional role of ERR{alpha} in breast cancer, we performed gene ontology (GO) analysis on the ERR{alpha} regulated genes identified above. This analysis yielded 12 overrepresented GO terms, the majority of which are composed of genes involved in aerobic metabolism (Supplementary Table S3). Notably, we found that the majority of enzymes of TCA and electron transport chain are induced by ERR{alpha}. Several of these genes have been shown previously to be physiologically relevant ERR{alpha} targets in other tissues, and their potential roles in tumor pathophysiology are a matter of active research (24).

Whereas GO analysis revealed a major effect of ERR{alpha} signaling on genes involved in aspects of energy metabolism, inspection of the list of ERR{alpha}-regulated genes also provided insight into its role in tumor biology. Considering both the intrinsic and treatment-induced metabolic stress in the tumor microenvironment, it is notable that several enzymes involved in the response to oxidative stress, including superoxide dismutase (SOD2) and detoxification enzyme glutathione S-transferase M1 (GSTM1), were induced by ERRspPGC1 (Fig. 2A ). Using a knockdown approach to compliment activation of ERR{alpha} by PGC-1{alpha}, we verified that these genes were regulated by ERRspPGC1 in an ERR{alpha}-dependent manner in both ER{alpha}-positive and ER{alpha}-negative cell lines (Fig. 2A). Furthermore, and of particular relevance to cancer biology, ERRspPGC1 induced expression of the highly angiogenic vascular endothelial growth factor (VEGF), which has recently been identified as a direct ERR{alpha} transcriptional target in murine skeletal muscle (26). We found that the basal level of many ERRspPGC1-induced genes, including VEGF, SOD2, and the key TCA cycle enzyme IDH3A (isocitrate dehydrogenase 3A), is dependent on ERR{alpha} expression in the ER{alpha}-positive MCF-7 and BT474 cell lines and in the ER{alpha}-negative AU565 and MDA-MB-231 cell lines (Fig. 2B and data not shown).


Figure 2
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  		Figure 2. The regulation of genes involved in the response to oxidative stress by ERR{alpha} and the dependence of basal levels of ERR{alpha} target genes on ERR{alpha} expression. A, PGC-1{alpha} induction of SOD2 and GSTM1, ERR{alpha} target genes identified in the MCF-7 array, is dependent on ERR{alpha} expression. MCF-7 and MDA-MB-231 cells were serially infected with the siRNA to ERR{alpha} (siERR{alpha}) or scrambled control (siCON) adenovirus indicated, followed by ERRspPGC1 or the control β-galactosidase virus. mRNA levels are assessed by RT-qPCR, normalized to the expression of 36B4 and represented ± SE. B, basal levels of ERR{alpha} target genes in breast cancer cell lines MCF-7 and BT474 (ER{alpha}-positive) and AU565 (ER{alpha}-negative) cell line analyzed by RT-qPCR after adenoviral infection with siRNA to ERR{alpha} (siERR{alpha}) or scrambled control (siCON).

 
Taken together, our data suggest that ERR{alpha} is critical for expression of the detoxification enzymes SOD2 and GSTM1 and the angiogenic factor VEGF, and that it can regulate the expression of numerous genes involved in energy metabolism. Furthermore, ERR{alpha} plays a role in the basal expression of these genes in both ER{alpha}-positive and ER{alpha}-negative breast cancer cell lines. Encouraged that endogenous ERR{alpha} contributes to the basal expression of these potentially cancer-associated genes, we sought to determine the phenotypic effects of manipulating ERR{alpha} activity. Because of the limited cross-talk found in the MCF-7 microarray, as well as the potential significance of the many ERR{alpha}-regulated genes unrelated to ER{alpha} signaling, we focused on delineating the role of ERR{alpha} in ER{alpha}-negative breast cancer.

