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Loss o CADM1 exp ession is associa ed wi h poo p ognosis and
b ain me as asis in b eas cance pa ien s
Ha ie Wikman1,*, Lau a Wes phal1,*, Felici as Schmid1,2, Si kku Polla i3, Jolan he
K opidlowski1, Be ina Siela -F impong1, Ma kus Gla zel4, Jakob Ma schke4,
Man ed Wes phal5, K is iina Iljin3, Heini Huh ala6, Luigi Te acciano7, Anne
Kallioniemi8, Guido Sau e 9, Volkma Mülle 10, Isabell Wi zel10, Ka in Lamszus5,
Di k Kemming11, Klaus Pan el1
1 Ins i u e o Tumo Biology, Uni e si y Medical Cen e Hambu g-Eppendo , Hambu g, Ge many
2 Max-Delb ück Cen e o Molecula Medicine, Be lin, Ge many
3 Medical Bio echnology, VTT Technical Resea ch Cen e o Finland and Tu ku Cen e o Bio echnology, Uni e si y o Tu ku,
Tu ku, Finland
4 Ins i u e o Neu opa hology, Uni e si y Medical Cen e Hambu g-Eppendo , Hambu g-Eppendo , Hambu g, Ge many
5 Depa men o Neu ological Su ge y, Uni e si y Medical Cen e Hambu g-Eppendo , Hambu g-Eppendo , Hambu g,
Ge many
6 School o Heal h Sciences, Uni e si y o Tampe e, Tampe e, Finland
7 Depa men o Pa hology, Basel Uni e si y Clinics, Basel, Swi ze land
8 Ins i u e o Biomedical Technology, Uni e si y o Tampe e and BioMediTech, Tampe e, Fimlab Labo a o ies, Tampe e, Finland
9 Ins i u e o Pa hology, Uni e si y Medical Cen e Hambu g-Eppendo , Hambu g, Ge many
10 Depa men o Gynecology, Uni e si y Medical Cen e Hambu g-Eppendo , Hambu g, Ge many
11 Eu opean Labo a o y Associa ion, Ibbenbue en, Ge many
* These au ho s sha e i s -au ho ship
Co espondence o: Ha ie Wikman, email: [email p o ec ed]
Keywo ds: b eas cance , b ain me as ases, CADM1, me hyla ion
Recei ed: Janua y 22, 2014 Accep ed: Ma ch 16, 2014 Published: Ma ch 16, 2014
This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use,
dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
ABSTRACT:
B eas cance b ain me as ases (BCBM) a e de ec ed wi h inc easing incidence.
In o de o de ec po en ial genes in ol ed in BCBM, we i s sc eened o genes
down- egula ed by me hyla ion in cell lines wi h si e-speci ic me as a ic abili y. The
exp ession o i e genes, CADM1, SPARC, RECK, TNFAIP3 and CXCL14, which we e also
ound down- egula ed in gene exp ession p o iling analyses o BCBM issue samples,
was e i ied by qRT-PCR in a la ge pa ien coho . CADM1 was chosen o u he
down-s eam analyses. A highe incidence o CADM1 me hyla ion, co ela ing wi h
lowe exp ession le els, was ound in BCBM as compa ed o p ima y BC. Loss o
CADM1 p o ein exp ession was de ec ed mos commonly among BCBM samples as
well as among p ima y umo s wi h subsequen b ain elapse. The p ognos ic ole o
CADM1 exp ession was inally e i ied in ou la ge independen b eas cance coho s
(n=2136). Loss o CADM1 p o ein exp ession was associa ed wi h disease s age,
lymph node s a us, and umo size in p ima y BC. Fu he mo e, all analyses e ealed
a signi ican associa ion be ween loss o CADM1 and sho e su i al. In mul i a ia e
analyses, su i al was signi ican ly sho e among pa ien s wi h CADM1-nega i e
umo s. Loss o CADM1 exp ession is an independen p ognos ic ac o especially
associa ed wi h he de elopmen o b ain me as ases in b eas cance pa ien s.
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INTRODUCTION
B eas cance (BC) is he mos common non-skin
malignancy in women a ec ing abou 1.2 million women
in he wo ld each yea . The sp ead o malignan cells om
he p ima y umo o dis an o gans such as he b ain is
he main cause o BC ela ed dea hs. B eas cance is
he second mos common cause o he de elopmen
o cen al ne ous sys em (CNS) me as ases, which
a e, i espec i ely o he p ima y umo o igin, mo e
commonly diagnosed han p ima y b ain umo s [1, 2].
CNS-me as ases a e diagnosed in 15-20% o pa ien s wi h
me as a ic BC and a e usually occu ing wi h a median
ime pe iod o 31 mon hs a e he diagnosis o b eas
cance [2]. Due o imp o ed sys emic ea men op ions
o b eas cance , pa ien s bene i om p olonged su i al
a es. Howe e , consecu i ely o he longe su i al and
an in ensi ied use o sensi i e de ec ion me hods such as
con as -enhanced magne ic esonance imaging o he
b ain (cMRI), he incidence a es o b ain me as ases a e
ising.
