Fundamentals of Cancer Metabolism

REVIEW

ONCOLOGY 2016 © The Authors, some rights reserved;

exclusive licensee American Association for
Fundamentals of cancer metabolism the Advancement of Science. Distributed

under a Creative Commons Attribution
NonCommercial License 4.0 (CC BY-NC).
Ralph J. DeBerardinis1* and Navdeep S. Chandel2* 10.1126/sciadv.1600200
Tumors reprogram pathways of nutrient acquisition and metabolism to meet the bioenergetic, biosynthetic,
and redox demands of malignant cells. These reprogrammed activities are now recognized as hallmarks of
cancer, and recent work has uncovered remarkable flexibility in the specific pathways activated by tumor cells
to support these key functions. In this perspective, we provide a conceptual framework to understand how and
why metabolic reprogramming occurs in tumor cells, and the mechanisms linking altered metabolism to tumor-
igenesis and metastasis. Understanding these concepts will progressively support the development of new strat-
egies to treat human cancer.
INTRODUCTION AND OVERARCHING PRINCIPLES

Cancer metabolism is one of the oldest areas of research in cancer nated by systemic administration of asparaginase. Ultimately, effective
biology, predating the discovery of oncogenes and tumor suppressors metabolic therapy will require defining the stage of tumor progression
by some 50 years. The field is based on the principle that metabolic in which each pathway provides its benefit to the cancer cell. Some
activities are altered in cancer cells relative to normal cells, and that activities first become essential very early in tumorigenesis as the nas-
these alterations support the acquisition and maintenance of malig- cent tumor begins to experience nutrient limitations (6). In other cases,
nant properties. Because some altered metabolic features are observed altered pathways may be dispensable in primary tumors but essential
quite generally across many types of cancer cells, reprogrammed me- for metastasis (7, 8). Because new therapeutic targets are nominated
tabolism is considered a hallmark of cancer (1, 2). Precisely how me- from simple experimental models like cultured cells, it will be essential
tabolism becomes reprogrammed in cancer cells, whose functions or to define their context-specific roles in biologically accurate models of
malignant properties are enabled by these activities, and how to ex- tumor initiation and progression.
ploit metabolic changes for therapeutic benefit are among the key
questions driving research in the field.
This review covers several fundamental principles in cancer metab- METABOLIC REPROGRAMMING AND ONCOMETABOLITES
Downloaded from https://www.science.org on July 29, 2022

olism, with the goal of introducing non-experts to the concepts mo- IN CANCER
tivating ongoing research. With the explosion of research in cancer
metabolism over the past decade, no single review can possibly cover Altered metabolic activity supports anabolic growth during nutrient-
it all. The sections below highlight some of the essential, recent papers replete conditions, catabolism to support cell survival during nutrient
supporting these core principles. An overarching theme in cancer me- limitation, and fortification of redox homeostatic systems to counter-
tabolism is that reprogrammed activities improve cellular fitness to act the metabolic effects of oncogene activation, tumor suppressor
provide a selective advantage during tumorigenesis. Most of the clas- loss, and other stresses (9). Discovery and characterization of repro-
sical examples of reprogrammed activities either support cell survival grammed activities may provide opportunities to image tumor tissue
under stressful conditions or allow cells to grow and proliferate at path- noninvasively, predict tumor behavior, and prevent tumor progression
ologically elevated levels. Three of these—altered bioenergetics, by blocking essential pathways. It is important to differentiate “meta-
enhanced biosynthesis, and redox balance—are discussed at length be- bolic reprogramming” from “oncometabolites,” two terms widely used
low. It logically flows that if these activities provide benefit to the main the recent cancer metabolism literature (10). We propose that the
lignant cell, then some of them might be suitable therapeutic targets. term metabolic reprogramming be used to describe conventional
This rendering of cancer metabolism is supported by many examples metabolic pathways whose activities are enhanced or suppressed in
in which inhibition of an enhanced metabolic activity results in im- tumor cells relative to benign tissues as a consequence of tumorigenic
paired growth of experimental tumors (3, 4). In some cases, the par- mutations and/or other factors. Oncometabolite is a relatively new
ticular metabolic liabilities of cancer cells have been translated into term that refers to metabolites whose abundance increases markedly
effective therapies in human cancer. Asparaginase, an enzyme that in tumors. We suggest that this term be reserved for metabolites for
converts the amino acid asparagine to aspartic acid and ammonia, is which (i) there is a clear mechanism connecting a specific mutation in
an essential component of treatment for acute lymphoblastic leukemia the tumor to accumulation of the metabolite, and (ii) there is
(ALL) (5). Because of their high rates of protein synthesis and poor compelling evidence for involvement of the metabolite in the develop-
ability to synthesize asparagine de novo, ALL cells require a constant ment of malignancy.
supply of asparagine from the plasma. This supply is essentially elimi- The classical example of a reprogrammed metabolic pathway in
cancer is the Warburg effect or aerobic glycolysis (11). Glycolysis is
a physiological response to hypoxia in normal tissues, but Otto Warburg
1
Children’s Medical Center Research Institute, University of Texas Southwestern Med- in the 1920s observed that tumor slices and ascites cancer cells
ical Center, Dallas, TX 75390, USA. 2Department of Medicine, Northwestern University constitutively take up glucose and produce lactate regardless of oxygen
Feinberg School of Medicine, Chicago, IL 60611, USA.
*Corresponding author. Email: [email protected] (R.J.D.); nav@ availability, an observation that has been seen in many types of cancer
northwestern.edu (N.S.C.) cells and tumors (12). The increase in glycolytic flux allows glycolytic
DeBerardinis and Chandel Sci. Adv. 2016; 2 : e1600200 27 May 2016 1 of 18

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intermediates to supply subsidiary pathways to fulfill the metabolic contain mutations that allow the PI3K-AKT-mTOR network to achieve
demands of proliferating cells (11). Like glycolytic intermediates, tri- high levels of signaling with minimal dependence on extrinsic stimula-
carboxylic acid (TCA) cycle intermediates are also used as precursors tion by growth factors (17). Many of the best-characterized oncogenes
for macromolecule synthesis (13). Their utilization in biosynthetic and tumor suppressors reside in the PI3K-AKT-mTOR network, and
pathways requires that carbon be resupplied to the cycle so that inter- aberrant activation of this pathway is among the most frequent altera-
mediate pools are maintained; pathways that “refill” the cycle are termed tions seen in a diverse set of cancers.
anaplerotic pathways, and they generate TCA cycle intermediates that Another commonly deregulated pathway in cancer is gain of func-
can enter the cycle at sites other than acetyl-CoA (coenzyme A) (14). Two tion of MYC by chromosomal translocations, gene amplification, and
activities that provide anaplerotic fluxes in cancer cells are glutaminolysis, single-nucleotide polymorphisms. MYC increases the expression of
which produces a-ketoglutarate from glutamine, and pyruvate carboxyl- many genes that support anabolic growth, including transporters and
ation, which produces oxaloacetate from glucose/pyruvate. Oxidation of enzymes involved in glycolysis, fatty acid synthesis, glutaminolysis,
the branched-chain amino acids (BCAAs) isoleucine and valine also pro- serine metabolism, and mitochondrial metabolism (18). Oncogenes
vides an anaplerotic flux in some tissues. like Kras, which is frequently mutated in lung, colon, and pancreatic
Despite the incredible genetic and histological heterogeneity of tu- cancers, co-opt the physiological functions of PI3K and MYC pathways
mors, malignancy seems to involve the common induction of a finite to promote tumorigenicity. Aside from oncogenes, tumor suppressors
set of pathways to support core functions like anabolism, catabolism, such as the p53 transcription factor can also regulate metabolism (19).
and redox balance (15). The general induction of these pathways may The p53 protein–encoding gene TP53 (tumor protein p53) is mutated
reflect their regulation by signaling pathways that are commonly per- or deleted in 50% of all human cancers. The tumor-suppressive func-
turbed in cancer cells (Fig. 1). Normal cells, upon stimulation by tions of p53 have been ascribed to execution of DNA repair, cell cycle
growth factors, activate phosphatidylinositol 3-kinase (PI3K) and its arrest, senescence, and apoptosis. However, recent studies indicate that
downstream pathways AKT and mammalian target of rapamycin p53 tumor-suppressive actions might be independent of these canonical
(mTOR), thereby promoting a robust anabolic program involving p53 activities but rather dependent on the regulation of metabolism and
increased glycolytic flux and fatty acid synthesis through activation oxidative stress (20, 21). Loss of p53 increases glycolytic flux to pro-
of hypoxia-inducible factor–1 (HIF-1) and sterol regulatory element– mote anabolism and redox balance, two key processes that promote
binding protein (SREBP), respectively (16). Tumor cells very frequently tumorigenesis (19).
Fig. 1. Signaling pathways that regulate cancer metabolism. Tumor cells have aberrant activation of mTORC1 that induces an anabolic growth
program resulting in nucleotide, protein, and lipid synthesis. Loss of tumor suppressors like p53 or activation of oncogenes like MYC further promotes
anabolism through transcriptional regulation of metabolic genes. Metabolism controls signaling through regulating reactive oxygen species (ROS), acet-
ylation, and methylation. PPP, pentose phosphate pathway; G6P, glucose-6-phosphate; 3-PG, 3-phosphoglycerate; ATP, adenosine 5´-triphosphate;
mTORC1, mTOR complex 1; a-KG, a-ketoglutarate; RTK, receptor tyrosine kinase.

