Tumor Suppress Lec

7/31/2019 Tumor Suppress Lec

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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-oncogenes

andexplaintheirrolesincancer.

DescribeKnudsonstwo-hithypothesisforthepathogenesisofretinoblastoma.

Listthecommonclinicalfeatures,evolutionandtreatmentforretinoblastoma.

Explainwhyloss-of-heterozygosityofaparticularchromosome/chromosomalregionin

tumorDNAsuggeststheexistenceofatumorsuppressorgeneinthatregion.

ExplainwhylossofeitherRborp16drivesacelltoproliferate.

ExplainhowtheproteinsencodedbytheE6andE7genesoftheoncogenichuman

papillomavirusesfunctiontopromotetumorformation.

DescribehowtheHER2/neuoncogeneisactivatedinbreastcancer.

DescribethenormalfunctionofRasproteinsandthemolecularmechanismbywhich

mutationsinRasgenesleadtocancer. DescribethreepathwaysbywhichthecyclinDgeneisactivatedintumors.

ExplainwhyactivationoftheBcl-2genepromotescancer.

Describethemoleculareventsassociatedwithdifferentstagesinthedevelopmentof

coloncancer.

Describethesixhallmarkfeaturesofcancercellsandthemolecularbasisofeach.

KEY WORDS

Adenomatouspolyposiscoli(APC) multi-steptumorgenesis

Bcl2 mutatorphenotype

caretaker myc

chromosomalinversion neurobromatosis(NF1)chromosometranslocation oncogene

cyclinD promoters

E6protein p16

E7protein p53

EMSY promoters

gain-of-function proto-oncogene

geneamplication Rasprotein

HER2/neu Rbgene

humanpapillomavirus(HPV) retinoblastoma

loss-of-function tumorsuppressorgene

loss-of-heterozygosity(LOH) two-hithypothesis

Li-Fraumenisyndrome

OPTIONAL READING

Albertsetal.Molecular Biology of the Cell;5thEdition,GarlandScience,2008

Chapter20;Cancer,pp.1230-1256

KumarV,AbbasA,FaustoN:Robbins and Cotran Pathologic Basis of Disease7thed,Elsevier/

Saunders,2005;Chapter7:OncogenesandTumorSuppressorGenespp.292-306


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Tumor Suppressor Genes and Oncogenes

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I. INTRODUCTION : TYPES OF GENES INVOLVED IN CANCER

Canceriscausedbytheaccumulationofgeneticandepigeneticmutationsingenesthatnormally

playaroleintheregulationofcellproliferation(asdescribedintheCellProliferationlecture),

thusleadingtouncontrolledcellgrowth.Cellsacquiremutationsinthesegenesasaresultof

spontaneousandenvironmentally-inducedDNAdamage.Thosecellswithmutationsthatpromote

agrowthandsurvivaladvantageovernormalcellsareselectedforthroughaDarwinianprocess,

leadingtotheevolutionofatumor.Genesinvolvedintumorigenesisincludethosewhoseprod-

ucts:1)directlyregulatecellproliferation(eitherpromotingorinhibiting),2)controlprogrammed

celldeathorapoptosis,and3)areinvolvedintherepairofdamagedDNA.Dependingonhow

theyaffecteachprocess,thesegenescanbegroupedintotwogeneralcategories:tumor suppres-

sor genes(growthinhibitory)andproto-oncogenes(growthpromoting).

Mutantallelesofproto-oncogenesarecalledoncogenes.Sincemutationinasinglealleleofa

proto-oncogenecanleadtocellulartransformation,suchmutationsareconsidereddominant.In

contrast,typicallybothallelesofatumorsuppressorgenemustbealteredfortransformationto

occur.Genesthatregulateapoptosismaybedominant,aswithproto-oncogenes,ortheymaybe-

haveastumorsuppressorgenes.Tumorsuppressorgenesmaybedividedintotwogeneralgroups:

promotersandcaretakers.Promotersarethetraditionaltumorsuppressors,likep53andRB.

Mutationofthesegenesleadstotransformationbydirectlyreleasingthebrakesoncellularpro-liferation.Caretakergenesareresponsibleforprocessesthatensuretheintegrityofthegenome,

suchasthoseinvolvedinDNArepair.Althoughtheydonotdirectlycontrolcellproliferation,

cellswithmutationsinthesegenesarecompromisedintheirabilitytorepairDNAdamageand

thuscanacquiremutationsinothergenes,includingproto-oncogenes,tumorsuppressorgenes

andgenesthatcontrolapoptosis.AdisabilityinDNArepaircanpredisposecellstowidespread

mutationsinthegenome,andthustoneoplastictransformation.Cellswithmutationsincaretaker

genesarethereforesaidtohaveamutator phenotype.Thefactthatpatientswithdefectsin

DNArepairarecancerproneprovidesoneofthemoststrikingpiecesofevidencethatmutations

inDNAlieattheheartoftheneoplasticprocess.(Moreaboutgeneswhoseproductsareinvolved

inDNArepairintheMutationandCancerlecture.)

