Regulation of tumor metabolism by post translational modifications on metabolic enzymes

Abstract

Metabolic reprogramming is a hallmark of cancer development, progression, and metastasis. Several metabolic pathways such as glycolysis, tricarboxylic acid (TCA) cycle, lipid metabolism, and glutamine catabolism are frequently altered to support cancer growth. Importantly, the activity of the rate limiting metabolic enzymes in these pathways are specifically modulated in cancer cells. This is achieved by transcriptional, translational, and post translational regulations that enhance the expression, activity, stability, and substrate sensitivity of the rate limiting enzymes. These mechanisms allow the enzymes to retain increased activity supporting the metabolic needs of rapidly growing tumors, sustain their survival in the hostile tumor microenvironments and in the metastatic lesions. In this review, we primarily focused on the post translational modifications of the rate limiting enzymes in the glucose and glutamine metabolism, TCA cycle, and fatty acid metabolism promoting tumor progression and metastasis.

Introduction

Tumors reprogram metabolic pathways to support the demand for rapid cellular proliferation. This event of metabolic reprogramming is triggered by various stress factors in the tumor microenvironment (TME) during the process of cancer progression (1). Alterations in the tumor metabolic pathways are dependent on both intrinsic and extrinsic stimuli (2, 3). Availability of nutrients in the TME stimulates intracellular nutrient-sensing cascades that shift nutrients from catabolic pathways designed for metabolite breakdown to anabolic biosynthetic pathways(4). These cellular alterations in response to nutrient availability allow tumors to sustain high demands for energy and macromolecule synthesis required for rapid proliferation.

Regulation of altered metabolism in tumors

Reprogramming of metabolic pathways in tumors are regulated by a variety of factors. Oncogenic induction or loss of tumor suppressors can directly stimulate signaling pathways to modulate rate-limiting enzymes. These signaling cascades converge on transcriptional or translational regulators that can modulate enzyme expression, enzyme stability, and activity. Importantly, methylation and acetylation marks on histones modulate chromatin accessibility regulating expression of rate-limiting metabolic enzymes (5–8). In addition, germline mutations can directly alter the enzyme kinetics by modulating substrate or cofactor binding efficiency. Substrate availability and nutrient-sensing also play a vital role in modulating conformational changes in the enzyme structure inducing changes in metabolic activity. We will specifically focus on the post-translational modifications (PTMs) on metabolic enzymes that regulate the biochemical properties of tumors. Although PTMs such as acetylation, ubiquitination, phosphorylation, and glycosylation have been actively studied over the years, in the last decade or so, several new modifications such as succinylation, nitrosylation, palmitoylation and ADP-ribosylation have been found to play critical roles in cancer progression (9–12). These enzymes catalyzing PTMs are activated in response to oncogenic signals, which then add specific chemical groups on certain amino acid residues. Proteins are acetylated usually at lysine residues by lysine acetyltransferases (KAT), which transfer acetyl groups from acetyl CoA. Succinyl groups are added to lysine residues as well, and both acetyl and succinyl groups are removed by the deacetylases such as sirtuin 5 (SIRT5) (13). Ubiquitination is the process of adding either mono or multiple ubiquitin residues to the target proteins that in turn regulate protein stability and degradation (14). Kinases mediate the addition of phosphate groups to serine, tyrosine, or threonine residues to phosphorylate the metabolic enzymes regulating their enzymatic activities. Glycosylation of a protein occurs in the endoplasmic reticulum (ER) and Golgi apparatus, where glycosyltransferases add glycans either on asparagine nitrogen-residue (N-GlcNAc) or on serine/threonine oxygen-residue (O-GlcNAc) (15). Similarly, nitrosylation is a covalent modification of cysteine residue using a nitrogen monoxide group that affects protein-protein interactions, subcellular localization, and protein degradation (16). In addition, several lipid moieties are also added to proteins such as isoprenyl, geranylgeranyl, and palmitoyl groups that regulate membrane localization, protein function and stability (17). In this review, we will categorize the importance of PTMs functioning as a critical regulator of enzymes in the glycolytic, tricarboxylic acid cycle (TCA) or Krebs cycle, glutamine, and fatty acid metabolism promoting cancer progression.

Regulation of enzymes driving Warburg effect

Otto Warburg in the 1920s demonstrated that cultured cancer cells have an increased rate of glucose uptake and lactate secretion even in presence of oxygen, eventually being referred to as the “Warburg Effect” (18). Subsequently, Carl Cori and Gerty Cori in their 1925 landmark publications also demonstrated increased glucose uptake and lactate secretion in tumor-bearing living animals, which was analogous to Warburg’s in vitro findings (19). These publications set up the importance of glucose uptake and metabolism as a dominant biochemical property of tumors. However, it remained unexplained for many years how increased glycolysis favors cell proliferation. In the last two decades, several publications highlighted the importance of glycolytic intermediates and the rate-limiting enzymes in the pathway that can divert glucose carbons and contribute towards macromolecule synthesis (20–22). The regulation of these enzymes in the glycolytic pathway is critical to balance between energy production and macromolecule synthesis to sustain the rapid proliferation of cancer cells (Figure. 1).

