TheImportanceofConstraintsandControlinBiologicalMechanisms:InsightsfromCancerResearch

WilliamBechtel

DepartmentofPhilosophyUniversityofCalifornia,SanDiego

LaJolla,CA92093-0119Email:bechtel@ucsd.edu

Abstract

Researchondiseasessuchascancerrevealsthatprimarymechanisms,whichhavebeenthefocusofstudybythenewmechanistsinphilosophyofscience,areoftensubjecttocontrolbyothermechanisms.Cancercellsemploythesameprimarymechanismsashealthycells,butcontrolthemdifferently.Iusecancerresearchtohighlightjusthowwidespreadcontrolisinindividualcells.Toprovideaframeworkforunderstandingcontrol,Ireconceptualizemechanismsasimposingconstraintsonflowsoffreeenergy,withcontrolmechanismsoperatingonflexibleconstraintsinprimarymechanisms.Controlmechanismsthemselvesoftenformcomplex,integratednetworks.

1.IntroductionThenewmechanistsinphilosophyofscienceemphasizetheroleofmechanismsingeneratingbiologicalphenomena.Theyhavecommonlyconstruedmechanismsasdiscreteentitiesthatoperateindividuallytogenerateagivenphenomenon(e.g.,thesynthesisofproteins).Buttheseprimarymechanismsareoftensubjecttocontrolbyothermechanisms.Theactivityofsomemechanismsexercisingcontroloverothermechanismsoftenbecomesapparentinthecontextofdiseaseinwhichchangesincontrolmechanismscauseprimarymechanismstobehaveinaberrantways.Thisisespeciallytrueinthecaseofcancer.Cancercellsmaintainthemselvesaslivingsystemsinaradicallytransformedstatenotbycreatingnewprimarymechanismsbutbytakingadvantageofalreadyexistingprimarymechanismsandcontrollingtheminnewways.1Bydirectingresearcherstothecontrolmechanismsthatarealteredincancer,cancerresearchhashighlightedjusthowabundantandcomplexthesecontrolmechanismsare.InthispaperIdrawuponcancerresearchtodevelopthedistinctionbetweenprimaryandcontrolmechanisms,characterizehowthetwotypesof 1 InthispaperIwillfocusonthealterationswithinindividualcancercells,butitisimportanttorecognizethatcancerisnotjustadiseaseofindividualcellsbutultimatelyinvolvestissuesandorgansofmulti-cellularorganisms.Withintumorstherearecomplexintercellularcontrolsystemsthatregulatethebehaviorofindividualcells.Butmuchofwhatisnowknownaboutthealteredfunctioningincancerconcernscontrolmechanismswithinindividualcells.Focusingonintracellularcontrolissufficienttomakethepointthatunderstandingbiologicalmechanismsrequiresafocusnotjustontheirparts,operations,andorganization,butontheextensivecontrolnetworksthatoperateontheconstraintsthatenableeachmechanismtoproduceitsphenomenon.

mechanismsarerelated,andtoarguefortheimportanceofunderstandingcontrolmechanismswithinanetworkperspective.Tounderstandtheroleofmechanismsperformingcontroloperationsonothermechanismsrequiresexpandingthecharacterizationofmechanismsaspartsandoperationsorganizedtogenerateaphenomenon.Insection2Iofferaframeworkforunderstandingthecomponentsofmechanismsasconstraints,someofwhichareflexibleandabletobeoperatedonandalteredbyothermechanisms.Insection3,Iprovideacharacterizationofcancerasmanifestinthealteredoperationsofordinarycellmechanisms,drawingonwhatHanahanandWeinberg(2000,2011)termedthehallmarksofcancer.Insection4Idevelophowthesehallmarksstemfromalterationsincontrolsystemsoperativeincellsthatinturnactontheconstraintsinprimarymechanisms.Insection5Ifocusingreaterdetailontheparticularcontrolsystemsaffectingglucosemetabolismincancercells.Theindividualcontrolpathwaysdiscussedinsections4and5interactwitheachotherinnumerousways.Thus,insection6Iturntohowthispointstoaneedtoshiftfromafocusoncontrolpathwaysthatareindividuallyinterpretableasmechanismstoafocusoncontrolnetworksinwhichmultiplecontrolmechanismsareembedded.2.RethinkingBiologicalMechanisms:ConstraintsandControlThenowstandardviewofbiologicalmechanismsconstruesthemasconsistingofentitiesorparts,eachperforminganactivityoroperation,organizedsoastoproduceaphenomenon(Machamer,Darden,andCraver2000;BechtelandAbrahamsen2005).Onthisview,abiologicalmechanism,muchlikehuman-mademachines,isanenduringsystemthatawaitsitsstartupconditionsandthencarriesoutitsactivitiesuntilitreachesitsterminationcondition.Theonlychangesaretheregularchangesthatoccuralongthewayformstarttoterminationconditions.Inpreviouswork(BechtelandAbrahamsen2010,2013),Ihaveemphasizedthedynamiccharacterofmechanisms—theyarenotstaticsystemsawaitinginputbutendogenouslychangestateduetothenon-linear,non-sequentialorganizationimplementedinthem.Noneofthesediscussions,however,haveemphasizedthatbiologicalmechanismsarehighlycontrolledsystems.Someaccountsofmechanismshavedescribedcontrolmechanisms,suchasneuralmechanisms(Craver2007)orcircadianclocks(BechtelandAbrahamsen2009),buttheseaccountshavesimplylookedatcontrolmechanismsintheirownrightandhavenotemphasizedhowtheyexercisecontroloverothermechanisms.Tocharacterizecontrol,itishelpfultobeginwithamodifiedperspectiveonmechanisms.Mechanismsperformwork,andthisrequiresbothasourceoffreeenergyandwaystodirectitsoastocarryoutwork.Adoptingthelanguageofclassicalmechanics,severaltheoristshavecharacterizedwhatdirectstheflowoffreeenergyasconstraints(PatteeandRączaszek-Leonardi2012;Hooker2013).Theconceptofconstraintswasintroducedintoclassicalmechanicstoaccountforthebehaviorofmacroscaleobjects.Constraints,generallyintheformofchemicalbondsbetweenparticles,reducethedegreesoffreedominwhichindividualparticlescanmove,eitherbyeliminatingmotionalongoneormoredegreesoffreedomorbycouplingvaluesthatcanbetakenontwoormoredegreesof

