Accepted Manuscript Title: Nuclear receptors as regulators of stem cell and cancer stem cell metabolism Author: Zoltan Simandi Ixchelt Cuaranta-Monroy Laszlo Nagy PII: DOI: Reference:
S1084-9521(13)00107-9 http://dx.doi.org/doi:10.1016/j.semcdb.2013.10.002 YSCDB 1470
To appear in:
Seminars in Cell & Developmental Biology
Please cite this article as: Simandi Z, Cuaranta-Monroy I, Nagy L, Nuclear receptors as regulators of stem cell and cancer stem cell metabolism, Seminars in Cell and Developmental Biology (2013), http://dx.doi.org/10.1016/j.semcdb.2013.10.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nuclear receptors as regulators of stem cell and cancer stem cell metabolism
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Zoltan Simandi1, Ixchelt Cuaranta-Monroy1 and Laszlo Nagy1,2#
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Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, University of Debrecen, Medical and Health Science Center MTA-DE “Lendulet” Immunogenomics Research Group
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E-mail: [email protected] Phone: +36-52-416-432
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Fax: +36-52-314-989
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Contact Information:
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Egyetem tér 1., Debrecen, Hungary, H-4012
Highlights
Nuclear receptors (NRs) are key links between metabolism and cell fate decisions. ESRRB, DAX-1, LRH-1, TR4, NGF1-B, LXRb and RARs are NRs expressed in pluripotent embryonic stem cells. ESRRB, DAX-1 and LRH-1 are regulators of metabolism in embryonic stem cells. TR4, NGF1-B, LXRb and RARs are putative regulators of stemness. The discussed NRs play a role in tumor progression and might be metabolic regulators of cancer stem cells.
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Abstract
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Cellular metabolism is underpinning physiological processes in all cells. These include housekeeping functions as well as specific activities unique to a particular cell type. A growing number of studies in various experimental models indicate that metabolism is tightly connected to embryonic development as well. It is also emerging that metabolic processes have regulatory roles and by changing metabolism, cellular processes and even fates can be influenced. Nuclear receptors (NRs) are transcription factors, responding to changes in metabolites and are implicated in diverse biological processes such as embryonic development, differentiation, metabolism and cancer. Therefore, NRs are key links between metabolism and cell fate decisions. In this review, we introduce ESRR , DAX-1 and LRH-1 as putative regulators of metabolism in pluripotent embryonic stem cells. We also discuss the role of TR4, NGF1 , LXR and RARs in stemness. In addition, we summarize our current understanding of the potential roles of NRs in cancer stem cells.
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Key words: metabolism, nuclear receptors, pluripotency, embryonic stem cells, cancer stem cells
Inroduction
Unicellular organisms or cells within multicellular organisms need to make decisions throughout their lifespan. The constantly changing environment dictating whether a cell stays quiescent, proliferate or differentiate [1]. It is well established in yeast that nutrient levels are the major determinants of cell fate. Recent evidence suggests that cellular metabolism determines the cell fate specification of mammalian cells as well [2-4]. Murine embryonic stem cells (ESCs) provide a unique model system to understand the importance of metabolic regulation in cell fate decisions in vertebrates. ESCs are derived from the inner cell mass (ICM) of developing blastocysts and display an almost unlimited proliferation capacity in vitro [5]. Moreover, this cell type retains the ability to contribute to all cell lineages. The rate of cell division of mouse ESCs is very high; a cell doubling takes place in every 4-5 hours. This is even faster than any fast growing cancer cell. Therefore, ESCs have a high
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requirement for carbon, hydrogen and nitrogen to support biosynthesis of cell building blocks required for replication. Accordingly, metabolic features of ESCs are significantly different from that of terminally differentiated cell types (reviewed in [6]).
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Metabolism in pluripotent stem cells
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One of the major differences is the way these cells can produce sufficient energy. Oxidative consumption of glucose does not enable a fast rate of ATP production. In contrast, partial breakdown of glucose through glycolysis and shunting of intermediates through the pentose phosphate pathway provide sufficient NADPH and energy for rapid cell proliferation. Oxidative capacity is reduced and glycolysis-dependent anabolic pathways are enriched in stem cells (Figure 1). This underlines the not so critical role of mitochondria in these cells. In fact, ESCs have a low number of mitochondria and harbor a sparse mitochondrial infrastructure with immature cristae and limited perinuclear localization [2]. ESCs are routinely cultured under atmospheric (20%) oxygen tensions; however these cells are originally derived from embryos which reside in a 3-5% oxygen (hypoxic) environment. However the mechanism is just partly understood, emerging pieces of evidence suggest that such hypoxic conditions in cell culturing increase the quality of stemness [7, 8] and promotes the reprogramming of differentiated cells to induced pluripotent stem cells [9]. Under low oxygen tension hypoxia-inducible factors (HIFs), key transcription factors in hypoxia, are activated and directly regulate transcription of genes involved in self-renewal [10]. Different isoforms of the HIF-family are also known to regulate the transcription of glycolytic enzymes. For instance, HIF1 upregulates the expression of pyruvate dehydrogenase kinase (PDK), which is in turn, inhibits glucose oxidation [11]. Indeed, human ESCs cultured at 5% O2 consume significantly more glucose, less pyruvate and produce more lactate compared to those maintained at 20% O2 [12]. Comparison of ESCs and cells from early stages of differentiation has identified differences in lipid metabolism as well. Stem cells show a global enrichment of unsaturated fatty acid metabolites in the pluripotent state. While high levels of unsaturated fatty acids preserve pluripotency, addition of saturated metabolites to the ESCs is known to accelerate the differentiation program [13]. A recent study nicely demonstrates that lipid biosynthesis underlies the proliferative capacity of stem and progenitor cells in the brain [14]. Another recently discovered example of unique metabolic feature of stem cell is related to the distinct mode of amino acid catabolism. Threonine dehydrogenase (Tdh), an enzyme that catalyzes threonine oxidation (Thr), shows more than 200 times higher expression in ESCs. The restriction of Thr or inhibition of Tdh results in decreased accumulation of SAdenosylMethionine (SAM) that leads to decreased trimethylation of histone 3 lysine 4 (H3K4me3) and thus abolished stem cell growth [15].
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Are nuclear receptors regulating metabolism in stem cells?
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The above listed examples (summarized in Figure 1) strongly suggest that metabolic signals most likely play an essential role in regulation of stemness, pluripotency and cell fate decisions and the regulation of these signaling pathways are essential for proper embryonic development. A central question is how embryonic stem cells sense, regulate and respond to these environmental cues. Several lines of evidence suggest that a family of transcription factors, called nuclear receptors, might play important roles in recognizing different signals and convert it to transcriptional outcome and epigenetic changes. A goal of this review is to provide insight into the metabolic regulatory role of nuclear receptors in stem cell biology with an outlook to cancer stem cells.
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The human and mouse genomes encode 48 and 49 nuclear receptors, respectively. Intense research on NRs in the last two decades has uncovered diverse roles of these receptors in developmental biology and metabolism [16].
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Nuclear receptors are ligand activated transcription factors. With a few exceptions, such as DAX1 and SHP1, all NRs expressed in vertebrates share a common domain structure. A highly conserved DNA-binding domain (DBD) ensure the binding to receptor-specific DNA sequences, while a less conserved ligand-binding domain (LBD) determines the signal specific action. Several small lipophilic molecule are known to function through NRs, however still a large fraction of nuclear receptors, often called orphan receptors, has no identified ligand. Based on their ligands and mechanistic action NRs are diveded into four groups: 1, steroid receptors 2, retinoid X receptor heterodimers 3, dimeric orphan receptors 4, monomeric orphan receptors (for detailes on grouping and nomenclature visit the website of NURSA (www.nursa.org)). DAX-1, LRH-1, SF1 and ESRR are linked directly to the maintenance of pluripotency in mESCs (reviewed in [17]) and recent genome-wide studies allow now a deeper insight into their regulatory role. A systematic profiling of NRs [18] and our independent gene expression characterization of mESCs (re-analysis of microarray data published by [19]) reveal in addition the high expression of TR4, NGF1 , LXR , RAR and RAR in undifferentiated ESCs (Figure 2). The importance of these nuclear receptors in stem cell biology is largely unknown. We will here summarize how DAX-1, LRH-1 and ESRR might regulate pluripotency and differentiation from a metabolic point of view. To further enhance the possible connection between these NRs, metabolism and pluripotency, we re-analyzed available genome-wide chromatin-immunoprecipitation data (ChIP-chip and ChIP-seq) and included several putative metabolic targets, that are likely to be regulated by these factors. Moreover, we sum up our recent knowledge on the TR4, NGF1 , LXR and RARs in the context of development and metabolism.
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DAX-1 (NR0B1) DAX-1 is a unique member of the NR family. Instead of binding to regulatory DNA sequences directly it appears to bind to other transcription factors and act mainly as a co-repressor [20].