B ain me as ases a e o en associa ed wi h he
agg essi e iple nega i e b eas cance (TNBC), being
ho mone ecep o (HR) and HER2 nega i e, and also
wi h HER2 posi i e p ima y b eas cance s [3, 4]. Pa ien s
su e ing om b eas cance b ain me as ases (BCBM)
ha e an ex emely poo p ognosis wi h a median su i al
o only 7.8 mon hs [3]. This b ings o h o in es iga e
new p ognos ic ma ke s, which may be help ul o
he iden i ica ion o pa ien s wi h an inc eased isk
o subsequen CNS-in il a ion and o de elop new
he apeu ic app oaches.
The loss o umo supp esso gene (TSG) exp ession
is known o cons i u e a c ucial s ep in cance o ma ion.
Fo he dissemina ion and ou g ow h o me as ases,
ano he se o me as asis supp esso and ac i a o genes is
needed. Me as asis supp esso genes (MSGs) usually do
no in luence umo g ow h a he p ima y si e, bu con ol
he umo cells’ capaci y o escape om he p ima y
umo and o m o e me as ases a dis an si es [5].
MSGs con ibu e o do mancy con ol and/o ou g ow h
a seconda y si es and hus egula e he inal s ep o
me as asis, he me as a ic coloniza ion. In e es ingly, in
con as o TSGs, MSGs seem o be mo e o en cance
ype speci ic. Fu he mo e, MSGs a e a ely mu a ed, and
epigene ic e en s, such as me hyla ion, a e hus likely he
main cause o hei loss o unc ion [6]. Since epigene ic
e en s a e e e sible, do mancy con ol by MSGs is
po en ially a new o m o a ge ed gene he apy ( e iewed
in [6, 7 ]).
The aim o his s udy was o iden i y no el genes
in ol ed in BCBM o ma ion. We i s sc eened o genes
down- egula ed by me hyla ion in b eas cance cell lines
(pa en al MDA-MB-231 cell line and he b ain- and bone-
speci ic sub cell lines) known o si e-speci ic me as asis
[8]. Gene exp ession p o iling and quan i a i e eal- ime
RT-PCR (qRT-PCR) alida ion o he candida e genes
we e he ea e conduc ed on umo issues om p ima y
BC and BCBM in o de o iden i y he mos clinically
ele an genes. The me hyla ion pa e n o CADM1
was u he mo e cha ac e ized, and CADM1 p o ein
exp ession was alida ed in wo la ge independen p ima y
umo coho s as well as in BCBM samples and co ela ed
wi h clinico-pa hological pa ame e s.
RESULTS
Me hyla ion a ay sc eening o b ain me as ases
ela ed genes
A subclone o MDA-MB-231 wi h a high me as a ic
po en ial o he b ain, MDA-MB-231 BR, was compa ed o
he pa en al MDA-MB-231 and o a bone-seeking a ian
MDA-MB-231 SA in o de o iden i y genes, which migh
be speci ically in ol ed in b ain me as asis o ma ion.
The cell lines we e ea ed wi h 5-Aza-2’-deoxycy idine,
a deme hyla ing agen , in o de o ind genes po en ially
down- egula ed by me hyla ion. Mic oa ay analysis was
pe o med on pooled iplica e expe imen s and he non-
umo igenic epi helial cell line MCF 10A was used o
con ol o s ess esponse a e he ea men wi h 5-Aza-
2’-deoxycy idine.
The gene exp ession p o iling a e 5-Aza-2’-
deoxycy idine ea men e ealed 914 di e en ansc ip s,
which we e signi ican ly up- egula ed in one o he MDA-
MB-231 cell lines bu no al e ed in MCF 10A (Figu e
1A). The la ges numbe o up- egula ed genes (691
ansc ip s) and cell line-speci ic up- egula ed genes
(20%) was ound in he MDA-MB-231 BR cell line. Mos
o he genes we e, howe e , up- egula ed in all o hese
cell lines (30%, 279/914). In gene al, he mo e agg essi e
subclones BR and SA we e mo e simila o each o he han
o he pa en al cell line, indica ing a di e en ia ion in o
gene ally mo e agg essi e o ms.
Gene exp ession sc eening o me hyla ion- ela ed
genes in p ima y and me as a ic b eas umo s
Gene exp ession p o iling o p ima y non-
me as asized b eas umo s (n=32) and b ain me as ases
(n=9) was pe o med o ind he mos ele an genes
among he 690 po en ially b ain me as ases de e mining
genes de ec ed in me hyla ion a ay sc eening. 110
ansc ip s (16%) o hose up- egula ed in he MDA-
MB-231 BR subclone in esponse o 5-Aza2’-
deoxycy idine ea men we e ound signi ican ly lowe
exp essed among he BCBM samples as compa ed o
he samples om non- elapsed p ima y BC pa ien s
(Supplemen a y Table 2).
