Cancer Science. 2019;00:1–11.  | 1wileyonlinelibrary.com/journal/cas

Received:18April2019  |  Revised:16May2019  |  Accepted:28May2019DOI: 10.1111/cas.14081

O R I G I N A L A R T I C L E

Carcinogen‐induced tumors in SFN‐transgenic mice harbor a characteristic mutation spectrum of human lung adenocarcinoma

Yunjung Kim1 | Aya Shiba‐Ishii1  | Karina Ramirez2 | Masafumi Muratani3 | Noriaki Sakamoto1 | Tatsuo Iijima4 | Masayuki Noguchi1

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution-NonCommercialLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycitedandisnotusedforcommercialpurposes.©2019TheAuthors.Cancer SciencepublishedbyJohnWiley&SonsAustralia,LtdonbehalfofJapaneseCancerAssociation.

1DepartmentofPathology,FacultyofMedicine,UniversityofTsukuba,Tsukuba,Japan2Ph.D.PrograminHumanBiology,SchoolofIntegrativeandGlobalMajors,UniversityofTsukuba,Tsukuba,Japan3DepartmentofGenomeBiology,FacultyofMedicine,UniversityofTsukuba,Tsukuba,Japan4DepartmentofPathology,IbarakiPrefecturalCentralHospital,Kasama,Japan

CorrespondenceAyaShiba-Ishii,DepartmentofPathology,FacultyofMedicine,UniversityofTsukuba,1-1-1Tennodai,Tsukuba,Ibaraki305-8575,Japan.Email:aya_shiba@md.tsukuba.ac.jp

AbstractThe landscapeofgeneticalterations indiseasemodels suchas transgenicmiceormicewithcarcinogen-induced tumorshasprovidedahugeamountof informationthathasshedlightontheprocessoftumorigenesisinhumannon-small-celllungcan-cer(NSCLC).Wehavepreviouslyidentifiedstratifin(SFN)asapotentoncogene,andgeneratedSFN-transgenic (Tg-SPC-SFN+/−)mice,whichexpresshumanSFN(hSFN)only in the lung.Here,wehave found thatcarcinogennicotine-derivednitrosami-noketone (NNK)-induced tumorsdeveloping inTg-SPC-SFN+/− mice show a similar histologytohumanlungadenocarcinomaandexhibithighhSFNexpression.InordertocomparethegeneticcharacteristicsofTg-SPC-SFN+/−tumorsandhumanlungad-enocarcinoma,theformerweresubjectedtowhole-exomesequencing.Interestingly,Tg-SPC-SFN+/− tumors showed the distinct distribution of exonic mutations andhighnumberofmutatedgenesandtransversion.Moreover,Tg-SPC-SFN+/−tumorsshowed73genesthatwerecommonlydetectedinmorethan2tumors,mutationsofwhichwerealsofoundinhumanlungadenocarcinoma.Theexpressionlevelsofsomeofthesegenesweresignificantlyassociatedwiththeclinicaloutcomeoflungadenocarcinoma patients. Additionally, mutated genes in Tg-SPC-SFN+/− tumorswere closely associatedwith key canonical pathways such as PI3K/AKT signalingandapoptosissignaling.TheseresultssuggestthatSFNoverexpressionisauniversalabnormalityinhumanlungadenocarcinogenesisandTg-SPC-SFN+/−tumorsrecapit-ulatekeyfeaturesofmajorhumanlungadenocarcinoma.Therefore,Tg-SPC-SFN+/− miceprovideausefulmodelforclarifyingthemolecularmechanismunderlyinglungadenocarcinogenesis.

K E Y W O R D S

lungadenocarcinoma,mutationalsignature,nicotine-derivednitrosaminoketone,stratifin,transgenicmice

2  |     KIM et al.

1  | INTRODUC TION

Lungcanceristhemostfrequentcauseofcancer-relatedmortalityworldwide1,2duetoitsunsatisfactorycurerateandpoorprogno-sis,andisdividedinto2majorhistologicalsubtypes:small-celllungcancer and non-small-cell lung cancer (NSCLC).3 Lung adenocar-cinoma, the predominant type ofNSCLC, is considered to showamultistepprocessofcarcinogenesis.Atypicaladenomatoushy-perplasiaprogressestoadenocarcinomainsitu,andsubsequentlyto invasive adenocarcinoma with specific molecular alterationsthat are responsible for both the initiation and maintenance oftumorigenesis.4

Previously,we examined the gene expression profiles of earlystage lung adenocarcinomas5 and identified stratifin (SFN, 14-3-3sigma),whichshowedsignificantlyhigherexpressioninsmallbutin-vasivelungadenocarcinoma(tumorsizelessthan3cmindiameter)thaninadenocarcinomainsituornormallung.5,6OverexpressionofSFN isdue toDNAhypomethylation in itspromoter region7,8 and is associatedwithpoorpatientoutcome in lungadenocarcinoma.9 Wehave found that suppressionofSFN reduces cell proliferationinvitro9,10andtumorformationormetastasis invivo,11suggestingthatSFNfacilitatestumorprogression.Consistently,SFN-transgenicmice(Tg-SPC-SFN+/−),whichexpresshumanSFN(hSFN)onlyinthelungundercontroloftheSPCpromoter,alung-specificenhancer,de-veloplungtumorsspontaneously,11implyingthatSFNhasoncogenicpotential in thecontextof lungadenocarcinogenesis.Additionally,overexpressionofSFNdysregulatessignaltransductionassociatedwithcellularproliferation,thecellcycle,apoptosis,andproteintraf-fickingbyinteractingwithphosphorylatedserineorthreonineinthebindingpartnerproteins.9,10,12