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DAX-1 has a restricted expression pattern to the hypothalamic-pituitary-adrenal/gonadal-axis and was shown as an important regulator of steroidogenesis, gonadal development and sex determination [21]. Forming a pair with SF-1 (steroidogenic factor-1), an other NR family member, DAX-1 is playing a role in the transcriptional regulation of enzymes involved in conversion of cholesterol to steroid hormones [22]. The first indication of an essential role of DAX-1 in embryogenesis and pluripotency was revealed by the difficulty encountered in establishing DAX-1 knockout mice [23]. DAX-1 was identified as a highly expressed gene in ESCs and gene-silencing of DAX-1 was found to result loss of pluripotency of these cells [24].
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A recent analysis identified DAX-1 binding genomic regions in ESCs as part of the transcriptional network of pluripotency [25]. This data is valuable to get information how DAX-1 can regulate pluripotency. The list of putative DAX-1 regulated genes are available as a Supplement of the above manuscript. In agreement with the repressor function of DAX-1, several differentiation-related genes can be find on the list, such as members of the Hox-genes (Hoxa2, Hoxa9, Hoxa11, Hoxb4, Hoxc10 and Hoxc12), Wnt-signal (Wnt11, Wnt16, Wnt8a) and early lineage-specific markers (Pax6, Stra8, Foxa1, Foxd3, Lefty1, T, Rest). However, promoter region of stem cell marker Oct3/4 and Nanog are also bound by DAX-1, indicating its potential contextdependent activator function as well. The list contains several genes which are known to be involved in metabolic processes, such as Apoa2, Apoe, Atp5a1, Pgm2, Pgam1, Fabp3, etc. Importantly, members of stem cell related metabolic pathways are also represented in force. Glucose transporter (Slc2a3), much of glycolytic enzymes (Hk3, Gpi1, Pfkp, Gapdh, Pgk1, Ldhb, Pdhb), enzyme in pentose phosphate pathway (Pgls), nucleotide synthesis (Prps1), metabolism of threonine (Tdh, Gcat) and fatty acid synthesis (Hmgcr, Dhcr7) are all putative DAX-1 targets (Figure 1, Table 1). Based on these findings it is secure to say that DAX-1 has a largely uncharacterized metabolic regulatory function in stem cells.
LRH-1 (NR5A2)
Liver receptor homolog-1 (LRH-1) has been implicated in diverse biological processes, including bile acid metabolism, steroidogenesis and cell proliferation [26]. In adult, LRH-1 is known to be highly expressed in liver and promotes expression of genes involved in hepatic cholesterol uptake and efflux, HDL formation and fatty acid synthesis [27]. LRH-1 also controls the first step of hepatic glucose uptake through direct transcriptional regulation of the glucokinase gene. This suggests that LRH-1, at least in hepatocytes, acts as a critical regulatory component of the glucose-sensing system [28].
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ESRR
(NR3B2)
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Importantly, LRH-1 is also expressed during early mouse embryogenesis. LRH-1-/- embryos display growth retardation, epiblast disorganization, a mild embryonic-extraembryonic constriction, as well as abnormal thickening of the proximo-posterior epiblast [29]. LRH-1 has been identified as a key transcription factor in cellular reprogramming [30]. Similar to DAX-1, there is available genome-wide study for LRH-1, in which the LRH-1 occupied regions have been identified [31]. We re-analyzed this dataset and annotated the LRH-1 binding sites to the closest genes. Considering the limitations of this type of analysis, we performed a prediction of enriched pathways (Table 2). Several genes, involved in various metabolic processes, seem to be regulated directly by LRH-1. Genes encoding enzymes of glycolysis and gluconeogenesis (Hk2, Pfkm, Pgam2, Ldha, Ldhc, Aldh3a2, Aldh9a1), pentose phosphate pathway (H6pd, Rpia), fatty acid biosynthesis (Acacb, Acaca) or fatty acid metabolism (Aldh3a2, Aldh9a1, Acsl1, Cyp4a31, Hadh) are all possess LRH-1 occupied region in the close proximity (Figure 1, Table 1). Geneexpression studies combined with LRH-1 ChIP data will complete our understanding on the LRH-1-dependent regulatory mechanisms in ESCs. Inverse agonists that have been recently described for the LRH-1 [32], might also accelerate discoveries related to biological function of this nuclear receptor.
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There are three isoforms of the ESRR genes in mouse and human cells: ESRR , and . ESRRs share a homology with estrogen receptors (ERs), but ESRRs do not bind estrogen or other known physiological ligands [33]. ESRRs are typically expressed in tissues with high metabolic demands and they have a predominant role in orchestrating mitochondrial biogenesis and cellular energy metabolism such as oxidative phosphorylation (OxPhos), tricarboxylic acid (TCA) cycle, fatty acid oxidation and ATP synthesis. ESRRs directly acivate transcription of genes involved in these metabolic processes [34, 35]. In ESCs only one isoform, ESRR shows high expression (Figure 2). However, this one seems to be indispensable for pluripotency [36]. Expression of ESRR is regulated by Nanog, a key transcription factor of ES cells [37]. Loss of ESRR results spontaneous differentiation of ESCs while the overexpression of ESRR has been shown to promote reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells [38]. Only limited information is available why ESRR is so important in the maintenance of pluripotency. Recent genome-wide studies identified the binding sites of ESRR in mouse embryonic stem cells. The analysis revealed that ESRR -binding sites partly overlap with the Nanog-Oct3/4-Sox2 transcription factor binding sites [39], however large fraction of binding sites are independent from the core complex. ESRR was previously suggested to bind as a monomer [40]. In contrast, a detailed analysis identified three equally enriched full-site motifs, DR0, DR5, and DR8, but no half-site enrichment, suggesting that ESRR is likely to bind in a form of dimer to DNA [41]. So far, ESRR ChIP-seq data has not been analyzed in a deeper biological context. To get a preliminary
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insight into the ESRR -mediated functions in ESCs, we re-analyzed an available ChIP-seq data [39] and in Table 3 summarized the top biological functions identified for the putative ESRR regulated genes. This clearly indicates that ESRR beside the regulation of pluripotency, plays a role in the regulation of metabolic processes. Indeed, much of the glycolytic, lipid- and amino acid-related enzymes that are important in stemness, are seems to be regulated by ESRR (Figure 1, Table 1). So far, this is the first indication that ESRR regulate the metabolic state of ESCs and raise the possibility of an ESRR -dependent metabolic regulation of stem cell pluripotency. However, these preliminary indications of ESRR mediated regulation of stem cell metabolism will requires additional analysis and validation.
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TR4 (NR2C2)
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Our genome-wide gene expressional data suggest that TR4 is expressed at a higher level in stem cells (Figure 2) and a recent study implicated also its functional importance in pluripotency [42]. Importantly, TR4 -/- mice display significant growth retardation [43]. In addition, gene disruption results in defects of spermatogenesis, in testis and in cerebellar development. Importantly, TR4 is expressed in metabolic tissues, including liver, muscle, brown and white adipose tissues and regulate metabolic genes, such as apolipoprotein E, enoyl-CoA hydratase, acyl-CoA oxidase [44, 45]. Moreover, TR4 also regulates phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme controlling the rate of gluconeogenesis [46]. Thus, its high expression in stem cell might be essential in the regulation of metabolism via controlling mainly carbohydrate metabolism.
NGF1
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A recent study has determined the TR4 binding regions in four different cell types [47]. The majority of TR4 targets are shared between the different cell types and responsible for RNA metabolism and protein translation. However, cell-type specific TR4 binding could be also observed and these results further confirmed that TR4 is regulate genes involved in cellular metabolism. Genome-wide studies of TR4 binding in ESCs are not available yet. Loss of function experiments will be also required to identify the TR4 regulated genes in ESCs and in early embryonic differentiation. This might be helpful to understand the phenotypic alterations of the TR4-/- animals as well.
(NR4A1)
The NR4A family includes three highly homologous isoforms, NR4A1, NR4A2 and NR4A1, also known as Nur77, Nurr1 and Nor1, respectively. X-ray crystallography studies suggest that the ligand-binding pocket of these receptors is unable to accommodate ligands [48]. Previous studies have pointed to NR4A1 as a transcriptional regulator of glucose metabolism in liver and skeletal muscle [49]. Moreover, later studies demonstrated that Nur77 also regulates hepatic cholesterol metabolism [50]. Nur77 modulates lipid metabolism in hepatocytes by 7
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decreasing the expression of SREBP1c, which is a master regulator of triglyceride metabolism. In the context of developmental biology, Nur77 has been shown to be involved in cellular differentiation, apoptosis, proliferation, T-cell function.
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Higher expression level of NGF1 in ESCs is also indicated by our data (Figure 2). However, it is still very tentative to suggest NR4A1 as a metabolic regulator of stemness. ChIP-seq experiments in combination with transcriptome analyses will establish how Nur77 modulates the expression profile of a specific cell-type under defined conditions.
