FEBS Letters xxx (2014) xxx–xxx

journal homepage: www.FEBSLetters.org

Review

Pyruvate kinase M2 and cancer: an updated assessment Mohd Askandar Iqbal a,1, Vibhor Gupta a,2,3, Prakasam Gopinath a,3, Sybille Mazurek b, Rameshwar N.K. Bamezai a,⇑ a b

National Centre of Applied Human Genetics, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India Institute of Veterinary Physiology and Biochemistry, University of Giessen, Frankfurter Strasse 100, 35392 Giessen, Germany

a r t i c l e

i n f o

Article history: Received 5 March 2014 Revised 6 April 2014 Accepted 7 April 2014 Available online xxxx Edited by Shairaz Baksh, Giovanni Blandino and Wilhelm Just Keywords: PKM2 Cancer metabolism Warburg effect

a b s t r a c t Cancer cells are characterized by high glycolytic rates to support energy regeneration and anabolic metabolism, along with the expression of pyruvate kinase isoenzyme M2 (PKM2). The latter catalyzes the last step of glycolysis and reprograms the glycolytic flux to feed the special metabolic demands of proliferating cells. Besides, PKM2 has moonlight functions, such as gene transcription, favoring cancer. Accumulating evidence suggests a critical role played by the low-activity-dimeric PKM2 in tumor progression, supported by the identification of mutations which result in the down-regulation of its activity and tumorigenesis in a nude mouse model. This review discusses PKM2 regulation and the benefits it confers to cancer cells. Further, conflicting views on PKM2’s role in cancer, its therapeutic relevance and future directions in the field are also discussed. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Cancer metabolism and PKM2 The metabolism of cancer cells differs significantly from normal cells. As observed by Otto Warburg in 1924, cancer cells take up high amounts of glucose and utilize it differently from normal cells by producing lactate even in the presence of oxygen [1,2]. This phenomenon is termed ‘‘Warburg effect’’ or ‘‘aerobic glycolysis’’. At his time, Otto Warburg postulated a defect in mitochondrial respiration as a cause for the increased aerobic glycolysis in tumor cells. However, as will be discussed later in this review, aerobic glycolysis is now evaluated as a sophistication of cancer cells [3]. Glycolytic energy regeneration is independent of oxygen supply and allows tumor cells to grow in areas with varying oxygen concentrations. In addition, cancer cells use large amounts of glucose carbons for anabolic processes [4]. The high rates of glucose conversion have been exploited in the clinical detection of tumors by 18fluorodeoxyglucose–positron emission tomography (fDG–PET) and hyperpolarized 13C magnetic resonance imaging

⇑ Corresponding author E-mail address: [email protected] (R.N.K. Bamezai). Present address: Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 2 Present address: Abramson Family Cancer Research Institute, Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA 19104, USA 3 These two authors contributed equally. 1

(MRI) [5–9]. Production of lactate, during aerobic glycolysis, has the consequence of reduced pyruvate channelled into oxidative phosphorylation [10]. The lactate produced is secreted out of the cells to keep aerobic glycolysis active. Studies suggest a role of secreted lactate in immune escape, migration, metastasis, tumor vascularization and radioresistance [11–13]. Acute dependency of tumors on aerobic glycolysis has kept cancer biologists wondering about its advantage to tumor cells. Gatenby et al. in their review ‘‘Why do cancer cells have high aerobic glycolysis?’’ propose aerobic glycolysis as an adaptation to hypoxic microenvironment in pre-malignant lesions [14]. Later reviews discuss the evolutionary conserved preference for nonoxidative metabolism for production of biomass and reason that aerobic glycolysis is vital in maintaining macromolecular synthesis [10,15]. M2 isoform of pyruvate kinase (PKM2) is a key regulator of the metabolic fate of the glycolytic intermediates and has the unique ability to shift glucose metabolism in favor of cancer cells [10,16,17]. In accordance, cancerous state correlates with high PKM2 expression in a variety of tumor tissues and cell lines [18]. Besides a crucial metabolic role, a large body of data have unravelled the role of PKM2 in gene transcription and its ability to act as protein kinase [19–23]. Due to the soaring number of papers dealing with the role of PKM2 in cancer, PKM2 has gained enormous attention and, here, we discuss PKM2 biology, in the light of new developments, to comprehend its therapeutic relevance.

http://dx.doi.org/10.1016/j.febslet.2014.04.011 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Please cite this article in press as: Iqbal, M.A., et al. Pyruvate kinase M2 and cancer: an updated assessment. FEBS Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.011

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2. The isoforms of pyruvate kinase The mammalian pyruvate kinase (PK) family is comprised of two genes that encode four isoforms (L, R, M1 and M2). L and R isoforms, present in liver and erythrocytes, respectively [24], are encoded by PKLR gene using different promoters. M1 and M2 isoforms are alternative splicing products of the PKM gene. M1 is mainly found in adult skeletal muscle and brain, while M2 is the dominant isoform in proliferating cells [25]. The distribution of the pyruvate kinase isoforms is tissue specific depending on the metabolic necessities of the tissues. Constitutively active M1 isoform is expressed in muscle and brain tissues that need a large supply of ATP. The isoform L is expressed in tissues with gluconeogenesis, such as liver, kidney and intestine. Cells and tissues with a high rate of nucleogenesis are characterized by the expression of the isoform M2. The existence of different PK isoforms reflects the importance of the last step of glycolysis to cope with the differential metabolic requirements of cells. The presence of PKM2 in embryonic tissues is a first sign of its importance for proliferating cells. PKM2 is the prototypic isoform of pyruvate kinase and is gradually replaced by other tissue specific isoforms during differentiation [17]. The replacement of PKM2 by other isoforms during tissue differentiation is indicative of a mechanism that ensures the strict regulation of PKM2 expression during development. Several studies have reported an isoform shift back to PKM2 during tumor formation. L to M2 shift is reported to be a late event in hepatocarcinogenesis [26]. Christofk et al. demonstrated that the switch from M1 to M2 is required for aerobic glycolysis [27]. They further described that tumor growth diminished when PKM2 was replaced by PKM1, thereby concluding that the switch to PKM2 is necessary for cancer metabolism and tumor growth. However, these results do not correlate with the presence of PKM2 in some differentiated tissues, i.e. lung, adipose, pancreatic islets, retina, distal renal tubules, Henle´s loops and renal collecting tubules, blood vessel walls, bile duct epithelium, and sinusoidal lining cells in the liver [28,29]. Using the cancer genome atlas, RNA-Seq, and exon array datasets, Desai et al. showed elevated PKM2 expression in sixteen different tumor types compared to their normal counterparts [30]. They observed an isoform switch from PKM1 to PKM2 (at mRNA level) only in glioblastomas and not in other cancer types. However, in this study, PK isoform expression among the different cell types within the particular tissues, i.e. the tubules system in the kidney or the different cell types in the liver [29,31] have not been taken into account. 3. PKM gene and its expression Understanding the regulation of PKM2 expression is of paramount importance for it is how PKM2 integrates into the cellular scenario. The PKM locus encodes for the mutually exclusive M1 and M2 isoforms of pyruvate kinase [25]. These isoforms result from alternative splicing of the PKM pre mRNA into the M1 (with exon 9) and M2 (with exon 10) isoforms. Both isoforms differ only in 22 out of 531 amino acids and the exon that is exchanged encodes for 56 amino acids [25]. This region forms the intersubunit contact domain (ISCD) of PKM2, which is involved in the association of dimers into tetramers [32]. Since the metabolic implications of M1 and M2 are different [17], PKM gene splicing is tightly regulated to ensure appropriate isoform expression. Oncogenic c-Myc in cancer cells has been shown to dictate splicing machinery for inclusion of exon 10 to ensure high PKM2/PKM1 ratio and thus regulate PKM splicing in favor of PKM2 [33]. Recently, it is reported that growth factor signaling via PI3K/Akt/mTOR upregulates the expression of PKM2 through HIF1a/c-Myc axis to promote Warburg effect and tumor

