Hallmarks of Cancer: New Dimensions - AACR Journals

The hallmarks of cancer conceptualization is a heuristic tool for distilling the vast complexity of cancer phenotypes and genotypes into a ... SkiptoMainContent Advertisement Close AACRJournals BloodCancerDiscovery CancerDiscovery CancerEpidemiology,Biomarkers&Prevention CancerImmunologyResearch CancerPreventionResearch CancerResearch CancerResearchCommunications ClinicalCancerResearch MolecularCancerResearch MolecularCancerTherapeutics ForAuthors InformationforAuthors AuthorServices JournalAwards Bestof:AuthorProfiles AboutUs AACRJournals Advertising Permissions&Reprints Subscriptions Submit ArticleCollections JournalCollections HotTopics 'BestOf'Collection Editors'Picks COVID-19 Webinars SearchDropdownMenu headersearch searchinput Searchinputautosuggest Search AdvancedSearch UserToolsDropdown Register SignIn ToggleMenuMenu About TheJournal AACRJournals Subscriptions PermissionsandReprints Articles OnlineFirst Issues MeetingAbstracts Collection:PrecisionMedicineandTherapeuticResistance Collection:ClinicalTrials Collection:Immuno-oncology Collection:Editors'Picks 'Best'Of'Collection ForAuthors InformationforAuthors AuthorServices Bestof:AuthorProfiles Submit Alerts News COVID-19 Webinars 10thAnniversary SkipNavDestination ArticleNavigation Closemobilesearchnavigation Articlenavigation Volume12,Issue1 1January2022 Abstract Introduction UnlockingPhenotypicPlasticity NonmutationalEpigeneticReprogramming PolymorphicMicrobiomes SenescentCells ConcludingRemarks Author'sDisclosures Acknowledgments References ArticleNavigation Review| January122022 HallmarksofCancer:NewDimensions DouglasHanahan DouglasHanahan * *CorrespondingAuthor:DouglasHanahan,AgoraTranslationalCancerResearchCenter,RueduBugnon25A,LausanneCH-1011,Switzerland.Phone:41-21-545-1119;E-mail:[email protected] Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar Author&ArticleInformation *CorrespondingAuthor:DouglasHanahan,AgoraTranslationalCancerResearchCenter,RueduBugnon25A,LausanneCH-1011,Switzerland.Phone:41-21-545-1119;E-mail:[email protected] CancerDiscov2022;12:31–46 Received: August042021 RevisionReceived: November172021 Accepted: November182021 OnlineIssn:2159-8290 PrintIssn:2159-8274 ©2022AmericanAssociationforCancerResearch2022AmericanAssociationforCancerResearch CancerDiscov(2022)12(1):31–46. https://doi.org/10.1158/2159-8290.CD-21-1059 Articlehistory Received: August042021 RevisionReceived: November172021 Accepted: November182021 Split-Screen ViewsIcon Views Articlecontents Figures&tables Video Audio SupplementaryData PeerReview PDF ShareIcon Share Twitter LinkedIn ToolsIcon Tools GetPermissions CiteIcon Cite SearchSite ArticleVersionsIcon Versions VersionofRecord January122022 Citation DouglasHanahan;HallmarksofCancer:NewDimensions.CancerDiscov1January2022;12(1):31–46.https://doi.org/10.1158/2159-8290.CD-21-1059 Downloadcitationfile: Ris(Zotero) ReferenceManager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbarsearch SearchDropdownMenu toolbarsearch searchinput Searchinputautosuggest Search AdvancedSearch Abstract Thehallmarksofcancerconceptualizationisaheuristictoolfordistillingthevastcomplexityofcancerphenotypesandgenotypesintoaprovisionalsetofunderlyingprinciples.Asknowledgeofcancermechanismshasprogressed,otherfacetsofthediseasehaveemergedaspotentialrefinements.Herein,theprospectisraisedthatphenotypicplasticityanddisrupteddifferentiationisadiscretehallmarkcapability,andthatnonmutationalepigeneticreprogrammingandpolymorphicmicrobiomesbothconstitutedistinctiveenablingcharacteristicsthatfacilitatetheacquisitionofhallmarkcapabilities.Additionally,senescentcells,ofvaryingorigins,maybeaddedtotherosteroffunctionallyimportantcelltypesinthetumormicroenvironment.Significance:Cancerisdauntinginthebreadthandscopeofitsdiversity,spanninggenetics,cellandtissuebiology,pathology,andresponsetotherapy.Evermorepowerfulexperimentalandcomputationaltoolsandtechnologiesareprovidinganavalancheof“bigdata”aboutthemyriadmanifestationsofthediseasesthatcancerencompasses.Theintegrativeconceptembodiedinthehallmarksofcancerishelpingtodistillthiscomplexityintoanincreasinglylogicalscience,andtheprovisionalnewdimensionspresentedinthisperspectivemayaddvaluetothatendeavor,tomorefullyunderstandmechanismsofcancerdevelopmentandmalignantprogression,andapplythatknowledgetocancermedicine. Introduction TheHallmarksofCancerwereproposedasasetoffunctionalcapabilitiesacquiredbyhumancellsastheymaketheirwayfromnormalcytoneoplasticgrowthstates,morespecificallycapabilitiesthatarecrucialfortheirabilitytoformmalignanttumors.Inthesearticles(1,2),BobWeinbergandIenumeratedwhatweimaginedweresharedcommonalitiesthatunitealltypesofcancercellsatthelevelofcellularphenotype.Theintentwastoprovideaconceptualscaffoldthatwouldmakeitpossibletorationalizethecomplexphenotypesofdiversehumantumortypesandvariantsintermsofacommonsetofunderlyingcellularparameters.Initiallyweenvisagedthecomplementaryinvolvementofsixdistincthallmarkcapabilitiesandlaterexpandedthisnumbertoeight.Thisformulationwasinfluencedbytherecognitionthathumancancersdevelopasproductsofmultistepprocesses,andthattheacquisitionofthesefunctionalcapabilitiesmightbemappedinsomefashiontothedistinguishablestepsoftumorpathogenesis.Certainly,thediversityofmalignantpathogenesisspanningmultipletumortypesandanincreasingplethoraofsubtypesincludesvariousaberrations(andhenceacquiredcapabilitiesandcharacteristics)thataretheresultoftissue-specificbarriersnecessarilycircumventedduringparticulartumorigenesispathways.Whileappreciatingthatsuchspecializedmechanismscanbeinstrumental,welimitedthehallmarksdesignationtoparametershavingbroadengagementacrossthespectrumofhumancancers. Theeighthallmarkscurrentlycomprise(Fig.1,left)theacquiredcapabilitiesforsustainingproliferativesignaling,evadinggrowthsuppressors,resistingcelldeath,enablingreplicativeimmortality,inducing/accessingvasculature,activatinginvasionandmetastasis,reprogrammingcellularmetabolism,andavoidingimmunedestruction.Inthemostrecentelaborationofthisconcept(2),deregulatingcellularmetabolismandavoidingimmunedestructionweresegregatedas“emerginghallmarks,”butnow,elevenyearslater,itisevidentthatthey,muchliketheoriginalsix,canbeconsideredcorehallmarksofcancer,andareincludedassuchinthecurrentdepiction(Fig.1,left). Figure1.ViewlargeDownloadslideInessence:theHallmarksofCancer,circa2022.Left,theHallmarksofCancercurrentlyembodyeighthallmarkcapabilitiesandtwoenablingcharacteristics.Inadditiontothesixacquiredcapabilities—HallmarksofCancer—proposedin2000(1),thetwoprovisional“emerginghallmarks”introducedin2011(2)—cellularenergetics(nowdescribedmorebroadlyas“reprogrammingcellularmetabolism”)and“avoidingimmunedestruction”—havebeensufficientlyvalidatedtobeconsideredpartofthecoreset.Giventhegrowingappreciationthattumorscanbecomesufficientlyvascularizedeitherbyswitchingonangiogenesisorbyco-optingnormaltissuevessels(128),thishallmarkisalsomorebroadlydefinedasthecapabilitytoinduceorotherwiseaccess,principallybyinvasionandmetastasis,vasculaturethatsupportstumorgrowth.The2011sequelfurtherincorporated“tumor-promotinginflammation”asasecondenablingcharacteristic,complementingoverarching“genomeinstabilityandmutation,”whichtogetherwerefundamentallyinvolvedinactivatingtheeighthallmark(functional)capabilitiesnecessaryfortumorgrowthandprogression.Right,thisreviewincorporatesadditionalproposedemerginghallmarksandenablingcharacteristicsinvolving“unlockingphenotypicplasticity,”“nonmutationalepigeneticreprogramming,”“polymorphicmicrobiomes,”and“senescentcells.”ThehallmarksofcancergraphichasbeenadaptedfromHanahanandWeinberg(2).Figure1.ViewlargeDownloadslideInessence:theHallmarksofCancer,circa2022.Left,theHallmarksofCancercurrentlyembodyeighthallmarkcapabilitiesandtwoenablingcharacteristics.Inadditiontothesixacquiredcapabilities—HallmarksofCancer—proposedin2000(1),thetwoprovisional“emerginghallmarks”introducedin2011(2)—cellularenergetics(nowdescribedmorebroadlyas“reprogrammingcellularmetabolism”)and“avoidingimmunedestruction”—havebeensufficientlyvalidatedtobeconsideredpartofthecoreset.Giventhegrowingappreciationthattumorscanbecomesufficientlyvascularizedeitherbyswitchingonangiogenesisorbyco-optingnormaltissuevessels(128),thishallmarkisalsomorebroadlydefinedasthecapabilitytoinduceorotherwiseaccess,principallybyinvasionandmetastasis,vasculaturethatsupportstumorgrowth.The2011sequelfurtherincorporated“tumor-promotinginflammation”asasecondenablingcharacteristic,complementingoverarching“genomeinstabilityandmutation,”whichtogetherwerefundamentallyinvolvedinactivatingtheeighthallmark(functional)capabilitiesnecessaryfortumorgrowthandprogression.Right,thisreviewincorporatesadditionalproposedemerginghallmarksandenablingcharacteristicsinvolving“unlockingphenotypicplasticity,”“nonmutationalepigeneticreprogramming,”“polymorphicmicrobiomes,”and“senescentcells.”ThehallmarksofcancergraphichasbeenadaptedfromHanahanandWeinberg(2).Closemodal