SREBF1 Activity Is Regulated by an AR/mTOR Nuclear Axis in ... · such as aerobic glycolysis, mitochondrial respiration, and steroid metabolism (8–13). Reprogrammed energy metabolism,

Metabolism

SREBF1 Activity Is Regulated by an AR/mTORNuclear Axis in Prostate Cancer�Etienne Audet-Walsh1, Mathieu Vernier1, Tracey Yee1, Chlo�e Laflamme1,Susan Li1, Yonghong Chen, and Vincent Gigu�ere1,2,3

Abstract

Reprogramming of cellular metabolism is an importantfeature of prostate cancer, including altered lipid metabolism.Recently, it was observed that the nuclear fraction of mTOR isessential for the androgen-mediated metabolic reprogram-ming of prostate cancer cells. Herein, it is demonstrated thatthe androgen receptor (AR) and mTOR bind to regulatoryregions of sterol regulatory element-binding transcription fac-tor 1 (SREBF1) to control its expression, whereas dual activa-tion of these signaling pathways also promotes SREBF1 cleav-age and its translocation to the nucleus. Consequently, SREBF1recruitment to regulatory regions of its target genes is inducedupon treatment with the synthetic androgen R1881, an effectabrogated upon inhibition of themTOR signaling pathway. Inturn, pharmacologic and genetic inhibition of SREBF1 activityimpairs the androgen-mediated induction of the key lipogenicgenes fatty acid synthase (FASN) and stearoyl-CoA desaturase(SCD1). Consistent with these observations, the expression of

the SREBF1, FASN, and SCD1 genes is significantly correlatedin human prostate cancer tumor clinical specimens. Function-ally, blockade of SREBF1 activity reduces the androgen-drivenlipid accumulation. Interestingly, decreased triglyceride accu-mulation observed upon SREBF1 inhibition is paralleled byan increase inmitochondrial respiration, indicating a potentialrewiring of citrate metabolism in prostate cancer cells.Altogether, these data define an AR/mTOR nuclear axis, inthe context of prostate cancer, as a novel pathway regulatingSREBF1 activity and citrate metabolism.

Implications: The finding that an AR/mTOR complexpromotes SREBF1 expression and activity enhances ourunderstanding of the metabolic adaptation necessary forprostate cancer cell growth and suggests novel therapeuticapproaches to target metabolic vulnerabilities in tumors.Mol Cancer Res; 16(9); 1396–405. �2018 AACR.

IntroductionAndrogens and their receptor (AR) are nowwell established as a

key oncogenic pathway in the development and progression ofprostate cancer. As such, most tumors respond favorably toandrogen deprivation therapy. However, this favorable responsein treatment is temporary, and the disease will eventually evolveto castration-resistant prostate cancer (1, 2). Several mechanismshave been shown to promote resistance to testicular androgensblockade, most of them involving AR hyperactivation, such as ARgenomic amplification, AR gene mutation, and de novo steroidbiosynthesis by tumor cells (1, 3–7). Thus, identification ofdownstream effectors of AR is important for future developmentand design of novel therapeutic approaches.

A major function of AR in prostate cancer cells is to fuel cancercell metabolism, notably by inducing key metabolic pathways

such as aerobic glycolysis, mitochondrial respiration, and steroidmetabolism (8–13). Reprogrammed energy metabolism, consid-ered as a hallmark of cancer (14), is crucial to androgen signaling-driven prostate cancer development and progression. Anotherimportant metabolic pathway rewired in prostate cancer andstimulated by androgens is de novo lipid synthesis, notably viaupregulation of the gene encoding fatty acid synthase (FASN;refs. 9, 12, 13, 15).

mTOR signaling, a major regulator of cell metabolism andproliferation (16, 17), is also frequently activated in prostatecancer, most notably by loss of function of negative upstreamregulators such as PTEN (3, 7, 18, 19). Recently, we demonstratedthat androgens induce mTOR relocalization to the nucleus whereit acts as a transcriptional integrator of AR function, and that itsactivity is essential for the androgen-mediated metabolic repro-gramming of prostate cancer cells (13). The identification oftranscription factors downstream of the AR/mTOR axis partici-pating in the metabolic reprogramming of prostate cancer cellswould provide additional opportunities for further control of thisregulatory pathway.

