Recent characterizations of metabolic enzymes as tumor suppressors and oncogene-driven
metabolic reprogramming have reinvigorated interest in cancer metabolism. Although
therapies targeting metabolic processes have long been a staple in cancer treatment
(e.g. inhibition of folate metabolism via methotrexate), the focused therapeutic potential
surrounding these findings have generated a renewed appreciation for Otto Warburg’s
work almost a century ago. Warburg observed that tumor cells ferment much of the glucose
taken up during growth to lactate, thus using glycolysis as a major means of adenosine
triphosphate (ATP) regeneration 1]. However, the observation of decreased respiration in cancer cells and idea that
“the respiration of all cancer cells is damaged†belies the critical role of mitochondria in biosynthesis and cell survival 1]. On the contrary, functional mitochondria are present in all proliferative cells
within our body (including all tumors), as they are responsible for converting the
diverse nutrients available to cells into the fundamental building blocks required
for cell growth. These organelles execute numerous functions in cancer cells to promote
tumor growth and survival in response to stress. Here, we outline the critical biosynthetic
functions served by mitochondria within tumors (Figure 1). Although many of these functions are similarly important in normal, proliferating
cells, we have attempted to highlight potential points where mitochondrial metabolism
may be therapeutically targeted to slow cancer growth. This review is organized by
specific metabolic pathways or processes (i.e., glucose metabolism and lipogenesis,
amino acid metabolism, and nucleotide biosynthesis). Tumors or cancer cell types where
enzymes in each pathway have been specifically observed to by dysregulated are described
within the text and summarized in Table 1.
Figure 1. Biosynthetic nodes within mitochondria. Metabolic pathways within mitochondria that contribute to biosynthesis in cancer
and other proliferating cells. TCA metabolism and FOCM enable cells to convert carbohydrates
and amino acids to lipids, non-essential amino acids, nucleotides (including purines
used for cofactor synthesis), glutathione, heme, and other cellular components. Critical
biosynthetic routes are indicated by yellow arrows. Enzymatic reactions that are dependent
on redox-sensitive cofactors are depicted in red.
Table 1. Overview of mitochondrial biosynthetic enzymes important in cancer
Glucose anaplerosis
Glucose is the most widely available nutrient in our body; not surprisingly, most
tumor cells consume this carbohydrate (or analogs) at high rates. This phenotype allows
for detection and imaging of some cancers and metastatic lesions using the glucose
analog 2-deoxy-2-[18?F]fluoro-D-glucose (FDG), which accumulates in tumors (and some other tissues) and
can be noninvasively observed when using positron emission tomography integrated with
computed tomography (FDG-PET/CT) 2]. While FDG-PET/CET tracks cells with high glucose uptake and phosphorylation only,
the use of isotope tracers, mass spectrometry (MS), and nuclear magnetic resonance
(NMR) have enabled researchers to more closely examine the fate of glucose within
cancer cells 3]-5]. Indeed, flux through glycolysis and lactate secretion remains a hallmark of many
tumor cells, presumably to facilitate biosynthesis of ribose, purines (via serine
and glycine), and lipid headgroups (via glycerol-3-phosphate and serine). However,
increasing evidence now indicates that cancer cells transport a significant portion
of glucose-derived pyruvate into mitochondria where it serves as an anaplerotic substrate
to replenish tricarboxylic acid (TCA) cycle intermediates used for biosynthesis. For
example, conditions of limited glutamine availability or glutaminase (GS) suppression
drive cancer cells to increasingly rely on glucose carbon flux through pyruvate carboxylase
(PC) to maintain oxaloacetate (OAC) production and downstream TCA cycle activity 6]. Furthermore, NMR analysis of mice bearing three distinct human orthotopic tumors
and infused with [3,4-13C2]glucose indicated these glioblastoma lines used glucose as a mitochondrial anaplerotic
substrate 7]. Although glutamine is one of the most abundant amino acids present in plasma, typical
in vitro culture media used for cell line expansion contain relatively high concentrations
(2–4 mM) of glutamine. Thus, as tumor cells are increasingly analyzed prior to “adaptation/selectionâ€
in vitro, we are beginning to better appreciate the importance of glucose-derived pyruvate
as an anaplerotic substrate in tumors.
