Post-translational modification (PTM) is an important step of signal transduction that transfers chemical groups such as phosphate, acetyl and glycosyl groups from one protein to another protein. As most of the PTMs are reversible, normal cells use PTMs as a ‘switch’ to determine the resting and proliferating state of cells that enables rapid and tight regulation of cell proliferation. In cancer cells, activation of oncogenes and/or inactivation of tumor suppressor genes provide continuous proliferative signals in part by adjusting the state of diverse PTMs of effector proteins that are involved in regulation of cell survival, cell cycle and proliferation, leading to abnormally fast proliferation of cancer cells. In addition to dysregulated proliferation, ‘altered tumor metabolism’ has recently been recognized as an emerging cancer hallmark. The most common metabolic phenotype of cancer is known as the Warburg effect or aerobic glycolysis that consists of increased glycolysis and enhanced lactate production even in the presence of oxygen. Although Otto Warburg observed aerobic glycolysis nearly 90 years ago, the detailed molecular mechanisms how increased glycolysis is regulated by oncogenic and/or tumor suppressive signaling pathways remain unclear. In this review, we summarize recent advances revealing how these signaling pathways reprogram metabolism through diverse PTMs to provide a metabolic advantage to cancer cells, thereby promoting tumor cell proliferation, tumorigenesis and tumor growth.
It was almost 90 years ago when Otto Warburg first described a common metabolic phenotype of cancers, which has been well-known nowadays as ‘the Warburg effect’ or aerobic glycolysis.1 This phenomenon describes that cancer cells prefer to uptake more glucose compared with normal cells but use most of the glucose for fermentative lactate production by glycolysis instead of oxidative phosphorylation in the mitochondria even in the presence of oxygen. The importance of aerobic glycolysis in cancers had been overlooked for a long time, but recently there has been a significant amount of emerging evidence, which demonstrates that upregulated glycolysis is correlated to activated oncogenes and/or mutated tumor suppressor genes, making aerobic glycolysis as a newly recognized hallmark of cancer.2 In addition to Warburg’s initial observations that include a high rate of glucose uptake and lactate production in cancers compared with normal cells, recent studies further emphasize the important role of diverse glycolytic intermediates as precursors for anabolic biosynthesis besides ATP production in cancer cell proliferation and tumor growth.3, 4, 5, 6, 7 Glycolytic intermediates can be metabolized for anabolic biosynthesis of riboses, amino acids such as serine and glycine and fatty acids. These molecules are important for cells to make macromolecules as ‘building blocks’ for cell proliferation, which include DNA/RNA, proteins and lipids. In particular, cancer cells require such building blocks more than normal cells do to fulfill the request of their rapid proliferation rate. Despite the fact that enhanced glucose uptake is correlated with increased biosynthetic metabolism, the detailed molecular mechanisms by which upregulated glycolysis and anabolic biosynthesis are co-ordinated by oncogenic events are unclear.
Dysregulation of protein kinase signaling is one of the most common oncogenic events in cancer cells. Among ∼500 protein kinases identified in the human genome, nearly 30 protein kinases are either overexpressed or mutated in human cancers,8, 9 leading to continuous phosphorylation of downstream effector proteins to induce cancer cell transformation and to promote cancer cell proliferation. Although phosphorylation of glycolytic enzymes including enolase, phosphoglycerate mutase and lactate dehydrogenase in response to treatment of chick embryo fibroblasts with Rous sarcoma virus was reported over 25 years ago,10, 11 the significance of such phosphorylation on enzyme function and subsequent metabolic properties has long been questioned owing to the relatively low stoichiometry of phosphorylation of these enzymes observed in other transformed cells.12 However, recent studies have shed new insights into a mechanistic link between protein kinase signaling and metabolic pathways, showing that phosphorylation of diverse metabolic enzymes is important for cancer cell metabolism, cell proliferation and tumor growth. In addition, other studies have shown that lysine acetylation, glycosylation and cysteine oxidation are also important to link cell signaling pathways to metabolic pathways in cancer cells. This review article highlights the recent advances in the understanding of how post-translational modifications (PTMs) of diverse metabolic enzymes contribute to the Warburg effect and subsequent reprogram of cell metabolism in human cancers.
