tumor suppressor genes and oncogenes: genes that prevent and

Katherine M. Hyland, PhD,

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Tumor Suppressor Genes and Oncogenes: Genes that Prevent and Cause Cancer

(Biochemistry/Molecular Biology Lecture)OBJECTIVES

• Describethenormalcellularfunctionsoftumorsuppressorgenesandproto-oncogenesandexplaintheirrolesincancer.

• DescribeKnudson’stwo-hithypothesisforthepathogenesisofretinoblastoma.• Listthecommonclinicalfeatures,evolutionandtreatmentforretinoblastoma.• Explainwhyloss-of-heterozygosityofaparticularchromosome/chromosomalregionin

tumorDNAsuggeststheexistenceofatumorsuppressorgeneinthatregion.• ExplainwhylossofeitherRborp16drivesacelltoproliferate.• ExplainhowtheproteinsencodedbytheE6andE7genesoftheoncogenichuman

papillomavirusesfunctiontopromotetumorformation.• DescribehowtheHER2/neuoncogeneisactivatedinbreastcancer.• DescribethenormalfunctionofRasproteinsandthemolecularmechanismbywhich

mutationsinRasgenesleadtocancer.• DescribethreepathwaysbywhichthecyclinDgeneisactivatedintumors.• ExplainwhyactivationoftheBcl-2genepromotescancer.• Describethemoleculareventsassociatedwithdifferentstagesinthedevelopmentof

coloncancer.• Describethesixhallmarkfeaturesofcancercellsandthemolecularbasisofeach.

KEY WORDSAdenomatouspolyposiscoli(APC) multi-steptumorgenesisBcl2 mutatorphenotypecaretaker mycchromosomalinversion neurofibromatosis(NF1)chromosometranslocation oncogenecyclinD promotersE6protein p16E7protein p53EMSY promotersgain-of-function proto-oncogenegeneamplification RasproteinHER2/neu Rbgenehumanpapillomavirus(HPV) retinoblastomaloss-of-function tumorsuppressorgeneloss-of-heterozygosity(LOH) two-hithypothesisLi-Fraumenisyndrome

OPTIONAL READINGAlbertsetal. Molecular Biology of the Cell;5thEdition,GarlandScience,2008 Chapter20;Cancer,pp.1230-1256KumarV,AbbasA,FaustoN:Robbins and Cotran Pathologic Basis of Disease7thed,Elsevier/ Saunders,2005;Chapter7:OncogenesandTumorSuppressorGenespp.292-306

Tumor Suppressor Genes and Oncogenes

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I. INTRODUCTION : TYPES OF GENES INVOLVED IN CANCERCanceriscausedbytheaccumulationofgeneticandepigeneticmutationsingenesthatnormallyplayaroleintheregulationofcellproliferation(asdescribedintheCellProliferationlecture),thusleadingtouncontrolledcellgrowth.Cellsacquiremutationsinthesegenesasaresultofspontaneousandenvironmentally-inducedDNAdamage.ThosecellswithmutationsthatpromoteagrowthandsurvivaladvantageovernormalcellsareselectedforthroughaDarwinianprocess,leadingtotheevolutionofatumor.Genesinvolvedintumorigenesisincludethosewhoseprod-ucts:1)directlyregulatecellproliferation(eitherpromotingorinhibiting),2)controlprogrammedcelldeathorapoptosis,and3)areinvolvedintherepairofdamagedDNA.Dependingonhowtheyaffecteachprocess,thesegenescanbegroupedintotwogeneralcategories:tumor suppres-sor genes(growthinhibitory)andproto-oncogenes(growthpromoting).

Mutantallelesofproto-oncogenesarecalledoncogenes.Sincemutationinasinglealleleofaproto-oncogenecanleadtocellulartransformation,suchmutationsareconsidereddominant.Incontrast,typicallybothallelesofatumorsuppressorgenemustbealteredfortransformationtooccur.Genesthatregulateapoptosismaybedominant,aswithproto-oncogenes,ortheymaybe-haveastumorsuppressorgenes.Tumorsuppressorgenesmaybedividedintotwogeneralgroups:promotersandcaretakers.Promotersarethetraditionaltumorsuppressors,likep53andRB.Mutationofthesegenesleadstotransformationbydirectlyreleasingthebrakesoncellularpro-liferation.Caretakergenesareresponsibleforprocessesthatensuretheintegrityofthegenome,suchasthoseinvolvedinDNArepair.Althoughtheydonotdirectlycontrolcellproliferation,cellswithmutationsinthesegenesarecompromisedintheirabilitytorepairDNAdamageandthuscanacquiremutationsinothergenes,includingproto-oncogenes,tumorsuppressorgenesandgenesthatcontrolapoptosis.AdisabilityinDNArepaircanpredisposecellstowidespreadmutationsinthegenome,andthustoneoplastictransformation.Cellswithmutationsincaretakergenesarethereforesaidtohavea“mutator phenotype”.ThefactthatpatientswithdefectsinDNArepairarecancerproneprovidesoneofthemoststrikingpiecesofevidencethatmutationsinDNAlieattheheartoftheneoplasticprocess.(MoreaboutgeneswhoseproductsareinvolvedinDNArepairintheMutationandCancerlecture.)

II. TUMOR SUPPRESSOR GENESTumorsuppressorgenescanbedefinedasgeneswhichencodeproteinsthatnormallyinhibittheformationoftumors.Theirnormalfunctionistoinhibitcellproliferation,oractasthe“brakes”forthecellcycle.Mutationsintumorsuppressorgenescontributetothedevelopmentofcancerbyinactivatingthatinhibitoryfunction.Mutationsofthistypearetermedloss-of-functionmutations.Aslongasthecellcontainsonefunctionalcopyofagiventumorsuppressorgene(expressingenoughproteintocontrolcellproliferation),thatgenecaninhibittheformationoftumors.Inactivationofbothcopiesofatumorsuppressorgeneisrequiredbeforetheirfunctioncanbeeliminated.Therefore,mutationsintumorsuppressorgenesarerecessiveatthelevelofanindividualcell.Aswewillsee,theinactivationoftumorsuppressorgenesplaysamajorroleincancer.

