Androgen-Dependent Repression of Errγ Reprograms Metabolism in Prostate Cancer

Published OnlineFirst November 7, 2016; DOI: 10.1158/0008-5472.CAN-16-1204 Cancer Molecular and Cellular Pathobiology Research

Androgen-Dependent Repression of ERRg Reprograms Metabolism in Prostate Cancer Etienne Audet-Walsh1, Tracey Yee1, Shawn McGuirk1,2, Mathieu Vernier1, Carlo Ouellet1, Julie St-Pierre1,2, and Vincent Giguere 1,2,3

Abstract

How androgen signaling contributes to the oncometabolic state paralleled the loss of ERRg expression. It occurred in both state of prostate cancer remains unclear. Here, we show how the androgen-dependent and castration-resistant prostate cancer and estrogen-related receptor g (ERRg) negatively controls mitochon- was associated with cell proliferation. Clinically, we observed an drial respiration in prostate cancer cells. Sustained treatment of inverse relationship between ERRg expression and disease sever- prostate cancer cells with androgens increased the activity of ity. These results illuminate a mechanism in which androgen- several metabolic pathways, including aerobic glycolysis, mito- dependent repression of ERRg reprograms prostate cancer cell chondrial respiration, and lipid synthesis. An analysis of the metabolism to favor mitochondrial activity and cell proliferation. intersection of gene expression, binding events, and motif anal- Furthermore, they rationalize strategies to reactivate ERRg signal- yses after androgen exposure identiﬁed a metabolic gene expres- ing as a generalized therapeutic approach to manage prostate sion signature associated with the action of ERRg. This metabolic cancer. Cancer Res; 77(2); 1–12. 2016 AACR.

Introduction in the induction of glucose consumption and lactate production (4, 5). Moreover, long-term androgen treatment leads to a repro- Prostate cancer is one of the most common cancers in men, and gramming of energy metabolism that increases mitochondrial the central role of androgens in the development and progression biogenesis and activity (6). However, mapping genome-wide AR- of this disease is well established. Most prostate cancer will binding sites using chromatin immunoprecipitation (ChIP) respond favorably to androgen deprivation therapy (ADT) ini- coupled with deep sequencing (ChIP-seq) revealed that metabolic tially (1) but will later evolve into castration-resistant prostate genes are underrepresented in AR direct target genes (4, 5). cancer (CRPC), for which only palliative treatments are available. Therefore, the androgen signaling pathway likely requires mod- Several mechanisms are involved in the development of castration ulation of effectors downstream of AR to promote metabolic gene resistance, including hyperactivation of the androgen signaling expression in prostate cancer. pathway via gene ampliﬁcation, mutation, and alternative splic- Estrogen-related receptors a and g (ERRa and g) are orphan ing of the androgen receptor (AR; ref. 2). Identifying downstream members of the nuclear receptor superfamily (7). The ERRs are effectors of the AR would lead to a better understanding of the now considered master regulators of energy metabolism (8, 9), course of prostate cancer and, most importantly, identify novel controlling the vast majority of nuclear-encoded mitochondrial therapeutic targets. genes (10–12). The ERR transcriptional pathway has also been The prostate gland produces high levels of citrate due to a extensively linked with the cancer phenotype (13, 14). In breast blunted tricarboxylic acid (TCA) cycle inhibited by high levels of cancer, ERRa and g appear to play opposite roles in disease zinc (3). Through the process of malignant transformation, citrate progression (15). ERRa expression correlates with more aggres- accumulation ceases, which is thought to be the consequence of sive tumors, whereas the presence of ERRg correlates with better restored mitochondrial function and increased lipid synthesis outcome and is associated with oxidative metabolism (15–19). In from citrate production (3). In prostate cancer cells, androgens prostate cancer, ERRa expression is also considered as a marker of have been shown to modulate metabolic gene networks, resulting poor survival, as its expression is higher in cancerous lesions than in benign prostate hyperplasia (20). Conversely, there was a trend toward lower survival of patients with low ERRg expression in a 1Goodman Cancer Research Centre, McGill University, Montreal, Quebec, limited immunohistochemical study (21). Given the high poten- 2 Canada. Department of Biochemistry, McGill University, Montreal, Quebec, tial for druggability of the ERRs and their prominent role in cell Canada. 3Departments of Medicine and Oncology, McGill University, Montreal, metabolism, it is key to deﬁne their respective role in the context of Quebec, Canada. prostate cancer. Note: Supplementary data for this article are available at Cancer Research In this study, we show that activated AR directly inhibits the Online (http://cancerres.aacrjournals.org/). expression of ERRg and that loss of its expression contributes to an Corresponding Author: Vincent Giguere, Goodman Cancer Research Centre, androgen-dependent metabolic reprogramming in prostate can- McGill University, 1060 Pine Avenue West, Montreal, QC, Canada H3A 1A3. cer. Unexpectedly, ERRg acts as a repressor of oxidative metabo- Phone: 514-398-5899; Fax: 514-398-8578; E-mail: [email protected] lism in prostate cancer cells whereby AR-mediated ERRg inhibi- doi: 10.1158/0008-5472.CAN-16-1204 tion leads to a derepression of mitochondrial activity, thus favor- 2016 American Association for Cancer Research. ing an oncometabolic state associated with proliferation. The

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biologic significance of these findings is supported by a strong the 11,053 AR peaks identified by Massie and colleagues (4) and inverse relationship between ERRg expression and prostate cancer after processing them with the LiftOver tool of the UCSC Genome aggressiveness in several independent clinical studies. Bioinformatics toolbox from hg18 to hg19.

