AnalysisofIntcrfcron-Iudu cihlcGene Express ionFollowing l{as/l{af iMEKpathway Inhihitionil/!'i\'(}
by ArczooAlcmzadehMchrizi
A Thesis submittedtothe School ofG raduate Studies inparliall'lIllillmcnlol'lh cn :qllircmcnlsl'orlhctlcgrcc ol'
MusterofSciencein Medicine DivisionofBasic Medical Sciences.FacultyofMedic ine
MemorialUnivcrsitvofNcwfoundlund
.luly21112
St.Tohns Newfo undland
ABSTRACT
TypeI interferonshavebeenusedtotreat viral infcctionsandcanccrr howcvcr, IFNtreatment isnotalwayseffec t ivedueto the presence ofcellularsuppressorsofthe IFNpathwaysuchasRas signallingactivation.Wehaveinvestigatedwhether inhibition 01'Rassignallingat thelevel oft he MEK promotestheexpression01'IFN-inducib legenes illvivo, Bi\Ll 3/cmice were injectedintrapcritoncallywith eitherDMSO/I'BS.an inhibitorI()('MEK (SL327.100rug/kgBW).IFN-u(IOxIO~U/mouse)or SL327/IFN-u combined.At S hrsaftc r injection.thebrain.intestineandlungwere harvestedfor confi rmationofMliKinhibition andsemi-quantitative expressionanalysisfor thrccIFN- induciblegenes:Chll].Rig-/a ndS IIII] ,Western blot analysisofproteinsisolated from SL327treated lung showed reducedlevelsofp hosphorylatedsubstrate.indicatingthat the drugworkedeffective lytoinhibit MEK invivo,IFN significantlyinduced theexpression ofallgenesinintestineandRig-linthebrain;however.there wasno signilicant induction ofgene expressionfollowing the SL327treatmentand nosynergisticeffec twasobserved followingtheco-injection01'SL327and IFN-It,Theresultsofthisworksuggestthat MEKinhibitionc annote l'ICctivelypromotet ranscriptionol' lFN-induciblegenesunder physiologicalconditionsillvivo,
Acknow lcdgmcnt
I wouldlike tothankmy supervisor Dr. KcnuskcIlirasawafor givingmethe opportunity tostudy cancerbiologyfromthe immunologicalpcrspcctivc .formuIhiswas ancw and inlcrcstingavcnllco f rcscarch.
lwouidlikctotakclhisorrortunitylocxrrc ssm ysinccrc arrrccialionlomy surcrvisorycommillcc mcmbcrsDr. AnnDorwardand Dr.Mani Larijaniforguiding me throughallaspectsofmyp rogramofstudy.Thankyou forIistcning.cncourugingand lcadingmc in thc rigludircction.
Nomatter where youarcliving.therearc alwayspeoplewhomakccvcrywhcrc likchomc !lhavcm adcncwli'icndshir swithwondc rfulrcorlcwhorosscssdignity.
generosity.andlovein the heart.Thankyou tomynew friends:Firouzch Na far.Parisa Modirrousta,Ghazal Assadipour,MahdisMonajcmi and PeyvandAmini.
I wholeheartedly sendout mytrcrncndousthunksnnd appreciationtomyb eloved rarcnlsf()r thcirsinccrc and cndlcsslovc.cncouragcmcnta ndsur ro rt.la mso gratcful forthcirctlon s10provideme with the bestenvironmentforgrowingup, and inspiringme withtheconfidence thatalldrcamsarc possihlc.Finally.Iwish todedicatethiswork10 mybeloved Grandma. whomIloveand miss.
iii
Tabl('ofContcnt s
ANALYSIS OFINT ER FERO N-I NI>L'CIBL E(;EfE EXPRESSI ONFO L L O W I'(;
RA SIR A F/l\IEKPATIIWA YINIIIBITIONINVIV O I
J\ I~STRA CT 11
ACKN O \V LE I>(;l\lENT 11I
TABLE OF CON TEN TS IV
LIST OF TABL ES VI I I
LIST OF FIGUR ES IX
LIST OF ABBREV IA T IONS X
CI IAI'T ER I: INTROI> UCTION I
1.1INTERFERONS... . 1
1.1.17)'I }('I III /n .1<'/'(III.I'... . ...3
1.1TlPEIIFNPIWIJUCT/ON1':lT/III:,')'S 6
1.1.1RIG-lele{Jel/llell/I}(//IIlI·I~I'./i),.If·'Ninduction / 1.1.1TU ?-ele{Jel/l lell/ {Jl/ /IIlI'l/.l'.liJrIFNill ellll'l io ll 9 1.3T)'PE I IFNSIGNAU JNGPAT/IW.J),... .... ...11 1../.MUI.T/FUCT/ON,I/.ISGSWIT/I /'IVOTAL ROU'SIN7'l'PEIIFN SIGNAUJ NCi
1.5.IFNTlIERAI'Y AND17:\'HI//7/IT/ONS Ili
1.5.IIFN/!Jemp l 18
1.5.2l.itnitations ofI l-Ntherapy 20
1.6.CELLULAR SUI'I'RE\'SORS OFIFNS/GNALUNG 22
1. 6.1Ra.l'.I'igllolillg 25
1.6.1.1TheHiclogynfRas 25
1.6.1.2Ras/RaliMEKpathway 26
1.6. 1.3Manipulation ofno rmalRas/Raf/MlKpathwayactivityduringviral
infection s... .., 29
1.6.1.4 Exploitingor constitutiveRas/Ral/MEKpathwayforpromotingoncolytic
virusesinfecti on 30
1,6.1.5Antagonisticcrosstalkofthe Ras/Ral/Ml-Kpathwayandlntcrfcron
pathways 30
1.7.IIYI'OTl IESIS,.INf)O/J.JI,'CTlVI:'S 35
1.7.1"..""""."., ,'J
1.7.20 /Jjec/il·e.\...
CI IAI'T ER2:MATI~R IA LAND METII ODS 36
2.2WESTERN/JUJTANALYSIS 36
2.2.ll'rel){f/·o/iolloj'f!I'II/eill.l'{f/l/ple.I... ....36
2.2.2Ql/olI/i/iw/iOIl (!/j }f'o/eill 37
2.2.3 Elecirofl horesisilll d lrll llsji'r o{flroleills 37
2.2.-Il lI/lIlIl1lOhlOl/ illg... . 38
... ... ... ...39 2.3.Rl~/'CR....
2.3.1RNAc.xtrucu on >0'
2.3.2eDNASynthesis.: .. -111
2.3.3SI{//IIIi1rdl'CR... . -111
2.3.-1Eleclroflho resiso jPCRl lI'odllcls... ...-12
2.-1DENSITOMETRYANALySIS..... .. 42
2.5 STATlSTlCA LAN"IL 1'SIS 43
CI IAPT ER3:RESUL T S ..
3.1.D·TICACYO FSU27TREATMElvTONINI IIIJITlONOFR.·J.\//{ ·I/·/MD{
1'"ITlflVA YINVIVO... ... ...,44 3.2.EFFEC lS0 FI' I IJ'SIOUJG/CAI.U:'VELS OFAC'TIVI:'R.J.\/R.u/MD (1'"ITlflVAr ON EXI' RESSf()NI.EVD.S OFMDIIGDVESINTlIE IJRAIN,INn'STlNI:·. AN/)
CI I APT ER ..:DISCUSSIO N 5S
-1.1AI'I' UCA TION OFM I:'KINIIIIJITORSASASTRA TH; Y TO1'R(h\lO n'IFN EFFICACY...
vi
...5X
-1.2EFFI:'C7SOFII''N-,IANDSU27('OMBINAT/ONSONMDII(lENI:'S
EXI'RE',SION... . 59
-1.31:'XI'RIM EN7/1I,D/:'SIGN:INVI 7'lWVSINVIVO 60
-1.-1T/M I:'ANDDOSE DI:'/'/:'NDI:'NCYOFMD{INIIIIUTORS ANIJHI'I:'I IFNs 62 -1.5MODEL SYSTEMVARIAIUUTY..
-1.6CONCI,W ;f()N ...
-1.7FUT URE DIRI:'CT/ONS....
.. 64
. 65
.. 65
BIBLIOGRAPII Y 67
vii
Listof Tables
Table1.1GeneontologyclassificationsoI'ISCls...
Table1.2Expressionpatternoftype IIFNsin mammals 4
Table 1.3RNA viruses sensed byRIG-I I.>
Table1.4I'v1DIIgenesexpression in Rasvl2cellslullowingIFNtrcatmcntM l.K inhibition orlh e colllbinedtreatlllentilll'itl'O... ...33
Table2.1Gcncspccificprimerscqucnccs .41
viii
Listof Figures
Figure 1.1 RIG-Idependentproduction oftypeIIFNs... . X Figure 1.2TbeTl.Rvdcpcndcnt productionoftypeIII''s 10
Figure1.3TypeI IFNsignallingpathway... ...12
Figure1.'1Suppressors orlFNsignalling 23
Figure1.5 Overviewofphysiologicalactivationoft heRas/Rali'I'v1EK pathway 27 Figure3.1 Wcsrcrnblotunalysisofp-lR kinthelung... ...--15 Figure3.2Densitometryanalysisor p-ERK in thelung .'1(,
Figure3.3 RT-I'CRanalysisorthebrainsamples --IX
Figure3.'1Densitometryanalysis ofthe RT-I'CRresultsofthebrainsamples .'19
Figure3.5RT-I'CR analysisoft heintestinesamples 51
Figure3.6Densitometry analysisofthe RT-I'CR results ofthe intestinesamples 52
Figure3.7RT-I'CRanalysisort helungsamples 56
Figure3.XDensitometry analysisoft he RT-I'CRresults ofthelungsamples 57
n"-OAS AI'S BCA bp 13W
c-Myc CARD ChI' CNS CML
em
DMSO DC dsR NA EMCV ERK GAP Gapdh
ListofAbbrev ia tio ns
2"S"-0Iigoadcnylatcsynthctasc Ammoniumpcrsulfaic Bicinchonini c acid Basepair llodywcight
Centigrade
Myclocytom atosisviraloncogene homolog Caspaserecruitmentdom ain Chromosome Centra l nerv ous system
Chronicmyeloidleukemia Cvtc rm i naldomain Dimethylsul foxide
Dendriticcells Double-strandedRNA Enccphal omyocarditisvirus Extracellularsignal-regulatedkinase G'lPusc ucti vatingprot cin s
MouscGlyccraldchydc3-rhosrhatcdch ydrogcnasc (G apdh :l'rotcin:Gapdh:Gene )
GAI'DII
Gbp2
CJ!31'2
GDI' CIT
GTI'
IIIW IICL IICV IIIV hrs
IISV IgG IFNs IFNR IL-IR II' IPS-I IRF
IlulllanGlyceraldehyde 3-phosphate dehydrogenase (GAI'DII:Protein;GAI'D!!:Gene) MouseGianylatcbindingprotein2 (Gbp2:Protein: Gh!J2:Gene) lIuman Guanylate bindingprotein2 (GBI'2:Protein: G1J1'2:Gene) Guanylate diphosphate Guaninenucleotide exchangefactor Gram
Guanylatetriphosphate Iiour
IlepatitisB virus llairyc cllleukcmia
HepatitisCvirus Human immunodeficiencyvirus Ilours
l lcrpcs simplcxvirus ImmunoglobulinG lntcrfcrons IFN-ureceptor
Toll-intcrlcukinlreceptor Intraperitoneally
IFN-[lprollloterstilllulator-I Interferonregulatoryfactor
xi
ISGs ISGF3 lSRE Jak .Iak-ST!\ T
kDa Kg
LRR M MDlIgcncs
Mile min mg
nil mM MMl'9
My88 Mx N!\
ng
lntcrfcronstimulatcdgcncs
lFN-stimlilatcd gcnclilctor 3 lFN-stimlilatcd rcsponsc clcmcnt Junusfamilyott yro sinckinascs
Janusactivated kinase-signaltransduce rand activationor transcription
Kilodalton
Kilogram l.itcr
Leucine-rich-repeat Molar
MliKvmitogcn-acti vatcdprotcin/cxtru cellularsignal- regulatedkinase
Majorhistocompatibilitycomplex Minutes
Milligram Millilitrc Millimolar Matrixmc.allop rutcin9
Mycloiddirrcrcntiationprimaryrcsponscgcnc 88 Myxovirusresistancegene
Notassessed Nanogram
xii
NK
NTI' p-E RK 1'53 PBS I'IAS I'KR 1'1'1' q-I'CR Ras RCL RD Rig-I
RIG-I
RNAascL RNAi RSV In-I'CR RTK SDS-I'AGE
Natural killcrc clls Nanomctrc
NucleotideTriphosphate I'hospho-I:RK protcin53 Phosphatebuff eredsaline
Protein inhibitor ol'activatcdSTA T dsRNA-activatcdproteinkinase Triphosph.uc Ouuntitarivcl'CR
Rat Sarcomavirus oncogcnc Renalcel lcarcinoma Repressor domai n
MouseRctinoic acidinducible gene- I (Rig-I :Protein:Rig-t:gene)
Latentribonuclease RNAinterf erence Respiratorysyncytial virus Realtime-polymerase chain reaction Receptortyrosinekinascs Sodium dodccylsulfate-polyacrylamidegel Electro phoresis
xiii
SEM SII2 Sill'
sacs
Slat2
STAT2
ssRNA
T13K T13ST TEMED Th- I TIR TLR TNF TRIF Tris-IICI
VSV xg
Standarderror oft he mean Src-homology?