A. RetinoblastomaRetinoblastoma (RB) isararechildhoodtumoroftheeye(seeclinicalcorrelate).Mostcases(60-70%)aresporadic(asopposedtoinherited),occurunilaterally(affectingoneeye),andpresentinchildren1-4yearsofage.Theremaining30-40%ofpatientshaveahereditaryformofretinoblastomaandthushaveinheritedagermlinecancerpredisposingmutation(seebelow).Thesechildrentendtoacquiretumorsearlierthanthosewithsporadicdiseaseandaremorelikelytohavemultipletumorsinone(unilateral)orboth(bilateral)eyes.Infamilieswiththeinheritedformofretinoblastoma,thediseaseshowsanautosomaldominantinheritancepattern.


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CLINICAL CORRELATION OF RETINOBLASTOMA

Retinoblastoma is a malignant tumor of immature neuroectodermal cells of the developing

retina that occurs almost exclusively in young children. Approximately 175 cases of

retinoblastoma are diagnosed in the US each year. It accounts for 3% of malignant disease in

children younger than age 15. It is the most common intraocular tumor in pediatric patients and

causes 5% of cases of childhood blindness.

Retinoblastoma may gradually fill the eye and extend through the optic nerve to the brain and

less commonly, along the emissary vessels and nerves in the sclera to the orbital tissues.

Occasionally, it grows diffusely in the retina, discharging malignant cells into the vitreous or

anterior chamber, thereby producing a pseudoinflammatory process that may mimic other

ocular inflammatory conditions.

Inheritance

Retinoblastoma occurs in retinal cells that have cancer-predisposing mutations in both copies of

the RB1 gene. About 60-70% of patients have the non-hereditary or “sporadic” form of the

disease, in which the initial mutation affecting one copy of the RB1 gene arises in a somatic

retinal cell (or a precursor of a retinal cell). The remaining 30-40% of patients have inherited a

cancer-predisposing mutation in one copy of their RB1 gene from one parent, or have a new

germline mutation. Sporadic cases tend to be unilateral (one eye) and unifocal (one tumor), and

typically present by 24-30 months of age. Heritable cases tend to be bilateral (both eyes) and

multifocal (multiple tumors), and generally present earlier, in the first year of life. Second

primary malignant tumors, the most common of which is osteosarcoma, develop in large

numbers of survivors of the heritable form of retinoblastoma after a period of many years.

Clinical Presentation

The most common presenting sign is leukocoria (white pupillary reflex). A crossed eye or

strabismus is the second most common symptom of retinoblastoma. The child's eye may turn

towards the ear (exotropia) or towards the nose (esotropia). Other symptoms may include red

painful eye, glaucoma, pseudohypopyon (appearance of purulent material in the anterior

chamber of the eye), or poor vision.

Evaluation

The clinical diagnosis of retinoblastoma is usually established by examination of the fundus of

the eye using indirect ophthalmoscopy to directly visualize the intraocular tumor(s). Imaging

studies (CT or ultrasound) can be used to support the diagnosis by detecting intrinsic

calcification within the mass, a finding highly suggestive of retinoblastoma. High-resolution

MRI of the orbits can help to stage the tumor, and determine any extraocular spread.

Retinoblastoma usually remains unnoticed until it grows large enough to produce leukocoria or

strabismus (inflammation is a much rarer presentation – orbital cellulitis represents about 1% of

all presentations of RB) with 90% of cases diagnosed before age 5. All children with poor

vision, strabismus, or intraocular inflammation should be evaluated for the presence of

retinoblastoma. The earlier the discovery and treatment of the tumor, the better the chance to

prevent spread through the optic nerve and orbital tissues. Retinoblastoma can lead to loss of

eyesight and, if not detected early enough, death, since systemic and CNS metastasis is almost

impossible to treat.

Individuals with a germline mutation in RB1 are also at increased risk of developing tumors

outside the eye over their lifetime. Most of the second primary cancers are osteosarcoma, soft

tissue sarcomas or melanomas. These tumors usually manifest in adolescence or adulthood. To

detect second non-ocular tumors in individuals with retinoblastoma, physicians and parents

should promptly evaluate complaints of bone pain or lumps because of the high risk of

sarcomas; however, no specific screening protocols currently exist.


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1. Two-hit hypothesisAfterstatisticalanalysisofmanypatientswithretinoblastoma,Dr.AlfredKnusonproposedin1971thatsporadiccasesofthisdiseaseinvolvetheinactivationofbothcopiesofaparticulargene,whichhecalledtheretinoblastomagene(RB1).Heproposedthatthisoccursintwosteps:A“firsthit”inactivatesoneofthetwocopiesofRB1inoneretinoblast.Latera“secondhit”inactivatestheremainingfunctionalcopyofRB1inthesamecelloroneofitsprogeny(Figure1).Toexplaintheinheritedformofthedisease,heproposedthattheaffectedpatientsinheritedonedefectivecopyofRB1fromoneparentandafunctionalcopyfromtheotherparent.Becausethe“firsthit”isinheritedandispresentinallretinalcells,andinfactallofthecellsofthebody,atumorariseswhena“secondhit”occursinanyretinoblast.Becauseittakesonlyoneadditionalmutational(orepigenetic)eventforanyofthesecellstodevelopintoatumor,inheritedretinoblastomawouldbemorelikelytooccurearlier

Figure 1. Knudson’s two-hit hypothesis for retinoblastoma.In sporadic Rb, both copies of RB1 (RB1) must be inactivated. This requires two mutational events “hits” which each inactivate one copy of RB1. In inherited Rb, the first hit is inherited.

Rb Rb Rb Rb Rb Rb

Rb Rb Rb Rb

�rst hit second hit

second hit

X X X

X X X

Tumor

Tumor

Sporadic Rb: Two hits required

Inherited Rb: First hit is inherited; only one additional hit required

Treatment

Large tumors in eyes with no salvageable vision are often treated by enucleation (surgical

removal of the eye). Smaller tumors can be treated with plaque or external beam radiotherapy,

cryotherapy (use of liquid nitrogen to freeze and destroy a lesion or growth), or

photocoagulation (use of laser to destroy a small tumor). Chemotherapy can be used to reduce

initial tumor size prior to applying other modes of therapy. Combined therapy using

chemotherapy and coordinated laser treatment can often preserve vision and spare the patient

enucleation and radiation that may lead to disfigurement and the induction of secondary tumors.

Eradication of tumor before infiltration into the optic nerve or choroid carries an excellent

prognosis for survival.

