Molecular Biology of Cancer - Angelfire

50 Pages·2003·0.43 MB·English

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