Glucose oxidation and lipogenesis
Beyond flux through PC or analogous reactions, the more predominant fate of mitochondrial
pyruvate is oxidation by the pyruvate dehydrogenase (PDH) complex to form acetyl-coenzyme
A (AcCoA). AcCoA is subsequently converted to citrate via condensation with OAC by
citrate synthase. In turn, citrate is either converted to isocitrate in the TCA cycle
or transported out of mitochondria and metabolized by ATP citrate lyase to yield cytosolic
AcCoA, which is the substrate for de novo lipogenesis and acetylation. Mitochondrial activity within this pyruvate-citrate
shuttle is therefore critical for the biosynthesis of fatty acids and cholesterol
as well as protein acetylation. With some notable exceptions (e.g. hypoxia, discussed
below), most cancer cells derive the majority of their lipogenic AcCoA from glucose-derived
pyruvate through PDH 8]-10]. Numerous oncogenic pathways stimulate glucose-derived carbon atom flux through the
citrate shuttle to promote lipogenesis and TCA metabolism. Specific mutations in Kirsten
rat sarcoma viral oncogene homolog (KRAS) stimulate flux of glucose through PDH to generate fatty acids 11],12]. Alternatively, active Akt promotes glucose-mediated fatty acid synthesis downstream
of PDH 8],13]. This Akt-dependent lipogenesis occurs by activation of mammalian target of rapamycin
complex 1 (mTORC1) and sterol regulatory element-binding protein 1 (SREBP1), which
are key regulators of cellular growth and lipid homeostasis, respectively 14]. Interestingly, SREBPs have also been shown to coordinate lipid and protein biosynthesis
as well as protect cancer cells from saturated fatty acid-induced lipotoxicity 15],16]. On the other hand, inactivation of mTORC1 reduces mitochondrial fluxes that supply
the citrate and AcCoA which fuel these pathways 17],18]. Finally, overexpression of the HER2 oncogene or epidermal growth factor (EGF) stimulation both activate MEK/ERK signaling
to suppress the inhibitory PDH kinase 4 (PDK4) and maintain glucose oxidation in mammary
epithelial cells 19].
These above concepts and results contrast the established role of PDH kinase 1 (PDK1)
in supporting tumor growth downstream of hypoxia-inducible factor (HIF) signaling
by suppressing PDH activity 20]-22]. Indeed, inhibition of PDK1 activity using dichloroacetate (DCA) forces glucose oxidation
under hypoxic conditions 9] and inhibits the growth of xenograft tumors 23]. Limited mitochondrial glucose metabolism due to hypoxic or pseudohypoxic stabilization
of HIFs is a hallmark of some renal carcinomas 9],24],25], and normalization of HIF levels (thus increasing glucose oxidation) in such cells
abrogates tumor formation in xenografts 26]. Suppression of PDK1 to activate PDH flux also contributes to BRAF(V600E)-induced
oncogene senescence 27], further suggesting that limiting glucose oxidation is important for tumor growth.
Furthermore, some tumors downregulate expression of the mitochondrial pyruvate carrier
(MPC), and acute inhibition of the MPC in cancer cells significantly decreases glucose
oxidation but has no effect on growth or respiration 28]-30]. Tumor cells are clearly able to compensate for this lack of glucose-mediated biosynthesis
under these conditions through extramitochondrial pathways, scavenging acetate 31],32], unsaturated lipids 8],33], or proteins 34] when required. Therefore, the relative importance of glucose-driven biosynthesis
through mitochondrial pathways may be tumor specific. Alternatively, there may be
a particular level of glucose flux into mitochondria that supports biosynthesis while
limiting oxidative TCA metabolism and potentially deleterious byproducts (e.g. reactive
oxygen species; ROS). Further mechanistic studies are required to characterize the
mechanisms through which cancer cells balance mitochondrial energetic (catabolic)
and biosynthetic (anabolic) metabolism.