freedom.Incontextsinwhichthereisasourceoffreeenergy,suchconstraintscanservetochanneltheflowoffreeenergy.Forexample,apipecanrestrictwater,whichwouldotherwisespread,toflowinonedirection.IfapipeisdirecteddownwardstowardstheEarth’ssurfacethenthefreeenergyavailableinwaterenteringthetopofthepipecanperformtheworkofcarvingoutagulleyatthebottom.AsHooker(2013)emphasizes,althoughthetermconstraintconnotesrestrictionorlimitation,constraintsalsocreatepossibilities.Inthepreviousexample,afurtherseriesofpipescanconstraintheflowofwatersoastorotateamillwheelandperformtheworkofgrindinggrain.Likewise,theconstraintsthatfixthestructureofaproteinenableittocatalyzespecificreactionsbybringingreactantsintocloseproximitysothatavailablefreeenergycreatesorbreakschemicalbonds.Ingeneral,componentsofbiologicalmechanisms(aswellashuman-builtmachines)servetoconstraintheflowofavailablefreeenergysothatworkisperformed.WhenenzymesbindwithATP,forexample,theyhydrolyzeitanddirectthefreeenergythatisreleasedtocarryoutcoupledreactions.Linkingtothemoretraditionalvocabularyintroducedbythemechanistsinphilosophyofscience,theorganizationofcomponentsintomechanismsconstrainsfreeenergysoastoperformtheworkrequiredtogenerateparticularphenomena.Machinesandbiologicalmechanismsconstrainfreeenergytoperformwork,butunlessthisactivitycanbecontrolled,theworkwillnotbeuseful.Ifausercannotturnoffamachine,itwillcontinuetoperformitsactivity,evenifthatisnolongeruseful,untilitdepletesthesourceoffreeenergy.Tocontrolamachine,someconstraintsinitmustbeflexible,capableofbeingoperatedonbysomethingexternal.Mostmachinesincludeswitchesthatallowuserstoturnthemoffbystoppingtheflowoffreeenergy.Aswitchisaconstraintthatcanbealteredbyworkperformedonit.Inhuman-mademachines,thehumanusertypicallyperformstheworkofflippingaswitch.Someconstraintsmaytakeacontinuumofvaluesasworkisperformedonthem,leadingtovariableactivityofthemachines.Forexamples,howfarthedriverofacardepressestheacceleratorpedaldeterminestheopeningofthevalvethatconstrainstheflowofgasolineintotheengineandconsequentlyhowfastthecarmoves.Insomehuman-builtmachinesthecontrolprocessisinternalized.ThegovernorWattdesignedforthesteamengine,forexample,reliesonnegativefeedbacktoaltertheflexibleconstraintrealizedinvalveinthesteampipe,reducingorincreasingtheflowofsteamsoastokeepaflywheeloperatingatthesamespeed.Control,then,requiresasecondmechanism(e.g.,thedriverofthecarortheWattgovernor)tooperateonaflexibleconstraintintheprimarymechanismthatisdirectingtheflowoffreeenergy.Thecontrolmechanismitselfrequiresconstraintsthatdirectfreeenergytoperformitswork,althoughthisgenerallyrequiresmuchlessenergythantheprimarymechanismemploystodoitswork.Asimportantascontrolmechanismsareinhuman-builtmachines,theyareevenmoreimportantinthecaseofbiologicalmechanisms.Organismsarefar-from-equilibriumsystemsthat,unlesstheyperformtheworkrequiredtobuildandrepairtheirownparts,willdissipate(MorenoandMossio2015).Specializedmechanismscarryouttheconstructionandrepairprocesses.Iftheyaretokeeptheorganismfunctioning,thesemechanismsmustbecontrolledsoastoperformtheiractivitieswhentheyareneeded.

Doingsoatothertimescanbejustasbadasnotperformingthemwhentheyarerequired.Aneffectivecontrolmechanism,accordingly,doesnotjustoperateonaflexibleconstraintinanothermechanismbutmustdosoinresponsetoaconditionthatrequirestheprimarymechanismtooperateinaparticularmanner.Acontrolmechanismthuscontainsadetectorthatconstrainsitsoperation.Animportantchallengeinunderstandingthefunctioningofbiologicalmechanismsistofigureouthowtheyarecontrolledbyothermechanismsthatperformworkontheflexibleconstraintswithinthem.However,identifyingcontrolmechanismsisoftendifficult.2Therearewellworkedoutheuristicsfordecomposingandexplainingthefunctioningofprimarymechanisms(CraverandDarden2013)andmechanisticphilosophershavediscussedtheseintheiraccountsofdiscoveringthemechanismsofproteinsynthesis(DardenandCraver2002)andmetabolism(Bechtel2006).Theseaccounts,however,havebeensilentaboutcontrolprocesses,inpartbecausetheyaretypicallynotactiveintheexperimentalsetupsusedtostudyprimarymechanisms.Theseprocedureskeepconditionsconstantandpreventcontrolfromalteringthemechanism’soperation.Diseases,however,arecontextsinwhichthesecontrolmechanismsareoftenmanifestasadiseasedcellistypicallyoneinwhichacontrolsystemhasbeenaltered.Recentresearchoncancerisunusuallyinformativeinthisregard,soIturntoitinthenextsection.3.CancerandItsHallmarksTheroleofcontroloperatingonconstraintsleadingtoabnormaloperationofcellmechanismscanbeseenbyexaminingwhatHanahanandWeinberg(2000,2011)characterizeas“sixhallmarksofcancer—distinctiveandcomplementarycapabilitiesthatenabletumorgrowthandmetastaticdissemination”:

• sustainingproliferativesignaling• evadinggrowthsuppressors• resistingcelldeath• enablingreplicativeimmortality• inducingangiogenesis• activatinginvasionandmetastasis

Eachhallmarkinvolvesaphenomenonexhibitedinnormalcellsbutwhichoccurswithincreasedordecreasedfrequencyincancercellsduetoalteredcontroloperatingontheresponsiblemechanism.

