Review Article PKM2, a Central Point of Regulation in Cancer Metabolismdownloads.hindawi.com/journals/ijcb/2013/242513.pdf · of proliferation. e M isoform of pyruvate kinase (PKM)

Hindawi Publishing CorporationInternational Journal of Cell BiologyVolume 2013 Article ID 242513 11 pageshttpdxdoiorg1011552013242513

Review ArticlePKM2 a Central Point of Regulation in Cancer Metabolism

Nicholas Wong1234 Jason De Melo1234 and Damu Tang1234

1 Division of Nephrology Department of Medicine McMaster University Hamilton ON Canada L8S 4L82Division of Urology Department of Surgery McMaster University Hamilton ON Canada L8S 4L83 Father Sean OrsquoSullivan Research Centre St Josephrsquos Healthcare Hamilton Hamilton ON Canada L8N 4A64The Hamilton Center for Kidney Research St Josephrsquos Healthcare Hamilton Hamilton ON Canada L8N 4A6

Correspondence should be addressed to Damu Tang damutmcmasterca

Received 2 December 2012 Revised 11 January 2013 Accepted 13 January 2013

Academic Editor Claudia Cerella

Copyright copy 2013 Nicholas Wong et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Aerobic glycolysis is the dominant metabolic pathway utilized by cancer cells owing to its ability to divert glucose metabolitesfrom ATP production towards the synthesis of cellular building blocks (nucleotides amino acids and lipids) to meet the demandsof proliferation The M2 isoform of pyruvate kinase (PKM2) catalyzes the final and also a rate-limiting reaction in the glycolyticpathway In the PK family PKM2 is subjected to a complex regulation by both oncogenes and tumour suppressors which allowsfor a fine-tone regulation of PKM2 activity The less active form of PKM2 drives glucose through the route of aerobic glycolysiswhile active PKM2directs glucose towards oxidativemetabolism Additionally PKM2possesses protein tyrosine kinase activity andplays a role in modulating gene expression and thereby contributing to tumorigenesis We will discuss our current understandingof PKM2rsquos regulation and its many contributions to tumorigenesis

1 Introduction

Metabolism lies in the heart of cell biology Understandinghow cancer cells cope with metabolic needs for their uniquebiology has been a focus of cancer research for many yearsIt began with the landmark observation reported more than80 years ago by Otto Warburg that cancer cells consumedmore glucose and produced a large amount of lactate evenin a well-oxygenized environment a process known asaerobic glycolysis or the Warburg effect [1 2] While normaldifferentiated cells maximize ATP production by mitochon-drial oxidative phosphorylation of glucose under normoxicconditions cancer cells generate much less ATP from glucoseby aerobic glycolysis Despite being less efficient in ATPproduction glycolysis is a much more rapid process [3 4]Cancers commonly deregulate pathways that enhance glycol-ysis including activation of the PI3 K-ATK-mTOR pathwayand upregulation of HIF-1 and c-Myc [5 6] The increasein aerobic glycolysis together with its dynamic process incancer cells enables glycolytic intermediates to be redirectedfor the biosynthesis of cellular building blocks (nucleotidesamino acids and lipids) while also producing ATPTherefore

the Warburg effectaerobic glycolysis meets the demands ofcancer and proliferating cells for macromolecular synthesisand energy production [7 8] As a result cancer cellsdisplay enhanced glucose uptake and produce higher levelsof lactate [1 2] The Warburg effect was explored for thecommon clinical detection of tumors by fluorodeoxyglu-cose (2-deoxy-2-(18F)fluoro-D-glucose) positron emissiontomography (FDG-PET) [7] In the glycolytic process pyru-vate kinase (PK) catalyzes the last reaction transfer of ahigh-energy phosphate group from phosphoenolpyruvate(PEP) to ADP producing ATP and pyruvate [9] Pyruvateis then either reduced to lactate by lactate dehydrogenase(LDH) in the cytosol or enters the mitochondria to produceATP through the tricarboxylic acid (TCA) cycle (Figure 1)Along the glycolysis pathway intermediate metabolites canbe channeled to synthesize amino acids nucleotides andlipids (Figure 1) if the rate of flux through the pathway iscontrolled PK is an ideal candidate for this control [10]because (1) PK catalyzes the last reaction of the pathway(Figure 1) and (2) the reaction is essentially irreversible(Figure 1) [9 11] Therefore lowering PK activity is expectedto produce less pyruvate (Figure 1) or prevent complete

2 International Journal of Cell Biology

GLUT

Glucose

GlucoseCytosol

Glucose-6-P

Fructose-6-P

Fructose-6-P2 (FBP)

Glyceraldehyde-3-P

3-Phosphoglycerate

Phosphoenolpyruvate (PEP)

HK

LDHA

PKM2 (tetramer)PKM2(dimer)

PyruvatePyruvate

Lactate

ADP

ATP

Pyruvate

Acetyl-CoAPDH

PDK1

6-Phosphogluconolactone 6-P-Gluconate

Ribose-5-phosphate

Pentosephosphatepathway

NADP

NADPH

G6PD

Nucleotide andamino acid synthesis

Serine synthesis

Citrate

Citrate

Lipid synthesis

MitochondriaCytosol

Glutamine

Glutamine

HIF-1

HIF-1

HIF

-1

Gly

coly

sis

Figure 1 Schematic illustrating the cancer utilization of the metabolic pathways Pyruvate kinase catalyzes the last step of glycolysis byconverting PEP and ADP to pyruvate and ATP respectively PKM2 dimers and tetramers possess low and high levels of Pyruvate kinaseactivity respectively With reduced enzymatic activity PKM2 dimer drives aerobic glycolysis which allows the intermediate metabolitesto be used for the synthesis of nucleotides amino acids and lipids and the production of reduced NADPH (see the pentose phosphatepathway) HIF-1 upregulates the indicated proteins GLUT glucose transporter HK hexokinase G6PD glucose-6-phosphate dehydrogenaseHIF-1 hypoxia-inducible factor 1 LDHA lactate dehydrogenase A PDK1 pyruvate dehydrogenase kinase isoenzyme 1 and PDH pyruvatedehydrogenase

conversion of glucose to pyruvate (1 molecule of glucose to 2molecules of pyruvate) This enables the upstream glycolyticintermediates to accumulate and thus contribute to the shiftof metabolism towards the anabolic phase for amino acidsnucleotides and lipid production (Figure 1) Cancer cellsexplore this logic by predominantly using PKM2 an isoform

of PK as its activity can be dynamically regulated between theless active PKM2 dimer and the highly active PKM2 tetramer[12]

PK consists of four isoforms the L (PKL) and R (PKR)isoforms encoded by the PKLR (1q22) gene and the M1(PKM1) and M2 (PKM2) isoforms encoded by the PKM2

International Journal of Cell Biology 3

(15q23) gene The PKLR gene is regulated by tissue-specificpromoters The full-length PKR isoform is expressed inred blood cells while the PKL isoform missing exon 1 isdetected in liver and kidney [5 13 14] PKM1 and PKM2are produced from the PKM2 gene by alternative splicing[15] The highly active PKM1 is expressed in tissues thatconsistently need high levels of energy like skeletal muscleheart and brain [5 10] PKM2 is expressed in most cellsexcept adult muscle brain and liver [12 16 17] and is thepredominant PK in proliferating and cancer cells [18] WhilePKL PKR and PKM1 form stable tetramers (the active formof PK) PKM2 exists as both dimers and tetramers [18 19]The PKM2 dimer has a higher 119870

119898towards PEP than the

tetramer and thus is less active in converting PEP to ATPand pyruvate [19 20] While tetrameric PKM2 favors ATPproduction through the TCA cycle dimeric PKM2 plays acritical role in aerobic glycolysis (Figure 1) [19] Thereforethe dynamic equilibrium between dimer and tetramer PKM2allows proliferating cells to regulate their needs for anabolicand catabolic metabolismThis not only explains why cancercells predominantly express PKM2 but also reveals theexistence of mechanisms that regulate this dynamic equilib-rium To ensure PKM2 expression cancer cells also developmechanisms for alternative splicing to produce PKM2 ratherthan PKM1 These mechanisms are regulated by oncogenesand tumor suppressors [21ndash25] Surprisingly dimeric PKM2has additional functions in regulating gene expression in thenucleus [26]

