Transcriptional Control of the ERBB2 Amplicon by Errα and PGC-1Β Promotes Mammary Gland Tumorigenesis

Author Manuscript Published OnlineFirst on October 20, 2010; DOI: 10.1158/0008-5472.CAN-10-2840 AuthorPublished manuscripts OnlineFirst have been onpeer October reviewed and20, accepted2010 as for 10.1158/0008-5472.CAN-10-2840 publication but have not yet been edited.

Transcriptional control of the ERBB2 amplicon by ERRα and PGC-1β promotes mammary gland tumorigenesis

Geneviève Deblois1,2, Ghada Chahrour1,2, Marie-Claude Perry1,2, Guillaume Sylvain-Drolet1,2, William J. Muller1,2 and Vincent Giguère1,2,3

1Goodman Cancer Research Centre, 1160 Pine Avenue West, McGill University, Montréal, Québec H3A 1A3, Canada. 2Department of Biochemistry, 3655 Promenade Sir William Osler, McGill University, Montréal, Québec H3G 1Y6, Canada. 3Department of Medicine and Department of Oncology, 687 Pine Avenue West, McGill University, Montréal, Québec H3A 1A1, Canada.

Running Title: ERRα regulation of the ERBB2 amplicon

Keywords: ChIP; estrogen receptor; GRB7; nuclear receptor; Pax2; tamoxifen.

Corresponding author, Vincent Giguère, Goodman Cancer Research Centre, McGill University, 1160 Pine avenue West, Montréal Québec, H3A 1A3, Canada. Tel.: (514) 398-5899; Fax: (514) 398-8578 E-mail: [email protected]

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2010 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 20, 2010; DOI: 10.1158/0008-5472.CAN-10-2840 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Overexpression of ERBB2 and its neighboring genes on chromosome 17 occurs in approximately 25% of breast tumors and is associated with poor prognosis. While amplification of the 17q12-21 chromosomal region often correlates with an increase in the transcriptional rates of the locus, the molecular mechanisms and the factors involved in the coordinated expression of genes residing within the ERBB2 amplicon remain largely unknown. Here we demonstrate that estrogen-related receptor α

(ERRα, NR3B1) and its coregulator PGC-1β are key effectors in this process. Using a mouse model of ERBB2-initiated mammary tumorigenesis, we first show that ablation of ERRα significantly delays ERBB2-induced tumor development and lowers the levels of amplicon transcripts. Chromosome 17q-wide binding site location analyses in human breast cancer cells show preferential recruitment of

ERRα to DNA segments associated with the ERBB2 amplicon. Furthermore, ERRα directs the co-recruitment of the coactivator PGC-1β to segments in the 17q12 region and the recruitment of RNA polymerase II to the promoters of the ERBB2 and co-amplified genes. ERRα and PGC-1β also participate in the de-repression of

ERBB2 expression through competitive genomic cross-talk with estrogen receptor α

(ERα) and, as a consequence, influence tamoxifen sensitivity in breast cancer cells.

Taken together, our results suggest that ERRα and PGC-1β are key players in the etiology of malignant breast cancer by coordinating the transcriptional regulation of genes located in the 17q12 region, a process that also involves interference with the repressive function of ERα on ERBB2 expression.

Introduction

Breast cancer is a complex disease implicating distinct cell types and multiple signaling pathways that together engender a multiplicity of tumor subtypes (1). The molecular heterogeneity of breast tumor subtypes dictates their intrinsic response to specific therapeutic approaches and has therefore become an important aspect in the clinical management of the disease (2). Amplification of the 17q12-21 region leads to concomitant over-expression of ERBB2 and several co-amplified genes and occurs in approximately 20 to 30% of breast tumors (3). The ERBB2-amplified breast cancer subtype is strongly associated with poor prognosis (4). Although over-expression of

ERBB2 is usually linked to amplification of the 17q12 region, this region is not amplified in some ERBB2-positive breast tumors. However, in both instances, an increase in the transcriptional regulation rate of the locus is observed (5-8).

Loss of repression of ERBB2 has been linked to tamoxifen resistance in breast cancer cells (9, 10). It is believed that transcriptional repressors including FOXP3 (11), PAX2

(10), GATA4 (12), PEA3 (13), and MYB (14) act to quench the expression of ERBB2 in

ERBB2-negative tumors. Notably, it has also been shown that estrogen receptor α (ERα,

NR3A1), in cooperation with the transcription factor PAX2, can repress ERBB2 expression through binding to a cis-regulatory element in the presence of either 17β- estradiol (E2) or 4-hydroxy-tamoxifen (OHT) (10). Indeed, ERα-positive tumors with the worst prognosis that do not respond to tamoxifen therapy often express high levels of

ERBB2 (15-18).