SI12 coniainingphosphaiases Suppresso rsolcyto kincsignalling
MOlise Signaltransdllcerand activalion oft ranscriplion (Stat2:I'mtein: SlaI2: Gene)
II1Iman Signaitransdllcer andactivation ofiranscription (STAT2:Protein:S/:·I'l'2:Gene)
Singled-strandedRNA Total- ERK TANK-bindingkinase1 Tris-bulfcrcdsalinewithtween Tctrarncthvlcthylcncdiami nc Tbclpcr-l
Toll-intcrlcukinl receptorhomology Tol l- Ii kcrcccptor
Tumornecrosisfactor
TIR-domain-conta ining adaptor-indllcingIFN Tris(hydroxymcthyl)aminomcthanc-hydrochloridc Volts
Vesicularst omatitisvirus Timesgravity Microgram
xiv
!II Microliter
Chapter I:Introduction
/.//NT/:'RFDWNS
Interf erons(IFs) arecytokinc sbestrecognizedfixthei rpivotalrolein antiviral defenceinlllallllllals(I.2).IFswcrc discovcred byIsaacsand l.indenlllann (1957) while theywerestudyingthcmcchan ism sof'nmiv iral defence,andrecognitionoftheir ahilityi n inrcrfcringwithviralin iectioniedlo their nolllinationas"inlcr iero ns"bythcsc scientists(3).Incontrast to antiviraldrugsused atthattime,llNshadalmostnotoxic ity and acted against u widcrange ofviruses. Theseuniqueproperticsmadcthemsupcrior overcommonantiv iralsubstances. whichencouragedvirologiststofurthcr invcstigntc and expandtheir knowledgeoflFNbiok'gy(l.2 .cJ).IFswcrc thcfirstcytokinc s identified.purified.cloned.sequenced.andproducedin rccombin ant Ionnforclinical use (3.5 -7).
IFlsare agroupof pleiotropiccyto kincswithantiviral. antitumor.and inuuunomodulatoryfunctions(1-3).Basedon therangeandimportance oft heir activities.
IFNshave beenusedlorthetreatmentof variouspathologicalcond itions.including certa in viralinfections,scvcral typcsolcancc randuuroimmuncdiscnscstx-lZj.Thc biologicalfunctionsandsubsequent iniervcntionalproperties ofl FNsarellled iatedbylhe IFN stimulatedgenes (ISGs).whichcomprise alargegroupofgeneswithgreatdiversity in Iunctiontlnhlc1.1) (13).Theyareclaisiliedintothreegroupsaccordingto their receplorspec ilici ly and alllinoac idsequcnce holllologya stypel.typella lld type l ll IFNs(l cJ).
Tahlc1.1Gcnco nto logyclassilica tionsof lSGs
Fu uct ionnlCate uorics Adhesio n Anuioucncsis Antigenpresentation Antigenprecessing Antiviral Apopt osis Cancer Cel l-cel ladhesion Ccllcvcle Chcmokinc Cvtoskclcton Dcvclopncnt DNAreplicati on/repair Extracellularma trix Gvprotcinsignalling Growth factor G'lP vbindinu I lost dcI~:usc Immunemodul ati on ln flamm..uion Lvmnhocvteadhesion Oncogene Protease Proteaseinhibitor RNA splicing Transcriptionfactor
~~t ion alact i vator 'I'ranscriptional rcprcssor Translation Tumor sunnrcssor Ubiquitination Vesicletransport
Adaptcdtrom rcfcrcn cctlJ)
1.1.2Type lIntcrfcrons
TypeIIFNs are a multi-genellullily comprisedol'eight subclasscsinmanuua ls:
IFN-u,IFN-[l.IFN-E,IFN-" .IFN-Ul.IF1\-T.I FN-o. andIFN-scra ble1.2)(15.16).Each geneiscncodcdbya singlccxonon humanchromosome(ChI')9.andmouse Chr4(17).
Thesizeoftype IIFNsrangesfrom165-200al11ino aeidsand theirsequence homology rangesli·om 30-S0 %withinspeeies(6.15,16).IFNsactivateintraeellularsignalling networksin anautocrincandparacrincmanner bybindingtoherero-dimcricccllsurfacc receptors.IFN-ureceptor(IFNR)IandIFNR2.whicharc distributedinall humancell types(I. IS,19).Antiviral.immunomodulutory, andunt it umoractionsnrcthcmain functionsofthetypeIIFNs(I.2).
Inmammals.typeI IFNsarcconsideredthe principalantiviralcytokincswith significantregulatoryrolesin innateandadaptive im111uncresponses.Toinduceinnate immune responses.they act directlyonuniu fcctcdorvirallyinlcctcdcclls.xvh ichlcndsto inhibitionofviralnucleicacidreplicntion and mltNzvtranslation (20).Type IIFNs accomplish thisby signalling theup-regulationol'lSGsthat mcdiatcthcantiviralattack.
Examples ofantiviral ISGs arc:2' 5 '-Oli~:oadenylatesynthetase (Oi\ S). dsRNi\ -activaled proteinkinase(I'KR)and Myxovirusresistance gene (Mxr. whichplnyimportuntrolesin typcl llNvinduccdinnateimmunity(2).Theantiviralactivity oI'2'S'-Oi\S targetsviral RNi\.whileI'KRtargetsviral translation.and M:--:targetsviralreplication.
Tahlc 1.2ExpressionpatternoftypeIll'sinIIIaIII 111aIs
Exp rcss lonpattcrn T\'(lCI IFNs llhtYllCS u l,n2.u3.u4.u5.u6.a], Ub iquitouslyex pressed uS.ul O.u13.u14.u\6.
uI7.u2J. 11
Leukocytes (J)
Uterus. Ovarv c
Eiidcrmalkcratinocvt cs K
'I'mhoblast
,'.o r
Snlccnt hvmus.Jvmnh nodc Z
"Absent in mice:bRuminantsonly:"Pigsundcuulconly Adaptcdli'olll referenccs(15)and( 16)
2'S'-OAS activatesalatentribonucleasetermedRNAascL.lead ingtodegradation01' hostand viralRNA(21),I'KRinhibitsviraltranslationthroughphosphorylationand subsequent inactivationofcukaryotic initiationfactor2u(22),Mxblocksviral rcplication viai nhibilionol' viral translocalionacros ithc nuclcar mcmbranc(23).
TypeI IFN-induccd adaptivc immunityismediated through their immunomodulatoryl'unctionsandsubscqucnt promotionol'antigcnp rcscnlationand pathogcnclcaranccstagcs(24. 25).TypcIIFNscnhancc thc proccssol'antigcn presentation throughinductionofdi ffe rcntiarionoI'T-hclpcr(Th)-1cclls(26).
dilkrc nliationandactivalionol'dcndriticc clls(DC) andincrcascd cxprcssionol'major histocompatibility complex (Mi le) ClassIandMIICII(27. 28),Their promotionalrole to stimulatetheformationandactivityofcytotoxic Tlymphocytcs(29.30).i nductionol' macrophagc (31)and naturalkillercells(NK)activity(32. 33)facilita teselimination01' virullyi nfcctcd cclls.NOlably.thcscimmllnoll1odulatory l'unctionshighlightthe importance oftypeIIFNst obridgc thc innatcandadaptivcimmunc rcsponscs.which cmphasizcsthcir qualityasstronganliviralcncctors.
TypeIIFNsinducc anlitumorrcsponscsthroughdi rccta nd indirecteff ectson tumorcellgrowthand progression.Dircctly. jhcyrcduccoucogcnc expressionsuchas Myclocytomatosisviralo ncogcnchomolog(c-Myc).andthcy inducclumorsupprcssor geneexpressionsuchasthetumor nccros sfact or(TNF) receptorfam i lyandprotein53 (1'53)(4.21).lndircctly.thcantitlimor clkctsol'typc I IFNs arcmcdiatcd byincreased immllnc-mcdiatcdtumorsurvcillancca nd down-rcgulationol' proangiogcnicmolcculcs (34. 35).Thcimmunomodulatoryl'unctionsol'lypc I IFNsalsocaliscstimulationol'
adaptive illllllunityrcsponscsagainsttul1lor cclls,which promotehostimIIIunc recognition and eradicationofemergingturnorc c l ls.Tnaddition, bothIFN-u andIFN-I\
down-rcgulatcscvcralilllportant proangiJgcnic lllolcculcs suchaSlllatrix lllctalioprotcin (MMI'9),vascular cndothclialgrowth filCtor.a nd basic fibroblastgrowth factorto restrict bloodvesselsupply (36).Throughtheir broad illlillunorcgulatoryandantiangiogcnic activitics.jypc IIFNs suppresstumorinitiationand progrcssion.Thisi sthcbasisfor clinical researchinitiativesthat incorporatetypeIIFNsintoantitumor thcrupcutic stratcgies(34).