2 PKM2 Contributes to Tumorigenesis

A large body of evidence supports the notion that cancerspredominantly express PKM2 [14] Immunohistochemicalanalysis revealed that PKM2 is commonly expressed incolon cancer [12] renal cell carcinoma (RCC) [27] and lungcancer [28] PKM2 has been suggested to be a marker forRCC [29 30] and testicular cancer [31] Elevation of serumPKM2 levels was reported in patients with colon cancer [32]breast cancer [33] urological tumors [34] lung carcinomacervical cancer and gastrointestinal tumor [18] PKM2 wasdetected in the feces of patients with gastric and colorectalcancers [35] Recently mass spectrometry has demonstratedincreases in PKM2 and the predominant presence of PKM2was confirmed in RCC bladder carcinoma hepatocellularcarcinoma colorectal cancer lung carcinoma and follicularthyroid adenoma [16]

PKM2 expression correlates with tumorigenesis Highlevels of PKM2 associate with poor prognosis for patientswith signet ring cell gastric cancer [36] A unique patternof four expressed genes including PKM2 was reportedto predict outcomes for mesothelioma patients undergoingsurgery [37] Events that negatively impact tumorigenesis canalso reduce PKM2 function Vitamins K3 and K5 inhibittumorigenesis along with potently inhibiting PKM2 activity[38] Butyrate displays anticolon cancer effects along withthe inhibition of PKM2 expression in neoplastic but notnontumor colon tissues [39] Shikonin a derivative of aChinese herb with antitumor activities induces necrosis and

inhibits PKM2 expression in cancer cell lines [40] A reversecorrelation was observed between antitumor microRNA-326andPKM2 in glioma [41] Finally the Spry2 tumor suppressorwas reported to inhibit hepatocarcinogenesis via the MAPKand PKM2 pathways [42]

Furthermore PKM2 possesses activities that directly pro-mote tumorigenesis Overexpression of PKM2 upregulatesBcl-xL in gastric cancer and promotes the proliferation andmigration of colon cancer cells [43 44] Knockdownof PKM2using specific siRNA inhibited cancer cellrsquos proliferation andinvasion in vitro and the formation of xenograft tumors invivo [41 45]

3 PKM2 Promotes Tumorigenesis viaRegulating the Warburg Effect

The needs of energy production (ATP) and synthesis ofcellular building blocks for proliferating cancer cells dictatethe shift from oxidative to glycolytic metabolism even undernormoxic conditions the Warburg effect or aerobic glycol-ysis [2 7 8] Under hypoxic conditions cells metabolizeglucose by anaerobic glycolysis a process that is regulatedby two master transcription factors hypoxia-inducible factor(HIFs) and c-Myc [46] Both transcriptional factors are alsocritical for aerobic glycolysis in cancer cells Consistent withPKM2 being essential for aerobic glycolysis a relationshipexists among HIF-1 c-Myc and PKM2 We will discuss thecurrent understanding of these relationships

31 Positive Feedback Regulation between PKM2 andHIF-1 Itwas first demonstrated by Christofk and colleagues in 2008that knockdown of PKM2 in a panel of cancer cell linesdecreased the rate of glycolysis and proliferation IntroducingPKM2 but not PKM1 to the knockdown cells not onlyenhanced glycolysis but also increased the ability to formxenograft tumors [12] This research elegantly revealed thatPKM2 is important and that the level of PK activity is essen-tial as the defects in PKM2 knockdown cells in supportingtumorigenesis could not be corrected by overexpression ofthe more active isoform PKM1 Furthermore in comparisonto PKM1 rescued cells reintroducing PKM2 into knockdowncells rescued the deficiency of cell proliferation under hypoxicconditions

This investigation also suggests that PKM2 may con-tribute to the adaptive response (hypoxia response) of cellsto hypoxia which is specifically relevant to tumorigenesisas solid cancers consistently face hypoxia intratumorallyIt is thus a typical characteristic that cancers consistentlyexecute hypoxia response In the heart of this response liesthe master transcription factor hypoxia-inducible factor 1(HIF-1) [47] HIF-1 is a heterodimeric transcription factorconsisting of HIF-1120572 and HIF-1120573 The 120573 subunit is consti-tutively expressed while the 120572 subunit is directly regulatedby oxygen (O

2) levels [48 49] Under normoxic conditions

HIF-1120572 is hydroxylated at prolines (P) 402 and 564 by threeprolyl hydroxylase domain proteins (PHD1-3) in the presenceof oxygen 120572-ketoglutarate iron and ascorbate [50] Thisresults in the ubiquitination of prolyl-hydroxylated HIF-1120572


by the von Hippel-Lindau (VHL) tumor suppressor and thesubsequent degradation of HIF-1120572 [51 52] Under hypoxicconditions HIF-1120572 is stabilized as a result of inhibiting prolylhydroxylation allowing HIF-1120572 to dimerize with HIF-1120573 inthe nucleus This leads to transcription of a set of genesto cope with reduced O

2availability [53ndash55] These target

genes include those responsible for promoting glycolysis[56] HIF-1 transactivates the glucose transporters GLUT1and GLUT3 hexokinase (the first kinase in the glycolysispathway) lactate dehydrogenase A (LDHA) and pyruvatedehydrogenase kinase 1 which phosphorylates and inhibitspyruvate dehydrogenase (PDH) [57] (Figure 1) Consistentwith the Warburg effectrsquos association with synthesis of cel-lular building blocks HIF-1 also transactivates glucose-6-phosphate dehydrogenase (G6PD) to channel glucose-6-Pinto the pentose phosphate shunt for nucleotide and aminoacid synthesis (Figure 1) [56]Therefore the collective actionsof HIF-1 transcription activity seem to shift cells from oxida-tive metabolism to glycolysis (Figure 1) In line with theseobservations PKM2 shares an intimate connectionwithHIF-1 The first intron of the PKM2 gene contains the functionalhypoxia-response element (HRE) thus alsomaking it a targetof HIF-1 [21]

PKM2 also possesses a positive feedback regulationtowards HIF-1 PKM2 interacts with HIF-1120572 a process thatrequires the prolyl hydroxylase 3 (PHD3) PHD3 binds to andcauses hydroxylation of PKM2 at P303408 This associationand hydroxylation induces PKM2 to interact with HIF-1120572which plays a role inHIF-1-mediated transactivation of targetgenes including the LDHA PDK1 and VEGFA (encoding thevascular endothelial growth factor) genes [21] AdditionallyPKM2binds to p300 and enhances its recruitment to theHREsites of HIF-1 target genes Taken together PKM2 functionsas a HIF-1 coactivator by enhancing the Warburg effect incancers [21 22]

The regulation between HIF-1 and PKM2 also occursunder normoxic conditions by changes in other signallingevents which act to stabilize HIF-1120572 in cancer cells HIF-1 is stabilized by mTOR and induced for degradation byVHL Activation of mTOR is inhibited by tumor suppressorsTSC1TSC2 and facilitated by the PI3 K-AKT pathway [57]Consistent with this knowledge abnormal activation of thePI3 K-AKT-mTOR pathway and loss of function of tumorsuppressors VHL TSC12 and PTEN have been demon-strated to stabilize HIF-1120572 [57 58] Activation of mTORby downregulation of TSC12 and PTEN induced PKM2expression via stabilization of HIF-1120572 [59] PKM2 makesessential contributions tomTOR-mediated aerobic glycolysisas knockdown of PKM2 reduced glucose consumption andlactate production in cells with elevated mTOR activationFurthermore downregulation of PKM2 also suppressedmTOR-mediated tumorigenesis [59]