Glucose uptake is the first step of glycolysis, providing carbon source for glycolysis (Figure 1). Human glucose transporter (GLUT) family consists of 14 members, which are transmembrane proteins and transfer glucose into the cells. GLUT1 is believed to be responsible for basal glucose uptake as it is the most ubiquitously expressed GLUT family member in diverse tissues. A number of studies correlate GLUT1 overexpression with the increased glycolysis in cancer cells and insulin-stimulated adipocytes.13 In the late 90s, it was reported that expression of Akt induces translocation of another GLUT family member GLUT4 to the cell membrane in adipocytes, suggesting a potential role of Akt in glucose uptake.14, 15, 16, 17, 18 Akt is a serine/threonine kinase and a major downstream effector of phosphoinositide 3-kinase in growth factor-mediated cell survival. Overexpression of Akt has been frequently observed in many types of cancers, which exhibit aerobic glycolysis.19 Indeed, Plas et al.20 and Rathmell et al.21 found that expression of a constitutively active form of Akt also promotes translocation of GLUT1 to the cell membrane in hematopoietic FL5.12 cells, leading to increased glucose uptake, intracellular ATP levels and glycolytic rates and preventing cell death from growth factor withdrawal. Furthermore, FL5.12 cells expressing constitutively active Akt are tumorigenic and highly glycolytic.22 These reports were among the first studies that suggest a potential link between Akt-dependent protein kinase signaling and upregulated glucose uptake/glycolysis in tumorigenesis. Further studies are warranted to explore whether Akt directly promotes GLUT membrane translocation and subsequent glucose uptake by phosphorylating GLUT family members or indirectly through phosphorylation of other related protein effectors.
Hexokinase 2 (HK2) is the first glycolytic enzyme that converts glucose to glucose-6-phosphate (G-6-P) with the use of ATP in the cytosol (Figure 1). Although the detailed molecular mechanisms are not clear, it was reported that the expression of constitutively active Akt in fibroblast Rat1a cells causes translocation of HK2 to mitochondria, where HK2 binds to voltage-dependent anion channels to inhibit cytochrome c release and to protect cells from apoptosis induced by growth factor withdrawal.23 Such HK2–voltage-dependent anion channel association is dynamic and inhibited by the HK2 product G-6-P in a feedback manner.19 As HK2 that is regulated by both intracellular G-6-P and ATP levels is the first of three rate-limiting glycolytic enzymes, a proposed advantage for the mitochondrial localization of HK2 could be that mitochondrial HK2 gains better access to mitochondrial ATP to catalyze its enzymatic reaction more efficiently compared with the cytosolic HK2, which is crucial for controlling glycolytic rate to fulfill the bioenergetics and biosynthetic request of proliferating cells. Recently, Akt was reported to directly phosphorylate HK2 at T473 and promote mitochondrial translocation of HK2 in cardiomyocytes,24 it is possible that Akt similarly phosphorylates HK2 in cancer cells to enhance the efficiency of HK2 catalytic reaction and subsequently promotes glycolysis.
Recent studies reveal that glycolysis is important for not only generating ATP but also for providing glycolytic intermediates as precursors for anabolic biosynthesis pathways, which are important in cancer cell metabolism and tumor growth (Figure 1). These include the oxidative pentose phosphate pathway (PPP) that uses glycolytic intermediate G-6-P to produce NADPH and nucleotides, and the serine biosynthesis pathway that uses 3-phosphoglycerate (3-PG) from glycolysis to produce serine and glycine3, 4, 5, 6, 7 (Figure 1). The products of these anabolic biosynthesis pathways are important for cancer cell growth. For example, NADPH is the most important reductive power in the reduction of glutathione to prevent cancer cells from oxidative stress, and another product of oxidative PPP, ribose-5-P, is used for de novo synthesis of nucleotides. Serine and glycine from the serine synthesis pathway can be used for de novo synthesis of proteins as well as phospholipids. Thus, cancer cells appear to co-ordinate glycolysis and anabolism to provide an overall metabolic advantage to cancer cell proliferation and tumor development. However, the detailed mechanisms remain unknown.
