The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis
2011; Springer Nature; Volume: 30; Issue: 13 Linguagem: Inglês
10.1038/emboj.2011.158
ISSN1460-2075
AutoresCharles Massie, Andy G. Lynch, Antonio Ramos‐Montoya, Joan Boren, Rory Stark, Ladan Fazli, Anne Y. Warren, Helen E. Scott, Basetti Madhu, Naomi L. Sharma, Hélène Bon, Vinny Zecchini, Donna-Michelle Smith, Gina M. DeNicola, Nik Mathews, Michelle Osborne, James Hadfield, Stewart MacArthur, Boris Adryan, Scott K. Lyons, Kevin M. Brindle, John R. Griffiths, Martin Gleave, Paul S. Rennie, David E. Neal, Ian G. Mills,
Tópico(s)Estrogen and related hormone effects
ResumoArticle20 May 2011free access The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis Charles E Massie Charles E Massie CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Andy Lynch Andy Lynch CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Antonio Ramos-Montoya Antonio Ramos-Montoya CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Joan Boren Joan Boren CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Rory Stark Rory Stark CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Ladan Fazli Ladan Fazli The Vancouver Prostate Centre, Vancouver, British Columbia, Canada Search for more papers by this author Anne Warren Anne Warren Department of Pathology, Addenbrookes Hospital, Cambridge, UK Search for more papers by this author Helen Scott Helen Scott CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Basetti Madhu Basetti Madhu CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Naomi Sharma Naomi Sharma CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Helene Bon Helene Bon CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Vinny Zecchini Vinny Zecchini CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Donna-Michelle Smith Donna-Michelle Smith CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Gina M DeNicola Gina M DeNicola CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Nik Mathews Nik Mathews CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Michelle Osborne Michelle Osborne CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author James Hadfield James Hadfield CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Stewart MacArthur Stewart MacArthur CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Boris Adryan Boris Adryan Cambridge Systems Biology Centre and Department of Genetics, University of Cambridge, Cambridge, UK Search for more papers by this author Scott K Lyons Scott K Lyons CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Kevin M Brindle Kevin M Brindle CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author John Griffiths John Griffiths CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Martin E Gleave Martin E Gleave The Vancouver Prostate Centre, Vancouver, British Columbia, Canada Search for more papers by this author Paul S Rennie Paul S Rennie The Vancouver Prostate Centre, Vancouver, British Columbia, Canada Search for more papers by this author David E Neal David E Neal CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Ian G Mills Corresponding Author Ian G Mills CRUK Cambridge Research Institute, Cambridge, UK Centre for Molecular Medicine Norway, Nordic European Molecular Biology Laboratory Partnership, University of Oslo, Oslo, Norway Search for more papers by this author Charles E Massie Charles E Massie CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Andy Lynch Andy Lynch CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Antonio Ramos-Montoya Antonio Ramos-Montoya CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Joan Boren Joan Boren CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Rory Stark Rory Stark CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Ladan Fazli Ladan Fazli The Vancouver Prostate Centre, Vancouver, British Columbia, Canada Search for more papers by this author Anne Warren Anne Warren Department of Pathology, Addenbrookes Hospital, Cambridge, UK Search for more papers by this author Helen Scott Helen Scott CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Basetti Madhu Basetti Madhu CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Naomi Sharma Naomi Sharma CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Helene Bon Helene Bon CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Vinny Zecchini Vinny Zecchini CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Donna-Michelle Smith Donna-Michelle Smith CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Gina M DeNicola Gina M DeNicola CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Nik