The canonical cell has significant bioenergetic needs for maintenance and homeostasis.6 Protein synthesis and maintenance of cellular membrane potentials consume most of the ATP produced under homeostatic conditions.7 In many differentiated or quiescent cells, it is believed that fatty acid oxidation provides the bulk of the energy, followed by the use of glucose. In this regard, mitochondrial respiration is essential for adult tissues and cells. It is notable, however, that specialized cell functions in the various organs could require different metabolic pathways. Glucocorticoid hormone-producing cells, for example, express specialized metabolic pathways. Although cardiac muscle cells depend heavily on fatty acid oxidation, skeletal muscle cells use glucose. The brain depends largely on glucose, but it can feed on ketone bodies under starved states. For differentiated cells, which are the bulk of cells in mammals, homeostasis drives the demand for nutrients, to “the availability of which are determined by feeding and interorgan (liver, muscle, and endocrine tissues) metabolic interplays. For example, lactate produced by exercising muscle circulates back to the liver and is processed via the Cori cycle to produce glucose.3 Glucose plasma level is tightly controlled by the pancreas, which produces insulin and glucagon, and by the liver (as well as kidney) that can produce glucose through gluconeogenesis.
CELL GROWTH: SIGNALING, NUTRIENTS, AND METABOLISM
Normal cell growth and proliferation are triggered by extracellular cues. Yeast cells, for example, only require the presence of nutrients to initiate cell growth or an increase in cell size.6 During this growth phase, nutrients are imported and channeled into biomass, which is largely comprised of ribosomes. It is estimated that ribosomes constitute more than 50 percent of cellular dry mass, and hence ribosome biogenesis is highly regulated and vitally important for cell growth and proliferation. Once a critical cell size (mass) is reached with a balanced nucleotide pool, DNA synthesis begins. Yeast cells sense nutrients, particularly glucose and glutamine, through pathways involving RAS and target of rapamycin complex 1 (TORC1), which silence transcriptional repressors of ribosome biogenesis.8 With nutrient deprivation, activation of these transcriptional repressors provides a metabolic checkpoint that restrains cells from growing in the absence of adequate bioenergetic support. Hence, the normal feedback loops couple nutrient availability with cell growth: no nutrients, no growth. The normal feedback loops can be artificially disrupted by deletion of transcriptional repressors of ribosomal biogenesis, rendering yeast mutants constitutively activated for growth. These mutants resemble mammalian cancer cells, which have mutations that drive autonomous cell growth with disregard for nutrient availability. The severance of nutrient sensing from growth signaling causes addiction of these yeast mutants to nutrients, such that deprivation of glucose or glutamine results in nonviable mutants. Similarly, cancer cells are addicted to nutrients.6
Cell growth in multicellular organisms requires additional cues in addition to the availability of nutrients. Mammalian cells live in a community of cells and are constantly bathed in nutrients derived from the circulation, but they do not proliferate unless there are appropriate cues from growth factors and the extracellular matrix. Mammalian cells can be envisioned as bioreactors that require at least two signals to grow: (1) growth factor and (2) nutrients.6 Cell growth is arrested in the absence of either growth factor or nutrients. Similar to yeast cells, metabolic checkpoints are critical to the growth of normal mammalian cells.