Recent studies reveal that diverse PTMs of glycolytic enzymes may contribute to such a metabolic co-ordination between glycolysis and anabolic biosynthesis. Deprez et al.25 reported that phosphorylation of phosphofructokinase-2 at S466 by Akt enhances phosphofructokinase-2 enzyme activity, leading to allosteric activation of the second rate-limiting glycolytic enzyme, phosphofructokinase-1 (PFK1) by phosphofructokinase-2 product, fructose 2,6-bisphosphate, suggesting an additional molecular mechanism by which Akt promotes glycolysis (Figure 1). Interestingly, PFK1 is found to be modified by O-linked β-N-acetylglucosamine (O-GlcNAcylation) at S529, which inhibits PFK activity in response to hypoxia.26 Using lung cancer H1299 cells in which endogenous PFK1 wild type is replaced with a glycosylation-deficient PFK1 S529A mutant, Yi et al.26 showed that S529 glycosylation of PFK1 redirects glucose flux toward oxidative PPP, leading to increased NADPH production and subsequently elevated glutathione concentration that prevents reactive oxygen species-mediated cell death and promotes tumor growth. Future studies are warranted to determine how hypoxia-induced inhibition of PFK1 via O-GlcNAcylation and indirect activation of PFK1 by Akt co-ordinate to modulate PFK1 activity to strike an optimized balance between glycolytic and oxidative PPP fluxes, providing an overall metabolic advantage to tumor development under hypoxia.
Recent studies on phosphoglycerate mutase 1 (PGAM1) provide another example to explain how PTMs including phosphorylation of a glycolytic enzyme contributes to co-ordination between glycolysis and anabolic biosynthesis. PGAM1 is located at the branching point of glycolysis and serine biosynthesis and converts 3-PG to 2-phophoglycerate (2-PG). PGAM1 is activated by the binding of cofactor 2,3-bisphosphoglycerate (2,3-BPG) at its catalytic site, which ‘phosphorylates’ histidine 11 (H11) of PGAM1 by transferring the phosphate group of C3 from 2,3-BPG to H11.27, 28, 29, 30 During the conversion of 3-PG to 2-PG by PGAM1, phospho-H11 of PGAM1 is positioned to facilitate the transfer of phosphate from phospho-H11 to C-2 of the substrate 3-PG. This creates a 2,3-BPG intermediate in the catalytic pocket, which in turn ‘re-phosphorylates’ H11 by transferring the C3 phosphate group back to H11 to return the enzyme to its initial H11-phosphorylated, fully activated state and allow for release of product 2-PG. Such a ‘ping-pong’ mechanism of PGAM1 activation involving catalytic H11 phosphorylation by 2,3-BPG had been known for decades, and is supported by kinetic studies27, 30 and isolation of the phospho-H11-containing peptide.31 However, the structural mechanisms underlying 2,3-BPG binding and how H11 phosphorylation activates PGAM1 had remained unknown.
PGAM1 is believed to be upregulated owing to the loss of TP53 in cancer cells because TP53 negatively regulates PGAM1 level.32, 33, 34 Hitosugi et al.4 recently showed that PGAM1 is important for glycolysis and tumor growth. Attenuation of PGAM1 in cancer cells results in decreased glycolytic rate and reduced lactate production, leading to accumulated 3-PG (PGAM1 substrate) levels but decreased 2-PG (PGAM1 product) levels in cancer cells. Interestingly, PGAM1 is also crucial to co-ordinate glycolysis and anabolic biosynthesis. 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the oxidative PPP as a competitive inhibitor, whereas 2-PG activates 3-phosphoglycerate dehydrogenase in serine biosynthesis pathways to provide feedback control of 3-PG levels. Thus, PGAM1 use 3-PG and 2-PG as signaling molecules to control anabolic biosynthesis.
In addition, Hitosugi et al.35 also reported that PGAM1 is commonly tyrosine phosphorylated at Y26 in cancer cells by diverse oncogenic tyrosine kinases (OTKs), which enhances 2,3-BPG binding and stabilizes H11-phosphorylated, active conformation of PGAM1. Structural analysis of H11-phosphorylated active form of PGAM1 suggests that Y26 phosphorylation releases the negatively charged E19 that blocks the active site of PGAM1, thus promoting cofactor 2,3-BPG binding and consequently H11 phosphorylation. Moreover, the expression of a phosphorylation-deficient PGAM1 Y26F mutant in cancer cells leads to decreased oxidative PPP flux and biosynthesis of lipids and RNA as well as reduced cell proliferation and tumor growth. These observations suggest that OTK signaling co-ordinates glycolysis and anabolic biosynthesis through phosphorylation and activation of PGAM1 (Figure 1).