Mathews Nik Mathews CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Michelle Osborne Michelle Osborne CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author James Hadfield James Hadfield CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Stewart MacArthur Stewart MacArthur CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Boris Adryan Boris Adryan Cambridge Systems Biology Centre and Department of Genetics, University of Cambridge, Cambridge, UK Search for more papers by this author Scott K Lyons Scott K Lyons CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Kevin M Brindle Kevin M Brindle CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author John Griffiths John Griffiths CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Martin E Gleave Martin E Gleave The Vancouver Prostate Centre, Vancouver, British Columbia, Canada Search for more papers by this author Paul S Rennie Paul S Rennie The Vancouver Prostate Centre, Vancouver, British Columbia, Canada Search for more papers by this author David E Neal David E Neal CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Ian G Mills Corresponding Author Ian G Mills CRUK Cambridge Research Institute, Cambridge, UK Centre for Molecular Medicine Norway, Nordic European Molecular Biology Laboratory Partnership, University of Oslo, Oslo, Norway Search for more papers by this author Author Information Charles E Massie1, Andy Lynch1, Antonio Ramos-Montoya1, Joan Boren1, Rory Stark1, Ladan Fazli2, Anne Warren3, Helen Scott1, Basetti Madhu1, Naomi Sharma1, Helene Bon1, Vinny Zecchini1, Donna-Michelle Smith1, Gina M DeNicola1, Nik Mathews1, Michelle Osborne1, James Hadfield1, Stewart MacArthur1, Boris Adryan4, Scott K Lyons1, Kevin M Brindle1, John Griffiths1, Martin E Gleave2, Paul S Rennie2, David E Neal1,‡ and Ian G Mills 1,5,‡ 1CRUK Cambridge Research Institute, Cambridge, UK 2The Vancouver Prostate Centre, Vancouver, British Columbia, Canada 3Department of Pathology, Addenbrookes Hospital, Cambridge, UK 4Cambridge Systems Biology Centre and Department of Genetics, University of Cambridge, Cambridge, UK 5Centre for Molecular Medicine Norway, Nordic European Molecular Biology Laboratory Partnership, University of Oslo, Oslo, Norway ‡These authors contributed equally to this work *Corresponding author. Department of Uro-Oncology, CRUK Cambridge Research Institute, Robinson Way, Cambridge CB2 0RE, UK. Tel.: +44 122 340 4463; Fax: +44 122 340 4199; E-mail: [email protected] or [email protected] The EMBO Journal (2011)30:2719-2733https://doi.org/10.1038/emboj.2011.158 There is a Have you seen? (July 2011) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach. Introduction The androgen receptor (AR) is a ligand activated transcription factor and is the main therapeutic target in prostate cancer. Androgen deprivation therapy (chemical castration) is an effective first-line therapy for prostate cancer, but despite good initial responses the recurrence of castrate-resistant disease is common and ultimately fatal. Functional studies have shown that the AR is essential for cell viability, proliferation and invasion in both hormone-sensitive and castrate-resistant prostate cancer (Haag et al, 2005; Hara et al, 2008; Snoek et al, 2009). These findings are supported by clinical studies reporting the sensitivity of castrate-resistant prostate cancer to second-generation AR antagonists and hormone synthesis blockade (Attar et al, 2009; Attard et al, 2009; Tran et al, 2009). In castrate-resistant disease, where tumours are less sensitive to androgen depletion, AR activity is maintained by gene amplification (Visakorpi et al, 1995), activating mutations (Veldscholte et al, 1990; Steinkamp et al, 2009) or signalling cross talk with other oncogenic pathways (Craft et al, 1999). All of these mechanisms suggest a strong selective pressure to maintain AR-regulated signalling pathways in castrate-resistant disease, although the nature of these important pathways has remained unclear. Previous studies have aimed to identify androgen-regulated genes or AR genomic binding sites (Wang et al, 2007; Jia et al, 2008; Yu et al, 2010). However, no single study has identified the transcriptional networks, which underlie AR dependency in both hormone naive and castrate-resistant prostate cancer. Individual studies have focussed on the use of a single model of prostate cancer (LNCaP or its in vitro-derived subclones) (Velasco et al, 2004; Massie et al, 2007; Wang et al, 2007, 2009), been limited by genome coverage on microarrays (Massie et al, 2007; Wang et al, 2007; Jia et al, 