Dysregulated metabolism contributes to oncogenesis

Dysregulated metabolism contributes to oncogenesis

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Citation

Hirschey, Matthew D. et al. “Dysregulated Metabolism Contributes

to Oncogenesis.” Seminars in Cancer Biology 35 (2015): S129–S150.

© 2015 Elsevier Ltd

As Published

http://dx.doi.org/10.1016/j.semcancer.2015.10.002

Publisher

Elsevier

Version

Final published version

Citable link

http://hdl.handle.net/1721.1/105828

Terms of Use

Creative Commons Attribution-NonCommercial-NoDerivs License

ContentslistsavailableatScienceDirect

Seminars

in

Cancer

Biology

jou rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / s e m c a n c e r

Review

Dysregulated

metabolism

contributes

to

oncogenesis

Matthew

D.

Hirschey

_,

_Ralph

_J.

_DeBerardinis

_,

_Anna

_Mae

_E.

_Diehl

_,

Janice

E.

Drew

_,

_Christian

_Frezza

_,

_Michelle

_F.

_Green

_,

_Lee

_W.

_Jones

_,

Young

H.

Ko

_,

_Anne

_Le

_,

_Michael

_A.

_Lea

_,

_Jason

_W.

_Locasale

_,

_Valter

_D.

_Longo

_,

Costas

A.

Lyssiotis

_,

_Eoin

_McDonnell

_,

_Mahya

_Mehrmohamadi

_,

Gregory

Michelotti

_,

_Vinayak

_Muralidhar

_,

_Michael

_P.

_Murphy

_,

Peter

L.

Pedersen

_,

_Brad

_Poore

_,

_Lizzia

_Raffaghello

_,

_Jeffrey

_C.

_Rathmell

_,

Sharanya

Sivanand

_,

_Matthew

_G.

_Vander

_Heiden

_,

Kathryn

E.

Wellen

_,

_Target

_Validation

_Team

a_{DepartmentofMedicine,DukeUniversityMedicalCenter,Durham,NC27710,USA}

b_{DukeMolecularPhysiologyInstitute,DukeUniversityMedicalCenter,Durham,NC27701,USA} c_{DepartmentofPharmacology&CancerBiology,DukeUniversityMedicalCenter,Durham,NC27710,USA}

d_{Children’sMedicalCenterResearchInstitute,UniversityofTexas–SouthwesternMedicalCenter,Dallas,TX75390,USA} e_{RowettInstituteofNutritionandHealth,UniversityofAberdeen,Aberdeen,Scotland,UnitedKingdom}

f_{MRCCancerUnit,UniversityofCambridge,Hutchison/MRCResearchCentre,Cambridge,UnitedKingdom} g_{DepartmentofMedicine,MemorialSloan-KetteringCancerCenter,NewYork,NY10065,USA}

h_{UniversityofMarylandBioPark,KoDiscovery,Baltimore,MD20201,USA}

i_{TheSolGoldmanPancreaticCancerResearchCenter,DepartmentofPathology,JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MD21231,USA} j_{NewJerseyMedicalSchool,RutgersUniversity,Newark,NJ07103,USA}

k_{DivisionofNutritionalSciences,CornellUniversity,Ithaca,NY14850,USA}

l_{FieldofGenetics,Genomics,andDevelopment,CornellUniversity,Ithaca,NY14850,USA}

m_{AndrusGerontologyCenter,DivisionofBiogerontology,UniversityofSouthernCalifornia,LosAngeles,CA90089,USA}

n_{DepartmentofMolecularandIntegrativePhysiologyandDepartmentofInternalMedicine,UniversityofMichigan,AnnArbor48109,USA} o_{KochInstituteforIntegrativeCancerResearch,MassachusettsInstituteofTechnology,Cambridge,MA02139,USA}

p_{Harvard-MITDivisionofHealthSciencesandTechnology,HarvardMedicalSchool,Boston,MA02115,USA} q_{MRCMitochondrialBiologyUnit,WellcomeTrust-MRCBuilding,Cambridge,UnitedKingdom}

r_{DepartmentofBiologicalChemistryandDepartmentofOncology,JohnsHopkinsUniversity,Baltimore,MD21205,USA} s_{LaboratoryofOncology,IstitutoGianninaGaslini,Genoa,Italy}

t_{DepartmentofCancerBiology,AbramsonFamilyCancerResearchInstitute,UniversityofPennsylvania,Philadelphia,PA19104,USA} u_{Dana-FarberCancerInstitute,Boston,MA02115,USA}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received12March2014 Receivedinrevisedform 30September2015 Accepted5October2015 Availableonline8October2015 Keywords: Cancermetabolism Mitochondria Warburg Hostmetabolism Cancertherapy

a

b

s

t

r

a

c

t

Cancerisadiseasecharacterizedbyunrestrainedcellularproliferation.Inordertosustaingrowth, can-cercellsundergoacomplexmetabolicrearrangementcharacterizedbychangesinmetabolicpathways involvedinenergyproductionandbiosyntheticprocesses.Therelevanceofthemetabolictransformation ofcancercellshasbeenrecentlyincludedintheupdatedversionofthereview“HallmarksofCancer”, wheredysregulationofcellularmetabolismwasincludedasanemerginghallmark.Whileseverallinesof evidencesuggestthatmetabolicrewiringisorchestratedbytheconcertedactionofoncogenesandtumor suppressorgenes,insomecircumstancesalteredmetabolismcanplayaprimaryroleinoncogenesis. Recently,mutationsofcytosolicandmitochondrialenzymesinvolvedinkeymetabolicpathwayshave beenassociatedwithhereditaryandsporadicformsofcancer.Together,theseresultsdemonstratethat aberrantmetabolism,onceseenjustasanepiphenomenonofoncogenicreprogramming,playsakeyrole inoncogenesiswiththepowertocontrolbothgeneticandepigeneticeventsincells.Inthisreview,we

∗ Correspondingauthorat:DukeMolecularPhysiologyInstitute,300N.DukeStreet,Durham,NC27701,USA. E-mailaddress:matthew.hirschey@duke.edu(M.D.Hirschey).

1 _{Equalcontribution;authorslistedalphabetically.}

2 _{Thecompletelistofcontributingauthorsonthetargetvalidationteamarelistedalphabeticallyintheacknowledgements.}

http://dx.doi.org/10.1016/j.semcancer.2015.10.002

discusstherelationshipbetweenmetabolismandcancer,aspartofalargerefforttoidentifya broad-spectrumoftherapeuticapproaches.Wefocusonmajoralterationsinnutrient metabolismandthe emerginglinkbetweenmetabolismandepigenetics.Finally,wediscusspotentialstrategiestomanipulate metabolismincancerandtradeoffsthatshouldbeconsidered.Moreresearchonthesuiteofmetabolic alterationsincancerholdsthepotentialtodiscovernovelapproachestotreatit.

©2015ElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

A non-profit organization called Getting to Know Cancer launchedaninitiativeentitled“TheHalifaxProject”in2011,which waschargedwithidentifyingsynergisticmoleculartargetsand/or smallmoleculesforeachoftheareasthatarewidelyconsidered tobehallmarksofcancer[1].Therationaleforthisapproach is basedontheideathatcancersharborsignificantgenetic hetero-geneity[2],whichisoftennotaddressedwithmonotherapeutic approaches.Whileeffortshavebeenmadetocombinetherapiesto overcomeresistance,risingdrugcosts,significantlevelsoftoxicity, andalackofoverallsuccesshavestymiedeffortstoeffectivelytreat cancerwithmulti-drugcombinations[3].

Thus,thefirstaimoftheHalifaxProjectwastoproduceaseries ofreviews,includingthisreviewoncancermetabolism,tobroadly assesscurrentknowledgeonthebiologyofcancer.Theoverallgoal oftheHalifaxProjectistoidentifybiologicaltargetsand prospec-tiveleadcompoundsthatcouldpotentiallybeusedtoreacheach prioritizedarea, andsynergisticallytargetmultiplehallmarksof cancer.Bybuildingthisrationaleintotheapproachapriori,the problemofheterogeneitymightbeovercome.Intheory,multiple lowtoxicityapproachescouldbeexperimentallycombined,which thenmightleadtosynergismwithinagivenhallmark,suchas can-cermetabolism.Futurestudieswillbuilduponthesefindingsand testthesehypotheses,aswellasintegratetheseconceptsintothe approachesrecommendedinotherhallmarkareasinthisspecial issue.

In this review, we first discuss the relationships between metabolismandcancer.Wefocusonmajoralterationsinnutrient metabolism,aswellastheemerginglinksbetweenmetabolismand epigenetics.Next,wediscusspotentialtherapeuticstrategiesthat couldbeusedtomanipulatemetabolismincancercellsorto manip-ulate host metabolism thereby influencing cancer metabolism. Finally, we describe tradeoffs that should be considered when leveragingtheseapproaches.Together,thisinformationwillbethe basisofsignificantfutureresearchtofullyrealizethepotentialof targetingmetabolismincancer.

