The role of glutaminase in cancer

Increased glutamine metabolism (glutaminolysis) is a hallmark of cancer and is recognised as a key metabolic change in cancer cells. Breast cancer is a heterogeneous disease with different morphological and molecular subtypes and responses to therapy, and breast cancer cells are known to rewire glutamine metabolism to support survival and proliferation. Glutaminase isoenzymes (GLS and GLS2) are key enzymes for glutamine metabolism. Interestingly, GLS and GLS2 have contrasting functions in tumorigenesis. In this review, we explore the role of glutaminase in cancer, primarily focusing on breast cancer, address the role played by oncogenes and tumour suppressor genes in regulating glutaminase, and discuss current therapeutic approaches to targeting glutaminase.


Introduction
An established hallmark of cancer is cellular metabolic reprogramming to maintain the high demand for energy needed to sustain proliferation and survival. 1 Cancer cells alter their glucose and glutamine metabolism to acquire sufficient energy and cellular building blocks needed to support this unremitting growth. Some consume more glucose, showing aerobic glycolysis or the 'Warburg effect', whereby glucose is converted mainly to lactic acid instead of engaging in mitochondrial oxidative phosphorylation to allow proper respiration. [2][3][4] Other cancer cell types fail to grow or proliferate in the absence of glutamine, and show 'glutamine addiction' 5 , which helps the cells to sustain high proliferative rates under conditions of hypoxia and glucose depletion [6][7][8] (Figure 1).
The intracellular processing of glutamine begins with its catalysis by glutaminase. In this review, we discuss the role, regulation and relevance of glutaminase and its isozymes and splice variants in cancer, particularly focusing on breast cancer (BC). We also highlight the opportunities that exist to utilise glutaminase as a therapeutic target.

Glutamine Metabolism And Addiction In Cancer
Glutamine metabolism plays an important role in normal cell metabolism and generating energy for rapidly proliferating cells and tissues. As the most abundant amino acid in the blood circulation, glutamine serves directly or indirectly, via its metabolic products glutamate and a-ketoglutarate (a-KG), as a source of carbon and nitrogen for the biosynthesis of nucleic acids, fatty acids, and proteins. Some cancers can become highly dependent on glutamine, 1,4,9 such that the demand for glutamine outpaces the supply. Additionally, some tumour cells in vitro are unable to survive in the absence of an exogenous supply of glutamine. 8,10 Consequently, cells develop a metabolic strategy to provide a source of carbon other than glucose to derive the carbon that is necessary to fuel the tricarboxylic acid (TCA) cycle. 11 Recently, it has been shown that glutamine can enhance cancer progression independently of its metabolic role, as it can act as a signalling agent to activate the transcription factor STAT3, which is required to mediate the proliferative effects of glutamine on cancer cells. 12 Furthermore, glutamine can indirectly activate other signalling pathways, such as the mammalian target of rapamycin complex 1 (mTORC1) pathway; mTORC1 is a critical kinase that regulates cell growth and proliferation, as glutamine-derived a-KG is required for GTP loading of RagB and subsequent activation of mTORC1. 13 Additionally, glutamine efflux through SLC7A5 is coupled to leucine uptake. The latter is known as a potent activator of mTORC1. 14 The maintenance of redox homeostasis in cancer is important, because the highly proliferating cells that encounter increased reactive oxygen species (ROS) production due to enhanced glutamine metabolism need a defence mechanism to avoid apoptosis. 15 Glutamine has a role in maintaining the redox balance through different mechanisms. Metabolites produced during the TCA cycle serve as precursors for the reducing agent NADPH. Furthermore, exchange of intracellular glutamate through the transporter SLC7A11 mediates cystine uptake. Cystine is then reduced to cysteine which is the rate-limiting product for glutathione (GSH) biosynthesis. 16 Both NADPH and GSH act as key regulators of cellular redox status. 11 Figure 1. Glutamine metabolism in cancer cells. Glutamine is an essential amino acid that serves as a carbon and nitrogen source for energy production and nucleotide biosynthesis. Amino acid transporters regulate glutamine supply into the intercellular space. Glutamine is transported across the plasma membrane mainly by transporters: SLC1A5 and SLC7A5. In the mitochondrion, either glutaminase, GLS or GLS2 converts glutamine to glutamate. The latter is converted into a-ketoglutarate and enters the tricarboxylic acid cycle for processing. Up-regulation of the oncogene c-Myc is responsible for direct promotion of the expression of glutamine transporters, enhancing glutamine entry into the cell and up-regulation of GLS. The tumour suppressor p53 regulates the transcription for GLS2, increasing its expression under both stressed and non-stressed conditions. GLS inhibitors used to target GLS directly are 6-diazo-5-oxy-L-norleucine (DON), 968, bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulphide (BPTES), and 2-(pyridin-2-yl)-N-(5-(4-(6-(2-(3-(trifluoromethoxy) phenyl)acetamido)pyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)acetamide (CB-839). 1,58,71 Glutaminase Isoforms In humans, glutaminase exists as two isoforms, i.e. kidney-type glutaminase (GLS) and liver-type glutaminase (GLS2), which differ not only in kinetic properties, but also in protein structure and tissue distribution. 17 GLS and GLS2 are encoded by GLS and GLS2, respectively, 18 and both can undergo alternative splicing to produce several variants (Figure 2). GLS (KGA; long transcript), GLS C (GAC; short transcript) and GAM are encoded by GLS. However, GAM is significantly shorter than KGA or GAC, and shows no measurable catalytic activity, whereas GAC has greater catalytic activity and is frequently up-regulated in cancer cells. 19,20 GLS2 (LGA; shorter transcript) and GAB (long-transcript isoform) splice variants encoded by GLS2 also exist. 17,18,21

