T-cell-expressed proprotein convertase FURIN inhibits DMBA/TPA-induced skin cancer development

ORIGINAL RESEARCH T-cell-exp essed p op o ein con e ase FURIN inhibi s DMBA/TPA-induced skin cance de elopmen Ma ia V€ ah€ a upa a , *, Saa a Ai om€ aki b , *, Zuze Ma inez Co do a b , *, Ul ike May a , S ua P ince a , Hannele Uusi alo-J€ a inen c , Te o A. J€ a inen a , d , # , and Ma ko Pesu b , e , # a School o Medicine, Depa men o Ana omy and Cell Biology, Uni e si y o Tampe e, Tampe e, Finland; b Immuno egula ion, BioMediTech, Uni e si y o Tampe e, Tampe e, Finland; c Depa men o Oph halmology, Tampe e Uni e si y Hospi al, Tampe e, Finland; d Depa men o O hopedics & T auma ology, Tampe e Uni e si y Hospi al, Tampe e, Finland; e Depa men o De ma ology, Tampe e Uni e si y Hospi al, Tampe e, Finland ARTICLE HISTORY Recei ed 25 July 2016 Re ised 22 Sep embe 2016 Accep ed 3 Oc obe 2016 ABSTRACT P op o ein con e ases (PCSK) ha e a c i ical ole in he body homeos asis as enzymes esponsible o p ocessing p ecu so p o eins in o hei ma u e o ms. FURIN, he fi s cha ac e ized membe o he mammalian PCSK amily, is o e exp essed in mul iple malignancies and he inhibi ion o i s ac i i y has been conside ed po en ial cance ea men . FURIN has also an impo an unc ion in he adap i e immuni y, since i s deficiency in T cells causes an impai ed pe iphe al immune ole ance and accele a es immune esponses. We add essed whe he dele ing FURIN om he immune cells would s eng hen an icance esponses by subjec ing mouse s ains lacking FURIN om ei he T cells o mac ophages and g anulocy es o he DMBA/TPA wo-s age skin ca cinogenesis p o ocol. Unexpec edly, deficiency o FURIN in T cells esul ed in enhanced and accele a ed de elopmen o umo s, whe eas FURIN dele ion in mac ophages and g anulocy es had no e ec . The epide mises o T-cell-specific FURIN deficien mice we e significan ly hicke wi h mo e p oli e a ing Ki67Ccells. In con as , he e we e no di e ences in he numbe s o he T cells. The flow cy ome ic analyses o T-cell popula ions in skin d aining lymph nodes showed ha FURIN T-cell KO mice ha e an inhe en up egula ion o ea ly ac i a ion ma ke CD69 as well as mo e CD4 C CD25 C Foxp3 C posi i e T egula o y cells. In he ea ly phase o umo p omo ion, T cells om he T-cell-specific FURIN knockou animals p oduced mo e in e e on gamma, whe eas a la e s age he p oduc ion o Th2- and Th17- ype cy okines was mo e p ominen han in wild- ype con ols. In conclusion, while PCSK inhibi o s a e p omising he apeu ics in cance ea men , ou esul s show ha inhibi ing FURIN specifically in T cells may p omo e squamous skin cance de elopmen . Abb e ia ions: CTL, cy o oxic T lymphocy e; dLN, d aining lymph node; DMBA, 7,12-Dime hylbenz[a]an h acene; IFNg, in e e on gamma; IL, in e leukin; KO, knockou ; LysM, lysozyme-M; OVA, o albumin; PCSK, p op o ein con e - ase; TCR, T-cell ecep o ; TGF-b1, ans o ming g ow h ac o -b1; TPA, 12-O- e adecanoylpho bol-13-ace a e; T eg, egula o y T cell; VEGF, ascula endo helial g ow h ac o KEYWORDS FURIN; p op o ein con e ase; squamous skin cance ; T cells; ans o ming g ow h ac o -b1 In oduc ion The mammalian p op o ein con e ase (PCSK) amily consis s o nine membe s. The p ima ily iden ified se en PCSKs (PCSK1-2, FURIN, PCSK4-7) a e closely ela ed and e olu ion- a ily conse ed sub ilisin/kexin-like se ine p o eases ha con- e hei imma u e subs a es in o unc ional end-p oduc s by clea ing basic amino acid mo i s ((K/R)-(X)n-(K/R)#, whe e n is 0, 2, 4, o 6 and X is any amino acid). 1 PCSKs ope a e mainly in he sec e o y pa hway, on he cell su ace and in he endo- somes. Mo e ecen ly iden ified and dis an ly ela ed PCSK amily membe s MBTPS1 and PCSK9 di e om he se en o he membe s in hei a ge sequence specifici ies. MBTPS1 a ge s a consensus mo i (R/K)-X-(hyd ophobic)-X#and PCSK9 has only au oca aly ic clea age ac i i y. 2 PCSK enzymes play an ins umen al ole in main aining homeos asis in he body bu also in a numbe o pa hological con- di ions. 2 Va ious PCSK a ge p o eins a e in ol ed in malignan ans o ma ion and p og ession. PCSKs ac i a e o example cell su ace-exp essed ecep o s (e.g., in eg ins), issue-modi ying enzymes like ma ix me allop o einases 3-5 and g ow h ac o s needed o suppo umo angiogenesis, including ascula endo- helial g ow h ac o s (VEGF) C and D. 6,7 Many human cance s show high le els o PCSK exp ession ( e iewed in e . 