32 Positive Feedback Regulation between PKM2 and c-MycThe PKM2 gene produces both M1 andM2 isoforms throughalternative splicing The difference between these is theinclusion of exon 9 and exclusion of exon 10 for PKM1and vice versa for PKM2 (Figure 2) [5 15] This mutuallyexclusive pattern of splicing is mediated by members of the

heterogeneous nuclear ribonucleoprotein (hnRNP) familyhnRNPA1 hnRNPA2 and hnRNP1PTB (polypyrimidinetrack binding protein) [23 60] Binding of these proteins tothe DNA sequence flanking exon 9 prevents its inclusionresulting in the inclusion of exon 10 [23 60 61] In order toachieve predominant expression of the M2 isoform cancercells have a strategy to preferentially splice the M2 isoformover M1 through c-Myc-mediated upregulation of hnRNPA1hnRNPA2 and PTB (Figure 2) [23 61] This finding issupported by the discovery that cells with high levels ofc-Myc activity also demonstrated high PKM2PKM1 ratios[23 62] These observations are well in line with a large bodyof evidence indicating that c-Myc stimulates glycolysis andis required to coordinate with HIF-1 to regulate the cellularresponse to hypoxia [24 46] Thus evidence suggests thatPKM2 plays a role in c-Myc-mediated cancer metabolismand in c-Mycrsquos communication with HIF-1 Adding to thisattractive possibility is a recent demonstration that PKM2also upregulates c-Myc transcription [63 64] suggestinganother positive feedback loop involving PKM2 in regulatingthe Warburg effect Taken together PKM2 is an integratedpiece in the network of glycolysis regulation together withHIF-1 and c-Myc The importance of hnRNPA1 hnRNPA2and PTB in splicing PKM2 has also been explored bytumour suppression activity The microRNAs mir-124 mir-137 and mir-340 inhibit colorectal cancer growth by repress-ing the expression of these hnRNAs favouring PKM1 splicingthereby inhibiting aerobic glycolysis or the Warburg effect[65]

4 Regulation of PKM2 in the Warburg Effectduring Tumorigenesis

Cancers have developed a complex regulation of PKM2 tomeet the needs for energy and synthesis of nucleotides aminoacids and lipids These mechanisms center on regulatingPKM2rsquos expression allosteric regulation and modificationsThe latter twomechanisms directly or indirectly affect PKM2activity through physical interaction and by regulating thePKM2 dimer-tetramer dynamic

41 TranscriptionRegulation In addition to the above discus-sion of HIF-1 and c-Myc-mediated transcription and splicingof PKM2 transcription of the PKM2 gene is also regulated bythe SP1 and SP3 transcription factors [5 22 66]The networkof PI3 K-AKT-mTOR (mammalian target of rapamycin) playsa critical role in cell metabolism proliferation and survivaland is one of the most frequently activated pathways incancer owing to the activation of kinases and the inactivationof tumor suppressors TSC12 (tuberous sclerosis 12) andPTEN [67] Nutrient status is well known tomodulate mTORactivation [68] Under normoxic conditions mTOR activityinduces PKM2 expression through the combination of HIF-1120572 and c-Myc [59 69] Inhibition of mTOR has been foundto reduce glycolysis and PKM2 expression [70] Elevationin PTEN function reduces glucose uptake and the Warburgeffect and inhibits PKM2 expression [25] In a feedback


Exon 10 Exon 11 Exon 9 Exon 8 PKM2

PKM1

hnRNPA1hnRNPA2

PTB

PKM2

c-Myc

Figure 2 Schematic illustration of alternative splicing of PKM1 and PKM2 The proportion of the PKM2 gene is shown c-Myc upregulatesthe indicated complex which inhibits the splicing for exon 9 resulting in its exclusion in PKM2 PTB polypyrimidine track binding proteinhnRNPA1 and hnRNPA2 heterogeneous nuclear ribonucleoprotein 1 and 2

manner PKM2 is able to sustain mTOR activation in serine-depleted medium by enhancing endogenous serine synthesis[71] Taken together evidence supports that the upregulationof PKM2 plays an important role in the mTOR-mediatedWarburg effect in tumors

42 Regulation of the Dimer-Tetramer Dynamics Tumor cellsexpress high levels of dimer PKM2 [14 32] Among thefour PK isoforms PKM2 is the only one to be allosteri-cally regulated between a less active dimer and an activetetramer [18 19] These different forms of PKM2 regulateglucose metabolism through either the TCA cycle or gly-colysis Accumulating evidence supports the concept thatthe less active PKM2 dimer drives aerobic glycolysis whilethe active PKM2 tetramer produces pyruvate for oxidativephosphorylation (Figure 1) [12 72ndash74] PKM2 is regulatedby fructose-16-biphosphate (FBP) an upstream interme-diate of glycolysis which when bound to PKM2 activatestetramerization through high affinity association [75ndash77]Binding of tyrosine-phosphorylated peptides dissociates FBPfrom the PKM2 tetramer resulting in conversion to thePKM2 dimer [72] The less active PKM2 dimer is critical inmediating aerobic glycolysis in tumor cells based on highlevels of lactate production and lower oxygen consumption[72] Disrupting the binding of the phosphotyrosine peptidein a PKM2 mutant (M2KE) increased PKM2 kinase activitywhich was associated with reduction in lactate productionand elevation of oxygen consumption [72] In supporting thelow levels of cellular pyruvate kinase activity being criticalfor aerobic glycolysis replacing PKM2 with PKM1 led to anincrease in cellular pyruvate kinase activity decreasing lactateproduction and elevating oxygen consumption [12 74]

Collectively evidence supports that the PKM2 dimer iscritical in mediating aerobic glycolysis In addition to theabove mechanism regulating PKM2 activity PKM2 was alsocontrolled by tyrosine phosphorylation [73] It was observedin 1988 that PKM2 was tyrosine-phosphorylated in v-Src-transformed chicken embryo cells This phosphorylationreduced the affinity of PKM2 towards its substrate phospho-enolpyruvate (PEP) [78] In vitro v-Src was able to directlyphosphorylate PKM2 [78] Although this investigation

suggested that v-Src phosphorylated PKM2 the sites ofphosphorylation remain unknown Recent developmentdemonstrated that PKM2 was phosphorylated at severaltyrosine residues including Y105 by fibroblast growth factorreceptor type 1 (FGFR1) [73] Phosphorylation at Y105 causesFBP to dissociate from the PKM2 tetramer which resultsin PKM2 dimers and promotes the Warburg effect basedon the production of lactate [73] Conversely abolishingY105 phosphorylation by substitution with phenylalanine(Y105F) elevated the kinase activity resulting in decreasedlactate production and increased oxygen consumption [73]Taken together evidence demonstrates that phosphorylationat Y105 plays a role in the conversion of PKM2 tetramers todimers

More importantly regulation of PKM2 dimer andtetramer conversion is critical for tumorigenesis While theless active PKM2dimer enhances xenograft tumor formationenforced formation of active PKM2 (KE and Y105F muta-tions) and replacing PKM2 with PKM1 inhibited the forma-tion of xenograft tumors [72 73 79] In line with this conceptthe conversion between dimer and tetramer PKM2 is alsoused in tumour suppression to inhibit tumorigenesis Thedeath-associated protein kinase (DAPK) tumor suppressoractivates PKM2 by stabilizing the PKM2 tetramer via a directassociation This reduces cancer metabolism or the Warburgeffect which may be one aspect of DAPK-mediated tumorsuppression [80 81]

In line with these observations several small moleculePKM2 activators have been identified Among them DASA-58 (the substitutedNN1015840-diarylsulfonamideNCGC00185916)and TEPP-46 (the thieno-[32-b]pyrrole [32-d]pyridazinoneNCGC00186528) activate PKM2 by inducing PKM2tetramerization Unlike FBP-induced activation the tetramerinduced by these compounds is resistant to tyrosine-phosphorylated peptide-mediated conversion to the PKM2dimer This suggests that FBP and these small moleculeactivators bind PKM2 at distinct sites but importantly allinhibit tumorigenesis [74 82 83] Additionally a new set ofchemical platform bases the quinolone sulfonamide-basedPKM2 activators have recently been reported Similar toDASA-58 and TEPP-46 these activators also stabilize thePKM2 tetramer via binding to a pocket distinct from FBP


PKM2 dimer

Oct

4

Tran

sact

ivity

EGFRSrc

P-Y3

33P-

Y333 Cyclin D

c-Myc

Kinase activity Stat

3

P-Y7

05

HIF

-1120572

120573-C

aten

in

Figure 3 Diagram showing the nuclear function of PKM2 PKM2 dimers in the nucleus bind to Oct 4 and HIF-1120572 and enhance theirtranscription activity EGFR signal activates Src tyrosine kinase which phosphorylates 120573-catenin at tyrosine (Y) 333 (P-Y333) PKM2 bindsY333-phosphorylated 120573-catenin contributing to 120573-catenin-mediated transcription of cyclin D and c-Myc PKM2 dimer possesses kinaseactivity that phosphorylates Stat 3 at Y705 which enhances Stat 3rsquos transcriptional activity

binding and thus prevent the PKM2 tetramer from tyrosine-phosphorylated peptide-mediated disruption Quinolonesulfonamide-based PKM2 activators reduce carbon flowtowards the serine biosynthetic pathway rendering cells toserine auxotrophy [84]