Twen y- ou (22%) o hese genes we e ound
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exclusi ely in he BR subclone, implica ing a b ain
speci ic down- egula ion o hese genes. 28 genes we e
in common be ween he highly me as a ic BR and SA
a ian s, indica ing ha hese genes may media e a mo e
agg essi e beha io in gene al.
Valida ion o po en ial me as asis-supp essing
genes in p ima y BC and BCBM issue samples
The exp ession o i e genes, which we ound
down- egula ed in he b eas cance da a se and in he
cell line analyses, was u he in es iga ed by qRT-PCR in
39 p ima y BC samples wi hou b ain me as ases and 20
BCBM issue samples.
SPARC, RECK, TNFAIP3 and CXCL14 we e down-
egula ed (p < 0.05) in BCBM as compa ed o p ima y
BC samples i espec i ely o he cance sub ype, while
o CADM1, his co ela ion was ound in HR posi i e
and TNBC samples (Figu e 1B and Supplemen a y Figu e
1). Wi hin he p ima y BC samples, 84% o he HER2
nega i e pa ien s showed an ele a ed CADM1 mRNA
exp ession ( op 75% pe cen ile), whe eas only 29% o he
HER2 posi i e pa ien s had a high CADM1 exp ession (p
= 0.003) (Supplemen a y Figu e 1). Among he p ima y
umo s, high RECK exp ession was also associa ed wi h
HR posi i e s a us, whe eas he highes SPARC exp ession
was s a is ically signi ican ly linked o iple nega i e
(TNBC) samples. CXCL14 exp ession was no associa ed
wi h a sub ype, bu i s low exp ession was associa ed wi h
bo h posi i e lymph node s a us (p = 0.011) and high g ade
(p = 0.029) (Supplemen a y Table 3).
F equency o CADM1 me hyla ion in p ima y
b eas cance and BCBM issue samples
The me hyla ion s a us o CADM1 was de e mined
by MSP in 17 BCBM and 14 p ima y BC samples.
CADM1 was homozygously me hyla ed in 17.5% (3/17),
Figu e 1:A) Gene exp ession changes in esponse o 5-Aza-2’-deoxycy idine ea men in pa en al MDA-MB-231 b eas cance cell line
and MDA-MB-231 BR and MDA-MB-231 SA a ian s. The numbe be o e b acke s de ines he genes up- egula ed in each cell line a e
5-Aza-2’-deoxycy idine ea men . The numbe in b acke s de ines he numbe o genes also down- egula ed in he b ain me as ases as
compa ed o non- elapsed p ima y umo s. Figu e 1B) Exp ession o CADM1, RECK, CXCL14, SPARC and TNFAIP3 in BCBM samples
ela i e o p ima y b eas umo s (PT). HR pos.: es ogen and p oges e one posi i e ecep o , TNBC: iple nega i e b eas cance . P- alues
we e de e mined by he log ank es .
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he e ozygously me hyla ed in 17.5% (3/17) and no
me hyla ed in 65% (11/17) o he BCBM samples. In
con as , p ima y BC samples showed no homozygous
CADM1 me hyla ion, only 7% (1/14) he e ozygous and
93% (13/14) WT CADM1 s a us (Figu e 2). Due o he low
equency o me hyla ion in he p ima y BC samples, no
associa ion be ween clinico-pa hological ac o s and he
me hyla ion s a us o CADM1 could be ound.
CADM1 p o ein exp ession in p ima y and
me as a ic b eas cance
CADM1 p o ein exp ession could be assessed in
a la ge numbe o p ima y b eas umo s (TMA I, n=
1331) and 27 BCBM samples (Table 1, and Figu e 3).
No mal b eas duc showed an in ensi e memb anous
s aining o CADM1, whe eas he umo s oma was
CADM1 nega i e. The CADM1 exp ession in he plasma
memb ane was los in 42% o he p ima y BC and in 68%
o he BCBM samples. S ong exp ession was eco ded in
22% o he p ima y BC and in 11% o he BCBM cases.
The CADM1 p o ein exp ession di e ed be ween p ima y
umo and BCBM wi h mo e nega i e s aining among he
BCBM samples (p = 0.011).
Su p isingly, a nuclea s aining o CADM1 was seen
in a small subse o pa ien s. Nuclea s aining has been
p e iously epo ed o occu in ce ical epi helium wi h
he localiza ion o s aining being dependen on epi helial
o igin [9]. 86 (6.5%) o he p ima y BC samples on he
p ognos ic TMA (TMA I) had a clea CADM1 s aining
in he nucleus. The nuclea s aining highly co ela ed
wi h he memb anous s aining (p < 0.001). In 70 o he
86 pa ien s wi h nuclea s aining, also he memb ane was
s ained o CADM1 (81% conco dance).
In addi ion, 43 ma ched pai s o p ima y umo s and
lymph node me as asis samples (TMA III) we e analyzed
o CADM1 p o ein exp ession. 70% (30/43) o he
ma ched pai s showed a conco dan CADM1 exp ession
wi h 51% (22/43) o he p ima y umo and lymph node
samples being CADM1 nega i e and 19% (8/43) CADM1
posi i e in bo h ypes o issues. Only 7% (3/43) o he
samples had a highe CADM1 exp ession in he lymph
nodes as compa ed o he ma ched p ima y umo s, while
down- egula ion o CADM1 exp ession in he lymph node
me as ases was obse ed in 23% (10/43) o he ma ched
pai s, indica ing a loss o CADM1 p o ein exp ession
du ing he me as a ic cascade.