Amino acid metabolism
In addition to carbohydrates, amino acids are critical substrates fueling mitochondrial
metabolism and the biosynthesis of proteins, lipids, and other molecules. Of particular
interest in cancer are key mitochondrial enzymes in the metabolism of glutamine, glutamate,
proline, aspartate, and alanine (Figure 2). Glutamine is one of the most critical nutrients required for cell proliferation,
as the amido nitrogen of this amino acid is the obligate substrate for hexosamine
and nucleotide biosynthesis in the cytosol. Furthermore, the carbon backbone of glutamine
is an important anaplerotic substrate fueling TCA cycle metabolism (Figure 1). Upon conversion to glutamate via glutaminase (GLS) activity, N-acetyl-glucosamine
production, or nucleotide biosynthesis, glutamine carbons enter the TCA cycle as alpha-ketoglutarate
(aKG) downstream of glutamate dehydrogenase (GDH) or transaminase activity 35],36]. The GLS (rather than GLS2) isoform is commonly expressed in tumors and is regulated
downstream of the MYC oncogene 37]. Pharmacological inhibition of GLS is being investigated as a potential means of
therapy for a number of different tumor types 38]-40]. Indeed, GLS facilitates oxidative glutaminolytic flux in tumor cells derived from
gliomas, lymphomas, breast cancers, prostate cancers, pancreatic cancers, and melanomas
38],40]-44]. Recent flux studies in tumor cells bearing isocitrate dehydrogenase 1 (IDH1) mutations
indicate that these cells may be particularly dependent upon glutamine to fuel oxidative
mitochondrial metabolism and thus may be responsive to inhibition of GLS or respiration
39],45]. GLS-derived glutamate is also important for glutathione synthesis, which is abundant
at mM levels in cells and plays an important role in redox homeostasis and tumor cell
survival in response to oxidative stress 46].
Figure 2. Coordination of carbon and nitrogen metabolism across amino acids. Glutamate and aKG are key substrates in numerous transamination reactions and can
also serve as precursors for glutamine, proline, and the TCA cycle. Mitochondrial
enzymes catalyzing these reactions are highlighted in blue, and TCA cycle intermediates
are highlighted in orange (pyruvate enters the TCA cycle as acetyl-CoA or oxaloacetate).
Glutaminolysis and reductive carboxylation
Glutamine carbon can also fuel AcCoA generation for lipid biosynthesis when metabolized
by malic enzymes (MEs) through glutaminolysis or alternatively via reductive carboxylation.
The former pathway, by which glutamine-derived malate is converted to pyruvate and
subsequently lactate or AcCoA, is active in some tumor cells that express high levels
of cytosolic ME1 or the other mitochondrial isozymes ME2 and ME344],47]. Leukemic cells under hypoxia have been observed to employ this pathway for biosynthesis
and ATP regeneration 38]. Glutaminolysis is also specifically activated in proliferating cells upon inhibition
of MPC function, which may contribute to the sensitivity of cancer cells to inhibitors
of glutamine metabolism 29],30].
