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CHAPTER ONE Molecular Biology of Cancer JESSED.MARTINEZ MICHELETAYLORPARKER KIMBERLYE.FULTZ NATALIAA.IGNATENKO EUGENEW.GERNER DepartmentsofRadiationOncology/CancerBiologySection MolecularandCellularBiology BiochemistryandMolecularBiophysics CancerBiologyGraduateProgram TheUniversityofArizona Tuscon,Arizona Contents 1Introduction,2 2Tumorigenesis,2 2.1Normal-Precancer-CancerSequence,2 2.2Carcinogenesis,3 2.3GeneticVariabilityandOtherModifiersof Tumorigenesis,5 2.3.1GeneticVariabilityAffectingCancer,5 2.3.2GeneticVariabilityin c-myc–DependentExpressionof OrnithineDecarboxylase,7 2.4EpigeneticChanges,7 3MolecularBasisofCancerPhenotypes,10 3.1Immortality,10 3.2DecreasedDependenceonGrowthFactorsto SupportProliferation,11 3.3LossofAnchorage-DependentGrowthand AlteredCellAdhesion,12 3.4CellCycleandLossofCellCycleControl,14 3.5ApoptosisandReducedSensitivityto Apoptosis,16 3.6IncreasedGeneticInstability,19 3.7Angiogenesis,20 4Cancer-RelatedGenes,21 4.1Oncogenes,21 4.1.1GrowthFactorsandGrowthFactor Receptors,21 4.1.2GProteins,23 4.1.3Serine/ThreonineKinases,24 Burger’sMedicinalChemistryandDrugDiscovery 4.1.4NonreceptorTyrosineKinases,24 SixthEdition,Volume5:ChemotherapeuticAgents 4.1.5TranscriptionFactorsasOncogenes, EditedbyDonaldJ.Abraham 25 ISBN0-471-37031-2 ©2003JohnWiley&Sons,Inc. 4.1.6CytoplasmicProteins,26 1 2 MolecularBiologyofCancer 4.2TumorSuppressorGenes,26 5.3.4LimitationsofMicroarray 4.2.1Retinoblastoma,27 Technologies,37 4.2.2p53,27 5.4ModifyingCellAdhesion,37 4.2.3AdenomatousPolyposisColi,29 5.4.1MMPInhibitors,37 4.2.4PhosphataseandTensinHomologue, 5.4.2Anticoagulants,38 30 5.4.3InhibitorsofAngiogenesis,38 4.2.5TransformingGrowthFactor-(cid:1),30 5.5ProspectsforGeneTherapyofCancer,39 4.2.6HeritableCancerSyndromes,32 5.5.1GeneDeliverySystems,39 5Interventions,32 5.5.1.1ViralVectors,40 5.1PreventionStrategies,32 5.5.1.2Non-ViralGeneDelivery 5.2Targets,33 Systems,42 5.2.1BiochemicalTargets,33 5.6GeneTherapyApproaches,43 5.2.2Cyclooxygenase-2andCancer,33 5.6.1Immunomodulation,43 5.2.3OtherTargets,35 5.6.2SuicidalGeneApproach,44 5.3Therapy,35 5.6.3TargetingLossofTumorSuppressor 5.3.1ImportanceofStudyingGene FunctionandOncogene Expression,35 Overexpression,44 5.3.2cDNAMicroarrayTechnology,35 5.6.4AngiogenesisControl,45 5.3.3DiscoveriesfromcDNAMicroarray 5.6.5MatrixMetalloproteinase,45 Data,37 6Acknowledgments,46 1 INTRODUCTION optosis, are now known to contribute to cer- taintypesofcancer.Cancerisdistinctivefrom Cancer is a major human health problem othertumor-formingprocessesbecauseofits worldwide and is the second leading cause of ability to invade surrounding tissues. This deathintheUnitedStates(1).Overthepast chapter will address mechanisms regulating 30 years, significant progress has been the important cancer phenotypes of altered achievedinunderstandingthemolecularbasis cellproliferation,apoptosis,andinvasiveness. of cancer. The accumulation of this basic Recently,ithasbecomepossibletoexploit knowledgehasestablishedthatcancerisava- thisbasicinformationtodevelopmechanism- riety of distinct diseases and that defective based strategies for cancer prevention and genescausethesediseases.Further,genede- treatment.Thesuccessofbothpublicandpri- fectsarediverseinnatureandcaninvolveei- vate efforts to sequence genomes, including therlossorgainofgenefunctions.Anumber humanandotherorganisms,hascontributed of inherited syndromes associated with in- tothiseffort.Severalexamplesofmechanism- creasedriskofcancerhavebeenidentified. basedanti-cancerstrategieswillbediscussed. Thischapterwillreviewourcurrentunder- Finally, potential strategies for gene therapy standingofthemechanismsofcancerdevelop- ofcancerwillalsobeaddressed. ment,orcarcinogenesis,andthegeneticbasis of cancer. The roles of gene defects in both 2 TUMORIGENESIS germlineandsomaticcellswillbediscussedas they relate to genetic and sporadic forms of 2.1 Normal-Precancer-CancerSequence