SHP2 Is Overexpressed and Inhibits pSTAT1-Mediated APM Component Expression, T-cell ...

12 Pages·2013·0.79 MB·English

Published OnlineFirst January 30, 2013; DOI: 10.1158/1078-0432.CCR-12-1517
Clinical
Cancer
Human Cancer Biology Research
SHP2 Is Overexpressed and Inhibits pSTAT1-Mediated APM
Component Expression, T-cell Attracting Chemokine
Secretion, and CTL Recognition in Head and Neck Cancer
Cells
MichaelS.Leibowitz1,RaghvendraM.Srivastava2,PedroA.AndradeFilho2,AnnMarieEgloff2,LinWang3,
RajaR.Seethala3,SoldanoFerrone1,4,andRobertL.Ferris1,2,5
Abstract
Purpose: Human leukocyte antigen (HLA) class I antigen processing machinery (APM) component
downregulationpermitsescapeofmalignantcellsfromrecognitionbycytotoxicTlymphocytes(CTL)and
correlateswithpoorprognosisinpatientswithheadandneckcancer(HNC).ActivatedSTAT1(pSTAT1)is
necessary for APM component expression in HNC cells. We investigated whether an overexpressed
phosphatasewasresponsibleforbasalsuppressionofpSTAT1andsubsequentAPMcomponent-mediated
immuneescapeinHNCcells.
Experimental Design: Immunohistochemical staining and reverse transcription PCR of paired HNC
tumorswasperformedforthephosphatasessrchomologydomain-containingphosphatase(SHP)–1and
SHP2. Depletion of phosphatase activity in HNC and STAT1(cid:1)/(cid:1) tumor cells was achieved by siRNA
knockdown.HLAclassI–restricted,tumorantigen-specificCTLwereusedinIFN-gELISPOTassaysagainst
HNCcells.ChemokinesecretionwasmeasuredafterSHP2depletioninHNCcells.
Results:SHP2,butnotSHP1,wassignificantlyupregulatedinHNCtissues.InHNCcells,SHP2depletion
significantlyupregulatedexpressionofpSTAT1andHLAclassIAPMcomponents.OverexpressionofSHP2
innonmalignantkeratinocytesinhibitedIFN-g–mediatedSTAT1phosphorylation,andSHP2depletionin
STAT1(cid:1)/(cid:1)tumorcellsdidnotsignificantlyinduceIFN-g–mediatedAPMcomponentexpression,verifying
STAT1 dependence of SHP2 activity. SHP2 depletion induced recognition of HNC cells by HLA class
I–restrictedCTLandsecretionofinflammatory,T-cellattractingchemokines,RANTESandIP10.
Conclusion:ThesefindingssuggestforthefirsttimeanimportantroleforSHP2inAPM-mediatedescape
ofHNCcellsfromCTLrecognition.TargetingSHP2couldenhanceT-cell–basedcancerimmunotherapy.
ClinCancerRes;19(4);798–808.(cid:2)2012AACR.
Introduction absoluteTlymphocytecounts(3).Atthetargetcelllevel,a
majormechanismisrepresentedbydownregulationofthe
In the majority of cases, T-cell–based cancer immu-
antigenprocessingmachinerycomponents;thisabnormal-
notherapieshaveyieldeddetectableimmuneresponsesin
ityleadstopoortumorantigenprocessingandpresentation
the absence of appreciable clinical activity (1). Several
(4–7). As a result, HNC cells are poorly recognized by
immune escape mechanisms have been identified in
cognateHLAclassIantigen-restricted,tumorantigen–spe-
patients with head and neck squamous cell carcinoma
cific cytotoxic T lymphocytes (CTL; refs. 4,7, 8).Interest-
(HNC).Attheeffectorphaselevel,theyincluderecruitment
ingly, antigen processing machinery (APM) component
of regulatory T cells andmyeloid-derived suppressor cells
downregulationcorrelateswithpoorprognosisinpatients
into the tumor microenvironment (2) and reduction of
with HNC (6), showing that this escape mechanism is
clinically significant. The molecular mechanism for this
Authors'Affiliations:1DepartmentsofImmunologyand2Otolaryngology, poor tumor antigen presentation has been characterized
UniversityofPittsburgh;and3DepartmentsofPathologyand4Surgeryand onlytoalimitedextent.
5CancerImmunologyProgram,UniversityofPittsburghCancerInstitute,
Previous work has shown that treatment of HNC cells
Pittsburgh,Pennsylvania
with IFN-g upregulates APM components and restores
CorrespondingAuthor:RobertL.Ferris,HillmanCancerCenterResearch
Pavilion,5117CentreAvenue,Room2.26b,Pittsburgh,PA15213.Phone: tumorantigen–specificCTLlysisinvitro(4).Inpriorstudies,
412-623-0327;Fax:[phone];E-mail:[email protected] wehaveshownthatAPMexpressionandT-cellrecognition
doi:10.1158/1078-0432.CCR-12-1517 require IFN-g-STAT1 pathway activation (4, 9). The low
(cid:2)2012AmericanAssociationforCancerResearch. basal pSTAT1 levels in HNC cells led us to investigate
798 ClinCancerRes;19(4)February15,2013
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Published OnlineFirst January 30, 2013; DOI: 10.1158/1078-0432.CCR-12-1517
SHP2CausespSTAT1-DependentImmuneEscape
Cytokines
Translational Relevance
Human IFN-g was purchased from InterMune. IFN-g
T-cell–based cancer immunotherapyis an encourag- concentrationincellculturesupernatantswasdetermined
ingareaofinvestigationwithemergingclinicalefficacy. byhumanIFN-gELISAkit(R&Dsystems)accordingtothye
ThefailuretoaddressthetumorasadefectivetargetforT- manufacturer’sinstructions.Intheexperimentalconditions
cellrecognitionhasbeenamajorobstacletoitssuccess. used,100U/mLofrecombinantIFN-gequatedto1,464pg/
Here,wereportanoveltherapeutictargetinheadand mLusingthisELISAkit.