Growing cells largely depend on glucose, glutamine and other amino acids.9 Indeed, the core metabolic pathways including glycolysis, glutaminolysis, and the tricarboxylic acid (TCA) cycle link amino acid and glucose metabolism to lipogenesis and nucleotide synthesis (Fig. 14–1). Glycolysis starts with the transport of glucose into cells through several transporters, known as GLUTs, with SLC2A1 (GLUT1) being one that is coupled with cell growth stimulation. Once inside the cell, glucose is phosphorylated in an ATP-consuming step by hexokinases (HK) with HKII being stimulated by many growth signals and directly regulated by the MYC oncogene or the hypoxia inducible factor 1 alpha (HIF-1α). Glucose-6-phosphate (G6P) is used by the pentose phosphate pathway to produce ribose for nucleotide synthesis (Fig. 14–1) or converted to fructose phosphate via an isomerization reaction catalyzed by glucose phosphate isomerase (GPI).9 Fructose-6-phosphate is further phosphorylated with consumption of a second ATP through a rate-limiting step catalyzed by phosphofructokinase (PFK) to fructose-1,6-bisphosphate (F1,6BP). F1,6BP is converted by aldolase and an isomerase to the three-carbon phosphorylated molecule, glyceraldehyde 3-phosphate (GAP), which is oxidized and phosphorylated using inorganic phosphate by the dehydrogenase, GAPDH, to 1,3-bisphosphoglycerate. The energy gained by nicotinamide adenine dinucleotide (NAD+)-mediated oxidation and phosphorylation is released from 1,3-bisphosphoglycerate by phosphoglycerate kinase, which transfers the high-energy phosphate bond to adenosine diphosphate (ADP) to form ATP. The resulting 3-phosphoglycerate (3-PG) provides a substrate for serine and glycine synthesis, for the production of glycerol, or for the production of phosphoenol pyruvate (PEP) in glycolysis. Pyruvate kinase mediates the transfer of the high-energy phosphate bond from PEP to ADP producing ATP and pyruvate, which is the terminal substrate of glycolysis. Collectively, glycolysis uses ATP to charge up several intermediates for their transformations and uses NAD+ to oxidize intermediates and generate energy through new high-energy phosphate bonds with inorganic phosphate. Each glucose molecule results in the production of a net two ATP molecules from ADP through glycolysis.
Central metabolic pathways involving glycolysis, glutaminolysis, and the tricarboxylic acid (TCA) cycle. Glucose is shown metabolized to pyruvate, which can be converted to lactate, alanine, or acetyl-coenzyme A (acetyl-CoA). Upstream of pyruvate, glucose carbons are shunted toward the pentose phosphate pathway for ribose synthesis, glycine, and glycerol synthesis. Acetyl-CoA (2-carbon unit) combines with oxaloacetate (4-carbon) to form citrate (6-carbon), which is subsequently metabolized in the TCA cycle to generate isocitrate, α-ketoglutarate, and other intermediates as depicted. Glutamine is shown to enter the TCA cycle via α-ketoglutarate, after being converted to glutamate. Glucose gives rise to glycerol and citrate, which contributes 2-carbon units for fatty acid synthesis, and contributes to lipid synthesis. Glutamine and glucose are depicted to contribute to nucleotide synthesis.
Pyruvate, derived from glucose through glycolysis, from malate through malic enzyme or from alanine through transamination, could enter the mitochondria through specific transporters and be converted to acetyl-coenzyme A (CoA) by pyruvate dehydrogenase (PDH) (see Fig. 14–1).10 PDH activity can be attenuated by phosphorylation, mediated by PDH kinase (PDK), which is activated by hypoxia to divert glucose carbons away from the TCA cycle toward lactate production. Under aerobic conditions, acetyl-CoA combines with oxaloacetate coming from a complete turn of the TCA cycle to produce citrate, which can be extruded into the cytoplasm to participate in lipid synthesis or which can be converted to isocitrate in the TCA cycle. Isocitrate is further oxidized to α-ketoglutarate by isocitrate dehydrogenase (IDH) with the production of either nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH) and release of a carbon dioxide molecule. There are three IDH isozymes with IDH1 being located in the cytosol, while IDH2 and IDH3 are in the mitochondrion. NADH in the mitochondrion contributes to the high-energy electrons that drive production of ATP through the electron transport chain. NADPH produced by cytosolic IDH1 or mitochondrial IDH2 could participate in reductive biosynthesis of fatty acids or nucleobases.