Figure 1:

Post-translational modifications on glycolytic enzymes modulate glucose metabolism and regulates flow of metabolic flux towards several biosynthetic pathways. Glucose transporter 1 (GLUT1), Hexokinase (HK), Phosphofructokinase (PFK1), PFKFB4 (Phosphofructokinase 2/ Fructose-2-6-bisphosphatase 4), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Phosphoglycerate mutase-1 (PGAM), Pyruvate kinase (PKM), and Lactate dehydrogenase (LDH).

1.1. Glucose transporter 1 (GLUT1)

GLUT1 is a transmembrane protein that regulates the first step of glycolysis by facilitating uptake of glucose from the microenvironment. Glucose uptake is tightly regulated through growth factor signaling in normal cells. To hijack that regulation, cancer cells increase GLUT1 expression by transcriptional activation, and by increasing membrane localization of GLUT1 to enhance the glycolytic rate in cancer cells. Phosphorylation of serine 226 on GLUT1 enhances its membrane translocation to promote increased uptake of glucose (23, 24). S-palmitoylation of cysteine 207 on GLUT1 by Zinc Finger (Asp-His-His-Cys Domain) DHHC-Type Palmitoyltransferase 9 (ZDHHC9) adds lipid tails to GLUT1 allowing it to anchor to the cell membrane (25). Upstream signaling by insulin can induce both serine 274 phosphorylation and cysteine 233 palmitoylation to increase GLUT4 trafficking to the plasma membrane (26, 27). Interestingly, phosphorylation of serine 490 on GLUT1 by hypoxia-induced ataxia-telangiectasia mutated (ATM) kinase in cancer associated fibroblasts (CAF) enhances glucose metabolism resulting in increased lactate secretion in the TME, which promotes cancer invasiveness and metastatic progression (29, 30).

1.2. Hexokinase (HK1/2)

HK1/2 catalyzes the first step of glycolysis through irreversible conversion of glucose to glucose-6-phosphate (G6P) using ATP as a phosphate donor and prevents efflux of glucose. Apart from transcriptional upregulation, post-translational modifications enhance HK2 enzymatic activity to increase the glycolytic capacity of cancer cells. Growth factor dependent activation of tyrosine kinase c-Src phosphorylates tyrosine 732 on HK1 and tyrosine 686 on HK2 increasing enzymatic activity (31). Phosphorylation of threonine 473, serine 124, and serine 364 by various kinases stabilize HK2 protein with sustained activity (32–34). Interestingly, HK2 can directly interact with mitochondria through voltage-dependent anion channel (VDAC) and decrease cytochrome c release inhibiting apoptosis to facilitate tumor proliferation (35, 36). Various PTM modifications modulate HK2 interaction with the mitochondria. Phosphorylation at threonine 473, ubiquitination at lysine 63, and de-SUMOylation (SUMO, small ubiquitin-like modifier) at lysine 315 and 492 enhance interactions of HK2 with the mitochondria to promote glycolysis and inhibit apoptosis that in turn promotes cancer progression (33, 37–39).

1.3. Phosphofructokinase (PFK1)

PFK1 catalyzes the conversion of fructose-6-phosphate (F6P) to Fructose 1–6-bisphosphate (Fru-1-6-BP) using ATP. Glycolytic intermediates G6P and F6P are precursor molecules connecting glycolysis with pentose phosphate pathway (PPP) to synthesize ribose-5-phosphate required for nucleotide synthesis, and NADPH, which provides reducing power for anabolic reactions. Upregulation of the PPP pathway is a prerequisite for tumorigenesis. Post-translational regulation of the PFK1 enzyme plays a key role in diverting glycolytic flux towards PPP in tumors. O-GlcNAcylation of PFK1 at serine 529 under hypoxia inhibits PFK1 enzyme activity, which then diverts glucose flux towards the PPP pathway (40). Similarly, under limiting conditions of amino acid and growth factor, Unc-51 like autophagy activating kinase (ULK1/2) mediated phosphorylation of PFK1 at serine 74 and serine 762 decreases PFK1 enzyme activity to enhance PPP and NADPH production (34). To coordinate the balance between glucose catabolism and anabolic biosynthetic pathways, the enzyme activity of PFK1 is tightly regulated not only by PTM but also through ATP/AMP ratio and Fructose 2–6-bisphosphate (Fru-2-6-BP) levels. High ATP inhibits the enzymatic activity of PFK1, whereas build-up of AMP or Fru-2-6-BP increase PFK1 affinity towards F6P to enhance glucose catabolism (41–43). Fru-2-6-BP levels are regulated by bifunctional enzyme PFKFB4 (Phosphofructokinase 2/ Fructose-2-6-bisphosphatase 4). Kinase domain of PFKFB4 enzyme phosphorylates F6P using ATP to synthesize Fru-2-6-BP, whereas phosphatase domain catalyzes the hydrolysis Fru-2-6-BP into F6P. Thus, PFKFB4 with its kinase and phosphatase functional domains regulate glucose flux towards biosynthetic pathways. In breast cancer cells, PFKFB4 can also function as a protein kinase by phosphorylating steroid receptor coactivator-3 (SRC-3) at serine 857 to enhance its transcriptional activity, driving glucose flux towards PPP biosynthetic pathway (44–46). Thus, modulation of PFK1 enzymatic activity helps cancer cells to divert a large proportion of glycolytic intermediates towards biosynthetic pathways to sustain uncontrolled proliferation.

