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THEJOURNALOFBIOLOGICALCHEMISTRYVOL.289,NO.21,pp.14520–14533,May23,2014
©2014byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Pancreatic Cancer Stem-like Cells Display Aggressive
Behavior Mediated via Activation of FoxQ1□S
Receivedforpublication,November5,2013,andinrevisedform,March25,2014Published,JBCPapersinPress,April9,2014,DOI10.1074/jbc.M113.532887
BinBao‡,AsfarS.Azmi‡,AmroAboukameel§,AamirAhmad‡,AlicciaBolling-Fischer§,SeemaSethi‡,ShadanAli¶,
YiweiLi‡,DejuanKong‡,SanjeevBanerjee‡,JessicaBack(cid:2),andFazlulH.Sarkar‡§1
Fromthe‡DepartmentofPathology,§GenomicsCoreFacility,¶DepartmentofOncology,and(cid:2)FlowCytometryCoreFacility,
KarmanosCancerInstitute,WayneStateUniversitySchoolofMedicine,Detroit,Michigan48201
Background:Cancerstemcells(CSCs)correlatetopoorerclinicaloutcomesofmanytumors.
Results:Weidentifiedahighlyaggressive(CD44(cid:2)/CD133(cid:2)/EpCAM(cid:2))CSC-likefractionfrompancreaticcancer(PC)celllines
withdifferentialexpressionofmanygenes,includingFoxQ1.
Conclusion:FoxQ1knockdowninhibitedCSCaggressiveness.
Significance:TargetingdifferentiallyexpressedCSC-relatedgenescouldprovideanovelapproachforbettertreatmentout-
comesofaggressivePCtumors.
Subpopulationsofcancerstemcells(CSCs)orcancerstem- Cancerstem-likecells(CSLCs)2reminiscentofcancerstem
like cells (CSLCs) have been identified from most tumors, cells(CSCs)ortumor-initiatingcellsdefinedbyCD133(cid:2)were
includingpancreaticcancer(PC),andtheexistenceofthesecells isolatedandcharacterizedfromthebonemarrowofacutemye-
is clinically relevant. Emerging evidence suggests that CSLCs loidleukemiapatientsin1997(1),whichmarkedthediscovery
participate in cell growth/proliferation, migration/invasion, oftheroleofCSLCsintumorigenesisandtumorprogression.
metastasis,andchemo-radiotherapyresistance,ultimatelycon- AlthoughCSLCsaresimilartonormaladultstemcellstosome
tributingtopoorclinicaloutcome.However,thepathogenesis extent,especiallytheirself-renewalanddifferentiationcapac-
and biological significance of CSLCs in PC has not been well ity, CSLCs have unique characteristics, such as their highest
characterized. In the present study, we found that isolated capacityofself-renewal,differentiationintomultiplecelllineages
triple-marker-positive (CD44(cid:2)/CD133(cid:2)/EpCAM(cid:2)) cells of with greater proliferative potential, prolonged life span, and
humanPCMiaPaCa-2andL3.6plcellsbehaveasCSLCs.These tumor-initiatingpotentialinvivo(2–4).Alargenumberofstudies
haveshownclearevidenceinsupportoftheexistenceofCSLCs
CSLCs exhibit aggressive behavior, such as increased cell
andtheirclinicalimplicationsbecausetheraresubpopulationsof
growth, migration, clonogenicity, and self-renewal capacity.
CSLCshavebeenidentifiedfrommosttumors,suchasprostate,
The mRNA expression profiling analysis showed that CSLCs
(CD44(cid:2)/CD133(cid:2)/EpCAM(cid:2)) exhibit differential expression of lung,breast,pancreas,brain,gastric,andcolorectaltumors.These
CSLCsareinvolvedincellgrowth,migration/invasion,andapo-
morethan1,600mRNAs,includingFoxQ1,comparedwiththe
triple-marker-negative (CD44(cid:3)/CD133(cid:3)/EpCAM(cid:3)) cells. The ptosisresistance,attributingtotreatmentresistanceandmetasta-
sis,leadingtopoorclinicaloutcome(2–4).However,thepatho-
knockdown of FoxQ1 by its siRNA in CSLCs resulted in
genesisofCSLCsduringtumorigenesisandtumorprogressionhas
theinhibitionofaggressivebehavior,consistentwiththeinhibi-
notbeenwelldocumented.
tionofEpCAMandSnailexpression.Mousexenografttumor
Although significant advances have been made in the fight
studiesshowedthatCSLCshavea100-foldhigherpotentialfor
againstcancers,pancreaticcancer(PC)remainsoneofthemost
tumorformationandrapidtumorgrowth,consistentwithover-
aggressive and lethal malignant diseases in the world, and
expression of CSC-associated markers/mediators, including
remains the 4th leading cause of cancer-related death in the
FoxQ1,comparedwithitsparentalMiaPaCa-2cells.Theinhi-
United States (5). For example, it was estimated that 45,220
bitionofFoxQ1attenuatedtumorformationandgrowth,and
peoplewouldbenewlydiagnosedwithPC,and38,460patients
expressionofCSCmarkersinthexenografttumorderivedfrom
would die in 2013 (5). Due to the lack of specific signs and
CSLCsofMiaPaCa-2cells.Thesedataclearlysuggesttheroleof
symptomsandthelackofearlydetectiontechniquesforPC,the
differentiallyexpressedgenesintheregulationofCSLCcharac- majorityofpatientsarediagnosedatanadvancedstage((cid:3)80%
teristics,furthersuggestingthattargetingsomeofthesegenes of newly diagnosed cases). The conventional treatments,
couldbeimportantforthedevelopmentofnoveltherapiesfor includingsurgicalresectionsandchemo-radiotherapyarenot
achievingbettertreatmentoutcomeofPC. effective, which is in part due to therapeutic resistance and
greater potential for locally advanced and metastatic disease.
