Reprogramming of Isocitrate Dehydrogenases Expression and Activity by the Androgen
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Author Manuscript Published OnlineFirst on May 8, 2019; DOI: 10.1158/1541-7786.MCR-19-0020 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Reprogramming of isocitrate dehydrogenases expression and activity by the androgen 2 receptor in prostate cancer 3 4 Kevin Gonthier1,2, Raghavendra Tejo Karthik Poluri1,2, Cindy Weidmann1, Maude Tadros1, and 5 Étienne Audet-Walsh1,2,3 6 7 1 Endocrinology - Nephrology Research Axis, Centre de recherche du CHU de Québec - 8 Université Laval, Québec City, Canada 9 2 Department of molecular medicine, Faculty of Medicine, Université Laval, Québec City, 10 Canada 11 3 Centre de recherche sur le cancer de l’Université Laval, Québec City, Canada 12 13 Keywords: steroid; metabolism; glioma; nuclear receptor; castration-resistant prostate cancer, 14 IDH1, oncometabolism 15 16 Abbreviated title: Reprogramming of IDH activity by AR in PCa 17 18 Corresponding Author: Étienne Audet-Walsh, Centre de recherche du CHU de Québec, 2705 19 Boulevard Laurier, room R-4714, Québec City, QC, Canada, G1V 4G2 20 Phone: 1-418-525-4444 ext. 48678 ; e-mail : [email protected] 21 22 Disclosure statement: the authors declare no potential conflicts of interest. 1 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 8, 2019; DOI: 10.1158/1541-7786.MCR-19-0020 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 23 Abstract 24 Mutations of the isocitrate dehydrogenase genes IDH1 and IDH2, key enzymes involved in 25 citrate metabolism, are important oncogenic events in several cancer types, including in 1-3% of 26 all prostate cancer (PCa) cases. However, if IDH1 and other IDH isoforms are associated with 27 PCa progression, as well as the regulatory factors controlling their expression and activity, 28 remain mostly unknown. Using publicly available datasets, we showed that PCa harbors the 29 highest IDH1 expression across the human cancer spectrum and that IDH1 expression is altered 30 during PCa progression. We showed that the androgen receptor (AR), a key oncogene in PCa, 31 controls multiple IDH isoforms in both in vitro and in vivo models, predominantly positively 32 regulating IDH1. Chromatin immunoprecipitation experiments confirmed the recruitment of AR 33 at several regulatory regions of IDH1 and enzymatic assays demonstrated that AR significantly 34 induces IDH activity. Genetic blockade of IDH1 significantly impaired PCa cell proliferation, 35 consistent with IDH1 having a key function in these cancer cells. Importantly, knockdown of 36 IDH1 blocked the AR-mediated induction in IDH activity, indicating that AR promotes a 37 mitochondrial to cytoplasmic reprogramming of IDH activity. Overall, our study demonstrates 38 that IDH1 expression is associated with PCa progression, that AR signaling integrates one of the 39 first transcriptional mechanisms shown to regulate IDH1, and that AR reprograms PCa cell 40 metabolism by selectively inducing extra-mitochondrial IDH activity. 41 Implications: The discovery that AR reprograms IDH activity highlights a novel metabolic 42 reprogramming necessary for PCa growth and suggests targeting IDH activity as a new 43 therapeutic approach for PCa treatment. 44 2 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 8, 2019; DOI: 10.1158/1541-7786.MCR-19-0020 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 45 Introduction 46 Prostate cancer (PCa) is an androgen-dependent disease that relies on the androgen receptor (AR) 47 for cancer cell proliferation and survival, and thus inhibiting AR activity is currently the 48 cornerstone therapy in clinical settings (1, 2). Despite the positive response of most cancer cases 49 to AR blockage, evolution of the disease to castration-resistant PCa (CRPC) is almost inevitable 50 (1-4), highlighting the need to develop novel therapies for PCa. In >80% of all cases, resistance 51 involves AR reactivation (5-8). Cancer cells can also evolve into AR-negative PCa, which are 52 often referred to as neuroendocrine PCa (NEPC)(9). 53 Glucose, amino acids, and fatty acids can fuel mitochondrial activity through the 54 tricarboxylic acid (TCA) cycle for ATP production and to sustain biosynthesis of essential 55 cellular components (e.g., nucleotides and lipids). The prostate is metabolically unique in human 56 in that epithelial prostate cells have a blunted TCA cycle to produce high levels of citrate (10, 57 11). One of the earliest events in PCa, which occurs in virtually all cancer cases, is the 58 reprogramming of this unique metabolic program (10, 11). Given its major oncogenic functions 59 in PCa, it is not surprising that AR was identified as a major orchestrator of energy metabolism 60 and that PCa progression was linked to specific metabolic gene signatures (12-18). Moreover, AR 61 was shown to alter citrate metabolism, most notably by inducing mitochondrial respiration and 62 dysregulating lipid metabolism, contributing to the rising characterization of AR functions in the 63 transcriptional control of energy metabolism (12, 14, 15). However, what are the key limiting 64 enzymatic steps under the AR-driven metabolic program remain unknown. 