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HNMT Upregulation Induces Cancer Stem Cell Formation and Confers Protection against Oxidative Stress through Interaction with HER2 in Non‐Small‐Cell Lung Cancer

Authors:
Narpati Wesa Pikatan at Taipei Medical University
Narpati Wesa Pikatan
Vijesh K Yadav at Translational Research Laboratory Shuang-Ho Hospital, Taipei Medical University
Vijesh K Yadav
  • Translational Research Laboratory Shuang-Ho Hospital, Taipei Medical University
Chi-Tai Yeh
Chi-Tai Yeh
Chi-Tai Yeh
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Abstract

Background: The treatment of non-small-cell lung cancer (NSCLC) involves platinum-based chemotherapy. It is typically accompanied by chemoresistance resulting from antioxidant properties conferred by cancer stem cells (CSCs). Human epidermal growth factor receptor 2 (HER2) enhances CSCs and antioxidant properties in cancers, including NSCLC. Methods: Here, we elucidated the role of histamine N-methyltransferase (HNMT), a histamine metabolism enzyme significantly upregulated in NSCLC and coexpressed with HER2. HNMT expression in lung cancer tissues was determined using quantitative reverse transcription PCR (RT-qPCR). A publicly available dataset was used to determine HNMT's potential as an NSCLC target molecule. Immunohistochemistry and coimmunoprecipitation were used to determine HNMT-HER2 correlations and interactions, respectively. HNMT shRNA and overexpression plasmids were used to explore HNMT functions in vitro and in vivo. We also examined miRNAs that may target HNMT and investigated HNMT/HER2's role on NSCLC cells' antioxidant properties. Finally, how HNMT loss affects NSCLC cells' sensitivity to cisplatin was investigated. Results: HNMT was significantly upregulated in human NSCLC tissues, conferred a worse prognosis, and was coexpressed with HER2. HNMT depletion and overexpression respectively decreased and increased cell proliferation, colony formation, tumorsphere formation, and CSCs marker expression. Coimmunoprecipitation analysis indicated that HNMT directly interacts with HER2. TARGETSCAN analysis revealed that HNMT is a miR-223 and miR-3065-5p target. TBHp treatment increased HER2 expression, whereas shHNMT disrupted the Nuclear factor erythroid 2-related factor 2 (Nrf2)/ hemeoxygenase-1 (HO-1)/HER2 axis and increased reactive oxygen species accumulation in NSCLC cells. Finally, shHNMT sensitized H441 cells to cisplatin treatment in vitro and in vivo. Conclusions: Therefore, HNMT upregulation in NSCLC cells may upregulate HER2 expression, increasing tumorigenicity and chemoresistance through CSCs maintenance and antioxidant properties. This newly discovered regulatory axis may aid in retarding NSCLC progression and chemoresistance.
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Int.J.Mol.Sci.2022,23,1663.https://doi.org/10.3390/ijms23031663www.mdpi.com/journal/ijms
Article
HNMTUpregulationInducesCancerStemCellFormationand
ConfersProtectionagainstOxidativeStressthrough
InteractionwithHER2inNon‐Small‐CellLungCancer
Kuang‐TaiKuo
1,2
,Cheng‐HsinLin
3,4,5
,Chun‐HuaWang
6,7
,NarpatiWesaPikatan
8
,VijeshKumarYadav
9
,
Iat‐HangFong
9
,Chi‐TaiYeh
9,10,
*,Wei‐HwaLee
11
andWen‐ChienHuang
12,13,
*
1 DivisionofThoracicSurgery,DepartmentofSurgery,SchoolofMedicine,CollegeofMedicine ,
TaipeiMedicalUniversity,Taipei110,Taiwan;ktkuo@tmu.edu.tw
2 DivisionofThoracicSurgery,DepartmentofSurgery,ShuangHoHospital,TaipeiMedicalUniversity,
NewTaipeiCity235,Taiwan
3 TaipeiHeartInstitute,TaipeiMedicalUniversity,Taipei110,Taiwan;chlin99025@tmu.edu.tw
4 DivisionofCardiovascularSurgery,DepartmentofSurgery,ShuangHoHospital,
TaipeiMedicalUniversity,NewTaipeiCity235,Taiwan
5 DivisionofCardiovascularSurgery,DepartmentofSurgery,SchoolofMedicine,CollegeofMedicine,
TaipeiMedicalUniversity,Taipei110,Taiwan
6 DepartmentofDermatology,TaipeiTzuChiHospital,BuddhistTzuChiMedicalFoundation,
NewTaipeiCity231,Taiwan;10205@s.tmu.edu.tw
7 SchoolofMedicine,BuddhistTzuChiUniversity,Hualien970,Taiwan
8 DivisionofUrology,DepartmentofSurgery,FacultyofMedicine,UniversitasGadjahMada/Dr.Sardjito
Hospital,Yogyakarta55281,Indonesia;narpatiwp@gmail.com
9 DepartmentofMedicalResearch&Education,ShuangHoHospital,TaipeiMedicalUniversity,
NewTaipeiCity235,Taiwan;vijeshp2@gmail.com(V.K.Y.);impossiblewasnothing@hotmail.com(I.‐H.F.)
10 DepartmentofMedicalLaboratoryScienceandBiotechnology,YuanpeiUniversityofMedicalTechnology,
Hsinchu300,Taiwan
11 DepartmentofPathology,TaipeiMedicalUniversity—ShuangHoHospital,NewTaipeiCity235,Taiwan;
whlpath97616@s.tmu.edu.tw
12 DepartmentofMedicine,MacKayMedicalCollege,NewTaipeiCity252,Taiwan
13 DivisionofThoracicSurgery,DepartmentofSurgery,MacKayMemorialHospital,Taipei104,Taiwan
*Correspondence:ctyeh@s.tmu.edu.tw(C.‐T.Y.);wjhuang0@gmail.com(W.‐C.H.);
Tel.:+886‐2‐2490088(ext.8881)(C.‐T.Y.);+886‐2‐2490088(ext.2919)(W.‐C.H.);
Fax:+886‐2‐2248‐0900(C.‐T.Y.);+886‐2‐2248‐0900(W.‐C.H.)
Abstract:Background:Thetreatmentofnon‐small‐celllungcancer(NSCLC)involvesplatinum‐
basedchemotherapy.Itistypicallyaccompaniedbychemoresistanceresultingfromantioxidant
propertiesconferredbycancerstemcells(CSCs).Humanepidermalgrowthfactorreceptor2
(HER2)enhancesCSCsandantioxidantpropertiesincancers,includingNSCLC.Methods:Here,
weelucidatedtheroleofhistamineN‐methyltransferase(HNMT),ahistaminemetabolismenzyme
significantlyupregulatedinNSCLCandcoexpressedwithHER2.HNMTexpressioninlungcancer
tissueswasdeterminedusingquantitativereversetranscriptionPCR(RT‐qPCR).Apubliclyavail‐
abledatasetwasusedtodetermineHNMT’spotentialasanNSCLCtargetmolecule.Immunohisto‐
chemistryandcoimmunoprecipitationwereusedtodetermineHNMT–HER2correlationsandin‐
teractions,respectively.HNMTshRNAandoverexpressionplasmidswereusedtoexploreHNMT
functionsinvitroandinvivo.WealsoexaminedmiRNAsthatmaytargetHNMTandinvestigated
HNMT/HER2′sroleonNSCLCcells’antioxidantproperties.Finally,howHNMTlossaffects
NSCLCcells’sensitivitytocisplatinwasinvestigated.Results:HNMTwassignificantlyupregu‐
latedinhumanNSCLCtissues,conferredaworseprognosis,andwascoexpressedwithHER2.
HNMTdepletionandoverexpressionrespectivelydecreasedandincreasedcellproliferation,col‐
onyformation,tumorsphereformation,andCSCsmarkerexpression.Coimmunoprecipitationanal‐
ysisindicatedthatHNMTdirectlyinteractswithHER2.TARGETSCANanalysisrevealedthat
HNMTisamiR‐223andmiR‐3065‐5ptarget.TBHptreatmentincreasedHER2expression,whereas
shHNMTdisruptedtheNuclearfactorerythroid2‐relatedfactor2(Nrf2)/hemeoxygenase‐1(HO‐
Citation:Kuo,K.‐T.;Lin,C.‐H.;
Wang,C.‐H.;Pikatan,N.W.;
Yadav,V.K.;Fong,I.‐H.;Yeh,C.‐T.;
Lee,W.‐H.;Huang,W.‐C.HNMT
HNMTUpregulationInduces
CancerStemCellFormationand
ConfersProtectionagainstOxidative
StressthroughInteractionwith
HER2inNon‐Small‐CellLung
Cancer.Int.J.Mol.Sci.2022,23,1663.
https://doi.org/10.3390/ijms23031663
AcademicEditors:PinarUysal
OnganerandRichardW.E.
Clarkson
Received:22December2021
Accepted:28January2022
Published:31January2022
Publisher’sNote:MDPIstaysneu‐
tralwithregardtojurisdictional
claimsinpublishedmapsandinstitu‐
tionalaffiliations.
Copyright:©2022bytheauthors.Li‐
censeeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsandcon‐
ditionsoftheCreativeCommonsAt‐
tribution(CCBY)license(https://cre‐
ativecommons.org/licenses/by/4.0/).
Int.J.Mol.Sci.2022,23,16632of21
1)/HER2axisandincreasedreactiveoxygenspeciesaccumulationinNSCLCcells.Finally,shHNMT
sensitizedH441cellstocisplatintreatmentinvitroandinvivo.Conclusions:Therefore,HNMTup‐
regulationinNSCLCcellsmayupregulateHER2expression,increasingtumorigenicityandchemo‐
resistancethroughCSCsmaintenanceandantioxidantproperties.Thisnewlydiscoveredregulatory
axismayaidinretardingNSCLCprogressionandchemoresistance.
Keywords:non‐small‐celllungcancer;cancerstemcells;humanepidermalgrowthfactorreceptor
2;NRF2/HO‐1/HER2axis;HNMT/HER2′srole
1.Introduction
Lungcancerisoneofthemostcommoncausesofcancer‐associatedmortalityworld‐
wide,accountingforalmost1.76milliondeathcasesperyear.Non‐small‐celllungcancer
(NSCLC)islinkedwithapproximately80%ofalllungcancercases[1].Radiotherapy
aloneorwithchemotherapyandadjuvantdurvalumabarethemostcommontreatments
forpatientswithlocallyadvancedNSCLC.Platinum‐basedchemotherapyremainsthe
standardfirst‐linedefensetreatmentformetastaticNSCLCbutisfrequentlyaccompanied
bychemoresistance.Manyotherchemotherapeuticsdrugshavebeentriedasasecond‐
linetreatmentbutthepan‐drugresistanceacquiredbymostNSCLCresultsintreatment
failureanduninhibiteddiseaseprogression.Therefore,developingpredictivebiomarkers
toidentifypatientsandtargetedtherapiestotreatpatientslikelytobenefitfromthese
therapiesiscritical[2].
Tumormetastasisandchemoresistancehavebeeninvestigatedseparatelyinthepast,
butthesearefrequentlyobservedtogetherclinicallyandarelinkedbiologically.Inastudy
onbreastcancer,theinteractionbetweenthecancercellsandthehostmicroenvironment
wascharacterized,andtheconnectingchemotherapyfailurewithmetastaticrelapsewas
observed[3].Nevertheless,thekeymolecularmechanismsinvolvedintheassociationbe‐
tweenmetastasisandchemoresistance—thatmightdifferincancertypesandclinicalset‐
tings—remainunknown.Cancerstemcells(CSCs)contributetotumorrelapse,cancercell
propagation,andchemo–radioresistance[4].Theyalsoconferprotectiveantioxidant
propertiestocancers,leadingtochemoresistance[5].