Susan Hung, Curriculum Ambassador 2007


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inchildhoodandmorelikelytocausebilateraldisease(multipleprimarytumors).SubsequentresearchidentifiedRB1onchromosome13andconfirmedKnudson’shypothesis,thusmarkingthediscoveryofthefirsttumorsuppressorgene.

Inbothinheritedandsporadicretinoblastoma,thesecondalleleofRB1canbeinactivatedbyseveralmechanisms.Inadditiontoepigeneticsilencing(seeMutationandCancerlecture),thepossibilitiesincludepointmutation,largedeletionsthatremoveRB1andmanyadjacentgenes,orerrorsinchromosomesegregationleadingtolossoftheentirewild-typechromosome.Thelattertwomechanismsaremorecommon.

2. Loss-of-heterozygosity (LOH)Howaretumorsuppressorgenesfound?AsdiscussedintheGeneticslecturesinPrologue,inthegenomethereareperiodicvariationsintheDNAsequencebetweenthetwohomologouschromosomes(oneinheritedfromeachparent).Variantsthatarecommonlyusedasgeneticmarkersincludeshorttandemrepeatpolymorphisms(STRPs)andsinglenucleotidepolymorphisms(SNPs).Thesevariantscanbevisualizedbymoleculartechniques.Ifweexamineanyregionofthegenome,wewillfindthatmostpeopleareheterozygousforcertainpolymorphicgeneticmarkers.Althoughthesevariantsoftenoccurbetweengenesratherthaninthem,theycanbeusedtotrackthepresenceofadjacentgenes.(SeeGeneticVariationlectureinPrologueandLinkageAnalysisILMinOrgansCV.)

AsshowninFigure2,tumorsuppressorgeneslikeRB1canbefoundbylookingforloss-of-heterozygosity(LOH)inatumor.LOHmeansthatpre-tumor cellsareheterozygousforallelesofatumorsuppressorgene(e.g.onenormalandonemutantallele),orallelesofgeneticmarkersthatsurroundthetumorsuppressorgene,butthetumor cellshavelostthenormaltumorsuppressorallele(andthesurroundingmarker

Figure 2: Loss of heterozygosity.The diagram represents chromosome 13 homologs (circle = centro-mere). The RB1 locus is indicated between two marker loci, with alleles A/a and B/b. Either a mutation in RB1 is inherited, or a sporadic mutation occurs inactivating one copy of RB1 in a somatic cell. A second mutation, in this case loss of the portion of chromosome 13 that contains RB1, occurs in the same cell, resulting in complete lack of a functional RB1, leading to tumorigenesis. Thus the pre-tumor cells are heterozygous for a mutation in RB1, but tumor cells are no longer heterozygous, having lost the functional copy of RB1.

Rb Rb

deletion removinga, Rb, and B

X

Tumor Cells

ARbXAa

b B b

Second hit:

Pre-Tumor Cells

alleles),sotheyarenolongerheterozygous.


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TheexampleillustratedinFigure2involvesasporadiccaseofretinoblastoma.Shownischromosome13afterthefirstcopyofRB1hasbeeninactivated.Inthisexample,A(a)andB(b)representtwomarkerlocithatlieoneithersideofRB1.Eachhasoneoftwoalleles(e.g.,Aanda).TheRB1allelethatisinactivatedfirstisflankedbytheAandballeles,whereastheremainingfunctionalcopyofRB1isflankedbytheaandBalleles.ThesecondhitinRB1ofteninvolvesdeletionofalargeregionorlossofanentirechromosome.Asaresult,thefunctionalcopyofRB1islostasaretheflankingaandBmarkers.Consequently,onlytheAandballelesareleftandthecellisnolongerheterozygousforthesemarkers(i.e.onlyAexists,notA/aandonlybexists,notB/b)inthetumor.Acellthatloosesbothcopiesofaparticulartumorsuppressorgenemayhaveaproliferativeadvantage,andthusbeselectedforduringtumorprogression.Therefore,ifloss-of-heterozygosityisfrequentlyseeninaspecificregionofthegenomeinaparticulartumortype,thissuggeststhatatumorsuppressorgenethatplaysanimportantroleindevelopmentofthattumormaybepresentinthatregion.

3. Mutation or loss of RB1 removes a brake on the cell cycleHowdoesthelossofRB1promotetumorformation?RecallthattheRbproteinplaysakeyroleinregulatingthecellcycle.Itisexpressedineverycelltype,whereitexistsinanactivehypophosphorylatedandinactivehyperphosphorylatedstate.Initsactivestate,RbservesasabrakeontheadvancementofcellsfromtheG1totheS-phaseofthecellcycle.IfRbislostormadenonfunctionalthroughmutation,thisbrakeonthecellcycleisreleasedandcellsmoveintoS-phaseunrestrained.Specifi-cally,RbnormallybindstoandinactivatestheE2Ftranscriptionfactor.LossofRbresultsinactivationofE2F.E2FbindsthepromoterofthecyclinEgeneandinturncausesincreasedexpressionofthecyclinEgeneandsynthesisofCdk2-cyclinEcomplexes,whichthendrivesthecellcycle(Figure3).RecallthatCdk2-cyclinEac-tivityrepresentsthetransitionfrommitogen-dependenttomitogen-independentcellcycleprogression,soinactivationofRbcanlockcellsinaproliferatingstate.

Aspreviouslymentioned,individualswithgermlinemutationofRB1areatincreasedriskofdevelopingsecondprimarynon-oculartumorsovertheirlifetime-mostoftenosteoscercomas,softtissuesarcomas,ormelanomas.Inaddition,somaticallyac-quiredmutationsinRB1 havebeendescribedinbreastcancers,glioblastomas,smallcelllungcancers,andbladdercancers.SinceRbispresentineverycellandplaysanimportantroleincellcyclecontrol,acouplequestionscometomind.First,whydopatientswithgermlinemutationofRB1developprimarilyretinoblastomas?ItisnotcompletelyclearwhytumorsaretypicallyrestrictedtotheretinainpatientswhoinheritadefectivealleleofRB1,thoughevidencesuggeststhathomozygouslossofRB1triggersapoptosis,andthatunrestrainedactionofE2Fproteins(aswouldoccurwithlossofbothRB1alleles)notonlydrivesthecellcycle,butalsotriggersapopto-sis.Thereforeitisplausiblethatalthoughinmosttissues,homozygouslossofRB1inducescelldeath,theretinoblastsarerelativelyresistanttotheapoptosis-inducingeffect.Inthesecells,therefore,dysregulatedE2Fgivesrisetotumors.Inaddition,therearelikelyparallelregulatorypathwaysindifferentcelltypes.Whilesomecelltypes,e.g.breast,lungorbladderepithelialcells,requireadditionallossofothertumorsuppressorgenesoractivationofoncogenes,theproliferationofretinoblastsinearlychildhoodmaybeuniquelycontrolledbyRb,suchthatfewothergeneticchangesarerequiredfortumorformation.Thismaybebecauseretinoblaststermi-nallydifferentiatebytheageofsixyears,andthusdonotneedextensivesafeguards