Genetic imbalancesofDNAormRNAsequences inthecancergenometriggervariousgeneticalterationsduringtheprocessoftu-morigenesis.Unlikethesituationinearlystagelungadenocarcinoma,many studies have revealed comprehensive somatic mutations inadvanced lung adenocarcinomas using whole-exome sequencing(WES),13whichhashighaccuracy, high sensitivity, and cost effec-tivenessforidentifyingdrivermutations14,15andcanpredicttheef-ficacyof anticancer agents such asmolecular targetingdrugs andimmunecheckpoint inhibitors.16Transgenicmiceormicewithcar-cinogen-inducedtumorscanbeusedtoassesspotentialmolecularalterationsrelatedtohumantumorigenesis.Transgenicmousemod-els of oncogenetic activation show frequent aneuploidy and copynumber alteration, but few somatic single-nucleotide variants.17 However,carcinogenssuchasmethyl-nitrosoureaandnicotine-de-rivednitrosaminoketone (NNK) included in tobacco smoke initiatetumorigenesisthroughinductionofnumerousdeleteriousmutationsatthelociofoncogenessuchasKRAS17,18ortumorsuppressorgenessuch as TP53.19,20Inthisregard,wehavebeenabletorecapitulatetheearlystageofhumanlungadenocarcinomausingTg-SPC-SFN+/− mice treatedwithNNKto trigger tumorigenesis.ThishasclarifiedthegeneticprofileoflungtumorsdevelopinginTg-SPC-SFN+/−mice,thushelpingtoidentifythegenealterationscontributingtohumanlungadenocarcinogenesis.

In the present study, we investigated the genetic landscapeofNNK-induced lungadenocarcinomas inaTg-SPC-SFN+/−back-ground. Paired samples of tumor tissue and normal lung weresubjectedtoWES,andmutatedgenesassociatedwithSFNover-expression in lung adenocarcinoma were examined. Here, weemphasizetheimportanceofSFNoverexpressioninlungadeno-carcinogenesisandshowthatTg-SPC-SFN+/− mice provide a valu-ablemodel for clarifyingearlygeneticalterations inhuman lungadenocarcinoma.

2  | MATERIAL S AND METHODS

2.1 | SFN transgenic mouse model and pathological analysis

WegeneratedtheTg-SPC-SFN+/−strainfromICRmicethathadbeenpurchasedfrom(CharlesRiverLaboratories,Tsukuba,Japan),asde-scribed previously.11Wild-type(WT)andTg-SPC-SFN+/− mice were givenasinglei.p.doseof4mgNNK(TorontoResearchChemicals,NorthYork,ON,Canada) dissolved in saline to induce pulmonarytumors.Twentyweeks afterNNK treatment, all of themicewerekilled, and the induced tumorswere resected from the lungs.Theresected tumorswerepartially fixedwith formalin forhistologicalanalysis including hematoxylin and eosin (H&E) staining and im-munohistochemistry for hSFN, as describedpreviously,11 and also frozeninliquidnitrogenforWESandstoredat−80°CuntilDNAex-traction.AllanimalexperimentswereapprovedbytheInstitutionalAnimal Experiment Committee of the University of Tsukuba(Tsukuba,Japan).

2.2 | Library preparation and WES

TheWES librarieswerepreparedwith theNEBNextDNALibraryPrepMasterMixSet(NewEnglandBiolabs,Ipswich,MA,USA)andaSeqCapEZDeveloperLibrarykit(NimbleGen;Roche,Pleasanton,CA,USA)from0.5μgofgenomicDNA.ThequalityoftheDNAlibrarywasevaluatedusingAgilent2100Bioanalyzer(AgilentTechnologies,SantaClara,CA,USA).SequencingofthemultiplexedlibraryusingaNextSeq500sequencer (Illumina,SanDiego,CA,USA)wascar-riedoutandthedatawereimportedtoCLCGenomicsWorkbenchsoftware(version10.1.1;Qiagen,Hilden,Germany).Sequencesweremapped to the GRCm38/mm10 mouse reference and annotatedusingEnsemble,Hanava,orEnsembleandHanavamergedtranscriptdatabase.(http://www.ensembl.org/index.html)

Possible single-nucleotidepolymorphismsor possible ICRdriftmutationswere excluded from thedata as follows: (a) singlebasesubstitutions common to each normal lung samplewere removedfromdatasetsoftumorsamples,(b)singlebasesubstitutionsfoundinnormallungsamplesfromWTorTg-SPC-SFN+/−micetreatedwithsalinewerealsoremovedfromeachdataset.Inaddition,weunder-tookfilteringbasedonthefollowingcriteria:singlenucleotidevari-anttypewithnonsynonymousmutation;coverageofreads10%orhigher;frequency10%orhigher;variantatleast5basesawayfrom

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theendof theread;andanaveragevariantqualityscoreof20ormore.