Reactiveoxygenspecies(ROS),includingdifferentspeciesofsecondarymessengers,
suchashydrogenperoxide(H2O2),superoxideanion(O2−),hypochlorousacid(HOCl),
singletoxygen(1O2),andhydroxylradical(OH),areinvolvedincellsignalingforvarious
biologicalprocessesinbothnorms.Wealandcancercells[6].ROSregulatecriticalrecep‐
tortyrosinekinasesignalingtargets,includinghumanepidermalgrowthfactorreceptor
2(HER2),whichalsodirectlyinteractswithNRF2toregulateDNAbindingactivity.Fur‐
thermore,HER2downstreameffectorsincludePI3K,andmitogen‐activatedproteinki‐
nasesignalingpromotestheNrf2bindingtoDNAandresultsinthetranscriptionalacti‐
vationoftargetgenes[7,8].
Histamine,throughitsinteractionswithH1–4receptorsubtypes,stimulatesvarious
signalingpathways.AllthesereceptorsaremembersoftheheptahelicalGprotein‐cou‐
pledreceptor(GPCR)family[9–11].Highhistamine‐N‐methyltransferase(HNMT)ex‐
pressionhasbeennotedinpatientswithductalbreastcancercomparedwithhealthycon‐
trols[12].AnotherstudyreportedthatahistamineH3receptor(H3R)antagonistinhibited
proliferationandtriggeredcaspase‐dependentapoptosisinbothestrogenreceptor‐posi‐
tiveand‐negativebreastcancercells[13].However,theexactmechanismunderlyingthis
overexpressionanditseffectinNSCLCremainsunexplored.
Inthisstudy,bigdata,clinicalpatients,andinvitroandinvivoapproacheswereused
toinvestigatetheroleofHNMTinNSCLC.WeevaluatedHNMTexpressioninNSCLC
anditsinteractionorcoexpressionwithHER2.Ourfindingsprovidethefirstevidence
thattheCSCspopulationinNSCLChasalowermiR3065/223expression,resultingina
higherexpressionoftheirtargetgene,HNMT.This,inturn,resultsinafeedbackloop
Int.J.Mol.Sci.2022,23,16633of21
mechanisminwhichHNMTupregulationaffectsHER2expressionandsubsequentlyin‐
creaseschemoresistanceinNSCLCthroughtumorsphereformationandtheantioxidant
responsesystem.
2.Results
2.1.HNMTUpregulationinNSCLCTissuesIsRelatedtoWorsenedPrognosisand
SignificantlyCoexpressedwithHER2
ToassesstheroleofHNMTintheprogressionanddevelopmentofhumanNSCLC,
wefirstexaminedHNMTmRNAexpressionbyusingabioinformaticsapproach.HNMT
mRNAlevelsincreasedsignificantlyinpatientswithNSCLCcomparedwithpairednor‐
maladjacenttissue(Figure1A).Wethenextrapolatedourpreviousfindingtoevaluateits
prognosticsignificancebyusingthePrognoscandatabase[14].Weobservedthatpatients
withhigherHNMTexpression(n=74)hadworseprognosesthandidpatientswithlower
HNMTexpression(n=37;Figure1B).HER2mutationwasnotedtoleadtotheworst
prognosisinNSCLC.Inaddition,otherstudieshaveindicatedapotentialinteractionbe‐
tweenHNMTandHER2[15].Therefore,weinvestigatedtheinteractionbetweenthemin
NSCLC.WeusedtheR2database[16]andthreedatasets—namelyPeitsch(n=121),EXPO
(n=150),andBild(n=114)—anddetectedthatHNMTandHER2weresignificantlyco‐
expressed(Figure1C–E).WeverifiedHER2coexpressionwithHNMTintissuesobtained
frompatientswithNSCLC(Figure1F).Moreover,HER2wasnotmoresignificantlyex‐
pressedinNSCLCtumortissues,whereasHNMTwasstronglyexpressedinthetumor
tissuecomparedwiththenormaladjacenttissue(Figure1G).Notably,accordingtoour
bioinformaticsfindings,bothHER2andHNMTweresignificantlycoexpressedintheclin‐
icalsamples(Figure1H).Furthermore,wevalidatedthestandardtocomparetheclinico‐
pathologicparameters,asshowninTable1(below),wefoundthattumordifferentiation,
tumorsize,lymphnodemetastasis(LMN)status,andpathologicalclinical‐stagere‐
mainedsignificantlydifferentbetweenHNMThighandlowexpressiongroup.Addition‐
ally,thecelltypewasalsoasignificantfactor,withHNMThighexpressionlessfrequent
inNSCLC.
Subsequentunivariate(UA)andmultivariateanalysis(MA)wasusedtoevaluatethe
significanceoftheexpressionofHNMTandHer2,age,gender,tumorsize,LNM,clinical‐
stage,subtypesonNSCLC‐specificsurvival.OverexpressionofHNMT(hazardratio
0.294,95%confidenceinterval:0.108to0.806,p<0.017forUAandhazardratio0.152,95%
confidenceinterval:0.047to0.495;p<0.002forMA,Table2)couldbeconsideredasinde‐
pendentprognosticfactorsinNSCLCpatients.Takentogether,theseresultsimplythat
HNMTisassociatedwithHER2expressionandisapotentialtherapeutictargetinNSCLC.
Int.J.Mol.Sci.2022,23,16634of21
Figure1.AssociationofHNMTwithworseprognosisinNSCLCanditscorrelationwithHER2
expression.(A)HNMTmRNAexpressionwasevaluatedusingRT‐qPCR.Samplesweregrouped
asNSCLCandpairedwithnormaladjacent.(B)Kaplan–Meiersurvivalanalysisofpatientswith
NSCLCexpressingHNMTbasedonthePrognoscandatabase(GSE3141).HNMTandHER2
(ERBB2)genecorrelationanalysisdeterminedusingR2:GenomicsAnalysisandVisualizationPlat‐
form(http://r2.amc.nl;accessedon21Nov2021)fromthedataset(C)Peitsch(n=121),(D)EXPO(n
=150),and(E)Bild(n=114).(F)RepresentativeimmunohistochemistrystainingfromthreeNSCLC
cases.(G)J‐scorecomparisonbetweennontumorandtumortissuestakenfromNSCLCclinicaltis‐
suesamples.(H)HNMTandHER2proteinexpressioncorrelationanalysisinNSCLCclinicaltissue
samples.Scalebar:100μm.
Int.J.Mol.Sci.2022,23,16635of21
Table1.ThesummaryofclinicopathologicalfeaturesanddistributionofHNMTstatusin60pa‐
tientswithNSCLCpatients.
ClinicopathologicalVariables
 HNMT
N=60HighLowx2p‐Value
ExpressionExpression
Age,years
≤602911181.1330.287
>60311615
Gender    
Male3718190.5190.471
Female23914
Differentiation
Well/Moderately3813254.8750.027
Poor22148
TumorSize(cm)    
≤53912279.1180.003
>521156
Lymphnodemetastasis
N03611257.5870.006
N1‐N224168
ClinicalStage    
Early‐stage(I‐II)3072311.38<0.001
Late‐stage(III‐IV)302010
Subtypes
Adeno3720173.1970.074
Squamous23716
HER2Qscore    
Low3511246.2510.012
High25169
Table2.UnivariateandmultivariateanalysisofHNMTexpressionintheNSCLCcohort.
ParameterUnivariateMultivariate
HR95%CIpHR95%CIp
Gender(Femalevs.male)0.6790.2741.6830.4030.5940.1791.9710.395
age_60(<60vs.>60)0.8570.3602.0370.7260.6240.2111.8400.392
tumor_50(<50vs.>50)0.7210.2971.7530.4710.2960.0541.6320.162
LN_12(1,2vs.0)0.6750.2781.6370.3840.4270.1251.4580.174
Subtype_c(squavs.adeno)0.9990.4132.4120.9980.7380.2242.4310.618
stage(latevs.early)1.1400.4792.7110.7681.0110.3163.2350.986
Differentiation_c(well,moderatelyvs.
poor)0.9520.4002.2670.9110.4720.0992.2550.347
HNMT(lowvs.high)0.2940.1080.8060.0170.1520.0470.4950.002
HER2(lowvs.high)1.0610.4422.5460.8941.3460.4084.4450.626
2.2.HNMTInteractswithHER2andAffectsNSCLCCellLineDevelopment
WenextexaminedHNMTexpressioninsixNSCLCcelllines(BEAS‐2B,CL1‐0,CL1‐
5,H838,A549,andH441)throughWesternblotting.TheresultsrevealedthatHNMTpro‐
teinlevelsweresignificantlyupregulatedinA549andH441cellsbutweremostlyunder‐
expressedinotherNSCLCcelllines.Notably,HNMTexpressionwasaccompaniedby
HER2andHER3expression.ThecellswithhigherHNMTexpressionalsohadhighervi‐
mentinbutlowerN‐cadherinexpression,implyingHNMT’sroleinEMTinNSCLC
Int.J.Mol.Sci.2022,23,16636of21
(Figure2A).Therefore,weemployedH441andCL1‐0cellsforloss‐of‐functionandgain‐
of‐functionteststoelucidatetheroleofHNMTexpressioninNSCLC.Figure2B,Cdepicts
theeffectivenessoftheclonesusedforHNMTknockdownoroverexpressioninH441and
CL1‐0cells,respectively.BecausewedetectedHER2coexpressionwithHNMTinNSCLC
celllines,weinvestigatedtheassociationbetweenthetwo.Reciprocalimmunoprecipita‐
tionwithHNMTorHER2antibodiesindicatedapowerfulinteractionbetweenendoge‐
nousHNMTandHER2intheH441cells(Figure2D).HNMTalsoaffectedNSCLCcell
proliferation(Figure2E,F);itsknockdownwasassociatedwithreducedcellproliferation,
anditsoverexpressionwasassociatedwithanincreasedcellproliferationrate.Rawdata
ofwesternblotwithfull‐sizeblotshowninsupplementaryFigureS1.Theresultsofthese
experimentsfurtherconfirmtheinteractionbetweenHNMTandHER2andtheirrolein
NSCLCcelldevelopment.
Figure2.MolecularmanipulationeffectivenessofHNMTinlungcancercelllines.(A)Westernblot‐
tingofNSCLCcelllines.CelllysatesfromsixNSCLCcelllineshavebeensubjecttoWesternblot‐
ting.HNMT,HER2,HER3,vimentin,andN‐cadweredetectedusingtheircorrespondingantibod‐
ies.TheexperimentswerecarriedoutaftercalibrationusingGAPDHasaninternalcontrol.(B)
WesternblottingfortheHNMTandHER2expressioninH441cellstransfectedwithshScramble,
shHNMT#1,orshHNMT#2.(C)WesternblottingforHNMTandHER2expressionofCL1‐0cells
transfectedwithcontrolvector,HNMT‐overexpressingplasmid#1(pCMV6‐HNMT,HNMT#1),or
Int.J.Mol.Sci.2022,23,16637of21
HNMT‐overexpressingplasmid#2(pCMV‐HNMT‐N1,HNMT#2).Densitometryanalysisforthe
proteinexpressionofbothcelllinesisprovided.(D)Coimmunoprecipitationwasusedtodetect
HNMT–HER2interactionandβ‐actinwereusedasahousekeepingcontrol.(E)Cellproliferation
growthcurvecomparisonofH441cellstransfectedwithshScramble,shHNMT#1,orshHNMT#2,
and(F)CL1‐0cellstransfectedwithvectorcontrolorcontrolvector,HNMT‐overexpressingplasmid
#1(HNMT#1),orHNMT‐overexpressingplasmid#2(HNMT#2).***p<0.001.