43 Factors Affecting PKM2 Activity via Physical Associa-tion In addition to the above two small molecule PKM2activators a third activator was recently reported by thesame research group based on modifications to one of theirprevious compounds [85] The mechanism underlying thisactivation remains to be defined A series of PKM2 activators(1-(sulfonyl)-5-(arylsulfonyl)indoline) were also reportedvery recently [86] In contrast to these potent small moleculePKM2 inhibitors which may in part induce cell death byinhibiting PKM2 activity have also been developed [87]Furthermore the pyruvate kinase activity of PKM2 canbe inhibited by association with several distinct proteinsWhile the nuclear promyelocytic leukemia (PML) proteinfunctions as a tumor suppressor cytosolic PML was reportedto specifically inhibit tetrameric but not dimeric PKM2activity thereby contributing to the Warburg effect [88]Prolactin signal promotes cell proliferation by inducing itsreceptor to associate with PKM2 leading to PKM2 activityreduction [89] The MUC1-C oncoprotein was reported topromote breast cancer tumorigenesis in part via inhibitingPKM2 activity Although interaction of MUC1-C Cys3 withPKM2 C-domain Cys474 results in activation of PKM2oncogenic signals from EGFR (epidermal growth factorreceptor) can alter the association of MUC1-C and PKM2thereby leading to inhibition of PKM2 activity [90] EGFRphosphorylates MUC1-C at tyrosine 46 causing MUC1-Cto interact with PKM2 at Lys433 This association inhibitstetrameric PKM2 activity and thereby increases aerobic gly-colysis along with glucose uptake [90] PKM2 was also foundto interact with human papillomavirus 16 (HPV16) proteinE7 which may contribute to HPV16-induced cervical cancer[91] A potential therapeutic protein TEM8-Fc consistingof a portion of the tumor endothelial marker 8 (TEM8)

and the Fc domain of human IgG1 was found to associatewith PKM2 [92] Whether this interaction contributed toTEM8-Fc-associated tumor suppression was not clear [92]Consistent with the knowledge that PKM2 plays a critical rolein regulating aerobic glycolysis and biosynthesis for cellularbuilding blocks PKM2 is activated by serine but inhibited byalanine and phenylalanine when bound to these amino acids[93]

44 Posttranslational Modifications of PKM2 A reduction inactivity was reported by acetylation of PKM2 at lysine (K)305 in response to high levels of glucose This modificationreduces PKM2 activity and its affinity towards the PEPsubstrate resulting in PKM2 degradation via chaperone-mediated autophagy [94] As a result acetylation enhancescell proliferation by increasing the availability of glycolyticintermediates for anabolic synthesis [94 95]

PKM2 also plays a role in cell survival to oxidative stressAcute increases in intracellular levels of ROS (reactive oxygenspecies) induce oxidation of PKM2 at Cys358 This reducesPKM2 activity which allows the accumulation of glucose-6-phosphate and thus shifts glucose flux through the pentosephosphate pathway (PPP) to generate reduced NADPH(Figure 1) As PPP is the major pathway of generatingreduced NADPH oxidation-mediated inhibition of PKM2is therefore a mechanism of detoxification during oxidativestress Consistent with this notion substitution of C358 withS358 to produce oxidation-resistant mutants sensitized cellsto oxidative stress and inhibited xenograft tumor formation[96] A similar antioxidative stress function of PKM2 is alsomediated through binding to CD44 a major cell adhesionmolecule Cancer stem cells are known to be CD44 posi-tive so this interaction is consistent with CD44 promotingcancer progression metastasis and chemoresistance [97 98]CD44rsquos tumorigenic function is in part also attributableto its association with EGFR [99] Consistent with theseobservations PKM2 was reported to bind CD44 resulting inreceptor tyrosine kinase-mediated phosphorylation of PKM2and inhibition of PKM2 activity This enhanced glucose flux


through the PPP pathway to generate reduced NADPH andcounteract oxidative stresses through detoxification [61 100]

5 The Nuclear Function of PKM2

PKM2 displays intriguing nonglycolytic functions in thenucleus In addition to its cytoplasmic presence to regulateaerobic glycolysis PKM2 was also detected in the nucleusin response to interleukin-3 and apoptotic signals [101 102]Nuclear PKM2 binds Oct 4 through its C-terminal region(residues 307ndash531) enhancing Oct-4-mediated transcription[103] (Figure 3) Nuclear PKM2 was also reported to be acoactivator of HIF-1 [21] (Figure 3) EGFR signaling wasreported to activate Src tyrosine kinase which in turnphosphorylates 120573-catenin at Y333 PKM2 binds to tyrosine-phosphorylated 120573-catenin in the nucleus and contributesto 120573-catenin-mediated transactivation of cyclin D andc-Myc thereby promoting both cell proliferation and tumorprogression (Figure 3) This process requires the kinaseactivity of PKM2 [63 104] Since the binding of tyrosine-phosphorylated peptides maintains PKM2 in its dimer sta-tus [72] these observations suggest that dimerized PKM2binds and enhances 120573-catenin function in which a newkinase activity rather than pyruvate kinase activity mightbe involved Indeed it was very recently reported that thePKM2dimer contributes to its nuclear function andpossessesprotein tyrosine kinase activity Surprisingly instead of usinghigh-energy ATP PKM2 uses the high-energy phosphatefrom PEP as a phosphate donor to phosphorylate its proteinsubstrates [26] The PKM2 dimer phosphorylates Stat 3 atY705 in the nucleus and thus enhances Stat 3 transcriptionactivity [26 105] (Figure 3) Taken together while tetramerPKM2 is a pyruvate kinase dimer PKM2 can also act as aprotein tyrosine kinase [26]

6 Concluding Remarks andFuture Perspectives

The last decade has seen a high reemergence of interest inthe Warburg effect the typical cancer cell metabolism thatwas reported almost 90 years ago The detailed molecularand genetic knowledge accumulated in the last few decadesof extensive cancer research has rapidly advanced our under-standing of cancermetabolismMutations in several enzymesof the TCA-cycle were discovered including isocitrate dehy-drogenases 1 and 2 (IDH1 and IDH2) succinate dehydroge-nase (SDH) and fumarate hydratase (FH) [106ndash109] Thesemutations collectively reduce TCA-cycle-mediated oxidativephosphorylation resulting in an accumulation ofmetabolitesfor the biosynthesis of amino acids nucleotides and lipidsas well as increases in glucose uptake [110] The increases inglucose uptake together with aerobic glycolysis yield a robustelevation of lactate productionAlthough recent developmentsuggests that the by-product of aerobic glycolysis (lactate)contributes to overall tumorigenesis [111 112] it is also criticalfor cancer cells to efficiently export lactate to maintain theflux of glycolysis and to prevent cellular acidification [112]Cancer cells accomplish this task in part by upregulation

of the monocarboxylate transporters (MCTs) [112] Anotherstrategy to reduce the cellular burden of lactate accumulationduring aerobic glycolysis may be the prevention of a completeconversion of glucose to lactate (1 glucose for every 2 lactatemolecules) by reducing the conversion of PEP to pyruvateThis would allow the glycolytic intermediates to be used formacromolecular synthesis Therefore predominantly usingthe less active PKM2 dimer fits this logic

Accumulating evidence obtained in the last 10 yearsdemonstrates that PKM2rsquos glycolytic enzyme activity is regu-lated by oncogenes and tumor suppressors [21ndash25]These reg-ulations center on modulation of aerobic glycolysis Favoringa shift of the dimer-tetramer dynamic towards dimerizationis critical for PKM2 to promote theWarburg effect leading tocell proliferation and tumorigenesis

Surprisingly in addition to its glycolytic pyruvate kinaseactivity in the cytosol the PKM2 dimer also displays proteintyrosine kinase activity in the nucleus and nuclear PKM2pro-motes the transcriptional activities of HIF 120573-catenin STAT 3and Oct 4 [21 26 63 103ndash105] This all indirectly contributesto cancer metabolism and other aspects of tumorigenesisIn light of this new development future research shoulddetermine the contributions of the cytosolic versus nuclearPKM2 dimer to aerobic glycolysis

