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illustrationbyatelierolschinsky
When cells divide, chromosomes aren’t always doled out equally. The consequences can be dire.
bymeganscudellari
14 Fall 2014 / HHMI Bulletin
AAngelikaAmonhaswitnessedplentyofcelldivisionerrorsinhercareerasayeastgeneticist.Inyeast,theprocessofmeiosis—wheregermcellsdividetoformreproductivegametes—iserrorprone.Ascellsdivide,thread-likespindlefibersaresupposedtopullstubbychromosomepairsapartandsendthetwochromosomestooppositeendsofthecell.Sometimes,however,apairofchromosomesismistakenlydeliveredtoasingleside.Afterthecytoplasmdivides,theresultingtwodaughtercellsareaneuploid:onecarriesanextrachromosome;theotherismissingachromosome.
SittingatamicroscopetoexamineasetofaneuploidcellsinherlabattheMassachusettsInstituteofTechnology(MIT),Amonknewthecellswouldbesickly.Aneuploidyeastrarelyreplicate;theyoftendie.That’sbecauseorganisms—whethertheyhaveonesetofchromosomes(haploid,likesomeyeast),two(diploid,likemammals),or12(dodecaploid,liketheUgandaclawedfrog)—relyonbalancedgenomes.Anequalnumberofchromosomesassuresabalancedratioofgeneproducts.Whenthatbalanceisoutofwhack—whenanucleushasoneormoreextraormissingchromosomes—thecellalmostalwaysfailstodeveloporfunctionproperly.
Almostalways.Cancercells,whichareoverwhelminglyaneuploid,areanexception.Theythriveanddividewithafury.ThecontradictionnaggedAmon,anHHMIinvestigator,foryears.Howcouldaneuploidy—thekissofdeathformostcells—beahallmarkinthosecellswithanunparalleledabilitytoproliferate?Itdidn’tmakesense.
Whatwasworsewasthat,outsideofDownsyndrome(awell-studiedgeneticdisordercausedbyanextrachromosome21),aneuploidywasamystery.Despitetheprevalenceofaneuploidyincancercells—morethan90percentofsolidtumorsandabout75percentofblood
cancersdisplayit—scientistshadlittleunderstandingofhowaneuploidyaffectsthephysiologyofacell.
“Italwaysbuggedme,”saysAmon.“SoIdecidedtolookintoit.”Startingin2002,Amonsetouttodeterminethecellularimpactofhavinganabnormalnumberofchromosomesandtoresolvetheaneuploidyparadox.Beforelong,shehadalotofcompany.
Today,arosterofexperiencedcancerresearchersandmolecularbiologistsistacklinganeuploidy’seffectoncellsanditsroleincancer.Theireffortsarenotpurelyacademic:aneuploidycouldbeausefultargetfornewcancerdrugs.
Leaningforwardinachairinherfifth-storyoffice,Amonpropsherelbowsatopatable,firmlyclaspsherhands,anddeclares,“Iwanttoidentifygeneticorevenchemicalcompoundsthatkillaneuploidcellspreferentially.”Then,hervoicegrowingsoft,AmondescribeswatchingheryoungersisterwithbreastcancerperseverethroughnumerousroundsoftreatmentwithTaxol,acommonanti-cancerdrug.“It’sbrutal.Ifwecouldcombinepotentialaneuploidy-selectivecompoundswithTaxol,wecouldbemoreeffectiveandlowerthedoseofTaxol.Wecouldmakethelivesofpeoplewithcancersomuchbetter.”
Extra Gain, Extra PainAmon’sfirstforayintoaneuploidywasashotinthedark.“IhadatalentedtechniciannamedMonicaBoselli.SheandIusedtodocrazyexperimentsallthetime,”saysAmon.In2002,sheaskedBosellitomakeafewaneuploidcelllinessotheycouldgetagoodlookatthem—easiersaidthandone.First,Bosellilearnedanarcanechromosometransferstrategyusingmolecularmarkerstoselectandmoveindividualchromosomesfromoneyeastcelltoanother.Shecreated13strainsofhaploidyeast,eachwithoneortwoextrachromosomes.Then,Bosellicloselymonitoredthetransplantedchromosomes,asyeasthaveatalentforriddingthemselvesofextraneousgeneticmaterial.
Boselli’seffortsworked,andsheandAmon,alongwithAmon’sthenpostdocEduardoTorres,immediatelyobservedthattheextrachromosomewasnotdormant.Ineverycase,thechromosomewasactive:hundredsofgeneswerebeingexpressedfromitsDNA,and,Amonlaterconfirmed,thosegenetranscriptsweretranslatedintoproteins.