2.3.HNMTPromotesCSCDevelopmentinNSCLCCellLines
ToevaluatewhetherHNMTalsoplayedaroleintheoriginofcancerstemnessin
NSCLC,wefirstassessedthecolonyformationabilityofH441withHNMTknockdown
andofCL1‐0withHNMToverexpression.WeobservedthatHNMTknockdownnega‐
tivelyinfluencedH441colonyformation.Itsoverexpression,however,significantlyin‐
creasedthecolonyformationabilityofCL1‐0cells(Figure3A).Similarly,HNMTknock‐
downandoverexpressionresultedinreducedandincreased,respectively,tumorsphere
formationabilityinH441andCL1‐0cellslines(Figure3B).HNMTmanipulationalsoaf‐
fectedstemnessmarkerexpression,whichwasevaluatedthroughWesternblotting.In
H441cells,shHNMTledtosignificantdownregulationofKLF4,NANOG,OCT4,and
CD133comparedwithshScramble.HNMToverexpression,inturn,increasedKLF4,
NANOG,OCT4,andCD133expressioninCL1‐0cells(Figure3C),rawdataofwestern
blotwithfull‐sizeblotillustratedinsupplementaryFigureS2.
Int.J.Mol.Sci.2022,23,16638of21
Figure3.EffectsofHNMTonCSCsmaintenanceinlungcancercelllines.(A)H441andCL1‐0cells
transfectedwithknockdownandexpressionvectorswereseededon6‐wellplates.Subsequentto2
weeks,thecolonieswerestainedwithcrystalviolet,andthenumberofcolonieswascounted.(B)
TumorsphereformationpotentialofNSCLCcells.BothH441andCL1‐0cellscouldformtu‐
morspheres;HNMTknockdownandoverexpressionsignificantlyreducedandincreasedtheirtu‐
morsphere‐formingpotential,respectively.(C)CelllysatesfromtheH441andCL1‐0cellswith
HNMTknockdownandoverexpressionweresubjectedtoWesternblotting,respectively.HNMT,
HER2,HER3,vimentin,andN‐cadweredetectedusingtheirrespectiveantibodies.Theexperiments
wereconductedfollowingcalibrationusingβ‐actinastheinternalhousekeepingcontrol.Theexper‐
imentswereanalyzedandnormalizedacrossthreeseparateexperiments.**p<0.01,***p<0.001.
Scalebar:100μm.
2.4.HNMTIstheGeneTargetofMiR‐223andMiR‐3065‐5p
WenextexaminedwhethermiRNAmaytargetHNMTusingTARGETSCAN(ver‐
sion6.2)andobservedthatHNMTcontainedtwocomplementary7mertargetsitesfor
miR‐223andonesiteformiR‐3065‐5p(Figure4A).WeevaluatedmiR‐223andmiR‐3065‐
5pexpressioninNSCLCtumorspheresandfoundbothexpressionsweresignificantly
Int.J.Mol.Sci.2022,23,16639of21
decreasedinNSCLCtumorspherescomparedwiththatincontroladherentcells(Figure
4B).Furthermore,theH441cellsweretransfectedwiththemimicsofmiR‐223,miR‐3065‐
5p,orboth.ThecellswereprobedforHNMTandHER2expressionanalysisthroughim‐
munofluorescence.Asexpected,asinglemiRNAmimicforeithermiR‐223ormiR‐3065‐
5pdecreasedHNMTexpressionandcombiningboththemimicsfurtherdecreased
HNMTexpression(Figure4C).Next,wetransfectedH441cellswiththewild‐type(WT)
ormutated(mut)HNMT3′UTR‐directedluciferasereporter,andwethencotransfected
thecellswithmiR‐223,miR‐3065‐5pmimic,orboth.Throughthisexperiment,wedemon‐
stratedthatWTHNMT3′UTRluciferaseactivitywassuppressedwithmiRNAmimics
cotransfection,butmutHNMT3′UTRdidnotsubstantiallyaffectmiRNAmimics.This
resultfurtheraffirmedthatHNMTisthetargetgeneofmiR‐223ormiR‐3065‐5p(Figure
4D).WesternblottingalsoyieldedsimilarresultsforHNMT,andHER2expressionsig‐
nificantlydecreasedwitheithermiR‐223ormiR‐3065‐5pmimictransfection,withfurther
reductionwithtransfectionofbothmimics(Figure4E),Rawdataofwesternblotwithfull‐
sizeblotdisplayedinsupplementaryFigureS3.MiR‐223andmiR‐3065‐5pmimictrans‐
fectionalsonegativelyinfluencedH441colonyformation,whichfurtherreduced
shHNMTcotransfection(Figure4F).
Int.J.Mol.Sci.2022,23,166310of21
Figure4.HNMTisoneofthekeytargetgenesofmiR‐3065andmiR‐223inlungcancer.(A)Com‐
plementarysitesformiR‐3065‐5pandmiR‐223inthe3′UTRregionofHNMTaccordingtoTAR‐
GETSCAN(version6.2).(B)miR‐3065‐5pandmiR‐223expressioninadherentversussphereH441
cells.RNU6‐2wasusedascontrol.BothqPCRresultswerenormalizedtoadherentH441value.(C)
ImmunofluorescencestainingofHNMTandHER2inH441cellstransfectedwithNCormiR‐3065
and/ormiR‐223inhibitordemonstrateddifferentiallocalization.DAPIstaining(blue)wasusedto
labelDNAinallcells.Scalebar=100μm.(D)H441cellswerecotransfectedwithmiR‐3065,miR‐
223,orcontrolmimic(miR‐NC)andwild‐type(WT)ormutated(mut)HNMT3′UTR‐directedlucif‐
erasereporter.Luciferaseactivitywasmeasuredusingdual‐luciferasereporterassays.(E)Western
blottingofHNMTandHER2differentialproteinexpressioninH441cellstransfectedwithNCor
miR‐3065and/ormiR‐223inhibitor.(F)H441cellstransfectedwithNCormiR‐3065and/ormiR‐223
Int.J.Mol.Sci.2022,23,166311of21
mimicsshowingdifferentialcolonyformationpotential.Graphbarsaremean±SDsofthreeinde‐
pendentexperiments.*p<0.05,**p<0.01,***p<0.001.Scalebar:100μm.
2.5.HNMTRegulatestheAntioxidativeStressPathwayinNSCLCCells
ROSaccumulationhasbeenemployedasacancer‐killingmechanisminchemother‐
apeuticdevelopment.HER2expressionhasbeenshowntoconferprotectionagainstROS
damageincancercells;thus,targetingHER2activityincancercellsmayincreasetheir
susceptibilitytochemotherapy.WefirstobservedincreasedHER2promoteractivityafter
treatmentwithvariousdosagesofTBHp(50,100,or200μM).Next,wetransfectedH441
cellswithshHNMTandtreatedthemwith200μMTBHp.Asexpected,HER2promoter
activitysignificantlydecreasedincellswithHNMTknockdown(Figure5A).Next,tocon‐
firmthatHER2andHER3expressionincreasesunderoxidativestressconditions,we
treatedH441cellswith200μMTBHpandfoundthatTBHpincreasedHER2andHER3
expressioncomparedwithuntreatedcells(Figure5B).Wethenevaluatedintracellular
ROSlevelsbyusingDCFDA,afluorescenceprobe,andfoundthatHNMTknockdown
significantlyincreasedROSgeneration(Figure5C).HNMTknockdownalsosignificantly
disruptedtheactivityofHER2viaNRF2/HO‐1signalingasanalyzedthroughWestern
blotting(Figure5D),rawdataofwesternfull‐sizeblotshowninsupplementaryFigure
S4.
Figure5.ThenegativeinfluenceofHNMTknockdownonHER2‐dependentantioxidantresponse
mechanisminlungcancercells.(A)tBHQ‐inducedHER2promotertranscriptionalactivity
Int.J.Mol.Sci.2022,23,166312of21
inductioninaconcentration‐dependentmanner.TheH441celllinewastransfectedandthentreated
for4hwithvariousconcentrationsoftBHQ,asindicated.(B)FollowingtBHqtreatment,immunob‐
lotanalysisshowedtotalproteininductionofbothHER2andHER3.Cellsweretreatedwith200
μMtBHQfor4h,lysedtoextracttheprotein,andanalyzedthroughWesternblotting.(C)Flow
cytometricanalysisoftBHQ‐inducedROSformationinH441cellswithorwithoutshHNMTtrans‐
fectionusingtheROS‐sensitivefluorometricprobeDCFDA.RelativeROSproductioninallthe
testedcellswasnormalizedtotheTBH1+shHNMTgroup.(D)Immunoblotanalysisofcellstreated
with200μMtBHqwithorwithoutshHNMTtransfection.NRF2,HO‐1,pHER2,andHER2were
detectedwiththeirrespectiveantibodies.*p<0.05,**p<0.01,***p<0.001.
2.6.HNMTInhibitionSensitizesNSCLCtoCisplatinChemotherapyBothatInVivoandIn
Vitro
OurinvitroassaysindicatedthatHNMT/HER2signalingplaysavitalroleincancer
stemnessandantioxidantpathways.Thus,wehypothesizedthattargetingHNMTexpres‐
sionsensitizesNSCLCcellstoconservativechemotherapy.Wefirstcomparedtheviabil‐
ityofH441cellstransfectedwithshScrambleandshHNMTandtreatedwithanincreasing
cisplatindosage.HNMTknockdownsensitizedH441cellstocisplatintreatment(Figure
6A).WethenevaluatedtheapoptosisofcellsbyusingAnnexinV/PIstainingandnoted
thatH441cellstransfectedwithshHNMThadahighercellapoptosisratecomparedwith
thosetransfectedwithshScramble(Figure6B).Subsequently,theH441cellstransfected
withshHNMTandtreatedwith4μMcisplatinhadgreatlyreducedcolonyformationabil‐
itycomparedwitheithercisplatin‐onlyorshHNMT‐onlycells(Figure6C).Next,weex‐
trapolatedoursinvitroresultstotheconductedinvivotrials.Afterall,miceweresacrificed
onday22itwasdiscoveredthatshHNMTsensitizedthetumortocisplatintreatment
(Figure6D).However,weidentifiednosignificantweightdifferencebetweenthegroups,
implyingthatshHNMTknockdownwas,atleastpartially,safe(Figure6E).Wetheneval‐
uatedthetumortissuethroughtheTUNELassay.WenotedthattumorswithHNMT
knockdownandtreatedwithcisplatinhadanincreasedlevelofTUNELstaining,suggest‐
inganincreasedapoptoticrate(Figure6F).Combined,thesedatasuggestthatshHNMT
hasavitalroletoplayinconferringchemoresistancetoNSCLCcells.
Int.J.Mol.Sci.2022,23,166313of21
Figure6.AssociationofHNMTwithcisplatinresistanceinlungcancer.(A)Cellviability(SRB)as‐
sayperformedonA‐549celllinewithorwithoutshHNMTtransfectionfollowedcloselybytreat‐
mentwithvariousdosagesofcisplatinfor48h.(B)AnnexinVdetectionofapoptosisinH441cells
Int.J.Mol.Sci.2022,23,166314of21
transfectedwithshScrambleorshHNMTandincubatedwithorwithout4μMcisplatinfor48h.