Effort is currently underway to target PKM2 for cancertherapy which is part of the current attempt in targetingcancer metabolism Several small molecule PKM2 inhibitorsand activators have been developed [61] As nearly completeknockdown of PKM2 does not completely inhibit cancercell proliferation the utility of PKM2 inhibition in targetingcancer should be cautious [61] On the other hand smallmolecule activators might be an attractive approach How-ever several factors call for precautions in targeting PKM2(1) PKM2 is also expressed in normal tissue [16 17] andthe function of PKM2 in normal tissues has not yet beendetermined (2) genetic changes in PKM2 have not beenreported in primary cancers (3) despitemodulation of PKM2which affects formation of xenograft tumors whether tissue-specific manipulation of PKM2 impacts tumorigenesis is stillon the waiting list (4) as PKM2 was detected in cancerstroma [113 114] whether it plays a role in tumorigenesisby affecting cancer-associated fibroblasts is not clear (5)while aerobic glycolysis has been a hot topic in the lastdecade its impact on cancer stem cells (CSCs) has not beenaddressed As it is becoming increasingly clear that CSCsplay a critical role in tumorigenesis especially in tumorprogression and metastasis [115] it would appear critical tounderstand whether targeting cancer metabolism in generaland PKM2 in particular will have an inhibitory effect onCSCs This knowledge became important as it was suggestedthat glioma CSCs (GSCs) may not use aerobic glycolysis tothe same degree as differentiated cancer cells Thus targetingPKM2 or cancer metabolism may still spare GSCs [116]

Acknowledgments

This work was in part supported by a Grant from Kid-ney Foundation of Canada (KFOC110017 2011ndash2013) to D


Tang and by St Josephrsquos Healthcare Hamilton ON Canadathrough financial support to the Hamilton Centre for KidneyResearch (HCKR)

References

[1] O Warburg F Wind and E Negelein ldquoThe metabolism oftumors in the bodyrdquo The Journal of General Physiology vol 8no 6 pp 519ndash530 1927

[2] OWarburg ldquoOn the origin of cancer cellsrdquo Science vol 123 no3191 pp 309ndash314 1956

[3] R Curi P Newsholme and E A Newsholme ldquoMetabolism ofpyruvate by isolated rat mesenteric lymphocytes lymphocytemitochondria and isolated mouse macrophagesrdquo BiochemicalJournal vol 250 no 2 pp 383ndash388 1988

[4] T Pfeiffer S Schuster and S Bonhoeffer ldquoCooperation andcompetition in the evolution of ATP-producing pathwaysrdquoScience vol 292 no 5516 pp 504ndash507 2001

[5] B Chaneton and E Gottlieb ldquoRocking cell metabolism revisedfunctions of the key glycolytic regulator PKM2 in cancerrdquoTrends in Biochemical Sciences vol 37 no 8 pp 309ndash316 2012

[6] P E Porporato S Dhup R K Dadhich T Copetti and PSonveaux ldquoAnticancer targets in the glycolytic metabolism oftumors a comprehensive reviewrdquo Frontiers in Pharmacologyvol 2 p 49 2011

[7] P P Hsu and D M Sabatini ldquoCancer cell metabolismWarburgand beyondrdquo Cell vol 134 no 5 pp 703ndash707 2008

[8] M G V Heiden L C Cantley and C B Thompson ldquoUnder-standing the warburg effect the metabolic requirements of cellproliferationrdquo Science vol 324 no 5930 pp 1029ndash1033 2009

[9] L Stryer Glycolysis in Biochemistry WH Freemen New YorkNY USA 1995

[10] M E Munoz and E Ponce ldquoPyruvate kinase current statusof regulatory and functional propertiesrdquo Comparative Biochem-istry and Physiology B vol 135 no 2 pp 197ndash218 2003

[11] G Valentini L Chiarelli R Fortini M L Speranza AGalizzi and A Mattevi ldquoThe allosteric regulation of pyruvatekinase a site-directed mutagenesis studyrdquo Journal of BiologicalChemistry vol 275 no 24 pp 18145ndash18152 2000

[12] H R Christofk M G Vander Heiden M H Harris et al ldquoTheM2 splice isoform of pyruvate kinase is important for cancermetabolism and tumour growthrdquoNature vol 452 no 7184 pp230ndash233 2008

[13] T Noguchi K Yamada H Inoue T Matsuda and T TanakaldquoThe L- and R-type isozymes of rat pyruvate kinase are pro-duced from a single gene by use of different promotersrdquo Journalof Biological Chemistry vol 262 no 29 pp 14366ndash14371 1987

[14] K Yamada and T Noguchi ldquoNutrient and hormonal regulationof pyruvate kinase gene expressionrdquo Biochemical Journal vol337 no 1 pp 1ndash11 1999

[15] T Noguchi H Inoue and T Tanaka ldquoThe M1- and M2-type isozymes of rat pyruvate kinase are produced from thesame gene by alternative RNA splicingrdquo Journal of BiologicalChemistry vol 261 no 29 pp 13807ndash13812 1986

[16] K Bluemlein N M Gruning R G Feichtinger H Lehrach BKofler andMRalser ldquoNo evidence for a shift in pyruvate kinasePKM1 to PKM2 expression during tumorigenesisrdquo Oncotargetvol 2 no 5 pp 393ndash400 2011

[17] K Imamura and T Tanaka ldquoMultimolecular forms of pyruvatekinase from rat and othermammalian tissues I electrophoreticstudiesrdquo Journal of Biochemistry vol 71 no 6 pp 1043ndash10511972

[18] S Mazurek C B Boschek F Hugo and E EigenbrodtldquoPyruvate kinase type M2 and its role in tumor growth andspreadingrdquo Seminars in Cancer Biology vol 15 no 4 pp 300ndash308 2005

[19] S Mazurek ldquoPyruvate kinase type M2 a key regulator of themetabolic budget system in tumor cellsrdquo International Journalof Biochemistry andCell Biology vol 43 no 7 pp 969ndash980 2011

[20] W Luo and G L Semenza ldquoPyruvate kinase M2 regulatesglucosemetabolismby functioning as a coactivator for hypoxia-inducible factor 1 in cancer cellsrdquo Oncotarget vol 2 no 7 pp551ndash556 2011

[21] W Luo H Hu R Chang et al ldquoPyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1rdquo Cell vol145 no 5 pp 732ndash744 2011

[22] W Luo and G L Semenza ldquoEmerging roles of PKM2 in cellmetabolism and cancer progressionrdquo Trends in Endocrinologyand Metabolism vol 23 no 11 pp 560ndash566 2012

[23] C J David M Chen M Assanah P Canoll and J L ManleyldquoHnRNP proteins controlled by c-Myc deregulate pyruvatekinase mRNA splicing in cancerrdquoNature vol 463 no 7279 pp364ndash368 2010

[24] DMMiller S DThomas A IslamDMuench andK Sedorisldquoc-Myc and cancer metabolismrdquo Clinical Cancer Research vol18 no 20 pp 5546ndash5553 2012

[25] I Garcia-Cao M S Song R M Hobbs et al ldquoSystemicelevation of PTEN induces a tumor-suppressive metabolicstaterdquo Cell vol 149 no 1 pp 49ndash62 2012

[26] X Gao H Wang J J Yang X Liu and Z R Liu ldquoPyruvatekinase M2 regulates gene transcription by acting as a proteinkinaserdquoMolecular Cell vol 45 no 5 pp 598ndash609 2012

[27] U Brinck E Eigenbrodt M Oehmke S Mazurek and GFischer ldquoL- and M2- pyruvate kinase expression in renal cellcarcinomas and their metastasesrdquoVirchows Archiv vol 424 no2 pp 177ndash185 1994

[28] J Schneider K Neu H Grimm H G Velcovsky G Weisseand E Eigenbrodt ldquoTumor M2-pyruvate kinase in lung cancerpatients immunohistochemical detection and diseasemonitor-ingrdquo Anticancer Research vol 22 no 1A pp 311ndash318 2002

[29] H W Wechsel E Petri K H Bichler and G Feil ldquoMarker forrenal cell carcinoma (RCC) the dimeric formof pyruvate kinasetype M2 (Tu M2-PK)rdquo Anticancer Research vol 19 no 4A pp2583ndash2590 1999

[30] G M Oremek S Teigelkamp W Kramer E Eigenbrodt andK H Usadel ldquoThe pyruvate kinase isoenzyme tumor M2 (TuM2-PK) as a tumor marker for renal carcinomardquo AnticancerResearch vol 19 no 4A pp 2599ndash2601 1999

