Oncogene (2014) 33, 4279–4285 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

REVIEW Post-translational modifications and the Warburg effect

T Hitosugi1 and J Chen2

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.

Oncogene (2014) 33, 4279–4285; doi:10.1038/onc.2013.406; published online 7 October 2013 Keywords: the Warburg effect; post-translational modifications; cancer metabolism

INTRODUCTION kinases identified in the human genome, nearly 30 protein kinases 8,9 It was almost 90 years ago when Otto Warburg first described are either overexpressed or mutated in human cancers, leading a common metabolic phenotype of cancers, which has been well- to continuous phosphorylation of downstream effector proteins to known nowadays as ‘the Warburg effect’ or aerobic glycolysis.1 induce cancer cell transformation and to promote cancer cell This phenomenon describes that cancer cells prefer to uptake proliferation. Although phosphorylation of glycolytic enzymes more glucose compared with normal cells but use most of the including enolase, phosphoglycerate mutase and lactate dehydro- glucose for fermentative lactate production by glycolysis instead genase in response to treatment of chick embryo fibroblasts 10,11 of oxidative phosphorylation in the mitochondria even in the with Rous sarcoma virus was reported over 25 years ago, the presence of oxygen. The importance of aerobic glycolysis in significance of such phosphorylation on enzyme function and cancers had been overlooked for a long time, but recently there subsequent metabolic properties has long been questioned owing has been a significant amount of emerging evidence, which to the relatively low stoichiometry of phosphorylation of these 12 demonstrates that upregulated glycolysis is correlated to activated enzymes observed in other transformed cells. However, recent oncogenes and/or mutated tumor suppressor genes, making studies have shed new insights into a mechanistic link between aerobic glycolysis as a newly recognized hallmark of cancer.2 In protein kinase signaling and metabolic pathways, showing that addition to Warburg’s initial observations that include a high rate phosphorylation of diverse metabolic enzymes is important for of glucose uptake and lactate production in cancers compared cancer cell metabolism, cell proliferation and tumor growth. In with normal cells, recent studies further emphasize the important addition, other studies have shown that lysine acetylation, role of diverse glycolytic intermediates as precursors for anabolic glycosylation and cysteine oxidation are also important to link biosynthesis besides ATP production in cancer cell proliferation cell signaling pathways to metabolic pathways in cancer cells. and tumor growth.3–7 Glycolytic intermediates can be metabolized This review article highlights the recent advances in the under- for anabolic biosynthesis of riboses, amino acids such as serine standing of how post-translational modifications (PTMs) of diverse and glycine and fatty acids. These molecules are important for metabolic enzymes contribute to the Warburg effect and cells to make macromolecules as ‘building blocks’ for cell subsequent reprogram of cell metabolism in human cancers. 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 GLUCOSE TRANSPORTER rate. Despite the fact that enhanced glucose uptake is correlated Glucose uptake is the first step of glycolysis, providing carbon with increased biosynthetic metabolism, the detailed molecular source for glycolysis (Figure 1). Human glucose transporter (GLUT) mechanisms by which upregulated glycolysis and anabolic family consists of 14 members, which are transmembrane proteins biosynthesis are co-ordinated by oncogenic events are unclear. and transfer glucose into the cells. GLUT1 is believed to be Dysregulation of protein kinase signaling is one of the most responsible for basal glucose uptake as it is the most ubiquitously common oncogenic events in cancer cells. Among B500 protein expressed GLUT family member in diverse tissues. A number

1Department of Oncology, Division of Oncology Research, Mayo Clinic, Rochester, MN, USA and 2Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory, Emory University School of Medicine, Atlanta, GA, USA. Correspondence: Dr T Hitosugi or Dr J Chen, Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory, Emory University School of Medicine, 1365-C Clifton Road NE, Room C-3002, Atlanta, GA 30322, USA. E-mail: [email protected] or [email protected] Received 3 July 2013; revised 13 August 2013; accepted 26 August 2013; published online 7 October 2013 Post-translational modifications and the Warburg effect T Hitosugi and J Chen 4280