Aswenotedatthetime,thesehallmarktraits,ontheirown,failtoaddressthecomplexitiesofcancerpathogenesis,thatis,theprecisemolecularandcellularmechanismsthatallowevolvingpreneoplasticcellstodevelopandacquiretheseaberrantphenotypiccapabilitiesinthecourseoftumordevelopmentandmalignantprogression.Accordingly,weaddedanotherconcepttothediscussion,portrayedas“enablingcharacteristics,”consequencesoftheaberrantconditionofneoplasiathatprovidemeansbywhichcancercellsandtumorscanadoptthesefunctionaltraits.Assuch,theenablingcharacteristicsreflecteduponmolecularandcellularmechanismsbywhichhallmarksareacquiredratherthantheaforementionedeightcapabilitiesthemselves.Thesetwoenablingprocessesweregenomeinstabilityandtumor-promotinginflammation. Wefurtherrecognizedthatthetumormicroenvironment(TME),hereindefinedtobecomposedofheterogeneousandinteractivepopulationsofcancercellsandcancerstemcellsalongwithamultiplicityofrecruitedstromalcelltypes—thetransformedparenchymaandtheassociatedstroma—isnowwidelyappreciatedtoplayanintegralroleintumorigenesisandmalignantprogression. GiventhecontinuedinterestintheseformulationsandourenduringintenttoencourageongoingdiscussionandrefinementoftheHallmarksscheme,itisappropriatetoconsiderafrequentlyposedquestion:arethereadditionalfeaturesofthisconceptualmodelthatmightbeincorporated,respectingtheneedtoensurethattheyarebroadlyapplicableacrossthespectrumofhumancancers?Accordingly,Ipresentseveralprospectivenewhallmarksandenablingcharacteristics,onesthatmightinduecoursebecomeincorporatedascorecomponentsofthehallmarksofcancerconceptualization.Theseparametersare“unlockingphenotypicplasticity,”“nonmutationalepigeneticreprogramming,”“polymorphicmicrobiomes,”and“senescentcells”(Fig.1,right).Importantly,theexamplespresentedinsupportofthesepropositionsareillustrativebutbynomeanscomprehensive,asthereisagrowingandincreasinglypersuasivebodyofpublishedevidenceinsupportofeachvignette. UnlockingPhenotypicPlasticity Duringorganogenesis,thedevelopment,determination,andorganizationofcellsintotissuesinordertoassumehomeostaticfunctionsisaccompaniedbyterminaldifferentiation,wherebyprogenitorcells—sometimesirrevocably—stopgrowinguponculminationoftheseprocesses.Assuch,theendresultofcellulardifferentiationisinmostcasesantiproliferativeandconstitutesaclearbarriertothecontinuingproliferationthatisnecessaryforneoplasia.Thereisincreasingevidencethatunlockingthenormallyrestrictedcapabilityforphenotypicplasticityinordertoevadeorescapefromthestateofterminaldifferentiationisacriticalcomponentofcancerpathogenesis(3).Thisplasticitycanoperateinseveralmanifestations(Fig.2).Thus,nascentcancercellsoriginatingfromanormalcellthathadadvanceddownapathwayapproachingorassumingafullydifferentiatedstatemayreversetheircoursebydedifferentiatingbacktoprogenitor-likecellstates.Conversely,neoplasticcellsarisingfromaprogenitorcellthatisdestinedtofollowapathwayleadingtoend-stagedifferentiationmayshort-circuittheprocess,maintainingtheexpandingcancercellsinapartiallydifferentiated,progenitor-likestate.Alternatively,transdifferentiationmayoperate,inwhichcellsthatwereinitiallycommittedintoonedifferentiationpathwayswitchtoanentirelydifferentdevelopmentalprogram,therebyacquiringtissue-specifictraitsthatwerenotpreordainedbytheirnormalcells-of-origin.Thefollowingexamplessupporttheargumentthatdifferingformsofcellularplasticity,whentakentogether,constituteafunctionallydistincthallmarkcapability. Figure2.ViewlargeDownloadslideUnlockingphenotypicplasticity.Left,phenotypicplasticityisarguablyanacquiredhallmarkcapabilitythatenablesvariousdisruptionsofcellulardifferentiation,including(i)dedifferentiationfrommaturetoprogenitorstates,(ii)blocked(terminal)differentiationfromprogenitorcellstates,and(iii)transdifferentiationintodifferentcelllineages.Right,depictedarethreeprominentmodesofdisrupteddifferentiationintegraltocancerpathogenesis.Byvariouslycorruptingthenormaldifferentiationofprogenitorcellsintomaturecellsindevelopmentallineages,tumorigenesisandmalignantprogressionarisingfromcellsoforigininsuchpathwaysisfacilitated.ThehallmarksofcancergraphichasbeenadaptedfromHanahanandWeinberg(2).Figure2.ViewlargeDownloadslideUnlockingphenotypicplasticity.Left,phenotypicplasticityisarguablyanacquiredhallmarkcapabilitythatenablesvariousdisruptionsofcellulardifferentiation,including(i)dedifferentiationfrommaturetoprogenitorstates,(ii)blocked(terminal)differentiationfromprogenitorcellstates,and(iii)transdifferentiationintodifferentcelllineages.Right,depictedarethreeprominentmodesofdisrupteddifferentiationintegraltocancerpathogenesis.Byvariouslycorruptingthenormaldifferentiationofprogenitorcellsintomaturecellsindevelopmentallineages,tumorigenesisandmalignantprogressionarisingfromcellsoforigininsuchpathwaysisfacilitated.ThehallmarksofcancergraphichasbeenadaptedfromHanahanandWeinberg(2).Closemodal Dedifferentiation Coloncarcinogenesisexemplifiesdisrupteddifferentiation,inthatthereisateleologicalnecessityforincipientcancercellstoescapefromtheconveyerbeltofterminaldifferentiationandexfoliation,whichcouldinprincipleoccurviadedifferentiationofnotyetirrevocablyterminallydifferentiatedcolonicepithelialcells,orviablockeddifferentiationofprogenitor/stemcellsinthecryptsthatspawnthesedifferentiatingcells.Bothdifferentiatedcellsandstemcellshavebeenimplicatedascell-of-originforcoloncancer(4–6).Twodevelopmentaltranscriptionfactors(TF),thehomeoboxproteinHOXA5andSMAD4,thelatterinvolvedinBMPsignaltransmission,arehighlyexpressedindifferentiatingcolonicepithelialcells,andtypicallylostinadvancedcoloncarcinomas,whichcharacteristicallyexpressmarkersofstemandprogenitorcells.FunctionalperturbationsinmousemodelshaveshownthatforcedexpressionofHOXA5incoloncancercellsrestoresdifferentiationmarkers,suppressesstemcellphenotypes,andimpairsinvasionandmetastasis,providingarationaleforitscharacteristicdownregulation(7,8).SMAD4,bycontrast,bothenforcesdifferentiationandtherebysuppressesproliferationdrivenbyoncogenicWNTsignaling,revealedbytheengineeredlossofSMAD4expression,providinganexplanationforitslossofexpressionsoastoenablededifferentiationand,subsequently,WNT-drivenhyperproliferation(5).Notably,thelossofbothofthese“differentiationsuppressors”withconsequentdedifferentiationisassociatedwithacquisitionofotherhallmarkcapabilities,asareotherhallmark-inducingregulators,whichcomplicatesthestrictdefinitionofthisprovisionalhallmarkasseparableandindependent. AnotherlineofevidenceinvolvessuppressedexpressionoftheMITFmasterregulatorofmelanocytedifferentiation,whichisevidentlyinvolvedinthegenesisofaggressiveformsofmalignantmelanoma.LossofthisdevelopmentalTFisassociatedwiththereactivationofneuralcrestprogenitorgenesandthedownregulationofgenesthatcharacterizefullydifferentiatedmelanocytes.Thereappearanceoftheneuralcrestgenesindicatesthatthesecellsreverttotheprogenitorstatefromwhichmelanocytesarisedevelopmentally.Moreover,alineagetracingstudyofBRAF-inducedmelanomasestablishedmaturepigmentedmelanocytesasthecellsoforigin,whichundergodedifferentiationduringthecourseoftumorigenesis(9).Ofnote,themutantBRAFoncogene,whichisfoundinmorethanhalfofcutaneousmelanomas,induceshyperproliferationthatprecedesandhenceismechanisticallyseparablefromthesubsequentdedifferentiationarisingfromdownregulationofMITF.AnotherstudyfunctionallyimplicatedupregulationofthedevelopmentalTFATF2,whosecharacteristicexpressioninmouseandhumanmelanomasindirectlysuppressesMITF1,concomitantwithmalignantprogressionoftheconsequentlydedifferentiatedmelanomacells(10).Conversely,expressioninmelanomasofmutantformsofATF2thatfailtorepressMITFresultsinwell-differentiatedmelanomas(11). Additionally,arecentstudy(12)hasassociatedlineagededifferentiationwithmalignantprogressionfrompancreaticisletcellneoplasiasintometastasis-pronecarcinomas;theseneuroendocrinecellsandderivativetumorsarisefromadevelopmentallineagethatisdistinctfromtheonegeneratingthefarlargernumberofadjacentcellsthatformtheexocrineandpancreasandtheductaladenocarcinomasthatarisetherefrom.Notably,themultistepdifferentiationpathwayofisletprogenitorcellsintomatureβcellshasbeenthoroughlycharacterized(13).Comparativetranscriptomeprofilingrevealsthatadenoma-likeislettumorsaremostsimilartoimmaturebutdifferentiatedinsulin-producingβcells,whereastheinvasivecarcinomasaremostsimilartoembryonicisletcellprecursors.Theprogressiontowardpoorlydifferentiatedcarcinomasinvolvesafirststepofdedifferentiationthatdoesnotinitiallyinvolveincreasedproliferationorreducedapoptosiswhencomparedwiththewell-differentiatedadenomas,bothofwhichratheroccurlater.Thus,thediscretestepofdedifferentiationisnotdrivenbyobservablealterationsinthehallmarktraitsofsustainedproliferationandresistancetoapoptosis.Rather,upregulationofamiRNApreviouslyimplicatedinspecifyingtheisletprogenitorstate,onethatisdownregulatedduringterminaldifferentiationofβcells,hasbeenshowntoorchestratetheobserveddedifferentiationoccurringduringmalignantprogression(12). BlockedDifferentiation Whiletheaboveexamplesillustratehowsuppressionofdifferentiationfactorexpressioncanfacilitatetumorigenesisbyenablingmorewell-differentiatedcellstodedifferentiateintoprogenitors,inothercasesincompletelydifferentiatedprogenitorcellscansufferregulatorychangesthatactivelyblocktheircontinuedadvanceintofullydifferentiated,typicallynonproliferativestates. Acutepromyelocyticleukemia(APL)haslongbeendocumentedtoresultfromachromosomaltranslocationthatfusesthePMLlocuswiththegeneencodingtheretinoicacidαnuclearreceptor(RARα).Myeloidprogenitorcellsbearingsuchtranslocationsareevidentlyunabletocontinuetheirusualterminaldifferentiationintogranulocytes,resultingincellstrappedinaproliferative,promyelocyticprogenitorstage(14).Proof-of-conceptofthisschemecomesfromtreatingculturedAPLcells,mousemodelsofthisdisease,aswellasafflictedpatients,withretinoicacid,theligandofRARα;thistherapeutictreatmentcausestheneoplasticAPLcellstodifferentiateintoostensiblymaturenonproliferatinggranulocytes,short-circuitingtheircontinuingproliferativeexpansion(14–16). Avariationonthisthemeinvolvesanotherformofacutemyeloidleukemia,thisonecarryingthet(8;21)translocation,whichproducestheAML1–ETOfusionprotein.Thisproteincan,onitsown,transformmyeloidprogenitors,atleastinpartbyblockingtheirdifferentiation.Therapeuticinterventioninmousemodelsandinpatientswithapharmacologicinhibitorofachromatin-modifyinghistonedeacetylase(HDAC)causesthemyeloidleukemiacellstorecommencetheirdifferentiationintocellswithamorematuremyeloidcellmorphology.Concomitantwiththisresponseisareductioninproliferativecapacity,therebyimpairingtheprogressionofthisleukemia(17,18). Athirdexample,inmelanoma,involvesadevelopmentalTF,SOX10,whichisnormallydownregulatedduringmelanocytedifferentiation.Gain-andloss-of-functionstudiesinazebrafishmodelofBRAF-inducedmelanomahavedemonstratedthataberrantlymaintainedexpressionofSOX10blocksdifferentiationofneuralprogenitorcellsintomelanocytes,enablingBRAF-drivenmelanomastoform(19). Otherexamplesofdifferentiationmodulatorsinvolvethemetabolitealpha-ketoglutarate(αKG),anecessarycofactorforanumberofchromatin-modifyingenzymes,whichisdemonstrablyinvolvedinstimulatingcertaindifferentiatedcellstates.Inpancreascancer,thetumorsuppressorp53stimulatestheproductionofαKGandmaintenanceofamorewell-differentiatedcellstate,whereasprototypicallossofp53functionresultsinreductionsinαKGlevelsandconsequentdedifferentiationassociatedwithmalignantprogression(20).Inoneformoflivercancer,mutationofanisocitratedehydrogenasegene(IDH1/2)resultsintheproductionnotofdifferentiation-inducingαKGbutratherarelated“oncometabolite,”D-2-hydroxygluterate(D2HG),whichhasbeenshowntoblockhepatocytedifferentiationfromliverprogenitorcellsbyD2HG-mediatedrepressionofamasterregulatorofhepatocytedifferentiationandquiescence,HNF4a.TheD2HG-mediatedsuppressionofHNF4afunctionelicitsaproliferativeexpansionofthehepatocyteprogenitorcellsintheliver,whichbecomesusceptibletooncogenictransformationuponsubsequentmutationalactivationoftheKRASoncogenethatdrivesmalignantprogressiontolivercholangiocarcinoma(21).MutantIDH1/2andtheironcometaboliteD2HGarealsooperativeinavarietyofmyeloidandothersolidtumortypes,whereD2HGinhibitsαKG-dependentdioxygenasesnecessaryforhistoneandDNAmethylationeventsthatmediatealterationsinchromatinstructureduringdevelopmentallineagedifferentiation,therebyfreezingincipientcancercellsinaprogenitorstate(22,23). Anadditional,relatedconceptis“circumventeddifferentiation,”whereinpartiallyorundifferentiatedprogenitor/stemcellsexitthecellcycleandbecomedormant,residinginprotectiveniches,withthepotentialtoreinitiateproliferativeexpansion(24),albeitstillwiththeselectivepressuretodisrupttheirprogrammeddifferentiationinonewayoranother. Transdifferentiation Theconceptoftransdifferentiationhaslongbeenrecognizedbypathologistsintheformoftissuemetaplasia,whereincellsofaparticulardifferentiatedphenotypemarkedlychangetheirmorphologytobecomeclearlyrecognizableaselementsofanothertissue,ofwhichoneprominentexampleisBarrett'sesophagus,wherechronicinflammationofthestratifiedsquamousepitheliumoftheesophagusinducestransdifferentiationintoasimplecolumnarepitheliumthatischaracteristicoftheintestine,therebyfacilitatingthesubsequentdevelopmentofadenocarcinomas,andnotthesquamouscellcarcinomasthatwouldbeanticipatedtoarisefromthissquamousepithelium(3).Now,moleculardeterminantsarerevealingmechanismsoftransdifferentiationinvariouscancers,bothforcaseswheregrosstissuemetaplasiaisevidentandforotherswhereitisrathermoresubtle,asthefollowingexamplesillustrate. Oneilluminatingcasefortransdifferentiationasadiscreteeventintumorigenesisinvolvespancreaticductaladenocarcinoma(PDAC),whereinoneoftheimplicatedcellsoforigin,thepancreaticacinarcell,canbecometransdifferentiatedintoaductalcellphenotypeduringtheinitiationofneoplasticdevelopment.TwoTFs—PTF1aandMIST1—govern,viatheirexpressioninthecontextofself-sustaining,“feed-forward”regulatoryloops,thespecificationandmaintenanceofthedifferentiatedpancreaticacinarcellstate(25).BothoftheseTFsarefrequentlydownregulatedduringneoplasticdevelopmentandmalignantprogressionofhumanandmousePDAC.FunctionalgeneticstudiesinmiceandculturedhumanPDACcellshavedemonstratedthatexperimentallyforcedexpressionofPTF1aimpairsKRAS-inducedtransdifferentiationandproliferation,andcanalsoforcetheredifferentiationofalreadyneoplasticcellsintoaquiescentacinarcellphenotype(26).Conversely,suppressionofPTF1aexpressionelicitsacinar-to-ductalmetaplasia,namelytransdifferentiation,andtherebysensitizestheduct-likecellstooncogenicKRAStransformation,acceleratingsubsequentdevelopmentofinvasivePDAC(27).Similarly,forcedexpressionofMIST1inKRAS-expressingpancreasalsoblockstransdifferentiationandimpairstheinitiationofpancreatictumorigenesisotherwisefacilitatedbytheformationofpremalignantduct-like(PanIN)lesions,whereasgeneticdeletionofMIST1enhancestheirformationandtheinitiationofKRAS-drivenneoplasticprogression(28).LossofeitherPTF1orMIST1expressionduringtumorigenesisisassociatedwithelevatedexpressionofanotherdevelopmentalregulatoryTF,SOX9,whichisnormallyoperativeinthespecificationofductalcells(27,28).ForcedupregulationofSOX9,obviatingtheneedtodownregulatePTF1aandMIST1,hasalsobeenshowntostimulatetransdifferentiationofacinarcellsintoaductalcellphenotypethatissensitivetoKRAS-inducedneoplasia(29),implicatingSOX9asakeyfunctionaleffectoroftheirdownregulationinthegenesisofhumanPDAC.