1.4. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

GAPDH catalyzes the synthesis of glycerate 1,3-bisphosphate by phosphorylation and oxidation of glyceraldehyde 3-phosphate using NAD+ as a cofactor. GAPDH expression is increased in multiple cancer types to support the Warburg effect. Upon glucose stimulation, P300/CBP-associated factor (PCAF) mediated acetylation of GAPDH at lysine 254 can further increase GAPDH enzymatic activity and promote tumor proliferation (47). Interestingly, translocation of GAPDH in the nucleus activates non-canonical functions of GAPDH that can modulate gene transcription, mRNA stabilization, autophagy, telomeric DNA protection, and DNA repair (48–50). Src kinase mediated tyrosine 41 phosphorylation on GAPDH upon DNA damage induces nuclear translocation of GAPDH, which increases base excision repair (BER) efficiency and protects cancer cells from DNA damage (48). During a low energy state, AMP-activated protein kinase (AMPK) phosphorylates cytosolic GAPDH at serine 122 causing nuclear translocation of GAPDH where it activates sirtuin 1 (SIRT1) to promote autophagy (51). In addition, the build-up of nitric oxide (NO) cause S-nitrosylation of GAPDH at cysteine 150, initiating an apoptotic cell death cascade in the brain and leading to neurodegeneration (52, 53). In the brain cells, GAPDH acetylation at lysine 160 by p300/CBP acetyltransferase can also activate the p53 mediated cell death pathway (54). Activation of macrophages induce GAPDH malonylation on lysine 213 which not only increase glycolytic flux but also enhance cytokine production to drive the pro-inflammatory function of macrophages (55). Thus, GAPDH promotes tumorigenesis by enhancing glycolytic rate as well as by other non-canonical nuclear functions.

1.5. Phosphoglycerate mutase-1 (PGAM1)

PGAM1 catalyzes the reversible conversion of 3-phosphoglycerate (3-PG) to 2-phosphoglycerate (2-PG) with 2,3-bisphosphoglycerate (2,3-BPG) as an intermediate (56). PGAM1 enzymatic activity regulates substrate (3-PG) and product (2-PG) ratio, modulating oxidative pentose phosphate and serine biosynthesis pathway in cancer. The build-up of PGAM1 product 2-PG allosterically activates phosphoglycerate dehydrogenase (PHGDH), a rate-limiting enzyme in the serine biosynthesis pathway. Inhibition of PGAM1 cause accumulation of 3-PG, which inhibits 6-phosphogluconate dehydrogenase (6PGD) in the PPP pathway and decrease tumor proliferation (56, 57). Under high glucose conditions, PGAM1 enzyme activity can be increased by lysine acetylation of 251, 253, and 254 residues, which is reversed by SIRT1 mediated deacetylation under low glucose conditions (58). PGAM1 phosphorylation at tyrosine 26 stabilizes 2,3-BPG intermediate binding and enhances histidine 11 phosphorylation at the catalytic site increasing PGAM1 enzymatic activity to promote tumor growth (59). Interestingly, decreased enzymatic activity of pyruvate kinase (PKM2) in cancer cells causes the accumulation of phosphoenolpyruvate, which is used as a phosphate donor for phosphorylation of histidine 11 on PGAM1 and enables cancer cells to generate pyruvate independent of PKM2 (60).

