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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5186_Библиотеки_им_академика_М_И_Перельмана.pdf
Figure 1.2 Schematic illustration of the phosphoinositide 3-kinase or
phosphatidylinositol3-kinase(PI-3K)signalingcascade.PI-3Kisdividedintofourclasses.
MostisknownaboutclassI,whichisfurthersubclassifiedintoclassIAandIB.Theschematic
shown here summarizes some of the functions of class IA whose role in cancer is best
described. The PI-3K pathway is activated by various growth factor (GF) receptor tyrosine
kinases (RTKs) or G protein–coupled receptors (not shown). Class I PI-3K proteins are
recruitedtotheplasmamembranebyadapterproteinsandarecomposedofacatalyticsubunit
andaregulatorysubunit(notshown).Gaininfunctionofthecatalyticsubunit,forexample,the
phosphatidylinositol 3-kinase, catalytic subunit α (PIK3CA), and loss in function of the
regulatory subunit, for example, the phosphatidylinositol 3-kinase regulatory subunit α
(PIK3R1), can result in overactivity of the PI-3K pathway. Activated class I PI-3K
phosphorylates phosphatidylinositol 4,5-bisphoshate (PIP2) to phosphatidylinositol 3,4,5-
trisphosphate(PIP3).Thephosphataseandtensinhomolog(PTEN)dephosphorylatesPIP3to
PIP2andtherebycounteractsPI-3K.PIP3activatesAKTkinases,whichinturnphosphorylate
tuberous sclerosis protein 1 (TSC1) and 2 (TSC2). Phosphorylation of TSC results in
dissociationoftheTSC1–TSC2complex.Theintact TSC complex inhibits the kinase mTOR.
Thus,activationofAKTresultsinactivationofthemTORcomplex1(mTORC1).Rashomolog
enrichedinbrain(Rheb)in its GTP-bound form activates mTORC1. mTORC1 is involved in
negativefeedbackto prevent overactivationofAKT. Depictedistheinteractionandactivation
of the TSC1–TSC2 complex by serine/threonine kinase 11(STK11)/liver kinase B1 (LKB1)
and adenosine monophosphate-activated protein kinase (AMPK). Not depicted here are
interactionswiththeMAPKpathway,whichondifferentlevelscanresultinactivationofthePI3Kpathway.ThePI3K-AKT-mTORsignalingisinvolvedinmultiplecellularprocessesincluding
metabolism,motility,proliferation,growth,andcellsurvival.
NuclearOncogenes
Myc(Myelocytomatosis)
The myelocytomatosis (Myc) proteins, c-myc, l-myc, and n-myc, constitute a family of
transcription factors. C-myc is located on chromosome 8 and reported to regulate the
expressionof 15% of all genes. In mouse models, tissue-specific overexpression of myc
proteinscausescancerdevelopmentinthosetissues,demonstratingthe potencyof themyc
proteins. Myc proteins are activated by various mitogenic stimuli including the Wnt
(derivedfromwinglessandint-1)andMAPK(Fig.1.1)signalingpathway.Myc proteins
bindtoDNAviaabasichelix-loop-helixmotif(bHLH);withtheleucinezippermotif,they
heterodimerize with another bHLH transcription factor. It is thought that myc proteins
activategeneexpressionby binding to enhancer box sequences (E-boxes), that is, directly
through activation of transcriptions, and recruiting histone acetyltransferases, that is,
indirectlythroughthespatialreorganizationofnucleosomesthatmakeDNAmoreaccessible
forthetranscriptionmachinery.
CDKs(Cyclin-DependentKinases)
Cyclin-dependent kinases (CDKs) are highly conserved serine/threonine kinases and small
proteinsof30to40kDathatcompriseonlyakinasedomain.CDKsregulatetheprogression
throughthe cellcycle. WhileCDK expressionlevelsremain relativelyconstant throughout
thecellcycle,itsactivityismainlyregulatedbypost-translationalmechanisms.Asimplied
byitsname,aCDKneedstobindacyclintoformanactiveproteinkinase.Thebindingof
cyclinsin turn optimizes the accessibilityandbindingof ATPtothekinasedomain of the
CDK. Phosphorylation, that is, activating and inhibitory phosphorylation, and binding of
CDKinhibitorysubunitsareadditionalmechanismsofCDKactivityregulation.
