Neoplasia2020/21lecture6Dr Heyam AwadMD,FRCPath

ILOS

• 1.understandthebasicconceptsofcellaging.• 2.realizetheimportanceofavoidingsenescenceasahallmarkofcancer.• 3.understandevadingapoptosisasahallmarkofcancer.• 4.listingchangesintheapoptoticpathwaysthatcanleadtocancer.• 5.applythisknowledgeinclinicalscenarios,likefollicularlymphoma.• 6.understandthemetabolicchangesoccurringduringtumorigenesis• 7.understandthebasicconceptsbehindPETscanexamination• 8.understandtheeffectofautophagyincancerdevelopment• 9.understandtheconceptofoncometabolitesandtheirtargetedtherapy.

Thirdhallmark:limitlessreplicativepotential

• Normalcells:limitedcapacitytoduplicate(usually60-70doublings).• Afterthesedoublingscellslosethecapacitytoreplicateandbecomesenescent.• Thisisbecauseofprogressiveshorteningoftelomeres.

telomeres

• Eachcellhasalimitedreplicativepotential.• Thisisbecausechromosomeshaverepeatednucleotidesequencesattheendsofeachchromosome.• Witheachcellreplication,telomeresshorten,till theybecometooshortandthechromosomalendsfusetogetherwhichcausescelldeathbyapoptosis.• StemcellshavelimitlessreplicativepotentialbecausetheyhavetelomeraseenzymewhichusesitsRNAnucleotidesequencetoreplacethelosttelomeres.• Cancercellsupregulatetelomerasetranscriptionandbecomeimmortal.

CellSenescence&Telomeres

• Cellsavoidsenescencebyactivatingtelomerase.• Telomerelengthismaintainedinallcancercells..MainlybyupregulationoftelomerasebutalsobyothermechanismslikeDNArecombinations

Fourthhallmark

• Evasionofcelldeathbyevadingapoptosis.

Apoptosis:areminder!

• Apoptosis:programmedcelldeathinwhichcellsactivateenzymesthatdegradethecells’ownnuclearDNAandnuclearandcytoplasmicproteins• Sothecellscommitsuicide!• Thecellsfragmentandthefragmentsarephagocytosedwithoutelicitinginflammatoryresponse

Apoptosis

Extrinsicpathway

• Fasligand• Fasreceptor• FADD• Caspase8.• Caspase 3

• Decreaseanyoftheabove…..Evasionofcelldeath

FLIP

extrinsicpathway

• Triggerwhichstartsapoptosisisoutsidethecells.• ThepathwaystartswhenFasligandbindstoFasreceptor• Uponthisthereceptorisactivated;ittrimerizesanditscytoplasmicpart(deathdomain)isactivated.• Activationofthereceptorattractsacytoplasmicprotein=FADD• FADDrecruitsprocaspase8• Procaspasecleavedtoactivecaspase8(initiationcaspase)• Caspase8activatescaspase3(executioner)whichcleavesDNAandcellularprotein

Extrinsicpathway

• FLIP isacaspase 8antagonist• SoifFLIPisincreasedcellscanevadeapoptosis

• FLIP-similarproteinsareproducedbysomeviruses..Helpingthemtokeepinfectedcellsalive.

Intrinsicpathway=mitochondrialpathway

• ThispathwayisstimulatedifthereisDNAdamagesecondarytostress,radiation,chemicalsorduetowithdrawalofsurvivalfactors• Thispathwayisintrinsic..Sonotinitiatedbymembranereceptors…insteaditisinitiatedbyincreasedmitochondrialpermeability• Whenmitochondrialpermeabilityincreases..cytochromecleaksoutandinitiatesapoptosis• Nowcytochromecisinthecytosol..SoitbindsAPAF1• Thisbindingactivatescaspase 9• Caspase 9activatescaspase 3

Intrinsicpathway

InternalstresseswithincellsIncreasemitochondrialpermeabilityCytochromecleaksoutsidethemitochondriaCytochromecbindstoAPAF1Caspase 9activatedCaspase 3activated

Again:decreaseanyoftheseandthecellcanavoidapoptosis

IAP

Mitochondrialpermeability

• MitochondrialpermeabilityiscontrolledbyBH3proteins(BAD,BID,PUMA)• WhenBH3proteinssenseinternalstress..Stimulateproapoptoticproteinsandinhibitantiapoptotic ones• Proapoptotic:BAX,BAK• Antiapoptotic:BCL2,BCL- Xl

• SodecreaseBAD,BID,PUMA,BAX,BAK…NOAPOPTOSIS• IncreaseBCL2ANDBCL-Xl….Noapoptosis

Mitochondrialpermeability

• MitochondrialpermeabilityiscontrolledbyBH3proteins(BAD,BID,PUMA)• WhenBH3proteinssenseinternalstress..Stimulateproapoptoticproteinsandinhibitantiapoptotic ones• Proapoptotic:BAX,BAK• Antiapoptotic:BCL2,BCL- Xl

• SodecreaseBAD,BID,PUMA,BAX,BAK…NOAPOPTOSIS• IncreaseBCL2ANDBCL-Xl….Noapoptosis

note

• IAP=inhibitorofapoptoticprotein,inhibitscaspase 9• SoincreaseIAPandapoptosiscanbeavoided.