neckcancer(HNC)thatalterstheimmunogenicityofthe
tumor. Src homology-2 domain-containing phospha- Antibodies
tase (SHP2) was found to be overexpressed in HNC, Anti-HLA-A,B,C mAb (W6/32; EBiosciences) and anti-
whichwhendepleted,restoredrecognitionofHNCcells HLA-DR (L243) mAb (Biolegend) were used in ELISPOT
by cytotoxic T lymphocytes and induced secretion of assays. LMP2-specific mAb SY-1 (17), TAP1-specific mAb
proinflammatory,T-cellattractingchemokines,RANTES NOB-1,TAP2-specificmAbNOB-2,andcalreticulin-specific
andIP10.Thesefindingsprovidetherationaleforfuture mAbTO-11,weredevelopedandcharacterizedasdescribed
investigation of the effects of small-molecule SHP2 (17,18).Fluoresceinisothiocynate(FITC)-conjugatedIgG
inhibitors on tumor growth in vivo, functioning as a anti-mousemAb(LifeTechnologies)wasusedasasecond-
potential adjuvant to enhance T-cell–based immuno- aryantibodyforAPMcomponents.TheintracellularpSTAT
therapyofcancer. stainingwasconductedusingphycoerythrin(PE)-conjugat-
ed irrelevant IgG2a mAb isotype control, PE-conjugated
phosphorylatedtyrosine701STAT1mAb(pSTAT1Tyr701).
AlexaFluor647-conjugatedtotalSTAT1mAb,FITC-conju-
regulatorymechanismsthatdisruptSTAT1activation,APM
gatedphosphorylatedserine727STAT1mAb(pSTAT1Ser
componentexpression,T-cellrecruitment,andsubsequent
727),PE-conjugatedpSTAT1Tyr701,pSTAT3Tyr705mAb,
recognitionofHNCcells.
and FITC-conjugated phosphorylated serine 727 STAT3
SincewehaverecentlyfoundthatHNCcellsexpressbasal
mAb (pSTAT3 Ser 727) were purchased from BD Bios-
total(unphosphorylated)STAT1,butnotactivatedpSTAT1
ciences. Anti-pSTAT1 Tyr701 mAb (Cell Signaling Tech),
(9), we hypothesized that a negative regulator of STAT1
anti-totalSTAT1(C-24)polyclonal(pAb;SantaCruzBio-
phosphorylationmightberesponsibleforlowAPMcompo-
tech), anti-b-actin mAb (Sigma-Aldrich Inc.), anti-rabbit
nentexpressionandCTLescapeinHNCcells.Srchomology-
IgG-HRP (Promega), anti-mouse IgG-HRP (Bio-Rad),
2 domain-containing phosphatase (SHP)-2 has been sug-
anti-phosphorylated JAK-1 (Tyr1022/1023) mAb, anti-
gestedasanegativeregulatoroftheJAK-STATsignaltrans-
phosphorylatedJAK-2(Tyr1007/1008)mAb(Cellsignal-
ductionpathway(10,11)butithasnotbeenimplicatedin
ingTech),anti-totalJAK-1mAb,andanti-totalJAK-2mAb
tumor immune escape. Furthermore, SHP2 overexpression
(SantaCruzBiotech)wereusedinimmunoblotanalyses.
and/orhyperactivityhavebeenshowedinleukemia,breast,
andbladdercancers(12,13).Thus,weinvestigatedwhether
Immunohistochemistry
overexpressionofSHP2mightprovideanovelmechanism
Proteinlevelswereevaluatedbyimmunohistochemical
for CTL escape through dephosphorylation of pSTAT1 in
(IHC)stainingoftumorandadjacentmucosalspecimens
cancercells,reducingexpressionofHLAclassIAPMcompo-
arrayedinapreviouslydescribedtissuemicroarray(TMA;
nentsaswellasproinflammatorycytokinesandchemokines.
ref. 19). For the studies presented here, the maximum
numberofevaluabletumorspecimenswas46;16ofthese
Materials and Methods
tumorshadarrayedadjacentmucosaltissuesavailablefor
Celllines analysis.TMAqualityassessmentandmorphologicconfir-
The HLA-A(cid:3)0201þ HNC cell lines, PCI-13 and SCC-90 mationoftumorornormalhistologywasconductedusing
(14)andHLA-A(cid:3)0201(cid:1)celllines,SCC-4andPCI-15Bwere onehematoxylinandeosin(H&E)-stainedslideforevery10
characterizedanddescribedpreviously(15).Alltumorcell tissuesections.
lineswereculturedinDulbecco’smodifiedEagle’smedium Arrayedtissueswereimmunohistochemicallystainedfor
(DMEM;Sigma-AldrichInc)supplementedwith10%FBS SHP1andSHP2andtissuelevelswereevaluatedsemiquan-
(Mediatech), 2% L-glutamine, and 1% penicillin/strepto- titatively. Before incubation with anti-SHP1 (Santa Cruz
mycin (Life Technologies). Keratinocytes were described Biotech)oranti-SHP2(SantaCruzBiotech)antibodiesfor
previously(16)andgrowninkeratinocyteserum-freemedi- 60 minutes at room temperature, antigen retrieval was
um (Keratinocyte-SFM; Life Technologies) supplemented conducted using citrate pH 6 buffer (Dako) followed by
with bovine pituitary extract. Parental 2fTGH(STAT1þ/þ) incubation with a 3% hydrogen peroxide solution for 5
andU3A(STAT1(cid:1)/(cid:1))fibrosarcomacells (akindgiftfrom minutes at room temperature. The tissue specimens were
Dr.GeorgeStark,ClevelandClinicFoundation,Cleveland, thenblockedwithcalfserumblock(LifeTechnologies)for
OH)wereculturedinIscove’sModifiedDulbecco’sMedium 10minutesatroomtemperature.SHP1stainingwasdevel-
(LifeTechnologies) supplemented with 10%FBS (Media- opedusingDakoDualEnvisionþfor30minutesatroom
tech), 2% L-glutamine, and 1% penicillin/streptomycin temperaturefollowedbyincubationwithSubstrateChro-
(LifeTechnologies). magen for 5 minutes at room temperature. Slides were
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Leibowitzetal.
counterstainedwithHarrisHematoxylin,andcytoplasmic FACSanalysiswasperformedonthesamedayasstaining.
