Folate deficiency and aberrant expression of cell adhesion molecule 1 are potential indicators of prognosis in laryngeal squamous cell carcinoma

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1 4510 Folte deficiency nd berrnt expression of cell dhesion molecule 1 re potentil indictors of prognosis in lryngel squmous cell crcinom HAO CHANG 1, MIN MA 1, RUI MA 2, CHAO ZHANG 1, WEI ZENG 1 nd LU QI XING 3 1 Deprtment of Otolryngology, The First Affilited Hospitl, College of Clinicl Medicine of Henn University of Science nd Technology, Luoyng, Henn ; 2 Deprtment of Centrl Lbortory, Eye & ENT Hospitl of Fudn University, Shnghi ; 3 Deprtment of Pthology, The First Affilited Hospitl nd College of Clinicl Medicine of Henn University of Science nd Technology, Luoyng, Henn , P.R. Chin Received July 10, 2015; Accepted August 19, 2016 DOI: /ol Abstrct. The etiology of lryngel squmous cell crcinom (LSCC) hs not yet been dequtely exmined. Therefore, the present study imed to investigte the ssocition between serum folte deficiency nd bnorml expression of the cell dhesion molecule 1 (CADM1) protein in the progression of LSCC. Smples were collected from 60 ptients with LSCC nd 30 helthy people. Rdioimmunossys nd immunohistochemicl stining were performed to mesure serum folte levels nd CADM1 protein expression, respectively. The results demonstrted tht CADM1 expression in LSCC specimens ws significntly lower thn in djcent norml tissues (χ 2 =28.229, P<0.001), which ws ssocited with histologicl differentition nd clinicl stge (P=0.010 nd 0.020, respectively). Levels of serum folte in ptients with LSCC were significntly lower thn those observed in helthy individuls (P=0.002). Furthermore, TSLCl expression nd serum folte levels were positively correlted in LSCC (r=0.642, P=0.001). Thus, the present study determined tht decresed CADM1 protein expression nd low levels of serum folte were correlted with n incresed severity of LSCC. Introduction Lryngel squmous cell crcinom (LSCC) is one of the most common mlignnt tumors identified in the hed nd neck. LSCCs ccount for 90% of ll lrynx crcinoms, ~90% Correspondence to: Dr Min M, Deprtment of Otolryngology, The First Affilited Hospitl, nd College of Clinicl Medicine of Henn University of Science nd Technology, 24 Jing Hu Rod, Luoyng, Henn , P.R. Chin E mil: minm33@sin.com Key words: folte, cell dhesion molecule 1, lryngel squmous cell crcinom of which re in mles. Furthermore, the mjority of ptients re ged between 40 nd 60 yers old (1). At present, there is no specific biomrker for LSCC dignosis nd prognosis. Thus, the current study imed to exmine potentil biomrkers tht could improve the erly dignosis of LSCC. The cell dhesion molecule 1 (CADM1) gene encodes membrne protein, which is silenced in ~44% of ptients with non smll cell lung cncer nd 30 60% of those with other types of cncer (2). Previous studies hve identified two mechnisms of CADM1 inctivtion: Promoter hypermethyltion nd loss of heterozygosity (3 5). Furthermore, it hs been demonstrted tht the CADM1 protein prticiptes in number of biologicl functions, including synpse formtion, cell dhesion nd tumor suppression (6). In severl types of tumors, the bsence of CADM1 expression is closely relted to invsive tumor behvior (6 9). However, the role of CADM1 in LSCC remins lrgely unknown. To investigte the ssocition between CADM1 protein expression nd the development of LSCC, 60 surgiclly resected specimens were collected nd ssessed for loss of the CADM1 protein using immunohistochemistry. Subsequently, the ssocition of CADM1 protein expression with LSCC clinicopthologicl prmeters nd the histologicl growth pttern of the tumors were investigted. Folte is wter soluble B vitmin tht medites one crbon metbolism in vivo nd serves multiple functions in physiologicl processes. Folte provides one crbon groups for DNA repliction, epigenetic modifictions nd DNA muttion prevention, thus protecting ginst tumorigenesis (10). Additionlly, tetrhydrofolte synthesis in vivo requires folte for blood cell development nd mturtion (11). Folte deficiency is closely ssocited with glossitis, nemi nd, in pregnncy, fetl neurl tube defects (12). Previous studies hve suggested tht folte my id cncer prevention (13,14), nd severl studies hve demonstrted tht reduction in serum folte levels my be closely relted to severl types of cncer, including colorectl cncer (15 17). However, no similr report in LSCC ws found in the literture. Therefore, it is necessry to investigte the reltionship between serum folte levels nd