II. TUMOR SUPPRESSOR GENESTumorsuppressorgenescanbedenedasgeneswhichencodeproteinsthatnormally

inhibittheformationoftumors.Theirnormalfunctionistoinhibitcellproliferation,oract

asthebrakesforthecellcycle.Mutationsintumorsuppressorgenescontributetothe

developmentofcancerbyinactivatingthatinhibitoryfunction.Mutationsofthistypeare

termedloss-of-functionmutations.Aslongasthecellcontainsonefunctionalcopyofagiven

tumorsuppressorgene(expressingenoughproteintocontrolcellproliferation),thatgene

caninhibittheformationoftumors.Inactivationofbothcopiesofatumorsuppressorgene

isrequiredbeforetheirfunctioncanbeeliminated.Therefore,mutationsintumorsuppressor

genesarerecessiveatthelevelofanindividualcell.Aswewillsee,theinactivationoftumor

suppressorgenesplaysamajorroleincancer.

A. RetinoblastomaRetinoblastoma (RB) isararechildhoodtumoroftheeye(seeclinicalcorrelate).Most

cases(60-70%)aresporadic(asopposedtoinherited),occurunilaterally(affectingone

eye),andpresentinchildren1-4yearsofage.Theremaining30-40%ofpatientshavea

hereditaryformofretinoblastomaandthushaveinheritedagermlinecancerpredisposing

mutation(seebelow).Thesechildrentendtoacquiretumorsearlierthanthosewith

sporadicdiseaseandaremorelikelytohavemultipletumorsinone(unilateral)orboth

(bilateral)eyes.Infamilieswiththeinheritedformofretinoblastoma,thediseaseshows

anautosomaldominantinheritancepattern.


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CLINICAL CORRELATION OF RETINOBLASTOMARetinoblastoma 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

theRB1 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 theirRB1 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) andmultifocal (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). Imagingstudies (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 inRB1 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 hypothesis

Afterstatisticalanalysisofmanypatientswithretinoblastoma,Dr.AlfredKnuson

proposedin1971thatsporadiccasesofthisdiseaseinvolvetheinactivationofboth

copiesofaparticulargene,whichhecalledtheretinoblastomagene(RB1).He

proposedthatthisoccursintwosteps:ArsthitinactivatesoneofthetwocopiesofRB1inoneretinoblast.Laterasecondhitinactivatestheremainingfunctional

copyofRB1inthesamecelloroneofitsprogeny(Figure1).Toexplaintheinherited

formofthedisease,heproposedthattheaffectedpatientsinheritedonedefective

copyofRB1fromoneparentandafunctionalcopyfromtheotherparent.Because

thersthitisinheritedandispresentinallretinalcells,andinfactallofthecells

ofthebody,atumorariseswhenasecondhitoccursinanyretinoblast.Because

ittakesonlyoneadditionalmutational(orepigenetic)eventforanyofthesecellsto

developintoatumor,inheritedretinoblastomawouldbemorelikelytooccurearlier

Figure 1. Knudsons 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 rst 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 patientenucleation and radiation that may lead to d isfigurement 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).

SubsequentresearchidentiedRB1onchromosome13andconrmedKnudsons

hypothesis,thusmarkingthediscoveryofthersttumorsuppressorgene.

Inbothinheritedandsporadicretinoblastoma,thesecondalleleofRB1canbe

inactivatedbyseveralmechanisms.Inadditiontoepigeneticsilencing(seeMutation

andCancerlecture),thepossibilitiesincludepointmutation,largedeletionsthatremoveRB1andmanyadjacentgenes,orerrorsinchromosomesegregationleading

tolossoftheentirewild-typechromosome.Thelattertwomechanismsaremore

common.

2. Loss-of-heterozygosity (LOH)

Howaretumorsuppressorgenesfound?AsdiscussedintheGeneticslecturesin

Prologue,inthegenomethereareperiodicvariationsintheDNAsequencebetween

thetwohomologouschromosomes(oneinheritedfromeachparent).Variantsthat

arecommonlyusedasgeneticmarkersincludeshorttandemrepeatpolymorphisms

(STRPs)andsinglenucleotidepolymorphisms(SNPs).Thesevariantscanbe

visualizedbymoleculartechniques.Ifweexamineanyregionofthegenome,we

willndthatmostpeopleareheterozygousforcertainpolymorphicgeneticmarkers.

Althoughthesevariantsoftenoccurbetweengenesratherthaninthem,theycan

beusedtotrackthepresenceofadjacentgenes.(SeeGeneticVariationlecturein

PrologueandLinkageAnalysisILMinOrgansCV.)

AsshowninFigure2,tumorsuppressorgeneslikeRB1canbefoundbylooking

forloss-of-heterozygosity(LOH)inatumor.LOHmeansthatpre-tumor cellsare

heterozygousforallelesofatumorsuppressorgene(e.g.onenormalandonemutant

allele),orallelesofgeneticmarkersthatsurroundthetumorsuppressorgene,butthe

tumor cellshavelostthenormaltumorsuppressorallele(andthesurroundingmarker

Figure 2: Loss of heterozygosity.The diagram represents chromosome 13 homologs (circle = centro-mere). TheRB1 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 functionalRB1, leading to tumorigenesis. Thus the pre-tumor cells areheterozygous for a mutation in RB1, but tumor cells are no longer heterozygous, having lost the functional

copy ofRB1.

Rb Rb

deletion removing

a, Rb, and B

X

Tumor Cells

A

RbX

Aa

b B b

Second hit:

Pre-Tumor Cells

alleles),sotheyarenolongerheterozygous.


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TheexampleillustratedinFigure2involvesasporadiccaseofretinoblastoma.

