From Krebs to clinic: glutamine metabolism to cancer therapy

Abstract

The resurgence of research into cancer metabolism has recently broadened interests beyond glucose and the Warburg effect to other nutrients, including glutamine. Because oncogenic alterations of metabolism render cancer cells addicted to nutrients, pathways involved in glycolysis or glutaminolysis could be exploited for therapeutic purposes. In this Review, we provide an updated overview of glutamine metabolism and its involvement in tumorigenesis in vitro and in vivo, and explore the recent potential applications of basic science discoveries in the clinical setting.

Figure 1

Timeline of key discoveries in mammalian glutamine metabolism and cancer α-KG, α-ketoglutarate; GLUD, glutamate dehydrogenase.

Figure 2. Major metabolic and biosynthetic fates of glutamine

Glutamine enters the mammalian cell through transporters such as SLC1A5 (also known as ASCT2) . Glutamine itself can contribute to nucleotide biosynthesis and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis for support of protein folding and trafficking , or is converted to glutamate by glutaminase (GLS or GLS2) . Glutamate can contribute to the synthesis of glutathione , and has many other metabolic fates in the cell that impact on several inborn errors of metabolism, which were recently reviewed . Glutamate is converted to α-ketoglutarate (αKG) through one of two sets of enzymes, glutamate dehydrogenase (GLUD1 or GLUD2, henceforth referred to collectively as GLUD) or aminotransferases . While the byproduct of GLUD is NH₄⁺, the byproduct of aminotransferase reactions is other amino acids. Note that aminotransferases may be present either in the cytoplasm or the mitochondria. α-ketoglutarate enters the tricarboxylic acid (TCA) cycle and can provide energy for the cell. Malate exiting the TCA cycle can produce pyruvate and NADPH for reducing equivalents , and oxaloacetate (OAA) can be converted to aspartate to support nucleotide synthesis . These two pathways are illustrated in more detail in Figure 4. Alternately, α-KG can proceed backwards through the TCA cycle, in a process called reductive carboxylation (RC) to produce citrate, which supports synthesis of acetyl-CoA and lipids .

Figure 3. Glutamine control of amino acid pools and ROS

Glutamate acts as a nitrogen donor for the transamination involved in the production of ‘dispensable amino acids’ alanine, aspartate, and serine through the actions of glutamic-oxaloacetic transaminase (GOT), glutamic pyruvate transaminase (GPT) and phosphoserine aminotransferase 1 (PSAT1), respectively. Glutamine can also act as a nitrogen donor for asparagine through asparagine synthetase (ASNS). In a reaction independent of transamination, proline can be synthesized by conversion of glutamate to pyrroline-5-carboxylate (P5C) by pyrroline-5-carboxylate synthase (P5CS; also known as aldehyde dehydrogenase 18 family member A1, (ALDH18A1)) and subsequently to proline by pyrroline-5-carboxylate reductase 1 (PYCR1) and PYCR2. Glutamine also contributes to the tripeptide glutathione (composed of glutamate, cysteine and glycine), which neutralizes the ROS H₂O₂ . The first step in glutathione synthesis is the condensation of glutamate and cysteine through glutamate-cysteine ligase (GCL; not shown in the figure). Glutamine input directly contributes to the availability of cysteine and glycine for production of glutathione. Glutamate can be exchanged for cystine (which is quickly reduced to cysteine inside the cell) through the xCT antiporter (a heterodimer of SLC7A11 and SCL3A2), which has been shown to be important in a variety of cancers and has been considered as a drug target ^, . Glycine is next added by glutathione synthetase (GSS; not shown in the figure). Additionally, glutamate can contribute to glycine through transamination by PSAT1 into phosphoserine (pSer) and α-ketoglutarate (αKG) and subsequent conversion to glycine through serine hydroxymethyltransferase (SHMT; not shown in the figure) as part of the one-carbon metabolism pathway, which has been shown in numerous studies to be critical in cancer metabolism and is also reviewed in this Focus Issue by Dr. Karen Vousden ^{, ,} . GLS, kidney-type glutaminase; GLS2, liver-type glutaminase; GLUD, glutamate dehydrogenase; OAA, oxaloacetate.

