Transcriptional Control of the ERBB2 Amplicon by Erra and PGC-1B Promotes Mammary Gland Tumorigenesis

Published OnlineFirst October 20, 2010; DOI: 10.1158/0008-5472.CAN-10-2840

Cancer Therapeutics, Targets, and Chemical Biology Research

Transcriptional Control of the ERBB2 Amplicon by ERRa and PGC-1b Promotes Mammary Gland Tumorigenesis

Genevieve Deblois1,2, Ghada Chahrour1,2, Marie-Claude Perry1,2, Guillaume Sylvain-Drolet1,2, William J Muller1,2, and Vincent Giguere 1–3

Abstract 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 a (ERRa, NR3B1) and its coregulator PGC-1b are key effectors in this process. Using a mouse model of ERBB2-initiated mammary tumorigenesis, we first show that ablation of ERRa 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 ERRa to DNA segments associated with the ERBB2 amplicon. Furthermore, ERRa directs the corecruitment of the coactivator PGC-1b to segments in the 17q12 region and the recruitment of RNA polymerase II to the promoters of the ERBB2 and coamplified genes. ERRa and PGC-1b also participate in the de-repression of ERBB2 expression through competitive genomic cross-talk with estrogen receptor a (ERa) and, as a consequence, influence tamoxifen sensitivity in breast cancer cells. Taken together, our results suggest that ERRa and PGC-1b 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 ERa on ERBB2 expression. Cancer Res; 70(24); 10277–87. 2010 AACR.

Introduction tumors. However, in both instances, an increase in the transcriptional regulation rate of the locus is observed (5–8). Breast cancer is a complex disease implicating distinct cell Loss of repression of ERBB2 has been linked to tamoxifen types and multiple signaling pathways that together engender resistance in breast cancer cells (9, 10). It is believed that a multiplicity of tumor subtypes (1). The molecular hetero- transcriptional repressors including FOXP3 (11), PAX2 (10), geneity of breast tumor subtypes dictates their intrinsic GATA4 (12), PEA3 (13), and MYB (14) act to quench the response to specific therapeutic approaches and has therefore expression of ERBB2 in ERBB2-negative tumors. Notably, it become an important aspect in the clinical management of the has also been shown that estrogen receptor a (ERa, NR3A1), disease (2). Amplification of the 17q12–21 region leads to in cooperation with the transcription factor PAX2, can repress concomitant overexpression of ERBB2 and several coamplified ERBB2 expression through binding to a cis-regulatory element genes and occurs in approximately 20% to 30% of breast tumors in the presence of either 17b-estradiol (E2) or 4-hydroxy- (3). The ERBB2-amplified breast cancer subtype is strongly tamoxifen (OHT; ref. 10). Indeed, ERa-positive tumors with associated with poor prognosis (4). Although overexpression of the worst prognosis that do not respond to tamoxifen therapy ERBB2 is usually linked to amplification of the 17q12 region, often express high levels of ERBB2 (15–18). this region is not amplified in some ERBB2-positive breast A positive increase in the transcriptional control of the locus can contribute to relieve the transcriptional repression that takes place on ERBB2, and overexpression of ERBB2 is Authors’ Affiliations: 1Goodman Cancer Research Centre; 2Department thought to drive the amplification of the 17q12 locus (19). of Biochemistry; and 3Department of Medicine and Department of Oncol- Transcription factors such as AP-2 (8), YY1 (20), ETS (21), YB-1 ogy, McGill University, Montreal, Quebec, Canada (22), and EGR2 (23) have been shown to be recruited to the Note: Supplementary data for this article are available at Cancer Research promoter of ERBB2 and play a role in its overexpression in Online (http://cancerres.aacrjournals.org/). breast cancer cells. Therefore, understanding the mechanisms Corresponding Author: Vincent Giguere, Goodman Cancer Research ERBB2 Centre, McGill University, 1160 Pine Avenue West, Montreal, Quebec, governing the repressive and positive regulation of Canada H3A 1A3. Phone: (514)-398-5899; Fax: (514)-398-8578. E-mail: expression and its neighboring genes in the amplified locus is [email protected] of considerable importance in identifying the transcription doi: 10.1158/0008-5472.CAN-10-2840 factors and molecular events involved in the establishment of 2010 American Association for Cancer Research. ERBB2-positive tumors.

