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