2 Anexceptionisthenervoussystem,whichisparadigmaticallyinvolvedincontrollingotheractivitiesintheanimal.Yet,eveninexaminingneuralmechanisms,philosophershavefocusedprimarilyontheirrolein,forexample,creatingmentalrepresentationsofspace(Craver2007)orvisualinputs(Bechtel2008)andnotontheirroleincontrollingprimarymechanismsinvolvedin,forexample,metabolismormusclecontraction(see,however,Keijzer,vanDuijn,andLyon2013,,whohypothesizethattheearliestfunctionofneuronswastointegratetheactivityofmuscles).

Thefirsthallmarkisperhapsthemostwidelyrecognizedcharacteristicofcancer—cancercellsproliferate,replicatinginanuncontrolledfashion.Normalsomaticcellsdivideaswell,butafterthedevelopmentalstage,theyreachahomeostaticstate.Controlmechanismsshutdowndivisionunlessthecellreceivesasignal,suchasTGF-α,indicatinganeedforthecelltoreplicate.Incancercellsthiscontrolisremovedandcellscontinuetoproliferateindependentlyofsuchsignals.Thesecondhallmarkiscloselyrelated.Cellsinnormaltissuedonotjustfailtoreceivesignalstoproliferatebutactivelysuppressproliferationinresponsetoothersignals,suchasTGF-β.Antigrowthsignalscanblockproliferationthroughacontrolmechanismthatforcescellsintoaquiescentstateinwhichthecellcyclestopsexceptwhenthecellreceivesasignaltoproliferate.Byalteringcontrolmechanisms,cancercellsnotonlyinitiativedivisionontheirown,butalsoescapetheeffectsofmechanismsthatwouldnormallysuppressproliferation.Thethirdhallmarkisthatcancercellsshutdownnormalcellprocessesthatenablerecyclingofdamagedorunneededbiologicalstructures.Whenacellistoodisruptedtocontinuenormalfunction,controlmechanismstypicallyactivateprimarymechanismsthatperformapoptosisorprogrammedcelldeath.Overthecourseof30to120minutes,abevyofthesemechanismswithinthecellareturnedontodismantlethecellbydisruptingitsmembrane,breakingdownthecytoplasmicandnuclearskeletons,extrudingthecytosol,degradingthechromosomes,andfragmentingthenucleus.Nearbycellsthenengulftheremains.Whileapoptosisandnecrosiseliminatecells,theprocessofautophagyrecyclescomponentswithincellseitherwhentheyarenolongerneededorwhentheyaredamaged(Fengetal.2014).Inthecourseofautophagy,intracellularvesicles,autophagosomes,enveloporganellessuchasmitochondriaandribosomesandthenmergewithlysosomesthatdegradetheenclosedorganelleintoitsmolecularcomponents,whicharethenreusedinbiosyntheticprocesses.Byinterferingwiththecontrolmechanismsthatactivatetheseprocesses,cancercellsavoidthesefatesthatnormallybefalldisruptedcellsandcellcomponentsandcontinuetomaintainthemselvesandproliferate.Thefourthandfifthhallmarksinvolvealteringcontrolmechanismstoreactivatecellularprocessesthatarenormallydown-regulatedinmaturesomaticcells.Inmulticellularorganisms,HayflickandMoorhead(1961)discoveredthathumanembryoniccellsonlydividealimitednumberoftimesandthenenterintoasenescentstate.ThenumberofpossibledivisionsisknownastheHayflicklimit,whichwassubsequentlyfoundtocorrelatewithanalreadyknownprocessoftelomereshortening.Ineachcellreplicationtelomererepeatsareremoved.Whenthelasttelomeresareremoved,theendsofchromosomalDNAfuse.Stemcellsprovideanexceptiontotheprogressiveremovaloftelomeres.Instemcells,theenzymetelomeraseaddsratherthanremovestelomeres.Telomeraseisdown-regulatedinmostsomatictissue,butincancer,asinstemcells,telomeraseisactivated,allowingcellstoacquirereplicativeimmortality.Thefifthhallmarkinvolvesinhibitingthecontrolmechanismthatinmaturecellsturnsofftheembryonicprocessesofgeneratingnewvasculature,neededtoprovideoxygenandnutrientsandremovewasteproducts.Oneoftheseprocesses,angiogenesis,involvessproutingnewvesselsfromonesalreadygenerated.Oncedevelopmentiscomplete,controlmechanismsgenerallyblockangiogenesis,allowingittooccuronlytransientlyincontexts

suchaswoundhealingandfemalereproductivecycling.Whenthissuppressionisinhibitedincancer,themechanismofangiogenesiscontinuallygeneratesnewvesselstosupporttheongoingproliferationofcells.Thelasthallmarkofcancer,invasionofothertissueandmetastasis,reversesthecontrolmechanismsthatkeepsomaticcellsinaquiescentstateadheringtoepithelialcellsheets.Thisenablesthecancercelltoreactivatemechanismsthatarenormallyactiveonlyindevelopment.Sincethesecontrolmechanismsweremostlywerediscoveredbydevelopmentalbiologists,notcancerresearchers,Iwillnotfocusonthishallmark.4.CancerHallmarksRevealControlMechanismsinNormalCellsSofarIhaveappealedtothehallmarksofcancertoidentifytheexistenceofcontrolmechanismsthatarealteredincancerwithoutgoingintoanydetailsabouthowtheyoperate.Butcancerresearchhasalsorevealedmanyofthepartsandoperationsconstitutingthesecontrolmechanisms.InthissectionIdiscusshow,startingfromidentificationofgenesthatareoftenmutatedincancer,researchersidentifiedthepartsandoperationsofnumerousofthesecontrolmechanisms.(Thecontrolmechanismsconsistofproteinscodedforbythegenes,butresearchersoftenfinditconvenienttoskipoverthestepsoftranscriptionandtranslationandsimplyslidebetweenreferringtogenesandreferringproteins.Ifollowthatpractice.)Asareferencepointforsubsequentdiscussions,Ireproduceawiringdiagramofthemostprominentcontrolcircuits(fromHanahanandWeinberg2000)thatpresentsthelargelysequentialpathwaysofreactionsthatconstitutethecontrolmechanismsthatregulateparticularprimarycellmechanismsinnormalcellsthatarealteredinthehallmarksofcancer.Discoveryofthesemechanismsbeganwiththediscoveryofafewgenesthatwerefrequentlymutatedincancercells.Researchinthe1960sthroughthe1980sresultedintheidentificationofHrasandKrasasthefirstoncogenes—genesthatwhenmutatedinitiatetheprogressionintocancer(Ellisetal.1981).Otherresearchduringthesametimeidentifieddifferentgenes,startingwithRb,whoseproductsnormallyservetosuppressdevelopmentofcancerbutwhenmutatedallowcancertodevelop(MurphreeandBenedict1984).Thesecametobeknownastumorsuppressorgenes.Intheattempttounderstandhowthosegenesfunctionedincontrolmechanisms,researchrevealedmanyothergenes/proteinswithwhichtheyinteracted,ultimatelyidentifyingpathways—sequencesofreactions,eachproducingaproductthatisaninputtothenextreaction.Thesepathwayscorrespondcloselytophilosophicalaccountsthatviewmechanismsasproceedingfrom“startorset-uptofinishorterminationconditions”(Machamer,Darden,andCraver2000,3).Researchonotherbiologicalmechanisms,suchasthoseinvolvedinextractingenergyfrommetabolites,alsorevealedpathways.Thepathwaysconsistingoftheproteinscodedforbygenesmutatedincancer,however,arenotinvolveddirectlyinmetabolizingfoodstuffsorsynthesizingbiologicalstructuresbutincontrollingtheseprimarymechanisms.