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Although most studies on the 17q12 amplicon have been (39). ERRa chromatin-immunoprecipitation (ChIP) assays were centered on the expression and activity of ERBB2 itself, it is performed using an anti-hERRa polyclonal antibody raised and now clear that coamplified genes not only contribute but validated in our laboratory (32, 40). Other antibodies used were could also be essential to the progression of ERBB2-positive anti-RNA-Polymerase II (8WG16), anti-ERRa (Millipore), anti- breast tumors (24, 25). The minimal 17q12 amplicon includes PGC-1b, and anti-ERa (Santa Cruz Biotechnologies). siRNAs genes that are involved in signal transduction (GRB7, PERLD1, against ERa,ERRa, PGC-1b, and control (ON-Target-Plus siRNA PPP1R1B), transcription (MED1, IKZF3, NEUROD2, PNMT), cell pool) were obtained from Dharmacon. migration and invasion (C17orf37, GRB7), inhibition of apo- ptosis (MED1), genomic instability (PERLD1), and tamoxifen Transgenic mouse model study resistance (STARD3, GRB7; refs. 26–28). Although transcrip- The derivation of the conditionally activated NeuNT tional regulation of the coamplified genes in relation to the (erbB2NT) has been described in detail (41). To generate mice ERBB2 subtype is now considered an important aspect in the that expressed erbB2NT in the mammary glands of animals NT establishment of ERBB2-positive breast tumors, the molecular carrying a null allele for Esrra, erbB2 and MMTV-Cre mice mechanisms associated with this phenomenon have yet to be were first bred with mice heterozygous for null alleles of Esrra investigated in detail. (42). All mice were previously derived in a pure FVB genetic The orphan nuclear receptor estrogen-related receptor a background. These mice were then bred to generate mice null NT (ERRa, NR3B1) shares both structural and functional features for Esrra also carrying one copy of erbB2 and one copy of NT with ERa (29). The expression of ERRa is inversely correlated MMTV-Cre (e.g., Ersra / /erbB2 /MMTV-Cre). The resulting to that of ERa but positively associates with that of ERBB2 and mice were examined for the presence of the excised recombi- NT with poor prognosis in breast cancer (30, 31). A recent nant erbB2 allele through Southern blot analysis. The levels genome-wide binding sites location analysis in breast cancer of amplification of the locus were determined by quantitative cell lines intersected with gene expression data from breast RT-PCR (qRT-PCR) using primers listed in Supplementary tumors has shown that ERRa signaling contributes to known Table S1. Female mice were examined twice a week for pathways linked to breast cancer progression, including those mammary tumor development by palpation. involving ERa and ERBB2 (32). Conversely, the transcriptional activity of ERRa can be modulated by the epidermal growth ChIP assays and ChIP-on-chip on chr.17q tiled arrays factor (EGF)/ERBB2 signaling pathway in breast tumors (33, ChIP was performed as described previously (38). Quanti- 34). ERRa preferentially acts in concert with the coregulators fication of ChIP enrichment by real-time qRT-PCR was carried peroxisome proliferator-activated receptor g, coactivator-1a out using the LightCycler480 instrument (Roche). ChIP-on- (PGC-1a), and PGC-1b, whose combined roles have been chip was carried out on custom chr.17q Agilent tiled arrays extensively studied in the context of the regulation of bioe- (150-bp resolution). Chromatin was prepared from SKBr3 cells nergetic pathways (35, 36). Gene expression and binding (for ERRa and PGC-1b ChIP-on-chip) or from MCF-7 cells sites location analyses in breast cancer cell lines have also exposed to 10 nmol/L E2 for 45 minutes (for ERa ChIP-on- demonstrated that ERa and ERRa display strict binding site chip). The primers used for standard ChIP are listed in specificity and maintain independent mechanisms of tran- Supplementary Table S1. scriptional activation, suggesting a prevailing ERa-independent role for ERRa in breast tumor development (32, 37). Computational motif discovery Nevertheless, a significant number of genes that are common De novo and known motif discovery was performed with targets of the 2 nuclear receptors are also important players in MEME Suite (http://meme.sdsc.edu/meme4_4_0/intro.html). breast tumor development (32). Motif discovery was also confirmed using the Genomatix Here we show that ERRa is required for the full potential of Software Suite (http://www.genomatix.de/en/produkte/ ERBB2-driven mammary tumorigenesis in mice and that genomatix-software-suite.html). tumors lacking ERRa express lower levels of most amplicon genes. Chromosome-wide identification of regulatory regions siRNA occupied by ERRa and PGC-1b as well as gene expression data siRNA for ERa and control were transfected in MCF-7 cells identify both factors as key regulators of the expression of cultured in phenol-red-free DMEM media supplemented with ERBB2 and several coamplified genes in the 17q12 region in hormone-deprived serum using the HyperFect reagent breast cancer cells. Finally, we demonstrate that an antag- (Qiagen). Similarly, siRNAs for ERRa, PGC-1b, and control onistic interaction between ERa and the ERRa/PGC-1b com- were transfected in SKBr3 cells using the HyperFect reagent plex at the ERBB2 locus is an important determinant of ERBB2 for 48 to 60 hours. expression involved in the development of tamoxifen resistance in breast cancer cells. Expression analysis mRNA from SKBr3 cells transfected with the ERRa or PGC- Materials and Methods 1b siRNA were reverse-transcribed into cDNA using Super- script (Invitrogen) and analyzed by qRT-PCR with SYBR- Cell culture, reagents, and antibodies green–based RT-PCR (Roche). Alternatively, RNA was MCF-7 and SKBr3 cells were cultured as described previously extracted from mice mammary gland tumors using the (38). Tam-R-MCF-7 cell line was derived as described previously RNA tissue extraction kit (Qiagen) and reverse-transcribed