2 CHANG et l: CADM1 PREDICTS THE PROGNOSIS OF LARYNGEAL SQUAMOUS CELL CARCINOMA 4511 cncer progression nd prognosis. The present study imed to investigte the underlying ssocition between folte deficiency nd CADM1 protein expression in LSCC. Ptients nd methods Ptients nd tissue specimens. A totl of 60 pirs of LSCC nd precncerous specimens were collected from ptients treted t the Deprtment of Otolryngology, Hed nd Neck Surgery t the First Affilited Hospitl of Henn University of Science nd Technology (Luoyng, Chin) between September 2011 nd September The control group consisted of 30 helthy volunteers whose blood ws collected during routine helth exmintions. The rtio of ptients to helthy volunteers ws 1:2. Precncerous tissues were defined s the lryngel mucos 2 cm from the edge of the cncerous site. Ptients hd undergone preopertive prtil or totl lryngectomy without rdicl nd chemicl therpies. Ptients with megloblstic nemi or other types of nemi, upper gstrointestinl trct diseses, nd those treted with ny other surgicl procedures, were excluded. Ptients with history of smoking, orl contrceptive use or those who hd tken B vitmins in the pst 6 months were lso excluded from the current study. The Ethics Committee of the First Affilited Hospitl of Henn University of Science nd Technology (Luoyng, Chin) pproved the use of humn tissues for the current study. All ptients nd volunteers provided written informed consent to prticipte. Immunohistochemistry. Expression of the CADM1 protein ws tested using n Immunohistochemicl S P kit ginst CADM1 (Kngwei Bio, Ltd., Beijing, Chin), ccording to the mnufcturer's protocol. A rbbit polyclonl ntibody ginst CADM1 (1:100 dilution, ct. no. Rs 1147R) ws purchsed from Kngwei Bio, Ltd. Tissues were fixed with 10% formldehyde solution, cut into 3 µm sections, embedded in prffin blocks nd subsequently incubted with the polyclonl ntibody t 4 C overnight (>14 h). Sections were subsequently incubted with biotinylted got nti rbbit immunoglobulin G (1:100 dilution, ct. no. CW2035S; Kngwei Bio, Ltd.) for 40 min t room temperture. Phosphte buffered sline ws used s negtive control. 3,3' diminobenzidine ws used to stin the sections nd hemtoxylin ws used s counterstin to highlight the cytoblsts nd bckground color. Ech section ws observed in 10 rndom fields under x200 mgnifiction (18). The score ws clculted by dding the scores for intensity (wek=1, moderte=2, intense=3) nd proportion (5%=1, 6 24%=2, 25 49%=3, 50 74%=4, 75%=5) of positively stined cells (19). Scores of 2 were considered positive. Two independent pthologists ssessed ech slide. Rdioimmunossy. Whole blood smples (6 ml) were drwn from peripherl vein of ech prticipnt fter overnight fsting. Following the centrifugtion of whole blood smples t 300 x g for 15 min, serum smples were collected nd stored t 80 C until nlysis. Serum folte levels were tested using rdioimmunossy quntifiction with commercil kit for mesurement of folte (Shnghi Ruiqi Bio Technology, Co., Ltd., Shnghi, Chin), ccording to the mnufcturer's instructions. Tble I. Expression of the CADM1 protein in lryngel squmous cell crcinom nd djcent norml tissues. CADM1 Group n Positive Negtive χ 2 P vlue Tumors Adjcent norml tissues P<0.01; n, number of smples; CADM1, cell dhesion molecule 1. Sttisticl nlysis. Dt were nlyzed using Student's t test, χ 2 test, one wy nlysis of vrince (ANOVA), Student Newmn Keuls test, Fisher's exct test nd Spermn's rnk correltion coefficient. SPSS version 16.0 for Windows (SPSS Inc., Chicgo, IL, USA) ws used to nlyze ll results. P<0.05 ws considered