2008), did not map sites of transcriptional activity (Massie et al, 2007; Wang et al, 2009) or assessed only a limited number of time points following androgen stimulation (Velasco et al, 2004). These studies have provided important insights into the upstream mechanisms which direct the transcriptional activities of the AR and identified a number of transcription factors which cooperate with or antagonize AR activity (Wang et al, 2007; Jia et al, 2008; Yu et al, 2010). However, efforts to identify the key downstream targets of the AR in prostate cancer have identified indirect links to anabolic pathways (Heemers et al, 2001, 2004; Xu et al, 2006) or focussed on cell-cycle regulators (Knudsen et al, 1998; Wang et al, 2009), some of which appear only to be AR targets in models of castrate-resistant prostate cancer (Wang et al, 2009). Therefore, a detailed map of AR-regulated genes in diverse models of prostate cancer is needed to better understand the essential signalling pathways downstream of the AR in both hormone-sensitive and castrate-resistant stages of the disease. Results Identifying a core set of direct AR-regulated genes To identify direct AR-regulated genes, we combined genome-wide AR binding profiles with detailed transcript profiling. We also integrated androgen-stimulated recruitment of the transcriptional machinery to identify a core set of AR binding sites, which regulate gene expression in prostate cancer cells. First, we mapped AR binding profiles in two cell lines which represent distinct molecular subtypes of prostate cancer: one harbouring an AR ligand binding domain mutation (LNCaP); one harbouring an AR gene amplification (VCaP). Using chromatin immunoprecipitation with direct Solexa sequencing (ChIP-seq), we identified 11 053 AR binding sites in LNCaP cells and 51 811 androgen-dependent AR binding sites in VCaP cells (Supplementary Tables S1 and S2). VCaP cells harbour a copy number gain of the AR gene locus resulting in elevated AR expression (Supplementary Figure S1; Makkonen et al, 2011), which may at least in part explain the larger number of AR binding sites found in VCaP compared with LNCaP cells. Despite the difference in total number of identified binding sites, over 90% of the LNCaP AR binding sites were also found in the VCaP cells (Figure 1A; Supplementary Figure S2; Supplementary Table S3), suggesting that these core binding sites are commonly occupied by the AR even in distinct molecular subtypes of prostate cancer. The common AR binding sites between LNCaP and VCaP cells included all established AR target genes (Figure 1A; Supplementary Figure S2), had significant correlations with other published data sets (Supplementary Figure S3) and identified thousands of AR targets not identified in previous AR ChIP studies (Supplementary Table S3). As a resource we have compiled all published AR ChIP-chip studies together with our data in Supplementary Table S3. Figure 1.Mapping transcriptional targets of the AR. (A) ChIP-seq enrichment profiles for AR and RNAP II with or without androgen treatment and in LNCaP or VCaP cells, as indicated (cells cultured in steroid depleted media and treated with 1 nM R1881 or 0.01% ethanol for 4 h). Location of the PSA gene is indicated below enrichment plots, arrow indicates the direction of transcription. Androgen-stimulated expression is shown at the bottom, represented as a heatmap showing expression changes with time following androgen stimulation (1 nM R1881, data from Illumina beadarray gene expression time course). (B) Venn diagram showing the overlap between AR binding sites and androgen-dependent RNAP II enriched genomic regions (1 nM R1881, 4 h; data from intersects of all peaks overlapping by >1 bp). (C) Gene set enrichment analysis (GSEA) of androgen-regulated genes (Illumina beadarray androgen stimulation time course), using gene sets identified within genomic windows from 1 to 500 kb from AR binding sites. (D) Combined analysis of AR binding sites, androgen-dependent RNAP II enriched regions and androgen-regulated genes. Genes are grouped by expression changes early ( 4 h), up, down or no change in response to androgen stimulation (left). Pie charts indicate the proportion of genes with adjacent AR, RNAP II or overlapping AR-RNAP II sites in each set (<25 kb from gene boundaries; groups indicated above). (E) Sequence logos for 15 and 6 bp AR/GR binding motifs (P-values indicate enrichment in AR-RNAP II overlapping regions). (F) Venn diagram showing the occurrence of 15 and 6 bp AR binding motifs in androgen-dependent AR-RNAP II overlapping