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A salient feature of many tumors is that they reside in a low-oxygen by the inheritance of biallelic mutations in the gene encoding L2HGDH
environment (hypoxia) ranging from 0 to 2% O2 because the tumor cell (45). Affected children have seizures, mental retardation, white matter
proliferation rate often exceeds the rate of new blood vessel formation abnormalities of the brain, and systemically elevated levels of L2HG.
(angiogenesis) (22). The metabolic adaptation to hypoxia is coordinated Remarkably, a number of these children have developed malignant
by HIF-1, which induces metabolic genes involved in increasing glyco- brain tumors (46), providing an early clue to the significance of D2HG
lytic flux (23). Some tumors display constitutive activation of HIF-1 un- in IDH1/IDH2-mutant gliomas and raising the question of whether
der normoxic conditions through a variety of mechanisms, including L2HG is also an oncometabolite. L2HG and D2HG exhibit different
(i) hyperactivation of mTORC1, (ii) loss of von Hippel–Lindau, (iii) effects on dioxygenase function (38), suggesting that the sensitivity of a
accumulation of ROS, and (iv) accumulation of the TCA cycle metabo- particular tissue to the presence of either metabolite may depend on
lites succinate or fumarate due to cancer-specific mutations in succinate which dioxygenases are expressed. Recent work has demonstrated mod-
dehydrogenase (SDH) or fumarate hydratase (FH), respectively (24). est accumulation of L2HG in cells experiencing hypoxia or electron
The robust coordinated induction of metabolic pathways that transport chain (ETC) dysfunction (42, 47) and in human renal cell
support tumorigenesis by combination of deregulation of PI3K-AKT- carcinomas, which frequently display epigenetic silencing of L2HGDH
mTOR signaling pathways, loss of tumor suppressors, and activation of (48). It is unknown whether reducing L2HG levels in these settings will
oncogenes alleviates the necessity of having mutations or amplifications promote cellular differentiation or suppress tumor progression.
in metabolic enzymes per se. Thus, examples of metabolic enzyme de- The principle that oncometabolites exert their effects outside of the
regulation through genetic mutation are rare. One example is the ele- conventional metabolic network also applies to the other two molecules
vated expression of phosphoglycerate dehydrogenase (PHGDH) due that can reasonably be called oncometabolites: fumarate and succinate
to amplification in a fraction of breast cancer and melanoma (25, 26). (28). Both are TCA cycle intermediates found throughout the body, but
PHGDH catalyzes the conversion of the glycolytic intermediate 3- some tumors accumulate massive levels of fumarate and/or succinate as
phosphoglycerate to 3-phosphohydroxypyruvate in the first step of a consequence of loss-of-function mutations in FH or the SDH complex,
the serine biosynthesis pathway. Serine metabolism supplies methyl respectively (49–51). Although these mutations markedly reprogram
groups to the one-carbon and folate pools contributing to nucleotide metabolism by impairing TCA cycle flux, the extent to which fumarate
synthesis, methylation reactions, and NADPH (reduced nicotinamide and succinate participate in cancer development likely involves their
adenine dinucleotide phosphate) production (27). Inhibiting serine nonmetabolic functions (28). Like D2HG, evidence indicates that suc-
biosynthesis by silencing PHGDH in cells with high levels of this en- cinate and fumarate interfere with dioxygenase activity, supporting the
zyme results in growth suppression, and the enzyme displays oncogenic notion that a general property of oncometabolites is the ability to regulate
properties in gain of function assays (25, 26). epigenetics (52, 53). PHGDH overexpression and mutations in IDH1/
The other examples of mutational deregulation of metabolic en- IDH2, SDH, and FH all alter metabolite levels that control epigenetics

zymes are those that generate oncometabolites. The current list of true (54). Several other metabolites, including acetyl-CoA, a-ketoglutarate,
oncometabolites is short (28). The term is most commonly and appro- and S-adenosylmethionine also participate in epigenetic reprogramming,
priately applied to D-2-hydroxyglutarate (D2HG), a reduced form of the and time will tell whether genetically specific alterations of these metab-
TCA cycle intermediate a-ketoglutarate. D2HG is scarce in normal tis- olites in tumors promote tumorigenesis (54). Some metabolites, notably
sues but rises to millimolar concentrations in tumors with mutations in fumarate, covalently bind to sulfhydryl groups in glutathione, proteins,
isocitrate dehydrogenase 1 or 2 (IDH1 or IDH2). These mutations oc- and peptides, altering their function and perhaps accounting for anoth-
cur commonly in gliomas, acute myelogenous leukemias (AMLs), and er mechanism by which oncometabolites promote or perpetuate malig-
other types of cancer (29–31). D2HG and its relationship to mutant nant phenotypes (55–58).
IDH1 and IDH2 have been reviewed extensively elsewhere (32). The
most relevant point here is that D2HG production requires a neomor-
phic enzyme activity imparted to IDH1/IDH2 by specific active-site BIOENERGETICS
mutations (33, 34). High levels of D2HG interfere with the function
of dioxygenases requiring a-ketoglutarate as a cosubstrate. These in- Otto Warburg’s hypothesis that cancer cells take up glucose and gen-
clude prolyl hydroxylases, cytosine hydroxylases, and histone demethylases, erate a substantial amount of lactate in the presence of ambient oxygen
whose inhibition influences gene expression in part via an altered epi- due to impaired mitochondrial function led to the widely held miscon-
genetic state characterized by a failure to express differentiation pro- ception that cancer cells rely on glycolysis as their major source of ATP
grams (35–41). Thus, although D2HG arises from an alteration of the (59, 60). Today, it is clear that cancer cells exhibit aerobic glycolysis due
metabolic network, its role in cancer seems to depend on nonmetabolic to activation of oncogenes, loss of tumor suppressors, and up-regulation
effects. Currently, D2HG is being used as a biomarker for disease mo- of the PI3K pathway, and that one advantage of high glycolytic rates is
nitoring, and inhibitors specific to mutants IDH1/IDH2 are in clinical the availability of precursors for anabolic pathways (2, 61). Warburg’s
trials for AML and solid tumors. observation that tumors display a high rate of glucose consumption has
The metabolite 2HG also exists as the enantiomer L-2HG (L2HG), been validated in many human cancers with fluorodeoxyglucose posi-
which is not produced by mutant forms of IDH1/IDH2. This metabolite tron emission tomography, which uses a radioactive glucose analog to
arises from the noncanonical activity of various dehydrogenases, in- image glucose uptake in tumors and adjacent normal tissue. Neverthe-
cluding malate dehydrogenase and lactate dehydrogenase, whose pro- less, many studies have demonstrated that the great majority of tumor
miscuous behavior reduces a-ketoglutarate to L2HG (42–44). L2HG cells have the capacity to produce energy through glucose oxidation
may be oxidized back to a-ketoglutarate by a FAD-linked enzyme, (that is, the process by which glucose-derived carbons enter the TCA cy-
L2HG dehydrogenase (L2HGDH). L2HGDH deficiency, also called cle and are oxidized to CO2, producing ATP through oxidative phospho-
L2HG aciduria, is a rare neurometabolic disease of childhood caused rylation). Furthermore, limiting glycolytic ATP production by inhibiting