to indicte sttisticlly significnt difference. Results Expression of the CADM1 protein in LSCC. CADM1 protein expression ws detected vi immunohistochemicl nlysis of 60 mlignnt squmous cells of lrynx tumors nd 60 corresponding helthy djcent non neoplstic tissues, which were exmined s comprison. The results demonstrted tht 47 specimens (78%) of djcent norml tissues expressed CADM1 protein. By contrst, only 12 tumor specimens (20%) expressed the CADM1 protein (P<0.001; Tble I). The ssocitions mong CADM1 expression ptterns in the tumors, serum folte levels nd ptient clinicl chrcteristics re presented in Tble II. Positive stining indicted tht CADM1 ws primrily locted in the cytoplsm in the djcent norml tissues, but ws bsent in the tumor tissues (Fig. 1A nd B, indicted by shpes). Expression of the CADM1 protein ws not correlted with ptient ge or ntomic locliztion (P=1.000 nd 0.386, respectively), but ws correlted with histologicl differentition nd clinicl stge (P=0.010 nd 0.020, respectively; Tble II). Associtions mong ptient chrcteristics, expression of CADM1 nd serum folte levels. One wy ANOVA ws used to determine differences in the serum folte levels mong LSCC tissues with different clinicl chrcteristics, including ge, clinicl stge, histologicl differentition nd ntomic locliztion. The differences were sttisticlly significnt mong tumor tissues with different clinicl stges, histologicl differentition nd ntomic locliztions (P=0.001, nd 0.001, respectively; Tble II). To further clrify the reltionship between serum folte levels nd stge of LSCC, Student Newmn Keuls test ws used for multiple comprisons of clinicl stge, histologicl differentition nd ntomic locliztion. The results indicted tht serum folte levels decresed s tumor mlignncy incresed (Tble III). Additionlly, serum folte levels were significntly lower in ptients with LSCC compred with helthy control subjects (P=0.002; Tble IV). The spermn correltion nlysis identified

3 4512 Tble II. Associtions mong ptient clinicl chrcteristics, protein expression of CADM1 nd serum folte levels. Folic cid P vlue: P vlue: Chrcteristic n P N (ng/ml) Fisher's exct test One wy ANOVA Age (yers) ±1.53 < ± TNM stge I ±1.45 II ±1.57 III ±1.32 IV ± b Histologicl differentition Well ±1.33 Modertely ±1.66 Poorly ± b Antomic locliztion Suprglottic ±0.96 Glottic ±1.45 Subglottic ± b P<0.05; b P<0.01; n, number of ptients; P, CADM1 positive cells; N, CADM1 negtive cells; CADM1, cell dhesion molecule 1; ANOVA, nlysis of vrince; TNM, tumor node metstsis. Figure 1. Cell dhesion molecule 1 (CADM1) expression in lryngel squmous cell crcinom tumor tissue compred with djcent norml tissue. (A) CADM1 expression in severe typicl hyperplsi tissue nd the bsence of its expression in the tumor (mgnifiction, x100). =Severe typicl hyperplsi tissue, =tumor tissue. (B) CADM1 expression in mild typicl hyperplsi tissue nd the bsence of its expression in the tumor (mgnifiction, x100). =Mild typicl hyperplsi tissue. =tumor tissue. significnt correltion between CADM1 protein expression nd serum folte levels (r=0.642, P=0.001). Therefore, folte deficiency my decrese the methyltion of CADM1 promotor nd regulte CADM1 expression. However, further studies re required to confirm this. Discussion In the present study, ll included ptients were mle nd most ptients ged <50 yers were TNM stge III or IV with higher serum folte levels, wheres ptients ged >50 yers were stges I II with lower serum folte levels. The criticl fctor in the prognosis of LSCC is tumor metstsis, however, the etiopthogenesis of tumor metstsis remins lrgely unknown nd my involve immune dysregultion. CADM1, locted on chromosome 11q23.2, encodes trnsmembrne protein from the immunoglobulin superfmily (20). Previous studies hve demonstrted tht CADM1 is humn tumor suppressor nd muttions in its cytoplsmic domin hve been linked to lung tumor cell metstsis, ggrvted histologicl differentition, clinicl stge clssifiction nd poor prognosis (6,21,22). The results of the present study were consistent with previous studies (23,24) nd estblished tht, s compred with djcent non neoplstic tissue, tumor tissues express lower levels of CADM1. This is importnt s CADM1 my serve potentil role in the dignosis nd prognosis of LSCC. Duthie et l (25) ffirmed the effect of folte deficiency on chromosome breks nd the risk of crcinogenesis. Furthermore, it hs been determined tht folte deficiency

