Biochimica et Biophysica Acta 1850 (2015) 1781–1785
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Review
IDH1, lipid metabolism and cancer: Shedding new light on old ideas Elena Bogdanovic ⁎ Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Ave, Room 988, Toronto, ON M5G 1X5, Canada
a r t i c l e
i n f o
Article history: Received 6 March 2015 Received in revised form 22 April 2015 Accepted 30 April 2015 Available online 7 May 2015 Keywords: IDH1R132 Lipid Peroxisome Plasmalogen Cancer
a b s t r a c t Background: Since the initial discovery of mutations in the isocitrate dehydrogenase 1 (IDH1) gene in a large subset of human low-grade gliomas and acute myelogenous leukemia (AML), much interest focused on the function of IDH1 and on the relationship between mutations in IDH1 and tumor progression. To date, mutations in the IDH1 gene have been found in numerous cancers with the highest frequencies occurring in gliomas, chondrosarcomas/enchondromas and cholangiocarcinomas. Scope of review: IDH1 was first described in the scientific literature as early as 1950. Early researchers proposed that the enzyme likely functions in cellular lipid metabolism based on the observation that the enzymatic reaction produces NADPH and partially localizes to peroxisomes. This article highlights the studies implicating IDH1 in cytoplasmic and peroxisomal lipid metabolism from the early researchers to the recent studies examining mutant IDH1R132, the most common IDH1 mutation found in cancer. Major conclusions: While a role for IDH1 in lipid biosynthesis in the liver and adipose tissue is now established, a role in lipid metabolism in the brain and tumors is beginning to be examined. The recent discoveries that IDH1R132H interferes with the metabolism of phospholipids in gliomas and that IDH1 activity could participate in the synthesis of acetyl-CoA from glutamine in hypoxic tumors highlight roles for IDH1 in lipid metabolism in a broad spectrum of tissues. General significance: Interferences in cytoplasmic and peroxisomal lipid metabolism by IDH1R132 may contribute to the more favorable clinical outcome in patients whose tumors express mutations in the IDH1 gene. © 2015 Elsevier B.V. All rights reserved.
1. Introduction With the discovery of heterozygous mutations in the IDH1 gene in a large subset of human low-grade gliomas and in AML [1–3], much interest focused on the function of IDH1 and on the role of mutant IDH1 in the development of cancer. Mutations in IDH1 have now been reported in numerous cancers, most notably in gliomas (N70%), chondrosarcomas (~50%), cholangiocarcinomas (~20%) and AML (~10%) [1–5]. In gliomas and cholangiocarcinomas mutations in the IDH1 gene predict longer survival and a favorable prognosis [1,2,6]. IDH1 belongs to the family of isocitrate dehydrogenase enzymes which is comprised of 3 members: IDH1, IDH2 and IDH3 [7]. IDH1 is localized within the cytoplasm and peroxisomes [8,9]. IDH2 and IDH3 are located exclusively within the mitochondria [10]. All IDH enzymes catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate with Abbreviations: IDH1, isocitrate dehydrogenase 1; AML, acute myelogenous leukemia; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; 2-HG, 2 hydroxyglutarate; RCDP, rhizomelic chondrodysplasia punctata; NADP, nicotinamide adenine dinucleotide phosphate; NAD, nicotinamide adenine dinucleotide; TCA, tricarboxylic acid; DHAP, dihydroxyacetone phosphate ⁎ Tel.: +1 416 586 4800x8268. E-mail address: [email protected].
http://dx.doi.org/10.1016/j.bbagen.2015.04.014 0304-4165/© 2015 Elsevier B.V. All rights reserved.
the reduction of either NADP+ or NAD+ to generate NADPH or NADH respectively [7,11]. IDH1 and IDH2 utilize NADP+ as a cofactor and IDH3 utilizes NAD+ and functions in the tricarboxylic acid (TCA) cycle [7,10]. IDH1 and IDH2 can also catalyze the reverse reaction where αketoglutarate (in the presence of NADPH and CO2) is converted to isocitrate and NADP+ via reductive carboxylation [10,11]. The most frequent mutation in IDH1 found in cancer occurs at the substrate binding site at arginine 132 (R132) where R132 is replaced by either histidine (R132H), cysteine (R132C), serine (R132S), glycine (R132G), leucine (R132L) or glutamine (R132Q) [2,12]. The frequency with which each amino acid substitution at R132 occurs varies and depends on the type of cancer; the IDH1R132H mutation is the most frequent IDH1 mutation found in gliomas, whereas IDH1R132C is more often observed in cholangiocarcinomas and chondrosarcomas/enchondromas [1,2,4,5]. Mutation of R132 at the substrate binding site abolishes the wild-type activity of the enzyme [1,2] and induces a novel enzymatic activity which catalyzes the conversion of α-ketoglutarate to 2-hydroxyglutarate (2HG) utilizing NADPH as a cofactor [13]. The accumulation of 2-HG produced by mutant IDH1R132 may contribute to the development of cancer by inhibiting collagen maturation, upregulating HIF1α and inhibiting histone demethylases leading to increased histone methylation and altered gene expression [14–18].