RAR alpha and gamma (NR1B1 and NR1B3)
LXR
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Three genes code for receptors that functionally bind retinoic acid (RA): RAR , and . Retinoic acid (RA) acts as an important signaling molecule during embryonic development and is commonly used for induction of in vitro differentiation of ESCs [51]. Ligands for RAR activation are being synthetized upon LIF removal and induction of differentiation [52]. These formally suggest that RARs, present in ESCs, should fulfill rather an inhibitory role than activator function in transcription. Indeed, a well-accepted model is that in the absence of ligand RAR and RXR dimers bind to DNA and interact with co-repressors to repress transcriptional events. Active repression by unliganded RARs has been proposed in development [53]. To understand the function of unliganded RARs is stem cells, genome-wide characterization of RAR genomic binding has been carried out [54]. Analysis of the RAR-bound sites shows that among the enzymes summarized in Figure 1 and Table 1, only Ldhb and Pdk1 seem to have RAR occupied promoter region. Validation of these targets will be required to confirm the putative role of RARs in the metabolic regulation of undifferentiated ESCs.
(NR1H2)
The liver X receptors form permissive heterodimers with retinoid X receptors (RXRs) and are important regulators of lipid metabolism [55, 56]. The LXR family consists of the two subtypes: LXR and LXR . LXR and LXR are detected in the liver starting at 11.5 days postcoitum [57]. Later, LXR expression remains high in organs involved in lipid homeostasis, such as liver, intestine, and brown adipose tissue, whereas LXR is more ubiquitously expressed and enriched in tissues of neuronal and endocrine origin. LXR -deficient mice are viable and show minor metabolic differences as compared to the wild-type [58]. As a remarkable difference, reduced fertility has been shown [59]. Oxysterols, oxidative derivatives of cholesterol has been identified as physiological ligands for LXRs, thus LXRs seems to act as a cholesterol sensors. Treatment of rodents with synthetic LXR ligands results in decreased hepatic gluconeogenesis and increased lipogenesis, indicating that
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LXR serves as a transcription factor that integrates liver carbohydrate and lipid metabolism [60]. A recent genome-wide study has identified LXR and RXR binding regions in mouse liver [61].
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Taken together these results suggest that however Lxr is expressed in ESCs and might regulate metabolic pathways, it is unlikely that would be essential for pluripotency. In the same time, experimental evidence is required to confirm this statement.
Cancer stem cells and metabolism
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Numerous studies have revealed the existence of crosstalk between various nuclear receptor signaling pathways. One interesting example in the context of metabolism and differentiation is the potential link between LXR-dependent cholesterol metabolism and retinoic acid biosynthesis. Cholesterol metabolites, predominantly the oxysterols were identified that induces the expression of retinal dehydrogenases (RALDHs) via upregulation of sterol regulatory element binding protein-1c (SREBP1c) [62]. RALDHs synthesize retinoic acid (RA) from retinal [63]. RA is known to critically important for embryogenesis and also involved in proliferation, differentiation and apoptosis. However this crosstalk was mainly characterized in liver cells results in the same study also raise a similar mechanism in embryonic carcinoma cells. Existence of such a crosstalk between LXR and RAR signaling might play important roles in the metabolic switch when stem cell differentiation starts.
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The pluripotent nature of various cancers has been recognized early on [64]. Overall, there are two major cancer development theories as far as stem cell involvement is concerned: origin from cancer stem cells (CSCs) and de-differentiation of mature cells. The first one has been published by Furth et al., suggesting that a single cell from a tumor can initiate a new tumor in a recipient mouse [64]. Since then several studies have been performed to investigate the theory, which in principle states that the different tumor populations arise from a delimitated number of cells which conserves the same characteristics than stem cells in adult tissues [65]. CSCs can occur in diverse solid tumors, such as glioblastomas and breast cancer, but their existence has been reported in acute myeloid leukemias (AML) as well [65, 66]. A number of markers has been used for the isolation of CSCs, such as CD133 or SSEA-1, however difficulties of isolating pure populations of CSCs are still limiting our understanding of these cell populations. Elucidation of the pathways that regulate the maintenance and survival of CSCs is important for the development of novel therapies. Metabolic pathways have recently been implicated in governing the function of CSCs. In general, cancer cells have very similar metabolic features compared to ESCs [67]. Glucose metabolism is typically characterized by a shift from aerobic, oxidative metabolism to a glycolytic pathway. This aerobic glycolysis phenotype is known as the Warburg effect [68]. Metabolic switch to aerobic glycolysis is driven primarily by oncogenic signals and transcription factors. HIF1 is one of the most studied one, either the intratumoral hypoxic condition or genetic defects results in its stabilization and downstream regulatory
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function [69]. Identification of additional factors that play a role in metabolic switch of cancer cell is one of the hot topics in research.
Nuclear receptors as potential targets in cancer (stem) cell metabolism
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NRs have been extensively studied and linked directly to pathogenesis and treatment of various cancers; however their expression profile and putative role in cancer stem cells remained completely unknown. Importantly, CSCs have ESC-like transcriptional programs [70], thus detection of the above discussed pluripotency-related NRs, such as ESRR , LRH-1, DAX-1 are not unexpected in CSCs.
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Interestingly, many of these NRs have been already linked to poor prognosis of cancer. LRH-1 was shown to promote motility and invasiveness in breast cancer cells [71] and regulate steroidogenic gene transcription in endometrial cancer cells [72]. LRH-1 transcription is also activated in human pancreatic cancer cells [73]. The other stem cell specific nuclear receptor DAX-1 has been also recognized as a putative target in cancer treatment [74]. It is a direct target of the EWS/FLI1 oncoprotein, and found to be highly expressed in Ewing’s tumors. Genes implicated in cell cycle progression are regulated by DAX-1. ESRR , and are identified coactivating factors of HIF and thus might directly modulate HIF-mediated gene transcription of cancer cells. Further studies are required to helpus understand which pathways are regulated by NRs in cancer cells and CSCs. A recent study applied ChIP-seq to map the in vivo genome-wide binding (cistrome) of NRs in both normal and cancer cells [41]. Such studies combined with gene expression data will bring us closer to understand how NRs mechanistically contribute to cancer development. The above stated important role of NRs in cancer biology and their well established role in metabolism pose many interesting questions: most importantly whether NRs play any role in cancer cells to sense and regulate the level of nutrients to facilitate tumor development and progression. There are growing number of pieces of evidence indicating a direct relationship between NRs, metabolism and cancer. Anti-estrogen therapy for breast cancer was the first targeted therapy used in any type of cancer. Since its discovery, several metabolic pathways affected by anti- estrogens in cancer cells has been identified [75]. Activation of LXR signaling by oxysterols is an other example of how NR ligands can be used to target metabolic pathways in the treatment of various cancers [76]. Oxysterols are known to interfere with proliferation and initiate cell death of many cancer cells. Oxysterols and LXRsignaling might also help to treat “incurable” conditions, such as glioblastoma multiforme (GBM). GBM, a WHO grade IV astrocytoma, is the most aggressive and common primary brain tumor in adults. The median survival of GBM patients is approximately 1 year. Importantly, LXR
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agonists have been shown to inhibit GBM growth and promote tumor cell death, and LXR was initiated as a novel antitumor agents in glioblastoma [77, 78]. Role of NR agonists has been also studied and their roles confirmed in the metabolism and gene expression regulation of breast cancer stem cells [79, 80].
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A proof of principle for NR-mediated cancer therapy is the use of all-trans retinoic acid (atRA) treatment in AML. In AML the presence of leukemia stem cells has been demonstrated. It has been shown that they are hierarchically organized due to the heterogeneity in their self-renewal capacity and in longevity of the produced clone [81-83]. In this context, Alcalay et al., found that expressing some fusion proteins in myeloid precursors, previously observed in murine models of AML which includes the transcription factor RAR , the gene expression pattern was congruent with the induction of genes related to the maintenance of the stem cell phenotype and repression of DNA repair genes. Importantly, metabolism was one of the functional classifications of AMLfusion protein target genes and some of the identified genes could be validated in patient samples [84].
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These examples provide evidence that targeting metabolic pathways through the activation of NRs may represent effective strategies for eradicating CSCs.
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Recent studies are just beginning to show the importance of metabolism in cell fate determination, including cancer development. Role of nuclear receptors are still poorly characterized in the metabolic regulation of pluripotency. The recent development of highthroughput sequencing methodologies such as RNA-seq and ChIP-seq combined with metabolomics analyses holds the promise to provide more accurate gene expression measurements and deeper mechanistic insights into the regulatory role of nuclear receptors in cellular metabolism. This knowledge can be potentially turned into novel therapeautic approaches later.
Acknowledgement
The authors would like to acknowledge Erik Czipa and Dr. Endre Barta for the contibution in data analyzis, and the Nagy laboratory for discussions and comments on the manuscript. L.N is supported by a grant from the Hungarian Scientific Research Fund (OTKA K100196), and TÁMOP422_2012_0023 VÉD-ELEM implemented through the New Hungary Development Plan co-financed by the European Social Fund and the European Regional Development Fund. Z.S is a recipient of TÁMOP-422/B10/1_2010_0024 grant.
