In . J. Mol. Sci. 2015, 16, 4372-4378; doi:10.3390/ijms16034372
In e na ional Jou nal o
Molecula Sciences
ISSN 1422-0067
www.mdpi.com/jou nal/ijms
Communica ion
Cell Memb ane CD44 6 Le els in Squamous Cell Ca cinoma o
he Lung: Associa ion wi h High Cellula P oli e a ion and High
Concen a ions o EGFR and CD44 5
Ál a o Ruibal 1,2,3, Pablo Aguia 1,2,*, Ma ía Ca men Del Río 4, Ma ilde Isabel Nuñez 2,
Vi ginia Pubul 2 and Michel He anz 1,2
1 Molecula Imaging G oup, Facul y o Medicine, Uni e si y o San iago Compos ela, R/de San
F ancisco, s/n., San iago de Compos ela 15782, Spain; E-Mails: al a[email p o ec ed] (A.R.);
[email p o ec ed] (M.H.)
2 Nuclea Medicine Depa men , Uni e si y Hospi al San iago Compos ela (CHUS), R/Choupana, s/n.,
San iago de Compos ela 15706, Spain; E-Mails: m[email p o ec ed] (M.I.N.);
[email p o ec ed] (V.P.)
3 Fundación Teje ina, C/José Abascal, 40, Mad id 28003, Spain
4 Gene al Lab, Hospi al A qui ec o Ma cide, Fe ol, A Co uña 15405, Spain;
E-Mail: ma ia.del.ca m[email p o ec ed]
* Au ho o whom co espondence should be add essed; E-Mail: pablo.aguia[email p o ec ed].
Academic Edi o : William Chi-shing Cho
Recei ed: 18 No embe 2014 / Accep ed: 3 Feb ua y 2015 / Published: 18 Feb ua y 2015
Abs ac : Memb anous CD44 6 le els in umo s and su ounding samples ob ained om
94 pa ien s wi h squamous cell lung ca cinomas we e s udied and compa ed o clinical s age,
cellula p oli e a ion, memb anous CD44 5 le els, epide mal g ow h ac o ecep o EGFR
and cy oplasma ic concen a ions o CYFRA 21.1. CD44 6 posi i e alues we e obse ed
in 33/38 non- umo samples and in 76/94 umo samples, bu he e we e no s a is ically
signi ican di e ences be ween bo h subg oups. In CD44 6 posi i e umo samples,
CD44 6 was no associa ed wi h clinical s age, his ological g ade, ploidy and lymph node
in ol emen , bu signi ican associa ion was ound wi h high cellula p oli e a ion. Likewise,
CD44 6 posi i e umo s had signi ican ly highe le els o EGFR and CD44 5. In pa ien s
wi h squamous cell lung ca cinomas and clinical s age I, posi i e CD44 6 cases we e
associa ed wi h he same pa ame e s. Fu he mo e, posi i e CD44 5 squamous umo s we e
associa ed signi ican ly wi h his ological g ade III and lowe le els o CYFRA21.1. Ou
OPEN ACCESS
In . J. Mol. Sci. 2015, 16 4373
indings suppo he alue o CD44 6 as a possible indica o o poo ou come in pa ien s
wi h squamous lung ca cinomas.
Keywo ds: lung cance ; squamous lung ca cinomas; CD44 6; CD44 5; EGFR; p oli e a ion
1. In oduc ion
CD44 is a su ace adhesion molecule. I consis s o a ansmemb ane glycop o ein exp essed in many
no mal issues by cells o di e en o igin and in ol ed in some physiological p ocesses such as
cell-cell and cell-ma ix adhesion, hema opoiesis, lymphocy e homing and ac i a ion, and umo
dissemina ion [1,2]. CD44 exis s in a s anda d o m (CD44s) and in mul iple iso o ms, gene a ed by
al e na i e splicing o a leas 10 a ian exons (CD44 ) encoding pa s o he ex acellula domain and
ela ed wi h some ea u es o he umo e olu ion [3]. One o hem is CD44 6, associa ed wi h he
me as a ic po en ial o some human malignan umo s, egula ing umo in asion, p og ession and
me as asis [4–7]. Classical s udies in non-small cell lung cance s ha e demons a ed ha CD44 6
exp ession was highe in squamous han in adenoca cinoma sub ype [8–10] and ha i was associa ed
wi h a poo e su i al, included s age I [3]. Ne e heless, i s associa ion wi h lympha ic o ascula
in asion, as well as he ou come is no unanimous [8], whe eas he e was no any ela ion nei he wi h
his ological g ade no clinical s age [10].