In addition to being a key TCA cycle intermediate at the crossroads of several metabolic pathways, a-ketoglutarate (or oxoglutarate) serves as a cofactor for many important oxygenases, such as those involved in the hydroxylation and degradation of the hypoxia inducible factors (HIFs), modification of ribosomes, or those involved in demethylation of DNA and histones.11,12 Notably, glutamine can enter the TCA cycle at this junction. α-Ketoglutarate is further oxidized by oxoglutarate dehydrogenase (OGDH) to produce succinyl-CoA and carbon dioxide. Succinyl-CoA, which is also used for heme synthesis, is then converted to succinate with the production of a guanosine-5′-triphosphate (GTP) from guanosine-5′-diphosphate (GDP). Succinate is then converted to fumarate by succinate dehydrogenase (SDH), which is mutated in certain familial cancer syndromes. Fumarate hydratase (FH), which is also mutated in cancer syndromes, converts fumarate to malate that is, in turn, converted to oxaloacetate. Oxaloacetate can serve as a substrate for glutamate oxaloacetate transaminase (GOT) for the production of aspartate for nucleotide synthesis, or it can further cycle forward into the TCA cycle by combining with acetyl-CoA to form citrate, thus completing the TCA (citric acid or Krebs) cycle (see Fig. 14–1).
Glutamine also serves as a key metabolic substrate for growing cells (see Fig. 14–1). Glutamine is imported by membrane transporters, such as SLC1A5 or ASCT2.13,14 Once in the cytosol, glutamine can contribute to protein synthesis or glucosamine or nucleobase biosynthesis by donating its nitrogen. Glutamine is further imported into the mitochondrion and converted to glutamate by glutaminase (GLS) with the release of ammonia. Glutamate is converted to α-ketoglutarate by either glutamate dehydrogenase (primarily in nongrowth states) or aminotransferases (GOT or glutamate pyruvate transaminase [GPT]). In this manner, glutamine serves as a major growth substrate for growing cells. Hence, the TCA cycle is a metabolic roundabout that uses carbons from glucose, glutamine, and fatty acids to generate carbon skeletons for biosynthesis, NADH for the production of ATP, or α-ketoglutarate for catalyzing key oxygenase reactions.
Oxidation of glucose, glutamine, and fatty acids produces energy for growing cells. On the other hand, synthesis of fatty acids and other building blocks require the reductive power of NADPH for bond formation. NADPH is produced from several well-characterized pathways, including the pentose phosphate pathway, malic enzyme, IDH, and the folate pathway.15 Glucose-6-phosphate dehydrogenase (G6PD) is well-known for its role in oxidation of G6P to 6-phosphogluconolactone and the concurrent reduction of NADP+ to NADPH, which contribute to an antioxidant state through maintaining reduced glutathione. Specifically, loss of G6PD function is associated with severe hemolytic anemia in patients who inherit hypomorphic alleles of G6PD (see Chap. 47). Malic enzyme mediates the oxidation of malate to pyruvate using nicotinamide adenine dinucleotide phosphate (NADP+), which is reduced to NADPH. IDH1 oxidizes isocitrate to α-ketoglutarate with the production of NADPH from NADP+. Lastly, it was recently documented that the folate pathway plays a major role in NADPH production through the oxidation of methylene-tetrahydrofolate (THF) to formyl-THF.15 The largest consumer of NADPH, on the other hand, involves fatty acid synthesis with reduction of glutathione following closely behind. Thus production of NADPH is critical for both biosynthesis and for redox homeostasis.