growth [34]. Glucose is al‘so reported to favor PKM2 expression in rat hepatoma cell line and isolated adipocytes [35,36]. Furthermore, hormones like insulin, triiodothyronine (T3), and glucocorticoids also regulate PKM gene expression in order to attain the required metabolism in the cell [37–39]. We have shown that insulin up-regulates PKM2 expression but decrease its activity to promote cancer metabolism [40]. Since insulin signaling has been implicated in cancer [41], it could therefore be conjectured that insulin, at least in part, contributes to cancer metabolism by regulating PKM2 state [40]. Hypoxia, a common feature of most tumors, induces upregulation of PKM gene expression via stabilization of transcription factor HIF1a [42]. Besides hypoxic induction, HIF1a accumulation is also induced by glycolytic metabolites in aerobic conditions, eventually resulting in induction of HIF1 target genes [43]. Hypoxic induction of PKM2 may underlie the resistance of hypoxic tumor cells to chemo and radiotherapy [44,45]. Epigenetic alterations by hypomethylation of intron 1 in the PKM gene are also reported to correlate with elevated PKM2 expression in tumors [30]. The complex regulation of the PKM gene ensures the expression of PKM2 upon requirement, especially during neonatal stages. Cancer cells appear to exploit this very regulation of PKM2 in their favor through oncogenic and dysregulated growth factor signaling. 4. Bifunctional role of PKM2 in glycolysis Glycolysis (glucose lysis) is a central metabolic pathway in both prokaryotes and eukaryotes. It feeds energy regeneration and anabolism. In differentiated tissues, such as muscle, pyruvate, the end product of glycolysis, is channelled into the Krebs cycle followed by oxidative phosphorylation during respiration [46]. Further, in several cell types, the intermediates of glycolysis like glucose-6-phosphate, fructose-6-phosphate, glyceraldehyde3-phosphate, dihydroxyacetone-P, glycerate 3-P are required for biosynthetic pathways of nucleic acid, phospholipid, amino acid and sphingolipids synthesis. [47]. Pyruvate kinase M2 catalyzes the last step of glycolysis; the conversion of phosphoenolpyruvate (PEP) to pyruvate with concomitant production of ATP [48]. The ATP generation by PKM2, unlike mitochondrial respiration, is independent of oxygen and thus allows tumor cells to grow in hypoxic conditions. Also, the position of PKM2 in the glycolytic sequence is instrumental in regulating the metabolic fate of the glycolytic intermediates. PKM2 activity, if high, results in rapid conversion of PEP to pyruvate with regeneration of energy [49]. In contrast, decreased PKM2 activity leads to an expansion of all glycolytic intermediates above the pyruvate kinase reaction, which are then available as precursors for synthetic processes that offshoot from glycolysis. The ability of PKM2 to redirect glucose flux in glycolysis is controlled by its allosteric activator fructose 1,6-bisphosphate (FBP) [50]. Binding of FBP to PKM2 increases its affinity towards its substrate PEP [51]. Thus, the position of PKM2 within the glycolytic sequence makes it a ‘‘glycolytic valve’’ in deciding the direction of glucose flux in glycolysis. 5. Inactive and active oligomeric forms of PKM2: activity regulation PKM2 exists in two oligomeric forms- tetrameric and dimeric. The tetrameric form of PKM2 has a high affinity to PEP and is highly active whereas the dimeric form is characterized by a low PEP affinity and is nearly inactive, under physiological conditions [17] On requirement, the tetramer dissociates into less active dimers or the dimers associate to the tetrameric form. An

Please cite this article in press as: Iqbal, M.A., et al. Pyruvate kinase M2 and cancer: an updated assessment. FEBS Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.011

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important metabolic regulator of the tetramer: dimer ratio is FBP. At high levels, FBP induce the association of two dimers into a highly active tetrameric PKM2. The ratio between the tetrameric and dimeric form of PKM2 decides the overall activity of PKM2 in cellular milieu [48] and thus the glycolytic flux towards catabolism or anabolism. The tetramer: dimer ratio is also regulated by post-translational modifications as well as interactions with oncoproteins which favor the inactive dimeric form of the enzyme [17]. Post-translational modifications in the A-domain of PKM2 play a key role in subunit dissociation thereby resulting in reduced activity (Fig. 1A). The tyrosine-105-phosphorylation of PKM2 by oncogenic tyrosine kinases inhibits its activity by causing the release of its allosteric activator FBP (Fig. 1B) [52]. Xenograft tumor model studies have shown that tyrosine-105-phosphorylationinduced decline in PKM2 activity promoted tumor growth [52]. Lv et al. described that acetylation of PKM2 at lysine-305 is responsible for a downregulation of its activity (Fig. 1B). Interestingly,

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high glucose concentrations induced K305 acetylation to funnel the glucose into anabolic synthesis [53]. Furthermore, the authors demonstrated that the acetylation-induced decrease in PKM2 activity promotes glycolytic pooling, NADPH synthesis, and eventually tumor growth [53]. Study by Anastasiou et al. revealed a remarkable role of oxidized PKM2 in reducing oxidative stress in cancer cells. In this paper, the authors report that intracellular reactive oxygen species (ROS)-induced oxidation of cysteine-358 decreased its activity to divert glucose flux into anabolic pentose phosphate pathway, thereby generating sufficient reducing potential for detoxification of ROS (Fig. 1B) [54]. Moreover, the authors show that oxidized-PKM2, through its lower activity, promotes tumor growth, which was inhibited when mice were fed on N-acetyl-L-cysteine (NAC; a potent scavenger of reactive oxygen species, ROS). This report extended the relevance of PKM2 to cancer and also emphasized the important role of this enzyme in cancer. Besides post-translational modifications, numerous viral

Fig. 1. Post-translational modifications in PKM2 and the metabolic implications. (A) Schematic organization of the various domains of the glycolytic enzyme pyruvate kinase M2; The interface of the A and B domain shows the presence of an active site (ACS); the C domain contains a nuclear localization signal sequence (NLS) and the binding site for the allosteric activator FBP. (B) Scheme of how different factors dictate various post-translational modifications in PKM2, followed by the downstream consequences on tumor metabolism. Briefly, the phosphorylation of tyrosine 105 carried out by tyrosine kinases-FGFR1, BCR-ABL, JAK-2 and FLT-3; acetylation of lysine-305 residue due to high glucose concentration; and oxidation of cysteine-358 residue by intracellular ROS, facilitates the generation of enzymatically inactive dimers of PKM2, eventually contributing to aerobic glycolysis in tumor. Abbreviations: FGFR1 – fibroblast growth factor receptor-1, BCR-ABL – (breakpoint cluster region-abelson murine leukemia viral oncogene homolog 1), JAK2 – janus kinase 2, FLT3 – fms like tyrosine kinase 3, ROS – reactive oxygen species.