Role Of Glutaminase In Cancer
In cancer, the two glutaminase isozymes have opposing roles in tumorigenesis. GLS correlates with tumour growth rate and malignancy, and is regulated by the oncoprotein c-Myc, whereas GLS2 tends to have tumour suppressive features, and is regulated by p53. 17,[22][23][24] Up-regulation of GLS is observed in cancers, including BC, liver cancer, colorectal cancer, brain cancer, cervical cancer, lung cancer, and melanoma. 20,25 Rapidly growing malignant cells have elevated GLS mRNA levels and enhanced GLS expression, 24,26-28 and GLS enzymatic activity correlates with poor disease outcome in patients with liver, lung, colorectal, breast and brain tumours. 24,25,29-32 (refer Table 1) However, it is the GAC variant that is a key enzyme for cancer cell growth. 20,28,33,34 It appears that post-translational phosphorylation of GAC at specific regions of the enzyme by different signalling pathways can alter GLS activity. 28,33,35 GAC is elevated as a result of phosphorylation at Ser314 by the oncogenic protein RhoC which is regulated by protein kinase Ce. 35 . In contrast, Ser95 phosphorylation at the GLS N-terminal region leads to decreased GLS activity. 33 The expression levels of GLS2 variants are markedly increased in tumour cells that are well differentiated, and this is associated with a significantly prolonged survival time 24,25,36,37 (Table 1). GLS2 negatively regulates the activity of the phosphoinositide 3-kinase-AKT signalling pathway 38 and Rac1 by mediating p53 function in hepatocellular carcinoma, resulting in the inhibition of migration, invasion and metastasis of cancer cells. 25,27 Glutamine Dependency in BC The need for glutamine varies according to different BC molecular subtypes, 34,35,39 some of which only require an exogenous supply of glutamine and show glutamine dependence. 38 For example, triple-negative BC (TNBC) and HER2+ cell lines are highly glutamine-dependent, whereas luminal tumours have variable amounts of glutamine dependence. 32,40,41 Luminal A tumours are primarily glutamine-independent, as they show only moderate effects on growth and viability in a glutamine-deprived environment, whereas luminal B cells show much higher glutamine metabolic activity. 39 Glutaminase In BC Interrogation of Breast Cancer Gene-expression Miner v4.3 (http://bcgenex.centregauducheau.fr) showed that GLS mRNA expression and GLS2 mRNA expression are negatively correlated (Figure 3). GLS is associated with high-grade tumours (P = 0.006), whereas GLS2 is associated with low-grade tumours (P < 0.0001). In a relatively small study of breast tumours, a high GLS level was associated with high tumour grade and high grade metastatic BC, but not with tumour size or nodal stage. 20,42 In terms of BC patient outcome, GLS mRNA expression predicts poor patient survival ( Figure 4A) and high GLS2 mRNA expression predicts better patient survival ( Figure 4B).
Among biological subtypes, GLS mRNA expression and/or GLS expression are higher in basal-like/TNBC and HER2+ tumours, and are associated with poor disease-free survival in patients with positive lymph node metastasis. 41 Luminal B tumours have higher expression of GLS than luminal A tumours, and this is predictive of poor patient outcome 30,[42][43][44][45] (Figure 3).
In contrast, GLS2 mRNA expression is significantly higher in luminal A tumours than in luminal B, HER2+ and TNBC tumours ( Figure 3). However, there is very little published information on GLS2 expression in cancer, including BC. Interestingly, patients with tumours expressing GLS but not GLS2 have worse survival ( Figure 4C), although this is observed only in patients with luminal B tumours ( Figure 4D). Immunohistochemical expression of GLS and GLS2 in invasive ductal breast cancers of no special type. Cytoplasmic GLS shows homogeneous and granular immunoreactivity ( Figure 5A, 5), and GLS2 shows homogeneous immunoreactivity ( Figure 5C, 5).