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Figure 1.
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Figure 1. Metabolism in pluripotent embryonic stem cells (ESCs). Rapidly proliferating pluripotent stem cells has unique metabolism. I. ATP production: ATP synthesis is decoupled from O2 consumption and depends on glycolysis. Gylcolytic enzymes: hexokinase (HK), glucose6-phosphate isomerase (GPI), phosphofructokinase (PFK), aldolase (ALDO), triosephosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), pyruvate dehydrogenase (PDH), lactate dehydrogenase (LDH). II. Nucleotide synthesis: Pentose phosphate pathway (Pentose PP) is active in ESCs and by converting glucose6-phospate (G6P) into ribose-5-phosphate for nucleotide synthesis, it also produces NADPH. Pentose PP enzymes among others: hexose-6-phosphate dehydrogenase (H6PD), 6phosphogluconolactonase (PGLS), ribose 5-phosphate isomerase A (RPIA), transketolase (TKT) Phosphoribosyl pyrophosphate synthetase 1 (PRPS1) catalyzes the phosphoribosylation of ribose 5-phosphate and necessary for purine metabolism. CAD encodes a trifunctional protein that is involved in pyrimidine nucleotide synthesis. III. Amino acid metabolism: ESCs requires the catabolism of L-threonine (Thr). The degradation of L-threonine to glycine consists of a two-step biochemical pathway involving the enzymes L-threonine dehydrogenase (Tdh) and 2-amino-3ketobutyrate coenzyme A ligase (GCAT). Glycine dehydrogenase (GLDC) is involved in the degradation of glycin. IV. Lipid metabolism: Unsaturated fatty acids preserve the pluripotency. 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and 7-dehydrocholesterol reductase (DHCR7) are enzymes for cholesterol synthesis. V. Epigenetic regulation: Acetyl-CoA (AcCoA) and S-AdenosylMethionine (SAM) are substrates for histone acetylation and methylation, respectively VI. Hypoxia: hypoxic culture conditions results the stabilization of hypoxia inducible factor 1 alpha (HIF1 ) and enhance the transcription of pyruvate dehydrogenase kinase (Pdk). Pdk inhibits glucose oxidation. Out of all the shown enzymes, only the marked (*) ones are not regulated by DAX-1, LRH-1 and/or ESRR (see Table 1).
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Figure 2.
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Figure 2. Per chip normalized signal intensity data of nuclear receptors in undifferentiated ESCs. Values of all entities and some known embryonic stem cell markers are also shown. Red line indicates the median value of all entities. One dot represents one gene. Gene symbols on the top of the figure indicate the dominantly expressed genes from each group and are considered to be expressed in stem cells. Nuclear receptors shown in red are discussed in the text.
Table 1. Summary of DAX-1, LRH-1 and ESRR Gene symbol
GLUT1/3
HK GPI
regulated metabolic genes DAX-1
LRH-1
ESRR
Yes
Yes
(Slc2a1)
(Slc2a1, a3)
Yes
Yes
Yes
(Hk3)
(Hk2)
(Hk1, 2, 3)
Facilitated glucose transporter, member Yes 1 (Slc2a3) Hexokinase glucose-6-phosphate isomerase
Yes
Yes
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(Gpi1)
(Gpi1)
Yes
Yes
Yes
(Pfkp)
(Pfkl, m, p)
(Pfkl, m, p)
phosphofructokinase
PFK
Yes
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aldolase
ALDO
(Aldoa) Yes
PGK
phosphoglycerate kinase
Yes
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glyceraldehyde-3-phosphate dehydrogenase
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GAPDH
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triosephosphate isomerase 1
TPI1
LDH
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Yes
lactate dehydrogenase
PGLS RPIA* TKT PRPS1
CAD*
hexose-6-phosphate dehydrogenase
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H6PD
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(Ldh )
6-phosphogluconolactonase
Yes
(Tpi1)
Yes Yes (Pgk1)
Yes
Yes
(Ldh , Ldh )
(Ldh , Ldh )
Yes
Yes Yes
ribose 5-phosphate isomerase A transketolase
Yes
phosphoribosyl pyrophosphate synthetase 1
Yes
carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase Yes
PDH
pyruvate dehydrogenase complex (Pdhb)
PDK
pyruvate dehydrogenase kinase
Yes
Yes
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(Pdk1)
(Pdk1, 2, 4)
Yes
Yes
GLDC*
glycine dehydrogenase
TDH
L-threonine dehydrogenase
Yes
HMGCR
3-hydroxy-3-methylglutaryl-CoA reductase
Yes
DHCR7
7-dehydrocholesterol reductase
Yes
HIF
hypoxia inducible factor
Yes
Yes
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glycine C-acetyltransferase
Yes
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GCAT
Yes
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Yes
(HIF1 )
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(HIF1 )
Yes
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Table 2. Top 20 Biological Process GO Terms for genes annotated to LRH-1 (NR5A2) binding site(s). Enrichme nt
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Term
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TermID
Genes in Term
Target Genes in Term
Fraction of Targets in Term
GO:003132 3
regulation of cellular metabolic 4.43E-34 process
3995
832
0.27
GO:001921 9
regulation of nucleobasecontaining compound metabolic process
1.91E-33
3072
673
0.22
GO:005117 1
regulation of nitrogen compound metabolic process
3.68E-33
3113
679
0.22
GO:000727 5
multicellular organismal development
5.78E-33
3492
743
0.24
GO:006025 5
regulation of macromolecule metabolic process
6.76E-33
3904
812
0.26
GO:008009 0
regulation of primary metabolic process
9.81E-33
3956
820
0.27
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positive regulation of cellular process
3.78E-32
3118
676
0.22
GO:001046 8
regulation of gene expression
6.55E-32
3135
678
0.22
GO:200011 2
regulation of cellular macromolecule biosynthetic process
1.69E-31
2820
622
GO:005125 2
regulation of RNA metabolic process
3.69E-31
2644
GO:003132 6
regulation of cellular biosynthetic process
8.12E-31
GO:003250 2
developmental process
1.42E-30
GO:001055 6
regulation of macromolecule biosynthetic process
1.56E-30
GO:000988 9
regulation of biosynthetic process
GO:004851 8
positive regulation of biological process
GO:000635 5
regulation of transcription, DNA-dependent
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GO:004852 2
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590
0.20
0.19
654
0.21
3854
794
0.26
2893
631
0.20
3058
659
0.21
2.04E-30
3452
726
0.24
2.07E-30
2580
576
0.19
GO:001922 2
regulation of metabolic process 3.02E-30
4667
926
0.30
GO:200114 1
regulation of RNA biosynthetic 3.20E-30 process
2584
576
0.19
GO:004885 6
anatomical structure development
5.91E-30
3404
716
0.23
GO:001604 3
cellular component organization
4.43E-28
3172
669
0.22
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3020
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2.00E-30
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Table 3. Top 20 Biological Process GO Terms for genes annotated to ESRR site(s).
Term
P-value
GO:004423 7
cellular metabolic process
1.21E-141
6331
GO:000815 2
metabolic process
3.70E-129
7476
GO:004423 8
primary metabolic process
1.17E-120
GO:003132 3
regulation of cellular metabolic 1.24E-112 process
GO:003250 2
developmental process
GO:004426 0
cellular macromolecule metabolic process
GO:004885 6
anatomical structure development
GO:000727 5
multicellular organismal development
Number in common
5120
Fraction of List
0.36
5912
0.42
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TermID
Numbe r in Term
(NR3B2) binding
5162
0.36
3813
3184
0.22
3670
3062
0.22
4637
3768
0.27
1.22E-99
3222
2707
0.19
5.24E-99
3323
2782
0.20
GO:001922 2
regulation of metabolic process 6.62E-99
4482
3648
0.26
GO:008009 0
regulation of primary metabolic process
1.19E-98
3772
3119
0.22
GO:004851 8
positive regulation of biological process
1.41E-89
3258
2710
0.19
GO:005117 1
regulation of nitrogen compound metabolic process
2.49E-88
2971
2490
0.18
GO:001921 9
regulation of nucleobasecontaining compound t b li
3.05E-88
2937
2464
0.17
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6468
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1.31E-106
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6.93E-101
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metabolic process cellular component organization
1.10E-87
2977
2493
0.18
GO:004317 0
macromolecule metabolic process
1.93E-86
5167
4114
0.29
GO:004873 1
system development
1.15E-85
2770
2331
GO:004852 2
positive regulation of cellular process
3.13E-85
2936
GO:005117 9
localization
3.16E-84
GO:007184 0
cellular component organization or biogenesis
2.48E-83
GO:006025 5
regulation of macromolecule metabolic process
5.49E-82
cr 2456
0.17
us
0.16
2767
0.19
3109
2582
0.18
3762
3067
0.22
an
3352
Ac ce pt e
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GO:001604 3
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S1084-9521(13)00107-9 http://dx.doi.org/doi:10.1016/j.semcdb.2013.10.002 YSCDB 1470
To appear in:
Seminars in Cell & Developmental Biology
Please cite this article as: Simandi Z, Cuaranta-Monroy I, Nagy L, Nuclear receptors as regulators of stem cell and cancer stem cell metabolism, Seminars in Cell and Developmental Biology (2013), http://dx.doi.org/10.1016/j.semcdb.2013.10.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nuclear receptors as regulators of stem cell and cancer stem cell metabolism
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Zoltan Simandi1, Ixchelt Cuaranta-Monroy1 and Laszlo Nagy1,2#
1
Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, University of Debrecen, Medical and Health Science Center MTA-DE “Lendulet” Immunogenomics Research Group
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2
E-mail: [email protected] Phone: +36-52-416-432
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Fax: +36-52-314-989
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Contact Information:
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#
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Egyetem tér 1., Debrecen, Hungary, H-4012
Highlights
Nuclear receptors (NRs) are key links between metabolism and cell fate decisions. ESRRB, DAX-1, LRH-1, TR4, NGF1-B, LXRb and RARs are NRs expressed in pluripotent embryonic stem cells. ESRRB, DAX-1 and LRH-1 are regulators of metabolism in embryonic stem cells. TR4, NGF1-B, LXRb and RARs are putative regulators of stemness. The discussed NRs play a role in tumor progression and might be metabolic regulators of cancer stem cells.