The majority of patients will die within an average of 5–6
□S ThisarticlecontainssupplementalTable1. monthsafterdiagnosis.Theoverall5-yeardisease-freesurvival
ThedatareportedinthispaperhavebeendepositedintheGeneExpressionOmnibus
(GEO)database,www.ncbi.nlm.nih.gov/geo(accessionno.GSE51971).
1Towhomcorrespondenceshouldbeaddressed:Dept.ofPathology,Karma- 2Theabbreviationsusedare:CSLC,cancerstem-likecell;CSC,cancerstem
nosCancerInstitute,WayneStateUniversitySchoolofMedicine,740Hud- cell;PC,pancreaticcancer;PI,propidiumiodide;MTT,3-(4,5-dimethylthi-
sonWebberCancerResearchCenter,4100JohnRSt.,Detroit,MI48201. azol-2-yl)-2,5-diphenyltetrazoliumbromide;EMT,epithelial-to-mesen-
Tel.:[phone];Fax:[phone];E-mail:[email protected]. chymaltransition.
This is an open access article under the CC BY license.
14520 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER21•MAY23,2014
DeregulatedExpressionofGenesinCancerStem-likeCells
rateis1–4%.Ithasbeenreportedthatverysmallsubpopula- Cell Growth Assay—The 3-(4,5-dimethylthiazol-2-yl)-2,5-
tions of CSCs (CSLCs), positive for CD133, can be identified diphenyltetrazolium bromide (MTT) assay was conducted to
fromPCtissues(6).TheseCSLCsexhibitmoreaggressivephe- assessthecellsurvivalorgrowth.Briefly,5,000cells/wellwere
notypes,suchasincreasedtumorigenicandmetastaticpoten- seeded in a 96-well plate and incubated in 5% FBS-DMEM
tialsinvitroandinvivo,respectively(6,7).Therefore,thedis- mediumovernight.Afterchangingthemedium,thecellswere
covery,mechanisticunderstanding,andtargetingapproachfor continued for the incubation. After 3 days of incubation, the
killingofCSLCscouldopenneweravenuesforthetreatment cellswereharvestedforthestandardMTTassay,asdescribed
and/orpreventionoftumoraggressivenessforPC. previously(8,11).
Inthepresentstudy,weisolatedandcharacterizedCD44(cid:2)/ Colony Formation Assay—The colony formation assay was
CD133(cid:2)/EpCAM(cid:2)(triple-marker-positive)cellsfromhuman conducted to assess clonogenic potential of the cells, as
PC cells (MiaPaCa-2 and L3.6pl cells) as the CSLCs. These described previously (8, 11). Briefly, 1,000 single viable cells
CSLCs(triple-marker-positivecells)displayaggressivebehav- wereseededin10mlof5%FBS-DMEMin10-cmPetridishes.
ior,suchasenhancedcellgrowth,clonogenicity,cellmigration, Thecellswerethenincubatedat37°Cinatissuecultureincu-
and CSC self-renewal capacity, which was consistent with batorfor14days.Colonieswerestainedwith2%crystalviolet,
increasedexpressionofCSCsignatures/mediators,compared washedwithwater,andcounted.
withcellsthataretriple-marker-negative.Thegeneexpression WoundHealingAssay—Thewoundhealingassaywascon-
profiling analysis showed that CSLCs (triple-marker-positive ductedtoassessthemigrationcapacityofthecellsunderdif-
cells) of MiaPaCa-2 cells display differential expressions of ferentexperimentalconditions,asdescribedpreviously(8,11).
more than 1,600 genes, including Bmi1, BMP4, BST2, BTG1, Thecellswereseededinthe6-wellplatesin3mlof5%FBS-
FOLR1,FoxQ1,PRKAR1A,Sox4,TACSTD2,andWnt3a.The DMEMmediumineachwell.Whentheconfluenceofthecells
knockdownofFoxQ1byitssiRNAledtoattenuateaggressive- grewto95%orabove,theywerethenscratchedwitha200-(cid:2)l
nessandCSLCcharacteristics,whichwasconsistentwithinhi- pipettetip.Thewoundhealingcapacitywasassessedandpho-
bitionofEpCAMandsnailexpression.Theinvivostudiesusing tographedwithacamera-equippedinvertedmicroscopeafter
a mouse xenograft tumor model showed that CSLSs derived 20and48hofincubation.
from MiaPaCa-2 cells display a 100-fold higher potential for Cell Cycle Analysis—A propidium iodide (PI) staining/flow
tumorformationandalsofastertumorgrowth,whichwasconsis- cytometricassaywasconductedtoassessthecellcycleunderdif-
tentwithoverexpressionofCSC-associatedmarkers/mediators, ferentexperimentalconditions.Afterwashing,thecellswerefixed
includingFoxQ1,comparedwithitsparentalcells.Theinhibition with4mlof75%ethanolat(cid:4)20°Covernight.Thefixedcellswere
ofFoxQ1byitssiRNAattenuatedtumorformationandgrowth, washedandstainedwithPI-RNaseAsolution(Invitrogen)at4°C
consistentwiththedown-regulationofCSCmarkers/mediatorsin for3h,followedbyastandardflowcytometricassay.
xenograft tumor derived from CSLCs of MiaPaCa-2 cells. Our Apoptosis Assay—The annexin V/PI staining and flow
observation suggests that pathways that are activated in CSLCs cytometry assay was performed to evaluate apoptosis in the
couldbetargetedasnoveltherapiesforPC. cellsbyusingtheFITCannexinVapoptosisdetectionkit(BD
Pharmingen),followingthemanufacturer’sinstructions.