Interestingly, it has been shown that PGAM1 is phosphorylated at H11 and activated by the glycolytic intermediate phosphoenolpyruvate (PEP), which is substrate of the third rate-limiting glycolytic enzyme pyruvate kinase M2 (PKM2).7 PKM2 enzyme activity is known to be suppressed, although PKM2 expression is typically high in many types of cancer; the replacement of PKM2 with another splice variant form pyruvate kinase M1 that is constitutively active in cancer cells significantly increases oxygen consumption and reduces lactate production as well as tumor growth in mice.3 Compared with cells with endogenous PKM2 replaced by catalytically more active pyruvate kinase M1, cancer cells expressing PKM2 have an accumulation of PEP. Such excess amount of PEP enables to transfer one phosphate group of PEP to the H11 residue of PGAM1 catalyzed by unknown histidine phosphotransferase(s), leading to PGAM1 activation.7 This finding suggests an alternative way to produce pyruvate in cancer cells in which pyruvate kinase activity is downregulated, whereas high glycolytic flux is maintained. Moreover, the authors showed that the HPLC fraction of cell lysates that contains H11-phosphorylated PGAM1 but no pyruvate kinase activity produces 50% of pyruvate from PEP compared with the total cell lysates. This suggests that 50% of the total pyruvate pool from PEP could be provided through an unidentified alternative pathway other than pyruvate kinase in cells.
In addition to phosphorylation, Hallows et al.36 reported that PGAM1 is also lysine acetylated and activated in HEK293 cells treated with deacetylase inhibitors tricostain A and nicotinamide, which are commonly used deacetylase inhibitors. Glucose deprivation results in deacetylation of PGAM1 that is likely mediated by deacetylase Sirt1. Overexpression of an acetylation-mimetic PGAM1 triple mutant (K251/253/254Q) in HEK293 cells results in enhanced lactate production, suggesting that Sirt1-dependent deacetylation of PGAM1 may regulate aerobic glycolysis. However, it is not clear whether lysine acetylation of PGAM1 provides a metabolic advantage to cancer cell proliferation and/or tumor growth. Moreover, although protein expression and Y26 phosphorylation levels of PGAM1 have been found to be upregulated in human primary leukemia cells and increased PGAM1 protein levels were detected in primary tissue samples of tumors from head and neck cancer patients compared with control tissue samples,4, 35 it is not clear whether lysine acetylation of PGAM1 is actually upregulated in cancer/leukemia cells and in tumor tissues and contributes to disease development.
Furthermore, these studies also suggest that PGAM1 could be a therapeutic target for anticancer treatment due to its crucial role in cancer metabolism. Indeed, Evans et al.37, 38 identified a compound MJE3 from a natural product-inspired small-molecule library using cell-based screening, which inhibits PGAM1 activity in intact cells and results in decreased breast cancer cell proliferation. Hitosugi et al.4 also identified alizarin and its derivatives including PGAM inhibitor (PGMI)-004 as novel PGAM1 inhibitors, which are efficacious in the treatment of tumor xenograft mice in vivo as well as in diverse cancer cells including primary leukemia cells from human patients in vitro with minimal toxicity to and no obvious off-target effect. These studies provide ‘proof-of-principle’ that suggests PGAM1 as a promising anticancer target.
