Glutamate, the product of the GLS reaction,
is a precursor of
glutathione, the major cellular antioxidant. It is also the source of amino
groups for nonessential amino acids like alanine, aspartate, serine, and
glycine, all of which are required for macromolecular synthesis. In glutamine-consuming
cells, glutamate is also the major source of α-ketoglutarate, a TCA cycle
intermediate and substrate for dioxygenases that modify proteins and DNA. These
dioxygenases include prolyl hydroxylases, histone demethylases, and
5-methylcytosine hydroxylases. Their requirement for α-ketoglutarate, although
likely accounting for only a small fraction of total α-ketoglutarate
utilization, makes this metabolite an essential component of cell signaling and
Conversion of glutamate to
α-ketoglutarate occurs either through oxidative deamination by glutamate
dehydrogenase (GDH) in the mitochondrion or by transamination to produce nonessential amino acids in either
the cytosol or the mitochondrion. During avid glucose metabolism, the
transamination pathway predominates (17).
When glucose is scarce, GDH becomes a major pathway to supply
glutamine carbon to the TCA cycle, and is required for cell survival (17, 18).
Metabolism of glutamine-derived α-ketoglutarate in the TCA
cycle serves several purposes: it generates reducing equivalents for the
electron transport chain (ETC) and oxidative phosphorylation, becoming a major source of energy (19);
and it is an important anaplerotic nutrient, feeding net
production of oxaloacetate to offset export of intermediates from the cycle to
supply anabolism (20).
Glutamine oxidation also
supports redox homeostasis by supplying carbon to malic enzyme, some isoforms
of which produce NADPH (Figure 1).
pancreatic adenocarcinoma cells, a pathway involving glutamine-dependent NADPH
production is essential for redox balance and growth (21).
In these cells, glutamine is used to produce aspartate in the
mitochondria. This aspartate is then trafficked to the cytosol, where it is
deaminated to produce oxaloacetate and then malate, the substrate for malic
Recent work has uncovered
an unexpected role for glutamine in cells with reduced mitochondrial function.
Despite glutamine’s conventional role as a respiratory substrate, several studies
demonstrated a persistence of glutamine dependence in cells with permanent
mitochondrial dysfunction from mutations in the ETC or TCA cycle, or transient
impairment secondary to hypoxia (22–25).
Under these conditions,
glutamine-derived α-ketoglutarate is reductively carboxylated by
NADPH-dependent isoforms of isocitrate dehydrogenase to produce isocitrate,
citrate, and other TCA cycle intermediates (Figure 1).
These conditions broaden glutamine’s utility as a carbon source
because it becomes not only a major source of oxaloacetate, but also generates
acetyl-CoA in what amounts to a striking rewiring of TCA cycle metabolism.
the hallmarks of malignancy
Deregulated energetics. One hallmark of cancer cells is aberrant bioenergetics (26).
Glutamine’s involvement in the pathways outlined above
contributes to a phenotype conducive to energy formation, survival, and growth.
In addition to its role in mitochondrial metabolism, glutamine also suppresses
expression of thioredoxin-interacting protein, a negative regulator of glucose
contributes to both of the energy-forming pathways
in cancer cells: oxidative phosphorylation and glycolysis. Glutamine also modulates hallmarks not traditionally thought to
be metabolic, as outlined below. These interactions highlight the complex
interplay between glutamine metabolism and many aspects of cell biology.
proliferative signaling. Pathological cancer cell growth relies on maintenance of
proliferative signaling pathways with increased autonomy relative to
non-malignant cells. Several lines of evidence argue that glutamine reinforces activity
these pathways. In some cancer cells, excess glutamine is exported in exchange
for leucine and other essential amino acids. This exchange facilitates
activation of the serine/threonine kinase mTOR, a major positive regulator of
cell growth (28).
glutamine-derived nitrogen is a component of amino sugars, known as
hexosamines, that are used to glycosylate growth factor receptors and promote
their localization to the cell surface. Disruption of hexosamine synthesis
reduces the ability to initiate signaling pathways downstream of growth factors
replicative immortality. Some aspects of glutamine
metabolism oppose senescence and promote replicative immortality in cultured
cells. In IMR90 lung fibroblasts, silencing either of two NADPH-generating
isoforms of malic enzyme (ME1, ME2) rapidly induced senescence, while malic
enzyme overexpression suppressed senescence (30).