Please cite this article in press as: Iqbal, M.A., et al. Pyruvate kinase M2 and cancer: an updated assessment. FEBS Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.011

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encoded and endogenous oncogenic proteins, i.e. pp60v-src kinase, HPV 16 E7, are known to interact with PKM2 and modulate its tetrameric structure (reviewed in detail [16,48]). Recently, CD44 adhesion molecule, a surface marker of cancer stem cells, has been shown to negatively regulate the PKM2 activity and thus contributes to aerobic glycolysis [55,56]. Evidently, decrease in PKM2 activity helps tumor cells in fulfilling their metabolic needs. 6. Non-metabolic functions of PKM2 – unorthodox roles Apart from its essential metabolic role, PKM2 also contributes to the process of tumorigenesis through its ‘‘non-metabolic’’ attributes. PKM2 is reported to interact with the C-terminal residues of

Oct-4 and enhances its transactivation [57]. Oct-4 protein is important in maintaining pluripotency in embryonic stem cells. In a study published in Cell by Luo et al., nuclear PKM2 has been shown to interact with its transcriptional activator HIF1a in a prolyl hydroxylase 3 (PHD3) dependent manner. This interaction increases transcriptional activity of HIF1a which results in higher expression of its (HIF1a) target genes e.g. GLUT1 (glucose transporter), LDHA (lactate dehydrogenase A) and PKM2 (Fig. 2). The authors, thus, provide a possible explanation to the previous observations that a switch to PKM2 correlated with increased glucose uptake and lactate production (aerobic glycolysis). Further, they unravel a ‘‘positive feedback loop’’ mechanism that reprograms glucose metabolism [19]. Wang et al. identified that Jumonji C

Fig. 2. Nuclear PKM2 and its functions. The illustration shows the factors responsible for nuclear localization of PKM2 and the non-metabolic implications. In short, PKM2 acts as a transcriptional co-activator or as a protein kinase inside the nucleus to facilitate the expression of genes important for cell division. EGF-induced ERK1/2 phosphorylates PKM2 at serine 37 residue (S37), enabling its import into the nucleus to aid the transactivation of b-catenin target genes such as c-Myc. Likewise, PHD3 catalyzed 403/408 proline hydroxylated PKM2 and JMJD5–PKM2 heterodimer complex interacts with HIF1a and initiate its transcriptional activation of glycolytic genes; and self transactivate to establish a positive feedback loop. Furthermore, lysine 433 acetylation of PKM2 by P300 localizes it to the nucleus, which enables the PKM2 dimer to act as a protein kinase by phosphorylating Stat3 and histone H3, thus, facilitating transcription of genes that are important for tumor cell proliferation. Abbreviations: PHD3 – prolyl hydroxylase 3, ERK1/2 – extracellular signal regulated kinase 1/2, EGF – endothelial growth factor, P300 – histone acetyltransferase, LCF/TCF – lymphoid enhancing factor/T-cell factor, PTB – polypyrimidine tract-binding protein, JMJD5 – jumonji C domain-containing dioxygenase, LDHA – lactate dehydrogenase A, PKM2 – pyruvate kinase M2, GLUT1 – glucose transporter 1, PDK1 – pyruvate dehydrogenase kinase, H3 – histone 3, HIF – hypoxic inducible factor, STAT 3 – signal transducer and activator of transcription 3.

Please cite this article in press as: Iqbal, M.A., et al. Pyruvate kinase M2 and cancer: an updated assessment. FEBS Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.011

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domain-containing dioxygenase (JMJD5) directly interacts with PKM2, disrupts its tetrameric structure, inhibits activity and promotes its nuclear translocation. Inside the nucleus, JMJD5–PKM2 heterodimer promotes HIF1a – mediated transactivation for metabolic reprogramming [58]. Another letter in Nature by Yang et al. identified the role of PKM2 in transcriptional activation in response to epidermal growth factor (EGF). The authors report that PKM2 associates with p-Tyr peptides of b-catenin in response to EGF stimulation, and that this complex translocates into the nucleus where it localizes to the cyclin D1 (CCND1) and c-Myc promoters (Fig. 2) [20]. PKM2-b-catenin complex enhances cyclin D1 and c-Myc expression through dissociation of HDAC3, a histone deacetylase, from the CCND1 promoter. PKM2-dependent b-catenin transactivation is involved in EGFR promoted tumor cell proliferation and development. Upregulation of c-Myc expression by PKM2 forms a feed-forward loop, since c-Myc has been shown to upregulate transcription of heterogeneous nuclear ribonucleoproteins (hnRNPs) that promote the alternative splicing of PKM2 over PKM1 [33]. Later, Yang et al. found that PKM2 interacts and phosphorylates histone H3 at threonine 11 residue upon EGF stimulation, which favors dissociation of HDAC from the cyclin D1 and c-Myc promoters and facilitates the H3-K9 acetylation (Fig. 2). This paper reports PKM2 as a protein kinase involved in histone modifications to control the expression of c-Myc and cyclin D1 in cancer cells upon EGF stimulation [59]. Further, nuclear PKM2 levels were found to be correlated with H3/T11 phosphorylation status in glioma malignancy grades. However, the mechanism controlling the switch between pyruvate kinase and protein kinase functions of PKM2 remained unclear. A recent article in Molecular Cell reported that PKM2 dimer is a protein kinase and that it regulates gene transcription [21]. The authors demonstrated that PKM2 dimer is an active protein kinase (with PEP as phosphate donor), while PKM2 tetramer is a pyruvate kinase. Further, they showed that expression of a PKM2 mutant, which exists as dimer, promotes cellular proliferation indicating that its protein kinase activity is essential for cellular proliferation. This study exposed an important link between metabolic transformation and gene expression. Lv et al. described that acetylation at K433 dictates PKM2 to act as a protein kinase rather than pyruvate kinase by inducing its nuclear accumulation. Upon mitogenic or oncogenic stimulation, p300 acetyltransferase catalyzes K433 acetylation of PKM2. Consequently, K433 acetylated PKM2 fails to respond to its allosteric activator FBP. This study further postulates the involvement of K433 acetylated PKM2 in the promotion of cancer cell proliferation by phosphorylation of STAT3 and Histone-3 and initiating a downstream transcriptional network (see Fig. 2) [60]. Besides PKM2’s role in regulating the expression of the cell cycle protein cyclin D1, direct involvement of PKM2 in the regulation of cell cycle progression has also been identified. PKM2 is shown to phosphorylate spindle-checkpoint protein Bub3 at Y207, to assist formation of Bub3-Bub1-Blinkin complex for correct kinetochore microtubules attachment and proper chromosomal segregation [61]. This study highlighted yet another protein kinase function of PKM2. Most recently, SAICAR has been reported to bind and induce protein kinase activity of PKM2. Moreover, SAICAR–PKM2 complex is shown to phosphorylate several proteins (e.g. EGFR/MAPK signaling proteins) that are important in proliferation [62]. Apart from protein kinase function, PKM2 is also involved in generation of cAMP via interaction with soluble adenylyl cyclase (sAC). The cAMP produced induces EPAC/RAP1 mediated upregulation of b1 integrin pathway resulting in loss of cell polarity [63]. The complex modifications of PKM2 along with its pyruvate and protein kinase activity orchestrate the events necessary for cancer development.