In the present study, we show that the expression and activity ofsterol regulatory element-binding transcription factor 1 (SREBF1,also known as SREBP1), a key transcriptional regulator of lipidmetabolism, is contingent on the dual activation of the AR/mTORsignaling pathways. There are two major SREBF1 transcriptsarising fromdistinct transcriptional start site (TSS) and producingSREBF-1a and SREBF-1c proteins (20). SREBF-1c is expressedubiquitously and generally considered as the main SREBF1 iso-form in human cells (20) and is the focus of the present study

1Goodman Cancer Research Centre, McGill University, Montr�eal, Qu�ebec,Canada. 2Departments of Medicine and Oncology, McGill University, Montr�eal,Qu�ebec, Canada. 3Department of Biochemistry, McGill University, Montr�eal,Qu�ebec, Canada.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Vincent Gigu�ere, McGill University, 1060 Pine AvenueWest, Montr�eal, QC H3A 1A3, Canada. Phone: 514-398-5899; Fax: 514-398-8578;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-17-0410

�2018 American Association for Cancer Research.

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(hereafter referred as SREBF1). Acting as an effector of this AR/mTOR axis, SREBF1 contributes to the androgen-mediatedincrease in de novo lipid synthesis in prostate cancer cells. Inaddition, SREBF1 also serves as a gatekeeper of citrate metabo-lism, regulating the balance between citrate consumption formitochondrial ATP production and as a carbon source for de novolipid synthesis in the cytoplasm.

Materials and MethodsCell culture, treatments, and transfections

LNCaP and 22rv1 cells were originally obtained from theAmerican Type Culture Collection and reauthenticated in July2016. Cells were grown in phenol red–free RPMI supplementedwith 10% FBS, penicillin, streptomycin, and sodium pyruvate at37�C and 5% CO2. For androgen stimulation, cells were culturedin media with charcoal-stripped serum (CSS) for 48 hours andthen treated with 10 nmol/L R1881 (Steraloids), 10 mmol/L fato-statin (Tocris Bioscience), 80 nmol/L rapamycin (Calbiochem),and/or 100 nmol/L torin 1 (Toronto Research Chemicals). ForsiRNA transfections, cells were trypsinized, seeded in media withCSS, and transfected with either a nontargeted pool of siRNA or apool of siRNA specific against SREBF1 (SMARTpoolTM) withHiperfect transfection reagent according to the manufacturer'sinstructions (QIAGEN). For shRNA against SREBF1 stable trans-fection (TRCN0000020605 and TRCN0000020606, from Sig-ma), viruses were first produced by transfecting HEK293T cells.Media were changed the day after transfection, and fresh mediawere added for another 24 hours. The media containing theviruses were then used to infect LNCaP cells, and selection wasperformed by adding puromycin antibiotic to the culture media.

RNA purification and analysisRNAwas extractedwith theRNeasyMini Kit (Qiagen), andfirst-

strand cDNA synthesis was performed with ProtoScript II reversetranscriptase (New England Biolabs). cDNA expression was thenquantified by SYBR green–based qPCR techniques using theLightCycler480 instrument (Roche). Relative expressionwas stan-dardized to the expression of 2 housekeeping genes. Gene-specificprimers for qRT-PCR can be found in Supplementary Table S1.

Chromatin immunoprecipitation-qPCRAfter steroid deprivation, cells were treated for 48 hours with

R1881, torin 1, or vehicles for chromatin immunoprecipitation(ChIP)-qPCR studies with AR (sc-816X from Santa Cruz Biotech-nology), SREBF1 (Abcam; 3259), RNA polymerase II (05-623from Millipore), H3K27Ac (ab4729 from Abcam), or mTOR(Abcam; ab32028). After crosslinking with 1% formaldehyde atroom temperature, cells were lysed, nuclei were enriched bysequential centrifugations, and ChIPs were performed as previ-ously described (12, 21). Two or more negative regions were usedfor ChIP normalization along with IgG control for antibodyspecificity. Gene-specific primers for ChIP-qPCR can be found inSupplementary Table S2.

Protein analysisWhole-cell lysates were harvested with buffer K supplemented

with protease and phosphatase inhibitors or separated intonuclear and cytoplasmic fractions by differential centrifugationas described previously (13). Relative protein levels were assessedusing ImageJ and normalized over tubulin and Lamin B1 for

cytoplasmic and nuclear extracts, respectively. Primary antibodiesusedwere as follows: AR (SantaCruzBiotechnology, sc-816), P-S6(S235/236, Cell Signaling Technology, 2211), S6 (Santa CruzBiotechnology, sc-74459), Lamin B1 (Cell Signaling Technology,12586), SREBF1 (Abcam, 3259) and anti–a-Tubulin (Cedarlane,CLT-9002).