cancer.Specificexamplesofoncogenes,orcan- cer-causing genes, and tumor suppressor Insight into tumor development first came genes will be presented, along with descrip- from epidemiological studies that examined tionsoftherelevantpathwaysthatsignalnor- therelationshipbetweenageandcancerinci- malandcancerphenotypes. dence that showed that cancer incidence in- While cancer is clearly associated with an creaseswithroughlythefifthpowerofelapsed increaseincellnumber,alterationsinmecha- age (2). Hence, it was predicted that at least nismsregulatingnewcellbirth,orcellprolif- fiverate-limitingstepsmustbeovercomebe- eration,areonlyonefacetofthemechanisms foreaclinicallyobservabletumorcouldarise. ofcancer.Decreasedratesofcelldeath,orap- Itisnowknownthattheserate-limitingsteps 2Tumorigenesis 3 aregeneticmutationsthatdysregulatetheac- humans as the paradigm. They suggest that tivitiesofgenesthatcontrolcellgrowth,reg- malignant colorectal tumors (carcinomas) ulate sensitivity to programmed cell death, evolve from preexisting benign tumors (ade- andmaintaingeneticstability.Hence,tumor- nomas)inastepwisefashionwithbenign,less igenesisisamultistepprocess. aggressive lesions giving rise to more lethal Although the processes that occur during neoplasms.Intheirmodel,bothgenetic[e.g., tumorigenesis are only incompletely under- adenomatouspolyposiscoli(APC)mutations] stood,itisclearthatthesuccessiveaccumula- and epigenetic changes (e.g., DNA methyl- tionofmutationsinkeygenesistheforcethat ation affecting gene expression) accumulate drives tumorigenesis. Each successive muta- overtime,anditistheprogressiveaccumula- tion is thought to provide the developing tu- tionofthesechangesthatoccurinapreferred, mor cell with important growth advantages but not invariable, order that are associated that allow cell clones to outgrow their more withtheevolutionofcolonicneoplasms.Other normalneighboringcells.Hence,tumordevel- important features of this model are that at opmentcanbethoughtofasDarwinianevolu- least four to five mutations are required for tion on a microscopic scale with each succes- theformationofamalignanttumor,inagree- sivegenerationoftumorcellmoreadaptedto ment with the epidemiological data, with overcomingthesocialrulesthatregulatethe fewerchangesgivingrisetointermediatebe- growth of normal cells. This is called clonal nign lesions, that tumors arise through the evolution(3). mutational activation of oncogenes and inac- Given that tumorigenesis is the result of tivationoftumorsuppressorgenes,andthatit mutationsinaselectsetofgenes,mucheffort isthesumtotaloftheeffectofthesemutations bycancerbiologistshasbeenfocusedoniden- on tumor cell physiology that is important tifying these genes and understanding how ratherthantheorderinwhichtheyoccur. theyfunctiontoaltercellgrowth.Earlyefforts Animportantimplicationofthemultistep inthisareawereleadbyvirologistsstudying model of tumorigenesis is that lethal neo- retrovirus-induced tumors in animal models. plasms are preceded by less aggressive inter- Thesestudiesledtocloningofthefirstonco- mediate steps with predictable genetic alter- genes and the realization that oncogenes, in- ations.Thissuggeststhatifthegeneticdefects deed all cancer-related genes, are aberrant whichoccurearlyintheprocesscanbeidenti- formsofgenesthathaveimportantfunctions fied, a strategy that interferes with their inregulatingnormalcellgrowth(4).Insubse- functionmightpreventdevelopmentofmore quent studies, these newly identified onco- advancedtumors.Moreover,preventivescreen- geneswereintroducedintonormalcellsinan ing methods that can detect cells with the efforttoreproducetumorigenesisinvitro.Im- early genetic mutations may help to identify portantly, it was found that no single onco- theselesionsintheirearliestandmostcurable genecouldconferallofthephysiologicaltraits stages. Consequently, identification of the ofatransformedcelltoanormalcell.Rather genes that are mutated in cancers and