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7. PKM2 as a therapeutic target Targeting metabolic behavior of cancer cells is emerging as a new avenue in cancer therapeutics [64,65]. Several metabolic targets are under preclinical and clinical trials [66,67]. Owing to the array of benefits PKM2 provides to tumor cells, it is emerging as an attractive target in cancer therapy [68]. This section will review the approaches targeting PKM2 expression and activity along with their advantages and pitfalls. 7.1. RNAi (targeting the expression of PKM2) siRNA specifically targeting PKM2 was shown to attenuate growth and induce caspase-dependent apoptosis in several cancer cell lines. Additionally, intra-tumoral injection of siRNA reduced tumor growth of established tumor xenograft [69]. Further, Knockdown of PKM2 by RNAi synergized with the efficacy of the anticancer drugs- docetaxel and cisplatin in A549 lung cancer cell line and mouse model [70,71]. Since PKM2 is also present in normal proliferating and adult stem cells, chances of non-specific targeting in normal cell could be a drawback in this approach [16,72]. Also, silencing of PKM2 may mimic inactive PKM2 state, which is rather beneficial to proliferating cells. A decrease in PKM2 expression and activity has been associated with drug resistance in various cancer cell lines [73–76]. A plausible explanation for this correlation could be that low PKM2 level results in the pooling and shunting of glycolytic intermediates to the pentose pathway to produce reducing equivalent (NADPH and GSH), thus keeping the anti-oxidative system active. 7.2. Inhibition of PKM2 activity The idea behind this approach is that proliferating cells strongly depend on energy and that inhibition of PKM2 inhibits energy regeneration. Vander Heiden et al. performed a high throughput screen and identified small molecules selectively inhibiting PKM2. One of the identified compounds, (N-(3-carboxy-4hydroxy) phenyl 1-2,5,-dimethylpyrole), inhibited PKM2 activity with IC50 in lM range and induced apoptosis in H1299 cells. Allosteric activation of PKM2 by FBP was also significantly inhibited by this compound, suggesting its interference with the allosteric FBP binding loop in PKM2 [77]. Another compound named Arava, which is known for inhibiting key enzyme in pyrimidine synthesis, binds to PKM2 and decreases its activity by inducing M2 dimerization in rat Novikoff hepatoma cells [78]. Natural drugs like Shikonin and its analog Alkanin selectively inhibit PKM2 activity and induce cell death in PKM2, but not PKM1, expressing cells [79]. However, the PKM2 inhibition by these drugs is partially reversed by addition of FBP to protein lysates. The effects of these drugs on the tumor promoting non-glycolytic functions of inactive nuclear PKM2 are not known and should be studied. PKM2 inhibition may result in glycolytic accumulation required for anabolic synthesis. In addition, glycolysis is not the only source for energy regeneration in tumor cells. Most tumor cells are characterized by high glutaminolytic capacities, thus, glutaminolysis may be an escape mechanism for drugs inhibiting PKM2 [80]. 7.3. PKM2 activation Besides energy, proliferating cells strongly depend on the synthesis of cell building blocks that is favored by the less active dimeric PKM2. Thus, activation and fixation of PKM2 in the highly active tetrameric form may also inhibit cell proliferation due to the resultant deficiency of precursors for the synthesis of cell building

Please cite this article in press as: Iqbal, M.A., et al. Pyruvate kinase M2 and cancer: an updated assessment. FEBS Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.011

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blocks. In a recent study by Anastasiou et al., two representative compounds, namely- thieno [3,2-b] pyrrole [3,2 d] pyridazinone and substituted N,N-diarylsulfonamide, were found to be potent activators of PKM2 [81,82]. These chemical entities are cell permeable analogs that largely resemble the FBP-allosteric activator of PKM2. These compounds activated PKM2 with AC50 value of 26–99 nM and reduced the Km for substrate PEP by 10-fold without affecting the Vmax. Interestingly, the activation of PKM2 altered cancer metabolism in vitro and reduced xenograft tumor growth. Another study by Walsh et al. showed that 2-Oxo-N-aryl-1,2,3, 4-tetrahydroquinoline-6-sulfonamides activated PKM2, with AC50 in the range of 90 nM and 5-fold reduced Km for PEP [83]. The irreversible inactivation of PKM2 by modifications like Y105 or by mutations could pose a problem in targeting modified PKM2 for activation by chemical compounds. Alternatively, upstream factors, which facilitate the formation of dimeric PKM2, could be targeted in place of PKM2 itself (inhibition of formation of dimeric PKM2 would result in high activity PKM2 tetramers). Studies have shown that targeting the oncogenic tyrosine kinase BCR–ABL with Imatinib, JAB2 by AG490 and FLT3 by TKI258 inhibit tyrosine-105 phosphorylation of PKM2, thereby preventing its inactivation [52]. Taken together, a multitude of studies indicate that PKM2 is a promising target for therapeutic approaches. However, presence of PKM2 in normal cells and existence of metabolic escape mechanisms could limit the efficacy of these approaches. Hence, there is a need for the development of therapeutic strategies based on metabolic individualization of tumors. 8. Evidences challenging the role of PKM2 in cancer In the past 5 years, extensive research has unravelled a notable involvement of PKM2 in cancer via its canonical and non-canonical functions. However, some recent studies revealed controversial observations about the role of PKM2 in cancer. For example, knock-down of PKM2 resulted in a modest inhibitory effect on in vitro proliferation of cancer cells and the xenograft tumors without any signs of inhibition, suggesting that PKM2 is not an obligatory requirement for tumor growth [84]. A latest Cell paper describes that breast tumors in a PKM2 conditional mouse model progressed faster despite PKM2 deletion, and that proliferation within the PKM2 null mice was associated with PKM1 expression [85], which is not consistent with the earlier finding that PKM1 expression and pyruvate kinase activators can suppress tumor growth [85,86]. The authors discuss that following PKM exon 10 deletion, the tumors may create analogous states of high and low pyruvate kinase activity by titrating the expression of PKM1. The presence of loss-of-function heterozygous PKM2 mutations in human cancers further supports this notion. In addition, the authors refer to a possible pyruvate kinase independent mechanism for conversion of PEP to pyruvate. The results may also imply that the non-metabolic functions of PKM2, i.e. nuclear functions, are not absolutely necessary for tumor cell proliferation [85]. Accordingly, nuclear translocation of PKM2 induced by somatostatin analogues has been linked with apoptosis long before the documentation of the tumor promoting consequences of nuclear PKM2 [87]. 9. Conclusion and future directions In the last decade, a bursting number of publications revealed a manifold role of PKM2 within tumor cells. However, there are still voids that need to be filled in before we completely understand its role in cancer. The recent evidence of the presence of loss-of-function PKM2 mutations in some tumors, as well as our observation that natural heterozygous mutations in PKM2 (observed in Bloom