Compa ison o gene exp ession, me hyla ion
pa e n and p o ein exp ession s a us o CADM1
in p ima y BC and BCBM
In o de o ind ou how much o he silencing
o exp ession is go e ned by p omo e me hyla ion,
we compa ed he esul s om he qRT-PCR, IHC and
me hyla ion analyses. In 22 samples esul s om he gene
exp ession, me hyla ion and p o ein exp ession s a us o
CADM1 we e a ailable (Table 2).
Homozygous me hyla ion was associa ed wi h a
nega i e CADM1 s aining and low o in e media e mRNA
exp ession. He e ozygo e me hyla ion pa e n showed a
nega i e IHC esul in 50% o he cases and o he es
Figu e 2: P omo e CpG me hyla ion o CADM1. A)
Gel elec opho esis pic u e o CADM1 in 5 ep esen a i e cases,
MW= molecula weigh ma ke , UM= unme hyla ed PCR,
M= me hyla ed PCR. B) Tables o MSP esul s o CADM1 o
p ima y umo s and b eas cance b ain me as ases. WT= wild
ype, HET= he e ozygous me hyla ion, MET= homozygous
me hyla ion.
Figu e 3: CADM1 immunos aining in p ima y b eas
cance and BCBM samples. A) P ima y BC sample
wi h homogenous posi i e CADM1 memb ane and nega i e
nuclea s aining, B) P ima y BC sample wi h nega i e CADM1
memb ane and weak nuclea s aining, C) BCBM sample wi h
nega i e CADM1 memb ane and nuclea s aining wi h posi i ely
s ained e y h ocy es, D) BCBM sample wi h weak he e ogeneous
CADM1 memb ane and nega i e nuclea s aining.
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Table 1: CADM1 memb ane p o ein exp ession in co ela ion o clinical pa ame e s in p ima y BC (TMA I) and BCBM.
CADM1 nega i e CADM1 weak CADM1 s ong p-
alue
n % n % n %
B ain me as ases
All 19 67.9 6 21.4 3 10.7
P ima y umo s
All 557 42.1 488 36.9 286 21.6
His ology n.s.
Duc al 408 42.1 361 37.2 201 20.7
Lobula 71 46.4 51 33.3 31 20.3
o he s 78 37.7 76 35.2 54 27.1
Tumo s age <0.001
pT1 118 27.6 185 43.0 125 29.0
pT2 290 44.8 224 34.6 133 20.6
pT3 40 52.6 25 32.9 11 14.5
pT4 106 60.6 52 29.7 17 9.7
n.a. 3 2 0
Lymph node s a us 0.003
pN0 193 35.8 219 40.6 127 23.6
pN1 207 44.6 159 34.3 98 21.1
pN2 43 53.8 28 35.0 9 11.3
n.a. 114 82 52
G ade n.s.
1 130 42.6 113 37.0 62 20.3
2 201 40.4 188 37.8 108 21.7
3 203 45.8 151 34.1 89 20.1
n.a. 23 36 27
Tumo size <0.001
< 2.0 cm 120 27.9 185 43.0 125 29.1
> 2.0 cm 421 48.4 290 33.3 159 18.3
n.a. 16 2.9 13 2.7 2 0.7
Ho mone ecep o n.s.
nega i e 126 44.3 87 32.8 65 22.9
posi i e 410 41.0 379 37.9 210 21.0
n.a. 21 22 11
HER2 n.s.
nega i e 438 40.4 403 37.2 242 22.3
posi i e 93 45.6 73 35.8 38 18.6
n.a. 26 12 6
Sub ype 0.036
ER/PR pos. 350 40.4 332 38.3 185 21.3
TNBC 81 42.4 55 28.8 55 28.8
HER2 pos. 93 45.6 73 35.8 38 18.6
n.a. 33 28 8
Age 0.028
< 50 yea s 85 37.1 85 37.1 59 25.8
> 50 yea s 379 44.8 310 36.6 157 18.6
n.a. 93 93 70
Cou se o disease <0.001
dead 238 51.7 145 31.7 76 16.6
ali e 319 36.6 343 39.3 210 24.0
n.a. = no a ailable; n.s. = no signi ican

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a weak posi i e CADM1 p o ein s aining was obse ed.
Nega i e CADM1 p o ein exp ession was always
associa ed wi h ei he low/ nega i e o in e media e
mRNA exp ession.
Clinical signi icance o CADM1 p o ein exp ession
The clinical signi icance o CADM1 exp ession
was assessed in wo publicly a ailable mRNA exp ession
da a se s (GSE3494 and GSE6532) and on wo p ognos ic
TMAs o p ima y BC samples (Figu e 4). In bo h
exp ession a ay da a se s a signi ican associa ion be ween
low CADM1 exp ession and bad p ognosis was ound (p =
0.033 and p = 0.001).
