senescence and immortalization - cancer treatment

SenescenceandImmortalizationLecture18 November52015Assignedreading Chapter10

Contact:[email protected]

Normal Development

Adult stem/progenitors

functional

maturation

PCD (apoptosis)

Differentiation

Death

Senescence

ESCs

chronological aging

SlideCredit:Dr.DeanGTang

Immortalization,Senescence,Telomerase,andCancer

1. CellSenescence:Characteristics2. Telomerase,Senescence,andCancer

WhatisSenescence??

Astateofcellularbeingcharacterizedby:a) metabolicactivitybutb) irreversiblelossofthecapacitytoenteractivecellcyclec) Growthfactorshelpsustainviabilitybutd) Areunabletoelicitusualproliferativeresponse

LeonardHayflickandPaulMoorhead(1961)firstshowedthephenomenonthatcellswouldstopgrowingafteracertainnumberofdivisions.

PhaseICellsdivideactivelytocovertheculturedishPhaseIICellsproliferaterobustlyPhaseIIICellsremainviablebutdonotproliferate

NormalcellshavepredeterminedpopulationdoublingsProliferativecapacitydecreaseswithage

Thenumberofgrowthanddivisioncyclesisdictatedbythespeciesandtissueoforiginandageofthedonor.

Regenerativecapacitydeclineswithage.

Characteristicsofsenescentcells

• Permanentgrowtharrest–cannotbereversedbyphysiologicalstimuli• Increasedcellsize–flatcellswithhugecytoplasm(appearanceofafriedegg)• Increasedcytoplasmicgranularity• Expresssenescenceassociated(SA)betagalactosidase• Metabolicallyactive

Influenceofcultureconditions

HigherOxygenLevels ReactiveOxygenSpecies Oxidativedamage

SenescenceHypoxia

PhysiologicalLaboratoryincubator

Senescentcellsexhibitinabilitytoenteractivecellcycle


Cyclindependentkinaseinhibitorsareinducedduringsenescence

Overexpressionofp16(middle)caninducesenescencephenotypeasseenincellsthathaveenteredsenescenceafterextensivepropagationinvitro.

TheINK4locus


Twomajortumorsuppressorsp53andpRbregulatesenescence

Inactivationofbothp53andpRbisneededtoescapefromsenescenceBothp53andpRbareinactivatedinmajorityofhumancancers

DNAdamageresponsecanactivatecellsenescence

MarkerisgammaH2AX

Normal Development

Adult stem/progenitors

functional

maturation

PCD (apoptosis)

Differentiation

Death

Senescence

ESCs

chronological aging


Comparisonbetweenterminallydifferentiatedandsenescentcellsinculture

Differentiated Senescent

Morphology generallybig big&flatSA-βgalpositivity - +Motility littleorlow littleSaturationdensity low lowDifferentiationmarkers + +/-Differentiationfunctions + -Cellcycle “permanent”G0/G1 “permanent”G0/G1Responsetomitogens low lowKaryotypicstability high lowResistancetoapoptosis +/- +Essence “permanent”cell “permanent”cell cyclearrestto cyclearrestwith evolveintofunc- continuedgrowth tionalcelltypes


Senescenceassociatedheterochromaticfoci(SAHFs)

Markersare:H3K9meHP1gamma

Senescence&SenescentCells1) Bigandflat,prominentcytoplasmic/nuclearvacuolization,lessmotile,decreasedsaturation

density,andpositiveforSA-βgal.2) Multiplealterationsingeneexpression,e.g.,overexpressionofcollagenaseand

underexpressionofTIMPs(tissueinhibitorofmetalloproteinase).3) Attenuatedproliferativeresponsetomitogens(EGF,PDGF,IGF-1)andunabletoinducec-fos

(butmycandrasinductionok).4) “Irreversible”cell-cyclearrestatG1/Swith2Nnuclearcontent(butincreasednuclearsize);<1

PDin2weeks.5) Decreaseinpositiveregulators(cyclinD/Cdk4,cyclinE/Cdk2,etc)andincreaseinnegative

regulators(p16,p21,p19ARF,hypo-phosphorylatedRB,etc).6) Resistancetoapoptosisinduction.7) Senescentcells,toadegree,resembleterminallydifferentiatedcells.8) PresenescentcellsoftenshowtelomeredysfunctionasrevealedbymarkersATMactivationand

formationofnuclearfocicontainingH2AX-γ,53BP1,MDC1,NBS1,whichdisappearinfullysenescentcells.*

9) Fullysenescentcellsoftenpossesskaryotypicinstability:tetraploidy,endoreduplication,aneuploidy,andotherabnormalkaryotypes.