2. Classicmetabolicderangements

Thefirstrealizationthatmetabolismisalteredin cancercan traceitsroots totheworkof OttoWarburg. During the1920s, Warburgfoundthatunlikemostnormaltissues,cancertissues fer-mentedglucosetolactateathighratesregardlessofthepresence ofoxygen[4,5].ThiswasincontrasttotheresultsthatPasteurhad obtainedpreviouslystudyingfermentationinyeast,wherebyO2 wasfoundtoinhibitfermentation[6,7].Tostudythemetabolismof cancerinvivo,WarburgusedJensensarcomacellstoformtumors withintheabdomensofrats.Bycomparingarterialglucoseand lactateconcentrations tovenousglucoseand lactate concentra-tions,Warburgwasabletoinfertheglucoseuptakeand lactate excretionbythetumor.Whereasnormaltissuestookup2–18%of arterialglucose,tumorsconsumed47–70%.Lactatewasnot signif-icantlychangedinbloodafterperfusionofnormaltissues,butby Warburg’scalculations,tumorsconverted66%oftheirconsumed glucoseintolactate.Thus,Warburgsurmisedthattumorstakeup

muchmoreglucosethannormaltissuesandconvertamuchlarger percentageofittolactate[4].

Warburg’sworkonrespirationandfermentationincancercells ultimatelyledhimtoproposethat“therespirationofallcancer cellsisdamaged”[8].Infact,hereasonedthatknowncarcinogens, suchasarsenicandhydrogensulfide,likelyworkedbyinhibiting respiration.Hesuggestedthattheprimaryoncogenicinsultwas aninabilityof cellstooxidizeglucose carbons,andthat X-rays werecarcinogenicmainlyduetotheireffectonmitochondria[8], whichbythistimehadbeenshowntobetherespiratorycenterof cells.

Theexactmolecularmechanismsleadingtoalteredmetabolism incancerandtheWarburgeffectremainamajorunsolvedquestion; forareview,see[9].Subsequentstudieshaveshownthatwhile changesinmitochondrialrespirationaresometimesseenincancer cells,thesealterationsarenotlikely thedrivinglesionformost cancercells. For example,Warburg’s follow-up worksuggested thatoxidativerespirationwasimportantinmalignanttumors,and reportedthatplacingratsin5%O2for40hresultedinthedeathof mostcancercells,suggestingthatoxygenwasneededforviability ofthosecancercells[4].Similarly,theworkofhiscontemporaries showedthatoxygenconsumptionisintactinmanycancers,thereby decouplingtheWarburgeffectfromdefectiveoxygen consump-tion [10,11]. However, oxygenconsumption cannot be a direct measurement of intact respiration, becausemitochondrial cou-pling/uncouplinginfluencestheefficiencyofoxygenconsumedto ATPproduced.Nevertheless,manycancercellsdisplayincreased glucose uptake and elevated lactate production, irrespective of oxygenavailability–alsocalled“aerobicglycolysis”orthe War-burgeffect[12],andthisobservationremainsahallmarkofaltered metabolismincancercells.

3. Emergingmetabolicderangements

While the mechanisms leading to the Warburg effect are under intense investigation, thegeneral consensus of the field is that dysregulated metabolism and altered mitochondrial structure–function [13] is consistently found in several cancer celltypes.Thesechangesmayoccurbefore,asa resultof,orin combinationwith,thegeneticchangesdrivingcancer,including oncogeneexpressionortumorsuppressorloss;forrecent compre-hensivereviewsontheseconcepts,see[14,15].Forexample,one well-studiedlinkbetweenoncogenesisandglucosemetabolismis thephosphoinositide3-kinase(PI3K)signalingpathway. Activat-ingmutationsin PI3Koroverexpression oftheAKT oncogenes, whichliedownstreamofPI3K,caninducehighratesofaerobic gly-colysisinnon-transformedcells.Thisoccursinpartbyincreasing expressionandlocalizationofthehigh-affinityglucosetransporter, GLUT1, on the plasma membrane [16,17]. In addition, activa-tionofthePI3K pathwaycanacceleratefluxthrough glycolysis byincreasing theactivityof hexokinase-2, phosphfructokinase-1(PFK1),or phosphofructokinase-2(PFK-2)[18–20].Thetumor suppressorp53, which hasa welldescribed role in DNA dam-agesensing, cellcycle control,and control ofapoptosis, isalso abletoopposetheWarburgeffectbystimulatingrespirationand reducingglycolyticflux[21–23].Thus,lossofp53incancercells

Table1

Prioritizedpathwaystotargetcancermetabolism.

Target Knownpathways Predictedmanipulation Hexokinase2(HK2) Glucosemetabolism Reduceglucoseuptake

andmetabolism

6-Phosphofructo-2- Kinase/Fructose-2,6-Biphosphatase3 (PFKFB3)

Glucosemetabolism Reduceglycolysis

Pyruvatekinaseisoform M2(PKM2)

Glucosemetabolism Activatepyruvate oxidation Glutaminolysis Aminoacid

metabolism

Inhibitglutamine anaplerosis Reductivecarboxylation Aminoacid

metabolism

Inhibitreductive carboxylation O-GlcNAcylation Epigenetics Unknown Methylation/one-carbon metabolism Epigenetics Unknown Acetylation/sirtuin deacylases(SIRTs) Epigenetics Unknown

Oncometabolites Epigenetics Inhibitoncometabolite formationand signaling

Lactate Glucosemetabolism Inhibituseoflactateas afuel

is anothereventthat canimpactglucose metabolismin cancer cells.

Giventheinextricablerelationshipbetweenoncogenes,tumor suppressors, and the regulation of glycolysis, metabolic alter-ations including the Warburg effect could provide a selective advantage to rapidly proliferating cells [24]. Although fermen-tationproduces almost20 timesless adenosine5-triphosphate (ATP) perglucose moleculethanoxidative glucosemetabolism, ATP is never limiting in dividing cells [24,25]. Instead, prolif-erating cells require macromolecular precursors and reducing power in the form of reduced nicotinamide adenine dinucleo-tide phosphate(NADPH)tosynthesizenewbiomass. Therefore, one possibleadvantage of theWarburg effect is that highflux through glycolysis allows for more efficient use of glycolytic intermediates for NADPH production and biosynthetic path-ways includinglipidsynthesis, nucleotidesynthesis,and amino acid synthesis, which would be permissive for rapid cellular proliferation.

Although glucose metabolism is important for proliferating cells,othernutrientsourcescontributeaswell.TCAcycle inter-mediates are required forbiosynthetic processes, includingthe generationofcitrateforlipidsynthesisandaspartatefornucleotide synthesis.WhentheseintermediatesareremovedfromtheTCA cycle,theymustbereplenishedinaprocessknownas anaplero-sis. Glutamine has been shown to be a key contributor to anaplerotic flux in many cancer cells [26,27]. Glutamine car-bonentryintotheTCAcyclesupportsboth ATPgenerationand biosynthesis, and a widevariety of cancercelltypes are sensi-tivetoglutaminewithdrawal[28,29].Catabolismofextracellular protein can also contribute carbon to the TCA cycle [30], and lipidsarescavengedtosupportproliferationofsomecancercell types[31].

Collectively, metabolic shifts that enable the generation of biosynthetic precursors are a key feature in cancer ini-tiation, development, and/or growth. Below, we discuss how alterationsinkeymetabolicpathwayscontributetobiomass gen-eration (Table 1). We discuss some prioritized targets within these pathways, their therapeutic potential, and strategies to manipulate metabolism for the prevention or treatment of cancer.

4. Glucosemetabolism 4.1. Hexokinase2(HK2)

Regulation of glycolysis is exerted by the three important kinasesthatcatalyzediscretephosphorylationreactions(Fig.1). Hexokinasesareafamilyofenzymesthatcatalyzethefirst phos-phorylationof glucosetoglucose-6-phosphate.Earlyworkona highly malignant AS-30D hepatoma cell line showed that one isozymeofhexokinasewasuniquelyboundtotheouter mitochon-drialmembrane[32,33],whichwaslateridentifiedasHexokinase 2(HK2)[34].

In a seriesofexperiments inthis system,removal of malig-nanttumormitochondriacontainingboundhexokinasefromthe cytoplasmbycentrifugationmarkedlydecreasedtherateof glycol-ysis.Then,whentumormitochondriacontainingboundhexokinase were added back to the tumor cytosol, the original glycolytic capacityofthecytoplasmwasrestored.Finally,whensolubilized hexokinase alone was added to the liver cytosol, it markedly enhanced the glycolytic rate [34]. Therefore, HK2 could be a majorcontributortohighglycolysisandlactateproductioneven inthepresenceofoxygen.OnemechanismbywhichHK2binds tomitochondriaisviatheoutermembraneproteinknownasthe voltage-dependentanionchannelVDAC[35].Thisbinding interac-tionfacilitatestheimmortalizationofcancercells[36,37].