1.2TYPEI/FNI'!?OJ)UCT/ONPATIIW,/YS
Inlllallllllalianorgan isllls,thefirsl stcpt o control viralinf cctionisdclcclion of thesepathogens,whichsubsequentlytriggersinduction of innate immuneresponses.The scnsing of'viruscsandsubscq ucnt induclionoflypcI IFNsi slllcdiatcd bytwo pathways:
theRctinoic acidinduciblegene-I(RIC-I)-dcpcndcnt pathwayandthe Toll-like receptor (Tl.Rj- dcpcndcntpathw ay(37,38).Thesepathways showsome cellspecificity;lor example. typeI IFNproductionin macrophagesand DCismed iatedthroughTLR- dependent pathways(39-4 1)andinothertypesofcells,includingbothimmuneand non- immune,is stimulated byRlGvl-dcpcndcutpalhway(39, 41-43).Severaltranscription factorscan beactivated byRIG-Iand TLR pathwaysthroughdifferentmechanisms,but thep roduction oftypeII FNsi s stilllulatcdby activation oflntcrtCronrcgulatoryfactor (IRF)3andIRF7 (44, 45).
1.2.1RICJ-Idependent pathwayforIFNinduction
RIG-I(DDX58) functionsasa cytoplasmicsensor lordifferenttypesorRNA viruses,includingsingled-stranded RNA(ssRNA)virusesharbouring a 5'-T riphosphatc (5'-1'1'1')group (46-48)anddouble-strandedRNA (dsRNA) viruses(49, 50).RICJ-I belongstoDEAD (Asparaginc-Glutaminc-Alanine-l listaminclAsparaginc)-boxRNA hclicascfamily(43).Thisfamilyofproteinsusc nuclcotidctriphosphatc(NTI')sas source ofenergytomediate a varictyof'func tionssuchastranslation.transcription. mRNA splicing and RNAdecay(51.52).RIG-Iconsistsoffo urfunctionaldomains:twoamino- terminalcaspascrecruitment domains(C.i\RD)(43.53).a centralDEAD-boxhclicasc domain (43.54.55) and aC -terminaldomain(CTD)alsoknownasthe repressordomain (RD) (50.53.56. 57).TheCARD domainsare responsiblefortransmittingthesignal fromRIG-Iro downstrcamsignallingmolecules:the DEAD-boxhclicasc domainis responsibleforboth RNArecognitionand providingenergyforconfonnatioual changcs throughthe hydrolysisor ATI':theCTD/RD domain isresponsible1(11'both inhibitionor RICJ-Isignalling andrecognitionorRNA viruses,RIG-Iisinactivated in theabsence or viral RNA. and in thisstate theCARD domain ismasked by CTD/RD domaintorepress signallingtodownstreammolecules(Figirc1.1) (58).Viralbindingto CTD/RDor DEAD-boxdomainsactivatesA'l'Pascactivityleadingtothe unmasking orC ARD domainsand activationorsignalling(50. 59).TheCARD domainor activatedRICJ-I interactswith theCARDdomainora mitochondrial antiviralsignallingprotein (MA VS) alsotermcdIFN-[lpromoterstimulatorIliPS- I)or VISAor CARDIF(25.60).
Viral RNA
FigUl"C 1.1I~IG-Idependent productiun of tYIICI IFNs.BindingofvirustoRIG-I inducesconformationalchanges anditssubsequentactivation.Activated RIG-Iinduces activationof MA VSwhichin tumactivatesTBKIandIKKl:throughphospho rylation AfterphosphorylationbyTBK Iand IKKl:,IRF3andIRF7 translo catcto thenucleusand inducc transcriptionofTypcl lFNs
Activated IPS-Irelaysthesignaltothe TANK-bindingkinaseI(T I3K I) and inducible Iid~kinase(IKK-i:also known asIKKc).whichphosphorylateIRF3andIFR7(61.62).
Phosphcr ylatcd IRF3 andIRF7transioeatetothcnu elcusto aetivatctranseript ion o rt ype I IFNs(44. 45).
1.2.2TLR-dcpcndcnt pathwaylorIFNinduction
TUbarc anUllilyor trans-mcmbranc protcins thatscnscd iflc rcnttypcsor pathogensat the cellsurfaceor in the endosome(63-66).AmongTLRs. TLR3. TLR7.
TLRXandTLR9arclocalizedin theendosomeandarcable toinduce Type 1 IFN dependentantiviral responses(37.67).The bestknownligand1'01'TLR3isdsRNA.lor TUn/Xis ssRNAandforTLR9 isviralDNA or bacterialDNAwitha CpG moti r( 6X- 75).InallTLRs.rccognition ofv iral particlesismediated by the leucine-rich-repeat (LRR)domain. and they alsopossesthe Toll-intcrlcukin I receptor (11.-1R)homology (TIR)domain. whichisa dockingsitefordownstrcumadaptor proteins(Figure 1.2) (76- 79). The TIR-domain-containingadaptor-inducingIFN(TRIF)servesasanadaptor proteinlor TLR3 signalling. while Myeloiddifferent iationprimaryresponse GeneXX (MyXX)servesas anadaptor proteinforTLR7. TLRXandTLR9signalling(XO.XI).
Theseadaptor proteinsactivate TBKlaneIKKf;(X2.X3).which in turn phosphorylate IRF3andIRF7 (61. 62).Phosphorylatcd IRF3 and IRF7subscqucntlytranslocatcto the nucleusto activate transcripti on oftypeIIFNs(44. 45).
FigUH'1.2Till' TLH.-dI' IIl'IIlII'Jll producti on oftype I IF Ns. Bindingofviralproducts (ssR A.dsRNA.CpG) tothe LRRdomain oft he TLRsresultedin theiractivati on.Upon activation.TLR3activateditsdownstreamadaptor proteinTRIF while MyDXXisthe downstreamtargelof T LR7.TLRX.and TLR'J.Uponactivation.TRIFand 1\1IDXX activate TBKI.andIKKl:which phosphor, lateIRF3and IRF7.Following phosphorylation.theytranslocatetothenucleusand inducetranscripti onofTypeIll's
1.3TYPE IIFN SIGNAL/JNe; I'ATlIWA Y
llNs scrvc toestablishan antiviralstatein thehostorganism II)Ilowin g viral infectio n.Followin gtheirsecretionfromvirallyin fectcdcclls.H'Nsactin bothan autocrincand paracrincmannertoup-rcgulatcISGsinbothinfectedandnon-infe cted ncighboring cclls(84).Awellrecognizedsignaling pathway importantforrcl ayo ftypcI lIONactionsistheJanus activated kinase-signaltransduccrundactivationoftra nscription (Jak-ST AT) pathway (2.85-87).Members oft he janusfamilyottyrosinc kinascst.luk) andsignal transduccr andactivator oftranscription(STAT)proreinsarc theprincipal componcnlsof.lak-STAT pathway thatc;I n bc induccdbyscvcralcyrokincsbcsidcslIONs.
Jakl(88) andTyk-2(89-9 1)oft he Jakfamily.andSTAT I (92.(3) andSTAT2 (94-96) ofthcS'I'A'lfamilyp layccntrulrolesintypeIlIONsignaling (20).
Autocrincorp aracrinc llN signalingistriggeredbybin ding oftype IlIO Nsto thcir hctcro-dimcricccllsurfacereceptors comprisedofoneIFNR Iandone IFNR2 subunit.lntraccllulurly.IFNR Iisassociated with Tyk-Zand IFNR2isassociated with Jakl(97-99).such thatlIONbindingstimlilatcstransphosphorylationandsubscqucnt activationo f Tyk-2a nd .lak l. rcspcctivc1y( Figurc IJ)(9 8.1(0).ActivatcdJakland Tyk2phosphorylatc thcirassoci atcd rcccptorson thcir tyrosinc rcsiducsin thc cyloplasmicdomain.l' hospholyrosylrcsiducs acl asadockingsitClorccruitSTAT2 (9().
1( 1) leading to STAT2phosphorylationbyJak land Tyk-Lwhich accclcratc phosphorylationofSTAT I bythese kinascs(2. 102). Oncephosphorylated.STAT I and STAT21IJr111 hctcrodimers, which in turnbind toIRF9andIIJrl11atrimcric complcx termed IFN-stimulalcdgcncIllCtor 3 (ISG F3) (20.1(3).
IFNSti mulatedGenes
\
ISGs ISGF3 \\
ISGF3rr: I
Fignn '1.3 TYIll' IIFNsignalli ngpathway.TypeIIFNsbind andactivateIFNR Iand IFNR2 whichin turn activateTyk2 and JakI,leadingtophosphorylation ofSTAT2 and STATLPhospho rylatedSTAT2:U1dSTIlTI formhctcrodimcrswhichbindstoIRF9mill formsatrimericcomplexnamedISG FJ.ISG FJtranslocatestothe nucleusandactivates transcriptionofIFN-induciblcgenes
12
ISCiF3lra nslocalcstolhcnuclcusand bindslothc lFN-stimulatcd rcsponscclcmcnt (ISRI:) in thcpromotcrofI St.lstoinducetheirexpression (2,104),
1.-1. MUL7'lFUC7'lONAI,ISGSWI7'lII'fII07>1I.UOI,ESINTJ'f'I~'I IFNSI(i N/IU IN(i
IA.I CiBI'2
GBI'2 isone member oftheCiBI'family,which arconeclassoft hetypeIIFN- induced guanosinetriphosphatase.Their naming asGll l' isbasedon thcircapacitytobind ugarosc-immobilizcdguanine nuclcotidcs(105-107).GBI'2isacytoplasmic proteinthat wasfirst identifi edli'om a cD NAlibraryobtainedfromtypelandtype ll ll-N-trcatcd humanIi brohlastcel lsin 1991 (l OX).GBI'2 'shighrateofi nduction(>6 fold)((lilowing ex ogenoustypeIandtypeIIIFNhasledtoitsrccogllition asonc orth cmarkcrsorlFN rcsponsivcncsstlO'i,106,109-1 12).Thereisevidencelorbothantiv ira landantitumor propertiesforGBI'2.The antiv iral activities orGBI'2werefirstidentified byCarter<,Iill.
in2005.Thcy showcdG Bl'2hasanliviralactivityagainstvcsicularstomatitisvirus (VSV)and cncc phalomyoca rd itisvirus(I:MCV)(109).GBI'21llcd iatcditsantiviral activitiesagainst VSV inaG'I'Pusc-dcpcndcntmanner andagainstI:MCVina G'lPasc independe nt lllann cr (109).lnaddition,CiBI'2wasoncorth cup-rcgulatcd gcncsin lungs ofp igsinfectedwith l'orcin c Ci rcovirustypc2(113).Fernandes<,Iill.suggestedthe antiviralucriv uyofGtsl'zagnmst thisvirusismediatedthroughT-cc!lactivation,which impliesanimmunomodulatoryrolcandGBI'2involvementinada ptive immuni ty(1 13).