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Abstract
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Cellular metabolism is underpinning physiological processes in all cells. These include housekeeping functions as well as specific activities unique to a particular cell type. A growing number of studies in various experimental models indicate that metabolism is tightly connected to embryonic development as well. It is also emerging that metabolic processes have regulatory roles and by changing metabolism, cellular processes and even fates can be influenced. Nuclear receptors (NRs) are transcription factors, responding to changes in metabolites and are implicated in diverse biological processes such as embryonic development, differentiation, metabolism and cancer. Therefore, NRs are key links between metabolism and cell fate decisions. In this review, we introduce ESRR , DAX-1 and LRH-1 as putative regulators of metabolism in pluripotent embryonic stem cells. We also discuss the role of TR4, NGF1 , LXR and RARs in stemness. In addition, we summarize our current understanding of the potential roles of NRs in cancer stem cells.
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Key words: metabolism, nuclear receptors, pluripotency, embryonic stem cells, cancer stem cells
Inroduction
Unicellular organisms or cells within multicellular organisms need to make decisions throughout their lifespan. The constantly changing environment dictating whether a cell stays quiescent, proliferate or differentiate [1]. It is well established in yeast that nutrient levels are the major determinants of cell fate. Recent evidence suggests that cellular metabolism determines the cell fate specification of mammalian cells as well [2-4]. Murine embryonic stem cells (ESCs) provide a unique model system to understand the importance of metabolic regulation in cell fate decisions in vertebrates. ESCs are derived from the inner cell mass (ICM) of developing blastocysts and display an almost unlimited proliferation capacity in vitro [5]. Moreover, this cell type retains the ability to contribute to all cell lineages. The rate of cell division of mouse ESCs is very high; a cell doubling takes place in every 4-5 hours. This is even faster than any fast growing cancer cell. Therefore, ESCs have a high
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requirement for carbon, hydrogen and nitrogen to support biosynthesis of cell building blocks required for replication. Accordingly, metabolic features of ESCs are significantly different from that of terminally differentiated cell types (reviewed in [6]).
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Metabolism in pluripotent stem cells
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One of the major differences is the way these cells can produce sufficient energy. Oxidative consumption of glucose does not enable a fast rate of ATP production. In contrast, partial breakdown of glucose through glycolysis and shunting of intermediates through the pentose phosphate pathway provide sufficient NADPH and energy for rapid cell proliferation. Oxidative capacity is reduced and glycolysis-dependent anabolic pathways are enriched in stem cells (Figure 1). This underlines the not so critical role of mitochondria in these cells. In fact, ESCs have a low number of mitochondria and harbor a sparse mitochondrial infrastructure with immature cristae and limited perinuclear localization [2]. ESCs are routinely cultured under atmospheric (20%) oxygen tensions; however these cells are originally derived from embryos which reside in a 3-5% oxygen (hypoxic) environment. However the mechanism is just partly understood, emerging pieces of evidence suggest that such hypoxic conditions in cell culturing increase the quality of stemness [7, 8] and promotes the reprogramming of differentiated cells to induced pluripotent stem cells [9]. Under low oxygen tension hypoxia-inducible factors (HIFs), key transcription factors in hypoxia, are activated and directly regulate transcription of genes involved in self-renewal [10]. Different isoforms of the HIF-family are also known to regulate the transcription of glycolytic enzymes. For instance, HIF1 upregulates the expression of pyruvate dehydrogenase kinase (PDK), which is in turn, inhibits glucose oxidation [11]. Indeed, human ESCs cultured at 5% O2 consume significantly more glucose, less pyruvate and produce more lactate compared to those maintained at 20% O2 [12]. Comparison of ESCs and cells from early stages of differentiation has identified differences in lipid metabolism as well. Stem cells show a global enrichment of unsaturated fatty acid metabolites in the pluripotent state. While high levels of unsaturated fatty acids preserve pluripotency, addition of saturated metabolites to the ESCs is known to accelerate the differentiation program [13]. A recent study nicely demonstrates that lipid biosynthesis underlies the proliferative capacity of stem and progenitor cells in the brain [14]. Another recently discovered example of unique metabolic feature of stem cell is related to the distinct mode of amino acid catabolism. Threonine dehydrogenase (Tdh), an enzyme that catalyzes threonine oxidation (Thr), shows more than 200 times higher expression in ESCs. The restriction of Thr or inhibition of Tdh results in decreased accumulation of SAdenosylMethionine (SAM) that leads to decreased trimethylation of histone 3 lysine 4 (H3K4me3) and thus abolished stem cell growth [15].
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Are nuclear receptors regulating metabolism in stem cells?
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The above listed examples (summarized in Figure 1) strongly suggest that metabolic signals most likely play an essential role in regulation of stemness, pluripotency and cell fate decisions and the regulation of these signaling pathways are essential for proper embryonic development. A central question is how embryonic stem cells sense, regulate and respond to these environmental cues. Several lines of evidence suggest that a family of transcription factors, called nuclear receptors, might play important roles in recognizing different signals and convert it to transcriptional outcome and epigenetic changes. A goal of this review is to provide insight into the metabolic regulatory role of nuclear receptors in stem cell biology with an outlook to cancer stem cells.
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The human and mouse genomes encode 48 and 49 nuclear receptors, respectively. Intense research on NRs in the last two decades has uncovered diverse roles of these receptors in developmental biology and metabolism [16].
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Nuclear receptors are ligand activated transcription factors. With a few exceptions, such as DAX1 and SHP1, all NRs expressed in vertebrates share a common domain structure. A highly conserved DNA-binding domain (DBD) ensure the binding to receptor-specific DNA sequences, while a less conserved ligand-binding domain (LBD) determines the signal specific action. Several small lipophilic molecule are known to function through NRs, however still a large fraction of nuclear receptors, often called orphan receptors, has no identified ligand. Based on their ligands and mechanistic action NRs are diveded into four groups: 1, steroid receptors 2, retinoid X receptor heterodimers 3, dimeric orphan receptors 4, monomeric orphan receptors (for detailes on grouping and nomenclature visit the website of NURSA (www.nursa.org)). DAX-1, LRH-1, SF1 and ESRR are linked directly to the maintenance of pluripotency in mESCs (reviewed in [17]) and recent genome-wide studies allow now a deeper insight into their regulatory role. A systematic profiling of NRs [18] and our independent gene expression characterization of mESCs (re-analysis of microarray data published by [19]) reveal in addition the high expression of TR4, NGF1 , LXR , RAR and RAR in undifferentiated ESCs (Figure 2). The importance of these nuclear receptors in stem cell biology is largely unknown. We will here summarize how DAX-1, LRH-1 and ESRR might regulate pluripotency and differentiation from a metabolic point of view. To further enhance the possible connection between these NRs, metabolism and pluripotency, we re-analyzed available genome-wide chromatin-immunoprecipitation data (ChIP-chip and ChIP-seq) and included several putative metabolic targets, that are likely to be regulated by these factors. Moreover, we sum up our recent knowledge on the TR4, NGF1 , LXR and RARs in the context of development and metabolism.
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DAX-1 (NR0B1) DAX-1 is a unique member of the NR family. Instead of binding to regulatory DNA sequences directly it appears to bind to other transcription factors and act mainly as a co-repressor [20].