In contrast to the oxidative glutaminolytic pathway, reductive carboxylation involves
the “reverse†or reductive activity of NADP+-dependent IDHs to generate isocitrate and citrate from aKG, a pathway which becomes
important in cells proliferating in hypoxic microenvironments or those with dysfunctional
mitochondria 9],25],42],48]. HIFs stabilized by low oxygen levels or loss of the von Hippel Lindau tumor suppressor
reduce PDH activity 20],21], leading cells to use alternate substrates for AcCoA generation such as glutamine
or aKG 24]. In this manner, mass action and/or altered mitochondrial redox states induce proliferating
cells to reductively metabolize aKG via NADP+-dependent IDHs and subsequently generate isocitrate and ultimately AcCoA 49]. Evidence also suggests that aKG-dehydrogenase (OGDH) and nicotinamide nucleotide transhydrogenase (NNT) expression are both required for activation of this pathway 48],50]. Indeed, hypoxic cells maintain and even upregulate oxidative glutamine metabolism
in mitochondria despite the observed increase in reductive carboxylation activity
38],45],51]. Thus, some mitochondrial functions are required to allow conversion of glutamine
to AcCoA through this pathway. On the other hand, cells with heterozygous mutations
in IDH1 are specifically compromised in their ability to use reductive carboxylation
for fatty acid synthesis 45], suggesting that the cytosolic isozyme catalyzes reductive carboxylation. While the
specific contributions and functions of mitochondrial IDH2 and cytosolic IDH1 in this
pathway must be definitively characterized (both in vitro and in vivo), increased exchange of aKG and isocitrate/citrate occurs in the context of perturbed
redox states when fatty acid biosynthesis is maintained, a common occurrence in the
tumor microenvironment. Ultimately, this pathway may effectively allow cancer cells
to maintain biosynthesis, transfer reducing equivalents between compartments, or both
to support growth and survival in hypoxic microenvironments.
Glutamine synthesis
Many amino acids are not extremely abundant in plasma or the tumor microenvironment
and therefore must be synthesized de novo. Mitochondrial metabolism plays a definitive role in the production of many non-essential
amino acids and their further utilization in biosynthetic pathways. Although glutamine
is relatively abundant in plasma, de novo glutamine synthesis in the liver and surrounding tissues is likely critical for tumor
cell growth. Glutamine biosynthesis requires a supply of aKG from mitochondrial metabolism
to generate glutamate (a critical precursor for most non-essential amino acids) and
subsequently glutamine via glutamine synthetase (GS). De novo glutamine biosynthesis in tumors has been detected in vivo using infusions of [13C]glucose into mice bearing human glioblastoma orthotopic tumors 7]. Furthermore, some breast epithelial cells can mediate glutamine independence via
expression of GS52]. Finally, glutamine as well as other amino acids may be scavenged via protein catabolism
when it is not available in sufficient quantities 34].
Proline metabolism
Mitochondrial proline metabolism and synthesis are critically important for tumor
cells, at least in part due to the unique, modifiable chemical properties it provides
to proteins. Proline is synthesized from glutamine or urea-cycle-derived ornithine
via the intermediate pyrroline-5-carboxylate (P5C). P5C is then converted to proline
via the NAD(P)H-dependent enzyme pyrroline-5-carboxylate reductase (PYCR), which exists
in three isoforms: PYCR1, PYCR2, and PYCRL (Figure 2). Mitochondrial PYCR1 and PYCR2 are upregulated in multiple types of cancer, including
prostate, lymphoma, and others 41],53],54]. Overexpression of c-Myc in P493 human Burkitt lymphoma and PC3 human prostate cancer induced an upregulation
of PYCR1 expression as well as the P5C biosynthetic enzyme delta-1-pyrroline-5-carboxylate
synthase (P5CS), resulting in higher levels of intracellular proline 41]. In line with this observation, expression of both PYCR1 and PYCR2 was increased in a panel of melanoma cell lines but was undetectable in normal melanocytes
53]. Furthermore, a recent large-scale comparative analysis of published mRNA microarray
datasets found that PYCR1 was one of the most commonly overexpressed metabolic enzyme genes in comparison to
normal tissue among the 19 represented cancer types 54]. Although the functional advantages provided to cancer cells by modulating proline
metabolism are not completely clear, the importance of proline in extracellular matrix
proteins (e.g. collagen) could play a role in tumorigenesis. Alternatively, interconversions
of proline and P5C in the cytosol and mitochondria have been proposed as a means of
transferring reducing equivalents between these compartments 55], though more detailed functional analyses are required to elucidate how proline metabolism
contributes to cancer progression.
