This decade has seen renewed interest in understanding the metabolic activities that support tumor cell survival and growth. The abundance of data showing that mutations in oncogenes and tumors suppressors influence glucose metabolism, coupled with the utility of 18FDG-PET, have raised the expectation that metabolic therapies directed against glycolysis will be effective in cancer. However, recent studies showing imperfect correlation between improvement on 18FDG-PET and patient survival have tempered this hope. Such studies suggest that tumors are capable of surviving periods of reduced glucose metabolism by using alternative metabolic pathways. Finding and targeting these survival pathways may ultimately improve the long-term efficacy of treatments that interfere with tumor glucose metabolism. Here we identified such a survival pathway, catalyzed by GDH, in a highly glycolytic gliobastoma cell line.
The emerging picture of tumor cell metabolism is that a complex network links signal transduction with the metabolism of glucose, glutamine and other nutrients. Understanding this network will enhance efforts to develop metabolically-targeted cancer therapies. This work emphasizes how distinct signaling pathways can influence glucose and glutamine metabolism in the same cells. In the glioblastoma cells we studied, Akt facilitates glucose metabolism but exerts no effect on total glutamine consumption (9
). Rather, c-Myc stimulates glutamine metabolism by increasing the expression of amino acid transporters and glutaminase, resulting in increased glutamine catabolism and increased sensitivity to glutamine withdrawal (8
). c-Myc activates a metabolic program in which glucose-replete cells require transamination reactions to produce α-KG (9
). Thus, while Akt directs the delivery of glucose-derived acetyl-CoA to the TCA cycle, c-Myc directs the delivery of glutamine-derived α-KG and ultimately OAA for anaplerosis and cell growth. Glucose-replete cells can tolerate suppression of GDH because there are other sources of both α-KG and acetyl-CoA.
The current work adds to this picture by describing an alternative pathway that is activated in glucose-starved cells. The new pathway involves a large increase in GDH activity compared to ALT, which is the most active transaminase when glucose is present (5
). Unlike transamination reactions, GDH has the advantage of delivering α-KG to the TCA cycle without the expenditure of a keto-acid, which could otherwise be oxidized to supplement ATP production. For example, the cells we studied displayed aspartate aminotransferase activity during glucose withdrawal, as shown by the transfer of 13
C from glutamine to aspartate (). This reaction converts glutamate to α-KG, but consumes OAA in the process. Thus, the pathway serves as a “mini-cycle” that generates aspartate but does not produce OAA for traditional TCA cycling (20
). The concomitant induction of GDH would yield net OAA and therefore support ongoing citrate synthesis and function of the TCA cycle. Moreover, under these conditions glutamine metabolism also generated acetyl-CoA for the TCA cycle in a GDH-dependent manner. This may explain how the cells maintained their ATP content in the absence of glycolysis (Supplementary Fig. 1
The effects of glucose withdrawal on both ALT and GDH were reversed by CH3-Pyr (). Presumably this molecule stimulates ALT by supplying a large intracellular pyruvate pool for transamination. Since both ALT and GDH consume glutamate, competition between the two enzymes might contribute to CH3-Pyr's negative effect on GDH. However, the large discrepancy between GDH suppression (~60 nmol/hr/million cells) and ALT induction (only ~10 nmol/hr/million cells) suggest the involvement of additional mechanisms.
GDH has not received much attention in cancer cell metabolism, probably because its activity is suppressed during robust glycolysis in vitro
. GDH is a widely-expressed homohexameric enzyme localized to the mitochondrial matrix, where it coordinates carbon and nitrogen metabolism. It determines the rate of oxidative degradation of glutamate and other nonessential amino acids, and thus is uniquely poised to respond to glucose deprivation. Regulation of GDH activity is extremely complex and involves allosteric effects, post-translational modifications and other levels of control (21
). Suppression of GDH activity by glucose is not restricted to glioblastoma, since we also observed it in mouse embryonic fibroblasts (Supplementary Fig. 3
), and another study observed it in myeloma and hybridoma cells (23
). The mechanism for GDH activation during glucose withdrawal is independent of changes in mRNA and protein levels (data not shown and ). Because the forward reaction (oxidative deamination of glutamate) uses NAD+
as a cofactor, it is possible that the low cellular NAD+
/NADH ratio of highly glycolytic cells holds GDH activity in check. A similar phenomenon has been observed in brain slices, where glucose deprivation increased the cytoplasmic and mitochondrial NAD+
/NADH ratio and ammoniagenesis (24
). It is significant that in the cells studied here, there was no toxicity associated with the increased production of ammonia when glucose was withdrawn. Rather, the additional GDH activity sustained cell viability. GDH serves a similar compensatory role in plants. Mutant strains of Arabidopsis
lacking GDH activity grew normally in carbon-replete conditions, but could not survive carbon starvation induced by prolonged growth in the dark. Viability was restored to the mutants simply by providing an exogenous carbohydrate source (25
). Thus, we conclude that de-repression of GDH is necessary for some cells to adapt to and survive low-glucose conditions.
It is not known to what extent the metabolism of cultured tumor cells parallels tumor metabolism in vivo
, but evidence suggests that many core metabolic activities are shared between settings. First, 18
FDG-PET and 1
H magnetic resonance spectroscopy reveal robust glucose consumption and lactate production in aggressive tumors. Second, expression studies have shown that enzymes involved in glucose and glutamine metabolism are abundantly expressed in tumor tissue and in some cases predict patient outcome (26
). Third, inhibition of some of these enzymes impairs tumor growth in animal models (30
). Fourth, some mechanisms of metabolic compensation, particularly autophagy, allow cells to survive nutrient stress in vitro
and in vivo
). Likewise, GDH inhibition may increase the efficacy of treatments that interfere with glucose metabolism. There are many such treatments already in use that could benefit from concomitant blockade of GDH. Alkylators and other DNA damaging agents suppress glycolysis in vitro
and in vivo
through PARP-dependent depletion of cytoplasmic NAD (37
). The kinase inhibitors imatinib and rapamycin inhibit glycolysis in vitro
by reversing the effects of oncogenic signaling pathways on glucose metabolism, and they suppress 18
FDG-PET signal in some tumors (15
A major challenge in cancer therapeutics is to develop strategies that antagonize tumor cell survival without causing dose-limiting toxicity. Previous efforts to inhibit tumor glutamine metabolism were complicated by nausea, mucositis and pancytopenia (43
). In this regard, tea polyphenols are intriguing because they are consumed in large quantities by millions of people worldwide and because a number of epidemiological studies have demonstrated the benefits of green tea in preventing the initiation or progression of cancer (44
). EGCG in particular has a number of pharmacological properties that could independently suppress tumor cell growth (45
). The relevance of EGCG's activity as a GDH inhibitor is unknown, although this has been proposed as a mechanism to explain the protective effect of green tea against diabetes (19
). In the glioblastoma cells we studied, the effect of EGCG perfectly mimicked GDH knockdown in that it was enhanced during decreased glucose metabolism and reversed by providing GDH-dependent nutrients. Thus it will be interesting to test whether genetic or pharmacological impairment of GDH activity can suppress tumor growth or enhance the effect of conventional cancer therapies.