Livecells(lowerleftsquare),deadcells(upperleftsquare),cellsinearlyapoptosis(lowerright
square),andlateapoptoticcells(upperrightsquare).(C)ColonyformationassayofH441cellstrans‐
fectedwitheithershScrambleorshHNMTandincubatedwithorwithout1μMcisplatinfor10days.
Cellswerethenstainedwithcrystalvioletsolution.(D)Tumorvolumeofmicexenograftedwith
H441cellswitheithershScrambleorshHNMTwasevaluatedevery3daysafterday8oftumor
implantationtoevaluatecisplatintreatmenteffectiveness.(E)Mousebodyweighthasbeenevalu‐
atedevery3days.(F)RepresentativeimagesoftheTUNELassay(redfluorescenceforapoptotic
cellsandbluefluorescenceforcellnucleihavebeendetectedusingafluorescencemicroscope;mag‐
nification,400×).Theapoptosisrateisdepictedinthebottompanel.*p<0.05,**p<0.01,***p<
0.001.Scalebar:100μm.
3.Discussion
Here,wediscoveredthattheHNMTwassubstantiallyupregulatedinNSCLCtis‐
sues,inaccordancewithourfindingsobtainedthroughtheuseofthePrognoscandata‐
basedata.Furthermore,patientswithhighHNMTexpressionhadtheworstprognosis.
WeexaminedthreedatasetsusingtheR2platform,andwenotedthatHNMTwassignif‐
icantlyassociatedwithHER2expression.WeconfirmedthisfindingintheclinicalNSCLC
samples,whichexhibitedhigherHNMTandHER2expressioninthetumortissuethanin
thenontumortissue.TofurtherelucidatetheroleofHNMTinNSCLCtumorigenesis,we
employedNSCLCcelllinesforloss‐of‐andgain‐of‐functionstudies.Ourco‐immunopre‐
cipitationdataindicatedthatHNMTinteractedwithHER2,anditsmanipulationaffected
NSCLCcellgrowthrates.IdentifyingtheroleofHNMTintheCSCssubpopulationin
NSCLC,weassessedthecolonyformationabilityofNSCLCcelllineswithHNMTknock‐
downoroverexpression.HNMTlossorgainsignificantlyreducedorincreasedNSCLC
colonyformation,respectively.Consistently,thecelllines’tumorsphereformationwas
similarlyaffectedbyHNMTloss/gain.CSCsmarkers,suchasKLF4,NANOG,OCT4,and
CD133,wereincreasedanddecreasedfollowingHNMTknockdownandoverexpression,
respectively.Next,weexploredtheregulationofHNMTexpressioninNSCLCbyusing
TARGETSCANdatatodeterminewhichmiRNAmaytargetthismolecule.Resultsindi‐
catethatthe3′UTRregionofHNMThasbeenacomplementarysiteofmiR‐3065‐5pand
miR‐223.Notably,miR‐3065andmiR‐223hadrelativelylowexpressionintheNSCLC
tumorspherepopulation.UsinginhibitorsandmimicsofbothmiR‐3065andmiR‐223,we
observedthatHNMTwasthetargetofbothmiRNAs.
HER2endowscancercellswithrobustantioxidantproperties[8,17].Giventhe
HNMT–HER2interaction,weassessedtheeffectofTBHp‐inducedoxidativedamageon
NSCLCcellswithHNMTknockdown.Ourluciferasereporterassayconfirmedthatvari‐
ousTBHpdosagesincreasedHER2andHER3signalintensity,butHNMTknockdown
significantlyreducedtheirintensity.WealsodetectedahigherrateofcellswithDCFDA
staining,implyinghigherROSaccumulation,inH441cellswithHNMTknockdown.Fi‐
nally,ourWesternblottingresultsindicatedthatshHNMTreducedtheHER2/NRF2/HO‐
1signalingpathwayactivity.CSCsandcancercells’antioxidativepropertiescontributeto
chemoresistance.HavingproventhatHNMTplaysasignificantroleinCSCsmaintenance
andoxidativestressresponse,wesubsequentlytreatedWTandshHNMTH441cellswith
varieddosagesofcisplatinandevaluatedtheirresponse.HNMTknockdownincreased
H441cells’sensitivitytocisplatintreatment,asevidencedbytheirlowerviabilityand
higherapoptosisrate.Inaddition,cisplatinsignificantlyreducedshHNMTH441colony
formationcomparedwithshScrambleH441.Wethenextrapolatedtheseinvitrofindings
bytransfectingshScrambleandshHNMTH441cellsintoNOD/SCIDmiceandevaluating
theirresponsetocisplatintreatment.MicebearingshHNMTH441cellsweremoresensi‐
tivetocisplatintreatmentasindicatedbyadelayedgrowthrateandincreasedTUNEL
stainingintensitycomparedwiththeshScramblegroup.
Histaminehasnumerousbiologicalactivities,includingrolesincellproliferationand
differentiation,gastrointestinalfunctionregulation,andimmuneresponsemodulation
[18].Itisalow‐molecular‐weightaminegeneratedsolelybyL‐HDCandisfoundin
Int.J.Mol.Sci.2022,23,166315of21
variouscellsinthebody,includingthegastricmucosaandparietalandmastcells[19].
HistamineactsbyinteractingwithitsGPCRs—namelyH1HR,H2HR,H3HR,andH4HR.
Whenthesereceptorsareactivatedorinhibited,downstreamsignalingpathwaysaretrig‐
gered,elicitingimmune‐modulatoryandproinflammatorycellresponses[20].Afterhis‐
tamineisproducedbyHDC,itisrapidlystoredordegradedbyHNMTandmonoamine
oxidaseB[21,22].
TheroleofHNMTincancerremainsunclear.Amicroarray‐basedstudyonesopha‐
gealsquamouscellcarcinomadeterminedthatHNMTisoneofthecrucialplayersincon‐
trollingsignaltransductionnetworks[23].Inpediatricacutelymphoblasticleukaemia,75
keygeneswereidentified,allofwhichwereconsiderablyenrichedin25GOfunctions
andthechronicmyeloidleukaemiapathway.Subsequently,27diseaserisksubpathways
wereidentifiedwithHNMTasakeygeneenrichedinthesesubpathways[24].B[a]Pisa
well‐knownpolycyclicaromatichydrocarbonandacommonpollutantintheatmosphere
thatcancausecancerinbothanimalsandhumans.AstudyconductedinHepG2cells
revealedthatHNMTgeneexpressionsignificantlyincreasedwithB[a]Ptreatment;how‐
ever,itspreciseroleincarcinogenesisremainsunclear[25].
Inthisstudy,weelucidatedtheroleofHNMTincancer,specificallyinNSCLCcar‐
cinogenesis.HistaminemetabolismmayplayamajorroleinNSCLCcarcinogenesis
[26,27].ThedirectinteractionofHNMTwithHER2atleastpartiallyexplainsthecarcino‐
genicroleofHNMT.HER2overexpressionhasbeenobservedinpatientswithmanysolid
cancers,includingNSCLC.AlthoughtheexactmechanismunderlyingHER2–HNMTin‐
teractionremainsunclear,HNMTmaybeassociatedwithHER2homodimerization,as
indicatedbyYoshiokaetal.[28].TheauthorsalsodemonstratedthatSETandMYND
domain‐containingprotein3(SMYD3),aproteinlysinemethyltransferase,canaffect
HER2homodimerizationandtheactivationofitsdownstreampathwaysbyinducingthe
trimethylationoflysine175residuesinHER2[28].
HER2isawell‐knownprognosticandpredictivefactorinbreastcancer;however,its
functioninlungcancerwarrantsfurtherclarification[15].HER2mutationscanbefound
inasmallpercentageofpatientswithlungcancer.Theseshiftscanbeinterpretedason‐
cogenicdriversaswellasamechanismofacquiredresistancefollowingtargetedtherapy.
Similarchangeshavebeenobservedinothercancers,suchasbreastandgastriccancer,
andtheyhavebeenlinkedtopoorprognosisandshortoverallsurvival[29–32].Inaddi‐
tion,HER2‐alteredstageIVNSCLCleadstoarelativelyshortoverallsurvival,presuma‐
blyduetointrinsicresistancetochemotherapy[33].Consistentwithourfindings,HER2
expressionhasalsobeenimplicatedinCSCs’development.Accordingtopreviousdata,
HER2mayenhancecarcinogenesis,invasion,andmetastasisinHER2‐positivebreastcan‐
cers,atleastinpart,bysustainingandincreasingCSCs[34,35].Honkanenetal.found
that,inpatientswithNSCLC,HER2modulatestheCSLCphenotypeinALK‐translocated
lungcancers,andthismodulationisprimarilyorchestratedbyHER2/HER3heterodimers
[36].HER2wasalsoimplicatedinCSCs‐inducedchemoresistance[37].Wangetal.[37]
investigatedtheeffectofHER2ontheinductionofCSCsandthedrugsusceptibilityof
ovariancancercelllines.HER2expressionwascorrelatedwithtumorsphereformation
efficiency,andNF‐κβwasresponsibleforthemediationofHER2‐inducedCSCs.Further‐
more,HER2inhibitionsignificantlyincreasedthesensitivityofovariancancercells[37].
Inaddition,CSCsmayconferantioxidantpropertiesthatmayinterferewithchemothera‐
peuticresponsesincancers[38].Asmentionedabove,weobservedthatHNMTknock‐
downdisruptstheHER2/NRF2/HO‐1signalingaxis.Thisisinaccordancewithprevious
studiesthatdemonstratedHER2–NRF2interactioninducesoxaliplatinresistanceincolon
andbreastcancercells[5,8].
CSCscanundergosignificantchangesthatmayaltertheirbiologicalsignature,in‐
cludingtheirmiRNAexpression.MiRNAsareoftenfoundtobedysregulated,andthey
maystronglyaffecttumorigenicity.Inourstudy,miR‐223andmiR‐3065weresignifi‐
cantlydownregulatedinH441CSCspopulations.Thisfindingvalidatedourpreliminary
findingsbecausewealsodemonstratedthroughTARGETSCANdatabaseminingthat
Int.J.Mol.Sci.2022,23,166316of21
thesetwomiRNAswerehighlycorrelatedwithHNMT.Therefore,thesedata—atleast
partially—explainedwhyHNMTismorehighlyexpressedinsphere‐enrichedH441cells.
TheroleofmiRNAsinHNMTregulationremainsunderexplored.Astudydemonstrated
thatmiR‐223mightindirectlyupregulateHNMTexpressioninatopicdermatitispatho‐
genesis[39].Bycontrast,weobservedthatmiR‐223inhibitedHNMTexpression.Arecent
reviewelaboratedthecomplexitiesofthismiRNA:itcanactaseitheranoncomiroron‐
cosuppressor[40].UnlikemiR‐223,dataconcerningtheroleofmiR‐3065incancerhave
beenrelativelylimited;however,miR‐3065wasrecentlyfoundtobeapotentialpredictor
ofdiseaseseverityinovariancancer,alongwithitstargetADH7[41].MiR‐3065isalso
associatedwithrenalcancercelltumorigenicity[42].Ourstudyaddstoexistingevidence
foramiR‐3065′sroleasanoncosuppressorthatinteractswithHNMTinlungcancer.