eluci- thisrequiredthatatleasttwooncogenesact- dationoftheirmechanismofactionisimpor- ingcooperativelytogiverisetocellswiththe tantnotonlytoexplainthecharacteristicphe- fully transformed phenotype (5). This obser- notypes exhibited by tumor cells, but also to vation provides important insights into tu- provide targets for development of therapeu- morigenesis.First,themultistepnatureoftu- ticagents. morigenesiscanberationalizedasmutations 2.2 Carcinogenesis indifferentgeneswitheacheventprovidinga selectivegrowthadvantage.Second,oncogene Carcinogenesisistheprocessthatleadstoge- cooperativity is likely to be cause by the re- neticmutationsinducedbyphysicalorchem- quirementfordysregulationofcellgrowthat icalagents.Conceptually,thisprocesscanbe multiplelevels. divided into three distinct stages: initiation, FearonandVogelstein(6)haveproposeda promotion,andprogression(7).Initiationin- linearprogressionmodel(Fig.1.1)todescribe volvesanirreversiblegeneticchange,usually tumorigenesis using colon carcinogenesis in amutationinasinglegene.Promotionisgen- 4 MolecularBiologyofCancer DNA hypomethylation Mutation of Mutation of Other genetic APC K-ras Loss of DCC Loss of p53 alterations Normal Hyper- Early Intermediate Late colon proliferation adenoma adenoma adenoma Carcinoma Metastasis cell Figure1.1. Adenoma-carcinomasequence.FearonandVogelstein(6)proposedthisclassicmodel forthemultistageprogressionofcolorectalcancer.AmutationintheAPCtumorsuppressorgeneis generallyconsideredtobetheinitiationevent.Thisisfollowedbythesequentialaccumulationof otherepigeneticandgeneticchangesthateventuallyresultintheprogressionfromanormalcellto ametastatictumor. erally associated with increased proliferation Promotionisareversibleprocessinwhich ofinitiatedcells,whichincreasesthepopula- chemicalagentsstimulateproliferationofini- tionofinitiatedcells.Progressionistheaccu- tiated cells. Typically, promoting agents are mulationofmoregeneticmutationsthatlead nongenotoxic,thatistheyareunabletoform totheacquisitionofthemalignantorinvasive DNA adducts or cause DNA damage but are phenotype. abletostimulatecellproliferation.Hence,ex- Inthebest-characterizedmodelofchemical posure to tumor promoting agents results in carcinogenesis, the mouse skin model, initia- rapid growth of the initiated cells and the tionisanirreversibleeventthatoccurswhena eventualformationofnon-invasivetumors.In genotoxicchemical,oritsreactivemetabolite, themouseskintumorigenesismodel,applica- causes a DNA mutation in a critical growth tionofasingledoseofaninitiatingagentdoes controlling gene such as Ha-ras (8). Out- not usually result in tumor formation. How- wardly,initiatedcellsseemnormal.However, ever, when the initiation step is followed by theyremainsusceptibletopromotionandfur- repeated applications of a tumor promoting ther neoplastic development indefinitely. agent, such as 12-O-tetradecanoyl-phorbol- DNA mutations that occur in initiated cells 13-acetate(TPA),numerousskintumorsarise can confer growth advantages, which allow andeventuallyresultininvasivecarcinomas. them to evolve and/or grow faster bypassing Consequently, tumor promoters are thought normalcellulargrowthcontrols.Thedifferent tofunctionbyfosteringclonalselectionofcells types of mutations that can occur include with a more malignant phenotype. Impor- point mutations, deletions, insertions, chro- tantly, tumor formation is dependent on re- mosomal translocations, and amplifications. peatedexposuretothetumorpromoter.Halt- Three important steps involved in initiation ing application of the tumor promoter are carcinogen metabolism, DNA repair, and preventsorreducesthefrequencywithwhich cellproliferation.Manychemicalagentsmust tumorsform.Thesequenceofexposureisim- bemetabolicallyactivatedbeforetheybecome portantbecausetumorsdonotdevelopinthe carcinogenic.Mostcarcinogens,ortheiractive absence of an initiating agent even if the tu- metabolites,arestrongelectrophilesandbind mor promoting agent is applied repeatedly. toDNAtoformadductsthatmustberemoved