Glutaminase Regulation In Cancer-Role Of Oncogenes And Tumour Suppressor Genes
c-Myc plays a key role in the induction of glutamine dependence, as it can enhance glutamine influx and metabolism. There is evidence that both splice variants of GLS, i.e. KGA and GAC, are positively regulated by c-Myc and are strongly expressed in c-Mycinduced tumours. 26,34,46,47 Tumours showing overexpression of c-Myc with elevated GLS expression, together with a high influx of glutamine into the cells, consequently become glutamine-addicted. 10,48 c-Myc transcription stimulates GLS expression through different mechanisms. 34,48-50 GLS is partly up-regulated by microRNAs, whereby its translation is repressed by miR-23a/b through the mTORC1 pathway. Moreover, cancer cells that are dependent on Rho GTPase signalling via nuclear factor-jB activity for progression of malignancy have activated GAC and consequently elevated levels of GLS activity. 28,51,52 Conversely, GLS2 is induced by p53, in response to oxidative stress, to engage antioxidant responses in order to decrease ROS levels and participate in DNA damage repair processes. 53  LGA long/GAB GLS2 intron lengths (kb) LGA short GLS2 is a p53 target gene containing p53 DNA-binding elements in the promoter region, and that, in turn, GLS2 mediates p53 function in the regulation of energy metabolism and antioxidant defence in cells. 22,54 The differences between the catalytic activities of GLS and GLS2 could be due to the underlying regulatory mechanisms. The deamination of glutamate by GLS results in the release of ammonia, which is essential to support cell survival processes through providing a-KG and intermediates for biosynthesis. However, glutamate produced from GLS2 activity supports the antioxidant machinery/mechanism (GSH) in the cell cycle.

Potential Therapeutic Uses
As glutaminase is critical for tumour growth, is predominantly up-regulated in highly proliferating breast tumours, and is a key enzyme in the first step of glutamine catabolism, it has the potential to be a target for therapy.