Syndrome background) impair its activity [88] and promote tumor growth [89], uphold the notion that lowered PKM2 activity is critical for tumor growth. Observations of dimeric PKM2 acting as a protein kinase in regulation of gene expression further indicate its importance to proliferating tumor cells [21]. However, it is yet to be known if dimeric PKM2 promotes processes like migration and metastasis, to further assist cancer. Also, it seems pertinent to explore if the genetic variations in PKM2 that are responsible for hindering its tetrameric structure increase cancer risk. It has been known for a long time that dimeric PKM2 (M2-PK) is present in plasma of cancer patients and that the dimeric M2-PK plasma concentrations correlate with staging [90–92]. Besides, PKM2 is also detectable in the stool from patients of lower gastrointestinal tract cancer and is used for colorectal cancer (CRC) screening [93]. In CRC patients high sera M2-PK values correlate with poor response to 5-FU-based chemotherapy [94]. In the follow-up studies, PKM2 plasma levels have been shown to drop after complete remission and increase in case of a local relapse [93,95]. Further, Duan et al. have shown that PKM2 binds to tumor endothelial marker 8 (TEM8), which is located on the cell surface of endothelial cells [96]. They discuss that PKM2 released from the tumor might stimulate angiogenesis by binding to TEM8. These studies warrant the investigation of extracellular PKM2 and the potential role it might play in promoting cancer. Most recent evidence showing that some tumor cells may not require PKM2 for their growth are noteworthy in emphasizing the need of a better understanding of metabolic heterogeneity in tumor cells to envisage improved therapeutic strategies. Further, possible heterogeneity among tumor cells with regards to oligomeric status of PKM2 may be challenging in therapeutic targeting of its activity. Abnormal vascularization in tumors gives rise to regions with low nutrient access; cells in this region, being essentially quiescent, might express active tetrameric PKM2 which is rather fit for generation of survival ATP through glycolysis. However, at some point during tumorigenesis, changes in nutrient availability and vascularization may result in conversion of active PKM2 into inactive one; for instance, high glucose is known to induce PKM2 acetylation thereby decreasing its activity. Active PKM2 in quiescent cells may be contributory to chemoresistance and relapse, since these cells are resistant to standard chemotherapy regimens, and therefore, cause tumor relapse [97]. Regardless, therapeutic activation of PKM2 seems promising at least in inhibiting the metabolism of proliferating tumor cells; however, it is yet to be known if this holds true for most tumor types.

References [1] Warburg, O., Posener, K. and Negelein, E. (1924) Über den Stoffwechsel der Carcinomzelle. Biochem. Ztschr. 152, 309–344. [2] Warburg, O. (1956) On the origin of cancer cells. Science 123, 309–314. [3] Dang, C.V. and Semenza, G.L. (1999) Oncogenic alterations of metabolism. Trends Biochem. Sci. 24, 68–72. [4] Hsu, P.P. and Sabatini, D.M. (2008) Cancer cell metabolism: Warburg and beyond. Cell 134, 703–707. [5] Gambhir, S.S. (2002) Molecular imaging of cancer with positron emission tomography. Nat. Rev. Cancer 2, 683–693. [6] Hawkins, R.A. and Phelps, M.E. (1988) PET in clinical oncology. Cancer Metastasis Rev. 7, 119–142. [7] Weber, W.A., Avril, N. and Schwaiger, M. (1999) Relevance of positron emission tomography (PET) in oncology. Strahlenther. Onkol. 175, 356–373. [8] Golman, K., Zandt, R.I., Lerche, M., Pehrson, R. and Ardenkjaer-Larsen, J.H. (2006) Metabolic imaging by hyperpolarized 13C magnetic resonance imaging for in vivo tumor diagnosis. Cancer Res. 66, 10855–10860. [9] Kurhanewicz, J., Vigneron, D.B., Brindle, K., Chekmenev, E.Y., Comment, A., Cunningham, C.H., Deberardinis, R.J., Green, G.G., Leach, M.O., Rajan, S.S., Rizi, R.R., Ross, B.D., Warren, W.S. and Malloy, C.R. (2011) Analysis of cancer metabolism by imaging hyperpolarized nuclei: prospects for translation to clinical research. Neoplasia 13, 81–97. [10] Vander Heiden, M.G., Cantley, L.C. and Thompson, C.B. (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029–1033.

Please cite this article in press as: Iqbal, M.A., et al. Pyruvate kinase M2 and cancer: an updated assessment. FEBS Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.011

M.A. Iqbal et al. / FEBS Letters xxx (2014) xxx–xxx [11] Hirschhaeuser, F., Sattler, U.G. and Mueller-Klieser, W. (2011) Lactate: a metabolic key player in cancer. Cancer Res. 71, 6921–6925. [12] Husain, Z., Huang, Y., Seth, P. and Sukhatme, V.P. (2013) Tumor-derived lactate modifies antitumor immune response: effect on myeloid-derived suppressor cells and NK cells. J. Immunol. 191, 1486–1495. [13] Fischer, K., Hoffmann, P., Voelkl, S., Meidenbauer, N., Ammer, J., Edinger, M., Gottfried, E., Schwarz, S., Rothe, G., Hoves, S., Renner, K., Timischl, B., Mackensen, A., Kunz-Schughart, L., Andreesen, R., Krause, S.W. and Kreutz, M. (2007) Inhibitory effect of tumor cell-derived lactic acid on human T cells. Blood 109, 3812–3819. [14] Gatenby, R.A. and Gillies, R.J. (2004) Why do cancers have high aerobic glycolysis? Nat. Rev. Cancer 4, 891–899. [15] Lunt, S.Y. and Vander Heiden, M.G. (2011) Aerobic glycolysis: meeting the metabolic requirements of cell proliferation. Annu. Rev. Cell Dev. Biol. 27, 441–464. [16] Mazurek, S. (2011) Pyruvate kinase type M2: a key regulator of the metabolic budget system in tumor cells. Int. J. Biochem. Cell Biol. 43, 969–980. [17] Mazurek, S., Boschek, C.B., Hugo, F. and Eigenbrodt, E. (2005) Pyruvate kinase type M2 and its role in tumor growth and spreading. Semin. Cancer Biol. 15, 300–308. [18] Bluemlein, K., Gruning, N. M., Feichtinger, R. G., Lehrach, H., Kofler, B. & Ralser, M. (2011) No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis, Oncotarget. [19] Luo, W., Hu, H., Chang, R., Zhong, J., Knabel, M., O’Meally, R., Cole, R.N., Pandey, A. and Semenza, G.L. (2011) Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell 145, 732–744. [20] Yang, W., Xia, Y., Ji, H., Zheng, Y., Liang, J., Huang, W., Gao, X., Aldape, K. and Lu, Z. (2011) Nuclear PKM2 regulates beta-catenin transactivation upon EGFR activation. Nature 480, 118–122. [21] Gao, X., Wang, H., Yang, J.J., Liu, X. and Liu, Z.R. (2012) Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Mol. Cell 45, 598– 609. [22] Jiang, Y., Li, X., Yang, W., Hawke, D.H., Zheng, Y., Xia, Y., Aldape, K., Wei, C., Guo, F., Chen, Y. and Lu, Z. (2014) PKM2 regulates chromosome segregation and mitosis progression of tumor cells. Mol. Cell 53, 75–87. [23] Yang, W., Xia, Y., Hawke, D., Li, X., Liang, J., Xing, D., Aldape, K., Hunter, T., Alfred Yung, W.K. and Lu, Z. (2012) PKM2 phosphorylates histone H3 and promotes gene transcription and tumorigenesis. Cell 150, 685–696. [24] Noguchi, T., Yamada, K., Inoue, H., Matsuda, T. and Tanaka, T. (1987) The L- and R-type isozymes of rat pyruvate kinase are produced from a single gene by use of different promoters. J. Biol. Chem. 262, 14366–14371. [25] Noguchi, T., Inoue, H. and Tanaka, T. (1986) The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. J. Biol. Chem. 261, 13807–13812. [26] Hacker, H.J., Steinberg, P. and Bannasch, P. (1998) Pyruvate kinase isoenzyme shift from L-type to M2-type is a late event in hepatocarcinogenesis induced in rats by a choline-deficient/DL-ethionine-supplemented diet. Carcinogenesis 19, 99–107. [27] Christofk, H.R., Vander Heiden, M.G., Harris, M.H., Ramanathan, A., Gerszten, R.E., Wei, R., Fleming, M.D., Schreiber, S.L. and Cantley, L.C. (2008) The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452, 230–233. [28] Bluemlein, K., Gruning, N.M., Feichtinger, R.G., Lehrach, H., Kofler, B. and Ralser, M. (2011) No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis. Oncotarget 2, 393–400. [29] Steinberg, P., Klingelhoffer, A., Schafer, A., Wust, G., Weisse, G., Oesch, F. and Eigenbrodt, E. (1999) Expression of pyruvate kinase M2 in preneoplastic hepatic foci of N-nitrosomorpholine-treated rats. Virchows Arch. 434, 213– 220. [30] Desai, S., Ding, M., Wang, B., Lu, Z., Zhao, Q., Shaw, K., Yung, W.K., Weinstein, J.N., Tan, M. and Yao, J. (2013) Tissue-specific isoform switch and DNA hypomethylation of the pyruvate kinase PKM gene in human cancers. Oncotarget. [31] Brinck, U., Eigenbrodt, E., Oehmke, M., Mazurek, S. and Fischer, G. (1994) Land M2-pyruvate kinase expression in renal cell carcinomas and their metastases. Virchows Arch. 424, 177–185. [32] Dombrauckas, J.D., Santarsiero, B.D. and Mesecar, A.D. (2005) Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis. Biochemistry 44, 9417–9429. [33] David, C.J., Chen, M., Assanah, M., Canoll, P. and Manley, J.L. (2010) HnRNP proteins controlled by c-Myc deregulate pyruvate kinase mRNA splicing in cancer. Nature 463, 364–368. [34] Sun, Q., Chen, X., Ma, J., Peng, H., Wang, F., Zha, X., Wang, Y., Jing, Y., Yang, H., Chen, R., Chang, L., Zhang, Y., Goto, J., Onda, H., Chen, T., Wang, M. R., Lu, Y., You, H., Kwiatkowski, D. & Zhang, H. Mammalian target of rapamycin upregulation of pyruvate kinase isoenzyme type M2 is critical for aerobic glycolysis and tumor growth, Proc. Natl. Acad. Sci. U.S.A. 108, 4129–4134. [35] Schafer, D., Hamm-Kunzelmann, B. and Brand, K. (1997) Glucose regulates the promoter activity of aldolase A and pyruvate kinase M2 via dephosphorylation of Sp1. FEBS Lett. 417, 325–328. [36] Traxinger, R.R. and Marshall, S. (1992) Insulin regulation of pyruvate kinase activity in isolated adipocytes. Crucial role of glucose and the hexosamine biosynthesis pathway in the expression of insulin action. J. Biol. Chem. 267, 9718–9723.