The antitumor functionsorGB1'2,whichwerediscoveredin thecontextoftypeII IFN
13
signalling.arc alsomediatedthroughdownregulation ofan angiogenic1;\ctor.1\11\11'9.
anddo\\'n rcgulationo raccll migration l;lctor. Rac (114.115).Ovcrall. cv idcnce suggests a broadrangeofactivitieslor GBl'2 .pivotal forantiviralandantitumorresponses inducedbyIFNs.
104.2RIG-I
In 1997.RIG-I \\'asidcnlilicdas oncorthegcncslhatmediatcsall-transretinoic acid-induccddilkre ntiationoracutc promyc locyticlcukcmiace lls(116).ln2004. Yoncya macr o/, recognizcd antiviral propertiesorRI G-I(117).Thcyl i.JulHl that it triggeredproduction oft ypeIIFNsthroughsensingorRNA viruses,lntcrcstinglv, its express ionisalsounderregulati onbyIF-ls:therefore,RIG-Iiscatego rizedas anISG
\\'hichc nablesRIG-lto rurther promotc innatc immuncrcsponscsina positivc fecdhack 100p (117).RIG-I isu kcycomponcntofH'Nproduction.asmany positiveandnegative regulatorsofthe11'1pathwa yexerttheire ffe ctsatthele vel oft hisprot ein(39.ll S. 119 ).
RIG-Iisubiquitously expressedin most celltypesand recognizesRNA viruseswith specificmolecularfeatures.including: 5'-1'1'1'.ssR A. dsR NAforms.Thisis hypothesizedtobea practicalstrategy Iorccllstodiscriminatcscl fRbl/vmol ecul esfrom non-sclrRNAandthu sprcv cntautoimmllncrc actions(42. 54.5 S.ll S),Morethan t\\'cnty RNA viruscsarckno\\'n lohcreccgnizcdbyRIC-I.includingsomethathave severe effectsonhumanhealth. including:hcpatit isC virus(IICY).human immunodcficicncyvirus (IIIY)and influenzavirustlablcl.J)(39.117.120).
14
Table 1.3RNAviruses sensed byRIG-I
Familv Viruses
Rhahdoviridae Rcoviridac,Rabiesvirus Scndai virus,NewCastle diseasevirus, Paramyxo virid ae Respiratorys yncyticalvirus,
Measlesvirus,Ninahvirus Orthotnvxo viridac lnflu cnzaAvirus.InfluenzaIlvirus
Ebola virus,llcpatitisCvirus,Japanese Filoviridae encephalitis virus,Denguevirus,
West Nile virus Arcna viridae LIssavirus BIIII)'I/\' irit/lI l' Rift Valle'feverv irus
Reoviridae Orthorc ovirus
Adaptedfrom reli:renees( 39),(117),(1 20)
15
Since the activation ofinnate immunityisacriticalstep tormountin glongterm adaptive immunity. RIG-Ican beconsideredaninjirectstimulalorora daptive immunity.ln additiontothisindirectinfluence.theinununom odulatoryfunct ionsor RIG-Ion DC (121).'I- cells (56).NKcells(122)andMIIC Iexpression(123).directlyinduce adaptive immunity againstviral lyinfectedcellsand tumor cells.Theanti tumorpotentialor RIG-1 wasIirst identified byZhongSuct1/1.inW07(124).Theyobservedlowerexpressionor thisproteininseveralcancermodels.includingprostate.breast.melanoma andmalignant glioma cancer cell lineswhencoll1pared ton ormalcelllincsofcquivalenttissue.The antitumorfunctionsorRIG-Iarcmediated throughapoptotic(122.125- 127)and anti- proli ferat ive(12X)activities.thusreduced expressionorRIG-llI'ouldpermitapoptosis evasionand excessiveproli ferati on.C0ll11110ncharacteristics0t'tumorcclls.
Overall.RIG-Ihasasignificantimpactoncellularsignalling integration withamajor impacton hosthealth.whichhasencour agedbothvirologistsandcancer researchersto focusonthisproteinforthe developmentofintcrvcntionaltherapc uticapproachcstIv.
118.119.127).
104.3STAn
STAT2wasfirstidentifiedbyFu"II/ I..whiletheywere exploring thepolypeptide compositionorthelSG F3complex inl9l)Othat was alreadyrecognizedas a key componentof the IFNresponse(129).Several lossor runctionstudiesdemonstratedt he potenti alor ST AT2asanessentialcompcncntofty peIIFNsignalling. Leungi'll/I.round thatccllslackingS'lA'lZhavcdclcctivctypcIIFNrcsponsc(lJ5).Intll'o othcr
16
independentstudies, itwasfoundthal SIII/2knockout mice arc morevulnerabletomany typc sof'viruscstlJfl,13l).Thcrc arct wothcmcsol"typcIIFNsignallingpathway.in bothol"whichSTAT2 isa keytranscriptionfactor:I) the classic pathway.described at section1.3. includesSTAT2.STAT Iandlkl'):2) the non-classicpathway includes STAT2 andothertranscriptionfactorsrathe rthan STATIandIRF9.Intcrmsofthe classicsignalingpalhway.thcrc arctwok atllrcs alrcadydcli ncd:ljrcquircd lcvclof STAT2 phosphorylationorthresholdthat isrequired forphosphorylation ol"STATIand subsequentform ationofactiveISG F3(102);2)requirementol"STAT2forintcracting withadditional transcriptionbc torsandrcgulatorsol"typcI IFNsignalling( 132-134).
There arcsometypeI IFN-induccdnon-classic.lak-STATsignallingpathways.lorwhich STAT2 isapivotaltranscriptionfactor:I)STATZmed iates signallingina STATI- independent manncrthroughanactiveSTAT2:IRF9complex(135-137); 2) STAT2 medi atessignallinginanIRF9- indcpclltk nt manncr througha STAT2:STATIcomplex (138);3)STAT2mediates signallingin a STAT I/IRF9 independentmanncr (STAT2:STAT3complex ,STAT2:STATScom p lex)(110.139). Allaboveexamples suggcstthedependencyoftypeIIFNsignallingfunction toSTAT2rather than to STAT I andIRF9.
LikeRIG-I.Vanet al.(1995)foundthatSTAT2 isalsoanISG. andthereby amplifi estypeIIFNrcsponscs(9I).STAT2 isc sscntial forndaptivc immunityagainst viralandtumo rcellsofm acrophage(31).DC(140).and Thcclls(14I).T hc lirsl obscrvationol"antitumoractivity ol"STAT2comcstromastudyin2003.whichshowed S/a/2knockout mice developed brain tumors(142).Inaddition. loss ol"STAT2
17
accompanied tumorprogression character isticincolorcctal.renal,andmelanomacancer cells(143.144).STAT2 also plays critical rolesforinducing apoptosisof tumor cells (1-1-1-1-16).inhibition ofc ellgrowth (140.147) anddown-regulation ofanangiogenic fllctorivlMI'9(14li).Ol'crall.byitsdctcrminantro lc in typcIII' signallingand modulatingvitalbiological process. STAT2 canbeconsideredoneofthemostprominent ISGs.important lorlidclityo ftypcIII' signalling andhculth.
/.5./FNT//I:'RA/')',INDtts/Jill/TAT/ ONS
1.5.IIFNthcrapy
Basedontheirpivotalphysiological roles.JlNs.especiallytype Ill' s.have achieveda notableroleinclinicalmed icinefollowingthcir rccombinautproductionin 19liO(I.2).Thcir lirstcntry inc linicsoccurrcdin 19li51lJr trcatmcntofh airy ccil Icukcmia(lICL ). 2li yearsafter theirdiscovery(1-19).Sincethentheyhave been approvcdas a trcatmcnt fl)fv iralinfcctions.c anccr.and multiplcsclerosis(i'vIS)as an autoimmuncdiscasc(20.149 ).
Ofviral discascs.f ypeIII' swcrc approvcd fortrcutmcntofllljVtl Ju).
IICY(151).herpessimplexvirus(HSY)(152).lilY (153.1 54).herpeszoster(155).and commoncoldviruscs(156.157). but nowthey arc appliedjustIorrrcuuucutofllllvand IICY.IIBY isaseriousintcrnational publichealthproblem,withupproximatclv400 miliionpco plc infcctcdlhroughout thc \\'crld(15li).This inlCclionc auscsscvcrc symptomsincluding livercirrhosisand hcpatoccllulurcurcinoma, therebyleadingto
lli
chronic and deadly d iseaseo utcollles ( 159- 16 1).IFN-u was introducedas the fi rst interventi on for IIBYin 1991 (162).lnlhepasttwodecades.IFN-uha sbeen recognized asaneffective treatment.asitcausesareductionin disease severityin about 25-40'X,o f patients(163.1(4 ).IICY currentlyinfects175lllilli onpeopleworld-wid e and inducesthe samesym ptomsandoutco m esasIIBY(165).Thetreatmentofchronic IICYwithIFNs began withsoleapplicat ion of IFN-u. towhich20%of patientsrcsponded ( 166).Co- ndminisuutio notllN vuj with lowdose (lflh e antiviraldrugribavirinl edto asynergistic encc la ndi ncrease d response rates to4 0%of patients( 167 ).Current f(lI'Illo f treatlllents includesapplicationof long-acting.pegylated IFN-u2withribavirinwhichhasledto improvelllents-over60% of patients arecured(168). l'egylated IFN-u2 consistso f polyethyleneglycol addedtotheIFN-u2. a chelllicallll odilicationwhi ch increases IFN-u2halllife and duration of activit y(169).
Type I IFNsare an illlportant treallllento ption l()r MS.w hich isana utoimmune disease.Inthisdisease.neuraltransmissionin the centralnervo ussystem(CNS)becollles aberrantasaresult of demyelination ofCNS.Thisisa consequenceofauto i mm une reactionsagainstmyelin(170- 172).Mxputicnts xuffcr frommusclcwcukncssmusclc spasmsor d if'ficultyin llloving.d if'ficuiticswithcoordinationand balanccandvisua l problems(173.174 ).IF N-[l isthelllainilllmunolllodulalorymedicationl()rthisdisease.
and itsclinica l benefitsareobservedin50-75'Yopatients(175).It redu cesCNS inflammation. and thereb yreduc essymptomsofp hysicul disability(176).Thelllolecular mechanismsthroughwhichIFN-[lexerlsilSciinical benefil sarenolweliund erslood.ll is
19
hypothesisedthatt hoseelkc tsarc mcdiatedt hroughi mmunomodulatorytilllction ofIFN-[1( 176).
Even though typeI IFNsarcbestrecognizedtor theirunique antiviralproperties, theyfirstentered intothe clinicasanantitumortherapeuticagent lo rIICL in 19R5(177).