The wo p ognos ic TMAs (TMA I and TMA II)
o p ima y BC ga e highly consis en esul s. Clinico-
pa hologic examina ion o bo h independen pa ien
coho s consis ing o 1718 p ima y b eas umo samples
e ealed a signi ican associa ion be ween nega i e
CADM1 s a us and ad anced umo s age, posi i e lymph
node s a us and la ge umo size (all p < 0.05; Table 1
and Supplemen a y Table 4). In he TMA I a signi ican
associa ion be ween CADM1 and age, sub ype and cou se
o disease was also de ec ed. The equency dis ibu ion o
CADM1 was compa able on he TMA II bu signi icance
was no eached due o smalle sample numbe s. G ade
Figu e 4: Kaplan–Meie analysis o CADM1 mRNA and p o ein exp ession in p ima y b eas cance . A) Su i al
analyses in wo publicly a ailable exp ession da a se s. B) Su i al analyses o he p ognos ic TMA (TMA I) o he whole s udy popula ion
and among HR-posi i e pa ien s. C) Su i al analyses in he second TMA (TMA II) o he whole s udy popula ion and among HR-posi i e
pa ien s. Su i al di e ences we e analyzed by he log ank es .
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was associa ed wi h CADM1 loss on he second TMA,
whe eas no di e ence be ween he di e en his ological
sub ypes could be ound on ei he TMA.
The si e o elapse was eco ded o 353 p ima y
umo pa ien s (TMA II). Among pa ien s wi hou elapse
a loss o CADM1 was seen in 58.6%, whe eas he highes
equency o CADM1 loss was seen among pa ien s
su e ing om b ain elapse wi h 81.1%. Also o he
ypes o elapse had a highe equency o CADM1 loss
compa ed o he non- elapsed (bone 73.8% (p = 0.023),
li e 69.7% and lung 65.5%; Supplemen a y able 4).
Consis en wi h he memb anous s aining, a
s a is ically signi ican associa ion be ween loss o
CADM1 in he nucleus and mo e agg essi e o ad anced
umo s age and size, as well as o he ho mone ecep o
s a us and he cou se o disease (p < 0.05) was ound (only
assessed on TMA I, Supplemen a y Table 5). In addi ion,
he majo i y o cases wi h nuclea CADM1 s aining was
ound among lobula umo s (11% s. 6% in duc al, p =
0.042).
A highly signi ican associa ion be ween wo se
pa ien ou come (o e all su i al and disease ee su i al)
and nega i e CADM1 p o ein exp ession could be ound
in bo h da a se s (p < 0.001 and p = 0.006) (Figu e 4).
Loss o CADM1 was signi ican ly associa ed wi h wo se
pa ien ou come among duc al ca cinomas (p = 0.001
and p = 0.001), whe eas a signi ican associa ion was
ound o lobula ca cinomas only in TMA II (p = 0.026;
TMA I p = 0.068) (da a no shown). Fu he mo e, loss
o CADM1 exp ession was signi ican ly associa ed wi h
sho e su i al in HR posi i e (bo h da a se s p = 0.001
and p = 0.022, Figu e 4), bu did no ha e a p ognos ic
ele ance in HER2 posi i e pa ien s, indica ing a umo
supp essing e ec o CADM1 in HER2 nega i e pa ien s
only (da a no shown). Mul i a ia e analysis showed ha
loss o CADM1 exp ession was a signi ican independen
nega i e p ognos ic ac o (TMA I p = 0.045 and TMA
II p = 0.01). Fo he i s la ge s udy coho (TMA I) he
i e-yea su i al was 64.8% (median 105 mon hs) o
pa ien s wi h no CADM1 p o ein exp ession and 75.9%
(154 mon hs) and 77.7% o pa ien s wi h weak and s ong
CADM1 exp ession espec i ely. Fo TMA II he i e-yea
su i al was 84.7% (median 179.7 mon hs) o pa ien s
wi h no CADM1 p o ein exp ession and 92.3% (189
mon hs) and 95.2% (221 mon hs) o pa ien s wi h weak
and s ong CADM1 exp ession, espec i ely.
DISCUSSION
The incidence o b ain me as ases in b eas cance
pa ien s is inc easing, wi h no s anda d diagnos ic
managemen o hese pa ien s applied in clinical ou ine.