Thus,threeTFsthatregulatepancreaticdifferentiationcanbevariouslyalteredtoinduceatransdifferentiatedstatethatfacilitates—inthecontextofmutationalactivationofKRAS—oncogenictransformationandtheinitiationoftumorigenesisandmalignantprogression. AdditionalmembersoftheSOXfamilyofchromatin-associatedregulatoryfactorsareontheonehandbroadlyassociatedbothwithcellfatespecificationandlineageswitchingindevelopment(30),andontheotherwithmultipletumor-associatedphenotypes(31).AnothersalientexampleofSOX-mediatedtransdifferentiationinvolvesamechanismoftherapeuticresistanceinprostatecarcinomas.Inthiscase,lossoftheRBandp53tumorsuppressors—whoseabsenceischaracteristicofneuroendocrinetumors—inresponsetoantiandrogentherapyisnecessarybutnotsufficientforthefrequentlyobservedconversionofwell-differentiatedprostatecancercellsintocarcinomacellsthathaveenteredadifferentiationlineagewithmolecularandhistologicfeaturesofneuroendocrinecells,whichnotablydonotexpresstheandrogenreceptor.InadditiontolossofRBandp53,theacquiredresistancetoantiandrogentherapyrequiresupregulatedexpressionoftheSOX2developmentalregulatorygene,whichisdemonstrablyinstrumentalininducingtransdifferentiationofthetherapy-responsiveadenocarcinomacellsintoderivativesthatresideinaneuroendocrinecellstatethatisrefractorytothetherapy(32). Athirdexamplealsorevealstransdifferentiationasastrategyemployedbycarcinomacellstoavoideliminationbyalineage-specifictherapy,inthiscaseinvolvingbasalcellcarcinomas(BCC)oftheskintreatedwithapharmacologicinhibitoroftheHedgehog-Smoothened(HH/SMO)oncogenicsignalingpathwayknowntodrivetheneoplasticgrowthofthesecells(33).Drug-resistantcancercellsswitch,viabroadepigeneticshiftsinspecificchromatindomainsandthealteredaccessibilityoftwosuperenhancers,toadevelopmentallyrelatedbutdistinctcelltype.ThenewlygainedphenotypicstateoftheBCCcellsenablesthemtosustainexpressionoftheWNToncogenicsignalingpathway,whichinturnimpartsindependencefromthedrug-suppressedHH/SMOsignalingpathway(34).Asmightbeanticipatedfromthistransdifferentiation,thetranscriptomeofthecancercellsshiftsfromagenesignaturereflectingtheimplicatedcell-of-originofBCCs,namelythestemcellsofhairfolliclebulge,tooneindicativeofthebasalstemcellsthatpopulatetheinterfollicularepidermis.Suchtransdifferentiationtoenabledrugresistanceisbeingincreasinglydocumentedindifferentformsofcancer(35). Developmentallineageplasticityalsoappearstobeprevalentamongthemajorsubtypesoflungcarcinomas,thatis,neuroendocrinecarcinomas[small-celllungcancer(SCLC)]andadenocarcinomas+squamouscellcarcinomas[collectivelynon–smallcelllungcancer(NSCLC)].Single-cellRNAsequencinghasrevealedremarkablydynamicandheterogeneousinterconversionamongthesesubtypesaswellasdistinctvariationsthereofduringthestagesinlungtumorigenesis,subsequentmalignantprogression,andresponsestotherapy(36–38).Thus,ratherthanthesimpleconceptualizationofapureclonalswitchfromonelineageintoanother,thesestudiespaintamuchmorecomplexpicture,ofdynamicallyinterconvertingsubpopulationsofcancercellsexhibitingcharacteristicsofmultipledevelopmentallineagesandstagesofdifferentiation,asoberingrealizationinregardtolineage-basedtherapeutictargetingofhumanlungcancer.Regulatorydeterminantsofthisdynamicphenotypicplasticityarebeginningtobeidentified(37,39,40). Synopsis Thethreeclassesofmechanismdescribedabovehighlightselectiveregulatorsofcellularplasticitythatareseparable—atleastinpart—fromcoreoncogenicdriversandotherhallmarkcapabilities.Beyondtheseexamplesliesaconsiderablebodyofevidenceassociatingmanyformsofcancerwithdisrupteddifferentiationconcomitantwiththeacquisitionoftranscriptomesignaturesandotherphenotypes—forexample,histologicmorphology—associatedwithprogenitororstemcellstagesobservedinthecorrespondingnormaltissue-of-originorinothermoredistantlyrelatedcelltypesandlineages(41–43).Assuch,thesethreesubclassesofphenotypicplasticity—dedifferentiationofmaturecellsbacktoprogenitorstates,blockeddifferentiationtofreezedevelopingcellsinprogenitor/stemcellstates,andtransdifferentiationtoalternativecelllineages—appeartobeoperativeinmultiplecancertypesduringprimarytumorformation,malignantprogression,and/orresponsetotherapy.Thereare,however,twoconceptualconsiderations.First,dedifferentiationandblockeddifferentiationarelikelyintertwined,beingindistinguishableinmanytumortypeswherethecell-of-origin—differentiatedcellorprogenitor/stemcell—iseitherunknownoralternativelyinvolved.Second,theacquisitionormaintenanceofprogenitorcellphenotypesandlossofdifferentiatedfeaturesisinmostcasesanimprecisereflectionofthenormaldevelopmentalstage,beingimmersedinamilieuofotherhallmark-enablingchangesinthecancercellthatarenotpresentinnaturallydevelopingcells.Inaddition,yetanotherformofphenotypicplasticityinvolvescellsenescence,discussedmoregenerallybelow,whereincancercellsinducedtoundergoostensiblyirreversiblesenescenceareinsteadabletoescapeandresumeproliferativeexpansion(44).Finally,aswithotherhallmarkcapabilities,cellularplasticityisnotanovelinventionoraberrationofcancercells,butratherthecorruptionoflatentbutactivatablecapabilitiesthatvariousnormalcellsusetosupporthomeostasis,repair,andregeneration(45). Collectively,theseillustrativeexamplesencourageconsiderationofthepropositionthatunlockingcellularplasticitytoenablevariousformsofdisrupteddifferentiationconstitutesadiscretehallmarkcapability,distinguishableinregulationandcellularphenotypefromthewell-validatedcorehallmarksofcancer(Fig.2). NonmutationalEpigeneticReprogramming Theenablingcharacteristicofgenome(DNA)instabilityandmutationisafundamentalcomponentofcancerformationandpathogenesis.Atpresent,multipleinternationalconsortiaarecatalogingmutationsacrossthegenomeofhumancancercells,doingsoinvirtuallyeverytypeofhumancancer,atdifferentstagesofmalignantprogression,includingmetastaticlesions,andduringthedevelopmentofadaptiveresistancetotherapy.Oneresultisthenowwidespreadappreciationthatmutationsingenesthatorganize,modulate,andmaintainchromatinarchitecture,andtherebygloballyregulategeneexpression,areincreasinglydetectedandfunctionallyassociatedwithcancerhallmarks(46–48). Thereis,inaddition,acasetobemadeforanotherapparentlyindependentmodeofgenomereprogrammingthatinvolvespurelyepigeneticallyregulatedchangesingeneexpression,onethatmightbetermed“nonmutationalepigeneticreprogramming”(Fig.3).Indeed,thepropositionofmutation-lesscancerevolutionandpurelyepigeneticprogrammingofhallmarkcancerphenotypeswasraisedalmostadecadeago(49)andisincreasinglydiscussed(46,50–52). Figure3.ViewlargeDownloadslideNonmutationalepigeneticreprogramming.Muchasduringembryogenesisandtissuedifferentiationandhomeostasis,growingevidencemakesthecasethatinstrumentalgene-regulatorycircuitsandnetworksintumorscanbegovernedbyaplethoraofcorruptedandco-optedmechanismsthatareindependentfromgenomeinstabilityandgenemutation.ThehallmarksofcancergraphichasbeenadaptedfromHanahanandWeinberg(2).Figure3.ViewlargeDownloadslideNonmutationalepigeneticreprogramming.Muchasduringembryogenesisandtissuedifferentiationandhomeostasis,growingevidencemakesthecasethatinstrumentalgene-regulatorycircuitsandnetworksintumorscanbegovernedbyaplethoraofcorruptedandco-optedmechanismsthatareindependentfromgenomeinstabilityandgenemutation.ThehallmarksofcancergraphichasbeenadaptedfromHanahanandWeinberg(2).Closemodal Theconceptofnonmutationalepigeneticregulationofgeneexpressionisofcoursewellestablishedasthecentralmechanismmediatingembryonicdevelopment,differentiation,andorganogenesis(53–55).Intheadult,forexample,long-termmemoryinvolveschangesingeneandhistonemodification,inchromatinstructure,andinthetriggeringofgeneexpressionswitchesthatarestablymaintainedovertimebypositiveandnegativefeedbackloops(56,57).Growingevidencesupportsthepropositionthatanalogousepigeneticalterationscancontributetotheacquisitionofhallmarkcapabilitiesduringtumordevelopmentandmalignantprogression.Afewexamplesarepresentedbelowinsupportofthishypothesis. MicroenvironmentalMechanismsofEpigeneticReprogramming