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2. Lipid metabolism in the liver and adipose tissue One of the earliest functions ascribed to wild-type IDH1 was a role in lipid metabolism based on the observation that the enzymatic reaction of IDH1 produces NADPH [19]. IDH1 was discovered as early as 1950 when Hogeboom and Schneider [20] identified isocitrate dehydrogenase activity in the cytoplasm of liver tissue obtained from adult mice. These investigators noted the increased production of NADPH following the addition of isocitrate to rat liver cytosolic preparations. Early investigators hypothesized that the function of IDH1 may be to provide high levels of NADPH for lipid biosynthesis since NADPH is an obligatory reducing equivalent for the synthesis of fatty acids and lipids [7,19]. It was also hypothesized that IDH1 participates in a biochemical pathway in the rat liver where carbons derived from glutamate become incorporated into fatty acids [11]. Further suggestion that IDH1 plays a role in lipid metabolism came from the observation that a small (≤ 25%) pool of IDH1 is localized within rat liver peroxisomes [8]. Peroxisomes are organelles found in all mammalian cells with the exception of erythrocytes [21]. Their size (0.1–1.0 μm) and number varies depending on the tissue being examined [22]. Tissues that are highly active in the metabolism of lipids such as the liver have large and more numerous peroxisomes [22,23]. Peroxisomes contain over 50 enzymes; half of which participate in functions related to lipid metabolism such as βoxidation of very long chain fatty acids, α-oxidation of branched-chain fatty acids and the synthesis of ether-linked (plasmalogen) phospholipids [21,23,24]. Other functions of peroxisomes include purine and polyamine catabolism, amino acid metabolism and the synthesis of bile [21,23]. The name peroxisome was derived from the observation that hydrogen peroxide is produced and degraded within this organelle [25]. Hydrogen peroxide is formed during the first reaction in the βoxidation pathway and is then reduced to water by catalase [21,25]. For the early researchers, the hypothesis that IDH1 participates in cellular lipid metabolism was primarily based on correlative data obtained from tissues with high lipogenic activity such as the liver, adipose tissue and mammary gland. The advent of techniques in molecular biology which allowed genetic manipulation provided a more direct link between IDH1 and cellular lipid metabolism. Koh et al. [26] generated transgenic mice overexpressing IDH1 in the liver and adipose tissue under the rat phosphoenolpyruvate carboxykinase promoter. These
mice displayed a 35% increase in body weight, increased fat mass and adipocyte size, had fatty livers and increased serum cholesterol and triacylglycerols. The authors also showed that decreasing IDH1 mRNA in 3T3-L1 preadipocytes impaired adipocyte differentiation and reduced the lipid content of these cells. Conversely, mice transduced with IDH1 shRNA gained less weight when fed a high fat diet, had reduced fat mass and lower circulating triacylglycerols compared to control mice [27]. Consistent with these observations, Shechter et al. [28] showed that the IDH1 promoter can be activated by the lipogenic transcription factors SREBP-1a and SREBP-2 in HepG2 cells. 3. Lipid biosynthesis in tumors Studies examining the metabolism of glutamine in various cultured tumor cells have shown that IDH1 and IDH2 participate in the synthesis of acetyl-CoA from glutamine to support lipid biosynthesis in normoxia, hypoxia and when mitochondrial function is impaired [29–31]. Tumor cells avidly take up glutamine to sustain their growth and survival [32]. In both the cytoplasmic (IDH1) and mitochondrial (IDH2) pathways, glutamine is converted to glutamate and subsequently to αketoglutarate (Fig. 1) [29–31]. IDH1 and IDH2 convert α-ketoglutarate to isocitrate via reductive carboxylation. Isocitrate undergoes further conversion to citrate and subsequently to acetyl-CoA and oxaloacetate. Acetyl-CoA is then used as a building block for the synthesis of fatty acids (Fig. 1). In the mitochondria, this metabolic pathway is induced by the reversal of the TCA cycle (Fig. 1) [29–31]. Although acetyl-CoA derived from glutamine can be used for de novo synthesis of fatty acids and lipids, this may not be the only mechanism to increase lipid biosynthesis in tumors. Instead, tumor cells may increase the uptake of fatty acids [33]. Mutant IDH1R132H lacks the ability to convert αketoglutarate to isocitrate via reductive carboxylation [34]. 3.1. Phospholipid and peroxisomal metabolism in the brain and gliomas The brain is an organ rich with lipids where lipids comprise up to 50% of the brain's dry weight [35]. The most abundant lipids in the brain are the phospholipids, sphingolipids and sterols (cholesterol) with each lipid class containing numerous lipid species. The phospholipids are structural components of the plasma membrane and membranes of
Fatty acids
Acetyl-CoA + Oxaloacetate
Acetyl-CoA Oxaloacetate
Citrate
Malate
Citrate
Isocitrate
TCA Cycle
IDH3
Isocitrate
IDH2
α-Ketoglutarate
Fumarate
Succinate
Lipids
Succinyl CoA
Mitochondria
IDH1
IDH1-R132H
α -Ketoglutarate
Glu
Gln
Cytoplasm
Fig. 1. IDH1 and IDH2 mediate reductive carboxylation of α-ketoglutarate to isocitrate in the mitochondria and cytoplasm for the synthesis of lipids [29–31]. Carbons derived from glutamine (Gln) are incorporated into acetyl-CoA for fatty acid synthesis. In the TCA cycle, IDH3 catalyzes the conversion of isocitrate to α-ketoglutarate. IDH2 reverses the metabolite flux of the TCA cycle for the production of citrate from α-ketoglutarate. Citrate and α-ketoglutarate may exit the mitochondria and enter the cytoplasmic pathway. Due to the loss of wild-type IDH1 activity, mutant IDH1R132H (indicated in red) lacks the ability to interconvert isocitrate to α-ketoglutarate [34]. Glu = glutamate.
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intracellular organelles and also have roles in cell signaling. Phospholipids comprise the phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin. The PCs and PEs are the most abundant phospholipids in cellular membranes [36]. Recently, Esmaeili et al. [37] examined the changes in PCs and PEs by the expression of mutant IDH1R132H in glioma xenografts, U251 glioma cells and human glioma surgical specimens. Expression of mutant IDH1R132H was associated with significant increases in PCs and decreases in PEs in all three models. Similar results were obtained when the expression of IDH1 was knocked down using siRNA in E18.5 mouse astrocytes [38]. We examined the PCs and PEs in mouse brains expressing IDH1R132Q [12]. The wild-type activity of IDH1R132Q is abolished and similar to IDH1R132H, IDH1R132Q catalyzes the formation of 2-HG. Mice engineered to express IDH1R132Q heterozygously within the brain (in both neurons and glia) die during embryonic development as early as E15.5 and present with massive hemorrhaging and cell death [15]. In the brain, the majority (up to 70%) of PEs are in the form of ethanolamine plasmalogens [36]. Plasmalogen lipids are abundant in mammalian tissues and are either PEs or PCs that contain a fatty acyl chain attached to the sn-1 position of glycerol via a vinyl ether linkage [39]. In the brain, the ethanolamine plasmalogens are the predominant plasmalogen species [40]. The biosynthesis of plasmalogens initiates within peroxisomes and is completed in the endoplasmic reticulum [40]. Previous researchers hypothesized that IDH1 within peroxisomes participates in the synthesis of plasmalogen lipids [21]. Fig. 2 depicts the initial steps of plasmalogen biosynthesis in peroxisomes and the hypothesized involvement of IDH1. In mice expressing IDH1R132Q, early alterations in brain lipid composition were observed at E13.5 with up to 50% reductions in the ethanolamine plasmalogens (Table 1). The phosphatidylcholines (PCs) tended to be proportionally increased by the expression of IDH1R132Q (Table 2) consistent with the findings by Esmaeili et al. [37] obtained using various glioma samples expressing IDH1R132H. Changes in the levels of PCs, PEs and ethanolamine plasmalogens could be expected to affect the structure and function of the plasma membrane and those of intracellular organelles.