10) Cellularsenescence,likeaging,isdominant.Therefore,immortalityresultsfromrecessivechangesinnegativeregulators(tumorsuppressivegenes).

11) Senescentcellsaccumulatesenescence-associatedheterochromatinfoci(SAHFs),inwhichHMG-Aproteinsaccumulate.

12) Senescentcellsreleasepro-inflamatorycytokines(interleukins,IGFBPs,andTGF-beta)thatactinanautocrinemannertopromotesenescenceandinaparacrinefashiontorecruitpro-inflamatorycellstopromotetumorigenesis.

*BakkenistCJ,DrissiR,WuJ,KastanMB,DomeJS.Disappearanceofthetelomeredysfunction-inducedstressresponseinfullysenescentcells.CancerRes.2004Jun1;64(11):3748-52. SlidemodifiedfromDr.DeanGTang

Sohowdoweexplaintheseeffectsseeninsenescentcells?

Telomeres


**1978:Telomerewasfirstfoundasanunusualrepeatedsequencemotif(GGGGTT)atchromosometerminiintheciliateTetrahymena(Blackburn&Gall,J.Mol.Biol.120,33-53,1978).**Tremendousvariability:<50bpinthehypotrichousciliatesbutaslongas5100kbinmice.**1985-1989:Telomeraseactivityandtelomeraseuncovered(GreiderCWandBlackburnEH,Cell43,405-413,1985;Cell51,887-898,1987;Nature337,331-337,1989).

**Inhumans,telomeresaremadeupofanaverageof5,000-15,000bpofG-rich (TTAGGG)nrepeatsandtelomere-bindingproteins.**Eachcelldivisionloses50-100bpoftelomeres**Whenatelomerelosesacriticalnumberofbasepairs,ittriggersaDNAdamagesignaltostopcelldivisionandinitiatesenescence.

Telomerefunctions:

---formspecificcomplexeswithtelomerebindingproteins---protectchromosomeendsfromexonucleasedigestion---preventaberrantrecombination---preventthechromosomeendsfromactivatingcell-cycleandDNAdamagecheckpoints

Telomereandthechromosome

TelomereSynthesis

Endreplicationproblem:WatsonJD.NatureNewBiol.239,197-201,1972.OlovnikovAM.J.Theoret.Biol.41,181-190,1973. SlideCredit:Dr.DeanGTang

TelomereandTelomerase


TelomereandTelomeraseAssays**DirectmeasurementoftelomerelengthA.TRA(terminalrepeatassay)1.DigestDNAwithAluIorHinfI.2.PerformSouthernwithradiolabeled(TTAGGG)n

B.Q-FISHorflowcytometryusingfluorescently-labeledprobes1.MetaphasespreadsarehybridizedwithCy3-labeledPNA(CCCTAA)3telomericoligonucleotide.2.TelomerefluorescenceintensityanalyzedbyTFL-Telo.3.1TFU=1kbtelomere(PNAS94,7423-7428,1997).4.Flowcytometryisperformedusingsimilarprocedures.

**TRAP(telomericrepeatamplificationprotocol)(Kimetal.,Science266,2011-2015,1994;KimandWu,NAR,25,2595-2597,1997)1.Preparecelllysates(inCHAPSbuffer).2.Addanend-labeledtelomere-specificoligonucelotidesubstrate (TSprimer)tothelysates.3.Iftelomeraseispresent,itaddsTTAGGGrepeatstothesubstrates.4.PCRamplificationoftheextensionproductsusingTSprimerand reverseprimer(ACTorACX).


WT TRF2deprived

TRF2isakeyproteininmaintainingnormaltelomerestructure

LossofTRF2leadstoextensiveendtoendfusion

---TelomeresofculturedsomaticcellscontinuouslyerodeuntilM1---Telomeresderivedfromelderlyindividualstendtobeshorterthanthosederivedfromyoungdonors---Telomeresderivedfromconstantself-renewingtissuessuchasliverandGIsystemstendtobeshorterthanmostothertissuesandorgans.---Telomerelengthisapredicatorofproliferativepotential---IfM1isovercomebytransformationwithviraloncogenes,telomerescontinuetodecreaseinsizeuntilM2,aprocessthatmaybedictatedbytelomerelengthitself.---Whereastelomeresizecontinuouslydecreasesduringreplicativesenescence,immortalizedcellsreachanequilibrium,albeitatshorter-than-wild-typelength

Telomere,senescenceandtumorigenesis


Telomere,TelomeraseandtheHayflicklimit(HarleyCB,Oncogene21:494,2002)