Next,Amonexaminedtheappearanceandactivityoftheaneuploidyeast.All13aneuploidcelllines,regardlessofwhichoftheyeast’s16chromosomeswasaddedastheextra,sharedcommontraits.Theyallhaddefectivecellcycles:theymultipliedmoreslowly—somecouldn’tevenformcolonies—andmanywere10-20minutesslowertoinitiatecellcycledivisionthantheirnormal,wild-typecounterparts.Thisconfirmedacommonassumptionthataneuploidyreducesacell’sabilitytoreproducenormally.Theyeastalsoexhibitedincreasedglucoseuptakeand
15HHMI Bulletin / Fall 2014A
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sensitivitytogrowthconditionsthathinderedproteinfoldingandthebreakdownofproteins.
Allinall,thefindingssuggestedthataneuploidcellsrequireextraenergyforsurvival,andthattheystruggletofoldanddegradeproteins.Theproteindegradationpathwaysappearedtobeworkingatfulltilttryingtoridthecellofunfoldedandmisfoldedproteinsandpartsofproteins.Thesubunitsofmanyproteincomplexesareencodedacrossmultiplechromosomes,soanextraormissingchromosomewillresultinisolatedsubunitswithnopartners.Infact,workpublishedthisAprilinCellbyHHMIInvestigatorJonathanWeissman,attheUniversityofCalifornia,SanFrancisco,showedthathealthycellsproduceequalamountsoftheproteinsthatfunctiontogetherinacomplex.Thus,anychangeinthosecarefullymaintainedratiosislikelytocauseproblemsinacell.
“Itwasprettyobvious[aneuploidcells]hadmajorissuesintheproteinqualitycontroldepartment,”saysAmon.She,Torres,andBosellipublishedtheresultsin2007.Afewyearslater,Amonfollowedupwithimagesofaneuploidyeastchokedwithclumpsoffluorescentlylabeledproteinaggregatesshininglikegreenwartsinthecells.
Amonlovesyeast:thesmellofyeast,thepinkandivorycolonies,theteenybubble-likeyeastspores.Butafter
onetoomanycolleaguesinsinuatedthatheraneuploidyfindingswereapplicableonlytoyeast,Amonturnedtomousecellstodetermineifwhattheyfoundinyeastwasalsotrueinmammals.
Assheexpected,themousecellshadcellcycledefectsandstressedproteincontrolpathways.“Itwasthesame,”saysAmonofthemammalianworkpublishedin2008inScience.“Definitelythesame.”ApaperpublishedthisyearbyZuzanaStorchovaandcolleaguesattheMaxPlanckInstituteofBiochemistryinGermanyconfirmsAmon’sfindings,thistimeinhumancells:aneuploidhumancells,fromavarietyofsources,upregulatemetabolicandproteindegradationpathways.
Tug of WarThreemilessouthandacrosstheCharlesRiver,DavidPellman,alsoanHHMIinvestigatorandyeastaficionado,wassimultaneouslytryingtofigureouthowaneuploidyaffectsthefunctionofcellsandobtainmoredetailsabouthowitoccursinthefirstplace.
Inadditiontoanabnormalnumberofchromosomes,mostaneuploidcellshaveanabnormalnumberofcentrosomes—smallorganellesthatcontrolthepositionandactionofchromosomesonspindlefibersduringcelldivision.Healthycellshavejusttwocentrosomesduringmitosis—divisionofsomatic,ornon-germ,cells—butaneuploidcellsoftenhavethreeorfour.In2008,attheDana-FarberCancerInstituteandBostonChildren’sHospital,Pellmanandcolleaguesbegangeneratingcelllines(avarietyofmouseandhuman)differingonlyincentrosomenumber.Atthetime,itwaswellestablishedthatextracentrosomesgenerateaneuploidy,butthemechanismbywhichthatoccurredhadnotbeendirectlyobserved.
Angelika Amon is searching for the link between aneuploidy and cancer.
“It was pretty obvious [aneuploidy cells] had major issues in the protein quality control department.”—angelikaamon
16 Fall 2014 / HHMI Bulletin
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Watchingthecellsbyusingaliveimagingsystem,Pellman’steamfoundthatcellswithextracentrosomesdivideintotwo,justlikenormalcells,yettheextracentrosomesproduceadditionalspindlefibersthatpullchromosomesinthewrongdirections.Thistugofwarcanresultinlaggingchromosomesthatdon’tmakeittothecorrectsideofthecellpriortodivision.Thedaughtercells,therefore,areaneuploid.“Itwasanunexpectedmechanism,”saysPellman,butonethatconfirmedthatcentrosomesareactiveparticipantsinchromosomesegregationerrorsthatleadtoaneuploidy.