A positive increase in the transcriptional control of the locus can contribute to relieve

the transcriptional repression that takes place on ERBB2 and over-expression of ERBB2 is thought to drive the amplification of the 17q12 locus (19). Transcription factors such as

AP-2 (8), YY1 (20), ETS (21), YB-1 (22) and EGR2 (23) have been shown to be recruited to the promoter of ERBB2 and play a role in its over-expression in breast cancer cells. Therefore, understanding the mechanisms governing the repressive and positive regulation of ERBB2 expression and its neighboring genes in the amplified locus is of considerable importance in identifying the transcription factors and molecular events involved in the establishment of ERBB2-positive tumors.

While most studies on the 17q12 amplicon have been centered on the expression and activity of ERBB2 itself, it is now clear that co-amplified genes not only contribute but could be essential to the progression of ERBB2-positive breast tumors (24, 25). The minimal 17q12 amplicon includes genes that are involved in signal transduction (GRB7,

PERLD1, PPP1R1B), transcription (MED1, IKZF3, NEUROD2, PNMT), cell migration and invasion (C17orf37, GRB7), inhibition of apoptosis (MED1), genomic instability

(PERLD1) and tamoxifen resistance (STARD3, GRB7) (26-28). Although transcriptional regulation of the co-amplified genes in relation to the ERBB2 subtype is now considered an important aspect in the establishment of ERBB2-positive breast tumors, the molecular mechanisms associated with this phenomenon have yet to be investigated in detail.

The orphan nuclear receptor estrogen-related receptor α (ERRα, NR3B1) shares both structural and functional features with ERα (29). The expression of ERRα is inversely correlated to that of ERα but positively associates with that of ERBB2 and with poor prognosis in breast cancer (30, 31). A recent genome-wide binding sites location analysis

in breast cancer cell lines intersected with gene expression data from breast tumors has shown that ERRα signaling contributes to known pathways linked to breast cancer progression, including those involving ERα and ERBB2 (32). Conversely, the transcriptional activity of ERRα can be modulated by the EGF/ERBB2 signaling pathway in breast tumors (33, 34). ERRα preferentially acts in concert with the coregulators peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and β

(PGC-1β) whose combined roles have been extensively studied in the context of the regulation of bioenergetic pathways (35, 36). Gene expression and binding sites location analyses in breast cancer cell lines have also demonstrated that ERα and ERRα display strict binding site specificity and maintain independent mechanisms of transcriptional activation, suggesting a prevailing ERα-independent role for ERRα in breast tumor development (32, 37). Nevertheless, a significant number of genes that are common targets of the two nuclear receptors are also important players in breast tumor development (32).

Here we show that ERRα is required for the full potential of ERBB2-driven mammary tumorigenesis in mice and that tumors lacking ERRα express lower levels of most amplicon genes. Chromosome-wide identification of regulatory regions occupied by

ERRα and PGC-1β as well as gene expression data identify both factors as key regulators of the expression of ERBB2 and several co-amplified genes in the 17q12 region in breast cancer cells. Finally, we demonstrate that an antagonistic interaction between ERα and the ERRα/PGC-1β complex at the ERBB2 locus is an important determinant of ERBB2

expression involved in the development of tamoxifen resistance in breast cancer cells.

Materials and Methods

Cell culture, reagents and antibodies. MCF-7 and SKBr3 cells were cultured as described previously (38). Tam-R-MCF-7 cell line was derived as described previously

(39). ERRα ChIP assays were performed using an anti-hERRα polyclonal antibody raised and validated in our laboratory (32, 40). Other antibodies used were anti-RNA-

Polymerase II (8WG16), anti-ERRα (Millipore), anti-PGC-1β and anti-ERα (Santa Cruz

Biotechnologies). siRNAs against ERα, ERRα, PGC-1β and control (ON-Target-Plus siRNA pool) were obtained from Dharmacon.

Transgenic mice model study. The derivation of the conditionally activated NeuNT

(erbB2NT) has been described in detail (41). To generate mice that expressed erbB2NT in the mammary glands of animals carrying a null allele for Esrra, erbB2NT and MMTV-Cre mice were first bred with mice heterozygous for null alleles of Esrra (42). All mice were previously derived in a pure FVB genetic background. These mice were then bred to generate mice null for Esrra also carrying one copy of erbB2NT and one copy of MMTV-

Cre (e.g. Ersra-/-/erbB2NT/MMTV-Cre). The resulting mice were examined for the presence of the excised recombinant erbB2NT allele through Southern blot analysis. The levels of amplification of the locus were determined by Q-RT-PCR using primers listed in Supplementary Table S1. Female mice were examined twice a week for mammary tumor development by palpation.

ChIP assays and ChIP-on-chip on chr.17q tiled arrays. Chromatin- immunoprecipitation (ChIP) was performed as described previously (38). Quantification of ChIP enrichment by real-time Q-PCR was carried out using the LightCycler®480

instrument (Roche). ChIP-on-chip was carried out on custom chr.17q Agilent tiled arrays

(150 bp resolution). Chromatin was prepared from SKBr3 cells (for ERRα and PGC-1β

ChIP-on-chip) or from MCF-7 cells exposed to 10 nM E2 for 45 min (for ERα ChIP-on- chip). The primers used for standard ChIP are listed in Supplementary Table S1.