4.Methods
4.1.ClinicalSamples
Themicroarraygeneexpressiondatasetsofthelungcancerpatientswereanalyzed
byusingtheonlinePrognoscandatabase[Duke(n=111)]togeneratetheKaplan–Meier
survivalcurve[14].GenecorrelationanalysisofHNMTandHER2wasanalyzedusingR2
GenomicAnalysisandVisualizationPlatform(https://hgserver1.amc.nl/cgi‐
bin/r2/main.cgi;accessedon21Nov2021)byusingthePeitsch(n=121),EXPO(n=150),
andBild(n=114)lungcancerdatasets.Furthermore,thepatients’clinicalsampleswere
alsocollectedfromMacKayMemorialHospital,TaipeiCity,Taiwan.Allpatientspro‐
videdwrittenconsentfortheirtissuetobeusedforscientificresearch.Thestudyofpa‐
tients’sampleswasapprovedbytheMacKayMemorialHospital(Approvalno.:
IRB:20MMHIS500e)andcompliedwiththerecommendationsoftheDeclarationofHel‐
sinkiforBiomedicalResearch.Inallpatients,lungcancertissuesandnormaltissues>3
cmawayfromthecancerwereobtained.TissuearraysofNSCLCsamplesweresubjected
toimmunohistologicalanalysisafterincubatingwithprimaryantibodiesagainstthe
HNMT(1:100dilution,SC‐81159;SantaCruzBiotechnology,USA)andHER2(1:100dilu‐
tion,SC‐81159;SantaCruzBiotechnology,USA)at4°Covernight.HRPandDABstaining
withhematoxylincounterstainingwereperformedasperthestandardimmunohisto‐
chemistryprotocolwerefollowedbyimagingandestimationoftheexpressionofthepro‐
tein.
4.2.CellsandCultureMedium
Thelungcancercelllines,suchasBEAS‐2B,CL1‐0,CL1‐5,H838,A549,andH441,
werepurchasedfromtheAmericanType‐Culture‐Collection(ATCC,Manassas,VA,
USA).ThecellswereculturedinDulbecco’smodifiedEagle’smedium(#12491023;
GIBCO,LifeTechnologies,Carlsbad,CA,USA)supplementedwith10%fetalbovinese‐
rum(GIBCO,LifeTechnologies,Carlsbad,CA,USA),penicillin(100IU/mL),andstrepto‐
mycin(100g/mL;#15140122,GIBCO,LifeTechnologies,Carlsbad,CA,USA)growninthe
humidified,5%CO2incubatorat37°C.
4.3.CellStableTransfection
TheinformationoftheHNMTexpressionplasmid(OriGeneRC204676)wasusedto
designpolymerasechainreaction(PCR)primers.OligonucleotidesHindIII‐HNMT‐F(5′‐
AATTAAGCTTATGGCATCTTCCATGAGGAG‐3′)andMluI‐HNMT‐R(5′‐AAT
TACGCGTTGCCTCAATCTCTATG‐3′)weredesignedforPCRamplificationof
HNMTgenesequences.ThesegmentofHNMTwasborneonanemptyplasmid(pCMV‐
MCS‐N1EmptyvectorcontrolplasmidDNA,GenBankaccessionU55762).Cell
transfectionwasperformedusingLipofectamine2000(Invitrogen),followingthe
manufacturer’sprotocols.
Eightmicrogramsofemptyplasmid(pCMV6‐EntryvectorcontrolplasmidDNA,
OriGeneaccessionPS100001)orHNMTexpressionplasmid(pCMV6‐HNMT,OriGene
Int.J.Mol.Sci.2022,23,166317of21
RC204676,HNMT#1;pCMV‐HNMT‐N1,GenBankaccessionU55762,HNMT#2)were
used.TheDNA‐lipofectaminereagentcomplexesstayatroomtemperaturefor30min.
Themixturewasaddedtothewellandmixedgentlybyrockingtheplatebackandforth.
Reagentcomplexesdidnothavetoberemovedfollowingtransfection.Thecellswere
incubatedat37℃inaCO2incubatorfor48handassayedfortransgeneexpression.
4.4.Co‐Immunoprecipitation(Co‐IP)
Co‐immunoprecipitationwasusedtodetectHNMT–HER2interactioninvitro.The
standardCo‐IPprotocolwasthesameasthatdescribedforIP.Non‐denaturinglysis
buffer(20mMTrisHClpH8,137mMNaCl,1%NonidetP‐40,2mMEDTA)wasstored
at4°C,andimmediatelybeforeuse,proteaseinhibitorswereadded.Thecellculturewas
placedinadishonice,andthecellswerewashedwithice‐coldPBS.Thenice‐coldlysis
bufferwasadded.Afterthat,cellswerecompletelylysedundernon‐denaturingcondi‐
tions,andproteinsthatboundtogetherwerekept.Irrelevant,non‐bindingproteins,anti‐
gens,andanyproteinsthatwereboundwereelutedbyaseriesofwashes.Then,the
boundproteinswhichelutedwereanalyzedbySDS‐PAGE/immunoblotting.
4.5.WesternBlotting
Lungcancercellswereextractedandlysedaftertrypsinization.Afterthetotalpro‐
teinslysateswereextractedandthesamplewasprepared,itwasseparatedusingtheSDS‐
PAGEusingMini‐ProteanIIIsystem(Bio‐Rad,Taiwan)andtransferredontoPVDFmem‐
branesusingTrans‐BlotTurboTransferSystem(Bio‐Rad,Taiwan).Membraneswerein‐
cubatedovernightat4°CintheprimaryantibodiesshowninSupplementaryTableS1.
SecondaryantibodieswerepurchasedfromSantaCruzBiotechnology(SantaCruz,CA,
USA),andanECLdetectionkitwasusedforthedetectionoftheproteinofinterest.Im‐
ageswerecapturedandanalyzedusingtheUVPBioDoc‐Itsystem(Upland,CA,USA).
4.6.TotalRNAIsolationandQunatitiaveReverse‐TranscriptionPolymeraseChainReaction
(qRT‐PCR)
ThetotalRNAwasisolatedandpurifiedusingTRIzol‐basedprotocol(Invitrogen,
ThermoFisherScientific,Waltham,MA,USA)accordingtotheprotocolprovidedbythe
manufacturer.TwomicrogramoftotalRNAwasreversetranscribedusingQIAGEN
OneStepRT‐PCRKit(QIAGEN,Taiwan),andthePCRreactionwasperformedusinga
Rotor‐GeneSYBRGreenPCRKit(400,QIAGEN,Taiwan).HNMTmRNAexpressionwas
detectedinlungcancerandnormaltissues.Theprimersequencesusedwereasfollows:
HNMTamplification(452bp),5′‐TACGTCCAAGGTCGGGCAGGAAGA‐3′;upstream,
5′‐CACTGATAGGCAGTTCTC;downstream,5′‐GGTTCTCAGTTGGTGCTTC.Glycer‐
aldehyde‐3‐phosphatedehydrogenase(GAPDH)wasusedasaninternalreferencetode‐
tectHNMTmRNAexpressionlevels.TherelativeexpressionlevelofHNMTmRNAwas
calculatedusing2−∆∆Cqformulae.
4.7.ClonogenicAssay
Fortheassessmentofthesensitivityofcancercellstowardsanytreatment,the“Clon‐
ogenicAssay”isagoldstandard.Atotalof2.7×104cellsperwellwereseededina6‐well
plateandincubatedat37°Cfor2days.Further,thecellswereculturedforanadditional
24hinmediaandincubatedat37°Cfor2daysin5%CO2treated.Thecellswerethen
subculturedandseededat350cellsperwellintonew6‐wellplatesandkeptforincubation
for10daysat37°Cinahumidifiedincubatorwith5%CO2.Thecellswerefixedanddried
afterbeingsetandstainedwith0.1%crystalviolet.Theexperimentswereconductedin
triplicate.
Int.J.Mol.Sci.2022,23,166318of21
4.8.SulforhodamineBAssay
CellularviabilitywasdeterminedusingthesulforhodamineB(SRB)assayasperthe
protocolsuggestedbyourlabprotocol.Briefly,lungcancercellswereseededin96‐well
plates(3.5×105cells/well),followedbyincubationat37°C,inahumidified5%CO2incu‐
bator.Thecellswerefixedwiththegentleadditionof50mLofcold10%w/vtricarboxylic
acid(TCA)andincubatedat4°Cfor60min.Next,50μLof0.4%w/vSBRsolutionin1%
CH3COOHwasaddedtoeachwell,followedbyincubationatroomtemperaturefor20
min.Unbounddyewasrecoveredafterstaining,andresidualdyewasremovedbythor‐
oughlywashingwellplateswith1%CH3COOHandairdrying.Theboundstainwas
dissolvedin10mMTrizmabase,andtheabsorbancewasmeasuredat515nmonan
ELISAplatereader(690nmreferencewavelength).
4.9.ImmunofluorescenceAssay
H441cellswereculturedonglasscoverslipsbeforebeingtransfected,asmentioned
previously.Afterincubation,cellswerefixedfor15minat4°Cwith4%formaldehyde,
permeabilizedfor5minwith0.01%TritonX‐100andblockedfor30minatroomtemper‐
aturewith1%bovineserumalbumin.Thecellswerethenincubatedfor24hat4°Cwith
primaryantibodiesagainstGSK‐3(#12456,1:100,CellSignalingTechnology,Danvers,
MA,USA)andβ‐catenin(#8814,1:100,CellSignalingTechnology).Thecellswerestained
withanisotype‐specificsecondaryantibody(AlexaFluor594‐AffiniPuredonkeyanti‐
rabbitIgG;JacksonImmunoResearch,WestGrove,PA,USA)for1hthefollowingday.
4.10.ROSProductionMeasurement
ForROSproductionevaluation,cancercellswereplatedin96‐wellplatesat20,000
cellsperwellinafinalvolumeof80μLofthemedium.Next,10μLof50μMDCFDAwas
addedtoeachwell,andthecellswereincubatedfor30min.Onamicroplatefluorometer
(Tecan,Seestrasse,Männedorf,Switzerland),fluorescencewasmeasuredwithanexcita‐
tionfiltersetat488nmandanemissionfiltersetat530nm.
4.11.TumorXenograftStudy
Four‐to‐six‐week‐oldfemaleNOD/SCIDmice(meanweight=17.4±2.1g)werepur‐
chasedfromBioLASCOTaiwan(Taipei,Taiwan).Theinvivostudieswereapprovedby
theInstitutionalAnimalCareandUseCommittee(IACUC)oftheMacKayMemorialHos‐
pital(Approvalno.:MMH‐A‐S‐109‐10).Themice(n=10each)wererandomlyassigned
totheshScramble,shScramble+cisplatin,shHNMT,orshHNMT+cisplatingroupsafter
receivinganinjectionof2×106H441cellswithscrambleshRNA(shScramble)orHNMT
shRNA(shHNMT)intheirhindflanks.Whenthetumorswerepalpableonday8,2mg/kg
cisplatinwasadministeredintraperitoneallyevery72hfor12days.Tumorsizeswerede‐
terminedwithcallipersevery3daysondays6,9,12,15,and18,andtumorvolumes(v)
wereestimatedusingtheformulalength(l)×(width(w))2×0.5.Thetumor‐bearingmice
werehumanelysacrificedattheendofthetrialonday18,andthetumorswereremoved,
tested,photographed,andweighedagain.
4.12.StatisticalAnalysis
Themeansandstandarddeviations(SDs)wereusedtopresentallresults.Formul‐
tiplecomparisonsorrepeatedmeasurements,Student’st‐testwasused.Formultiplecom‐
parisonsorrepeatedmeasurements,ANOVAorrepeatedANOVAaccompaniedby
Tukey’sposthoctestwasused.Statisticalsignificancewasdefinedasp<0.05.GraphPad
Prism(version7;GraphPadSoftware,SanDiego,CA,USA)wasusedforallstatistical
analyses.