SIGNAL TRANSDUCTION: ONCOGENES, TUMOR SUPPRESSORS AND METABOLISM
Growth factors and nutrients drive the growth and proliferation of cells (Fig. 14–2 and Chap. 17). Growth factor engagement of a (usually dimeric) growth factor receptor triggers allosteric alterations that lead to autophosphorylation, in the case of the receptor tyrosine kinase family, or phosphorylation by Janus kinase in the cases of the hematopoietic cytokine receptor family. The phosphorylated receptor then recruits adaptor molecules that initiate a cascade of phosphorylation events, which culminate in the activation of concurrent pathways through phosphoinositol 3′-kinase (PI3K)/PTEN/AKT and RAS-RAF-ERK (extracellular regulated kinase) (Fig. 14–2). These cascades relay signals to mTOR complex, which is a vital hub for metabolic sensing and short-term post-transcriptional control of cell growth.16 mTOR, potentially coupled with additional outputs of the RAS-RAF-ERK pathway, also activates a genomic response to the growth signal. In essence, the mTOR pathway provides an immediate response to growth stimulus and nutrients followed thereafter by a transcriptional response that provides an increase in specific mRNAs needed for the production of new building blocks for the growing cell. The initial growth response occurs in cells that have a basal number of ribosomes, which serve to translate delayed early response genes. The cascade down the ERK pathway also activates the expression of early response genes such as FOS and MYC. The activation of MYC is probably mediated through the ERK-activation of Ets transcription factors, whose regulatory motifs are found in the MYC gene.17 Furthermore, ERK phosphorylates and stabilizes the MYC protein, enhancing ERK’s ability to drive a transcriptional response to growth.18,19
Canonical signal transduction pathways emanating from a receptor tyrosine kinase and their connections to metabolism. The phosphoinositol 3′-kinase (PI3K)-PTEN-Akt and Ras-Raf-ERK (extracellular regulated kinase) pathways are shown with connections to MYC and hypoxia-inducible factor 1 (HIF-1α), which trigger metabolic transcriptional programs. All of these pathways coordinate a response to growth factors and drive nutrients into the cell to generate ATP and building blocks for lipid, nucleotide, and protein synthesis. The net result is cell growth and generation of waste products, such as reactive oxygen species (ROS), lactate and carbon dioxide. Tumor suppressors (red octagons) and protooncogenes (green bursts) are highlighted.
The immediate sensing of nutrients is mediated through the adenosine monophosphate kinase (AMPK) and mTOR pathways, which, in turn, modulate cellular responses through phosphorylation of regulatory proteins (see Fig. 14–2).9,16,20 In the presence of nutrients, the import of branched-chain amino acids is sensed through lysosomes to activate mTOR complex 1 (mTORC1). Import of glutamine, which is converted to glutamate, is thought to play an important role in activating mTOR through the antiporter, SLC7A1, which exports glutamate in exchange for leucine. Leucine is one of the most potent activators of mTORC1, which, in turn, phosphorylates key regulatory proteins to increase protein translation, mitochondrial biogenesis and respiration, glycolysis, and lipogenesis. Many of these effects are also mediated through mTORC1’s regulation of transcription factors such as PGC1α (mitochondrial biogenesis), HIF-1α (glycolysis), and SREBP (lipogenesis). mTORC1 also phosphorylates and inactivates the transcription factor TFEB, which is a master positive regulator of lysosome biogenesis.16 Presumably, inhibition of TFEB would also diminish the machinery involved in autophagy. Although mTORC1 also stimulates ribosome biogenesis, the mechanism by which this occurs is not yet known. Activation of the mTORC2 complex is less well defined, but mTORC2 is responsible for the subsequent activation of AKT that plays a critical role in activating glycolysis. Thus, growth factor signaling stimulates nutrient uptake that in turn activates mTOR to stimulate cell growth.
In nutrient-deprived states, the AMPK pathway regulates cellular responses that optimize energy production and diminish energy utilization pathways (see Fig. 14–2).20,21 AMPK is allosterically altered by binding to adenosine monophosphate (AMP), which makes AMPK permissive for phosphorylation and activation by the tumor suppressor LKB1. The phosphorylated AMPK in turn phosphorylates and regulates many pathways involved in energy regulation. One of the earliest discoveries was that AMPK phosphorylates and inactivates acetyl-CoA carboxylase, which is involved in fatty acid synthesis. Thus, by diminishing lipogenesis, AMPK is able to inhibit an energy consuming process as well as inhibit cell growth by curbing lipogenesis. AMPK also attenuates protein synthesis through phosphorylation of RNA polymerase I, which is required for ribosome biogenesis. On the other hand, AMPK increases energy yield by stimulating glycolysis through phosphorylation and activation of PFK-2. AMPK stimulates mitochondrial biogenesis through phosphorylation of PGC1α and increases autophagy to recycle energy by phosphorylating ULK-1.21 Thus, increased AMPK activity conserves energy and maximizes energy production.