2.3 | Bioinformatics analysis

Mutationswerefirstcategorized into6typesaccordingtothese-quenceonthecodingstrand(C:G>A:T,C:G>G:C,C:G>T:A,T:A>A:T,T:A>C:G, or T:A>G:C). Here, we used pyrimidines to denote eachgroupsuchasC>A,C>G,C>T,T>A,T>C,andT>G,andcountedthenumber ofmutations detected in tumor samples fromWT or Tg-SPC-SFN+/−mice.Theywere then further subdivided into16pos-siblepairsof5′and3′nucleotideneighbors.ThetrinucleotidebaseswereverifiedusingtheUniversityofCaliforniaSantaCruzCancerGenomicsBrowser (https://genome.ucsc.edu/).Wecomparedmu-tationalsignaturesamongWT,Tg-SPC-SFN+/−,andalsohumancan-cers,whichweobtainedfromtheCatalogueofSomaticMutationsinCancer (COSMIC)database (https://cancer.sanger.ac.uk/cosmic).

Tocomparemutatedgenelistsamong4tumorsthathaddevelopedinTg-SPC-SFN+/−mice,4-wayVenndiagramswereproducedusingtheonlineVenndiagramplottingtool(http://bioinfogp.cnb.csic.es/tools/venny/),Venny2.1.0.ToidentifysignificantlymutatedgenesandcanonicalpathwaysinducedbyoverexpressionofhSFN,weusedIngenuity Pathway Analysis (IPA; Qiagen) or Kyoto Encyclopediaof Genes andGenomes (KEGG) pathway analysis throughDAVIDBioinformaticsResources6.8.(https://david.ncifcrf.gov/)

2.4 | Expression analysis of mutated genes in human lung adenocarcinoma

Mutation profiles, gene expression profiles, and clinical data for482casesoflungadenocarcinomafromTheCancerGenomeAtlas(TCGA) database (https://portal.gdc.cancer.gov/)were used to ex-ploremutationfrequencyand itscorrelationwithpatientsurvival.Thepercentageoflungadenocarcinomapatientshavingamutation

F I G U R E 1  HistologicalanalysisoftumorsthatdevelopedinTg-SPC-SFN+/− mice.A,Schematicoverviewforexperimentaldesignofnicotine-derivednitrosaminoketone(NNK)-inducedlungtumorigenesisinTg-SPC-SFN+/− and WT mice.At8wkafterbirth,allmiceweregivensalineorNNKbyi.p.injection.Themicewereobservedfor20wkafteradministrationwithNNKorsalineandkilledat28wkofagetocollectlungtumors.B,AlltumorsthatdevelopedinTg-SPC-SFN+/− or WT mice were subjectedtoH&Estainingtoexaminetheirhistology.Tg-SPC-SFN+/−tumorsweremoredifferentiatedadenomathanWTtumor,whichresemblesthehistologyofhumanlungadenocarcinoma.C,H&EandhumanSFN(hSFN)immunohistochemistry(IHC)imagesofrepresentativepairedsamplesofnormallungandtumortissueinTg-SPC-SFN+/− andWTmice.hSFNwashighlyexpressedintumors,typeIIalveolarepithelialcells,andbasalcellsinthebronchialepitheliumofthenormallunginTg-SPC-SFN+/− mice

4  |     KIM et al.

ineachgenewascalculatedas:numberofcaseswhereeachgeneismutated/totalnumberofcasestestedforsimplesomaticmutations.SurvivalplotsforeachgenewereexaminedusingtheKaplan-MeiermethodthroughOncoLnc (http://www.oncolnc.org),awebtoolforinvestigatingtheassociationbetweenpatientoutcomeandmRNAexpressiondataforlungadenocarcinomaobtainedfromTCGA.21

2.5 | Statistical analysis

For the significant canonical pathways associated with mutatedgenes in WT or Tg-SPC-SFN+/− tumors, we used the Benjamini-HochbergmethodandadjustedtheFisherexactPvaluethresholdto<.05.Additionally,weusedthelog-rankPvalueforsignificanceofthesurvivalratebetweenlowandhighgeneexpression.

3  | RESULTS

3.1 | Histological examination of tumors attributed to NNK treatment in Tg‐SPC‐SFN+/− mice

ToinvestigatetheuniquefunctionofhSFNontumorigenesisspe-cificallyinthelung,wegeneratedtransgenicmice(Tg-SPC-SFN+/− mice)withoverexpressionofhSFNratherthanmouseSFN(mSFN).AshSFNandmSFNshow98%homologyinaminoacidsequences(FigureS1A),wesupposedthatoverexpressionofhSFNmightshownotoxicityagainstmice,andneededtodistinguishhSFNandmSFNtounderstandwhethertheresultingtumorigenesisisduetohSFNoverexpressionorendogenousmSFNexpression.11Toexaminetu-morigenicactivityofhSFN,wei.p.injectedNNKorsalineasacon-trol intoTg-SPC-SFN+/− andWTmice (Figure1A). Twentyweeksafterthetreatment,wekilledallmiceandcollectedthelungsanddeveloping tumors,whichwere divided into 2 parts: histologicalexaminationsandWES.First,weundertookH&Estainingtoexam-ine thehistologyof tumors thatdeveloped inTg-SPC-SFN+/− and WTmice. Although 10 tumors developed in Tg-SPC-SFN+/− mice