Int.J.Mol.Sci.2022,23,166319of21
5.Conclusions
OurresultsrevealedthatHNMTupregulationinNSCLCcellsleadstoHER2upreg‐
ulation,whichinturnincreasestumorigenicityandchemoresistancethroughCSCs
maintenanceandantioxidantproperties.ThisCSCsmaydownregulatemiR‐3065‐5pand
miR‐223expression,thusreducingtheinhibitionoftheirtargetgene,HNMT,therebyre‐
sultinginafeedbackloopthatmayaidinmaintainingtheCSCspopulationofNSCLC
andconferringchemoresistance(Figure7).Theseresultsprovidenovelinsightintothe
rolesandinteractionsofHNMT,HER2,andmiRNAsinNSCLCpathogenesisandbehav‐
iour.TargetingthisnewlydiscoveredregulatoryaxismayaidinretardingNSCLCpro‐
gressionandcombatingchemoresistance.
Figure7.Graphicalsummary.HNMTinteractswithHER2toinduceCSCsphenotypedevelopment
andenhanceNSCLCantioxidantproperties,whichinturnattenuatemiR‐223/3065transcription
thatloopsbacktoincreasedHNMTexpression.HNMT/HER2‐inducedCSCsandantioxidanten‐
hancementsubsequentlyresultedincisplatinchemoresistanceinNSCLCs.
SupplementaryMaterials:Thefollowingsupportinginformationcanbedownloadedat:
www.mdpi.com/article/10.3390/ijms23031663/s1.SupplementaryTableS1.primaryantibodiesof
Westernblots.WesternBlotRawData:SupplementaryFigureS1.Full‐sizeblotsofFigure2.A,B,
C,andD,SupplementaryFigureS2.Full‐sizeblotsofFigure3C,SupplementaryFigureS3.Full‐size
blotsofFigure4E,SupplementaryFigureS4.Full‐sizeblotsofFigure5B,andD.
AuthorContributions:K.‐T.K.,C.‐H.L.,C.‐H.W.,N.W.P.,andV.K.Y.:Studyconceptionanddesign,
collection,andassemblyofdata,dataanalysisandinterpretation,andmanuscriptwriting.I.‐H.F.:
Dataanalysisandinterpretation.C.‐T.Y.,W.‐H.L.,andW.‐C.H.:Studyconceptionanddesign,data
analysisandinterpretation,finalmanuscriptapproval.Allauthorshavereadandagreedtothepub‐
lishedversionofthemanuscript.
Int.J.Mol.Sci.2022,23,166320of21
Funding:Pleaseadd:ThisresearchreceivednoexternalfundingorThisresearchwasfundedby
Dr.Kuang‐TaiKuograntnumber[MinistryofScienceandTechnology:MOST108‐2314‐B‐038‐114
‐MY3].
InstitutionalReviewBoardStatement:Allenrolledlungcancerpatientsprovidedwrittenin‐
formedconsentfortheirtissuestobeusedforscientificresearch.Thestudywasapprovedbythe
MacKayMemorialHospital(Approvalno.:IRB:20MMHIS500e)andcompliedwiththerecommen‐
dationsoftheDeclarationofHelsinkiforBiomedicalResearch.Theinvivostudieswereapproved
bytheInstitutionalAnimalCareandUseCommittee(IACUC)oftheMacKayMemorialHospital
(Approvalno.:MMH‐A‐S‐109‐10).
InformedConsentStatement:Anyresearcharticledescribingastudyinvolvinghumansshould
containthisstatement.Writteninformedconsenthasbeenobtainedfromthepatient(s)topublish
thispaper.
DataAvailabilityStatement:Thedatasetsusedandanalyzedinthecurrentstudyarepubliclyac‐
cessible,asindicatedinthemanuscript.
Acknowledgments:TheauthorsthankallresearchassistantsoftheCancerTranslationalResearch
LaboratoryandCoreFacilityCenter,TaipeiMedicalUniversityShuangHoHospital,fortheirassis‐
tancewiththemolecularandcell‐basedassays.
ConflictsofInterest:Theauthorsdeclarethattheyhavenopotentialfinancialcompetinginterests.
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... Recent work suggests that histamine N-methyltransferase (HNMT), which is regulated by ROS, is co-expressed with human epidermal growth factor receptor (HER2) [95]. The expression of both HER2 and HNMT is increased in breast and lung cancer [95,96]. ...
... Recent work suggests that histamine N-methyltransferase (HNMT), which is regulated by ROS, is co-expressed with human epidermal growth factor receptor (HER2) [95]. The expression of both HER2 and HNMT is increased in breast and lung cancer [95,96]. HNMT directly interacts with HER2 to regulate NRF2 DNA binding activity and target gene expression, which is in part mediated by phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) [95]. ...
... The expression of both HER2 and HNMT is increased in breast and lung cancer [95,96]. HNMT directly interacts with HER2 to regulate NRF2 DNA binding activity and target gene expression, which is in part mediated by phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) [95]. In non-small-cell lung cancer (NSCLC) CSCs, expression levels of the HNMTinhibiting miRs, miR3065/223, are low, leading to elevated expression/activity of NRF2. ...
Redox-Regulation in Cancer Stem Cells
Article
Full-text available
  • Sep 2022
Cancer stem cells (CSCs) represent a small subset of slowly dividing cells with tumor-initiating ability. They can self-renew and differentiate into all the distinct cell populations within a tumor. CSCs are naturally resistant to chemotherapy or radiotherapy. CSCs, thus, can repopulate a tumor after therapy and are responsible for recurrence of disease. Stemness manifests itself through, among other things, the expression of stem cell markers, the ability to induce sphere formation and tumor growth in vivo, and resistance to chemotherapeutics and irradiation. Stemness is maintained by keeping levels of reactive oxygen species (ROS) low, which is achieved by enhanced activity of antioxidant pathways. Here, cellular sources of ROS, antioxidant pathways employed by CSCs, and underlying mechanisms to overcome resistance are discussed.
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... Although several therapies such as chemotherapy and surgical resection are used to decrease mortality, there are still dilemmas including late detection for approximately 75% of LUAD patients, as well as high recurrence rates and poor prognoses [5,6]. Therefore, it is imperative to explore potential biomarkers and develop novel targeted cancer therapies for future application [7][8][9][10][11]. ...
Penetrating Exploration of Prognostic Correlations of the FKBP Gene Family with Lung Adenocarcinoma
Article
Full-text available
  • Dec 2022
The complexity of lung adenocarcinoma (LUAD), the development of which involves many interacting biological processes, makes it difficult to find therapeutic biomarkers for treatment. FK506-binding proteins (FKBPs) are composed of 12 members classified as conservative intracellular immunophilin family proteins, which are often connected to cyclophilin structures by tetratricopeptide repeat domains and have peptidyl prolyl isomerase activity that catalyzes proline from residues and turns the trans form into the cis form. Since FKBPs belong to chaperone molecules and promote protein folding, previous studies demonstrated that FKBP family members significantly contribute to the degradation of damaged, misfolded, abnormal, and foreign proteins. However, transcript expressions of this gene family in LUAD still need to be more fully investigated. In this research, we adopted high-throughput bioinformatics technology to analyze FKBP family genes in LUAD to provide credible information to clinicians and promote the development of novel cancer target drugs in the future. The current data revealed that the messenger (m)RNA levels of FKBP2, FKBP3, FKBP4, FKBP10, FKBP11, and FKBP14 were overexpressed in LUAD, and FKBP10 had connections to poor prognoses among LUAD patients in an overall survival (OS) analysis. Based on the above results, we selected FKBP10 to further conduct a comprehensive analysis of the downstream pathway and network. Through a DAVID analysis, we found that FKBP10 was involved in mitochondrial electron transport, NADH to ubiquinone transport, mitochondrial respiratory chain complex I assembly, etc. The MetaCore pathway analysis also indicated that FKBP10 was involved in "Ubiquinone metabolism", "Translation_(L)-selenoaminoacid incorporation in proteins during translation", and "Transcription_Negative regulation of HIF1A function". Collectively, this study revealed that FKBP family members are both significant prognostic biomarkers for lung cancer progression and promising clinical therapeutic targets, thus providing new targets for treating LUAD patients.
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... In addition, HNMT upregulation Frontiers in Nutrition 13 frontiersin.org causes cancer stem cell formation and protect it from oxidative stress by interaction with HER2 in NSCLC (41). TFEB (Transcription Factor EB) enables DNA-binding transcription factor activity. ...
Prediction of prognosis and immunotherapy response of amino acid metabolism genes in acute myeloid leukemia
Article
Full-text available
  • Dec 2022
Background Amino acid (AA) metabolism plays a crucial role in cancer. However, its role in acute myeloid leukemia (AML) is still unavailable. We screened out AA metabolic genes, which related to prognosis, and analyzed their correlation with tumor immune microenvironment in AML. Methods We evaluated 472 amino acid metabolism-related genes in 132 AML patients. The predictive risk model was developed according to differentially expressed genes, univariate Cox and LASSO analyses. We validated the risk signature by survival analysis and independence tests. Single-sample gene set enrichment analysis (ssGSEA), tumor immune microenvironment (TME), tumor mutation burden (TMB), functional enrichment, and the IC50 of drugs were assessed to explore the correlations among the risk model, immunity, and drug sensitivity of AML. Results Six amino acid metabolism-related genes were confirmed to develop the risk model, including TRH, HNMT, TFEB, SDSL, SLC43A2, and SFXN3. The high-risk subgroup had an immune “hot” phenotype and was related to a poor prognosis. The high-risk group was also associated with more activity of immune cells, such as Tregs, had higher expression of some immune checkpoints, including PD1 and CTLA4, and might be more susceptible to immunotherapy. Xenobiotic metabolism, the reactive oxygen species (ROS) pathway, fatty acid metabolism, JAK/STAT3, and the inflammatory response were active in the high-risk subgroup. Furthermore, the high-risk subgroup was sensitive to sorafenib, selumetinib, and entospletinib. ssGSEA discovered that the processes of glutamine, arginine, tryptophan, cysteine, histidine, L-serine, isoleucine, threonine, tyrosine, and L-phenylalanine metabolism were more active in the high-risk subgroup. Conclusion This study revealed that AA metabolism-related genes were correlated with the immune microenvironment of AML patients and could predict the prognosis and immunotherapy response of AML patients.
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... Zhong and their colleagues revealed that the TM6SF1 was related to the NSCLC tumor Microenvironment (34). Kuo and their colleagues found that the upregulation of HNMT could induce tumor stemness in NSCLC (35). MRO and CAPZA2 have not been reported in NSCLC. ...