MATERIALSANDMETHODS
QuantitativeReal-timeRT-PCRofmRNAs—ThemRNAlev-
Cell Lines and Culture Conditions—CD44(cid:2)/CD133(cid:2)/Ep- elsofthecellsweremeasuredbythereal-timeRT-PCRassay,as
CAM(cid:2) (triple-marker-positive cells) were isolated as the describedpreviously(8,11).TotalRNAswereisolatedbyusing
CSLCsfromhumanpancreaticcancercelllineMiaPaCa-2and TRIzolsolution(Invitrogen).Reversetranscription(RT)reac-
L3.6pl cells by the fluorescence-activated cell sorting (FACS) tionsofmRNAswerethenconductedbyusingtheHighCapac-
technique and cultured in the serum-free sphere formation ityRNA-to-cDNAassaykit(AppliedBiosystems),followingthe
medium(1:1DMEM/F-12KmediumplusB27andN2supple- manufacturer’s instructions. RT-PCRs of mRNAs were con-
ments, Invitrogen) to maintain its undifferentiated status. ductedbyusingtheCYBRGreenPCRMasterMixkit(Applied
Moreover, triple-marker-negative (CD44(cid:4)/CD133(cid:4)/EpCAM(cid:4)) Biosystems).TheprimersofspecificcDNAsweresynthesized
cells were isolated from MiaPaCa-2 and L3.6pl cells by the from Invitrogen. The C values were used for data analysis.
t
FACS technique and were also cultured in 5% fetal bovine GAPDHmRNAwasusedasthecontroltonormalizethedataof
serum(FBS)-DMEMat37°Cinstandardcultureconditions,as mRNAsineachsample.
describedpreviously(8,9).CD44(cid:2),CD133(cid:2),andEpCAM(cid:2)are Immunostaining and Confocal Imaging Microscopy—The
knownasstemcellsurfaceproteins,whichhavebeenconsid- proteinexpressionsinthecellswereassessedbyanimmuno-
eredasthepancreaticCSLC(CSC)markers(3,6,10). staining/confocal imaging microscopic assay. After the cells
Sphere Formation Assay—The sphere formation assay was reached 50–70% confluence on the 4-chamber culture glass
conducted to assess the CSLC self-renewal capacity, as slides in 0.5 ml of 5% FBS-DMEM, the cells were then har-
describedpreviously(8,9).Briefly,1,000singlesuspendedcells vested,washed,fixedwith4%formaldehydePBSsolution,and
wereseededontheultralowattachmentwellsofCostar6-well permeabilized with 0.05% Tween 20 solution. Antibodies
plates(CorningInc.)in2mlofsphereformationmedium.After againstCD44,EpCAM,Notch-1,EZH2,andSnail(CellSig-
7 days of incubation, the sphere cells termed “pancreato- naling) were applied for immunostaining, following the
spheres”wereharvestedbycentrifugation(300(cid:5)gfor5min). manufacturer’s protocol. DAPI solution (Invitrogen) was
The number of pancreatospheres was counted under a con- used to stain the nucleus of the cells as the control. The
vertedmicroscope. antibody-labeled cells were examined and photographed
MAY23,2014•VOLUME289•NUMBER21 JOURNALOFBIOLOGICALCHEMISTRY 14521
DeregulatedExpressionofGenesinCancerStem-likeCells
FIGURE1.ThecharacteristicsofCSLCs(triple-marker-positive;CD44(cid:2)/CD133(cid:2)/EpCAM(cid:2)cells).A,recoveryoffournumbersofCSLCs(triple-marker-
positivecells)isolatedfromhumanpancreaticcancerMiaPaCa-2cellsbyFACSanalysisafter4weeksofincubationinsphereformationmedium(A,a–d).
eandf,photographofsphereformationinCSLCs(triple-marker-positivecells)after7daysofincubationinsphereformationmedium.B,thepercent-
ages of CD44(cid:2)/CD133(cid:2)/EpCAM(cid:2) cells in MiaPaCa-2, triple-marker-negative CD44(cid:4)/CD133(cid:4)/EpCAM(cid:4), and triple-marker-positive CD44(cid:2)/CD133(cid:2)/
EpCAM(cid:2)cellsbyflowcytometry.C,higherself-renewalcapacityofCSLCs(triple-marker-positivecells)asassessedbythesphereformationassay.D,the
CSCself-renewalcapacityofcellswithdifferentcombinationsofCSLCtriplemarkersisolatedfromMiaPaCa-2cellsbytheFACStechnique.Thesphere
formationassaywasperformedtoevaluatetheCSCself-renewalcapacityofcellswithdifferentcombinationsofCSLCtriplemarkersisolatedfrom
MiaPaCa-2cellsbytheFACStechnique.Errorbars,S.D.(n(cid:6)3).*,p(cid:6)0.05(n(cid:6)3)comparedwithCSLCtriple-marker-positivecells.