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the activity of pyruvate kinase fails to prevent tumorigenesis, suggest- activates catabolic pathways like fatty acid oxidation to stimulate ATP
ing that the major role of glycolysis is not to supply ATP (62). Moreover, production (84). In conditions of metabolic stress, certain Ras-driven
mitochondrial metabolism is necessary for cancer cell proliferation and cancer cells scavenge lipids to support ATP production (85). Ovarian
tumorigenesis (63–65). Thus, despite their high glycolytic rates, most can- cancer cells use fatty acids from neighboring adipocytes to fuel mito-
cer cells generate the majority of ATP through mitochondrial function, chondrial ATP production (86). Thus, there are multiple mechanisms
with the likely exception of tumors bearing mutations in enzymes involved by which cancer cells maintain their ATP/ADP ratio to sustain viability
in mitochondrial respiration (for example, SDH and FH) (66). Neverthe- in nutrient- and oxygen-poor environments.
less, cells harboring mutations in FH or SDH still rely on mitochondrial
metabolism by metabolically rewiring themselves to provide the neces-
sary TCA cycle intermediates and ROS for cell proliferation (55, 67–70). BIOSYNTHESIS OF MACROMOLECULES
In addition to pyruvate derived from glycolysis, fatty acids and amino
acids can supply substrates to the TCA cycle to sustain mitochondrial Biosynthetic or anabolic pathways are an essential aspect of cancer me-
ATP production in cancer cells (Fig. 2). The breakdown of fatty acids tabolism because they enable cells to produce the macromolecules re-
(b-oxidation) in the mitochondria generates acetyl-CoA and the reducing quired for replicative cell division and tumor growth. As a general theme,
equivalents NADH and FADH2, which are used by the ETC to produce these pathways involve the acquisition of simple nutrients (sugars, es-
mitochondrial ATP. The amino acid glutamine can generate glutamate sential amino acids, etc.) from the extracellular space, followed by their
and subsequently a-ketoglutarate to fuel the TCA cycle through a series conversion into biosynthetic intermediates through core metabolic
of biochemical reactions termed glutaminolysis (71). Furthermore, the pathways like glycolysis, the PPP, the TCA cycle, and nonessential amino
BCAAs isoleucine, valine, and leucine, which are elevated in plasma of acid synthesis, and finally the assembly of larger and more complex mol-
patients with pancreatic cancers, can be converted into acetyl-CoA and ecules through ATP-dependent processes (Fig. 3). The three macro-
other organic molecules that also enter the TCA cycle (72). The metabolic molecular classes most commonly studied in cancer metabolism are
flexibility afforded by multiple inputs into the TCA cycle allows cancer proteins, lipids, and nucleic acids, which comprise approximately 60,
cells to adequately respond to the fuels available in the changing micro- 15, and 5% of the dry mass of mammalian cells, respectively. Evidence
environment during the evolution of the tumor (9). A combination of the indicates that biosynthesis of all three classes is under the control of the
local tumor microenvironment and oncogenic lesions is likely to dictate same signaling pathways that govern cell growth and are activated in
the fuel used by mitochondria to sustain tumor growth. cancer via tumorigenic mutations, particularly PI3K-mTOR signaling.
Solid tumors contain significant heterogeneity of perfusion, such Protein biosynthesis is highly regulated and requires access to a full
that many tumor cells reside in nutrient- and oxygen-poor environments. complement of essential and nonessential amino acids. Cancer cells
Cancer cells have therefore adapted multiple mechanisms to sustain and other cells under the influence of growth factor signaling express sur-

mitochondrial function for survival. For example, the mitochondrial face transporters that allow them to acquire amino acids from the extra-
ETC can function optimally at oxygen levels as low as 0.5% (73). More- cellular space (87). This not only provides cells with the raw materials
over, hypoxic tumor cells (<2% O2) can continue to cycle and use gluta- needed for protein synthesis but also allows them to maintain activity
mine as a fuel for oxidative ATP production (74–76). Kras-driven of the mTOR signaling system, specifically mTORC1. mTORC1 is stim-
pancreatic cancer cells in nutrient-depleted conditions use proteins ulated by the presence of amino acids and activates protein synthesis via its
scavenged from the extracellular space to produce glutamine and effects on translation and ribosome biogenesis (80). Most nonessential
other amino acids to fuel the TCA cycle for cell survival and growth amino acids are produced through transamination reactions, which trans-
(Fig. 2) (77). Furthermore, if pyruvate oxidation to acetyl-CoA is com- fer the amino group from glutamate to a ketoacid. Proliferating cancer cells
promised by hypoxia or ETC impairment, glutamine can provide acetyl- take up glutamine and convert it to glutamate through a variety of de-
CoA as a biosynthetic precursor to sustain tumor growth (69, 78, 79). amidation and transamidation reactions, most notably the mitochondrial
When tumor cells become nutrient-deprived, they adapt to the amidohydrolase glutaminase (71). Together, these enzymes generate a large
microenvironment by decreasing their demand for ATP. The result- intracellular glutamate pool available for nonessential amino acid syn-
ant increase in ATP availability maintains an adequate ATP/ADP thesis. Both glutamine uptake and glutaminase activity are stimulated by
(adenosine 5´-diphosphate) ratio to drive unfavorable biological re- mTORC1, providing glutamate for transamination reactions and/or main-
actions. The anabolic kinase mTOR, discussed in greater detail below, tenance of the TCA cycle, which also contributes to amino acid synthesis.
drives the energetically demanding growth of tumor cells. This kinase Furthermore, when the intracellular glutamine supply exceeds the cell’s
is inhibited when amino acids and oxygen levels are diminished (80). demands, glutamine can be exported in exchange for essential amino acids
Furthermore, decreased mTOR activity increases autophagic flux. In to stimulate mTORC1 and protein synthesis (88). Thus, growth conditions
oncogenic Kras- or Braf-driven non–small-cell lung cancer (NSCLC) in which glutamine and essential amino acids are abundant enable
cells, autophagy provides an intracellular glutamine supply to sustain mTORC1-mediated activation of protein synthesis.
mitochondrial function (81, 82). To survive the hypoxic tumor micro- When nutrients are scarce, cells have access to a number of catabolic
environment, cancer cells also diminish their demand for ATP by de- pathways to degrade macromolecules and resupply key pools of intra-
creasing highly demanding ATP-dependent processes, such as running cellular metabolic intermediates. Protein degradation pathways have
the Na/K-dependent adenosine triphosphatase. If diminishing ATP been characterized extensively as mechanisms to supply amino acids
demand is not sufficient to maintain ATP/ADP ratio, the rise in ADP in cancer cells. Intracellular proteins and other macromolecules can be
activates adenylate kinase, a phosphotransferase enzyme that buffers recycled through autophagy, a highly regulated process through which
the fall in ATP levels by converting two ADP molecules into adenosine proteins and organelles are delivered to the lysosome and degraded
5´-monophosphate (AMP) and ATP (83). The rise in AMP during nu- (89). Autophagy is an essential survival pathway during nutrient or growth
trient deprivation triggers the activation of AMP kinase (AMPK), which factor deprivation, and genetic studies demonstrate that it contributes to

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Fig. 2. Metabolic pathways under nutrient-replete and nutrient-deprived conditions. Accessibility to nutrients within solid tumors is regulated
by proximity to the vasculature. Cells located adjacent to the vasculature use nutrients and oxygen to fuel anabolic pathways that support proliferation.
However, cells distant from the vasculature have diminished accessibility to nutrients and oxygen and may engage in alternative forms of metabolism
including oxidation of fatty acids and BCAAs as well as macromolecular degradation through autophagy and macropinocytosis to support cell viability.
some forms of cancer in mice (90, 91). However, because autophagy cell lines (97). The relative importance of these nutrients for fatty
supplies amino acids through protein degradation, it does not serve acid synthesis in vivo is unknown, although early studies suggested that
as a source of net protein synthesis. It is also potently suppressed by most fatty acyl carbon in experimental tumors is derived from glucose
mTORC1. Macropinocytosis allows cells to internalize proteins and (98, 99). Isotopic tracing experiments designed to assess the cytosolic
other components of the extracellular milieu and deliver them for NADPH pool have recently suggested that most NADPH used for fatty
degradation via the endocytic pathway. Under conditions of nutrient acid synthesis arises from the PPP (100, 101).
depletion, macropinocytosis supplies both nitrogen and carbon to Transcription of genes involved in fatty acid synthesis is regulated by
central metabolic pathways (92). Evidence indicates that extracellular the SREBP-1 transcription factor (102). SREBP-1 regulates not only the
protein degradation, like autophagy, is suppressed by mTORC1 (93). enzymes needed to convert acetyl-CoA into fatty acids but also the
Suppressing pathways of protein degradation may help maximize rates enzymes of the PPP and pathways required to convert acetate and
of net protein synthesis when extracellular amino acids are available glutamine into acetyl-CoA (103). This transcription factor therefore
and mTORC1 is active. regulates genes encoding proteins that catalyze or facilitate fatty acid
Tumor cells rapidly produce fatty acids for membrane biosynthesis, synthesis. In lipid-replete conditions, SREBP-1’s transcriptional activity
lipidation reactions, and cellular signaling. Fatty acid synthesis requires is suppressed by its sequestration in the endoplasmic reticulum. Under
sources of acetyl-CoA and reducing power in the form of cytosolic conditions of sterol depletion, proteolytic cleavage releases the tran-
NADPH; effective fatty acid synthesis therefore requires integration scriptionally active domain, which travels to the nucleus and binds to
with other pathways of carbon and redox metabolism. In most cultured sterol response elements in the promoters of lipogenic genes (104).
cells, glucose is the most prominent acetyl-CoA source for fatty acid In cancer cells with constitutively high rates of fatty acid synthesis,
synthesis (94, 95). Glutamine and acetate have been demonstrated to additional mechanisms help keep SREBP-1 in a transcriptionally active
provide alternative carbon sources when access to glucose-derived state. mTORC1 signaling, via its effector S6 kinase (S6K), activates a
acetyl-CoA is impaired by hypoxia or mitochondrial dysfunction transcriptional program that includes both SREBP-1 and the related pro-
(69, 78, 79, 96). Leucine degradation can also supply acetyl-CoA in some tein SREBP-2, which regulates transcription of genes in sterol biosynthesis

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Fig. 3. Anabolic pathways that promote growth. Glucose metabolism generates glycolytic intermediates that can supply subsidiary pathways including
the hexosamine pathway, PPP, and one-carbon metabolism, all of which support cell growth. Mitochondrial TCA cycle intermediates such as oxaloacetate
(OAA) and citrate are used to generate cytosolic aspartate and acetyl-CoA for nucleotide and lipid synthesis, respectively. Mitochondria also generate H2O2
and acetyl-CoA for redox signaling and acetylation, respectively. NADPH is used to drive anabolic reactions and to maintain antioxidant capacity. Cytosolic
sources of NADPH include the oxidative PPP, IDH1, and enzymes from one-carbon metabolism including MTHFD1. Mitochondrial sources of NADPH include
MTHFD2, MTHF2L, and IDH2. HK2, hexokinase 2; G6PDH, glucose-6-phosphate dehydrogenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LDH, lactate
dehydrogenase; ACLY, ATP citrate lyase; GLS, glutaminase; SHMT, serine hydroxymethyltransferase; MTHFD2, methylenetetrahydrofolate dehydrogenase 2;
MTHFD2L, MTHFD2-like; ACSS2, acyl-CoA synthetase short-chain family member 2; THF, tetrahydrofolate.

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(103). Both SREBP-1 and SREBP-2 are required for mTORC1-mediated points to a distinct mechanism by which activation of the signaling
cell proliferation. The mechanism of SREBP activation by mTORC1 is pathway enables nucleotide biosynthesis. The mTORC1 effector ribo-
incompletely understood but involves nuclear entry of the phosphatidic somal S6K1 phosphorylates the trifunctional enzyme CAD (carbamoyl-
acid phosphatase Lipin-1, which enhances nuclear SREBP abundance phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase),
and activity on the promoters of lipogenic genes (105). which catalyzes the first three steps of pyrimidine synthesis (113). Phos-
Both fatty acids and lipids can also be acquired from the extracellular phorylation on CAD S1859 is required for mTORC1-dependent stim-
space to supply membrane biosynthesis. PI3K signaling activates fatty ulation of pyrimidine biosynthesis (113). Additional work is needed to
acid uptake and suppresses fatty acid oxidation, thereby maximizing determine how other aspects of de novo nucleotide synthesis, purine
lipogenesis in proliferating cells under the control of growth factors and pyrimidine salvage pathways, and accessory activities like folate me-
(106). Lipid uptake may acquire further importance during conditions tabolism are regulated in cancer cells in support of cell proliferation.
of metabolic stress, when the ability to meet oncogene-driven demands
for biosynthesis is compromised. The ability to scavenge lysophospho-
lipids (lipid intermediates containing a glycerophosphate backbone REDOX BALANCE
with one acyl chain) is required for maximal cancer cell growth during
hypoxia, which suppresses de novo fatty acid synthesis from glucose ROS are intracellular chemical species that contain oxygen and include
(85). Furthermore, in cancer cells with constitutive mTORC1 signaling, the superoxide anion (O2−), hydrogen peroxide (H2O2), and the hy-
hypoxia induces a state of dependence on access to extracellular desatu- droxyl radical (OH·) (114). The mitochondria and cytosolic NADPH
rated fatty acids to maintain endoplasmic reticulum integrity in support oxidases (NOXs) produce O2− from the one-electron reduction of ox-
of protein biosynthesis (107). Notably, SREBP-1 was first identified as the ygen (115). O2− is converted into H2O2 by the enzymatic activity of
transcription factor responsible for expression of the low-density lipo- superoxide dismutase 1 or 2, which are localized to the cytosol or mito-
protein receptor (LDLR) (108), implying that enhanced de novo lipo- chondrial matrix, respectively. H2O2 is subsequently detoxified to water
genesis occurs concomitantly with enhanced import of lipids from the by the enzymatic activity of mitochondrial and cytosolic peroxiredoxins
extracellular space. These parallel pathways appear to be essential (PRXs), which, as a consequence, undergo H2O2-mediated oxidation of
in glioma, where oncogenic activation of epidermal growth factor re- their active-site cysteines (116). Thioredoxin (TXN), thioredoxin reduc-
ceptor (EGFR) signaling stimulates SREBP-1 and expression of LDLR tase (TrxR), and the reducing equivalent NADPH reduce oxidized PRXs
(109). These cancer cells are highly sensitive to inhibitors of fatty acid to complete the catalytic cycle (117). Glutathione peroxidases (GPXs)
and cholesterol biosynthesis. Inhibition of the EGFR-PI3K signaling can also convert H2O2 to water in the mitochondrial matrix and cytosol
axis but not of mTORC1 suppresses nuclear translocation of SREBP-1 through H2O2-mediated oxidation of reduced glutathione (GSH)
in glioma cells with oncogenic EGFR, suggesting an alternate, mTORC1- (118). Glutathione reductase (GR) and NADPH reduce oxidized gluta-