sites. Download figure Download PowerPoint Next, we integrated the location of the transcriptional machinery on the prostate cancer genome together with detailed expression profiling. We identified 15 761 androgen-dependent RNAP II (serine 5 phosphorylated RNA polymerase II) regions in LNCaP cells using ChIP-seq (Supplementary Table S4), 1283 of which overlapped with androgen-stimulated AR binding sites (Figure 1B). The regions enriched for RNAP II alone (n=14 478) represent sites of paused, primed or active transcription (Bernstein et al, 2006; Core et al, 2008); however, sites to which the AR and RNAP II are dynamically co-recruited (n=1283) are candidate regions for androgen-stimulated transcriptional initiation (explored in detail below). Using Illumina BeadArrays, we made a detailed study of androgen-regulated gene expression with samples taken every 30 min for 4 h and then every hour up to 24 h following androgen stimulation of LNCaP cells. This detailed expression array time course made it possible to identify androgen-regulated gene expression changes based on trends with time following androgen stimulation (using autocorrelation), allowing detection of early and even small gene expression changes (see Materials and methods for details). In total, we found 3319 transcripts with altered expression in response to androgens, 1556 (47%) transcripts were upregulated and 1763 (53%) were downregulated (Supplementary Table S5). We made a combined analysis of our treatment contrast ChIP and detailed gene expression profiling to identify AR binding sites which recruit the transcriptional machinery and direct AR-regulated genes. Such combined analyses require a predefined genomic distance between transcript factor binding sites and genes, which is often set arbitrarily. To address this issue, we took a more empirical approach using gene set enrichment analysis (GSEA) to define the optimal genomic distance between AR binding sites (peaks) and androgen-regulated genes (gene boundaries). We found that genes located within 25 kb of an AR binding site were the most significantly enriched for androgen-regulated genes; the maximal enrichment score at 25 kb suggests that smaller genomic windows would include a greater proportion of false negatives and that larger genomic windows would include a greater proportion of false positives (Figure 1C; Supplementary Figure S3; Supplementary data). Therefore, using this 25 kb window we integrated the genomic locations of AR binding sites, androgen-stimulated recruitment of the core transcriptional machinery and detailed gene expression profiling (Figure 1D). This integrated analysis revealed that loci bound by either the AR or RNAP II alone contain genes which are upregulated, downregulated or not affected by AR signalling (Figure 1D). This finding is consistent with previous studies showing that the AR can directly activate or repress transcription (Margiotti et al, 2007; Prescott et al, 2007), that only a proportion of transcription factor binding sites are active in a given cellular context (Carroll et al, 2006) and that RNAP II enriched regions of the genome include both sites of active transcription and also sites where transcription has paused or stalled (Bernstein et al, 2006; Core et al, 2008). In contrast, the core overlap of androgen-stimulated AR binding sites and RNAP II recruitment showed specific enrichment for androgen upregulated transcripts (Figure 1D). Therefore, our combined analysis has identified a core set of AR binding sites that recruit the transcriptional machinery and upregulate the transcription of adjacent genes. These core AR targets may help to identify the features of transcriptionally active AR binding sites. Analysis of the genomic sequences underlying all AR binding sites found in LNCaP, VCaP or those identified in both cell lines revealed evolutionary conservation, enrichment of AR binding sequences and motifs for previously reported AR interacting transcription factors, including forkhead and NF-1 (Supplementary Figure S1; Supplementary Table S6) (Wang et al, 2007; Jia et al, 2008). The overlapping AR and RNAP II sites showed significant enrichment of 6 bp and 15 bp AR binding motifs (Figure 1E and F), but also showed enrichment for CREB and AHR binding motifs, in contrast to the full set of AR binding sites (Supplementary Figure S1; Supplementary Table S6). De novo motif analysis revealed an inverted-repeat 15 bp ARE similar to the in vitro-derived consensus binding motif (Roche et al, 1992) and also a 6 bp motif consisting of one half of the consensus 15 bp