1.6. Pyruvate kinase (PKM)

PKM regulates the last rate-limiting step of glycolysis by transferring phosphate from phosphoenolpyruvate to ADP generating ATP and pyruvate (61). Most cancer cells have increased expression of PKM2, which favors anabolic metabolism to support cancer progression. PKM2 enzymatic function is regulated through intracellular signaling and post-translational modifications, which modulate reversible conformational change from low activity dimer into high activity tetramer (62). The binding of FBP (fructose-1-6 bisphosphate), produced by PFK1, stabilizes the tetrameric form of PKM2. However, intracellular signaling in cancer cells regulates FBP interactions with PKF2 to enhance the diversion of glycolytic intermediates towards the PPP biosynthetic pathway. Direct phosphorylation of PKM2 at tyrosine 105 by Fibroblast Growth Factor Receptor 1 (FGFR1) disrupts FBP binding, inhibiting the formation of active PKM2 tetrameric form (63). Under high glucose concentration, PKM2 is acetylated at lysine 305, which decreases PKM2 enzymatic activity and promotes lysosomal-dependent degradation (64). Similarly, PKM2 lysine 433 acetylation, serine 37 phosphorylation, and threonine 405 and serine 406 O-GlcNAcylation inhibit FBP interaction with PKM2 and enhance nuclear translocation where it acts as a nuclear protein kinase (65–67). Nuclear PKM2 phosphorylates STAT3 at tyrosine 705, histone H3 at threonine 11, BuB3 at tyrosine 207, and MLC2 at tyrosine 118 driving cell proliferation and tumorigenesis (68–71). These studies indicate negative regulation of PKM2 enables cancer cells to divert glycolytic intermediates towards biosynthetic pathways as well as directly stimulate various transcriptional factors in the nucleus.

1.7. Lactate dehydrogenase (LDH)

LDH has two subunits LDHA and LDHB which perform opposite reactions. LDHA subunit catabolizes pyruvate to lactate and regenerates NAD+ using NADH, whereas, LDHB subunit allows cancer cells to use lactate as a nutrient source by oxidizing lactate to pyruvate that is used for mitochondrial oxidation and gluconeogenesis (72). Although lactate was considered a waste product, it plays a vital role in cancer development, progression, and metastasis. Export of lactate by cancer cells acidifies the TME, initiating the inflammatory response and blocking the anti-tumor immunity that promotes tumorigenesis (72). LDHA enzymatic activity can be enhanced by multiple tyrosine kinases through its phosphorylation at tyrosine 83 and tyrosine 10 in various cancer types (73, 74). LDHA enzymatic activity is negatively regulated by lysine 5 acetylation that promotes its lysosomal degradation, which could be reversed by SIRT2 deacetylase restoring its enzymatic activity (75). LDHA succinylation at lysine 222 by carnitine palmitoyltransferase 1A (CPT1A) inhibits lysosomal degradation of LDHA and promote gastric cancer proliferation and invasiveness (76). Interestingly, the reverse activity of LDHB, catabolizing pyruvate to lactate, can be induced by serine 162 phosphorylation that removes substrate inhibition by pyruvate and promotes cancer growth (77).

2. Impact of Tricarboxylic Acid (TCA) cycle enzymatic regulation in cancer

Glycolysis derived pyruvate enters mitochondria for further catabolism through TCA cycle to generate the reducing equivalents NADH and FADH2 that are used for ATP synthesis by oxidative phosphorylation. Although Warburg described the increased rate of aerobic glycolysis is due to impaired mitochondrial oxidative metabolism, his own experiments revealed continuous oxygen usage at a relatively low level (78). Recent studies have strongly indicated the importance of respiration and mitochondrial metabolism in cancer progression (22, 79, 80). In the last decade, the use of carbon isotopes (C¹³) in tracing techniques has revealed the importance of mitochondrial metabolism in a variety of tumors, supporting bioenergetic needs and macromolecule synthesis. TCA cycle intermediates are used as a precursor molecule to synthesize fatty acids as well as non-essential amino acids such as asparagine and aspartate. TCA cycle metabolites also play critical role in gene transcription by providing substrates and cofactors required for altering chromatin accessibility thereby altering cell fate and functions. We will describe here post-translational modifications of the TCA cycle enzymes that regulate tumorigenesis (Figure. 2).

Figure 2:

Critical post-translational modifications of metabolic enzymes regulating mitochondrial TCA cycle, glutamine metabolism, and de novo fatty acid biosynthesis. Glutaminase (GLS), Glutamate dehydrogenase (GDH), Pyruvate dehydrogenase complex (PDH), Citrate synthase (CS), Aconitase 2 (ACO2), Isocitrate dehydrogenase 2 (IDH2), α ketoglutarate dehydrogenase (αKGDH), Succinate dehydrogenase (SDH), Fumarate hydratase (FH), ATP citrate lyase (ACLY), Acetyl-CoA carboxylase (ACC), Fatty acid synthase (FASN), Stearoyl-CoA desaturase-1 (SCD1), Carnitine palmitoyl transferase 1 (CPT1).