TumorSuppressorGenes(Anti-Oncogenes)
Tumor suppressorgenes are genes whose loss or reduction of function may resultin
transformationof anormal cellintoa tumorcell. Tumorsuppressorgenes with distinct
featureshavebeendescribedandclassifiedasfollows:gatekeepergenesareabletorepress
cell cycle progression and regulate apoptosis; caretaker genes encode DNA repair
mechanisms; landscaper genes promote cell adhesion formation and extracellular matrix
interaction. Mutant germline tumor suppressor gene alleles are typically autosomal
recessiveandfollowKnudson’s2“two-hithypothesis”(25), which impliesthatboth alleles
must be affected to show an effect. Knudson observed that the age of onset for
retinoblastomafollowedsecond-orderkineticsimplyingtwoindependentgeneticevents.The
first hit can be a genetic or an epigenetic event, which inactivates one gene copy. In
hereditarysyndromes,patientsarebornwiththefirst hit,butstillownthefunctional
gene allele on the sister chromatid. This heterozygosity,however, can be lost (loss of
heterozygosity[LOH])leavingonlynonfunctioningallelesof atumorsuppressorgene.
While the second hit is generally assumed to be a gene deletion, it can be due to mitotic
recombination,geneconversion,uniparentaldisomy, or other copy-number neutral genetic
events. Twenty to 80% of LOH in human cancers is copy-number neutral and cannot be
detected using fluorescence in situ hybridization (FISH) or gene copy-number counting
methodssuchascomparativegenomichybridization(CGH).
Important exceptions to Knudson’s two-hit rule are certain mutations of the tumor
suppressorgeneTP53thatcanmakemutatedTP53actasadominant-negativemutant,
thatis,themutationsononeTP53alleleinhibitthefunctionoftheotherwildtypeallele
(26). Other tumor suppressor genes may be haploinsufficient with only one mutated (and
therebylost)genealleleandanotherwildtypeallelethathasnotbeensilencedbutisnotable
to produce sufficient functional protein (27). Examples for haploinsufficiency in tumor
suppressor genes include phosphatase and tensin homolog (PTEN), TP53, p27, and
transforminggrowthfactor-βreceptor1-2(TGFβR1-2).
CellMembraneReceptors
TGF-β(TransformingGrowthFactor-β)
Thevarioustransforminggrowth factor-β (TGF-β) ligands arecategorizedinto four major
subfamilies:(i)theTGF-βsubfamily,(ii)bonemorphogenicproteins,(iii)theactivin/inhibin
subfamily,and(iv)left-rightdeterminationfactors.Theligandsareonlyactiveashomo-or
heterodimers. Thus far, 13 TGF-β receptors have been described and grouped into three
types.TypeIIreceptorsbindligandsand thenactivatetypeIreceptorsbyphosphorylation,
whichinturnautophosphorylate,bind,andphosphorylateSmad2/3.Smadsarenamedafter
theirhomologsinCaenorhabditiselegans(sma=smallbodysize)andDrosophila(Mad=
mothers against dpp). Activated Smads shuttle from the plasma membrane through the
cytoplasmintothecellnucleuswheretheyregulatetargetgenetranscription.
ThecytostaticprogramofTFG-βincludestheupregulationofCDKinhibitorsthatblockthe
progressionthroughthecellcycleandthedownregulationofc-myc.TGF-βligandsboundto
typeIIIreceptorformareservoirofTGF-βligands.ThehallmarkofTGF-β,however,isits
pleiotropicsignalingpathway.Thereby,TGF-βplaysadualroleincancerdevelopment.
In the early stages of cancer, the TGF-β pathway acts as a tumor suppressor gene,
inducing cell differentiation, cell cycle arrest, and apoptosis as described above. In
advanced-stagecancer,however,variousmutationswithintheTGF-βpathwaychange
its effects so that TGF-β in fact acts as an oncogene promoting cancer cell growth,
neoangiogenesisandimmunesuppression(28–30).
CytoplasmicTumorSuppressorGenes
PTEN(PhosphataseandTensinHomolog)
Phosphatase and tensin homolog (PTEN) is the phosphatidylinositol-3,4,5-triphosphate 3phosphatase, a tyrosine phosphatase, which—unlike other tyrosine phosphatases—
preferentiallydephosphorylatesphosphoinositidesubstrates.Assuch,itdephosphorylatesthe
phosphatidylinositol (3,4,5)-trisphosphate (PIP3), specifically the 3′ phosphate of the
inositolring in PIP3, resultinginphosphatidylinositol (4,5)-bisphosphate (PIP2). Thereby,
PTEN negatively regulates the PI-3K signaling pathway (Fig. 1.2) and functions as an
importanttumorsuppressor gene. PTEN comprises a phosphatase domain andC2domain,
which binds the phospholipid membrane and brings the active domain in proximity to the
membrane-boundPIP3.PTENisoneofthemostcommonlylosttumorsuppressorgenes
incancer.