Figure 1. PTM-dependent regulation of enzymes related to the Warburg effect. GLUT1, glucose transporter 1; HK2, hexokinase 2; LDH-A, lactate dehydrogenase-A; PDHA1, pyruvate dehydrogenase A1; PDHK1, pyruvate dehydrogenase kinase 1; PFK1, phosphofructokinase-1; PFK2, phosphofructokinase-2; PGAM1, phosphoglycerate mutase 1; PHGDH, 3-phosphoglycerate dehydrogenase; and PKM2, pyruvate kinase M2.

of studies correlate GLUT1 overexpression with the increased cytosol (Figure 1). Although the detailed molecular mechanisms glycolysis in cancer cells and insulin-stimulated adipocytes.13 In the are not clear, it was reported that the expression of constitutively late 90s, it was reported that expression of Akt induces active Akt in fibroblast Rat1a cells causes translocation of HK2 to translocation of another GLUT family member GLUT4 to the cell mitochondria, where HK2 binds to voltage-dependent anion membrane in adipocytes, suggesting a potential role of Akt in channels to inhibit cytochrome c release and to protect cells glucose uptake.14–18 Akt is a serine/threonine kinase and a major from apoptosis induced by growth factor withdrawal.23 Such HK2– downstream effector of phosphoinositide 3-kinase in growth voltage-dependent anion channel association is dynamic and factor-mediated cell survival. Overexpression of Akt has been inhibited by the HK2 product G-6-P in a feedback manner.19 As frequently observed in many types of cancers, which exhibit HK2 that is regulated by both intracellular G-6-P and ATP levels is aerobic glycolysis.19 Indeed, Plas et al.20 and Rathmell et al.21 found the first of three rate-limiting glycolytic enzymes, a proposed that expression of a constitutively active form of Akt also promotes advantage for the mitochondrial localization of HK2 could be that translocation of GLUT1 to the cell membrane in hematopoietic mitochondrial HK2 gains better access to mitochondrial ATP to FL5.12 cells, leading to increased glucose uptake, intracellular catalyze its enzymatic reaction more efficiently compared with the ATP levels and glycolytic rates and preventing cell death from cytosolic HK2, which is crucial for controlling glycolytic rate to growth factor withdrawal. Furthermore, FL5.12 cells expressing fulfill the bioenergetics and biosynthetic request of proliferating constitutively active Akt are tumorigenic and highly glycolytic.22 cells. Recently, Akt was reported to directly phosphorylate HK2 These reports were among the first studies that suggest a potential at T473 and promote mitochondrial translocation of HK2 link between Akt-dependent protein kinase signaling and in cardiomyocytes,24 it is possible that Akt similarly phospho- upregulated glucose uptake/glycolysis in tumorigenesis. Further rylates HK2 in cancer cells to enhance the efficiency of HK2 studies are warranted to explore whether Akt directly promotes catalytic reaction and subsequently promotes glycolysis. GLUT membrane translocation and subsequent glucose uptake by phosphorylating GLUT family members or indirectly through phosphorylation of other related protein effectors. PHOSPHOFRUCTOKINASE Recent studies reveal that glycolysis is important for not only generating ATP but also for providing glycolytic intermediates as HEXOKINASE precursors for anabolic biosynthesis pathways, which are impor- Hexokinase 2 (HK2) is the first glycolytic enzyme that converts tant in cancer cell metabolism and tumor growth (Figure 1). These glucose to glucose-6-phosphate (G-6-P) with the use of ATP in the include the oxidative pentose phosphate pathway (PPP) that uses