and we selected 4 tumors that were sufficient size to examineWES, most WT tumors were small and detected in the centralareaofthelungwhenweundertookhistologicalexaminations.WeobtainedfreshmaterialfromonlyoneWTtumortobeappliedtoWES. Interestingly,tumorsthatdevelopedinTg-SPC-SFN+/− mice showed papillary or lepidic differentiation, which are major his-tologic subtypes of human lung adenocarcinoma,whereas thoseinWTmice showed the tendency to have poorer differentiatedhistology (Figure 1B,C).Moreover, by immunohistochemistry,weconfirmedthattumorsdeveloping inTg-SPC-SFN+/− mice showed positivity for hSFN (Figure 1C). These results indicated that thetransgenicmicehadbeengeneratedsuccessfullywithappropriategenerecombination.AsoverexpressionofSFNisacommoneventfacilitating tumor progression inmost human invasive adenocar-cinomas,5,9we speculated that genetic alterations in tumors thatdevelopedinTg-SPC-SFN+/−micemightmimicthoseinhumanlungadenocarcinoma.

3.2 | Whole‐exome sequencing reveals exonic mutational signatures in Tg‐SPC‐SFN+/− tumors

We undertook WES using paired samples of normal lung andtumor tissuedescribed inTable1 to identifygeneticalterationsduetooverexpressionofhSFN(TablesS1andS2).Afterexclud-ing single-nucleotide polymorphisms, the mean number of so-maticmutationsin4samplesofTg-SPC-SFN+/−tumorswas373.8(FigureS1B).Amongnonsynonymoussomaticmutations,7.3%ofthem in averagewerenonsensemutations inTg-SPC-SFN+/− tu-mors, which included several tumor suppressor genes, such asBanp, Smarcc1, Epha3, Ccdc154,andIng5(FigureS1C).Therefore,weexpectthatthesetumorsuppressorgenesharboringnonsensemutationsmight produce incomplete forms and possibly inducetumorprogression.

Moreover, according to previous reports, frequent somaticmutation of T>C or T>G in mice is caused by spontaneous point

Sex Treatment Strain Tissue type Genotype

WT-CON Male Saline ICR Normal Wildtype

Tg-CON Male Saline ICR Normal Tg-SPC-SFN+/−

WT Male NNK ICR Normal Tg-SPC-SFN+/−

Adenoma

Tg1 Male NNK ICR Normal Tg-SPC-SFN+/−

Adenoma

Tg2 Male NNK ICR Normal Tg-SPC-SFN+/−

Adenoma

Tg3 Male NNK ICR Normal Tg-SPC-SFN+/−

Adenoma

Tg4 Female NNK ICR Normal Tg-SPC-SFN+/−

Adenoma

Abbreviations:CON,control;NNK,nicotine-derivednitrosaminoketone;Tg,SFN-transgenicmouse;WT,wild-typemouse

TA B L E 1  Detailsofsamplesusedforwhole-exomesequencinganalysis

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mutations,unlikethesituationinhumans.22Consistently,asshowninFigure2A,WTtumorshowedahighfrequencyofT>CandT>G.However,Tg-SPC-SFN+/− tumors showedahigh frequencyofC>A(Figure2A),afeaturethatisknowntobeassociatedwithsmokinginhumans,23suggestingthatTg-SPC-SFN+/−tumorsmighthaveacloseresemblancetohumantumors.

Wealsoexamined themutational signaturesofTg-SPC-SFN+/− andWTtumor.OnthebasisoftheCOSMICdatabase,thesignaturesofTg-SPC-SFN+/−tumorsweresimilartotheCOSMICsignatures4and 24, which show a high frequency of C>A.24 Consistent withourresults,TCGAalsoindicatedthatsignatures4and24werefre-quentlyobservedinhumanlungadenocarcinomaandshowedahighfrequencyofC>AandC>Tsubstitutions(FigureS2A,B).TheTg-SPC-SFN+/−tumorstypicallyhadahighfrequencyoftrinucleotidevari-antssuchasACA,ACC,GCA,andGCCinC>AtypeandGCCinC>Ttype(Figure2B).However,GTCshowedthehighestpercentageofmutationsinT>G,bothinWTtumorandTg-SPC-SFN+/−tumors,in-dicatingthatGTCmightbeassociatedwithNNKtreatmentorspon-taneousmutationsinmice(Figure2B).

3.3 | Exonic mutational profiles of Tg‐SPC‐SFN+/− tumors

Next,weanalyzedassociationsbetweenthenumberofmutationsand SFNgeneexpressioninlungadenocarcinomapatientsinTCGAandlungcancercelllinesintheCOSMICdatabase.Thepatientswithhigh mutation number showed significantly higher expression ofSFN thanthosewith lowmutationnumber (FigureS3A).Similartolungadenocarcinomapatients, lungcancercell lineswithhighmu-tationnumber,suchasHCC827,A549,H1975,Calu-3,H1755,andH1573,hadsignificantlyhigherexpressionofSFN thanthosewithlowmutationnumber,suchasCOR-L105andCalu-6 (FigureS3B).Interestingly,thecell lineswithhighnumberofmutations,suchasA549,H1755,andH1573showedhighfrequencyofC>AandC>Tsubstitutions, a similar mutational signature of Tg-SPC-SFN+/− tu-mors(Figures2BandS3C).