Computational motif discovery. De novo and known motif discovery was performed with MEME Suite1. Motif discovery was also confirmed using the Genomatix Software

Suite2.

siRNA. siRNAs for ERα and control were transfected in MCF-7 cells cultured in phenol-red-free DMEM media supplemented with hormone-deprived serum using the

HyperFect reagent (QIAGEN). Similarly, siRNAs for ERRα, PGC-1β and control were transfected in SKBr3 cells using the HyperFect reagent (QIAGEN) for 48 to 60 hrs.

Expression analysis. mRNA from SKBr3 cells transfected with the ERRα or PGC-1β siRNA were reverse-transcribed into cDNA using Superscript (Invitrogen) and analyzed by Q-RT-PCR with SYBR-green based RT-PCR (Roche). Alternatively, RNA was extracted from mice mammary gland tumors using the RNA tissue extraction kit

(QIAGEN) and reverse-transcribed using Superscript (Invitrogen). Primer pairs used for

Q-RT-PCR are listed in Supplementary Table S2.

Proliferation assay assessed by 3H-thymidine incorporation. SKBr3 cells were transfected with the appropriate siRNA for 60 hrs. Cells were incubated in the presence of 1 μCi 3H-thymidine for 4 hrs prior to fixation and harvesting. Likewise, MCF-7 and

1 http://meme.sdsc.edu/meme4_4_0/intro.html 2 http://www.genomatix.de/en/produkte/genomatix-software-suite.html Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research 8

Tam-R-MCF-7 cells were grown in phenol-red-free DMEM supplemented with hormone- deprived serum. Upon siRNA transfection, the media was supplemented with 100 nM

OHT (Sigma) or vehicle for 60 hrs prior to harvesting and isotope counting.

Results

Ablation of ERRα delays ERBB2-induced mammary gland tumorigenesis. To initiate our study on the functional relationship between ERRα and ERBB2 in mammary gland tumorigenesis, we first used the well-characterized Neu-NT knock-in transgenic mouse model of mammary tumorigenesis (41) to generate ERRα-deficient transgenic mice conditionally expressing the activated Neu under the control of the endogenous

Erbb2 promoter. The choice of this model was guided by the previous observation that these mice develop focal mammary tumors with high frequency after a long latency period that bear amplified copies of the activated Erbb2 allele on chromosome 11 (41,

43). In addition, this model is ideal to study the impact of alteration in the gene regulatory machinery as expression of the Erbb2 locus remains under the control of endogenous regulatory elements. We observed that ablation of ERRα significantly delayed ERBB2- induced mammary gland tumorigenesis (p<0.05) (Fig. 1A), indicating that the presence of

ERRα is required for optimal development of ERBB2-driven tumors. The ERRα-null mice lactate normally and the development of the mammary gland is not affected by the lack of ERRα expression (Fig. 1B). As expected, amplification of the Erbb2 locus was observed in the tumors derived from this model but the absence of ERRα did not significantly affect the level of amplification of the locus as compared to wild-type (Fig.

1C). To investigate the possible contribution of ERRα in the transcriptional regulation of Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research 9

Erbb2 and neighboring genes in the Erbb2 amplicon, we next assessed their levels of expression in the tumors. We found that the relative transcript levels of most of the amplicon genes tested, including Erbb2, were either significantly decreased or show a similar downward trend in tumors arising from ERRα knock-out mice compared to the wild-type (Fig. 1D). Overall, these results suggest that ERRα might play a role in the development of ERBB2-driven mammary tumors through transcriptional regulation of

Erbb2 and other genes located within the amplicon.

ERRα is recruited to specific sites at chr17q12 and regulates the expression of

ERBB2 and co-amplified genes in human breast cancer cells. In order to investigate the role of ERRα in the transcriptional regulation of the genes located within the ERBB2 amplicon in human breast cancer cells, we first performed genomic location analysis of

ERRα in the ERα-negative SKBr3 cell line using a custom tiled array covering the q-arm of human chromosome 17. ChIP-on-chip analysis of ERRα on the chr.17q region revealed 92 segments bound by ERRα and validated by standard ChIP analyses

(Supplementary Table S3 and Fig. S1A). De novo DNA motif discovery confirmed the enrichment of the ERRE motif within the bound segments (Supplementary Fig. S1B).

Remarkably, a significant enrichment of ERRα-bound segments is observed within the amplicon region (32 out or 92 segments) and 13 segments map to the minimal region steadily amplified in ERBB2-positive tumors (Fig. 2A, shaded region). ERRα binding events were observed in ERBB2 itself as well as in the transcriptional unit of genes that are consistently co-amplified with ERBB2, such as PERLD1, C17orf37 and GRB7 (Fig.

To relate the binding profile of ERRα to the transcriptional regulation of target genes, we then monitored modulation in the recruitment of RNA-PolII by ChIP to the promoters of genes located within the ERBB2 amplicon upon depletion of ERRα in SKBr3 cells.