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DAX-1 has a restricted expression pattern to the hypothalamic-pituitary-adrenal/gonadal-axis and was shown as an important regulator of steroidogenesis, gonadal development and sex determination [21]. Forming a pair with SF-1 (steroidogenic factor-1), an other NR family member, DAX-1 is playing a role in the transcriptional regulation of enzymes involved in conversion of cholesterol to steroid hormones [22]. The first indication of an essential role of DAX-1 in embryogenesis and pluripotency was revealed by the difficulty encountered in establishing DAX-1 knockout mice [23]. DAX-1 was identified as a highly expressed gene in ESCs and gene-silencing of DAX-1 was found to result loss of pluripotency of these cells [24].
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A recent analysis identified DAX-1 binding genomic regions in ESCs as part of the transcriptional network of pluripotency [25]. This data is valuable to get information how DAX-1 can regulate pluripotency. The list of putative DAX-1 regulated genes are available as a Supplement of the above manuscript. In agreement with the repressor function of DAX-1, several differentiation-related genes can be find on the list, such as members of the Hox-genes (Hoxa2, Hoxa9, Hoxa11, Hoxb4, Hoxc10 and Hoxc12), Wnt-signal (Wnt11, Wnt16, Wnt8a) and early lineage-specific markers (Pax6, Stra8, Foxa1, Foxd3, Lefty1, T, Rest). However, promoter region of stem cell marker Oct3/4 and Nanog are also bound by DAX-1, indicating its potential contextdependent activator function as well. The list contains several genes which are known to be involved in metabolic processes, such as Apoa2, Apoe, Atp5a1, Pgm2, Pgam1, Fabp3, etc. Importantly, members of stem cell related metabolic pathways are also represented in force. Glucose transporter (Slc2a3), much of glycolytic enzymes (Hk3, Gpi1, Pfkp, Gapdh, Pgk1, Ldhb, Pdhb), enzyme in pentose phosphate pathway (Pgls), nucleotide synthesis (Prps1), metabolism of threonine (Tdh, Gcat) and fatty acid synthesis (Hmgcr, Dhcr7) are all putative DAX-1 targets (Figure 1, Table 1). Based on these findings it is secure to say that DAX-1 has a largely uncharacterized metabolic regulatory function in stem cells.
LRH-1 (NR5A2)
Liver receptor homolog-1 (LRH-1) has been implicated in diverse biological processes, including bile acid metabolism, steroidogenesis and cell proliferation [26]. In adult, LRH-1 is known to be highly expressed in liver and promotes expression of genes involved in hepatic cholesterol uptake and efflux, HDL formation and fatty acid synthesis [27]. LRH-1 also controls the first step of hepatic glucose uptake through direct transcriptional regulation of the glucokinase gene. This suggests that LRH-1, at least in hepatocytes, acts as a critical regulatory component of the glucose-sensing system [28].
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ESRR
(NR3B2)
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Importantly, LRH-1 is also expressed during early mouse embryogenesis. LRH-1-/- embryos display growth retardation, epiblast disorganization, a mild embryonic-extraembryonic constriction, as well as abnormal thickening of the proximo-posterior epiblast [29]. LRH-1 has been identified as a key transcription factor in cellular reprogramming [30]. Similar to DAX-1, there is available genome-wide study for LRH-1, in which the LRH-1 occupied regions have been identified [31]. We re-analyzed this dataset and annotated the LRH-1 binding sites to the closest genes. Considering the limitations of this type of analysis, we performed a prediction of enriched pathways (Table 2). Several genes, involved in various metabolic processes, seem to be regulated directly by LRH-1. Genes encoding enzymes of glycolysis and gluconeogenesis (Hk2, Pfkm, Pgam2, Ldha, Ldhc, Aldh3a2, Aldh9a1), pentose phosphate pathway (H6pd, Rpia), fatty acid biosynthesis (Acacb, Acaca) or fatty acid metabolism (Aldh3a2, Aldh9a1, Acsl1, Cyp4a31, Hadh) are all possess LRH-1 occupied region in the close proximity (Figure 1, Table 1). Geneexpression studies combined with LRH-1 ChIP data will complete our understanding on the LRH-1-dependent regulatory mechanisms in ESCs. Inverse agonists that have been recently described for the LRH-1 [32], might also accelerate discoveries related to biological function of this nuclear receptor.
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There are three isoforms of the ESRR genes in mouse and human cells: ESRR , and . ESRRs share a homology with estrogen receptors (ERs), but ESRRs do not bind estrogen or other known physiological ligands [33]. ESRRs are typically expressed in tissues with high metabolic demands and they have a predominant role in orchestrating mitochondrial biogenesis and cellular energy metabolism such as oxidative phosphorylation (OxPhos), tricarboxylic acid (TCA) cycle, fatty acid oxidation and ATP synthesis. ESRRs directly acivate transcription of genes involved in these metabolic processes [34, 35]. In ESCs only one isoform, ESRR shows high expression (Figure 2). However, this one seems to be indispensable for pluripotency [36]. Expression of ESRR is regulated by Nanog, a key transcription factor of ES cells [37]. Loss of ESRR results spontaneous differentiation of ESCs while the overexpression of ESRR has been shown to promote reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells [38]. Only limited information is available why ESRR is so important in the maintenance of pluripotency. Recent genome-wide studies identified the binding sites of ESRR in mouse embryonic stem cells. The analysis revealed that ESRR -binding sites partly overlap with the Nanog-Oct3/4-Sox2 transcription factor binding sites [39], however large fraction of binding sites are independent from the core complex. ESRR was previously suggested to bind as a monomer [40]. In contrast, a detailed analysis identified three equally enriched full-site motifs, DR0, DR5, and DR8, but no half-site enrichment, suggesting that ESRR is likely to bind in a form of dimer to DNA [41]. So far, ESRR ChIP-seq data has not been analyzed in a deeper biological context. To get a preliminary
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insight into the ESRR -mediated functions in ESCs, we re-analyzed an available ChIP-seq data [39] and in Table 3 summarized the top biological functions identified for the putative ESRR regulated genes. This clearly indicates that ESRR beside the regulation of pluripotency, plays a role in the regulation of metabolic processes. Indeed, much of the glycolytic, lipid- and amino acid-related enzymes that are important in stemness, are seems to be regulated by ESRR (Figure 1, Table 1). So far, this is the first indication that ESRR regulate the metabolic state of ESCs and raise the possibility of an ESRR -dependent metabolic regulation of stem cell pluripotency. However, these preliminary indications of ESRR mediated regulation of stem cell metabolism will requires additional analysis and validation.
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TR4 (NR2C2)
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Our genome-wide gene expressional data suggest that TR4 is expressed at a higher level in stem cells (Figure 2) and a recent study implicated also its functional importance in pluripotency [42]. Importantly, TR4 -/- mice display significant growth retardation [43]. In addition, gene disruption results in defects of spermatogenesis, in testis and in cerebellar development. Importantly, TR4 is expressed in metabolic tissues, including liver, muscle, brown and white adipose tissues and regulate metabolic genes, such as apolipoprotein E, enoyl-CoA hydratase, acyl-CoA oxidase [44, 45]. Moreover, TR4 also regulates phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme controlling the rate of gluconeogenesis [46]. Thus, its high expression in stem cell might be essential in the regulation of metabolism via controlling mainly carbohydrate metabolism.
NGF1
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A recent study has determined the TR4 binding regions in four different cell types [47]. The majority of TR4 targets are shared between the different cell types and responsible for RNA metabolism and protein translation. However, cell-type specific TR4 binding could be also observed and these results further confirmed that TR4 is regulate genes involved in cellular metabolism. Genome-wide studies of TR4 binding in ESCs are not available yet. Loss of function experiments will be also required to identify the TR4 regulated genes in ESCs and in early embryonic differentiation. This might be helpful to understand the phenotypic alterations of the TR4-/- animals as well.
(NR4A1)
The NR4A family includes three highly homologous isoforms, NR4A1, NR4A2 and NR4A1, also known as Nur77, Nurr1 and Nor1, respectively. X-ray crystallography studies suggest that the ligand-binding pocket of these receptors is unable to accommodate ligands [48]. Previous studies have pointed to NR4A1 as a transcriptional regulator of glucose metabolism in liver and skeletal muscle [49]. Moreover, later studies demonstrated that Nur77 also regulates hepatic cholesterol metabolism [50]. Nur77 modulates lipid metabolism in hepatocytes by 7
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decreasing the expression of SREBP1c, which is a master regulator of triglyceride metabolism. In the context of developmental biology, Nur77 has been shown to be involved in cellular differentiation, apoptosis, proliferation, T-cell function.
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Higher expression level of NGF1 in ESCs is also indicated by our data (Figure 2). However, it is still very tentative to suggest NR4A1 as a metabolic regulator of stemness. ChIP-seq experiments in combination with transcriptome analyses will establish how Nur77 modulates the expression profile of a specific cell-type under defined conditions.