8 ). Fo exam- ple, FURIN exp ession is ele a ed in non-small cell lung CONTACT Ma ko Pesu ma ko.pesu@u a.fiImmuno egula ion, BioMediTech, Uni e si y o Tampe e, Lääkä inka u 1, FI-33520 Tampe e, Finland; Te o A. J€ a inen e o.ah.ja inen@u a.fiSchool o Medicine, Depa men o Ana omy and Cell Biology, Uni e si y o Tampe e, Lääkä inka u 1, FI-33520 Tampe e, Finland. Supplemen al da a o his a icle can be accessed on he publishe ’s websi e. *These au ho s con ibu ed equally. # Equal con ibu ion and co esponding au ho s. Published wi h license by Taylo & F ancis G oup, LLC © Ma ia V€ ah€ a upa, Saa a Ai om€ aki, Zuze Ma inez Co do a, Ul ike May, S ua P ince, Hannele Uusi alo-J€ a inen, Te o J€ a inen and Ma ko Pesu. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-Non-Comme cial License (h p://c ea i ecommons.o g/licenses/by-nc/3.0/), which pe - mi s un es ic ed non-comme cial use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. The mo al igh s o he named au ho (s) ha e been asse ed. ONCOIMMUNOLOGY 2016, VOL. 5, NO. 12, e1245266 (11 pages) h p://dx.doi.o g/10.1080/2162402X.2016.1245266 ca cinoma as well as in human head and neck squamous cell ca - cinomas, and he up egula ed FURIN ac i i y co ela es wi h accele a ed umo p og ession. 9-11 T ansgenic o e exp ession o FURIN in mouse epide mal basal laye esul ed in inc eased papil- loma and squamous cell ca cinoma de elopmen and enhanced umo g ow h when he mice we e subjec ed o a wo-s age chem- ical ca cinogenesis p o ocol 12 . In line wi h ha da a, dele ing FURIN om mouse sali a y gland cells also delayed PLAG1- induced sali a y gland umo igenesis. 13 PCSK enzymes a e also key egula o s o he immune sys em. By using a condi ional, T-cell-specificFURINdeficien mouse (CD4c e- u flox/flox )weha eshown ha T-cell-exp essedFURIN is c i ical o main aining pe iphe al ole ance. 14 T-cell-specific FURIN dele ion causes age- ela ed au oimmuni y, wi h expan- sion and o e ac i a ion o e ec o T cells, excessi e p oduc ion o p o-inflamma o y cy okines, and unc ionally de ec i e egula- o y T cells (T eg). The au oimmune pheno ype in FURIN T- cell KO mice could be a leas pa ially a ibu ed o educed le - els o bioac i e, an i-inflamma o y cy okine ans o ming g ow h ac o -b1(TGF-b1), which is a known FURIN subs a e mole- cule. 15 Fu he mo e, we ha e ecen ly demons a ed ha FURIN di ec ly egula es T-cell ac i a ion by modi ying he TCR- induced ansac i a ion s eps. 16 The implica ion o PCSKs as egula o s o umo p og ession and me as asis has p o oked an in e es o use hem as a ge s o no el an icance agen s. Based on ou p e ious findings, inhibi- ion o FURIN ac i i y in immune cells could be an e ec i e way o boos ing an i umo hos esponses. Thus, we in es iga ed how he immune-cell-exp essed FURIN egula es umo igenesis by using mouse s ains wi h a condi ional dele ion o FURIN ei he in T cells o in myeloid cells (CD4c e and LysMc e, espec i ely) in he wo-s age chemical DMBA/TPA-induced skin ca cinoma model. Ou da a demons a e ha FURIN exp ession in T cells, bu no in myeloid cells, cons ains he DMBA/TPA-induced de elopmen o squamous skin cance . Resul s T-cell-exp essed FURIN inhibi s skin umo induc ion Ta ge ing PCSK, and in pa icula , FURIN ac i i y, has been conside ed a p omising cance ea men . 17 Howe e , he ge m-line dele ion o FURIN causes emb yonic le hali y, which con ounds he s udies on i s in i o unc ions in cance esea ch. 18 The e o e, he cell- ype-specific unc ion o FURIN in ca cinogenesis has emained incomple ely unde s ood. To in es iga e i he immune-cell-exp essed FURIN con ols skin umo o ma ion, we ea ed he back skin o adul mice defi- cien o FURIN gene exp ession ei he in mac ophages and g anulocy es (designa ed LysMc e KO 19,40 ) o in CD4 C and CD8 C T cells (designa ed CD4c e KO, 14 ) and hei espec i e wild- ype li e ma es (LysM WT and CD4 C WT) once wi h a local applica ion o he mu agen DMBA, and hen wi h he g ow h-p omo ing agen TPA, wice weekly o a pe iod o 16 and 21 weeks. This ea men induces papillomas de i ed om he in e ollicula epide mis. 20 FURIN p o ein exp ession was de ec ed in un ea ed and DMBA/TPA- ea ed skin in CD4 C WT mice (Fig. S1). In no - mal skin, FURIN was exp essed abundan ly in he epide mis and some esiden cells in he de mis we e also posi i e o FURIN exp ession. DMBA/TPA applica ion induced FURIN mRNA exp ession and esul ed in a s ong accumula ion o FURIN exp essing cells in he de mal pa o he skin (Fig. S1). Unexpec edly, dele ion o FURIN specifically om T cells esul ed in he de elopmen o mo e papillomas (p<0.0001, Fig. 1A). The fi s papillomas we e obse ed in he CD4c e KO mice 8 weeks a e he beginning o he DMBA/TPA ea men , and a e 9 weeks, all o he CD4c e KO mice had de eloped papillomas on hei back skin. The fi s papillomas we e iden i- fied in bo h WT con ol s ains as well as in he LysMc e FURIN KO mice a e 10–12 weeks o ea men (Fig. 1A). Fu he mo e, he CD4c e KO mice also de eloped significan ly mo e umo s on hei back skin han he o he s ains (p<0.001, Fig. 1B). P io o eu haniza ion (a 17 weeks due o e hical easons), he CD4c e