Together with posttranscriptional responses to growth signaling and nutrients, the nuclear transcriptional response is necessary to sustain the growth program through production of components of the ribosome and mRNAs that give rise to all other components of the cell. mTOR through its direct activation of specific transcription factors contributes to lipogenesis and mitochondrial biogenesis. Growth signaling also activates the MYC protooncogene, that regulates gene expression broadly to support cell growth and proliferation (see Figs. 14–2 and 14–3).19 Loss of function of Drosophila dMYC results in decreased cell and body size, a phenotype that underscores MYC’s role in cell growth.22 This phenotype mimics the loss of ribosome protein gene function in a group of mutant flies termed Minutes. Hence, Drosophila genetics links MYC to cell growth control. Furthermore, MYC is the only transcription factor capable of stimulating the activity of RNA polymerases I, II, and III, all of which are involved in ribosome biogenesis.
Diagram depicting fates of growth factor-stimulated normal cells and MYC transforming cell. Upon engaging a growth factor (pink square), the stimulated cell reacts as a bioreactor, which grows and duplicates itself. MYC overexpression triggers checkpoints via p53, which induces cell death. With loss of p53 MYC’s full transforming potential is unleashed through its transcriptional activities.
MYC dimerizes with its partner Max to bind a specific DNA sequence, termed E-box (CACGTG), and activate transcription.23 It can also inhibit transcription partly through direct binding to Miz-1 and diminishing the expression of Miz-1 target genes, including the cyclin-dependent kinase (CDK) inhibitor p21 and genes involved in autophagy. Upon MYC activation, it is binding to proximal promoters accounts for most of it is transcriptional function in normal cells. When MYC is experimentally expressed at levels comparable to those found in cancers,19 excess MYC triggers p53-dependent checkpoints (see Fig. 14–3) that cause cell growth arrest or apoptosis. In multistep tumorigenesis, therefore, p53 is often lost, unleashing MYC’s full oncogenic potential. A high, unchecked level of MYC allows it to alter the transcriptome by amplifying selected target genes.24 MYC was first shown to directly regulate genes involved in glycolysis, thereby linking an oncogenic transcription factor to metabolism.6,19 Since these initial observations, high-throughput methods have mapped MYC to a broad swath of metabolic enzyme genes involved in glycolysis, glutaminolysis, and lipogenesis. MYC also directly regulates genes involved in mitochondrial biogenesis and the production of ribosomes. Specifically, genes highly induced by MYC include those involved in nucleolar function and ribosome biogenesis, such as Ncl, NPM1, fibrillarin, and NOP52. Collectively, these studies uncover MYC’s role as a central regulator of cell growth through coupling of energy metabolism with cellular biosynthetic processes.
Ribosome biogenesis is a critically important process for cell growth or cell mass accumulation.25,26 Ribosomes are produced through RNAs that are transcribed by RNA polymerases I (rRNA [ribosomal RNA]), II (mRNA), and III (tRNAs [transfer RNAs] and small RNAs). rRNA is synthesized in the nucleolus from high copy numbers of rDNA, whose chromatin structure and transcription depends on nutrient availability. Under nutrient limitation, rDNA chromatin becomes less accessible, thereby restricting ribosome biogenesis.26 Ribosomal proteins produced from mRNAs reenter the nucleolus, where components of ribosomes are assembled into mature ribosomal particles, which are exported to the cytosol. The production of rRNAs and proteins also provides an opportunity for bioenergetic sensing of adequate nutrients to support nucleic acid and protein synthesis required for growth. In this regard, specific ribosomal protein subunits (RPL5, RPL11, and others) can bind and inhibit MDM2 (mouse double minute 2 homolog), which binds to and mediates the degradation of p53.25 Thus, it is surmised that ribosomal proteins in excess of rRNAs would activate p53, triggering checkpoints that block progression through the cell cycle, presumably in response to nutrient limitation sensed as an imbalance in rRNA and ribosomal protein synthesis.