independent mode of SREBP-1 activation in glioma cells (109). This thione (GSSG) back to GSH. Additionally, catalase, an abundant antiox-
transcriptional program includes LDLR expression and induces reliance idant in peroxisomes, can detoxify H2O2 to water without any cofactors.
on cholesterol uptake to maintain the intracellular pool (110). Impairing However, in the presence of ferrous or cuprous ions, H2O2 can become
intracellular cholesterol availability by activating liver X receptor induced OH· and quickly cause the oxidation of lipids, proteins, and DNA, re-
cell death both in culture and in vivo, suggesting a pharmacological ap- sulting in cellular damage. NADPH is required to maintain multiple anti-
proach to silence lipogenic programs in glioma (110). oxidant defense systems. The cytosol has multiple sources of NADPH
Purine and pyrimidine nucleotides are required for synthesis of generation, including the oxidative PPP, malic enzyme 1, IDH1, and
RNA and DNA. De novo biosynthesis of nucleotides is complex, re- one-carbon metabolism. NADPH generation in the mitochondria, in
quiring input from several pathways in a coordinated fashion. The part, is controlled by one-carbon metabolism and IDH2.
phosphoribosylamine backbone of these molecules is produced from Historically, ROS have been thought of as lethal metabolic by-
ribose-5-phosphate, an intermediate of the PPP, and an amide donation products of cellular respiration and protein folding. However, studies
reaction using glutamine as a substrate (111). The purine and pyrimidine over the past two decades have unveiled a previously underappreciated
bases are constructed from various nonessential amino acids and methyl role of ROS in cellular signaling. Low levels of ROS, particularly H2O2,
groups donated from the one-carbon/folate pool. The TCA cycle contrib- can reversibly oxidize the cysteine residues of proteins to positively reg-
utes oxaloacetate, which is transaminated to aspartate, an intermediate ulate cell proliferation and cellular adaptation to metabolic stress (119).
required to synthesize both purine and pyrimidine bases. Conversion As H2O2 levels increase, however, cell death signaling pathways are ini-
of ribonucleotides to deoxynucleotides by ribonucleotide reductase re- tiated, and H2O2 is converted to OH·, which can directly damage DNA,
quires a source of NADPH. Well-characterized mechanisms of feedback proteins, and lipids. Cancer cells have an increased rate of spatially lo-
inhibition exist to prevent excessive accumulation of nucleotides, and calized mitochondria- and NOX-dependent ROS production compared
mutations interrupting these mechanisms can produce pathological ac- to normal cells. This allows for the proximal activation of signaling
cumulation of nucleotide intermediates (for example, precipitation of uric pathways [PI3K and mitogen-activated protein kinase/extracellular
acid crystals in gout). signal–regulated kinase (MAPK/ERK)] and transcription factors [HIF
Clearly, nucleotide biosynthesis is a targetable vulnerability in cancer and nuclear factor kB (NF-kB)] necessary for tumorigenesis. The can-
cells because nucleoside analogs and antifolates have been a mainstay of cer cell–specific increased rate of spatially localized ROS production is
chemotherapeutic regimens for decades (112). However, relatively little due to a combination of oncogenic lesions and the tumor micro-
is known about how oncogenic signaling interfaces with nucleotide bio- environment. For example, the activation of oncogenes, PI3K signaling
synthesis. It is likely that the effects of numerous signaling pathways on pathway induction, and hypoxia (low-oxygen levels) stimulate the
glucose and amino acid metabolism influence the availability of pre- increased rate of ROS production from the mitochondria and NOXs
cursors for purines and pyrimidines. In the case of mTORC1, evidence in cancer cells (120–122). Thus, mitochondria-targeted antioxidants

REVIEW
and NOX inhibitors can prevent cancer cell proliferation, hypoxic acti- bolic enzymes is likely to be systemically toxic because of their physio-
vation of HIF, tumorigenesis, and metastasis (64, 123–125). logical functions in normal tissues (139). The feasibility of targeting
The increased localized ROS in cancer cells, which activates sig- these pathways therapeutically depends on whether systemic blockade
naling pathways and transcription factors to promote tumorigenesis, of the pathway can be tolerated. Normal proliferating cells, such as im-
needs to be buffered from reaching levels of ROS that incur cellular mune cells and stem cells, also reprogram their metabolism in a manner
damage by the increased expression of antioxidant proteins (126). Thus, similar to cancer cells (140, 141). Metabolic inhibitors should likely not
cancer cells have higher levels of ROS scavenging enzymes than normal interfere with the adaptive immune system. Nevertheless, there are ex-
cells, preventing ROS-mediated activation of death-inducing pathways cellent examples of pathways whose reprogramming does provide an
like c-Jun N-terminal kinase (JNK) and p38 MAPK and oxidation of adequate therapeutic window in cancer. Enhanced nucleotide and DNA
lipids, proteins, and DNA, resulting in irreversible damage and cell synthesis in tumor cells is targeted by antifolates (methotrexate, peme-
death. One mechanism by which cancer cells increase their antioxidant trexed, and others) (112). Although these drugs do produce toxicity in
capacity is by activating the transcription factor nuclear factor (erythroid- normal proliferative tissues like the intestinal epithelium and bone mar-
derived 2)–related factor-2 (NRF2) (127). Specifically, NRF2 is activated row, they are essential components of highly successful chemotherapeutic
following disruption of the interaction of NRF2 with its binding partner regimens. Thus, it is critical to elucidate in normal cells any toxic effects
Kelch-like ECH-associated protein 1 (KEAP1). Critical cysteine residues of metabolic enzyme inhibition. To circumvent this challenge, one ap-
within KEAP1 can undergo oxidation, succination, and glutathionylation, proach is to target a metabolic enzyme in a deregulated pathway specific
thereby inhibiting the KEAP1-NRF2 interaction, leading to the proteasomal to cancer cells. To date, many of the genetic and pharmacologic inter-
degradation of NRF2. Additionally, NRF2 activation can occur indepen- ventions on metabolic enzymes have been conducted using human
dently of KEAP1 (128). Once activated, NRF2 induces the transcription cancer cells subcutaneously injected into athymic mice. Therefore, it will
of many antioxidant proteins including GPXs and TXNs as well as en- be important for future experiments to not only use patient-derived xeno-
zymes involved in GSH synthesis and cysteine import through the cystine/ graft (PDX) models but also make use of genetically engineered mouse
glutamate antiporter. Furthermore, to maintain the antioxidant capacity cancer models and syngeneic mouse models that have intact immune
of GPXs and TXNs, NADPH is required. NRF2 plays an important role systems, especially given the promising results from immunotherapy.
in activating enzymes that increase cytosolic NADPH levels. NRF2 also An emerging theme is that cancer cells display metabolic plasticity and
regulates the serine biosynthesis pathway, generating NADPH in the can alter their metabolic profile during the course of tumorigenesis and
mitochondria, which is critical for redox balance under hypoxic condi- metastasis. Thus, it is conceivable that cancer cells could develop resist-
tions (129, 130). Therefore, inactivating NRF2 or disabling antioxidant ance to inhibition of a particular metabolic pathway by expressing alter-
proteins in cancer cells would allow for the accumulation of excessive nate protein isoforms or up-regulating compensatory pathways. Therefore,
amounts of ROS to levels that initiate toxicity and reduce tumorigenesis a rational cancer therapeutic strategy should involve targeting multiple