Apoptosisassociatedwithcellpopulationsincrisis

BFB(Breakage-Fusion-Bridge)Cycle

Karyotypicchaosseeninhumanbladdercancercellline

CriticalDifferencesinHumanvsRodentCells

Mouse HumanPopulation

Doublings (PD) 10-30 60-80Telomere length 60-100 kb 10-15 kbInvolvement of

ARF-mdm2-p53 Yes YesInvolvement of

p16-cyclin D-pRb No YesTelomerase activity

in somatic cells Yes No/lowRate of spontaneous

immortalization (10-4-10-5) Low (10-7)Conclusion Prematur e senescence Related to

Induced by inappr o- telomerepr iate culture conditions shortening

Veryhigh


Istelomereshorteningreallyimportant:ThemTR-/-mousemodel

1mTR-/-micelackeddetectabletelomeraseactivityyetwereviableforthe6generationsanalyzed.Telomerase-deficientcellscouldbeimmortalizedinculture,transformedbyviraloncogenes,andgeneratedtumorsinnudemicefollowingtransformation.Cellsfromthe4thmTR-/-generationonwardpossessedchromosomeendslackingdetectabletelomererepeats,aneuploidy,andchromosomalabnormalitiesincludingend-to-endfusions.

2Late-generationmTR-/-miceshowdefects(decreasedproliferationandincreasedapoptosis)inhigh-renewableorgansystemssuchasspermatogenesisandhematopoieticcellsinbonemarrowandspleen.

3mTR-/-EScellsslowdowntheirproliferationafter~300divisionsandcompletelystopproliferationafter450divisions. 4Late-generationmTR-/-micedemonstrateshortenedtelomereandgeneticinstability,shortenedlifespanandreducedcapacitytorespondtostressessuchaswoundhealingandhematopoieticablation.Therewasincreasedincidenceofspontaneousmalignancies.

1.Blascoetal.,Cell91,25-34,1997.2.Leeetal.,Nature392,569-574,1998.3.Niidaetal.,NatureGenetics,19,203-206,1998.4.Rudolphetal.,Cell96,701-712,1999.


Istelomereshorteningreallyimportant:ThemTR-/-mousemodel

1mTR-/-significantlyreducestumorformationinp16INK4A/p19ARFnullmice.ReintroductionmTRintocellsrestoredtheoncogenicpotential,suggestingthattelomeraseactivationisacooperatingeventinthemalignanttransformationofcellscontainingcriticallyshorttelomeres.Lossoftelomerefunctionimpairs,butdoesnotpreventtumorformation.

2Late-generationmTR-/-cellsshowseveretelomereshortening,genomicinstability,andp53activation,leadingtocell-cyclearrestand/orapoptosis.ThemTR-/-p53-/-miceshowedsignificantlyincreasedrateofepithelialcancerformation.

3mTR-/-miceshowrapidlivercirrhosiswhensubjectedtogenetic,chemical,andsurgicalablation.Telomerasegenedeliveryalleviatedcirrhoticpathologyandrestoredliverfunction. 4Telomeredysfunctioninlate-generationmTR-/-miceimpairsDNArepairandenhancessensitivitytoionizingradiation.

5Telomeredysfunction,togetherwithp53deficiency,promotesnon-reciprocaltranslocationsandepithelialcancersinmice.

1.Greenbergetal.,Cell97,515-525,1999.2.Chinetal.,Cell97,527-538,1999.3.Rudophetal.,Science287,1253-1258,2000.4.Wongetal.,NatureGenetics26,85-88,2000.5.Artandietal.,Nature406,641-644,2000.


HumanvsMouseTumors

1.Themajorityofmousetumorsaresarcomasandleukemiaswhereas 80%ofthehumantumorsarecarcinomas-cancerofepithelia whererapidcellturnoveroccurs.2.Mostoftheexperimentaltherapeuticsthatworkinmousefailinhuman, why???3.Theanswermaypartlylieinthebehavioroftelomeres,andtherelation- shipbetweentelomereshortening,replicativecellsenescence, andgeneticinstability.4.Inhuman,telomeraseissuppressedorshutdownandtelomereshortening leadstoreplicativecellsenescence.Inmice,cellshavelongtelomeres andretaintelomeraseactivity,thusnotelomere-dependentreplicative senescence.However,inthe5-6thgenerationofTERC-/-cells,themice begintoshowvariousabnormalities,includingincreasedincidence ofcancer,raisingthepossibilitythatnaturaltelomereshorteninghelps toengendermanyhumantumors.(TERCistelomeraseRNAcomponent).