Evaluation of the role of ERR{alpha} on proliferation and migration in a cellular model of ER{alpha}-negative breast cancer. We generated a series of derivatives of the MDA-MB-231-luciferase cell line, which is a well-validated model of ER{alpha}-negative breast cancer that can be studied in vitro or as a xenograft (27). Derivative stable cell lines were constructed by retroviral-mediated expression of siRNA directed against ERR{alpha} or scrambled control siRNA and transformants selected using a coexpressed GFP protein (11). The resultant polyclonal populations (a) MDA-MB-231-luc siERR{alpha} and (b) MDA-MB-231-luc siCON (called MDA231 siERR{alpha} and MDA231 siCON) were then subjected to further characterization. We showed quantitative inhibition of ERR{alpha} expression in the MDA231 siERR{alpha} cells compared with the control cell line (Fig. 3A ). The efficacy of siRNA was confirmed as expression of ERR{alpha} target genes was decreased in cells with ERR{alpha} knockdown compared with control cells (Fig. 3A).


Figure 3
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  		Figure 3. Generation and characterization of MDA-MB-231-luciferase cells expressing ERR{alpha} short hairpin RNA. A, RT-qPCR of ERR{alpha} and target genes in MDA231 siERR{alpha} cells versus MDA231 siCON cells. Data normalized to 36B4 and shown as fraction of siCON mRNA ± SE. B, results of in vitro proliferation assays showing no statistically significant differences between cell lines. Data from three biological replicates plotted as fold over cells plated ± SE. C, down-regulation of ERR{alpha} results in significantly reduced ability to migrate, whereas re-expression of ERRsiR restores migration. The indicated stable MDA-MB-231 cell lines were serum starved for 18 h and allowed to migrate toward serum in a Boyden chamber. Migrated cells were stained, imaged, and counted. Cells were plated in triplicate, and data of biological replicates plotted as normalized ratio to cells migrated in the MDA231 siCON YFP cell line. Western blot analysis of the protein levels of ERR{alpha} in whole cell extracts from each MDA-MB-231–derived cell line with GAPDH shown as a loading control.

 
We next evaluated the effects of ERR{alpha} knockdown on in vitro proliferation using the MDA231 siERR{alpha} and MDA231 siCON cell lines. Under optimal growth conditions, we determined that altering ERR{alpha} activity does not affect the basal rate of cell growth in these cells (Fig. 3B). Comparable results were observed in other breast cancer cell lines similarly manipulated (data not shown). We next addressed whether ERR{alpha} is required for the invasive phenotype of MDA-MB-231 cells. Using an in vitro transwell migration assay, we observed that ERR{alpha} knockdown resulted in a 65% reduction in cell migration (Fig. 3C). Importantly, re-expression of ERR{alpha} using ERRsiR resulted in reversion to the control level of migration. Although the molecular basis for this change in migratory ability remains unknown, these results indicate that ERR{alpha} can affect in vitro migration and would suggest that ERR{alpha} may alter, in vivo correlate, tumor metastasis.

Decreased ERR{alpha} expression compromises the growth of ER{alpha}-negative tumors in athymic nude mice. Given that the majority of genes regulated by ERR{alpha} in breast cancer cells are involved in metabolism, we hypothesized that if ERR{alpha} had a role in cell growth, it might be evident only under the metabolic pressures of in vivo tumor growth. Therefore, we proceeded to evaluate the role of ERR{alpha} in cells propagated as murine xenografts, a model that would allow us to evaluate the effect of tumor microenvironment on ERR{alpha} function. Specifically, MDA231 siERR{alpha} or MDA231 siCON cells were injected orthotopically into the mammary fat pads of athymic nude mice (10 mice per group). Although the initial take rate of each cell line was equivalent, the growth rate of the resultant tumors was significantly lower in the ERR{alpha} knockdown cells versus control (Fig. 4A ). Intriguingly, the MDA231 siERR{alpha}-derived tumors exhibited a lag phase before resumption of tumor growth, such that 7 of 10 tumors reached over 500 mm3 by day 46. Analysis of the growth curves for each tumor showed that the doubling time of the siERR{alpha} tumors was 12.8 days versus 7.8 days for the tumors arising from control siCON cells (Fig. 4B). We hypothesize that under the selective pressure of the tumor environment, the siERR{alpha} tumors eventually develop alternative mechanisms to adapt to the metabolic conditions in vivo. The development of resistance to ER{alpha} inhibition is similarly manifested in a growth curve lag phase and increased doubling time when MCF-7 cells are grown as xenografts in the presence of fulvestrant (28).