Figure1.HanahanandWeinberg’srepresentationofpathwaysregulatingnormalcellsthataremutatedincancer.Theproteinscodedbythebest-knownoncogenes(Ras,Myc)andtumorsuppressorgenes(p53andPTEN)areshowninred.ReprintedfromCell,Vol.100,Hanahan,D.andWeinberg,R.A.,TheHallmarksofCancer,Figure2,©2000,withpermissionfromElsevier.

Ibeginwithcontrolmechanismsthatnormallypreventcelldivisionexceptwhenthecellreceivessignalsfromtheirenvironmentthatindicateaneedfordivision.MutationstothetwooncogenesHrasandKrasledtoproliferation.Todeterminehow,researchersfirstidentifiedanumberofotherproteinswithwhichtheyinteract:Raf,MEK,MAPK,etc.TheseinteractsequentiallyinthepathwayshownontheleftinFigure1.Forsignalingalongthispathwaytoresultinproliferation,thesignalmustbemaintained.ThischallengedresearcherstodeterminehowtheRasproteinsgenerateasustainedsignal.TheydiscoveredthatRasproteinsactivelytransducesignalswhentheyformcomplexeswithGTP.NormalRasproteinsfunctionasGTPasesandhydrolyzeboundGTPintoGDP.WhenGDPreplacedGTP,Rasnolongertransducessignals.ThisrevealedthatRasnormallyexercisescontrolbyfunctioningasaswitchthatturnsitselfoffbyhydrolyzingGTPtoGDP,thusinsuringthattheproliferationsignalisactiveforonlyashortperiod(VetterandWittinghofer2001).ThemutationsthatturnRasproteinsintooncogenesimpairtheirabilitytofunctionasGTPases,thusblockingtheswitchingoperationthatwouldnormallyabortsignaling.Aswitchoperatesonaflexibleconstraintthroughwhichtheworkofamechanismcanbechanged.Thisisthefirstofseveralswitchesrevealedbycancerresearchwhich,whendisrupted,resultinthehallmarksofcancer.

AnotherexampleofaswitchisfoundinthecontrolmechanismshowninthelowerleftinFigure1.WhensurvivalfactorssuchasIGF1bindtoRTKs,theyactivatePhosphoinositide3-kinase(PI3-kinase),whichinturninitiatesseparateproteolyticcascadesleadingtoreplicationandangiogenesis.NormallyPTEN(phosphataseandtensinhomologue)degradestheimmediateproductofPI3-kinase,phosphatidylinositol(3,4,5)trisphosphate(PIP3)toPIP2,switchingoffthesignaling.WhenPTENismutated,however,thisswitchingdoesnotoccur,leadingtocontinualreplicationandangiogenesis.Researchontumorsuppressorgenes—geneswhoseproductsnormallypreventdevelopmentoftumorsbutwhenmutatedallowtumorstodevelop—revealedothercontrolmechanismsdisruptedincancer.Twoofthebest-studiedtumorsuppressorproteinsareRbretinoblastoma-associated)andTP53.Rb(showncentertopofthenucleusinFigure1)integratessignalsmostlyfromextracellularsources(especiallygrowthinhibitorysignals)andgateswhetherthecellcanproceedthroughthecellcycle(BurkhartandSage2008).TP53(rightsideofthenucleusinFigure1),ontheotherhand,respondstostressandindicatorsofabnormaloperationofcellmechanismsand,dependingonseverity,blocksfurtherprogressionofthecellcycle(senescence)orinitiatesapoptosis.Toillustratethisprocessinmoredetail,Ifocusoncontroloverapoptosis,forwhichresearchhasrevealedacomplexcontrolsystem.Thecomponentsoftheapoptosismechanism(theproteasescaspase3-7)arealreadyinplaceinmitochondriainnormalcellsbutlimitedbyaflexibleconstraintfrominitiatingtheirapoptoticactivitiesuntiltheybindwitheitherCaspase8orCaspase9.TheavailabilityofCaspase9isfurthercontrolledbyanotherflexibleconstraint,an“apoptotictrigger”involvingcounterbalancedpro-andanti-apoptoticmembersoftheBcl-2familyofregulatoryproteins.MembersoftheBcl-2familyinhibitCaspase9bybindingtoandsuppressingtheactivityofBaxandBak.BaxandBakareembeddedintheoutermitochondrialmembraneandwhennotsuppressed,disrupttheintegrityoftheoutermitochondrialmembrane.ThiscausesthereleaseofcytochromecandApaf-1thattogetheractivateCaspase9.Morerecentresearchhasrevealedevenmorecomplexityinthemechanism.BothBcl-2,whichsuppressesthetrigger,andBaxandBak,whichactivatethetrigger,possessprotein-proteininteractiondomainsknownasBH3motifs(Lowe,Cepero,andEvan2004).TheproteinsthatdeterminethesettingoftheswitchandthuswhetherapoptosisoccurseachcontainasingleBH3thatenablesthemtobindeitherwithBcl-2orwithBaxandBak(AdamsandCory2007;WillisandAdams2005).Controlisthusachievedbyproteinsoperatingonflexibleconstraintsinthecontrolmechanismsthatsubsequentlyaffectflexibleconstraintsinthemechanismscarryingoutapoptosis.Whereasapoptosisdismantleswholecellstoprovideresourcestoothercells,autophagydismantlesandrecyclesorganellessuchasmitochondriaandribosomeswithincells.Likeapoptosis,itisaprocessthatcancontributetofightingcancer.Theautophagymechanismnormallyoperatesatalowlevel,onlydismantlingpoorlyfunctioningorganellesbyenvelopingthemandthenfusingwithalysosometodismantlethem.Controlmechanismoperateontheautophagymechanismtoincreaseitactivityunderstressconditionssuchasnutrientdeprivation(Mizushima2007).Beclin-1,amemberoftheBH3-onlyfamilydiscussedaboveascontrollingapoptosis,isalsoakeyelementincontrollingautophagy.It