Oncogene (2014) 4279 – 4285 & 2014 Macmillan Publishers Limited Post-translational modifications and the Warburg effect T Hitosugi and J Chen 4281 glycolytic intermediate G-6-P to produce NADPH and nucleotides, reduced lactate production, leading to accumulated 3-PG (PGAM1 and the serine biosynthesis pathway that uses 3-phosphoglyce- substrate) levels but decreased 2-PG (PGAM1 product) levels in rate (3-PG) from glycolysis to produce serine and glycine3–7 cancer cells. Interestingly, PGAM1 is also crucial to co-ordinate (Figure 1). The products of these anabolic biosynthesis pathways glycolysis and anabolic biosynthesis. 3-PG binds to and inhibits are important for cancer cell growth. For example, NADPH is the 6-phosphogluconate dehydrogenase in the oxidative PPP as a most important reductive power in the reduction of glutathione to competitive inhibitor, whereas 2-PG activates 3-phosphoglycerate prevent cancer cells from oxidative stress, and another product dehydrogenase in serine biosynthesis pathways to provide of oxidative PPP, ribose-5-P, is used for de novo synthesis of feedback control of 3-PG levels. Thus, PGAM1 use 3-PG and nucleotides. Serine and glycine from the serine synthesis pathway 2-PG as signaling molecules to control anabolic biosynthesis. can be used for de novo synthesis of proteins as well as phos- In addition, Hitosugi et al.35 also reported that PGAM1 is pholipids. Thus, cancer cells appear to co-ordinate glycolysis and commonly tyrosine phosphorylated at Y26 in cancer cells anabolism to provide an overall metabolic advantage to cancer by diverse oncogenic tyrosine kinases (OTKs), which enhances cell proliferation and tumor development. However, the detailed 2,3-BPG binding and stabilizes H11-phosphorylated, active mechanisms remain unknown. conformation of PGAM1. Structural analysis of H11-phospho- Recent studies reveal that diverse PTMs of glycolytic enzymes rylated active form of PGAM1 suggests that Y26 phosphorylation may contribute to such a metabolic co-ordination between releases the negatively charged E19 that blocks the active site glycolysis and anabolic biosynthesis. Deprez et al.25 reported of PGAM1, thus promoting cofactor 2,3-BPG binding and that phosphorylation of phosphofructokinase-2 at S466 by Akt consequently H11 phosphorylation. Moreover, the expression of enhances phosphofructokinase-2 enzyme activity, leading to a phosphorylation-deficient PGAM1 Y26F mutant in cancer cells allosteric activation of the second rate-limiting glycolytic leads to decreased oxidative PPP flux and biosynthesis of lipids enzyme, phosphofructokinase-1 (PFK1) by phosphofructokinase- and RNA as well as reduced cell proliferation and tumor growth. 2 product, fructose 2,6-bisphosphate, suggesting an additional These observations suggest that OTK signaling co-ordinates molecular mechanism by which Akt promotes glycolysis (Figure 1). glycolysis and anabolic biosynthesis through phosphorylation Interestingly, PFK1 is found to be modified by O-linked b-N- and activation of PGAM1 (Figure 1). acetylglucosamine (O-GlcNAcylation) at S529, which inhibits Interestingly, it has been shown that PGAM1 is phosphorylated PFK activity in response to hypoxia.26 Using lung cancer H1299 at H11 and activated by the glycolytic intermediate phosphoe- cells in which endogenous PFK1 wild type is replaced with a nolpyruvate (PEP), which is substrate of the third rate-limiting glycosylation-deficient PFK1 S529A mutant, Yi et al.26 showed that glycolytic enzyme pyruvate kinase M2 (PKM2).7 PKM2 enzyme S529 glycosylation of PFK1 redirects glucose flux toward oxidative activity is known to be suppressed, although PKM2 expression is PPP, leading to increased NADPH production and subsequently typically high in many types of cancer; the replacement of PKM2 elevated glutathione concentration that prevents reactive oxygen with another splice variant form pyruvate kinase M1 that is species-mediated cell death and promotes tumor growth. constitutively active in cancer cells