Toidentifycompoundsthatcouldselectivelyinhibitthetwo main energy-producingpathways(glycolysisand mitochondrial oxidativephosphorylation),alimitedscreenwasperformedin can-cercells.Thesmallmolecule3-bromopyruvate(3BP)wasidentified asaninhibitorofglycolysisandalsoasaninhibitorof mitochon-drialenergyproduction,which couldbeexplained byinhibiting HK2[38].Follow-upworkshowedthat3BPhaspotentanticancer activity and has the capacity to eradicate cancers in different animal models[39].Furthermore,3BP wasrecentlytested in a singlehumancase studyandshowedpromisingresults[40,41]. FutureworkwillbedirectedattheroleofotherHKisoformsin cancer, aswell asthespecificity of 3BPtoward HK2. However, theseearlystudiesareoneproof-of-principlethattargetingenergy metabolismofcancercellscanbeaneffectivetherapeuticapproach. 4.2. 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase3 (PFKFB3)

Anothergluco-regulatorykinaseisphosphofructokinase(PFK), whichcatalyzes thephosphorylationoffructose-6-phosphateto fructose-1,6-bisphosphate (Fig. 1). PFK is regulated by several metabolites, includinginhibition by highconcentrations ofATP and activation byfructose 2,6-bisphosphate. Earlystudies indi-catedthatphosphofructokinaseactivitycorrelatedwiththegrowth rateofMorrishepatomastransplantedinratsandalsocorrelated withlactateproductionbyslicesofthosetissues[42].Those obser-vationssuggestedthatinhibitionofphosphofructokinaseactivity represented a logical target for inhibition of malignant tumor growth.Withthediscoverythatfructose 2,6-bisphosphateisan activatorofphosphofructokinase1[43],theenzymeactivity cat-alyzing the formation of fructose 2,6-bisphosphate became an alternativetargetfortheinhibitionofglycolysisandcancergrowth. Steadystatelevelsoffructose2,6-bisphosphateareregulatedby bifunctionalenzymesthathavebothphosphofructo-2-kinaseand fructose-2,6-bisphosphatase(PFKFBs).Oftheseenzymes,PFKFB3 hasthehighestphosphofructo-2-kinaseactivity[44].Interestingly, PFKFB3expression,butnototherPFKFBs,ismarkedlyelevatedin multipleaggressiveprimarycancers,includingcolon,breast, ovar-ianandthyroidcarcinomas[45].

TheroleofPFKFB3intheregulationofglucosemetabolismin cancercellshasbeenreviewedpreviously[46].Anearlyindication

Fig.1. Schematicofglycolysis.Enzymesalteredincancerareshowninbold.Colorscheme:carbon,gray;oxygen,blue;phosphate,yellow;adenosine,A;NAD+_{/NADH,orange;}

singlebonds,thin;doublebonds,bold.

thatPFKFB3mightbearegulatoryenzymewastheidentificationof multiplecopiesoftheAUUUAinstabilitymotifinits3untranslated region[44].ExpressionofthePFKFB3geneisinducedbyhypoxia through hypoxia-inducible factor-1 [47]. Low pH, a common featureinmalignanttumors,isanotherfactorthatresultsin upre-gulationofPFKFB3.Thismaybemediatedthroughthemetabolic stresssensorAMP-activatedproteinkinase(AMPK)resultinginan increaseinserinephosphorylation[48].Inbreastcancercells, syn-theticprogestinsactivatePFKFB3isoenzymephosphorylationanda long-termsustainedactionduetoincreasedPFKFB3proteinlevels.

AnimmediateearlyresponseoccursthroughtheERK/RSKpathway leadingtophosphorylationonS461followedbyactivationof tran-scriptionviacis-actingsequencesonthePFKFB3promoter[49].In myeloproliferativeneoplasms,PFKFB3expressionwasupregulated viaactiveJAK2andSTAT5[50],suggestingthatspecificinhibitors ofPFKFB3mightinhibitJAK2/STAT5-dependentmalignancies.

LevelsofPFKFB3areregulatedbyubiquitinylationduringthe cellcycle.PFKFB3accumulatesinmidtolateG1andbreakdown inSphaseoccursspecificallyviaa distinctS273residuewithin theconservedrecognitionsiteforSCF-beta-TrCP[51].Theactivity

ofPFKFB3isshortlasting,coincidingwithapeakinglycolysisin midtolateG1incontrasttoglutaminemetabolism,whichremains highthroughoutSphase[52].Glycolysisischaracteristically asso-ciatedwithcytosolicfractionsofcellsbut PFKFB3hasanuclear localizationsignal.Furthermore,nuclearlocalizationofPFKFB3was associatedwithincreasedproliferationbyincreasedexpressionof cyclin-dependentkinasesanddecreasedexpressionofthecellcycle inhibitorp27[53].Thisbringstomindthemorerecentobservation withthepyruvatekinaseisoenzymePKM2(describedbelow)that hasbeenshowntohavepotentialtranscriptionregulatoryaction inadditiontoitsrole inglycolysis[54].Therefore,a dual func-tionmaybeassociatedwithsomeregulatoryenzymesinvolved inglycolysis.

Molecularmodelingstudieswerethebasisfortheidentification ofthefirstpublishedreportofasmallmoleculeinhibitorofPFKFB3

[55]. This molecule, 3-(3-pyridinyl)-1-(4-pyridnyl)-2-propen-1-one known as 3PO became commercially available through Calbiochem,EMDMilliporein2013.3POwasshowntosuppress glucoseuptakeandglycolyticfluxandwasselectivelycytostaticto Ras-transformedhumanbronchialepithelialcellsrelativeto nor-malcells.Growthofhumanerythroleukemiacellswasinhibited by3PO[50].Treatmentoftumor-bearingmicereducedthe intra-cellularconcentrationoffructose2,6-bisphosphate,glucoseuptake andtumorgrowth.Theobservationthat3POsuppressesT-cell acti-vationindicatesapotentialusefor smallmoleculeinhibitorsof PFKFB3inthetreatmentofautoimmuneconditionsbutmightbe problematicincombinationtreatmentswithimmunosuppressive cancerchemotherapeuticagents[56].Amorepotentderivativeof 3POdesignatedPFK15hasbeenidentifiedandaphaseIclinicaltrial isplanned[57].

4.3. PyruvatekinaseisoformM2(PKM2)

Pyruvatekinase(PK)catalyzes thefinal rate-limitingstep in glycolysis(Fig.1),transferringaphosphategroupfrom phospho-enolpyruvate(PEP)toADPandtherebygeneratingpyruvateand ATP[58].HumanshavefourPKenzymes:PKRisrestrictedto eryth-rocytes;PKLisfoundpredominantlyinliverandkidney;PKM1is expressedindifferentiatedsomaticcells(e.g.muscleandbrain)and PKM2isfoundinfetaltissuesandproliferatingcells.Incancercells, expressionofPKM2isup-regulatedsuchthatitbecomesthemost predominantPKisoform[59].Accumulatingevidencesuggeststhat PKM2expressioniselevatedincancercells,asitsenzymatic activ-ity canbe regulatedby various metabolicand signaling inputs

[24,60,61].PKM2influencesthefateofglucosecarbons(Fig.1). Ingeneral,PKM2activityislowinproliferatingcells,which cre-atesabottleneckattheterminalstepinglycolysis,whichresults inelevatedconcentrationsofupstreamglycolyticmetabolites.As aconsequence,suchintermediatesareavailableforthe biosyn-theticreactionsthatbranchoffofglycolysis,therebyincreasingthe generationofcellularbuildingblocksneededforproliferation[24]. Current models describing the function of PKM2 in cellu-lar proliferation focus mainly on mechanisms that regulate its pyruvate-generatingactivityasacytoplasmiccomponentof gly-colysis[61,62].However,recentstudieshaveprovidedevidence for activities of PKM2 that extend beyond this canonical role. Namely,severalnuclearactivitiesforPKM2,andmechanismsthat enablethe shuttlingofPKM2 intothenucleus, have nowbeen described [54,63–70]. Nuclear activity promotes tumor growth throughthedirecttranscriptionalactivationofgenesinvolvedin cancermetabolism,includingPKM2itselfandtheRNAsplicing fac-torsthatrepressPKM1[54,65,68].Inthisway,PKM2actsinafeed forwardlooptopromotebothitsnuclearactivitiesanditsmetabolic roleincancermetabolism.Ongoingstudiesseektoexaminethe relative contributionsof thesetwo functionsinoncogensis and growth.

ThecentralprinciplepresentedaboveassertsthatinhibitingPK activityfacilitatesproliferation.Thiscanbeachievedthroughthe expressionofthelessactiveand‘regulatable’PKenzyme,PKM2. Alternatively, PKM2 might be upregulated to eliminate PKM1 expression;PKM1isaconstitutivelyactiveenzyme,whichreduces thegenerationofcarbonforanabolicreactionsandfacilitatesthe generationofATPinthemitochondria.Recentsupportforthisline ofthinkingcomesfromtheobservationthatPKM2isnotrequired for tumormaintenance, where short hairpin (sh)RNA-mediated depletiondoesnotaffecttumorgrowth[71].Furthermore,genetic experiments,inwhichthePKM2-specificexonwasdeletedina mousemodelofbreastcancerusingCre-loxtechnology, demon-strated that the absence of PKM2 increased oncogenesis and negativelyaffectedsurvival.Strikingly,thesetumorsshowedno tissue-specificPKexpression.Theydid,however,stillproduce lac-tatefromglucose,suggestingthatalternativeandnon-canonical methods of pyruvate generation werefunctioning [72]. Collec-tively,theseexperimentsdemonstratethatPKM2isnotrequired foroncogenesisorgrowth,andsupportfortheconceptthatcancer cellshaveagrowthadvantagebyremovingPKM1.

Despitepotentialmechanisticdifferencesbetweenthesetwo modelsontheroleofPKincellularproliferation,theyconvergeon acommonpoint:constitutivePKactivityisdetrimentalfor prolif-eration.Consistentwiththisconcept,thefirststudytoexamine theactivity ofPK directlyin an isogeniccontext demonstrated that cancercell linesengineered toexpressPKM1produceless lactate,consumemoreoxygenandarelesstumorigenicinnude mousexenograftsthanthoseexpressingPKM2[73].Importantly, theseresultsdemonstratedthatlessPKactivityprovidedaselective growthadvantageforcancercellsinvivo.