2.1. Pyruvate dehydrogenase (PDH)

PDH complex regulates the entry of glucose carbon into the TCA cycle by catalyzing the decarboxylation of pyruvate into acetyl-CoA. Phosphorylation of several serine residues on PDH E1 Alpha-subunit (PDHA1) by pyruvate dehydrogenase kinase (PDK) inactivates the PDH enzyme, whereas dephosphorylation by pyruvate dehydrogenase phosphatase (PDP) restores activity (81, 82). Oncogenic fibroblast growth factor receptor 1 (FGFR1) mediated phosphorylation at tyrosine 243 on PDK increases its kinase activity resulting in PDH inhibition (83). In cancer cells, PDP phosphorylation on tyrosine 381 recruits Acetyl-CoA Acetyltransferase 1 (ACAT1) to acetylate PDHA1 (lysine 321) and PDP (lysine 202), resulting in PDHA1 dissociation from PDP and recruitment of PDK (81). PDK increases inhibitory serine 293 phosphorylation on PDHA1, facilitating cancer cells to become more glycolytic (81). Under hypoxic conditions, hypoxia-inducible factor-α (HIF1α) inhibits mitochondrial glucose oxidation by transcriptionally activating PDK to reduce PDH activity (84). Interestingly, nuclear translocation of PDH complex generates acetyl-CoA for histone acetylation independent of mitochondria (85). Currently, dichloroacetate (DCA) is being investigated as an anti-cancer drug that blocks PDK mediated inhibition of PDH complex, leading to reactivation of mitochondrial oxidation and induction of cancer cell death (86).

2.2. Citrate synthase (CS)

CS catalyzes irreversible condensation of oxaloacetate (OAA) with acetyl-CoA to form citrate, the first committed step of the TCA cycle. CS activity is mainly regulated by the availability of substrates acetyl-CoA and oxaloacetate and inhibited by citrate and succinyl-CoA. Recently, it was reported that CS is trimethylated at Lysine 395 in the active site by mitochondrial methyltransferase (METTL12) which decreases CS enzyme activity, however, its role in cancer remains unknown (87, 88).

2.3. Aconitase 2 (ACO2)

ACO2, iron-sulfur-containing dehydratase, catalyzes the reversible stereospecific isomerization of citrate to isocitrate via cis-aconitate intermediate. ACO2 enzyme activity depends on an intact cubane [4Fe-4S]²⁺ cluster that is susceptible to ROS-induced inactivation (89). Mitochondrial iron chaperone protein frataxin modulates ACO2 activity by inhibiting oxidants and reactivation by active [4Fe-4S]²⁺ cluster formation. ACO2 can be enzymatically inhibited by fumarate-dependent succination (2-succinylcysteine, 2SC) at three cysteine residues required for iron-sulfur binding at the active site (90). A high fat diet induced acetylation on lysine 144 and 689 increases Vmax and enzyme activity of ACO2 (91). In prostate cancer, ACO2 enzymatic activity is enhanced by Lysine 258 acetylation and negatively regulated by sirtuin 3 (SIRT3) deacetylase. SIRT3 is transcriptionally repressed by androgen receptor (AR) and steroid receptor coactivator 2 (SRC-2), facilitating increased ACO2 acetylation and enzymatic activity. This stimulates reductive carboxylation of glutamine carbon through the TCA cycle to promote de novo fatty acid synthesis and drive prostate cancer metastasis (92, 93). ACO2 catalyzed cis-aconitate intermediate is used for the synthesis of itaconate, a potent immunomodulator of macrophages (94, 95). Tumor associated macrophage derived itaconate boosts ROS production and promotes tumorigenesis (96).

2.4. Isocitrate dehydrogenase 2 (IDH2)

IDH2 catalyzes NADP+ dependent reversible oxidative decarboxylation of isocitrate generating α-ketoglutarate and NADPH. Acetylation of IDH2 at lysine 413 inhibits enzyme activity by decreasing IDH2 dimer formation, which is reversibly regulated by the SIRT3 deacetylase. The combination of increased IDH2 acetylation with low SIRT3 levels was found in higher frequency in luminal B breast cancer patients compared to luminal A patients (97–99). IDH2 synthesized α-ketoglutarate is an important cofactor for various dioxygenases, such as Jumonji domain-containing histone demethylases (JHDM), ten-eleven translocation (TET1/2) DNA demethylases and prolyl hydroxylases (PHD). Cancer-associated IDH2 mutants synthesize potent oncometabolite 2-hydroxyglutarate (2-HG) that inhibits JHDM and TET1/2 leading to the hypermethylation of histone and DNA severely impacting cellular differentiation (100) 2-HG also increase the stability of HIFα by blocking PHD mediated HIFα hydroxylation and degradation, resulting in increased glycolysis and lactate secretion (22, 100).