and nuclear staining intensity (0–3) and percentage of Isotype control antibody staining was set at a mean fluo-
tumorcellstothenearest5%weredeterminedseparately rescence intensity (MFI) of 5 on an EPICS XL cytometer
byaheadandneckcancerpathologist.AnIHCscorewas (BeckmanCoulter)orsubtractedfromeachconditionand
derivedfromtheproductoftheintensityandpercentageof celllinetested.Aminimumof10,000cellswerecollected
tumorstained,andIHCscoresforeachcoreofaspecimen per test. Analyses were conducted using EXPO32 ADC
wereaveraged. software (Beckman Coulter). pSTAT1, pSTAT3, and APM
componentexpressionwasdeterminedonthebasisofMFI
Quantitativereal-timereversetranscriptionPCR andwasexpressedasamean(cid:6)SEMoftheresultsobtained
TotalRNAfromtumorsamplesandnormalmucosawas inatleast3independentexperiments.
extracted using Trizol reagent (Invitrogen) and purified
usingRNAcleanup(Qiagen),followedbyDNasedigestion TransfectionofcellswithsiRNA
(Ambion) according to manufacturer’s instructions. The HNCcelllinesweretransfectedat30%to40%confluence
concentrationandpurityofRNAwasdeterminedbymea- with SHP1, SHP2, or a nontargeting siRNA control
suringabsorbanceat260and280nm.RNAwasusedfor (Ambion), Lipofectamine RNAi max (Life Technologies),
first strand cDNA synthesis using random hexamers and andOptimemI(LifeTechnologies)accordingtotheLipo-
MuLVreversetranscriptase(AppliedBiosystems)according fectamineRNAimaxinstructions.
to the manufacturer’s instructions. PCR probes for SHP2
(Hs00743983_s1) and b-actin (Hs99999903_m1) were SHP1:50-GGUGACCCAUAUUCGGAUCTT’(s)and
purchased from Applied Biosystems for TaqMan Gene 50-GAUCCGAAUAUGGGUCACCTG-30 (as)
Expression Assay. Real-time PCR cycling was conducted SHP2:50-GGAGAACGGUUUGAUUCUUTT-30 (s)and
(7700Real-TimePCRSystem;AppliedBiosystems)under 50-AAGAAUCAAACCGUUCUCCTC-30 (as)
the following conditions: denaturation at 95(cid:4)C for 10 Non-targeting:50-AGUACAGCAAACGAUACGGtt-30 (s)
seconds,annealingat60(cid:4)Cfor15seconds,andextension
and
at72(cid:4)Cfor30seconds.Aninitialdenaturationstepat95(cid:4)C
for 5 minutes and a final extension step at 72(cid:4)C for 10 50-CCGUAUCGUUUGCUGUACUtt-30 (as)
minutes were also included. PCR was conducted for 40
cycles.b-Actinwasamplifiedasaninternalcontrol.Allof
SHP2plasmids
the experiments were carried out in duplicates. Relative
expression of the SHP2 gene to endogenous control gene Keratinocytesweretransfectedat80%to90%confluence
(b-actin) was calculated using the DC method: relative with wild-type SHP2 (pcDNA3.1-myc-6xHis-SHP2) or
T
expression¼2(cid:1)DCT,whereDCT¼CT(shp2)(cid:1)CT(b-actin). mutant SHP2 (C4593) plasmid constructs (10 mg/mL; a
kind gift from Dr. Gibbes Johnson, U.S. Food and Drug
Administration, Silver Spring, MD). Lipofectamine 2000
Immunoblotanalysis
reagent (Life Technologies) was used at a 1:2 dilution in
Cellswerelysedin10mmol/LTrisHCl,5mmol/LEDTA,
OptimemI(LifeTechnologies)accordingtotheLipofecta-
50mmol/LNaCl,30mmol/LNa P O ,50mmol/LNaF,1
2 2 7
mine2000instructions.
mmol/L NaVO , 1% Triton X-100, 1 mmol/L phenyl-
4
methylsulfonylfluoride(PMSF)andvortexedforatleast1
hourat4(cid:4)C,sonicated for6secondsat20%ofthemaxi- ELISPOTassay
mumpotency(ColeParmerInstrument)andcentrifugedat ELISPOTassayswereconductedasdescribed(20).Brief-
4(cid:4)C,16,100(cid:5) gfor 15minutes. The supernatant protein ly,multiscreen -HAfilterplates(Millipore)werecoated
HTS
was normalized and 40 to 60 mg of protein were size withanti-humanIFN-gmAb1-D1K(Mabtech;10mg/mLin
fractionated through a 4 to 12% SDS-PAGE gel (Lonza), PBS) overnight at 4(cid:4)C. Plates were washed with PBS and
transferred to a polyvinylidene difluoride (PVDF) mem- thenblockedfor1hourat37(cid:4)CwithDMEMsupplemented
brane (Millipore) and immunoblotted with theindicated with 10% human serum. EGFR (20) and p53
853–861 65–73
antibodies. (21,22)specificCTLwereaddedtowellsintriplicate(5(cid:5)
104)andthenHNCcells(5(cid:5)104).Followingan18to24
IntracellularflowcytometryforAPMcomponent, hour incubation at 37(cid:4)C, plates were washed with PBS/
pSTAT1,andtotalSTAT1expression 0.05%Tween20(PBS-T),andincubatedwithbiotinylated
Briefly,thecellswerefixedusing1.5%paraformaldehyde anti-IFN-g mAb(Mabtech;2mg/mL)for4hoursat37(cid:4)C.
for 10 minutes at room temperature and permeabilized PlateswerewashedwithPBS-Tavidin-peroxidasecomplex
with100%methanolforatleast24hoursat(cid:1)20(cid:4)C.Cells (Vectorlaboratories)for1houratroomtemperatureand
werethenwashedin2%fetalcalfserum(FCS)/PBS(FACS then peroxidase staining was conducted with 3,3,50-tetra-
buffer), and stained with either a PE-conjugated primary methylbenzidine(VectorLaboratories)for4minutes.Spots
STAT-specific mAb or sequentially by incubation with an wereenumeratedintriplicatewellsasamean(cid:6)SEusing
unconjugatedprimaryAPMcomponent–specificmAband computer-assistedvideoimageanalysissoftware(Cellular
then with a FITC-conjugated anti-mouse secondary mAb. Technology Ltd.). HLA class I–restricted recognition of
Bothincubationswerefor30minutesatroomtemperature. thetargetcellsbyCTLwasassessedbyusingtheanti-HLA
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SHP2CausespSTAT1-DependentImmuneEscape
classI–specificmAbW6/32(10mg/mL)andthespecificity despiteabundantexpressionoftotalSTAT1protein(9).To
ofthe inhibition wasassessed using theHLA-DR–specific identifyamechanismforthisphenotype,wetestedwhether
mAbL243(10mg/mL). aproteintyrosinephosphatasemightberesponsibleforthe
dephosphorylation of activated STAT1. First, a panel of
Chemokineexpressionassay HNC cells, PCI-13, SCC-90, SCC-4, and PCI-15B, were
HNC cells were assayed for expression of various cyto- treatedwithabroadphosphataseinhibitor,sodiumortho-
kines and chemokines using a human cytokine 30-plex vandate(SOV).After24hoursoftreatmentwithSOV(100
panel (Life Technologies) as previously described (23), mmol/L), pSTAT1 levels were found to be significantly
48 hours after siRNA knockdown of SHP2 or irrelevant upregulated in these cells by immunoblot analysis (data
target. not shown). These data indicated that protein tyrosine
phosphatasesmaycontributetothebasaldephosphoryla-
Statisticalanalysis tionofSTAT1inHNCcells.