Therefore,thegeneticmutationcausedbythe byDNArepairmechanisms(9).Hence,DNA initiating agent is essential for further neo- repairisessentialtoreverseadductformation plasticdevelopmentundertheinfluenceofthe andtopreventDNAdamage.Failuretorepair promotingagent. chemical adducts, followed by cell prolifera- Progressionreferstotheprocessofacquir- tion,resultsinpermanentalterationsormu- ing additional mutations that lead to malig- tation(s)inthegenomethatcanleadtoonco- nancyandmetastasis.Manyinitiatingagents gene activation or inactivation of tumor canalsoleadtotumorprogression,strongsup- suppressorgenes. portforthenotionthatfurthermutationsare 2Tumorigenesis 5 Metabolic activation Procarcinogen Carcinogen Detoxification DNA binding Excretion of Figure 1.2. Possible outcomes of metabolites carcinogen metabolic activation. Once a carcinogen is metabolically Formation of activated it can bind to DNA and carcinogen-DNA adduct formcarcinogen-DNAadducts.These DNA repair adductswillultimatelyleadtomuta- DNA Cell tionsiftheyarenotrepaired.IfDNA death Normal cell replication repairdoesnotoccur,thecellwillei- ther undergo apoptosis or the DNA willbereplicated,resultinginanini- Initiated cell tiatedcell. needed for cells to acquire the phenotypic play important roles in the metabolic activa- characteristicsofmalignanttumorcells.Some tionanddetoxificationofcarcinogenicagents. of these agents include benzo(a)pyrene, The phase I enzymes include monooxygen- (cid:1)-napthylamine, 2-acetylaminofluorene, ases, dehydrogenases, esterases, reductases, aflatoxinB ,dimethylnitrosamine,2-amino-3- andoxidases.Theseenzymesintroducefunc- 1 methylimidazo(4,5-f)quinoline (IQ), benzi- tionalgroupsonthesubstrate.Themostim- dine, vinyl chloride, and 4-(methylnitros- portant superfamily of the phase I enzymes amino)-1-(3-pyridyl)-1-butanone (NNK) (10). are the cytochrome P450 monooxygenases, Thesechemicalsareconvertedintopositively whichmetabolizepolyaromatichydrocarbons, charged metabolites that bind to negatively aromaticamines,heterocyclicamines,andni- chargedgroupsonmoleculeslikeproteinsand trosamines. Phase II metabolizing enzymes nucleicacids.Thisresultsintheformationof areimportantforthedetoxificationandexcre- DNA adducts which, if not repaired, lead to tion of carcinogens. Some examples include mutations (9) (Fig. 1.2). The result of these epoxide hydrase, glutathione-S-transferase, mutationsenablesthetumorstogrow,invade and uridine 5(cid:1)-diphosphate (UDP) glucuro- surroundingtissue,andmetastasize. nide transferase. There are also some direct DamagetoDNAandthegeneticmutations acting carcinogens that do not require meta- thatcanresultfromthemareacentraltheme bolicactivation.Theseincludenitrogenmus- in carcinogenesis. Hence, the environmental tard, dimethylcarbamyl chloride, and (cid:1)-pro- factors that cause DNA damage are of great piolactone. interest.Environmentalagentsthatcancause DNAdamageincludeionizingradiation,ultra- 2.3 GeneticVariabilityandOtherModifiers violet (UV) light, and chemical agents (11). ofTumorigenesis Some of the DNA lesions that can result in- clude single-strand breaks, double-strand 2.3.1 Genetic Variability Affecting Cancer. breaks,basealterations,cross-links,insertion Differenttypesofcancers,aswellastheirse- of incorrect bases, and addition/deletion of verity,seemtocorrelatewiththetypeofmu- DNA sequences. Cells have evolved several tation acquired by a specific gene. Mutation differentrepairmechanismsthatcanreverse “hotspots”areregionsofgenesthatarefre- thelesionscausedbytheseagents,whichhas quentlymutatedcomparedwithotherregions beenextensivelyreviewedelsewhere(12). within that gene. For example, observations Themetabolicprocessingofenvironmental thatthemajorityofcolonadenomasareasso- carcinogensisalsoofkeyimportancebecause ciated with alterations in the adenomatous thiscandeterminetheextentanddurationto polyposis coli (APC) have been based on im- whichanorganismisexposedtoacarcinogen. munohistochemical