Indeed, a significant decrease in both ERRα and RNA-PolII recruitment was observed at the promoter of CRKRS, a target gene at which ERRα binding occurs directly at the promoter (Fig. 3A). Furthermore, a decrease in RNA-PolII recruitment was also observed at promoters of amplicon genes for which ERRα binding takes place far upstream of the transcriptional start sites or in intronic regions of genes such as ERBB2, GRB7 and

PERLD1 (Fig. 3A). Depletion of ERRα had no effect on the occupancy of RNA-PolII at the promoter of the gene, LZTS2, that is not a target of ERRα (Fig. 3B). These results demonstrate that the recruitment of ERRα at both near and distant sites from transcriptional start sites contributes to the recruitment of RNA-PolII at the promoters of target genes. We next assessed the effect of ERRα depletion on the expression of amplicon genes in human breast cancer cells. In agreement with the observation made in

ERBB2-induced tumors developed in the ERRα knock-out mice, depletion of ERRα in

SKBr3 cells leads to a significant decrease in the relative expression of most amplicon genes (Fig. 3C). These results indicate that ERRα positively regulates the expression of

ERBB2 and numerous genes that co-amplify with it on chr17q12.

The coactivator PGC-1β is recruited to ERRα-bound segments in the chr17q12 amplicon. We have previously shown that the coactivator PGC-1β can be co-recruited with ERRα at specific genomic locations in SKBr3 cells (32). To assess whether PGC-1β

contributes more broadly in the transcriptional regulation of amplicon genes, we performed PGC-1β ChIP-on-chip in SKBr3 cells hybridized on the chr.17q tiled array.

The ChIP-on-chip experiment identified 73 segments significantly bound by PGC-1β on chr.17q (Supplementary Table S4 and Supplementary Fig. S2A) of which 24 were segments common to ERRα (Fig. 4A). Interestingly, PGC-1β recruitment was observed at sites shared with ERRα in regions located within key genes of the ERBB2 amplicon including ERBB2, PERLD1, GRB7 and NR1D1 (Supplementary Table S4 and Fig. S2B).

De novo DNA motif discovery identified the ERRE as the most enriched motif within the common segments shared by both factors (p=7.8e-05), indicating that PGC-1β recruitment to the common segments occurs through ERRα (Fig. 4A and Supplementary

Fig. S3). Indeed, depletion of ERRα in SKBr3 cells using specific siRNAs leads to a significant reduction in PGC-1β recruitment to the common sites located in chr.17q amplicon genes as assessed by standard ChIP (Fig. 4B). We further tested the effect of

PGC-1β recruitment on the expression of the amplicon transcripts. As observed for

ERRα, depletion of PGC-1β using specific siRNAs leads to a significant decrease in the relative levels of transcripts of several amplicon genes (Fig. 4C). Since ERBB2 and the co-amplified genes are potent inducers of breast tumor growth, we next evaluated the impact of loss of ERRα and/or PGC-1β on cell proliferation. Specific knock-down of either ERRα, PGC-1β or of both factors using specific siRNAs led to a significant decrease in SKBr3 cell proliferation as assayed by 3H-thymidine incorporation (Fig. 4D).

Taken together, these results demonstrate that ERRα contributes to the co-recruitment of

PGC-1β to the chr.17q region and that both factors regulate the expression of amplicon genes and aggressive growth of SKBr3 breast cancer cells.

ERRα competes with ERα for recruitment to common segments in ERBB2 and

GRB7 and contributes, along with PGC-1β, to tamoxifen resistance in MCF-7 cells.

In ER-positive tumors that are tamoxifen-sensitive, the expression of ERBB2 is low due to transcriptional repression of the gene inflicted by various transcription factors, including ERα and the co-repressor PAX2. It has been suggested that loss of this repression and consequential increase in ERBB2 expression and signaling contribute to acquired tamoxifen resistance (10). Over-expression of other co-amplified genes such as

GRB7 have also been implicated in resistance to endocrine therapy. We next explored the possibility that ERRα could interfere with ERα signaling in the regulation of specific genes within the ERBB2 amplicon and thus take part in the development of tamoxifen resistance. In order to assess a potential crosstalk at the genomic level, we performed an

ERα ChIP-on-chip experiment in E2-treated MCF-7 cells using the same chr.17q arm tiled array (Supplementary Table S5). We identified 194 segments significantly bound by

ERα, of which 10 overlapped with ERRα-bound loci and 5 overlapped with PGC-1β- bound loci (Supplementary Fig. S4). Notably, the overlapping ERα/ERRα segments include a site downstream of GRB7 as well as a site located in the first intron of ERBB2.

Of particular interest, the intronic ERBB2 site was previously identified as bound by the

ERα/PAX2 repressor complex and shown to play a role in tamoxifen sensitivity of MCF-

7 cells (Fig. 5A).