Metabolic analysisBriefly, cells were first seeded inmedia with CSS for 48 hours to

ensure steroid deprivation. LNCaP and 22rv1 cells were thentrypsinized and seeded at 400,000 cells/well in 2 mL of mediacontaining CSS in 6-well plates, with or without treatment. Forlipid quantification using the Abcam triglyceride quantificationKit (ab65336), cells were treated for a total of 4 days, with newtreatment added after 48 hours (13). The oxygen consumptionrate (OCR) and extracellular acidification rateweremeasuredwitha Seahorse XFe24 or XFe96 instrument (Seahorse Bioscience),with cells prepared as per themanufacturer's protocol and treatedfor a total of 72 hours for LNCaP and 48 hours for 22rv1 cells, aspreviously described (12, 13).

Clinical data and statistical analysesExpression data from clinical specimens were retrieved and

analyzed from The Cancer Genome Atlas (TCGA) Research Net-work cbioportal web platform (22, 23). Microarray expressiondata from LNCaP cells treated with R1881, torin 1, or vehicles for48 hours were retrieved from GSE93603 (13). The Student t testwas used to assess statistical significance for expression dataanalyses.

ResultsmTOR integrates AR-mediated transcriptional activation ofSREBF1

Activity of the mTOR signaling pathway is induced followingAR activation (24), and a functionalmTOR is required to integratemultiple transcriptional signatures associated with the androgen-mediated metabolic reprogramming of prostate cancer cells (13).To identify players that contribute to transcription regulationdownstream the AR/mTOR axis, we further analyzed the previ-ously publishedmicroarrays of LNCaP cells treatedwithR1881, inthe presence or absence of cotreatment with the mTOR inhibitortorin 1 (GSE93603; ref. 13). Ingenuity Pathway Analysis was usedto pinpoint transcription factors whose activity are predicted to beassociated with genes sensitive to R1881 treatment in an mTOR-dependent manner. As expected, AR was the transcription factorfound most strongly associated with genes sensitive at the mRNAlevels after R1881 stimulation (Fig. 1A). Cotreatment of R1881with torin 1 slightly decreased the predicted role of AR, but it wasnevertheless the most enriched upstream regulator of genes mod-ulated by R1881 in LNCaP cells (Fig. 1A). These results areconsistent with the observation that mTOR inhibition does notblock the "androgen response" gene signature, characterized bygenes bound by AR and highly modulated upon AR activation(13). Accordingly, it seems that blocking mTORmostly interfereswith androgen-sensitive genes indirectly regulated by AR and/ornecessitating cooperative genomic dynamics with other transcrip-tional regulators. The SREBF1 pathway, comprised of SREBF1itself, its positive regulator SCAP, and its negative regulatorsINSIG1 and INSIG2, was also predicted to be activated byR1881 (Fig. 1A and B). Importantly, mTOR inhibition by torin

Lipid Metabolism Regulation by an AR/mTOR/SREBF1 Axis

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1 completely reversed the transcriptional signatures associatedwith components of the SREBF1 pathway (Fig. 1A). In agreementwith these findings, gene set enrichment analyses of gene signa-tures associatedwith SREBF1activity (Fig. 1C), particularly relatedto fatty acid metabolism, were significantly enriched after R1881stimulation (red color), but abrogated after inhibition of mTOR(blue color). In line with bioinformatics predictions (Fig. 1A),SREBF1 expression was significantly induced by R1881 (Fig. 1D).The mRNA expression of both FASN and SCD1, two key SREBF1target genes associated with lipid metabolism, also showed pos-

itive regulation after androgen stimulation (Fig. 1D). Treatmentwith 2 mTOR inhibitors, rapamycin or torin 1, significantlyimpaired or completely blocked this androgen-mediated regula-tion of SREBF1, FASN, and SCD1 (Fig. 1D). This is similar to ourprevious study, in which both mTOR inhibitors blocked andro-gen-sensitive transcriptional regulation of specific genes (13).Data analyses from RNA-seq experiments performed by Kach andcolleagues (25) in 22rv1 cells also indicated that R1881 treatmentinduces SREBF1 expression (Fig. 1E). In addition, the two keySREBF1 target genes associated with lipid metabolism, FASN and

Figure 1.