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using Superscript (Invitrogen). Primer pairs used for qRT-PCR amplicon in human breast cancer cells, we first performed are listed in Supplementary Table S2. genomic location analysis of ERRa in the ERa-negative SKBr3 cell line using a custom tiled array covering the q-arm of Proliferation assay assessed by 3H-thymidine human chromosome 17. ChIP-on-chip analysis of ERRa on the incorporation chr.17q region revealed 92 segments bound by ERRa and SKBr3 cells were transfected with the appropriate siRNA for validated by standard ChIP analyses (Supplementary Table S3 60 hours. Cells were incubated in the presence of 1 mCi 3H- and Fig. S1A). De novo DNA motif discovery confirmed the thymidine for 4 hours prior to fixation and harvesting. Simi- enrichment of the ERRE motif within the bound segments larly, MCF-7 and Tam-R-MCF-7 cells were grown in phenol– (Supplementary Fig. S1B). Remarkably, a significant enrich- red-free DMEM supplemented with hormone-deprived serum. ment of ERRa-bound segments is observed within the ampli- Upon siRNA transfection, the media was supplemented with con region (32 out of 92 segments) and 13 segments map to 100 nmol/L OHT (Sigma) or vehicle for 60 hours before the minimal region steadily amplified in ERBB2-positive harvesting and isotope counting. tumors (Fig. 2A, shaded region). ERRa binding events were observed in ERBB2 itself as well as in the transcriptional unit Results of genes that are consistently coamplified with ERBB2, such as PERLD1, C17orf37, and GRB7 (Fig. 2B). Ablation of ERRa delays ERBB2-induced mammary To relate the binding profile of ERRa to the transcriptional gland tumorigenesis regulation of target genes, we then monitored modulation in To initiate our study on the functional relationship between the recruitment of RNA-PolII by ChIP to the promoters of ERRa and ERBB2 in mammary gland tumorigenesis, we first genes located within the ERBB2 amplicon upon depletion of used the well-characterized Neu-NT knock-in transgenic mouse ERRa in SKBr3 cells. Indeed, a significant decrease in both model of mammary tumorigenesis (41) to generate ERRa- ERRa and RNA-PolII recruitment was observed at the pro- deficient transgenic mice conditionally expressing the activated moter of CRKRS, a target gene at which ERRa binding occurs Neu under the control of the endogenous Erbb2 promoter. The directly at the promoter (Fig. 3A). Furthermore, a decrease in choice of this model was guided by the previous observation that RNA-PolII recruitment was also observed at promoters of these mice develop focal mammary tumors with high frequency amplicon genes for which ERRa binding takes place far after a long latency period that bear amplified copies of the upstream of the transcriptional start sites or in intronic activated Erbb2 allele on chromosome 11 (41, 43). In addition, regions of genes such as ERBB2, GRB7, and PERLD1 this model is ideal to study the impact of alteration in the gene (Fig. 3A). Depletion of ERRa had no effect on the occupancy regulatory machinery as expression of the Erbb2 locus