under a confocal imaging microscope (EVOS Imaging Sys- cturer’s instructions. Total RNA quantity and quality was
tems,(cid:5)100magnifications). examinedbyanalysisusingNanoDropandtheAgilentBioana-
WesternBlotAnalysis—Therelativelevelsofproteinsinthe lyzer(AgilentTechnologies).AlloftheRNAsampleshadRNA
cells/tumortissuesweremeasuredbyWesternblotanalysis,as Integrity Number (RIN) scores of (cid:5)7. The whole genome
describedpreviously(9).Theproteinlysisofthesampleswas expression profiling was analyzed by a two-color microarray-
prepared as described previously (9). The antibodies against basedapproach.TheRNAsampleswerehybridizedtoAgilent
EpCAM, EZH2, Notch-1, Snail, p65, phospho-Akt, and hyp- 4(cid:5)44khumanarraysandscannedwiththeAgilentG2505B
oxia-inducible factor-1(cid:3)were obtained from Cell Signaling scannersystem.AllofthedatawereanalyzedbyAgilentFea-
Technology. The antibodies against FoxQ1, IL-6, VEGF, and tureExtractionsoftwarethatgeneratedexpressiondataparam-
Bmi1wereobtainedfromSantaCruzBiotechnology.Theanti- eters,includingLogRatiopvalues,LogRatioerror,andpvalue
bodyagainst(cid:4)-actinwasobtainedfromSigma. LogRatio.Thefeaturesincludedinfurtheranalysiswereanno-
TheGeneExpressionProfiling—ThemRNAmicroarrayassay tated, and gene level passed a p value LogRatio cut-off of
was conducted for gene expression profiling analysis. Briefly, (cid:6)0.001.Analysisofvarianceandmultipletestcorrection(Ben-
purified total RNAs were isolated by using the Vana mRNA jamin-Hochberg p (cid:6) 0.05) was conducted using Partek soft-
isolationkit(AmbionInc.,Austin,TX),followingthemanufa- waretocomparethetwosetsoffourtwo-colorarrayedrepli-
14522 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER21•MAY23,2014
DeregulatedExpressionofGenesinCancerStem-likeCells
FIGURE2.Thecellgrowth,migration,andclonogenicityofCSLCs(triple-marker-positivecells)comparedwithtriple-marker-negativecells.A,thecell
growthwasevaluatedbyanMTTassay.B,morphologyofCSLCs(triple-marker-positivecells)derivedfromMiaPaCa-2cellscomparedwithtriple-marker-
negativecellswasassessedat0,20,and48hofincubationandphotographedusingaconvertedmicroscope((cid:5)20magnification).C,cellmigrationcapacity
ofCSLCs(triple-marker-positivecells)comparedwithtriple-marker-negativecellswasevaluatedbyawoundhealingassay((cid:5)10magnification).D,clonogenic
potentialsofCSLCs(triple-marker-positivecells)comparedwithtriple-marker-negativecellsasassessedbyacolonyformationassay.E,cellmigrationof
triple-marker-negativecells(CD44(cid:4)/CD133(cid:4)/EpCAM(cid:4))andtheirparentalMiaPaCa-2cells.F,clonogenicityoftriple-marker-negativecells(CD44(cid:4)/CD133(cid:4)/
EpCAM(cid:4))andtheirparentalMiaPaCa-2cells.Errorbars,S.D.*,p(cid:6)0.05(n(cid:6)3).
TABLE1 TABLE2
ApoptosisoftheCSLCtriple-marker-positivecellsversusthetriple- CellcyclesofCSLCtriple-marker-positivecellsversustriple-marker-
marker-negativecellsderivedfromMiaPaCa-2cells negativecells
CellswereincubatedinFBS-freesphere-formingmediumfor7days,followedby Cellcycleswereanalyzedbypropidiumiodide(PI)stainingandflowcytometric
annexinVstaining,andassessedbyflowcytometricanalysis.Means(cid:4)S.D.are assay.Means(cid:4)S.D.areshown.n(cid:6)3independentexperiments.
shown.n(cid:6)3independentexperiments.
Triple-marker- CSLCtriple-marker-
Triple-marker- CSLCtriple-marker- negativecells positivecells p
negativecells positivecells p (n(cid:5)3) (n(cid:5)3) value
(n(cid:5)3) (n(cid:5)3) value G (%) 84.50(cid:4)2.70 73.67(cid:4)1.12 0.0002
Alive(%) 50.60(cid:4)1.85 59.43(cid:4)0.40 0.003 G1(%) 4.74(cid:4)2.11 8.98(cid:4)2.30 0.0174
Earlyapoptotic(%) 22.47(cid:4)2.30 12.17(cid:4)0.63 0.004 2
Lateapoptotic(%) 14.03(cid:4)1.35 22.50(cid:4)0.30 0.001 Sphase(%) 10.77(cid:4)4.80 17.35(cid:4)3.32 0.0325
Non-apoptoticdead(%) 12.93(cid:7)1.86 5.91(cid:7)1.08 0.009
Farmsandmaintainedbyfeedingregularanimaldiets(LabDiet
catesfortheidentificationofthegeneexpressionlevelchanges 5021(PurinaMills,Inc.)).2(cid:5)106MiaPaCa-2cellsor2(cid:5)104
((cid:5)2-foldchanges). CSLC triple-marker-positive cells (pancreatosphere cells)
TransfectionsofFoxQ1siRNA—FoxQ1siRNA(AppliedBio- under different conditions (control and FoxQ1 siRNA treat-
systems)wastransfectedintotheCSLCs(triple-marker-posi- ment)wereimplantedasdescribedpreviously(9).Thetrans-
tive cells) by using the DharmaFECT transfection reagent fectionofsiRNAswasdescribedasabove.Thetumorformation
(ThermoScientific)followingthemanufacturer’sinstructions, andgrowthweremonitoredeveryotherday.After34daysof
asdescribedpreviously(8,11).ThescrambledsiRNA(negative inoculation, all of the animals were sacrificed, and tumor
control 2, Applied Biosystems) was used as the negative con- tissues from all of the animals were collected for further
trol.Thetransfectedcellswereusedforeitherthespherefor- analysis,suchasRT-PCR,Westernblotanalysis,andhisto-
mationassay,woundhealingassay,orreal-timeRT-PCRassay logicalexamination.