Fromatherapeuticstandpoint,activationofPKmayserveas a promising strategy toslow cancergrowth.Indeed, numerous research teamsin the publicand privatesectors have recently developeddrug-likesmallmoleculeactivatorsofPKM2thatmake itbehavemorelikePKM1[74–79].Studiesusingsuchcompounds haveshownthatactivationofPKM2resultsindecreased accumu-lationofbiosyntheticcarbonbuildingblocksandreducedcancer growth[74].Interestingly,allcompoundsdescribedthusfarshare ahighdegreeofstructuralsimilarityandbindatthesameinterface inthePKM2multimer.Thesmallmoleculeactivatorbindingpocket isdistinct fromthefructosebisphosphate(FBP)bindingpocket, wherePKM2activatorsovercomenegativeregulationby phospho-tyrosinepeptides.Together,thesetwoactivitiesenablecompounds toovercomemechanismsthatnegativelyregulatePKM2.

Severalofthecompoundsdescribed abovehavebeen inves-tigated in animal models [74,75]. In one study, a compound calledTEPP-46activatedPKM2andimpairedtumorseedingand growth. Subsequentmetabolic analysessupport thehypothesis thatimpairedanabolicmetabolismisresponsibleforthegrowth inhibition.Futurestudiesarenowaimedatexploringtheuseof theseagentsincombinationwithcytotoxicchemotherapiesthat generateoxidativestress.Supportforthisconceptcomesfromthe observationthatactivatingPKM2impairsthemetaboliccontrol ofredox–specificallythegenerationofreducingequivalentsin theformofNADPHandGSH[80]–whichsensitizescancercells tofurtheroxidativestress.Finally,PKM2activatorsmayproveto bedoublyeffective,asmultimerformationpreventsthenuclear translocationofPKM2andthusitsactivityasaproteinkinaseand activatorofgeneexpression[67].

5. Aminoacidmetabolism

In addition to the well-established role for altered glucose metabolism in cancer, recent research highlights the involve-mentofaminoacidmetabolismin cancer,especiallyglutamine.

Inproliferatingcells,thetricarboxylicacid(TCA)cyclefunctions asasourceofprecursorsformacromolecularsynthesisinaddition togeneratingreducingequivalentsforoxidativephosphorylation

[81].Citrate,forexample,isboththecanonicalentrypointofthe TCAcycleandaprecursorfortheacetyl-CoAusedinfattyacid/sterol synthesisandacetylationreactions.Normally,thewithdrawalof citratetosupplytheseotherpathwaysismatchedbyaninfluxof carbonintothecycletoyieldoxaloacetate,refillingthepoolsofTCA cycleintermediatesandmaintainingfunctionofthecycleduring growth[26].

Inculture,manycancercellsmeet theiranapleroticdemand throughtheoxidative metabolismof glutamineto oxaloacetate (Fig. 2)[26]. Glutamine is anadvantageous anaplerotic precur-sor because its conversion to ␣-ketoglutarate (␣-KG) has the potentialtodispersenitrogentohexosamines,nucleotides, and non-essentialaminoacids,allofwhicharerequiredforgrowth. Fur-thermore,oxidativemetabolismof␣-KGtooxaloacetateproduces reducing equivalentsthat canbe usedtogenerate energy [82]. Alternativeanapleroticpathways,suchaspyruvatecarboxylation, alsoproduceoxaloacetatebutdonotsatisfytheseotherdemands, positioningglutamineasakeyplayerinmitochondrialmetabolism. 5.1. Glutaminolysis

Glutamineisanon-essentialaminoacidwithanamine func-tionalgroupandisthemostabundantaminoacidincirculation

[83].Glutaminesuppliesnitrogenfornucleobasesynthesisand car-bonfortheTCAcycle,lipidsynthesisandnucleotidesynthesis[84]. Glutamineis involvedin both anabolicandcatabolic processes. Thecatabolismofglutamineiscalledglutaminolysis,whichcan beconvertedintoglutamate,aspartate,CO2,pyruvate,lactate, ala-nineandcitrate.Thefirststepofglutaminolysisistheconversionof glutaminetoglutamateandammoniaviaglutaminase(GLS).After glutamineisconvertedintoglutamate,theglutamateisoxidized into_␣-KG.Thismostoftenoccursthroughtheenzymeglutamate dehydrogenase(GLDH),concomitantwiththegenerationof mito-chondrial NADHor NADPHand ammonia [85].This is thefirst energy-yieldingstepinglutaminolysisandisthelinktotheTCA cycle(Fig.2).

Whileglutaminolysisisanormalprocessformanycells,such aslymphocytesandadipocytes,manycancercellshaveelevated glutamineflux.Incellculture,humangliomaandHeLacellswere foundtobecompletelydependentuponglutaminefortheir sur-vival.The cells diedin theabsenceofglutamine, despitebeing in glucose-rich media, which is now known as the complete dependenceonglutamineforcellsurvival(a.k.a.glutamine addic-tion)[28,86].Interestingly,notallcancercellsexhibitglutamine addiction.Whilethecompletemolecularmechanismofglutamine addictionisnotknown,severaloncogenicmutationsoralterations havebeenfoundtoexplainglutamine-dependenceincancers.For example,Mycisabletoincreaseglutaminemetabolismby upregu-latingGLSexpression,whichleadstoglutamateentryintotheTCA cycleas␣-KG[87].

The ability touse exogenous glutamine is enhanced by the upregulation of glutamine transporters [88]. For example, one studyfoundtheincreaseinglutaminolysiswassoprofoundthat cancercellsaccumulatedmoreglutaminethanwasnecessaryto meet theenergy and anabolic requirementsof the cell; excess glutamine-derivedcarbonwassecretedfromthecellsintheform oflactateor,toalesserextent,alanine[28].Furtherevidence sug-geststhatoverexpressionofMycisenoughtoinduceglutamine addiction,duetothefactthatthismutationcausesthemetabolism of themitochondria tobealtered in sucha manner asto rely onglutamine despiteavailableglucose[28].Interestingly,these glutamine-addictedcellswerefoundtobekeenlysensitiveto elec-trontransportchaininhibitors.Thisobservationcouldindicatethat

eitherthepotentialenergyinglutamineisbeingusedinthe pro-ductionofATP[89]orthatmitochondrialglutamateuptakebythe glutamate/aspartatemitochondrialtransporterrequiresa proton-motiveforce.

In human pancreatic ductal adenocarcinoma (PDAC), the oncogene KRAScontributed toglutamine dependencyby using glutamine-derivedaspartate toproduce oxaloacetate via aspar-tatetransaminase(GOT1)inthecytoplasm.Oxaloacetatewasin turnconvertedintomalateandthenpyruvatetomaintainahigh NADPH/NADP+_{ratioforredoxhomeostasis.Therefore,disrupting} thesereactions,andultimatelyglutaminemetabolism,ledto sup-pressionofPDACgrowthinvitroandinvivo[90].

In a recent study, the use of nuclear magnetic resonance (NMR)spectroscopyandmassspectrometry(MS),aswellas sta-bleisotope-resolvedmetabolomics,allowedthefateof13_Cand15_N fromlabeledglutamineinBlymphomacellstobetracedunder aer-obicandhypoxicglucose-deprivedconditions[91].Inthisstudy, glutamine is thefuel that drives theTCA cycle,which is com-pletelyindependentoftheglucosesupply.Thisreprogrammingof theTCAcycleisparticularlyadvantageoustocancercellsunder glucose-deprivedand/orhypoxicconditions.Whereglucoseis pref-erentiallyconvertedtolactateunderhypoxicconditions,glutamine metabolismservestosustainATPproductionandredox homeosta-sisinordertosupportcancercellgrowthandsurvival.Thisimplies thatevenhypoxiccancercellscanoxidizeglutaminethroughthe TCAcycle,andintheabsenceofglucose,glutaminemetabolism alonecansustaintheTCAcycleandtherebymeettheanapleurotic andenergeticdemandsoftheproliferatingcancercells.

Supportingthisidea,Myc-transformedcellswerefoundtorely ona meansof␣-KGsynthesis otherthan thatinvolvingGLDH. Aspartateandalaninetransaminasesreversiblyconvertglutamate into␣-KG, along withoxaloacetate toaspartate or pyruvateto alanine,respectively(Fig.2).Thesereactionsoccurdespitethe pres-enceofGLDHinthemitochondriaofcancercells,whichcatalyzes the direct conversion of glutamate to _␣-KG [92]. This is espe-ciallyimportantconsideringitwasfoundthatMyc-transformed breastcancercellsundergoapoptosiswhenaspartate transami-naseisinhibited.Thisimpliesthatthiscancerisheavilyreliantupon transaminasereactionstoproduce␣-KG[93,94].

Duetothecentralnatureofglutaminolysisinmanycancers,it isbecominganincreasinglyprominenttargetincancertherapy. Oneoftheearlystrategiestosuppressglutaminemetabolismwas toreducetheamountofavailableglutaminebyusingglutamine analogs. Compounds such as 6-diazo-5-oxo-l-norleucine (DON) and acivicinshowedcytotoxiceffects againstseveralmalignant tumorstypes,includingleukemiaandcolorectalcancers;however theseanalogsarenolongerclinicallyavailableduetopatient tox-icity[95].