In iew o he inc easing success o a ge ed he apies in
ea ing BC pa ien s wi h dis an me as ases, imp o ed
insigh s in o he pheno ype o b ain me as ases could
ha e impo an he apeu ic implica ions. In he p esen
s udy, we i s sc eened o genes silenced by me hyla ion
in a b eas cance cell line wi h a high po en ial o o m
b ain me as ases. Fi e o he iden i ied genes, also down-
egula ed in clinical samples, CADM1, RECK, SPARC,
CXCL14 and TNFAIP3, we e chosen o u he exp ession
analyses. These genes ha e p e iously no been associa ed
wi h b ain me as asis o ma ion. Howe e , hey ha e been
implica ed as cance - ele an genes in epi helial umo s
[10-14]. Fu he mo e, he me hyla ion s a us o CADM1
in p ima y BC and BCBM was in es iga ed and CADM1
p o ein exp ession was examined in co ela ion wi h
clinico-pa hological pa ame e s in BC and compa ed
o BCBM issue samples. We iden i ied CADM1 as
an impo an p ognos ic ac o , whose loss was no
only associa ed wi h mo e agg essi e p ima y BC wi h
wo se ou come, bu also wi h an inc eased isk o b ain
me as asis o ma ion.
Table 2: Combined esul s om he qRT-PCR,
MSP, and IHC analyses o CADM1
CADM1
Pa . No. Sample qPCR me hyla ion IHC- esul
B M-9 BCBM INT MET NEG
B M-10 BCBM LOW MET NEG
B M-8 BCBM INT HET NEG
B M-7 BCBM LOW HET NEG
B M-5 BCBM LOW WT NEG
B M-25 BCBM LOW WT NEG
B M-19 BCBM LOW WT NEG
B M-1 BCBM INT WT NEG
B M-16 BCBM INT WT NEG
B M-18 BCBM INT WT NEG
B M-22 BCBM INT WT NEG
B M-2 BCBM INT WT NEG
PT-95 PT INT WT NEG
PT-103 PT INT WT NEG
B M-13 BCBM n.d. WT NEG
B M-21 BCBM INT HET WEAK
PT-88 PT INT HET WEAK
B M-15 BCBM INT n.d. WEAK
B M-14 BCBM HIGH n.d. WEAK
B M-24 BCBM LOW WT WEAK
PT-101 PT INT WT STRONG
B M-11 BCBM HIGH WT STRONG
BCBM: b eas cance b ain me as ase; PT: p ima y umo ;
HIGH: CT exp ession alue in uppe qua ile in all pa ien s analyzed;
LOW: CT exp ession alue in bo om qua ile in all pa ien s analyzed
n.d.: no de ined; WT: wild ype; HET: he e ozygous me hyla ion;
MET: homozygous me hyla ion; NEG: nega i e p o ein s aining
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CADM1/TSLC1 (cell adhesion molecule 1) is
a memb ane-spanning glycop o ein belonging o he
supe amily o immunoglobulin cell adhesion molecules.
I was i s ecognized as a umo supp esso in non-small
cell lung cance (NSCLC) [10]. I has been sugges ed
ha he dis up ion o cell adhesion h ough he loss o
CADM1 is a mechanism leading o cance cell in asion
and me as asis [15, 16]. CADM1 is known o be
in ol ed in inhibi ion o cell p oli e a ion and induc ion
o apop osis wi h a epo ed loss o exp ession in a
a ie y o cance s o epi helial cell o igin such as b eas ,
p os a e, panc ea ic, hepa ocellula and colo ec al cance ,
bu also in neu oblas oma ( e iewed in [16]). Recen ly,
CADM1 was iden i ied by Fa aji e al. (2012) o be a
me as asis suscep ibili y gene, i.e. an inhe i ed ac o
ha supp esses me as asis by sensi izing umo cells o
immunosu eillance by CD8+ T-cells [17].
We ound a down- egula ion o CADM1 bo h on
mRNA and p o ein le el in BCBM issue samples as
compa ed o p ima y BC. I was p e iously epo ed
ha CADM1 p o ein is de ec ed on he cell memb ane
in no mal epi helial cells o he b eas and ha nega i e
CADM1 s aining, de ec ed in mo e han hal o he
p ima y BCs, is associa ed wi h ad anced disease s ages
[18, 19]. He e, we ound ha in p ima y BC, loss o
CADM1 was mos commonly seen among TNBC and
HER2 posi i e pa ien s, wo sub ypes mo e closely
associa ed wi h b ain me as ases. Fu he mo e, loss o
CADM1 p o ein exp ession was associa ed wi h isk
ac o s such as high umo s age, posi i e lymph node
s a us and la ge umo size (all p < 0.05) in wo la ge
independen sample coho s consis ing o mo e han
1 700 umo samples, indica ing a p ognos ic ole o
CADM1 in p e en ing b eas cance p og ession. Loss o
CADM1 exp ession was ound mo e commonly among
p ima y umo pa ien s wi h lung, li e , bone o b ain
elapse compa ed o pa ien s wi h no elapse. Impo an ly,
loss o CADM1 exp ession was mos commonly seen
among pa ien s wi h b ain elapse indica ing a special
bu no exclusi e ole o CADM1 in b ain me as asis
o ma ion. Simila ly, ma ched pai s o p ima y BC and
lymph node (LN) me as asis samples we e addi ionally
analyzed o CADM1 p o ein exp ession, showing mo e
equen ly a down- egula ion o CADM1 exp ession
in he LN me as ases. The obse ed down- egula ion o
CADM1 no only in BCBM bu also in LN me as ases
as compa ed o he p ima y umo suppo s he ole o
CADM1 as a me as asis suscep ibili y gene in e ms o
educing he me as a ic capabili y (25). This hypo hesis
is u he suppo ed by ou esul s showing an associa ion
be ween CADM1 posi i e s aining in p ima y umo s and
longe o e all su i al. Mul i a ia e analysis showed ha
CADM1 exp ession is an independen p ognos ic ma ke ,
s eng hening he hypo hesis ha CADM1 may play an
impo an ole in p e en ing he me as a ic p og ession.