Investigation of M2 macrophage-related gene affecting patients prognosis and drug sensitivity in non-small cell lung cancer: Evidence from bioinformatic and experiments
Article
Full-text available
  • Dec 2022
Background The progression process of lung cancer can be accelerated by M2 macrophages. However, genes that affect M2 macrophage polarization remain unidentified. Methods The Cancer Genome Atlas, Gene Expression Omnibus, and Arrayexpress databases were used to obtain open-access data. The analysis of public data was mostly performed with R studio. The RNA levels of specific genes were detected using quantitative real-time PCR. The proliferation ability of the cells was assessed by CCK8, colony formation, and EdU assays. Results Based on the multiple datasets, we noticed a poor prognosis in patients with high M2 macrophage infiltration. There were 114 genes differentially expressed between high and low M2 macrophages infiltrated samples, regarded as M2 macrophage-related genes. Subsequently, a prognosis prediction signature consisting of ABHD5, HS3ST2, TM6SF1, CAPZA2, LEPROT, HNMT, and MRO was identified and presented a satisfactory performance. The pathway enrichment results revealed a positive correlation between riskscore and enrichment scores for most immunotherapy-related positive terms. Also, there might be an increase in genomic instability among patients at high risk. Interestingly, low risk patients are most likely to benefit from PD-1 therapy, while high risk patients may benefit from CTLA-4 therapy. Meanwhile, the estimated IC50 of seven drugs differs significantly between two risk groups, including Cisplatin, Docetaxel, Doxorubicin, Gefitinib, Paclitaxel, Sunitinib and Vinorelbine. Moreover, further experiments indicated that HNMT was overexpressed and can enhance the proliferation ability in lung cancer cells. Conclusions In summary, our study identified the molecules significantly affecting M2 macrophage infiltration and identified a prognosis signature that robustly indicated patients prognosis. Moreover, we validated the cancer-promoting effect of HNMT using in vitro experiments.
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... Once cell-cell adhesion is disturbed, adhesionrelated pathways are subsequently interfered with. Disruption of cadherin signaling has determining influence on tumor progression and tumor immune responses [99][100][101][102][103][104]. ...
Comprehensive analysis of prognostic significance of cadherin (CDH) gene family in breast cancer
Article
Full-text available
  • Oct 2022
Breast cancer is one of the leading deaths in all kinds of malignancies; therefore, it is important for early detection. At the primary tumor site, tumor cells could take on mesenchymal properties, termed the epithelial-to-mesenchymal transition (EMT). This process is partly regulated by members of the cadherin (CDH) family of genes, and it is an essential step in the formation of metastases. There has been a lot of study of the roles of some of the CDH family genes in cancer; however, a holistic approach examining the roles of distinct CDH family genes in the development of breast cancer remains largely unexplored. In the present study, we used a bioinformatics approach to examine expression profiles of CDH family genes using the Oncomine, Gene Expression Profiling Interactive Analysis 2 (GEPIA2), cBioPortal, MetaCore, and Tumor IMmune Estimation Resource (TIMER) platforms. We revealed that CDH1/2/4/11/12/13 messenger (m)RNA levels are overexpressed in breast cancer cells compared to normal cells and were correlated with poor prognoses in breast cancer patients' distant metastasis-free survival. An enrichment analysis showed that high expressions of CDH1/2/4/11/12/13 were significantly correlated with cell adhesion, the extracellular matrix remodeling process, the EMT, WNT/beta-catenin, and interleukin-mediated immune responses. Collectively, CDH1/2/4/11/12/13 are thought to be potential biomarkers for breast cancer progression and metastasis.
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... Moreover, HNMT has a positive relationship with HER2 that could improve the features of CSCs. The knockdown of HNMT could decrease the tumorsphere formation ability, and reduce the expression of CSC markers such as NANOG, CD133, OCT4, and KLF4 through the Nrf2/HO-1/ HER2 signaling pathway increasing the accumulation of reactive oxygen species (ROS) (79). The stemness markers ALDH and CD133 are well-verified in LCSCs; p53 is a cancer suppressor, the mutation which is found in 47% of NSCLC cases, and the knockdown of the three genes could reduce the CSC characteristics and prolong the survival of NSCLC patients (80). ...
Current treatments for non-small cell lung cancer
Article
Full-text available
  • Aug 2022
Despite improved methods of diagnosis and the development of different treatments, mortality from lung cancer remains surprisingly high. Non-small cell lung cancer (NSCLC) accounts for the large majority of lung cancer cases. Therefore, it is important to review current methods of diagnosis and treatments of NSCLC in the clinic and preclinic. In this review, we describe, as a guide for clinicians, current diagnostic methods and therapies (such as chemotherapy, chemoradiotherapy, targeted therapy, antiangiogenic therapy, immunotherapy, and combination therapy) for NSCLC.
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Novel Potential Therapeutic Targets of PTPN Families for Lung Cancer
Article
Full-text available
  • Nov 2022
Despite the treatment of lung adenocarcinoma (LUAD) having partially improved in recent years, LUAD patients still have poor prognosis rates. Therefore, it is especially important to explore effective biomarkers and exploit novel therapeutic developments. High-throughput technologies are widely used as systematic approaches to explore differences in expressions of thousands of genes for both biological and genomic systems. Recently, using big data analyses in biomedicine research by integrating several high-throughput databases and tools, including The Cancer Genome Atlas (TCGA), cBioportal, Oncomine, and Kaplan–Meier plotter, is an important strategy to identify novel biomarkers for cancer therapy. Here, we used two different comprehensive bioinformatics analysis and revealed protein tyrosine phosphatase non-receptor type (PTPN) family genes, especially PTPN1 and PTPN22, were downregulated in lung cancer tissue in comparison with normal samples. The survival curves indicated that LUAD patients with high transcription levels of PTPN5 were significantly associated with a good prognosis. Meanwhile, Gene Ontology (GO) and MetaCore analyses indicated that co-expression of the PTPN1, PTPN5, and PTPN21 genes was significantly enriched in cancer development-related pathways, including GTPase activity, regulation of small GTPase-mediated signal transduction, response to mechanical stimuli, vasculogenesis, organ morphogenesis, regulation of stress fiber assembly, mitogen-activated protein kinase (MAPK) cascade, cell migration, and angiogenesis. Collectively, this study revealed that PTPN family members are both significant prognostic biomarkers for lung cancer progression and promising clinical therapeutic targets, which provide new targets for treating LUAD patients.
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Circadian rhythm-related factors of PER and CRY family genes function as novel therapeutic targets and prognostic biomarkers in lung adenocarcinoma
Article
Full-text available
  • Nov 2022
The period (PER) and cryptochrome (CRY) families play critical roles in circadian rhythms. The imbalance of circadian factors may lead to the occurrence of cancer. Expressions of PER and CRY family members decrease in various cancers. Nevertheless, expression levels, genetic variations, and molecular mechanisms of PER and CRY family members in lung adenocarcinoma (LUAD) and their correlations with prognoses and immune infiltration in LUAD patients are still unclear. In this study, to identify their biological functions in LUAD development, comprehensive high-throughput techniques were applied to analyze the relationships of expressions of PER and CRY family members with genetic variations, molecular mechanisms, and immune infiltration. The present results showed that transcription levels of PER1 and CRY2 in LUAD were significantly downregulated. High expression levels of PER2, PER3, CRY1, and CRY2 indicated longer overall survival. Some cancer signaling pathways were related to PER and CRY family members, such as cell-cycle, histidine metabolism, and progesterone-mediated oocyte maturation pathways. Expressions of PER and CRY family members significantly affected the infiltration of different immune cells. In conclusion, our findings may help better understand the molecular basis of LUAD, and provide new perspectives of PER and CRY family members as novel biomarkers for LUAD.
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Immune Checkpoint Inhibitors combined with HER-2 targeted therapy in HER-2 positive Gastroesophageal cancer
Article
  • Nov 2022
  • CRIT REV ONCOL HEMAT
GEC is one of the most common cancers and has become a significant health burden worldwide. HER-2 is a proto-oncogene, amplified or overexpressed in different tumors, and associated with a worse prognosis. Trastuzumab plus chemotherapy is the current standard treatment for advanced HER-2 positive GEC. However, it still does not benefit well all patients. HER-2 targeted therapy can up-regulate the expression of PD-1, CTL-4, and TAMs in the tumor microenvironment, strengthen the ADCC process, and enhance the efficacy of immunotherapy. While immune checkpoint inhibitors could reduce drug resistance to anti-HER-2 drugs. Moreover, immunotherapy combined with HER-2 targeted therapy in HER-2 positive GEC has shown perceptible efficacy and acceptable side effects in clinical trials and is regarded as a new therapeutic strategy for GEC. However, there remain significant challenges and requires more research to improve the survival benefit of this therapeutic approach.
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Analysis of the multi-physiological and functional mechanism of wheat Alkylresorcinols based on reverse molecular docking and network pharmacology
Article
  • Aug 2022
Alkylresorcinols (ARs) are phenolic lipids present in the bran part of whole grain wheat and rye, which possess antioxidant, anti-inflammatory, anti-cancer and anti-tumor properties. The physiological activities of ARs have been proven to be diverse; however, the specific molecular mechanisms are still unclear. In this study, reverse virtual screening and network pharmacology were used to explore the potential molecular mechanisms of the physiological function of ARs and their endogenous metabolites. The Metascape database was used for GO enrichment and KEGG pathway analysis. Furthermore, molecular docking was used to investigate the interactions between active compounds and potential targets. The results showed that the bioavailability of most ARs and their endogenous metabolites was 0.55 and 0.56, while the bioavailability of certain endogenous metabolites was only 0.11. Multiplex analysis was used to screen 73 important targets and 4 core targets (namely, HSP90AA1, EP300, HSP90AB1 and ERBB2) out of the 163 initial targets. The important targets involved in the key KEGG pathway were pathways in cancer (hsa05200), lipid and atherosclerosis (hsa05417), Th17 cell differentiation (hsa04659), chemical carcinogenesis-receptor activation (hsa05207), and prostate cancer (hsa05215). The compounds involved in the core targets were AR-C21, AR-C19, AR-C17, 3,5-DHPHTA-S, 3,5-DHPHTA-G, 3,5-DHPPTA, 3,5-DHPPTA-S, 3,5-DHPPTA-G, 3,5-DHPPTA-Gly and 3,5-DHPPA-G. The interaction force between them was mainly related to hydrogen bonds and van der Waals. Overall, the physiological activities of ARs are not only related to their multiple targets, but may also be related to the synergistic effect of their endogenous metabolites.