element, as previously reported in other AR ChIP studies (Massie et al, 2007; Wang et al, 2007) (Figure 1E; Supplementary Figure S1). Identifying cellular processes regulated by the AR Our combined analysis of AR, RNAP II and detailed expression profiling also identified a core set of direct androgen-regulated genes for further investigation. We defined direct AR targets as those genes induced by androgen treatment and for which there were overlapping hormone-induced AR binding sites within 25 kb of the genes. This definition identified a sufficient number of genes to allow pathway enrichment analysis and as a resource we have included a table noting these genes together with their androgen-stimulated expression profile, annotated with the location of adjacent AR and RNAP II binding sites (Supplementary Table S7). Interestingly, this core set of direct AR-regulated genes gave overlapping but distinct functional enrichment by gene ontology (GO) analysis compared with gene expression data alone (Figure 2A; Supplementary Tables S8 and S9). Figure 2.Functional annotation of direct AR-regulated genes. (A) Gene ontology (GO) network of direct AR-regulated genes (androgen upregulated genes within 25 kb of AR binding site, Cytoscape BiNGO analysis). (B) Gene set enrichment analysis (GSEA) plots for direct AR-regulated genes, showing enrichment for carbohydrate metabolism GO and the curated peroxisome proliferator-activated receptor γ co-activator 1-α (PPARGC1A) metabolic gene set. (C) Gene expression heatmaps, showing androgen-regulated genes within 25 kb of an AR binding site, grouped by functional categories (indicated above; data from Illumina beadarray time course in LNCaP cells; pathway annotations from GO annotations, KEGG pathways and literature reviews). (D) Schematic showing the locations of direct AR-regulated genes in metabolic and cell-cycle pathways. Red boxes indicate direct AR upregulated genes, blue boxes represent direct AR downregulated genes and yellow boxes indicate proteins not found to be regulated by the AR. Dashed lines indicate intermediate steps not shown. (E–I) Levels of (E) glucose, (F) lactate, (G) citrate, (H) succinate and (I) oxygen consumption rates were measured following growth of LNCaP prostate cancer cells in steroid depleted media with and without androgen stimulation (1 nM R1881). Expressed as μM lactate, μM citrate, μM succinate and % glucose consumption in cell culture media and nmol/ml/min oxygen consumption rate, all normalized to cell number (represented as mean±s.e.m.; each data point represents triplicate measurements). Download figure Download PowerPoint While many established classes of AR target gene were represented within the data sets as a whole, including cell-cycle regulators (e.g., CDC25, CDK6 and E2F1) and signalling molecules which have been implicated in prostate cancer (e.g., WWP1, ERBB2, MEK5, SGK1 and IGF1R) (Figure 2C; Craft et al, 1999; Hellawell et al, 2002; Mehta et al, 2003; Chen et al, 2007; Sherk et al, 2008), the combined analysis of direct AR targets and androgen-regulated genes also revealed a significant enrichment of metabolic targets (Figure 2A–C; Supplementary Table S9). GO analysis highlighted central metabolism and biosynthetic pathways among the direct AR targets (Figure 2A) and GSEA also revealed significant enrichment of central metabolism and metabolic gene signatures (Mootha et al, 2003) among direct AR-regulated genes (Figure 2B). Unlike other biological processes, metabolism has been subjected to decades of detailed biochemical study and consists of a series of interlinked and well-characterized processing steps. This makes the functional consequences of changes in enzyme expression uniquely predictable for metabolic pathways and made these pathways an ideal testing ground for the linkages established by our genomics data. We found that the AR directly upregulated expression of key steps in glucose uptake and glycolysis including glucose transporter 1 (GLUT1/SLC2A1), hexokinase I and II (HK1 and HK2), phosphofructokinase (PFK2/PFKFB2) and also many anabolic enzymes at both the transcript and protein level (Figure 2C and D; Supplementary Figures S4 and S5). Based on the annotation of these direct metabolic AR target genes within well-defined metabolic pathways we predicted that the AR may facilitate cell growth by promoting glucose uptake and anabolic metabolism (Figure 2D). To test these predictions from our genomics data, we undertook comprehensive metabolomic profiling to assess the effects of AR signalling on glucose consumption and lactate