Dataareexpressedasamean(cid:6)SEoftheresultsobtained BothSHP1(25,26)andSHP2(10)havebeensuggested
inatleast3independentexperiments.A2-tailedttestwas to be negative regulators of STAT1 phosphorylation. To
usedtocalculatewhetherobserveddifferenceswerestatis- assess SHP1 and SHP2 expression in human HNC, IHC
ticallysignificant,definedasP<0.05.Thesignedranktest staining of a clinically annotated TMA cohort of HNC
wasusedtoevaluatedifferencesinSHP1andSHP2levels patientswasperformedonpairedtumorandnormalmuco-
between paired tumor and adjacent mucosal tissues and sa. Interestingly, IHC analysis revealed that SHP2 was
statisticallysignificancewasdefinedasP<0.05. significantly overexpressed in tumor cells compared with
normal mucosa (Fig. 1A; P ¼ 0.002, signed rank test).
Results
Moreover quantitative reverse transcription PCR analysis
SHP2butnotSHP1isoverexpressedinHNCtissues also showed higher levels of SHP2 transcript in tumor
PreviousworkshowedthatpSTAT1isrequiredforAPM samples compared with normal mucosa (Fig. 1B; P <
componentexpressioninHNCcells(9,24),andthatthe 0.05).Thisdifferenceisselective,asnosignificantdifference
STAT1agonist,IFN-g,inducedAPMcomponentupregula- inSHP1expressionwasdetectedbetweentumorandnor-
tionandCTLrecognition(4).Interestingly,wefoundthat malmucosa(SupplementaryFig.S1).Therefore,weinves-
basal levels of pSTAT1 are undetectable in HNC cells, tigatedtheroleofoverexpressedSHP2inHNCcells.
A SHP2 B
250 P = 0.02
n = 14 0.150
200 P = 0.002
e
r
sco150 els 0.125
C ev
P2 IH100 ript l 0.100
SH 50 nsc 0.075
a
r
2 t
0 P
H 0.050
Tumor Mucosa S
e
v
ati 0.025
el
R
0.000
SCCHN Tumor Normal mucosa
N = 33 N = 11
SCCHN tumor Normal mucosa
Figure1. SHP2isoverexpressedinHNCtissuebutnotinnormalmucosa.A,HNCtumorsandnormaladjacentmucosawerestainedwithananti-SHP2mAb
(asdescribedinMaterialsandMethods).TheIHCsemiquantitativescorewasderivedby2independentpathologists,bymultiplyingthestaining
intensity(scoredas0–3)bythepercentoftumorcellsstained(0%–100%),tothenearest5%.IHCscoresforeachcoreofaspecimenwereaveraged(n¼14)
andstatisticallyanalyzed.Representativeexamplesat(cid:5)200magnificationofSHP2fromHNCtissueandnormalmucosaareprovided.B,cDNAfromHNC
tumors(n¼33)andnormaladjacentmucosa(n¼11)weresubjectedtoquantitativereal-timePCRanalysis(asdescribedinMaterialsandMethods).
Relativeexpressionofshp2transcripttoendogenouscontrolgene(b-actin)wascalculatedusingtheDCTmethod:relativeexpression¼2(cid:1)DCT,
whereDCT¼CT(shp2)(cid:1)CT(b-actin).Two-tailedunpairedttestwasconductedforstatisticalanalysis.
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Leibowitzetal.
A B C
Control siRNA - + - 4,000
SHP2 siRNA - - + 20
SHP2 FI * P < 0.0001 3,000 * P < 0.0001
β-Actin 01M MFI
Tyr 7 AT1 2,000
%) 100 1 10 ST
ockdown ( 50 76% pSTAT Total 1,000
n
P2 k
H
S
0 0 0
Untreated Control SHP2 Untreated Control SHP2 IFN-γ Untreated Control SHP2 IFN-γ
siRNA siRNA siRNA siRNA siRNA siRNA
D E F
10
NS
Control siRNA - + - - Control siRNA - + - -
MFI SHP2 siRIFNNAγ -- -- -+ +- SHP2 siRIFNNAγ -- -- -+ +-
7
72 pSTAT1 Tyr 701 pJAK1
er
S 5
T1 Total STAT1 Total JAK1
A
T
S
p β-Actin pJAK2
Total JAK2
0
Untreated Control SHP2 IFN-γ
siRNA siRNA
Figure2. SHP2depletionupregulatespSTAT1,pJAK-1/2,andtotalSTAT1.(A–F)PCI-13and(G–L)SCC-90cellsweretransfectedwithSHP2siRNA
(100nmol/L,48hours),whichsignificantlyreducedSHP2expressioninHNCcellsbyWesternblotanalyses.SHP2depletionalsosignificantlyupregulated
pSTAT1anditstargetgeneproductSTAT1asdeterminedbyintracellularflowcytometryandimmunblottingcomparedwithcontrolsiRNAin(B,C,E)
PCI-13and(H,I,K)SCC-90cells.SHP2depletionalsoupregulatedpJAK1(Tyr1022/1023)andpJAK2(Tyr1007/1008)comparedwithcontrolsiRNAin(F)
PCI-13and(L)SCC-90cells.ThecellsweretreatedwithIFN-g(100U/mL¼1,464pg/mL,48hours)asapositivecontrolforSTAT1phosphorylation.Data
represent3independentexperimentsanderrorbarsindicateSE.