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againstuncontrolledproliferation.Whereasinothercelltypes,manymoresafeguards(includingtriggeringapoptosis)haveevolvedtoprotectagainstinappropriateprolif-eration,andthesesafeguardsmustbeovercometodevelopcancer.

Asecondquestionthatcomestomind:WhyareinactivatingmutationsofRbnotmorecommonlyseeninhumancancer?Theanswertothisquestionismorestraight-forward.MutationsinothergenesthatcontrolRbphosphorylationcanmimictheeffectofRbloss.SuchgenesaremutatedinmanycancersthatseemtohavenormalRB1genes.Thus,forexample,mutationalactivationofcyclinDorCDK4wouldfavorcellproliferationbyfacilitatingRbphosphorylation,thusmaintainingitinaninactivatestate.Canyouthinkofotheranswerstothesequestions?

B. Genes encoding Cdk inhibitors are tumor suppressor genesMutationalinactivationofCDKinhibitorsalsodrivesthecellcyclebyunregulatedactivationofcyclinsandCDKs.Onesuchinhibitor,encodedbythep16gene,isacommontargetofdeletionormutationalinactivationinhumantumors.Recallthatp16isaninhibitorofCdk4-cyclinDcomplexes.Germlinemutationsofp16areassociatedwithasubsetofhereditarymelanomas.Somaticallyacquireddeletionorinactivationofp16isseenin75%ofpancreaticcancers;40-70%ofglioblastomas;50%ofesophagealcancers;and20%ofnon-smallcelllungcancers,softtissuesarcomas,andbladdercancers.Thelossofp16leadstoincreasedCdk4-cyclinDactivity.ThisresultsinphosphorylationandinactivationofRb,leadingtoactivationofE2FandcyclinEtranscription.Infact,incellsthatharbormutationsineitherp16,CDK4,orcyclinD,thefunctionofRB1isdisruptedevenifRB1itselfisnotmutated.

C. p53: a key tumor suppressorp53,locatedonchromosome17p13.1,isthesinglemostcommontargetforgeneticalterationinhumantumors.Infact,morethan50%ofhumantumorscontainmutationsinthisgene!Thusitisamongthemostimportant“brakes”ontumorformation.Homozygouslossofthep53geneisfoundinvirtuallyeverytypeofcancer,includingcarcinomasofthebreast,colon,andlung–thethreeleadingcausesofcancerdeaths.Inmostcases,theinactivatingmutationsaffectingbothp53allelesareacquiredinsomaticcells.Insomecases,althoughitisrare,individualsinheritamutantp53allele.AswithRB1,inheritanceofonemutantallelepredisposestheseindividualstodevelopmalignanttumorsbecauseonlyoneadditional“hit”isneededtoinactivatethesecond,normal,allele.Inactivationofthesecondp53alleleleadstoincreasedcellproliferation,decreasedapoptosis,andtumordevelopment.TheseindividualshaveararecancerpredispositionsyndromecalledLi-Fraumeni syndrome,andhavea25-foldgreaterchanceofdevelopingamalignanttumorbyage50,comparedwiththegeneralpopulation.IncontrasttopatientswhoinheritamutantRB1allele,thespectrumoftumorsthatdevelopinpatientswithLi-Fraumenisyndromeisquitevaried.Themostcommontypesoftumors

Cdk4-cyclin D Rb E2Fcyclin E

transcription Cdk2-cyclin E proliferation

OFF ON ON ON ON

X

Figure 3. How inactivation of Rb leads to proliferation.Loss of both copies of Rb results in the activation of E2F, increased cyclin E transcription, formation of the Cdk2-cyclin E complex and thus cell proliferation (See “Cell Proliferation” lecture).


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aresarcomas,breastcancer,leukemia,braintumors,andcarcinomasoftheadrenalcortex.Ascomparedwithsporadictumors,thosethatafflictpatientswithLi-Fraumenisyndromeoccuratayoungerage,andagivenindividualmaydevelopmultipleprimarytumors.

p53restrainstumorformationbytwodifferentmechanisms(Figure4).Inthefirst,p53activatesthep21CdkinhibitorgeneinresponsetoDNAdamageandstress.Lossofp53incellspreventsthep21genefrombeingtranscribed,leadingtotheincreasedactivityofthemultipleCdksnormallyturnedoffbyp21andresultinginincreasedcellproliferation.Asecondwayinwhichp53restrainstumorformationisbyinducingapoptosis.

D. Caretaker Genes that Function as Tumor SuppressorsBRCA1,locatedon17q21,andBRCA2,locatedon13q12,aretumorsuppressorgenesassociatedwithbreastandovariancancer,alongwithseveralothercancers.About10%ofallcasesofbreastandovariancancerarehereditarycancers,andmostofthesecasesareduetoinheritanceofagermlinemutationineitherBRCA1orBRCA2(see Familial and Hereditary Cancer Syndromes lecture).Aswithothertumorsupporessorgenes,theremainingalleleisinactivatedorlostduringthecourseoftumorformation(LOH).TheproteinsencodedbyBRCA1andBRCA2areexpressedinmosttissuesandcelltypes(indicatingthatgeneexpressiondoesnotaccountfortherestrictedphenotypeofbreastandovariancancer),andshareanumberoffunctionalsimilarities.