significantly increases oxygen Future studies are warranted to determine how hypoxia-induced consumption and reduces lactate production as well as tumor inhibition of PFK1 via O-GlcNAcylation and indirect activation growth in mice.3 Compared with cells with endogenous PKM2 of PFK1 by Akt co-ordinate to modulate PFK1 activity replaced by catalytically more active pyruvate kinase M1, cancer to strike an optimized balance between glycolytic and oxidative cells expressing PKM2 have an accumulation of PEP. Such excess PPP fluxes, providing an overall metabolic advantage to tumor amount of PEP enables to transfer one phosphate group of PEP to development under hypoxia. 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 PHOSPHOGLYCERATE MUTASE which pyruvate kinase activity is downregulated, whereas high Recent studies on phosphoglycerate mutase 1 (PGAM1) provide glycolytic flux is maintained. Moreover, the authors showed that another example to explain how PTMs including phosphorylation the HPLC fraction of cell lysates that contains H11-phosphorylated of a glycolytic enzyme contributes to co-ordination between PGAM1 but no pyruvate kinase activity produces 50% of pyruvate glycolysis and anabolic biosynthesis. PGAM1 is located at the from PEP compared with the total cell lysates. This suggests that branching point of glycolysis and serine biosynthesis and converts 50% of the total pyruvate pool from PEP could be provided 3-PG to 2-phophoglycerate (2-PG). PGAM1 is activated by the through an unidentified alternative pathway other than pyruvate binding of cofactor 2,3-bisphosphoglycerate (2,3-BPG) at its kinase in cells. catalytic site, which ‘phosphorylates’ histidine 11 (H11) of PGAM1 In addition to phosphorylation, Hallows et al.36 reported that by transferring the phosphate group of C3 from 2,3-BPG to PGAM1 is also lysine acetylated and activated in HEK293 cells H11.27–30 During the conversion of 3-PG to 2-PG by PGAM1, treated with deacetylase inhibitors tricostain A and nicotinamide, phospho-H11 of PGAM1 is positioned to facilitate the transfer of which are commonly used deacetylase inhibitors. Glucose phosphate from phospho-H11 to C-2 of the substrate 3-PG. This deprivation results in deacetylation of PGAM1 that is likely creates a 2,3-BPG intermediate in the catalytic pocket, which in mediated by deacetylase Sirt1. Overexpression of an acetylation- turn ‘re-phosphorylates’ H11 by transferring the C3 phosphate mimetic PGAM1 triple mutant (K251/253/254Q) in HEK293 cells group back to H11 to return the enzyme to its initial H11- results in enhanced lactate production, suggesting that Sirt1- phosphorylated, fully activated state and allow for release of dependent deacetylation of PGAM1 may regulate aerobic product 2-PG. Such a ‘ping-pong’ mechanism of PGAM1 activation glycolysis. However, it is not clear whether lysine acetylation of involving catalytic H11 phosphorylation by 2,3-BPG had been PGAM1 provides a metabolic advantage to cancer cell known for decades, and is supported by kinetic studies27,30 and proliferation and/or tumor growth. Moreover, although protein isolation of the phospho-H11-containing peptide.31 However, the expression and Y26 phosphorylation levels of PGAM1 have been structural mechanisms underlying 2,3-BPG binding and how H11 found to be upregulated in human primary leukemia cells phosphorylation activates PGAM1 had remained unknown. and increased PGAM1 protein levels were detected in primary PGAM1 is believed to be upregulated owing to the loss of tissue samples of tumors from head and neck cancer patients TP53 in cancer cells because TP53 negatively regulates PGAM1 compared with control tissue samples,4,35 it is not clear whether level.32–34 Hitosugi et al.4 recently showed that PGAM1 is lysine acetylation of PGAM1 is actually upregulated in cancer/ important for glycolysis and tumor growth. Attenuation of leukemia cells and in tumor tissues and contributes to disease PGAM1 in cancer cells results in decreased glycolytic rate and development.