We confirmed that treatment of MCF-7 cells with E2 leads to a down-regulation of

ERBB2 and GRB7 expression (Supplementary Fig. S5A). Examination of the bound sequences in ERBB2 and GRB7 revealed the presence of a mixed ERE/ERRE binding site as previously defined for shared ERα/ERRα binding sites (32), indicating that ERα and

ERRα should compete for binding at these common sites. Using specific siRNAs and standard ChIP, we indeed observed that depletion of ERα from MCF-7 cells was accompanied by an increase in ERRα recruitment to the common sites that were otherwise not bound by ERRα in MCF-7 cells (Fig. 5B). Depletion of ERα had no effect on ERRα recruitment to ERRα-specific sites (Supplementary Fig. S5B). These results indicate that the concomitant presence of both nuclear receptors yields to competitive transcriptional regulation of ERBB2 and GRB7 gene expression.

We next asked whether this transcriptional crosstalk could play a role in endocrine resistance using a tamoxifen resistant MCF-7 cell line generated in our laboratory (Tam-

R-MCF-7). As expected, the expression of ERBB2 and GRB7 is increased in the Tam-R-

MCF-7 cell line compared to the parental MCF-7 cells (Fig. 5C). We further show that depletion of either ERRα or PGC-1β (Supplementary Fig. S5C and S5D) leads to a significant decrease in ERBB2 and GRB7 transcripts in Tam-R-MCF-7 cells while having no effect on the expression of these genes in the parental MCF-7 cell line (Fig. 5C). We then assessed the effect of loss of ERRα or PGC-1β on the proliferation of parental MCF-

7 and Tam-R-MCF-7 cells in the absence or presence of OHT. As shown in Fig. 5D, the proliferation rate of MCF-7 cells is decreased upon OHT treatment whereas depletion of either ERRα or PGC-1β has no additional effect on the proliferation of these cells. In Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research 14

contrast, while OHT does not influence the proliferation rate of the Tam-R-MCF-7 cells, depletion of either ERRα or PGC-1β reinstates the anti-proliferative effect of OHT in these cells. Taken together, these results suggest that both factors may play a role in the development of OHT resistance in breast cancer cells.

Discussion

Amplification of the ERBB2 locus and neighboring loci on chromosome 17q12, which is accompanied by over-expression of the amplified genes, plays an important role in the development of tamoxifen resistance and a more aggressive breast cancer phenotype (44).

The outcome of this work, derived from a functional genomic approach and validated in well-characterized in vivo and in vitro models of human breast cancer, demonstrates that the orphan nuclear receptor ERRα and its coregulator PGC-1β are key elements in the transcriptional regulation of genes located within the 17q amplicon, including ERBB2 itself. In addition, this study identifies molecular mechanisms through which these two factors can contribute to the development of tamoxifen resistance in breast cancer cells.

The observation that ERRα regulates the expression of several genes located within the ERBB2 amplicon denotes the importance of ERRα signaling in the progression of the

ERBB2-driven breast tumor subtype. This is further emphasized by the observation that the presence of ERRα is required to observe the full oncogenic potential of ERBB2 in a well-established mouse model of human breast cancer (Fig. 1). Notably, we have shown that ERRα regulates the expression of most genes present in the minimal ERBB2 amplicon. The co-amplified genes are mainly involved in signal transduction and transcription influencing various biological processes including cell migration, invasion and survival as well as resistance to tamoxifen (9, 10, 24-28). It can therefore be envisioned that, by acting as a global transcriptional regulator of the ERBB2 amplicon,

ERRα contributes to establish the ERBB2-positive tumor subtype which is characterized not only by increased ERRB2 signaling, but also by various cellular processes controlled

by co-amplified genes. Mechanisms that trigger the amplification of the locus are not well understood but it has been observed that an increase in the transcription levels of

ERBB2 often precedes locus amplification (5, 45). ERRα could inflict positive transcriptional pressure on the 17q12 region and thus participates actively in the establishment of ERBB2-positive aggressive tumors.

Our results are in agreement with previous observations demonstrating that expression of ERRα positively correlates with that of ERBB2 in breast tumors and that ERRα transcriptional activity is positively modulated by EGFR/ERBB2 signaling in breast cancer cells (30, 33, 34). Such interactions can be integrated in a model of positive feed- forward regulatory loops whereby all the processes involved further enhance the build up of their own stimulus (46). In ERBB2-negative tumors, the expression of ERBB2 is maintained at low levels by various sequence-specific transcription factors such as PAX2,

FOXP3, PEA3, GATA4, MYB as well as by ERα. It is thus likely that the positive feed- forward regulatory loop involving ERRα and PGC-1β favors escape from the negative regulation inflicted on ERBB2 by ERα during progression of breast cancer (Fig. 6). This model suggests a possible role for ERRα in mediating the transition of a subset of ER- positive luminal tumors towards the more aggressive ERBB2-expressing subtype.