Basedontheseresults,wehypothesizedthatTg-SPC-SFN+/−tu-morsmightbeassociatedwithhighmutationburden,whichisacrit-ical indicatorof theefficacyof immune checkpoint inhibitors.16,25

F I G U R E 2  DistributionofnucleotidealterationsinTg-SPC-SFN+/−tumorsandcomparisonwiththehumancancermutationalsignatureintheCOSMICdatabase.A,Sixclassesofsomaticsinglenucleotidemutationsareindicatedonthehorizontalaxisandthepercentagesofeachmutationtypearedisplayedontheverticalaxis.Thenumbersbelowthechartindicatethemutationnumbersdetectedineachtumor.B,Eachsignatureisdisplayedaccordingtothe96-substitutionclassificationdefinedbythesubstitutionclassandsequencecontextimmediately5′and3′tothemutatedbase.ThefrequencyofflankingbasepairsinTg-SPC-SFN+/−tumorsissimilartoCOSMICsignatures4and24,whicharecloselyassociatedwithsmokingandaflatoxininhumancancer,respectively

6  |     KIM et al.

Similartothemutationburdeninhumanlungcancer,whichrangesfrom0.1to100nonsynonymousmutationsperMb,26bothTg-SPC-SFN+/−andWTtumorshowedapproximately7.8perMb(Figure3A).However, thenumberofmutatedgeneswashigher in all Tg-SPC-SFN+/−tumorsthanWTtumor,indicatingthattheformerhadmuta-tionsinvariousgeneswhereasWTtumortendedtohavemultipointmutationsinparticulargenes(Figure3B).Additionally,thetransitionto transversion (Ts/Tv) ratio isknowntobewidelyvariableacrosstumor types depending on the mutagens involved or the mech-anisms responsible forDNA repair.26As themeanTs/Tv ratio forTg-SPC-SFN+/−tumorswas0.34,beingsimilartothatforhumanlungadenocarcinoma(LUADTs/Tv=0.44),Tg-SPC-SFN+/−tumorsmighthavecharacteristicscomparabletothoseofhumanlungadenocarci-noma(Figures3CandS2C).

Next,weanalyzedthemutationfrequencyineachchromosome.TheTg-SPC-SFN+/−andWTtumorsshowedthehighestfrequencyofmutatedgenesinchromosome7,includingthelociofBlm, Herc2, Nlrp2,andSmg1inTg-SPC-SFN+/−tumors(Figure3DandTableS3).

3.4 | Frequently mutated genes in Tg‐SPC‐SFN+/− tumors

To identify commonmutated genes among 4 Tg-SPC-SFN+/− tu-mors,whichwerepredictedtobeassociatedwithtumorigenesis,wedrewVenndiagramsusingsingle-nucleotidevariantsdetectedbyWES(Figure4A).Wedetected73genesthatwerecommonly

detectedin2ormoresamplesofTg-SPC-SFN+/−tumorsbutnotinWTtumor(Figure4BandTableS4).Consistentwiththepreviousstudy,whichexaminedsomaticmutationsinNSCLC,18,27wefoundthat Blm, Brd4, Dnmt3b, Herc2, Nlrp2, and Smg1 were common mutated genes in 3 samples of Tg-SPC-SFN+/− tumors, whereas67othergenes, includingCdc20, Cdh23, Cul9, Fgf7, Itga6, Kmt2c, Mki67, Tbc1d9,andTsc2,werepresentintwosamplesofTg-SPC-SFN+/− tumors (Figure 4B).Notably, 8 genes contained identicalmutationsitesamongTg-SPC-SFN+/− tumors, includingG235E inBlm, I108V and R124C in Smg1, I92V inCtbs, S283G inCyb5r2,S153A in Fgf7, D665E/H in Kmt2c, H366R inOvch2, and L575Iin Tsc2 (Table S2). However, according to TCGA, thesemutatedgenesdonothavemutationalhotspotsinhumancancers.Similartothepreviousstudy,inwhichlungtumorswereinducedinmicebyexposuretocarcinogenssuchasurethane,17wedetectedKras mutation(Q61R)onlyinWTtumor,indicatingthatKrasmutationcouldworkasadrivermutationcontributingtoWTlungtumori-genesis(TableS2).

Moreover, Tg-SPC-SFN+/− tumors showed accumulation ofpassenger gene and tumor suppressor gene mutations such asBlm, Dnmt3b, Cyb5r2, Dapk1, Foxp3, Kmt2c,andTsc2 rather thanaspecificdriveroncogenemutation,suchasKrasmutationinWTtumor (Figure 4B and Table S2). From these results, we expectthatoverexpressionofSFNwouldbeenoughtoinducetumorini-tiationandprogressionwithoutanydrivermutations,suchasKras mutation.