SREBF1 is activated by R1881 in an AR- and mTOR-dependent manner. A, Bioinformatics analyses of transcriptomics data from LNCaP cells treated withR1881, with and without torin 1, predict activation of SREBF1 by R1881 stimulation and blockade of that activation by torin 1 cotreatment. B, Schematicrepresentation of the SREBF1 pathway downstream the AR/mTOR regulatory axis. C, Gene set enrichment analysis (GSEA) plot for the fatty acid metabolismgene signature following R1881 or torin 1 treatments. Only genes identified as "core genes" are shown in the gene expression heatmaps. Note that colors indicatehigher expression (red) or lower expression (blue) as defined by GSEA. D, qRT-PCR analysis of SREBF1, FASN, and SCD1 following a 48-h treatment withR1881, torin 1, rapamycin (rapa), and/or vehicles. Values representmean� SEMof three independent experiments performed at least in duplicate. E, Fold induction ofSREBF1, FASN, and SCD1 after R1881 treatment in 22rv1 cells. Data were retrieved from the RNA-seq study by Kach and colleagues (25). F, qRT-PCR analysisof SREBF1, FASN, and SCD1 following 48-hour treatment with R1881, torin 1, and/or vehicles in 22rv1 cells. Values represent mean � SEM of one representativeexperiment performed in triplicate. G, ChIP-qPCR of AR and mTOR in LNCaP cells after treatment with R1881 or vehicle (n ¼ 3). H, Relative fold enrichmentof RNA polymerase II (pol II) and H3K27Ac at the dual AR/mTOR DNA-binding sites identified in SREBF1 in LNCaP cells. Values represent mean � SEMof three independent experiments. � , P < 0.05; �� , P < 0.01; and �� , P < 0.001.

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SCD1, were also induced in that cell lines (Fig. 1E). These resultswere further validated (Fig. 1F). As seen in LNCaP cells, mTORinhibition with torin 1 significantly impaired the induction ofSREBF1, FASN, and SCD1 in 22rv1 cells (Fig. 1F).

We next sought to test if the expression of SREBF1 is directlyregulated by the AR/mTOR axis. Analysis of AR and mTOR ChIP-sequencing datasets (GSE93845) identified two AR-binding sitesat SREBF1, located approximately 1.5 kb upstream and approx-imately 825 bp downstream of the SREBF1 TSS. AR was indeedrecruited to these two binding sites in the presence of R1881 (Fig.1G). As described in our previous study, mTOR can be found inthe absence of androgen in the nucleus of prostate cancer cells(13), and we were able to detect mTOR DNA binding to SREBF1gene without R1881 (Fig. 1G). Moreover, mTORDNA binding tothe same genomic loci as ARwas increased in response to the sameandrogen stimulation (Fig. 1G). Corecruitment of AR and mTORwas also increased uponR1881 treatment at FASN and SCD1 (Fig.1G). In addition, we detected a significant increase in RNApolymerase II enrichment following androgen stimulation atthese two AR/mTOR-binding sites (Fig. 1H). Moreover, theH3K27ac mark, an indicator of active genomic region, alsoshowed a significant increase at these loci after R1881 treatment(Fig. 1H). Altogether, these data indicate that the AR/mTOR axisdirectly participates to the transcriptional regulation of SREBF1mRNA expression, and that blockade of mTOR signaling issufficient to impair the AR-mediated induction of lipid metabo-lism genes.

The AR–mTOR axis promotes SREBF1 processing and activityAlthough we observed increased mRNA levels of SREBF1, its

transcriptional activity relies on its proteolytic cleavage, allowingits nuclear translocation andbinding toDNA. As shown in Fig. 2A,treatment of LNCaP cells with androgens not only enhancedglobal SREBF1 protein levels but also stimulated its cleavage toan active form and its nuclear accumulation. Conversely, mTORinhibition robustly impaired the observed R1881-induced pro-cessing and nuclear translocation of SREBF1 (Fig. 2A). To furtherunderstand if androgen-dependent regulation of both SREBF1expression increase and protein processing were required for theincrease in processed SREBF1 protein levels, we performed experi-ments with the RNA pol II inhibitor a-amanitin in LNCaP cells.Importantly, a-amanitin blocks transcription, but do not inhibitmTOR complex 1 activity, as shown by phosphorylation of S6after vehicle and R1881 treatment (Fig. 2B). However, itcompletely blocks the R1881-mediated induction of processedSREBF1 levels in LNCaP cells (Fig. 2B). Together, these dataindicate that the AR/mTOR axis is required to induce SREBF1expression and processing, and that both are required to obtainmaximal active SREBF1 levels in LNCaP cells.