BRCA1andBRCA2functionas“caretaker”genes,likep53,whichservetomaintaingenomicintegrity.ThegeneproductsencodedbyBRCA1andBRCA2arenuclearproteinsthatco-localizewithRAD-51atsitesofDNAdamage,andplayaroleinhomologousrecombinationrepairofdouble-strandedbreaks(see Mutation and Cancer lecture).ThereisalsoevidencethatBRCA1andBRCA2interactwiththep53-mediatedDNAdamagecheckpoint(see Mutation and Cancer lecture).LossofBRCA1orBRCA2leadstotheaccumulationofothergeneticdefects,whichcanthenleadtocancerformation.InadditiontotheirrolesinDNArepair,BRCA1andBRCA2havebeenimplicatedinavarietyofcellularprocesses,includingDNAsynthesis,regulationofgenetranscription(similartop53,onetargetofBRCA1transcriptionalactivationistheCdkinhibitorp21),cellcyclecheckpointcontrol,centrosomeduplicationandubiquitination.

MostBRCA1andBRCA2mutationsleadtoframeshiftsresultinginmissingornon-functionalprotein,or,inthecaseofBRCA2,tononsensemutationsleadingtoprematuretruncationoftheprotein.Thesemutationsareallconsistentwiththelossoffunctionexpectedwithtumorsuppressorgenes.

p53transcription

factor

p21 Cdk inhibitor Cdk-cyclincomplexes

proliferation

apoptosis

Figure 4. p53 tumor suppressor functions.p53 antagonizes tumor formation by activating the p21 Cdk inhibitor (which blocks proliferation) and by promoting apoptosis.


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ApuzzleinbreastcancerhasbeenwhymutationsinBRCA1andBRCA2arenotfoundasofteninsporadic(i.e.non-familial)casesofbreastcancer.Recentstudiessuggestpartialanswer.AproteincalledEMSYhasbeenfoundthatbindsandinhibitsBRCA2.Remarkably,EMSYisfrequentlyoverexpressedinsporadicbreastcancersduetogeneamplification,andthisisapparentlyamuchmorelikelyeventthatthelossofthetwonormalcopiesofeithertheBRCA1ofBRCA2genes.IncreasedEMSYexpressioncorrelateswithpoorclinicaloutcome.

E. Regulators of Signal TransductionAnotherpotentialwaythatproductsoftumorsuppressorgenesmayoperateisbydownregulatinggrowth-promotingsignals.TheproductsoftheAPCgene(5q21)andtheNF–1gene(17q11.2)fallintothiscategory.Germlinemutationsofthesegenesareassociatedwithbenigntumorsthatareprecursorsofcarcinomasthatdeveloplater.

InthecaseoftheAPC (adenomatous polyposis coli)gene,individualsbornwithonemutantalleleinvariablydevelophundredsoreventhousandsofadenomatouspolypsinthecolonduringtheirteensor20s(familialadenomatouspolyposis,orFAP;seeFamilialandHereditaryCancerSyndromeslecture).Almostinvariably,oneormoreofthesepolypsundergomalignanttransformation,givingrisetocancer.Aswithothertumorsuppressorgenes,bothcopiesoftheAPCgenemustbelostfortumordevelopment.Whenthisoccurs,adenomasformed.InadditiontocancersarisinginthesettingofFAP,themajority(70-80%)ofnon-familialcolorectalcarcinomasandsporadicadenomasalsoshowhomozygouslossoftheAPCgene,thusfirmlyimplicatingAPClossinthepathogenesisofcolonictumors.(SeelectureonColonCancer.)AdescribedintheCellProliferationlecture,animportantfunctionoftheAPCproteinistocausedegradationofbeta-catenin,thusmaintaininglowlevelsinthecytoplasm.InactivationoftheAPCgene,andconsequentlossoftheAPCprotein,increasesthecellularlevel’sofbeta-catenin,which,inturn,translocatestothenucleusandup-regulatescellularproliferation.ThusAPCisanegativeregulatorofbeta-cateninsignaling.Beta-cateninitselffunctionsasaproto-oncogene(seebelow).Interestingly,casesofcolorectalcancerthatdonotshowlossofAPCmayinfacthaveactivatingmutationsinbeta-catenin.DysregulationoftheAPC/beta-cateninpathwayisnotrestrictedtocoloncancer;mutationsineitherAPCorbeta-cateninhavealsobeenfoundinnearly30%ofmelanomacelllines.

TheNF1genebehavessimilartotheAPCgene.Individualswhoinheritonemutantalleledevelopnumerousbenignneurofibromas,presumablyasaresultoftheinactivationofthesecondcopyoftheNF1gene.Thisconditioniscalledneurofibromatosistype1,orNF1.Someoftheneurofibromasmayundergomalignanttransformationintoneurofibrosarcomas.ChildrenwithNF1alsoareatincreasedriskfordevelopingacutemyeloidleukemia.Thefunctionofneurofibromin,theproteinproductoftheNF1gene,istoregulatesignaltransductionviatheRasprotein.NeurofibrominisaGTP-aseactivatingproteinthatfacilitatesconversionofactiveRas(GTPfound)toinactiveRas(GDPbound).WithlossofNF1,Rasistrappedinanactive,signal-emittingstate.

III. VIRUSES AND CANCERVirusesareinfectiousagentsthatmustreplicateinsideahostcell.Virusescontaintheirowngenome(whichcanbeeitherDNAorRNA)protectedbyacoatmadeupofproteinorproteinpluslipid.Differentvirusesusethehostcell’smachineryindifferentways,butallvirusesrequirethetranslationapparatus(e.g.ribosomes)ofthehostcellstotranslatetheirmRNAs.Bacteria,incontrast,cangenerallygrowoutsidethehostandhavetheirowntranslation


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machinery.SomevirusespromotecancerinhumansincludingHIV,Epstein-BarrVirus(EBV),andhuman papilloma viruses (HPVs).Thefundamentalsofviraltumorgenesis,includingsomeofthehistory,werecoveredintheI3block.HerewewillmerelyreviewhowHPVscausecancerinordertoconnecttotwoofthemaintumorsuppressorgenesdiscussedinthissection.

Humanpapillomaviruses(transmittedthroughsexualcontact)playapathogenicroleinmostcasesofcervicalcancer(seeinformationoncervicalcancerinthePathologicalCharacteristicsofBenignandMalignantNeoplasmslab,theCancerScreeninglecture,andtheClinicalandTranslationalResearchonlinemodule).Thereareatleast77subtypesofHPVthataredistinguishedbyvariationsintheirDNAsequences.HPV-16orHPV-18DNAisfoundin70%ofcervicaltumors.Anadditional20%oftumorscontainHPVDNAcorrespondingtooneof20othercancer-associatedsubtypes.HPVshaveadouble-strandedDNAgenome.Thereare7viralgenesthatareexpressedearlyduringinfection(E1-E7)and2genesthatareexpressedlate(L1-L2).