Shownischromosome13aftertherstcopyofRB1hasbeeninactivated.Inthis

example,A(a)andB(b)representtwomarkerlocithatlieoneithersideofRB1.Each

hasoneoftwoalleles(e.g.,Aanda).TheRB1allelethatisinactivatedrstisanked

bytheAandballeles,whereastheremainingfunctionalcopyofRB1isankedby

theaandBalleles.ThesecondhitinRB1ofteninvolvesdeletionofalargeregionor

lossofanentirechromosome.Asaresult,thefunctionalcopyofRB1islostasaretheankingaandBmarkers.Consequently,onlytheAandballelesareleftandthe

cellisnolongerheterozygousforthesemarkers(i.e.onlyAexists,notA/aandonly

bexists,notB/b)inthetumor.Acellthatloosesbothcopiesofaparticulartumor

suppressorgenemayhaveaproliferativeadvantage,andthusbeselectedforduring

tumorprogression.Therefore,ifloss-of-heterozygosityisfrequentlyseeninaspecic

regionofthegenomeinaparticulartumortype,thissuggeststhatatumorsuppressor

genethatplaysanimportantroleindevelopmentofthattumormaybepresentinthatregion.

3. Mutation or loss ofRB1 removes a brake on the cell cycle

HowdoesthelossofRB1promotetumorformation?RecallthattheRbproteinplays

akeyroleinregulatingthecellcycle.Itisexpressedineverycelltype,whereit

existsinanactivehypophosphorylatedandinactivehyperphosphorylatedstate.In

itsactivestate,RbservesasabrakeontheadvancementofcellsfromtheG1tothe

S-phaseofthecellcycle.IfRbislostormadenonfunctionalthroughmutation,this

brakeonthecellcycleisreleasedandcellsmoveintoS-phaseunrestrained.Speci-

cally,RbnormallybindstoandinactivatestheE2Ftranscriptionfactor.LossofRb

resultsinactivationofE2F.E2FbindsthepromoterofthecyclinEgeneandinturn

causesincreasedexpressionofthecyclinEgeneandsynthesisofCdk2-cyclinE

complexes,whichthendrivesthecellcycle(Figure3).RecallthatCdk2-cyclinEac-

tivityrepresentsthetransitionfrommitogen-dependenttomitogen-independentcell

cycleprogression,soinactivationofRbcanlockcellsinaproliferatingstate.

Aspreviouslymentioned,individualswithgermlinemutationofRB1areatincreased

riskofdevelopingsecondprimarynon-oculartumorsovertheirlifetime-mostoften

osteoscercomas,softtissuesarcomas,ormelanomas.Inaddition,somaticallyac-

quiredmutationsinRB1 havebeendescribedinbreastcancers,glioblastomas,small

celllungcancers,andbladdercancers.SinceRbispresentineverycellandplays

animportantroleincellcyclecontrol,acouplequestionscometomind.First,why

dopatientswithgermlinemutationofRB1developprimarilyretinoblastomas?Itis

notcompletelyclearwhytumorsaretypicallyrestrictedtotheretinainpatientswho

inheritadefectivealleleofRB1,thoughevidencesuggeststhathomozygouslossof

RB1triggersapoptosis,andthatunrestrainedactionofE2Fproteins(aswouldoccur

withlossofbothRB1alleles)notonlydrivesthecellcycle,butalsotriggersapopto-

sis.Thereforeitisplausiblethatalthoughinmosttissues,homozygouslossofRB1inducescelldeath,theretinoblastsarerelativelyresistanttotheapoptosis-inducing

effect.Inthesecells,therefore,dysregulatedE2Fgivesrisetotumors.Inaddition,

therearelikelyparallelregulatorypathwaysindifferentcelltypes.Whilesomecell

types,e.g.breast,lungorbladderepithelialcells,requireadditionallossofother

tumorsuppressorgenesoractivationofoncogenes,theproliferationofretinoblasts

inearlychildhoodmaybeuniquelycontrolledbyRb,suchthatfewothergenetic

changesarerequiredfortumorformation.Thismaybebecauseretinoblaststermi-

nallydifferentiatebytheageofsixyears,andthusdonotneedextensivesafeguards


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againstuncontrolledproliferation.Whereasinothercelltypes,manymoresafeguards

(includingtriggeringapoptosis)haveevolvedtoprotectagainstinappropriateprolif-

eration,andthesesafeguardsmustbeovercometodevelopcancer.

Asecondquestionthatcomestomind:WhyareinactivatingmutationsofRbnot

morecommonlyseeninhumancancer?Theanswertothisquestionismorestraight-

forward.MutationsinothergenesthatcontrolRbphosphorylationcanmimicthe

effectofRbloss.Suchgenesaremutatedinmanycancersthatseemtohavenormal

RB1genes.Thus,forexample,mutationalactivationofcyclinDorCDK4would

favorcellproliferationbyfacilitatingRbphosphorylation,thusmaintainingitinan

inactivatestate.Canyouthinkofotheranswerstothesequestions?