Furtherinvestigationsrevealed their antitumoractivityagainst somc othcrtypcsof cancer,which ultimalelylcdto lheir applicationl'ortrealmento I'chronic myeloid leukemia (CML),13-andT-cellLymphomas,melanoma,renalcellcarcinoma(RCL) and Kaposi'ssarcoma (149).Although fixsome cancerssuch asCML and lfCL .typeIIFNs arc delegating their placetosomenew anticancerdrugs,theyarcstillwidelyused1'01' treatment ofso me malignancies suchasmelanoma(34).Since 1995,IFN-uhasbeen used asan adjuvanttherapyforpatientswith high risk melanoma (17R,179).It benefitsthese palientswithincreaseddisease-li'eesurvival llx about9months,and overallsurvivalby R-9%(IRO,IRI).
1.5.2l.imitationsofI lN therapy
Typel llNshavcshown promiseforthetrcauncntforsomeli fethreaten ingand deb ilitatingdiseases;however,theiroverall therapeutic effieacyisonlymoderate (I.2).
Oncobstaclc tothcsucccss ofllN therapyisthe phenomenonofpaticntresistance toIFN treatment.Given thepotential ol'lFNtherapy.understandingthc obstaclcs to succcss of IFN therapymight improvetherapeuticoutcomes,Threefacto rsarc associatedwith IFN resistance:I)genetic alterationsofc omponentsinvolved inlFNproductionor IFN signallingpath ways(IR2- IR4);2)anti-IFNproteinsencoded byviralgcnomcs (IR5.
20
186):and3) elevatedactivitiesorendogcnouscellularsuppressorsorlFNsignalling (187-190).
MutationsorpolymorphismsorlFN signalingc omponentscanchangclFN susccptibility li'om responsivetonon-responsivephenotype. Forcxumplc. xlcfcctivc STATIexpressioncorrelateswith IFN-(lresistance inCML andacutaneousT-ccll lymphoma ccli linc(183.184).Inaddition. Shang i'11I1.lcllll1dthata bolishedJa kl.Tyk2 and Stat lcxpressionlca ds toimpairmentor Jak-STATsignalling.whichunderlieslFN rcsistanccin RC Lceli lines(182).Recognitionofimpactsor mutations ofdilfcrcnttypeI IFN signalling willopenanavenuefordevelopingmarkersforfuturepersonalized therapydecisions.
Viruseshavedevelopedstrategiestoshutdown lFNsignallinginorder toevade innateimmune responsesandsupportviralpropagation.Virusesmaycounteract IFN-mediated antiviraldefencesineitheradirector indirectmanner(185.187).Directly.
virusesencode proteinswhichdisrupttheIFNsignalling pathwaythroughtarget ingits components. Forinstance.Dengue virusencodesaproteinwhichbindst oSTAT21eading toitsubiquitinationandsubsequentdcgradutiontl vl).Anotherexamp leis suppress ionor RIG-IsignallingbyIICV;thisvirus encodes aprotease whichcleavesIPS-I(rom mitochondria(192).Indirectly.virusesexploit cellularsuppressorsofllNsignallingto attenuateIFNresponse (193.1(4).Viruscslacking anti- H'N proteinspropagate selectivelyincellswithelevatedactivityofthesecellular inhibitors.thercby causingmore severediseasesinhumans andanimals(I'J5-197).Theup-rcgulat io nofcndogcnous supprcss orsofllNsignallingwasalso observedin cancercellsresistant toIFN(189.
21
190).Thisevide ncesuggested these areseriousbarrierstolIO Ntherapyefficacyandthe suppressormechanismshavebeenthelo cus01"recent research.
1.6.CEUULARSUPI'R/:'SSORSOFIFNSIGNAUJNCi
Theprima ryfunct ionofendogenouscellularsuppressorsol"lFNsignallingisto maintainthchomc ostasisofI l-Nsignal transduction (187). asprolongedorconstitutive acti vationofIlNsleadstodestructivebiologicalconscquenccssuchasustlnua.futiguc.
and depression(198.1(9 ).Thr eem ajorl:ulliliesol"lypeIlIONsignallingpathway inhibilorsinc lude:S rc-homology2(SII2;'-containingphosphatases(SI II').lhe supprcssors ofc ytokincsignalling(SOCS)family and proteininhibitoro fa ct ivatcd S'lA'!
(I'IAS) (Figure 1.4) (187).Activation01"l~as/Rali'M EKpathw ayalsowasrecognizedas inhibitorofl l-Nsignallinginour laboralory(I 97 ).
PTP
PTP
PTP
Transcription
Figure 1.4Suppressors of IFNsignalling.The
sacs
fami ly memb ers suppressJak kinasesactivity,oract as a competi to rofIFN inbindi ngto recepto rs.PTPfamily membersde-phospho rylateJaksandSTATI to induceubiqu itin-media ted deg radation of Jaks.PIASfam ilymemb erspreventtranscription al activ ityof STA T dimer s.23
Phosphorylation is anintegralfeat ureoft he activated Jak-STATsignalling cascade.suchthat de-phosphorylating eventsoff erthecellasignificautlcvclofcontrol.
SIIPIandSI1P2are tyrosinephosphatascsinvolvedin thenegative regulationofty peI lIONsignalling(200).SIIPIattenuatestypeIlION signalingthrough de-phosphorylationor JaKI (201.202).whileSIIP2inducesthesamesuppressionlhroughde-phosphorylation or STATI(202.203).Sodiumstibogluconatcisaninhibitorof'bothSll!'!andS IIP2and itscombination with IFN-ushowedsignificantsynergisticcllccton IFN-uresponsiveness inbothillvitroandill I"i\"()experiments(:~04 .2(5).Thiscombinationentered intophaseI trialsforc valu utingsafetyand target inhibitionfortreatment ormelanoma (206).
Thesacsfamilyiscomprisedofeightmembe rs.including:cyto k incinducible SI12containing protein andsacs1-7(207).Generally.their inhibitoryeff ectsare mediatedviathreemechanisms:asantagonistsforSTATbindingtophosphorylation target sitesonreceptors.inducers ofprotcosomaldegradationofboundsignallingproteins andsuppressorsolJ a kactivity througha.i indinginteraction(20S).AmongtheSOCS family,sacsIand SaCS3are involvedinthenegative regulationoft ypeI IF signalling(209).Up-rcgularionofS tX' Sdisoneoft hestrategies usedbylntlucnzaA virusandIIiVtosuppresstypeI IFsignalling (210.21I).Thesamemechanism isused byIISV.asitinducessacsIorsaCS3torevadingllN vinduccdimmunity (193. 212).
Inaddition.over expressionorS aC S3wasobservedinmyelogenousleukemiacells resistant toIFN-u(IS9).ThelevelsofbothsacsIandSOCS3activityaredeterminant l:tctorsorIF N-susceptibilityin melanoma(213).Furthermore.thelevelofS OCSIand
24
SOCS3 activityinfluence the interferon r,:sponsiveness orn eu roendocrine tumorcells.
andRCLcelis. res pectively(190.214).
TheI'IAS familyincludesI'IASI.I'IAS2.I'IASxand I'IASy(215).I'II\ SI and I'IASy areinvolvedin negat iveregulationor typeI lIONsignalling (216).BothI'IASIand I'IASybind toSTATIanddisrupt its[)'Abind ingcapability (217.21S). IIwasobserved that overexpressionorl'lASy in mouseembryonicfibroblastsil/l'itro diminished the antiviralactiv ity orlFN-uand lION-IIaga inst VSV.EMCCiV.andscndaivirus (219 ).
1.6.1Ras signaling
1.6.1.1TheBiologyofRas
Theratsarcoma virus oncogene (Ras)superfamily comprisesproteinsthatsharea proteinstructure thatbindstoGTI'andpossessesG'l'Pasc activi ty(220).They switch between two states.anactive(GTI'boun).andinactive (GDI'bound )form(221).This transition iscontrolled byintrinsicGTl'aseactivityorRas.guaninenucleotideexchange facto r(GEF)sandG'IPascactivatingprotein(GAI')s(222.223) .Rasprote inshave low intrinsicG'lPascactivity.such thatGEl's andGAl'sactinasupplementarymanner to increasethe rateoI'exchangeand hydrolysis.respectively.CiI:Fsfac ilitateRasactivation bypromotingthe release or GDI'and exchangeforactivatingGTI'.whileGAl'sIacili tatc RASdeactivationbypromoting GTI'hydrolysistoGDI'(222.223). Thebalancebetween activation anddeactivationisveryim port.uuforn onna ltuncrion sof'Ras (224-226 ).
Whenactivatedint heGT I'-boundcon format ion,Rasinteractswithman yeffectorsto
25
control numerousbiologicalresponses.includin g:prolifcratiou.ditfcrcmiarioucc!l adhesion. apoptosis, actin cytoskclctulimcgrityand ccll migration(220 ).Characterized Raseffectorsarephosphatidylinositol 3-kinase.RaIguanine nucleotideexchangefactors and the Serine/Threonineprote inkinasel{ar(227.22S).
1.6.1.2Ras/Rati'MEKpathway
The Ras/Raf/Mli},path way includesa cascadeorphosphorylationevents performedbymembersor threekeykinase (amilies:serine/threonineproteinkinaseRal:
mitogen-activa tedprotein/cxtrace llularsigna l-regulatedkinase (MI:K). andExtracellular Signal-Reg ulatedKinase(ERK).Thispathwaycan beactivatedin two ways. oneis regulatedor physiologicalactivationandtheothertypeisconstitutive oroncogenic activation(220).
Physiologica lactivationor the Raspathways occu rsupon activationofr eceptor tyrosinekinascs (RT K) by growth factorsand mitogens (Figure 1.5) (225.226).
Appropriate ligandbindingisrequired to activate theRT K.which stimulmcsthe activationofRasbyinducing the GTI'-bcundstate (226).Rafkinascisrecruitedtothe plasmamembrane byactivated Ras.whichleadstophosphorylationandactivationol'Raf (229).Phosphorylation cascades ensue.whcrcphosphoryl utcdRafphosphorylatcsMEl'. whichphospho rylatcsandactivatesERK(230-232).Acti vatedERKhasa widevariety or targetsin the cytoplasmand nucleusinclud ingkinascs.phosphatascs.transcriptionfactors andcytos kclctul protc ins.
26
MEK
.~
EHK
.~
Proliferat ion Growth Diff erent iat ion Migration
Figure 1.5 Overview of physiologicalaetivation of the Ras/RaflMEKpathway.
Followi ngmitogenbinding,a phos phory latio ncascadestarts.Thisultimatelyleads to stimulationofproli feration,gro wth,diffe rent iation or mig ration.
27
Thefinaloutcomeor physiologicalactivr.tcd Ras/Ral/MlKpathwayisinduction or prolife ratio n.growth.diff erentiationandmigration(233).