[31] T PottekMMuller T Blum andMHartmann ldquoTu-M2-PK inthe blood of testicular and cubital veins in men with testicularcancerrdquo Anticancer Research vol 20 no 6D pp 5029ndash50332000

[32] E Eigenbrodt D Basenau S Holthusen S Mazurek and GFischer ldquoQuantification of tumor type M2 pyruvate kinase (TuM2-PK) in human carcinomasrdquoAnticancer Research vol 17 no4B pp 3153ndash3156 1997

[33] D Luftner J Mesterharm C Akrivakis et al ldquoTumor typeM2 pyruvate kinase expression in advanced breast cancerrdquoAnticancer Research vol 20 no 6D pp 5077ndash5082 2000

[34] J Roigas G Schulze S Raytarowski K Jung D Schnorr and SA Loening ldquoTumor M2 pyruvate kinase in plasma of patientswith urological tumorsrdquo Tumor Biology vol 22 no 5 pp 282ndash285 2001


[35] P D Hardt M Toepler B Ngoumou J Rupp and H U KloerldquoMeasurement of fecal pyruvate kinase type M2 (Tumor M2-PK) concentrations in patients with gastric cancer colorectalcancer colorectal adenomas and controlsrdquoAnticancer Researchvol 23 no 2A pp 851ndash853 2003

[36] J Y Lim S O Yoon S Y Seol et al ldquoOverexpression ofthe M2 isoform of pyruvate kinase is an adverse prognosticfactor for signet ring cell gastric cancerrdquo World Journal ofGastroenterology vol 18 no 30 pp 4037ndash4043 2012

[37] G J Gordon L Dong B Y Yeap et al ldquoFour-gene expres-sion ratio test for survival in patients undergoing surgery formesotheliomardquo Journal of the National Cancer Institute vol 101no 9 pp 678ndash686 2009

[38] J Chen Z Jiang B Wang Y Wang and X Hu ldquoVitamin K(3)and K(5) are inhibitors of tumor pyruvate kinase M2rdquo CancerLetters vol 316 no 2 pp 204ndash210 2012

[39] F Jahns A Wilhelm K O Greulich et al ldquoImpact of butyrateon PKM2 and HSP90120573 expression in human colon tissuesof different transformation stages a comparison of gene andprotein datardquo Genes and Nutrition vol 7 no 2 pp 235ndash2462012

[40] J Chen J Xie Z Jiang BWang YWang and X Hu ldquoShikoninand its analogs inhibit cancer cell glycolysis by targeting tumorpyruvate kinase-M2rdquo Oncogene vol 30 no 42 pp 4297ndash43062012

[41] B Kefas L Comeau N Erdle E Montgomery S Amos and BPurow ldquoPyruvate kinase M2 is a target of the tumorsuppressivemicroRNA-326 and regulates the survival of glioma cellsrdquoNeuro-Oncology vol 12 no 11 pp 1102ndash1112 2010

[42] C Wang S Delogu C Ho et al ldquoInactivation of Spry2accelerates AKT-driven hepatocarcinogenesis via activation ofMAPK and PKM2 pathwaysrdquo Journal of Hepatology vol 57 no3 pp 577ndash583 2012

[43] O H Kwon T W Kang J H Kim et al ldquoPyruvate kinaseM2 promotes the growth of gastric cancer cells via regulationof Bcl-xL expression at transcriptional levelrdquo Biochemical andBiophysical Research Communications vol 423 no 1 pp 38ndash442012

[44] C F Zhou X B Li H Sun et al ldquoPyruvate kinase type M2is upregulated in colorectal cancer and promotes proliferationand migration of colon cancer cellsrdquo International Union ofBiochemistry and Molecular Biology Life vol 64 no 9 pp 775ndash782 2012

[45] M S Goldberg and P A Sharp ldquoPyruvate kinase M2-specificsiRNA induces apoptosis and tumor regressionrdquo Journal ofExperimental Medicine vol 209 no 2 pp 217ndash224 2012

[46] J D Gordan C B Thompson and M C Simon ldquoHIF and c-Myc sibling rivals for control of cancer cell metabolism andproliferationrdquo Cancer Cell vol 12 no 2 pp 108ndash113 2007

[47] W Liu S M Shen X Y Zhao and G Q Chen ldquoTargetedgenes and interacting proteins of hypoxia inducible factor-1rdquoInternational Journal of Biochemistry and Cell Biology vol 3 no2 pp 165ndash178 2012

[48] G LWang BH Jiang E A Rue andG L Semenza ldquoHypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimerregulated by cellular O

2tensionrdquo Proceedings of the National

Academy of Sciences of the United States of America vol 92 no12 pp 5510ndash5514 1995

[49] G L Semenza ldquoHypoxia-inducible factor 1 master regulator ofO2homeostasisrdquoCurrent Opinion in Genetics andDevelopment

vol 8 no 5 pp 588ndash594 1998[50] A C R Epstein J M Gleadle L A McNeill et al ldquoC

elegans EGL-9 and mammalian homologs define a family of

dioxygenases that regulate HIF by prolyl hydroxylationrdquo Cellvol 107 no 1 pp 43ndash54 2001

[51] P Jaakkola D R Mole Y M Tian et al ldquoTargeting of HIF-120572 tothe vonHippel-Lindau ubiquitylation complex by O

2-regulated

prolyl hydroxylationrdquo Science vol 292 no 5516 pp 468ndash4722001

[52] M Ivan K Kondo H Yang et al ldquoHIF120572 targeted for VHL-mediated destruction by proline hydroxylation implications forO2sensingrdquo Science vol 292 no 5516 pp 464ndash468 2001

[53] W G Kaelin and P J Ratcliffe ldquoOxygen sensing by metazoansthe central role of theHIF hydroxylase pathwayrdquoMolecular Cellvol 30 no 4 pp 393ndash402 2008

[54] L P Song J Zhang S F Wu et al ldquoHypoxia-induciblefactor-1120572-induced differentiation ofmyeloid leukemic cells is itstranscriptional activity independentrdquo Oncogene vol 27 no 4pp 519ndash527 2008

[55] R HWenger andM Gassmann ldquoOxygen(es) and the hypoxia-inducible factor-1rdquo Biological Chemistry vol 378 no 7 pp 609ndash616 1997

[56] T W Meijer J H Kaanders P N Span and J BussinkldquoTargeting hypoxia HIF-1 and tumor glucose metabolism toimprove radiotherapy efficacyrdquoClinical Cancer Research vol 18no 20 pp 5585ndash5594 2012

[57] G L Semenza ldquoHIF-1 upstream and downstream of cancermetabolismrdquoCurrentOpinion inGenetics andDevelopment vol20 no 1 pp 51ndash56 2010

[58] E Laughner P Taghavi K Chiles P C Mahon and G LSemenza ldquoHER2 (neu) signaling increases the rate of hypoxia-inducible factor 1120572 (HIF-1120572) synthesis novel mechanism forHIF-1-mediated vascular endothelial growth factor expressionrdquoMolecular and Cellular Biology vol 21 no 12 pp 3995ndash40042001

[59] Q Sun X Chen J Ma et al ldquoMammalian target of rapamycinup-regulation of pyruvate kinase isoenzyme type M2 is criticalfor aerobic glycolysis and tumor growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 10 pp 4129ndash4134 2011

[60] M Chen J Zhang and J LManley ldquoTurning on a fuel switch ofcancer hnRNPproteins regulate alternative splicing of pyruvatekinase mRNArdquo Cancer Research vol 70 no 22 pp 8977ndash89802010

[61] M Tamada M Suematsu and H Saya ldquoPyruvate kinase m2multiple faces for conferring benefits on cancer cellsrdquo ClinicalCancer Research vol 18 no 20 pp 5554ndash5561 2012

[62] C V Clower D Chatterjee Z Wang L C Cantley M G VHeidena and A R Krainer ldquoThe alternative splicing repressorshnRNP A1A2 and PTB influence pyruvate kinase isoformexpression and cell metabolismrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no5 pp 1894ndash1899 2010

[63] W Yang Y Xia H Ji et al ldquoNuclear PKM2 regulates 120573-catenintransactivation upon EGFR activationrdquo Nature vol 480 no7375 pp 118ndash122 2011

[64] I Harris S McCracken and T W Mak ldquoPKM2 a gatekeeperbetween growth and survivalrdquo Cell Research vol 22 no 3 pp447ndash449 2012