Both malic enzyme isoforms are repressed at the transcriptional
level by p53 and contribute to enhanced levels of glutamine consumption and
NADPH production in p53-deficient cells. The ability of p53-replete cells to
resist senescence required the expression of ME1 and ME2, and silencing either
enzyme reduced the growth of TP53+/+and,
to a lesser degree, TP53–/– tumors
These observations position malic enzymes as potential therapeutic
cell death. Although many cancer cells
require glutamine for survival, cells with enhanced expression of Myc
oncoproteins are particularly sensitive to glutamine deprivation (8, 12, 16).
In these cells, glutamine deprivation induces depletion of TCA
cycle intermediates, depression of ATP levels, delayed growth, diminished
glutathione pools, and apoptosis. Myc drives glutamine uptake and catabolism by
activating the expression of genes involved in glutamine metabolism, including GLS and SLC1A5,
which encodes the Na+-dependent amino acid transporter ASCT2 (12, 16).
Silencing GLS mimicked some of the effects of
glutamine deprivation, including growth suppression in Myc-expressing cells and
tumors (10, 12). MYCN amplification
occurs in 20%–25% of
neuroblastomas and is correlated with poor outcome (31).
In cells with high N-Myc levels, glutamine deprivation
triggered an ATF4-dependent induction of apoptosis that could be prevented by
restoring downstream metabolites oxaloacetate and α-ketoglutarate (15).
In this model, pharmacological activation of ATF4, inhibition
of glutamine metabolic enzymes, or combinations of these treatments mimicked
the effects of glutamine deprivation in cells and suppressed growth of MYCN-amplified subcutaneous and
transgenic tumors in mice.
The PKC isoform PKC-ζ also
regulates glutamine metabolism. Loss of PKC-ζ enhances glutamine utilization
and enables cells to survive glucose deprivation (32).
This effect requires flux of carbon and nitrogen from glutamine
into serine. PKC-ζ reduces the expression of phosphoglycerate dehydrogenase, an
enzyme required for glutamine-dependent serine biosynthesis, and also
phosphorylates and inactivates this enzyme. Thus, PKC-ζ loss, which promotes
intestinal tumorigenesis in mice, enables cells to alter glutamine metabolism
in response to nutrient stress.
and metastasis. Loss
of the epithelial
cell-cell adhesion molecule E-cadherin is a component of the
epithelial-mesenchymal transition, and is sufficient to induce migration,
invasion, and tumor progression (33, 34).
Addiction to glutamine may oppose this process because
glutamine favors stabilization of tight junctions in some cells (35).
Furthermore, the selection of breast cancer cells with the
ability to grow without glutamine yielded highly adaptable subpopulations with
enhanced mesenchymal marker expression and improved capacity for
anchorage-independent growth, therapeutic resistance, and metastasis in vivo (36).
It is unknown whether this result reflects a primary role for
glutamine in suppressing these markers of aggressiveness in breast cancer, or
whether prolonged glutamine deprivation selects for cells with enhanced fitness
across a number of phenotypes.
Figure 2: Model
for inter-organ glutamine metabolism in
health and cancer. Organs
that release glutamine into the bloodstream are shown in green, and those that
consume glutamine are in red; the shade denotes magnitude of
consumption/release. For some organs (liver, kidneys), evidence from model
systems and/or human studies suggests that there is a change in net glutamine
flux during tumorigenesis.
occurs largely in the gut and kidney. The organs of the gastrointestinal tract
drained by the portal vein, particularly the small intestine, are major
consumers of plasma glutamine in both rats and humans (37, 38, 49, 50). Enterocytes oxidize more
than half of glutamine carbon to CO2, accounting for a third of the
respiration of these cells in fasting animals (51).