In addition to sensing ribosome biogenesis, p53 also responds to genotoxic stresses by directly regulating metabolism (see Fig. 14–2). P53, in general, activates oxidative phosphorylation and inhibits glycolysis.27 P53 can activate HK, which phosphorylates glucose in the first step of glycolysis, and stimulate TIGAR that shunts glucose to the pentose phosphate pathway through decreasing the levels of fructose-2,6-bisphosphate (F2,6BP), which allosterically activates PFK. P53 also increases the efficiency of mitochondrial function through induction of cytochrome c oxidase (SCO2).28 Overall, it appears that the normal function of p53 is to rewire metabolism to mitigate oxidative stress through increased production of NADPH and the antioxidant glutathione. Gain of p53 function through specific mutations, on the other hand, appears to alter metabolism through specific target genes that are involved in cholesterol biosynthesis or phospholipase function.29,30
Other tumor suppressors are also involved in metabolism (see Fig. 14–2). PTEN negatively modulates PI3K, and hence its loss stimulates the PI3K pathway that is a potent regulator of cell metabolism through stimulation of glycolysis and activation of mTOR, AKT, MYC, and HIF.31 The tumor suppressor Lkb1, which is lost in some lung cancers, normally activates the AMPK pathway and diminishes lipogenesis.21 Loss of the von Hippel-Lindau (VHL) tumor suppressor activates HIF, which transcriptionally regulates glucose metabolism.32 In addition to any direct roles they play in regulating the cell-cycle machinery, tumor suppressors—similar to protooncogenes—also regulate metabolism. By coopting cellular responses to growth factor stimulation in the presence of nutrients, activation of oncogenes and disablement of tumor suppressors achieve coordinated posttranscriptional and transcriptional mobilizations that drive nutrients into ATP production and the building blocks for growing cells.
Growth factor stimulation also results in the production of metabolic wastes and toxins, including carbon dioxide, lactate, and reactive oxygen species (ROS) (Fig. 14–2). In this regard, various mechanisms have evolved to eliminate these wastes that accumulate as cells discard entropy into the environment after consuming “negative entropy” (macromolecules) to survive and grow. Carbon dioxide and protons are neutralized by carbonic anhydrase. Lactate is exported by monocarboxylate transporters.33 Reactive oxygen species (ROS) are generated by the mitochondria and other cellular reaction pathways, such as via NADPH oxidases or disulfide bond formation.34 ROS participates in signaling at ambient levels; however, very high levels of ROS result in oxidative cellular stress.34,35 In particular, electrons leaking from the mitochondrial electron transport chain (ETC) contribute to a large fraction of cellular ROS.35 Electrons are donated to the chain by NADH or succinate at mitochondrial complexes I, II, and III, which all generate ROS. Complexes I and II release ROS into the mitochondrial matrix, whereas complex III releases ROS into space on both sides of the inner mitochondrial membrane. Complex I accepts electrons from NADH, which is generated from TCA cycle oxidation, and passes them on to ubiquinone or coenzyme Q that also accepts electrons from succinate via complex II (SDH). Coenzyme Q then passes electrons to complex III, which, in turn, passes them onto cytochrome c. Finally, electrons are passed from cytochrome c to complex IV or cytochrome c oxidase that generates water from electrons, protons, and oxygen, which serves as the final electron acceptor. Upon accepting electrons at complexes I, III, and IV, a proton is pumped into the intermembrane space, creating a proton gradient across the inner mitochondrial membrane. The proton gradient is dissipated through complex V or ATP synthase with the generation of ATP from ADP. During the process of making ATP, leakage of electrons from complexes I, II, and III generates superoxide from oxygen.