[65] Y Sun X Zhao Y Zhou and Y Hu ldquomiR-124 miR-137 andmiR-340 regulate colorectal cancer growth via inhibition of theWarburg effectrdquo Oncology Reports vol 28 no 4 pp 1346ndash13522012

[66] D J Discher N H Bishopric X Wu C A Peterson and K AWebster ldquoHypoxia regulates 120573-enolase and pyruvate kinase-Mpromoters by modulating Sp1Sp3 binding to a conserved GC


elementrdquo Journal of Biological Chemistry vol 273 no 40 pp26087ndash26093 1998

[67] J A McCubrey L S Steelman W H Chappell et al ldquoMuta-tions and deregulation of RasRafMEKERK and PI3KPTENAktmTOR cascades which alter therapy responserdquoOncotargetvol 3 no 9 pp 954ndash987 2012

[68] A Efeyan and D M Sabatini ldquoMTOR and cancer many loopsin one pathwayrdquo Current Opinion in Cell Biology vol 22 no 2pp 169ndash176 2010

[69] J P Bayley and P Devilee ldquoTheWarburg effect in 2012rdquoCurrentOpinion in Oncology vol 24 no 1 pp 62ndash67 2012

[70] M A Iqbal and R N Bamezai ldquoResveratrol inhibits cancercell metabolism by down regulating pyruvate kinase M2 viainhibition of mammalian target of rapamycinrdquo PLoS ONE vol7 no 5 Article ID e36764 2012

[71] J Ye A Mancuso X Tong et al ldquoPyruvate kinaseM2 promotesde novo serine synthesis to sustain mTORC1 activity and cellproliferationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 18 pp 6904ndash69092012

[72] HRChristofkMGVanderHeidenNWu JMAsara andLC Cantley ldquoPyruvate kinase M2 is a phosphotyrosine-bindingproteinrdquo Nature vol 452 no 7184 pp 181ndash186 2008

[73] T Hitosugi S Kang M G Vander Heiden et al ldquoTyrosinephosphorylation inhibits PKM2 to promote the warburg effectand tumor growthrdquo Science Signaling vol 2 no 97 p ra73 2009

[74] D Anastasiou Y Yu W J Israelsen et al ldquoPyruvate kinase M2activators promote tetramer formation and suppress tumorige-nesisrdquoNature Chemical Biology vol 8 no 10 pp 839ndash847 2012

[75] K Ashizawa P McPhie K H Lin and S Y Cheng ldquoAn invitro novel mechanism of regulating the activity of pyruvatekinase M2 by thyroid hormone and fructose 16-bisphosphaterdquoBiochemistry vol 30 no 29 pp 7105ndash7111 1991

[76] K Ashizawa M C Willingham C M Liang and S YCheng ldquoIn vivo regulation of monomer-tetramer conversion ofpyruvate kinase subtypeM2 by glucose is mediated via fructose16-bisphosphaterdquo Journal of Biological Chemistry vol 266 no25 pp 16842ndash16846 1991

[77] J D Dombrauckas B D Santarsiero andADMesecar ldquoStruc-tural basis for tumor pyruvate kinase M2 allosteric regulationand catalysisrdquo Biochemistry vol 44 no 27 pp 9417ndash9429 2005

[78] P Presek M Reinacher and E Eigenbrodt ldquoPyruvate kinasetype M2 is phosphorylated at tyrosine residues in cells trans-formed by Rous sarcoma virusrdquo FEBS Letters vol 242 no 1 pp194ndash198 1988

[79] C V Dang ldquoPKM2 tyrosine phosphorylation and glutaminemetabolism signal a different viewof thewarburg effectrdquo ScienceSignaling vol 2 no 97 p pe75 2009

[80] A Erol ldquoDeath-associated proliferation kinetic in normal andtransformed cellsrdquo Cell Cycle vol 11 no 8 pp 1512ndash1516 2012

[81] I Mor R Carlessi T Ast E Feinstein and A Kimchi ldquoDeath-associated protein kinase increases glycolytic rate throughbinding and activation of pyruvate kinaserdquo Oncogene vol 31no 6 pp 683ndash693 2012

[82] M B Boxer J K Jiang M G Vander Heiden et al ldquoEvaluationof substituted 1198731198731015840-diarylsulfonamides as activators of thetumor cell specific M2 isoform of pyruvate kinaserdquo Journal ofMedicinal Chemistry vol 53 no 3 pp 1048ndash1055 2010

[83] J K Jiang M B Boxer M G Vander Heiden et al ldquoEvaluationof thieno[32-b]pyrrole[32-d]pyridazinones as activators of thetumor cell specificM2 isoformof pyruvate kinaserdquoBioorganicampMedicinal Chemistry Letters vol 20 no 11 pp 3387ndash3393 2010

[84] C Kung J Hixon S Choe et al ldquoSmall molecule activationof PKM2 in cancer cells induces serine auxotrophyrdquo ChemicalBiology vol 19 no 9 pp 1187ndash1198 2012

[85] M J Walsh K R Brimacombe H Veith et al ldquo2-Oxo-N-aryl-1234-tetrahydroquinoline-6-sulfonamides as activators of thetumor cell specificM2 isoformof pyruvate kinaserdquoBioorganicampMedicinal Chemistry Letters vol 21 no 21 pp 6322ndash6327 2011

[86] A Yacovan R Ozeri T Kehat et al ldquo1-(sulfonyl)-5-(aryl-sulfonyl)indoline as activators of the tumor cell specific M2isoform of pyruvate kinaserdquo Bioorganic amp Medicinal ChemistryLetters vol 22 no 20 pp 6460ndash6468 2012

[87] M G Vander Heiden H R Christofk E Schuman et alldquoIdentification of small molecule inhibitors of pyruvate kinaseM2rdquo Biochemical Pharmacology vol 79 no 8 pp 1118ndash11242010

[88] N Shimada T Shinagawa and S Ishii ldquoModulation of M2-type pyruvate kinase activity by the cytoplasmic PML tumorsuppressor proteinrdquo Genes to Cells vol 13 no 3 pp 245ndash2542008

[89] B Varghese G Swaminathan A Plotnikov et al ldquoProlactininhibits activity of pyruvate kinaseM2 to stimulate cell prolifer-ationrdquo Molecular Endocrinology vol 24 no 12 pp 2356ndash23652010

[90] M Kosugi R Ahmad M Alam Y Uchida and D KufeldquoMUC1-C oncoprotein regulates glycolysis and pyruvate kinaseM2 activity in cancer cellsrdquo PLoS ONE vol 6 no 11 Article IDe28234 2011

[91] S Mazurek ldquoPyruvate kinase type M2 a key regulator withinthe tumour metabolome and a tool for metabolic profiling oftumoursrdquo Ernst Schering Foundation symposium proceedingsno 4 pp 99ndash124 2007

[92] H F Duan X W Hu J L Chen et al ldquoAntitumor activitiesof TEM8-Fc an engineered antibody-like molecule targetingtumor endothelial marker 8rdquo Journal of the National CancerInstitute vol 99 no 20 pp 1551ndash1555 2007

[93] K H Ibsen and S W Marles ldquoInhibition of chicken pyruvatekinases by amino acidsrdquo Biochemistry vol 15 no 5 pp 1073ndash1079 1976

[94] L Lv D Li D Zhao et al ldquoAcetylation targets theM2 isoform ofpyruvate kinase for degradation through chaperone-mediatedautophagy and promotes tumor growthrdquoMolecular Cell vol 42no 6 pp 719ndash730 2011

[95] A N Macintyre and J C Rathmell ldquoPKM2 and the trickybalance of growth and energy in cancerrdquoMolecular Cell vol 42no 6 pp 713ndash714 2011

[96] D Anastasiou G Poulogiannis J M Asara et al ldquoInhibitionof pyruvate kinaseM2 by reactive oxygen species contributes tocellular antioxidant responsesrdquo Science vol 334 no 6060 pp1278ndash1283 2011

[97] M Zoller ldquoCD44 can a cancer-initiating cell profit from anabundantly expressed moleculerdquo Nature Reviews Cancer vol11 no 4 pp 254ndash267 2011

[98] ONagano andH Saya ldquoMechanism and biological significanceof CD44 cleavagerdquo Cancer Science vol 95 no 12 pp 930ndash9352004

[99] S J Wang and L Y W Bourguignon ldquoRole of hyaluronan-mediated CD44 signaling in head and neck squamous cellcarcinoma progression and chemoresistancerdquo The AmericanJournal of Pathology vol 178 no 3 pp 956ndash963 2011