P53andapoptosis

• DNAdamagecausesaccumulationofp53 incells• ItarrestscellsinG1phaseofcellcycletogivethecellachancetorepairitself• Ifnorepair,p53triggersapoptosisbystimulatingbax andbak• P53canbemutatedincancercells..Ifmutateditcannotinitiateapoptosis,sothecellsurvivesevenifitsDNAisdamaged..LongersurvivalofacellwithdamagedDNAincreasesthechancesofaccumulatingmoremutations..Sothiscellcanbecomemalignant

Warburgeffect

• AlthoughlessATPisproduced…theWarburgeffectensuresthatcarbonatomsinglucose(whichisconvertedtoPyruvate)areusedforsynthesisoforganiccompoundslikelipidsandproteinswhichareimportantinbuildingnewcellsinthehighlyproliferativetumor.• SO:Aerobicglycolysisprovidesrapidlydividingtumor cellswithmetabolicintermediatesthatareneededforthesynthesisofcellularcomponents,whereasmitochondrialoxidativephosphorylationdoesnot.

bcl2

• Follicularlymphomasareslowgrowing(indolent)tumorsthathaveatranslocationcausingincreasedbcl2• T(14;18)….Bcl2translocatedandoverexpressed• Inlymphocyteshavingthismutation…apoptosisisdecreased• Theselymphocyteslivelongerratherthanbeingtransformed…that’swhythistypeoflymphoma(follicularlymphoma)isindolent

Follicularlymphoma/notetheformationoffollicles

Fifthhallmark:changesincellmetabolism

• Thesechangesinclude• 1.reprogrammingofenergymetabolismtoaerobicglycolysis• 2.changesinautophagy• 3.formationofoncometabolites

Reprogrammingofenergymetabolism

Normalcellsobtainenergyby:• Oxidativephosphorylationifoxygenisavailable.Inthisprocesseachglucosemoleculeusedproduces36ATPmolecules.• Anaerobicrespirationifoxygenlevelsarelow.Inthisprocessglucoseisconvertedtolacticacidandforeachglucosemoleculeusedonly2ATPmoleculesareproduced.

Reprogrammingofenergymetabolism

• Cancercellshaveathirdway!• Theyconvertglucosetolacticacideveninthepresenceofadequateoxygen• Thisprocessiscalled:aerobicglycolysisorWarburgeffect.

Aerobicglycolysis

• Cancercellsdidn’tinventaerobicglycolysis!• Actually,rapidlyproliferatingnormalcells,likeinembryonictissuesandlymphocytesduringimmuneresponses,relyonaerobicfermentation(glycolysis).• So:“Warburgmetabolism”isnotcancerspecific,butinsteadisageneralpropertyofgrowingcells.

Note:

• Agrowingcellmustduplicateallofitscellularcomponents—DNA,RNA,proteins,lipid,andorganelles—beforeitcandivideandproducetwodaughtercells.• WhileoxidativephosphorylationyieldsabundantATP,itfailstoproduceanycarbonatomsthatcanbeusedtobuildthecellularcomponentsneededforgrowth(proteins,lipids,andnucleicacids).Evencellsthatarenotactivelygrowingmustshuntsomemetabolicintermediatesawayfromoxidativephosphorylationinordertosynthesizemacromoleculesthatareneededforcellularmaintenance.

Howdoescancercellsdothisswitchofmetabolism????• Metabolicreprogrammingisproducedbysignallingcascadesdownstreamofgrowthfactorreceptors,theverysamepathwaysthatarederegulatedbymutationsinoncogenesandtumorssuppressorgenesincancers.• Thus,whereasinrapidlydividingnormalcellsaerobicglycolysisceaseswhenthetissueisnolongergrowing,incancercellsthisreprogrammingpersistsduetotheactionofoncogenesandthelossoftumor suppressorgenefunction,includingTP53mutations.