& 2014 Macmillan Publishers Limited Oncogene (2014) 4279 – 4285 Post-translational modifications and the Warburg effect T Hitosugi and J Chen 4282 Furthermore, these studies also suggest that PGAM1 could be a model in which accumulated PEP due to low activity of PKM2 in therapeutic target for anticancer treatment due to its crucial role cancer cells activates PGAM1 via H11 phosphorylation, which in cancer metabolism. Indeed, Evans et al.37,38 identified a converts PEP to lactate but does not generate ATP. Further studies compound MJE3 from a natural product-inspired small-molecule are warranted to clearly elucidate the molecular mechanisms library using cell-based screening, which inhibits PGAM1 activity in underlying such a puzzling metabolic phenomenon in cancer cells. intact cells and results in decreased breast cancer cell pro- In addition to its role in glycolysis, PKM2 was also reported to liferation. Hitosugi et al.4 also identified alizarin and its derivatives translocate to the nucleus and regulate gene transcription.42–48 including PGAM inhibitor (PGMI)-004 as novel PGAM1 inhibitors, Yang et al.47 found that EGFR-activated ERK binds to and which are efficacious in the treatment of tumor xenograft mice phosphorylates PKM2 at S37 in human glioblastoma multiforme in vivo as well as in diverse cancer cells including primary leukemia cells. S37 phosphorylation recruits the peptidyl-proline isomerase cells from human patients in vitro with minimal toxicity to and no protein interacting with never in mitosis A 1 (PIN1) and sub- obvious off-target effect. These studies provide ‘proof-of-principle’ sequent importin alpha5 to PKM2, leading to nuclear translocation that suggests PGAM1 as a promising anticancer target. 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 PYRUVATE KINASE translocate into the nucleus results in attenuated aerobic PK catalyzes the production of pyruvate and ATP from PEP and glycolysis and tumor growth in mice. Nuclear PKM2 has been ADP. Pyruvate is one of the most important metabolites produced suggested to diverse functions, including as a Ser/Thr kinase by glycolysis. Pyruvate can either be further metabolized to lactate to phosphorylate histone H3 at T11 to mediate EGF-induced that is the final product of aerobic glycolysis or transported into transactivation of b-catenin to induce c-Myc and cyclin D1 the mitochondria and converted to acetyl-CoA, which participates expression,46,48 a co-activator for hypoxia inducible factor-1a in the tricarboxylic acid cycle and oxidative phosphorylation (HIF-1a) to increase glycolytic gene expression, and a tyrosine (Figure 1). Thus, the fate of pyruvate to be metabolized in kinase to phosphorylate and activate Stat3 to promote Stat3 glycolysis or mitochondria may determine whether cells rely on target gene expression42,44 (Figure 1). It remains unclear how more glycolysis or oxidative phosphorylation, respectively. With PKM2 carries out these differential functions in regulation of gene this said, tight regulation of PK activity is important to cancer cell expression in cancer cells, and how these nuclear functions of metabolism and proliferation. There are four mammalian PK PKM2 are regulated and co-ordinated during reprogramming of isoenzymes including M1, M2, L and R that exist in different cell cancer metabolism and tumor development. types. Pyruvate kinase M1 is a constitutively active form of PK that In addition to phosphorylation, Lv et al.49 reported that PKM2 is is expressed in normal adult cells. In contrast, PKM2 as an also acetylated at K305 upon the treatment of cells with high alternative slice variant is predominantly expressed in the fetus concentration glucose. Acetylation of K305 inhibits PKM2 enzyme and also in tumor cells, where the abundance of other isoforms of activity and promotes its binding with the lysosomal chaperon PK is low. PKM2 can exist either in active tetramers or inactive protein HSC70, leading to protein degradation of PKM2 via dimers but, in tumor cells, it predominantly occurs in dimers with chaperon-mediated autophagy. They also identified p300/CBP- low activity. Recent seminal