NuclearTumorSuppressorGenes
pRb(RetinoblastomaProtein)
Ifbothretinoblastomaprotein(pRb)allelesaremutatedearlyinlife,retinoblastomaarising
from retinal cells develops. The retinoblastoma protein belongs to the so-called pocket
proteins,thatis,itsmolecularstructureexhibitsapocketforthefunctionalbindingofother
proteins. Most importantly, pRb binds and inhibits E2 promotor-binding-proteindimerizationpartner(E2F-DP).Thereby,pRbkeepsthecell inthe G1phase andprevents
progressionthroughthecellcycle.pRbisabletorepresstranscriptionbyinterferingwiththe
preinitiation complex, and by recruiting histone deacetylases (HDACs). Histone
deacetylationleadstotightercompactionoftheDNAandnucleosomeformationpreventing
access of transcription factors to the DNA. DNA damage can induce pRb activation and
therebypreventacellfromreplicatingdamagedDNA.ThephosphorylationstatusofpRb
determines its activation. In general, hyperphosphorylation inactivates pRb, while
monophosphorylationmayfinetuneitsfunction(31).Furthermore,pRbcanbeboundand
inactivated itself by gene products of the high-risk human papilloma viruses (HPVs)
(seesectiononcervicalcancer).
TP53(GeneEncodingp53TumorProtein)
TP53isacriticaltumorsuppressorgenetopreventcancerdevelopmentinmulticellular
organisms and hence has been described as the “guardian of the genome.” The p53
proteinwasfirstreportedin1979independentlybyfourdifferentgroupsasaproteinofabout
53 kDa in size on an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel
electrophoresis)—andwasthusnamedp53(32–35).Todate,16humanisoformswithasize
distributionfrom3to43.7kDahavebeenidentified.TheencodinggeneTP53isthemost
frequentlymutated gene in human cancer. It is mutated in morethanhalfofhuman
cancers.Itislocatedontheshortarmofchromosome17(17p).Thep53proteincontainsthe
N-terminalactivation domains1(AD1) and 2(AD2). The latterisspecifically involvedin
regulatingproapoptoticgenes. p53furthercomprisesaproline-rich domainimportantinits
proapoptotic activity, a central DNA-binding domain (DBD), a homo-oligomerization
domainallowingtetramerization,whichiscrucialforp53function,andaC-terminaldomain,
which is involved in the regulation of DNA binding through the central DNA-binding
domain.
Inresponsetovariouscell stresses, p53 is phosphorylated and activated by two groups of
protein kinases, (i) MAPK protein kinases that respond to heat shock, osmotic shock,
oxidativestress,and(ii)DNAdamagecheckpointkinases.Mediatedbymultiplemechanisms
including deubiquitination and prevention of ubiquitination, cell stress results in increased
p53half-life,whichinturnleadstohigherp53expressionlevels.PhosphorylationoftheNterminaldomainsresultsinconformationalchangeofp53,whichenhancesitstranscriptional
activity.The“guardianofthegenome”fulfillsmultiplefunctionsinregulatinggenomic
stability,thecellcycle,andapoptosis.ItcanactivateDNArepairproteins,arrestthecell
cycletoincreasetimeforrepairmechanisms,andinduceapoptosisifdamagecannotbe
repaired. p53 has been shown to be involved in the regulation of cellular metabolism
(36,37).
Cancer-associatedTP53mutationsmayresultinlossoffunction,butmayprovidep53with
oncogenic properties promoting cell proliferation, invasion, and metastasis (38). In fact,
manyof the TP53 mutations are missense mutationsthatresultinfull-length p53 exerting
oppositeeffectsthanthewild-typeTP53(39).