B. Genes encoding Cdk inhibitors are tumor suppressor genes

MutationalinactivationofCDKinhibitorsalsodrivesthecellcyclebyunregulated

activationofcyclinsandCDKs.Onesuchinhibitor,encodedbythep16gene,isa

commontargetofdeletionormutationalinactivationinhumantumors.Recallthatp16is

aninhibitorofCdk4-cyclinDcomplexes.Germlinemutationsofp16areassociatedwith

asubsetofhereditarymelanomas.Somaticallyacquireddeletionorinactivationofp16is

seenin75%ofpancreaticcancers;40-70%ofglioblastomas;50%ofesophagealcancers;

and20%ofnon-smallcelllungcancers,softtissuesarcomas,andbladdercancers.The

lossofp16leadstoincreasedCdk4-cyclinDactivity.ThisresultsinphosphorylationandinactivationofRb,leadingtoactivationofE2FandcyclinEtranscription.Infact,

incellsthatharbormutationsineitherp16,CDK4,orcyclinD,thefunctionofRB1is

disruptedevenifRB1itselfisnotmutated.

C. p53: a key tumor suppressor

p53,locatedonchromosome17p13.1,isthesinglemostcommontargetforgenetic

alterationinhumantumors.Infact,morethan50%ofhumantumorscontainmutations

inthisgene!Thusitisamongthemostimportantbrakesontumorformation.

Homozygouslossofthep53geneisfoundinvirtuallyeverytypeofcancer,including

carcinomasofthebreast,colon,andlungthethreeleadingcausesofcancerdeaths.In

mostcases,theinactivatingmutationsaffectingbothp53allelesareacquiredinsomatic

cells.Insomecases,althoughitisrare,individualsinheritamutantp53allele.AswithRB1,inheritanceofonemutantallelepredisposestheseindividualstodevelopmalignant

tumorsbecauseonlyoneadditionalhitisneededtoinactivatethesecond,normal,

allele.Inactivationofthesecondp53alleleleadstoincreasedcellproliferation,decreased

apoptosis,andtumordevelopment.Theseindividualshaveararecancerpredisposition

syndromecalledLi-Fraumeni syndrome,andhavea25-foldgreaterchanceof

developingamalignanttumorbyage50,comparedwiththegeneralpopulation.In

contrasttopatientswhoinheritamutantRB1allele,thespectrumoftumorsthatdevelop

inpatientswithLi-Fraumenisyndromeisquitevaried.Themostcommontypesoftumors

Cdk4-cyclin D Rb E2Fcyclin E

transcriptionCdk2-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,andcarcinomasoftheadrenal

cortex.Ascomparedwithsporadictumors,thosethatafictpatientswithLi-Fraumeni

syndromeoccuratayoungerage,andagivenindividualmaydevelopmultipleprimary

tumors.

p53restrainstumorformationbytwodifferentmechanisms(Figure4).Intherst,p53

activatesthep21CdkinhibitorgeneinresponsetoDNAdamageandstress.Lossofp53

incellspreventsthep21genefrombeingtranscribed,leadingtotheincreasedactivityof

themultipleCdksnormallyturnedoffbyp21andresultinginincreasedcellproliferation.

Asecondwayinwhichp53restrainstumorformationisbyinducingapoptosis.

D. Caretaker Genes that Function as Tumor Suppressors

BRCA1,locatedon17q21,andBRCA2,locatedon13q12,aretumorsuppressorgenes

associatedwithbreastandovariancancer,alongwithseveralothercancers.About10%

ofallcasesofbreastandovariancancerarehereditarycancers,andmostofthesecases

areduetoinheritanceofagermlinemutationineitherBRCA1orBRCA2(see Familial

and Hereditary Cancer Syndromes lecture).Aswithothertumorsupporessorgenes,the

remainingalleleisinactivatedorlostduringthecourseoftumorformation(LOH).The

proteinsencodedbyBRCA1andBRCA2areexpressedinmosttissuesandcelltypes

(indicatingthatgeneexpressiondoesnotaccountfortherestrictedphenotypeofbreastandovariancancer),andshareanumberoffunctionalsimilarities.

BRCA1andBRCA2functionascaretakergenes,likep53,whichservetomaintain

genomicintegrity.ThegeneproductsencodedbyBRCA1andBRCA2arenuclearproteins

thatco-localizewithRAD-51atsitesofDNAdamage,andplayaroleinhomologous

recombinationrepairofdouble-strandedbreaks(see Mutation and Cancer lecture).

ThereisalsoevidencethatBRCA1andBRCA2interactwiththep53-mediatedDNA

damagecheckpoint(see Mutation and Cancer lecture).LossofBRCA1orBRCA2leads

totheaccumulationofothergeneticdefects,whichcanthenleadtocancerformation.

InadditiontotheirrolesinDNArepair,BRCA1andBRCA2havebeenimplicatedina

varietyofcellularprocesses,includingDNAsynthesis,regulationofgenetranscription

(similartop53,onetargetofBRCA1transcriptionalactivationistheCdkinhibitorp21),cellcyclecheckpointcontrol,centrosomeduplicationandubiquitination.

MostBRCA1andBRCA2mutationsleadtoframeshiftsresultinginmissingornon-

functionalprotein,or,inthecaseofBRCA2,tononsensemutationsleadingtopremature

truncationoftheprotein.Thesemutationsareallconsistentwiththelossoffunction

expectedwithtumorsuppressorgenes.

p53

transcription

factor

p21 Cdk inhibitor Cdk-cyclin

complexesproliferation

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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ApuzzleinbreastcancerhasbeenwhymutationsinBRCA1andBRCA2arenotfound

asofteninsporadic(i.e.non-familial)casesofbreastcancer.Recentstudiessuggest

partialanswer.AproteincalledEMSYhasbeenfoundthatbindsandinhibitsBRCA2.

