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
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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
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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
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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
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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