KO mice had de eloped almos 20 papillomas pe ani- mal, whe eas he WT con ols had less han fi e papillomas on a e age (Fig. 1B). In addi ion, bo h LysMc e KO and LysM WT mice had a simila numbe o umo s a 17 weeks as CD4 C WT mice. The ea men o LysMc e KO and WT s ains was con in- ued o addi ional 5 weeks, bu no di e ences in umo o ma- ion could be de ec ed (Fig. 1B). The umo s we e inciden in CD4c e KO animals a a a e on a e age 4.6- old g ea e han in CD4 C WT mice du ing he cou se o expe imen s (nega i e binominal eg ession analysis:incidence a e a io (IRR) D4.6; 95% confidence in e al (CI) 1.97, 10.79). Despi e CD4c e KO mice we e de eloping skin umo s significan ly as e and in g ea e numbe s han he o he s ains, he papillomas in he CD4c e KO mice did no con- inue o g ow in size (Fig. S2A). Ins ead, a la ge numbe o small papillomas isibly disappea ed and some con e ed in o ch onic ulce s (Fig. S2B). We could no de ec simila ulce s in CD4 C WT, LysMc e KO, and LysM WT s ains (Fig. 1C and Fig. S2). The his ological analysis e ealed ha he ulce s in CD4c e KO mice had papilloma o ma ions, bu also up u ed epide mis and clus e s o neu ophils as a sign o comp omised physical in eg i y o he skin (Fig. S2F). In conclusion, he lack o FURIN in T cells, bu no in LysM C myeloid cells, p omo es umo induc ion and o ma- ion in he DMBA/TPA-induced skin cance . This p omp ed us o u he cha ac e ize he ole o T-cell-exp essed FURIN in squamous skin cance immunosu eillance. The suscep ibili y o umo o ma ion in CD4c e FURIN KO mice is associa ed wi h enhanced cell p oli e a ion, bu no wi h ascula iza ion To unde s and he mechanism o he skin umo -inhibi ing unc ion o T-cell-exp essed FURIN, we fi s pe o med his olog- ical analyses o de e mine he epide mal and de mal hicknesses, cell p oli e a ion (Ki67) and apop osis (TUNEL) equency in he back skin o DMBA/TPA ea ed and un ea ed mice. In un ea ed mice, loss o FURIN om T cells esul ed in a sig- nifican hickening o he epide mis (Fig. S3A). T ea men wi h DMBA/TPA induced a subs an ial inc ease (p<0.001) in he epi- de mal hickness in bo h geno ypes (bo h epide mises p<0.0001 o e un ea ed skin), and app oxima ely a 30% inc ease in he hickness o he epide mis pe sis ed in he CD4c e KO mice o e he CD4 C WT mice (p<0.0001, Fig. S3A). In con as , significan e1245266-2 M. V € AH € ATUPA ET AL. di e ences we e no de ec ed in he de mal hicknesses o un ea ed o ea ed animals (Fig. S3B). CD4c e KO mice had also significan ly mo e p oli e a ing cells (as de e mined by Ki67-posi i i y) han WT mice in he epide mis o he un ea ed skin (p<0.0001, Fig. S3C). A e 17 weeks o DMBA/TPA ea men , he CD4c e KO mice had mo e p oli e a ing cells in bo h epide mal and de mal pa s o he skin (p<0.001 and pD0.0056, Figs. S3C and D). In con- as , he e was no di e ence in he cell p oli e a ion a e in he papilloma issue (o he de mis benea h i ) (Figs. S3C and D). TUNEL s aining demons a ed ha CD4c e KO mice had less apop o ic cells in un ea ed de mis (p<0.05, Fig. S4A), bu a p olonged DMBA/TPA- ea men (17 weeks) educed signifi- can ly he numbe s o dying cells in bo h geno ypes (epide mis, p<0.0001, de mis p<0.0001, Fig. S4). As he a ailabili y o ascula supply is a limi ing ac o o umo g ow h and FURIN has a subs an ial influence on angio- genesis, 6,7 we also examined he ascula u e in he skin. Angiogen- esis is u ned on al eady in he ea lies s ages o papilloma o ma ion and in la e s ages he blood essels s a o inc ease in size. 21 The ewe esligh lymo eblood esselsinCD4c eKOmice in he un ea ed skin compa ed o WT as de e mined by an immunohis ological analysis o endo helial cell ma ke CD31 (Fig. S5A). A 17-week ea men wi h DMBA/TPA induced a 2- old inc ease in he ascula densi y o he skin only in CD4 C WT mice (p<0.0001), whe eas in CD4c e KO he ascula densi y was significan ly smalle (p<0.0001, Fig. S5A). Despi e he ac ha non- umo ous WT skin had significan ly mo e blood essels hanin heCD4c eKOmicea e heDMBA/TPA ea men , he umo o ma ion equi ed ascula supply in bo h geno ypes as e idenced by he inc eased numbe o blood essels benea h he umo s (p<0.0001, Fig. S5). In conclusion, he accele a ed umo igenesis in CD4c e KO mice was no ound o be associa ed wi h inc eased angiogenesis. Mice lacking FURIN in T cells ha e an a enua ed mac ophage ex a asa ion o di e en ia ion esponse o DMBA/TPA ea men P e ious wo k has shown ha umo igenesis in he DMBA/ TPA model is p omo ed upon he induc ion o acu e inflamma ion in he skin a he si es o chemical applica- ion. 22,23 To in es iga e i he CD4c e KO mice had an enhanced skin inflamma o y esponse, we fi s quan ified skin CD3 C T cells as well as he numbe s o infil a ing F4/ 80Cmac ophages and elas ase posi i e neu ophils. The e