**Asignificantnumberofimmortalortumorcelllineshavenodetectabletelomeraseactivityandalsonodefectinproliferationandgrowth.**Thesecellshaveunusuallylongtelomeres(upto50kb;~30kblongerthanthatobservedinthelongesttelomerase-positivecelllines).**TheALT(alternativelengtheningoftelomeres)pathwayof telomeremaintenance(EMBOJ.,14,4240-4248,1995;NatureGenetics,26,447-450,2000).ALToccursbymeansofhomologousrecombinationandcopyingswitching(i.e.,DNAsequencesarecopiedfromtelomeretotelomere).

Telomerase-independenttelomeremaintenance


1. Telomeraseisanti-apoptotic(Caoetal.,Oncogene21,3130-3138,2002).2. Telomerasecontributestotumorigenesisbyatelomerelength-independent

mechanism(Stewartetal.,PNAS99,12606,2002;ChangandDePinho,PNAS99,12520-12522,2002).

3. TelomeraseenhancesDNArepairandgenomicstability(Oncogene22,131-146,2003).

4. TERTpromotescellularandorganismalsurvivalindependentlyoftelomeraseactivity.LeeJ,SungYH,CheongC,ChoiYS,JeonHK,SunW,HahnWC,IshikawaF,LeeHW.Oncogene.2008Jun12;27(26):3754-60.

Telomere-independentfunctionsoftelomerase


1) TelomeraseactivityandhTERT(telomerasereversetranscriptase)expressionare loworabsentinmostsomaticcellsandprimarytissues,dueto

a)transcriptionalrepressionbyWT1andMad,b)transcriptionalrepressionbyhistonedeacetylation.

2) Inimmortalizedorcancercells,hTERTactivityis‘reactivated’dueto a)transcriptionalupregulationbymyc,E2F1etc, b)geneamplification, c)varioussignalingpathwayssuchasc-Abl,bFGF,14-3-3,Hsp90,Akt,PKC,etc, d)epigeneticchromatinremodeling.

3) Telomeraseactivityisnormallyassociatedwithproliferation:cyclingcellshavehighwhiledifferentiatingcellshavelowtelomeraseactivity.Duetothis correlation,normalcellshaverelativelylongertelomeresthantumorcells becausethelatterhaveundergonemorecelldivisions.

DysregulationofTelomeraseduringTumorigenesis


ReplicativevsPrematureCellSenescence

LloydAc.NatureCellBiol.4,E25-E27,2002.

p21-inducedcellsenescenceappearstodependonROSproduction(Macipetal.,EMBOJ.21,2180-2188,2002).

InadequatecultureconditionsHighO2levelsTang, D.G., et al. Lack of replicative senescence in

cultured rat oligodendrocyte precursor cells. Science 291: 868-871, 2001.


Senescence,Aging,andCancer“Becausecancerincidenceriseswithage,itseemsthatcellularsenescenceisanimperfect

tumor-suppressivemechanismthatfailsincreasinglywithage.Infact,lateinlife,accumulationofsenescentcellsprobablyevencontributestotumorigenesisbecausesenescentcellsoverexpress,e.g.,collagenasesandunderexpresstheirinhibitors”

---JudyCampisi

1) Senescentfibroblastspromotepremalignantandmalignantcellproliferationinvitroandtumordevelopmentinvivo(KrtolicaAetal.,PNAS98,12072-12077,2001)

2) Drug-inducedsenescenttumorcellssecreteanti-apoptotic,mitogenic,andangiogenicfactorsthatpromotetumordevelopment(ChangBDetal.,PNAS99,389-394,2002)

3) Ionization-induced,senescent-likefibroblastspromotebreastcancerdevelopment(TsaiKKetal.,CancerRes.65,6734-6744,2005)

4) Stromalfibroblastsfromold(63-81yr)prostatesexpressandsecretehigherlevelsofCXCL12chemokine,whichstimulatesCXCR4-mediatedepithelialcellproliferation(BegleyLetal.,AgingCell4,291-298,2005)

5) Senescenthumanprostatefibroblastspromotenormalandcancerousprostateepithelialcellproliferation(BavicCetal.,CancerRes.66,794-802,2006)

6) Senescenthumanprostateepithelialcellspromotetumordevelopment(Bhatiaetal Int.J.Cancer,2008)


MartinRaff:in“MolecularBiologyoftheCell”,the4thedition,pp1324,2002.