Figure 4
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  		Figure 4. Silencing of ERR{alpha} decreases the growth rate of MDA-MB-231-luciferase cells grown as xenografts. Stable cell lines were injected into the mammary fat pad of nude mice. A, tumor growth rate of the cell lines after orthotopic injection. Points, mean volume for 10 mice per cell line; bars, SE (*, P < 0.05). B, doubling time of each tumor was calculated by best-fit exponential curve and plotted as the average ± SE (*, P < 0.05). C, the expression of ERR{alpha} and VEGF are decreased in ERR{alpha} knockdown tumors compared with control tumors, whereas the control GAPDH is not. mRNA was collected, and target gene expression was measured by RT-qPCR. Data are normalized to cyclophilin expression and represented ± SE (*, P < 0.05).

 
Real-time PCR analysis of the primary tumors harvested from mice revealed that ERR{alpha} remained silenced in the siERR{alpha} xenografts (Fig. 4C). Given the regulation of VEGF by ERR{alpha} in vitro and the prominent role of VEGF in tumor angiogenesis, we examined VEGF expression in the xenograft tumors. Although VEGF mRNA was significantly decreased in the MDA231 siERR{alpha}-derived tumors (Fig. 4C), there was no detectable difference in blood vessel density at the time of tumor excision (as indicated by CD31 staining; Supplementary Fig. S5). This may be due to excision timing, as all surgeries were performed when a significant tumor burden was reached (500–1,500 mm3) rather than at the same number of days postinjection. We speculate that while ERR{alpha} regulation of VEGF may play a role in the delayed growth kinetics of the MDA231 siERR{alpha} xenografts, activation of compensatory pathways may obscure any effect on vessel development by the time the tumors reach maximal size. Finally, whole-animal imaging for luciferase expression after primary tumor excision did not yield evidence of metastatic spread. Although the MDA-MB-231 cell line is characterized as highly aggressive, we hypothesize that the lack of metastases in our experiment may be due to heterogeneity of MDA-MB-231 cell lines and the particular subline used. Additional models, such as cardiac and tail vein injection, as well as alternative breast cancer cell lines, will be required to test the effects of ERR{alpha}, manifested as a decrease in migration in vitro, on tumor metastasis in vivo.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
Since the first correlative study was performed demonstrating a relationship between ERR{alpha} expression and breast cancer prognosis (1), additional associations between the expression of this receptor and patient prognosis have been observed in several types of tumors (29–31). However, a causal relationship between ERR{alpha} activity and disease outcomes has not been established. In the present study, we have investigated the role of ERR{alpha} signaling in breast cancer by identifying ERR{alpha}-regulated genes and examining the effect of altering ERR{alpha} activity in vitro and in vivo. Our initial characterization of ERR{alpha} target genes in relation to estrogen-regulated genes revealed that the majority of ERR{alpha} activity is unrelated to ER{alpha} signaling and led us to focus further phenotypic characterization on the role of ERR{alpha} in ER{alpha}-negative tumors. In vivo studies revealed that ERR{alpha} knockdown had a dramatic inhibitory effect on tumor growth in mice, a finding of pivotal importance in the validation of this NR as a therapeutic target in breast cancer.

One of the primary hypotheses we sought to address in this study was that the role of ERR{alpha} in breast cancer is that of a modulator of estrogen signaling. This hypothesis was based on the observation that ER{alpha} and ERR{alpha} exhibit similar DNA-binding site preferences and the finding that both receptors are capable of activating ERE-mediated gene transcription in transient transfection assays. It was not surprising, therefore, that our studies in MCF-7 cells revealed that a large number of ER{alpha}-regulated genes were modified by ERR{alpha} activity. One particularly interesting example of ERR{alpha}-ER{alpha} cross-talk is CXCL12, a chemokine previously identified as an ER{alpha} target, that is thought to be involved in mediating the mitogenic actions of estrogen and that has also been implicated in the metastasis of breast cancer to bone (32, 33). Unlike CXCL12, the majority of the genes coregulated by ERR{alpha} and ER{alpha} are positively regulated by estradiol and negatively regulated by ERR{alpha} after its activation by PGC-1{alpha}. This is consistent with reporter gene assays in which ERR{alpha} expression led to decreased estrogen-responsiveness of several promoters in MCF-7 cells (34). Previous studies have attributed this finding to competition for DNA binding by the two receptors or to transcriptional squelching subsequent to the sequestration of limiting coactivators (7, 10). Whole genome studies, such as chromatin immunoprecipitation, will enable an evaluation of the extent to which ER{alpha} and ERR{alpha} binding sequences overlap in endogenous genes and the relative importance of this mechanism of receptor cross-talk in breast tumor pathology.