DHAP
Peroxisome α-KG IDH1-R132 Isocitrate
1-Acyl-DHAP
1-Alkyl-DHAP NADPH
IDH1
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Table 1 Ethanolamine plasmalogens are decreased in brains expressing IDH1R132Q. Total lipids were extracted from E13.5 brains obtained from wild-type (WT) and IDH1R132Q expressing mice and analyzed by LC/MS/MS. Chromatographic intensities (counts per second) were obtained for each of the lipids listed and expressed as a percentage of the value from WT brains. The intensities from WT brains were given a value of 100%. Values are means ± SD from n=5 samples. * Indicates statistical significance p b 0.05 (WT vs. IDH1R132Q). Plasmalogen
Wild-type
IDH1R132Q
16:0/18:1 16:0/18:2 16:0/22:6 18:0/16:0 18:0/18:1 18:0/18:2 18:0/22:6 18:1/16:0
100 100 100 100 100 100 100 100
63 ± 11⁎ 69 ± 12⁎ 69 ± 10⁎ 51 ± 17⁎ 64 ± 10⁎ 68 ± 19⁎ 65 ± 14⁎ 63 ± 13⁎
3.2. IDH1 and other lipid pathways in the brain and astrocytes Fig. 3 summarizes the major lipid classes in astrocytes. Astrocytes have a higher lipid content compared to neurons [41]. The phospholipids are the most abundant lipids in astrocytes and comprise up to 70% of the total lipids [41]. PC is the major phospholipid (constituting 36% of the total lipids) followed by PE (20% of the total cellular lipids). PI, PS and sphingomyelin are minor components (3–5%) [41]. Cholesterol is also abundant in astrocytes; making up 14% of the total lipid content. Cholesterol is a major lipid constituent of cellular membranes in the central nervous system and its synthesis is higher in astrocytes [36,41]. The generation of acetyl-CoA from glutamine via IDH1 [29–31] or from TCA cycle intermediates can be used to synthesize cholesterol (Fig. 3). The expression of IDH1 is regulated by SREBP — a transcription factor that upregulates genes involved in fatty acid and cholesterol biosynthesis [28]. Future studies should determine whether IDH1 participates in the synthesis of cholesterol from acetyl-CoA in astrocytes (Fig. 3). This can be determined by measuring cholesterol levels in tissues/cells expressing mutant IDH1R132 or silencing wild-type IDH1 expression using siRNA. The sphingolipids are a large group of lipids highly abundant in the brain particularly in myelin although in astrocytes they are a minor component. The sphingolipids comprise the ceramides, sphingomyelin (which is also a phospholipid), cerebrosides, sulfatides and gangliosides [36]. In astrocytes, low levels of sphingomyelin and gangliosides have been reported (b5% of the total lipids) [41]. We examined the levels of ceramides and sphingomyelin in E13.5 mouse brains expressing IDH1R132Q. There were large fluctuations in the levels of these lipids in brains expressing IDH1R132Q (unpublished observations) indicating that IDH1 may not directly regulate their synthesis/degradation. The varying levels of ceramides and sphingomyelin may be the consequence of alternate routing of lipids from other pathways affected by IDH1. For
NADP+ 1-Alkyl-G3P
Plasmalogen Synthesis in ER Fig. 2. IDH1 located in peroxisomes may provide NADPH for the synthesis of plasmalogen lipids [21]. The synthesis of plasmalogens begins in peroxisomes with the attachment of an acyl group (fatty acid) to the sn-1 position of dihydroxyacetone phosphate (DHAP) to generate 1-acyl-DHAP. In the subsequent step, the acyl group is exchanged for an alkyl group (fatty alcohol) to generate 1-alkyl-DHAP [39,40]. NADPH generated by IDH1 via the oxidative decarboxylation of isocitrate reduces 1-alkyl-DHAP to 1-alkyl-glycerol-3phosphate (1-alkyl-G3P) which is then further processed in the endoplasmic reticulum (ER) to generate choline and ethanolamine plasmalogens [21]. Due to the loss of wildtype IDH1 activity, mutant IDH1R132 (indicated in red) would not contribute NADPH for the reduction of 1-alkyl-DHAP. α-KG = α-ketoglutarate. Figure concept adapted from Lazarow [21].