production, O2 consumption and detailed metabolite profiling of intracellular and extracellular metabolites using proton nuclear magnetic resonance (1H NMR) and glucose flux using carbon-13-labelled glucose with gas chromatography–mass spectrometry (GC/MS). The AR regulates aerobic glycolysis and anabolism in prostate cancer cells Androgen stimulation of prostate cancer cells increased glucose uptake and increased lactate production in normoxia, but had no effect on oxygen consumption, showing that AR signalling does indeed stimulate aerobic glycolysis (Christofk et al, 2008; Vander Heiden et al, 2009) (Figure 2E–I; Supplementary Figure S4F). Citrate levels were also increased following androgen stimulation (Figure 2G) while the levels of the tricarboxylic acid (TCA) cycle metabolite succinate remained unchanged (Figure 2H), further underscoring androgen stimulation of glycolysis and highlighting the truncated TCA cycle in prostate epithelial cells (Costello et al, 1997). We also found that AR signalling significantly stimulated anabolic synthesis, by measuring the flux from carbon-13-labelled glucose to amino acids (e.g., glutamine) and RNA (ribose) in response to androgen stimulation (Supplementary Figure S6; Figure 4F and G). Therefore by specifically upregulating these rate-limiting steps in glycolysis, the AR stimulates energy production and provides carbon needed for macromolecule synthesis. In addition, we found that the AR stimulated the expression of key anabolic enzymes, which utilize glucose metabolites (e.g., fatty acid synthase (FASN) and acetyl-CoA carboxylase α (ACACA)) and master regulators of biosynthesis (e.g., MTOR, encoded by FRAP1; Supplementary Figure S5). Therefore, AR signalling appears to coordinately regulate energy production and biosynthesis at multiple levels (Figure 2D), highlighting these metabolic pathways as potential targets to inhibit the growth of prostate cancer cells (Migita et al, 2009). CAMKK2 is a metabolic master regulator downstream of the AR in prostate cancer cells Identifying cancer-specific alterations that affect central metabolism is a major challenge for drug discovery in cancer biology. Therefore, we used clinical gene expression data (Rhodes et al, 2004) to assess cancer-specific expression of AR targets (Supplementary Table S7). Among the direct AR target genes, we identified calcium/calmodulin-dependent kinase kinase 2 (CAMKK2, Figure 3A) as consistently overexpressed in prostate cancer in nine independent clinical gene expression studies (Rhodes et al, 2004; Varambally et al, 2005), showing a similar pattern to the established prostate cancer marker AMACR (Jiang et al, 2001, 2002) (Figure 3B; Supplementary Figure S5). CAMKK2 was only highlighted by integrating AR genomic targets with clinical expression data, since we identified a core set of 1164 direct AR-regulated genes and on average CAMKK2 was ranked only 40th over the nine clinical studies (Figure 3B). CAMKK2 has previously been linked to central metabolism as an essential regulator of the metabolic sensor AMP kinase (AMPK) in the hypothalamus (Anderson et al, 2008), implicating CAMKK2 also as a regulator of cellular metabolism. The AR was recruited to the CAMKK2 promoter in both androgen-dependent and castrate-resistant prostate cancer cell lines (Supplementary Figure S7), suggesting that CAMKK2 is an AR target in both stages of the disease. CAMKK2 transcript and protein were upregulated early (<4 and <12 h, respectively) in response to androgen stimulation and downregulated in response to the AR antagonist bicalutamide, underscoring the direct regulation of CAMKK2 by the AR (Figure 3C and D; Supplementary Figure S7i). In clinical samples, we found CAMKK2 protein overexpression in two independent prostate cancer cohorts (Figure 3E; Supplementary Figure S7; Supplementary Table S10) and confirmed that CAMKK2 was a direct AR target in a panel of clinical prostate cancer samples using ChIP from human tissue (Figure 3F). These data highlight CAMKK2 as an AR-regulated gene in prostate cancer and in combination with the published role of CAMKK2 as a metabolic regulator (Anderson et al, 2008), prompted us to investigate the functional effects of AR-CAMKK2 signalling. Figure 3.The regulation and expression of CAMKK2 in prostate cancer. (A) ChIP-seq enrichment profiles for the AR (LNCaP and VCaP) and RNAP II (LNCaP), as indicated. The CAMKK2 gene position is indicated below, arrow indicates the direction of transcription. Androgen-regulated expression of CAMKK2 is sh
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