65 It is now well recognized that mutations of isocitrate dehydrogenase genes IDH1 and 66 IDH2 lead to the establishment of an oncometabolic environment that causes cancer, notably by 67 altering the epigenome (19, 20). These mutations, which induce a novel enzymatic activity 3 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 8, 2019; DOI: 10.1158/1541-7786.MCR-19-0020 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 68 catalyzing the formation of 2-hydroxyglutarate, interfere with α-ketoglutarate (αKG)-dependent 69 epigenetic regulatory mechanisms and lead to the hyper-methylation of the genome (20-23). 70 Although frequent in gliomas and other cancer types, IDH1 mutations occur in only 1–3% of PCa 71 and are believed to be the underlying oncogenic drivers in these cases (24-28). On the basis that 72 the prostate harbors a distinct citrate metabolic program, we hypothesized that an alteration in the 73 regulation of IDH enzymes could be linked to PCa development and progression, even in PCa 74 cases without IDH1 mutation. 75 IDHs are encoded by five different genes leading to three possible functional IDH 76 enzymatic complexes (IDH1, IDH2, and IDH3, the latter comprising IDH3A, IDH3B, and 77 IDH3G). In the mitochondria, either the IDH2 or the IDH3 complexes catalyze the 78 decarboxylation of isocitrate, produced from citrate, into αKG using respectively NADP+ and 79 NAD+ as co-substrates. This represents a key step in the mitochondrial TCA cycle. In addition, 80 cytoplasmic IDH1 promotes the NADP+-dependent production of extra-mitochondrial αKG, 81 which is required for the proper activity of several enzymes involved in epigenetic regulation 82 (20-23). Newly synthesized NADPH can be used for anabolic processes such as lipogenesis. 83 However, what are the molecular mechanisms controlling IDH gene expression and how these 84 dynamics are altered in PCa cell metabolism are still unknown. 85 In the current study, we observed that wild-type IDH1 is predominantly expressed in PCa 86 cells and that its expression is altered throughout PCa progression. We identified AR as a key 87 regulator of several IDH isoforms, most notably IDH1. We showed that androgen stimulation 88 reprograms IDH activity, favoring cytoplasmic over mitochondrial metabolism. Importantly, 89 knockdown of IDH1 decreased cancer cell proliferation and a blockade of the IDH metabolic 90 reprogramming, possibly highlighting IDH1 as a potential new therapeutic target for PCa. 4 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 8, 2019; DOI: 10.1158/1541-7786.MCR-19-0020 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 91 92 Material and Methods 93 Cell Culture 94 LNCaP, LAPC4, 22rv1, PC3, VCaP, and DU145 cells were grown in RPMI 95 supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin, and sodium pyruvate 96 at 37°C and 5% CO2. All cells were kept in culture for no more than 3 months after resuscitation 97 and were re-authenticated using the ATCC cell line authentication service during summer 2016. 98 For hormonal treatments with 10nM of the synthetic androgen R1881, steroid deprivation was 99 first performed for 48h in phenol red-free RMPI 1640 with 5% charcoal-stripped serum (CSS) as 100 described previously (13). 101 102 Cellular proliferation 103 AR+ PCa cells and AR- PCa cells were seeded in 96-well plates in media with 5% CSS, with 104 fresh media changed every 48h. For cell counting, culture medium was discarded and 50ul of 105 0.5% crystal violet staining solution was added to each well before being incubated at room 106 temperature on a bench rocker. After 30 minutes, plates were rinsed 4 times with water and plates 107 were dried overnight at room temperature. 200ul of SDS 2% were then added to each well and 108 plates were incubated at room temperature for 3h. Optical density (OD) was then measured at 109 570 nm using a spectrophotometer. OD values were then converted into cell numbers using 110 established standard curves for each cell line tested. 111 112 IDH enzyme activity 5 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 8, 2019; DOI: 10.1158/1541-7786.MCR-19-0020 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.