asdescribedabove. Immunohistochemistry—Freshtumortissueswerefixedwith
Animal Experiment—The protocol for the animal experi- zincformalinsolution(ThermoScientific)overnightandeval-
mentwasapprovedbytheAnimalInvestigationCommitteeof uated by hematoxylin and eosin staining, and Immunohisto-
WayneStateUniversity.Four-week-oldCB17severecombined chemistrywasperformedbyalicensedlaboratorytechnicianin
immunodeficient (SCID) mice were obtained from Taconic theCoreFacilityoftheDepartmentofPathology,WayneState
MAY23,2014•VOLUME289•NUMBER21 JOURNALOFBIOLOGICALCHEMISTRY 14523
DeregulatedExpressionofGenesinCancerStem-likeCells
FIGURE3.TheexpressionofCSLCmarkersintriple-marker-positivecellscomparedwithtriple-marker-negativecells.Confocalimagingmicroscopywas
performedtoevaluatetheproteinexpressionofCD44,EpCAM,EZH2,Notch-1,andSnail.DAPIwasusedtostainthenucleusofthecells.Magnification,(cid:5)100.
University/KarmanosCancerInstitute,asdescribedpreviously CSLC Self-renewal Capacity of Triple-marker-positive
(9).TheantibodiesagainstCD44,EpCAM,Notch-1,andEZH2 Cells—Thesphereformationassaywasconductedtoassessthe
wereobtainedfromCellSignalingTechnology.Thestainedslides CSLCs self-renewal capacity. The results show that the triple-
were examined by a licensed pathologist in the Department of marker-positivecellshadsignificantlyincreasednumbersofpan-
Pathology,WayneStateUniversity/KarmanosCancerInstitute. creatospheresafter7daysofincubationintheserum-freesphere
StatisticalAnalysis—Thedatawerepreparedandpresented formation medium, compared with triple-marker-negative
asmeanandS.D.byusingGraphPadPrismandExcelsoftware. cells (Fig. 1, A (e and f) and C). We also examined CSC self-
Student’s t test was performed to test significant difference renewalcapacityusingdifferenttriple-markercombinedphe-
between two groups. The p value equal or less than 0.05 was notypesofMiaPaCa-2cells,suchasCD44(cid:4)/CD44(cid:4)/EpCAM(cid:4),
consideredasstatisticallysignificant. CD44(cid:2)/CD133(cid:4)/EpCAM(cid:4), CD44(cid:2)/CD133(cid:2)/EpCAM(cid:4), CD44(cid:2)/
CD133(cid:4)/EpCAM(cid:2), and CD44(cid:2)/CD133(cid:2)/EpCAM(cid:2) cells iso-
RESULTS
lated by the FACS technique. The results showed that CSC
Isolations of CSLC Triple-marker-positive Cells and Triple-
triple-marker-positive cells had significantly higher levels of
marker-negative Cells—The CSLCs (triple-marker-positive
sphere formation, compared with their parental MiaPaCa-2
cells)wereisolatedandestablishedfromfourCD44(cid:2)/CD133(cid:2)/
cellsandotherphenotypesoftriple-markercombinations(Fig.
EpCAM(cid:2)(triple-marker-positive)cellsofhumanPCMiaPaCa-
1D).Therewasnosignificantdifferenceinthesphereforma-
2cellsbyusingtheFACStechnique.Thetriple-marker-nega-
tioncapacitybetweenMiaPaCa-2cellsandtriple-marker-neg-
tive cells were also isolated and established from 786,353
CD44(cid:4)/CD133(cid:4)/EpCAM(cid:4) (triple-marker-negative) cells of ativecells(p(cid:8)0.05;n(cid:6)3;Fig.1D).Thesefindingssuggestthat
the CSLCs (triple-marker-positive cells) have a remarkably
MiaPaCa-2cellsbytheFACStechnique.After4weeksofincu-
higherpotentialofself-renewal,suggestingthattriple-marker-
bation in the serum-free sphere formation medium, four iso-
positiveCSLCsaresimilartoCSCs.
latedCSLCs(triple-marker-positivecells)survivedandunder-
Cell Growth of CSLCs (Triple-marker-positive Cells)—The
wentself-renewaltobecomemorethanonemillionofthecells
MTTassaywasconductedtoassessthecellgrowth/prolifera-
(Fig.1A,bandd).However,fourisolatedtriple-marker-nega-
tion of the CSLCs (triple-marker-positive cells) in 5% FBS-
tivecellsfailedtosurviveandgrow(Fig.1A,aandc).Aflow
DMEMmedium.Theresultsshowthatthetriple-marker-pos-
cytometricassaywasconductedtoassessthepercentageoftri-
ple-marker-positive cells in each subpopulation of cells. The itive cells (CSLCs) had a remarkably increased cell growth
resultsshowedthatonly0.18%ofcellsweretriple-marker-pos- capacityafter3daysofincubation,comparedwiththetriple-
itive(CD44(cid:2)/CD133(cid:2)/EpCAM(cid:2))inMiaPaCa-2cells(Fig.1B). marker-negative cells (p (cid:9) 0.05; n (cid:6) 3) (Fig. 2A). The triple-
Triple-marker-negativecellscontainedlessthan0.01%triple- marker-negative cells exhibited more morphological changes
marker-positive (CD44(cid:2)/CD133(cid:2)/EpCAM(cid:2)) cells (Fig. 1B). similartothoseinapoptoticcellsthandidthetriple-marker-
However, the CSLCs contained 56% triple-marker-positive positivecells(Fig.2B).