Table 2 Increased phosphatidylcholine (PC) content in mouse brains expressing IDH1R132Q. Total lipids were extracted from E13.5 brains obtained from wild type (WT) and IDH1R132Q expressing mice and analyzed by LC/MS/MS. For each lipid listed, the chromatographic intensity obtained from one WT sample was designated 100%. All other values are expressed relative to the WT sample. Results are means ± SD from n=3–4 samples. * Indicates statistical significance p b 0.05 (WT vs. IDH1R132Q). PC
Wild-type
IDH1R132Q
16:0 30:0 32:1 32:2 34:0 34:4 36:4
100 ± 20 100 ± 17 100 ± 24 100 ± 10 100 ± 20 100 ± 15 100 ± 5
145 ± 5⁎ 142 ± 16⁎ 130 ± 16 119 ± 24 135 ± 10⁎ 124 ± 23 100 ± 12
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Fatty acids Cell Membrane
? Cholesterol
IDH1
Acetyl-CoA
Phospholipids
Fatty acids IDH1-R132
PC PE
PS PI
Citrate Ceramides Gangliosides
Sphingomyelin
Isocitrate IDH1 α-KG Fig. 3. The major lipid classes in astrocytes. The phospholipids are the most abundant lipids in astrocytes followed by cholesterol and the sphingolipids (ceramides, gangliosides and sphingomyelin). Sphingomyelin is both a sphingolipid and phospholipid. Acetyl-CoA generated from glutamine or TCA cycle intermediates can be used for the synthesis of cholesterol and fatty acids. Fatty acids may also be taken up from outside the cell and are incorporated into the phospholipids and sphingolipids. Mutant IDH1R132 (shown in red) increases the levels of PCs and decreases the PEs. Dashed arrows indicate intermediate steps have been removed for simplicity.
example, one pathway for the synthesis of sphingomyelin is from PC and ceramide (Fig. 3) [36]. Although PCs were higher in mouse brains expressing IDH1R132Q the levels of sphingomyelin were either increased or decreased compared to controls possibly due to the availability of ceramide (unpublished observations). 4. Peroxisomal metabolism and bone development It was recently reported that in mice the expression of IDH1R132Q in chondrocytes resulted in dwarfism and cartilaginous dysplasia of long bones, ribs and tracheal cartilage leading to death of these mice at birth or prior to weaning [12]. This phenotype is similar to mice lacking the Pex7 gene [42]. Pex7 is a peroxisomal receptor important for the translocation of proteins into the peroxisomal matrix. Mice lacking Pex7 are defective in importing enzymes involved in plasmalogen biosynthesis into peroxisomes and consequently have significant reductions in plasmalogen lipids (up to 90%) [42]. Hallmarks of Pex7 deficiency are skeletal and central nervous system abnormalities. The majority of Pex7−/− mice die at birth or prior to weaning and exhibit dwarfism and delayed or absent ossification [42]. In humans, mutations in the PEX7 gene and peroxisomal dysfunction are the underlying cause of rhizomelic chondrodysplasia punctata (RCDP), an autosomal recessive disease [43–45]. Patients with RCDP have significantly reduced plasmalogen levels and are characterized by dwarfism, skeletal dysplasia, abnormal mineralization of cartilage and central nervous system impairments [40,46]. The majority of RCDP patients do not survive beyond childhood [46]. Expression of mutant IDH1R132Q in chondrocytes may impair the synthesis of plasmalogen lipids and/or interfere with peroxisomal metabolism which may be important during growth and development. 5. Conclusions and future directions Since the first report of IDH1 in 1950, a role in lipid metabolism has been hypothesized particularly in tissues such as the liver, mammary gland and adipose tissue. The recent discoveries that IDH1R132H interferes with the metabolism of phospholipids in gliomas and that IDH1 activity could participate in the synthesis of acetyl-CoA from glutamine in hypoxic tumors highlights roles for IDH1 in lipid metabolism in a broad spectrum of tissues. Future studies will reveal what roles IDH1 may have in lipid metabolism in other tissues and cancers such as AML and cholangiocarcinomas. Future studies will also further our
understanding of the function of peroxisomal IDH1 and on the role of peroxisomal IDH1 in tumors. Recently, small molecule inhibitors targeting mutant IDH1R132H which specifically block the ability of mutant IDH1R132H to produce 2-HG have been generated and show promise in inhibiting tumor growth [47,48]. When U87 glioma cells were transfected with IDH1R132H and treated with the IDH1R132H inhibitor compound AGI-5198, the production of 2HG was suppressed but the metabolic alterations that accompanied the expression of mutant IDH1R132H were not reversed [49]. Forced expression of mutant IDH1R132H may interfere with the activity of endogenous wild-type IDH1. These observations suggest the possibility that the loss/ interference with the activity of wild-type IDH1 may underlie the changes in metabolites that were observed. Similarly, the deregulation of phospholipids in gliomas expressing mutant IDH1R132H could be observed when the expression of wild-type IDH1 was knocked-down using siRNA [37,38]. Future studies using small molecule inhibitors that block 2-HG production by mutant IDH1R132H should reveal whether cellular accumulation of 2-HG alters the metabolism of lipids within cells. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements The author is grateful to Dr. Tak Mak at the University of Toronto for providing E13.5 mouse brains expressing IDH1R132Q and for insightful discussion. References [1] D.W. Parsons, S. Jones, X. Zhang, J.C. Lin, R.J. Leary, P. Angenendt, P. Mankoo, H. Carter, I.M. Siu, G.L. Gallia, A. Olivi, R. McLendon, B.A. Rasheed, S. Keir, T. Nikolskaya, Y. Nikolsky, D.A. Busam, H. Tekleab, L.A. Diaz, J. Hartigan, D.R. Smith, R.L. Strausberg, S.K.N. Marie, S.M.O. Shinjo, H. Yan, G.J. Riggins, D.D. Bigner, R. Karchin, N. Papadopoulos, G. Parmigiani, B. Vogelstein, V.E. Velculescu, K.W. Kinzler, An integrated genomic analysis of human glioblastoma multiforme, Science 321 (2008) 1807–1812. [2] H. Yan, D.W. Parsons, G. Jin, R. McLendon, B.A. Rasheed, W. Yuan, I. Kos, I. BatinicHaberle, S. Jones, G.J. Riggins, H. Friedman, A. Friedman, D. Reardon, J. Herndon, K.W. Kinzler, V.E. Velculescu, B. Vogelstein, D.D. Bigner, IDH1 and IDH2 mutations in gliomas, N. Engl. J. Med. 360 (2009) 765–773. [3] E.R. Mardis, L. Ding, D.J. Dooling, D.E. Larson, M.D. McLellan, K. Chen, D.C. Koboldt, R.S. Fulton, K.D. Delehaunty, S.D. McGrath, L.A. Fulton, D.P. Locke, V.J. Magrini, R.M.