SHP2depletionupregulatespSTAT1,pJAK1/2,and pJAK1 (pTyr1022/1023) and pJAK2 (pTyr1007/1008) in
totalSTAT1inHNCcells SHP2-depletedPCI-13(Fig.2F)andSCC-90cells(Fig.2L)
UsingsiRNA(100nmol/L,48hours),weobtainedabout incomparisonwithcontrolsiRNA.IFN-gtreatmentdidnot
70%knockdownofSHP2proteininPCI-13(Fig.2A)and alterSHP2levelsundertheseconditions(datanotshown).
SCC-90 cells (Fig. 2G). SHP2 knockdown significantly To confirm that SHP1 was not involved in STAT1 acti-
upregulatedpSTAT1tyrosine(Tyr)701inPCI-13(Fig.2B vation, we investigated whether SHP1 depletion could
andE;P<0.002,2-tailedttest)andSCC-90cells(Fig.2H upregulate pSTAT1 and APM component expression in
andK;P<0.002,2-tailedttest),totalSTAT1inPCI-13(Fig. HNC cells. Under conditions in which SHP1 siRNA (100
2CandE)andSCC-90cells(Fig.2IandK)butdidnotaffect nmol/L,48hours)achievedmorethan70%knockdownof
pSTAT1serine(Ser)727ineitherPCI-13(Fig.2D)orSCC- SHP1proteincomparedwithsiRNAcontrol(Supplemen-
90 cells (Fig. 2J). Also, SHP2 depletion did not affect taryFigs.S3AandS3B),wedidnotobserveupregulationof
expression ofpSTAT3 Tyr705 orpSTAT3 Ser727 inPCI- pSTAT1orAPMcomponents(SupplementaryFigs.S3Cand
13 cells (Supplementary Figs. S2A and S2B) or SCC90 S3D).ThesedatapromptedustofocusonSHP2activityas
cells (Supplementary Figs. S2C and S2D) compared with theprimarynegativeregulatorofSTAT1phosphorylation,
cells transfected with control siRNA. SHP2-depleted cells APMexpression,andCTLrecognitionofHNCcells.
showed similar levels of STAT1 phosphorylation as com- Next,weinvestigatedhowSTAT1becomesactivatedafter
paredwithIFN-g(100U/mL,10minutes)treatedcells(Fig. SHP2depletion.WetestedwhetherSHP2depletioninduces
2B,E,H,andK).Interestingly,wealsodetectedinductionof secretion of a STAT1 agonist cytokine. To explore this
802 ClinCancerRes;19(4)February15,2013 ClinicalCancerResearch
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Published OnlineFirst January 30, 2013; DOI: 10.1158/1078-0432.CCR-12-1517
SHP2CausespSTAT1-DependentImmuneEscape
G H I
1,500
Control siRNA - + - 20 *P < 0.002 * P < 0.0001
SHP2 siRNA - - + 1,250
n (%)β-1SA0Hc0Ptin2 1 Tyr 701 MFI 10 al STAT1 MFI 1,070500
P2 knockdow 50 66% pSTAT Tot 250500
H
S 0 Untreated Control SHP2 0 Untreated Control SHP2 IFN-γ 0Untreated Control SHP2 IFN-γ
siRNA siRNA siRNA siRNA siRNA siRNA
J 6 NS K L
5 Control siRNA - + - - CoSnHtrPo2l ssiiRRNNAA -- +- +- --
MFI 4 SHP2 siRIFNNAγ -- -- +- +- IFNγ - - - +
27 pJAK1
1 Ser7 3 pSTAT1 Tyr 701 Total JAK1
T Total STAT1
A
ST 2 pJAK2
p β-Actin
Total JAK2
1
0
Untreated Control SHP2 IFN-γ
siRNA siRNA
Figure2. (Continued)
potentialmechanism,HNCcellsweretreatedwithcondi- ponents,SHP2depletionbysiRNA(100nmol/L,48hours)
tionedmediafromthesamecelllinesthatwerepreviously alsosignificantlyupregulatedHLAclassImoleculeexpres-
treated with IFN-g (100 U/mL, 48 hours), or transfected sioncomparedwithcontrolsiRNA-treatedHNCcells(Fig.
with control or SHP2 siRNA (100 nmol/L, 48 hours). 3CandD;P<0.02,2-tailedttest).
Interestingly, HNC cells treated with SHP2 siRNA condi-
tioned media induced STAT1 (Tyr) 701 phosphorylation SHP2regulatesTAP1/2expressioninaSTAT1-
(datanotshown). dependentfashion
We then examined whether transfection of SHP2 con-
SHP2depletionupregulatesHLAclassIAPM ferred a similar phenotype of APM component down-
componentexpressioninHNCcells regulation in nonmalignant keratinocytes, and whether
Next,weinvestigatedwhetherSHP2depletionandsub- this effect was dependent on STAT1. Interestingly, cul-
sequentenhancementofpSTAT1ledtoanupregulationof tured keratinocytes have significantly higher basal acti-
HLA class I APM component expression in HNC cells. vatedSTAT1andIFN-g–inducedpSTAT1comparedwith
Indeed,SHP2depletionbysiRNA(100nmol/L,48hours) HNC cell lines, PCI-13 and SCC-90 (data not shown).
induced significant upregulation of APM components in Furthermore, overexpression of wild-type (WT) SHP2 in
both PCI-13 and SCC-90 cells compared with control keratinocytes decreased both basal and IFN-g–induced
siRNA-treated cells (Fig. 3A–B; P < 0.003, 2-tailed t test). STAT1 phosphorylation compared with dominant nega-
STAT1phosphorylationinducedbySHP2depletiondidnot tive, mutant SHP2 showing that SHP2 overexpression
upregulatealloftheAPMcomponents,asnochangewas suppressed STAT1 activation in epithelial cells (Fig.
detected in the expression of calreticulin, a non-IFN- 4A). In addition, IFN-g–induced TAP1/2 expression in
g-pSTAT1–inducible APM component (Fig. 3A and B). STAT1þ/þ fibrosarcoma cells were significantly increased
IFN-g (100U/mL,48hours),awell-characterizedagonist after SHP2 depletion compared with both IFN-g treat-
of HLA class I APM component expression was used to mentaloneandcontrolsiRNA,andthiseffectwasabro-
compare relative APM component induction following gated in STAT1(cid:1)/(cid:1) fibrosarcoma cells (Fig. 4B and C).
siRNAtransfections.InadditiontoupregulatingAPMcom- These data verify that SHP2 mediates APM component
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Leibowitzetal.
