Download Interplay between cellular senescence and plasticity at the origin of

Interplaybetweencellularsenescenceandplasticityattheoriginof
lungcancer
LeadInvestigator:
DanielMuñoz-Espín
DepartmentofOncology
http://www.cambridgecancercentre.org.uk/users/dm7427210
Thisproposalcombinesoriginality,interdisciplinarysynergy,andacompetitivePhDpositionwiththe
EarlyDetectionProgrammeandinaworld-classcancerresearchinstitution.Wearelookingfor
highlymotivatedstudentswithanacademicdegreeinMedicine,BiologyorBiomedicalSciences.Our
locationintheCambridgeBiomedicalCampusprovidesapowerfullinkbetweenfundamentaland
translationalresearch.Awardedapplicantswillbenefitfromextensivecollaborationswithour
partnerinstitutionsintheCambridgeCancerCentre,includingtheCRUKCambridgeInstitute,clinical
andresearchdepartmentsintheSchoolofClinicalMedicine,otherMRClaboratoriesandthe
InstituteofMetabolicSciences,aswellascommercialpartnerssuchasAstraZenecaand
GlaxoSmithKline.Researcherswillhaveaccesstoallcentralfacilitiesandstate-of-the-art
infrastructuresandtechnologies.
MycollaborationswithDrsRobertRintoul(AerodigestiveProgrammeofCambridgeCancerCentre)
andManuelRodríguez-Justo(ConsultantPathologistatUCLHospitals)willprovideallsupportive
datainclinicaloncology.Inaddition,wewillinteractwithinternationalcentresofexcellencefor
cancerresearch,includingthelaboratoryofDrManuelSerrano(SpanishNationalCancerResearch
Centre).
ProjectDescription
Background
Identificationofthecelloforiginofcancerremainsachallengeinmedicine.Importantly,recent
advancesinlineagetracingareconcludinginvarioustumourmodelsthatthecelloforiginisa
differentiatedcellthatupondamage,includingtheactivationofoncogenesand/orthelossof
tumoursuppressors,undergoesde-differentiationandacquiresanaberrantplasticstatethat
initiatescancer(forreviewsee1).Examplescanbefoundinglioblastomas,intestinaltumoursand
pancreaticcancers,wheredamage-inducedplasticity(a“gainofcellularplasticity”inresponseto
damage)leadstoaberrantcancerinitiation.
Cellularsenescenceisacommoncellautonomousresponsetodamageandoncogenicstress
characterisedbyastableproliferativearrestandanintenseparacrinesecretion,termedSASP,
affectingnearbytissue.IhaverecentlyreportedinCellthatcellularsenescenceplaysanactiverole
inorchestratingtissueremodelling(2).Besidesbeingrelevantforageing,cellularsenescenceis
associatedwithawidevarietyofage-relateddisorders,includingcancer,playingantagonisticroles
(forreviewsee3).Cellularsenescenceis,likeapoptosis,acrucialbarrieragainstcancerasitarrests
theexpansionofpremalignantcells.Thisiswell-knownbothinhumanandinmice,andgaveriseto
theconceptof“oncogene-inducedsenescence”(OIS)(reviewedin4).However,inthelongterm,
accumulatedsenescentcellsthatarenotremovedbytheimmunesystemcanpromotemalignant
phenotypesbysecretingpro-inflammatoryandpro-tumorigenicfactors(reviewedin4).Additionally,
areasenrichedinsenescentcellsinresponsetochemotherapy(therapy-inducedsenescence),may
actuallycontributetotheemergenceofsecondarycancers(therapy-inducedcancers).
Hypothesis
Duringthelastfewyearsevidenceisaccumulatingofaninterconnectionbetweencellulardamage,
theinflammatorymicroenvironmentandcellularplasticity(reviewedin5).Ithasbeenconvincingly
reportedbyseveralgroupsthattheinflammatoryresponsetodamagefavourscellularplasticityand
reprogramming-likeprocesses.Theconceptofcellulardamagecreatingamicroenvironmentthat
favourscellde-differentiationandplasticityplacessenescenceasanintriguingkeyprocess.Onthe
onehand,cellularsenescenceisaresponsetodamageand,ontheotherhand,senescentcellscan
increasetheinflammatorymilieuthroughtheSASP.
Ourworkinghypothesisisthattheaccumulationofsenescentcellscanpromote,throughtheSASP,
a“maladaptivegainofplasticity”innearbycellsfavouringcancer.Thisnegativeeffectmayoccur
uponpersistentdamageoroncogenicstress,duringageing,inchronicpathologicdisorders,andin
responsetochemotherapeuticdrugs.Insupportofthisweknowthattheconditionedmediumof
senescentcellsstronglyenhancestheinvitroreprogrammingefficiencyofmouseembryonic
fibroblastsexpressingYamanakafactors.Interestingly,wehaveobservedthat,inmousemodelsof
bleomycin-inducedpulmonaryfibrosisandK-rasdrivenlungcancer,senescentcellscoexistinclose
vicinitytocellsexpressingpluripotencymarkers(includingOct4).
Objectives
Thisprojectfocusesontheprocessesandmechanismsthatlieattheoriginoflungcancer.In
particular,ontheroleofcellularsenescenceandtheSASP.WewilluseaK-rasdrivenlung
adenoma/adenocarcinomamousemodelthatrecapitulateshumanlungcanceraccuratelyasithas
similargeneexpressionprofilesandphenotype.Cellularsenescenceisadefiningfeatureoflung
adenomas(earlytumours)butnotoflungadenocarcinomas(advancedtumours).Wemayalso
exploremodelsofchronicobstructivepulmonarydisease(COPD),knowntobeassociatedwith
cellularsenescenceandwithahigherlungcancerincidence.