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Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen
Review
IDH1, lipid metabolism and cancer: Shedding new light on old ideas Elena Bogdanovic ⁎ Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Ave, Room 988, Toronto, ON M5G 1X5, Canada
a r t i c l e
i n f o
Article history: Received 6 March 2015 Received in revised form 22 April 2015 Accepted 30 April 2015 Available online 7 May 2015 Keywords: IDH1R132 Lipid Peroxisome Plasmalogen Cancer
a b s t r a c t Background: Since the initial discovery of mutations in the isocitrate dehydrogenase 1 (IDH1) gene in a large subset of human low-grade gliomas and acute myelogenous leukemia (AML), much interest focused on the function of IDH1 and on the relationship between mutations in IDH1 and tumor progression. To date, mutations in the IDH1 gene have been found in numerous cancers with the highest frequencies occurring in gliomas, chondrosarcomas/enchondromas and cholangiocarcinomas. Scope of review: IDH1 was first described in the scientific literature as early as 1950. Early researchers proposed that the enzyme likely functions in cellular lipid metabolism based on the observation that the enzymatic reaction produces NADPH and partially localizes to peroxisomes. This article highlights the studies implicating IDH1 in cytoplasmic and peroxisomal lipid metabolism from the early researchers to the recent studies examining mutant IDH1R132, the most common IDH1 mutation found in cancer. Major conclusions: While a role for IDH1 in lipid biosynthesis in the liver and adipose tissue is now established, a role in lipid metabolism in the brain and tumors is beginning to be examined. The recent discoveries that IDH1R132H interferes with the metabolism of phospholipids in gliomas and that IDH1 activity could participate in the synthesis of acetyl-CoA from glutamine in hypoxic tumors highlight roles for IDH1 in lipid metabolism in a broad spectrum of tissues. General significance: Interferences in cytoplasmic and peroxisomal lipid metabolism by IDH1R132 may contribute to the more favorable clinical outcome in patients whose tumors express mutations in the IDH1 gene. © 2015 Elsevier B.V. All rights reserved.
1. Introduction With the discovery of heterozygous mutations in the IDH1 gene in a large subset of human low-grade gliomas and in AML [1–3], much interest focused on the function of IDH1 and on the role of mutant IDH1 in the development of cancer. Mutations in IDH1 have now been reported in numerous cancers, most notably in gliomas (N70%), chondrosarcomas (~50%), cholangiocarcinomas (~20%) and AML (~10%) [1–5]. In gliomas and cholangiocarcinomas mutations in the IDH1 gene predict longer survival and a favorable prognosis [1,2,6]. IDH1 belongs to the family of isocitrate dehydrogenase enzymes which is comprised of 3 members: IDH1, IDH2 and IDH3 [7]. IDH1 is localized within the cytoplasm and peroxisomes [8,9]. IDH2 and IDH3 are located exclusively within the mitochondria [10]. All IDH enzymes catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate with Abbreviations: IDH1, isocitrate dehydrogenase 1; AML, acute myelogenous leukemia; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; 2-HG, 2 hydroxyglutarate; RCDP, rhizomelic chondrodysplasia punctata; NADP, nicotinamide adenine dinucleotide phosphate; NAD, nicotinamide adenine dinucleotide; TCA, tricarboxylic acid; DHAP, dihydroxyacetone phosphate ⁎ Tel.: +1 416 586 4800x8268. E-mail address: [email protected].
http://dx.doi.org/10.1016/j.bbagen.2015.04.014 0304-4165/© 2015 Elsevier B.V. All rights reserved.
the reduction of either NADP+ or NAD+ to generate NADPH or NADH respectively [7,11]. IDH1 and IDH2 utilize NADP+ as a cofactor and IDH3 utilizes NAD+ and functions in the tricarboxylic acid (TCA) cycle [7,10]. IDH1 and IDH2 can also catalyze the reverse reaction where αketoglutarate (in the presence of NADPH and CO2) is converted to isocitrate and NADP+ via reductive carboxylation [10,11]. The most frequent mutation in IDH1 found in cancer occurs at the substrate binding site at arginine 132 (R132) where R132 is replaced by either histidine (R132H), cysteine (R132C), serine (R132S), glycine (R132G), leucine (R132L) or glutamine (R132Q) [2,12]. The frequency with which each amino acid substitution at R132 occurs varies and depends on the type of cancer; the IDH1R132H mutation is the most frequent IDH1 mutation found in gliomas, whereas IDH1R132C is more often observed in cholangiocarcinomas and chondrosarcomas/enchondromas [1,2,4,5]. Mutation of R132 at the substrate binding site abolishes the wild-type activity of the enzyme [1,2] and induces a novel enzymatic activity which catalyzes the conversion of α-ketoglutarate to 2-hydroxyglutarate (2HG) utilizing NADPH as a cofactor [13]. The accumulation of 2-HG produced by mutant IDH1R132 may contribute to the development of cancer by inhibiting collagen maturation, upregulating HIF1α and inhibiting histone demethylases leading to increased histone methylation and altered gene expression [14–18].