isnormallyboundtoBcl-2,butwhenitsstresssensorsareactivated,ituncouplesfromBcl-2toinitiateautophagy.Thusstressconditionsinthecellresultingfromearlyprogressiontocancercantriggerautophagyasadefensemechanism.Accordingly,oneofthecommonstepsinthedevelopmentofcancerinvolvesdisablingthecontrolmechanismsthatinitiateautophagy(Whiteetal.2010).Butironically,autophagycanalsobeinvokedtoprotectcancercellswhentheyareplacedinstressconditionsasaresultofnutrientdeprivation,radiotherapy,orcytotoxicdrugs.Thiscanresultincancercellsshrinkingintoastateofreversibledormancy,enablingtumorstogrowagainaftertreatmentwithanticancerdrugshasceased(WhiteandDiPaola2009).InthissectionIhaveidentifiedseveralcontrolmechanismsofnormalcellsthat,whenaltered,resultinparticularhallmarksofcancer.Exceptforthelast,eachoftheseinvolvescontrolpathwaysshowninFigure1.Twocentralpointsemergefromthisdiscussion.Ineachcasethemechanismsbeingregulatedresideinnormalcellsandareactiveinappropriateconditionsinthesecells.Turningthesemechanismsonincancerrequiresmodifyingthecontrolmechanismsthatnormallyturnthemoff.Second,ineachcasetherearemultiplecontrolfactors,typicallyoperatingsequentiallyinpathwaysthatresultinalteringthefunctioningoftheprimarycellmechanisms.5.CancerandMetabolicRegulationSeveralofthecontrolmechanismsIhavediscussedsofaroperateseveralstepsremovedfromtheprimarymechanismstheycontrol.Glucosemetabolismprovidesausefulexampleinwhichresearchhasrevealedhowcontrolmechanismsactonprimarymechanisms.Theprimarymechanismformetabolizingglucosewaslargelycharacterizedbythe1930sthroughtheworkofEmbden,Meyerhof,andothers(Needham1971;Fruton1972;Bechtel2006).(ThekeyoperationsareshownintheverticalpathwayofreactionsinFigure3below.)CluestoitsalteredfunctioningincancerwerealreadyavailablewhenWarburg(1930,1956)reportedthatcancercellsexhibitamarkedincreaseinglucoseconsumptioncomparedwithnormalcells.Moreover,Warburgnotedthatcancercellsoftensecretelargequantitiesoflactate,aproductofglucosemetabolismthatisusuallydepletedthroughthesubsequentmechanismofoxidativemetabolism.3Sinceglycolysisonlygenerates2moleculesofATPpermoleculeofglucosecomparedwiththe36moleculesofATPgeneratedinoxidativemetabolism,theenergyharvestedismassivelyreducedincancercells.Warburgassumedthatmitochondria,inwhichoxidativemetabolismiscarriedoutthroughthetricarboxylicacidcycle(TCA),aredamagedincancercellsandthatthisforcedcancercellstorelytotallyonglycolysis.However,subsequentstudiesfoundthatthemitochondriainmostcancercellsarefullyfunctional.Thisrevealedthattherelianceonglycolysiswithoutcontinuingtooxidativemetabolismwasduetoalteredcontrolinthecaseofcancer.4 3 Lactate is then released into the intercellular matrix where, through the Cori Cycle, it is transported back to the liver and resynthesized to glucose, at considerable expense of ATP that is procured from normal oxidative metabolism in healthy cells. 4 In addition to glucose metabolism, glutamine metabolism is altered, providing access to needed amino acids (Pavlova and Thompson 2016).

Cancerresearchershaveidentifiedtwopointsatwhichtheglycolyticmechanismisalteredincancer.AtthebeginningofthepathwaytheuptakeofglucosebythecellisincreasedandattheendoftheprocesstheproductsofglucosemetabolismareredirectedawayfromtheTCAcycle.Thesearetwopointsatwhichthereareflexibleconstraintsthatarethetargetofcontrolprocesses.Ifocusnowoncontrolatthebeginningofthepathwayandreturntocontrolattheendofthepathwaybelow.Glucosetransporter1(GLUT1)istheenzymethattransportsglucoseacrossthecellularmembrane.Therateatwhichitdoessoiscontrolledbyhypoxiainduciblefactor-1(HIF-1),whichalterstheratesbothofthetranscriptionofGLUT1andthetranslocationoftheGLUT1proteinfromtheendomembranetothecellsurface.This,however,isnottheonlypointatwhichHIF-1altersconstraintsinglycolysis.AsshowninFigure2,italsobindstoandsoalterstheoperationofmanyoftheenzymesintheglycolyticpathwayitself(Semenza2010).(IreturntootherfunctionsofHIF-1showninFigure2below.)Innormalcellsoxygen,whenitisavailable,rendersHIF-1unstablesothatitisbrokendownbytheubiquitin–proteasomepathway.Asaresult,glucoseuptakeisnormallyreducedwhenoxygenisavailable.ThelackofoxygeninhypoxialeavesHIF-1inplace,resultinginanincreaseinglycolysis.Thus,HIF-1operatesasaswitchcontrolledbyoxygen,yieldingincreasedglycolysisinconditionsinwhichitistheonlysourceoffreeenergy.IncancerthiscontrolprocessisdisruptedsothatHIF-1isnolongerdegradedbyoxygenandcontinuestomaintainhighratesofglycolysis.