RAR alpha and gamma (NR1B1 and NR1B3)
LXR
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Three genes code for receptors that functionally bind retinoic acid (RA): RAR , and . Retinoic acid (RA) acts as an important signaling molecule during embryonic development and is commonly used for induction of in vitro differentiation of ESCs [51]. Ligands for RAR activation are being synthetized upon LIF removal and induction of differentiation [52]. These formally suggest that RARs, present in ESCs, should fulfill rather an inhibitory role than activator function in transcription. Indeed, a well-accepted model is that in the absence of ligand RAR and RXR dimers bind to DNA and interact with co-repressors to repress transcriptional events. Active repression by unliganded RARs has been proposed in development [53]. To understand the function of unliganded RARs is stem cells, genome-wide characterization of RAR genomic binding has been carried out [54]. Analysis of the RAR-bound sites shows that among the enzymes summarized in Figure 1 and Table 1, only Ldhb and Pdk1 seem to have RAR occupied promoter region. Validation of these targets will be required to confirm the putative role of RARs in the metabolic regulation of undifferentiated ESCs.
(NR1H2)
The liver X receptors form permissive heterodimers with retinoid X receptors (RXRs) and are important regulators of lipid metabolism [55, 56]. The LXR family consists of the two subtypes: LXR and LXR . LXR and LXR are detected in the liver starting at 11.5 days postcoitum [57]. Later, LXR expression remains high in organs involved in lipid homeostasis, such as liver, intestine, and brown adipose tissue, whereas LXR is more ubiquitously expressed and enriched in tissues of neuronal and endocrine origin. LXR -deficient mice are viable and show minor metabolic differences as compared to the wild-type [58]. As a remarkable difference, reduced fertility has been shown [59]. Oxysterols, oxidative derivatives of cholesterol has been identified as physiological ligands for LXRs, thus LXRs seems to act as a cholesterol sensors. Treatment of rodents with synthetic LXR ligands results in decreased hepatic gluconeogenesis and increased lipogenesis, indicating that
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LXR serves as a transcription factor that integrates liver carbohydrate and lipid metabolism [60]. A recent genome-wide study has identified LXR and RXR binding regions in mouse liver [61].
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Taken together these results suggest that however Lxr is expressed in ESCs and might regulate metabolic pathways, it is unlikely that would be essential for pluripotency. In the same time, experimental evidence is required to confirm this statement.
Cancer stem cells and metabolism
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Numerous studies have revealed the existence of crosstalk between various nuclear receptor signaling pathways. One interesting example in the context of metabolism and differentiation is the potential link between LXR-dependent cholesterol metabolism and retinoic acid biosynthesis. Cholesterol metabolites, predominantly the oxysterols were identified that induces the expression of retinal dehydrogenases (RALDHs) via upregulation of sterol regulatory element binding protein-1c (SREBP1c) [62]. RALDHs synthesize retinoic acid (RA) from retinal [63]. RA is known to critically important for embryogenesis and also involved in proliferation, differentiation and apoptosis. However this crosstalk was mainly characterized in liver cells results in the same study also raise a similar mechanism in embryonic carcinoma cells. Existence of such a crosstalk between LXR and RAR signaling might play important roles in the metabolic switch when stem cell differentiation starts.
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The pluripotent nature of various cancers has been recognized early on [64]. Overall, there are two major cancer development theories as far as stem cell involvement is concerned: origin from cancer stem cells (CSCs) and de-differentiation of mature cells. The first one has been published by Furth et al., suggesting that a single cell from a tumor can initiate a new tumor in a recipient mouse [64]. Since then several studies have been performed to investigate the theory, which in principle states that the different tumor populations arise from a delimitated number of cells which conserves the same characteristics than stem cells in adult tissues [65]. CSCs can occur in diverse solid tumors, such as glioblastomas and breast cancer, but their existence has been reported in acute myeloid leukemias (AML) as well [65, 66]. A number of markers has been used for the isolation of CSCs, such as CD133 or SSEA-1, however difficulties of isolating pure populations of CSCs are still limiting our understanding of these cell populations. Elucidation of the pathways that regulate the maintenance and survival of CSCs is important for the development of novel therapies. Metabolic pathways have recently been implicated in governing the function of CSCs. In general, cancer cells have very similar metabolic features compared to ESCs [67]. Glucose metabolism is typically characterized by a shift from aerobic, oxidative metabolism to a glycolytic pathway. This aerobic glycolysis phenotype is known as the Warburg effect [68]. Metabolic switch to aerobic glycolysis is driven primarily by oncogenic signals and transcription factors. HIF1 is one of the most studied one, either the intratumoral hypoxic condition or genetic defects results in its stabilization and downstream regulatory
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function [69]. Identification of additional factors that play a role in metabolic switch of cancer cell is one of the hot topics in research.
Nuclear receptors as potential targets in cancer (stem) cell metabolism
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NRs have been extensively studied and linked directly to pathogenesis and treatment of various cancers; however their expression profile and putative role in cancer stem cells remained completely unknown. Importantly, CSCs have ESC-like transcriptional programs [70], thus detection of the above discussed pluripotency-related NRs, such as ESRR , LRH-1, DAX-1 are not unexpected in CSCs.
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Interestingly, many of these NRs have been already linked to poor prognosis of cancer. LRH-1 was shown to promote motility and invasiveness in breast cancer cells [71] and regulate steroidogenic gene transcription in endometrial cancer cells [72]. LRH-1 transcription is also activated in human pancreatic cancer cells [73]. The other stem cell specific nuclear receptor DAX-1 has been also recognized as a putative target in cancer treatment [74]. It is a direct target of the EWS/FLI1 oncoprotein, and found to be highly expressed in Ewing’s tumors. Genes implicated in cell cycle progression are regulated by DAX-1. ESRR , and are identified coactivating factors of HIF and thus might directly modulate HIF-mediated gene transcription of cancer cells. Further studies are required to helpus understand which pathways are regulated by NRs in cancer cells and CSCs. A recent study applied ChIP-seq to map the in vivo genome-wide binding (cistrome) of NRs in both normal and cancer cells [41]. Such studies combined with gene expression data will bring us closer to understand how NRs mechanistically contribute to cancer development. The above stated important role of NRs in cancer biology and their well established role in metabolism pose many interesting questions: most importantly whether NRs play any role in cancer cells to sense and regulate the level of nutrients to facilitate tumor development and progression. There are growing number of pieces of evidence indicating a direct relationship between NRs, metabolism and cancer. Anti-estrogen therapy for breast cancer was the first targeted therapy used in any type of cancer. Since its discovery, several metabolic pathways affected by anti- estrogens in cancer cells has been identified [75]. Activation of LXR signaling by oxysterols is an other example of how NR ligands can be used to target metabolic pathways in the treatment of various cancers [76]. Oxysterols are known to interfere with proliferation and initiate cell death of many cancer cells. Oxysterols and LXRsignaling might also help to treat “incurable” conditions, such as glioblastoma multiforme (GBM). GBM, a WHO grade IV astrocytoma, is the most aggressive and common primary brain tumor in adults. The median survival of GBM patients is approximately 1 year. Importantly, LXR
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agonists have been shown to inhibit GBM growth and promote tumor cell death, and LXR was initiated as a novel antitumor agents in glioblastoma [77, 78]. Role of NR agonists has been also studied and their roles confirmed in the metabolism and gene expression regulation of breast cancer stem cells [79, 80].
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A proof of principle for NR-mediated cancer therapy is the use of all-trans retinoic acid (atRA) treatment in AML. In AML the presence of leukemia stem cells has been demonstrated. It has been shown that they are hierarchically organized due to the heterogeneity in their self-renewal capacity and in longevity of the produced clone [81-83]. In this context, Alcalay et al., found that expressing some fusion proteins in myeloid precursors, previously observed in murine models of AML which includes the transcription factor RAR , the gene expression pattern was congruent with the induction of genes related to the maintenance of the stem cell phenotype and repression of DNA repair genes. Importantly, metabolism was one of the functional classifications of AMLfusion protein target genes and some of the identified genes could be validated in patient samples [84].
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These examples provide evidence that targeting metabolic pathways through the activation of NRs may represent effective strategies for eradicating CSCs.
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Recent studies are just beginning to show the importance of metabolism in cell fate determination, including cancer development. Role of nuclear receptors are still poorly characterized in the metabolic regulation of pluripotency. The recent development of highthroughput sequencing methodologies such as RNA-seq and ChIP-seq combined with metabolomics analyses holds the promise to provide more accurate gene expression measurements and deeper mechanistic insights into the regulatory role of nuclear receptors in cellular metabolism. This knowledge can be potentially turned into novel therapeautic approaches later.
Acknowledgement
The authors would like to acknowledge Erik Czipa and Dr. Endre Barta for the contibution in data analyzis, and the Nagy laboratory for discussions and comments on the manuscript. L.N is supported by a grant from the Hungarian Scientific Research Fund (OTKA K100196), and TÁMOP422_2012_0023 VÉD-ELEM implemented through the New Hungary Development Plan co-financed by the European Social Fund and the European Regional Development Fund. Z.S is a recipient of TÁMOP-422/B10/1_2010_0024 grant.
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Figure 1.