Constitutiveactivationoft he Ras/Raf/Ml.Kpathw ayleadstotumordevelopment . andaberrantactivityofthispathwayisalmostroundinone thirdofa ll human cancers (224).Thistypeofac tivation isaconsequenceor activatingmutationsintheearly componentsof thispathwayincludingRT Ks. Ras,or Ral:or amplificationand dcrcgulation or itsdownstrcamcflcc tors(220).Thcoutcomc is signallingpathway activationthat is autonomousfromthepresenceorligand. andoveral llossofrcgulutory control. The mostabundant mutationsinvolved in constitutivcactiva tionofRas arc mutationsatami noacidrcsiduc 12.whiclJrcprcsscsGAI'-mcdiatcd inactivation of'Ras (234.235).and mutationsinres idues13and61whichresultinrcpressi onott hcintrinsic G'lPaseactivit y orRas (234.235).Forpathway componentsdownstreamorRas.B-Ra r mutationshavebeenfoundin manytypesor malignancicsincIuding malignant melanomas,coloncancer, papillarythyroid cancers andserousovariancancer(173).
Overa ll, mutationsresponsiblefor over-activityofRassignalling pathwa yc ompone nts havebeenfoundin20-25%or allhumantumorsandup to 90%inspccitictumortypcs such as colorcctalcanccr (226):thcrcli.)rc.thcsc protcinshavcbccn thc li.lcuso r che mo the ra pe uticdrugslor cancer(236).Oneoft hecommon Ras/RaI/MI:Kpathway inhibitors arc MEKinhibitors.becauseMEK isan importantregulationstcplorthe pathway(237).Thcir potcntialthcrapcuti:bcnclitshavc Icd todevelopingdiff erenttypes or MI:Kinhibitors with dilk rcntstrUclur.ll andchcmicalpropcrtics. ina way thatthcy targetdifferentMI:Kswithdiffere ntefficiencies(238).Twowidelyused experimental
28
inhibitorsor MEKI and MEK2 arcUO126 andSL327.UO126isused broadlyforillvitru studies andSL3271i.Jrill vivostudies(239-24 1).The obstac les suchasnotbeing sunicientlysoluble ors unicientlybioav,tilable and inability o rpassingblood-brain barriersrestrictedtheillI'i\'llapplicationorUO l26(236).resultingin broad applicationor SL327illvivo,especiallylorbehavioralstud ies(240-243).Schc rlc1'1al.applied both UOl26 andSL327in their invivostudies.andide ntifi edsupcriorityofSl.Ll ?over UOl26 lort he inhibitionofMl. K (244 ).
1.6.1.3Manipulationofnor mal Ras/Ral/Ml -Kpathwayactivity during viral infections Manyvirusesevade the hostdefe nsesystem byactivatingtheRas/Raf/MlK pathway.which ismediatedthroughdirect bindingor thevirustoitsreceptor or interactionorviral proteinswithc ellularsignalling components.Forexa mple. lhe coxsackie virusinducesabiphasic activationortheRas/Rali'MEKpathwayto enhanceits proteinsynthesisand progenyproduction (244.245). UOl26treauncntofthc infcctcd cel lssignificantly decreased viralproduc.ionand protcinsynthcsis(244.245).In theearly phascofborna virusinfecti on.Ras/Rali'i\lEKpathwayactivation facilitatesviral propagation.butUOl26treatmentreducesthespreadoft hisvirusup to 99'Yo(23X).Rap id transientactivationoft heRas/RaIi'MEKpathwayalsooccursuponrespiratory syncytial virus(RSY) infection.Earlyactivationolt hispathwayplaysan important rolein RSY- inducedearlygene expression.since treatmentwith MEKinhibitorl'D9 X059resuiledina sig ni fi cantreduct ionin theRSYinlection(246).l-urthcrm orcvisnavirusinfection resulted inactivationor Ras/Rali'MEK pathwayin the earlyphase ofi nfection,and this
29
activation rcmaincd during all phascsorinlection.Sincc l' D98059 treatment resultedina signilicant reductionofrc plic.uion.itcould bc concludcdthat activationorRas/Ral/Ml.K pathwayisncccssarylC:Jrvisnavi rusrcplication (247).!\swcll.activationo r Ras/Ratil'v1EKpathwayhasbeenobserved formany other virusessuchasIIIV-1.
cyromcgalovln ras imianvirus.intlucnza virus.IIBV. IICV.andvacina virus(245. 246).
Collcctivcly.thcscrcports dcmonstratc thats cvcral viruscs usctransientactivationor Ras/Rat/l'v1E K pathwa yasa tactic toevade innatcimmuneresponse s.
1.6.1.4 Exploitingorconstitutive Ras/Raf/MlKpathwaylorpromoting oncolyticviruses infec tion
l'v1anyoncolyticvirusespreferablyreplicatein canccr ccllsharboring constitutivc activationorRas/Ratil'v1EKpathwuytocvadc innateimmuneresponsesandspreadthei r progeny.l.xamplcsoft hosc oncolytic virusesarc:reovirus(248).adenovirus(249).
polivirus(250).IISV (251).VSV (252)..md influenzavirus (de INS Istrain) (224).
Constitutive Ras/Ratil'v1EKpathwayfacilitatesviraloncolysisgenerallythrough promotingthreev iralsteps.including:uucoatingorvirus(253. 254).lranslationorviral proteins(242)and relea seofne w progeny(254. 255).
1.6.1.5Antagonisticcrosstalkor the Ras/Ratil'v1EKpathwayandInterferonpathways Ampleevidenceshowsthatmanipulation of Ras/Ralil'v1l:Ksignalling.either in phy siol ogicalorinconstitutivestates. isauniversal tacticbywhichdifferentlyr esof
30
viruscssupport thciro wn propagation.Our laboratory prc viously rcportcd thatt hc Ras/Rali'MEKpathwayisacellularsuppressoroft he typeI IFN- induccd antiviral rcsponsc (197).lnthis stud y.it was shown thatIFNsensi ti vevirusescanin fectNII13T3 cellswithactivated Ras(Rasv 12)in thepresenceor IFN-u.whereas contro lce llswith norma l Rasactivity were protectedfrominfectionbyIFN-u.MEKinhibitionby chemical inhibitorsand knockd ownofR usbyRN,\interference(RNAi )resto redIFNsensitivity in thcRasactivatcdcel!s. Asexternal validat ion.Noserr/a!.1(IUndthat IFN-induccd antivira lresponseswererestored in human cancercell linesfollowin gRas/Ruf/Ml.K pathway inhibition (256).The evidenceputs forwardtwokeypoints:I)that viruses benefit from activated Ras signallingtor.void IFN-stimul atcdiuunuucsupprcssioun nd 2)thatreversa lofRasacti vation couldscnsitizccciistothcIFNresponse, Ex perim entaleffo rtstoidentifyIFNsignallingcom po nentstargetedbythc Ras/Ral i'MEK pathway.revealed reducedtota lSTAT2protcincx prcssiona nd rcduccd phosphorylat ion or STAT2 andSTAT I in RasVI2cells(:~57).UOl26restoredtheIFNresponse comp lete lyb utre-cx prcssio nofS'lA'I?in RasVI2 ccll srcsultcdin part ialrecoveryof IFN-induccd antivira l rcsponscs(257).I\lthough STAT2 isrccognizcd asa Ras /Rafi'MEKpath waystarget.evide ncesuch aseffec tive antiviralresponsesbyIFN treatm ent foll ow edbyMEKinhibition.andpartialrecov eryorIFNresponsesfol low i ng over expressionorSTA T2suggested thatRas/Rali'MEKpathw aytargctsmultiple com poncntsofllNsignalling. notjustSTAT2.T hcrc l(lrc.furtherexperimentswere pCrl(lI'Illcd to dctcrmincothcr c!cmcntsorthc IFNsignallingtargetedbythe Ras/Raf/Ml.K pathway.Globalgeneexpressionprofilin gbymicroarrayana lysiswas
31
conducted toident ifythell-Nvinduciblcgenestargetedbythe Ras/Rali'MEKpathway (Christian. unpublished). NII13T3cells transformedwithconstitutivelyactiveRas(Ras V12) were treated with DMSO(control)IFN-u or UO126.RNAwas extracted6hrspOSI treatme ntandsubjected tomicroarra yan al ysis.Of41960geneisofo rmsrepresented on thearray.1883were up-regul atedin the RasVI2 celistreatedwithUOl26comparedto contro l,witha threshold criteriaset loa>2.5foldincrease.while IFNtrc.umcntalone induced 1877 genes.Ofthese candidategenes.619 werefound10be up-regulatedbyboth UOf26 and IFNtreatm ent and the group wasnamedMEK-downregulatedIFN-indu cib le genes (MDIIgenes).Someoft hegeneswere knowntohave antiv iralfuncti ons.suchas Cih/)2.Rig-I ,andS11I12.Accordin gtomicroarray analysis data.the UOl261reatmenl resulted in 9.381()ld increase1()r Cih/)2. 2.55f()rR (l!,-I.and6.41 f()r SI1I1 2 crab le1.4).To confi nnmicroarrayanalysis.the same RNAsamples weresubjected toquantit ative- polymerasechainreaction(q- PCR) I(JrGbp], Rig-I,andS11I12.Theresultsfromq-PCR analysis showed that MEK inhibitioninducedtheexpressionof thesetested genes significantly andco-treatmentofUO I26/IFN-u inducedsynergisticeff ects(Tab le1.4) (Christian:unpublished).
Furtherex perimentswereco nducled toi nvestigate lhei nfluenceo f the Ras/Rafi'ME K pathway on IFNresponsivenessin human cance rcell lines. asmany
32
TableI..tMDIIgenesexpression inRa~;vI 2cells follow ingIF treatment.i\IEK inhibitionor thecombined treatment invitro
GE NE UU126 IFN UUI26/IFN
MieruarruvAna lvsis (R asV I 2Cells)
GhJ] 9.83 97.52 Ni\
Ril!,-/ 2.55 25 A 6 Ni\
su»:
6.41 16 .77 Ni\q-I'CRAn al vsis (Ra S VI 2Cells)
Gh/J] 5.81 78.16 218.51
Rig-/ 2.78 20.10 24.64
SIal] 3.74 10.61 24 .25
onco lytic virusesexploitpathologicalactivity of thispathwayasa mean toreplicatein cancercells(Christian ct.al:unpublishec ).Sixtccncclllincsofdi lfcrcnt origin (3breast, Icervica l.4colon.I fibrosarcoma. 2melanoma.3 ovarian and2prostatecell lines)were testedforthe irsensitivitytoIFN-Cltreatment.Threeof themwereIFNsensitive.90f thcm modcratclyrcsistant.und-lof them werecompletelyres istant.Treatment ofUOl26 improvedthe IFNresponsivenessinIOoutofl3moderalelyand compiete ly resistant ceil lines.Furtherstepsweretakentoexplorethe mechanismswhich undcrlie Ras/Raf/Ml.K pathwaymedia ted IFNresistancein thcsc cxamincdcunccrccll lines.Globa lgene expressionprofi l ingbymicroarray analysiswasperformed onone IFNsensiti ve and one moderately IFN resistantcell linetreated with DMSO (contro l).IFN-l( orUO126.RNA wasextracted6 hrsand l2hrsposttrealrnenta ndsubjectedtomi croarraya nalysis.
UOl26treatmenlincrea sedexpressionofa groupoflSGs such asGh112. Rig-l.and S/aI2.
and theseresultswereconfirmed byq-Pt.Ranalysis.