PETscan

• Becauseofthisreprogramming,tumorcellsare“glucosehungry”,theytakeloadsofglucose,andthispropertyisusedinPETscans• PET:positronemissiontomography• Patientisinjectedwithaglucosederivative..Tumorcellstakethisderivativemorethannormalcellsandassuchdetectedwiththescan• Themoreproliferativethetumoris…moreuptakeandmorepositivitywithPETscan

PETscan

PETscan

PETscan

IMPORTANTNOTE

• NotethatweagreedthatALLthephenotypes(cancerhallmarks)areneededtotransformcells.• But,itshouldbeclearnowthatwedon’tneed8mutationsforthe8hallmarks!• Example:p53mutationscancauseinsensitivitytogrowthsignals,evasionofapoptosis,andreprogrammingofenergymetabolism:threehallmarksfromonemutation!

autophagy

• Autophagyisacatabolicprocessthatbalancessynthesis,degradationandrecyclingofcellularproducts• Therecyclingofthecell’sorganellescanproduceenergyneededforthestressedcells.• Thisprocesscansignalcelldeathifthecellcannotberescuedbytherecyclingprocess

autophagy

• Autophagyisastateofseverenutrientdeficiencyinwhichcellsnotonlyarresttheirgrowth,butalsocannibalizetheirownorganelles,proteins,andmembranesascarbonsourcesforenergyproduction).• Ifthisadaptationfails,thecellsdie.• Tumor cellsgrowundermarginalenvironmentalconditionswithouttriggeringautophagy,suggestingthatthepathwaysthatinduceautophagyarederanged.• Inkeepingwiththis,severalgenesthatpromoteautophagyaretumorsuppressors.

note

• Althoughautophagyisananti-tumorprocess…..Lateronifthereisatumormassformed,autophagycanhelpthetumortosurviveifit’susedtorecycleorganellestobeusedasanenergysource.• Autophagycanhelptumorcellstosurviveduringunfriendlyclimates:forexampleduringchemotherapytreatment.

oncometabolism

• Thisisanewconcept,whichwasdiscoveredthroughfindingcertainmutationsinenzymesthatparticipateintheKrebscycle.• Ofthese,mutationsinisocitratedehydrogenase(IDH) isthemoststudied.

HowamutationinIDHcausescancer?

• IDHacquiresamutationinvolvingtheactivesiteoftheenzyme,soitlosesitsabilitytofunctionasanisocitratedehydrogenaseandinsteadacquiresanewenzymaticactivitythatcatalyzes theproductionof2-hydroxglutarate(2-HG).• 2-HGinturnactsasaninhibitorofseveralotherenzymesthataremembersoftheTETfamily,includingTET2.• TET2regulateDNAmethylation,whichisanepigeneticmodificationthatcontrolsnormalgeneexpression.• AbnormalDNAmethylationinturnleadstomis-expressionofcurrentlyunknowncancergenes,whichdrivecellulartransformationandoncogenesis.

Oncometabolite:ametabolicproductcausingoncogenesis.

• IDHmutationsarefoundingliomas,acutemyeloidleukaemia,andsarcomas.• themutatedIDHproteinshaveanalteredstructure,soithasbeenpossibletodevelopdrugsthatinhibitmutatedIDHandnotthenormalIDHenzyme.• Thesedrugsarenowbeingtestedincancerpatientsandhaveproducedencouragingtherapeuticresponses.

summary• Thethirdhallmarkofcancerisabilitytohavelimitlessreplications.Thisisacquiredviaupregulatingtelomeraseenzyme.

• Evadingapoptosisisanimportanttraitofcancercells.Thisoccursviablockingapoptoticorstimulatinganti-apoptoticmechanisms.

• Tumorcellshavealteredmetabolismthatenhancestheirsurvival.Theseinclude:reprogrammingofenergymetabolism,autophagychangesandoncometaboliteformation.

• Warburgphenomenon=aerobicmetabolism=fermentationevenwiththepresenceofoxygen.ThisreducesATPgeneratedpergramglucosebutprovidescarbonatomsneededforcelldivisionandgrowth.

• Thisswitchinmetabolismisachievedviaoncogenesoverexpressionandtumorsuppressorgenesinactivation.Inbothinstancesthereisshiftinglucosemetabolism.

• Autophagyisevadedintumors,butisupregulatedduringstress(chemotherapyorischemia)torecyclecellcomponentsandenhancesurvival.

• IDHmutationsaeaprototypeforoncometabolites,theyactbychangingenzymeactivityresultinginDNAmethylation.Newtherapiesarediscoveredtotargetthesemutatedenzymes.

Question

• A54yearoldmaledevelopedabraintumorwhichwasdiagnosedasanastrocytoma.ImmunohistochemicalstainsshowedpositiveIDHstainingimplyingamutationinIDH.Throughwhichofthefollowingmechanismsthismutationresultedincancer?• A.increasedmicroRNAs• B.oncogeneamplification• C.tumorsuppressorgenedownregulation• D.epigeneticchanges…(itactsviaDNAmethylation)• E.ImpairingDNArepairmechanisms