The genomic convergence between ERRα and the ERBB2 amplicon described in this study is even more relevant considering the finding of competitive ERRα recruitment at a site in ERBB2 targeted by ERα/Pax2. This site has been shown to be involved in maintaining the repressive state of ERBB2 in ERα-positive endocrine-responsive tumors

(10). We also observed competitive recruitment of ERRα/PGC-1β and ERα on a site Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research 17

proximal to GRB7. Interestingly, as it is the case for ERBB2, ERRα and ERα have opposite transcriptional effects on the expression of GRB7 in breast cancer cells. The loss of negative regulation leads to over-expression of ERBB2 and ERBB2-co-amplified genes such as GRB7 in luminal breast tumors and has been associated with a poor response to targeted endocrine therapy (47). The effect of ERRα/PGC-1β depletion on tamoxifen- mediated proliferation of Tam-R-MCF-7 cells suggests a mechanism whereby the transcriptional regulation of ERBB2 and co-amplified genes by these factors can contribute to the establishment of acquired tamoxifen resistance. Other ERBB2-co- amplified genes have been associated with the response to trastuzumab or antracycline- based chemotherapies in mono or combination therapies (48). It would therefore be of interest to assess how the transcriptional regulation of these genes by ERRα/PGC-1β affects the response to other targeted therapies.

Little is known about the expression and function of PGC-1β in breast cancer cells.

Here we show that PGC-1β induces the expression of ERBB2 in breast cancer cells. In line with this observation, we have recently shown that ERBB2 signaling also induces the expression of PGC-1β in breast cancer cells (49). Therefore, together with ERRα, our results suggest that PGC-1β contributes to the establishment of the aggressive ERBB2- positive tumors through a positive feed-forward regulatory loop (Fig. 6).

In conclusion, this study clearly demonstrates that, in addition to its primary role in the control of cellular energy metabolic pathways in both normal and cancer cells (32, 35,

49), the ERRα/PGC-1β complex promotes the development of the ERBB2-positive

tumor subtype and tamoxifen resistance in breast cancer via transcriptional control of the

ERRB2 amplicon.

Acknowledgements

This work was supported by grants from the Canadian Institutes of Health Research

(MOP-64275) and a Terry Fox Foundation Program Project Grant from the National

Cancer Institute of Canada. G.D. and M.-C.P. are recipients of studentships from the

Fonds de la Recherche en Santé du Québec. G.D. is also supported by a pre-doctoral traineeship award (W81XWH-10-1-0489) from the U.S. Department of Defense Breast

Cancer Research Program.

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Figure Legends

Figure 1. Ablation of ERRα delays mammary gland tumorigenesis in mice. A, Ablation of ERRα significantly delays Erbb2-induced mammary gland tumorigenesis in a mouse model conditionally expressing activated Neu under the transcriptional control of the intact endogenous Erbb2 promoter (p<0.05, log-rank test). Inset, a representative

Southern blot of tail DNA from wild type (WT) and ERRα-null mice heterozygous for the knock-in allele (arrow). B, Mammary whole mount staining showing normal development of the mammary gland in ERRα-null mice. C, The levels of normalized amplification of the Erbb2 locus detected using real-time Q-PCR are similar in wild-type and ERRα-null tumors (n=5). AMG, adjacent mammary gland. NS, not significant. D,

Tumors lacking the expression of ERRα express lower levels of amplicon gene transcripts (n=6). Error bars, SEM; *, p<0.05

Figure 2. ERRα is recruited to multiple segments in the ERBB2 amplicon region on human chromosome 17q12-21. A, Binding profile of ERRα from the ChIP-on-chip performed with SKBr3 cells on a high resolution tiled array covering the chromosome

17q arm. The minimal ERBB2 amplicon is represented by the grey region. B, Binding profile of ERRα on representative co-amplified gene regions located in the ERBB2 amplicon.

Figure 3. ERRα contributes to the recruitment of RNA-Polymerase II to the promoter of amplicon genes and induces their expression. A, Standard ChIP experiment in SKBr3 cells shows that siRNA-mediated depletion of ERRα leads to a significant decrease in

RNA-PolII recruitment to the promoters of ERRα target genes located in the ERBB2 amplicon. Inset, Western blot shows the level of ERRα in cells transfected with the siRNAs. B, Same experiment as in (A) on a negative control gene that is not affected by

ERRα. C, Relative expression of ERRα target genes in the ERBB2 amplicon upon siRNA-mediated depletion of ERRα in SKBr3 cells. The effect of ERRα depletion on the level of ERBB2 is shown by Western blot (inset). Grey region: minimal ERBB2 amplicon. (Error bars, sd; *, p<0.05; **, p<0.01; ***, p<0.001).

Figure 4. PGC-1β is recruited to the ERBB2 amplicon through ERRα and contributes to the regulation of amplicon genes. A, Venn diagram indicating the overlap between

ERRα-bound segments (green) and PGC-1β-bound segments in SKBr3 cells (blue). The sequence logo depicting the ERRα response element which is enriched in the common

ERRα/PGC-1β segments. B, Standard ChIP in SKBr3 cells upon siRNA-mediated depletion of ERRα shows that PGC-1β recruitment to the common sites is dependent on

ERRα. Inset, Western blot showing the level of ERRα and PGC-1β in cells transfected with siESRRA and with siPPARGC1B. C, Relative gene expression of common

ERRα/PGC-1β target genes located in the ERBB2 amplicon shows that the genes are regulated by PGC-1β. Inset, Western blot showing the level of PGC-1β in cells

transfected with siPPARGC1B. D, Depletion of ERRα (siESRRA), PGC-1β

(siPPARGC1B) or of both in SKBr3 cells for 60 hrs affects the proliferation rate of the cells as shown by a decrease in the relative 3H-thymidine incorporation over a 4 hr period. (Error bars, sd; *, p<0.05; **, p<0.01).