Ifnotsolelybyconsequenceofoncogenicmutations,howthenisthecancercellgenomereprogrammed?Agrowingbodyofevidenceindicatesthattheaberrantphysicalpropertiesofthetumormicroenvironmentcancausebroadchangesintheepigenome,fromwhichchangesbeneficialtothephenotypicselectionofhallmarkcapabilitiescanresultinclonaloutgrowthofcancercellswithenhancedfitnessforproliferativeexpansion.Onecommoncharacteristicoftumors(orregionswithintumors)ishypoxia,consequenttoinsufficientvascularization.Hypoxia,forexample,reducestheactivityoftheTETdemethylases,resultinginsubstantivechangesinthemethylome,inparticularhypermethylation(58).Insufficientvascularizationlikelyalsolimitsthebioavailabilityofcriticalblood-bornenutrients,andnutrientdeprivationhasbeenshownforexampletoaltertranslationalcontrolandconsequentlyenhancethemalignantphenotypeofbreastcancercells(59). Apersuasiveexampleofhypoxia-mediatedepigeneticregulationinvolvesaformofinvariablylethalpediatricependymoma.Likemanyembryonicandpediatrictumors,thisformlacksrecurrentmutations,inparticularadearthofdrivermutationsinoncogenesandtumorsuppressors.Rather,theaberrantgrowthofthesecancercellsisdemonstrablygovernedbyageneregulatoryprograminducedbyhypoxia(60,61).Notably,theputativecell-of-originofthiscancerresidesinahypoxiccompartment,likelysensitizingcellsresidentthereintotheinitiationoftumorigenesisbyasyetunknowncofactors. Anotherpersuasivelineofevidenceformicroenvironmentallymediatedepigeneticregulationinvolvestheinvasivegrowthcapabilityofcancercells.Aclassicexampleinvolvesthereversibleinductionofinvasivenessofcancercellsatthemarginsofmanysolidtumors,orchestratedbythedevelopmentalregulatoryprogramknownastheepithelial-to-mesenchymaltransition(EMT;refs.62–64).Notably,amasterregulatoroftheEMT,ZEB1,hasbeenrecentlyshowntoinduceexpressionofahistonemethyltransferase,SETD1B,thatinturnsustainsZEB1expressioninapositivefeedbackloopthatmaintainsthe(invasive)EMTregulatorystate(65).ApreviousstudysimilarlydocumentedthatinductionofEMTbyupregulatedexpressionofarelatedTF,SNAIL1,causedmarkedalterationsinthechromatinlandscapeconsequenttoinductionofanumberofchromatinmodifiers,whoseactivitywasdemonstrablynecessaryforthemaintenanceofthephenotypicstate(66).Furthermore,arosterofconditionsandfactorstowhichcancercellsatthemarginsoftumorsareexposed,includinghypoxiaandcytokinessecretedbystromalcells,canevidentlyinducetheEMTandinturninvasiveness(67,68). Adistinctiveexampleofmicroenvironmentalprogrammingofinvasiveness,ostensiblyunrelatedtotheEMTprogram,involvesautocrineactivation,inpancreascancercellsandothers,viainterstitialpressure–drivenfluidflow,ofaneuronalsignalingcircuitinvolvingsecretedglutamateanditsreceptorNMDAR(69,70).Notably,theprototypicalstiffnessofmanysolidtumors,embodiedinextensivealterationstotheextracellularmatrix(ECM)thatenvelopthecellswithin,hasbroadeffectsontheinvasiveandotherphenotypiccharacteristicsofcancercells.ComparedwiththenormaltissueECMfromwhichtumorsoriginate,thetumorECMistypicallycharacterizedbyincreasedcross-linkinganddensity,enzymaticmodifications,andalteredmolecularcomposition,whichcollectivelyorchestrate—inpartviaintegrinreceptorsforECMmotifs—stiffness-inducedsignalingandgene-expressionnetworksthatelicitinvasivenessandotherhallmarkcharacteristics(71). Inadditiontosuchregulatorymechanismsendowedbythephysicaltumormicroenvironment,paracrinesignalinginvolvingsolublefactorsreleasedintotheextracellularmilieubythevariouscelltypespopulatingsolidtumorscanalsocontributetotheinductionofseveralmorphologicallydistinctinvasivegrowthprograms(72),onlyoneofwhich—dubbed“mesenchymal”—seemstoinvolvetheaforementionedEMTepigeneticregulatorymechanism. EpigeneticRegulatoryHeterogeneity Agrowingknowledgebaseisheighteningappreciationoftheimportanceofintratumoralheterogeneityingeneratingthephenotypicdiversitywherethefittestcellsforproliferativeexpansionandinvasionoutgrowtheirbrethrenandhenceareselectedformalignantprogression.Certainly,onefacetofthisphenotypicheterogeneityisfoundedinchronicorepisodicgenomicinstabilityandconsequentgeneticheterogeneityinthecellspopulatingatumor.Inaddition,itisincreasinglyevidentthattherecanbenon–mutationallybasedepigeneticheterogeneity.AsalientexampleinvolvesthelinkerhistoneH1.0,whichisdynamicallyexpressedandrepressedinsubpopulationsofcancercellswithinanumberoftumortypes,withconsequentsequestrationoraccessibility,respectively,ofmegabase-sizeddomains,includingonesconveyinghallmarkcapabilities(73).Notably,thepopulationofcancercellswithrepressedH1.0werefoundtohavestem-likecharacteristics,enhancedtumor-initiatingcapability,andanassociationwithpoorprognosisinpatients. Anotherexampleofepigeneticallyregulatedplasticityhasbeendescribedinhumanoralsquamouscellcarcinomas(SCC),whereincancercellsattheinvasivemarginsadoptapartialEMT(p-EMT)statelackingtheaforementionedmesenchymalTFsbutexpressingotherEMT-defininggenesthatarenotexpressedinthecentralcoreofthetumors(74).Thep-EMTcellsevidentlydonotrepresentaclonalcompartmentalizationofmutationallyalteredcells:culturesofprimarytumor-derivedcancercellscontaindynamicmixturesofbothp-EMThiandp-EMTlocells,andwhenp-EMThi/locellswereFACS-purifiedandcultured,bothrevertedtomixedpopulationsofp-EMThiandp-EMTlocellswithin4days.Moreover,althoughparacrinesignalsfromtheadjacentstromacouldbeenvisagedasdeterministicforthep-EMThistate,thestablepresenceandregenerationofthetwoepigeneticstatesinculturearguesforacancercell–intrinsicmechanism.Notably,thisconclusionissupportedbyanalysisof198celllinesrepresenting22cancertypes,includingSCC,wherein12stablyheterogeneousepigeneticstates(includingthep-EMTinSCC)werevariouslydetectedinthecelllinemodelsaswellastheircognateprimarytumors(75).Again,theheterogeneousphenotypicstatescouldnotbelinkedtodetectablegeneticdifferences,andinseveralcasesFACS-sortedcellsofaparticularstatewereshowntodynamicallyreequilibrateuponculture,recapitulatingastablebalanceamongtheheterogeneousstatesseenintheoriginalcelllines. Additionally,technologiesforgenome-wideprofilingofdiverseattributes—beyondDNAsequenceanditsmutationalvariation—areilluminatinginfluentialelementsofthecancercellgenome'sannotationandorganizationthatcorrelatewithpatientprognosis,andincreasinglywithhallmarkcapabilities(76–78).Epigenomicheterogeneityisbeingrevealedbyincreasinglypowerfultechnologiesforprofilinggenome-wideDNAmethylation(79,80),histonemodification(81),chromatinaccessibility(82),andposttranscriptionalmodificationandtranslationofRNA(83,84).Achallengeinregardtothepostulatebeingconsideredhereinwillbetoascertainwhichepigenomicmodificationsinparticularcancertypes(i)haveregulatorysignificanceand(ii)arerepresentativeofpurelynonmutationalreprogramming,asopposedtobeingmutation-drivenandthusexplainablebygenomeinstability. EpigeneticRegulationoftheStromalCellTypesPopulatingtheTumorMicroenvironment Ingeneral,theaccessorycellsinthetumormicroenvironmentthatfunctionallycontributetotheacquisitionofhallmarkcapabilitiesarenotthoughttosuffergeneticinstabilityandmutationalreprogrammingtoenhancetheirtumor-promotingactivities;ratheritisinferredthatthesecells—cancer-associatedfibroblasts,innateimmunecells,andendothelialcellsandpericytesofthetumorvasculature—areepigeneticallyreprogrammedupontheirrecruitmentbysolubleandphysicalfactorsthatdefinethesolidtumormicroenvironment(2,85).Itcanbeanticipatedthemulti-omicprofilingtechnologiescurrentlybeingappliedtocancercellswillincreasinglybeusedtointerrogatetheaccessory(stromal)cellsintumorstoelucidatehownormalcellsarecorruptedtofunctionallysupporttumordevelopmentandprogression.Forexample,arecentstudy(86)suggeststhatsuchreprogrammingcaninvolvemodificationsoftheepigenomeinadditiontotheinductiveinterchangeofcytokines,chemokines,andgrowthfactorsthatalterintracellularsignalingnetworksinallofthesecelltypes:whenmousemodelsofmetastasistolungweretreatedwithacombinationofaDNAmethyltransferaseinhibitor(5-azacytidine)andaninhibitorofhistonemodification(anHDAC),theinfiltratingmyeloidcellswerefoundtohaveswitchedfromanimmature(tumor-promoting)progenitorstateintocellsresemblingmatureinterstitial(tumor-antagonizing)macrophages,which,incontrasttotheircounterpartsinuntreatedtumors,wereincapableofsupportingthehallmarkcapabilitiesnecessaryforefficientmetastaticcolonization(86).Itcanbeenvisagedthatmulti-omicprofilingandpharmacologicperturbationwillservetoelucidatethereprogrammedepigeneticstateinsuchmyeloidcellsaswellasotherhallmark-enablingaccessorycelltypespopulatingtumormicroenvironments. Synopsis