2.5. α-ketoglutarate dehydrogenase (αKGDH)

αKGDH complex catalyzes the oxidative decarboxylation of α-ketoglutarate to succinyl-CoA using NAD as a cofactor. Functional lipoylation of αKGDH complex by αβ-hydrolase domain-containing 11 (ABHD11) and lipoyl transferase (LIPT1) is essential for αKGDH enzyme activity and the maintenance of α-ketoglutarate in the mitochondria (101, 102). HIF1α inhibits mitochondrial glutamine oxidation by inducing Siah E3 Ubiquitin Protein Ligase 2 (SIAH2) mediated ubiquitination and proteolysis of αKGDH complex, which enhances reductive carboxylation and diverts glutamine carbon towards lipid synthesis (103). Interestingly, nuclear translocation of αKGDH complex generates local succinyl-CoA required for histone succinylation that promotes transcription (104).

2.6. Succinate dehydrogenase (SDH)

SDH complex (or mitochondrial complex II) is composed of catalytic subunits (SDHA and SDHB) and mitochondrial membrane anchorage subunits (SDHC and SDHD). SDH catalyzes the oxidation of succinate to fumarate using FAD as a cofactor and regulates oxidative phosphorylation by transferring an electron from succinate through its iron-sulfur cluster to ubiquinone (105). Loss of function mutations in SDH genes increases tumorigenesis to form paragangliomas, pheochromocytomas, gastrointestinal stromal tumors, and rare renal cell carcinomas (106–109). Inactivation of SDH results in the accumulation of succinate that inhibits HIFα prolyl hydroxylases (PHDs) and keeping HIFα activated in normoxic conditions (105, 107). Succinate accumulation also inhibits α-ketoglutarate dependent dioxygenases increasing histone and DNA methylation that represses gene transcription (110–112). Additionally, SDH deficiency enhances genomic instability due to increased production of ROS (105, 107). Enzymatic activity of SDH complex is also regulated through PTM modifications. SDHA flavination by SDH5 is required for the covalent binding of FAD to SDHA (108). SDHA phosphorylation on tyrosine 604 by Src-type tyrosine kinase Fgr increases the catalytic activity of mitochondrial complex II (113). SDHA acetylation at lysine 179, 485, 498, and 538 residues reduce substrate entry into its active site decreasing enzymatic activity, which can be reversibly regulated by SIRT3 mediated deacetylation (114, 115). Succinylation of SDHA increases enzymatic functions, whereas SIRT5 mediated desuccinylation represses the effects (116, 117).

2.7. Fumarate hydratase (FH)

FH is localized in the mitochondria as well as in cytosol. In mitochondria, FH catalyzes the reversible hydration of fumarate to malate, whereas, in the cytosol, FH metabolizes fumarate generated from arginine synthesis and purine cycle (118, 119). Loss of function germline mutation of FH is associated with renal cell cancer and hereditary leiomyomatosis (120). O-GlcNAcylation of FH by O-Linked N-Acetylglucosamine (GlcNAc) Transferase (OGT) blocks FH phosphorylation at serine 75 and enhances pancreatic tumorigenesis (121). FH can get phosphorylated at threonine 236 by DNA-PK, threonine 90 by TGFβ, or serine 75 by AMPK, all resulting in nuclear translocation of FH (121–124). Nuclear FH inhibits histone H3 demethylation at H3K36 through local production of fumarate, which promotes DNA repair and cell growth arrest (111, 112, 121–124). However, in lung cancer cells, PAK4 mediated FH phosphorylation at serine 46 blocks nuclear translocation of FH and is associated with poor prognosis in lung cancer patients (123). In summary, majority of the studies indicate FH as a tumor suppressor.

3. Glutaminolysis

Glutaminolysis is the conversion of glutamine or glutamate into α-ketoglutarate, replenishing TCA flux (Figure. 2) to promote synthesis of amino acids, nucleotides, and lipids (125–128). Glutaminolysis also generates antioxidants, such as glutathione and NADPH, that maintain redox homeostasis (129). Oncogenic transcription factor MYC as well as loss of tumor suppressor retinoblastoma (Rb1) promotes glutaminolysis in tumor cells (130, 131).

3.1. Alanine-serine-cysteine transporter 2 (ASCT2 or SLC1A5)

SLC1A5 is a cell surface Na+ dependent neutral amino acid transporter primarily responsible for glutamine uptake. N-glycosylation of SLC1A5 on asparagine 163 and 212 residue is required for trafficking of SLC1A5 from the ER to plasma membrane (132). SLC1A5 ubiquitination by E3 ubiquitin ligase (RNF5) under ER stress targets SLC1A5 for degradation reducing glutamine uptake (133). SLC1A5 is transcriptionally induced by c-MYC and repressed by tumor suppressors retinoblastoma protein (Rb) and liver kinase B1 (LKB1) (128). Increased expression of SLC1A5 is required for tumor cell proliferation in various cancer types such as breast cancer, prostate cancer, lung cancer, melanoma, and acute myeloid leukemia (134). In pancreatic cancer cells, HIF-2α dependent induction of SLC1A5 variant translocates the transporter to the mitochondrial membrane to enhance mitochondrial glutamine uptake (135).