Materials and Methods Metabolic analyses Levels of metabolites consumed and produced by prostate Cell culture cancer cells in the culture media were measured with the BioPro- LNCaP, LAPC4, 22rv1, and PC3 cells were originally obtained file Analyzer (Nova Biomedical). Cells were treated for a total of 4 from ATCC. All cells were kept in culture for no more than 3 days, with new treatment added after 48 hours. months after resuscitation and were reauthenticated using the The oxygen consumption rate (OCR) and extracellular acidi- ATCC cell line authentication service upon completion of the fication rate (ECAR) were measured using Seahorse XF24 and study (July 2016). For treatments with R1881 (10 nmol/L; Ster- XFe96 (Seahorse Bioscience). Forty-eight hours after siRNA trans- fi aloids) or enzalutamide (25 mmol/L; ApexBio), cells were rst fection, cells were treated with vehicle or R1881 for 48 hours, then deprived from androgens in media with 2% charcoal-stripped trypsinized, and seeded in Seahorse plates. After 24 hours, RPMI serum (CSS) for 48 hours, with media changed every 48 hours. For was replaced by Seahorse assay media and cells were incubated in siRNA transfections, cells were trypsinized, seeded in CSS media, aCO2-free incubator at 37 C for 1 hour prior to metabolic and transfected within the following hour with either a nontar- analysis. Following ECAR and OCR analyses, cells were trypsi- geted pool of siRNA (siC), or a pool of siRNA targeting AR, ERRa, nized for cell count and data normalization. or ERRg (SMARTpool, Dharmacon) with Hiperfect reagent TCA cycle intermediates were measured at the Metabolomics (Qiagen). Core Facility of the Goodman Cancer Research Centre using a standard protocol (24). For triglyceride quantification, lipids were RNA extraction and qRT-PCR then quantified using the Abcam Triglyceride Quantification fi RNA was extracted with the RNeasy Mini Kit (Qiagen), and rst- Assay Kit (colorimetric). Metabolic results were normalized for strand cDNA synthesis was performed with ProtoscriptII reverse cell number. transcriptase (New England Biolabs). cDNA expression was quantified by SYBR Green–based qPCR techniques using the Light- Microarrays Cycler 480 Instrument (Roche). Relative expression was standard- RNA was purified and sent for microarray analysis at Genome ized to the expression of housekeeping genes: PUM1 and TBP in Quebec and McGill University Genome Centre (Illumina LNCaP and Rplp0 in mouse tissues. Gene-specific primers can be HumanHT-12 Expression Beachip v4). FlexArray software was found in Supplementary Table S1. Two-tailed Student t test was used for microarray data normalization. Ingenuity Pathway Anal- used to determine statistical significance. ysis (IPA), using probes with P < 0.05, and Gene Set Enrichment Analysis (GSEA), using Hallmarks pathways, were used to analyze Protein analyses pathway enrichment. HOMER was used for motifs analysis (25). Cell lysates were harvested with buffer K or separated into Data were processed with Gene Cluster 3.0, using the hierarchical nuclear and cytoplasmic fractions by differential centrifugation as linkage method followed by manual curating on log-transformed described previously (22). For Western blot analyses or ChIP, data, and clusters were visualized using JavaTreeView (26). Micro- primary antibodies used were: AR (Santa Cruz, sc-816X), ERRa array data are available in the Gene Expression Omnibus (GEO; (Abcam, 2131X), ERRg (gift from Dr. Ronald Evans, Salk Institute, GSE86781). La Jolla, CA), lamin B1 (Cell Signaling, 12586), YY1 (Santa Cruz, sc281), and a-tubulin (Cederlane, CLT-9002). Clinical data and statistical analyses For ERRg expression data in clinically localized prostate cancer Animal procedures treated by radical prostatectomy and for association with recur- Animal procedures were done at the McGill Animal Facility on rence, we used data (GEO Series GSE25136) from Sun and 12-hour day/night cycle with water and food ad libitum. Mice were Goodison (27) and provisional The Cancer Genome Atlas either castrated or received a sham surgery and sacrificed 7 days (TCGA) data from February 3, 2016 (TCGA Research Network: after surgery by cervical dislocation. Local ethic committees have http//cancergenome.nih.gov/). For analysis of primary prostate approved all animal procedures. tumors compared with metastatic tumors, microarray data described previously were reanalyzed (GDS2545 and GSE3325; – – ChIP-qPCR refs. 28 30). For Kaplan Meier, log-rank test, and Cox regression After steroid deprivation, cells were treated for 16 hours with analyses, data from Taylor and colleagues (GSE21032; ref. 31) R1881 for AR ChIP-qPCR or 96 hours for ERRa or ERRg ChIP- were analyzed through the Project Betastasis webpage (www. qPCR. ChIPs were performed as described (23) using Dynabeads betastasis.com) and using XLSTAT. (Life Technologies). Enriched DNA was purified using a Qiagen Microarray data from Sun and colleagues (32) were analyzed PCR purification kit and analyzed by qPCR using LightCycler 480. for ERRa and ERRg expression in human LuCaP35 xenografts. Pten Nontargeted rabbit IgG ChIP was used as a control of antibody Expression data form the mouse model with loss of with or K-ras nonspecific binding, and 2 to 3 negative regions were used for without activation were taken from GSE34839 (33). ChIP normalization between samples. Gene-specific primers can be found in Supplementary Table S2. ChIP-seq data for AR in Results LNCaP cells were from publicly available published data, iden- Androgens reprogram prostate cancer cell metabolism tifying 2,473 unique genes with at least one AR-binding site 20 The androgen signaling pathway has been shown to regulate kb of the transcription start site (TSS) of known genes when using several metabolic pathways including both aerobic glycolysis and

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mitochondrial respiration (4, 6, 34). Accordingly, following sus- LAPC4, also induced glucose consumption (Fig. 1F), glycolysis tained treatment of the synthetic androgen R1881, we observed an (Fig. 1G and H), and OCR (Fig. 1I and J) in a similar manner. increase of about 400% in glucose uptake (Fig. 1A), as well as a These data indicate that sustained androgen stimulation pro- significant increase in lactate production (Fig. 1B) in LNCaP- motes global cellular energy metabolism. treated cells compared with controls. The extracellular acidifica- tion rate (ECAR), an indicator of glycolysis, was induced by more Downstream effectors of the AR transcriptional pathway are than 2-fold following androgen treatment (Fig. 1C). These results required for androgen-mediated metabolic reprogramming indicate that androgens are a driving force that increase glucose To study how androgens modulate metabolism, we investi- usage, consistent with the reported regulation of glycolytic genes gated global changes in transcriptional programs following such as hexokinases I and II (HK1 and HK2) by the AR (4). 72–hour treatment with R1881 in LNCaP cells. As expected, Androgens also stimulated mitochondrial respiration as demon- the most significantly enriched gene signature in treated cells strated by the increase in OCR of nearly 200% following 3-day was the "androgen response" (Supplementary Fig. S1B). Several stimulation by R1881 (Fig. 1D and E). Androgens also promoted gene signatures associated with cell metabolism were also lipid accumulation (Supplementary Fig. S1A). R1881 treatment significantly enriched in R1881-treated cells compared with of a second human androgen-dependent prostate cancer cell line, controls. Gene signatures related to glycolysis (Fig. 2A),

ABCED *** 20 300 200 40 0 *** Vehicle 35 R1881

−3 cells)

15 *** 4 30 200 cells) cells) −6 25 6 6 10 100 20

−9 15 100 % Over control 5 % Over control 10 Media metabolite (mmol/L/10 (mmol/L/10 −12 5 OCR P = 0.001 OCR (pMoles/min/10 *** nd ECAR P = 0.004 −15 Media metabolite accumulation 0 0 0 0 Glucose Lactate ECAR OCR 6420 ECAR (mpH/min/104 cells) Vehicle R1881

F G HJI 0 2.0 400 200 *** 40 *** *** 35 cells)

1.5 300 4 30 −0.5 cells) cells) 25 6 6 1.0 200 100 20

15 −1.0 % Over control 0.5 100 % Over control 10 Media metabolite (mmol/L/10 (mmol/L/10 5 OCR P < 0.001 Vehicle OCR (pMoles/min/10 ECAR P < 0.001 R1881

Media metabolite accumulation nd −1.5 *** 0 0 0 0 Glucose Lactate ECAR OCR 9630 ECAR (mpH/min/104 cells) Vehicle R1881

Figure 1. Androgens induce a metabolic reprogramming in prostate cancer cells. Glucose consumption (A) and lactate secretion (B) in extracellular media of LNCaP cells following treatment with R1881. One representative experiment performed in triplicate is shown (n ¼ 4). ECAR (C) and OCR (D) of LNCaP cells treated with R1881. E, Metabolic phenotype of LNCaP cells treated with R1881. One representative experiment of 5 independent experiments is shown. Glucose consumption (F) and lactate secretion (G) in extracellular media of LAPC4 cells following treatment with R1881. One representative experiment performed in triplicate is shown (n ¼ 3). ECAR (H) and OCR (I) of LAPC4 cells treated with R1881. J, Metabolic phenotype of LAPC4 cells treated with R1881. One representative experiment of 3 independent experiments is shown. , P < 0.05; , P < 0.01; , P < 0.001 compared with controls or as indicated. For all experiments, values were normalized for cell number following each experiment.