analysis of (cid:1)-catenin lo- Phase I and phase II metabolizing enzymes calization and formation of less than full 6 MolecularBiologyofCancer Armadillo Mutation Drosophilia repeats cluster DLG binding 453−766 region 2771−2843 APC∆716 Min APC∆1638 Min 0 2843 Homodimerization Microtubule region 1−71 binding 2143−2843 EB1 binding 2143−2843 Murine models Intestinal tumor number APC ∆716 200−600 Min (850 stopcodon) 60−80 APC∆1638 <10 Figure1.3. DiagramofAPCproteinregions,relatingriskofintestinalcarcinogenesistolengthof APCpeptidetranslated.APCcontains2833aminoacids.Mutationhotspotregionsarefoundinareas betweenaminoacids1500–2000.ThreegeneticallyalteredmousemodelsofAPC-dependentintes- tinalcarcinogenesishavebeendeveloped.Minmicehaveastopcodonmutationincodon850ofthe murine APC homolog. Two transgenic mice, APC(cid:2)716 and APC(cid:2)1635, also have been developed. IntestinaltumornumberinthesemodelsisinverselyrelatedtosizeoftheAPCpeptidetranslated. length APC protein production after in vitro resultsfromasinglebasemutationthatleads translation of colonic mucosal tissue RNA. to the substitution of one base for another. These studies have not documented specific SNPsoccurquitefrequently(aboutevery0.3– genemutationsinAPC.Thisisimportant,be- 1kbwithinthegenome)andcanbeidentified causeitisknownfromanimalstudiesthatthe by several different techniques. A common location of APC mutations can have a dra- method for the analysis of SNPs is based on maticeffectonthedegreeofintestinalcarci- the knowledge that single-base changes have nogenesis.Thus,itispossiblethatcolonade- the capability of destroying or creating a re- nomasize,andsubsequentriskofcoloncancer striction enzyme site within a specific region couldbedictatedbylocationofspecificmuta- ofDNA.DigestionofapieceofDNA,contain- tionsinAPC(Fig.1.3). ingthesiteinquestion,withtheappropriate AssuggestedbythemodeldepictedinFig. enzyme can distinguish between variants 1.3,highriskmightbeassociatedwithmuta- based on the resulting fragment sizes. This tionscausingstopcodonsintheaminotermi- type of analysis is commonly referred to as nalendoftheprotein.Lowriskmightbeas- restriction fragment length polymorphism sociatedwithmutationsresultinginpeptides (RFLP). ofgreaterlength.Currentresearchistesting TheimportanceofanalyzingSNPsrestson thehypothesisthatspecificgeneticalterations thepremisethatindividualswithanucleotide inAPCalonemaybesufficientasaprognostic at a specific position may display a normal factorforriskofadenomarecurrenceandsub- phenotype,whereasindividualswithadiffer- sequently,coloncancerdevelopment. ent nucleotide at this same position may ex- Onetypeofgeneticalterationthatisgain- hibitincreasedpredispositionforacertaindis- ing increasing attention is the single nucleo- ease or phenotype. Therefore, many studies tidepolymorphism(SNP).Thispolymorphism are being conducted to determine the fre- 2Tumorigenesis 7 ODC gene +300 e-box (1) e-box (2) e-box (3) G/A SNP E-box (1) SNP allele (Frequency) Promoter activity CACGTG G (90−95%) 1 CAGCTG G (90−95%) 0.5 CACGTG A (5−10%) 3−8 Figure1.4. InfluenceofspecificgeneticchangesonODCpromoteractivity.Thesedatawerederived fromtransienttransfectionexperimentsinhumancolontumor–derivedHT29cells.Thearrowin this figure 1.4 shows the SNP. The SNP occurs between two E-boxes that are located 3(cid:1) of the transcription start site. The effects of this genetic change are taken from Guo et al. (56). It is importanttopointoutthattheconstructsusedtoassessthepromoteractivityofthepolymorphic regioncontainingtheSNPandE-boxes2and3containedsomeofthe5(cid:1)promoterregion,butnot E-box1(56).TheconstructsusedtoassesstheroleofE-box1inHT-29containedthemajor,c-myc unresponsiveallelebetweenE-boxes2and3. quencyofspecificSNPsinthegeneralpopula- aminesynthesis,mayplayakeyroleintumor tionandtousethesefindingstoexplainphe- development. Therefore, elucidation of the notypicvariation. mechanismsbywhichODCisregulatedises- Forexample,arecentstudyfoundanasso- sential.TheliteratureindicatesthatODCisa