Untreated
A Control siRNA B
PCI-13 SHP2 siRNA SCC-90
100 IFN-γ 100 *
*P < 0.003 *P < 0.002
FI * FI
M 50 * M 50 *
*
*
0 0
Calreticulin TAP1 TAP2 LMP2 Calreticulin TAP1 TAP2 LMP2
C D
*P < 0.02 * P < 0.0002
400 700
FI FI
M M
s I s I
as200 as 350
cl cl
A A
L L
H H
0 0
Control SHP2 IFN-γ Control SHP2 IFN-γ
siRNA siRNA siRNA siRNA
Figure3. SHP2depletionupregulatesHLAclassIAPMcomponents.PCI-13andSCC-90cellswereuntreated,treatedwithIFN-g(100U/mL¼1,464pg/mL,48
hours),transfectedwithcontrolsiRNA(100nmol/L,48hours)orSHP2siRNA(100nmol/L,48hours).Flowcytometricanalysesofintracellularstained
cellsforTAP1,TAP2,andLMP2APMcomponentsin(A)PCI-13and(B)SCC-90cells.Calreticulin,anon-IFN-g–inducibleAPMcomponent,wasincludedto
controlforglobalchangesinproteinexpressionfollowingtransfection.MFIwasmeasuredanderrorbarsindicateSE.Datarepresent3independent
experimentsanderrorbarsindicateSE.PCI-13(C)andSCC-90(D)cellsweretreatedasdescribedabove,andHLAclassIexpressionwasmeasuredby
flowcytometericMFI.Datarepresent3independentexperimentsanderrorbarsindicateSE.
upregulationthroughSTAT1activation,andsupportsthe SHP2depletioninducesHNCcellrecognitionbytumor
notion that SHP2 overexpresssion confers an "escape" antigen-specificCTL
phenotype of APM component downregulation in HNC TheobservationthatSHP2depletionsignificantlyupre-
cells. gulated pSTAT1 and HLA class I APM components
A Isotype
Mutant-SHP2
WT-SHP2
No IFN-γ +IFN-γ Figure4. SHP2decreasesbasal
70 126 andIFN-ginducedpSTAT1and
60 110 TAP1/2expression.A,
50 94 keratinocytestransfectedwith
Cell #4300 764837 wanildd-ItFyNp-egS(1H0PU2/dmeLcr¼ea1s4e6d.4bapsga/l
20 31
10 15 mL,30minutes)inducedpSTAT1
0 0 comparedwithmutantSHP2.C,
100 101 102 100 101 102 103 parental2fTGH(STAT1þ/þ)and
pSTAT1 U3A(STAT1(cid:1)/(cid:1))fibrosarcoma
cellsweretreatedwithIFN-g
IUFnNtr-eγated (10U/mL¼146.4pg/mL)for48
B Control siRNA + IFN-γ C hoursaloneor24hoursafter
*P < 0.0001 SHP2 siRNA + IFN-γ *P < 0.009 transfectionwithSHP2(50nmol/L)
60 500
*P < 0.001 *P < 0.006 orcontrolsiRNA.Flowcytometric
analyseswereusedtodetermine
MFI NS MFI NS TthAePd1aatanwdeTrAePp2loettxepdrebsysMioFnI.aDndata
1 30 2 250 represent3independent
P P
A A experimentsanderrorbars
T T
NS NS indicateSE.
0 0
STAT1+/+ STAT1–/– STAT1+/+ STAT1–/–
804 ClinCancerRes;19(4)February15,2013 ClinicalCancerResearch
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Published OnlineFirst January 30, 2013; DOI: 10.1158/1078-0432.CCR-12-1517
SHP2CausespSTAT1-DependentImmuneEscape
A
Control
Anti-HLA-A,-B,-C mAb
siRNA *P < 0.006
Figure5. SHP2knockdown Anti-HLA-DR mAb
restoresHNCcellrecognitionby PCI-13
tumorantigen-specificCTL.IFN-g SHP2
Anti-HLA-A,-B,-C mAb
ELISPOTassayswereconductedto siRNA
detectrecognitionbytumorantigen- Anti-HLA-DR mAb
specificCTLof(A)PCI-13and(B)
SCC-90cellsfollowingSHP2
IFN-γ
knockdown.Cellswere Anti-HLA-A,-B,-C mAb
untreated,treatedwithIFN-g
Anti-HLA-DR mAb
(100U/mL¼1,464pg/mL,48hours),
controlsiRNA(100nmol/L,48 0 100 200
hours),orSHP2siRNA(100nmol/L, Spots/50k EGFR CTL
48hours).EGFR853–861andp5365–73
-specificCTLswereusedaseffector B
cells.TheHLAclassI–specificmAb Control
WmA6/b32L2a4n3dwtheereHuLsAe-dDtRo–sshpoewcifitchat SCC-90 siRNA AnAtin-HtiL HAL-AA,--DBR,- Cm mAbAb *P < 0.001
therecognitionoftargetcellsby
tumorantigen-specificCTLwasHLA SHP2 Anti-HLA-A,-B,-C mAb
classI–restricted.Datarepresent siRNA
Anti HLA-DR mAb
asingleexperimentconducted
intriplicateanderrorbars
indicateSE. IFN-γ Anit-HLA-A,-B,-C mAb
Anti-HLA-DR mAb
0 375 750
Spots/50k p53 CTL
promptedourinvestigationintowhetherSHP2depletion- IL-2R,IL-4,IL-5,IL-6,IL-7,IL-8,IL-10,IL-13,IL-15,IL-17,
inducedHNCcellrecognitionbyCTL.Indeed,SHP2deple- TNF-a,GM-CSF,G-CSF,Eotaxin,MCP-1,MIP-1a,MIP-1b,
tionsignificantlyincreasedPCI-13andSCC-90cellrecog- EGF, HGF, VEGF) were not detected (data not shown).
nitionbyEGFR andp53 -specificCTL(Fig.5A– Thus,SHP2overexpressionconfersanimmunosuppressive
853–861 65–73
B;p<0.006,two-tailedt-test).TherecognitionofHNCcells phenotypebyreducingpSTAT1-mediatedAPMcomponent
bytumorantigen-specificCTLisHLAclassI–restricted,asit expressionandCTLrecognitionofHNCcells,andinhibits
wasinhibitedbytheHLA-A,-B,-C–specificmAbW6/32,but some proinflammatory, T 1 cell recruiting chemokines
H
wasnotaffectedbyHLA-DR–specificmAbL243. fromthetumormicroenvironment.