Aneuploidycanalsobecausedbyaweakenedordamagedspindlecheckpoint,thesecuritysysteminthecellthatensureschromosomesareaccuratelyseparated,saysHongtaoYu,anHHMIinvestigatorattheUniversityofTexasSouthwesternMedicalCenterinDallas.Ahostofspindlecheckpointproteins,suchasMad2andBubR1,monitortheattachmentofspindlefiberstokinetochores,stickyproteinhubsatthecenterofchromosomes.Inthecaseofincorrectfiber-kinetochoreattachments,checkpointproteinsbindandinhibitaproteincomplexthatwouldactivatethenextstageofcelldivision.
Completelossofthespindlecheckpointwhollykillscells,saysYu.Butlesserdefects,suchaslowlevelsordecreasedactivityofMad2orBubR1,resultinirregularattachments,continuedcelldivision,andchromosomemis-segregation.“Subtlechangesofthe
Hongtao Yu studies how subtle changes in spindle fiber proteins can cause chromosome mis-segregation.
Mitotic checkpoint defects
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If extra chromosomes reduce cell viability, how and why do aneuploid cancer cells thrive?
Centrosome amplification
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As chromosomes are partitioned during cell division, errors can occur along several pathways, resulting in aneuploidy.
spindlecheckpointsystemallowcellstolive,butcauseaneuploidy,”saysYu.
Paradoxically,itturnsoutthattoomuchofaspindlecheckpointproteincanalsocauseaneuploidy.OverexpressionofMad2inmousecellscausesaneuploidy,afindingpublishedbyRocioSotillo,anHHMIinternationalearlycareerscientistattheEuropeanMolecularBiologyLaboratoryinMonterotondo,Italy,in2007.Theexcessproteininitiallyarrestscellsinthephaseofthecellcyclewhenthechromosomesarelinedupalongthecell’sequator.Afewcellsmanageto“escape”thatblock,saysSotillo,andproceedwithmitosis.Buttheyoverwhelminglyescapewithbaggage—extraorfewerchromosomes.
Othercelldivisiondefectsandspindlecheckpointerrorscauseaneuploidyaswell,andevidencefromorganismssuchasDrosophilaandplantsreaffirmsthattheresultsofaneuploidyareslowedcellproliferation,sickness,and
death.Unfortunately,theseobservationsstillfailedtoanswerthequestionthatnaggedatAmon,Pellman,andothers:ifextrachromosomesreducecellviability,howandwhydoaneuploidcancercellsthrive?
Elephant in the RoomIn1902,whileobservingtheabnormaldevelopmentofaneuploidcellsinseaurchinembryos,GermanzoologistTheodorBoveritheorizedthatatumormightdevelopfromsuchacell.Laterscientistsindeedfoundthatmosttumorsareaneuploid,butmanycastthefindingasideasanoddity.Fordecades,theroleofaneuploidyincancerwaslargelyignored.
“Ifyoulookatoldcancerreviews,theyneverputaneuploidyinthere,”saysStephenElledge,ageneticistandHHMIinvestigatoratHarvardMedicalSchool.“Ithasalwaysbeentheelephantintheroom.”Thatmaybebecause,duringthe1970sand1980s,itwassignificantlyhardertoidentifythecellulareffectsofawholeadditionalchromosome,asopposedtoasingleoncogene,Elledgesuggests.“Itjusthurtpeople’sheads.”
Withtheadventofnewimagingandsequencingtoolsbeginningintheearly1990s,aneuploidyreceivedrenewedattention—butsoonbecamestymiedbycontroversy.Leadingcancerresearcherssuggestedaneuploidywassimplyaby-productofcancer,anunimportantconsequence.Othersvehementlyarguedthatitcouldbethesolecauseofcancer.“Therewasa
18 Fall 2014 / HHMI Bulletin
hugedebate,”saysPellman.Butitdidn’tscarehimawayfromthefield.
Priortoworkingfulltimeinthelab,Pellmanwasapediatriconcologist.LikeAmon,aviewthroughamicroscopedrewhimtothestudyofaneuploidy:overandover,heobservedabnormalstructuresinthenucleusthatindicatedaneuploidyinthecellsofhisyoungcancerpatients.“Ispentalotoftimelookingattheseabnormalnuclearstructures,”saysPellman.“Therewasanobviousconnection.”