Twooftheearlygenes,E6andE7,promotetumorformation(Figure5).TheE6proteinas-sociateswithacellularproteincalledE6AP(E6-AssociatedProtein).TheE6-E6APproteincomplexcatalyzescovalentattachmentofasmallproteincalledubiquitintop53.Theattach-mentofapolyubiquitinchaintoaproteintargetsittotheproteasomefordegradation.Thus,E6inactivatesp53bycausingthedegradationoftheproteinviathecell’snormalproteindegradationmachinery.TheproteinencodedbyE7directlybindstoRbandinactivatesit.ThusRbcanbeinactivatedbylossofthegene(asinretinoblastomaandothertumors),bymutationsingenesthatcontrolRbPhosphorylation,orbyinactivationoftheproteinbyaviralprotein.AsingeneticlossofRb,inactivationoftheRbproteinleadstotheactivationoftheE2FtranscriptionfactorandtranscriptionofthecyclinEgene.Withoutthesafeguardsofbothp53andRb,thecellisstronglydriventowardcancer.

Figure 5. Inactivation of tumor suppressor genes by proteins encoded by Human Papilloma Virus. The E6 protein of oncogenic HPVs binds to a cellular protein called E6-AP (E6 associated protein), which is a ubiquitin ligase. The E6-E6AP protein complex catalyzes the attachment many copies of the small protein ubiquitin to p53. This leads to the degradation of p53 by a large cytoplasmic protease called the proteasome. The E7 protein promotes cancer by binding directly to the Rb protein, rendering it inactive.

p53

degradation viathe proteasome

E6 E6-AP

+ p53-Ub-Ub-Ub

ubiquitin (Ub)

+

activationof E2F

and cyclin Etranscription

E7 + Rb

active

E7 Rb

inactive


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IV. ONCOGENESCellscontain manynormalgenesthatareinvolvedinregulatingcellproliferation.Someofthesegenescanbemutatedtoformsthatpromoteuncontrolledcellproliferation.Thenormalformsofthesegenesarecalledproto-oncogenes, whilethemutated,cancer-causingformsarecalledoncogenes.Incontrasttotumorsuppressorgenes,whichputthebrakesoncellproliferation,oncogenesactivelypromoteproliferation(analogoustothegaspetalofthecellcycle).Mutationsthatconvertproto-oncogenestooncogenestypicallyincreasetheactivityoftheencodedproteinorincreasetheexpressionofthenormalgene.Suchmutationsaredominantorgain-of-function mutations.Therefore,onlyonecopyofthegeneneedstobemutatedinordertopromotecancer.

Oncogeneswerefirstidentifiedinoncogenicretrovirusesthathadpickedupacellularoncogene(c-onc)andincorporateditintotheviralgenometoproduceaviraloncogene(v-onc).J.MichaelBishopandHaroldVarmusofUCSFwereawardedthe1989NobelPrizeinMedicineforthediscoveryofthecellularoriginoftheviraloncogenes.

A. HER2/neu and breast cancerTheHER2/neugeneencodesareceptortyrosinekinasecloselyrelatedtotheepidermalgrowthfactor(EGF)receptor(theybothbelongtotheErbBfamilyofreceptors).Thisgeneisoverexpressedin~25%ofbreastcancers.BreastcancersthatoverexpressHER2/neutendtobemoreaggressiveclinically(moreonthisinupcominglecturesonBreastCancerandCancerTreatment).ThemechanismbywhichHER2/neuisoverexpressedisgene amplification,whichresultsinmultiplecopiesofthegeneaccumulatingintumorcells.TheincreaseinthelevelsoftheHER2/neutyrosinekinasereceptorontumorcells

Sos

Ras-GDP Ras-GTP

GTP

GDP

inactive active

oncogenic mutationsblock GTPase activiy

Figure 6. How mutations in the Ras oncogenes promote cancer. Oncogenic mutations in ras genes prevent the protein from hydrolyzing GTP to GDP. As a result, the protein remains always in its active GTP-bound form, continually activating the MAP kinase cascade, leading to proliferation.

resultsinincreasedsignalingviatheRas-MAPKpathway,drivingcellularproliferation.ThediscoveryofHER2/neuamplificationinbreastcancerhasledtothedevelopmentoftheanticancerdrugHerceptin.HerceptinisamonoclonalantibodythatbindstoHER2/neuonthecellsurfaceresultinginreceptordown-regulation(decreasednumbersofHER-2/neuatthecellsurface)andkillingofcellsbytheimmunesystem.Itwasapprovedin1998foruseintreatmentofbreastcancer(moreaboutHerceptinintheDrugDevelopmentlecture).

B. Activation of Ras: the oncogene most commonly activated in human tumorsThehumangenomeencodesthreeRas genes:H-ras,K-ras,N-ras.Alargefractionoftumorscontainmutationsinoneofthesethreegenes.Forexample,70-90%ofpancreaticcarcinomascontainamutationintheK-rasgene.RasoncogenesareactivatedbypointmutationsthatresultinproteinsunabletohydrolyzeGTP(Figure6).ThesemutantRasproteinsaretherefore“locked”intheGTP-bound(active)form,whichthereforecontinuallyactivatestheMAPkinasepathway,whichinturnleadstocellproliferation.


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C. The Myc oncogene is often amplified or overexpressed in cancersTheMycproteinactsinthenucleusasasignalforcellproliferationthroughseveralmechanisms.Onemechanism,asdiscussedintheCellProliferationlecture,isasatranscriptionfactorforthecyclinDgene.Mycisencodedbyaproto-oncogenethatisoverexpressedoramplifiedinmanycancers.ExcessquantitiesofMyccancausecellstoproliferateincircumstanceswherenormalcellswouldhalt.Insomecancers,ratherthanbeingamplified,Mycismadeactivebychromosomaltranslocation.(SeetheCancerCytogeneticssectionoftheMutationandCancerlectureforadescriptionofchromosomeabnormalitiesincancer.)Asaresultofthischromosomalrearrangement,astrongpromotersequenceisplacedinappropriatelynexttotheMycproteincodingsequence,producingunusuallylargeamountsoftheMycmRNA.Forexample,asyouwilllearnlaterinthiscourse,inBurkitt’slymphomaatranslocationbringstheMycgeneunderthecontrolofsequencesthatnormallydrivetheexpressionoflargeamountsofantibodiesinBcells.Asaresult,mutantBcellsproducelargeamountsoftheMycprotein,proliferatetoexcess,andformatumor.