Morerecentstrategieshavefocusedontargetingtheenzymes ofglutaminemetabolism.Forexample,GLSisa potentialtarget fortheinhibitionofglutaminolysisincancercells.Thekidney iso-form,GLS1,isfoundinmanymalignanttumors[96]whiletheliver isoform,GLS2,islessoftenexpressedincancers.Thecompound bis-2-(5-phenylacetimido-1,2,4,thiadiazol-2-yl)ethyl sulfide (BPTES) hasbeenshowntoinhibitgrowthofavarietyofcancersinmouse modelsandincellculture,includingBlymphoma,which allosteri-callyinhibitsGLS1byalteringtheconformationoftheenzyme[97]. Theeffectisenhancedunderhypoxicconditions,ofteninducing cancercelldeath[91].

GLDHisanotherpotentialtargetofglutaminolysis,whichwhen knocked-downbysiRNAresultedinamarkeddecreaseinthe pro-liferationofglioblastomacellsthatwereglutaminedependent[98]. Greenteapolyphenols,hexachlorophene,GW5074,andbithionol mayinhibitGLDHfunction.Theseinhibitorsworkbyrestricting enzymemovement,eitherbyformingringsaroundtheenzyme orwedgingbetweentheenzyme’ssubunits.Currently,greentea

Fig.2.SchematicoftheTCAcycleandglutaminolysis.Enzymesalteredincancerareshowninbold.ReductivedirectionoftheTCAcycleshownwithbluearrows.Color scheme:carbon,gray;oxygen,blue;phosphate,yellow;nitrogen,brown;adenosine,A;coenzymeA,lightgray;NAD+_{/NADH,orange;NADP}+_{/NADPH,brown;FAD/FADH}

2,

blue;singlebonds,thin;doublebonds,bold.

polyphenolshavebeenshowntoinhibitlung,colon,andprostate adenocarcinoma growth in xenograft models [99]. These com-poundsalsohadsignificanteffectsonglioblastoma,colon,lungand prostateadenocarcinomacellproliferation[100].

Anotherstrategytoinhibitglutaminolysisistotargetalanine transaminasethroughl-cycloserine[101]oraspartate transam-inase through amino oxyacetate [93] which nearly halted the growthofbreast cancerxenograftsin mice.Similarly,aspartate transaminase knockdown in pancreatic cancer is also dramat-ically growth inhibitory in vitro and in vivo and leads to a profounddisruptionofredoxhomeostasis[90].Inbothofthese cases,littletonotoxicitywasobservedwithtransaminase inhibi-tioninnon-neoplasticcells.Thesestudiessuggestthataspartate aminotransferaseisapromisingcancertarget.Finally,giventhat inhibitionofaspartatetransaminasealsoleadstoadisruptionof redoxhomeostasis,suchinhibitionmaysynergizewiththerapies thatincreasereactiveoxygenspecies(ROS),suchaschemotherapy andradiation[102].

5.2. Reductivecarboxylation

Notallcancercells have theability toperform glutaminoly-sis.Hypoxialimitstheoxidative capacity oftheTCA cycle,and in particular suppressesproduction of acetyl-CoA fromglucose viapyruvatedehydrogenase(PDH)[103,104].Furthermore,some

cancercellscontainseveremutationsinTCAcycleenzymes (suc-cinatedehydrogenase,fumaratehydratase)orincomponentsof theelectrontransportchainthatpreventefficientproductionof oxaloacetate fromglutamine [105].Yet, both hypoxic cells and cells withdefectivemitochondriarequireglutamine for growth

[106–108]. To address this paradox, metaboliclabeling experi-mentswere performedusing13_{C, and revealedthat thesecells} metabolizeglutaminethroughanunusualpathwaycharacterized by“reversal” ofisocitrate dehydrogenase(IDH)enzyme activity (Fig.2,bluearrows) [106,107,109,110].IDHtypicallyactsasan oxidative decarboxylase,convertingisocitrate to␣-KGand CO2 inthepresenceofanelectronacceptor.Indeed,IDH3,the mito-chondrialNAD+_{-dependentIDHisoform,functionsexclusivelyin} thismanner.However,thetwoothermammalianIDHisoforms, IDH1 and IDH2, use NADP+_{/NADPH as cofactors, and can act} eitheras oxidativedecarboxylases orreductive carboxylases.In thelatterreaction,␣-KGiscarboxylatedtoproduceisocitrate, con-vertingNADPHtoNADP+_{.Isocitrateisreadilyconvertedtocitrate,} whichcanthenbecleaved toproduceacetyl-CoA.Under condi-tionsof reductive carboxylation, glutamine becomes themajor sourceofacetyl-CoA forfattyacidsynthesis, greatlydecreasing theneedtoproduceacetyl-CoAfromglucose.Furthermore, cit-rate cleavage yields oxaloacetate, which is converted to other 4-carbonintermediates,meaningthatessentiallytheentire cellu-larpoolofTCAcycleintermediatescanbederivedfromreductive

carboxylation,inreverseordertotheirconventionalrouteof pro-duction(Fig.2).

Reductive carboxylationhad previously been observed as a minor source of isocitrate and citrate in a number of non-transformed mammalian tissues [111–114]. Its importance in cancercellbiologyisrelated toitsabilitytoserveasthemajor source of citrate and acetyl-CoA when cellular circumstances conspire to inactivate pathways that normally produce these metabolites, including hypoxia and genetic reprogramming of mitochondrialmetabolism.Forexample,theVonHippel–Lindau (VHL) tumor suppressor normally functions in the oxygen-dependent degradation of the alpha subunits of the hypoxia inducible factor (HIF) transcriptional activators. Thus, in cells expressingVHL,oxygenfacilitatesthedegradationofHIF1␣and HIF2␣sothatHIFtargetgenesarenotexpressed.Incontrast,in malignanttumorcellslackingVHL,HIFtargetgenesareexpressed regardlessofoxygenavailability.Thesegenes,whichinclude glu-cosetransportersandglycolyticenzymes,arepartofthemetabolic adaptationtohypoxia.Importantly,hypoxiastimulatesthe expres-sionofPDHkinase-1(PDK1),whichphosphorylatesandinactivates thePDHcomplex,impairingitsabilitytoprovideacetyl-CoAand citratefromglucose[103,104].CancercellslackingVHLproduce asubstantialfractionofcitrateandfattyacidsusing glutamine-dependentreductive carboxylation in culture, and a small but detectablefractionofcitrateinvivo[115].Heterologousexpression ofwild-typeVhl,orsilencingofPDK-1,partiallyrevertsmetabolism toaphenotypeinwhichcitrateandfattyacidsareproducedfrom glucose/PDH[115].

Regulationofreductivecarboxylationisanareaofactive inves-tigation.Importantly,thepathwayisstimulatedbymutationsin theelectrontransport chainthat impairrecyclingof mitochon-drialNADHtoNAD+_{,butimportantlyNADPHisthecofactorfor} reductivecarboxylation.Thissuggesteda modelinwhicha low NAD+_{/NADH ratioin mitochondriaprovidesa sourceof} reduc-ingequivalentswhicharetransmittedtoNADPHbynicotinamide nucleotidetranshydrogenase(NNT)[107].NNTisaninner mito-chondrialmembraneproteinthatusestheelectrochemicalproton gradienttotransferreducingequivalentsfromNADHtoNADPH

[116].SilencingNNTexpressioninSkMel5melanomacellsreduced thecontributionofglutaminecarbontoTCAcycleintermediates throughbothoxidativeandreductivemetabolism,anddecreased therate of growth of SkMel5-derived subcutaneousxenografts

[117].InVHL-deficient786-Orenalcarcinomacells,whichproduce alargefractionofcitrateviareductivecarboxylation,NNTsilencing significantlysuppressedreductiveglutamine metabolism.These findingssuggestedthatNADPHproducedbyNNTisrequiredfor reductivecarboxylationinVHL-deficientcells.

Reductivecarboxylation is alsoregulated by changes in the abundanceofTCAcyclemetabolites.Citratelevelstendtobelow incellswithactivereductivecarboxylation,andmaneuversto sup-pressformation ofcitrate fromglucoseenhance thefractionof citratederivedfromreductivecarboxylation[106,107,109,115].In contrast,supplyingcellswithexogenousacetateorcitrateincreases theratioofcitrate/␣-KGwhilereducingtheoverallcontributionof reductivecarboxylationtoTCAcyclemetabolism[115].