7

[37] Ashizawa, K., Fukuda, T. and Cheng, S.Y. (1992) Transcriptional stimulation by thyroid hormone of a cytosolic thyroid hormone binding protein which is homologous to a subunit of pyruvate kinase M1. Biochemistry 31, 2774–2778. [38] Saheki, S., Saheki, K. and Tanaka, T. (1979) Changes in pyruvate kinase isozymes of rat small intestine during development and the synergistic effect on them of thyroid and glucocorticoid hormones. Enzyme 24, 8–17. [39] Asai, Y., Yamada, K., Watanabe, T., Keng, V.W. and Noguchi, T. (2003) Insulin stimulates expression of the pyruvate kinase M gene in 3T3-L1 adipocytes. Biosci. Biotechnol. Biochem. 67, 1272–1277. [40] Iqbal, M.A., Siddiqui, F.A., Gupta, V., Chattopadhyay, S., Gopinath, P., Kumar, B., Manvati, S., Chaman, N. and Bamezai, R.N. (2013) Insulin enhances metabolic capacities of cancer cells by dual regulation of glycolytic enzyme pyruvate kinase M2. Mol. Cancer 12, 72. [41] Gallagher, E.J. and LeRoith, D. (2011) Minireview: IGF, insulin, and cancer. Endocrinology 152, 2546–2551. [42] Kress, S., Stein, A., Maurer, P., Weber, B., Reichert, J., Buchmann, A., Huppert, P. and Schwarz, M. (1998) Expression of hypoxia-inducible genes in tumor cells. J. Cancer Res. Clin. Oncol. 124, 315–320. [43] Lu, H., Forbes, R.A. and Verma, A. (2002) Hypoxia-inducible factor 1 activation by aerobic glycolysis implicates the Warburg effect in carcinogenesis. J. Biol. Chem. 277, 23111–23115. [44] Dewhirst, M.W., Cao, Y. and Moeller, B. (2008) Cycling hypoxia and free radicals regulate angiogenesis and radiotherapy response. Nat. Rev. Cancer 8, 425–437. [45] Lara, P.C., Lloret, M., Clavo, B., Apolinario, R.M., Henriquez-Hernandez, L.A., Bordon, E., Fontes, F. and Rey, A. (2009) Severe hypoxia induces chemoresistance in clinical cervical tumors through MVP over-expression. Radiat. Oncol. 4, 29. [46] Lehninger, A.L., Nelson, D.L. and Cox, M.M. (1993) Principles of Biochemistry, second ed, Worth, New York. [47] Deberardinis, R.J., Sayed, N., Ditsworth, D. and Thompson, C.B. (2008) Brick by brick: metabolism and tumor cell growth. Curr. Opin. Genet. Dev. 18, 54–61. [48] Gupta, V. and Bamezai, R.N. (2010) Human pyruvate kinase M2: a multifunctional protein. Protein Sci. 19, 2031–2044. [49] Mazurek, S., Grimm, H., Boschek, C.B., Vaupel, P. and Eigenbrodt, E. (2002) Pyruvate kinase type M2: a crossroad in the tumor metabolome. Br. J. Nutr. 87 (Suppl. 1), S23–S29. [50] Mazurek, S., Drexler, H.C., Troppmair, J., Eigenbrodt, E. and Rapp, U.R. (2007) Regulation of pyruvate kinase type M2 by A-Raf: a possible glycolytic stop or go mechanism. Anticancer Res. 27, 3963–3971. [51] Ashizawa, K., Willingham, M.C., Liang, C.M. and Cheng, S.Y. (1991) In vivo regulation of monomer-tetramer conversion of pyruvate kinase subtype M2 by glucose is mediated via fructose 1,6-bisphosphate. J. Biol. Chem. 266, 16842–16846. [52] Hitosugi, T., Kang, S., Vander Heiden, M.G., Chung, T.W., Elf, S., Lythgoe, K., Dong, S., Lonial, S., Wang, X., Chen, G.Z., Xie, J., Gu, T.L., Polakiewicz, R.D., Roesel, J.L., Boggon, T.J., Khuri, F.R., Gilliland, D.G., Cantley, L.C., Kaufman, J. and Chen, J. (2009) Tyrosine phosphorylation inhibits PKM2 to promote the Warburg effect and tumor growth. Sci. Signal. 2, ra73. [53] Lv, L., Li, D., Zhao, D., Lin, R., Chu, Y., Zhang, H., Zha, Z., Liu, Y., Li, Z., Xu, Y., Wang, G., Huang, Y., Xiong, Y., Guan, K.L. and Lei, Q.Y. (2011) Acetylation targets the M2 isoform of pyruvate kinase for degradation through chaperonemediated autophagy and promotes tumor growth. Mol. Cell 42, 719–730. [54] Anastasiou, D., Poulogiannis, G., Asara, J.M., Boxer, M.B., Jiang, J.K., Shen, M., Bellinger, G., Sasaki, A.T., Locasale, J.W., Auld, D.S., Thomas, C.J., Vander Heiden, M.G. and Cantley, L.C. (2011) Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science 334, 1278–1283. [55] Tamada, M., Suematsu, M. and Saya, H. (2012) Pyruvate kinase M2: multiple faces for conferring benefits on cancer cells. Clin. Cancer Res. 18, 5554–5561. [56] Tamada, M., Nagano, O., Tateyama, S., Ohmura, M., Yae, T., Ishimoto, T., Sugihara, E., Onishi, N., Yamamoto, T., Yanagawa, H., Suematsu, M. and Saya, H. (2012) Modulation of glucose metabolism by CD44 contributes to antioxidant status and drug resistance in cancer cells. Cancer Res. 72, 1438–1448. [57] Lee, J., Kim, H.K., Han, Y.M. and Kim, J. (2008) Pyruvate kinase isozyme type M2 (PKM2) interacts and cooperates with Oct-4 in regulating transcription. Int. J. Biochem. Cell Biol. 40, 1043–1054. [58] Wang, H.J., Hsieh, Y.J., Cheng, W.C., Lin, C.P., Lin, Y.S., Yang, S.F., Chen, C.C., Izumiya, Y., Yu, J.S., Kung, H.J. and Wang, W.C. (2014) JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1alpha-mediated glucose metabolism. Proc. Natl. Acad. Sci. U.S.A. 111, 279–284. [59] Yang, W., Xia, Y., Hawke, D., Li, X., Liang, J., Xing, D., Aldape, K., Hunter, T., Alfred Yung, W. and Lu, Z. (2012) PKM2 phosphorylates histone H3 and promotes gene transcription and tumorigenesis. Cell 150, 685–696. [60] Lv, L., Xu, Y.-P., Zhao, D., Li, F.-L., Wang, W., Sasaki, N., Jiang, Y., Zhou, X., Li, T.T., Guan, K.-L., Lei, Q.-Y. and Xiong, Y. (2013) Mitogenic and oncogenic stimulation of K433 acetylation promotes PKM2 protein kinase activity and nuclear localization. Mol. Cell 52, 340–352. [61] Jiang, Y., Li, X., Yang, W., Hawke, D.H., Zheng, Y., Xia, Y., Aldape, K., Wei, C., Guo, F., Chen, Y. and Lu, Z. (2013) PKM2 regulates chromosome segregation and mitosis progression of tumor cells. Mol. Cell. 53, 75–87. [62] Keller, K.E., Doctor, Z.M., Dwyer, Z.W. and Lee, Y.S. (2014) SAICAR induces protein kinase activity of PKM2 that is necessary for sustained proliferative signaling of cancer cells. Mol. Cell 53, 700–709.