DNARepairProteins
DNA repair proteins are usually classified as tumor suppressor genes and comprise
single-strand and double-strand repair mechanisms. Three single-strand repair
mechanisms,(i)baseexcisionrepair,(ii)nucleotideexcisionrepair,and(iii)mismatchrepair
(MMR)systems,andthreedouble-strandrepairmechanisms,(i)nonhomologousendjoining
(NHEJ), (ii) microhomology-mediated end joining (MMEJ), and (iii) homologous
recombination(HR)exist.Geneticalterationsintwooftheserepairmechanisms,onesinglestrandrepairandonedouble-strandrepairsystem,arerelevantforgynecologicmalignancies,
thatis,themismatchrepairproteins(MMR)andBRCA1-2.
MMR(MismatchRepairProteins)—Single-StrandRepair
Mismatchrepairisahighlyconservedmechanism.ItisDNAstrand–specificandrecognizes
and repairs erroneous nucleotide insertions, deletions, and misincorporations. Mismatch
repair proteins were first described in Streptococcus pneumoniae (40). The function of
mismatch repair proteins and the consequences of their inactivation have been further
analyzed in Escherichia coli. Inactivation was shown to result in hypermutable E. coli
strains. Because of the 50- to 100-fold increased mutation rate, these proteins have been
named“Mut”:MutS,MutH,MutL(41–43).Ineukaryoticcellsincludinghumancells,three
MutShomologs(MSH2,MSH3,andMSH6)andthreeMutLhomologs(MLH1,PMS1,and
PMS2) have been identified. There is no eukaryotic homolog for MutH. Furthermore, the
eukaryotic MMR proteins function as heterodimers and show slightly different repair
mechanisms.MSH2/MSH6heterodimersrepairbasesubstitutionsand smaller DNAloops,
whileMSH2/MSH3heterodimersrepairlargeDNAloops(44).
For base mismatch replication errors, MSH2/MSH6 heterodimers form a ring around the
DNAwiththe mismatchrecognition domainand recruitMLH1/PMS2heterodimersthatin
turn help assemble a larger protein machinery and repair the mismatch (Fig. 1.3). This
machineryincludes the scaffoldproteinproliferatingcellnuclear antigen (PCNA) and the
exonuclease EXO1. EXO1 excises the daughter strand after recognition of the DNA
mismatch.PCNA,aring-shapedhomotrimericcomplex,encirclestheDNAandfunctionsas
a sliding clamp to guarantee processivity of DNA polymerases. DNA mismatch repair is
ATP-dependent. Aftergenotoxinexposure, again the MSH2/MSH6 heterodimerrecognizes
the damaged site and interacts with MLH1/PMS2 heterodimer to signal cell cycle arrest.
DNAdamagesignalingdoesnotrequireATPandcanresultinDNArepairorapoptosis.
Alterations in mismatch repair proteinsresultin an increased mutation rate: genetic
instability in general and instability of short nucleotide repeats, so-called
microsatellites, in particular. Microsatellites are short repeats in the DNA sequence,
mostfrequentlydinucleotiderepeatsofcytosine(C)andadenine(A).Longertandemrepeats
arecalledmini-satellitesand,ifevenlonger,satellites.Microsatellitesaremainlylocatedin
nontranslatedDNAregions,specificallyso-calledintrons(from“intragenicregions”).The
individual length of microsatellites varies from person to person and contributes to the
individualDNAfingerprint.Whilemicrosatelliteinstability(MSI) isused forthe diagnosis
of mismatch repair protein defects in endometrial and colon cancer, it was originally
describedinthecontextofxerodermapigmentosa,whichresultsfromadefectinnucleotide
excision repair. MSI can be caused by germline mutations in MMR proteins, epigenetic
silencing by hypermethylation, and downregulation of MMR mRNA by microRNAs (45)
(seesectiononepigeneticchanges).
Figure 1.3 Schematic illustration of DNA single-strand mismatch repair (MMR).
MSH2/MSH6orMSH2/MSH3heterodimersrecognizeDNAbasepairmismatch(1),formaring
around the DNA with their mismatch recognition domains (2), and recruit a MLH1/PMS2
heterodimer (3). The MLH1/PMS2 heterodimer couples mismatch recognition to the
subsequent steps of DNA repair, which include strand discrimination by proliferating cell
nuclear antigen (PCNA), unwinding of DNA by a DNA helicase, excision of the mismatch
containing DNA portion by exonuclease 1 (EXO1) (4) and synthesis by DNA polymerases
(POLδ/ε)(5).