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A D Glycolysis AR Metabolic R1881Vehicle

NSDHL ChIP-seq pathways PRPS1 0.5 NES:1.28 ENO1 KIF20A (2473 genes) (139 genes) 0.4 SLC25A13 P < 0.001 ME1 GOT2 0.3 ERO1L PDK3 0.2 ABCB6 SLC37A4 0.1 GOT1 PMM2 0.0 PYGB RRAGD

POLR3K -0.1 PGM2

Enrichment score SAP30 -0.2 ALG1 B4GALT1 PLOD1 ALDH9A1

IRS2 SLC35A3 FAM126A

Hits TSTA3 BPNT1 SLC25A10 2,455 18 121 1.0 CAPN5 DPYSL4 PFKP 0.5 B3GAT3 CLN6 MIF 0.0 ISG20 SRD5A3

PPIA -0.5 HOMER1 CHPF MDH2 -1.0 PAXIP1 MP1

Ranked list metrics B3GALT6 0 5,000 10,000 15,000 20,000 25,000 SLC16A3

B E OXPHOS R1881Vehicle GLUD1 ACAA1 NES:1.34 GOT2 0.5 GRPEL1 NUDFV2 ETFA 0.4 IDH2 P < 0.001 TIMM10 MRPL34 0.3 ATP6V1G1 PDHX DLAT –5 ACAA1 0.2 UQCRFS1 ERRE P = 1×10 NDUFA6 VDAC1 0.1 MRPS30 ATP6V1D ATP5C1 0.0 SDHD COX17 DLD -0.1 NDUFS2 SUPV3L1 Enrichment score MRPS22 -0.2 FH MRPS15 NDUFA9 COX7A2L IDH3A PDHB ATP5B SLC25A5 TOMM70A NDUFS8 Hits CYC1 UQCRC2 TIMM9 OXA1L –2 1.0 NQO2 NRF1 P = 1×10 POR MDH2 UQCRC1 HSD17B10 0.5 ETFDH ACADSB ECH1 0.0 NDUFAB1 NDUFB5 SUCLG1 MFN2 MRPL15 -0.5 SLC25A12 TOMM22 RETSTAT SDHB -1.0 ATP6AP1 ECHS1 COX5A Ranked list metrics SLC25A11 0 5,000 10,000 15,000 20,000 25,000 MGST3

C Fatty acid metabolism R1881Vehicle HMCS2 DHCR4 0.7 ODC1 NES:1.56 ELOVL5 0.6 HMGCS1 HPGD FASN 0.5 IDI1 P < 0.001 HIBCH NSDHL 0.4 ACAA1 H2AFZ SETD8 0.3 ME1 HADH MCEE 0.2 ADH1C HSD17B4 0.1 YWHAH ALDH9A1 BMPR1B Enrichment score 0.0 CRYZ SDHD CPT2 -0.1 DLD CBR1 HSPH1 FH PRDX6 ADSL

Hits PDHB REEP6 D2HGDH TP53INP2 1.0 CPOX NTHL1 MIF UGDH 0.5 SLC22A5 MDH2 HSD17B10 0.0 ETFDH GRHPR MLYCD ECH1 -0.5 SUCLG1 CCDC58 RETSAT -1.0 ECHS1 SUCLG2 CA4

Ranked list metrics MDH1 0 5,000 10,000 15,000 20,000 25,000 PDHA1

Figure 2. Sustained androgen treatment induces a metabolic signature associated with the AR and the ERRs. GSEA of LNCaP cells following treatment with R1881 indicates a signiﬁcant enrichment of genes associated with glycolysis in A, oxidative phosphorylation in B, and fatty acid metabolism in C, compared with vehicle. Only genes identiﬁed as "core genes" are shown in heatmaps. D, Overlap between genes with at least one AR-binding sites 20 kb their TSS in ChIP-seq analysis and core metabolic genes induced by R1881 in LNCaP cells. E, DNA motif enrichment analysis in the promoter of the 121 metabolic genes not bound by AR.

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oxidative phosphorylation (Fig. 2B), and fatty acid metabolism of the 139 metabolic core genes had an identifiable AR peak, (Fig. 2C) were all increased following sustained androgen indicating that the vast majority of these genes are indirectly stimulation. When considering genes with at least one AR peak regulated by R1881 treatment. We thus hypothesized that other 20 kb of their TSS, of the 139 genes included in the core transcription factors may be required downstream of the AR to enrichment of these 3 androgen-modulated metabolic path- sustain the metabolic program induced by androgens. There- ways, only 18 and 11 (13% and 8%) were direct AR targets fore, we performed DNA motif search in the promoter region according to previously published ChIP-seq data generated of the 121 metabolic core genes not directly bound by AR from LNCaP cells and CRPC tumors (Fig. 2D and Supplemen- in LNCaP cells (Fig. 2E). Only 2 known DNA motifs were tary Fig. S1C; refs. 4, 5). Even when considering peaks over 1 significantly enriched in these promoters: the ERR response 106 bp away from TSS in LNCaP cells AR ChIP-seq data, only 36 element (ERRE; P ¼ 1 10 5) and the nuclear respiratory factor A B C 1.0 R1881 1.5 Castration 1.0 Castration NS ** 0.5 * Figure 3. * Androgens regulate ERRg expression in 0 1.0 vivo and in vitro in healthy and tumor 0.5 tissues. A, Relative mRNA expression of − ERRa and ERRg in LNCaP cells 0.5 0.5 following androgen treatment. Vehicle R1881 − + ERRα conditions were set at 0. Results are −1.0 shown as the average SEM of three ERRγ NS 0 independent experiments performed at Lamin B1 0 − least in duplicate. Inset, protein 1.5 expression of ERRa and ERRg following Relative expression (log2) *** Relative expression (log2) treatment with androgens in LNCaP −2.0 −0.5 Esrrg Relative expression (log2) −0.5 Ventral Dorso. cells. Lamin B1 is shown as a loading ESRRA ESRRG ESRRA ESRRG control. B, ERRg (ESRRG) expression is induced in vivo by castration in human D 100 kb ESRRG prostate cancer xenografts of LuCap35. Data are from publicly available ESRRG microarrays published by Sun and ESRRG colleagues (32). C, ERRg (Esrrg) RefSeq ESRRG expression is induced by castration in AR binding sites vivo in mouse ventral and dorsolateral 1 2 3 prostate lobes (n 5 mice per group). D, ChIP-qPCR analysis of AR binding R1881− + R1881− + R1881− + following treatment with R1881 at three LNCaP ** LNCaP * LNCaP * genomic regions in ESRRG in LNCaP, 22rv1 22rv1 22rv1 22rv1, and LAPC4 cells (n ¼ 3). Three ** * * transcriptional variants of ESRRG of LAPC4 * LAPC4 * LAPC4 different lengths are shown. E, Relative <2 2−5 5−10 10−15 >15 mRNA expression of ERRg with or without RNAi against the AR in LNCaP cells treated with R1881 (left). Control AR Enrichment (Fold) cells treated with vehicle were set at 0. Results are shown as the average E F SEM of three independent experiments *** performed in triplicate. Protein levels of 0.5 R1881 1.0 *** AR and ERRg with or without RNAi interference against the AR in LNCaP *** 0.5 *** cells treated with R1881 (right). Tubulin 0 and lamin B1 are shown as loading 0 controls. F, ERRg relative mRNA siC siAR repression in LNCaP cells following −0.5 treatment with R1881, with or without R1881 − + − + 0.5 treatment with the antiandrogen AR 1.0 MDV3100. Results are shown as the −1.0 Vehicle average SEM of three independent Tubulin experiments performed in triplicate. 1.5 R1881 − , P < 0.05; , P < 0.01; , P < 0.001 1.5 ERRγ compared with controls or as 2.0 indicated. NS, nonsignificant. Lamin B1 −2.0 *** 2.5 *** ESRRG Relative expression (log2) ESRRG Relative expression (log2) ** Control MDV siC siAR