(CD44(cid:2)/CD133(cid:2)/EpCAM(cid:2))cells(Fig.1B).Thesedatasuggest Apoptosis of CSLCs (Triple-marker-positive Cells)—The
thatthetriple-marker-negativeandtriple-marker-positivecells results from the apoptosis assay showed that CSLC triple-
havestablegenotypes. marker-positivecellsderivedfromMiaPaCa-2cellshadmore
14524 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER21•MAY23,2014
DeregulatedExpressionofGenesinCancerStem-likeCells
FIGURE4.A,pathwayenrichmentanalysisofthealteredexpressionofgenesinCSLCs(triple-marker-positivecells)ofMiaPaCa-2cellscomparedwithtriple-
marker-negativecells.B,heatmapofselectiveCSLCsandtumor-associatedgenesinthetriple-marker-positivecells.MicroarrayofmRNAswasperformedby
usingAgilent4(cid:5)44khumanarraychipsatleastintriplicates.Green,negative-foldchanges;red,positive-foldchanges,comparedwithparentalMiaPaCa-2and
triple-marker-negativecells.
aliveandfewerearlyapoptotic/deadcells,comparedwiththe asignificantlydecreasedG phaseandhadincreasedG phase
1 2
triple-marker-negative cells cultured in the FBS-free sphere andSphase,comparedwithtriple-marker-negativecells(Table
formationmedium(Table1),andCSLCswereabletoreplicate 2).ThesefindingssuggestthatCSLCs(triple-marker-positive
much faster, compared with the triple-marker-negative cells cells)haveadifferentpatternofcellcycle.
(Figs.1(CandD)and2B).ThesefindingssuggestthatCSLCs CellMigrationofCSLCs(Triple-marker-positiveCells)—The
haveincreasedresistancetoapoptosis. woundhealingassaywasconductedtoassessthecellmigration
Cell Cycle Patterns of CSLCs (Triple-marker-positive capacity of triple-marker-positive cells (CSLCs). The results
Cells)—ThePIstaining/flowcytometryassaywasconductedto showed that the triple-marker-positive cells (CSLCs) have a
assessthecellcycleoftheCSLCs(triple-marker-positivecells). greaterwoundhealingcapacityafter20hand24hofincubation
TheresultsshowthatCSLCs(triple-marker-positivecells)had in5%FBS-DMEM,comparedwiththetriple-marker-negative
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TABLE3
SelectivegeneexpressionprofilesinCSLCtriple-marker-positivecellsversustriple-marker-negativecellsderivedfromMiaPaCa-2cellsas
assessedbymicroarrayanalysis
GenBankTMID Genesymbol Genedescription -Foldchangea
ILMN_1700915 Bmi1 BMI1polycombringfingeroncogene 3.928
ILMN_1740900 BMP4 Bonemorphogeneticprotein4 40.432
ILMN_1723480 BST2 Bonemarrowstromalcellantigen2 14.311
ILMN_1775743 BTG1 B-celltranslocationgene1,anti-proliferative 8.577
ILMN_1661733 FOLR1 Folatereceptor1(adult) 3.103
ILMN_1669046 FoxQ1 ForkheadboxQ1 20.781
ILMN_2389590 PRKAR1A Proteinkinase,cAMP-dependent,regulatory,typeI,(cid:3) 13.915
ILMN_1815745 Sox4 SRY(sex-determiningregionY)-box4 3.08
ILMN_1739001 TACSTD2 Tumor-associatedcalciumsignaltransducer2 37.126
ILMN_1815700 Wnt3a Wingless-typeMMTVintegrationsitefamily,member3A 2.973
a -Foldchange(log)wascalculatedbasedonthedatacomparisonsofCSLCtriple-marker-positivecellsversustriple-marker-negativecells.
2
FIGURE5.ValidationofdifferentialexpressionofselectedCSC-andtumor-associatedgenesinCSLCs(triple-marker-positivecells)ofMiaPaCa-2cells.
Areal-timeRT-PCRassaywasperformedtomeasuretherelativemRNAlevelsinCSLCsandtumor-associatedmarkers.GAPDHmRNAwasusedasthecontrol.
Errorbars,S.D.*,p(cid:6)0.05(n(cid:6)3).
cells (Fig. 2C). There was no significant difference in wound ity between MiaPaCa-2 cells and triple-marker-negative cells
healing capacity between MiaPaCa-2 cells and triple-marker- (p(cid:8)0.05;n(cid:6)3;Fig.2F).ThesefindingssuggestthatCSLCs
negativecells(Fig.2E).Thesedatasuggestthattriple-marker- (triple-marker-positivecells)exhibitsignificantlyhigherclono-
positiveCSLCshaveahighercapacityofcellmigration. genicpotential.
ClonogenicityofCSLCs(Triple-marker-positiveCells)—The CSLCs (Triple-marker-positive Cells) Exhibit High Expres-
clonogenic potential of the cells was assessed by the colony sionofCSCSignatures/Markers—Theconfocalimagingmicro-
formation assay. The results showed that the triple-marker- scopicassaywasconductedtoexaminetheproteinexpression
positivecells(CSLCs)hadasignificantlyincreasedcapacityfor ofCSCsignatures/markersinthecells.Theresultsshowthat
the formation of colonies in 5% FBS-DMEM, compared with theCSLCs(triple-marker-positivecells)hadincreasedprotein
the triple-marker-negative cells (Fig. 2D) (p (cid:9) 0.05; n (cid:6) 3). expressionofCD44,EpCAM,EZH2,Notch-1,andSnailcom-
Therewasnosignificantdifferenceincolonyformationcapac- paredwithtriple-marker-negativecells(Fig.3).CD44,CD133,
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FIGURE6.TheexpressionofselectedgenesassociatedwithoncogenicsignalingpathwaysinCSLCs(triple-marker-positivecells).Areal-timeRT-PCRassay
wasperformedtomeasuretherelativemRNAlevelsinCSLCsandtumor-associatedmarkers.GAPDHmRNAwasusedasthecontrol.Errorbars,S.D.*,p(cid:6)0.05(n(cid:6)3).