we e significan ly mo e CD3 C T cells in he CD4c e KO mice han in li e ma e con ols in bo h un ea ed skin (p<0.0001) and a 43 h pos DMBA/TPA ea men (p< 0.05), bu a e 17 weeks T cell numbe s we e simila (Figs. 2A and B). In con as , he numbe s o mac ophages we e mo e eadily induced in KO mice a 43-h ime-poin , bu in e es ingly his was e e ed a la e s ages; a e a 17- week ea men CD4c e KO mice showed significan ly educed mac ophage coun s in non- umo ous skin (p< 0.001, Figs. 2C and D). The significan ly lowe numbe o mac ophages in he CD4c e KO skin was especially e iden unde nea h he papillomas, whe e he KO mice had only ca. wen y-fi e pe cen o he mac ophages seen in he WT de mis (Figs. 2C and D). No significan di e ences we e Figu e 1. T-cell-specific dele ion o FURIN accele a es skin umo o ma ion. Wild- ype (LysM WT and CD4 C WT), T-cell (CD4c e) and mac ophage and neu ophil-specific (LysMc e) knockou mice we e subjec ed o DMBA/TPA-induced skin ca cinogenesis. (A) The pe cen age o umo - ee animals a each ime poin is shown. Su i al plo was gene a ed and analyzed ia log- ank (Man el-Cox) es . (B) The mean numbe o umo s pe mouse a each ime poin is shown §s anda d e o o he mean. The da a we e analyzed using STATA 13.0 so wa e. A non-linea eg ession model was used o compa e he slopes o he da a. (C) Rep esen a i e pho og aph o a CD4 C WT mouse a week 13 o he DMBA/TPA ea men ial, alongside a hema oxylin-eosin s ained sec ion o skin a week 17 ( he black ba ep esen s 2 mm). (D) Rep esen a i e pho og aph o a CD4c e KO mouse a week 13 o he DMBA/TPA ea men ial, alongside a hema oxylin-eosin s ained sec ion o skin a week 17 ( he black ba ep e- sen s 2 mm). CD4 C WT n D6, CD4c e KO n D4, LysM WT nD5, and LysMc e KO n D5. ONCOIMMUNOLOGY e1245266-3 Figu e 2. Immune cell numbe s in he skin o WT and CD4c e FURIN KO mice. CD4 C WT and CD4c e KO mice we e subjec ed o DMBA/TPA-induced skin ca cinogenesis as desc ibed in me hods. Skin samples we e collec ed om un ea ed animals and om mice sac ificed a 43 h a e he second TPA applica ion, and a e 17 weeks o ea men ( wice weekly). The skin samples we e p ocessed o IHC as desc ibed in me hods. Skin sec ions we e IHC s ained o ma ke s o T cells (CD3), mac ophages (F4/80), and neu- ophils (neu ophil elas ase). Resul s a e shown as mean §95% confidencein e als.Da awe eanalyzedbyno mali y es sandunpai ed wo- ailedS uden ’s - es s (G aph- Pad P ism 6). The black ba in images ep esen s 200 mm. Nuclei a e s ained blue, and immune cells b own. (A) Rep esen a i e pho og aphs o T cell s aining in CD4 C WT and CD4c e KO skin. (B) Quan i a i e analyses o scanned slides we e pe o med as desc ibed in supplemen a y me hods. Da a is exp essed as % o o al nuclei. (C) Rep esen a i e pho og aphs o mac ophage s aining and (D) esul s o quan i a i e analyses o scanned slides. (E) Rep esen a i e pho og aphs o neu ophil s aining and (F) esul s o quan i a- i e analyses o scanned slides. The in o ma ion o animal numbe s and analyzed issue egions is shown in Table S1. (  p<0.0001;  p<0.001;  p<0.01;  p<0.05). e1245266-4 M. V € AH € ATUPA ET AL. obse ed in neu ophils be ween he geno ypes (Figs. 2E and F). Collec i ely hese findings sugges ha CD4c e KO mice may ha e accele a ed skin immune esponses a he ea ly s age o cance de elopmen , bu his is la e ollowed by educed mac ophage p esence in cance ous skin. The skin-d aining lymph node T cells lacking FURIN display an ac i a ed pheno ype To gain specific in o ma ion on he T-lymphocy e popula ions, he skin d aining lymph node (dLN) cells om CD4c e KO mice and hei WT li e ma e con ols we e subjec ed o flow cy ome ic analysis. The e we e no significan di e ences in he o al CD4 C T cell pe cen ages (o o al li e cells) be ween he un ea ed CD4c e KO and WT mice o be ween CD4c e KO and WT mice ea ed wi h DMBA/TPA o 17 weeks (Fig. 3). In un ea ed CD4c e KO mice, he e we e ewe dLN CD8 C T cells (pe cen o he li e cells) han in he un ea ed WT con ols, bu he di e ence was los a e he 17-week DMBA/TPA ea men . Howe e , he dLN T cells om he CD4c e KO animals dis- played a mo e ac i e pheno ype, as significan ly highe p opo - ion o bo h CD4 C and CD8 C T cells om KO animals was posi i e o CD69, a ma ke o ecen ly ac i a ed cells (Fig. 3). The analysis o T-cell memo y pheno ypes using CD44 and CD62L an ibodies demons a ed a significan ly highe pe cen - age o cen al memo y CD62L high CD44 high CD8 C T cells (o o al CD8 C T cells) in bo h un ea ed and 17-week- ea ed CD4c e KO animals (Fig. 3). This is in pa allel wi h a ecen epo showing ha blocking endogenous TGF-b1 signaling in CD8 C T cells enhances hei con e sion in o cen al memo y cells. 