The observed increased enrichment of SREBF1 nuclear levelsfollowing androgen stimulation indicates that its DNA-bindingactivity would also increase in this context. We next assessed itsDNA binding based on its best characterized targets from livertissue (26–29). As shown in Fig. 2C, although weak occupancy ofSREBF1 target genes was observed in the absence of androgens,addition ofR1881promoted SREBF1 recruitment toDNA, includ-ing to regulatory regions within FASN and SCD1, two target genesof mTOR and AR (but to distinct binding sites). In contrast,cotreatment with the mTOR inhibitor torin 1 abrogated orimpaired SREBF1 genomic binding to its target genes (Fig. 2C),in agreement with its negative effect on SREBF1 expression and

processing observed in Figs. 1 and 2. To further validate our ChIPdata, we proceeded to shRNA-mediated knockdown of SREBF1.R1881 alone induced SREBF1 recruitment to its target DNAregions (Supplementary Fig. S1). However, cells infected withindividual shRNA against SREBF1 completely loosed recruitmentof SREBF1 to DNA regulatory regions (Supplementary Fig. S1),mimicking mTOR inhibition (Fig. 2C).

Mostly characterized in hepatocytes, SREBF1 is well knownto control FASN and SCD1 expression, but how this occurs inother cell types, most notably in cancer cells, is much lessunderstood. To further link SREBF1 to FASN and SCD1 expres-sion in prostate cancer, we analyzed their relative mRNAexpression levels using cbioportal to access TCGA clinical dataset(22, 23). Interestingly, the two genes found most significantlyassociated with SREBF1 expression are FASN and SCD1 (Fig.2D). Next, we validated these results in the Broad/Cornelldataset (30) and confirmed that both FASN and SCD1 weresignificantly correlated to SREBF1 expression in prostate cancerprimary tumors (Fig. 2E). Finally, we sought to validate thesefindings in the Fred Hutchinson cohort, mostly comprised ofcastration-resistant prostate cancer tumors (18), thereforerepresenting a more aggressive clinical setting. In agreementto the findings obtained with the TCGA and the Broad/Cornelldatasets, FASN and SCD1were also significantly correlated withSREBF1 expression (Fig. 2F). Therefore, SREBF1 expression istightly linked with lipid metabolic gene expression not only inthe liver (31), but this relationship is also highly relevant inprostate cancer human tumor tissues.

Pharmacologic and genetic blockade of SREBF1 alters thebalance between lipid metabolism and mitochondrialrespiration

To demonstrate the importance of SREBF1 as an integrator ofthe AR/mTOR axis–mediated transcriptional control of lipidmetabolic genes, we first used an siRNA pool to repress SREBF1expression in LNCaP cells (Fig. 3A). Loss of SREBF1 expressionsignificantly impaired the androgen-induced expression of bothFASN and SCD1 (Fig. 3B). However, it did not completely blocktheir androgen sensitivity, consistent with AR andmTOR bindingto their regulatory regions (Fig. 1G). Importantly, the androgenresponse of theAR classic target geneKLK3, encoding PSA,was notaffected by SREBF1 blockade (Fig. 3B). Similarly, inhibition ofSREBF1 with 2 individual shRNAs also significantly impairedinduction of FASN and SCD1 following R1881 treatment (Fig.3C). In addition, pharmacologic inhibition of SREBF1 byfatostatin also impaired the induction of these two genesfollowing R1881 treatment (Fig. 3D). Again, the expression ofKLK3 expression, induced by androgens, was not affectedfollowing inhibition of SREBF1, indicating that SREBF1 activityis required for the specific regulation of a lipid gene signaturedownstream AR (Fig. 3D).

Next, we sought to validate the functional role of SREBF1 in themetabolic reprogramming induced by AR activation. Given thatSREBF1 functions have been consistently associated with lipidmetabolism, we first investigated its relationship with the R1881-mediated accumulation of triglycerides (TG). Treatment withR1881 resulted in a 2- to 5-fold induction in TG levels in LNCaPcells (Fig. 4A) and 22rv1 cells (Fig. 4B). However, repression ofSREBF1 decreased this induction by about 60% and 30%, respec-tively, denoting that SREBF1 is essential for maximal lipid accu-mulation following AR activation (Fig. 4A and B), in agreement






with the positive correlation of SREBF1 with FASN and SCD1expression in prostate cancer tumors (Fig. 2D–F).