studies by Chritofk et al.3,39 identified associate factor (PCAF) as an upstream acetyltransferase that PKM2 as a phosphotyrosine-binding protein using a SILAC (stable catalyzes the acetylation of PKM2 at K305. Expression of an acetyl- isotope labeling by/with amino acids in cell culture)-based mimetic PKM2 K305Q mutant in tumor cells results in accumulation proteomic approach. Binding of a phosphotyrosine peptide of glycolytic intermediates upstream of PKM2, which promotes inhibits PKM2 enzyme activity by dissociating cofactor fructose biosynthesis and consequently cancer cell proliferation as well as bisphosphate from the catalytic site of PKM2. They also found that tumor growth in mice. These observations are consistent with the expression of PKM2 but not pyruvate kinase M1 is important previous findings that the downregulation of PKM2 provides a for aerobic glycolysis and tumor growth. In consonance with these proliferative advantage to cancer cells and tumors.3,39,40 findings, Hitosugi et al.40 reported that PKM2 is inhibited via direct Moreover, Anastasiou et al.50 recently reported that PKM2 is phosphorylation at Y105 by diverse OTKs including fibroblast oxidized at C358 by intracellular reactive oxygen species, which growth factor receptor 1 (FGFR1) through the disruption of results in decreased PKM2 enzyme activity. Oxidized PKM2 tetramer formation of active PKM2 by releasing fructose with low activity similarly causes accumulation of glycolytic bisphosphate. This study provides evidence suggesting that one intermediates, which diverts G-6-P into oxidative PPP (Figure 1), tyrosine phosphorylated PKM2 sister molecule itself could leading to increased NADPH production that clears reactive function as an inhibitory binding partner to other PKM2 sister oxygen species to protect cancer cells from oxidative stress.50 molecules in an active PKM2 homotetramer. In addition, Y105 Replacement of endogenous PKM2 with a catalytically active phosphorylation of PKM2 is common in diverse cancer cells. and oxidation-resistant PKM2 C358S mutant in lung cancer A549 Cancer cells with stable knockdown of endogenous PKM2 and cells results in decreased intracellular reduced glutathione con- rescue expression of a catalytically active and phospho-deficient centration owing to reduced oxidative PPP flux, leading to PKM2 Y105F mutant demonstrate increased mitochondrial enhanced sensitivity to oxidative stress and reduced tumor respiration and decreased lactate production as well as formation in xenograft nude mice. These findings demonstrate decreased cell proliferation under hypoxia with reduced tumor that cysteine oxidation of PKM2 renders cancer cells resistance to growth in xenograft nude mice, suggesting that Y105 oxidative stress, providing an additional example to support the phosphorylation of PKM2 may contribute to a metabolic switch concept that diverse PTMs regulate PKM2 to regulate cancer to aerobic glycolysis from oxidative phosphorylation to promote metabolism and tumor growth. tumor growth. Given that PKM2 is commonly tyrosine Furthermore, these studies suggest that PKM2 may serve as an phosphorylated with low activity in human cancer cells, an interesting therapeutic target in cancer treatment, such that either interesting question is how attenuated PK activity contributes to inhibition or activation of PKM2 may affect cancer cell metabolism the Warburg effect in cancer cell with increased lactate and cause tumor regression. Indeed, Thallion Pharmaceuticals production, which is one-step downstream from PKM2 in (Alexander-Fleming, Montreal, Canada) developed a seven amino- glycolysis.7,41 One explanation could be that glutamate-pyruvate acid peptide-based PKM2 inhibitor TLN-232/CAP-232 and started a transaminase-mediated glutamine oxidation, a reaction that Phase II clinical trial for metastatic renal cell carcinoma. Moreover, consumes pyruvate, is decreased in cancer cells.41 Alternatively, recent reports show that treatment of cancer cells with synthetic the recent study from Vander Heiden et al.7 suggests a novel PKM2 activators results in decreased cancer cell proliferation and