24 Since FURIN p ocesses p o-TGF-b1, he au oc ine TGF- b1 signaling is likely o be de ec i e in FURIN-deficien CD8 C T cells. The e we e also significan ly mo e CD4 C CD25 C Foxp3 C T eg cells in he skin dLNs o CD4c e KO mice compa ed o hose o WT li e ma es, bo h om un ea ed mice and mice ea ed wi h DMBA/TPA o 17 weeks (Fig. 3). In conclusion, FURIN exp ession did no a ec he numbe s o T cells in he skin dLN a e a 17-week DMBA/TPA- ea - men , bu T cells om he dLNs o he CD4 C KO mice we e inhe en ly mo e ac i e and cons i u ed mo e o cells wi h a T eg pheno ype, i espec i e o he ea men . The e o e, he inc eased suscep ibili y o CD4c e KO mice o de elop papillo- mas is no due o educed dLN T-cell numbe s o lack o T-cell ac i a ion. T cells lacking FURIN p oduce mo e in e e on gamma in he ea ly phase o umo de elopmen To in es iga e he cy okine p oduc ion o T cells in he ea ly phase o umo de elopmen , we analyzed dLN T cells a 48 h pos he second TPA ea men by in acellula s aining and flow cy ome y. Bo h CD4 C and CD8 C e ec o T cells om he CD4c e KO mice p oduced mo e p o-inflamma o y in e e on gamma (IFNg)(Figs. 4B and C). On he o he hand, he e was no di e ence in he p oduc ion o ano he p o-inflamma o y cy okine, in e leukin (IL) 17A (IL-17A), o in he CD4 C T-cell- p oduced IL-10, which is conside ed an i-inflamma o y (Figs. 4B and C). In o de o gain in o ma ion on he p edomi- nan ype o immune esponse in he CD4c e KO mice and hei WT con ols, we analyzed he sys emic p oduc ion o a - ious chemokines, by pe o ming a mul iplex assay om he se a 43 h pos second TPA ea men . The e we e no ob ious di e ences in he le els o CCL2/MCP1, CXCL-1/GROa, CCL11/Eo axin, CXCL2/MIP-2, CCL7/MCP-3, CCL5/ RANTES, CCL3/MIP-1a, o CCL4/MIP-1b(da a no shown), bu he le el o CXCL10/IP-10 was ele a ed by 2- olds in CD4c e KO se a compa ed o hose o WT mice (pD0.0711, Fig. 4E). Collec i ely, hese findings imply ha in he ea ly Figu e 3. P ofiling o he skin d aining lymph node T-cell popula ions in WT and CD4c e KO mice. Cells we e isola ed om un ea ed mice and om animals ea ed wi h a single applica ion o DMBA and wice-weekly doses o TPA o 17 weeks, and su ace ma ke s we e analyzed by flow cy ome y. F equency o cells among o al li e cells o ei he CD4 C o CD8 C cell popula ions is shown, each symbol ep- esen ing an indi idual mouse, lines indica ing he mean. S a is ics we e calcula ed wi h unpai ed wo- ailed S uden ’s - es (wi h Welch co ec ion). Un ea ed mice: nD5 ( o bo h geno ypes), 17-week- ea ed CD4c e KO animals: n D3, WT li e - ma e con ols n D4. ONCOIMMUNOLOGY e1245266-5 phase o umo de elopmen he FURIN T-cell-specific knock- ou mice ha e a Th1- ype-skewed immune esponse. 25 We also s udied he p oduc ion o IFNg, IL-17A, and IL-10 by CD4 C Foxp3 C T eg cells isola ed om he dLNs o CD4c e KO and WT mice. The e was a end owa d a highe p oduc- ion o IFNgin he T eg cells om he CD4c e KO animals (p D0.0592), and a significan educ ion in he pe cen age o CD4c e KO T eg cells posi i e o IL-17A (pD0.0066, Fig. 4D). Howe e , he pe cen age o dLN T eg cells p oducing IL-10 was no di e en be ween he CD4c e KO and WT. Also he mean fluo escence in ensi y o he CD25 su ace s aining was simila in T eg cells isola ed om dLNs o bo h CD4c e KO and WT mice indica ing no mal T eg ac i a ion s a us. Collec i ely, he flow cy ome ic analyses sugges ha he endency o CD4c e KO animals o de elop mo e papillomas could be ela ed o accele a ed Th1- ype immune esponses. 26 In con as , despi e CD4c e KO animals showing an inc eased numbe o egula o y CD4 C CD25 C Foxp3 C cells, hese cells a e Figu e 4. T cells om he skin d aining lymph nodes o CD4c e KO mice show ele a ed IFNgp oduc ion in he ea ly phase o umo de elopmen . In acellula flow cy ome y om skin d aining lymph node cells. Mice we e ea ed wi h one dose o DMBA, wo doses o TPA on he back skin, hen sac ificed a e 48 h om he 2nd TPA dose, and he isola ed dLN cells we e s imula ed wi h TPA CCa-ionomycin o 4 h in i o. (A) Rep esen a i e plo s om CD4 C WT and CD4c e KO mice, showing li e-ga ed cell popula ions posi i e o CD4 C , Foxp3, IFNg, and IL-10. (B) Pe cen ages o CD4 C cells posi i e o IFNg, IL-17A, and IL-10. (C) CD8 C cells posi i e o IFNg and IL-17A. (D) CD4 C Foxp3 C T eg posi i e o IFNg, IL-17A, and IL-10, and he mean fluo escence in ensi y (MFI) o CD25 om he T eg popula ion. (E) CXCL10 le els om se a om mice ea ed wi h TPA o 43 h, de e mined by he P oCa aPlex Mouse Cy okine & Chemokine 26-plex assay and Bio-Plex 200 ins umen . B, C, D: CD4 C WT mice n D6, CD4c e KO mice n D7; E: CD4 C WT mice n D3, CD4c e KO mice n D3. Each symbol ep esen s an indi idual mouse, lines indica e he mean. S a is ics: Unpai ed wo- ailed S uden ’s - es wi h Welch co ec ion. e1245266-6 M. V € AH € ATUPA ET AL. likely de ec i e in hei supp essi e ac i