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[63] Onodera, Y., Nam, J.M. and Bissell, M.J. (2014) Increased sugar uptake promotes oncogenesis via EPAC/RAP1 and O-GlcNAc pathways. J. Clin. Investig. 124, 367–384. [64] Hamanaka, R. and Chandel, N. (2012) Targeting glucose metabolism for cancer therapy. J. Exp. Med. 209, 211–215. [65] Vander Heiden, M. (2011) Targeting cancer metabolism: a therapeutic window opens. Nat. Rev. Drug Discovery 10, 671–684. [66] Tennant, D.A., Duran, R.V. and Gottlieb, E. (2010) Targeting metabolic transformation for cancer therapy. Nat. Rev. Cancer 10, 267–277. [67] Vander Heiden, M.G. (2011) Targeting cancer metabolism: a therapeutic window opens. Nat. Rev. Drug Discovery 10, 671–684. [68] Gupta, V., Wellen, K., Mazurek, S. and Bamezai, R. (2013) Pyruvate kinase M2: regulatory circuits and potential for therapeutic intervention. Curr. Pharm. Des.. [69] Goldberg, M. and Sharp, P. (2012) Pyruvate kinase M2-specific siRNA induces apoptosis and tumor regression. J. Exp. Med. 209, 217–224. [70] Guo, W., Zhang, Y., Chen, T., Wang, Y., Xue, J., Zhang, Y., Xiao, W., Mo, X. and Lu, Y. (2011) Efficacy of RNAi targeting of pyruvate kinase M2 combined with cisplatin in a lung cancer model. J. Cancer Res. Clin. Oncol. 137, 65–72. [71] Shi, H.-S., Li, D., Zhang, J., Wang, Y.-S., Yang, L., Zhang, H.-L., Wang, X.-H., Mu, B., Wang, W., Ma, Y., Guo, F.-C. and Wei, Y.-Q. (2010) Silencing of PKM2 increases the efficacy of docetaxel in human lung cancer xenografts in mice. Cancer Sci. 101, 1447–1453. [72] Bluemlein, K., Grüning, N.-M., Feichtinger, R., Lehrach, H., Kofler, B. and Ralser, M. (2011) No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis. Oncotarget 2, 393–400. [73] Yoo, B., Ku, J.-L., Hong, S.-H., Shin, Y.-K., Park, S., Kim, H. and Park, J.-G. (2004) Decreased pyruvate kinase M2 activity linked to cisplatin resistance in human gastric carcinoma cell lines. Int. J. Cancer 108, 532–539. [74] Martinez-Balibrea, E., Plasencia, C., Ginés, A., Martinez-Cardús, A., Musulén, E., Aguilera, R., Manzano, J., Neamati, N. and Abad, A. (2009) A proteomic approach links decreased pyruvate kinase M2 expression to oxaliplatin resistance in patients with colorectal cancer and in human cell lines. Mol. Cancer Ther. 8, 771–778. [75] Li, S.-L., Ye, F., Cai, W.-J., Hu, H.-D., Hu, P., Ren, H., Zhu, F.-F. and Zhang, D.-Z. (2010) Quantitative proteome analysis of multidrug resistance in human ovarian cancer cell line. J. Cell. Biochem. 109, 625–633. [76] Shin, Y.-K., Yoo, B., Hong, Y., Chang, H., Jung, K., Jeong, S.-Y. and Park, J.-G. (2009) Upregulation of glycolytic enzymes in proteins secreted from human colon cancer cells with 5-fluorouracil resistance. Electrophoresis 30, 2182– 2192. [77] Vander Heiden, M., Christofk, H., Schuman, E., Subtelny, A., Sharfi, H., Harlow, E., Xian, J. and Cantley, L. (2010) Identification of small molecule inhibitors of pyruvate kinase M2. Biochem. Pharmacol. 79, 1118–1124. [78] Müllner, S., Stark, H., Niskanen, P., Eigenbrodt, E., Mazurek, S. and Fasold, H. (2006) From target to lead synthesisProteomics in Drug Research, pp. 187– 207, Wiley-VCH Verlag GmbH & Co. KGaA. [79] Chen, J., Xie, J., Jiang, Z., Wang, B., Wang, Y. and Hu, X. (2011) Shikonin and its analogs inhibit cancer cell glycolysis by targeting tumor pyruvate kinase-M2. Oncogene 30, 4297–4306. [80] Spoden, G.A., Rostek, U., Lechner, S., Mitterberger, M., Mazurek, S. and Zwerschke, W. (2009) Pyruvate kinase isoenzyme M2 is a glycolytic sensor differentially regulating cell proliferation, cell size and apoptotic cell death dependent on glucose supply. Exp. Cell Res. 315, 2765–2774. [81] Boxer, M., Jiang, J.-K., Vander Heiden, M., Shen, M., Skoumbourdis, A., Southall, N., Veith, H., Leister, W., Austin, C., Park, H., Inglese, J., Cantley, L., Auld, D. and Thomas, C. (2010) Evaluation of substituted N,N0 -diarylsulfonamides as activators of the tumor cell specific M2 isoform of pyruvate kinase. J. Med. Chem. 53, 1048–1055. [82] Jiang, J.-K., Boxer, M., Vander Heiden, M., Shen, M., Skoumbourdis, A., Southall, N., Veith, H., Leister, W., Austin, C., Park, H., Inglese, J., Cantley, L., Auld, D. and Thomas, C. (2010) Evaluation of thieno[3,2-b]pyrrole[3,2-d]pyridazinones as