andEpCAMareknownascancerstemcellsurfacemarkers(3, genesisinvolvedin21ofthemajorbiologicalfunctiongroups,
6,10).EZH2,Notch-1,andSnailareknowntoplayakeyrolein including cell cycle, polo-like kinase, tight junction, cell-cell
tumordevelopmentandprogressionandhavebeenconsidered junction,insulin-likegrowthfactor-1,PI3K/Akt,ERK/MAPK,
aspotentialCSC-positivemarkers(3,12,13).Ourdatasuggest and VEGF signaling (Fig. 4A). Fig. 4B and Table 3 show the
thattheCSLCs(triple-marker-positivecells)haveremarkably differentialexpressionof10selectedgenes,suchasBmi1(poly-
higherexpressionofCSCsignatures/markers. combringfingeroncogene),BMP4(bonemorphogeneticpro-
DifferentialExpressionProfilingofmRNAsinCSLCs(Triple- tein 4), BST2 (bone marrow stromal cell antigen 2), BTG1
marker-positiveCells)—Microarrayanalysiswasperformedto (B-celltranslocationgene1),FOLR1(folatereceptor1),FoxQ1
examine the differential expression of mRNAs in the CSLCs (forkhead box Q1), PRKAR1A (protein kinase, cAMP-depen-
(triple-marker-positive cells), compared with triple-marker- dent, regulatory, type I, (cid:3)), Sox4 (sex-determining region
negativecells.1,653mRNAswereidentifiedtobedifferentially Y-box 4), TACSTD2 (tumor-associated calcium signal trans-
expressed in CSLCs (triple-marker-positive) versus triple- ducer 2), and Wnt3a (Wingless-type MMTV integration site
marker-negative cells (supplemental Table 1). Among these family,member3A).
genes, 753 mRNAs were up-regulated, and 900 mRNAs were Real-timeRT-PCRAssayofSelectedmRNAs—Weselected10
down-regulated in the CSLCs (triple-marker-positive cells), differentially expressed mRNAs for validation of mRNA
compared with triple-marker-negative cells (supplemental microarray data in the CSLCs (triple-marker-positive cells)
Table 1). 1,581 mRNAs were identified to be differentially compared with triple-marker-negative cells by real-time RT-
expressedinCSLCs(triple-marker-positive)versustheirparen- PCR. The results confirmed that the CSLCs (triple-marker-
tal MiaPaCa-2 cells (data not shown here; see supplemental positive cells) have significantly increased expression of
Table1).Therewere1,216differentiallyexpressedgenesthat Bmi1, BMP4, BST2, BTG1, FOLR1, FoxQ1, PRKAR1A, Sox4,
overlapped between the CSLCs (triple-marker-positive cells) TACSTD2,andWnt3agenes,comparedwiththetriple-mark-
versus triple-marker-negative or MiaPaCa-2 cells (data not er-negative cells (Fig. 5), as shown by the microarray data
shown here; see supplemental Table 1). The pathway enrich- (Table 3). We also examined the gene expression of CSLCs
ment analysis shows that differential expression of selected signaturegenesandtumor-relatedmarkersintheCSLCs(tri-
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FIGURE 7. The expression of CSC-associated proteins in CSLC triple-marker-positive cells and the triple-marker-negative cells derived from
MiaPaCa-2cells.Confocalimagemicroscopy(A)andWesternblotanalysis(B)wereperformedtoevaluatetheexpressionofCSC-associatedmarkerproteins
inCSLCtriple-marker-positivecellsandthetriple-marker-negativecellsderivedfromMiaPaCa-2cells.Anindividualblotwasperformedforeachmarker.
ple-marker-positive cells) by real-time RT-PCR. The results levelsofFoxQ1intheCSLCs(Fig.8,AandB).Theknockdown
show that the CSLCs (triple-marker-positive cells) have of FoxQ1 decreased the relative mRNA and protein levels of
increasedexpressionofNanog,Oct4,Akt,p65,PLK(polo-like EpCAMandSnailinthesecells(Fig.8,AandB).Theknock-
kinase),TGF-(cid:4)(transforminggrowthfactor-(cid:4)),HIF-(cid:3)(hypox- down of FoxQ1 also resulted in a significant decrease in the
ia-induciblefactor-1(cid:3)),VEGF,IL-6(interleukin-6),andIGF-1 formationofpancreatospheresandwoundhealingcapacityof
(insulin-like growth factor-1), compared with triple-marker- thesecells(Fig.8,CandD).Similarly,CSLCtriple-markerpos-
negativecells(Fig.6). itive cells isolated from another human pancreatic cancer
The Expression of CSC-associated Proteins in CSLCs—The L3.6plcellsbyFACStechniquehavealsodisplayedaggressive
resultsfromconfocalimagemicroscopyandWesternblotanal- phenotypes, such as overexpression of FoxQ1, EpCAM, and
ysis showed that CSLC triple-marker-positive cells derived Snail, as well as increased capacity of CSC self-renewal and
from MiaPaCa-2 cells have remarkably higher expression of higher potential of cell migration, compared with the triple-
phospho-Akt,Bmi1,FOLR1,FoxQ1,hypoxia-induciblefactor-1(cid:3), marker-negative cells derived from L3.6pl cells (Fig. 9). The
IL-6,VEGF,Oct4,EZH2,Notch-1,EpCAM,p65,andSnail,com- knockdownofFoxQ1byitssiRNAinhibitedtheseaggressive
paredwiththetriple-marker-negativecells(Fig.7,AandB). phenotypes (Fig. 9), which were observed in CSLCs derived
TheRoleofFoxQ1intheRegulationofCSLCPhenotypesand from MiaPaCa-2 cells. These findings clearly suggest that
FunctionsinTriple-marker-positiveCells—Thealteredexpres- FoxQ1mayplayanimportantroleintheregulationofCSLC
sionofFoxQ1hasbeenreportedtocorrelatewithpoorerclin- phenotypes and functions, and thus targeting FoxQ1 could
ical prognosis of various tumors (14–16). The evidence from becomeanoveltherapeuticapproachforPC.