3.2. Glutaminase (GLS)

GLS enzyme catalyzes the conversion of glutamine to glutamate and ammonia. GLS1 isoform is transcriptionally activated by c-Myc, c-jun, HIF1, KRAS, and NF-κB (128). GLS1 enzyme activity is enhanced in presence of inorganic phosphate, which induces protein tetramerization and mediates substrate entry by competing with glutamate (136). GLS1 phosphorylation on serine 95 and serine 314 by protein kinase C (PKCε) increase catalytic activity (137, 138). On the other hand, GLS1 acetylation on lysine 311 inhibits enzyme tetramerization and glutaminase activity (139). GLS1 succinylation at lysine 164 induces lysine 158 ubiquitination, which targets it for degradation. However, SIRT5 mediated desuccinylation of GLS1 stabilizes protein expression facilitating cancer cell proliferation (140). GLS2 isoform expression is induced during DNA damage and oxidative stress by p53 to regulate intracellular ROS levels (141, 142). In multiple cancer types, GLS2 is repressed to promote tumor growth, which is speculated not to be associated with the glutaminolysis-related function (141–144). GLS2 enzymatic activity is also suppressed by a mitochondrial acetylase GCN5-like protein 1 (GCN5L1) by acetylation on lysine 279 in the catalytic domain (145). Thus, GLS1 promotes tumorigenesis by enhancing glutaminolysis, whereas GLS2 represses tumor growth.

3.3. Glutamate dehydrogenase (GDH)

GDH is localized in the mitochondrial matrix and catalyzes the reversible oxidative deamination of glutamate to α-ketoglutarate (146). Reductive carboxylation of α-ketoglutarate generates citrate for lipid synthesis in specific tumors (147–150). GDH catalytic activity is enhanced by ADP and leucine, whereas palmitoyl-CoA, GTP, and ATP inhibit GDH activity (127). GDH1 is ADP-ribosylated on cysteine 119 by SIRT4 reducing its catalytic activity (151–153). GDH1 is upregulated in breast cancer, lung cancer, colorectal cancer, glioblastoma, and gliomas to support glutaminolysis and maintain redox homeostasis, especially under nutrient-deprived conditions (154–158). Cytosolic GDH1 phosphorylation on serine 384 under low glucose condition activates NF-κB signaling to promote glucose uptake, which enhances tumor cell survival (159). However, in pancreatic ductal adenocarcinoma (PDAC), GDH is repressed to promote aspartate transaminase (GOT1) mediated conversion of glutamate into aspartate and maintain redox balance to facilitate PDAC cell growth (160).

4. Fatty acid metabolism

Dysregulation of lipid metabolism is a major hallmark of a variety of cancers that support cancer development, progression, and metastasis (161, 162). Cancer cells can engulf free fatty acids from the surrounding microenvironment or synthesize by de novo lipogenesis from glucose or glutamine carbon sources (161). Fatty acid contributes to various aspects of tumor biology by generating lipid species used for membrane biogenesis, molecules used for signal transduction, fatty acid oxidation for energy synthesis, and storage as lipid droplets (163, 164). Oncogenic PI3K-AKT signaling pathway and mTORC1 stimulates lipogenesis by inducing nuclear entry of Sterol regulatory element-binding transcription factor 1 (SREBP1) that transcriptionally activates lipogenic enzymes (161). Below we have listed important enzymes regulating lipogenesis in tumors (Figure. 2).

4.1. ATP citrate lyase (ACLY)

Mitochondrial citrate is exported into the cytosol for de novo synthesis of fatty acid (92, 93, 165). ATP citrate lyase (ACLY) catalyzes ATP-dependent conversion of citrate into acetyl-CoA and oxaloacetate using coenzyme A (CoA). Acetyl-CoA is the precursor molecule for fatty acid synthesis and is also required for acetylation of histones in the nucleus (166). In addition to transcriptional activation of ACLY, post-translational modifications on ACLY regulate enzyme activity in multiple cancer types (167). Akt-dependent ACLY serine 455 phosphorylation increases ACLY enzyme activity, which promotes fatty acid synthesis and global histone acetylation in cancer cells (167–171). Under high glucose conditions, ACLY acetylation at lysine 540, 546, and 554 by P300/calcium-binding protein (CBP)-associated factor (PCAF) acetyltransferase increase protein stability by inhibiting ubiquitin-mediated degradation of ACLY in lung cancer (172, 173). Upon DNA damage, ACLY is phosphorylated at serine 455 in the nucleus which then stimulates localized histone acetylation at double-strand break (DSB) sites mediating BRCA1 recruitment and homologous recombination (174). Thus, PTMs on ACLY modulates its enzymatic activity to regulate cellular acetyl-CoA levels controlling fatty acid synthesis and histone acetylation in tumors.