In prostate cancer cells, androgens have been described toinduce both lipid synthesis and mitochondrial respiration (10,

12, 13, 24). As citrate is required for de novo lipid synthesis, wehypothesized that impairment of this pathway following SREBF1knockdown would redirect citrate toward mitochondrial respira-tion. Treatment with R1881 induced by nearly 2-fold the OCR of

Figure 2.

SREBF1 processing and DNA binding are increased after AR activation in an mTOR-dependent manner. A, Protein expression of unprocessed and processedSREBF1 in LNCaP cells, with tubulin and Lamin B1, shown as loading controls, respectively. Phosphorylation status of S6, a downstream target of mTOR, isshown as a control of mTOR activation by R1881 and inhibition by torin 1. Relative protein levels for unprocessed (n ¼ 3) and processed (n ¼ 5) SREBF1 are shownnormalized to tubulin and Lamin B1 levels, respectively (right plot). � , P < 0.05 and �� , P < 0.01. B, Protein expression of processed SREBF1 in LNCaP cells,with and without treatment with the RNA pol II inhibitor a-amanitin (a-A). Tubulin is shown as loading control. Phosphorylation status of S6, a downstream targetof mTOR, is also shown. Relative protein levels for processed (n ¼ 3) SREBF1 are shown normalized to tubulin levels (right). C, ChIP-qPCR of SREBF1 inLNCaP cells after treatment with R1881, torin 1, and/or vehicles. Results are shown as the average of three independent experiments. Correlation analysis ofSREBF1 mRNA with FASN and SCD1 mRNAs in the TCGA cohort (D), the Broad/Cornell (E), and the Fred Hutchinson (F) clinical datasets. Both Pearson(P) and Spearman (S) correlation coefficients are indicated in insets, and all correlations are significant with P < 0.05.

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LNCaP cells (Fig. 4C). Likewise, R1881 treatment also inducedOCR in 22rv1 cells (Fig. 4D). In addition, SREBF1 knockdownled to a significant induction in OCR and further increased theOCR in R1881-treated LNCaP cells (Fig. 4C) and 22rv1 cells(Fig. 4D; Supplementary Fig. S2). Finally, we proceeded to anadditional validation using two distinct shRNAs targetedagainst SREBF1 to confirm that our siRNA results were not dueto off-target effects. Again, R1881 treatment induced a signif-icant accumulation of TGs in LNCaP cells (Fig. 4E), but withlower fold induction in these stably infected cells under puro-mycin selection compared with parental cells. As observed withsiRNA pools (Fig. 4A and B), knockdown of SREBF1 withshRNAs significantly decreased the androgen-mediated TGs'accumulation in LNCaP cells (Fig. 4E). Moreover, shRNA-mediated knockdown of SREBF1 also led to a significantinduction of OCR in R1881-treated cells (Fig. 4F).

DiscussionIn the present study, we identified SREBF1 as a downstream

target and integrator of the AR/mTOR transcriptional axis inprostate cancer cells. As such, both AR and mTOR are requiredto induce SREBF1 expression, and AR-mediated activation ofmTOR is essential to promote SREBF1 processing, activation,and genomic occupancy. Acting as a transcriptional regulator oflipid metabolism, SREBF1 expression and activity are requiredfor maximal androgenic stimulation of this cellular pathway.Indeed, even in prostate cancer tumor samples, SREBF1 expres-sion levels are significantly correlated with the expression of keylipid metabolism genes. By controlling citrate usage for lipidsynthesis, SREBF1 potentially acts as a gatekeeper of properbalance between lipid synthesis and mitochondrial respirationunder AR metabolic control. As such, SREBF1 operates as a key

Figure 3.