The kidney consumes net quantities of glutamine to maintain
acid-base balance (37, 38, 52, 53).
During acidosis, the kidneys substantially increase their
uptake of glutamine, cleaving it by GLS to produce ammonia, which is excreted
along with organic acids to maintain physiologic pH (52, 54).
also a major
metabolic substrate in lymphocytes and macrophages, at least during mitogenic
stimulation of primary cells in culture (55–57).
Importantly, cancer seems
to cause major changes in inter-organ glutamine trafficking (Figure 2).
Currently, much work in this area is derived from studies in
methylcholanthrene-induced fibrosarcoma in the rat, a model of an aggressively
growing, glutamine-consuming tumor. In this model, fibrosarcoma induces skeletal
muscle expression of glutamine synthetase and greatly increases the release of
glutamine into the circulation. As the tumor increases in
size, intramuscular glutamine pools are depleted in association with loss of
lean muscle mass, mimicking the cachectic phenotype of humans in advanced
stages of cancer (52). Simultaneously, both the
liver and the kidneys become net glutamine exporters, although the hepatic effect may be diminished as the tumor size
becomes very large (48, 49, 52).
Glutamine utilization by organs supplied by the portal vein is
diminished in cancer (48).
In addition to its
function as a nutrient for the tumor itself, and possibly for cancer-associated
immune cells, glutamine provides additional, indirect metabolic benefits to
both the tumor and the host. For example, glutamine was used as a
gluconeogenic substrate in cachectic mice with large orthotopic gliomas,
providing a significant source of carbon in the plasma glucose pool (58).
This glucose was taken up and metabolized by the tumor to
produce lactate and to supply the TCA cycle.
It will be valuable to
extend work in human inter-organ glutamine trafficking, both in healthy
subjects and in cancer patients. Such studies will likely produce a better
understanding of the pathophysiology of cancer cachexia, a major source of
morbidity and mortality. Research in this area should also aid in the
anticipation of organ-specific toxicities of drugs designed to interfere with
glutamine metabolism. Alterations of glutamine handling in cancer may induce a
different spectrum of toxicities compared with healthy subjects.
Tumors differ according to their need
One important consideration
is that not all cancer cells need an exogenous supply of glutamine. A panel of
lung cancer cell lines displayed significant variability in their response to
glutamine deprivation, with some cells possessing almost complete independence
Breast cancer cells also
demonstrate systematic differences in glutamine dependence, with basal-type
cells tending to be glutamine dependent and luminal-type cells tending to be
glutamine independent (60).
Resistance to glutamine
deprivation is associated with the ability to synthesize glutamine de novo
and/or to engage alternative pathways of anaplerosis (10, 60).
Tumors also display
variable levels of glutamine metabolism in vivo. A study of orthotopic gliomas
revealed that genetically diverse, human-derived tumors took up glutamine in
the mouse brain but did not catabolize it (58).
Rather, the tumors synthesized glutamine de novo and used
pyruvate carboxylation for anaplerosis. Cells
derived from these tumors did not require glutamine to survive or proliferate
when cultured ex vivo. Glutamine synthesis from glucose was also a
prominent feature of primary gliomas in human subjects infused with 13C-glucose
at the time of
surgical resection (61).
Furthermore, an analysis
of glutamine metabolism in lung and liver tumors revealed that both the tissue
of origin and the oncogene influence whether the tumor produces or consumes glutamine
tumors produced glutamine, whereas Myc-induced liver tumors catabolized it. In
the lung, however, Myc expression was associated with glutamine accumulation.