i y as sugges ed by up egula ed CD69 exp ession and IFNgp oduc ion in CD4 C , CD8 C e ec o T cells, and T egs. This conclusion is also sup- po ed by ou p e ious da a demons a ing impai ed pe iphe al T eg-dependen immune ole ance in CD4c e KO mice. 14 In he la e s age o ca cinogenesis T-cell esponses in he FURIN deficien T cells swi ch owa d Th2 and Th17 ype In addi ion o T-cell ac i a ion, FURIN egula es he T helpe cell balance o he immune sys em. 27,28 We p ofiled he le els o mul iple cy okines and chemokines om he cells isola ed om skin dLNs o he mice ha had been ea ed wi h DMBA/ TPA o 17 weeks. Su p isingly, he cells om CD4c e KO mice no longe p oduced ele a ed IFNgle els (like in he ea ly phase), bu he p oduc ion o Th2- ype cy okine IL-13 and Th17- ype cy okine IL-22 was highe (pD0.0219 and pD 0.0393, espec i ely, Fig. 5). Also, he le els o o he Th2/Th17 cy okines, IL-4, IL-17A, IL-9, o IL-10 showed a end owa d up egula ed p oduc ion in he CD4c e KO animals (Fig. 5). QRT-PCR pe o med om he back skins confi med he swi ch in Th esponses, showing an up egula ion o IFNgin he KO animals in he ea ly ime poin s and o IL-17a in he la e ime poin (Fig. S6). The le els o IL-2 and umo nec osis ac o a (TNFa) we e lowe in he dLN cells isola ed om CD4c e KO mice compa ed o hose isola ed om WT animals, as we ha e p e iously shown in he un ea ed animals. 14 Al oge he , he esul s sugges ha du ing he cou se o ca cinogenesis p og es- sion, he ea ly Th1-biased immune esponses o he T-cell-spe- cific FURIN deficien mice swi ch inc easingly owa d Th2 and Th17 ype esponses. Thus, FURIN has an inhe en ole in modula ing T helpe cell balance in a ch onic immune insul . Discussion Va ious s udies ha e shown ha FURIN o e exp ession is associa ed wi h accele a ed ca cinogenesis. Thus, i s inhibi ion could be a iable cance ea men . Howe e , he unc ion o Figu e 5. FURIN-deficien T cells show a Th2- and Th17- ype cy okine p ofile o in he la e phase o umo p og ession. CD4c e KO and WT mice we e ea ed once wi h DMBA, hen wi h TPA o 17 weeks ( wice a week) on he back skin as in Fig. 1. A 17 weeks, animals we e sac ificed and skin d aining lymph node cells we e cul u ed o 48 h in he p esence o pla e-bound an i-CD3 and soluble an i-CD28 an ibodies. Cy okine le els we e de e mined om he cul u e supe na an s wi h P oCa aPlex Mouse Cy okine & Chemokine 26-plex assay and Bio-Plex 200 ins umen , o o IFNg, wi h ELISA. S a is ics: Unpai ed wo- ailed S uden ’s - es wi h Welch co ec ion. CD4 C WT nD4, CD4c e KO n D3. Each symbol ep esen s an indi idual mouse, lines indica e he mean. ONCOIMMUNOLOGY e1245266-7 FURIN in di e en cell ypes ha con ibu e o umo o ma- ion is poo ly cha ac e ized. To s udy he ole o FURIN in immune cells in he con ex o squamous skin cance de elop- men , we u ilized wo mouse s ains ha lack FURIN specifi- cally ei he in mac ophages and g anulocy es o in T cells. As ou p e ious esul s ha e shown ha FURIN-deficien T cells and mac ophages display an o e ly ac i a ed pheno- ype, 14,19,40 we assumed ha he FURIN-deficien immune cells migh be mo e e ec i e in an icance immune esponses. How- e e , a e being subjec ed o he DMBA/TPA ea men , mice wi h he T-cell-specific knockdown o FURIN (CD4c e KO) de eloped mo e papillomas and hey appea ed as e in he knockou han hei WT li e ma es. The CD4c e KO mice p e- sen ed wi h a hicke epide mis, wi h mo e epide mal cell p o- li e a ion han he WT mice, and his di e ence be ween he geno ypes was main ained a e 17 weeks o DMBA/TPA ea - men . Unde no mal condi ions, he CD4c e KO skin ha bo ed mo e CD3 C T cells han he WT skin, bu upon ea men ha di e ence was los . Analysis o skin dLN cells showed no majo FURIN-dependen e ec s o he DMBA/TPA ea men on he numbe s o CD4 C o CD8 C T-cell popula ions. The highe numbe o CD4 C CD25 C Foxp3 C T egs p esen in CD4c e KO dLNs in he s eady s a e pe sis ed a e he ea men , as did he numbe o ac i a ed CD4 C CD69 C and CD8 C CD69 C T cells. In he ea ly phase o umo p omo ion, he CD4c e KO mice had mo e p o-inflamma o y IFNg-p oducing T cells, whe eas CD4c e KO dLN cells sec e ed mo e Th2- and Th17- ype cy o- kines a e 17 weeks o DMBA/TPA ea men (Fig. S7). The ac ha CD4c e KO, bu no LysMc e KO, mice had an inc eased suscep ibili y o papilloma de elopmen is in line wi h se e al s udies showing a umo -enhancing ole o ab T cells. 29,30 P e ious wo k has demons a ed ha IFNgp omo es umo de elopmen p ima ily in he ea ly s age o papilloma de elopmen . 26 IL-17A, in u n, has a ole in he p omo ion p o- cess in bo h human non-melanoma skin cance and mouse mod- els o skin cance , sugges ing ha Th2- and Th17- ype cy okine p ofile o he T cells lacking FURIN could be d i ing he ca cino- genesis p ocess a la e s ages. 31-33 LysMc e KO mice, in con as , display accele a ed inna e immuni y esponses, and a he e ozy- gous, inac i a ing mu a ion in he Fu inA gene esul s in enhanced inna e esponses in Mycobac e ium ma inum –in ec ed zeb afish. 