Figure 5. ERRα competes with ERα for recruitment to segments in ERBB2 and GRB7 and contributes, along with PGC-1β, to tamoxifen resistance in MCF-7 cells. A, Binding profile of ERα in E2-treated MCF-7 cells (red) and ERRα in SKBr3 cells (green) on the

ERBB2 locus. The arrows indicate the common ERα/ERRα-bound segment. B, Relative enrichment of ERα and ERRα as assayed by standard ChIP upon siRNA-mediated depletion of ERα in E2-treated MCF-7 cells at common sites in ERBB2 and GBR7. Inset,

Western blot showing the level of ERα in cells transfected with siESR1. C, Relative gene expression for ERBB2 and GRB7 by Q-RT-PCR in MCF-7 and Tam-R-MCF-7 cells upon siRNA-mediated depletion of ERRα or PGC-1β. (*, p<0.05 relative to the siC of the same cell line; ‡‡, p<0.01 relative to the MCF-7 control cell line). D, Effect of siRNA- mediated depletion of ERRα (siESRRA) or PGC-1β (siPPARGC1B) in Tam-R-MCF-7 cells and parental MCF-7 cells on the proliferation rate of the cells as shown by relative

3H-thymidine incorporation over a 4 hr period. (Error bars, sd; *, p<0.05).

Figure 6. ERRα and PGC-1β are involved in a positive feed-forward regulatory loop with ERBB2. In ER-positive/tamoxifen sensitive tumors, ERBB2 expression is maintained at low levels by transcriptional repressor signals such as the ERα/Pax-2 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research 27

complex. ERRα and PGC-1β mediate positive transcriptional regulation of ERBB2 expression that can prevail over the negative transcriptional down-regulation of ERBB2 by ERα and Pax-2, especially in ER-negative/tamoxifen resistant tumors or in tumors that have lost the expression of Pax-2 (10). In addition, the positive feed-forward loop is further enhanced through the positive auto-regulation of ERRα (40). Solid lines represent transcriptional regulation and the dashed line indicates the ERRB2-mediated signaling pathway influencing ERRα transcriptional activity (33, 34).

Figure 1

A B WT ESRRA-/- 100

60 C WT (n=55) 60 NS ESRRA-/- (n=22) 40

% tumour free C ESRRA-/- WT 40

20 5.8 20 4.0 p<0.05 0

Amplification level (fold) 0 0426810 12 14 16 18 20 22 24 AMG WT ESRRA-/- Months D Erbb2 Stard3 Ppp1r1b Perld1

2.5 1.2 8 2 1.0 2.0 6 0.8 1.5 * 0.6 * 4 1 1.0 0.4 * 2 * 0.5 0.2 0 0 0 0 WT ESRRA-/- WT ESRRA-/- WT ESRRA-/- WT ESRRA-/- Stac2 Rara C17orf37 Nr1d1 6 1.0 50 1.0

5 0.8 40 0.8 * 4 0.6 30 0.6 3 0.4 20 0.4 2 1 * 0.2 10 0.2

Relative gene expression0 Relative gene expression Downloaded0 from cancerres.aacrjournals.org0 on October 2,0 2021. © 2010 American Association for Cancer WT ESRRA-/- WT ESRRA-/- WT ESRRAResearch.-/- WT ESRRA-/- Author Manuscript Published OnlineFirst on October 20, 2010; DOI: 10.1158/0008-5472.CAN-10-2840 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 2

A chr.17

34560872 17q12-21.2 36960074

4 Binding Ratio 0

CACNB1 CRKRS ERBB2 CSF3 RAPGEFL1 CR597260 KRT10 KRTAP2-4 KRT33A KRT15 RPL19 NEUROD2 GBR7 THRAP4 WIPF2 CCR7 TMEM99 KRTAP4-14 KRT33B KRT19 STAC2 PPP1R1B IKZF3 THRA CDC6 SMARCE1 KRT12 KRTAP4-12 KRT34 FBXL20 STARD3 ZPBP2 NR1D1 RARA KRT222P KRT20 KRTAP4-5 KRT31 PPARBP TCAP GSDML MSL-1 GJC1 KRT24 KRT23 KRTAP4-4 KRT37 PNMT ORMDL3 CASC3 TOP2A KRT25 KRT39 KRTAP4-2 KRT38 PERLD1 GSDM1 IGFBP4 KRT26 KRT40 KRTAP4-10 KRT32 C17ORF37 PSMD3 TSN4 KRT27 KRTAP9-2 KRT35 DKFZp686J17211 KRT28 KRTAP9-3 KRT36 KRTAP3-3 KRTAP9-8 KRT13 KRTAP3-2 KRTAP9-4 KRTAP3-1 KRTAP17-1 KRTAP1-5 AK123843 KRTAP1-3 KRTAP1-1 B 8 10 PERLD1 6 C17orf37 ERBB2 6 GRB7 8 6 4 4 6 4 4 2 2 2 2 Binding Ratio 0 0 0 +1 0 +1 +1 +1 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2010+1 American Association for Cancer+1 Research. Author Manuscript Published OnlineFirst on October 20, 2010; DOI: 10.1158/0008-5472.CAN-10-2840 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 3