This variability makes it
imperative to develop ways to predict which tumors have the highest likelihood
of responding to inhibitors of glutamine metabolism. Methods to image or
otherwise quantify glutamine metabolism in vivo would be useful in this regard
pre-surgical subjects with isotopically labeled glutamine, followed by
extraction of metabolites from the tumor and analysis of 13C enrichment,
can be used
to detect both glutamine uptake and catabolism (58, 62).
However, this approach requires a specimen of the tumor to be
obtained. Approaches for glutamine-based imaging, which avoid this problem,
include a number of glutamine analogs compatible with PET. Although glutamine
could in principle be imaged using the radioisotopes 11C, 13N, or 18F, the relatively
half-life of the latter increases its appeal. In mice, 18F-(2S,
is avidly taken up by tumors derived from highly glutaminolytic cells, and by
glutamine-consuming organs including the intestine, kidney, liver, and pancreas
Labeled analogs of
glutamate are also taken up by some tumors (65, 66).
One of these, (4S)-4-(3-[18F] fluoropropyl)-L-glutamate
(18F-FSPG, also called BAY 94-9392), was evaluated in small clinical
trials involving patients with several types of cancer (65, 67).
This analog enters the cell through the cystine/glutamate
exchange transporter (xC–transport system), which is linked to
glutathione biosynthesis (68).
The analog was well tolerated, with high tumor detection rates
and good tumor-to-background ratios in hepatocellular carcinoma and lung
PET approaches detect
analog uptake and retention but cannot provide information about downstream
metabolism. Analysis of hyperpolarized nuclei can provide a real-time view of
enzyme-catalyzed reactions. This technique involves redistribution of the
populations of energy levels of a nucleus (e.g., 13C, 15N), resulting in a gain
in magnetic resonance signal that can temporarily exceed 10,000-fold (69).
This gain in signal enables rapid detection of both the labeled
molecule and its downstream metabolites. Glutamine has been hyperpolarized on 15N and 13C (70, 71).
In the latter case, the conversion of hyperpolarized glutamine
to glutamate could be detected in intact hepatoma cells (70).
If these analogs are translated to clinical studies, they might
provide a dynamic view of the proximal reactions of glutaminolysis in vivo.
Phamacological strategies to inhibit
glutamine metabolism in cancer
Efforts to inhibit
glutamine metabolism using amino acid analogs have an extensive history,
including evaluation in clinical trials. Acivicin, 6-diazo-5-oxo-L-norleucine,
and azaserine, three of the most widely studied analogs (Figure 1), all demonstrated variable degrees of gastrointestinal
toxicity, myelosuppression, and neurotoxicity (72). Because these agents non-selectively target
glutamine-consuming processes, recent interest has focused on developing
methods directed at specific nodes of glutamine metabolism. First, ASCT2, the
Na+-dependent neutral amino acid transporter encoded by SLC1A5,
is broadly expressed in
lung cancer cell lines and accounts for a majority of glutamine transport in
those cells (Figure 1). It has been shown that γ-L-glutamyl-p-nitroanilide (GPNA)
inhibits this transporter and limits lung cancer cell growth (73). Additional interest in GPNA lies in its ability to
enhance the uptake of drugs imported via the monocarboxylate transporter MCT1.
Suppressing glutamine uptake with GPNA enhances MCT1 stability and stimulates
uptake of the glycolytic inhibitor 3-bromopyruvate (3-BrPyr) (74, 75). Because enforced MCT1 overexpression is sufficient to
sensitize tumor xenografts to 3-BrPyr (76), GPNA may have a place in 3-BrPyr–based therapeutic
Two inhibitors of GLS
isoforms have been characterized in recent years (Figure 1). Compound 968, an inhibitor of the GLS-encoded splice isoform GAC,
inhibits the transformation of fibroblasts by oncogenic RhoGTPases and delays
the growth of GLS-expressing lymphoma xenografts (13). Bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl
sulfide (BPTES) also potently inhibits GLS isoforms encoded by GLS (77). BPTES impairs ATP levels and growth rates of P493
lymphoma cells under both normoxic and hypoxic conditions and suppresses the
growth of P493-derived xenografts (78).