ciationbetweenapolymorphismleadingtoan downstream mediator of APC and suggests amino acid substitution (aspartate to valine) thatODCmaybeanAPCmodifiergene.Thus, incodon1822oftheAPCgeneandareduced polymorphisms in the ODC promoter affect- riskforcancerinpeopleeatingalow-fatdiet ing c-myc–dependent ODC transcription (13). The variant valine had an allele fre- couldbeamechanismofgeneticvariabilityof quencyof22.8%inaprimarilyCaucasiancon- APC-dependentcarcinogenesis. trol population. This non-truncating muta- O’Brienandcolleagues(15)havemeasured tionhasnotyetbeenshowntohavefunctional theincidenceinseveralhumansubgroupsofa significance. If functional, such a polymor- SNPinaregionoftheODCpromoter,3(cid:1)ofthe phismcouldcooperatewithsinglealleletrun- transcriptionstartsite,thatisflankedbytwo cating mutations that occur with high fre- E-boxes(CACGTG)(Fig.1.4).TheE-boxisa quency in sporadic colon adenomas (14), to DNA sequence where specific transcription increasecoloncancerrisk.Thispolymorphism is especially interesting, because dietary fac- factors bind. The two resulting alleles are tors, specifically fat consumption, may con- identified by a polymorphic PstI RFLP. The tributetoriskinonlyspecificgeneticsubsets. minor allele (A at position (cid:3)317) is homozy- gousin6–10%ofindividuals,whereasthema- 2.3.2 Genetic Variability in c-myc–Depen- jorallele(Gatposition(cid:3)317)ishomozygous dent Expression of Ornithine Decarboxylase. or heterozygous in 90–94% of these groups. The proliferation-associated polyamines are Theyhavealsomeasuredfunctionalityofthe essentialforcellgrowthbutmaycontributeto polymorphisms. When ODC promoter-re- carcinogenesis when in excess. Various stud- porter constructs are expressed in rodent ies have shown that inhibition of polyamine cells, the minor allele confers 3–8 times the synthesis impedes carcinogenesis. Ornithine promoteractivitycomparedwiththemajoral- decarboxylase(ODC),thefirstenzymeinpoly- lele.Further,expressionoftheminoralleleis 8 MolecularBiologyofCancer enhancedbyc-mycexpressiontoagreaterex- GeneexpressionisinhibitedbyDNAmeth- tentthanthemajorallele. ylation. DNA methylation patterns dramati- callychangeatdifferentstagesofcelldevelop- 2.4 EpigeneticChanges ment and differentiation and correlate with Genefunctioncanbedisruptedeitherthrough changes in gene expression (18). Demethyl- genetic alterations, which directly mutate or ationreleasesgeneexpressioninthefirstdays deletegenes,orepigeneticalterations,which ofembryogenesis.Later,denovomethylation alterthestateofgeneexpression.Epigenetic establishes adult patterns of gene methyl- mechanisms regulating gene expression in- ation.Indifferentiatedcells,methylationsta- clude signal transduction pathways, DNA tus is retained by the activity of the Dnmt1 methylation, and chromatin remodeling. enzyme.Innormaltissues,DNAmethylation MethylationofDNAisabiochemicaladdition isassociatedwithgenesilencing,chromosome ofamethylgroupatposition5ofthepyrimi- X inactivation (19), and imprinting (20). Be- dineringofcytosineinthesequenceCG.This cause the most normal methylation takes modification occurs in two ways: (1) from a placewithinhighlyrepeatedtransposableele- preexistingpatternonthecodingstrandor(2) ments,ithasbeenproposedthatsuchmethyl- bydenovoadditionofamethylgrouptofully ation plays a role in genome defense by sup- unmethylated DNA. Cleavage of DNA with pressing potentially harmful effects of the restriction endonuclease HpaII, which expressionatthesesites. cannot cut the central C in the sequence Neoplasticcellsarecharacterizedbysimul- CCGG if it is methylated, allows detection of taneous global DNA hypomethylation, local- methylated sites in DNA. Small regions of ized hypermethylation that involves CpG is- DNA with methylated cytosine, called “CpG lands and increased HDAC activity (21). islands,”havebeenfoundinthe5(cid:1)-promoter Hypomethylationhasbeenlinkedtochromo- region of