remains of RNA-PolII at the promoter of the gene, LZTS2, that is not a under the control of endogenous regulatory elements. We target of ERRa (Fig. 3B). These results demonstrate that the observed that ablation of ERRa significantly delayed ERBB2- recruitment of ERRa at both near and distant sites from induced mammary gland tumorigenesis (P < 0.05; Fig. 1A), transcriptional start sites contributes to the recruitment of indicating that the presence of ERRa is required for optimal RNA-PolII at the promoters of target genes. We next assessed development of ERBB2-driven tumors. The ERRa-null mice the effect of ERRa depletion on the expression of amplicon lactate normally and the development of the mammary gland genes in human breast cancer cells. In agreement with the is not affected by the lack of ERRa expression (Fig. 1B). As observation made in ERBB2-induced tumors developed in the expected, amplification of the Erbb2 locus was observed in ERRa knockout mice, depletion of ERRa in SKBr3 cells leads the tumors derived from this model but the absence of ERRa to a significant decrease in the relative expression of most did not significantly affect the level of amplification of the amplicon genes (Fig. 3C). These results indicate that ERRa locus as compared with wild-type (Fig. 1C). To investigate the positively regulates the expression of ERBB2 and numerous possible contribution of ERRa in the transcriptional regula- genes that coamplify with it on chr.17q12. tion of Erbb2 and neighboring genes in the Erbb2 amplicon, we next assessed their levels of expression in the tumors. We The coactivator PGC-1b is recruited to ERRa-bound found that the relative transcript levels of most of the segments in the chr.17q12 amplicon amplicon genes tested, including Erbb2, were either signifi- We have previously shown that the coactivator PGC-1b can cantly decreased or show a similar downward trend in tumors be corecruited with ERRa at specific genomic locations in arising from ERRa knockout mice compared with the wild- SKBr3 cells (32). To assess whether PGC-1b contributes more typeones(Fig.1D).Overall,theseresultssuggestthatERRa broadly in the transcriptional regulation of amplicon genes, might play a role in the development of ERBB2-driven mam- we performed PGC-1b ChIP-on-chip in SKBr3 cells hybridized mary tumors through transcriptional regulation of Erbb2 and on the chr.17q tiled array. The ChIP-on-chip experiment other genes located within the amplicon. identified 73 segments significantly bound by PGC-1b on chr.17q (Supplementary Table S4 and Supplementary ERRa is recruited to specific sites at chr.17q12 and Fig. S2A) of which 24 were segments common to ERRa regulates the expression of ERBB2 and coamplified (Fig. 4A). Interestingly, PGC-1b recruitment was observed at genes in human breast cancer cells sites shared with ERRa in regions located within key genes of In order to investigate the role of ERRa in the transcrip- the ERBB2 amplicon including ERBB2, PERLD1, GRB7, and tional regulation of the genes located within the ERBB2 NR1D1 (Supplementary Table S4 and Supplementary