(128, 131, 132). metabolic pathways simultaneously or targeting a particular metabolic
During tumorigenesis and metastasis, redox homeostasis is required pathway in combination with therapies against oncogenic or signaling
(Fig. 4). An emerging model of redox balance is that as a tumor initiates, pathways. Here, we highlight a few promising metabolic targets in gly-
the metabolic activity of cancer cells is increased, resulting in an increase colytic, one-carbon, mitochondrial, and redox metabolism.
in ROS production and subsequent activation of signaling pathways Glycolysis was an early attractive target for cancer therapy given
that support cancer cell proliferation, survival, and metabolic adaptation the clinical observation that many tumors exhibit a significant increase
(126). Accordingly, to prevent toxic levels of ROS, tumor cells increase in glucose uptake compared with adjacent normal tissue (112). LDH-A,
their antioxidant capacity to allow for cancer progression (133). The harsh a metabolic enzyme that converts pyruvate (the final product of glycol-
tumor microenvironment increases ROS levels due to hypoxia, and the ysis) to lactate, was identified as the first metabolic target of the oncogene
low glucose levels limit flux through the cytosolic oxidative PPP, thus MYC (142). Genetic or pharmacologic inhibition of LDH-A has been
decreasing cytosolic NADPH levels. Cells in these nutrient-deprived con- shown to diminish MYC-driven tumors in xenograft models (143, 144).
ditions activate AMPK to increase NADPH levels by stimulating PPP- Furthermore, recent studies indicate that inhibition of LDH-A leads
dependent NADPH and diminishing anabolic pathways, such as lipid to the regression of established tumors in genetically engineered mouse
synthesis, that require high levels of NADPH (134, 135). ROS-dependent models of NSCLC without systemic toxicity (145). Genetic ablation of
signaling and increased mitochondrial respiration are also necessary for LDH-A also delays the progression of myeloid leukemia (146). Thus,
tumor metastasis (124, 136). However, when tumor cells detach from a the increased expression of LDH-A, specifically in MYC-mutant cancer
matrix, they encounter high levels of ROS that incur cellular damage cells, may prove to be an attractive target. Another potential therapeutic
and require activation of adaptive ROS-mitigating pathways to survive target is the glycolytic protein HK2. Many tumor cells overexpress HK2,
and grow (137, 138). The ability to up-regulate antioxidant proteins and and preclinical mouse models of genetically engineered NSCLC and breast
increase flux through NADPH-producing metabolic pathways enables cancer demonstrate that HK2 inhibition delays tumor progression (3).
distant metastasis to occur (8). These findings suggest that perhaps dis- Furthermore, systemic HK2 deletion in mice does not cause adverse
abling antioxidant capacity in cancer cells to raise ROS levels might be physiological consequences. However, the effect of LDH-A and HK2
beneficial in preventing metastasis. on the adaptive immune system is currently unknown. Lactate has been
shown to inhibit cytotoxic T cells; thus, LDH-A inhibition may coop-
erate with immune checkpoint inhibitors to unleash host inflammatory
TARGETING METABOLISM FOR CANCER THERAPY T cells that will specifically attack tumor cells (147). Lactate can also re-
program macrophages to promote tumorigenesis (148). Thus, it may be
There are a few things to consider when determining what makes a efficacious to target LDH-A or HK2 in highly glycolytic tumors that
good metabolic target for cancer therapy. First, inhibition of some meta- overexpress these proteins.

REVIEW
Fig. 4. Cancer cells maintain redox balance. Cancer cells have increased rates of ROS production due to activation of oncogenes and loss of tumor
suppressors that promote signaling pathways supporting proliferation and survival. However, cancer cells prevent the buildup of ROS to levels that incur
damage by increasing antioxidant capacity through induction of NRF2-dependent genes and, in glucose replete conditions, the use of PPP to generate

NADPH. As cells encounter hypoxia and low glucose due to limited vasculature accessibility, the levels of ROS further increase, requiring AMPK and one-
carbon metabolism to enhance NADPH production to raise antioxidant capacity. Loss of matrix attachment and escape of cancer cells into the blood for
dissemination to distant sites incur further increases in ROS levels, which require additional enhancements of antioxidant defenses to avoid cell death. It is
important to note that too little ROS or too high steady-state ROS levels within cancer cells result in failure for solid tumor progression and metastasis.
Another potential glucose-dependent target is PHGDH, an enzyme improved survival rate if cancer was already present (154). Laboratory-
in the de novo serine synthesis pathway. High levels of PHGDH have based studies have also provided evidence that metformin may serve as
been found in a subset of human melanoma and breast cancers, and an anticancer agent (155–157). Biochemists recognized that metformin
these cancer cells require PHGDH for their growth in vitro (25, 26). reversibly inhibits mitochondrial complex I (158–160). Recent studies
Serine starvation in mice diminishes tumorigenicity of p53-null indicate that metformin acts as an anticancer agent by inhibiting mito-
cancers (149). De novo synthesis or exogenous uptake of serine can chondrial ETC complex I (161). Specifically, metformin inhibits mito-
enter the mitochondria where SHMT2 converts it into glycine to gen- chondrial ATP production, inducing cancer cell death when glycolytic
erate folate intermediates (101, 150). In many cancer types, SHMT2 ATP levels diminish as a result of limited glucose availability. Metfor-
expression is elevated and correlates with a poor prognosis. Further- min also inhibits the biosynthetic capacity of the mitochondria to gen-
more, the transcription factors MYC and HIF induce SHMT2 under erate macromolecules (lipids, amino acids, and nucleotides) within
hypoxia to promote survival (130, 151). Currently, it is not known cancer cells (162). The remarkable safety profile of metformin is due
whether targeting PHGDH, SHMT2, or other enzymes in the one-carbon to its uptake by organic cation transporters (OCTs), which are only
metabolism pathway would be effective in delaying or regressing tumor present in a few tissues, such as the liver and kidney (163). Certain tumor
progression in genetically engineered, PDX, or syngeneic mouse models cells also express OCTs to allow the uptake of metformin (164). How-
of cancer without incurring systemic toxicity. However, given the im- ever, in the absence of OCTs, tumors would not accumulate metformin
portance of one-carbon metabolism in supporting the anabolic needs of to inhibit mitochondrial complex I. Ongoing clinical trials using met-
cancer cells and its up-regulation in cancer cells, it is likely that this formin as an anticancer agent should assess the expression levels of
pathway is needed for in vivo tumor progression (152). OCTs to identify the tumors with highest expression, which are likely
Mitochondrial metabolism has also emerged as a key target for to be susceptible to metformin. Moreover, it is not clear whether the
cancer therapy, in part, due to the revelation that the antidiabetic drug current antidiabetic dosing of metformin used in clinical trials allows
metformin is an anticancer agent (153). Numerous epidemiological stu- for metformin accumulation to levels necessary to inhibit mitochondri-
dies first suggested that diabetic patients taking metformin, to control al complex I in tumors. Thus, it is possible that metformin at doses high-
their blood glucose levels, were less likely to develop cancer and had an er than those currently used for diabetes might be more efficacious

