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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 [email protected] Forfurtherinformationorquestionsrelatingtothisprojectpleasecontact: DanielMuñoz-Espín [email protected]