Evidence also supports a
role for targeting the flux from glutamate to α-ketoglutarate, although no
potent, specific inhibitors yet exist to inhibit these enzymes in intact cells.
Aminooxyacetate (AOA) inhibits aminotransferases non-specifically, but
milliomolar doses are typically used to achieve this effect in cultured cells
(Figure 1). Nevertheless, AOA has demonstrated efficacy in both
breast adenocarcinoma xenografts and autochthonous neuroblastomas in mice (15, 79). Epigallocatechin gallate (EGCG), a green tea polyphenol,
has numerous pharmacological effects, one of which is to inhibit GDH (80). The effects of EGCG on GDH have been used to kill
glutamine-addicted cancer cells during glucose deprivation or glycolytic
inhibition (17, 18) and to suppress growth of neuroblastoma xenografts (15).
Paradigm to exploit glutamine metabolism in cancer
Recent advances in glutamine-based imaging,
coupled with the successful application of glutamine metabolic inhibitors in
mouse models of cancer, make it possible to conceive of treatment plans that
feature consideration of tumor glutamine utilization. A key challenge will be
predicting which tumors are most likely to respond to inhibitors of glutamine
metabolism. Neuroblastoma is used here as an example of a tumor in which
evidence supports the utility of strategies that would involve both
glutamine-based imaging and therapy (Figure 3). Neuroblastoma is the second most common
extracranial solid malignancy of childhood. High-risk neuroblastoma is defined
by age, stage, and biological features of the tumor, including MYCN amplification, which occurs in some 20%–25%
cases (31). Because MYCN-amplified tumor cells require glutamine
catabolism for survival and growth (15), glutamine-based PET at the time of standard
diagnostic imaging could help predict which tumors would be likely to respond
to inhibitors of glutamine metabolism. Infusion of 13C-glutamine coordinated with the diagnostic biopsy could
enable inspection of 13C enrichment in glutamine-derived metabolites from the tumor,
confirming the activity of glutamine catabolic pathways. Following on evidence
from mouse models of neuroblastoma, treatment could then include agents
directed against glutamine catabolism (15). Of note, some tumors were sensitive to the ATF4
agonist fenretinide (FRT), alone or in combination with EGCG. Importantly, FRT
has already been the focus of a Phase I clinical trial in children with solid
tumors, including neuroblastoma, and was fairly well tolerated (81).
A strategy to integrate glutamine metabolism into the diagnosis,
classification, treatment, and monitoring of neuroblastoma.
Neuroblastoma commonly presents in children as an abdominal mass.
A standard evaluation of a child with suspected neuroblastoma includes
measurement of urine catecholamines, a bone scan, and full-body imaging with
meta-iodobenzylguanidine (MIBG), all of which contribute to diagnosis and
disease staging. In animal models, a subset of these tumors requires glutamine
metabolism. This finding implies that approaches to image, quantify, or block
glutamine metabolism (highlighted in red) in human neuroblastoma could be
incorporated into the diagnosis and management of this disease. In particular,
glutamine metabolic studies may help predict which tumors would respond to therapies
targeting glutamine metabolism. HVA, homovanillic acid; VMA, vanillylmandelic
is a versatile nutrient required for the survival
and growth of a potentially large subset of tumors. Work over the next several
years should produce a more accurate picture of the molecular determinants of
glutamine addiction and the identification of death pathways that execute cells
when glutamine catabolism is impaired. Advancement of glutamine-based imaging
into clinical practice should soon make it possible to differentiate tumors
that take up glutamine from those that do not. Finally, the development of
safe, high-potency inhibitors of key metabolic nodes should facilitate
therapeutic regimens featuring inhibition of glutamine metabolism.
Citation for this article:J Clin Invest. 2013;123(9):3678–3684. doi:10.1172/JCI69600.
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