Followin gtheobservations thatthe cellular activity of theR/\S/Ra ll ·IEK pathwa yimposeda negativeinfluenceon ce llularsusceptibilitytoIF-induccdantiviral and antitumoreffect sviaa globaldownregulation ofISGsillvitro,the nextstep wasto assessthephysiologicalapplicati on of theselindingsinananimalmodel.Toour knowlcdgc.jhisisthcli rstsllldyt estingl he e rticaeyof MEK inhibitiontopromote IFN's transeriptionactivi lyilll'il'll.llisthe ullimategoalo f thei nveSligatio ntoexp lore intcrvc ntio nalstratcgicsthatimprovcthccfficucyofllNto cure diseasessuchasviral infccrion.Mxandcanccr.
34
I.7.IIYI'0 7'l1l~SISAND OB.fEC7JVES
1.7.l l lypothcsis
MEKinhibitionwill independentlypromotetheexpressionof MD11genes,and theco-administrationof lFN andaMEKinhibitorwill havesynergistic effectonMDII gcnc cxprcss ion undcr physiologicalco nditions illl'il'O.
1.7.2 0bjc ctivcs
I.Invcstigatcwhcthcrtrcatmcnt'.VithaMEKinhibitorpromotes expressionof rcprcscntativcM Dllgcncs(Ghp2.Rig-l.'11(112)in a mousemodel.
2.lnvcstigatcwhcthcrco -trcatmcl1\with a MEKinhibitor and IFN-uin duccs a synergisticeffectin theinduction of MDIIgenes(Cihp2.Rig-I,SIilI2)inamousemodel.
35
Chapter 2: Materia landMethods
2.1 MICE
Nine weekold femalemice of theHalb/cinbredstrain(CharlesRiver l.abs, BostonMA) wereused lix in vivoex periments.Twenty mieewered ivided intofour groups offivemiee receivingthelilllowil1gtreatments intraperitoneally(IP): control mice received80ulof Dimethylsulfoxide (Dt'vISO)(Sigma.St.LouisMO)plus1:20rLi PhosphateBuffered Safine( PBS)(80gsodiumchloride.:2gpotassiumchloride.11.59g disodiumhydrogen phosphate.:2g/l monIJpotassium dihydroge np hosphate(Sigma.Sl.
LouisMO));onegroup received100mg/kgBWorSU27(AscentScientific.Cambridge MA)dissolvedi n80rt!o f DMSO andthendilutedatl 20pi:anothergroupreceived 1000UIFN-u (PestkaBiomed icalLaboratoriesInterferon Sourcc. PiscatawayNl) dissolvedin 100u l PBS.andonegroupwas co-inj ected withSU27 (100 mg/kg) and 1000UIFN-u in theirrespective vehicles.Themiceweresacrificed8hrsafterinjection.
The brain.intestine and lungswere harvested to obtain proteinsamplesforwesternblot analysisand RNAsamples fixsemi-quantitativePCk-bascdgeneexpressionanalysis.
2.2 1V1:'STERNIJUJTANALl'S IS
:2.:2.1Preparationofproteinsamples
l'issucswere cut into about50mg piecesand homogen izedin I mlof radioimmuno precipitation assay(RIPA)lysisbuffcrt lXPBS pI17.4.I'X,n onidet1'-40 (Sigma. St.LouisMO).0.5%sodium deoxycholate(Sigma.St.LouisMO).and 0.2";(,
36
sodiumdodccyl sulphate (SDS) (USB.Cleveland 011)containing protease and phosphataseinhibitors(IOOng/mlphenylmethyl sulfonylll ouride(Sigma-Aldrich.
St.LouisMO).2flg/mlleupcptin(Sigmr.,St. LouisMO).30ug/mlaprotinin(Sigma.St.
LouisMO).100 mMsodium vanadate (Sigma.St. LouisMO).and500 mMsodium lluoride( Sigma.St.LouisMO)).Thelysatewascentrifugedat 12000 xgl(lrltl min at-l
°c. Supernatantsconlaining cellular prott:inswerethenseparated li'om debris and kept al -40°Cuntil the time of assay.
2.2.2Quantificationof protein
The amount of protein wasmeasuredwithbicinchon inic acid(BCA) Protein assay kit (Pierce BiotechnologyInc..Rockford.IL). BCAreagent(50:I.reagentA:reagentB) was added t05 0 ftlof proteinsamplesorstandardsprepared lrom bovincserumalbumin (BSA)(0.1.0.1.5.2.0 and2.5fig).and incubatedf(Jr30minat 37°C.Alterincubation.
absorbanceof proteinsampleswasmeasuredwith aspcctrophotomctc rtttcckmanCoulter .Hrca,CA) at562nm and the protein concentrationof experiment alsam p lcsdctcnnin cd fromthestandardcurvcttlcckmanlnstrumcntsInc.Fullerton.CAl.Atlcrstand ard izing the proteinsamples.theywerethenmixedwith3xbromophenolblue(Sigma.St.Louis MO).boileda t 100°Cfllr 5minandstored at-4 5°Cuntil theimmun oblottingprocedure.
2.2.3Electrophoresisand transIeI'of proteins
SDS-polyacrylamidegel electrophoresis(SDS-PAGE)wasusedtors eparati onof proteinsam pleson a 10% separating gellnadel rolll: 3.44SIllII120.I.S761ll11.5MTris
37
(hydroxymethyl)a minomethane-hydrochloride (Tris-IICI). pI18.8. 2.025 mI30%
acrylamidc(29(Acrylamidc) :1 (Bisacrylamidcj ), 3pi tctramcthylcthylcncd iaminc el'l:MED)(Bio Ri\D .HerculesCi\).75 pi 10%ammonium pcrsulfate(i\I'S) (Pharmucia Biotech.Baic-D' UrfcQC).The proteinwasinitiallyloaded ontoa4.5'Yostackinggel:
1.720mldll"O.760 pI 0.5 MT ris-llclpI16.8.500 pI30%aerylamide. 30 fLi!O'X, SDS.
30fLi 10%i\I'S. and3ul TEMED.Tcnul/wcllolcac hproteinsamplesorpre-stained proteinladder (Benchmarkcompany.NewYorkNY). ranged lroml0to220 kDa.was loaded and thenrun in mini-protein celleleetrophoresisehambers( Bio-Rad. lle rcules Ci\ )withgelrunningbuffer(500.2mM trisbase. 38.3mMglycine(Sigma.St.Louis MO).and7 0 mMS DS)a lI00 V Ilx 2hr;.Foliowing electrophoresis.theproteinsamples weretransferredfromgelstonitrocellulosemembranes(13io-Ri\D .Ilcrcul csCi\)in transferbuffer(20%methanol(i\C I'.MontrealQC). 39.82 mM glycine.47.9mM tris base.1.28mM SDS)l lxI haliOOV.
2.2.4lmmunoblotting
Membraneswereblockedwith5% skim milk in iris-buffe red saline withtween (20m M trisand 137mM sodium chlorideI'l l7.3containing O.I'X,tween 20er BST) (Sigma.St.LouisMO)forIh.and thenincubatedwithprimary antibodiesincluding:
anti-phospho-E RK(p-ERK)(I:I0(0) (Cell SignallingTechnology.DanvcrsMi\: Catalog number:910I)andanti-total-ERK(l-ERK)(I:3( 00)(SantaCruz Biotech. SantaCruz Ci\:Catalognumber:SC(4)overnightat4°C.Thenext daythey were washed3times lor5min wilh TB ST.and incubatedwithgoatanti-rabbitIgCj-horseradishperoxidase (I:50( 0) (SantaCruz Biotech. SantaCruzCi\:Catalognumber:K04( 8)whichwasused
38
asthcs cconuary antibouy.Foliowing incubationwithsccondaryantibody. mcmbrancs were washed 3timesfor5min withTBST.Bandswere visualized onFuji medicalX-ray fi lm(Fujili lm,TokyoJapan )usingAFI'imagingdeveloper (AF!'Imaging Corp.
ElmsfordNY) followingincubation ofthemembraneswithmillipore chcmilumincsccnt detectionkit(Millipore.Billerica MA).The band imageswerescannedusingancpson perfec t io n3170photoscanner(Epson,TokyoJapan)togetihcimagcin digitallormatIor subsequentdensitometryanalysis.
2.3.IU -PCR
2.3.1RNAExtraction
RNAwasextractedwithTRlzol(Invitrogen.BurlingtonON)accordingtothe manuf acturcrsinstruction.RNAextracti onconsistsor fiveprocedures:hom ogenization.
separation.precipitation.RA wash.andre-dissolvingtheRA. At thehomogenization stc p.fissue sam pleswith about50 mgweightwerehomogenizedin l mlofTl clzo! applyingu glass-Tc flo nhomogenizer.At theseparation phasehomogenized samples were incubatcu lor5 min at roomtemperature,andthen 0.2mlofchloroform(Sigma. Si.L ou is i
\IO) \\'as auucu tosamplcs. Tubcs \\'crcs hakcn vigorouslybyhandtor 15 scc and incubatcd atroomtcmpcraturcliJr3min.Ncxl.samplcswcrc ccntrifllgcu at l2000:\gfor 15m inat4°C.Aficrccntrifilgc. thcaq ucousphasc.which contained RNA was separated and placed in ncwtubcs. undrhcorganic phase waskept torfutu reDNAand protein extraction.ToprecipitateRNA fromuqucouspbusc. Doml isopropanol(Sigma.St.Louis MO)\\'a s auucutocach tubc. anu thcnt hcmi.'(turc \\'asincubatcua troo mtemperature lor
39
10 min.Afterincubation.samples were centrifugedat 12000xgI(Jr10min at 4°C.;\(the washinglevel,supernatant wasremoved.andIml ethanoI7S%(Sigma.St.LouisMO) wasadded to each pallet,The mixture wasthenvortcxcdandccntrifugcdat7S00xgI(JrS minat 40C.;\(the re-dissolving step.sampleswere airdriedaftercentr if ugeI(Jr10 min.
palletwasthendissolvedatRNAase-li-cC'water.I(JllowedbyincubatingthemixtureI(Jr IOminat600C.Thequantityand qualit;'of samplesweremeasured based on260/2S0 nmratiosb y Nanodrop 1000speelrophotometer (ThermoScient ilie.Waltham MA).
2.3.2eDNA Synthesis
ComplementaryDNA (cDNA)wassynthesizedusingRcvertAidP'II MinuslirSl strand cDNAsynthesiskit (Fcrmcnias.Burlington ON).TheRT mixtureincluded:
RcvcrtAid' "IIMinusI'vI-MuLVreversetranscriprasc.SX reactionbuffer,Ribolock RNaseinhibitor.10 mM dNT Pmix.random hcxamerprimer. and SOOngRNA dilutedin atotalvolumcofoul Dicthylpyrocarbonatc-trcutcdwater.Sampleswereincubated I(Jr S minat2S0Cfollowcdbyritlmin ut-lL'".'.lhcrcactionwasterminated byheatingat70
°CforSmin.