Specificaimsare:
(i)
Toaddresstheconnectionbetweencellularsenescenceandplasticityinlungtumorigenesis.
Wewilldeterminewhethersenescence-inducedplasticityisanactiveoncogenicpromoter.
(ii)
Toanalysethecomponentsofthesecretomeofsenescentcellsinabackgroundoflung
cancer,andtoidentifythespecificSASPfactorsthatmayinducea“maladaptivegainof
plasticity”innearbycells.
(iii)
Toisolate(de-differentiatedor“plastic”)tumourprecursorcellsandtocharacterisegene
expressionprofilesandepigenetics.
Methodology
Task1.ToexaminetheimpactofcellularsenescenceandtheSASP-associatedmicroenvironment
oncellplasticity.
Wewilldeterminethekineticsanddynamicsofthehistologicalorganisationofsenescent,
pluripotent,andproliferativecellsinearlylunglesionsandtumours.Todoso,wewillemployboth
genetic(K-rasG12V)andchemical(injectingmethyl-nitrosurea/urethane)modelsoflung
adenomas/adenocarcinomas.Collectedtissuesandtumoursatdifferenttimepointswillbe
subjectedtosenescence-associatedβ-galactosidase(SAβ-gal)assaysandimmunohistochemistry
analysestodetectothermarkersofsenescence(p53,p16),pluripotency(Oct4,Nanog)and
proliferation(Ki67,BrdU).
Toestablishacausallinkbetweencellularsenescenceandtheinductionofpluripotentfeaturesin
nearbycellsduringlungcancerinitiationwewillcomparewild-typemicewithdifferentsenescencealteredmousestrains(includingp53KO,p16ArfKOandaSASP-deficientmodel).Tumourprogression
willbemonitored,andsampleswillbecollectedtodeterminehowsenescenceaffectstumour
latency,malignancyandhistology.
Task2.Dissectingthesenescencesecretomeintheinitiationoflungcancer.
ProteinarrayswillbeemployedtoidentifySASPcomponents(cytokines,metalloproteases,etc.)in
thelungsatearlycancerstages.Simultaneouslytotheproteomicsapproachesinvivo,the
contributionoftheSASPtoamaladaptivegainofplasticityinnearbycellswillbecharacterisedin
culturesofcelllinessubjectedtooncogenicstressorisolatedfromourcancermodels.Gene
expressionprofilesandstableisotopelabellingwithaminoacidincellculture(SILAC)analyseswillbe
performedtocharacterisethesenescence-associatedsecretome.Thesestudieswillbe
complementedbymetabolomicsapproaches.
Task3.Isolationoflungtumoursprecursorcells.Geneexpressionprofilesandepigenetics.
WewillemployaconditionallineagetracingmousemodelforOct4thatactivatestheexpressionof
greenfluorescentproteinatthecellmembrane(mGFP).Thismodelwillbeusedtocapturestableor
transienteventsofOct4activationduringlungcancerinitiation,bothinwild-typeandsenescencedeficientmice.TheproportionoftumourcellsthatderivefromanOct4positiveprecursorinthe
differentgeneticbackgroundswillbedetermined.Incaseofevidenceofcancercellsderivingfrom
Oct4positivecellsinasenescence-dependentmanner,wewilltakeadvantageofthemGFP
expressiontoisolatetheseprecursorsbycellsorting.Tumourprecursorcellswillbesubjectedto
RNA-seqanalysesandalsotomethodsappliedtothestudyofhistonemodificationsandDNA
methylation.
ImpactandExpectedOutcomes
Weaimtointegrateintoaunifiedmodeloncogene-inducedsenescenceandoncogene-induced
plasticity,andtodemonstratethatsenescence-inducedplasticityisapotentoncogenicpromoter.
Theproposedstudywillexpandourknowledgeoftheroleofcellularsenescenceandplasticityin
tumorigenesis,anditwillopennewfrontiersontheprocessesandmechanismsthatlieattheorigin
oflungcancer.
References
(1) Friedmann-MorvinskiD,VermaIM.Dedifferentiationandreprogramming:originsofcancerstem
cells.EMBOReports.2014;15(3):244-253.
(2) Muñoz-EspínD,CañameroM,MaraverA,Gómez-LópezG,ContrerasJ,Murillo-CuestaS,
Rodríguez-BaezaA,Varela-NietoI,RuberteJ,ColladoM,SerranoM.Programmedcellsenescence
duringmammalianembryonicdevelopment.Cell.2013;155(5):1104-1118.
(3) Muñoz-EspínD,SerranoM.Cellularsenescence:fromphysiologytopathology.NatRevMolCell
Biol.2014;15(7):482-496.
(4) Pérez-MánceraPA,YoungAR,NaritaM.Insideandout:theactivitiesofsenescenceincancer.Nat
RevCancer.2014;14(8):547-548.
(5) JessenKR,MirskyR,Arthur-FarrajP.Theroleofcellplasticityintissuerepair:adaptivecellular
reprogramming.DevCell.2015;34(6):613-620.
Applications
Toapplyforthisstudentshippleaseseehttp://www.cambridgecancercentre.org.uk/studentships
ForgeneralenquiriespleasecontactTinaThorn tina.thorn@cruk.cam.ac.uk
Forfurtherinformationorquestionsrelatingtothisprojectpleasecontact:
DanielMuñoz-Espín
dm742@cam.ac.uk