F I G U R E 3  DistinctmutationalspectrumpatternsinTg-SPC-SFN+/− tumors.A,Exonicmutationfrequencieswerecalculatedasthenumberofmutationsdetectedpermillionbasessequenced,whichwereestimatedfromtheaveragecoverageandbasecoveragedatafromwhole-exomesequencing.B,MutatedgeneswerecountedineachTg-SPC-SFN+/−andWTtumor.C,Ratiosoftransitions(Ts;C>T,T>C)andtransversions(Tv;C>A,C>G,T>A,T>G)werecalculatedforTg-SPC-SFN+/− and WTtumors.D,MutationfrequencywascountedaccordingtothegenechromosomelocationineachTg-SPC-SFN+/−andWTtumor.Valuesindicatethepercentagenumberofmutatedgenesamongthetotalnumberofmutatedgenesdetectedintheeachchromosome

     |  7KIM et al.

3.5 | Impact of mutated genes in mice and human lung adenocarcinoma

ToclarifydistinctsignalingpathwaysrelatedtotumorigenesisinTg-SPC-SFN+/−andWTmice,wecarriedoutpathwayanalysesforexonicmutationsusingtheKEGGpathwayandIPAsoftware.First,wese-lectedparticularsignalingpathwaysrelatedtocellgrowthorcancer,suchasPI3K/AKTsignaling,ERK/MAPKsignaling,STAT3signaling,theDNAdamageresponseandrepairsystem,cellcyclesignaling,p53signaling,PTENsignaling,and14-3-3-mediatedsignaling,andcountedthenumberofmutatedgenescategorizedintothosepathwaysineachtumorusingtheKEGGpathwayandIPAsoftware.ThisrevealedthatallTg-SPC-SFN+/−tumorshadahighpercentageofmutatedgenesre-latedtoPI3K/AKTsignaling,suchasFgf7, Itga6, Synj2,andTsc2 and theapoptosissignalingpathway(Figure5AandTableS5).

Wethencollectedallmutatedgenesin4Tg-SPC-SFN+/−tumorsand investigated pathways that were significantly enriched usingtheKEGGpathwayandIPAsoftware.TheKEGGpathwayanalysisrevealed that mutated genes in Tg-SPC-SFN+/− tumors were sig-nificantlyassociatedwithpathways incancer,ubiquitinatedprote-olysis,small-cell lungcancer, thePI3K-AKTsignalingpathway,andtranscriptionalmisregulation in cancer (Table S6). In contrast, IPAsoftware analysis indicated that Tg-SPC-SFN+/− tumors harboredgene mutations that were significantly correlated with canonicalpathways such as 14-3-3-mediated signaling, apoptosis signaling,PTEN signaling, and DNA double-strand break repair by homolo-gous recombination (Table S6). Taken together, overexpression ofSFNinthelungappearedtoinducegenemutationsmainlyinvolvedinPI3K/AKTsignalingandapoptosissignalingpathwaysfacilitatingtumorigenesis.

F I G U R E 4  FrequentlymutatedgenesidentifiedinTg-SPC-SFN+/−tumorsbywhole-exomesequencing.A,Venndiagramswereusedtoidentifycommonlymutatedgenesamong4Tg-SPC-SFN+/− tumors.NumbersineachregionindicatethenumbersofmutatedgenesdetectedinTg-SPC-SFN+/−tumors.Percentagesineachregionindicatethepercentagenumberofgenesamongthetotalnumberofmutatedgenesdetectedinthe4Tg-SPC-SFN+/−tumors.M,male;F,female;Tg,SFN-transgenicmouse.B,SomaticgenescommonlymutatedamongTg-SPC-SFN+/−tumorswereselected.ThecountednumberindicatesthemutationfrequencyofeachgenedetectedinTg-SPC-SFN+/−tumorsbywhole-exomesequencing.Eachcolumnrepresentsanindividualtumor,andeachrowdenotesagene.Allmutationswerenonsynonymousmissensemutations

8  |     KIM et al.

Next, usingTCGA,we investigated themutation frequencyof73genesthatwerecommonlymutatedinTg-SPC-SFN+/−tumorsinhumanlungadenocarcinomas.Mostofthetop-rankedgeneswerenotwellknown,andlikelypassengermutationsthatdonotdirectlyconferaselectivetumorgrowthadvantage.Amongthem,titin(TTN),aso-calledgiantgenewith363codingexonsinthehumangenomeand tending to have frequentmutations becauseof randomDNArepairerror,28,29wasthetoprankedgene,harboredby314of487lungadenocarcinomapatients.Moreover,HERC2, SMG1,andNLRP2,whichweredetectedin3samplesofTg-SPC-SFN+/−tumors,alsohada high ranking formutation frequency in human lung adenocarci-noma(Figure5BandTableS7).

Inordertoclarifytheimpactofthesegenealterationsonprog-nosis,overallsurvivalwascomparedbetweenpatientsshowinghighexpressionandthoseshowinglowexpressionusingTCGA(TableS8).ThisrevealedthatpatientswithhighexpressionofBLM, DNMT3B, MKI67, ITGA6, orCDC20 had a significantly poorer outcome thanthosewithlowexpressionofthegenes(FigureS4A).Incontrast,pa-tientswithhighexpressionofNLRP2, CUL9, TBC1D9,orCDH23 had a significantlymore favorable outcome relative to thosewith lowexpressionofthesegenes(FigureS4B).