SohedecidedtoputoneaspectofBoveri’shypothesis—thatwholegenomeduplication,whichcauseshighratesofaneuploidy,mightcausecancer—tothetest.Pellmaninstructedoneofhispostdocs,TakeshiFujiwara,toproducetetraploid(twicethenormalchromosomenumber)mousecells,whichhaveahighrateofchromosomemis-segregationandthusrapidlybecomeaneuploid.Then,Fujiwarawastoinjectthosecellsintomice.AlthoughPellmanthoughtitwasimportanttotesttheidea,hefeltitwasalmosttoosimple,“toogoodto
betrue,”forthetetraploidcellstocausecancer.Fujiwara’sinitialresultsshowedthatthecellsindeedcausedaggressivetumorsinthemice.“Itwaswhatwewereafter,butitwasneverthelessverysurprisingthatitworkedsowell,”Pellmanrecalls.Thediscoveryprovidedstrongevidencethattetraploidy,andtheresultinganeuploidy,promotestumordevelopment.
Sincethen,Pellman’slabhasbeenworkingtodeterminehowaneuploidymightcausetumors.In2012,histeamshowedthatinsidemicronuclei—smallmembrane-boundbodiesthatpartitionextrachromosomesawayfromthecytoplasm—chromosomesaresubjecttoextensiveDNAdamage.Thissuggeststhatonewayaneuploidycausescanceristheold-fashionedway:throughmutationsthatactivatecancer-causinggenesorinactivatetumorsuppressors.Additionally,Pellman’slabrecentlydiscoveredevidenceshowingthatextracentrosomes,independentofgeneratinganeuploidy,canleadtotheactivationofRac1,aproteininvolvedincancercellsignalingandtumorinvasion(publishedJune5,2014,inNature).
Moreevidenceismountingthataneuploidypromotescancerbyalteringoncogenesandtumorsuppressors.LastyearinCell,ElledgeandcolleaguesatHarvardreportedthatcancergenomesselectforextrachromosomesthatcontainpotentoncogenes,andselectagainstextrachromosomesrichintumorsuppressors(see“TheMethodinCancer’sMadness,”Spring2014HHMI Bulletin).AttheMayoClinicinMinnesota,molecularbiologistJanvanDeursenandcolleagueshave
Aneuploidy—an abnormal number of chromosomes—appears to help a normal cell transform into a cancer cell. But not by giving it an edge to grow. Evidence suggests that aneuploidy actually slows cell division. So how exactly does aneuploidy benefit tumors?
Scientists have two main hypotheses. The first is that aneuploidy helps cancer cells adapt quickly under extreme pressure. In 2012, geneticist Yitzhak Pilpel and colleagues at the Weizmann Institute of Science in Israel tested this theory in yeast cells by exposing them to high heat. The yeast that acquired chromosome duplications—especially of chromosome III, which contains special proteins that aid in heat tolerance—survived better than
yeast that did not. When Pilpel reduced the temperature back to normal for these chromosome III aneuploids, the cells quickly lost the extra copy of the chromosome during subsequent replication.
Interestingly, however, when Pilpel kept the aneuploids at a high temperature, they also lost the extra copy, but only after evolving a different, more efficient way to express the heat-tolerance proteins over time. Pilpel and his team concluded that having an extra chromosome is a “quick fix,” but not a permanent solution, to deal with stress. That ability to quickly adapt would be a great benefit to cancer cells during metastasis. When a breast tumor cell spreads to the bone, for example, it must survive in a completely different
environmental niche. “As the disease progresses, adaptation potential provided by aneuploidy could be very beneficial,” says Angelika Amon, an HHMI investigator and yeast geneticist at MIT. The hypothesis has yet to be tested in cancer cells, however.
Another hypothesis is that aneuploidy promotes structural abnormalities known to cause cancer. In studies of yeast, Amon’s team has shown that aneuploidy causes genomic instability—a high frequency of mutations in the genome, from small mutations of two to three nucleotides to larger structural changes. Amon assayed 13 aneuploid yeast strains in a series of tests for different types of genomic instability, such as increased mutation rates and increased rates
of recombination. “It was really remarkable. Every aneuploid strain we looked at scored in at least one assay,” says Amon, and most tested positive for multiple types of genomic changes. The finding suggests that aneuploidy, no matter which chromosome is duplicated, causes genomic instability. And that instability predisposes the cell to mutations that cause cancer.
Aneuploidy may help cancer cells adapt quickly to stress in the body and nudge cells toward mutations that directly promote tumorigenesis, according to this recent work. “But so far, all this work is done in [cultured] cells, and that’s not good enough,” says Amon. Her team and others are planning to test these two hypotheses in mouse models of aneuploidy. — M.S.