Although potential points of ERR{alpha}-ER{alpha} cross-talk were identified, our microarray analysis showed that a large portion of ERR{alpha} activity in breast cancer may be completely unrelated to estrogen signaling. It should be noted, however, that in contrast with previously reported MCF-7 microarray data, the majority of genes significantly regulated by ER{alpha} in this study were induced rather than repressed by estrogen treatment (35, 36). We hypothesize that that is due to a combination of the specific experimental conditions and the variables used for microarray analysis (applied uniformly to select ERR{alpha} and ER{alpha} regulated genes). In total, we may have underestimated the ER{alpha}-ERR{alpha} cross-talk, particularly with respect to genes down-regulated by estradiol. Despite this potential limitation, we identified genes coregulated by these receptors in different patterns, which likely represent distinct mechanisms of receptor cross-talk. Furthermore, under the conditions of our analysis, the predominant regulation on the largest class of genes was due to ERR{alpha} activation. As assessed by GO term analysis, PGC-1{alpha} coactivation of ERR{alpha} had the largest effect on genes classified as regulators of metabolism, few of which were affected by ER{alpha}. This result led us to investigate the ER{alpha}-independent activity of ERR{alpha} with the hypothesis that this activity may contribute toward breast cancer pathology. We observed that ERR{alpha} knockdown in the ER{alpha}-negative MDA-MB-231 cell line significantly attenuated the growth rate of these cells as murine xenografts, suggesting that ERR{alpha} may be a viable therapeutic target for patients with ER{alpha}-negative tumors. Although the present studies are restricted to the ER{alpha}/PR/ERBB2-negative MDA-MB-231 cell line, a model for a particularly aggressive class of breast cancer, our preliminary and ongoing experiments using other cells suggest that the role of ERR{alpha} is not restricted to this type of breast cancer. Specifically, the observation that ERR{alpha} expression supports target gene expression in multiple cell lines lends credence to the hypothesis that ERR{alpha} has a general role in breast cancer biology (Fig. 2B). Additional in vivo experiments will be necessary to determine whether the similarities in ERR{alpha} function at the level of target gene regulation translate to the level of cell phenotype and tumor growth.

One of our most interesting findings was that ERR{alpha} knockdown did not affect the proliferation rate of MDA-MB-231 cells in vitro whereas the same cells exhibited compromised growth propagated as xenografts. This is not completely unexpected, given that the physiologic roles of ERR{alpha} have been primarily described in tissues with high metabolic demand. We hypothesize that the microenvironment of a rapidly growing tumor recapitulates these conditions and, thus, highlights the functional significance of ERR{alpha}. Indeed, an examination of the MCF-7 array data revealed that all of the TCA enzymes and at least one subunit of each complex of the mitochondrial respiratory chain were positively regulated by ERR{alpha}. Building on experiments performed in skeletal muscle, our preliminary data (not shown) indicates that oxygen consumption is increased in MCF-7 breast cancer cells with activated ERR{alpha} (13). However, whether or not increased OXPHOS provides tumor cells with a growth advantage is a matter of considerable debate. Although an increased rate of OXPHOS has been associated with the production of reactive oxygen species (ROS) and subsequent caspase-mediated apoptosis (37), recent evidence suggests that ERR{alpha} may enhance ROS detoxification. Specifically, breast cancer brain metastases express increased levels of ERR{alpha} and PGC-1{alpha} and exhibit enhanced protection against the deleterious effects of oxidative stress (38). Interestingly, we observed in several breast cancer cell lines that ERR{alpha} increases the expression of SOD2, which inactivates superoxide anions, and GSTM1, an enzyme that buffers cells from ROS (Fig. 2A). These antioxidant defenses may allow cancer cells to capitalize on the ability of ERR{alpha} to increase OXPHOS activity. This hypothesis is also supported by compelling studies that have defined a role for PGC-1{alpha} coactivation of ERR{alpha} in the response to oxidative stress in pathologic processes, such as neurodegeneration (39). In addition to providing substrates for further aerobic metabolism, the TCA cycle may contribute to tumor growth in an OXPHOS-independent manner. Indeed, it has been shown recently that a primary function of the TCA cycle is to provide the intermediates needed for the production of nonessential amino acids, a rate-limiting process in rapidly growing cells (17). Clearly, the strong link between ERR{alpha}, metabolism, and the growth and progression of breast tumors warrants further investigation.