In summary, overexpression of SFN might frequently induce alargenumberofmutations,mostofwhichareinvolvedinPI3K/AKT

signaling and apoptosis signaling, and altered expression of thosegenesaffectspatientoutcome.

4  | DISCUSSION

Recently,thegenomicalterationsdrivinglungadenocarcinogenesishavebeeninvestigatedtoclarifythemolecularmechanismsinvolved,discovernoveldrugtargets,andscreencandidatepatientsforclini-caltrialsusingnext-generationsequencing.27,30Extensivemolecularanalysishasshownthatnormalcellsprogressivelyacquireavarietyofgeneticimbalancesthatleadtotumorigenesis.31Tumorigenesisismediatedbyacombinationofeventsincludingupregulationofon-cogenessustainingproliferativesignaling,downregulationoftumorsuppressorgenes,andalsoresistancetoapoptoticcelldeath,thusenablingreplicativeimmortality.32

Inthepresentstudy,byanalyzingtumorsthatdevelopedinTg-SPC-SFN+/−miceafterexposuretoNNK,weshowedthatSFN had po-tentoncogenicfunctionscausingdistinctgeneticalterationsduringtheprocessoftumorigenesis.AsTg-SPC-SFN+/−micewithoutNNKtreatmentdevelopedfewbuthistologicallysimilarlytumorstoNNK-induced tumors inTg-SPC-SFN+/−mice,11weconsider thatoverex-pressionofhSFNinthelunginICRmicerobustlyinducesinitiation

F I G U R E 5  PathwayanalysisformutatedgenesinTg-SPC-SFN+/−tumorsandfrequenciesofcommonlymutatedgenesinlungadenocarcinomapatients.A,IPAandKEGGpathwayanalysiswereusedtoinvestigatethemolecularfunctionsofeachofthemutatedgenesinTg-SPC-SFN+/−andWTtumors.TheheatmapindicatesthepercentagesofmutatedgenesamongthetotalnumberofmutatedgenesdetectedintheeachTg-SPC-SFN+/−andWTtumorandwasdrawnusingMorpheus,awebtool(https://software.broadinstitute.org/morpheus/).CommonlymutatedgenesamongTg-SPC-SFN+/−tumors,whichareincludedineachpathway,areindicatedintherightcolumn.B,PercentagesoflungadenocarcinomapatientsharboringeachmutationcommonlyfoundinTg-SPC-SFN+/−tumorsaredisplayedinthebargraph.TCGAwasusedforthisanalysis.Arrowsdenotecommonlymutatedgenesin3Tg-SPC-SFN+/−tumorsapartfromWTtumor

     |  9KIM et al.

of tumor formation, similar toNNKexposure.Moreover, allNNK-inducedtumorsinTg-SPC-SFN+/−micehavehighsimilaritytomosthuman lung adenocarcinomas in terms of histology33 and geneticprofiles, includingahighfrequencyofpassengermutations,ahighfrequencyoftransversion,alargemutationburden,andsmoking-likesignatures (Figures 1-3, S2, and S3).Moreover, tumors that devel-oped inTg-SPC-SFN+/−micedidnotcontainKrasmutation,knownasadrivermutationformurinelungcancer.34Therefore,webelievethatthismousemodelcloselymimicsactualhumanadenocarcinoma.

WerevealedthatNNKinducedtumorformationaccompaniedbyfrequentC>TtransitionsubstitutioninbothTg-SPC-SFN+/− and WT mice(Figure2A),indicatingthatthehighfrequencyofthissubstitu-tioniscausedbyadefectiveDNArepairprocessattributedtoNNK.35 Consistentwithourresults,NNKisknowntobeastrongtobacco-de-rivedcarcinogenandundergoesmetabolismtoDNAadducts,whichthenbindtoDNA,resultinginmutationofKRAS and STK11,andthuslung tumor formation invariousanimalmodels.36,37Unlike tobaccosmoke,whichcontainsmorethan70carcinogeniccompoundssuchasbenzo[a]pyrene,whichpredominantlyinducesC>Atransversionmu-tation,treatmentofratswithNNKleadstofrequentC>TtransitionmutationcausedbyO6-mGua,whichisahighlypromutagenicadductinducedbyNNKandcanberemovedbyDNArepairproteinssuchasO-6-methylguanine-DNAmethyltransferase.35,38

Incontrast,mutatedgenesinTg-SPC-SFN+/−tumorshadahighfrequencyofC>Atransversion(Figure2B).Thissubstitutionmightbeduetofailureofthetranscription-couplednucleotideexcisionrepairmachinery,similartohumanmutationalsignaturesthatarecorrelatedwithsmokinghistory.39Suchmutationalsignaturescanindicate thecauseof somaticmutations suchasDNAreplicationinfidelity, defective DNA repair processes, and mutagen expo-sureoccurringthroughoutlife.40Among30mutationalsignaturesin theCOSMIC database, signature 4 is themost closely relatedto lung cancers associated with smoking,23,39 and signature 24is linked to liver cancer and exposure to aflatoxin, a carcinogenproduced by fungi in nature,39 both of which show similarity toTg-SPC-SFN+/−tumors.