Figure 4. Control by glutamine of the integrated stress response, protein folding and trafficking, and ER stress

GCN2, a serine-threonine kinase with a regulatory domain that is structurally similar to histidine-tRNA synthetase, is allosterically activated by uncharged tRNAs with amino acid deprivation (including glutamine deprivation) and in turn activates the integrated stress response (ISR) ^{, ,} . Glutamine can suppress GCN2 activation through its contribution to amino acid pools by aminotransferases ^{, –}. To control endoplasmic reticulum (ER) homeostasis, glutamine supports protein folding and trafficking through its contribution to uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) as part of the hexosamine biosynthesis pathway. Glutamine is the substrate for glutamine fructose-6-phosphate aminotransferase (GFAT), which is the key rate-limiting enzyme in the hexosamine pathway, and the downstream product UDP-GlcNAc is a substrate for O-linked glycosylation through O-linked β-N-acetylglucosamine transferase (OGT). Thus, glutamine deprivation can lead to improper protein folding and chaperoning and ER stress . A key output of both the ISR and of ER stress is activating transcription factor 4 (ATF4), which is induced via cap-independent translation downstream of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation by GCN2 or other kinases . α-KG, α-ketoglutarate; GLS, kidney-type glutaminase; GLS2, liver-type glutaminase.

Figure 5. Glutamine derived TCA cycle intermediates can be used via two pathways to produce NADPH and neutralize ROS through the malic enzyme

Reduced glutathione (GSH) neutralizes H₂O₂ with the glutathione peroxidase enzyme, and oxidized glutathione (GSSG) is reduced by NADPH and glutathione reductase to regenerate GSH. In the first pathway, glutamine-derived malate is transported out of the mitochondria, and is converted by malic enzyme 1 (ME1) to pyruvate, reducing one molecule of NADP+ to NADPH. In the malate-aspartate shuttle-related second pathway, found in mutant KRAS-transformed cells, aspartate that is produced from GOT2 mediated transamination of glutamine-derived oxaloacetate (OAA) is transported out of the mitochondria. Aspartate is then converted in the cytosol back to OAA by GOT1 and then to malate by malate dehydrogenase 1 (MDH1), which is in turn processed to pyruvate by ME1 to produce one molecule of NADPH . The fate of glutamine-derived pyruvate is similar to glucose-derived pyruvate in that much of it is expelled as lactate. α-KG, α-ketoglutarate; TCA, tricarboxylic acid; GOT, glutamic oxaloacetate transaminase.

Figure 6. Glutamine controls mTOR activity

Amino acids stimulate the mTOR pathway, and amino acid pools rely on glutamine to be maintained. Specifically, arginine and leucine are two amino acids that can together almost fully stimulate mTOR complex 1 (mTORC1) through activation of the RAS-related GTPase (RAG) complex, which in turn recruits mTORC1 to the lysosome and stimulates its activity ^{, ,} . Glutamine can contribute to mTORC1 activation by being exchanged for essential amino acids, including leucine, through the large neutral amino acid transporter 1 (LAT1; a heterodimer of SLC7A5 and SLC3A2) transporter . This RAG-dependent regulation of mTOR is likely dependent on the lysosomal amino acid transporter SLC38A9, which transports glutamine, arginine, and leucine as substrates^{, ,} , as well as the leucine sensor sestrin 2 (not shown in Figure) ^, . Although the mechanism is not well understood, α-ketoglutarate (α-KG) may regulate RAGB activity and mTOR activation downstream of glutamine metabolism . Several RAG-independent pathways of mTOR regulation by glutamine have also been identified. Glutamine promotes mTOR localization to the lysosome (and thus activity) through the RAS-family member ADP ribosylation factor 1 (ARF1) in a poorly understood mechanism, as well as the TTT-RUVBL1/2 complex (not shown in Figure) ^, . GLS, kidney-type glutaminase; GLS2, liver-type glutaminase; GLUD, glutamate dehydrogenase.

Figure 7. Two roads to α-ketoglutarate

Glutamate can be converted by one of two different pathways into α-ketoglutarate (α-KG), and the choice of which pathway is influenced both by oncogene input and cell proliferation and metabolic state. GLS, kidney-type glutaminase; GLS2, liver-type glutaminase; GLUD, glutamate dehydrogenase; ISR, integrated stress response; ROS, reactive oxygen species.

Figure 8. Differing requirements for glutamine in cancer based on oncogene and tissue of origin

The oncoproteins MET and MYC lead to differing dependence on glutamine in different cancer types, which is partially influenced by differential expression of glutamine synthetase (GLUL) or glutaminase (GLS). α-KG, α-ketoglutarate; OAA, oxaloacetate; Illustration is drawn from primary data originally presented in Yuneva et al..