PK catalyzes the production of pyruvate and ATP from PEP and ADP. Pyruvate is one of the most important metabolites produced by glycolysis. Pyruvate can either be further metabolized to lactate that is the final product of aerobic glycolysis or transported into the mitochondria and converted to acetyl-CoA, which participates in the tricarboxylic acid cycle and oxidative phosphorylation (Figure 1). Thus, the fate of pyruvate to be metabolized in glycolysis or mitochondria may determine whether cells rely on more glycolysis or oxidative phosphorylation, respectively. With this said, tight regulation of PK activity is important to cancer cell metabolism and proliferation. There are four mammalian PK isoenzymes including M1, M2, L and R that exist in different cell types. Pyruvate kinase M1 is a constitutively active form of PK that is expressed in normal adult cells. In contrast, PKM2 as an alternative slice variant is predominantly expressed in the fetus and also in tumor cells, where the abundance of other isoforms of PK is low. PKM2 can exist either in active tetramers or inactive dimers but, in tumor cells, it predominantly occurs in dimers with low activity. Recent seminal studies by Chritofk et al.3, 39 identified PKM2 as a phosphotyrosine-binding protein using a SILAC (stable isotope labeling by/with amino acids in cell culture)-based proteomic approach. Binding of a phosphotyrosine peptide inhibits PKM2 enzyme activity by dissociating cofactor fructose bisphosphate from the catalytic site of PKM2. They also found that the expression of PKM2 but not pyruvate kinase M1 is important for aerobic glycolysis and tumor growth. In consonance with these findings, Hitosugi et al.40 reported that PKM2 is inhibited via direct phosphorylation at Y105 by diverse OTKs including fibroblast growth factor receptor 1 (FGFR1) through the disruption of tetramer formation of active PKM2 by releasing fructose bisphosphate. This study provides evidence suggesting that one tyrosine phosphorylated PKM2 sister molecule itself could function as an inhibitory binding partner to other PKM2 sister molecules in an active PKM2 homotetramer. In addition, Y105 phosphorylation of PKM2 is common in diverse cancer cells. Cancer cells with stable knockdown of endogenous PKM2 and rescue expression of a catalytically active and phospho-deficient PKM2 Y105F mutant demonstrate increased mitochondrial respiration and decreased lactate production as well as decreased cell proliferation under hypoxia with reduced tumor growth in xenograft nude mice, suggesting that Y105 phosphorylation of PKM2 may contribute to a metabolic switch to aerobic glycolysis from oxidative phosphorylation to promote tumor growth. Given that PKM2 is commonly tyrosine phosphorylated with low activity in human cancer cells, an interesting question is how attenuated PK activity contributes to the Warburg effect in cancer cell with increased lactate production, which is one-step downstream from PKM2 in glycolysis.7, 41 One explanation could be that glutamate-pyruvate transaminase-mediated glutamine oxidation, a reaction that consumes pyruvate, is decreased in cancer cells.41 Alternatively, the recent study from Vander Heiden et al.7 suggests a novel model in which accumulated PEP due to low activity of PKM2 in cancer cells activates PGAM1 via H11 phosphorylation, which converts PEP to lactate but does not generate ATP. Further studies are warranted to clearly elucidate the molecular mechanisms underlying such a puzzling metabolic phenomenon in cancer cells.
In addition to its role in glycolysis, PKM2 was also reported to translocate to the nucleus and regulate gene transcription.42, 43, 44, 45, 46, 47, 48 Yang et al.47 found that EGFR-activated ERK binds to and phosphorylates PKM2 at S37 in human glioblastoma multiforme cells. S37 phosphorylation recruits the peptidyl-proline isomerase protein interacting with never in mitosis A 1 (PIN1) and subsequent importin alpha5 to PKM2, leading to nuclear translocation of PKM2 to regulate Myc and glycolytic gene expression. Replacement of endogenous PKM2 in glioblastoma multiforme cells with a phospho-deficient PKM2 S37A mutant that fails to translocate into the nucleus results in attenuated aerobic glycolysis and tumor growth in mice. Nuclear PKM2 has been suggested to diverse functions, including as a Ser/Thr kinase to phosphorylate histone H3 at T11 to mediate EGF-induced transactivation of β-catenin to induce c-Myc and cyclin D1 expression,46, 48 a co-activator for hypoxia inducible factor-1α (HIF-1α) to increase glycolytic gene expression, and a tyrosine kinase to phosphorylate and activate Stat3 to promote Stat3 target gene expression42, 44 (Figure 1). It remains unclear how PKM2 carries out these differential functions in regulation of gene expression in cancer cells, and how these nuclear functions of PKM2 are regulated and co-ordinated during reprogramming of cancer metabolism and tumor development.