Oncogene (2014) 4279 – 4285 & 2014 Macmillan Publishers Limited Post-translational modifications and the Warburg effect T Hitosugi and J Chen 4283 tumor growth in mice.51,52 Thus, it is of clinical interest to develop at multiple serine sites, leading to the inactivation of pyruvate PKM2 activators that abrogate the inhibitory PTMs of PKM2 as dehydrogenase complex (PDC). PDC is a huge mitochondrial promising anticancer agents. complex considered as a gatekeeper of pyruvate entry into the mitochondria, consisting of more than 60 subunits including PDHA1 (E1), dihydrolipoyl acetyltransferase (E2), dihydrolipoyl LACTATE DEHYDROGENASE dehydrogenase (E3), E3-binding protein (E3-bp), PDHK and Lactate dehydrogenase-A (LDH-A) converts pyruvate to lactate, pyruvate dehydrogenase phosphatase.56 Among these functional which is the last step of glycolysis that permits the regeneration of proteins, PDHA1 catalyzes the first irreversible and rate-limiting NAD þ . NAD þ is needed as an electron acceptor to maintain step in the conversion of pyruvate to acetyl-CoA, whereas cytosolic glucose catabolism. Therefore, most tumor cells are PDHK and pyruvate dehydrogenase phosphatase inhibits and reliant on lactate production for their survival. Fan et al.53 found activates PDC via serine phosphorylation and dephosphorylation, that phosphorylation of LDH-A at Y10 is common in diverse respectively.57 human cancer cells by multiple OTKs such as FGFR1, breakpoint In cancer cells, majority of pyruvate is converted into lactate cluster region–Abelson (BCR-ABL), Janus-activated kinase 2 (JAK2) instead of acetyl-CoA regardless of presence of oxygen. This may and FMS-like tyrosine kinase 3-internal tandem duplication be in part due to upregulation of PDHK activity and/or inhibition (FLT3-ITD), which activates LDH-A by promoting the formation of PDH in cancer cells. PDHK1 is believed to be upregulated by of active tetramers. Expression of a catalytic hypomorph LDH-A Myc and HIF-1 to achieve functional inhibition of the mitochondria Y10F mutant in cancer cells leads to decreased lactate production by phosphorylating and inactivating PDH in cancer cells. Hitosugi, and NADH/NAD þ ratio with enhanced mitochondrial respiration, et al.58 recently showed that PDHK1 is commonly tyrosine but does not affect a metabolic rate from glucose to PEP in phosphorylated at multiple sites in diverse cancer cells. Tyrosine normoxia. Once Y10F cells undergo hypoxia, or are treated with phosphorylation activates PDHK1 by promoting ATP and PDC complex I inhibitor rotenone, these cells exhibit increased NADH/ binding, leading to elevated inhibitory phosphorylation of PDHA1 NAD þ ratio with decreased glycolytic rate (glucose to PEP), ATP and subsequent inactivation of PDC. Such functional attenuation levels and cell proliferation compared with cells expressing wild of the mitochondria in cancer cells ensures the metabolic switch type LDH-A, which suggests that Y10 phosphorylation of LDH is to aerobic glycolysis from oxidative phosphorylation. Interestingly, important for regulation of NADH/NAD þ redox homeostasis, although the molecular mechanism remains unclear, a fraction of which, however, cannot be compensated by mitochondrial several OTKs including full-length and fusion FGFR1, BCR-ABL, complex I, to sustain metabolic reactions from glucose to PEP. JAK2 V617F and FLT3-ITD were found to localize in different In addition, Zhao, et al.54 recently showed that K5 acetylation mitochondrial compartments, where they phosphorylate and inhibits LDH-A. Treatment of HEK293 cells with deacetylase activate PDHK1 in different cancer cells. It would be interesting inhibitors results in elevated K5 acetylation of LDH-A that to study how these OTKs regulate mitochondrial metabolism via promotes LDH-A binding to the chaperon protein HSP70, tyrosine phosphorylation within the different mitochondrial leading to degradation of LDH-A via chaperon-mediated compartments, and how the functional PDC activity outside of autophagy. They identified Sirt2 as the upstream deacetylase matrix contributes to pyruvate decarboxylation and consequently that removes K5 acetylation of LDH-A. Notably, both PKM2 and mitochondrial function and metabolism. Of note, targeting PDHK1 LDH-A bind to HSP70 upon lysine acetylation and consequently by dichloroacetate, an orally available small-molecule inhibitor of are degraded through chaperon-mediated autophagy, suggesting PDHK, shifts cancer metabolism to oxidative phosphorylation from a potential common mechanism by which protein levels of glycolysis in cancer cells and inhibits tumor growth in mice as well metabolic enzymes might be regulated. Replacement of as in glioblastoma multiforme patients, which suggests PDHK1 as endogenous LDH-A in pancreatic cancer Bx-PC3 cells with an a promising therapeutic target for anticancer treatment.59,60 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 CONCLUSIONS reversely correlates with the protein expression level of Sirt2 in Accumulating evidence suggests that PTMs of diverse metabolic human tissue samples of tumors from pancreatic cancer patients. enzymes contribute to the Warburg effect and subsequently This finding supports their model in which Sirt2 negatively reprogram of cancer metabolism, which represents acute mole- regulates LDH-A activity via deacetylation at K5, which provides a cular mechanisms underlying cancer metabolism in addition to growth disadvantage to pancreatic cancer. Sirt2 has recently been chronic mechanisms that are believed to be regulated by recognized as a tumor suppressor, as Sirt2-deficient female and activation of HIF and Myc or inactivation of TP53, all of which male mice develop mammary tumors and hepatocellular result in increased gene expression of multiple metabolic enzymes carcinoma, respectively, and human breast cancers and related to glycolysis.34,61–67 As summarized in Table 1, different hepatocellular carcinoma samples exhibit decreased Sirt2 PTMs modulate properties of metabolic enzymes including (1) protein expression levels compared with normal tissue.55 It cofactor binding, substrate binding and oligomerization of would be interesting to study whether Sirt2-dependent metabolic enzymes that lead to upregulation or downregulation deacetylation of LDH-A also contributes to the development of of their catalytic activities; (2) degradation of metabolic enzymes; human breast cancer and hepatocellular carcinoma in addition to and (3) subcellular localization of metabolic enzymes. One the well-known effect of Sirt2 on histone acetylation. 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 PYRUVATE DEHYDROGENASE KINASE and FLT3-ITD are able to phosphorylate diverse metabolic As pyruvate is metabolized not only to lactate by LDH-A but also enzymes including PGAM1, PKM2, LDH-A and PDHK1 in cancer to acetyl-CoA that is further metabolized in tricarboxylic acid cycle cells, so how tyrosine phosphorylation of these enzymes in the mitochondria, one interesting question is whether pyruvate co-ordinate and/or crosstalk to each other in order to provide entry into the mitochondria is also regulated by PTMs (Figure 1). an ultimately optimized metabolic advantage to cancer cell Mitochondrial serine/threonine protein kinase pyruvate dehydro- proliferation and tumor growth? In fact, Y105 phosphorylation of genase kinase 1 (PDHK1) has been shown to contribute to the PKM2 was suggested to be important in regulation of PDHK1 gene upregulation of glycolysis in cancers. PDHK1 negatively regulates expression.58 This suggests a dual role for OTKs such as FGFR1 in pyruvate dehydrogenase A1 (PDHA1) by phosphorylating PDHA1 regulation of PDHK1 in cancer metabolism, which consists of a