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2. Lipid metabolism in the liver and adipose tissue One of the earliest functions ascribed to wild-type IDH1 was a role in lipid metabolism based on the observation that the enzymatic reaction of IDH1 produces NADPH [19]. IDH1 was discovered as early as 1950 when Hogeboom and Schneider [20] identified isocitrate dehydrogenase activity in the cytoplasm of liver tissue obtained from adult mice. These investigators noted the increased production of NADPH following the addition of isocitrate to rat liver cytosolic preparations. Early investigators hypothesized that the function of IDH1 may be to provide high levels of NADPH for lipid biosynthesis since NADPH is an obligatory reducing equivalent for the synthesis of fatty acids and lipids [7,19]. It was also hypothesized that IDH1 participates in a biochemical pathway in the rat liver where carbons derived from glutamate become incorporated into fatty acids [11]. Further suggestion that IDH1 plays a role in lipid metabolism came from the observation that a small (≤ 25%) pool of IDH1 is localized within rat liver peroxisomes [8]. Peroxisomes are organelles found in all mammalian cells with the exception of erythrocytes [21]. Their size (0.1–1.0 μm) and number varies depending on the tissue being examined [22]. Tissues that are highly active in the metabolism of lipids such as the liver have large and more numerous peroxisomes [22,23]. Peroxisomes contain over 50 enzymes; half of which participate in functions related to lipid metabolism such as βoxidation of very long chain fatty acids, α-oxidation of branched-chain fatty acids and the synthesis of ether-linked (plasmalogen) phospholipids [21,23,24]. Other functions of peroxisomes include purine and polyamine catabolism, amino acid metabolism and the synthesis of bile [21,23]. The name peroxisome was derived from the observation that hydrogen peroxide is produced and degraded within this organelle [25]. Hydrogen peroxide is formed during the first reaction in the βoxidation pathway and is then reduced to water by catalase [21,25]. For the early researchers, the hypothesis that IDH1 participates in cellular lipid metabolism was primarily based on correlative data obtained from tissues with high lipogenic activity such as the liver, adipose tissue and mammary gland. The advent of techniques in molecular biology which allowed genetic manipulation provided a more direct link between IDH1 and cellular lipid metabolism. Koh et al. [26] generated transgenic mice overexpressing IDH1 in the liver and adipose tissue under the rat phosphoenolpyruvate carboxykinase promoter. These
mice displayed a 35% increase in body weight, increased fat mass and adipocyte size, had fatty livers and increased serum cholesterol and triacylglycerols. The authors also showed that decreasing IDH1 mRNA in 3T3-L1 preadipocytes impaired adipocyte differentiation and reduced the lipid content of these cells. Conversely, mice transduced with IDH1 shRNA gained less weight when fed a high fat diet, had reduced fat mass and lower circulating triacylglycerols compared to control mice [27]. Consistent with these observations, Shechter et al. [28] showed that the IDH1 promoter can be activated by the lipogenic transcription factors SREBP-1a and SREBP-2 in HepG2 cells. 3. Lipid biosynthesis in tumors Studies examining the metabolism of glutamine in various cultured tumor cells have shown that IDH1 and IDH2 participate in the synthesis of acetyl-CoA from glutamine to support lipid biosynthesis in normoxia, hypoxia and when mitochondrial function is impaired [29–31]. Tumor cells avidly take up glutamine to sustain their growth and survival [32]. In both the cytoplasmic (IDH1) and mitochondrial (IDH2) pathways, glutamine is converted to glutamate and subsequently to αketoglutarate (Fig. 1) [29–31]. IDH1 and IDH2 convert α-ketoglutarate to isocitrate via reductive carboxylation. Isocitrate undergoes further conversion to citrate and subsequently to acetyl-CoA and oxaloacetate. Acetyl-CoA is then used as a building block for the synthesis of fatty acids (Fig. 1). In the mitochondria, this metabolic pathway is induced by the reversal of the TCA cycle (Fig. 1) [29–31]. Although acetyl-CoA derived from glutamine can be used for de novo synthesis of fatty acids and lipids, this may not be the only mechanism to increase lipid biosynthesis in tumors. Instead, tumor cells may increase the uptake of fatty acids [33]. Mutant IDH1R132H lacks the ability to convert αketoglutarate to isocitrate via reductive carboxylation [34]. 3.1. Phospholipid and peroxisomal metabolism in the brain and gliomas The brain is an organ rich with lipids where lipids comprise up to 50% of the brain's dry weight [35]. The most abundant lipids in the brain are the phospholipids, sphingolipids and sterols (cholesterol) with each lipid class containing numerous lipid species. The phospholipids are structural components of the plasma membrane and membranes of
Fatty acids
Acetyl-CoA + Oxaloacetate
Acetyl-CoA Oxaloacetate
Citrate
Malate
Citrate
Isocitrate
TCA Cycle
IDH3
Isocitrate
IDH2
α-Ketoglutarate
Fumarate
Succinate
Lipids
Succinyl CoA
Mitochondria
IDH1
IDH1-R132H
α -Ketoglutarate
Glu
Gln
Cytoplasm
Fig. 1. IDH1 and IDH2 mediate reductive carboxylation of α-ketoglutarate to isocitrate in the mitochondria and cytoplasm for the synthesis of lipids [29–31]. Carbons derived from glutamine (Gln) are incorporated into acetyl-CoA for fatty acid synthesis. In the TCA cycle, IDH3 catalyzes the conversion of isocitrate to α-ketoglutarate. IDH2 reverses the metabolite flux of the TCA cycle for the production of citrate from α-ketoglutarate. Citrate and α-ketoglutarate may exit the mitochondria and enter the cytoplasmic pathway. Due to the loss of wild-type IDH1 activity, mutant IDH1R132H (indicated in red) lacks the ability to interconvert isocitrate to α-ketoglutarate [34]. Glu = glutamate.
E. Bogdanovic / Biochimica et Biophysica Acta 1850 (2015) 1781–1785
intracellular organelles and also have roles in cell signaling. Phospholipids comprise the phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin. The PCs and PEs are the most abundant phospholipids in cellular membranes [36]. Recently, Esmaeili et al. [37] examined the changes in PCs and PEs by the expression of mutant IDH1R132H in glioma xenografts, U251 glioma cells and human glioma surgical specimens. Expression of mutant IDH1R132H was associated with significant increases in PCs and decreases in PEs in all three models. Similar results were obtained when the expression of IDH1 was knocked down using siRNA in E18.5 mouse astrocytes [38]. We examined the PCs and PEs in mouse brains expressing IDH1R132Q [12]. The wild-type activity of IDH1R132Q is abolished and similar to IDH1R132H, IDH1R132Q catalyzes the formation of 2-HG. Mice engineered to express IDH1R132Q heterozygously within the brain (in both neurons and glia) die during embryonic development as early as E15.5 and present with massive hemorrhaging and cell death [15]. In the brain, the majority (up to 70%) of PEs are in the form of ethanolamine plasmalogens [36]. Plasmalogen lipids are abundant in mammalian tissues and are either PEs or PCs that contain a fatty acyl chain attached to the sn-1 position of glycerol via a vinyl ether linkage [39]. In the brain, the ethanolamine plasmalogens are the predominant plasmalogen species [40]. The biosynthesis of plasmalogens initiates within peroxisomes and is completed in the endoplasmic reticulum [40]. Previous researchers hypothesized that IDH1 within peroxisomes participates in the synthesis of plasmalogen lipids [21]. Fig. 2 depicts the initial steps of plasmalogen biosynthesis in peroxisomes and the hypothesized involvement of IDH1. In mice expressing IDH1R132Q, early alterations in brain lipid composition were observed at E13.5 with up to 50% reductions in the ethanolamine plasmalogens (Table 1). The phosphatidylcholines (PCs) tended to be proportionally increased by the expression of IDH1R132Q (Table 2) consistent with the findings by Esmaeili et al. [37] obtained using various glioma samples expressing IDH1R132H. Changes in the levels of PCs, PEs and ethanolamine plasmalogens could be expected to affect the structure and function of the plasma membrane and those of intracellular organelles.