Remarkably,EMSYisfrequentlyoverexpressedinsporadicbreastcancersduetogene

amplication,andthisisapparentlyamuchmorelikelyeventthatthelossofthetwo

normalcopiesofeithertheBRCA1ofBRCA2genes.IncreasedEMSYexpression

correlateswithpoorclinicaloutcome.

E. Regulators of Signal Transduction

Anotherpotentialwaythatproductsoftumorsuppressorgenesmayoperateisbydown

regulatinggrowth-promotingsignals.TheproductsoftheAPCgene(5q21)andtheNF1

gene(17q11.2)fallintothiscategory.Germlinemutationsofthesegenesareassociated

withbenigntumorsthatareprecursorsofcarcinomasthatdeveloplater.

InthecaseoftheAPC (adenomatous polyposis coli)gene,individualsbornwithone

mutantalleleinvariablydevelophundredsoreventhousandsofadenomatouspolypsin

thecolonduringtheirteensor20s(familialadenomatouspolyposis,orFAP;seeFamilial

andHereditaryCancerSyndromeslecture).Almostinvariably,oneormoreofthese

polypsundergomalignanttransformation,givingrisetocancer.Aswithothertumor

suppressorgenes,bothcopiesoftheAPCgenemustbelostfortumordevelopment.

Whenthisoccurs,adenomasformed.InadditiontocancersarisinginthesettingofFAP,

themajority(70-80%)ofnon-familialcolorectalcarcinomasandsporadicadenomas

alsoshowhomozygouslossoftheAPCgene,thusrmlyimplicatingAPClossinthe

pathogenesisofcolonictumors.(SeelectureonColonCancer.)AdescribedintheCell

Proliferationlecture,animportantfunctionoftheAPCproteinistocausedegradationof

beta-catenin,thusmaintaininglowlevelsinthecytoplasm.InactivationoftheAPCgene,

andconsequentlossoftheAPCprotein,increasesthecellularlevelsofbeta-catenin,

which,inturn,translocatestothenucleusandup-regulatescellularproliferation.Thus

APCisanegativeregulatorofbeta-cateninsignaling.Beta-cateninitselffunctionsasa

proto-oncogene(seebelow).Interestingly,casesofcolorectalcancerthatdonotshowlossofAPCmayinfacthaveactivatingmutationsinbeta-catenin.Dysregulationofthe

APC/beta-cateninpathwayisnotrestrictedtocoloncancer;mutationsineitherAPCor

beta-cateninhavealsobeenfoundinnearly30%ofmelanomacelllines.

TheNF1genebehavessimilartotheAPCgene.Individualswhoinheritonemutant

alleledevelopnumerousbenignneurobromas,presumablyasaresultoftheinactivation

ofthesecondcopyoftheNF1gene.Thisconditioniscalledneurobromatosistype

1,orNF1.Someoftheneurobromasmayundergomalignanttransformationinto

neurobrosarcomas.ChildrenwithNF1alsoareatincreasedriskfordevelopingacute

myeloidleukemia.Thefunctionofneurobromin,theproteinproductoftheNF1gene,is

toregulatesignaltransductionviatheRasprotein.NeurobrominisaGTP-aseactivating

proteinthatfacilitatesconversionofactiveRas(GTPfound)toinactiveRas(GDPbound).WithlossofNF1,Rasistrappedinanactive,signal-emittingstate.

III. VIRUSES AND CANCER

Virusesareinfectiousagentsthatmustreplicateinsideahostcell.Virusescontaintheirown

genome(whichcanbeeitherDNAorRNA)protectedbyacoatmadeupofproteinorprotein

pluslipid.Differentvirusesusethehostcellsmachineryindifferentways,butallviruses

requirethetranslationapparatus(e.g.ribosomes)ofthehostcellstotranslatetheirmRNAs.

Bacteria,incontrast,cangenerallygrowoutsidethehostandhavetheirowntranslation


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machinery.SomevirusespromotecancerinhumansincludingHIV,Epstein-BarrVirus

(EBV),andhuman papilloma viruses (HPVs).Thefundamentalsofviraltumorgenesis,

includingsomeofthehistory,werecoveredintheI3block.Herewewillmerelyreviewhow

HPVscausecancerinordertoconnecttotwoofthemaintumorsuppressorgenesdiscussed

inthissection.

Humanpapillomaviruses(transmittedthroughsexualcontact)playapathogenicrole

inmostcasesofcervicalcancer(seeinformationoncervicalcancerinthePathological

CharacteristicsofBenignandMalignantNeoplasmslab,theCancerScreeninglecture,

andtheClinicalandTranslationalResearchonlinemodule).Thereareatleast77subtypes

ofHPVthataredistinguishedbyvariationsintheirDNAsequences.HPV-16orHPV-18

DNAisfoundin70%ofcervicaltumors.Anadditional20%oftumorscontainHPVDNA

correspondingtooneof20othercancer-associatedsubtypes.HPVshaveadouble-stranded

DNAgenome.Thereare7viralgenesthatareexpressedearlyduringinfection(E1-E7)and2

genesthatareexpressedlate(L1-L2).