SREBF1 knockdown or inhibition impairs androgen-mediated induction of lipid metabolism genes in LNCaP cells. A, Protein expression of processed SREBF1,with andwithout siRNA-mediated knockdown. Tubulin is shownas a loading control. Relative protein levels for SREBF1 are shown,with normalization to tubulin levels(n ¼ 3). B, qRT-PCR analysis of FASN, SCD1, and KLK3 following treatment with R1881 in cells transfected with control siRNA (siCtl) or a siRNA pool againstSREBF1 (siSREBF1). Values represent mean� SEM of three independent experiments performed in triplicate. C, qRT-PCR analysis of FASN, SCD1, KLK3, and SREBF1following treatment with R1881 in cells stably expressing control shRNA (shNTC) or against SREBF1 (shSREBF1 #1 and 2). Values represent mean � SEMof one representative independent experiment performed in triplicate. D, qRT-PCR analysis of FASN, SCD1, and KLK3 following a 48-hour treatment withR1881, with and without cotreatment with the SREBF1 inhibitor fatostatin. Values represent mean � SEM of three independent experiments performed intriplicate. � , P < 0.05; �� , P < 0.01; and �� , P < 0.001 compared with control or as indicated.






metabolic effector of AR and an important regulator of prostatecancer cellular metabolism.

The androgen signaling pathway has been shown to coordinatethe expression of lipogenic gene signatures, notably involvingSREBF1 processing (32, 33). Now, we provide direct evidence thatSREBF1 gene expression is also induced following AR activation,contributing to the overall increase in SREBF1 protein levels. Inaddition, we identify mTOR as a key regulator involved in boththe induction of SREBF1 expression and processing. In agreementwith these findings, mTOR was previously shown to regulateSREBF1 processing in embryonic stem cells of immortalizednoncancerous cell lines (34, 35). Activation of this mTOR func-tion by androgen action therefore most probably contributes tothe amplificationof the SREBF1pathway activation. In the presentstudy, we further demonstrate that AR and mTOR are required to

induce SREBF1 expression and promote its activation (Fig. 5A).Moreover, they also show collaborative gene regulation, withrecruitment of AR and mTOR in close vicinity to regulatoryregions of FASN and SCD1, and subsequent requirement ofSREBF1 binding to other regions for their maximal expression.

Consistent with its key role downstream AR and mTOR, twomajor oncogenic pathways in prostate cancer, pharmacologicinhibition of SREBF1 impairs prostate cancer cell proliferationin vitro and in vivo, whereas overexpression of SREBF1 on thecontrary promotes prostate cancer cell proliferation (36). Indeed,prostate cancer cells, like most cancer cells, have exacerbated denovo lipid synthesis pathway, notably through increased FASNexpression (15, 37–44). Accordingly, genetic or pharmacologicinhibition of FASN leads to cancer cell death and to a decrease inthe epithelial to mesenchymal transition/migratory phenotype

Figure 4.

SREBF1 regulates TGs andmitochondrial metabolism underandrogen stimulation.A,TGcontent ofLNCaP cells treated with R1881 orvehicle, with and without siRNA-mediated knockdown of SREBF1 (n ¼6). B, TG content of 22rv1 cells treatedwith R1881 or vehicle, with andwithoutsiRNA-mediated knockdown ofSREBF1 (n ¼ 6). C, Oxidative capacityand uncoupled respiration in LNCaPcells following treatment with R1881 orvehicle, with transfection of a pool ofcontrol siRNAs (siCtl) or siRNAsagainst SREBF1 (siSREBF1). Onerepresentative experiment is shown(n ¼ 5 samples/condition).Quantification of basal respirationonly is shown (right). D, Oxidativecapacity and uncoupled respiration in22rv1 cells following treatment withR1881 or vehicle, with transfection of apool of control siRNAs (siCtl) orsiRNAs against SREBF1 (siSREBF1).One representative experiment isshown (n¼ 20 samples/condition) outof three independent experiments.Quantification of basal respirationonly is shown (right). E, TG content ofLNCaP cells treated with R1881 orvehicle, with or without shRNA control(shNTC) or shRNA against SREBF1(n¼ 3). F,Oxidative capacity in LNCaPcells following treatment with R1881 orvehicle, with infection with a controlshRNA (shCtl) or individual shRNAagainst SREBF1 (shSREBF1). Onerepresentative experiment is shown(n¼ 7 samples/condition). � , P < 0.05;��, P < 0.01; and �� , P < 0.001compared with controls or asindicated.