19,40 Howe e , he p o-inflamma o y pheno ype due o lack o FURIN in myeloid cells does no seem o play a majo ole in he papilloma de elopmen in he DMBA/TPA-model. FURIN and o he PCSK amily membe s play an impo an ole in ca cinogenesis and me as asis, and he e has been con- side able in e es o de elop pha maceu ical inhibi o s o hei ac i i y o cance ea men . Inhibi ing FURIN/PCSK is ea- soned o di ec ly block he p ocessing o ac o s ha a e associ- a ed wi h umo in asion and me as a ic ac i i y, including ma ix me allop o einases, g ow h ac o s, and o he s. Fu he - mo e, FURIN is c i ical o he ac i a ion o an i-inflamma o y cy okine TGF-b1. Thus, specific blocking o FURIN is hough o suppo an i umo hos - esponses by p omo ing cance immunosu eillance. The assessmen o a bi unc ional GM- CSF-FURINshRNA cons uc (FANGTM, VigilTM,G adalis)has al eady en e ed he phase II clinical ials o ea men o mela- noma, o a ian cance , and colo ec al cance wi h li e me as a- sis. 17,34 Recen ly, he shRNA-media ed inhibi ion o FURIN oge he wi h dend i ic cell suppo ing GM-CSF exp ession was also ound o be e ficacious in me as a ic, ad anced Ewing’s sa - coma. 35 In con as , in li e cance s FURIN o e exp ession sup- p esses umo g ow h and p edic s be e pos ope a i e su i al. 36 The beneficial e ec o FURIN in hepa ocellula ca - cinoma was also epo ed in mice whe e FURIN was dele ed using li e specific Albumin CRE. 37 I seems he e o e plausible ha FURIN has umo ype-specific e ec s, and hus FURIN inhibi ion sys ema ically may no always be beneficial o oncol- ogy pa ien s. The ole o FURIN in T-cell-dependen immuni y is clea ly mul i ace ed. A majo unc ion o FURIN is o con- ol he bioa ailabili y o an i-inflamma o y TGF-b1, and T eg-dependen pe iphe al immune ole ance. 14 TGF-b1is a mul i unc ional g ow h ac o ha has oles in bo h p o- mo ing and supp essing umo igenesis. 38 Dele ing TGF-b1 specifically om ac i a ed CD4 C T cells and T eg cells educed me as a ic B16-OVA umo cell sp ead o he mouse lung, indica ing ha ac i a ed CD4 C T-cell-de i ed TGF-b1 inhibi s umo immunosu eillance. 39 Impo an ly, we ha e also shown ha FURIN is an impo an ac o in modula ing he T helpe cell balance. Mice ha we e ch on- ically in ec ed wi h in acellula Toxoplasma gondii pa asi e had less pa hogen-specificTh1- ypeimmunecells,and na€ ı e, OVA-specificFURINKOCD4 C Tcellsshowedan inc eased endency o pola ize in o he IL-4-p oducing Th2 cells. 27 The swi ch om Th1 in o Th2/Th17- ype esponses was also seen he e in CD4c e KO mice a e a 17-week DMBA/TPA ea men . Thus, inhibi ing FURIN does no jus p omo e T-cell-d i en adap i e immuni y, bu i also modula es he ype o T helpe cell esponses. I is also no ewo hy ha he egula o y ole o FURIN in CD8 C cy o oxic lymphocy es emains incomple ely unde s ood. Ou da a showed ha FURIN-deficien CTLs p oduced mo e IFNgand showed an up egula ion o CD69 ac i a ion ma ke , bu a ca e ul analysis o o example hei cy o oxic po en ial, g anzyme B, and pe o in exp essions needs u - he s udies. In conclusion, ou da a demons a e ha FURIN exp ession in T cells clea ly modula es adap i e immune esponses in bo h un ea ed mice and in animals su e ing om DMBA/TPA- induced skin papillomas. This leads o accele a ed umo de el- opmen accompanied wi h abe an T-cell cy okine p oduc ion in T-cell-specific FURIN KO mice. Ou findings sugges ha inhibi ing FURIN sys ema ically, o specifically in T cells, may p omo e he de elopmen o cance ypes whe ein a ch onic immune insul has a cance p o oking ole. This is an impo - an aspec when conside ing FURIN inhibi o s’ he apeu ic po en ial in human cance s. Ma e ials and me hods Mice T-cell-specific FURIN condi ional knockou (CD4c e KO) mice on a C57BL/6 backg ound ha e been desc ibed p e iously. 14,27 Mac ophage-specific FURIN condi ional knockou mice (LysMc e KO) we e gene a ed using a LysMc e C57BL/6 back- g ound. 19,40 Mice we e ed wi h s anda d labo a o y pelle s and e1245266-8 M. V € AH € ATUPA ET AL. wa e ad libi um. All animal expe imen s we e pe o med in acco dance wi h p o ocols app o ed by he Na ional Animal E hics Commi ee o Finland. Skin umo induc ion Bo h FURIN KO s ains, LysMc e and CD4c e, as well as hei espec i e li e ma e con ol C57BL/6 WT, LysM WT and CD4 C WT mice we e ea ed wi h DMBA and TPA o induce skin umo s as p e iously desc ibed. 22 In b ie , he backs o 8–14- week-old mice we e sha ed and 24 h la e 50 mg DMBA (7,12- Dime hylbenz[a]an h acene) (Sigma, Do se , UK) in 200 mL ace- one was applied opically on he sha ed a