Collectively,theseillustrativesnapshotssupportthepropositionthatnonmutationalepigeneticreprogramingwillcometobeacceptedasabonafideenablingcharacteristicthatservestofacilitatetheacquisitionofhallmarkcapabilities(Fig.3),distinctfromthatofgenomicDNAinstabilityandmutation.Notably,itcanbeanticipatedthatnonmutationalepigeneticreprogrammingwillprovetobeintegrallyinvolvedinenablingtheprovisionalnewhallmarkcapabilityofphenotypicplasticitydiscussedabove,inparticularbeingadrivingforceinthedynamictranscriptomicheterogeneitythatisincreasinglywelldocumentedincancercellspopulatingmalignantTMEs.Theadvanceofsinglecellmulti-omicprofilingtechnologiesisenvisagedtoilluminatetherespectivecontributionsofandinterplaybetweenmutation-drivenversusnonmutationalepigeneticregulationtotheevolutionoftumorsduringmalignantprogressionandmetastasis. PolymorphicMicrobiomes Anexpansivefrontierinbiomedicineisunfoldingviailluminationofthediversityandvariabilityoftheplethoraofmicroorganisms,collectivelytermedthemicrobiota,thatsymbioticallyassociatewiththebarriertissuesofthebodyexposedtotheexternalenvironment—theepidermisandtheinternalmucosa,inparticularthegastrointestinaltract,aswellasthelung,thebreast,andtheurogenitalsystem.Thereisgrowingappreciationthattheecosystemscreatedbyresidentbacteriaandfungi—themicrobiomes—haveprofoundimpactonhealthanddisease(87),arealizationfueledbythecapabilitytoauditthepopulationsofmicrobialspeciesusingnext-generationsequencingandbioinformatictechnologies.Forcancer,theevidenceisincreasinglycompellingthatpolymorphicvariabilityinthemicrobiomesbetweenindividualsinapopulationcanhaveaprofoundimpactoncancerphenotypes(88,89).Associationstudiesinhumanandexperimentalmanipulationinmousemodelsofcancerarerevealingparticularmicroorganisms,principallybutnotexclusivelybacteria,whichcanhaveeitherprotectiveordeleteriouseffectsoncancerdevelopment,malignantprogression,andresponsetotherapy.Sotoocantheglobalcomplexityandconstitutionofatissuemicrobiomeatlarge.Indeed,whilethegutmicrobiomehasbeenthepioneerofthisnewfrontier,multipletissuesandorganshaveassociatedmicrobiomes,whichhavedistinctivecharacteristicsinregardtopopulationdynamicsanddiversityofmicrobialspeciesandsubspecies.Thisgrowingappreciationoftheimportanceofpolymorphicallyvariablemicrobiomesinhealthanddiseasepositsthequestion:isthemicrobiomeadiscreteenablingcharacteristicthatbroadlyaffects,bothpositivelyandnegatively,theacquisitionofhallmarkcapabilitiesforcancer?Ireflectonthispossibilitybelow,illustratingevidenceforsomeoftheprominenttissuemicrobiomesimplicatedincancerhallmarks(Fig.4),beginningwiththemostprominentandevidentlyimpactfulmicrobiome,thatoftheintestinaltract. Figure4.ViewlargeDownloadslidePolymorphicmicrobiomes.Left,whileintersectingwiththeenablingcharacteristicsoftumor-promotinginflammationandgenomicinstabilityandmutation,thereisgrowingreasontoconcludethatpolymorphicmicrobiomesinoneindividualversusanother,beingresidentinthecolon,othermucosaandconnectedorgans,orintumorsthemselves,candiverselyinfluence—byeitherinducingorinhibiting—manyofthehallmarkcapabilities,andthusarepotentiallyaninstrumentalandquasi-independentvariableinthepuzzleofhowcancersdevelop,progress,andrespondtotherapy.Right,multipletissuemicrobiomesareimplicatedinmodulatingtumorphenotypes.Inadditiontothewidelystudiedgutmicrobiome,otherdistinctivetissuemicrobiomes,aswellasthetumormicrobiome,areimplicatedinmodulatingtheacquisition—bothpositivelyandnegatively—oftheillustratedhallmarkcapabilitiesincertaintumortypes.ThehallmarksofcancergraphichasbeenadaptedfromHanahanandWeinberg(2).Figure4.ViewlargeDownloadslidePolymorphicmicrobiomes.Left,whileintersectingwiththeenablingcharacteristicsoftumor-promotinginflammationandgenomicinstabilityandmutation,thereisgrowingreasontoconcludethatpolymorphicmicrobiomesinoneindividualversusanother,beingresidentinthecolon,othermucosaandconnectedorgans,orintumorsthemselves,candiverselyinfluence—byeitherinducingorinhibiting—manyofthehallmarkcapabilities,andthusarepotentiallyaninstrumentalandquasi-independentvariableinthepuzzleofhowcancersdevelop,progress,andrespondtotherapy.Right,multipletissuemicrobiomesareimplicatedinmodulatingtumorphenotypes.Inadditiontothewidelystudiedgutmicrobiome,otherdistinctivetissuemicrobiomes,aswellasthetumormicrobiome,areimplicatedinmodulatingtheacquisition—bothpositivelyandnegatively—oftheillustratedhallmarkcapabilitiesincertaintumortypes.ThehallmarksofcancergraphichasbeenadaptedfromHanahanandWeinberg(2).Closemodal DiverseModulatoryEffectsoftheGutMicrobiome Ithaslongbeenrecognizedthatthegutmicrobiomeisfundamentallyimportantforthefunctionofthelargeintestine(colon)indegradingandimportingnutrientsintothebodyaspartofmetabolichomeostasis,andthatdistortionsinthemicrobialpopulations—dysbiosis—inthecoloncancauseaspectrumofphysiologicmaladies(87).Amongthesehasbeenthesuspicionthatthesusceptibility,development,andpathogenesisofcoloncancerisinfluencedbythegutmicrobiome.Inrecentyears,persuasivefunctionalstudies,involvingfecaltransplantsfromcolontumor–bearingpatientsandmiceintorecipientmicepredisposedtodevelopcoloncancerhasestablishedaprinciple:therearebothcancer-protectiveandtumor-promotingmicrobiomes,involvingparticularbacterialspecies,whichcanmodulatetheincidenceandpathogenesisofcolontumors(90). Themechanismsbywhichmicrobiotaimpartthesemodulatoryrolesarestillbeingelucidated,buttwogeneraleffectsareincreasinglywellestablishedfortumor-promotingmicrobiomesandinsomecasesforspecifictumor-promotingbacterialspecies.Thefirsteffectismutagenesisofthecolonicepithelium,consequenttotheproductionofbacterialtoxinsandothermoleculesthateitherdamageDNAdirectly,ordisruptthesystemsthatmaintaingenomicintegrity,orstresscellsinotherwaysthatindirectlyimpairthefidelityofDNAreplicationandrepair.AcaseinpointisE.colicarryingthePKSlocus,whichdemonstrablymutagenizesthehumangenomeandisimplicatedinconveyinghallmark-enablingmutations(91). Additionally,bacteriahavebeenreportedtobindtothesurfaceofcolonicepithelialcellsandproduceligandmimeticsthatstimulateepithelialproliferation,contributinginneoplasticcellstothehallmarkcapabilityforproliferativesignaling(88).Anothermechanismbywhichspecificbacterialspeciespromotetumorigenesisinvolvesbutyrate-producingbacteria,whoseabundanceiselevatedinpatientswithcolorectalcancer(92).Theproductionofthemetabolitebutyratehascomplexphysiologiceffects,includingtheinductionofsenescentepithelialandfibroblasticcells.Amousemodelofcoloncarcinogenesispopulatedwithbutyrate-producingbacteriadevelopedmoretumorsthanmicelackingsuchbacteria;theconnectionbetweenbutyrate-inducedsenescenceandenhancedcolontumorigenesiswasdemonstratedbytheuseofasenolyticdrugthatkillssenescentcells,whichimpairedtumorgrowth(92).Inaddition,bacterial-producedbutyratehaspleiotropicandparadoxicaleffectsondifferentiatedcellsversusundifferentiated(stem)cellsinthecolonicepitheliuminconditionswheretheintestinalbarrierisdisrupted(dysbiosis)andthebacteriaareinvasive,affecting,forexample,cellularenergeticsandmetabolism,histonemodification,cell-cycleprogression,and(tumor-promoting)innateimmuneinflammationthatisimmunosuppressiveofadaptiveimmuneresponses(93). Indeed,abroadeffectofpolymorphicmicrobiomesinvolvesthemodulationoftheadaptiveandinnateimmunesystemsviamultifariousroutes,includingtheproductionbybacteriaof“immunomodulatory”factorsthatactivatedamagesensorsonepithelialorresidentimmunecells,resultingintheexpressionofadiverserepertoireofchemokinesandcytokinesthatcansculpttheabundanceandcharacteristicsofimmunecellspopulatingthecolonicepitheliaanditsunderlyingstromaanddraininglymphnodes.Inaddition,certainbacteriacanbreachboththeprotectivebiofilmandthemucusliningthecolonicepitheliaandproceedtodisrupttheepithelialcell–celltightjunctionsthatcollectivelymaintaintheintegrityofthephysicalbarrierthatnormallycompartmentalizestheintestinalmicrobiome.Uponinvadingthestroma,bacteriacantriggerbothinnateandadaptiveimmuneresponses,elicitingsecretionofarepertoireofcytokinesandchemokines.Onemanifestationcanbethecreationoftumor-promotingortumor-antagonizingimmunemicroenvironments,consequentlyprotectingagainstorfacilitatingtumorigenesisandmalignantprogression.Concordantly,themodulationbydistinctivemicrobiomesinindividualpatientsoftheintertwinedparametersof(i)eliciting(innate)tumorpromotinginflammationand(ii)escaping(adaptive)immunedestructioncanbeassociatednotonlywithprognosis,butalsowithresponsivenessorresistancetoimmunotherapiesinvolvingimmunecheckpointinhibitorsandothertherapeuticmodalities(89,94–96).Provisionalproof-of-concepthascomefromrecentstudiesdemonstratingrestoredefficacytoimmunotherapyfollowingtransplantsoffecalmicrobiotafromtherapy-responsivepatientsintopatientswithmelanomawhohadprogressedduringpriortreatmentwithimmunecheckpointblockade(97,98). Anongoingmysteryhasinvolvedthemolecularmechanismsbywhichparticularandvariableconstituentsofthegutmicrobiomesystemicallymodulatetheactivityoftheadaptiveimmunesystem,eitherenhancingantitumoralimmuneresponsesevokedbyimmunecheckpointblockade,orratherelicitingsystemicorlocal(intratumoral)immunosuppression.Arecentstudyhasshedsomelight:certainstrainsofEnterococcus(andotherbacteria)expressapeptidoglycanhydrolyasecalledSagAthatreleasesmucopeptidesfromthebacterialwall,whichcanthencirculatesystemicallyandactivatetheNOD2patternreceptor,whichinturncanenhanceT-cellresponsesandtheefficacyofcheckpointimmunotherapy(99).Otherimmunoregulatorymoleculesproducedbyspecificbacterialsubspeciesarebeingidentifiedandfunctionallyevaluated,includingbacteria-producedinosine,arate-limitingmetaboliteforT-cellactivity(100).Theseexamplesandothersarebeginningtochartthemolecularmechanismsbywhichpolymorphicmicrobiomesareindirectlyandsystemicallymodulatingtumorimmunobiology,aboveandbeyondimmuneresponsesconsequenttodirectphysicalinteractionsofbacteriawiththeimmunesystem(101,102). Beyondthecausallinkstocoloncancerandmelanoma,thegutmicrobiome'sdemonstrableabilitytoelicittheexpressionofimmunomodulatorychemokinesandcytokinesthatenterthesystemiccirculationisevidentlyalsocapableofaffectingcancerpathogenesisandresponsetotherapyinotherorgansofthebody(94,95).Anilluminatingexampleinvolvesthedevelopmentofcholangiocarcinomasintheliver:gutdysbiosisallowstheentryandtransportofbacteriaandbacterialproductsthroughtheportalveintotheliver,whereTLR4expressedonhepatocytesistriggeredtoinduceexpressionofthechemokineCXCL1,whichrecruitsCXCR2-expressinggranulocyticmyeloidcells(gMDSC)thatservetosuppressnaturalkillercellssoastoevadeimmunedestruction(103),andlikelyconveyotherhallmarkcapabilities(85).Assuch,thegutmicrobiomeisunambiguouslyimplicatedasanenablingcharacteristicthatcanalternativelyfacilitateorprotectagainstmultipleformsofcancer. BeyondtheGut:ImplicatingDistinctiveMicrobiomesinOtherBarrierTissues Virtuallyalltissuesandorgansexposed,directlyorindirectly,totheoutsideenvironmentarealsorepositoriesforcommensalmicroorganisms(104).Unliketheintestine,wherethesymbioticroleofthemicrobiomeinmetabolismiswellrecognized,thenormalandpathogenicrolesofresidentmicrobiotainthesediverselocationsisstillemerging.Thereareevidentlyorgan/tissue-specificdifferencesintheconstitutionoftheassociatedmicrobiomesinhomeostasis,aging,andcancer,withbothoverlappinganddistinctivespeciesandabundanciestothatofthecolon(104,105).Moreover,associationstudiesareprovidingincreasingevidencethatlocaltumor-antagonizing/protectiveversustumor-promotingtissuemicrobiomes,similarlytothegutmicrobiome,canmodulatesusceptibilityandpathogenesistohumancancersarisingintheirassociatedorgans(106–109). ImpactofIntratumoralMicrobiota? Finally,pathologistshavelongrecognizedthatbacteriacanbedetectedwithinsolidtumors,anobservationthathasnowbeensubstantiatedwithsophisticatedprofilingtechnologies.Forexample,inasurveyof1,526tumorsencompassingsevenhumancancertypes(bone,brain,breast,lung,melanoma,ovary,andpancreas),eachtypewascharacterizedbyadistinctivemicrobiomethatwaslargelylocalizedinsidecancercellsandimmunecells,andwithineachtumortype,variationsinthetumormicrobiomecouldbedetectedandinferredtobeassociatedwithclinicopathologicfeatures(110).Microbiotahavebeensimilarlydetectedingeneticallyengineereddenovomousemodelsoflungandpancreascancer,andtheirabsenceingerm-freemiceand/ortheirabrogationwithantibioticscandemonstrablyimpairtumorigenesis,functionallyimplicatingthetumormicrobiomeasanenableroftumor-promotinginflammationandmalignantprogression(111,112).Associationstudiesinhumanpancreaticductaladenocarcinomaandfunctionaltestsviafecaltransplantsintotumor-bearingmicehaveestablishedthatvariationsinthetumormicrobiome—andtheassociatedgutmicrobiome—modulateimmunephenotypesandsurvival(113).Animportantchallengeforthefuturewillbetoextendtheseimplicationstoothertumortypes,andtodelineatethepotentiallyseparablecontributionsofconstitutionandvariationinthetumormicrobiometothatofthegut(andlocaltissueoforigin)microbiome,potentiallybyidentifyingspecificmicrobialspeciesthatarefunctionallyinfluentialinonelocationortheother. Synopsis Amongthefascinatingquestionsforthefutureiswhethermicrobiotaresidentindifferenttissuesorpopulatingincipientneoplasiashavethecapabilitytocontributetoorinterferewiththeacquisitionofotherhallmarkcapabilitiesbeyondimmunomodulationandgenomemutation,therebyinfluencingtumordevelopmentandprogression.Therearecluesthatparticularbacterialspeciescandirectlystimulatethehallmarkofproliferativesignaling,forexample,incolonicepithelium(88),andmodulategrowthsuppressionbyalteringtumorsuppressoractivityindifferentcompartmentsoftheintestine(114),whereasdirecteffectsonotherhallmarkcapabilities,suchasavoidingcelldeath,inducingangiogenesis,andstimulatinginvasionandmetastasis,remainobscure,asdoesthegeneralizabilityoftheseobservationstomultipleformsofhumancancer.Irrespective,thereisanincreasinglycompellingcasetobemadethatpolymorphicvariationinmicrobiomesoftheintestineandotherorgansconstitutesadistinctiveenablingcharacteristicfortheacquisitionofhallmarkcapabilities(Fig.4),albeitintersectingwithandcomplementingthoseofgenomeinstabilityandmutation,andtumor-promotinginflammation. SenescentCells Cellularsenescenceisatypicallyirreversibleformofproliferativearrest,likelyevolvedasaprotectivemechanismformaintainingtissuehomeostasis,ostensiblyasacomplementarymechanismtoprogrammedcelldeaththatservestoinactivateandinduecourseremovediseased,dysfunctional,orotherwiseunnecessarycells.Inadditiontoshuttingdownthecelldivisioncycle,thesenescenceprogramevokeschangesincellmorphologyandmetabolismand,mostprofoundly,theactivationofasenescence-associatedsecretoryphenotype(SASP)involvingthereleaseofaplethoraofbioactiveproteins,includingchemokines,cytokines,andproteaseswhoseidentityisdependentonthecellandtissuetypefromwhichasenescentcellarises(115–117).Senescencecanbeinducedincellsbyavarietyofconditions,includingmicroenvironmentalstressessuchasnutrientdeprivationandDNAdamage,aswellasdamagetoorganellesandcellularinfrastructure,andimbalancesincellularsignalingnetworks(115,117),allofwhichhavebeenassociatedwiththeobservedincreaseintheabundanceofsenescentcellsinvariousorgansduringaging(118,119). Cellularsenescencehaslongbeenviewedasaprotectivemechanismagainstneoplasia,wherebycancerouscellsareinducedtoundergosenescence(120).Mostoftheafore-mentionedinstigatorsofthesenescentprogramareassociatedwithmalignancy,inparticularDNAdamageasaconsequenceofaberranthyperproliferation,so-calledoncogene-inducedsenescenceduetohyperactivatedsignaling,andtherapy-inducedsenescenceconsequenttocellularandgenomicdamagecausedbychemotherapyandradiotherapy.Indeed,therearewell-establishedexamplesoftheprotectivebenefitsofsenescenceinlimitingmalignantprogression(118,119).Tothecontrary,however,anincreasingbodyofevidencerevealsquitetheopposite:incertaincontexts,senescentcellsvariouslystimulatetumordevelopmentandmalignantprogression(119,121).Inoneilluminatingcasestudy,senescentcellswerepharmacologicallyablatedinagingmice,inparticulardepletingsenescentcellscharacteristicallyexpressingthecell-cycleinhibitorp16−INK4a:inadditiontodelayingmultipleage-relatedsymptoms,thedepletionofsenescentcellsinagingmiceresultedinreducedincidencesofspontaneoustumorigenesisandcancer-associateddeath(122). TheprincipalmechanismbywhichsenescentcellspromotetumorphenotypesisthoughttobetheSASP,whichisdemonstrablycapableofconveying,inparacrinefashiontoviablecancercellsinproximity,aswellastoothercellsintheTME,signalingmolecules(andproteasesthatactivateand/ordesequesterthem)soastoconveyhallmarkcapabilities.Thus,indifferentexperimentalsystems,senescentcancercellshavebeenshowntovariouslycontributetoproliferativesignaling,avoidingapoptosis,inducingangiogenesis,stimulatinginvasionandmetastasis,andsuppressingtumorimmunity(116,118,120,121). Yetanotherfacettotheeffectsofsenescentcancercellsoncancerphenotypesinvolvestransitory,reversiblesenescentcellstates,wherebysenescentcancercellscanescapefromtheirSASP-expressing,nonproliferativecondition,andresumecellproliferationandmanifestationoftheassociatedcapabilitiesoffullyviableoncogeniccells(44).Suchtransitorysenescenceismostwelldocumentedincasesoftherapyresistance(44),representingaformofdormancythatcircumventstherapeutictargetingofproliferatingcancercells,butmaywellprovetobemorebroadlyoperativeinotherstagesoftumordevelopment,malignantprogression,andmetastasis. Moreover,thehallmark-promotingcapabilitiesofsenescentcellsarenotlimitedtosenescentcancercells.Cancer-associatedfibroblasts(CAF)intumorshavebeenshowntoundergosenescence,creatingsenescentCAFsthataredemonstrablytumor-promotingbyvirtueofconveyinghallmarkcapabilitiestocancercellsintheTME(115,116,121).More-over,senescentfibroblastsinnormaltissuesproducedinpartbynaturalagingorenvironmentalinsultshavesimilarlybeenimplicatedinremodelingtissuemicroenvironmentsviatheirSASPsoastoprovideparacrinesupportforlocalinvasion(so-called“fieldeffects”)anddistantmetastasis(116)ofneoplasiasdevelopinginproximity.Additionally,senescentfibroblastsinagingskinhavebeenshowntorecruit—viatheirSASP—innateimmunecellsthatarebothimmunosuppressiveofadaptiveantitumoralimmuneresponsesanchoredbyCD8Tcells,andstimulatoryofskintumorgrowth(123),withthelattereffectpotentiallyreflectingparacrinecontributionsofsuchinnateimmunecells(myeloidcells,neutrophils,andmacrophages)tootherhallmarkcapabilities. Whilelesswellestablished,itseemslikelythatotherabundantstromalcellspopulatingparticulartumormicroenvironmentswillprovetoundergosenescence,andtherebymodulatecancerhallmarksandconsequenttumorphenotypes.Forexample,therapy-inducedsenescenttumorendothelialcellscanenhanceproliferation,invasion,andmetastasisinbreastcancermodels(124,125). Certainly,suchclueswarrantinvestigationinothertumortypestoassessgeneralityoffibroblastic,endothelial,andotherstromalcellsenescenceasadrivingforceintumorevolution.AlsocurrentlyunresolvedaretheregulatorymechanismsandfunctionaldeterminantsthroughwhichaparticularsenescentcelltypeinagivenTMEevokesatumor-promotingversusatumor-antagonizingSASP,whichcanseemingbealternativelyinducedinthesamesenescingcelltype,perhapsbydifferentinstigatorswhenimmersedindistinctivephysiologicandneoplasticmicroenvironments. Synopsis Theconceptthattumorsarecomposedofgeneticallytransformedcancercellsinteractingwithandbenefitingfromrecruitedandepigenetically/phenotypicallycorruptedaccessory(stromal)cellsiswellestablishedasinstrumentaltothepathogenesisofcancer.Theconsiderationsdiscussedaboveanddescribedinthereviewsandreportscitedherein(andelsewhere)makeapersuasivecaseforthepropositionthatsenescentcells(ofwhatevercellularorigin)shouldbeconsideredforadditiontotherosteroffunctionallysignificantcellsinthetumormicroenvironment(Fig.5).Assuch,senescentcellswarrantbeingfactoredintothequestfordeepknowledgeofcancermechanisms.Furthermore,therealizationoftheirimportancemotivatestheancillarygoaltotherapeuticallytargettumor-promotingsenescentcellsofallconstitutions,beitbypharmacologicorimmunologicablation,orbyreprogrammingtheSASPintotumor-antagonizingvariants(115,121,126). Figure5.ViewlargeDownloadslideSenescentcells.Heterogeneouscancercellsubtypesaswellasstromalcelltypesandsubtypesarefunctionallyintegratedintothemanifestationsoftumorsasoutlaworgans.CluesareincreasinglyimplicatingsenescentcellderivativesofmanyofthesecellularconstituentsoftheTME,andtheirvariableSASPs,inmodulatinghallmarkcapabilitiesandconsequenttumorphenotypes.ThehallmarksofcancergraphichasbeenadaptedfromHanahanandWeinberg(2).Figure5.ViewlargeDownloadslideSenescentcells.Heterogeneouscancercellsubtypesaswellasstromalcelltypesandsubtypesarefunctionallyintegratedintothemanifestationsoftumorsasoutlaworgans.CluesareincreasinglyimplicatingsenescentcellderivativesofmanyofthesecellularconstituentsoftheTME,andtheirvariableSASPs,inmodulatinghallmarkcapabilitiesandconsequenttumorphenotypes.ThehallmarksofcancergraphichasbeenadaptedfromHanahanandWeinberg(2).Closemodal ConcludingRemarks Whiletheeighthallmarksofcancerandtheirtwoenablingcharacteristicshaveprovedofenduringheuristicvalueintheconceptualizationofcancer,theconsiderationspresentedabovesuggestthattheremaybenewfacetsofsomegeneralityandhenceofrelevancetomorefullyunderstandingthecomplexities,mechanisms,andmanifestationsofthedisease.Byapplyingthemetricofdiscernableifnotcompleteindependencefromthe10coreattributes,itisarguablethatthesefourparametersmaywell—pursuanttofurthervalidationandgeneralizationbeyondthecasestudiespresented—becomeintegratedintothehallmarksofcancerschematic(Fig.6).Thus,cellularplasticitymaycometobeaddedtotherosterofhallmarkcapabilities.Notably,whiletheeightcoreandthisnouveaucapabilityareeach,bytheirdefinitionasahallmark,conceptuallydistinguishable,aspectsoftheirregulationareatleastpartiallyinterconnectedinsomeandperhapsmanycancers.Forexample,multiplehallmarksarecoordinatelymodulatedinsometumortypesbycanonicaloncogenicdrivers,including(i) KRAS(https://cancer.sanger.ac.uk/cosmic/census-page/KRAS),(ii) MYC(https://cancer.sanger.ac.uk/cosmic/census-page/MYC),(iii) NOTCH(https://cancer.sanger.ac.uk/cosmic/census-page/NOTCH1;ref.127),and(iv) TP53(https://cancer.sanger.ac.uk/cosmic/census-page/TP53), Figure6.ViewlargeDownloadslideHallmarksofCancer—newadditions.Depictedarethecanonicalandprospectivenewadditionstothe“HallmarksofCancer.”Thistreatiseraisesthepossibility,aimingtostimulatedebate,discussion,andexperimentalelaboration,thatsomeorallofthefournewparameterswillcometobeappreciatedasgenerictomultipleformsofhumancancerandhenceappropriatetoincorporateintothecoreconceptualizationofthehallmarksofcancer.ThehallmarksofcancergraphichasbeenadaptedfromHanahanandWeinberg(2).Figure6.ViewlargeDownloadslideHallmarksofCancer—newadditions.Depictedarethecanonicalandprospectivenewadditionstothe“HallmarksofCancer.”Thistreatiseraisesthepossibility,aimingtostimulatedebate,discussion,andexperimentalelaboration,thatsomeorallofthefournewparameterswillcometobeappreciatedasgenerictomultipleformsofhumancancerandhenceappropriatetoincorporateintothecoreconceptualizationofthehallmarksofcancer.ThehallmarksofcancergraphichasbeenadaptedfromHanahanandWeinberg(2).Closemodal highlightingtheimportantchallengetomorefullyelucidatetheregulatorynetworksgoverningtheseacquiredcapabilities. Inadditiontoaddingcellularplasticitytotheroster,nonmutationalepigeneticreprogrammingandpolymorphicvariationsinorgan/tissuemicrobiomesmaycometobeincorporatedasmechanisticdeterminants—enablingcharacteristics—bywhichhallmarkcapabilitiesareacquired,alongwithtumor-promotinginflammation(itselfpartiallyinterconnectedtothemicrobiome),aboveandbeyondthemutationsandotheraberrationsthatmanifesttheafore-mentionedoncogenicdrivers. Finally,senescentcellsofdifferentorigins—includingcancercellsandvariousstromalcells—thatfunctionallycontributetothedevelopmentandmalignantprogressionofcancer,albeitinmarkedlydistinctivewaystothoseoftheirnonsenescentbrethren,maybecomeincorporatedasgenericcomponentsoftheTME.Inconclusion,itisenvisagedthatraisingtheseprovisional“trialballoons”willstimulatedebate,discussion,andcontinuingexperimentalinvestigationinthecancerresearchcommunityaboutthedefiningconceptualparametersofcancerbiology,genetics,andpathogenesis. Author'sDisclosures Nodisclosureswerereported. 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