In he las yea s he clinical in e es o his molecule has inc eased. Recen ly, Zhao e al. [11] and
Jiang e al. [12] a e wo me a-analysis, showed ha CD44 6 exp ession in non-small cell lung cance
(NSCLC) is associa ed wi h squamous sub ype, lymph node me as asis, and a poo su i al, conside ing
a new p ognos ic ma ke in pa ien s wi h hese lung ca cinomas. Fu he mo e, Sun e al. [13] s udied he
beha io o CD44 6 and os eopon in, gene in ol ed in me as a ic p ocess o malignan umo s binding
o CD44 6 and in eg in, and conside ed ha bo h pa ame e s we e independen p edic o s o poo e
o e all su i al and disease ee su i al. These ac s and he lack o pape s di e en ia ing he mos
impo an sub ypes in non-small cell lung cance NSCLC, led us o s udy he possible associa ions o
CD44 6 wi h o he clinical and biological pa ame e s only in pa ien s wi h squamous cell lung cance .
2. Resul s and Discussion
No mal lung issues had highe le els o ca hepsin D han umo issues ( : 8.9–1332; median
74.6 pmol/mg p o . s. : 7.7–567; median 38.8; p: 0.0019), while umo issues had highe le els o
CD44s, CD44 5, CD44 6, EGFR, CA125 and neu on speci ic enolase (NSE). (See Table 1).
CD44 6 posi i e alues (>5 ng/mg p o .) we e obse ed in 33/38 non- umo samples (87%) and in
76/94 umo samples (81%). The e we e no s a is ically signi ican di e ences be ween bo h subg oups.
CD44 6 alues co ela ed signi ican ly wi h CD44 5 le els bo h in non- umo samples ( : 0.78) and in
umo samples ( : 0.89). Likewise, only in umo al issues, he e we e s a is ically signi ican co ela ions
be ween CD44 5 and CD44s ( : 0.64), be ween CD44 6 and CD44s ( : 0.57) and be ween CD44 6 and
cy osolic hyalu onic acid concen a ions ( : 0.26).
Finally, i should be men ioned ha CD44 6 le els ob ained in squamous umo samples
( ange: 5.1–1395; median 68.2) we e signi ican ly highe (p: 0.00006) han hose obse ed in 34
In . J. Mol. Sci. 2015, 16 4374
adenoca cinoma umo s ( : 5.8–454; median 28.8 ng/mg p o .). Ou indings we e simila o hose
desc ibed by o he au ho s as Miyoshi e al. [14], Wu e al. [15] and sligh ly highe han hose epo ed
by E en e al. [8].
Table 1. S a is ically signi ican di e ences in some biological pa ame e s be ween no mal
and squamous cell lung issues.
Pa ame e No mal Tumo Tissue p
CD44s ** 84.1–237 (127) 80.6–643 (153) 0.016
CD44 5 ** 3.5–47.6 (22.7) 3.5–1080 (33.8) 0.001
CD44 6 ** 8.6–100 (32.3) 5.1–1305 (68.2) <0.001
EGFR * 2.7–45.8 (19.2) 1–394 (34.7) <0.001
CA125 ** 1–51.7 (8.4) 1–576 (12.8) 0.005
NSE ** 56–657 (141) 4.5–2234 (267) <0.001
Range (median); *: mol/mg p o .; and **: ng/mg p o .
In he 76 CD44 6 posi i e umo samples, CD4 6 doesn’ associa e wi h clinical s age, his ological
g ade, ploidy and lymph node in ol emen bu signi ican associa ion (p: 0.015) be ween high CD44 6
le els and high cellula p oli e a ion (SP) (31/49 s. 2/13) measu ed by S-phase was ound (cu o : 14%,
which ep esen s, in ou expe ience, he pe cen ile 75 h o all alues ob ained wi h mo e han 200 lung
umo s. Posi i e CD44 6 umo s had highe le els o EGFR ( : 3.1–325; median 38.7 s. : 1–394;
median 20.5 mol/mg p o .; p: 0.0046), CD44 5 le els ( : 3.5–1080, median 42.2 s. : 6.9–45.8; median
17.5 ng/mg p o .; p: 0.0002), CA125, NSE and cy osolic hyalu onic acid concen a ions ( : 50–22591;
median 5154 s. : 817–9799; median 3101 ng/mg p o .; p: 0.008).
Same indings we e obse ed also in pa ien s wi h squamous cell lung ca cinomas in clinical s age I:
high SP (p: 0.021), highe CD44 5 (p: 0.001) and highe EGFR le els (p: 0.01) (see Table 2). I dese es
o be ou lined ha posi i e CD44 5 squamous umo s we e associa ed signi ican ly (p: 0.021) wi h
his ological g ade III and lowe le els o CYFRA 21.1( : 16.5–3306; median 119 s. : 113–3817;
median 383 ng/mg p o .; p: 0.0081).