Superoxide is highly reactive and could damage membranes and proteins if unattenuated. Hence, superoxide dismutases (SODs) have evolved to convert superoxide to hydrogen peroxide, which is, in turn, neutralized by catalases and converted to water and oxygen. In addition to enzymatic ROS neutralizers, the family of peroxiredoxins also plays an important role in titrating mitochondrial and cytosolic ROS by neutralizing hydrogen peroxide. Because oxidative stress imposed by ROS is a part of normal metabolism, a system of cellular response to this stress has evolved. Immediate response to ROS is mediated by SOD, catalase, peroxiredoxins, and glutathione. A sustained response to ROS is mediated chiefly through NRF2, which is a transcription factor that is negatively regulated by KEAP1, a protein that is directly inhibited by oxidative modification of sensitive cysteine residues.36 NRF2 activates many genes involved in redox homeostasis, including SODs and catalase. Intriguingly, KEAP1 has been identified as a tumor suppressor in human cancers, illustrating that increased NRF2 activity or antioxidant response is protumorigenic in the setting of heightened metabolic rates and oxidative stress.
METABOLISM AND THE EPIGENOME
Cells have evolved a genome that mediates posttranscriptional and transcriptional mechanisms to import nutrients and harness energy and building blocks for the growing cell. In turn, metabolic intermediates generated from various nutrients can modulate gene expression, seemingly as an adaptive response to the metabolic milieu.37,38,39 The epigenome is richly regulated by metabolic intermediates such as acetyl-CoA, S-adenosylmethionine (SAM), α-ketoglutarate, NAD+, and N-acetylglucosamine.40 Acetyl-CoA mediates histone acetylation and modulates gene expression by rendering the genome accessible to specific transcriptional factors. SAM permits methylation of histones and DNA to prevent access of the transcriptional machinery to certain DNA sequences. α-Ketoglutarate serves as a cofactor for histone and DNA demethylation reactions, thereby countering the modifications provided by SAM. NAD+ serves as a cofactor for sirtuins that play a key role in histone deacetylation. N-acetylglucosamine, which is produced from glucose and glutamine, serves to modify histones. Other metabolic intermediates such as propionate, butyrate, formate, and crotonate also play a role in modifying histones, which have emerged as the metabolic sensor for gene expression.40
The evolution of cancers is not only driven by hard-wired somatic DNA mutations and predisposing germline alleles but also by erasable covalent modifications of DNA and histones. A deregulated epigenetic regulatory system, which randomly silences or makes more accessible portions of the genome, could enhance the adaptability of cancer cells and thus provide a selection advantage that does not require DNA mutations. In this regard, deep sequencing of human cancers, particularly leukemias, has revealed that chromatin-modifying proteins, such as MLL2 (mixed-lineage leukemia protein 2), are frequently mutated at the somatic level.41,42 Thus, somatic mutations in chromatin modifiers are surmised to increase the degrees of freedom for cancer cell adaptation to the dynamic tumor microenvironment and permit tumor progression.