In line wi h he esul s om ou exp ession s udies,
he CADM1 p omo e egion was ound me hyla ed
in 35% o he BCBM samples, whe eas in p ima y BC
samples wi h good p ognosis, CADM1 p omo e was
only me hyla ed in 7% o he samples. Howe e , o he
mechanisms esul ing in a loss o gene unc ion, such
as dele ion, need o be u he in es iga ed. Clea ly, he
p edic i e alue o CADM1 in b ain me as ases o ma ion
needs o be es ed on la ge ma ched samples se s in u u e
s udies.
In addi ion o CADM1, we also analyzed he
exp ession o ou o he genes (RECK, SPARC, TNFAIP3
and CXCL14) in p ima y BC and BCBM. All ou
genes we e ound down- egula ed in BCBM pa ien s as
compa ed o p ima y BC. Fu he s udies will be needed
in o de o assess hei biological and clinical ole in
b ain me as ases o ma ion. RECK p o ein is capable
o inhibi ing ma ix me allop o einases and plays an
impo an ole in emb yogenesis and asculogenesis
[11]. RECK is o en down- egula ed in a ious p ima y
epi helial cance s and gliomas, and he down- egula ion
co ela es wi h poo p ognosis ( e iewed in [11, 20]).
Recen ly, Hill e al (2011) showed ha RECK down-
egula ion by me hyla ion in BC is associa ed wi h elapse
and poo su i al [21]. Fu he mo e, Hsu e al. showed
ha HER2 can ep ess RECK exp ession in o de o
p omo e cell in asion, which is consis en wi h ou esul s
showing he lowes exp ession o RECK among HER2
posi i e pa ien s [22].
SPARC/ os eonec in (sec e ed p o ein acidic and
ich in cys eine) is a ma icellula calcium-binding
glycop o ein in ol ed in cell adhesion, cell-ma ix
in e ac ions du ing issue emodelling, cell p oli e a ion,
mig a ion, angiogenesis and apop osis [12, 23]. Recen ly,
Nagai e al. [24] desc ibed a co ela ion be ween educed
SPARC exp ession and ad anced clinical s age and poo
ou come in p ima y BC. SPARC exp ession in me as a ic
issue has no been epo ed. Bo h CXCL14 (CXC mo i e
ligand 14) and TNFAIP3/A20 ( umo nec osis ac o
α induced p o ein 3) a e in ol ed in immuno egula o y
and in lamma o y p ocesses. CXCL14 is a p o-mig a o y
chemokine and plays an impo an ole in umo
ecogni ion by he immune sys em [13]. TNFAIP3 is a
zinc- inge p o ein in ol ed in he cy okine-media ed
immune and in lamma o y esponse mainly h ough he
inhibi ion o nuclea ac o NF-κB (NFκB) ac i a ion and
umo nec osis ac o (TNF)-media ed apop osis [25].
Down- egula ion o CXCL14 and TNFAIP3 has been
desc ibed in a ious p ima y umo en i ies including
b eas umo s [13, 14, 26, 27], bu o ou knowledge, hei
ole in me as asis has no been epo ed.
In conclusion, ou s udy shows a down- egula ion o
CADM1, RECK, SPARC, CXCL14 and TNFAIP3 in b ain
me as ases samples and especially implies CADM1 as an
impo an p ognos ic ac o , which is commonly los in BC
sub ypes wi h poo ou come and e e s o an inc eased isk
o b ain me as asis o ma ion. The mechanisms leading o
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a loss o CADM1 exp ession a e a leas pa ially induced
by p omo e hype me hyla ion, a mechanism po en ially
e e sible by he apeu ic in e en ion. As loss o CADM1
seems o ep esen in gene al a mo e agg essi e disease
wi h a highe isk o elapse especially o he b ain, hese
pa ien s migh need a mo e equen ollow-up including
diagnos ics such as egula clinical con ols and ea ly
cMRI o he b ain.
MATERIALS AND METHODS
Cell lines and pa ien ma e ial
Fo he me hyla ion sc eening analysis, he human
b eas cance cell line MDA-MB-231 (pa en al) and
i s bone- (SA) and b ain-seeking (BR) a ian s we e
used. MDA-MB-231 was ob ained om ATCC and
passaged o a maximum o 6 mon hs. MDA-MB-
231(SA) cells we e ob ained om P o . The esa Guise
and we e comp ehensi ely cha ac e ized by compa a i e
genomic hyb idiza ion and genome-wide gene exp ession
p o iling as desc ibed in Polla i e al. (2011) [28]. The
b ain me as a ic MDA-MB-231 a ian was ob ained
om P o . Toshiyuki Yoneda and was cha ac e ized in
[29]. A non- umo igenic epi helial cell line MCF 10A
(ATCC, USA) was used as a con ol (see supplemen a y
ma e ial). Au hen ica ions o all cell lines we e conduc ed
by sho andem epea (STR) p o iling o exclude c oss-
con amina ion be ween he cell lines.