BRCA1andBRCA2—Double-StrandBreakRepair
In1866,PaulBroca3(46)notedtheincreasedfrequencyofbreastcancer(notablynotovarian
cancer) in his wife’s family. In his book “Traite des Tumeurs,” he provided the first
descriptionofafamilialbreastcancersyndrome.Morethanacenturylater,in1990,thefirst
linkageoffamilialbreastcancertochromosome17q21wasmade(47).In1994,BRCA1was
cloned(48).Inthesameyear,BRCA2waslocatedtochromosome13q12(49).BRCA2was
clonedin1995(50).BothBRCA1andBRCA2arerathercomplexgeneswith24and27
exons,and encode large proteinsof 1,863 and 3,418aminoacids, respectively.In both
genes,exon1isnoncodingandexon11isunusuallylarge.
TheBRCAproteinsparticipateinDNAdouble-strandbreak(DSB)repair.DNAdoublestrand breaks can occur during DNA replication, and as a consequence of ionizing
radiation or genotoxic agents. Since both DNAstrands are affected, DSBs are the most
concerning kind of DNA damage. DSBs can be repaired by mechanisms that are
considered to be error-freesuch as homologous recombination (HR) and mechanisms
thatareconsiderederror-prone,suchasnon-homologousend-joining(NHEJ).HRusesthe
genome’sinherentredundancyofgeneticinformation.Itinvolvestheintactsisterchromatid
asatemplateforthecorrectrepairandthereforeoccursmainlyinSandG2phasewhenthe
intactsisterchromatidbecomesavailable.ImportantstepsforHRinclude(i)theresectionof
thebrokenDNAtocreate5’overhangingends,(ii)thesearchforhomologousDNAonthe
sister chromatid, (iii) the strand invasion and displacement-loop (D-loop) formation, (iv)
DNAsynthesis,andfinally(v)thesynthesis-dependentstrandannealing.Duringthisprocess
—as illustrated in Figure 1.4—Holliday4 junctions form that mostly resolve without
crossovereventsbetweendouble-strandDNA(dsDNA)ofthesisterchromatids.
BRCA1
TheBRCA1 protein consists of four protein domains: the aminoterminal really interesting
new gene (RING) domain, the serine cluster domain (SCD) and the two BRCA1 CTerminus(BRCT) domains.Furthermore,it shows nuclear localizationandnuclear export
signals.TheRINGdomainmediatesheterodimerizationofBRCA1with BARD1 (BRCA1associated RING domain protein 1). The RING domain exhibits a ubiquitin E3 ligase
function.TheadditionofubiquitintotheBRCA1proteinisthoughttohelptargetBRCA1to
sites of DNA damage. SCD includes multiple phosphorylation sites for the ataxia
telangiectasiamutated(ATM)/ataxiatelangiectasiaandRad3-relatedprotein(ATR)kinases,
whichareactivatedbyDNAdamage.Similartoubiquitination,phosphorylationofBRCA1is
thought to be involved in targeting BRCA1 to DNA damage sites. The BRCT domain
partakesintheregulationofgenetranscriptionandrepairofDNA(Fig.1.5).
BRCA2
BRCA2compriseseightcopiesof20to30aminoacidrepeats,“BRC”repeats,andtheDNAbinding domain. Both domains bind the recombinase RAD51 and thereby mediate the
recruitment of RAD51 to DSBs and specifically to single-strand DNA (ssDNA). The Nterminus of the BRCA2 protein binds the Partner and localizer of BRCA2 (PALB2). The
localization of RAD51 to DSBs requires the BRCA1-PALB2-BRCA2 complex. BRCA2
stimulatesandmaintainsDNAstrandinvasion,acriticalstepinhomologousrecombination
(Fig.1.5).
In summary,BRCA1 mainly acts as a DNA damage response protein for checkpoint
activation and DNA repair, while BRCA2 directly mediates DNA repair through
homologousrecombination.BothBRCAproteinsworknotonlytogetherbutinconcert
witharathercomplexmultiproteinmachinery,thedetailedmechanismofwhichisyet
tobeelucidated.
Figure 1.4 Schematic illustration of double-strand break (DSB) repair by homologous
recombination (HR). The first step in DNA double-strand repair is DSB recognition, mainly
through the ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3 related
protein (ATR) kinases. These kinases phosphorylate and activate checkpoint kinase 2
(CHEK2),TP53, BRCA1.BRCA1will serveasa scaffoldtoorganizerepairproteinsincluding
BRCA1-associated RING domain protein 1 (BARD1) and BRCA1-interacting protein1