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of various cancer cells (45, 46), including prostate cancer cells(47). FASN has also been shown to regulate the WNT/b-cateninpathway (44, 48), an oncogenic pathway often hyperactivatedin aggressive castration-resistant prostate cancer (3). Therefore,inhibition of de novo fatty acid synthesis, particularly throughrepression of FASN activity, has been studied extensively as anovel therapeutic approach for several cancer types (41, 42).Indeed, except for the liver and adipose tissues which displaysignificant de novo lipid synthesis, most tissues rely on exogenousdietary fatty capacities. As such, targeting this pathway shouldbe mostly harmful to cancer cells that are particularly addictedto it. Targeting SREBF1 itself, which controls several of theenzymes in this pathway, is also a promising target for prostatecancer, but the development of specific inhibitors is still requiredto pursue this approach. Accordingly, a recent study publishedafter the initial submission of this work identified a SREBF1-dependent lipogenic gene signature to be associated with themetastatic potential of prostate cancer cells (49). This studyshows that loss of PML and PTEN promotes SREBF1 activity inprostate cancer cells (49). Our findings provide a molecularmechanism by which SREBF1 can be activated to promote lipo-genesis via the AR–mTOR axis. Interestingly, apart from having ade novo lipogenesis program, prostate cancer is also characterizedby a reprogramming of mitochondrial metabolism, which isincreased in tumors compared with normal prostate epithelialcells (50, 51). It thus appears that both lipogenesis and respirationincrease prostate cancer cell growth, and that the balance betweenboth is important for cancer cell proliferation and survival. Itremains to be determined how to target prostate cancer cellmetabolism as a therapeutic approach for the treatment of pros-tate cancer.

In summary, AR and mTOR form a regulatory complex that,upon androgen stimulation, promotes SREBF1 expression, pro-teolytic cleavage, andDNAbinding.Once activated, SREBF1 playsthe role of an AR effector in the androgen-signaling pathway,allowing maximal transcriptional induction of lipid metabolismgenes. By doing so, it appears that SREBF1 controls citrate metab-olism, by regulating the balance between mitochondrial usage ofcitrate for ATP synthesis and de novo lipid synthesis from citrate,

twokeyprocesses in prostate cancer. It remains tobedetermined ifthis AR/mTOR/SREBF1 pathway is also required in vivo in humantumors to control prostate cancer cell metabolism. Further under-standing of this metabolic balance in cancer cells is required toeffectively design therapies aimed at targeting SREBF1 and de novolipid synthesis in cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: �E. Audet-Walsh, V. Gigu�ere

Development of methodology: �E. Audet-Walsh, V. Gigu�ereAcquisition of data (provided animals, acquired and managed patients,

provided facilities, etc.): �E. Audet-Walsh, M. Vernier, T. Yee, C. Laflamme,S. Li, Y. Chen, V. Gigu�ereAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,

computational analysis): �E. Audet-Walsh, M. Vernier, C. Laflamme, V. Gigu�ere

Writing, review, and/or revision of the manuscript: �E. Audet-Walsh,M. Vernier, V. Gigu�ereAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Yee, S. Li, V. Gigu�ere

Study supervision: �E. Audet-Walsh, V. Gigu�ere

AcknowledgmentsThis work was supported by a Terry Fox Research Institute Program Project

TeamGrant on Oncometabolism (116128) and an Innovation Award from the

Canadian Cancer Society Research Institute (to V. Gigu�ere). �E. Audet-Walsh wasa recipient of a postdoctoral fellowship from the Canadian Institutes of HealthResearch, the Fonds de Recherche duQu�ebec–Sant�e, and supported by aMcGillIntegrated Cancer Research Training Program (MICRTP) scholarship. T. Yee wasrecipient of a summerMICRTP studentship. The authors also thankCatherine R.Dufour for critical reading of the article.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 28, 2017; revised December 21, 2017; accepted May 9, 2018;published first May 21, 2018.

Figure 5.

Working model of the AR/mTOR/SREBF1 axis and its role in regulating prostate cancer cell metabolism. A, Depiction of how AR and mTOR control bothtranscription and activation of SREBF1 and lipid metabolism genes. B, It is hypothesized that by upregulating genes related to de novo lipid synthesis, SREBF1controls global citrate metabolism in prostate cancer cells. Modulation of SREBF1 functions, such as repression using siRNA, shRNA, and pharmacologicinhibition, will lead to an imbalance in citrate usage, thus promoting mitochondrial usage of citrate.






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2018;16:1396-1405. Published OnlineFirst May 21, 2018.Mol Cancer Res Étienne Audet-Walsh, Mathieu Vernier, Tracey Yee, et al. Prostate CancerSREBF1 Activity Is Regulated by an AR/mTOR Nuclear Axis in

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