about one-half of all human genes somalinstabilityinvitroanditseemstohave (includingmosthousekeepinggenes). thesameeffectincarcinogenesis(22).5-Meth- There are three DNA methyltransferases ylcytosineisarelativelyunstablebasebecause (Dnmt), Dnmt1, Dnmt3a, and Dnmt3b, that itsspontaneousdeaminationleadstothefor- havebeenidentifiedinmammaliancells(16). mation of uracil. Such changes can also con- The most abundant and ubiquitous enzyme, tribute to the appearance of germline muta- Dnmt1, shows high affinity for hemimethyl- tions in inherited disease and somatic atedDNA,suggestingaroleofDnmt1inthe mutationsinneoplasia.AberrantCpGisland inheritance of preexisting patterns of DNA hypermethylation in normally unmethylated methylationaftereachroundofDNAreplica- regionsaroundgenetranscriptionstartsites, tion. The other two enzymes, Dnmt3a and which results in transcriptional silencing of Dnmt3b, are tissue specific and have been genes,suggeststhatitplaysanimportantrole showntobeinvolvedindenovomethylation. as an alternate mechanism by which tumor DenovoCpGislandmethylation,however,is suppressor genes are inactivated in cancer notafeatureofproliferatingcells,andcanbe (21).Hypermethylatedgenesidentifiedinhu- consideredapathologiceventinneoplasia. man cancers include the tumor suppressor Over the years, a number of different genesthatcausefamilialformsofhumancan- methyl-CpGbindingproteins,suchasmethyl- cerwhenmutatedinthegermline,aswellas CpG-binding domain-containing proteins genes that are not fully documented tumor (MBD1-4) were identified (17) that compete suppressors(Table1.1).Someofthesegenes, with transcription factors and prevent them suchasAPC,thebreastcancergeneBRCA-1, from binding to promoter sequences. These E-cadherin, mismatch repair gene hMLH1, methyl-CpG binding factors can also recruit and the Von Hippel-Lindau gene can exhibit histone deacetylases (HDACs), resulting in thischangeinnon-familialcancers. condensation of local chromatin structure Recent studies indicate that promoter hy- (Fig. 1.5). This makes the methylated DNA permethylation is often an early event in tu- lessaccessibletotranscriptionfactorsandre- mor progression. It has been shown in the sultsingenesilencing. colon that genes that have increased hyper- 2Tumorigenesis 9 Active gene De novo methylation Active (?) gene Recruitment of MBP HDAC HDAC and HDAC Inactive gene Deacetylated histones Condensed chromatin- silenced gene Figure1.5. Effectofmethylationandhistonedeacetylationongeneexpression.Whenageneis active,thepromoterregionisoccupiedbytranscriptionfactorsthatdirectproductionofmessenger RNA. De novo methylation has minimal effects on gene expression. However, methylated DNA attractsmethyl-bindingproteins(MBP).Thesemethyl-bindingproteinsinturnattractaprotein complex that contains histone deacetylase (HDAC). This results in inhibition of messenger RNA synthesis,andnofunctionalproteincanbemadefromthegene.ThroughtheactionofMBPand HDAC,theDNAstructurechangestoacompact,“condensedchromatin”configuration,whichre- sultsinpermanentinhibitionofmessengerRNAandproteinsynthesis(silencing). methylationinthepromoterregioninnormal and gastric neoplasia, has been seen in early tissue as a function of aging are the same as stagesofcancerprogression(24).Finally,hy- geneswiththehighestrateofpromoterhyper- permethylation of the E-cadherin promoter methylationintumors(9).Interestingly,this frequentlyoccursinearlystagesofbreastcan- groupofgenesdoesnotincludeclassictumor cerandcantriggerinvasion(25). suppressorgenes.Somegenes,suchasthees- Loss of gene function through epigenetic trogenreceptorwhereage-relatedhypermeth- changesdiffersfromgeneticchangesinterms ylationinthecolonwasfirstdiscovered,may of its consequences for tumor biology. First, beimportantforthemodulationofcellgrowth gene function loss caused by aberrant pro- anddifferentiationinthecolonicmucosa. moter methylation may manifest in a more Promoter hypermethylation of genes, subtle, selective