SHP2depletioninducessecretionofchemokinesby
Discussion
HNCcells
To investigate downstream effects of SHP2 depletion Multiplemechanismsareusedbytumorcellstoescape
andpSTAT1activationinHNCcells,weconductedmulti- recognition and elimination by the immune system. We
plex ELISA assays (Luminex) to measure the secretion of describeanovelmechanismresponsibleforthephenotype
several cytokines and chemokines. Interestingly, signifi- of HLA class I APM component downregulation, namely
cantly elevated levels of RANTES and IP10 were detected suppression of STAT1 activation, leading to tumor cell
inthesupernatantsofHNCcellsafterSHP2depletionby escape from tumor antigen-specific CTL recognition and
siRNA(100nmol/L,48hours)comparedwithcellstrans- lysis, a widely observed phenomenon in many human
fected with control siRNA (Fig. 6A–B, P < 0.008, 2-tailed cancers(4,7,27).Thesedefectsseemtobedependenton
t test). Under similar conditions, monokine induced by overexpressionofSHP2,anSH2domain-containingtyro-
interferon-gamma (MIG) was not secreted by HNC cells sine phosphatase, which was found to be highly overex-
after SHP-2 depletion, whereas it was secreted after these pressed in vivo in HNC tumor tissues (Fig. 1), compared
cellswereincubatedwithIFN-gfor48hours(Fig.6AandB). with surrounding normal epithelium. SHP2 is overex-
Notably, cytokines that induce STAT1 phosphorylation pressed and/or hyperactive in multiple malignancies (12,
suchasIFN-g (Fig.6AandB)andIFN-a(datanotshown) 13, 28–30); however, few studies have investigated their
were not detected in the supernatants of HNC cells after impactonimmunecellmigrationandrecognitionoftumor
SHP2 depletion despite expression of IFN receptors on cells(31–34).Tothebestofourknowledge,thisisthefirst
HNCcells(9).Othercytokinesandchemokinesthatwere reporttoshowthatSHP2issignificantlyoverexpressedin
analyzedinthismultiplexELISApanel(IL-1b,IL-1RA,IL-2, HNCtissue(Fig.1)withimportantbiologicconsequences.
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Published OnlineFirst January 30, 2013; DOI: 10.1158/1078-0432.CCR-12-1517
Leibowitzetal.
Untreated
Control siRNA
SHP2 siRNA Figure6. SHP2siRNAinducedthe
IFN-γ secretionofRANTESandIP10
A B fromHNCcells.MultiplexELISA
PCI-13 SCC-90
6,000 * *P < 0.008 6,000 * * *P < 0.003 (oLnumsuinpeexrn)aatsasnatyssfrwoemre(Ac)oPnCduI-c1t3ed
and(B)SCC-90cellsfollowing
* transfectionwiththeindicated
siRNA(100nmol/L,48hours)
L ortreatmentwithIFN-g
m
g/ 3,000 3,000 (100U/mL¼1,464pg/mL,48
p hours).Datarepresent3
independentexperimentsand
errorbarsindicateSE.
0 0
MIG RANTES IP-10 IFN-γ MIG RANTES IP-10 IFN-γ
OurinvitroresultsshowthatSHP2negativelyregulates renalcellcarcinoma(38),colorectalcarcinoma(39),small
STAT1 phosphorylation in HNC cells. Expression of WT celllungcarcinoma(40),andmelanoma(41).Thus,inves-
SHP2decreasedbasalandinducedpSTAT1(Fig.4A),SHP2 tigation of the molecular regulators responsible for APM
depletion upregulated basal levels of pSTAT1 and total componentdefectsisclinicallyimportantinHNCandthe
STAT1 (Fig. 2B, C, E, H, I, and K) and significantly aug- information derived from these studies may be generally
mented TAP1/2 expression by IFN-g in STAT1-expressing applicable to a variety of other tumor types for which
cellsbutnotinSTAT1-deficientcells(Fig.4BandC).Wedid immunotherapy is currently being evaluated. As mechan-
not observe differences in TAP1 expression after SHP2 ismsofimmuneescapeusedbytumorcellsarecomplexand
depletion without pretreatment with IFN-g in STAT-1þ/þ notmutuallyexclusive,werecognizethatothersuppressive
andSTAT-1(cid:1)/(cid:1)fibrosarcomatumorcelllines,butdetailsof effectsonCTL,suchasbyTreg,mayalsocooperatetoreduce
theimmuneescapemechanismsintheseuniquefibrosar- eliminationoftumorcells.ThefindingthatSHP2depletion
comacellsisaninterestingquestionofinterestinourfuture enhancessomechemoattractingT 1typechemokinespro-
H
studies.Fibrosarcomatumorcellslikelyhaveothermechan- videssupportforthisapproachtoreversetheimmunosup-
isms that suppress APM expression beyond SHP2 activity pressivephenotypeinthemicroenvironment.
andthattheIFN-g overcomesthoseothermechanisms. Previously,weshowedthatIFN-gtreatmentcouldrestore
AspSTAT1homodimersareimportantregulatorsofAPM CTLrecognitionandlysisofHNCcells(4),indicatingthata
componentgenes(9,24),ourobservationthatpSTAT1was regulatory,asopposedtoageneticmechanismwasrespon-
upregulatedinresponsetoSHP2(butnotSHP1)depletion sible for this escape phenotype. Furthermore, HNC cells
inHNCcellssuggestedthatthiseffectcouldbesufficientto werefoundtoexpressabundantlevelsoftotalSTAT1,but
induceAPMcomponentexpression,atalevelcomparable lack phosphorylated STAT1 (9). These finding suggested
withthatinducedbyIFN-g treatment.Indeed,HLAclassI thatanegativeregulatorofSTAT1phosphorylationmight
APM component expression was upregulated after SHP2 be responsible for the low pSTAT1 phenotype observed.