[100] M Tamada O Nagano S Tateyama et al ldquoModulation ofglucose metabolism by CD44 contributes to antioxidant statusand drug resistance in cancer cellsrdquoCancer Research vol 72 no6 pp 1438ndash1448 2012


[101] A Hoshino J A Hirst and H Fujii ldquoRegulation of cellproliferation by interleukin-3-induced nuclear translocation ofpyruvate kinaserdquo Journal of Biological Chemistry vol 282 no24 pp 17706ndash17711 2007

[102] A Stetak R Veress J Ovadi P Csermely G Keri and AUllrich ldquoNuclear translocation of the tumor marker pyruvatekinase M2 induces programmed cell deathrdquo Cancer Researchvol 67 no 4 pp 1602ndash1608 2007

[103] J Lee H K Kim Y M Han and J Kim ldquoPyruvate kinaseisozyme type M2 (PKM2) interacts and cooperates with Oct-4in regulating transcriptionrdquo International Journal of Biochem-istry and Cell Biology vol 40 no 5 pp 1043ndash1054 2008

[104] F Canal and C Perret ldquoPKM2 a new player in the 120573-cateningamerdquo Future Oncology vol 8 no 4 pp 395ndash398 2012

[105] G Semenova and J Chernoff ldquoPKM2 enters the morpheeinacademyrdquoMolecular Cell vol 45 no 5 pp 583ndash584 2012

[106] D W Parsons S Jones X Zhang et al ldquoAn integrated genomicanalysis of human glioblastoma multiformerdquo Science vol 321no 5897 pp 1807ndash1812 2008

[107] H Yan D W Parsons G Jin et al ldquoIDH1 and IDH2 mutationsin gliomasrdquoThe New England Journal of Medicine vol 360 no8 pp 765ndash773 2009

[108] T Bourgeron P Rustin D Chretien et al ldquoMutation of anuclear succinate dehydrogenase gene results in mitochondrialrespiratory chain deficiencyrdquo Nature Genetics vol 11 no 2 pp144ndash149 1995

[109] I P M Tomlinson N A Alam A J Rowan et al ldquoGermlinemutations in FH predispose to dominantly inherited uterinefibroids skin leiomyomata and papillary renal cell cancer themultiple leiomyoma consortiumrdquo Nature Genetics vol 30 no4 pp 406ndash410 2002

[110] B A Teicher W M Linehan and L J Helman ldquoTargetingcancermetabolismrdquoClinical Cancer Research vol 18 no 20 pp5537ndash5545 2012

[111] P Sonveaux F Vegran T Schroeder et al ldquoTargeting lactate-fueled respiration selectively kills hypoxic tumor cells in micerdquoJournal of Clinical Investigation vol 118 no 12 pp 3930ndash39422008

[112] S Dhup R K Dadhich P E Porporato and P SonveauxldquoMultiple biological activities of lactic acid in cancer influenceson tumor growth angiogenesis and metastasisrdquo Current Phar-maceutical Design vol 18 no 10 pp 1319ndash1330 2012

[113] G Bonuccelli D Whitaker-Menezes R Castello-Cros et alldquoThe reverse Warburg effect glycolysis inhibitors prevent thetumor promoting effects of caveolin-1 deficient cancer associ-ated fibroblastsrdquo Cell Cycle vol 9 no 10 pp 1960ndash1971 2010

[114] B Chiavarina D Whitaker-Menezes U E Martinez-Outschoorn et al ldquoPyruvate kinase expression (PKM1 andPKM2) in cancer-associated fibroblasts drives stromal nutrientproduction and tumor growthrdquo Cancer Biology and Therapyvol 12 no 12 pp 1101ndash1113 2011

[115] I Baccelli and A Trumpp ldquoThe evolving concept of cancer andmetastasis stem cellsrdquo Journal of Cell Biology vol 198 no 3 pp281ndash293 2012

[116] E Vlashi C Lagadec L Vergnes et al ldquoMetabolic state ofglioma stem cells and nontumorigenic cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 38 pp 16062ndash16067 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


GLUT

Glucose

GlucoseCytosol

Glucose-6-P

Fructose-6-P

Fructose-6-P2 (FBP)

Glyceraldehyde-3-P

3-Phosphoglycerate

Phosphoenolpyruvate (PEP)

HK

LDHA

PKM2 (tetramer)PKM2(dimer)

PyruvatePyruvate

Lactate

ADP

ATP

Pyruvate

Acetyl-CoAPDH

PDK1

6-Phosphogluconolactone 6-P-Gluconate

Ribose-5-phosphate

Pentosephosphatepathway

NADP

NADPH

G6PD

Nucleotide andamino acid synthesis

Serine synthesis

Citrate

Citrate

Lipid synthesis

MitochondriaCytosol

Glutamine

Glutamine

HIF-1

HIF-1

HIF

-1

Gly

coly

sis

Figure 1 Schematic illustrating the cancer utilization of the metabolic pathways Pyruvate kinase catalyzes the last step of glycolysis byconverting PEP and ADP to pyruvate and ATP respectively PKM2 dimers and tetramers possess low and high levels of Pyruvate kinaseactivity respectively With reduced enzymatic activity PKM2 dimer drives aerobic glycolysis which allows the intermediate metabolitesto be used for the synthesis of nucleotides amino acids and lipids and the production of reduced NADPH (see the pentose phosphatepathway) HIF-1 upregulates the indicated proteins GLUT glucose transporter HK hexokinase G6PD glucose-6-phosphate dehydrogenaseHIF-1 hypoxia-inducible factor 1 LDHA lactate dehydrogenase A PDK1 pyruvate dehydrogenase kinase isoenzyme 1 and PDH pyruvatedehydrogenase

conversion of glucose to pyruvate (1 molecule of glucose to 2molecules of pyruvate) This enables the upstream glycolyticintermediates to accumulate and thus contribute to the shiftof metabolism towards the anabolic phase for amino acidsnucleotides and lipid production (Figure 1) Cancer cellsexplore this logic by predominantly using PKM2 an isoform

of PK as its activity can be dynamically regulated between theless active PKM2 dimer and the highly active PKM2 tetramer[12]

PK consists of four isoforms the L (PKL) and R (PKR)isoforms encoded by the PKLR (1q22) gene and the M1(PKM1) and M2 (PKM2) isoforms encoded by the PKM2





























PKM1

hnRNPA1hnRNPA2

PTB

PKM2

c-Myc









PKM2 dimer

Oct

4

Tran

sact

ivity

EGFRSrc

P-Y3

33P-

Y333 Cyclin D

c-Myc


3

P-Y7

05

HIF

-1120572

120573-C

aten

in

















Acknowledgments




References

























































2-regulated













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























PKM1

hnRNPA1hnRNPA2

PTB

PKM2

c-Myc









PKM2 dimer

Oct

4

Tran

sact

ivity

EGFRSrc

P-Y3

33P-

Y333 Cyclin D

c-Myc


3

P-Y7

05

HIF

-1120572

120573-C

aten

in

















Acknowledgments




References

























































2-regulated













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














PKM1

hnRNPA1hnRNPA2

PTB

PKM2

c-Myc









PKM2 dimer

Oct

4

Tran

sact

ivity

EGFRSrc

P-Y3

33P-

Y333 Cyclin D

c-Myc


3

P-Y7

05

HIF

-1120572

120573-C

aten

in

















Acknowledgments




References

























































2-regulated













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



PKM1

hnRNPA1hnRNPA2

PTB

PKM2

c-Myc









PKM2 dimer

Oct

4

Tran

sact

ivity

EGFRSrc

P-Y3

33P-

Y333 Cyclin D

c-Myc


3

P-Y7

05

HIF

-1120572

120573-C

aten

in

















Acknowledgments




References

























































2-regulated













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


PKM2 dimer

Oct

4

Tran

sact

ivity

EGFRSrc

P-Y3

33P-

Y333 Cyclin D

c-Myc


3

P-Y7

05

HIF

-1120572

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Acknowledgments




References

























































2-regulated













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Acknowledgments




References

























































2-regulated













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



References

























































2-regulated













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























2-regulated













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Review Article PKM2, a Central Point of Regulation in Cancer Metabolismdownloads.hindawi.com/journals/ijcb/2013/242513.pdf · of proliferation. e M isoform of pyruvate kinase (PKM)