Figure2.TheroleofHIF-1inregulatingcellprocess.ReprintedbypermissionfromSpringerNature:MolecularBiologyReports,CancerMetabolismandtheWarburgEffect:TheRoleofHif-1andPi3k,Courtnay,R.,Ngo,D.C.,Malik,N.,Ververis,K.,Tortorella,S.M.,andKaragiannis,T.C.©2015,Figure2.

Therelianceonthefarlessefficientprocessofglycolysisratherthanoxidativemetabolismincellsneedingenergytoproliferateatfirstseemscounterintuitive.Potter(1958)proposedanexplanation:theintermediatesofglycolysisalsofigureinmetabolicreactions

thatproduceothermoleculesusedtosynthesizevariousbiologicalcompounds(e.g.,fattyacids,cholesterol,nucleotides)neededinproliferatingcells.VanderHeiden,Cantley,andThompson(2009)haverevivedandfurtherdevelopedPotter’sproposal.Thearrowsprojectinghorizontallyfromtheintermediatesglucose-6-phosphate,fructose-6-phosphate,dihydroxyacetone-phosphate(DHA-P),and3-P-glycerateinFigure3indicatereactionsthatsynthesizeotherbiologicalcompounds.Forexample,thefirstreactionproductinthepathway,glucose-6-phosphate,feedsintothepentosephosphatepathway(PPP),inwhich

Figure3.TheglycolyticpathwayandtheTCAcycle.GLUT1transportsglucoseintothecytoplasmwhereitcanentertheglycolyticpathway.Redhorizontalarrowsidentifyalternativepathwaysfromkeyintermediatesinglycolysisthatareactivatedinaerobicglycolysis.OtherkeyregulatorssuchasPKM2areshowninblue.ReprintedfromCellMetabolism,Vol.23,Pavlova,N.N.andThompson,C.B.TheEmergingHallmarksofCancerMetabolism,Figure4,©2016,withpermissionfromElsevier.

glucose-6-phophatebecomespartiallyoxidizedtoyieldribose-5-phosphate,whichinturnisaconstituentofnucleotides.Thealterationofthecontrolmechanismsthatinitiateglycolysisalsoincreasethegenerationoftheseotherproducts(PavlovaandThompson2016).Cancer,onceagain,alterscontrolmechanismstoincreaserelianceonprimarymechanismsthatnormalcellsalsouse,albeitmoresparingly.Ihavefocusedononlyoneofseveralpointsofcontrolintheglycolyticmechanismthatarealteredincancer.Individualenzymesintheglycolyticpathwayarealsoregulated,includingphosphofructokinase,whichcatalyzesthekeyirreversiblestepfromfructose-6-phosphatetofructose-1,6-pyrophosphate.Ratherthandevelopingthese,Iturnbrieflytoregulationattheendofthepathway.Evenwithcontrolmechanismsthatincreasetheshuntingofglycolyticintermediatesintoalternativepathways,agooddealofphosphoenolpruvateisdephosphorylatedintopyruvate.Ifpyruvatedehydrogenasewerethentoconvertpyruvatetoacetyl-CoA,itwouldproceedthroughtheTCAcycle,generatingATP.Thiswouldinhibittheactionofphosphofructokinase,anallostericenzymethatisinhibitedbyATPwhileitalsoconsumedATPtophosphorylateFructose-6-phosphate.AfurtherfunctionofHIF-1,asshowninFigure2,istoupregulatepyruvatedehydrogenasekinase1(PDK1).AsshowninFigure3,PDK1inhibitspyruvatedehydrogenaseandstopsentryintotheTCAcycle.HIF-1alsoup-regulateslactatedehydrogenase(LDH),whichconvertspyruvatetolactate.Thisaccountsoftheaccumulationoflactateanditstransportoutofthecellincancer.ResearchonthecontrolprocessesthroughwhichglucosemetabolismisalteredinthecourseofcancerhasthusrevealedahostofflexibleconstraintsintheglycolyticmechanismonwhichcontrolcanbeexercisedbyothermoleculessuchasHIF-1.Innormalcellsthiscontrolservestoreducetherateofglycolysisandtodirecttheproduct,pyruvate,intotheTCAcycle.ThesecontrolmechanismsarealteredbythefailuretodegradeHIF-1inthepresenceofoxygen.Asaresult,therateofglycolysisisincreasedincancerandmanyofitsintermediatesareusedtosynthesizeothercompoundsrequiredincancercells.Byrevealingtheseflexibleconstraintsandthecontrolthatcanbeexercisedonthem,cancerresearchprovidesadifferentperspectiveontheglycolyticmechanism.Ratherthanoperatingfromstarttoterminationconditionswheneverglucoseisavailable(andnotsuppressedbyalreadyaccumulatedATP5),theglycolyticmechanismisseentocontainahostofflexibleconstraintsthatareoperatedonbyvariouscontrolprocesses.6.AnIntegratedNetworkofControllersTheresearchdiscussedsofarshowsthatinvestigatingcancerhasrevealedaplethoraofcontrolpathwaysthatwhendisruptedleadtohallmarksofcancer.Asiscommoninmechanisticresearch,theinvestigationstartedbyidentifyingindividualgenesthatarefrequentlymutatedincancer(BechtelandRichardson1993/2010)andthenreasoned 5 The control of phosphofructokinase by ATP had been discovered independently as a result of discovery of oscillations in the concentrations of NAD+ and other intermediates and determination that these oscillations resulted from the feedback of ATP on the mechanism (Ghosh and Chance 1964).