HEMATOLOGIC NEOPLASMS AND METABOLISM
Normal hematopoietic stem cells (HSCs) and committed multipotent progenitor cells appear to have different metabolic programs, which may be adopted in the neoplastic state. The HSC resides in a hypoxic environment, and hence low mitochondrial mass and high glycolytic rates appear favored for survival. One of the mechanisms by which the hypoxic HSC niche induces stem cell quiescence is through HIF-1α and inhibits, which transactivates genes involved in glycolysis and inhibits DNA replication.32 Two studies of HSCs documented that HIF-1α is essential for the quiescent state, such that deletion of HIF-1α resulted in HSC proliferation and depletion of the HSC compartment.43,44 Conversely, loss of VHL stabilized HIF-1α resulted in an expansion of the HSCs incapable of replenishing hematopoietic cells, resulting in cytopenia. Intriguingly, three studies showed that the Lkb1 tumor suppressor also plays a role in HSC quiescence; loss of Lkb1 resulted in cell proliferation and loss of the HSC compartment.45,46,47 Interestingly, loss of Lkb1 in HSCs does not seem to be mediated solely through AMPK, as loss of AMPK in HSCs did not phenocopy the HSC nonquiescent phenotype seen with Lkb1 loss. Instead, one study identified the mitochondrial biogenesis coregulators, PGC1α and PGC1β, as being central to the Lkb1 loss phenotype.45 Loss of Lkb1 in HSCs was associated with decreased expression of PGC1α and PGC1β and decreased mitochondrial DNA content and membrane potential. These studies collectively suggest that both HIF-1α and LKB1 are necessary for induction of quiescence by the hypoxic microenvironment. Loss of either HIF-1α or Lkb1 resulted in increased HSC proliferation and, presumably, commitment toward progenitors, thereby depleting the HSC pool. Although the hypoxic HSC microenvironment suggests that glycolysis predominates, it should be noted that hypoxic cells can still respire and consume oxygen. In fact, cytochrome c oxidase only ceases to function at oxygen tension well below 0.5 percent (as compared to the ambient 21 percent oxygen or approximately 6 percent oxygen found in perfused normal tissues). As such, the observation that loss of Lkb1 is associated with a mitochondriopathy in HSCs suggests that mitochondrial function is essential for HSC maintenance, and may resolve the paradox that HSCs seems to rely also on glutamine oxidation (Fig. 14–4).
Metabolic features of hematopoietic stem cells undergoing symmetric and asymmetric division. Some of these features appear to be preserved in leukemic stem cells.
The HSC uses symmetric commitment to replenish and maintain the stem cell pool and asymmetric division for the generation of committed progenitors (see Fig. 14–4). HIF-1α and Lkb1 appear to play a role in maintaining symmetric commitment. Fatty acid and glutamine oxidation, which requires mitochondrial function, may be required for asymmetric commitment toward progenitors. Surprisingly, despite anticipated HIF-mediated upregulation of glycolysis, the inducible glucose transporter GLUT1 is not highly expressed in HSCs and is only expressed upon differentiation.48 Instead, the glutamine transporter ASCT2 (SLC1A5) is more highly expressed in HSCs, suggesting that glutamine oxidation via the TCA cycle unexpectedly plays a role in hypoxic stem cell metabolism. Consistent with the notion that hypoxic cells continue to respire, recent studies with human B cells or fibroblasts illustrate this capacity to oxidize glutamine in hypoxia when glucose is largely shunted away from the TCA cycle as lactate.49,50 Interestingly, glutamine metabolism also appears to influence cell fate. For example, persistent glutamine metabolism in HSCs seems required for erythroid differentiation as glutamine deprivation blunts erythroid nucleotide synthesis and favors differentiation toward the myelomonocytic lineage even in the presence of erythropoietin.48 Fatty acid oxidation, on the other hand, appears to be necessary for asymmetric division. Activation of peroxisome proliferator-activated receptor (PPAR)-δ, which augments mitochondrial function and fatty acid oxidation through the promyelocytic leukemia (PML)-PPARδ–fatty acid oxidation (FAO) pathway, increases asymmetric stem cell division, whereas inhibition of FAO enhances symmetric stem cell commitment.51 These findings suggest that mitochondrial oxidation of fatty acids and glutamine may play a role in asymmetric division and lineage commitment, while hypoxia-promoted glycolysis and, surprisingly, glutamine oxidation may be associated with the quiescent HSC pool. How these metabolic cues may affect cellular states through the epigenome is not yet known, however.