Stressed-Out Cancer Cells: The Benefits of Aneuploidy
“We’re at the point where there’s pretty strong trust that aneuploidy is driving cancer.”—stephenelledge
19HHMI Bulletin / Fall 2014B
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foundthataneuploidypredisposesmicewithonlyoneworkingcopyofp53,insteadofanormalpair,tolosethatcopyofthesuppressorgeneanddeveloptumors.
Basedoncurrentunderstanding,aneuploidyappearstopromotetumorproductionthroughmultiplemechanisms,butnotbyincreasingacell’sabilitytomultiply.That,atleast,helpsresolvetheaneuploidyparadox.“Wethink[aneuploidy]offerstumorcellssomethingotherthanproliferation,somethingelsethatisgoodfortumorigenesis,”saysAmon.(Seesidebar,“Stressed-OutCancerCells:TheBenefitsofAneuploidy.”)
Butdoesaneuploidycausecanceronitsown?VanDeursen’sworksuggeststhatitdoesnot.Formorethanadecade,vanDeursenhasbeenengineeringmicetoproducelowlevelsofindividualspindlecheckpointproteins,includingBub1andBubR1(completelossofsuchproteinsislethaltoembryos).Someofthemicedevelopcancer,fromlungcancertolymphomatolivertumors,butnotall.What’smore,vanDeursencan’tpredictwhichcheckpointproteinthatheknocksdown—andhehastriedadozen—willresultinatumor.
“Ifitweremerelyaneuploidythatdrivesmalignanttransformation,thenwe’dexpectthatallthemicewouldhavecancers,andmanycancers.Butthatreallyisn’tthecase,”saysvanDeursen.“It’slikelytheanswerisaneuploidyplussomethingelse.Soyouwillhavetwo,three,orfourindependent,tumor-promotingactivities.”
Today,mostresearchers,includingAmon,agreethatoncogenesarethemaindriversofcancer,andaneuploidycontributesvariationandinstabilitytothetumorcells.Ifoncogenesarethespark,aneuploidyisthekindling.“We’reatthepointwherethere’saprettystrongtrustthataneuploidyisdrivingcancer,”saysElledge.“Thequestionis,howpotentisit?”
Targeting StressUntilthatquestionisanswered,mostaneuploidyresearchremainsfocusedonbasicmechanisms.Butafewinquisitivescientistsaredippingtheirtoesintotranslationalresearch,lookingforwaystotargetorexploitaneuploidytopreferentiallykillcancercells.
“Aneuploidyisdefinitelystressingthecells,andthecellsmustdependonstresssupportpathwaysmorethannormalcellsdo,”saysElledge.“Soifyoucantinkerwiththat,youcanpotentiallymakethecellvulnerableorkillit.Thiscouldcertainlyaugmentothertherapies.”
SomeofPellman’sworksuggeststhattargetingcellswithtoomanycentrosomesmightbeawaytoselectivelytargetcancercells.SeveraldrugcompaniesarepursuingsmallmoleculedrugstoinhibitaspecificmotorproteinthatPellmanidentifiedasessentialforthesurvivalofextracentrosome-containingcancercells.
AtUTSouthwestern,Yuandcolleaguesrecentlycompletedanexperimentinwhichtheysilencedeachofthe20,000+genesinhumancancercells,onebyone,andthentreatedtheresultingcellswithTaxol.TheirgoalwastoseewhichgeneshelpcellsavoiddeathbyTaxol,whichisbelievedtocausemassiveaneuploidy,andwhichgenespromoteTaxol-triggereddeath.
Manyofthegenestheyidentifiedencodecomponentsofthespindlecheckpoint,suggestingpotentialdrugtargetstohelpTaxolworkbetter,saysYu.ThosetargetscouldleadtothekindofdrugsAmonhaslongdreamedof,toimprovetheeffectivenessofcurrentchemotherapyregimensandreducesideeffects.
BackatMIT,Amon’stranslationalworkisinfullswing.IncollaborationwithaHarvardscreeningfacility,Amonhasbeensearchingthroughlibrariesofcompoundsforchemicalsthatpreferentiallypreventaneuploidcellsfrommultiplying.Shehasidentifiedseveral.Oneofthemisachemicalinvolvedintheformationoflipidsthatappearstotargethighlyaneuploidcells.
Amon’steamfoundthatbycombiningTaxolwiththenewcompoundatconcentrationswhereeachcouldkill10percentofcancercellsifgivenseparately,themixturewasabletodestroycancercellswithanefficacyof90percent.“Nowwe’reworkingtotreatmousemodelsofcanceranddeterminethemechanism,”shesays.“We’rereallyexcitedaboutit.”
Stephen Elledge has found evidence that aneuploidy alters the balance of tumor suppressors and promoters.