Twooftheearlygenes,E6andE7,promotetumorformation(Figure5).TheE6proteinas-

sociateswithacellularproteincalledE6AP(E6-AssociatedProtein).TheE6-E6APprotein

complexcatalyzescovalentattachmentofasmallproteincalledubiquitintop53.Theattach-mentofapolyubiquitinchaintoaproteintargetsittotheproteasomefordegradation.Thus,

E6inactivatesp53bycausingthedegradationoftheproteinviathecellsnormalprotein

degradationmachinery.TheproteinencodedbyE7directlybindstoRbandinactivatesit.

ThusRbcanbeinactivatedbylossofthegene(asinretinoblastomaandothertumors),by

mutationsingenesthatcontrolRbPhosphorylation,orbyinactivationoftheproteinbya

viralprotein.AsingeneticlossofRb,inactivationoftheRbproteinleadstotheactivationof

theE2FtranscriptionfactorandtranscriptionofthecyclinEgene.Withoutthesafeguardsof

bothp53andRb,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)

+

activation

of E2F

and cyclin E

transcription

E7 + Rb

active

E7 Rb

inactive


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IV. ONCOGENES

Cellscontainmanynormalgenesthatareinvolvedinregulatingcellproliferation.Someof

thesegenescanbemutatedtoformsthatpromoteuncontrolledcellproliferation.Thenormal

formsofthesegenesarecalledproto-oncogenes, whilethemutated,cancer-causingforms

arecalledoncogenes.Incontrasttotumorsuppressorgenes,whichputthebrakesoncell

proliferation,oncogenesactivelypromoteproliferation(analogoustothegaspetalofthecell

cycle).Mutationsthatconvertproto-oncogenestooncogenestypicallyincreasetheactivity

oftheencodedproteinorincreasetheexpressionofthenormalgene.Suchmutationsare

dominantorgain-of-function mutations.Therefore,onlyonecopyofthegeneneedstobe

mutatedinordertopromotecancer.

Oncogeneswererstidentiedinoncogenicretrovirusesthathadpickedupacellular

oncogene(c-onc)andincorporateditintotheviralgenometoproduceaviraloncogene

(v-onc).J.MichaelBishopandHaroldVarmusofUCSFwereawardedthe1989NobelPrize

inMedicineforthediscoveryofthecellularoriginoftheviraloncogenes.

A. HER2/neu and breast cancer

TheHER2/neugeneencodesareceptortyrosinekinasecloselyrelatedtotheepidermal

growthfactor(EGF)receptor(theybothbelongtotheErbBfamilyofreceptors).This

geneisoverexpressedin~25%ofbreastcancers.BreastcancersthatoverexpressHER2/neutendtobemoreaggressiveclinically(moreonthisinupcominglecturesonBreast

CancerandCancerTreatment).ThemechanismbywhichHER2/neuisoverexpressedis

gene amplication,whichresultsinmultiplecopiesofthegeneaccumulatingintumor

cells.TheincreaseinthelevelsoftheHER2/neutyrosinekinasereceptorontumorcells

Sos

Ras-GDP Ras-GTP

GTP

GDP

inactive active

oncogenic mutations

block GTPase activiy

Figure 6. How mutations in the Ras

oncogenes promote cancer. Oncogenic

mutations in ras genes prevent the

protein from hydrolyzing GTP toGDP. 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/neuamplicationinbreastcancerhasledtothedevelopment

oftheanticancerdrugHerceptin.Herceptinisamonoclonalantibodythatbindsto

HER2/neuonthecellsurfaceresultinginreceptordown-regulation(decreasednumbers

ofHER-2/neuatthecellsurface)andkillingofcellsbytheimmunesystem.Itwas

approvedin1998foruseintreatmentofbreastcancer(moreaboutHerceptinintheDrug

Developmentlecture).

B. Activation of Ras: the oncogene most commonly activated in human tumors

ThehumangenomeencodesthreeRas genes:H-ras,K-ras,N-ras.Alargefractionof

tumorscontainmutationsinoneofthesethreegenes.Forexample,70-90%ofpancreatic

carcinomascontainamutationintheK-rasgene.Rasoncogenesareactivatedbypoint

mutationsthatresultinproteinsunabletohydrolyzeGTP(Figure6).Thesemutant

RasproteinsarethereforelockedintheGTP-bound(active)form,whichtherefore

continuallyactivatestheMAPkinasepathway,whichinturnleadstocellproliferation.


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C. The Myc oncogene is often amplied or overexpressed in cancers

TheMycproteinactsinthenucleusasasignalforcellproliferationthroughseveral

mechanisms.Onemechanism,asdiscussedintheCellProliferationlecture,isasa

transcriptionfactorforthecyclinDgene.Mycisencodedbyaproto-oncogenethatis

overexpressedorampliedinmanycancers.ExcessquantitiesofMyccancausecells

toproliferateincircumstanceswherenormalcellswouldhalt.Insomecancers,rather

thanbeingamplied,Mycismadeactivebychromosomaltranslocation.(SeetheCancerCytogeneticssectionoftheMutationandCancerlectureforadescriptionofchromosome

abnormalitiesincancer.)Asaresultofthischromosomalrearrangement,astrong

promotersequenceisplacedinappropriatelynexttotheMycproteincodingsequence,

producingunusuallylargeamountsoftheMycmRNA.Forexample,asyouwilllearn

laterinthiscourse,inBurkittslymphomaatranslocationbringstheMycgeneunderthe

controlofsequencesthatnormallydrivetheexpressionoflargeamountsofantibodiesin

Bcells.Asaresult,mutantBcellsproducelargeamountsoftheMycprotein,proliferate

toexcess,andformatumor.