2.3.3 StandardPCR
TheexpressionlevelsofGapdh.U/JfJ2.Rig-!andSIII12mRNA weremeasured followinglimitedPCR amplilicalionu sir ggene-specilic oligonucleotide primers describedin(Tablc Llj.Thc PCRreaction mixture included:2ul lUXbuffer, Ipi MgCI2.
40
Tahle2.1Gcncspccificprimcrscqucnccs
Gene Primerforward Primerreverse size
hn CaJl/1I S'-( i(;( i l( i( i ACiCCAA M' (;( ;U rc,\ -,1'S'-(;(;A (i(iT(iCrCOTT( ;,\A(;TC( ;CA-3' 532
cua
S'- A(; (; ITAACCiCiAAAACC( ,(il('A·}' S'-CACACiTCCi('(;( iCT('ATTAAACi-}' 106nu-t
5'-( ,CiC,\( ii\ ('i\ AAC,ACi( ;;\ CiC,i\ Cii\·3' 5'-('(i( iACATCCiHi(;AACi!\ACi(i-3' 150 Stat2 S'-( iTCTTC ACi!\(,(,CCC!\TC!\(;/\- } ' S-ClC i CCT TCCI(;(;!\UrCTC!\C-}' 50741
(Fcrmcmas.Burlington ON).0.2fti lOmMdcoxynuclcotidctriphosphatemix(dNT I').
0.2ftlTaq polymerase (lnvitrogen. Burlington ON).0.5 ftlofIOmM forwardprimer.
0.5ulof10 mMreverse primer (IntegratedDNA Technologies.CoralvilleIA)and14.6 uldistillcdwater. The I'CRreact ionwascarriedoutasfollows:denaturation at94°Clor Imin.annea lingat63.5°C11.11'Imin.extensionat
n oc
11.)1'Imin11.11'32cycles11.11' GhIJ2.Rig -f .and Stat2.The annealingtemperaturewasset at63°C11.11'Gatidh and proceededforonlyZ')cyclesofamplilication.TheIinalextensionwas carriedoutat72°Clor10minlorall the reactions.
2.3.4Elcctrophoresisof l'C R products
Fiveu lof theI'CRproductsmixedwith0.5ftlofloadingbutler aswellas 3ulof 100bpladder (Fermentas.BurlingtonON)wererunon I'VoAgarosegel (lnvitrogen.
13urlingtonON) containingEthidiumBromide (Invitrogen. BurlingtonON)placed on a horizontalgel transferapparatus11.11'30min.Thegeltransfcr appuratuswaslillcdwithlx Trisacelate(50xstock/ml:242g trisbas':.5 7.l mlglocialacct ic acid.100m 11'11.XO.5m ethylenediarninctetraacetic acid).ThePCRproductswereobserved underUV light usingagellogic 200imagingsystem(Eastma nKodakCompany. NewYorkNY).
2../f)ENSIT(}AII~TRYANALYSIS
Densitometryanalysisonscannedprotein filmsand l.rhid iumstainedgelswas performcdwithlmngcJsofh varc (NlI l. llcthcsda.Maryland. USA.
42
hltp://imagej.nih.gov/ijl) .Proteinbands were normalizedtot-ERK and RT-PCRbands were normalizedto Gapdl».
2.5STATIS TICA LANALYSIS
Statistical analysisofthe proteinand RT-PCRdensitometryratioswasperformed usingOne-way AnalysisofVariance(ANaYA)byGraph l'adPrismVersion4 (Graphl'adSoftw are. LaJollaCali forniaUSA).
43
Chapter3:Result s
3./ ./:'FF/CIC TOF SL317TRE·/7~\I/:W7' ON/N/IIIJITION OF Rl/.I'/Rl/flMEK/'ATI/W·Il' /N V!r'O
Thelirst stepof the experimentalstrategywastodeterminewhether the MEK inhibitor SU27caninhibit theactivityofthe Ras/Rafi'MEKpathwayillvivo.Western blotanalysiswasperformedtoexaminetileactivation slateo fE RK kinase.the downstreamtarget of MEK.andthiswas representedbythe phosphorylation statusof I~R K(p-ERK).p-ERK was examined inproteinlysatcspreparedfrom the lungsof three miceinje ctedintrapcritoneallywithDM~,Oor SU27in DMSO(100rug/kgI3W).1\
specific antibody against p-E RKwasusedto assess thephosphorylationstate.asan indicator forthe efficacy ofthe SU27treatment. Intraperitoneal injection of S U27 reducedthe relativeratio of phosphorylatedtot-ERKinthe lungs(Figure 3.1andFigure 3.2).Thesame dose andsiteofd rugadministration wasusedtorsub sequent experiments.
3.1.EFFEC/:',OFPIIl'SIOU)Ci/CAL
u: 'vn,s
OFAC7'1/1J:' RII.I'IRl!/!J/EKPATI/ W / l' ON/:'XPR/:'SSIO N1.1:'11/:'1..\'OF MOIlCi/:'NESIN 7'IIEIJR/I/N.IN7'ESTIN/:'. ANDuit«,If thephysiologicalactivationoftheRas/Rafi'MEK pathwaysuppresses the expression ofthe MDIIgenes. inhibitionofRas/Rali'MEK wouldbe expectedtoincrease their expression.
44
p-ERK
Figun' 3.1 WcstCJ"IIblot analysi sof p-ERKin the lung Micewereinjected intrapcrito nca llywith80~Iof'DMSO(contro l)or SL327(100 mg/Kg) At 8hrsalter injcctio n,prot ein sampleswer eprepar edfrom thelungslor weste rn blotanalysisThe membraneswereblottedwithantibodiesagain stp-ERK.and t-ER K.Each lanenumber rcpr cscntsan ind ividua l mouscpcr trca tlllc nt
~2.5
~2.0 ] 1.5 .a:;
~1.0
.~0.5
(ij
c.:O. O . . L . . . . - - - - . - - -- - - mouse 1 mouse2 mouse3
Control 51327
Figlll·c3.2Densito me try ana lysisof II-ERKinthe lung.Pro teinsa mples wer eprepared fromthelungsofmiceintr aper iton eally injectedwith80 pIofDMSO (control)or SU2 7 (100mglKg) Followingweste rnblotting,densitometry ofthe band sW1L~measured with NllilmageJ. Theband densit yofp-ERKwasnormalizedto that oft- ERKand plott ed per mouse.Ho rizontallinesrep resenttheme anden sit y±SEM
46
Toexamine thishypothesis.Balblcfemalemiceweretreatedwith DMSO (control.n=5).
100mg/kg SL327 (n=5).1000U/mouscIFN-u(n=5).or the combinationoflFN-ul SL327 (n=5).RNAwasextractedfromthebrain.lung.and intestineatShrspost injectionandsubjected to semi-quantitativeRT-I'CRtomeasurethe expression ofthe ivlDllgenes: Ghp2.Rig-/ and S/a/2.
3.2.1Brain
Figure3.3showsthcrclarivcchangcsinclcctrophorcsisbandingpaucrnsfort hc MDIIgenesinthebrainofmicethat receivedcontrol DMSOinjection.SL327alone.
IFN-ualoneorSL327incombinationwithIFN-u.InFigure3.4. the densitometricratios forGbp],Rig -IandS/a/2relativeto Gapd ltare plotted forindividualmice acrossthelour diff erenttreatment groups.alongwiththemean expressionchange.
IF -utreatmentinduced expressionofCih/J2inall individualmice.butthe individualmouse responseswere quitevariablein SL327treatmentgroup(Figure3.3A).
For example.mouse Iand2weretheonlyresponderstoSL327treatmentbasedon this semi-quantitative analysis.Asa group, therewerenosignilicantchanges inCihp2relative expression relative tomice receivingthe vehiclecontrolin the brain(Figure3.4A).Co- inj ectionoflFN-u andSU 27induced no synergisticeffect(Figure3.3A.Figure3.-t A).
The individualmouseresponseswerequite variable ItlrR ig-/expression in the brainof lFN-u treatmentgroups(Figure3.3B).Forexample:mostrobust inductionof Rig-/wasobserved in mouse3.4.and5.as theinduction in mouseI.and2wasnot considerable.
47
[JJ
s,at2 ~~GaPdh~"""'"
Gapdh
rt' &mm!!!
Figlll'e3.3RT-PCRanalysisof thebraiusamples.RNAwasextrac tedfrom the brain of animalstreatedwithDMSO (cont,n=5),and 100mg/K gSU27 (n=5),1000U/mouse IFN-(l(n=5),andSU27/1FN-a(n=5),at:3hrsafterinjec tion.RT-PCRanalysisW,L~
perfor medlo r(;hfl2,Rtg -I,SIal2,and(;apdIJ.Eachnumber repr esentsan individual mousepertreatm ent
48
i . . ~ . ~~ f+
: hj . . . .
O.OCO:troI SL32~
Hj~.----,
~2.0 •~ ::~ ~~ ~- ~
~0.5 •
0.0Cont rol SL3;---'7- - - -IF-N--:-::-=
~
"tHI...ouse!';"l VlbeJFiglll'c3A DensitometryanalysisoftheRT-P CI{result softill'b...linsa m p lesRNA was extracted fromthebrainofanima lstreatedwithDMSO(cont,n=5),and100mg/Kg SU27(n=5),1000lJImouscIFN-(x(n=5),andSU27/IFN-IX(n=5),at8hrs after inj ection.Thebandintensity of MDIIgeneswasnormaliz edtothatof(;updhandplo tted permouse.*P'<0.05byone- way ana lys isof'Variancc(ANOVA).Horizont all incs repres entthemeandensity±SEM
49
Expressionor Rig- 1wasup-regulatedin none oft he micefollow ing SU27 administration.Asa group.thcrcwcrc oillysignilicant changcsin Rig-/rclati vc expressionin theIFN-atrcatmcnt grouprelative tomicereceivingthe vehiclecontrol (Figure3.4B). Co-injectionor IFN-u andSU27 induced nosynergisticeff ect(Figure3.3 B;Figure3.4B).
Similarly.individual mouse responseswere quite variable f(JI' SI(/I]exprcssionin the brainofall trcatment groups(Figurc3.3 C).Forexampl e:mouseIand2wc rc non- responders to anytreatment based on thisRT-PCR analysis.Mouse 3.4 and5showedan up-regulationOrsl (/l]expressionin thebrain followingIFN-n.butonly inmouse5SI(/I]
expression wasinducedfollowing SU27treatment.Asagroup.there werenosignificant changes inSI(/ 12relative expression inthebrain comparedtomice receivingthevehicle control(Figure3.4C).Co-inj ectionor IFN-u andSU27inducednosynergistic effect (Figure3.3C;Figure3.4 C).
3.2.2Intcstinc
Figurc3.5shows the relative changesin electrophoresisbandin gpaucrnsfor MDII genes in theintestine or micethat receivedcontrol DMSOinjection, SU27alone, IFN-u aloneorSU27incombinationwith IFN-u.In Figure3.6.the densitometric ratios It)rG!JIJ].Rig-la ndSI(/12relativetoGapdhare plottedlorindividualmice acrossthelour difkrc nt trcatmentgroups.along with thc l11ca nexpressionchangc.
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