In addition to phosphorylation, Lv et al.49 reported that PKM2 is also acetylated at K305 upon the treatment of cells with high concentration glucose. Acetylation of K305 inhibits PKM2 enzyme activity and promotes its binding with the lysosomal chaperon protein HSC70, leading to protein degradation of PKM2 via chaperon-mediated autophagy. They also identified p300/CBP-associate factor (PCAF) as an upstream acetyltransferase that catalyzes the acetylation of PKM2 at K305. Expression of an acetyl-mimetic PKM2 K305Q mutant in tumor cells results in accumulation of glycolytic intermediates upstream of PKM2, which promotes biosynthesis and consequently cancer cell proliferation as well as tumor growth in mice. These observations are consistent with previous findings that the downregulation of PKM2 provides a proliferative advantage to cancer cells and tumors.3, 39, 40
Moreover, Anastasiou et al.50 recently reported that PKM2 is oxidized at C358 by intracellular reactive oxygen species, which results in decreased PKM2 enzyme activity. Oxidized PKM2 with low activity similarly causes accumulation of glycolytic intermediates, which diverts G-6-P into oxidative PPP (Figure 1), leading to increased NADPH production that clears reactive oxygen species to protect cancer cells from oxidative stress.50 Replacement of endogenous PKM2 with a catalytically active and oxidation-resistant PKM2 C358S mutant in lung cancer A549 cells results in decreased intracellular reduced glutathione concentration owing to reduced oxidative PPP flux, leading to enhanced sensitivity to oxidative stress and reduced tumor formation in xenograft nude mice. These findings demonstrate that cysteine oxidation of PKM2 renders cancer cells resistance to oxidative stress, providing an additional example to support the concept that diverse PTMs regulate PKM2 to regulate cancer metabolism and tumor growth.
Furthermore, these studies suggest that PKM2 may serve as an interesting therapeutic target in cancer treatment, such that either inhibition or activation of PKM2 may affect cancer cell metabolism and cause tumor regression. Indeed, Thallion Pharmaceuticals (Alexander-Fleming, Montreal, Canada) developed a seven amino-acid peptide-based PKM2 inhibitor TLN-232/CAP-232 and started a Phase II clinical trial for metastatic renal cell carcinoma. Moreover, recent reports show that treatment of cancer cells with synthetic PKM2 activators results in decreased cancer cell proliferation and tumor growth in mice.51, 52 Thus, it is of clinical interest to develop PKM2 activators that abrogate the inhibitory PTMs of PKM2 as promising anticancer agents.
Lactate dehydrogenase-A (LDH-A) converts pyruvate to lactate, which is the last step of glycolysis that permits the regeneration of NAD+. NAD+ is needed as an electron acceptor to maintain cytosolic glucose catabolism. Therefore, most tumor cells are reliant on lactate production for their survival. Fan et al.53 found that phosphorylation of LDH-A at Y10 is common in diverse human cancer cells by multiple OTKs such as FGFR1, breakpoint cluster region–Abelson (BCR-ABL), Janus-activated kinase 2 (JAK2) and FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD), which activates LDH-A by promoting the formation of active tetramers. Expression of a catalytic hypomorph LDH-A Y10F mutant in cancer cells leads to decreased lactate production and NADH/NAD+ ratio with enhanced mitochondrial respiration, but does not affect a metabolic rate from glucose to PEP in normoxia. Once Y10F cells undergo hypoxia, or are treated with complex I inhibitor rotenone, these cells exhibit increased NADH/NAD+ ratio with decreased glycolytic rate (glucose to PEP), ATP levels and cell proliferation compared with cells expressing wild type LDH-A, which suggests that Y10 phosphorylation of LDH is important for regulation of NADH/NAD+ redox homeostasis, which, however, cannot be compensated by mitochondrial complex I, to sustain metabolic reactions from glucose to PEP.
In addition, Zhao, et al.54 recently showed that K5 acetylation inhibits LDH-A. Treatment of HEK293 cells with deacetylase inhibitors results in elevated K5 acetylation of LDH-A that promotes LDH-A binding to the chaperon protein HSP70, leading to degradation of LDH-A via chaperon-mediated autophagy. They identified Sirt2 as the upstream deacetylase that removes K5 acetylation of LDH-A. Notably, both PKM2 and LDH-A bind to HSP70 upon lysine acetylation and consequently are degraded through chaperon-mediated autophagy, suggesting a potential common mechanism by which protein levels of metabolic enzymes might be regulated. Replacement of endogenous LDH-A in pancreatic cancer Bx-PC3 cells with an acetylation-mimetic K5Q mutant results in decreased cell migration, proliferation and tumor growth in mice. Most importantly, they found that K5 acetylation status of LDH-A reversely correlates with the protein expression level of Sirt2 in human tissue samples of tumors from pancreatic cancer patients. This finding supports their model in which Sirt2 negatively regulates LDH-A activity via deacetylation at K5, which provides a growth disadvantage to pancreatic cancer. Sirt2 has recently been recognized as a tumor suppressor, as Sirt2-deficient female and male mice develop mammary tumors and hepatocellular carcinoma, respectively, and human breast cancers and hepatocellular carcinoma samples exhibit decreased Sirt2 protein expression levels compared with normal tissue.55 It would be interesting to study whether Sirt2-dependent deacetylation of LDH-A also contributes to the development of human breast cancer and hepatocellular carcinoma in addition to the well-known effect of Sirt2 on histone acetylation.