REVIEW
without causing toxicity. Like metformin, the biguanide phenformin NADP+ to NADPH. However, certain tumors rely on this pathway as
exhibits anticancer properties by inhibiting mitochondrial complex I a major source of cytosolic NADPH; therefore, it may be therapeutic
(165). In contrast to metformin, phenformin is readily transported into to disable this pathway and induce oxidative stress and diminish tumor
tumor cells and has been withdrawn from human use in most parts of growth. Moreover, RNA profiling of metabolic enzymes identified the
the world due to its clinical increase in the incidence of lactic acidosis. mitochondrial one-carbon metabolism protein MTHFD2, which can
However, it is worth considering phenformin as a possible cancer ther- generate NADPH, as being highly expressed in 19 different cancer types
apy because lactic acidosis can be monitored. Biguanide sensitivity can but not in normal adult proliferating cells (152). Loss of MTHFD2 in
be improved in mice starved for serine or in tumors that have lost p53 cancer cells increases ROS levels and sensitizes the cells to oxidant-induced
or LKB1 (155, 166, 167). Thus, biguanides, and possibly other mito- cell death in vitro. An interesting approach to depleting NADPH
chondrial complex I inhibitors, may be effective anticancer agents. levels and increasing ROS is to administer high doses of vitamin C
Another potential therapeutic strategy to inhibit mitochondrial me- (ascorbate). Vitamin C is imported into cells through sodium-dependent
tabolism in certain tumors would be to use autophagy or glutaminase vitamin C transporters, whereas the oxidized form of vitamin C, dehydro-
inhibitors. Autophagy provides amino acids, such as glutamine, that fu- ascorbate (DHA), is imported into cells through glucose transporters
el the TCA cycle in NSCLC and pancreatic cancers, and short-term such as GLUT1 (179). When the cell takes up DHA, it is reduced back to
autophagy inhibition has been shown to decrease tumor progression vitamin C by glutathione (GSH), which consequently becomes GSSG.
without incurring systemic toxicity in mouse models of NSCLC (168, 169). Subsequently, GSSG is converted back to GSH by NADPH-dependent
Some tumors are addicted to using glutamine to support TCA cycle GR. Because the blood is an oxidizing environment, vitamin C becomes
metabolism even in the absence of autophagy; thus, glutaminase inhib- oxidized to DHA before being taken up by the cell. Thus, high doses of
itors can reduce tumor burden in these models (4, 75, 170). An alterna- vitamin C diminish the tumorigenesis of colorectal tumors that harbor
tive approach is to target acetate metabolism. Although a major function oncogenic KRAS mutations and express high levels of GLUT1 by deplet-
of the mitochondria is to provide acetyl-CoA to the cell, cancer cells can ing the NADPH and GSH pools and consequently increasing ROS levels
also use acetate to support cell growth and survival during metabolic to induce cancer cell death (179, 180). Vitamin C administered at high
stress (hypoxia or nutrient deprivation) (96, 171). The cytosolic enzyme doses intravenously is safe in humans and, in conjunction with con-
acetyl-CoA synthase 2 (ACCS2), which converts acetate to acetyl-CoA, is ventional paclitaxel-carboplatin therapy, demonstrated a benefit in a
dispensable for normal development; thus, ACCS2 is a promising target small number of patients (181). Additional strategies to diminish GSH
of acetate metabolism. ACCS2 knockout mice do not display overt include the administration of buthionine sulfoximine, an irreversible
pathologies, but genetic loss of ACCS2 reduces tumor burden in models inhibitor of g-glutamylcysteine synthetase, which can be safely admin-
of hepatocellular carcinoma (171). Human glioblastomas can oxidize ac- istered to humans and is efficacious in preclinical tumor models (182).
etate and may be sensitive to inhibitors of this process (172). Thus, tar- Moreover, glutathione is a tripeptide consisting of cysteine, glutamate,

geting metabolism with inhibitors of autophagy, acetate metabolism, and and glycine. Thus, decreasing glutamate levels using glutaminase inhib-
other pathways that supply key metabolic intermediates may be efficacious itors or diminishing cysteine levels by preventing extracellular cystine
in some contexts. (two linked cysteine molecules) uptake can also raise ROS levels in can-
Because mitochondrial inhibitors are unlikely to be effective cancer cer cells to induce cell death.
therapies as single agents, combination therapy is likely the best ap- An important consideration is that normal stem cells are sensitive to
proach. For example, the use of metformin with the current clinical ROS levels; thus, it is important to stratify patients on the basis of their
PI3K inhibitors, which reduce glucose uptake and glycolysis (173), is expression levels of a particular antioxidant protein or antioxidant
one approach that would impair both sources of ATP within cells. Tar- pathway. It is critical to determine which antioxidant pathways are likely
geted therapies against oncogenes such as KRAS, BRAF, and NOTCH1 up-regulated as a result of the high rate of ROS production within cancer
kill a large majority of cancer cells but ultimately yield resistant cells that cells. Many cancer types use the NRF2 pathway to maintain redox balance;
exhibit an increased sensitivity to inhibitors that impair mitochondrial therefore, targeting this pathway may provide a viable therapeutic op-
metabolism (174–176). Cancer-initiating cells also have increased sensi- portunity (128). Additionally, superoxide dismutase 1 (SOD1) is over-
tivity to mitochondrial inhibitors, adding further evidence that inhibiting expressed in NSCLC, and its inhibition kills human NSCLC cells and
mitochondrial metabolism may suppress tumor recurrence (177, 178). decreases the tumor burden in mouse models of NSCLC (183). Because
Furthermore, to counterbalance the increased production of ROS NRF2 and SOD1 knockout mice develop normally, short-term inhibition
encountered during tumorigenesis and metastasis, cancer cells increase of these pathways might be an effective way to kill cancer cells.
their antioxidant capacity (126). Thus, an additional therapeutic ap-
proach is to target redox metabolism, that is, selectively disable the
antioxidant capacity of cancer cells causing ROS levels to rise and in- TECHNOLOGIES ENABLING DISCOVERY
duce cancer cell death (133). The reducing equivalent NADPH is re- IN CANCER METABOLISM
quired to maintain multiple antioxidant defense systems. The cytosol
has multiple sources of NADPH generation, including the oxidative Many recent advances in our understanding of cancer metabolism
PPP, malic enzyme 1, IDH1, and one-carbon metabolism. By contrast, have been propelled by advanced technologies to detect metabolites
NADPH generation in the mitochondria is controlled in part by one- and metabolic activities (184). A key concept is that quantifying metab-
carbon metabolism and IDH2. Many of these NADPH-generating olites (that is, metabolomics) is a more distinct form of metabolic analysis
systems are critical for normal cell survival and function. Nevertheless, than measuring the activities of metabolic pathways [that is, metabolic
there are two NADPH-generating systems that may serve as potential flux analysis (185)]. Although these two approaches can provide
therapeutic targets. It is estimated that 400 million people worldwide complementary types of information, they are not interchangeable. One
are deficient in G6PDH, an enzyme in the oxidative PPP that converts cannot infer metabolic activity from changes in metabolite levels, and

REVIEW
altered metabolic fluxes may or may not cause changes in metabolite levels substantial labeling of glycolytic and TCA cycle intermediates in tu-
(186). Both of these approaches have provided important recent insights mors. In mice bearing orthotopic transplants of high-grade human
into cancer metabolism, and using the two techniques together provides gliomas, continuous infusion of 13C-glucose was demonstrated to pro-
the most complete assessment of metabolic phenotypes. duce steady-state labeling of metabolites from the TCA cycle within the
Metabolomics experiments seek to characterize and quantify the tumor, enabling the assessment of several metabolic pathways (192).
metabolites in a biological sample, usually by nuclear magnetic reso- Here, tumors with diverse oncogenotypes oxidized glucose-derived
nance (NMR) or, more commonly, mass spectrometry. Depending pyruvate in the mitochondria and synthesized glutamine from glucose
on the methods of extraction, separation, and detection, metabolomics carbon. In contrast to most cultured glioma cell lines, these tumors did
experiments may focus on particular classes of metabolites or provide a not demonstrate significant levels of 13C-glutamine oxidation in vivo,
comprehensive analysis of as many metabolites as possible. Targeted and primary cell lines derived from the tumors did not require gluta-
approaches typically detect a few dozen to a few hundred molecules, mine for survival or proliferation. In another study, metabolism of
whereas untargeted analyses may detect more than 1000. Detecting al- 13
C-glucose and 13C-glutamine in autochthonous models of MYC- or
terations of metabolite levels in cancer can be extremely valuable. The MET-driven tumorigenesis revealed that metabolic phenotypes depend
massive accumulation of D2HG in IDH1-mutant gliomas was initially not only on the tumor’s genetic driver but also on the tissue or origin.
discovered through a metabolomics approach (33). Because altered MYC but not MET stimulated glutamine catabolism in liver tumors,
metabolite levels can be detected noninvasively using 1H magnetic whereas MYC-driven lung tumors expressed glutamine synthetase and
resonance spectroscopy (MRS), perturbed metabolite levels discovered accumulated glutamine (193). Thus, in vivo isotope tracing can detect
through metabolomics can sometimes be translated into clinical diag- metabolic activities of intact tumors and characterize some of the factors
nostic techniques. Elevated levels of lactate, choline, glycine, and other that specify the metabolic phenotype.
metabolites are detected by MRS in glioma. More recently, MRS techniques Administration of 13C-labeled nutrients has also proven to be valu-
have been developed to monitor specific metabolic states programmed able in human cancer (172, 194–197). Fan et al. (196) used 13C-glucose
by tumor-specific mutations in metabolic enzymes. Applications include to demonstrate that human non–small cell lung tumors metabolize
elevated 2HG in IDH1/IDH2-mutated gliomas (187) and elevated suc- glucose through glycolysis and the TCA cycle concurrently, with me-
cinate in SDH-deficient paragangliomas (188). tabolites from both pathways demonstrating higher levels of labeling
Metabolic flux studies use isotope tracers like 13C, 15N, and 2H to in tumors relative to adjacent lung tissue. In a subsequent study, these
track flow through metabolic pathways. Typically, a nutrient of inter- investigators demonstrated that the anaplerotic enzyme pyruvate car-
est is labeled by an isotope (for example, 13C-glucose) and supplied to boxylase (PC) was highly expressed in lung tumors and contributed to
13
cancer cells in the culture medium. Metabolites extracted from the C labeling in TCA cycle intermediates (195). Enhanced glucose oxi-
culture are analyzed for isotope enrichment using mass spectrometry dation involving both PC and pyruvate dehydrogenase (PDH) was de-