ReplicativecellsenescenceandcancerHypothesis1:Replicativesenescenceisanti-cancermechanismandanaddedbarrierthat cancercellshavetobreakthrough.Hypothesis2:Cellsinmosttissuesneveractuallyreachreplicativesenescenceinthe courseofahumanlifetime,andthe“immortality”ofcancercellsismerelya side-effectofselectionforsomeotherpropertytheyneedtohave.Hypothesis3:Manycellsinnormaltissueundergoreplicativesenescenceandslowdown orhalttheirproliferationasapersonages,andthiscreatescircumstances inwhichcancercellscanthriveallthebetterbycontinuingtodivideatfull throttle.Thus,replicativesenescence,farfromprotectingusfromthegrowth oftumors,createsabreedinggroundformutantcellsthatevadethenormal controlsandoverrunthetissuebecausetheyfacenocompetitionfromtheir senescentnormalneighbors.Themutationsthatallowcontinuedproliferation mayconferatthesametimeothercanceroustraits,suchasgeneticinstabilityor ageneraldisregardforcell-cyclecontrols,leadingonprogressivelymore disorderedbehavior.Inthisway,replicativecellsenescenceinself-renewing tissuesmightbeexpectedtofavorthegenesisofcancer;itcouldbeapartofthe reasonwhycancerispredominantlyadiseaseofoldage.


Maser&DePinho:Science,297,565,2002.

Replicativecellsenescenceandcancer

“TheHayflicklimitpresentsablocktonormalcellgrowthinculture,butbecausecancercellsinvariablyinactivateRbandp53pathways,ithasbeendifficulttodocumentadirectroleforshortenedtelomere-inducedsenescenceintumorsuppressioninvivo.Wefavorthehypothesisthatcrisisplaysamoreprominentrolethansenescenceintumorigenesis.Althoughcrisisisapotentbarriertoimmortalgrowthinculture,themassivegeneticinstabilityassociatedwiththisstatemaywellbethemechanismbywhichtherarecellssurvivingcrisisacquiretheconstellationofgeneticalterationsneededformalignanttransformation.Theserarecellsemergebyactivatingtelomeremaintenancemechanisms-mostcommonlybyactivatingtelomerase.”


Evidenceofsenescenceasatumorsuppressionmechanism

KangTWetal.,Senescencesurveillanceofpre-malignanthepatocyteslimitslivercancerdevelopment.Nature.2011Nov9;479(7374):547-51.

Upontheaberrantactivationofoncogenes,normalcellscanenterthecellularsenescenceprogram,astateofstablecell-cyclearrest,whichrepresentsanimportantbarrieragainsttumourdevelopmentinvivo.Senescentcellscommunicatewiththeirenvironmentbysecretingvariouscytokinesandgrowthfactors,anditwasreportedthatthis'secretoryphenotype'canhavepro-aswellasanti-tumorigeniceffects.Hereweshowthatoncogene-inducedsenescenceoccursinotherwisenormalmurinehepatocytesinvivo.Pre-malignantsenescenthepatocytessecretechemo-andcytokinesandaresubjecttoimmune-mediatedclearance(designatedas'senescencesurveillance'),whichdependsonanintactCD4(+)T-cell-mediatedadaptiveimmuneresponse.Impairedimmunesurveillanceofpre-malignantsenescenthepatocytesresultsinthedevelopmentofmurinehepatocellularcarcinomas(HCCs),thusshowingthatsenescencesurveillanceisimportantfortumoursuppressioninvivo.Inaccordancewiththeseobservations,ras-specificTh1lymphocytescouldbedetectedinmice,inwhichoncogene-inducedsenescencehadbeentriggeredbyhepaticexpressionofNras(G12V).WealsofoundthatCD4(+)Tcellsrequiremonocytes/macrophagestoexecutetheclearanceofsenescenthepatocytes.Ourstudyindicatesthatsenescencesurveillancerepresentsanimportantextrinsiccomponentofthesenescenceanti-tumourbarrier,andillustrateshowthecellularsenescenceprogramisinvolvedintumourimmunesurveillancebymountingspecificimmuneresponsesagainstantigensexpressedinpre-malignantsenescentcells.


TumorDevelopment

Normalcells(stem/progenitorcellsortheirprogeny)

Tumorcells

oncogenictransformation

malignanttransformation

Cancercells

Metastasesprogression

Immortalcells

immortalization

Cellsenescence


1. DNAdamageisabletoinducesenescenceinp53-wttumorcellsinvitroandinvivo.p53andp21appeartoplayacriticalroleintheonsetofsenescencewhilep16isinvolvedinmaintenanceofsenescence(tePoeleetal.,CancerRes.62,1876-1883,2002).

2. Senescenceinductionappearstocontributesignificantlytotheefficacyofanti-neoplasticdrugs(Schmittetal.,Cell109,335-346,2002;CancerCell1,289-296,2002;JCI,113,169-174,2004).

SenescenceasaTherapeuticAlternative


senescence and immortalization - cancer treatment

Documents