D. Cyclin D can be activated through several different mechanismsAsdiscussedintheCellProliferationlecture,cyclinDformspartoftheG1-Cdk,whichnormallyfunctionstoinactivetheRbtumorsuppressorproteinbyphosphorylatingit(Figure7).CyclinDisoftenoverexpressedincancersleadingtoreducedactivityofRb.Inbreastcancer,thecyclinDgeneisoftenamplified.AswithHER2/neuamplification,thisresultsinmorecyclinEproteinbeingproduced.

CyclinDalsoplaysaroleinparathyroidadenomas,tumorsoftheparathyroidglandthatresultinuncontrolledcalciummobilizationfrombones(Figure8).(Thenormalfunctionofparathyroidhormoneistoinducecalciumreleasefrombones).Inthesetumors,cyclinDcanbeactivatedbyadifferentmechanism.Achromosome inversionplacesthestrongtranscriptionalcontrolregionoftheparathyroidhormonegeneadjacenttothecyclinDgeneonchromosome11q.ThisresultsinthecontinuousexpressionofthecyclinDgene,whichinturnpromotesproliferation.

Inchroniclymphocyticleukemias,cyclinDisderegulatedbyyetanothermechanism.Inthesetumors,cyclinDisactivatedviaachromosometranslocationthatplacesthecyclinDgeneundercontroloftheimmunoglobulinheavychaintranscriptioncontrolregion.(ChromosomerearrangementsaredescribedintheCancerCytogeneticssectionoftheMutationandCancerlecture.)

Figure 7. How cyclin D gene amplification leads to proliferation.Increased numbers of cyclin D genes leads to increased levels of Cdk4-cyclin D complexes which activate cyclin E transcription via the Rb-E2F pathway.

Cdk4-cyclin D Rb E2Fcyclin E

transcription Cdk2-cyclin E proliferation

OFF ON ON ON ON

cyclin D gene ampli�cation

ON


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E. Bcl-2: apoptosis proteins and cancerAtranslocationbetweenchromosomes14and18(t(14:18)(q32;q21)isthemostcommontranslocationinhumanlymphoidmalignancies.Itisespeciallycommoninfollicularcelllymphomas(80-90%ofcaseshaveit).ThetranslocationresultsintheactivationofBcl-2becausethegeneisbroughtintoproximitywithpotenttranscriptionalregulatorysequencesfromtheimmunoglobulinheavychaingene.TheantiapopototicactivityofBcl-2resultsintheincreasedsurvivalofthecellsthatoverexpressBcl-2.Thisinturngivesthecellstheopportunitytoacquireadditionalmutationsthatleadtocancer.Thus,programmedcelldeathisanimportantmechanismthatnormallyrestrictstumorformation.

V. MULTISTEP TUMORIGENESIS: MOLECULAR EVENTS ASSOCIATED WITH THE GENESIS OF COLON CANCERColoncancerisunusualinthattheprecancerouslesionscanbeidentifiedearlyviacolonoscopy.Molecularanalysisofthevariousstagesofcoloncanceristhereforepossible.Thesestudieshaveidentifiedaroughsequenceofmoleculareventsthatcharacterizetumordevelopmentincolorectalcancer(Figure9).Becausewenowknowthemolecularfunctionmanytumorsuppressorgenesandoncogenes,wearebeginningtounderstandthegeneticchangesthatoccurduringcancerformation.

Theearlieststageofcoloncancerformationistheappearanceofahyperproliferativeepithelium(Figure9).ThisisassociatedwiththelossoftheAPCtumorsuppressor.RecallthatAPClimitsWntsignalinginthegutepitheliumwhereitisexpressedathighlevelsinnon-dividingcellsandlowlevelsinthestemcellsofthecrypts.LossoftheAPCgeneresultsinhyperactiveWntsignalingandproliferationofcellsbeyondthecrypts.SubsequenttolossofAPC,thereappearstobeaperiodofgenomicinstability,whichpermitsthecellstoacquiremutationsmorerapidly.ProgressionofadenomasisassociatedwithactivationofthemitogenicRas-MAPKpathwaythroughtheacquisitionofactivatingmutationsintheK-rasoncogene.Later,afurtherblowtogrowthcontroloccursviathelossofaSMADgenerequiredforsignalingthroughthegrowthinhibitoryTGF-betasignalingpathway.Finally,theprogressionfromlateademonastomalignantcarcinomsisassociatedwiththelossofthep53tumorsuppressor,whichreleasesthecellsfromDNAdamageandstressinducedapoptosisandcellcyclearrest.(Fordescriptionofcancernomenclature,seetheIntroductiontoCancerPathologylecture.)

Figure 8. Activation of cyclin D in parathyroid adenomas.In parathyroid adenomas, the cyclin D gene is activated by a DNA inversion. The inversion places the promoter of the parathyroid hormone gene (PTH gene) next to the cyclin D coding region.

PTH genepromoter

cyclin D gene promoter

cyclin D genePTH gene

PTH genepromoter

cyclin D gene promoter

cyclin D genePTH gene

inversion of DNA segmentbetween cyclin D gene and PTH gene


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Thereareadditionalchangesintumorsuppressorgenesandoncogenesthatoccurduringtumorprogressionthataredifferentindifferentcoloncancers,someofwhichhaveyettobeidentified.Nonetheless,thestudyofthemolecularbasisofcoloncancerhasgivenusapictureofthemulti-stepevolutionofatumorandthegeneticchangesthatdrivethem.Importantly,anunderstandingofthespecificgeneticchangesthatoccurduringthismulti-stepprocessallowsfortheidentificationofkeystepsforinterventionandthedevelopmentofspecifictherapeuticstrategies

VI. THE HALLMARKS OF CANCERIfweconsiderthehallmarkfeaturesofcancercellsthatwereintroducedintheintroductorylectureonCancerBiology,wecanbegintomapthemoleculardefectsinthegenesandprocesseswe’vediscussedinthelasttwolecturestooneofthesixacquiredcapabilitiesofcancercells.Letsconsiderthefirstfouracquiredcapabilitieshere(thelasttwowillbecoveredinlaterlectures).Acoupleexamplesofmoleculardefectsthatcanresultineachacquiredcapabilityareincludedbelow.Canyouthinkofothers?