Fo he qRT-PCR and me hyla ion speci ic PCR
(MSP) analyses, umo issue om 29 ea ly s age p ima y
BC pa ien s and 21 BCBMs we e collec ed om esh
ozen specimens (see supplemen a y ma e ial) [30].
Pa ien samples we e ob ained a e su gical esec ion
a he Uni e si y Medical Cen e , Hambu g-Eppendo
(UKE), Ge many. Fo he immunohis ochemis y (IHC)
analysis, h ee di e en se s o Tissue Mic o A ays
(TMA) we e used. The i s la ge p ognos ic a ay (TMA
I) consis ed o 2197 p ima y b eas cance specimens
ope a ed in Basel, Swi ze land [31]. Follow-up da a we e
a ailable o all pa ien s wi h a median ollow-up ime
o 61.0 mon hs ( ange 7-171 mon hs). The second TMA
(TMA II) consis ed o 243 in asi e duc al ca cinomas and
243 in asi e lobula ca cinomas ope a ed in Tampe e,
Finland, wi h a maximum ollow-up pe iod o 19.8 yea s
con aining in o ma ion on he si e and ime o umo
ecu ence and o e all su i al [32]. A hi d TMA om
UKE (TMA III) con ained 89 cases wi h ma ched p ima y
and lymph node me as asis samples [33]. In addi ion,
whole sec ion slides om 28 BCBM pa ien s we e also
analyzed by IHC (see supplemen a y ma e ial). The
REMARK c i e ia we e ollowed o he pa ien and s udy
se up, howe e no in o ma ion abou he ea men could
be collec ed. All sample dono s om UKE ga e w i en
in o med consen o biological esea ch in o hei samples
as app o ed by he e hics commi ee o he chambe o
physicians, Hambu g, Ge many. The use o he Finnish
umo samples and pa ien eco ds was app o ed by he
E hics Commi ee o he Pi kanmaa Hospi al Dis ic . All
clinical in es iga ions ha e been conduc ed acco ding o
he p inciples exp essed in he Decla a ion o Helsinki.
5-Aza-2’-deoxycy idine ea men o cells and
isola ion o o al RNA and DNA
Di e en concen a ions o 5-Aza-2’-deoxycy idine
we e es ed o minimal oxici y (MTT assay) bu ull
e e sion o me hyla ion. The concen a ion o 1 µM has
been ound as he mos e icien one. Cells we e cul u ed
in absence o p esence o 1 µM 5-Aza-CdR (n=3), and
he medium was changed daily un il he cells eached
a con luence o 90-100%. Expe imen s we e done in
iplica es using di e en cell passages (see supplemen a y
ma e ial).
Tumo issue sec ions we e manually dissec ed o
ob ain a umo cell con en o a leas 70% [34]. To al
RNA was isola ed wi h he RNeasy Mini Ki (Qiagen,
Hilden, Ge many). DNA was ex ac ed using InnuPREP
DNA Mic oki (Analy ik Jena, Jena, Ge many) (see
supplemen a y ma e ial). The success o 5-Aza-2’-
deoxycy idine ea men was e i ied by measu ing he
exp ession o me hyla ed MAGE-A1 and RUNX3 genes
[35, 36].
Genome-wide exp ession analysis
The gene exp ession analyses o he cell lines
and BCBM samples we e ca ied ou using he Whole
Human Genome Oligo Mic oa ay Ki , 4x44K (Agilen
Technologies) (see supplemen a y ma e ial). The non-
malignan cell line MCF 10A was used as a con ol o
geno oxic s ess esponse o he 5-Aza-2’-deoxycy idine
ea men . The MIAME guidelines we e ollowed in
sample, a ay and da a p ocessing (see supplemen a y
ma e ial). All a ay da a a e a ailable a h p://www.ncbi.
nlm.nih.go /geo GSE44354.
Po en ially me hyla ed genes in BC cells we e
de ined using he ollowing c i e ia: a) 2- old exp ession
di e ence be ween he ea ed and non- ea ed MDA-
MB-231 BR cells, b) minimum exp ession alue o 100
in one o he cell lines, and c) no exp ession change in he
con ol cell line MCF 10A a e he 5-Aza-CdR ea men .
The a ay da a om nine BCBM samples we e
compa ed o 32 un ea ed p ima y b eas umo s wi hou
elapse p esen in he GEO Da aSe GSE21974 [37].
Di e en ially exp essed genes we e selec ed using he
signi icance analysis o mic oa ays (SAM) algo i hm wi h
a alse disco e y a e o 5%. Genes wi h exp ession alues
abo e 100, which we e a leas 2- old down- egula ed in