forwardsandbackwards(CraverandDarden2013)toidentifyoperationsinthecontrolmechanisms.Researchersattemptedtoorganizetheseoperationsintopathwayscharacterizedbysequencesofreactionsandthentolinkthesepathwaystoflexibleconstraintsinprimarymechanisms.Butthisresearchalsorevealsthatthesepathwaysinterconnect.AlthoughFigures1-3showpathways,theyalsorevealnumerouspointsofconnection.Ifonefollowstheseout,itbecomesapparentthatinfactthecontrolsystemsarenotindependentbutintegratedintoasinglenetwork.InthissectionIwillnoteacoupleofthesepointsofintegrationandthendemonstratehownetworkanalysesarenowplayingcrucialrolesinadvancingtheunderstandingofbothcancerandcontrolinnormalcells.AboveIfocusedontherolesmutatedRasproteinsplayinpromotingproliferation,buttheyalsoregulatemanyothermechanismsresponsibleforhallmarksofcancer.Whenthesignaltheyproduceistoostrong,apoptosisisinducedbyactivatingRbandTP53.Inaddition,Rasproteins,aswellasotheroncogenes,upregulateGLUT1(Murakamietal.1992),therebyincreasingglycolysis.Rasproteinsalsofigureinthekeyalternativepathwaysinglycolysis—inthepentosephosphatepathway,theyupregulatetwokeyenzymes,transketolase-like1(TKTL1)andtransaldolase(TALDO).TheseenzymesareinturnsuppressedbyTP53,sowhenRasproteinsaresufficientlyactivesoastoactivateTP53,itdown-regulatestheseenzymes.Thus,Rasproteinsareimplicatedinproliferation,apoptosis,andalteredmetabolism.HIF-1alsofiguresincontrolofmanycellmechanisms.AboveIonlyaddressedthepathwaysontheleftsideofFigure2throughwhichHIF-1operatestocontrolglycolysis.ButthefigurealsoindicatesthatHIF-1playsaroleinregulatingvascularendothelialgrowthfactor(VEGF),whichencodesligandsthatcontrolnewbloodvesselgrowthduringembryonicandpostnataldevelopmentandisup-regulatedinhypoxiaandcancer(Ferrara2009).Thus,italsohelpsexplainthehallmarkofangiogenesis.AngiogenesisisalsocontrolledbylactateresultingfromthealteredcontrolHIF-1exertsoverglycolysisincancercells(Végranetal.2011).Finally,HIF-1up-regulateserythropoietin(EPO),whichstimulatesproductionofredbloodcells.Bycontrollingthesetwoprocesses,HIF-1functionstoincreaseoxygenandnutrientdeliverybeyondwhatisrequiredinnormalcellfunction.ThisresearchhasrevealedthatHIF-1performscontroloperationswithrespecttoadiverserangeofcellmechanisms.Yetanotherexampleinwhichresearchhaspointedtotheinteractionofcontrolprocessesisprovidedbytheproteinsubunitoftelomeres,telomerereversetranscriptase(TERT).InadditiontoitsroleinaddingtelomeresandthusdefeatingtheHayflicklimitoncellreplication,notedabove,TERTalsofunctionsasacofactorofβ-catenin/LEFtranscriptionfactorcomplexthatfiguresintheWntpathway(notdiscussedabovebutshownintheupperleftofFigure1).TheWntpathwayisimportantinregulatingthecellcycleasitactivatesreplicationinstemcells(Parketal.2009;Bryja,Červenka,andČajánek2017).TERThasalsobeenshowntohaveeffectsonregulatingapoptosis(Kangetal.2004)andDNA-damagerepair(Masutomietal.2005).ThesevariousfindingsillustratesomethingthatisalreadyapparentinFigure1:thedifferentpathwaysinvolvedincontrolofcellprocessesarenotindependent,butinteractat

numerouspoints.Althoughthenotionofpathwayplaysanimportantrolewhentracingouttheindividualstepsinspecificcontrolmechanisms,itdoesnotprovidethebestwaytounderstandtheseinteractions.Conceptualizingcontrolintermsofnetworksprovidesanalternativeframeworkthatisincreasinglybeingemployedincancerresearchtounderstandcontrolsystemsthatareoperativeinbothcancerandnormalcells.Networkdiagramsconsistofnodesandedges,inwhichnodesstandforvariouskindsofentities(genes,proteins,etc.)andedgesforvarioustypesofinteractionsbetweennodes.Pathwayanalysescanbetransformedintonetworkdiagramsbyabstractingfromspecificdetails(Figure1effectivelydoesthat),butthereareotherstrategiesforconstructingnetworkmodelsthatcanyieldadditionalinsightsintocontrolsystems.Researchersworkingonmodelorganismssuchasyeasthavecreatednetworkdiagramsbasedonavarietyoftypesofgeneandproteininteractiondata.Forexample,theyeasttwo-hybridtechniquerevealswhichproteinsinacellareabletoformcomplexeswhereasinvestigationsofsyntheticlethalityindicategenesthatinteractinthegenerationoftraits.InBechtel(2017,inpress)Ihaveshownhowanalysesofthesenetworkshaveprovidednewinsightsintothemechanismsinvolvedinyeastcells.6Similardataisnowbeinggeneratedforhumancells,butinthemeantimeinvestigatorshavealsofounditproductivetopredictgeneandproteininteractionsonthebasisofhomologybetweenmodelorganismsandhumans.Thesenetworksalonedonotprovideinformationaboutwhereproteinsareexpressedincellsorthebiologicalprocessestowhichtheycontribute,crucialforamechanisticunderstanding.However,networkresearchershavedevelopedstrategiesforannotatingthesenetworkswiththistypeofinformationusingresourcessuchasGeneOntology(GO)(Ashburneretal.2000).7Giventhenumberofinteractionsbetweengenesandproteinsthatthesetechniquesreveal,theresultingnetworkdiagramsinitiallyappearashairballsinwhichnointerpretablepatternscanbeidentified.Networkresearchershavedevelopedanumberofanalyticaltools,availableinnetworkrepresentationplatformssuchasCytoscape(Shannonetal.2003),tomakesenseofnetworks.AparticularlyusefultypeofanalysisthatCytoscapefacilitatesisidentifyingclustersinnetworks—setsofnodesthatareespeciallydenselyinterconnected.Networkresearchersofteninterpretdenseclustersofhighlyinterconnectednodesinprotein-proteininteractionnetworksasmechanismsengagedinparticulartaskswhereasconnectionsbetweenthesemodulesareinterpretedasvehiclesofcontrol.OthertoolsinCytoscapethathelpturnhairballsintointerpretablenetworksarelayoutalgorithms(suchasforce-basedonesthattreatedgeslikesprings,pullingconnectednodessituatedfarfromeachothertogetherandpushingthoseveryclosetoeachotherslightlyapart)andfilteringtoolstolookselectivelyatparticularnodesandedges. 6 There is disagreement as to whether network analyses complement (Matthiessen 2017) or compete with (Braillard 2010) mechanistic accounts. In Green et al. (2018), we discuss a range of examples of network analyses, ranging from those that integrate with mechanistic accounts to those that abstract from concrete mechanisms to focus on dynamics. 7 For a philosophical examination of the classification scheme employed in GO, see Leonelli (2010).