invitroandinvivoexperimentalstudiessuggeststhatFoxQ1 Tumor Formation and Growth in Mouse Xenograft Tumor
mayplayanimportantroleintumorigenesisandtumordevel- Models—Mouse xenograft tumor studies showed that the
opmentbytheregulationofvariousoncogenicsignalingpath- implantation of 2 (cid:5) 104 CSLSs (pancreatosphere cells) has a
ways(14–16).Inthepresentstudy,theCSLCs(triple-marker- 100-foldhigherpotentialfortumorformation,comparedwith
positivecells)ofMiaPaCa-2cellsshowedhigherexpressionof theimplantationof2(cid:5)106oftheirparentalMiaPaCa-2cells
FoxQ1, compared with triple-marker-negative cells, as (Table4).CSLCshavealsodisplayedrapidgrowthinthexeno-
describedearlier(Figs.4B,5,and7).Toexplorethepotential grafttumormodelafter34daysofinoculation(874.8(cid:7)504.4
roleofFoxQ1intheregulationofCSLCphenotypesandfunc- mg versus 333.5 (cid:7) 163.6 mg, comparing CSLCs versus
tions,weconductedthefunctionallossofexpressionstudyof MiaPaCa-2cells;Table4).TheunderexpressionofFoxQ1byits
FoxQ1 by transfection of its siRNA into the CSLCs (triple- siRNAattenuatedtumorformationandgrowth(Table4).The
marker-positivecells).Theresultsshowthatthetransfectionof histologicalstudyshowedthatxenografttumorsderivedfrom
FoxQ1 siRNA significantly decreased the mRNA and protein CSLCsdisplayedmorenecrosisandadedifferentiatedstateand
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FIGURE8.TheeffectofknockdownofFoxQ1onEpCAMandSnailmRNAs(A)andproteins(B),CSLCself-renewalcapacity(C),andclonogenicity(D)of
triple-marker-positivecellsderivedfromMiaPaCa-2cells.ThetransfectionofFoxQ1siRNAwasperformedinCSLCs(triple-marker-positivecells).Real-time
RT-PCRwasperformedtomeasureFoxQ1,EpCAM,andSnailmRNAsintriple-marker-positivecells.Confocalimagingmicroscopywasperformedtoevaluate
theproteinexpression.DAPIwasusedtostainthenucleusofthecells.Magnification,(cid:5)100.Sphereformationandwoundhealingassayswereperformedto
evaluatetheself-renewalandcellmigrationcapacitiesofCSLCs,respectively.Errorbars,S.D.*,p(cid:6)0.05(n(cid:6)3).
alsohadhigherexpressionofKi-67(tumorproliferationindex) rare population of CSLCs ((cid:9)0.1%) has been found in many
andCSC-associatedproteins,suchasFoxQ1,CD44,EpCAM, different tumors, including pancreatic cancer (PC), and is
EZH2, Notch-1, and Snail, compared with xenograft tumor related to the poorer clinical outcomes, such as reduced dis-
derivedfromtheirparentalMiaPaCa-2cells(Fig.10,AandB). ease-free survival rate and increased mortality (6). Different
The knockdown of FoxQ1 decreased the expression of CSC from tumor non-CSLCs, this minority fraction of CSLC sub-
markers, such as EpCAM and EZH2, in xenograft tumor populations exhibits distinct features, such as the highest
derivedfromCSLCs(Fig.10,CandD).Thesefindingssuggest capacity of self-renewal, prolonged survival potential, and
that CSLCs have very high potential of tumor formation and greater capacity of differentiation into multiple different cell
growth,consistentwithoverexpressionofCSCmarkers/medi- types of tumor cells or tumor-associated cells. Emerging evi-
ators, including FoxQ1, in vivo. The inhibition of FoxQ1 dencesuggeststhatthesefeaturesarebelievedtobeassociated
expressionattenuatedtumorformationandgrowth,consistent with increased cell growth/proliferation, cell migration/inva-
withthedown-regulationofCSCmarkers/mediators,suchas sion,treatmentresistance,andmetastasis,leadingtopoorclin-
EpCAMandEZH2,invivo. icaloutcomeinmosttumors,includingPC(17).Inthepresent
study, we found that CD44(cid:2)/CD133(cid:2)/EpCAM(cid:2) cells (triple-
DISCUSSION
marker-positive cells) of pancreatic cancer cells behave like
IthasnowbeenwellacceptedthatCSCs(CSLCs)playacrit- CSCs (CSLCs). These CSLCs that are characterized as triple-
icalroleduringtumorigenesisandtumorprogressionbecause marker-positive cells have aggressive phenotypes and func-
theexistenceofCSLCshassignificantclinicalimplications.The tions, which is consistent with overexpression of CSC signa-
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