advantage than gene muta- whicharenormallyunmethylatedatallages, tions during tumor progression. Second, al- has also been found early in tumorigenesis. though promoter hypermethylation causing These epigenetic alterations can produce the genesilencingisusuallystableincancercells, earlylossofcellcyclecontrol,alteredregula- thischange,unlikemutation,ispotentiallyre- tion of gene transcription factors, disruption versible. It has become evident that not only ofcell-cellinteractions,andmultipletypesof themutagens,butvariousfactorsinfluencing geneticinstability,whichareallcharacteristic cell metabolism, particularly methylation, lie of neoplasia. For example, hypermethylation attheoriginofcarcinogenesis. oftheAPCgenehasrecentlybeenreportedfor Silencing of gene expression by methyl- a subset of colon cancers (23). Hypermethyl- ationmaybemodulatedbybiochemicalorbi- ationofhMLH1,whichisassociatedwithmi- ologicalmanipulation.Ithasbeenshownthat crosatellite instability in colon, endometrial, pharmacological inhibition of methyltrans- 10 MolecularBiologyofCancer Table1.1 HypermethylatedGenesinCancer Gene Function TypeofTumor FamilialCancers APC Signaltransduction Coloncancer BRCA1 DNArepair Breastcancer E-cadherin Adhesionandmetastasis Multiplecancers hMLH1 DNAmismatchrepair Colon,gastric,andendometrial cancer p16/CDKN2A Cellcycleregulation Multiplecancers RB1 Cellcycleregulation Retinoblastoma VHL Cytoskeletalorganization,angiogenesis Renal-cellcancer inhibition OtherCancers Androgenreceptor Growthanddifferentiation Prostatecancer c-ABL Tyrosinekinase Chronicmyelogenousleukemia EndothelinreceptorB Growthanddifferentiation Prostatecancer Estrogenreceptor(cid:2) Transcription Multiplecancers FHIT Detoxification Esophagealcancer GST-(cid:3) Drugtransport Prostatecancer MDR1 Drugtransport Acuteleukemias O6-MGMT DNArepair Multiplecancers p14/ARF Cellcycleregulation Coloncancer p15/CDKN2B Cellcycleregulation Malignanthematologicdisease Progesteronereceptor Growthanddifferentiation Breastcancer Retinoicacidreceptor(cid:1) Growthanddifferentiation Colonandbreastcancer THBS1 Angiogenesisinhibition Coloncancer,glioblastoma multiforme TIMP3 Metastasis Multiplecancers ferasesresultedinreactivationofgeneexpres- properties that are characteristic of tumor sioninvitro(26)andpreventedtumorgrowth cellsandthatarenowknowntobethebasis in animal models (27). These studies gener- forthebehaviorsexhibitedbyneoplasticcells. atedinterestintheclinicalusesofhypomethy- Someofthefeaturesthatwillbediscussedin latingagentsinhumans. detail include immortality, decreased depen- denceongrowthfactorstosupportprolifera- tion,lossofanchorage-dependentgrowth,loss 3 MOLECULAR BASIS OF CANCER ofcellcyclecontrol,reducedsensitivitytoap- PHENOTYPES optoticcelldeath,andincreasedgeneticinsta- bility. Other morphological and biochemical Cancerisamultistepprocessthatrequiresthe characteristics used to identify the trans- accumulationofmultiplegeneticmutationsin formedphenotypearecytologicalchanges,al- asinglecellthatbestowfeaturescharacteris- tered enzyme production, and the ability to tic of a neoplastic cell. Typically, tumor cells producetumorsinexperimentalanimals(28). differ from normal cells in that they exhibit uncontrolled growth. Because features that 3.1 Immortality distinguish tumor from normal cells may be keytounderstandingneoplasticcellbehavior Normal diploid fibroblasts have a limited ca- andmayultimatelyleadtotherapiesthatcan pacitytogrowanddividebothinvivoandin target tumor cells, considerable effort has vitro. Even if provided with optimal growth been directed at identifying the phenotypic conditions,invitronormalcellswillceasedi- characteristics of in vitro–transformed cells viding after 50–60 population doublings and and of tumor cells derived from natural then senesce and die. In contrast, malignant sources. This work has resulted in a list of cellsthathavebecomeestablishedinculture

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