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A D SDHB FH ACADM LNCaP 0.6 * 1.0 1.0

ERRγ *** 0.8 0.8 ** R1881 -+ 0.4 * * 0.6 * 0.6 ACO2 P = 0.06 ATP5B ** 0.2 0.4 0.4 ATP5G3 ** CS * 0.2 0.2 SDHD * 0.0 ACADM * 0 0 ELAC1 * Relative expression (log2) RNF113B *** −0.2 −0.2 −0.2 Mir145 * IDH3A * siC siERRγ siC + R1881 IDH1 P = 0.06 SDHB * KEGG - Citrate cycle (TCA cycle) FH E siC siERRγ * 0.7 IDH3B P = 0.06 SDHD 0.6 NES:1.68 MDH1 0.5 SUCLG1 P < 0.001 IDH3A <2 2–5 5–10 10–15 >15 0.4 0.3 DLAT DLD 0.2 FH 0.1 PDHB

ERRγ Enrichment (fold) Enrichment score 0.0 SDHB LOC642502 MDH2 B Hits IDH2 γ SUCLG2 siC siERR 0.1 LOC283398 R1881 -+-+ 0.0 DLST -0.1 ACO2 -0.2 PDHA1 -0.3 ERRγ OGDHL -0.4 ACO1 Ranked list metrics 0 5,000 10,000 15,000 20,000 25,000 Lamin B1 F Cholesterol synthesis Acetyl-CoA synthesis Valine degradation C Glycine biosynthesis IPA Pathways enriched with siERRγ TCA Cycle Glycolysis EIF2 01 234 EIF4 and p70S6K AMPK Signaling OXPHOS Cardiac hypertrophy CXCR4 Signaling mTOR Signalling Estrogen signaling Valine degradation Thrombin Signaling Fatty acid β-oxidation RAR Activation Folate transformations 01234 AMPK Signalling siC siC <−0.08 >0.08 -log(P Value) 012345 6 7 siERRγ siERRγ Clustering weight -log (P Value) Control R1881

Figure 4. ERRg transcriptional control of metabolic gene programs. A, ChIP-qPCR of ERRg with or without androgens (n ¼ 3). B, ERRg repression following siRNA transfection at the protein level. Microarray analyses were performed from LNCaP cells first transfected with siERRg or siC and then treated for 72 hours with R1881 or vehicle. C, Most significantly enriched pathways (IPA) in LNCaP cells following ERRg inhibition by RNAi. A value over 1.3 indicates significance with P < 0.05. D, Relative mRNA expression of SDHB and FH in LNCaP cells transfected with siC or siERRg and then treated with R1881 or vehicle. , P < 0.05; , P < 0.01; , P < 0.001 compared with control. Results are shown as the average SEM of three independent experiments performed in triplicate. E, GSEA identifies the TCA cycle as upregulated in cells with ERRg inhibition under androgen deprivation. F, Cluster analysis of genes modulated by ERRg knockdown and with a similar modulation by R1881 treatment (left) with their associated pathways using IPA (right).

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A 120 150 * 150 125 150 ** 125 * siC siERRγ ** 100 100 Control 80 100 100 100 75 ** 75 ERRγ *** 50 50 OCR (%) ECAR (%) 40 50 50 50 OCR/ECAR (%) 25 25

0 0 0 0 0 0

B C Oligo FCCP Rotenone siERRγ 0.50 300 * * γ Control ERR γ 0.25 250 ERR R1881 200 ERRγ + R1881 0 150 -0.25 100 Relative OCR -0.50 50

Metaoblite level (log2) -0.75 * *** 0 *** 010 20304050607080 * ** ** e e e Time (min) Citrate Malate Control ERRγ Succinat Fumarate E Cis-aconitat 250 α-ketoglutarat ** D 200 250 * 150 200 * * 100 150 OCR (%)

100 Control 50 R1881 Relative OCR 50 ERRγ ERRγ + R1881 0 0 0210 030 Vehicle R1881 Vehicle R1881 Relative ECAR siC siERRγ F G H 300 250 200 * * ** 200 * 150 200 ** 150 ** * * 100 100 100 50 Rel. cell number (%) Rel. cell number (%) 50 Rel. cell number (%)

0 0 0 Vehicle R1881 Vehicle R1881 Vehicle R1881 siC siERRγ shNTC shERRγ-1 shERRγ-2 Control ERRγ