DHAP
Peroxisome α-KG IDH1-R132 Isocitrate
1-Acyl-DHAP
1-Alkyl-DHAP NADPH
IDH1
1783
Table 1 Ethanolamine plasmalogens are decreased in brains expressing IDH1R132Q. Total lipids were extracted from E13.5 brains obtained from wild-type (WT) and IDH1R132Q expressing mice and analyzed by LC/MS/MS. Chromatographic intensities (counts per second) were obtained for each of the lipids listed and expressed as a percentage of the value from WT brains. The intensities from WT brains were given a value of 100%. Values are means ± SD from n=5 samples. * Indicates statistical significance p b 0.05 (WT vs. IDH1R132Q). Plasmalogen
Wild-type
IDH1R132Q
16:0/18:1 16:0/18:2 16:0/22:6 18:0/16:0 18:0/18:1 18:0/18:2 18:0/22:6 18:1/16:0
100 100 100 100 100 100 100 100
63 ± 11⁎ 69 ± 12⁎ 69 ± 10⁎ 51 ± 17⁎ 64 ± 10⁎ 68 ± 19⁎ 65 ± 14⁎ 63 ± 13⁎
3.2. IDH1 and other lipid pathways in the brain and astrocytes Fig. 3 summarizes the major lipid classes in astrocytes. Astrocytes have a higher lipid content compared to neurons [41]. The phospholipids are the most abundant lipids in astrocytes and comprise up to 70% of the total lipids [41]. PC is the major phospholipid (constituting 36% of the total lipids) followed by PE (20% of the total cellular lipids). PI, PS and sphingomyelin are minor components (3–5%) [41]. Cholesterol is also abundant in astrocytes; making up 14% of the total lipid content. Cholesterol is a major lipid constituent of cellular membranes in the central nervous system and its synthesis is higher in astrocytes [36,41]. The generation of acetyl-CoA from glutamine via IDH1 [29–31] or from TCA cycle intermediates can be used to synthesize cholesterol (Fig. 3). The expression of IDH1 is regulated by SREBP — a transcription factor that upregulates genes involved in fatty acid and cholesterol biosynthesis [28]. Future studies should determine whether IDH1 participates in the synthesis of cholesterol from acetyl-CoA in astrocytes (Fig. 3). This can be determined by measuring cholesterol levels in tissues/cells expressing mutant IDH1R132 or silencing wild-type IDH1 expression using siRNA. The sphingolipids are a large group of lipids highly abundant in the brain particularly in myelin although in astrocytes they are a minor component. The sphingolipids comprise the ceramides, sphingomyelin (which is also a phospholipid), cerebrosides, sulfatides and gangliosides [36]. In astrocytes, low levels of sphingomyelin and gangliosides have been reported (b5% of the total lipids) [41]. We examined the levels of ceramides and sphingomyelin in E13.5 mouse brains expressing IDH1R132Q. There were large fluctuations in the levels of these lipids in brains expressing IDH1R132Q (unpublished observations) indicating that IDH1 may not directly regulate their synthesis/degradation. The varying levels of ceramides and sphingomyelin may be the consequence of alternate routing of lipids from other pathways affected by IDH1. For
NADP+ 1-Alkyl-G3P
Plasmalogen Synthesis in ER Fig. 2. IDH1 located in peroxisomes may provide NADPH for the synthesis of plasmalogen lipids [21]. The synthesis of plasmalogens begins in peroxisomes with the attachment of an acyl group (fatty acid) to the sn-1 position of dihydroxyacetone phosphate (DHAP) to generate 1-acyl-DHAP. In the subsequent step, the acyl group is exchanged for an alkyl group (fatty alcohol) to generate 1-alkyl-DHAP [39,40]. NADPH generated by IDH1 via the oxidative decarboxylation of isocitrate reduces 1-alkyl-DHAP to 1-alkyl-glycerol-3phosphate (1-alkyl-G3P) which is then further processed in the endoplasmic reticulum (ER) to generate choline and ethanolamine plasmalogens [21]. Due to the loss of wildtype IDH1 activity, mutant IDH1R132 (indicated in red) would not contribute NADPH for the reduction of 1-alkyl-DHAP. α-KG = α-ketoglutarate. Figure concept adapted from Lazarow [21].