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A B WT ESRRA-/- 100

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

% Tumor 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 0 0 0 WT ESRRA-/- WT ESRRA-/- WT ESRRA-/- WT ESRRA-/-

Figure 1. Ablation of ERRa delays mammary gland tumorigenesis in mice. A, ablation of ERRa 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 ERRa-null mice heterozygous for the knock-in allele (arrow). B, mammary whole mount staining showing normal development of the mammary gland in ERRa-null mice. C, levels of normalized amplification of the Erbb2 locus detected using real-time qRT-PCR are similar in wild-type and ERRa-null tumors (n ¼ 5). AMG, adjacent mammary gland. NS, not significant. D, tumors lacking the expression of ERRa express lower levels of amplicon gene transcripts (n ¼ 6). Error bars, SEM; *, P < 0.05.

Fig. S2B). De novo DNA motif discovery identified the ERRE as decrease in the relative levels of transcripts of several amplicon the most enriched motif within the common segments shared genes (Fig. 4C). Because ERBB2 and the coamplified genes are by both factors (P ¼ 7.8e-05), indicating that PGC-1b recruit- potent inducers of breast tumor growth, we next evaluated the ment to the common segments occurs through ERRa (Fig. 4A impact of loss of ERRa and/or PGC-1b on cell proliferation. and Supplementary Fig. S3). Indeed, depletion of ERRa in Specific knockdown of ERRa, PGC-1b, or of both factors, using SKBr3 cells using specific siRNAs leads to a significant reduc- specific siRNAs led to a significant decrease in SKBr3 cell tion in PGC-1b recruitment to the common sites located in proliferation as assayed by 3H-thymidine incorporation chr.17q amplicon genes as assessed by standard ChIP (Fig. 4B). (Fig. 4D). Taken together, these results demonstrate that ERRa We further tested the effect of PGC-1b recruitment on the contributes to the corecruitment of PGC-1b to the chr.17q expression of the amplicon transcripts. As observed for ERRa, region and that both factors regulate the expression of ampli- depletion of PGC-1b using specific siRNAs leads to a significant con genes and aggressive growth of SKBr3 breast cancer cells.

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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 02LXBF STARD3 ZPBP2 1D1RN 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 +1 +1

Figure 2. ERRa is recruited to multiple segments in the ERBB2 amplicon region on human chromosome 17q12–21. A, binding profile of ERRa 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 gray region. B, binding profile of ERRa on representative coamplified gene regions located in the ERBB2 amplicon.

ERRa competes with ERa for recruitment to common sion and signaling contribute to acquired tamoxifen resistance segments in ERBB2 and GRB7 and contributes, along (10). Overexpression of other coamplified genes such as GRB7 with PGC-1b, to tamoxifen resistance in MCF-7 cells have also been implicated in resistance to endocrine therapy. In ER-positive tumors that are tamoxifen-sensitive, the We next explored the possibility that ERRa could interfere expression of ERBB2 is low due to transcriptional repression of with ERa signaling in the regulation of specific genes within the gene inflicted by various transcription factors, including the ERBB2 amplicon and thus take part in the development of ERa and the corepressor PAX2. It has been suggested that loss tamoxifen resistance. In order to assess a potential cross-talk of this repression and consequential increase in ERBB2 expres- at the genomic level, we performed an ERa ChIP-on-chip

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A aCAAGGTCA CRKRS +1 ERBB2 +1 +22 kb 25 CRKRS 25 ERBB2 25 ERBB2 (promoter) siC (promoter) (intronic) 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

B C ESRRA LZTS2 +1 siC si 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 (iog 2) PERLD1 0 STARD3 Normalized fold enrichment CACNB1 C17orf37 PPP1R1B SMARCE1

α NEUROD2

ChIP: ctrl ERR PolII RAPGEFL1

Figure 3. ERRa 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 ERRa leads to a significant decrease in RNA-PolII recruitment to the promoters of ERRa target genes located in the ERBB2 amplicon. Inset, Western blot shows the level of ERRa in cells transfected with the siRNAs. B, same experiment as in (A) on a negative control gene that is not affected by ERRa. C, relative expression of ERRa target genes in the ERBB2 amplicon upon siRNA-mediated depletion of ERRa in SKBr3 cells. The effect of ERRa depletion on the level of ERBB2 is shown by Western blot (inset). Gray region, minimal ERBB2 amplicon. Error bars, SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

experiment in E2-treated MCF-7 cells using the same chr.17q loci (Supplementary Fig. S4). Notably, the overlapping ERa/ arm tiled array (Supplementary Table S5). We identified 194 ERRa segments include a site downstream of GRB7 as well as a segments significantly bound by ERa, of which 10 overlapped site located in the first intron of ERBB2. Of particular interest, with ERRa-bound loci and 5 overlapped with PGC-1b-bound the intronic ERBB2 site was previously identified as bound by