4.2. Acetyl-CoA carboxylase (ACC)

ACC catalyzes irreversible ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA, a rate-limiting step for de novo fatty acid synthesis (FAS). There are two isoforms of the ACC enzyme. ACC1 is the cytosolic isoform that supports de novo lipogenesis, whereas ACC2 is located on the mitochondrial membrane and generates malonyl-CoA to block carnitine palmitoyl transferase 1 (CPT1) activity to inhibit fatty acid oxidation (175, 176). ACC1 polymerization is prerequisite for optimal enzymatic activity, which is controlled by citrate levels as well as liver X receptor (LXR) dependent activation of midline-1-interacting G12-like protein (MIG12) (176–178). ACC2 hydroxylation at proline 450 by prolyl hydroxylase 3 (PHD3) under nutrient abundance conditions increases the catalytic activity of ACC2 (179). Under nutrient deprived conditions, AMPK and cAMP-dependent protein kinase (PKA) phosphorylates ACC1 on serine 80, 1201, and 1216 residues, blocking its dimerization to reduce catalytic activity (176, 180). Tumor suppressor gene BRCA1 binds to phosphorylated-ACC1 blocking dephosphorylation and subsequent activation (181–184). Germline mutations in BRCA1 that increase breast and ovarian cancer susceptibility disrupt BRCA1 interaction with ACC1 causing enhanced lipogenesis (181, 182). On the contrary, leptin and TGFβ mediated inhibitory phosphorylation of ACC1 increases breast cancer invasiveness by elevating acetyl-CoA levels and transcriptional activation of SMAD2 to support epithelial to mesenchymal transition (EMT) (185). These findings suggest that ACC1 plays various important function in cancer progression.

4.3. Fatty acid synthase (FASN)

FASN catalyzes the condensation of acetyl-CoA and malonyl-CoA in a series of biochemical reactions to synthesize saturated fatty acid, using NADPH as a cofactor. Post-translational modifications enhance FASN catalytic activity by increasing protein stability. S-nitrosylation of FASN at cysteine 1471 and 2091 increases its enzymatic activity by enhancing FASN dimerization (186). O-GlcNAcylation of FASN by O-GlcNAcase (OGA) also increases its activity and protein stability (187). In human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer, HER2 induces phosphorylation of FASN, increasing its activity favoring tumorigenesis (188). In prostate cancer, FASN activity is enhanced by ubiquitin specific protease (USP2a) in response to androgen signaling to promote tumor survival (189, 190).

4.4. Stearoyl-CoA desaturase-1 (SCD1)

SCD1 is a rate-limiting enzyme that catalyzes the conversion of saturated fatty acids into monounsaturated fatty acids (MUFA). SCD1 is transcriptionally activated by SREBP and carbohydrate response element binding protein (ChREBP) in response to growth factors and hormone signaling (191). EGFR mediated phosphorylation of SCD1 on tyrosine 55 increases protein stability and enhances MUFA synthesis (191). Apart from playing a critical role in de novo fatty acid synthesis, SCD1 also protects cancer cells from ferroptosis and cell death by generating anti-ferroptosis MUFA in gastric, ovarian, and hepatocellular carcinoma (192–195).

4.5. Carnitine palmitoyl transferase 1 (CPT1)

CPT1 is localized in the outer mitochondrial membrane and regulates transfer of fatty acids (C12-C18 chain length) into the mitochondria for fatty acid oxidation by conjugating with carnitine (196). CPT1 is allosterically inhibited by malonyl-CoA generated by ACC2, which maintains coordination between fatty acid catabolism and anabolism (196, 197). CPT1 is found to be nitrated at tyrosine 282 and 589 residues in cardiac muscle during sepsis and inflammation that decreases its enzymatic activity (198, 199). CPT1 phosphorylation on serine 741 and 747 enhances catalytic activity (200). Recently, CPT1A was found to have lysine succinyltransferase activity, modifying cytosolic proteins to increase glycolysis (76, 201). These modifications increase CPT1 activity to enhance tumorigenesis.

Conclusions

The enzymatic activity of metabolic enzymes is regulated through various types of PTM modifications that allow cancer cells to rapidly integrate extrinsic environmental signals with intrinsic cellular processes to promote cancer progression and metastasis. On one hand transcriptional activation of various metabolic enzymes increase their expression to support metabolic reprogramming, while on the other, PTM modifications fine tune the metabolic activities of the enzymes allowing them to divert the flux towards essential pathways creating metabolic flexibility and plasticity. Especially, metabolite mediated regulation of gene expression through histone and DNA modification depicts how metabolism can alter cellular functions and cell fate. PTM modifications on metabolic enzymes are also critical for the diversion of nutrients towards biosynthetic pathways to support cancer proliferation and survival. In addition, recent studies have just started to explore the importance of PTM modifications on non-canonical functions of metabolic enzymes which may alter their cellular localization to mediate unpredicted functions in cancer cells.