Taken together, our findings provide significant validation for ERR{alpha} as a therapeutic target in ER{alpha}-negative breast tumors. Pharmaceutical interest in ERR{alpha} as a drug target was initially stimulated by studies that highlighted the potential utility of agonists as treatments for metabolic disorders, such as diabetes and obesity. However, despite tremendous effort, ERR{alpha} has proved to be an intractable target with respect to the development of agonists (40). In contrast, several small molecules that function as antagonists or inverse agonists by altering ERR{alpha} in such a manner as to block coactivator interactions have been developed (41). Although there are no published studies that examine the in vivo activities of these compounds, the mild phenotype observed in the ERR{alpha} knockout mouse suggests that systemic administration of this class of ERR{alpha} antagonists may exhibit minimal significant side effects (42). A priority for future studies, therefore, will be the analysis in vivo of appropriate ERR{alpha} inverse agonists and antagonists for their ability to recapitulate the effects on tumor growth observed subsequent to siRNA-mediated knockdown of ERR{alpha}. In conjunction with the growing body of literature defining the role of ERR{alpha} in tumor pathology, our work highlights the activity of ERR{alpha} in cellular models of ER{alpha}/PR/ERBB2 triple-negative breast cancer and suggests that ERR{alpha} may be an attractive therapeutic target in this aggressive and treatment-resistant type of breast cancer.


   Disclosure of Potential Conflicts of Interest
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: NIH grants DK074652 and DK48807 (D.P. McDonnell), Department of Defense grant W81XWH-06-1-0045 (R.A. Stein), and Duke University Breast Cancer Specialized Programs of Research Excellence (M.W. Dewhirst).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank McDonnell laboratory members for helpful discussions and Drs. Chris Counter, Sally Kornbluth, Patrick Casey, and Patrick Kelly for technical advice and reagents.

This manuscript is dedicated to the memory of our colleague Alexander Ney for sharing with us his intellectual creativity and scientific insight.


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

Received 4/29/08. Revised 8/ 7/08. Accepted 9/ 3/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