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A B ERRα PGC-1β siC siESRRA 5 ESRRA siC si siPPARGC1B 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) (downstream) (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 (log 2) ** -1.5 Relative 0

GRB7 ERBB2 NR1D1 CRKRS PERLD1

Figure 4. PGC-1b is recruited to the ERBB2 amplicon through ERRa and contributes to the regulation of amplicon genes. A, Venn diagram indicating the overlap between ERRa-bound segments (green) and PGC-1b-bound segments in SKBr3 cells (blue). The sequence logo depicts the ERRa response element which is enriched in the common ERRa/PGC-1b segments. B, standard ChIP in SKBr3 cells upon siRNA-mediated depletion of ERRa shows that PGC-1b recruitment to the common sites is dependent on ERRa. Inset, Western blot showing the level of ERRa and PGC-1b in cells transfected with siESRRA and with siPPARGC1B. C, relative gene expression of common ERRa/PGC-1b target genes located in the ERBB2 amplicon shows that the genes are regulated by PGC-1b. Inset, Western blot showing the level of PGC-1b in cells transfected with siPPARGC1B. D, depletion of ERRa (siESRRA), PGC-1b (siPPARGC1B), or of both, in SKBr3 cells for 60 hours affects the proliferation rate of the cells as shown by a decrease in the relative 3H-thymidine incorporation over a 4-hour period. Error bars, SD; *, P < 0.05; **, P < 0.01.

the ERa/PAX2 repressor complex and shown to play a role in competitive transcriptional regulation of ERBB2 and GRB7 tamoxifen sensitivity of MCF-7 cells (Fig. 5A). gene expression. We confirmed that treatment of MCF-7 cells with E2 leads We next asked whether this transcriptional cross-talk could to a downregulation of ERBB2 and GRB7 expression (Supple- play a role in endocrine resistance using a tamoxifen-resistant mentary Fig. S5A). Examination of the bound sequences in MCF-7 cell line generated in our laboratory (Tam-R-MCF-7). ERBB2 and GRB7 revealed the presence of a mixed ERE/ERRE As expected, the expression of ERBB2 and GRB7 is increased in binding site as previously defined for shared ERa/ERRa the Tam-R-MCF-7 cell line compared with the parental MCF-7 binding sites (32), indicating that ERa and ERRa should cells (Fig. 5C). We further show that depletion of either ERRa compete for binding at these common sites. Using specific or PGC-1b (Supplementary Fig. S5C and D) leads to a sig- siRNAs and standard ChIP, we indeed observed that depletion nificant decrease in ERBB2 and GRB7 transcripts in Tam-R- of ERa from MCF-7 cells was accompanied by an increase in MCF-7 cells while having no effect on the expression of these ERRa recruitment to the common sites that were otherwise genes in the parental MCF-7 cell line (Fig. 5C). We then not bound by ERRa in MCF-7 cells (Fig. 5B). Depletion of ERa assessed the effect of loss of ERRa or PGC-1b on the pro- had no effect on ERRa recruitment to ERRa-specific sites liferation of parental MCF-7 and Tam-R-MCF-7 cells in the (Supplementary Fig. S5B). These results indicate that the absence or presence of OHT. As shown in Figure 5D, the concomitant presence of both nuclear receptors yields to proliferation rate of MCF-7 cells is decreased upon OHT

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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 MCF-7 Tam-R-MCF-7

Figure 5. ERRa competes with ERa for recruitment to segments in ERBB2 and GRB7 and contributes, along with PGC-1b, to tamoxifen resistance in

MCF-7 cells. A, binding profile of ERa in E2-treated MCF-7 cells (red) and ERRa in SKBr3 cells (green) on the ERBB2 locus. The arrows indicate the common ERa/ERRa-bound segment. B, relative enrichment of ERa and ERRa as assayed by standard ChIP upon siRNA-mediated depletion of ERa in

E2-treated MCF-7 cells at common sites in ERBB2 and GBR7. Inset, Western blot showing the level of ERa in cells transfected with siESR1. C, relative gene expression for ERBB2 and GRB7 by qRT-PCR in MCF-7 and Tam-R-MCF-7 cells upon siRNA-mediated depletion of ERRa or PGC-1b. *, P < 0.05 relative to the siC of the same cell line; zz, P < 0.01 relative to the MCF-7 control cell line. D, effect of siRNA-mediated depletion of ERRa (siESRRA) or PGC-1b (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-hour period. Error bars, SD; *, P < 0.05; NS, not significant.

treatment whereas depletion of either ERRa or PGC-1b has no reinstates the antiproliferative effect of OHT in these cells. additional effect on the proliferation of these cells. In contrast, Taken together, these results suggest that both factors may whereas OHT does not influence the proliferation rate of the play a role in the development of OHT resistance in breast Tam-R-MCF-7 cells, depletion of either ERRa or PGC-1b cancer cells.