Compartmentalizationofthereductivecarboxylationreactionis notwellunderstood.IDH1iscytosolicwhileIDH2islocalizedtothe mitochondria,anddatasuggestthateachisoformcanparticipatein reductivecarboxylation.IDH2wasfoundtobethecrucialisoform forreductivecarboxylationinhypoxicgliomacells[109],whereas othercelllinesrequiredIDH1underhypoxia[106].Genetic sup-pressionofpyruvatedehydrogenaseinlungcancercellsissufficient toinduceanetfluxofreductivecarboxylation,andunderthese circumstancesthefluxis completelydependentonIDH1[118]. Incellswithmutationsintheelectrontransportchain,silencing eitherisoformreducedactivityofthepathway,andsilencingboth

togetherhadamaximaleffect[107].TheinvolvementofNNTasa sourceofNADPHarguesformitochondriallocalizationofthe reduc-tivecarboxylationreaction,atleastinthosecellsthatrequireNNT expressiontomaintaincitratelevels.However,cytosolicsourcesof NADPH,includingtheoxidativebranchofthepentosephosphate pathway,mayplayamoreprominentroleincellswhereIDH1 cat-alyzesreductivecarboxylation.Presumably,thechoiceofisoformis alsorelatedtolocalizationofanavailablesourceofthesubstrate ␣-KG,meaningthatcompartmentalizationofglutaminemetabolism couldaddanotherdimensiontoregulationofthispathway. 6. Lipidmetabolism

Cancersmay usea wide variety of substratesand substrate sources to meet their catabolic and anabolic needs, including internally and externally derived fattyacids (FAs). Indeed, FAs areessentialforcellularproliferation.Inparticular,FAsareused as cellular building blocks for lipid membrane synthesis, for energystorageand production,aswellasfor cellularsignaling. Thus,limitingabundanceofFAscouldbeatherapeuticstrategy againstcancer.LimitingcellularFAscouldbeachievedby:blocking synthesis(lipogenesis),increasingbreakdown(oxidation), reduc-ingavailabilityfromstorage(lipolysis), orbyincreasingFAflux towardstorage (re-esterification);thesedistinctstrategies have beenconsidered as chemotherapeuticstrategies recently [119]. Aswithothermetabolicshiftsincancer,characterizingthestate oflipidmetabolisminuniquecancertypesandcells linesisan importantfirststep.Indeed,successfulchemotherapeutic strate-gieswillrequireunderstandingthespecificabnormalitiesinlipid metabolismforagivencancertype.Targetinglipidmetabolismin cancerisanemergingideathatwarrantsfurtherinvestigation. 7. Epigeneticsandoncometabolites

In addition to alterations in metabolism, metabolic repro-grammingmediatedbyspecificoncogenesandtumorsuppressors canimpactdynamicregulationofchromatinviapost-translational modifications.Indeed,increasingevidenceindicatesthataltered metabolismcanalsoleadtochangesinnutrient-sensitive post-translational modifications. These chemical modifications, such asO-GlcNAcylation,methylation,andacetylation,canimpactthe activities of metabolic enzymes, signaling components, trans-criptionalregulators,and chromatin-associatedproteinssuchas histones [120–124]. Furthermore, oncometabolites, such as 2-hydroxyglutarate (2-HG), can have profound consequences on theregulation of chromatinorganization, gene expression, and genomeintegrity. Improvedunderstandingofthelinks between metabolismandepigeneticsisexpectedtohaveimportant thera-peuticimplications,andintenseeffortsarecurrentlygearedtoward targetingbothalteredmetabolismandepigenetics[125,126]. 7.1. O-GlcNAcylation

The hexosamine biosynthetic pathway (HBP), a branch of metabolism that diverges from glycolysis at fructose-6-phosphate, generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a key donor substrate for glycosylation reac-tions,includingtheO-GlcNAcmodification,whichismediatedby O-GlcNActransferase(OGT)[122,127,128].O-GlcNAcylationis gen-erallyelevatedincancercellsandhasbeenlinkedtomalignant tumor growth through direct modification of phosphofructoki-nase(PFK1)andindirectregulationofFoxM1[129–131].Allfour histones (H2A, H2B, H3, and H4) are modified by O-GlcNAc, whichisdynamicallyregulatedinresponsetonutrient availabil-ity[132–134].O-GlcNAcylationofhistoneH3onSer10iscellcycle

regulated and suppresses mitosis-specific H3 phosphorylation

[135],whereasH2BSer112O-GlcNAcylationfacilitates monoubiq-uitylationofLys120ofhistoneH2B,amarkassociatedwithactive transcription, indicating a role for O-GlcNAcin gene regulation throughchromatinmodification[134].OGTcanalsoassociatewith Ten–Eleven-translocation(TET)family5-methylcytosine hydroxy-lasesandparticipateinTET-mediatedgeneregulation[136–139]. WhethercancercellsexhibitalteredhistoneO-GlcNAcylation pat-ternsduetometabolicalterationsisnotyetclear.Notably,TET2 function is frequently disrupted in hematopoietic malignancies

[140–142], and future studies will determinewhether changes inhistoneO-GlcNAcylationparticipateincancerdevelopmentin thesecases.

7.2. Methylation

AlterationsinhistoneandDNAmethylationarewidelyobserved incancer.ManycancertypesdisplayglobalDNAhypomethylation comparedwithnormaltissue,whilegenesregulatingcell-cycleand DNAdamage responseare frequentlyfoundtobe hypermethy-lated,andthussilenced[143].HistoneandDNAmethyltransferases utilizethemethyldonorS-adenosylmethionine(SAM),whichis synthesizedfrommethionineandATP.Hence,SAMavailabilityfor methylationreactionsmay besensitivetolevelsof methionine takenupfromtheenvironmentorsynthesizedthroughone-carbon metabolismpathways,discussedinmore detailbelow.Inyeast, SAMcanserve asa sensorof aminoacidavailability,inhibiting autophagyandpromotinggrowth[144].Whiletransformedcells require growth medium supplemented with methionine [145], knowledgeof howcancercells usemethionine toregulate his-toneandDNA methylationis stilllimited. Notably,theenzyme nicotinamideN-methyltransferase(NNMT),which depletesSAM bycatalyzingthetransferofSAM’smethylgrouptonicotinamide, is frequently overexpressed in human cancers. A recent study demonstratedthatNNMTlevelsimpactmethylationincancercells, withNNMToverexpressionassociatedwithreducedlevelsofSAM andhistonemethylation[146].Interestingly,metabolicregulation ofSAM productionhasalsobeenimplicated inmodulating his-tonemethylationlevelsandmaintainingpluripotency inmouse embryonic stem cells [147]. In addition tooverall cellular lev-els of SAM, localized pools of SAM may provide an additional layer of metabolic control. The enzyme methionine adenosyl-transferase (MATII␣), which produces SAM, hasbeen localized togenepromotersandimplicatedingeneregulation,indicating thatenzymaticproductionofmetabolitescanbetargetedto spe-cificlociandcoupledtotranscriptionalregulation[148].DNAand histonemethylationlevelsarealsocontrolledbyratesof demethy-lation.Metaboliccontrolofdemethylationisdiscussedbelowin the“Oncometabolites”sub-section.Giventhesubstantialevidence that DNA and histone methylation abnormalities contribute to cancerinitiationandgrowth,furtherinvestigationintotherole ofmetabolismin determiningmethylationchanges incanceris needed.

7.3. Acetylation

Lysine acetylation levels are determined by the combined actions of lysine acetyltransferases (KATs) and deacetylases (HDACs),both of which maybe influenced by metabolicstate. Histone proteins are acetylated at multiple lysines, and these modifications are involved in chromatin-dependent processes, includinggenetranscription,DNA replication,and DNAdamage repair.Inmammaliancells,nuclear-cytosolicacetyl-CoA,thedonor substrate for acetylation reactions, is generated either through the cleavage of citrate by ATP-citrate lyase (ACLY) or directly fromacetatebyacetyl-CoAsynthetases[122].Histoneacetylation

can be regulated by the availability of glucose or acetyl CoA

[149–152], and in mammalian cells glucose-dependent regula-tionofhistoneacetylationoccursinanACLY-dependentmanner

[149].Reciprocally,nuclear-cytoplasmiclysinedeacetylationcan be mediated by SIRT1, a member of the NAD+_-dependent sir-tuin familyof lysinedeacetylases [classIIIhistonedeacetylases (HDACs)] [153,154]. NAD+ _{levels rise under nutrient-restricted} conditions, in part mediated by AMP-activated protein kinase (AMPK)[155,156].Inaddition,theketonebody_{␤-hydroxybutyrate} (␤OHB) was recently demonstrated to function as an endoge-nousinhibitorof classIHDACs underketogenicconditionsand influencethestateofhistoneacetylationandgenetranscription

[157].

Metaboliccontrolofhistoneacetylationislikelytoimpact can-cergrowth,althoughthishasnotyetbeendirectlyshown.Inyeast, acetyl-CoAactsasagrowthsignal,promotinghistoneacetylation atandexpressionofgrowth-relatedgenes[151].LevelsofACLY arefrequently elevatedin cancer[158], althoughtheimpactof thisonoverallhistoneacetylationis notyetknown.Global his-toneacetylationlevelsarehighlyheterogeneousamongcancers, andseveralstudieshaveshowncorrelationswithcancerrecurrence andpatientsurvival[159–163].BothKATandHDACinhibitorshave shownpromiseincancertherapy[125],althoughmechanismsof actionremainpoorlyunderstood.

7.4. Sirtuindeacylases(SIRTs)

Thesirtuins(SIRT1-7)areaclassofconservedNAD+_-dependent deacylasesthatcontrolcellularmetabolicprocessesandprotect thecellagainstmetabolicandgenotoxicstresses.Notsurprisingly, altered regulation of the sirtuins is associated with many dis-easessuchasdiabetes,neurodegenerativediseases,obesity,and cancer[164].Rapidly proliferatingcancercells requireshifts in metabolismthatpromoteanabolicmetabolism;assuch,the sir-tuinsplayimportantrolesincontrollingcancerdevelopmentand progressionbymaintainingnormalcellularmetabolism.Ofthe7 mammaliansirtuins,SIRT1,SIRT3,SIRT4,andSIRT6havebeen asso-ciatedwithvarioustypesofcancer. SIRT1,SIRT3,andSIRT6are strongdeacetylases,whiletheenzymaticactivityofSIRT4isless clearbutithasbeenshowntobeaweakADP-ribosyltransferaseand possiblyaweakdeacetylase[165].SIRT1andSIRT6servetomainly deacetylatehistonesandtranscriptionfactorsinthenucleus,while SIRT3andSIRT4aremitochondrial,wheretheyactonmetabolic proteins[166].