O he g oups desc ibed an associa ion be ween CD44 6 posi i i y and lymph node me as asis [14,15],
poo ly di e en ia ed umo s, ad anced clinical s age and poo e su i al [12,15], being a p ognos ic
ac o wi h pTNM s age a e mul i a ia e analysis [15], e en in s age I non-small cell lung [3].
Hi a a e al. [3] desc ibed also a co ela ion be ween CD44 6 and p oli e a ing cell nuclea an igen
PCNA posi i e exp ession, ano he indica o o cellula p oli e a ion. We know ha cell p oli e a ion is
associa ed wi h a poo p ognosis in some umo s and wi h be e esponse o chemo he apy [16].
I has o be men ioned ha EGFR le els in NSCLC samples a e highe han hose obse ed in no mal
lung issues and hey a e ela ed wi h a poo umo di e en ia ion and a high p oli e a ion. Likewise,
hey a e associa ed wi h a poo e p ognos ic and ou come in squamous lung umo s [17,18]. Now,
we know he clinical in e es o he mu a ion in EGFR gene and he di e en beha io o hese al e a ions
acco ding o he age o he pa ien [19]. CYFRA 21.1, which e lec s agmen s o cy oke a in 19, is a
good se um umo ma ke o he diagnosis o lung cance , specially he squamous sub ype in addi ion
o compu e ized omog aphy, is associa ed wi h poo ou come in pa ien s wi h NSCLC ea ed wi h
adju an chemo he apy o e lo inib [20–23] and i is an use ul ool o me as asis de ec ion in lung cance
pa ien s wi hou symp oms o me as asis in pa ien s wi h hose lung umo s [24]. Ne e heless, he
In . J. Mol. Sci. 2015, 16 4375
CYFRA 21.1 beha io in lung umo issues is opposi e o wha happens in se um; so, he weak
exp ession o CK19, as de e mined by immunos aning in ensi y in umo issues and a high se um
concen a ion o CYFRA 21.1 was a signi ican p edic o o poo e disease-speci ic su i al in human
lung squamous cell ca cinoma [25].
Table 2. Dis ibu ion o he biological pa ame e s wi h s a is ically signi ican di e ences
in squamous cell ca cinomas o he lung (global and s age I) classi ied acco ding o
CD44 6 posi i i y.
Pa ame e CD44 6+ CD44 6− p
Global
EGFR * 3.1–325 (38.7) 1–394 (20.5) 0.005
CD44 5 ** 3.5–1080 (42.2) 6.9–45.8 (17.5) 0.0002
S age I
EGFR * 3.1–215 (35.8) 1–394 (21.3) 0.010
CD44 5 ** 9.5–1080 (45.3) 6.9–45.8 (17.6) 0.001
Range (median); *: mol/mg p o .; and **: ng/mg p o .
3. Expe imen al Sec ion
3.1. S udy Design
Memb anous CD44 6 le els in umo s and su ounding samples we e ob ained om pa ien s wi h
squamous cell ca cinomas o he lung.
3.2. Subjec s
The s udy g oup was 94 pa ien s (85 male and 9 emale; age: 36–72; median 62 yea s) wi h squamous
cell lung ca cinomas. Acco ding o clinical s age, he pa ien s we e classi ied as ollows: I = 69; II = 5;
III = 20 cases. Likewise, 38 lung no mal samples we e emo ed om a egion o su ounding issue a
leas 2 cm away om he umo s. They we e mac oscopically ee om neoplas ic g ow h. A s udy
g oup o 34 pa ien s wi h adenoca cinoma umo s we e also conside ed o compa ison.
3.3. Blood Samples and Me hods
Lung ca cinoma issue samples we e ob ained a he ime o su ge y. Immedia ely a e su gical
esec ion, samples we e p ocessed o pa hological examina ion while he emainde issue was washed
wi h cold saline solu ion, di ided in aliquo s, apidly anspo ed on ice o he labo a o y (−70 °C)
pending biochemical s udies. The specimens ob ained om neoplas ic issues we e pul e ized wi h a
mic odismemb a o (B aun Bio ech In e na ional, Melsungen, Ge many) a −70 °C and homogenized in
T is-hyd ochlo ide bu e (10 mM o T is, 1.5 mM o EDTA, 10% glyce ol, 0.1% o mono hioglyce ol).
Homogena es, kep a 4 °C, we e cen i uged a low speed (800× g o 10 min, a 4°C), and he
supe na an was ul acen i uged a 100,000× g o 60 min, a 4 °C. We ob ained a supe na an con aining
he cy sol and a p ecipi a e wi h he memb anes.