Downregulation of proline catabolism is complementary to its biosynthesis and commonly
observed in a number of tumor types. The first step of this process is catalyzed in
the mitochondria by proline oxidase (POX), and the expression of this enzyme is markedly
reduced in many cancers compared to normal tissue from the same patient 56]. POX expression is induced by the tumor suppressor p53, and ectopic expression of POX in DLD-1 colon cancer cells induces cell cycle arrest and reduces tumor burden in
xenograft models 56]. Furthermore, POX expression is inhibited by MYC via miR-23b* in lymphoma, renal, and prostate cancers
41],57]. The widespread repression of POX in cancer indicates that this enzyme may act as
a tumor suppressor; however, the specific mechanisms through which POX deficiency
promotes tumorigenesis are not yet clear.
Aspartate and asparagine metabolism
Aspartate can be generated from the TCA intermediate oxaloacetate by glutamate-mediated
transaminase activity (Figure 2); thus, the biosynthesis of aspartate and downstream metabolites is intimately tied
to mitochondrial activity. Aspartate transaminases (GOT1, cytosolic; GOT2, mitochondrial),
which bidirectionally convert aspartate and aKG to OAC and glutamate, are important
for the growth of human pancreatic adenocarcinoma (PDAC) 43]. Oncogenic KRAS, the most common mutation in PDAC, redirects glutamine metabolism toward aspartate
production in a number of settings 11],43],58]. This metabolic reprogramming is thought to facilitate regeneration of NADPH for
reductive biosynthesis and redox homeostasis as well as NAD+ for maintaining glycolysis 43]. Ablation of oncogenic KRAS in a mouse model of pancreatic cancer markedly reduced tumor size and also revealed
a subpopulation of surviving tumor cells which did not express KRAS. These surviving cells relied heavily on oxidative phosphorylation and were sensitive
to oligomycin treatment, providing evidence that inhibition of mitochondrial function
may effectively target cells that survive after suppression of oncogenic KRAS signaling 59]. Additionally, aspartate and glutamine are the precursors for asparagine, which is
synthesized in the cytosol by asparagine synthetase (ASNS). ASNS expression is required for the survival of cultured glioma and neuroblastoma cell
lines, and supplementation of exogenous asparagine can prevent apoptosis induced by
glutamine withdrawal, in part, by modulating ER stress 60]. Expression of ASNS is also correlated with drug resistance in childhood acute lymphoblastic leukemia
(cALL) and some forms of acute myeloblastic leukemia (AML), which are typically deficient
in their ability to synthesize asparagine de novo61],62]. Finally, aspartate is a key initiator of pyrimidine synthesis and donates nitrogen
for purine synthesis via adenylosuccinate synthetase (Figure 3), further highlighting the role of mitochondrial aspartate metabolism in tumor cell
biosynthesis.
Figure 3. Biosynthetic sources for purine and pyrimidine synthesis. Sources and fates of nitrogen, carbon, and oxygen atoms are colored as indicated.
Italicized metabolites can be sourced from the mitochondria or cytosol. The double
bond formed by the action of DHODH/ubiquinone is also indicated.
Alanine and BCAA metabolism
Alanine production via alanine transaminases (GPT1, cytosolic; GPT2, mitochondrial),
which transfer an amino group between glutamate and pyruvate to yield alanine and
aKG, not only provide proteinogenic alanine but also aKG for TCA cycle activity (Figure 2). Maintenance of glutamine anaplerosis and catabolism in cancer cells via increased
GPT2 activity is essential for oncogenic KRAS-induced anchorage independent growth, as demonstrated by knockdown of GPT2 expression in HCT116 colon cancer cells 35]. GPT activity may also facilitate disposal of excess nitrogen (such as that derived
from glutamine) via alanine secretion 63]. Indeed, secretion of alanine is higher in melanoma cell lines compared to normal
melanocytes and is quite significant in human colon carcinoma tumors 35],64].