SIRT1sharesthemosthomologytoSir2(Saccharomyces cere-visiae),thefirstsirtuinidentified,andhasbeenshowntoincrease lifespanandprotectagainstage-relateddiseases.Themaintargets of SIRT1 are the transcription factor peroxisome proliferator-activatedreceptorgammacoactivator1-alpha(PGC1␣),theFOXO familyoftranscriptionfactors,andthetumorsuppressorp53[167]. StudieshavefoundthatSIRT1expressioniselevatedincertain can-cersandrepressedinothers,makingtheroleofSIRT1incancer difficulttodefine[168].It seemsasthoughthemainconfusion underlyingtherole ofSIRT1incanceris thecomplexitybehind theSIRT1-mediatedinhibitorydeacetylationofp53.SIRT1 expres-sionisalsopositivelyregulatedbyp53inanapparentnegative feedbackloopwherelossofp53reducesSIRT1expression, lead-ingtoanincreaseinp53activity[169].SIRT1hasbeendescribed asatumorsuppressorthatpromotestheDNAdamageresponse, andSIRT1+/−p53+/−micedevelopcancerina varietyoftissues, particularlysarcomas.Furthermore,SIRT1overexpressingmiceare moremetabolicallyefficient,resistanttodiabetes,andnocancer phenotypehasbeenreportedinthesemice[170].SIRT1 expres-sionisreducedinvarioushumancancers,particularlybreastcancer andhepatocellularcarcinoma[171].ActivationofSIRT1hasalready provenbeneficialinBRCA1-associatedbreastcancersastreatment

withresveratrolactivatedSIRT1and inhibitedcell proliferation in vitro and tumor growth in vivo [172]. It seems as though activationofSIRT1mayhavetherapeuticpotentialincertain can-cersinordertonormalizecellularmetabolismandimprovethe DNAdamageresponse.

SIRT3isthemainmitochondrialdeacetylaseandassuchit con-trolstheactivitiesofmanymetabolicenzymesinthemitochondria. SIRT3deacetylatesalonglistofmitochondrialproteinsactingat severalnodesof mitochondrialmetabolism,includingfattyacid oxidation,glutaminemetabolism,andmitochondrialROS produc-tion.WhenSIRT3islostorablated,metabolicderangementsoccur

[173],whichmaybethesourceforthelinkbetweenSIRT3and cancer.ThefirstdescribedlinkbetweenSIRT3andcancerreported thatSIRT3KOmicespontaneouslydevelopmammarytumorsafter 2years[174].Furthermore,thisstudyidentifiedSIRT3asatumor suppressorbyshowingthatSIRT3KOMEFscouldbetransformed invitrobytheadditionofasingleoncogene,MycorRas[174].A laterstudyattributedtheelevatedlevelofcellularROSseenwith lossofSIRT3tothestabilizationofHIF-1␣andactivationof HIF-1␣glycolytictargetgenes,leadingtoalteredcellularmetabolism towardglycolysis[175].ItispossiblethatSIRT3deficiencyleadsto acancerpermissivestatebycoordinatingashiftinmetabolismto aWarburgphenotype.

Cancercellsalsorelyheavilyonthemetabolismofglutamineas anitrogensourceforproteinandnucleotidesynthesisnecessaryfor proliferation[28].SIRT4waspreviouslyfoundtoinhibittheactivity ofglutamatedehydrogenase(GDH)activitybyADP-ribosylation. Morerecently,onestudyshowedthatgenotoxicstresscausesan inductionofSIRT4expressionleadingtoarepressionofglutamine metabolism[176].SIRT4KOMEFshadhigherglutamineuptakein responsetoUVdamageandincreasedglutamine-dependent pro-liferationcomparedtowildtypecells.Finally,SIRT4KOmicehad increasedincidenceofvarioustypesof cancer,particularlylung cancercomparedtowildtypemice[176].

Finally,SIRT6controlscancermetabolismandSIRT6ablation activatesaerobicglycolysisandleadstooncogenesis,intheabsence ofoncogeneactivation[177].Thisstudyshowedthatwhen aero-bicglycolysiswasinhibitedbyinhibitionofPDK1,aninhibitorof pyruvatedehydrogenase(PDH),theSIRT6deficientcellsfailedto formtumors,showingarelianceonglycolyticmetabolismof glu-cose.Further,SIRT6expressionisalsolowinpancreatic,colon,and livercancersinhumans[177,178].Overall,itseemsasthoughSIRT6 suppressescancergrowthbyregulatingaerobicglycolysisandthat amutatorphenotypeisnotrequiredforSIRT6-dependenttumor formation.

Collectively,thesestudiesshowthatthelossofanindividual sir-tuincanpromoteoncogenesisbycreatingastatethatisfavorableto cancercellformationorsurvival,suchasincreasingDNAdamage, inhibitingDNArepairmechanisms,oralteringcellularmetabolism towardaWarburg-likephenotype.Modulatingsirtuinexpression maybeawaytoaltersubstrateusebycancercellsandslowor preventtheirgrowth.Manyofthesirtuinsareknowntobe regu-latedbynutrientstatus,thereforeitmaybebeneficialtomodulate sirtuinexpressionbydietand/orexercise.SIRT1andSIRT3 expres-sionareinduced withcalorierestrictionand fasting,and SIRT3 expressionisreducedwithhighfatdiet(HFD)feeding[179,180]. Modulatingsirtuinexpressionthroughdiet,andpossiblyexercise, maybeusefulincombinationwithconventionalcancertherapy. Bycombiningchemotherapywithcalorierestrictionorexercise (describedbelow),sirtuinexpressionmaybeelevatedleadingto amorenormalizedcellularmetabolismandbettercontrolofthe cancer,possiblyincreasingtheefficacyofthedrugtherapy.Along theselines,effortsaimedatidentifyingcalorierestrictionmimetics, similarinactiontoresveratrol,havetheoverallgoalof increas-ingtheexpressionoractivityofthesirtuins.However,resveratrol, otherCRmimetics,anddietarymanipulationsarenotselectivefor

aparticularsirtuinandmayhaveundesiredoroff-targetaffects, andmostimportantlymaynotactivatethesirtuinsrobustly.More investigationintothesirtuinsincancerisclearlyneeded,and bet-terwaystoselectivelytargetindividualsirtuinswillbekeymoving forward.

7.5. One-carbonmetabolism

Thefolatecycleincombinationwiththemethioninecycleare collectivelyreferredtoastheone-carbonmetabolism,since car-bon units are circulated through multipleenzymatic reactions. The one-carbon cycle forms a critical component of the cellu-larmetabolicnetwork,whichislinkedtonearlyallofthemajor biosyntheticpathways.Themainsourcesthatputcarbonunitsinto this cycleareserineand glycinebiosyntheticpathways. Impor-tantly, thefate of the carbons (i.e. outputs) of the one-carbon cycleconsistofavarietyofcriticalmetabolicpathways;for exam-ple,someoutputs includenucleotidemetabolism[181],protein translation[182],lipidmetabolism[183],methylationmetabolism

[121,123,184–186],proteinsulfhydration,andglutathione produc-tion.Therefore,theactivityoftheone-carboncycleisimportant inregulatingthebiosynthesis ofthebuildingblocksofacellas wellasitsepigeneticstatus.Furthermore,theredoxstateofthe cellis regulatedby this cycleat two levels: throughthe folate cyclebythefunctionoftetrahydrofolate(THF),andalsothrough theglutathioneproductionviathetransulfurationpathwaythat mediatesthelevelsofROSincells.Bothofthesemechanismsserve toregulatethebalanceofNADPH/NADP+_{levelsinthecell[24].} Recently,avarietyofcancerswereshowntodivertcarbonsfrom glycolyticmetabolismintotheone-carboncycle,providingalink fromtheWarburgeffecttotheactivityofone-carbonmetabolism

[187,188].

For example, phosphoglycerate dehydrogenase (PHGDH) is hyperactivatedinasubsetofcancers,resultinginover-production ofserineinthesecancers[189,190].Furthermore,thegene encod-ingPHGDHwasshowntohaveundergonesignificantcopynumber gain insome cancersand cancercelllines, andthese celllines weredependentonthePHGDHamplificationforrapidproliferation

[191].

Asecondinstancewhere one-carbonmetabolismhasshown correlations with cell transformation is the metabolism and generation of glycine. A metabolomics study on cancer cell linesreporteda strongassociationbetweenglycineuptake and catabolismwithcancercellproliferation[192].Alsoanotherstudy foundthat ectopic expressionof glycine dehydrogenase(GLDC, decarboxylating), phosphoserine aminotransferase (PSAT), and serinehydroxymethyltransferase(SHMT),allenzymesimportant inglycineuptakeandcatabolism,couldinducecellgrowthinNIH 3T3cells,thusconferringtothesecellspropertiesindicativeofthe hallmarksofcancer[193].