Pyruvate dehydrogenase kinase
As pyruvate is metabolized not only to lactate by LDH-A but also to acetyl-CoA that is further metabolized in tricarboxylic acid cycle in the mitochondria, one interesting question is whether pyruvate entry into the mitochondria is also regulated by PTMs (Figure 1). Mitochondrial serine/threonine protein kinase pyruvate dehydrogenase kinase 1 (PDHK1) has been shown to contribute to the upregulation of glycolysis in cancers. PDHK1 negatively regulates pyruvate dehydrogenase A1 (PDHA1) by phosphorylating PDHA1 at multiple serine sites, leading to the inactivation of pyruvate dehydrogenase complex (PDC). PDC is a huge mitochondrial complex considered as a gatekeeper of pyruvate entry into the mitochondria, consisting of more than 60 subunits including PDHA1 (E1), dihydrolipoyl acetyltransferase (E2), dihydrolipoyl dehydrogenase (E3), E3-binding protein (E3-bp), PDHK and pyruvate dehydrogenase phosphatase.56 Among these functional proteins, PDHA1 catalyzes the first irreversible and rate-limiting step in the conversion of pyruvate to acetyl-CoA, whereas PDHK and pyruvate dehydrogenase phosphatase inhibits and activates PDC via serine phosphorylation and dephosphorylation, respectively.57
In cancer cells, majority of pyruvate is converted into lactate instead of acetyl-CoA regardless of presence of oxygen. This may be in part due to upregulation of PDHK activity and/or inhibition of PDH in cancer cells. PDHK1 is believed to be upregulated by Myc and HIF-1 to achieve functional inhibition of the mitochondria by phosphorylating and inactivating PDH in cancer cells. Hitosugi, et al.58 recently showed that PDHK1 is commonly tyrosine phosphorylated at multiple sites in diverse cancer cells. Tyrosine phosphorylation activates PDHK1 by promoting ATP and PDC binding, leading to elevated inhibitory phosphorylation of PDHA1 and subsequent inactivation of PDC. Such functional attenuation of the mitochondria in cancer cells ensures the metabolic switch to aerobic glycolysis from oxidative phosphorylation. Interestingly, although the molecular mechanism remains unclear, a fraction of several OTKs including full-length and fusion FGFR1, BCR-ABL, JAK2 V617F and FLT3-ITD were found to localize in different mitochondrial compartments, where they phosphorylate and activate PDHK1 in different cancer cells. It would be interesting to study how these OTKs regulate mitochondrial metabolism via tyrosine phosphorylation within the different mitochondrial compartments, and how the functional PDC activity outside of matrix contributes to pyruvate decarboxylation and consequently mitochondrial function and metabolism. Of note, targeting PDHK1 by dichloroacetate, an orally available small-molecule inhibitor of PDHK, shifts cancer metabolism to oxidative phosphorylation from glycolysis in cancer cells and inhibits tumor growth in mice as well as in glioblastoma multiforme patients, which suggests PDHK1 as a promising therapeutic target for anticancer treatment.59, 60
Accumulating evidence suggests that PTMs of diverse metabolic enzymes contribute to the Warburg effect and subsequently reprogram of cancer metabolism, which represents acute molecular mechanisms underlying cancer metabolism in addition to chronic mechanisms that are believed to be regulated by activation of HIF and Myc or inactivation of TP53, all of which result in increased gene expression of multiple metabolic enzymes related to glycolysis.34, 61, 62, 63, 64, 65, 66, 67 As summarized in Table 1, different PTMs modulate properties of metabolic enzymes including (1) cofactor binding, substrate binding and oligomerization of metabolic enzymes that lead to upregulation or downregulation of their catalytic activities; (2) degradation of metabolic enzymes; and (3) subcellular localization of metabolic enzymes. One interesting question is how oncogenic signals determine the state and levels of different PTMs of distinct enzyme in cancer cells. For example, a similar set of OTKs including FGFR1, BCR-ABL and FLT3-ITD are able to phosphorylate diverse metabolic enzymes including PGAM1, PKM2, LDH-A and PDHK1 in cancer cells, so how tyrosine phosphorylation of these enzymes co-ordinate and/or crosstalk to each other in order