& 2014 Macmillan Publishers Limited Oncogene (2014) 4279 – 4285 Post-translational modifications and the Warburg effect T Hitosugi and J Chen 4284 Table 1. Functional effects of PTMs on metabolic enzymes

PTMs Tyrosine Serine Histidine Lysine Glycosylation and phosphorylation phosphorylation phosphorylation acetylation oxidation

Functional effects Upregulation of enzyme PDHK158 LDH-A53 PFK225 PGAM17,35 PGAM136 activity PGAM135 Downregulation of enzyme PKM239,40 PDHA156,57 PKM250 PFK126 activity Degradation of enzyme LDH-A54 PKM249 Localization of enzyme PKM247 GLUT120,21 HK223 Abbreviations: GLUT1, glucose transporter 1; HK2, hexokinase 2; LDH-A, lactate dehydrogenase-A; PTMs, post-translational modifications; PDHA1, pyruvate dehydrogenase A1; PDHK1, pyruvate dehydrogenase kinase 1; PFK1 and 2, phosphofructokinase-1 and 2; PGAM1, phosphoglycerate mutase 1; PHGDH, 3-phosphoglycerate dehydrogenase; PKM2, pyruvate kinase M2.

long-term mechanism by which FGFR1 may phosphorylate PKM2 improve proteomic methodologies, which will allow us to obtain to promote PDHK1 gene expression and a short-term mechanism comprehensive understanding of the role of diverse PTM in by which FGFR1 activates PDHK1 through tyrosine phospho- regulation of the Warburg effect and cancer metabolism. rylation 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 CONFLICT OF INTEREST and activation of PDHK1 in cancer cell metabolism. Future The authors declare no conflict of interest. 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 ACKNOWLEDGEMENTS the understanding of a signaling network regulated by OTKs that We apologize to authors whose contributions were not directly cited owing to space contributes to reprogram of cancer metabolism. limitations. This study is supported in part by NIH grants CA140515 (JC) and DoD In addition, many metabolic enzymes such as PKM2 have been grant W81XWH-12–1–0217 (JC). JC is a Georgia Cancer Coalition Distinguished suggested to be regulated by diverse PTMs. It would be interesting Cancer Scholar and a Scholar of the Leukemia and Lymphoma Society. to explore how differential PTMs occurring within the same metabolic enzyme interact and/or co-ordinate with each other to REFERENCES determine the final functional property of this enzyme that provides a metabolic advantage to cancer cell proliferation and 1 Warburg O. On the origin of cancer cells. Science 1956; 123: 309–314. 2 Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; tumor growth. Moreover, it is also interesting to study whether two 144: 646–674. different kinds of PTMs can crosstalk to control metabolic enzymes, 3 Christofk HR, Vander Heiden MG, Harris MH, Ramanathan A, Gerszten RE, Wei R and how such interplay of PTMs is regulated in human cancers. For et al. 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