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1 DNA-binding motif (NRF1; P ¼ 1 10 2). Similar results could function as an effector of the AR in the reprogramming of were also obtained using AR ChIP-seq data generated from cellular metabolism in prostate cancer. We first performed stan- CRPC tumors (Supplementary Fig. S1D). Both motifs are dard ChIP-qPCR experiments to validate that ERRg, as previously associated with transcription factors known to regulate nucle- observed in other cell types (17, 35–37), targets known ERR- ar-encoded mitochondrial genes. modulated metabolic genes in prostate cancer cells. As shown in Fig. 4A and Supplementary Fig. S3A, ERRg is strongly bound to ESRRG is a direct AR target the promoter of several metabolic genes in the absence of andro- As both ERRa and g isoforms recognize the same ERRE motif, gen. Notably, several of these genes such as ATP5B as well as FH, we first determined whether androgens could regulate expres- IDH3A, SDHB, and SDHD were previously identified in the sion of one or both ERR isoforms. ERRa expression was not metabolic signatures associated with sustained R1881 treatment affected by androgens, even after a 3-day treatment (Fig. 3A and (Fig. 2). ERRg binding to these metabolic target genes was sig- Supplementary Fig. S2A and S2B). In contrast, ERRg expression nificantly decreased upon R1881 exposure, which is consistent was significantly downregulated following exposure to R1881 with a reduction in ERRg protein levels (Figs. 3A and 4A). We at both the mRNA and protein levels (Fig. 3A). This decrease observed between 2- and 4-fold decreases in ERRg binding fol- was stable over time, lasting at least 4 days (Supplementary Fig. lowing androgen treatment on all targets (Fig. 4A and Supple- S2C). Conversely, decreased androgen availability through mentary Fig. S3A). We next tested whether an increase in ERRa serum starvation led to an induction of ERRg expression in binding, which recognizes the same response element present in LNCaP cells (Supplementary Fig. S2D). Androgen deprivation these genes, could compensate for the decreased ERRg binding to in vivo asaresultofcastrationalsoledtoasignificant increase of these regions. However, ERRa binding was unchanged following ERRg in human prostate cancer xenografts of LuCaP35 (32), R1881 (Supplementary Fig. S3B). whereas ERRa expression again remained unchanged (Fig. 3B). To understand the functional consequence of ERRg repression ERRg was also induced by castration in the mouse ventral and on cell metabolism, we conducted microarray experiments in dorsolateral prostate lobes (Fig. 3C) while being less responsive LNCaP cells following siRNA-mediated ERRg repression, with or in the anterior lobe (Supplementary Fig. S2E). We did not without R1881 treatment (Fig. 4B and Supplementary Fig. S3C). detect significant change in Esrra expression in various mouse In the absence of androgens, ERRg knockdown significantly prostate lobes (Supplementary Fig. S2E and S2F). Taken togeth- altered the expression of 3,726 genes. IPA revealed that genes er, these data indicate that androgens regulate the expression of enriched in our microarray analysis following ERRg knockdown ERRg in vitro and in vivo, in both the normal prostate and in the absence of androgens displayed enrichment for metabolic prostate cancer cells. pathways such as fatty acid oxidation (FAO) and oxidative phos- Our re-analysis of AR ChIP-seq data identified 3 AR-binding phorylation (OXPHOS), but also folate metabolism, a biochem- sites within the ESRRG locus (Fig. 3D). R1881 treatment led to a ical pathway recently identified as being regulated by ERRa (Fig. significant recruitment of AR at all 3 binding sites in LNCaP cells as 4C; ref. 38). GSEA also revealed that metabolic pathways were measured by ChIP-qPCR (Fig. 3D and Supplementary Fig. S2G). strongly affected. Notably, the TCA cycle pathway was enriched This observation was further validated in 2 other AR-positive following ERRg knockdown, with the expression of genes such as prostate cancer cell lines, 22rv1 and LAPC4 (Fig. 3D and Supple- FH and SDHB being increased significantly (Fig. 4D and E and mentary Fig. S2H and S2I). ESRRG is also a strong AR direct target Supplementary Fig. S3D). Importantly, the effect on transcrip- in CRPC tumors (5). AR knockdown using siRNAs significantly tional regulation of these genes by inhibition of ERRg was similar impaired ERRg repression by androgens at both the mRNA and to that of treatment with R1881. Genes associated with glycolysis protein levels in LNCaP cells (Fig. 3E and Supplementary Fig. S2J). were also enriched in cells with ERRg knockdown (Supplementary Treatment of LNCaP cells with the AR antagonist enzalutamide Fig. S3E). (MDV3100) led to a significant increase in ERRg expression and To further understand which ERRg targets were associated with completely abolished the androgen-mediated repression of ERRg the androgen response, we intersected mRNA targets that were (Fig. 3F). altered by either repression of ERRg or androgen treatment; the latter also exhibiting decreased ERRg levels and activity. By clus- ERRg regulates the expression of metabolic genes in prostate tering the probes correlating following R1881 treatment or ERRg cancer cells knockdown, we identified 886 repressed and 526 activated genes We next examined the possibility that modulation of the (Fig. 4F). Interestingly, genes induced following ERRg knockdown expression of ERR g, and consequently of its downstream targets, or androgen treatment were associated with cell metabolism and

Figure 5. Loss of ERRg promotes oxidative metabolism and proliferation in prostate cancer cells. A, Following ERRg knockdown by siRNA or its overexpression, ECAR, OCR, and the OCR/ECAR ratio were measured in LNCaP cells under steroid deprivation. B, Levels of TCA cycle intermediates following ERRg knockdown or overexpression. Log-transformed data are shown normalized to their respective controls. C, Oxidative capacity and uncoupling respiration of LNCaP cells stably overexpressing ERRg following 72-hour treatment with R1881. D, Metabolic organization of control cells or cells overexpressing ERRg following 72-hour treatment with R1881. E, OCR of LNCaP cells with or without siERRg in absence or presence of ERRg overexpression following a 72-hour treatment with R1881 or vehicle. F, Relative cell number of LNCaP cells transfected with siC or siERRg following a 72-hour treatment with R1881. G, Relative cell number of LNCaP cells stably expressing shRNA against ERRg or control (shNTC) following a 72-hour treatment with R1881. H, Relative cell number of LNCaP cells overexpressing ERRg or controls following 72-hour treatment with R1881. , P < 0.05; , P < 0.01; , P < 0.001. For metabolic analyses, values were normalized for cell number following each experiment. C and D are shown as a representative experiment performed with 5 samples per group; E is shown as the average SEM of two independent experiments (n 5 samples per group); for other panels, results are shown as the average SEM of at least three independent experiments performed at least in triplicate.

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Role of ERRg in Prostate Cancer Cell Metabolism