D. Cyclin D can be activated through several different mechanisms

AsdiscussedintheCellProliferationlecture,cyclinDformspartoftheG1-Cdk,which

normallyfunctionstoinactivetheRbtumorsuppressorproteinbyphosphorylatingit

(Figure7).CyclinDisoftenoverexpressedincancersleadingtoreducedactivityofRb.

Inbreastcancer,thecyclinDgeneisoftenamplied.AswithHER2/neuamplication,

thisresultsinmorecyclinEproteinbeingproduced.

CyclinDalsoplaysaroleinparathyroidadenomas,tumorsoftheparathyroidglandthat

resultinuncontrolledcalciummobilizationfrombones(Figure8).(Thenormalfunction

ofparathyroidhormoneistoinducecalciumreleasefrombones).Inthesetumors,cyclin

Dcanbeactivatedbyadifferentmechanism.Achromosome inversionplacesthestrong

transcriptionalcontrolregionoftheparathyroidhormonegeneadjacenttothecyclinD

geneonchromosome11q.ThisresultsinthecontinuousexpressionofthecyclinDgene,

whichinturnpromotesproliferation.

Inchroniclymphocyticleukemias,cyclinDisderegulatedbyyetanothermechanism.In

thesetumors,cyclinDisactivatedviaachromosometranslocationthatplacesthecyclin

Dgeneundercontroloftheimmunoglobulinheavychaintranscriptioncontrolregion.

(ChromosomerearrangementsaredescribedintheCancerCytogeneticssectionofthe

MutationandCancerlecture.)

Figure 7. How cyclin D gene amplication 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

transcriptionCdk2-cyclin E proliferation

OFF ON ON ON ON

cyclin D gene

amplication

ON


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E. Bcl-2: apoptosis proteins and cancer

Atranslocationbetweenchromosomes14and18(t(14:18)(q32;q21)isthemostcommon

translocationinhumanlymphoidmalignancies.Itisespeciallycommoninfollicularcelllymphomas(80-90%ofcaseshaveit).Thetranslocationresultsintheactivationof

Bcl-2becausethegeneisbroughtintoproximitywithpotenttranscriptionalregulatory

sequencesfromtheimmunoglobulinheavychaingene.Theantiapopototicactivity

ofBcl-2resultsintheincreasedsurvivalofthecellsthatoverexpressBcl-2.Thisin

turngivesthecellstheopportunitytoacquireadditionalmutationsthatleadtocancer.

Thus,programmedcelldeathisanimportantmechanismthatnormallyrestrictstumor

formation.

V. MULTISTEP TUMORIGENESIS: MOLECULAR EVENTS ASSOCIATED WITH

THE GENESIS OF COLON CANCER

Coloncancerisunusualinthattheprecancerouslesionscanbeidentiedearlyvia

colonoscopy.Molecularanalysisofthevariousstagesofcoloncanceristhereforepossible.Thesestudieshaveidentiedaroughsequenceofmoleculareventsthatcharacterizetumor

developmentincolorectalcancer(Figure9).Becausewenowknowthemolecularfunction

manytumorsuppressorgenesandoncogenes,wearebeginningtounderstandthegenetic

changesthatoccurduringcancerformation.

Theearlieststageofcoloncancerformationistheappearanceofahyperproliferative

epithelium(Figure9).ThisisassociatedwiththelossoftheAPCtumorsuppressor.Recall

thatAPClimitsWntsignalinginthegutepitheliumwhereitisexpressedathighlevelsin

non-dividingcellsandlowlevelsinthestemcellsofthecrypts.LossoftheAPCgeneresults

inhyperactiveWntsignalingandproliferationofcellsbeyondthecrypts.Subsequentto

lossofAPC,thereappearstobeaperiodofgenomicinstability,whichpermitsthecellsto

acquiremutationsmorerapidly.ProgressionofadenomasisassociatedwithactivationofthemitogenicRas-MAPKpathwaythroughtheacquisitionofactivatingmutationsinthe

K-rasoncogene.Later,afurtherblowtogrowthcontroloccursviathelossofaSMADgene

requiredforsignalingthroughthegrowthinhibitoryTGF-betasignalingpathway.Finally,the

progressionfromlateademonastomalignantcarcinomsisassociatedwiththelossofthep53

tumorsuppressor,whichreleasesthecellsfromDNAdamageandstressinducedapoptosis

andcellcyclearrest.(Fordescriptionofcancernomenclature,seetheIntroductiontoCancer

Pathologylecture.)

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 codingregion.