Table 2 Increased phosphatidylcholine (PC) content in mouse brains expressing IDH1R132Q. Total lipids were extracted from E13.5 brains obtained from wild type (WT) and IDH1R132Q expressing mice and analyzed by LC/MS/MS. For each lipid listed, the chromatographic intensity obtained from one WT sample was designated 100%. All other values are expressed relative to the WT sample. Results are means ± SD from n=3–4 samples. * Indicates statistical significance p b 0.05 (WT vs. IDH1R132Q). PC
Wild-type
IDH1R132Q
16:0 30:0 32:1 32:2 34:0 34:4 36:4
100 ± 20 100 ± 17 100 ± 24 100 ± 10 100 ± 20 100 ± 15 100 ± 5
145 ± 5⁎ 142 ± 16⁎ 130 ± 16 119 ± 24 135 ± 10⁎ 124 ± 23 100 ± 12
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Fatty acids Cell Membrane
? Cholesterol
IDH1
Acetyl-CoA
Phospholipids
Fatty acids IDH1-R132
PC PE
PS PI
Citrate Ceramides Gangliosides
Sphingomyelin
Isocitrate IDH1 α-KG Fig. 3. The major lipid classes in astrocytes. The phospholipids are the most abundant lipids in astrocytes followed by cholesterol and the sphingolipids (ceramides, gangliosides and sphingomyelin). Sphingomyelin is both a sphingolipid and phospholipid. Acetyl-CoA generated from glutamine or TCA cycle intermediates can be used for the synthesis of cholesterol and fatty acids. Fatty acids may also be taken up from outside the cell and are incorporated into the phospholipids and sphingolipids. Mutant IDH1R132 (shown in red) increases the levels of PCs and decreases the PEs. Dashed arrows indicate intermediate steps have been removed for simplicity.
example, one pathway for the synthesis of sphingomyelin is from PC and ceramide (Fig. 3) [36]. Although PCs were higher in mouse brains expressing IDH1R132Q the levels of sphingomyelin were either increased or decreased compared to controls possibly due to the availability of ceramide (unpublished observations). 4. Peroxisomal metabolism and bone development It was recently reported that in mice the expression of IDH1R132Q in chondrocytes resulted in dwarfism and cartilaginous dysplasia of long bones, ribs and tracheal cartilage leading to death of these mice at birth or prior to weaning [12]. This phenotype is similar to mice lacking the Pex7 gene [42]. Pex7 is a peroxisomal receptor important for the translocation of proteins into the peroxisomal matrix. Mice lacking Pex7 are defective in importing enzymes involved in plasmalogen biosynthesis into peroxisomes and consequently have significant reductions in plasmalogen lipids (up to 90%) [42]. Hallmarks of Pex7 deficiency are skeletal and central nervous system abnormalities. The majority of Pex7−/− mice die at birth or prior to weaning and exhibit dwarfism and delayed or absent ossification [42]. In humans, mutations in the PEX7 gene and peroxisomal dysfunction are the underlying cause of rhizomelic chondrodysplasia punctata (RCDP), an autosomal recessive disease [43–45]. Patients with RCDP have significantly reduced plasmalogen levels and are characterized by dwarfism, skeletal dysplasia, abnormal mineralization of cartilage and central nervous system impairments [40,46]. The majority of RCDP patients do not survive beyond childhood [46]. Expression of mutant IDH1R132Q in chondrocytes may impair the synthesis of plasmalogen lipids and/or interfere with peroxisomal metabolism which may be important during growth and development. 5. Conclusions and future directions Since the first report of IDH1 in 1950, a role in lipid metabolism has been hypothesized particularly in tissues such as the liver, mammary gland and adipose tissue. The recent discoveries that IDH1R132H interferes with the metabolism of phospholipids in gliomas and that IDH1 activity could participate in the synthesis of acetyl-CoA from glutamine in hypoxic tumors highlights roles for IDH1 in lipid metabolism in a broad spectrum of tissues. Future studies will reveal what roles IDH1 may have in lipid metabolism in other tissues and cancers such as AML and cholangiocarcinomas. Future studies will also further our
understanding of the function of peroxisomal IDH1 and on the role of peroxisomal IDH1 in tumors. Recently, small molecule inhibitors targeting mutant IDH1R132H which specifically block the ability of mutant IDH1R132H to produce 2-HG have been generated and show promise in inhibiting tumor growth [47,48]. When U87 glioma cells were transfected with IDH1R132H and treated with the IDH1R132H inhibitor compound AGI-5198, the production of 2HG was suppressed but the metabolic alterations that accompanied the expression of mutant IDH1R132H were not reversed [49]. Forced expression of mutant IDH1R132H may interfere with the activity of endogenous wild-type IDH1. These observations suggest the possibility that the loss/ interference with the activity of wild-type IDH1 may underlie the changes in metabolites that were observed. Similarly, the deregulation of phospholipids in gliomas expressing mutant IDH1R132H could be observed when the expression of wild-type IDH1 was knocked-down using siRNA [37,38]. Future studies using small molecule inhibitors that block 2-HG production by mutant IDH1R132H should reveal whether cellular accumulation of 2-HG alters the metabolism of lipids within cells. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements The author is grateful to Dr. Tak Mak at the University of Toronto for providing E13.5 mouse brains expressing IDH1R132Q and for insightful discussion. References [1] D.W. Parsons, S. Jones, X. Zhang, J.C. Lin, R.J. Leary, P. Angenendt, P. Mankoo, H. Carter, I.M. Siu, G.L. Gallia, A. Olivi, R. McLendon, B.A. Rasheed, S. Keir, T. Nikolskaya, Y. Nikolsky, D.A. Busam, H. Tekleab, L.A. Diaz, J. Hartigan, D.R. Smith, R.L. Strausberg, S.K.N. Marie, S.M.O. Shinjo, H. Yan, G.J. Riggins, D.D. Bigner, R. Karchin, N. Papadopoulos, G. Parmigiani, B. Vogelstein, V.E. Velculescu, K.W. Kinzler, An integrated genomic analysis of human glioblastoma multiforme, Science 321 (2008) 1807–1812. [2] H. Yan, D.W. Parsons, G. Jin, R. McLendon, B.A. Rasheed, W. Yuan, I. Kos, I. BatinicHaberle, S. Jones, G.J. Riggins, H. Friedman, A. Friedman, D. Reardon, J. Herndon, K.W. Kinzler, V.E. Velculescu, B. Vogelstein, D.D. Bigner, IDH1 and IDH2 mutations in gliomas, N. Engl. J. Med. 360 (2009) 765–773. [3] E.R. Mardis, L. Ding, D.J. Dooling, D.E. Larson, M.D. McLellan, K. Chen, D.C. Koboldt, R.S. Fulton, K.D. Delehaunty, S.D. McGrath, L.A. Fulton, D.P. Locke, V.J. Magrini, R.M.
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