Cell Membrane CD44v6 Levels in Squamous Cell Carcinoma of the Lung: Association with High Cellular Proliferation and High Concentrations of EGFR and CD44v5

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 . Re e ences 1. 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