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ERRa Regulation of the ERBB2 Amplicon

Discussion

Amplification of the ERBB2 locus and neighboring loci on chromosome 17q12, which is accompanied by overexpression of the amplified genes, plays an important role in the devel- ERRα opment of tamoxifen resistance and a more aggressive breast cancer phenotype (44). The outcome of this work, derived ERBB2+ from a functional genomic approach and validated in well- amplicon characterized in vivo and in vitro models of human breast PGC-β TAM resistant cancer, demonstrates that the orphan nuclear receptor ERRa and its coregulator PGC-1b are key elements in the transcriptional regulation of genes located within the chr.17q amplicon, ERBB2 including itself. In addition, this study identifies mole- ERBB2 cular mechanisms through which these 2 factors can con- and amplicon tribute to the development of tamoxifen resistance in breast cancer cells. The observation that ERRa regulates the expression of ER+ several genes located within the ERBB2 amplicon denotes ERα TAM sensitive the importance of ERRa signaling in the progression of the and Pax2 ERBB2-driven breast tumor subtype. This is further empha- sized by the observation that the presence of ERRa is required to observe the full oncogenic potential of ERBB2 in a well- Figure 6. ERRa and PGC-1b are involved in a positive feed-forward regulatory loop with ERBB2. In ER-positive/tamoxifen sensitive established mouse model of human breast cancer (Fig. 1). tumors, ERBB2 expression is maintained at low levels by transcriptional Notably, we have shown that ERRa regulates the expression of repressor signals such as the ERa/Pax2 complex. ERRa and PGC-1b most genes present in the minimal ERBB2 amplicon. The mediate positive transcriptional regulation of ERBB2 expression coamplified genes are mainly involved in signal transduction that can prevail over the negative transcriptional downregulation of ERBB2 and transcription influencing various biological processes by ERa and Pax2, especially in ER-negative/tamoxifen- resistant tumors or in tumors that have lost the expression of Pax2 (10). including cell migration, invasion, and survival as well as In addition, the positive feed-forward loop is further enhanced through resistance to tamoxifen (9, 10, 24–28). It can therefore be the positive autoregulation of ERRa (40). Solid lines represent envisioned that, by acting as a global transcriptional regulator transcriptional regulation and the dashed line indicates the of the ERBB2 amplicon, ERRa contributes to establish the ERRB2-mediated signaling pathway influencing ERRa transcriptional activity (33, 34). ERBB2-positive tumor subtype which is characterized not only by increased ERRB2 signaling but also by various cellular processes controlled by coamplified genes. Mechanisms that trigger the amplification of the locus are not well understood The genomic convergence between ERRa and the ERBB2 but it has been observed that an increase in the transcription amplicon described in this study is even more relevant con- levels of ERBB2 often precedes locus amplification (5, 45). sidering the finding of competitive ERRa recruitment at a site ERRa could inflict positive transcriptional pressure on the in ERBB2 targeted by ERa/Pax2. This site has been shown to 17q12 region and thus participates actively in the establish- be involved in maintaining the repressive state of ERBB2 in ment of ERBB2-positive aggressive tumors. ERa-positive endocrine-responsive tumors (10). We also Our results are in agreement with previous observations observed competitive recruitment of ERRa/PGC-1b and demonstrating that expression of ERRa positively correlates ERa on a site proximal to GRB7. Interestingly, as it is the with that of ERBB2 in breast tumors and that ERRa tran- case for ERBB2, ERRa, and ERa have opposite transcriptional scriptional activity is positively modulated by EGFR/ERBB2 effects on the expression of GRB7 in breast cancer cells. The signaling in breast cancer cells (30, 33, 34). Such interactions loss of negative regulation leads to overexpression of ERBB2 can be integrated in a model of positive feed-forward regu- and ERBB2-coamplified genes such as GRB7 in luminal breast latory loops whereby all the processes involved further tumors and has been associated with a poor response to enhance the buildup of their own stimulus (46). In ERBB2- targeted endocrine therapy (47). The effect of ERRa/PGC-1b negative tumors, the expression of ERBB2 is maintained at low depletion on tamoxifen-mediated proliferation of Tam-R- levels by various sequence-specific transcription factors such MCF-7 cells suggests a mechanism whereby the transcrip- as Pax2, FOXP3, PEA3, GATA4, and MYB, as well as by ERa.It tional regulation of ERBB2 and coamplified genes by these is thus likely that the positive feed-forward regulatory loop factors can contribute to the establishment of acquired involving ERRa and PGC-1b favors escape from the negative tamoxifen resistance. Other ERBB2-coamplified genes have regulation inflicted on ERBB2 by ERa during progression of been associated with the response to trastuzumab or anthra- breast cancer (Fig. 6). This model suggests a possible role for cycline-based chemotherapies in single or combination thera- ERRa in mediating the transition of a subset of ER-positive pies (48). It would therefore be of interest to assess how the luminal tumors toward the more aggressive ERBB2-expressing transcriptional regulation of these genes by ERRa/PGC-1b subtype. affects the response to other targeted therapies.