or NMR. The extent and distribution of labeling within informative monstrated in a separate cohort of non–small cell lung tumors, in which
metabolites encode information about which pathways are active in formal analysis of metabolic fluxes was used to complement mea-
the cells. Incorporating additional data (for example, definitive rates surements of 13C labeling (197). An important conclusion from these
of nutrient consumption, waste secretion, and biomass production) al- studies, and from a similar study in mice bearing KRAS-driven tumors
lows quantitative fluxes to be determined across a metabolic network. (198), is that non–small cell lung tumors demonstrate higher levels of
Isotope tracing studies provide information about metabolic altera- both glycolysis and glucose oxidation relative to adjacent, benign lung.
tions in cancer cells that cannot be detected by metabolite levels alone. This finding sharply contrasts with the frequently invoked “switch”
For example, hypoxia and mutations in the ETC induce a restructur- from oxidative metabolism to glycolysis in malignant tissue, commonly
ing of the TCA cycle in which many of the intermediates are produced used to explain the Warburg effect (Fig. 5A). Rather, the data support a
in the reverse order from the conventional form of the cycle. The model in which the amplitude of both pathways is increased simulta-
key reaction in this pathway involves the reductive carboxylation of neously, perhaps through increased substrate delivery and enzyme ex-
a-ketoglutarate to isocitrate in a NADPH-dependent carboxylation pression in tumor cells (Fig. 5B). It is also significant that human tumors
reaction catalyzed by IDH1 and/or IDH2. Although metabolomics exhibit substantial heterogeneity of metabolic phenotypes, both between
experiments can detect altered levels of TCA cycle metabolites in cells tumors and even within distinct regions of the same tumor (197). The
using the reductive carboxylation pathway or in cells with deficiencies extent of glucose-dependent labeling of TCA cycle intermediates is pre-
in pyruvate import into mitochondria, the marked restructuring of the dicted by noninvasive assessment of tumor perfusion by magnetic
cycle is apparent only through isotope tracing experiments, particular- resonance imaging, providing an approach to identify areas of regional
ly experiments using 13C-glutamine as the tracer (69, 78, 79, 189–191). metabolic heterogeneity in human cancer (197).
An example of the use of isotope tracers to identify metabolic liabilities Metabolomics and metabolic flux analysis can be integrated with
involves the surprising discovery that a significant fraction of cellular functional genomics to identify and understand metabolic vulnerabil-
NADPH, particularly in the mitochondria, is produced through folate ities in cancer cells. This approach has produced several good examples
metabolism (100, 101). These studies involved a sophisticated combi- of screens that identified potential therapeutic targets while stimulating
nation of 13C and 2H tracers, coupled with quantitative measurements entirely new lines of investigation in cancer cell biology. For example,
of metabolic flux. the serine biosynthetic enzyme PHGDH was first identified as a meta-
Several recent studies have begun to use stable isotopes to investigate bolic vulnerability in breast cancer cells through a large-scale in vivo
metabolism in intact tumors. Because these isotopes do not undergo short hairpin RNA screen targeting thousands of metabolic enzymes
radioactive decay, they are safe for administration to animals and hu- (25). PHGDH is frequently amplified at the genomic level in breast tu-
man subjects. Systemic administration of 13C-labeled nutrients through mors and melanomas and exhibits oncogene-like features in cell culture
either boluses or continuous infusions has been shown to generate (25, 26). Subsequent work on serine biosynthesis, much of it involving

REVIEW
metabolomics and metabolic flux analysis, has uncovered novel themes have emerged from this research (Box 1). First, metabolic re-
functions and liabilities of this pathway in cancer cell growth and stress programming is essential for the biology of malignant cells, particular-
resistance (129, 150, 151). Combining functional screens with metabolic ly their ability to survive and grow by using conventional metabolic
analysis can also identify context-specific vulnerabilities that may be pathways to produce energy, synthesize biosynthetic precursors, and
therapeutically actionable. A CRISPR (clustered regularly interspaced maintain redox balance. Second, metabolic reprogramming is the re-
short palindromic repeats)–based loss-of-function screen identified sult of mutations in oncogenes and tumor suppressors, leading to ac-
GOT1, the cytosolic aspartate aminotransferase, as conditionally essen- tivation of PI3K and mTORC1 signaling pathways and transcriptional
tial for survival during treatment with the ETC inhibitor phenformin networks involving HIFs, MYC, and SREBP-1. Third, alterations in
(199). Isotope labeling then demonstrated that ETC blockade caused metabolite levels can affect cellular signaling, epigenetics, and gene ex-
the direction of this enzyme to reverse from aspartate consumption pression through posttranslational modifications such as acetylation,
in untreated cells to aspartate synthesis during ETC blockade (200). methylation, and thiol oxidation. Fourth, taken together, studies on
In addition to the discovery of synthetic lethality between ETC and cultured cells have demonstrated a remarkable diversity of anabolic
GOT1 inhibition, these studies led to the novel biological concept that and catabolic pathways in cancer, with induction of autophagy and
a major function of the ETC in proliferating cells is to support the syn- utilization of extracellular lipids and proteins complementing the clas-
thesis of aspartate for nucleotide and protein synthesis (199, 200). sical pathways like glycolysis and glutaminolysis. We have exited the
period when cancer metabolism could be considered synonymous
with the Warburg effect.
CONCLUSIONS AND CURRENT CHALLENGES Several challenges will likely shape research over the next decade.
First, the studies cited above were performed primarily in cancer cell
Substantial progress has been made in the past decade toward under- lines rather than intact tumors. These straightforward experimental
standing the mechanisms, biological consequences, and liabilities as- models have been highly informative about the molecular mechanisms
sociated with metabolic reprogramming in cancer. Several common of metabolic reprogramming, particularly those linking aberrant

B
Fig. 5. Relationship between glycolysis and oxidative phosphorylation in cancer cells. (A) A common view of cancer cell metabolism invokes a
switch from glucose oxidation in normal tissues toward glycolysis and suppressed oxidative phosphorylation (OxPhos) in cancer. (B) Analysis of
metabolic activity in intact tumors from humans and mice argues against a switch. Rather, tumors appear to enhance both glycolysis and glucose
oxidation simultaneously relative to surrounding tissue.

REVIEW
signaling to altered metabolic fluxes. But it is challenging (perhaps pact on public health. It is clear that obesity and diabetes, both of which
impossible) to model an accurate tumor microenvironment in culture. are reaching epidemic proportions in the developed world, increase
Direct analysis of metabolic fluxes in intact tumors should begin to play cancer risk, but we lack insight into how to break these links.
a more prominent role in the field and may prove essential in deter-
mining precisely how to deploy metabolic inhibitors in clinical trials.
Along these lines, it is remarkable that some tumor cell metabolic vul- REFERENCES AND NOTES
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REVIEW
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Fundamentals of cancer metabolism
Ralph J. DeBerardinisNavdeep S. Chandel
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