Additionallevelsatwhich theone-carboncycleand its out-put pathways have been linked to cancer include nucleotide metabolism,epigeneticmodifications,polyaminemetabolism,and theregulationof theoxidative stateof thecell.Multiplegenes involvedinnucleotidemetabolismcausetransformationby reduc-ingthegenomicintegrity[194].Also,throughthetransferofmethyl groupstoproteins,DNA,andRNA,S-adenosylmethionineregulates proteinactivityaswellasepigeneticmarksonDNAandproteinsin cellswhichareallbroadlyimplicatedincancer.

Targetingone-carbonmetabolismusingfolateantagonistshas long been used as a major class of cancer therapeutic agents

[195].Historically,aminopterinwasthefirstoneofsuchdrugs, andmethotrexateandpemetrexedarestillbeingusedascommon chemotherapeuticagentsinavarietyofcancersactingtoinhibit di-andtetrahydrofolatereductaseactivities,therebydisruptingthe one-carboncycle.Inadditiontothisclassofcompounds,another

majorgroupofchemotherapeuticagents linkedtotheone car-boncycleareinhibitorsofnucleotidemetabolism.Theseinclude 5-fluorouracil(5-FU)andgemcitabine[196].

Furthermore,severalnovelanti-cancerdrugsthatarecurrently beingtestedinclinicaltrialsalsotargetspecificcomponentsofthe onecarboncycle.Theseagentsmostlytargettheepigeneticstateof thecancercellsthroughinhibitingSAMorDNAmethyltransferases or polyamine synthesis [197]. Difluromethylornithine (DFMO), methylglyoxalbis(guanylhydrazone)(MGBG),andaninhibitorof S-adenosylmethioninedecarboxylasecalledSAM486Aareexamples ofsuchagents.

Onecarbonmetabolismconsistsofenzymesthatareinprinciple druggable[198].Therefore,severalpromisingdrugtargetsinclude PHGDH,PSAT,PSPH,GCAT,GLDC,andglycineN-methyltransferase (GNMT).Arecentstudyhasshown thatreductioninserineand glycineintakeindietcansignificantlyreducecancercell prolifera-tioninmice,suggestingthatonecarbonmetabolismcanpotentially betargetedthroughdietaryadjustments[199].Finally,duetothe prevalenceofanti-metabolitechemotherapeuticagents thatare somehowinvolvedwiththeonecarboncycle,expressionlevelsof someofthecomponentsofthispathwaycouldpotentiallybeused asbiomarkersfordrugselectionandoutcomepredictioninseveral typesofcancers[200,201].

7.6. Oncometabolites

Isocitrate dehydrogenases (IDH1 and IDH2) are frequently mutatedinseveralcancertypes,includingglioma,acutemyeloid leukemia, and chondrosarcoma [202–204], resulting in signifi-cant changes to the epigenetic landscape in cancers harboring these mutations [120,141,205,206]. The products of wild type IDH1andIDH2enzymes,NADPHand␣-KG,playbroadfunctions inregulatingcellularmetabolism.ThemutantIDHproteinslose their wildtype function and instead convert ␣-KG into (R)-2-hydroxyglutarate[(R)-2HG],astructuralanalogof_␣-KG[207].IDH mutantcancercellscanaccumulatemillimolarlevelsofthis nor-mallyundetectablemetabolite[207,208].Recently,mousemodels withalteredIDH2activityhaveshownthecapacityofIDH muta-tionstofacilitatemalignanttumordevelopmentandmaintenance

[209,210].

(R)-2HGisthoughttoactbycompetitivelyinhibitingcertain enzymes that require ␣-KG as a cofactor, including Jumonji-C domainhistonedemethylases(JHDMs)andTETproteins,whichare implicatedinDNAdemethylation[211,212].IDHmutanttumors displayahypermethylationsignature,associatedwithablockin cellulardifferentiation[120,141,211].Asimilarhypermethylation signatureisassociatedwithIDHmutationsandTET2mutations, whichoccurinamutuallyexclusivemannerinAML,indicatingthat thesemutationslikelytargetthesamepathway[141].Moreover, eithertreatmentwith(R)-2HGorsilencingofTET2wassufficientto promotegrowthfactor-independentgrowthofTF-1leukemiacells

[213].However,theepigeneticalterationsmediatedby(R)-2HG maynotfullyexplainitscancerpromotingeffects,since(S)-2HG, whichisnotproducedbymutantIDHbutservesasanevenmore potentinhibitorofTET2than(R)-2HG,failstotransformTF-1cells. Onepossibleexplanationisthat(R)-2HG,butnot(S)-2HG,canactas anagonistforEGLNprolylhydroxylases,whichpromote hypoxia-induciblefactor(HIF)degradation;henceregulationofHIFproteins mayalsobeapartofthemalignanttumor-promotingactivityof (R)-2HG[213,214].Indeed,EGLN1silencingimpairedtheabilityof IDH1R132HtotransformTF-1cells.

2HGhasemergedasausefuldiagnosticmarkerforpatientswith IDHmutanttumors.PatientswithIDHmutantAMLexhibitelevated serum2HGlevels,andthosewiththehighestlevelsof2HGhad shorteroverallsurvival[215].Moreover,2HGcanbedetected non-invasivelybymagnetic resonancespectroscopyinpatientswith

IDHmutantglioma[216].Substantialinteresthasalsoarisenin therapeutictargetingofmutantIDHenzymes,asacancer-specific metabolicalteration.Inhibitors tomutantIDH1 and IDH2 were recentlyreported.Forexample,anIDH2/R140Qinhibitorpromoted thedifferentiationofleukemiacellscontainingthatmutation[217]. AnIDH1/R132Hinhibitor likewisepromoteddifferentiationand reduced histonemethylation in glioma cells. Growthof glioma xenograftswasalsoimpaired.Notably,thetumorgrowthinhibitory effectsofthedrugwereobservedatlowerdosesthanwererequired to stimulate histone demethylation and differentiation, further suggestingthatadditionalmechanismsmaycontributetomutant IDH-mediatedmalignanttumorgrowth[218].

Inadditionto(R)-2HG,succinateandfumaratealsohavethe potentialtoactasoncometabolites.Inspecificcancertypes, loss-of-functionfumaratehydratase(FH)andsuccinatedehydrogenase (SDH)mutationsareobserved,resultinginbuild-upoffumarate andsuccinate,respectively.Similarto2HG,fumarateandsuccinate cancompetitivelyinhibit␣-KG-dependentdioxygenases.Assuch, succinatehasbeenknownforseveralyearstopromoteHIF1␣ stabi-lizationviainhibitionofprolylhydroxylases[219].Morerecently,it hasbeenidentifiedthatsuccinateandfumaratealsoinhibithistone demethylasesandTETproteins[220].BothSDHmutant gastroin-testinalstromaltumorsandparagangliomawereshowntoexhibit ahypermethylationphenotype[221,222].

7.7. Lactate

Lactateismadefrompyruvatebytheenzymelactate dehydro-genase(LDH)duringnormalcellularmetabolism(Fig.1).Cancer cells produce high levels of lactate, as described above. More recently,anewroleforlactatehasbeendescribedwhereinsome cancercells uselactate toenable proliferation. For example,in a cancermicroenvironment,excesslactate issecreted and con-tributes to an extracellular environment that promotes cancer progression[223].Thus,lactate,whichwaspreviouslyconsidered awasteproductofcancercells,hasnowbeenidentifiedasakey metabolitethatplaysadirectroleinpromotingcellulargrowth. Theconceptoftheroleoflactateasasignalorasafuelincanceris oftencalledthe“reverseWarburgeffect”[224].

Lactatelevelsaregovernedbyanumberoffactors,including dif-ferentialexpressionofLDHisoforms,thelactatemonocarboxylate transporter(MCT)levels,andoxidativecapacityoftissues.LDHis theprimaryenzymecatalyzinglactateturnover,which intercon-vertspyruvateandlactate,withconcomitantinterconversionof NADHandNAD+_{,respectively.LDHisinvolvedinthemetabolism} ofbothglucoseandglutaminecarbon,aswellasindetermining malignanttumorpHandtheactivityoftheTCAcycle[225].LDHis classifiedintofivedifferentsubtypes(LDH1–5),whichstructurally conform intohomo- orhetero-tetramersof Mproteinsubunits encodedbyLDHisoformA(LDHA)andHproteinsubunitsencoded byLDHisoformB(LDHB)genes.Thesesubtypesvarybasedoftissue distribution.Forexample,innormaltissue,LDH1hashigh expres-sioninthebrain,heart,andkidney;LDH5isfoundinglycolytic tissues,suchasliverorskeletalmuscle[226].

Recently,LDHAwasshowntoberequiredforthemaintenance andprogressionofmanycancers[227–230],butthemechanisms bywhichLDHAfacilitatescancerprogressionremainpoorly under-stood[231].GiventhecorrelationbetweenLDHAlevelsandcancer outcomes,LDHAhasattractedattentionasacancertarget. Reduc-ingLDHAlevels(byshRNA)incellsleadstodecreasedproliferation and suppressed oncogenicity [227]. Furthermore, inhibition of LDHApharmacologicallyorbysiRNAreducesATPlevelsandresults in cellular death[228]. A follow-up study examiningthe com-bination treatment of the LDHA inhibitor FX11 and the NAD+

synthesis inhibitorFK866resultedinlymphomaregression ina xenograftmodel[228].AnotherstudyfoundthatLDHAplaysarole