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Figure 1. Metabolism in pluripotent embryonic stem cells (ESCs). Rapidly proliferating pluripotent stem cells has unique metabolism. I. ATP production: ATP synthesis is decoupled from O2 consumption and depends on glycolysis. Gylcolytic enzymes: hexokinase (HK), glucose6-phosphate isomerase (GPI), phosphofructokinase (PFK), aldolase (ALDO), triosephosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), pyruvate dehydrogenase (PDH), lactate dehydrogenase (LDH). II. Nucleotide synthesis: Pentose phosphate pathway (Pentose PP) is active in ESCs and by converting glucose6-phospate (G6P) into ribose-5-phosphate for nucleotide synthesis, it also produces NADPH. Pentose PP enzymes among others: hexose-6-phosphate dehydrogenase (H6PD), 6phosphogluconolactonase (PGLS), ribose 5-phosphate isomerase A (RPIA), transketolase (TKT) Phosphoribosyl pyrophosphate synthetase 1 (PRPS1) catalyzes the phosphoribosylation of ribose 5-phosphate and necessary for purine metabolism. CAD encodes a trifunctional protein that is involved in pyrimidine nucleotide synthesis. III. Amino acid metabolism: ESCs requires the catabolism of L-threonine (Thr). The degradation of L-threonine to glycine consists of a two-step biochemical pathway involving the enzymes L-threonine dehydrogenase (Tdh) and 2-amino-3ketobutyrate coenzyme A ligase (GCAT). Glycine dehydrogenase (GLDC) is involved in the degradation of glycin. IV. Lipid metabolism: Unsaturated fatty acids preserve the pluripotency. 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and 7-dehydrocholesterol reductase (DHCR7) are enzymes for cholesterol synthesis. V. Epigenetic regulation: Acetyl-CoA (AcCoA) and S-AdenosylMethionine (SAM) are substrates for histone acetylation and methylation, respectively VI. Hypoxia: hypoxic culture conditions results the stabilization of hypoxia inducible factor 1 alpha (HIF1 ) and enhance the transcription of pyruvate dehydrogenase kinase (Pdk). Pdk inhibits glucose oxidation. Out of all the shown enzymes, only the marked (*) ones are not regulated by DAX-1, LRH-1 and/or ESRR (see Table 1).
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Figure 2.
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Figure 2. Per chip normalized signal intensity data of nuclear receptors in undifferentiated ESCs. Values of all entities and some known embryonic stem cell markers are also shown. Red line indicates the median value of all entities. One dot represents one gene. Gene symbols on the top of the figure indicate the dominantly expressed genes from each group and are considered to be expressed in stem cells. Nuclear receptors shown in red are discussed in the text.
Table 1. Summary of DAX-1, LRH-1 and ESRR Gene symbol
GLUT1/3
HK GPI
regulated metabolic genes DAX-1
LRH-1
ESRR
Yes
Yes
(Slc2a1)
(Slc2a1, a3)
Yes
Yes
Yes
(Hk3)
(Hk2)
(Hk1, 2, 3)
Facilitated glucose transporter, member Yes 1 (Slc2a3) Hexokinase glucose-6-phosphate isomerase
Yes
Yes
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(Gpi1)
(Gpi1)
Yes
Yes
Yes
(Pfkp)
(Pfkl, m, p)
(Pfkl, m, p)
phosphofructokinase
PFK
Yes
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aldolase
ALDO
(Aldoa) Yes
PGK
phosphoglycerate kinase
Yes
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glyceraldehyde-3-phosphate dehydrogenase
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GAPDH
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triosephosphate isomerase 1
TPI1
LDH
M
Yes
lactate dehydrogenase
PGLS RPIA* TKT PRPS1
CAD*
hexose-6-phosphate dehydrogenase
Ac ce pt e
H6PD
d
(Ldh )
6-phosphogluconolactonase
Yes
(Tpi1)
Yes Yes (Pgk1)
Yes
Yes
(Ldh , Ldh )
(Ldh , Ldh )
Yes
Yes Yes
ribose 5-phosphate isomerase A transketolase
Yes
phosphoribosyl pyrophosphate synthetase 1
Yes
carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase Yes
PDH
pyruvate dehydrogenase complex (Pdhb)
PDK
pyruvate dehydrogenase kinase
Yes
Yes
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(Pdk1)
(Pdk1, 2, 4)
Yes
Yes
GLDC*
glycine dehydrogenase
TDH
L-threonine dehydrogenase
Yes
HMGCR
3-hydroxy-3-methylglutaryl-CoA reductase
Yes
DHCR7
7-dehydrocholesterol reductase
Yes
HIF
hypoxia inducible factor
Yes
Yes
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glycine C-acetyltransferase
Yes
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GCAT
Yes
us
Yes
(HIF1 )
an
(HIF1 )
Yes
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Table 2. Top 20 Biological Process GO Terms for genes annotated to LRH-1 (NR5A2) binding site(s). Enrichme nt
d
Term
Ac ce pt e
TermID
Genes in Term
Target Genes in Term
Fraction of Targets in Term
GO:003132 3
regulation of cellular metabolic 4.43E-34 process
3995
832
0.27
GO:001921 9
regulation of nucleobasecontaining compound metabolic process
1.91E-33
3072
673
0.22
GO:005117 1
regulation of nitrogen compound metabolic process
3.68E-33
3113
679
0.22
GO:000727 5
multicellular organismal development
5.78E-33
3492
743
0.24
GO:006025 5
regulation of macromolecule metabolic process
6.76E-33
3904
812
0.26
GO:008009 0
regulation of primary metabolic process
9.81E-33
3956
820
0.27
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positive regulation of cellular process
3.78E-32
3118
676
0.22
GO:001046 8
regulation of gene expression
6.55E-32
3135
678
0.22
GO:200011 2
regulation of cellular macromolecule biosynthetic process
1.69E-31
2820
622
GO:005125 2
regulation of RNA metabolic process
3.69E-31
2644
GO:003132 6
regulation of cellular biosynthetic process
8.12E-31
GO:003250 2
developmental process
1.42E-30
GO:001055 6
regulation of macromolecule biosynthetic process
1.56E-30
GO:000988 9
regulation of biosynthetic process
GO:004851 8
positive regulation of biological process
GO:000635 5
regulation of transcription, DNA-dependent
cr
ip t
GO:004852 2
us
590
0.20
0.19
654
0.21
3854
794
0.26
2893
631
0.20
3058
659
0.21
2.04E-30
3452
726
0.24
2.07E-30
2580
576
0.19
GO:001922 2
regulation of metabolic process 3.02E-30
4667
926
0.30
GO:200114 1
regulation of RNA biosynthetic 3.20E-30 process
2584
576
0.19
GO:004885 6
anatomical structure development
5.91E-30
3404
716
0.23
GO:001604 3
cellular component organization
4.43E-28
3172
669
0.22
M
an
3020
Ac ce pt e
d
2.00E-30
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Table 3. Top 20 Biological Process GO Terms for genes annotated to ESRR site(s).
Term
P-value
GO:004423 7
cellular metabolic process
1.21E-141
6331
GO:000815 2
metabolic process
3.70E-129
7476
GO:004423 8
primary metabolic process
1.17E-120
GO:003132 3
regulation of cellular metabolic 1.24E-112 process
GO:003250 2
developmental process
GO:004426 0
cellular macromolecule metabolic process
GO:004885 6
anatomical structure development
GO:000727 5
multicellular organismal development
Number in common
5120
Fraction of List
0.36
5912
0.42
us
cr
ip t
TermID
Numbe r in Term
(NR3B2) binding
5162
0.36
3813
3184
0.22
3670
3062
0.22
4637
3768
0.27
1.22E-99
3222
2707
0.19
5.24E-99
3323
2782
0.20
GO:001922 2
regulation of metabolic process 6.62E-99
4482
3648
0.26
GO:008009 0
regulation of primary metabolic process
1.19E-98
3772
3119
0.22
GO:004851 8
positive regulation of biological process
1.41E-89
3258
2710
0.19
GO:005117 1
regulation of nitrogen compound metabolic process
2.49E-88
2971
2490
0.18
GO:001921 9
regulation of nucleobasecontaining compound t b li
3.05E-88
2937
2464
0.17
an
6468
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1.31E-106
Ac ce pt e
d
6.93E-101
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metabolic process cellular component organization
1.10E-87
2977
2493
0.18
GO:004317 0
macromolecule metabolic process
1.93E-86
5167
4114
0.29
GO:004873 1
system development
1.15E-85
2770
2331
GO:004852 2
positive regulation of cellular process
3.13E-85
2936
GO:005117 9
localization
3.16E-84
GO:007184 0
cellular component organization or biogenesis
2.48E-83
GO:006025 5
regulation of macromolecule metabolic process
5.49E-82
cr 2456
0.17
us
0.16
2767
0.19
3109
2582
0.18
3762
3067
0.22
an
3352
Ac ce pt e
d
M
ip t
GO:001604 3
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