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Deblois et al.

Little is known about the expression and function of PGC- Disclosure of Potential Conflicts of Interest 1b in breast cancer cells. Here we show that PGC-1b induces the expression of ERBB2 in breast cancer cells. In line with this No potential conflicts of interest were disclosed. observation, we have recently shown that ERBB2 signaling also induces the expression of PGC-1b in breast cancer cells Grant Support (49). Therefore, together with ERRa, our results suggest that PGC-1b contributes to the establishment of the aggressive This work was supported by grants from the Canadian Institutes of Health ERBB2-positive tumors through a positive feed-forward reg- Research (MOP-64275) and a Terry Fox Foundation Program Project Grant from the National Cancer Institute of Canada. G. Deblois and M.-C. Perry are ulatory loop (Fig. 6). recipients of studentships from the Fonds de la Recherche en Santedu In conclusion, this study clearly demonstrates that, in Quebec. G. Deblois is also supported by a predoctoral traineeship award (W81XWH-10-1-0489) from the U.S. Department of Defense Breast Cancer addition to its primary role in the control of cellular energy Research Program. metabolic pathways in both normal and cancer cells (32, 35, The costs of publication of this article were defrayed in part by the payment 49), the ERRa/PGC-1b complex promotes the development of 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. the ERBB2-positive tumor subtype and tamoxifen resistance ERRB2 in breast cancer via transcriptional control of the Received 08/04/2010; revised 10/15/2010; accepted 10/15/2010; amplicon. published OnlineFirst 10/20/2010.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Cancer Correction Research Correction: Transcriptional Control of the ERBB2 Amplicon by ERRa and PGC-1b Promotes Mammary Gland Tumorigenesis

In this article (Cancer Res 2010;70:10277–87), which was published in the December 15, 2010 issue of Cancer Research (1), the article's abstract was repeated as the last paragraph of the Introduction section.

Reference 1. Deblois G, Chahrour G, Perry M-C, Sylvain-Drolet G, Muller WJ, Giguere V. Transcriptional control of the ERBB2 amplicon by ERRa and PGC-1b promotes mammary gland tumorigenesis. Cancer Res 2010;70:10277–87.

Published onlineFirst January 18, 2011. 2011 American Association for Cancer Research. doi: 10.1158/0008-5472.CAN-10-4571

1508 Cancer Res; 71(4) February 15, 2011 Published OnlineFirst October 20, 2010; DOI: 10.1158/0008-5472.CAN-10-2840

Transcriptional Control of the ERBB2 Amplicon by ERRα and PGC-1 β Promotes Mammary Gland Tumorigenesis

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

Cancer Res 2010;70:10277-10287. Published OnlineFirst October 20, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-10-2840

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