Iwillpresentoneexampleinwhichresearchersextendedapathwayanalysisintoanetworkanalysis.FromReactome,adatabaseofpathways,andavarietyofresourcesprovidingproteinandgeneinteractiondata,Wu,Feng,andStein(2010)developedalargenetworkof10,956proteinsand209,988interactions,whichtheytermedtheFunctionalInteraction(FI)network.TheyusedthisnetworktointerpretglioblastomadatacompiledinTheCancerGenomeAtlas’scharacterizationof206glioblastomas(CancerGenomeAtlasResearchNetwork2008).TheresearchersidentifiedthoseproteinsinFIthatcorrespondedtogenesidentifiedasmutatedinatleasttwoTCGAsamples.TheythenincludedthefewestadditionalproteinsinFItocreateaconnectednetworkincludingatleast70%ofthesealteredgenes.Theyproposedthattheresultingnetwork,showninFigure4,isthecoresubnetworkofmutationsinglioblastoma.SincetheyhadbegunwithapathwayanalysisfromReactome,theywereabletoidentifythoseproteinsintheirproposedcoresubnetworkthatbelongedtofourpathways—p53,focaladhesion,signalingbyPDGF,and

Figure4.CoresubnetworkWu,Feng,andSteinextractedfromTCGAglioblastomadatawithidentificationofgenesinfourpathwaysshowninshadedregions.Thecolorofthenodesindicatesgenessharedinanothersampleofglioblastomatumors(yellow)ornotshared(blue).Redindicatesthosenodesaddedtoconnectthenetwork.NodesizeindicatesfrequencyofmutationinTCGAsample.ThisappearsasFigure8AinWu,Feng,andStein(2010)andisreprintedfromBioMedCentralundertheCreativeCommonsAttribution(CC-BY)license.

cellcyclepathways.TheseareshowninshadinginFigure4.Thefigurerevealsboththatmanyoftheproteinsinthenetworkbelongtothesefourpathwaysandalsothatthesepathwaysarehighlyintertwined.Networkanalysesgobeyondthepathwayanalysesinrevealingtheinterconnectednatureofthecontrolmechanismsoperativeinnormalcellsanddisruptedincancer.Inparticular,theyprovideastrategytoavoidthinkingofcontrolinpurelyhierarchicalterms,withcontrollersresidingatahigherlevelthantheprocessestheycontrol.Asnotedabove,inhuman-mademachines,controlprocessesareorganizedhierarchicallytoenablehumanoperatorstodeterminetheactivityofthemachine.Butinbiologicalorganisms,controlmechanismsgenerallyterminatewithintheorganism.Differentcomponentsoftheorganisminitiatecontrolprocessesthatregulateothercomponents,butthisdoesnotleadtoatop-levelcontrolleroverseeingthewholeoperation.Instead,theresearchrevealsanetworkinwhichmultiplecontrolmechanismsareembeddedandthroughwhichtheyinteract.Asaresultoftheirinteractions,whenageneismutated,asincancer,itdoesnotalterthebehaviorofjustoneprimarymechanism,butoftenawiderange(givingrisetothemultiplehallmarksofcancerdiscussedabove).7.ConclusionsIhavepresentedarangeofexamplesofresearchoncancerasawaytomakeclearhowcomplexthecontrolprocessesoperatingonmechanismsinlivingcellsare.ThehallmarksofcanceridentifiedbyHanahanandWeinbergallinvolvealtereddeploymentsofprimarymechanismsthatfigureinnormalcelllife.Altereddeploymentresultsfromchangesincontrolmechanismsthatdeterminewhenthesevariousprimarymechanismsoperate.Thesecontrolmechanismsareproductivelyanalyzedintermsofpathwaysconsistingofcomponentsthatactsequentiallyoneachother.But,asIhavetriedtoillustrate,thesepathwaysofteninteractwithoneanother,constitutingnetworks.Inadditiontotheprimarymechanismsthatareresponsibleforphenomenasuchasenergyprocurementandproteinsynthesis,cellscontaincomplexnetworksofcontrolmechanismsthatoperateonflexibleconstraintsintheprimarymechanisms.Changesincontrolmechanismsresultintheredeploymentoftheprimarymechanismstomaintainthealteredlifeofacancercell.Philosophicalaccountsofbiologicalmechanismshavenotemphasizedcontrol,insteadtreatingmechanismsasoperatingwhenevertheirstart-upconditionsarerealized(Machamer,Darden,andCraver2000).InthispaperIhaveadvancedaperspectiveinwhichthepartsandoperationsofmechanismsprovideconstraintsthatdirecttheflowoffreeenergy,enablingthemechanismtoperformwork.Manyoftheconstraintsinmechanismsareflexible,capableofbeingalteredthroughworkperformedbyothermechanisms.Theseothermechanismsexercisecontrol.Theytooinvolveflexibleconstraintsthatarecapableofbeingoperatedonbyyetothermechanisms.Inhuman-builtmachines,suchahierarchyofcontrolmechanismstypicallytopsoutwhenhumanagentsalterconstraintstoexercisecontrol.Butinbiologicalorganismscontrolprocessesoriginatewithinorganismsandenabletheprimarymechanismstheycontroltooperateappropriatelytomaintaintheorganisms.Sincethesecontrolmechanismsareinterconnected,itishelpfultorepresenttheminanetwork,notastricthierarchy.

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