Glutaminase Inhibitors
The GLS inhibitor 6-diazo-5-oxo-L-norleucine is the earliest inhibitor of both GLS and GLS2 to be used in preclinical models. As a result of its non-selectivity and undesirable effects, due to it having a similar structure to glutamine and having reactive chemical compounds, other compounds were developed. 55 Recently, two small molecules that inhibit both GLS and GLS2 have been found: bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulphide (BPTES) and dibenzophenanthridine-968. In-vitro and mouse model xenograft studies have shown that BPTES inhibits GLS significantly more than it does GLS2 28,55,56 in various cancer types, whereas the dibenzophenanthridine-968 class of inhibitors inhibit GLS and GLS2 with similar potencies. 17,57   BPTES is a potent GLS inhibitor with minimal toxicity. The inhibitor does not have structural similarities to glutamate or glutamine. However, it forms an inactive tetramer complex site where it interacts with GLS, and this is not the site where glutamine is catalysed. Thus, there is no competition in the inhibition of GLS with the molecule. 58 BPTES inhibits GLS activity in glioma cells, in which glutamate and a-KG levels are decreased, leading to decreases in the levels of subsequent TCA cycle intermediates and their downstream products, and slowing of tumour growth. 56 BPTES also suppresses cell proliferation in HER2+ BC cells associated with increased GLS activity. 59 Although BPTES selectively inhibits GLS over GLS2, 53 it has been reported to have limitations in pharmacological application, owing to its poor metabolic stability, low solubility, and moderate potency. 57 Like BPTES, dibenzophenanthridine-968 is an allosteric inhibitor of GLS and inhibits the activity of KGA and GAC. 60 In-vitro and mouse xenograft model studies have shown antitumour activity of the compound in lymphoma, BC, ovarian and glioblastoma cells. 24,28,61 Very recently, evidence regarding the inhibition of glutaminase as a therapeutic approach in the treatment of cancer has resulted in the development of a The small molecule is a GLS inhibitor that regulates the enzymatic activity of KGA and predominantly the GAC splice variant isoenzyme 28,41 by targeting the allosteric site of GLS. The inhibitor works by binding to and stabilising an inactive tetrameric state of the enzyme, rather than by competition with glutamine for binding to the active site where glutamine is hydrolysed. 53,56 In terms of inhibition of GLS, CB-839 is a more potent compound than BPTES. 28,55,58 In addition, CB-839 has inhibitory concentrations 30fold and 50-fold lower than those of BPTES. 62 Preclinical models have demonstrated that CB-839 causes significant growth inhibition in certain subtypes of BC. Gross et al. 41 demonstrated that TNBC cells are more sensitive to CB-839 than are luminal A/oestrogen receptor-positive cells (MCF-7), mainly because of their high glutamine dependence and enhanced glutamine utilisation. Treatment of TNBC with CB-839 lowered glutamate levels, suggesting blockade of glutamine metabolism by inhibition of GLS. 8 Consistent with these findings, CB-839 inhibits signalling pathways in transformed cells via Rho GTPases, which are linked to the activation of GLS, hence inhibiting enzyme invasive activity. Treatment with CB-839 resulted in a reduction by half of TNBC growth in mouse models injected with tumour cells. However, the inhibitory effect of CB-839 on the growth of the other highly proliferative BC subtypes,  i.e. luminal B and HER2+, has yet to be comprehensively confirmed.

Future Perspectives
The GLS inhibitor CB-839 has already shown promising results in several solid cancers, including TNBC, and therefore has strong therapeutic potential, particularly in those tumours showing high glutamine dependency. 63 Phase I and II clinical trials currently being conducted are summarised in Table 2. A further GLS allosteric inhibitor, UPGL00004, shows similar potency in TNBC, with additional growth inhibition in combination with the anti-vascular endothelial growth factor antibody bevacizumab. 64 Although TNBCs have a high dependence on glutamine, it certainly appears that glutaminase, particularly GLS but potentially GLS2, also plays an important role in the aggressive subclass of luminal BC. Therefore, it is essential to elucidate the role of glutaminolysis in luminal B BC growth and progression, and whether GLS offers a potential new therapeutic option for these BC patients, who have an uncertain prognosis because of relapse and/or the development of resistance to current therapies.
Indeed, a very recent finding provided some initial evidence that a luminal B-like patient xenograft was sensitive to CB-839, 65 demonstrating its potential use against BC subtypes other than TNBC. GLS inhibition in the luminal B xenograft model resulted in reduction of the downstream metabolites proline and alanine, indicating that its sensitivity is, perhaps, linked to it not being able to adapt to a hypoxic environment through activation of proline mechanisms. It certainly suggests that BC subtypes might possibly be dependent on different glutamine metabolic characteristics.

Conclusion
Glutaminase plays a key role in various tumours, including BC that show deregulated glutaminolysis because of overexpression and/or regulation of glutaminase. Both GLS isoenzymes are expressed in BC, and GLS, particularly the GAC splice variant, is primarily linked to cancer progression and overexpression. Allosteric inhibitors, such as the small molecule CB-839, offer a unique opportunity to regulate this important metabolic enzyme. Clinical trials on TNBC and haematological malignancies are underway, and look promising. However, there is still a need to understand the roles of both GLS and GLS2 in other rapidly proliferating BC subtypes, including luminal B tumours, for which inhibition of BC could be a potential therapeutic approach, in addition to endocrine therapies, which have had limited success.