[83]

[84]

[85]

[86]

[87]

[88]

[89]

[90] [91]

[92]

[93]

[94]

[95]

[96]

[97]

activators of the tumor cell specific M2 isoform of pyruvate kinase. Bioorg. Med. Chem. Lett. 20, 3387–3393. Walsh, M., Brimacombe, K., Veith, H., Bougie, J., Daniel, T., Leister, W., Cantley, L., Israelsen, W., Vander Heiden, M., Shen, M., Auld, D., Thomas, C. and Boxer, M. (2011) 2-Oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamides as activators of the tumor cell specific M2 isoform of pyruvate kinase. Bioorg. Med. Chem. Lett. 21, 6322–6327. Cortés-Cros, M., Hemmerlin, C., Ferretti, S., Zhang, J., Gounarides, J., Yin, H., Muller, A., Haberkorn, A., Chene, P., Sellers, W. and Hofmann, F. (2013) M2 isoform of pyruvate kinase is dispensable for tumor maintenance and growth. Proc. Natl. Acad. Sci. U.S.A. 110, 489–494. Israelsen, W.J., Dayton, T.L., Davidson, S.M., Fiske, B.P., Hosios, A.M., Bellinger, G., Li, J., Yu, Y., Sasaki, M., Horner, J.W., Burga, L.N., Xie, J., Jurczak, M.J., DePinho, R.A., Clish, C.B., Jacks, T., Kibbey, R.G., Wulf, G.M., Di Vizio, D., Mills, G.B., Cantley, L.C. and Vander Heiden, M.G. (2013) PKM2 isoform-specific deletion reveals a differential requirement for pyruvate kinase in tumor cells. Cell 155, 397–409. Anastasiou, D., Yu, Y., Israelsen, W.J., Jiang, J.K., Boxer, M.B., Hong, B.S., Tempel, W., Dimov, S., Shen, M., Jha, A., Yang, H., Mattaini, K.R., Metallo, C.M., Fiske, B.P., Courtney, K.D., Malstrom, S., Khan, T.M., Kung, C., Skoumbourdis, A.P., Veith, H., Southall, N., Walsh, M.J., Brimacombe, K.R., Leister, W., Lunt, S.Y., Johnson, Z.R., Yen, K.E., Kunii, K., Davidson, S.M., Christofk, H.R., Austin, C.P., Inglese, J., Harris, M.H., Asara, J.M., Stephanopoulos, G., Salituro, F.G., Jin, S., Dang, L., Auld, D.S., Park, H.W., Cantley, L.C., Thomas, C.J. and Vander Heiden, M.G. (2012) Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis. Nat. Chem. Biol. 8, 839–847. Stetak, A., Veress, R., Ovadi, J., Csermely, P., Keri, G. and Ullrich, A. (2007) Nuclear translocation of the tumor marker pyruvate kinase M2 induces programmed cell death. Cancer Res. 67, 1602–1608. Gupta, V., Kalaiarasan, P., Faheem, M., Singh, N., Iqbal, M.A. and Bamezai, R.N. (2010) Dominant negative mutations affect oligomerization of human pyruvate kinase M2 isozyme and promote cellular growth and polyploidy. J. Biol. Chem. 285, 16864–16873. Iqbal, M.A., Siddiqui, F.A., Chaman, N., Gupta, V., Kumar, B., Gopinath, P. and Bamezai, R.N. (2014) Missense mutations in pyruvate kinase m2 promote cancer metabolism, oxidative endurance, anchorage independence, and tumor growth in a dominant negative manner. J. Biol. Chem. 289, 8098–8105. Hugo, F., Fischer, G. and Eigenbrodt, E. (1999) Quantitative detection of tumor M2-PK in serum and plasma. Anticancer Res. 19, 2753–2757. Wechsel, H.W., Petri, E., Bichler, K.H. and Feil, G. (1999) Marker for renal cell carcinoma (RCC): the dimeric form of pyruvate kinase type M2 (Tu M2-PK). Anticancer Res. 19, 2583–2590. Zhang, B., Chen, J.Y., Chen, D.D., Wang, G.B. and Shen, P. (2004) Tumor type M2 pyruvate kinase expression in gastric cancer, colorectal cancer and controls. World J. Gastroenterol. 10, 1643–1646. Tonus, C., Sellinger, M., Koss, K. and Neupert, G. (2012) Faecal pyruvate kinase isoenzyme type M2 for colorectal cancer screening: a meta-analysis. World J. Gastroenterol. 18, 4004–4011. Shin, Y.K., Yoo, B.C., Hong, Y.S., Chang, H.J., Jung, K.H., Jeong, S.Y. and Park, J.G. (2009) Upregulation of glycolytic enzymes in proteins secreted from human colon cancer cells with 5-fluorouracil resistance. Electrophoresis 30, 2182– 2192. Schneider, J., Neu, K., Grimm, H., Velcovsky, H.G., Weisse, G. and Eigenbrodt, E. (2002) Tumor M2-pyruvate kinase in lung cancer patients: immunohistochemical detection and disease monitoring. Anticancer Res. 22, 311–318. Duan, H.F., Hu, X.W., Chen, J.L., Gao, L.H., Xi, Y.Y., Lu, Y., Li, J.F., Zhao, S.R., Xu, J.J., Chen, H.P., Chen, W. and Wu, C.T. (2007) Antitumor activities of TEM8-Fc: an engineered antibody-like molecule targeting tumor endothelial marker 8. J. Natl Cancer Inst. 99, 1551–1555. Tannock, I.F. (2001) Tumor physiology and drug resistance. Cancer Metastasis Rev. 20, 123–132.

Please cite this article in press as: Iqbal, M.A., et al. Pyruvate kinase M2 and cancer: an updated assessment. FEBS Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.011