to provide an ultimately optimized metabolic advantage to cancer cell proliferation and tumor growth? In fact, Y105 phosphorylation of PKM2 was suggested to be important in regulation of PDHK1 gene expression.58 This suggests a dual role for OTKs such as FGFR1 in regulation of PDHK1 in cancer metabolism, which consists of a long-term mechanism by which FGFR1 may phosphorylate PKM2 to promote PDHK1 gene expression and a short-term mechanism by which FGFR1 activates PDHK1 through tyrosine phosphorylation at multiple sites. Y105 phosphorylation of PKM2, which is involved in FGFR1-regulated PDHK1 expression, appears to be an upstream event that precedes FGFR1-dependent phosphorylation and activation of PDHK1 in cancer cell metabolism. Future studies are also warranted to explore the interaction and crosstalk between other tyrosine phosphorylated metabolic enzymes in cancer cells, which may be informative to provide the understanding of a signaling network regulated by OTKs that contributes to reprogram of cancer metabolism.
In addition, many metabolic enzymes such as PKM2 have been suggested to be regulated by diverse PTMs. It would be interesting to explore how differential PTMs occurring within the same metabolic enzyme interact and/or co-ordinate with each other to determine the final functional property of this enzyme that provides a metabolic advantage to cancer cell proliferation and tumor growth. Moreover, it is also interesting to study whether two different kinds of PTMs can crosstalk to control metabolic enzymes, and how such interplay of PTMs is regulated in human cancers. For example, many acetyltransferases and deacetylases are found to be commonly phosphorylated in cancer cells (http://www.phosphosite.org/homeAction.do). This may suggest that upregulated protein kinase signaling in cancer cells may contribute to reprogramming of cancer metabolism by regulating acetylation state of metabolic enzymes. In addition, further studies are warranted to investigate whether PTM-dependent regulation of metabolic enzymes is affected by extracellular environments. For example, it is well known that glucose deprivation affects glycosylation and/or acetylation, leading to the alteration of metabolic enzyme activities, which may suggest a potential mechanism by which cancer cells use PTMs to control activities of metabolic enzymes in response to extracellular stimuli and nutrient availability and subsequently adjust their metabolism in an acute way, thereby providing a proliferative advantage to tumors.
Of last note, there are very few studies reporting PTMs other than the aforementioned ones to regulate metabolic enzymes, especially in cancer cells. Nevertheless, it was reported that nitric oxide generation causes S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to nuclear translocation and apoptosis in macrophages and neurons.68 Another study by Døskeland et al.69 showed that phenylalanine hydroxylase is a substrate for unknown ubiquitin ligase. In summary, although these studies imply potential involvement of PTMs other than popular phosphorylation and acetylation in regulation of cancer metabolism, it is still difficult to carry out large scale proteomic studies in cancers on PTMs such as S-nitrosylation, cysteine oxidation and glycosylation.70,71 Future studies are warranted to improve proteomic methodologies, which will allow us to obtain comprehensive understanding of the role of diverse PTM in regulation of the Warburg effect and cancer metabolism.
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We apologize to authors whose contributions were not directly cited owing to space limitations. This study is supported in part by NIH grants CA140515 (JC) and DoD grant W81XWH-12–1–0217 (JC). JC is a Georgia Cancer Coalition Distinguished Cancer Scholar and a Scholar of the Leukemia and Lymphoma Society.
The authors declare no conflict of interest.
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Hitosugi, T., Chen, J. Post-translational modifications and the Warburg effect. Oncogene 33, 4279–4285 (2014). https://doi.org/10.1038/onc.2013.406
- the Warburg effect
- post-translational modifications
- cancer metabolism
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