Finally, the branched chain amino acids (BCAAs) valine, leucine, and isoleucine are
also highly metabolized by transaminases in both the cytosol (via BCAT1) and mitochondria
(via BCAT2) (Figure 2) 65]. While cytosolic BCAT1 metabolism has been implicated in gliomas with wild-type IDH1
66], how BCAA catabolism contributes to cancer progression remains unclear. Ultimately,
by coordinating cellular bioenergetics and biosynthesis through the TCA cycle, amino
acid metabolism plays a critical role in tumor growth and survival.
Nucleotide biosynthesis
In addition to amino acid and lipid biosynthesis, nucleotide production is highly
dependent upon mitochondrial metabolism and associated intermediates. While the ribose
moiety of nucleotides is exclusively generated in the cytosol, many components that
contribute to both pyrimidine and purine bases are derived directly or indirectly
from mitochondria (Figure 3). Pyrimidine ring synthesis requires glutamine and aspartate, which can be supplied
by mitochondrial pathways as noted above. Pyrimidine synthesis also requires the activity
of dihydroorotate dehydrogenase (DHODH), a mitochondrial enzyme that converts dihydroorotate
to orotate coupled with the reduction of ubiquinone to ubiquinol. Importantly, oxidation
of ubiquinol in the electron transport chain (ETC) is necessary to maintain an adequate
supply of ubiquinone for DHODH activity. In fact, uridine must be supplemented to
culture media to allow proliferation of ?0 cells (i.e., cells lacking functional mitochondrial DNA) and other cell lines with
genetic modifications that compromise respiration 45],67]. Thus, DHODH links cellular respiration and pyrimidine synthesis. Elevated DHODH expression and increased activity have been observed in multiple types of cancers
(Table 1) 68]-71]. Inhibition of DHODH in human melanoma decreases growth both in vitro and in murine xenografts 70]. Doxorubicin, a common chemotherapeutic, induces a decrease in DHODH expression and acts synergistically with tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL) to selectively kill tumor cells 68]. DHODH is also suppressed by miR-502, which is expressed at significantly lower levels in
human colon tumors relative to normal tissue 71]. Finally, suppression of DHODH also impairs the function of complex III in the ETC,
causing accumulation of p53 and induction of apoptosis, which further relates mitochondrial
respiration to cancer growth and survival 72].
Purine nucleotide synthesis requires nitrogen from aspartate and glutamate as well
as glycine and formate for backbone synthesis (Figure 3). While enzymes involved in glycine and formate synthesis are present in both the
cytosol and mitochondria, increasing evidence suggests that the formate (and potentially
glycine) fueling this pathway is primarily derived from mitochondrial metabolism.
Formate is incorporated into purines via 10-formyl-tetrahydrofolate (10-CHO-THF) and
thymidine via 5,10-methylene-THF. These substrates can be generated in both the cytosol
and mitochondria via serine hydroxymethyltransferase (SHMT), methylenetetrahydrofolate
dehydrogenase (MTHFD), and downstream reactions in folate-mediated one carbon metabolism
(FOCM) 73]. We recently developed a system for quantifying the contribution of different substrates
to the mitochondrial and cytosolic NADPH pools using [2H] tracing and inducible expression of mutants IDH1 and IDH2 74]. Application of [2H]-labeled serine, glycine, and glucose tracers to non-small cell lung cancer cells
indicated that serine flux through SHMT2 and MTHFD2(L) operates primarily in the oxidative
direction to produce mitochondrial NAD(P)H in these cancer cells 74]. Additional evidence by others supports the concept that mitochondrial FOCM is an
important contributor of reducing equivalents and one carbon intermediates for nucleotide
biosynthesis 75],76]. While the cytosolic pathway may independently contribute to nucleotide biosynthesis
77], our results correlate with the recent demonstration that MTHFD2 expression is commonly
elevated in many cancers and associated with poor survival in breast cancer patients
54].