PTH genepromoter

cyclin D gene

promotercyclin D genePTH gene

PTH gene

promoter

cyclin D gene

promoter

cyclin D genePTH gene

inversion of DNA segment

between cyclin D gene and PTH gene


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Thereareadditionalchangesintumorsuppressorgenesandoncogenesthatoccurduring

tumorprogressionthataredifferentindifferentcoloncancers,someofwhichhaveyettobeidentied.Nonetheless,thestudyofthemolecularbasisofcoloncancerhasgivenus

apictureofthemulti-stepevolutionofatumorandthegeneticchangesthatdrivethem.Importantly,anunderstandingofthespecicgeneticchangesthatoccurduringthismulti-

stepprocessallowsfortheidenticationofkeystepsforinterventionandthedevelopmentof

specictherapeuticstrategies

VI. THE HALLMARKS OF CANCER

Ifweconsiderthehallmarkfeaturesofcancercellsthatwereintroducedintheintroductory

lectureonCancerBiology,wecanbegintomapthemoleculardefectsinthegenesand

processeswevediscussedinthelasttwolecturestooneofthesixacquiredcapabilities

ofcancercells.Letsconsidertherstfouracquiredcapabilitieshere(thelasttwowillbe

coveredinlaterlectures).Acoupleexamplesofmoleculardefectsthatcanresultineach

acquiredcapabilityareincludedbelow.Canyouthinkofothers?

1. Self-Sufciency in Growth Signals

Normalcellscannotproliferateintheabsenceofstimulatorysignals,butcancercells

can.Manyoncogenesactbymimickingnormalgrowthsignalingthroughoneofseveral

mechanisms.

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 APC

tumor suppresor

increased genetic

instability

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),manycancercellsacquiretheability

tosynthesizetheirowngrowthfactors,creatingapositivefeedbacksignalingloop

(autocrinestimulation),andobviatingthedependenceongrowthfactorsfromother

cells.TheproductionofPDGF(plateletderivedgrowthfactor)andTGF-(tumor

growthfactor)byglioblastomasandsarcomasaretwoexamples.

Cellsurfacereceptorsthattransducegrowth-stimulatorysignalsintothecell,for

exampleEGFRandHER2/neu,maybeoverexpressedorstructurallyaltered,leadingtoligand-independentsignaling.

Downstreamtargetsofthesignalingpathwaycanalsobealtered.Forexample,Ras

isfoundmutatedabout25%ofhumantumors,thusleadingtoligand-independent

activationoftheRas-Raf-MAPKsignalingpathway.

2. Insensitivity to antigrowth signals

Antigrowthsignalscanblockproliferationbytwomechanisms:1)forcingcellsoutofthe

activeproliferativecycleintothequiescent(G0)state,untilappropriategrowthsignalsput

thembackintothecellcycle;or2)inducingdifferentiation,whichpermanentlyremoves

theirproliferativepotential.Cancercellsevadetheseantiproliferativesignals.Atthe

molecularlevel,manyandperhapsallanti-proliferativesignalsarefunneledthroughthe

Rbprotein(anditstworelatives,p107andp130).DisruptionoftheRbpathwayliberates

E2Fsandthusallowscellproliferation,renderingcellsinsensitivetoanti-growthfactors

thatnormallyblockadvancethroughG1.TheRbsignalingpathway(regulatedbyTGF-

anti-proliferativesignalingandotherextrinsicfactors)canbedisruptedinavarietyof

waysindifferenttypesofhumantumors,forexample:

LossofTGF(normallyactstopreventphosphorylationthatinactivatesRb)

LossofSmad4(normallytransducessignalsfromligand-activatedTGFreceptorsto

downstreamtargets)

LossofCDKinhibitorsp16,p21,p53

InactivationofRbdirectlybyhyperphosphorylationorinactivatingmutation

Tumorcellsalsousevariousstrategiestoavoidterminaldifferentiation.OverexpressionofcMycisonesuchstrategy.

3. Evading Apoptosis

Asdiscussedearlier,theabilityoftumorcellpopulationstoexpandinnumberis

determinednotonlybytherateofcellproliferationbutalsobytherateofcellattrition.

Programedcelldeath,orapoptosis,representsamajorsourceofthisattrition.Resistance

toapoptosisascanbeacquiredbycancercellsthroughavarietyofstrategies.Examples

include;

Lossofp53(normallyactivatespro-apoptoticproteins;representsthemostcommon

lossofaproapoptoticregulator)

Activationorupregulationofanti-apoptoticBcl2

4. Limitless Replicative Potential

Therstthreeacquiredcapabilitieslackofrequirementforgrowthsignals,insensitivity

toantigrowthsignals,andresistancetoapoptosisallleadtoanuncouplingofthecells

growthprogramfromsignalsinitsenvironment.However,ithasbeenshownthatthis

uncouplingalonedoesnotensureexpansivetumorgrowth.Mosttypesofmammalian

cellscarryanintrinsicmechanismthatlimitstheirproliferationbykeepingtrackofthe

numberofcellgenerations.Thismechanismusesthenumberoftelomererepeatsatthe

endsofchromosomes,whicherodethroughsuccessivecyclesofreplication-eventually


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leadingtocelldeath.Asmallpercentageoftumorcellsavoidthisbyobtainingamutation

thatupregulatesexpessionofthetelomeraseenzyme,thusacquiringlimitlessreplicative

potentialbymaintainingtheirtelomeres.(Moreontelomeresandtelomeraseinthe

MutationandCancerlecture.)

5and6.The5thand6thacquiredcapabilities,SustainedAngiogenesisandTissueInvasion

andMetastasis,willbecoveredindetailinlaterlectures.

References

Albertsetal.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:

Signicantcontributionstothislectureweremadeby

HitenMadhani,PhD,DepartmentofBiochemistryandBiophysics,whooriginallydevelopedthislecture

(lecturer20022006);

Tumor Suppress Lec

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