depletion (Fig. 3), and potentially induced HNC cell rec- Othershaveshownthatproteintyrosinephosphatasescan
ognition by tumor antigen-specific CTL (Fig. 5). Interest- dephosphorylate and inactivate STAT1 (42); therefore we
ingly,knockdownofSHP2alsoinducedHNCcellsecretion investigatedwhetherproteintyrosinephosphatasesmight
ofproinflammatory,T 1-associatedchemokines,RANTES be responsible for low basal STAT1 phosphorylation in
H
and IP-10 (Fig. 6). These findings have potential clinical HNCcells.Indeed,treatmentofapanelofHNCcellswith
significancebecauseSHP2isupregulatedinHNCtumorsin abroadphosphataseinhibitor,SOV,inducedSTAT1phos-
vivo, suggesting that targeting SHP2 could increase the phorylation to levels comparable or greater than those
immunogenicityofHNCcellsthroughupregulationofHLA inducedbyIFN-g treatment(datanotshown).Thesedata
classIAPMcomponentswhilechemoattractingna€(cid:2)veand suggestedthataphosphatasecouldcontributetothelackof
þ þ
memory (CCR5 ) and T 1 (CXCR3 ) lymphocytes (35). pSTAT1expressioninHNCcells,maintainingabasaldefect
H
ThisstrategycouldovercomeamajorlimitationofT-cell– intheproapoptoticIFN-gsignaltransductionpathway,and
basedcancerimmunotherapy,whichistherelianceonthe promoting immune escape. Further studies will analyze
tumor cell to serve as a target for recognition by tumor whether SHP2 overexpressed by HNC cells also contain
antigen-specificTlymphocytes. activatingmutations(30)orcanberegulatedbyimmuno-
APM component downregulation as an escape mecha- suppressivecytokine(s),assuggestedbyothers(30,43).
nism(6,7)ofHNCcellsfromCTLrecognitionalsocorre- The unexpected observation that SHP2 depletion not
lateswithpoorprognosis(5,6,36,37),andisobservedin only reversed APM component downregulation and
806 ClinCancerRes;19(4)February15,2013 ClinicalCancerResearch
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SHP2CausespSTAT1-DependentImmuneEscape
restored tumor antigen-specific CTL recognition of HNC tumorantigen-specificimmuneeffectorcellstothetumor
cells (Figs. 3 and 5), but also induced the secretion of microenvironment. Future studies will investigate the
RANTESandIP-10bythetumorcells(Fig.6)haspotential effectofSHP2inhibitionontumorcellgrowthinvivousing
implications for T-cell–based immunotherapy. RANTES newly developed small-molecule targeted agents as adju-
strongly chemoattracts several immune cells, including vant efforts to enhance T-cell–based immunotherapeutic
eosinophils,basophils,mastcells,monocytes,naturalkiller approaches.
þ
(NK) cells, CTL, naive CD4 T cells, and memory T cells
(44). RANTES also induces intratumoral infiltration of DisclosureofPotentialConflictsofInterest
þ
dendritic cells, CD4 T cells, CTLs, and NK cells (45) Nopotentialconflictsofinterestweredisclosed.
resultingintumorgrowthinhibitioninvivo(46).IP-10has
also been found to have antitumor activity by attracting Authors'Contributions
þ
CD4 Tcells,CTLs,andNKcellstothetumormicroenvi- Conception and design: M.S. Leibowitz, P.A.A. Filho, R.R. Seethela, S.
ronment (47). Indeed, successful T-cell–based immuno- Ferrone,R.L.Ferris
Developmentofmethodology:M.S.Leibowitz,R.L.Ferris
therapyhasbeenlinkedtoCXCR3expressionasabiomark- Acquisitionofdata(providedanimals,acquiredandmanagedpatients,
er for active, tumor homing T 1 antitumor lymphocytes providedfacilities,etc.):R.M.Srivastava,P.A.A.Filho,A.M.Egloff,R.R.
H
Seethela,R.L.Ferris
(35,48).
Analysisandinterpretationofdata(e.g.,statisticalanalysis,biosta-
Interestingly,MIG,anotherCXCR3ligand,andMIP1-a tistics, computational analysis): M.S. Leibowitz, R.M. Srivastava, A.M.
(data not shown) and MIP1-b, CCR5 ligands, were not Egloff,L.Wang,R.R.Seethela,S.Ferrone,R.L.Ferris
Writing,review,and/orrevisionofthemanuscript:M.S.Leibowitz,R.M.
upregulatedafterSHP2siRNAtransfection,showingspec-
Srivastava,S.Ferrone,R.L.Ferris
ificityinchemokinesecretionbythetumorcellsafterSHP2 Administrative,technical,ormaterialsupport(i.e.,reportingororga-
depletion. Blockade of several STAT1 signaling receptors, nizingdata,constructingdatabases):M.S.Leibowitz,R.L.Ferris
Studysupervision:R.L.Ferris
suchasIFN-gR,IFN-aR,andIL-10R,didnotinhibitSHP2
siRNA-mediated STAT1 phosphorylation in HNC cells
Acknowledgments
(datanotshown).Furtherworkisongoingtoelucidatethe
TheauthorsthankDrs.AmyK.Wesa(Celsense,Inc)andYvonneK.Mburu
STAT1 agonist(s) secreted by HNC cells following SHP2 (UniversityofPittsburgh)forcriticallyreviewingthemanuscript.
knockdown that are responsible for STAT1 phosphoryla-
tion and induction of these chemokines, as well as their
GrantSupport
cognatereceptors.
This study was supported by research funding from the NIH, grants
In summary, SHP2 was investigated as a negative regu- DE019727, CA110249, CA097190, and facilities and cores of the UPCI
latorofSTAT1phosphorylationandAPMcomponentsin P30CA047904.
The costs of publication of this article were defrayed in part by the
HNC cells. These novel data strongly indicate that SHP2 payment of page charges. This article must therefore be hereby marked
overexpression in HNC tumors in vivo may contribute to advertisementinaccordancewith18U.S.C.Section1734solelytoindicate
thisfact.
APMcomponentdownregulationandCTLevasionbyHNC
cells.AsSHP2depletionincreasestheimmunogenicityof
ReceivedMay13,2012;revisedNovember13,2012;acceptedDecember2,
HNC tumor cells, and may also induce recruitment of 2012;publishedOnlineFirstJanuary30,2013.
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