Through interaction with the target proteins, 14-3-3 proteinsincludingSFNalter theactivity,modifications,and intracellular lo-calizationofligands.41-43Inparticular,SFNregulatesDNArepairfac-torssuchasDNA-PKcs,whenDNAisdamagedbychemotherapyorradiotherapyinhumancancer.44Basedonthepreviousreport,weassume that thehighnumberofgenemutations inTg-SPC-SFN+/− tumorsmight be due to bindingwith unknown factor(s), which isassociatedwiththeDNArepairsystem.SFNmightinhibitDNAre-pair-relatedfactors,resultingintheaccumulationofnumerousgenemutations. Indeed,we found somemutatedgenes involved in theDNAdamageresponseandrepairsystemsuchasBlm, Brd4, Herc2, Brca1, Pole, Ddb1, Msh6, and Xrcc1 only in Tg-SPC-SFN+/− tumors(Table S5), suggesting that these alterations could also indirectlyaccelerate dysfunction of the DNA repair system and thus thesemutatedgenesmightcauseadditionalmutationagain.Hightumormutationburdenisknowntoinducehighneoantigenexpression,45 whichenhancestumorgrowth,invasion,andmetastasisbyevasion

fromimmuneattack.46ItimpliesthatTg-SPC-SFN+/−mightserveasausefultooltoevaluatetheefficacyofimmunecheckpointinhibitors.

Additionally,ourpreviouspull-downassayandLC-MS/MSanaly-sishasidentifiedSFNbindingproteinsinlungadenocarcinomacellsincluding cell cycle-relatedproteins such as S-phase kinase tumorprogression(SKP1),10andproliferationrelatedproteinssuchasser-ine/threonine-proteinkinaseA-Raf(ARAF)47andubiquitin-specificprotein8 (USP8),whichparticularlyenhancesstabilityof receptortyrosinekinasesuchasepidermalgrowthfactorreceptorandMET.9 Wespeculatethat,asSFNcontrolsthoseproteins,tumorcellsmightpass through the cell cycle checkpoint even though they recog-nizea largenumberofmutationsduetoinsufficientDNArepair.48 Consequently,SFNmightinducealargenumberofmutations,whicharepredominantlyinvolvedinPI3K-AKTsignalingandapoptosissig-naling(Figure5A),andthosemutationsmightboostcanonicalsignal-ingforadenocarcinogenesis.

Moreover,werevealedthatTg-SPC-SFN+/−tumorsharboredmu-tationsofBlm, Dnmt3b, Mki67, Itga6, Cdc20, Nlrp2, Cul9, Tbc1d9,andCdh23 thatwerealsodetectable in lungadenocarcinomapatients,andthattheexpressionofthesegeneswassignificantlycorrelatedwithoverallsurvival(FigureS4).However,themutationalhotspotsandoncogenicactivityofthesemutatedgenesinlungcancershavenotyetbeenclarified.Althoughthereisapossibilitythatthesemu-tatedgenesmightbeinvolvedinlungadenocarcinogenesistriggeredby SFN, further verification and functional analysis using humanlungadenocarcinomaspecimenswillbeneeded inordertounder-stand the overall molecularmechanisms involved. Additionally, asWEShaslimitationstodetectinparticulargeneticalterationssuchasmutations,deletions,andinsertions,weareplanningtoundertakewholegenomeamplification-sequencingorRNA-sequencingto in-vestigatetheamplificationorexpressionofgenesforfurtherstudy.

Furthermore,weexaminedthemutationallandscapeinbothTg-SPC-SFN+/−tumorsandWTtumor.ComparedtotheTg-SPC-SFN+/− tumors,WT tumor showed poorer differentiated histology, higherfrequencyofT>CandT>G,andcontainedalowernumberofmutatedgenes.Additionally,onlyWTtumorcontainedKrasmutation.ThesedifferencesbetweenTg-SPC-SFN+/−andWTtumorsarevery inter-esting.Unfortunately,inthecurrentstudy,wecouldexamineonlyoneWTtumor,asthetumorigenicfrequencyisverylowinICRmicebyNNKtreatment.Large-scaleexaminationsshouldbeplanned inthefuturetomakeclearthecharacteristicsofTg-SPC-SFN+/−tumors.

Insummary,wecharacterizedtumorsthatdevelopedinTg-SPC-SFN+/−miceandfounddistinctexonicalterations,remarkablymim-ickingthefeaturesofhumanlungadenocarcinoma.WebelievethatSFNoverexpression is critical tohuman lungadenocarcinogenesisandthatourmousemodel,createdbybothgeneticengineeringandcarcinogen exposure,would be a valuable resource for preclinicalexaminationsoftherapyforlungadenocarcinoma.

DISCLOSURE

The authors have no financial conflicts of interest related to thismanuscript.

10  |     KIM et al.

ORCID

Aya Shiba‐Ishii https://orcid.org/0000-0001-6639-4135

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SUPPORTING INFORMATION

Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle.

How to cite this article:KimY,Shiba-IshiiA,RamirezK,etal.Carcinogen-inducedtumorsinSFN-transgenicmiceharboracharacteristicmutationspectrumofhumanlungadenocarcinoma. Cancer Sci. 2019; 00: 1–11. https://doi.org/10.1111/cas.14081