  1. Suzuki T, Miki Y, Moriya T, et al. Estrogen-related receptor {alpha} in human breast carcinoma as a potent prognostic factor. Cancer Res 2004;64:4670–6.[Abstract/Free Full Text]
  2. Ariazi EA, Clark GM, Mertz JE. Estrogen-related receptor {alpha} and estrogen-related receptor {gamma} associate with unfavorable and favorable biomarkers, respectively, in human breast cancer. Cancer Res 2002;62:6510–8.[Abstract/Free Full Text]
  3. Fujimoto J, Alam SM, Jahan I, Sato E, Sakaguchi H, Tamaya T. Clinical implication of estrogen-related receptor (ERR) expression in ovarian cancers. J Steroid Biochem Mol Biol 2007;104:301–4.[CrossRef][Medline]
  4. Garber K. Energy boost: the Warburg effect returns in a new theory of cancer. J Natl Cancer Inst 2004;96:1805–6.[Free Full Text]
  5. Bonnelye E, Aubin JE. Estrogen Receptor-Related Receptor {alpha}: a mediator of estrogen response in bone. J Clin Endocrinol Metab 2005;90:3115–21.[Abstract/Free Full Text]
  6. Vanacker JM, Pettersson K, Gustafsson JA, Laudet V. Transcriptional targets shared by estrogen receptor-related receptors (ERRs) and estrogen receptor (ER) {alpha}, but not by ERβ. EMBO J 1999;18:4270–9.[CrossRef][Medline]
  7. Zhang ZP, Teng CT. Estrogen receptor {alpha} and estrogen receptor-related receptor {alpha} 1 compete for binding and coactivator. Mol Cell Endocrinol 2001;172:223–33.[CrossRef][Medline]
  8. Yang NY, Shigeta H, Shi HP, Teng CT. Estrogen-related receptor, hERR1, modulates estrogen receptor-mediated response of human lactoferrin gene promoter. J Biol Chem 1996;271:5795–804.[Abstract/Free Full Text]
  9. Xie W, Hong H, Yang NN, et al. Constitutive activation of transcription and binding of coactivator by estrogen-related receptors 1 and 2. Mol Endocrinol 1999;13:2151–62.[Abstract/Free Full Text]
  10. Johnston SD, Liu XD, Zuo FG, et al. Estrogen-related receptor {alpha} 1 functionally binds as a monomer to extended half-site sequences including ones contained within estrogen-response elements. Mol Endocrinol 1997;11:342–52.[Abstract/Free Full Text]
  11. Schreiber SN, Knutti D, Brogli K, Uhlmann T, Kralli A. The transcriptional coactivator PGC-1 regulates the expression and activity of the orphan nuclear receptor estrogen-related receptor {alpha} (ERR{alpha}). J Biol Chem 2003;278:9013–8.[Abstract/Free Full Text]
  12. Huss JM, Torra IP, Staels B, Giguere V, Kelly DP. Estrogen-related receptor {alpha} directs peroxisome proliferator-activated receptor at signaling in the transcriptional control of energy metabolism in cardiac and skeletal muscle. Mol Cell Biol 2004;24:9079–91.[Abstract/Free Full Text]
  13. Mootha VK, Handschin C, Arlow D, et al. ERR {alpha} and Gabpa/b specify PGC-1{alpha}-dependent oxidative phosphorylation gene expression that is altered in diabetic muscle. Proc Natl Acad Sci U S A 2004;101:6570–5.[Abstract/Free Full Text]
  14. Huss JM, Imahashi K, Dufour CR, et al. The nuclear receptor ERR{alpha} is required for the bioenergetic and functional adaptation to cardiac pressure overload. Cell Metab 2007;6:25–37.[CrossRef][Medline]
  15. Kallinowski F, Schlenger KH, Runkel S, et al. Blood flow, metabolism, cellular microenvironment, and growth rate of human tumor xenografts. Cancer Res 1989;49:3759–64.[Abstract/Free Full Text]
  16. Mazurek S, Zwerschke W, Jansen-Durr P, Eigenbrodt E. Metabolic cooperation between different oncogenes during cell transformation: interaction between activated ras and HPV-16 E7. Oncogene 2001;20:6891–8.[CrossRef][Medline]
  17. DeBerardinis RJ, Mancuso A, Daikhin E, et al. Beyond aerobic glycolysis: transformed cells can engage in glutamine metabolism that exceeds the requirement for protein and nucleotide synthesis. Proc Natl Acad Sci U S A 2007;104:19345–50.[Abstract/Free Full Text]
  18. Gaillard S, Dwyer MA, McDonnell DP. Definition of the molecular basis for estrogen receptor-related receptor-{alpha}-cofactor interactions. Mol Endocrinol 2007;21:62–76.[Abstract/Free Full Text]
  19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–{Delta}{Delta}C(T)) method. Methods 2001;25:402–8.[CrossRef][Medline]
  20. Li C, Wong WH. Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc Natl Acad Sci U S A 2001;98:31–6.[Abstract/Free Full Text]