CD44 6 and CD44 5 we e assayed in cell su ace memb anes using an enzymoimmunoassay om
Bende MedSys ems (Vienna, Aus ia) wi h wo monoclonal an ibodies. The lowes limi o sensi i i y
In . J. Mol. Sci. 2015, 16 4376
was 0.13 ng/mL o bo h CD44 6 and CD44 5. The in aassay a ia ion coe icien o CD44 6 o
a mean alue o 0.52 and 6.4 ng/mL we e 8.6% and 5.1% espec i ely and he in aassay a ia ion
coe icien o a mean alue o 3.47 and 9.2 ng/mL we e 9.3% and 8.4% espec i ely. The in aassay
a ia ion coe icien o CD44 5 o a mean alue o 0.86 and 5.2 ng/mL we e 8.6% and 5.0%
espec i ely and he in e assay a ia ion coe icien o a mean alue o 0.95 and 7.4 ng/mL we e 9.3%
and 7.4% espec i ely. Each sample was dossi ied by duplica e. The cu -o o posi i i y was es ablished
in 5 ng/mg p o . o CD44 6 and 3 ng/mg p o . o CD44 5 espec i ely.
Epide mal g ow h ac o ecep o (EGFR) was assayed in cell su ace memb ane using a adioligand
me hod (ViennaLab, Vienna, Aus ia) wi h a lowes limi o sensi i i y o 1 mol/mg p o . CYFRA 21.1
was assayed using an immuno adiome ic assay (CIS In e na ional, Gi su Y e e, F ance) wi h wo
monoclonal an ibodies (KS19.1 and BM 19.21) and wi h a lowes limi o sensi i i y o 0.1 ng/mL,
CD44s wi h an enzymoimmunoassay (Bende MedSys ems, Vienna, Aus ia) wi h a lowe limi o
sensi i i y o 0.42 ng/mL, Hyalu onic Acid using a Radioligand me hod om Pha macia (Upjohn,
Sweden) wi h a lowe limi o sensi i i y o 1 ng/mL, CA125 wi h an immuno adiome os
assys (Cen oco , Mal e n, PA, USA) wi h a lowe limi o sensi i i y o 0.4 U/mL, NSE wi h an
immuno adiome is assay (CIS BioIn e na ional, F ance) wi h wo monoclonal an ibodies and a lowe
limi o sensi i i y o 0.3 ng/mL, and ca hepsin D was assayed using an immuno adiome ic assay
(CIS BioIn e na ional) wi h wo monoclonal an ibodies and a lowe limi o sensi i i y o 20 pmol/mL.
All esul s we e e e ed o mg o p o ein measu ed by B ad o d me hod [26]. DNA ploidy and
p oli e a i e ac i i y we e e alua ed by low cy ome y (Bec on Dickinson, San Jose, CA, USA), on
nuclei ob ained om esh samples and s ained wi h p opidium iodide, and calcula ed wi h he CellFi
so wa e p og am (Bec on Dickinson), acco ding o he DNA Cy ome y Consensus Con e ence
ecommenda ions [27].
3.4. S a is ics Analysis
A e analyzing he dis ibu ion o CD44 6 and CD44 5 alues by he Kolmogo o –Smi no es ,
non-pa ame ic ank me hods we e used because hose pa ame e s ha did no ollow a no mal dis ibu ion.
CD44 6 and Cd44 5 le els con en we e exp essed as median and ange. Compa ison o he CD44 6
le els be ween di e en subg oups g oups was made wi h he Mann–Whi ney and K uskal–Wallis es s.
Co ela ions be ween con inuous a iables we e calcula ed by he Spea man es . Di e eces in pe cen ages
we e calcula ed wi h he 2 es wi h Ya es co ec ion, i necessa y. A p- alue < 0.05 was conside ed as
s a is ically signi ican .
4. Conclusions
Ou esul s showed ha CD44 6 posi i e alues we e obse ed in 80% o squamous umo samples
and we e associa ed wi h high cellula p oli e a ion, high le els o EGFR and high CD44 5
concen a ions in cell memb anes. Fu he mo e, posi i e CD44 5 squamous umo s we e associa ed
signi ican ly wi h his ological g ade III and lowe le els o CYFRA 21.1 in cy osols.
Ou indings suppo he alue o CD44 6 as an indica o o poo ou come in pa ien s wi h squamous
lung ca cinomas.

In . J. Mol. Sci. 2015, 16 4377
Acknowledgmen s
This wo k was suppo ed by g an PI11/01806 om ISCIII (Spain).
Au ho Con ibu ions
Ál a o Ruibal designed, de eloped and w o e he wo k. Pablo Aguia and Michel He anz con ibu ed
o he design and he analysis o he esul s and p o ided da a. Ma ía Ca men Del Río, Vi ginia Pubul
and Ma ilde Isabel Nuñez con ibu ed o he discussion abou he clinical impac o he wo k.
Con lic s o In e es
The au ho s decla e no con lic o in e es .
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