included pathways related to cholesterol and acetyl-CoA synthe- was rescued (Fig. 5E and Supplementary Fig. S4H). These results sis, TCA cycle, and glycolysis (Fig. 4F). Accordingly, both ERRg nicely parallel the mRNA levels of ERRg target genes, ACADM and knockdown and R1881 treatment affected TCA cycle genes such as SDHB, whose expression is also rescued in this context (Supple- FH and SDHB by inducing their expression (Fig. 4D). Taken mentary Fig. S4I and S4J). together, these results suggest that androgens modulate metabolic We next investigated the effect of ERRg knockdown on LNCaP genes, in part, through direct repression of ERRg. cell proliferation. Knockdown of endogenous ERRg with siRNA or shRNA significantly increased cell proliferation, both in absence Loss of ERRg promotes oxidative metabolism in prostate cancer and presence of androgen (Fig. 5F and G). As both androgens and cells RNAi repressed ESRRG, we hypothesized that the additive effect To gain more insight into the function of ERRg on cell metab- on cell proliferation could be due to a more rapid metabolic olism, we next focused on its metabolic role under steroid reprogramming of prostate cancer cells. Indeed, ESRRG expres- deprivation, when it is expressed at high levels. Expression data sion was reduced more rapidly when combining siERRg and a 48- in prostate cancer cells showed increased expression of genes hour treatment with R1881 (Supplementary Fig. S5A). Consistent related to mitochondrial activity and TCA cycle following ERRg with these results, differences in mRNA expression levels of the knockdown (Fig. 4), suggesting that, in the context of prostate ERRg target gene ACADM and of OCR were more pronounced at cancer cells, ERRg represses mitochondrial activity. To validate 48 hours between siC and siERRg following androgen stimulation this hypothesis, both ECAR and OCR were measured in LNCaP (Supplementary Fig. S5A and S5B). On the other hand, over- cells lacking ERRg. Depletion of ERRg significantly decreased expression of ERRg significantly decreased cell number in LNCaP ECAR and increased OCR, resulting in an increase in the OCR/ (Fig. 5H) and PC3 cells (Supplementary Fig. S5C). Therefore, ECAR ratio (Fig. 5A). Knockdown of ERRg using siRNA also ERRg expression is inversely associated with cell proliferation. increased OCR in LAPC4 cells (Supplementary Fig. S4A). To further validate our results, we performed a stable infection ERRg expression decreases with prostate cancer progression of LNCaP cells with ERRg overexpression or control vector (Sup- We next assessed the relationship between ERRg mRNA plementary Fig. S4B and S4C). ERRg-overexpressing cells exhib- expression levels and prostate cancer aggressiveness by analyz- ited the opposite metabolic profile compared with cells exposed ing several publicly available expression datasets. First, we to siERRg, with a significant increase in ECAR paired with a reanalyzed data from a mouse model of prostate cancer with significant decrease in OCR and the OCR/ECAR ratio (Fig. 5A). loss of Pten,inwhichK-ras was activated to promote a more Consistent with these results, levels of TCA cycle intermediates aggressive phenotype (33). In this context, ERRg expression was such as citrate and a-ketoglutarate were significantly elevated significantly reduced in the more aggressive condition (Pten null upon ERRg knockdown whereas they were significantly reduced with K-ras activation; Fig. 6A). upon ERRg overexpression (Fig. 5B). These results indicate In human prostate cancer samples, ERRg expression was sig- that ERRg is a repressor of oxidative metabolism in prostate nificantly lower in metastasis than in primary tumors in 2 inde- cancer cells. pendent clinical datasets (Fig. 6B and C). Moreover, we found that To further understand the interplay between ERRg, androgens recurrent prostate cancer following radical prostatectomy had and prostate cancer metabolism, we investigated the impact of lower ERRg expression than nonrecurrent diseases (Fig. 6D). ERRg modulation following androgen stimulation. Treatment of Importantly, this was also validated using a large dataset from LNCaP cells with 72-hour treatment with R1881 concomitant the TCGA (n ¼ 429 patients with data on ERRg expression). Again, with ERRg knockdown did not further increase OCR when com- recurrent diseases were characterized by significantly lower levels pared with control cells with androgen (Supplementary Fig. S4D), of ERRg (Fig. 6E). In a third independent cohort, we further consistent with a strong repression of ERRg by R1881 (Figs. 3 validated that low expression of ERRg was significantly associated and 4). Repression of ERRg with 2 distinct shRNAs gave similar with a higher recurrence rate following radical prostatectomy (P ¼ results and significantly increased OCR in LNCaP cells in absence 3 10 5; Fig. 6F), whereas no association was observed for ERRa of androgens, mimicking R1881 treatment effect (Supplementary (Supplementary Fig. S6). In the data from Taylor and colleagues Fig. S4E and S4F). In cells stably transfected with the control vector (31), when considering several clinical parameters such as Glea- of our overexpression system, R1881 repressed ESRRG expression son score and age at diagnosis, low ERRg expression levels were as it did in parental cells (Supplementary Fig. S4B and S4C). not significantly associated with BCR, even though a trend for Importantly, ERRg rescue (overexpression; Supplementary Fig. increased risk was observed, with a risk ratio of 1.70 (Supple- S4C) significantly decreased the respiratory capacity of LNCaP mentary Table S3). Using backward stepwise Cox regression to cells compared to control cells, in both the absence and presence determine the best predictive model of recurrence, low ERRg of R1881 (Fig. 5C). ECAR, strongly induced by R1881, was also expression levels was retained and was significantly associated impaired by ERRg overexpression, but at a lesser extent than OCR, with an increased risk, with an HR of 2.5 (P ¼ 0.033), together indicating a dominant role of AR in modulating aerobic glycolysis with pathological Gleason score and tumor stage (Fig. 6G and capacity of prostate cancer cells (Fig. 5D). Overall, overexpression Supplementary Table S3). of ERRg impaired the ability of R1881 to induce global metabolism (Fig. 5D). Finally, we tested the biologic consequence of ERRg modulation in the CRPC cell line PC3. As observed in Discussion androgen-dependent cell lines, ERRg acted as a repressor of In this study, we have shown that, while androgens strongly mitochondrial respiration as its repression significantly increased influence the metabolic state of prostate cancer cells, differential OCR (Supplementary Fig. S4G). As an additional control, we regulation of the AR downstream effector ERRg is required to show that by lowering the levels of ERRg in prostate cancer cells achieve this phenotype. Indeed, intersection of gene expression, exogenously expressing ERRg the respiratory capacity of these cells binding events, and motif finding analyses following androgen

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A B C D E 10 100 1,200 300 8

8 * Figure 6. ERRg expression is a biomarker of 80 800 200 ** prostate cancer progression. A, Esrrg 6 * mRNA expression is lower in more * 7 aggressive prostate cancer mouse Expression Expression Expression models. Analysis of prostate tumors Expression

4 Expression (log2) from mouse lines with prostate-speciﬁc 60 400 100 Pten loss with or without K-ras activation (33). B, Relative expression Esrrg 2 *** ¼ ESRRG ESRRG ESRRG levels of ERRg (ESRRG) in primary (n 65) and metastatic (n ¼ 25) tumor ESRRG samples (28, 30). C, Relative expression 6 0 40 0 0 levels of ERRg in clinically localized / primary prostate tumors (n ¼ 7) and null metastatic tissue pools (n ¼ 6; ref. 29). Primary Primary Pten null K-ras D, Relative expression levels of ERRg in Recurrent Recurrent Pten Metastasis nonrecurrent (n ¼ 40) and recurrent (n

Non-recurrent Non-recurrent ¼ 39) prostate cancer tumor samples in Metastatic CRPC GEO proﬁle GSE25136 (27). E, Relative F G expression levels of ERRg in nonrecurrent and recurrent prostate 100 High ESRRG 30 *** cancer tumor samples in the provisional Low ESRRG TCGA prostate cancer cohort (n ¼ 429; 59 patients had recurrence). F, Kaplan– 80 Meier curves of patients with prostate 10 cancer following radical prostatectomy (n ¼ 141) from Taylor and colleagues 60 ** 8 * n.s. (31). ERRg expression is deﬁned as low

HR (lowest 25%) or high (highest 75%). The 6 log-rank test P value is shown. G, Cox 40 regression following backward 4 selection model of risk variables 20 modulating biochemical recurrence in Recurrence free (%) 2 data from Taylor and colleagues (31). Low ESRRG expression levels is the P = 0.00003 0 0 lowest 25% (highest 75% with an HR ﬁ 0 20 40 60 80 100 120 140 of 1.0). n.s., nonsigni cant. Time (Months) ESRRG

Stage T3A Gleason >7 Low Stage ≥ T3B

exposure identified a metabolic gene signature associated with the The AR acts as a strong positive modulator of mitochondrial action of a member of the ERR subfamily of nuclear receptors. We metabolism (Figs. 1 and 2; ref. 6). Accordingly, induction of further showed that, by directly and specifically suppressing the mitochondrial activity as reported herein is in agreement with expression of the ERRg isoform, the androgen signaling pathway prior findings that androgen-mediated metabolic reprogramming favors a metabolic state that is associated with the growth of is associated with prostate cancer progression (6, 42, 43). prostate cancer cells. Unexpectedly, we showed that ERRg acts as a It has been well demonstrated that ERRa and g function as repressor of mitochondrial respiration in that cellular context. The integral regulators of cellular energy metabolism (8, 15, 44). ERRg-dependent metabolic state was observed in both androgen- Indeed, the ERRs bind to and regulate most nuclear-encoded dependent and CRPC cells, and ERRg expression was found to be mitochondrial genes, as well as genes associated with other met- inversely associated with disease aggressiveness in several inde- abolic pathways such as glycolysis and lipid synthesis (10, 11, 22). pendent clinical datasets. Taken together, our results clearly Despite strong overlapping in their global DNA-binding site dis- identify ERRg both as a biomarker of disease progression and as tribution and functions (35), it is clear that each ERR isoform also a potential novel therapeutic target that, if reactivated, could plays independent roles in development, physiology, and disease reverse an oncogenic metabolic program enabling prostate cancer (15, 17, 36, 45–47). In addition, much remains to be learned on progression. how the expression of each receptor isoform is regulated in both Restoration of mitochondrial respiration is a key step of pros- normal and cancer cells. Here, we describe a regulatory mechanism tate carcinogenesis that contributes to increased ATP production that specifically governs ERRg activity. Activation of the androgen (3, 39). Augmentation of mitochondrial activity is also linked to signaling pathway significantly repressed ERRg expression in an several biosynthetic pathways that can favor tumor cell prolifer- AR-dependent manner in both normal and prostate cancer cells ation such as one-carbon metabolism for DNA synthesis (40, 41). without affecting ERRa expression. As ERRg represses these genes,