ea o he do sal skin. A e a week, he back skin o he mice was ea ed wice a week wi h 5 mg TPA (12-O- e adecanoylpho bol-13-ace a e) (Sigma) in 200 mL ace one o 16 o 21 weeks. The u excluding umo s was ca e ully sha ed e e y 2 weeks. Tumo s (1 mm in diame e o la ge ) we e coun ed wice a week and changes in umo de elopmen we e eco ded o each indi idual umo . Immunohis ochemical (IHC) and TUNEL s aining Samples o back skin om sac ificed, sha ed con ol mice o mice a 43 h o week 17 o he umo induc ion expe imen we e collec ed and fixed wi h 4% pa a o maldehyde and embed- ded in pa a fin acco ding o s anda d p o ocols. Hema oxylin/ eosin s aining and DAB immunohis ochemical s aining (IHC) was pe o med on 6-mm hickpa a fin sec ions as p e iously desc ibed. 22 The ollowing p ima y an ibodies we e used o IHC: A0452 abbi an i-CD3 (DakoCy oma ion, Glos up, Den- ma k), MF48000 BM8 a an i-F4/80 (Li e Technologies L d., Paisley, UK), and 68672 abbi an i-neu ophil elas ase (AbCam, Camb idge, UK). Mo e de ailed lis o eagen s,imaging,and quan i a ion a e desc ibed in he Supplemen a y me hods. Flow cy ome y Fo su ace ma ke s, he skin dLN cells we e s ained wi h an i- bodies agains mouse CD4, CD8, CD44, CD62L, and CD69 (all om eBioscience, San Diego, Cali o nia, USA). Fo in acellula s aining, isola ed dLN cells we e s imula ed wi h PMA and Ca- ionomycin o 4 h, and B e eldin A and Monensin we e applied o he las 2 h o he s imula ion. The cells we e s ained wi h su ace ma ke s and subsequen ly fixed o e nigh wi h Fixa- ion/Pe meabiliza ion solu ion ( om Foxp3/T ansc ip ion Fac- o S aining Bu e Se , eBioscience), pe meabilized wi h Pe meabiliza ion Bu e (eBioscience) and s ained wi h in a- cellula an ibodies (IL-10, IFNg, IL-17A, Foxp3; all om eBio- science), acco ding o he manu ac u e ’s ins uc ions. All cells we e analyzed wi h FACSCan o II (Bec on, Dickinson and Company, F anklin Lakes, New Je sey, USA), da a analysis pe - o med wi h FlowJo so wa e (Flowjo LLC, Ashland, O egon, USA). Luminex and ELISA assays Skin dLN cells om mice ea ed wi h DMBA/TPA o 17 weeks we e cul u ed o 48 h in he p esence o pla e-bound an i-CD3 an ibody (10 mg/mL, clone 17A2, eBioscience) and soluble an i-CD28 Ab (2 mg/mL, clone 37.51, eBioscience). Mul iplex cy okine/chemokine measu emen was done om he cul u e supe na an s using P oCa aPlex assay (Mouse Cy okine & Chemokine 26-plex, eBioscience) acco ding o he manu ac u e ’s ins uc ions, and wi h Bio-Plex 200 ins umen (Bio-Rad). IFNgle els we e de e mined om he cell cul u e supe na an s wi h a Ready-Se -Go! ELISA ki (eBioscience), acco ding o he manu ac u e ’s ins uc ions. S a is ical analysis Mean a e ages a e shown wi h 95% confidence in e als, in he case o Fig. 1B wi h SEM. Immunohis ochemis y da a we e analyzed o de e mine i i was no mally dis ibu ed (D’Agos- ino & Pea son omnibus and Shapi o–Wilk no mali y es s). Significance a a gi en ime poin was calcula ed by wo- ailed S uden ’s - es o no mally dis ibu ed da a. An ale el less han 0.05 was conside ed significan . Tumo - ee su i al plo da a we e analyzed by log- ank (Man el-Cox) es and non- no mally dis ibu ed ime cou se da a we e analyzed by non- linea eg ession. P ism 6 (G aphPad So wa e, La Jolla Cali o - nia, USA) was used o a majo i y o he analyses and STATA 13.0 (S a aCo p LP, College S a ion, Texas, USA:) s a is ical analysis so wa e was used o non-linea nega i e binomial eg ession analysis, as indica ed. Disclosu e o po en ial conflic s o in e es No po en ial conflic s o in e es we e disclosed. Acknowledgmen s We hank Ma ianne Ka lsbe g, Ma ja-Leena Koskinen, and Anni Lai inen o p ac ical suppo and Heini Huh ala o s a is ical ad ice. M s. Guille - mina Ga cia (San o d-Bu nham-P ebys Medical Disco e y Ins i u e, La Jolla, CA, USA) is hanked o he echnical expe ise and help wi h quan- i a i e mic oscopy. Funding The wo k was unded by he Sig id Juselius Founda ion, he Academy o Finland, P€ ai ikki and Saka i Sohlbe g Founda ion, Ins umen a ium Resea ch Founda ion, Finnish Medical Founda ion, Pi kanmaa Hospi al Dis ic Resea ch Founda ion, he Finnish Cul u al Founda ion, he Uni- e si y o Tampe e Founda ion, Biocen e Finland, Compe i i e Resea ch Funding o he Tampe e Uni e si y Hospi al, Cance Socie y o Finland, and Tampe e Tube culosis Founda ion. Au ho con ibu ions T.J., M.V., M.P., S.A., H.U.-J., and Z.M.C. designed he esea ch. M.V., S. A., Z.M.C., and U.M. pe o med he esea ch. M.V., S.P., S.A., Z.M.C., and U.M. analyzed he da a. T.J., M.V., U.M., S.A., and M.P. w o e he manu- sc ip . M.V., S.A., and U.M. made he figu es. M.V., S.P., U.M., S.A., H.U.- J., M.P., and T.J. e iewed and edi ed he pape . ORCID Te o A. J€ a inen h p://o cid.o g/0000-0002-4027-1759 ONCOIMMUNOLOGY e1245266-9