4 CHANG et l: CADM1 PREDICTS THE PROGNOSIS OF LARYNGEAL SQUAMOUS CELL CARCINOMA 4513 Tble III. Associtions between serum folic cid levels in ptients with lryngel squmous cell crcinom nd clinicl stge, histologicl differentition nd ntomic locliztion. Clinicl Histologicl stge differentition nd (TNM) P vlue ntomic locliztion P vlue I nd II nd I nd III nd b I nd IV b 2 nd II nd III A nd B b II nd IV b A nd C III nd IV B nd C P<0.05; b P<0.01; 1, well differentited; 2, modertely differentited; 3, poorly differentited; A, suprglottic; B, glottic; C, subglottic; TNM, tumor node metstsis. Tble IV. Serum folic cid levels in ptients with LSCC nd helthy controls. Group n Folic cid (ng/ml) Helthy controls ±1.47 Ptients with LSCC ±1.51 Dt re presented s the men ± stndrd devition. P= n, number of ptients; LSCC, lryngel squmous cell crcinom. my decrese thymidine synthesis nd stimulte urcil misincorportion into DNA (26,27). Homeostsis of folte is criticl to DNA stbility nd integrity, s well s the repir of dmged DNA, nd folte supplementtion my hve protective effect ginst cncer (15,16,28,29). The results of the current study demonstrted tht serum folte levels in ptients with LSCC were mrkedly lower thn those in helthy individuls nd decresed with the deteriortion in the degree of tumor mlignncy. This indictes tht low serum folte levels my be ssocited with the incresed mlignncy of LSCC. The results of the present study suggested tht there ws significnt ssocition between CADM1 protein expression nd serum levels of serum folte in ptients with LSCC (r=0.642, P=0.001). CADM1 expression vried mong different ntomic locliztions; therefore, this reltionship my result from the different mlignnt degrees nd crcinom progression. However, the unique microenvironments of different ntomicl locliztions my lso contribute to the difference. Therefore, further studies re required to understnd why these differences occur. The nlysis demonstrted tht the decline of serum folte levels my be the cuse of low CADM1 expression in LSCC tumor tissue. It ws observed tht significnt proportion of LSCC ptients hd low serum folte levels. Furthermore, s the severity of LSCC incresed, serum folic cid levels decresed. Therefore, folte deficiency my be ssocited with the excessive consumption of folic cid in the body (due to the incresed energy needs of ptients with cncer) during repir of the dmged CADM1 gene (17). Folte is criticl fctor of DNA methyltion (29) nd methyltion of promoter DNA hs been regrded s n importnt mechnism of CADM1 gene silencing (30). In the present study, CADM1 downregultion in LSCC tumor tissue ws observed; however, the underlying mechnism ws not investigted. Therefore, further clinicl studies re necessry. In future studies, we intend to evlute the effectiveness of serum folic cid supplementtion in ptients with LSCC nd to identify the intrinsic reltionship between folte nd promoter methyltion of the CADM1 gene. Acknowledgements This study ws supported by the Provincil Science nd Technology Foundtion of Henn Province, Chin (grnt no ). References 1. Hung XZ, Wng JB nd Kong WJ: Prctice of Otorhinolryngology Hed nd Neck Surgery. 2nd edition. People's Medicl Publishing House, Bei Jing, Chin, Murkmi Y, Nobukuni T, Tmur K, Mruym T, Sekiy T, Ari Y, Gomyou H, Tnigmi A, Ohki M, Cbin D, et l: Locliztion of tumor suppressor ctivity importnt in nonsmll cell lung crcinom on chromosome 11q. Proc Ntl Acd Sci USA 95: , Allinen M, Peri L, Kujl S, Lhti Domenici J, Outil K, Krppinen SM, Lunonen V nd Winqvist R: Anlysis of 11q21 24 loss of heterozygosity cndidte trget genes in brest cncer: Indictions of TSLC1 promoter hypermethyltion. 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