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Role of ERRg in Prostate Cancer Cell Metabolism

inhibition of ERRg activity would release these genes from this of ERRg would likely contribute substantial clinical benefits in negative regulation signal and allow them to be re-expressed. In the treatment of advanced prostate cancer. addition, loss of ERRg in this context may promote enrichment of ERRa homodimers at metabolic genes instead of ERRa/ERRg Disclosure of Potential Conflicts of Interest heterodimers, which are thought to be less active (35, 48). Such No potential conflicts of interest were disclosed. a change in ERRa occupancy would not be easily discernable using standard ChIP-qPCR. Nonetheless, modulation of ERRg Authors' Contributions expression by androgens clearly results in the alteration of a Conception and design: E. Audet-Walsh, J. St-Pierre, V. Giguere gene program highly associated with oxidative energy metab- Development of methodology: E. Audet-Walsh, T. Yee, M. Vernier olism, whereas AR had a dominant effect on aerobic glycolysis. Acquisition of data (provided animals, acquired and managed patients, Functional studies identified ERRg as a repressor of mitochon- provided facilities, etc.): E. Audet-Walsh, T. Yee, S. McGuirk, C. Ouellet drial respiration in prostate cancer cells. In contrast, ERRg has Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): E. Audet-Walsh, T. Yee, S. McGuirk, M. Vernier, been shown to act as an activator of mitochondrial activity in J. St-Pierre, V. Giguere muscles as well as in breast cancer cells (17, 47). The activity of Writing, review, and/or revision of the manuscript: E. Audet-Walsh, ERRg is likely dependent on functional interactions with cell- S. McGuirk, M. Vernier, J. St-Pierre, V. Giguere specific transcription factors and co-regulators. Development of Administrative, technical, or material support (i.e., reporting or organizing selective ERRg modulators could potentially be envisaged to data, constructing databases): E. Audet-Walsh, C. Ouellet induce a transition from a repressor to an activator state of the Study supervision: J. St-Pierre receptor in prostate cancer cells. In conclusion, we uncovered a mechanism by which AR- Acknowledgments ¸ dependent repression of ERRg contributes to the establishment We thank Dr. Michel Tremblay and Jean-Francois Theberge for PC3 cells. The authors also thank Christine Flageole, Ingrid Tam, and Catherine Rosa Dufour of an oncogenic metabolic phenotype in prostate cancer cells for fruitful discussions and Ryan Butler for technical assistance. that favors mitochondrial activity and cell proliferation. Although the expression of ERRg inthenormalprostateis Grant Support likely to be regulated according to developmental and physi- This work was supported by a Terry Fox Research Institute Program Project ologic needs, ERRg is expected to be constantly repressed under Team grant on Oncometabolism (116128; V. Giguere and J. St.-Pierre), the New constitutive AR signaling in prostate cancer cells. The biologic Innovation FundCFI 21875 (V. Giguere.) and the Canadian Institutes of Health fi fi and pathologic signi cance of these ndings is corroborated Research (CIHR; MOP-111144; V. Giguere). E. Audet-Walsh is recipient of a with the observation that lower expression of ERRg is associ- postdoctoral fellowship from CIHR, the Fonds de Recherche du Quebec–Sante ated with increased prostate cancer aggressiveness in a number (FRQS) and is supported by a McGill Integrated Cancer Research Training of preclinical and clinical studies (Fig. 6). Thus, ERRg expres- Program (MICRTP) scholarship. sion status emerges as a novel biomarker of prostate cancer 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 progression, whereas its reactivation represents a potential in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. therapeutic avenue for the management of prostate cancer. As ERRg expression is induced by antiandrogens such as enzalu- Received May 13, 2016; revised September 12, 2016; accepted October 16, tamide (Fig. 3F), AR silencing in combination with an activator 2016; published OnlineFirst November 7, 2016.

References 1. BollaM,deReijkeTM,VanTienhovenG,VandenBerghAC,OddensJ, 10. Charest-Marcotte A, Dufour CR, Wilson BJ, Tremblay AM, Eichner LJ, Arlow Poortmans PM, et al. Duration of androgen suppression in the treat- DH, et al. The homeobox protein Prox1 is a negative modulator of ERRa/ ment of prostate cancer. N Engl J Med 2009;360:2516–27. PGC-1a bioenergetic functions. Genes Dev 2010;24:537–42. 2. Watson PA, Arora VK, Sawyers CL. Emerging mechanisms of resistance to 11. Eichner LJ, Giguere V. Estrogen-related receptors (ERRs): a new dawn in the androgen receptor inhibitors in prostate cancer. Nat Rev Cancer 2015;15: control of mitochondrial gene networks. Mitochondrion 2011;11:544–52. 701–11. 12. Mootha VK, Handschin C, Arlow D, Xie X, St Pierre J, Sihag S, et al. ERRa 3. Costello LC, Franklin RB, Feng P. Mitochondrial function, zinc, and and GABPAa/b specify PGC-1a-dependent oxidative phosphorylation intermediary metabolism relationships in normal prostate and prostate gene expression that is altered in diabetic muscle. Proc Natl Acad Sci cancer. Mitochondrion 2005;5:143–53. U S A 2004;101:6570–75. 4. Massie CE, Lynch A, Ramos-Montoya A, Boren J, Stark R, Fazli L, et al. The 13. Deblois G, St-Pierre J, Giguere V. The PGC-1/ERR signaling axis in cancer. androgen receptor fuels prostate cancer by regulating central metabolism Oncogene 2013;32:3483–90. and biosynthesis. EMBO J 2011;30:2719–33. 14. Tam IS, Giguere V. There and back again: the journey of the estrogen- 5. Sharma NL, Massie CE, Ramos-Montoya A, Zecchini V, Scott HE, Lamb AD, related receptors in the cancer realm. J Steroid Biochem Mol Biol 2016; et al. The androgen receptor induces a distinct transcriptional program in 157:13–9. castration-resistant prostate cancer in man. Cancer Cell 2013;23:35–47. 15. Deblois G, Giguere V. Oestrogen-related receptors in breast cancer: control 6. Tennakoon JB, Shi Y, Han JJ, Tsouko E, White MA, Burns AR, et al. of cellular metabolism and beyond. Nat Rev Cancer 2013;13:27–36. Androgens regulate prostate cancer cell growth via an AMPK-PGC-1a- 16. Chang CY, Kazmin D, Jasper JS, Kunder R, Zuercher WJ, McDonnell DP. mediated metabolic switch. Oncogene 2014;33:5251–61. The metabolic regulator ERRa, a downstream target of HER2/IGF-1R, as a 7. Giguere V, Yang N, Segui P, Evans RM. Identiﬁcation of a new class of therapeutic target in breast cancer. Cancer Cell 2011;20:500–10. steroid hormone receptors. Nature 1988;331:91–94. 17. Eichner LJ, Perry M-C, Dufour CR, Bertos N, Park M, St-Pierre J, et al. mir- 8. Giguere V. Transcriptional control of energy homeostasis by the estrogen- 378 mediates metabolic shift in breast cancer cells via the PGC-1b/ERRg related receptors. Endocr Rev 2008;29:677–96. transcriptional pathway. Cell Metab 2010;12:352–61. 9. Audet-Walsh E, Giguere V. The multiple universes of estrogen-related 18. Ariazi EA, Clark GM, Mertz JE. Estrogen-related receptor a and estrogen- receptor alpha and gamma in metabolic control and related diseases. Acta related receptor g associate with unfavorable and favorable biomarkers, Pharmacol Sin 2015;36:51–61. respectively, in human breast cancer. Cancer Res 2002;62:6510–18.

www.aacrjournals.org Cancer Res; 77(2) January 15, 2017 OF11

Audet-Walsh et al.