1. Self-Sufficiency in Growth SignalsNormalcellscannotproliferateintheabsenceofstimulatorysignals,butcancercellscan.Manyoncogenesactbymimickingnormalgrowthsignalingthroughoneofseveralmechanisms.• Whilemostmitogenicgrowthfactorsaremadebyonecelltypeinordertostimulate

Figure 9. Multistep model for colorectal cancer. Shown is the sequence of pathological events that take place during the development of sporadic colon cancer and the genetic changes that occur during this sequence. The sequence of events is not absolute – this diagram describes what happens on average.

loss of APCtumor suppresor

increased geneticinstability

activation of K-ras

loss of SMAD4 andother tumor suppressors

loss of p53

normal epithelium

hyperproliferative epithelium

early adenoma

intermediate adenoma

late ademoma

carcinoma


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proliferationofanother(heterotypicsignaling),manycancercellsacquiretheabilitytosynthesizetheirowngrowthfactors,creatingapositivefeedbacksignalingloop(autocrinestimulation),andobviatingthedependenceongrowthfactorsfromothercells.TheproductionofPDGF(plateletderivedgrowthfactor)andTGF-α(tumorgrowthfactorα)byglioblastomasandsarcomasaretwoexamples.

• Cellsurfacereceptorsthattransducegrowth-stimulatorysignalsintothecell,forexampleEGFRandHER2/neu,maybeoverexpressedorstructurallyaltered,leadingtoligand-independentsignaling.

• Downstreamtargetsofthesignalingpathwaycanalsobealtered.Forexample,Rasisfoundmutatedabout25%ofhumantumors,thusleadingtoligand-independentactivationoftheRas-Raf-MAPKsignalingpathway.

2. Insensitivity to antigrowth signalsAntigrowthsignalscanblockproliferationbytwomechanisms:1)forcingcellsoutoftheactiveproliferativecycleintothequiescent(G0)state,untilappropriategrowthsignalsputthembackintothecellcycle;or2)inducingdifferentiation,whichpermanentlyremovestheirproliferativepotential.Cancercellsevadetheseantiproliferativesignals.Atthemolecularlevel,manyandperhapsallanti-proliferativesignalsarefunneledthroughtheRbprotein(anditstworelatives,p107andp130).DisruptionoftheRbpathwayliberatesE2Fsandthusallowscellproliferation,renderingcellsinsensitivetoanti-growthfactorsthatnormallyblockadvancethroughG1.TheRbsignalingpathway(regulatedbyTGF-βanti-proliferativesignalingandotherextrinsicfactors)canbedisruptedinavarietyofwaysindifferenttypesofhumantumors,forexample:• LossofTGFβ(normallyactstopreventphosphorylationthatinactivatesRb)• LossofSmad4(normallytransducessignalsfromligand-activatedTGFβreceptorsto

downstreamtargets)• LossofCDKinhibitors–p16,p21,p53• InactivationofRbdirectlybyhyperphosphorylationorinactivatingmutation

Tumorcellsalsousevariousstrategiestoavoidterminaldifferentiation.OverexpressionofcMycisonesuchstrategy.

3. Evading ApoptosisAsdiscussedearlier,theabilityoftumorcellpopulationstoexpandinnumberisdeterminednotonlybytherateofcellproliferationbutalsobytherateofcellattrition.Programedcelldeath,orapoptosis,representsamajorsourceofthisattrition.Resistancetoapoptosisascanbeacquiredbycancercellsthroughavarietyofstrategies.Examplesinclude;• Lossofp53(normallyactivatespro-apoptoticproteins;representsthemostcommon

lossofaproapoptoticregulator)• Activationorupregulationofanti-apoptoticBcl2

4. Limitless Replicative PotentialThefirstthreeacquiredcapabilities–lackofrequirementforgrowthsignals,insensitivitytoantigrowthsignals,andresistancetoapoptosis–allleadtoanuncouplingofthecell’sgrowthprogramfromsignalsinitsenvironment.However,ithasbeenshownthatthisuncouplingalonedoesnotensureexpansivetumorgrowth.Mosttypesofmammaliancellscarryanintrinsicmechanismthatlimitstheirproliferationbykeepingtrackofthenumberofcellgenerations.Thismechanismusesthenumberoftelomererepeatsattheendsofchromosomes,whicherodethroughsuccessivecyclesofreplication-eventually


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leadingtocelldeath.Asmallpercentageoftumorcellsavoidthisbyobtainingamutationthatupregulatesexpessionofthetelomeraseenzyme,thusacquiringlimitlessreplicativepotentialbymaintainingtheirtelomeres.(MoreontelomeresandtelomeraseintheMutationandCancerlecture.)

5and6.The5thand6thacquiredcapabilities,SustainedAngiogenesisandTissueInvasionandMetastasis,willbecoveredindetailinlaterlectures.

ReferencesAlbertsetal.MolecularBiologyoftheCell;5thEdition,GarlandScience,2008.

GeneReviewsontheNCBIGeneTestswebsite:http://www.ncbi.nlm.nih.gov/sites/GeneTests/?db=GeneTestsAndreferencestherein.• BRCA1andBRCA2HereditaryBreastandOvarianCancer• Retinoblastoma

Hanahan,D.andWeinberg,R.(2000).TheHallmarksofCancer.Cell100,57-70.

Mendelsohnetal.TheMolecularBasisofCancer,3rdEdition,Elsevier/Saunders,2008

OMIM(OnlineMendelianInheritanceinMan)http://www.ncbi.nlm.nih.gov/sites/entrez?db=omimAndreferencestherein.• BRCA1,BRCA2

Acknowledgements:SignificantcontributionstothislectureweremadebyHitenMadhani,PhD,DepartmentofBiochemistryandBiophysics,whooriginallydevelopedthislecture(lecturer2002–2006);

tumor suppressor genes and oncogenes: genes that prevent and

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