A aCAAGGTCA CRKRS +1 ERBB2 +1 +22 kb 25 CRKRS 25 ERBB2 25 ERBB2 (promoter) (promoter) (intronic) siC 20 20 20 siESRRA 15 15 15 ***

10 siC siESRRA 10 10 ** ERRα ** 5 *** α 5 5 -tubulin * 0 0 0 Normalized fold enrichment Normalized fold enrichment ChIP: ctrl ERRα PolII ChIP: ctrl ERRα PolII ctrl ERRα PolII

TCAAGGTCA TCAAGGaCA GRB7 +1 +1 PERLD1 +12 kb +10 kb 25 GRB7 25 GRB7 25 PERLD1 25 PERLD1 (promoter) (downstream) (intronic) (promoter) 20 20 20 20

15 15 15 15

10 * 10 10 10 *

5 5 5 ** 5 * 0 0 0 0 Normalized fold enrichment Normalized fold enrichment ChIP: ctrl ERRα PolII ctrl ERRα PolII ChIP: ctrl ERRα PolII ctrl ERRα PolII BC LZTS2 +1 siC siESRRA ERBB2 α-tubulin 10 LZTS2 1 (promoter) * 8 siC 0 siESRRA *** * 6 * * * ** ** *** * * -1 * * *** ** 4 * * -2 2 RARA MED1 GRB7 PNMT RPL19 KRT13 KRT15 KRT37 STAC2 NR1D1 ERBB2 KRT24 PSMD3 CRKRS Relative gene expression (Log2) PERLD1 0 STARD3 Normalized fold enrichment CACNB1 Downloaded from cancerres.aacrjournals.org on OctoberC17orf37 2, 2021. © 2010 American Association for Cancer PPP1R1B SMARCE1

α NEUROD2 ChIP: ctrl ERR PolII Research. RAPGEFL1 Author Manuscript Published OnlineFirst on October 20, 2010; DOI: 10.1158/0008-5472.CAN-10-2840 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 4

A α B ERR β siC siESRRA PGC-1 5 siC siESRRAsiPPARGC1B ERRα 4 PGC-1β 68 24 49 α-tubulin 3 *

2 ** ** * 1 *

Normalized fold enrichment 0 ChIP: ctrl CRKRS PERLD1 ERBB2 GRB7 NR1D1 PGC-1β (promoter) (intronic) (intronic) (downstr) (upstream)

C D 1.2 siC 1.0 siESRRA siPPARGC1B 1.0 siPPARGC1B 0.5 0.8 siESRRA+siPPARGC1B

0 0.6

-0.5 0.4 * * H-thymidine incorporation * 3 -1.0 0.2 * * ** Relative expression (Log2) ** -1.5 Relative 0

GRB7 DownloadedERBB2 from cancerres.aacrjournals.orgNR1D1 on October 2, 2021. © 2010 American Association for Cancer CRKRS PERLD1 Research. Author Manuscript Published OnlineFirst on October 20, 2010; DOI: 10.1158/0008-5472.CAN-10-2840 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 5

A B ERα siC ERα 8 siESR1 ß-actin 6 - siC siESR1 4 4 ERBB2 4 GRB7 2 (intronic) (downstream) Binding Ratio 0 3 3 * ERRα 8 ** 6 2 2 * 4 * 1 1 2 Normalized fold enrichment Binding Ratio 0 +1 0 0 ChIP: IgG ERα ERRα IgG ERα ERRα ERBB2 C ‡‡ * siC 1.8 ‡‡ 1.8 * siESRRA 1.5 1.5 siPPARGC1B 1.2 1.2

0.9 0.9

0.6 0.6

0.3 0.3

Relative Gene Expression 0 0 MCF-7 TamR-MCF-7 MCF-7 TamR-MCF-7 ERBB2 GRB7 D 1.4 1.4 NS * siC 1.2 1.2 * siESRRA 1.0 NS 1.0 siPPARGC1B 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 Relative cell proliferation 0 0 veh OHT veh OHT Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2010 American Association for Cancer MCF-7 Tam-R-MCF-7Research. Author Manuscript Published OnlineFirst on October 20, 2010; DOI: 10.1158/0008-5472.CAN-10-2840 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Transcriptional control of the ERBB2 amplicon by ERRα and PGC-1 β promotes mammary gland tumorigenesis

Genevieve Deblois, Ghada Chahrour, Marie-Claude Perry, et al.

Cancer Res Published OnlineFirst October 20, 2010.

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