19. Suzuki T, Miki Y, Moriya T, Shimada N, Ishida T, Hirakawa H, et al. metastasis initiated from prostate cancer stem/progenitor cells. Cancer Estrogen-related receptor a in human breast carcinoma as a potent prog- Res 2012;72:1878–89. nostic factor. Cancer Res 2004;64:4670–76. 34. Shi Y, Han JJ, Tennakoon JB, Mehta FF, Merchant FA, Burns AR, et al. 20. Fujimura T, Takahashi S, Urano T, Kumagai J, Ogushi T, Horie-Inoue K, Androgens promote prostate cancer cell growth through induction of et al. Increased expression of estrogen-related receptor a (ERRa)isa autophagy. Mol Endocrinol 2013;27:280–95. negative prognostic predictor in human prostate cancer. Int J Cancer 2007; 35. Dufour CR, Wilson BJ, Huss JM, Kelly DP, Alaynick WA, Downes M, et al. 120:2325–30. Genome-wide orchestration of cardiac functions by orphan nuclear recep- 21. Fujimura T, Takahashi S, Urano T, Ijichi N, Ikeda K, Kumagai J, et al. tors ERRa and g. Cell Metab 2007;5:345–56. Differential expression of estrogen-related receptors b and g (ERRb and 36. Alaynick WA, Kondo RP, Xie W, He W, Dufour CR, Downes M, et al. ERRg ERRg) and their clinical significance in human prostate cancer. Cancer Sci directs and maintains the transition to oxidative metabolism in the post- 2010;101:646–51. natal heart. Cell Metab 2007;6:16–24. 22. Chaveroux C, Eichner LJ, Dufour CR, Shatnawi A, Khoutorsky A, Bourque 37. Alaynick WA, Way JM, Wilson SA, Benson WG, Pei L, Downes M, et al. ERRg G, et al. Molecular and genetic crosstalks between mTOR and ERRa are key regulates cardiac, gastric, and renal potassium homeostasis. Mol Endocri- determinants of rapamycin-induced non-alcoholic fatty liver. Cell Metab nol 2010;24:299–309. 2013;17:586–98. 38. Audet-Walsh E, Papadopoli DJ, Gravel SP, Yee T, Bridon G, Caron M, 23. Langlais D, Couture C, Sylvain-Drolet G, Drouin J. A pituitary-specific et al. The PGC-1a/ERRa axis represses one-carbon metabolism and enhancer of the POMC gene with preferential activity in corticotrope cells. promotes sensitivity to anti-folate therapy in breast cancer. Cell Rep Mol Endocrinol 2011;25:348–59. 2016;14:920–31. 24. McGuirk S, Gravel S-P, Deblois G, Papadopoli D, Faubert B, Wegner A, et al. 39. Wu X, Daniels G, Lee P, Monaco ME. Lipid metabolism in prostate cancer. PGC-1a supports glutamine metabolism in breast cancer cells. Cancer Am J Clin Exp Urol 2014;2:111–20. Metab 2013;1:22. 40. Vazquez A, Tedeschi PM, Bertino JR. Overexpression of the mitochondrial 25. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, et al. Simple folate and glycine-serine pathway: a new determinant of methotrexate combinations of lineage-determining transcription factors prime cis-reg- selectivity in tumors. Cancer Res 2013;73:478–82. ulatory elements required for macrophage and B cell identities. Mol Cell 41. Zong WX, Rabinowitz JD, White E. Mitochondria and Cancer. Mol Cell 2010;38:576–89. 2016;61:667–76. 26. Saldanha AJ. Java Treeview–extensible visualization of microarray data. 42. Grupp K, Jedrzejewska K, Tsourlakis MC, Koop C, Wilczak W, Adam M, Bioinformatics 2004;20:3246–8. et al. High mitochondria content is associated with prostate cancer disease 27. Sun Y, Goodison S. Optimizing molecular signatures for predicting pros- progression. Mol Cancer 2013;12:145. tate cancer recurrence. Prostate 2009;69:1119–27. 43. Giannoni E, Taddei ML, Morandi A, Comito G, Calvani M, Bianchini F, 28. Chandran UR, Ma C, Dhir R, Bisceglia M, Lyons-Weiler M, Liang W, et al. Targeting stromal-induced pyruvate kinase M2 nuclear translocation et al. Gene expression profiles of prostate cancer reveal involvement of impairs oxphos and prostate cancer metastatic spread. Oncotarget 2015;6: multiple molecular pathways in the metastatic process. BMC Cancer 24061–74. 2007;7:64. 44. Villena JA, Kralli A. ERRa: a metabolic function for the oldest orphan. 29. Varambally S, Yu J, Laxman B, Rhodes DR, Mehra R, Tomlins SA, et al. Trends Endocrinol Metab 2008;19:269–76. Integrative genomic and proteomic analysis of prostate cancer reveals 45. Luo J, Sladek R, Carrier J, Bader J-A, Richard D, Giguere V. Reduced fat mass signatures of metastatic progression. Cancer Cell 2005;8:393–406. in mice lacking orphan nuclear receptor estrogen-related receptor a. Mol 30. Yu YP, Landsittel D, Jing L, Nelson J, Ren B, Liu L, et al. Gene expression Cell Biol 2003;23:7947–56. alterations in prostate cancer predicting tumor aggression and preceding 46. Luo J, Sladek R, Bader J-A, Rossant J, Giguere V. Placental abnormalities in development of malignancy. J Clin Oncol 2004;22:2790–9. mouse embryos lacking orphan nuclear receptor ERRb. Nature 1997;388: 31. Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y, Carver BS, et al. 778–82. Integrative genomic profiling of human prostate cancer. Cancer Cell 47. Narkar VA, Fan W, Downes M, Yu RT, Jonker JW, Alaynick WA, et al. 2010;18:11–22. Exercise and PGC-1a-independent synchronization of type I 32. Sun Y, Wang BE, Leong KG, Yue P, Li L, Jhunjhunwala S, et al. Androgen muscle metabolism and vasculature by ERRg. Cell Metab 2011;13: deprivation causes epithelial-mesenchymal transition in the prostate: impli- 283–93. cations for androgen-deprivation therapy. Cancer Res 2012;72:527–36. 48. Huppunen J, Aarnisalo P. Dimerization modulates the activity of the 33. Mulholland DJ, Kobayashi N, Ruscetti M, Zhi A, Tran LM, Huang J, et al. orphan nuclear receptor ERRg. Biochem Biophys Res Commun 2004;314: Pten loss and RAS/MAPK activation cooperate to promote EMT and 964–70.

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Androgen-Dependent Repression of ERRγ Reprograms Metabolism in Prostate Cancer

Étienne Audet-Walsh, Tracey Yee, Shawn McGuirk, et al.

Cancer Res Published OnlineFirst November 7, 2016.

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