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The many facets of miR-223 in cancer: Oncosuppressor, oncogenic driver, therapeutic target, and biomarker of response
Article
Full-text available
  • May 2021
  • WIREs RNA
Given their intrinsic pleiotropism, microRNAs (miR) play complex biological roles, in both normal and pathological conditions. Often the same miR can act as oncogene or oncosuppressor, depending on the biological process dysregulated in each specific tissue. miR‐223 does not represent an exception to this rule and its functions greatly differ in different contexts. miR‐223 has been widely studied in the hematopoietic compartment, where it plays a central role in innate immune response, regulating myeloid differentiation and granulocytes function. Accordingly, dysregulated expression of miR‐223 has been associated to different inflammatory disorders and tumors arising from the immune compartment. Most carcinomas, breast cancer being the most studied, display loss of miR‐223. However, in gastro‐esophageal cancers miR‐223 is frequently overexpressed and correlates with worse prognosis. A link between miR‐223 and response to CDK4/6‐inhibitors has been recently proposed, suggesting a role as biomarker of therapeutic response. The notion that one of the most commonly mutated protein in cancer, mutant p53, binds the promoter of miR‐223 and suppresses its transcription, adds a further level of complexity to the full understanding of miR‐223 in cancer. In this review, we will summarize the current knowledge on the molecular networks that alter or are altered by miR‐223, in different cancer types. We will discuss if the times are ready for the exploitation of miR‐223 as predictive biomarker of treatment response or, even, as therapeutic target, in specific settings. Finally, we will suggest which could be the next steps to be taken for a realistic clinical application of miR‐223. This article is categorized under: RNA in Disease and Development > RNA in Disease
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ADH7, miR‑3065 and LINC01133 are associated with cervical cancer progression in different age groups
Article
Full-text available
  • Jan 2020
The aim of the present study was to identify potential therapeutic targets that serve crucial roles in the progression of cervical cancer. Clinical data, RNA sequencing (RNAseq)-counts and micro (mi)RNA data regarding cervical squamous cell carcinoma were retrieved from The Cancer Genome Atlas, and analyses were performed using the University of California Santa Cruz database. RNAseq and miRNA data were stratified into 3 groups (according to the patients' age), and genes were re-annotated and preprocessed prior to Mfuzz time clustering analysis. Subsequently, enrichment analyses were performed in order to identify differentially expressed mRNAs (DEmRNAs) and a protein-protein interaction analysis network was constructed. miRNA-gene, miRNA-lncRNA, and long non-coding (lnc)RNA-mRNA pairs were collected and the lncRNA-miRNA-mRNA competing endogenous (ce)RNA network was established. Further enrichment analyses were performed in order to identify crucial mRNAs in the ceRNA network. Finally, survival and drug association analyses were implemented. A total of 269 DEmRNAs [including alcohol dehydrogenase 7 (ADH7), vestigial-like family member 3 (VGLL3) and cytochrome P450, family 26, subfamily B, polypeptide 1 (CYP26B1)], 274 DElncRNAs (including LINC01133) and 16 DEmiRNAs (including miR-3065 and miR-330) were identified. There were 102 lncRNAs, 15 miRNAs, 15 mRNAs and 522 interaction pairs in the ceRNA network. In particular, ADH7 was regulated by miR-3065, and miR-3065 interacted with LINC01133 in the ceRNA network. Furthermore, ADH7 and CYP26B1 were enriched in the retinoic acid metabolic process and the retinol metabolism pathway. ADH7 and VGLL3 were significantly associated with the cervical cancer survival rate. ADH7, VGLL3, CYP26B1, miR-3065, miR-330, miR-499a and LINC01133 play pivotal roles in the progression of cervical cancer in different age groups.
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HER2 decreases drug sensitivity of ovarian cancer cells via inducing stem cell-like property in a NFκB-dependent way
Article
Full-text available
  • Oct 2018
Increasing evidence shows that cancer stem cells are responsible for drug resistance and relapse of tumors. In breast cancer, human epidermal growth factor receptor 2 (HER2) induces Herceptin resistance by inducing cancer stem cells. In the present study, we explored the effect of HER2 on cancer stem cells induction and drug sensitivity of ovarian cancer cell lines. First, we found that HER2 overexpression (HER2 OE) induced, while HER2 knockdown (HER2 KD) decreased CD44+/CD24- population. Consistently, HER2 expression was closely correlated with the sphere formation efficiency (SFE) of ovarian cancer cells. Second, we found that NFκB inhibition by specific inhibitor JSH23 or siRNA targetting subunit p65 dramatically impaired the induction of ovarian cancer stem cells by HER2, indicating that NFκB mediated HER2-induced ovarian cancer stem cells. Third, we found that HER2 KD significantly attenuated the tumorigenicity of ovarian cancer cells. Further, we found that HER2 inhibition increased drastically the sensitivity of ovarian cancer cells to doxorubicin (DOX) or paclitaxel (PTX). Finally, we examined the correlation between HER2 status and stem cell-related genes expression in human ovarian tumor tissues, and found that expressions of OCT4, COX2, and Nanog were higher in HER2 positive tumors than in HER2 negative tumors. Consistently, the 5-year tumor-free survival rate of HER2 positive patients was dramatically lower than HER2 negative patients. Taken together, our data indicate that HER2 decreases drug sensitivity of ovarian cancer cells via inducing stem cell-like property.
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Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article
Full-text available
  • Nov 2017
  • INFLAMM RES
Objective: Recently, we characterized mouse monoclonal antibodies that allow the specific and sensitive detection of human histamine N-methyltransferase (HNMT). To understand differences in binding characteristics and recognition of enzyme variants we mapped the antibody binding sites. Methods: Fragments of human HNMT were expressed as glutathione S-transferase fusion proteins that were used for testing antibody binding on immunoblots. Combined information from species cross-reactivity, sequence comparison, protein structure, and binding site prediction software were used to localize the epitope recognized by each antibody. Results: All eight monoclonal HNMT antibodies bound to linear epitopes in the C-terminal domain of the 292 amino acid protein. Of the five antibodies cross-reacting with HNMT from other species, one bound region L(182)-T(223), three region M(224)-E(261), and one region L(262)-A(292). All three antibodies recognising only human HNMT bound the C-terminal region L(262)-A(292) that contains residues present only in the human protein. Conclusions: Our HNMT monoclonal antibodies bind in three different regions of the protein and those binding the same putative epitope exhibit similar binding characteristics and species cross-reactivity. Antibodies binding non-overlapping epitopes will facilitate analyses of all clinically relevant variants described for HNMT.
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Protein lysine methyltransferase SMYD3 is involved in tumorigenesis through regulation of HER2 homodimerization
Article
Full-text available
  • Jun 2017
HER2 is a receptor tyrosine kinase, which is amplified and overexpressed in a subset of human cancers including breast and gastric cancers, and is indicated in its involvement in progression of cancer. Although its specific ligand(s) has not been detected, HER2 homodimerization, which is critical for its activation, is considered to be dependent on its expression levels. Here, we demonstrate a significant role of HER2 methylation by protein lysine methyltransferase SMYD3 in HER2 homodimerization. We found that SMYD3 trimethylates HER2 protein at lysine 175. HER2 homodimerization was enhanced in the presence of SMYD3, and substitution of lysine 175 of HER2 with alanine (HER2-K175A) reduced the formation of HER2 homodimers. Furthermore, HER2-K175A revealed lower level of autophosphorylation than wild-type HER2. We also identified that knockdown of SMYD3 attenuated this autophosphorylation in breast cancer cells. Our results imply that SMYD3-mediated methylation of HER2 at Lysine 175 may regulate the formation of HER2 homodimer and subsequent autophosphorylation and suggest that the SMYD3-mediated methylation pathway seems to be a good target for development of novel anti-cancer therapy.
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Cancer Stem Cell (CSC) Inhibitors in Oncology—A Promise for a Better Therapeutic Outcome: State of the Art and Future Perspectives
Article
  • Dec 2020
Cancer stem cells (CSCs), a subpopulation of cancer cells endowed with self-renewal, tumorigenicity, pluripotency, chemoresistance, differentiation, invasive ability, and plasticity, reside in specialized tumor niches and are responsible for tumor maintenance, metastasis, therapy resistance, and tumor relapse. The new-age "hierarchical or CSC"model of tumor heterogeneity is based on the concept of eradicating CSCs to prevent tumor relapse and therapy resistance. Small-molecular entities and biologics acting on various stemness signaling pathways, surface markers, efflux transporters, or components of complex tumor microenvironment are under intense investigation as potential anti-CSC agents. In addition, smart nanotherapeutic tools have proved their utility in achieving CSC targeting. Several CSC inhibitors in clinical development have shown promise, either as mono- or combination therapy, in refractory and difficult-to-treat cancers. Clinical investigations with CSC marker follow-up as a measure of clinical efficacy are needed to turn the "hype"into the "hope"these new-age oncology therapeutics have to offer.
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Oxidative stress induces senescence in breast cancer stem cells
Article
  • May 2019
Cancer stem cells (CSCs) have been shown to be resistant to current anticancer therapies and the induction of oxidative stress is an important mechanism of action for many anticancer agents. However, it is still largely unknown how CSCs respond to hydrogen peroxide (H2O2)-induced oxidative stress. Here, we show that the levels of reactive oxygen species (ROS) are markedly lower in breast CSCs (BCSCs) than that in non-cancer stem cells (NCSCs). A transient exposure of breast cancer cells to sublethal doses of H2O2 resulted in a dose-dependent increase of the epithelium-specific antigen (ESA)⁺/CD44⁺/CD24⁻ subpopulations, a known phenotype for BCSCs. Although BCSCs survived sublethal doses of H2O2 treatment, they lost the ability to form tumor spheres and failed to generate colonies as demonstrated by mammosphere-formation and clonogenic assays, respectively. Mechanistic studies revealed that H2O2 treatment led to a marked increase of senescence-associated β-galactosidase activity but only minimal apoptotic cell death in BCSCs. Furthermore, H2O2 triggers p53 activation and promotes p21 expression, indicating a role for the p53/p21 signaling pathway in oxidative stress-induced senescence in BCSCs. Taken together, these results demonstrate that the maintenance of a lower level of ROS is critical for CSCs to avoid oxidative stress and H2O2-induced BCSC loss of function is likely attributable to oxidative stress-triggered senescence induction, suggesting that ROS-generating drugs may have the therapeutic potential to eradicate drug-resistant CSCs via induction of premature senescence.
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MicroRNA-223 is involved in the pathogenesis of atopic dermatitis by affecting histamine-N-methyltransferase
Article
  • Feb 2018
  • CELL MOL BIOL
Atopic dermatitis (AD) is one of the most prevalent skin diseases around the world. Excessive histamine plays a critical role as an inflammatory factor in the pathogenesis of AD. Deregulated microRNAs (miRNAs) were involved in atopic dermatitis by targeting various genes. MiR-223 had been reported to play a vital role in hematopoiesis. In this study, we identified upregulated miR-223 in the whole blood cells of a large group of AD patients. What's more, we found for the first time that one of the major histamine degradation enzymes, histamine-N-methyltransferase (HNMT), was increased in AD patients and AD model mice. Although there was one miR-223 binding site in the 3'- untranslated region of the HNMT gene, HNMT were not inhibited by miR-223. Taken together, it suggested that miR-223 participates in AD through upregulating HNMT indirectly to degrade the excessive histamine.
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EGFR, HER2 target based molecular docking analysis, in vitro screening of 2, 4, 5-trisubstituted imidazole derivatives as potential anti-oxidant and cytotoxic agents
Article
  • Sep 2017
In our endeavor towards the development of potent molecules for cancer diseases, we have designed and synthesized a series of 2,4,5-trisubstituted imidazole derivatives (B1-B24) and characterized by using various spectroscopic techniques. All these compounds are further evaluated for their in vitro anti-cancer, anti-oxidant activities and molecular docking studies against EGFR, HER2 protein receptors. The in vitro anti-cancer activity analysis reveals that compounds B11 and B16 were found to be effective scaffolds against the tested human cancer cell lines IMR-32, A549 and HeLa. Particularly, B16 and B11 showed effective cytotoxicity against A549 and IMR-32 with IC50 values of 09.521±0.54μM and 10.294±0.43μM, respectively. Moreover, compounds B17, B18 and B23 showed potent activity towards the anti-oxidant screening with IC50 values of 5.87±1.73μM, 6.29±1.27μM and 4.95±1.81μM, respectively compared to standard ascorbic acid. Molecular docking was performed against the EGFR, HER2 protein receptors to provide more insight into their mechanism of interaction by comparing with standard EGFR, HER2 inhibitors like Gefitinib (EFGR), Lapatanib (EGFR), Afitinib (HER2) and Canertinib (HER2). Compounds B15, B16, B11 and B10 were exhibiting their minimum binding energies. Out of the aforementioned docked molecules, B15 and B16 showed the best binding energies of -11.15kcalmol(-1), -10.70kcalmol(-1) and -10.49kcalmol(-1), -10.12kcalmol(-1) against EGFR and HER2 protein receptors, respectively. The molecular docking results are well corroborated with the in vitro anti-cancer activity finding.
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