  21. Kasper G, Reule M, Tschirschmann M, et al. Stromelysin-3 over-expression enhances tumourigenesis in MCF-7 and MDA-MB-231 breast cancer cell lines: involvement of the IGF-1 signalling pathway. BMC Cancer 2007;7:12.[CrossRef][Medline]
  22. Schreiber SN, Emter R, Hock MB, et al. The estrogen-related receptor {alpha} (ERR{alpha}) functions in PPAR{gamma} coactivator 1{alpha} (PGC-1{alpha})-induced mitochondrial biogenesis. Proc Natl Acad Sci U S A 2004;101:6472–7.[Abstract/Free Full Text]
  23. Lin J, Wu P-H, Tarr PT, et al. Defects in adaptive energy metabolism with CNS-linked hyperactivity in PGC-1a null mice. Cell 2004;119:121–35.[CrossRef][Medline]
  24. Gaillard S, Grasfeder LL, Haeffele CL, et al. Receptor-selective coactivators as tools to define the biology of specific receptor-coactivator pairs. Mol Cell 2006;24:797–803.[CrossRef][Medline]
  25. Lin J, Handschin C, Spiegelman BM. Metabolic control through the PGC-1 family of transcription coactivators. Cell Metab 2005;1:361–70.[CrossRef][Medline]
  26. Arany Z, Foo SY, Ma Y, et al. HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1{alpha}. Nature 2008;451:1008–12.[CrossRef][Medline]
  27. Mukhopadhyay R, Theriault RL, Price JE. Increased levels of {alpha}6 integrins are associated with the metastatic phenotype of human breast cancer cells. Clin Exp Metastasis 1999;17:325–32.[Medline]
  28. Macedo LF, Sabnis GJ, Goloubeva OG, Brodie A. Combination of anastrozole with fulvestrant in the intratumoral aromatase xenograft model. Cancer Res 2008;68:3516–22.[Abstract/Free Full Text]
  29. Sun P, Sehouli J, Denkert C, et al. Expression of estrogen receptor-related receptors, a subfamily of orphan nuclear receptors, as new tumor biomarkers in ovarian cancer cells. J Mol Med 2005;26:1699–706.
  30. Cavallini A, Notarnicola M, Giannini R, et al. Oestrogen receptor-related receptor {alpha} (ERR {alpha}) and oestrogen receptors (ER{alpha} and ERβ) exhibit different gene expression in human colorectal tumour progression. Eur J Cancer 2005;41:1487.[CrossRef][Medline]
  31. Cheung CP, Yu S, Wong KB, et al. Expression and functional study of estrogen receptor-related receptors in human prostatic cells and tissues. J Clin Endocrinol Metab 2005;90:1830–44.[Abstract/Free Full Text]
  32. Hall JM, Korach KS. Stromal cell-derived factor 1, a novel target of estrogen receptor action, mediates the mitogenic effects of estradiol in ovarian and breast cancer cells. Mol Endocrinol 2003;17:792–803.[Abstract/Free Full Text]
  33. Muller A, Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature 2001;410:50–6.[CrossRef][Medline]
  34. Kraus RJ, Ariazi EA, Farrell ML, Mertz JE. Estrogen-related receptor {alpha} 1 actively antagonizes estrogen receptor-regulated transcription in MCF-7 mammary cells. J Biol Chem 2002;277:24826–34.[Abstract/Free Full Text]
  35. Frasor J, Danes JM, Komm B, Chang KCN, Lyttle CR, Katzenellenbogen BS. Profiling of estrogen up- and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology 2003;144:4562–74.[CrossRef][Medline]
  36. Carroll JS, Meyer CA, Song J, et al. Genome-wide analysis of estrogen receptor binding sites. Nat Genet 2006;38:1289–97.[CrossRef][Medline]
  37. Hervouet E, Simonnet H, Godinot C. Mitochondria and reactive oxygen species in renal cancer. Biochimie 2007;89:1080–8.[CrossRef][Medline]
  38. Chen EI, Hewel J, Krueger JS, et al. Adaptation of energy metabolism in breast cancer brain metastases. Cancer Res 2007;67:1472–86.[Abstract/Free Full Text]
  39. St-Pierre J, Drori S, Uldry M, et al. Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators. Cell 2006;127:397–408.[CrossRef][Medline]
  40. Hyatt SM, Lockamy EL, Stein RA, et al. On the intractability of estrogen-related receptor {alpha} as a target for activation by small molecules. J Med Chem 2007;50:6722–4.[CrossRef][Medline]
  41. Busch BB, Stevens WC, Jr., Martin R, et al. Identification of a selective inverse agonist for the orphan nuclear receptor estrogen-related receptor {alpha}. J Med Chem 2004;47:5593–6.[CrossRef][Medline]
  42. Luo JM, Sladek R, Carrier J, Bader JA, Richard D, Giguere V. Reduced fat mass in mice lacking orphan nuclear receptor estrogen-related receptor {alpha}. Mol Cell Biol 2003;23:7947–56.[Abstract/Free Full Text]



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