Original Article CD40-1C>T polymorphism and the risk of lung cancer in a Chinese population

Transcription

1 Int J Clin Exp Pthol 2015;8(11): /ISSN: /IJCEP Originl Article CD40-1C>T polymorphism nd the risk of lung cncer in Chinese popultion Gng Zhou 1*, Ying Wng 1,2*, Ziyo Fng 1, Rongrong Liu 1, Anhui Wng 3, Feng Zho 4, Lihu Chen 1 Deprtments of 1 Immunology, 3 Preventive Medicine nd Helth Sttistics, Fourth Militry Medicl University, Xi n, Chin; 2 Deprtment of Stomtology, Affilited Hospitl of Acdemy of Militry Medicl Sciences, Beijing, Chin; 4 Deprtment of Respirtory Medicine, Xijing Hospitl, Fourth Militry Medicl University, Xi n, Chin. * Equl contributors. Received September 7, 2015; Accepted October 21, 2015; Epub November 1, 2015; Published November 15, 2015 Abstrct: Bckground: The co-stimultory molecule CD40 plys n importnt role in nti-tumor responses by promoting cytotoxic T lymphocyte (CTL) ctivity nd differentition of helper T cells. Growing evidence suggests tht single nucleotide polymorphisms (SNPs) in CD40 re ssocited with the susceptibility to cncer. This study investigted the ssocition between the CD40-1C/T SNP (rs ) nd lung cncer in Chinese popultion. Methods: We conducted hospitl-bsed cse-control study including 105 lung cncer ptients nd 109 helthy control subjects. The -1C/T SNP in CD40 ws genotyped by the polymerse chin rection restriction frgment length polymorphism (PCR-RFLP), nd its ssocition with lung cncer susceptibility ws evluted. Results: The distribution of the genotypes of CD40-1C/T ws significntly different between lung cncer ptients nd controls. The frequency of the TT genotype (djusted P = 0.017; overll risk [OR] = 2.94; 95% confidence intervl [CI] = ) nd TT/ CT genotype (djusted P = 0.020; OR = 1.95; 95% CI = ) were significntly higher in lung cncer ptients thn tht in controls. When the cses were ctegorized by tumor histology, the TT genotype ws ssocited with significntly incresed risk of squmous cell crcinom (djusted OR = 6.53; 95% CI = ; P = 0.002). Conclusion: Our findings suggest tht the CD40-1C/T SNP (rs ) is correlted with the susceptibility to lung cncer in Chinese, nd the TT genotype my further increse the risk of lung cncer. Keywords: CD40, lung cncer, single nucleotide polymorphism Introduction Lung cncer is the most commonly dignosed mlignncy worldwide nd the leding cuse of cncer-relted deth, with n incidence rte of 46.08/100,000 nd mortlity of 37/100,000 [1]. Seventy percent of ptients with lung cncer hve lredy reched n dvnced stge by the time they re dignosed. Although significnt progress hs been mde towrd understnding interindividul predispositions ssocited with lung cncer, there re few useful biomrkers to identify susceptive individuls in the generl popultion. Becuse of the frequent occurrence nd detectbility of SNP, mny studies hve investigted the reltionship between genetic polymorphisms nd the risk of lung cncers [2]. CD40 is member of the tumor necrosis fctor receptor (TNFR) fmily nd is expressed on the surfces of ntigen presenting cells (APC) such s B cells, monocytes, dendritic cells (DC), s well s non-immune cells such s endothelil cells, epithelil cells, nd mlignnt tumor cells. CD40 plys key role in stimulting cytotoxic lymphocytes nd interctions between CD40 nd its lignd, CD154, regulte ctivity of APC nd the immune response of T cells nd B cells [3]. Moreover, CD40 signling is criticl in ntitumor responses, cting to promote cytotoxic T lymphocyte (CTL) responses nd differentite helper T cells towrds Th1 cells [5, 6]. Yet some studies revel tht CD40 cn promote tumor prolifertion through prticulr pthwys [5]. For exmple, CD40 promotes tumor ngiogenesis by inducing the expression of vsculr endothelil growth fctor (VEGF) [7]. Other reserch shows tht CD40 promotes tumor growth by ffecting the inflmmtory microenvironment [8]. Due to the complex function of

2 Tble 1. Chrcteristics of the study popultion Chrcteristic informtion CD40, mny tumors nd utoimmune disorders re relted to CD40 expression [9-12]. In the Hn Chinese popultion, there re pproximtely 45 SNPs identified in the CD40 gene. The -1C>T SNP is ssocited with the expression of CD40 on the surfces of B cells nd DC [13], nd this polymorphism hs been studied in severl mlignncies, including lymphoms nd brest cncer [11, 14-17]. To further verify the role of the CD40-1C/T SNP in the risk of lung cncer, we conducted hospitlbsed cse-control study in Hn Chinese popultion from the ShnXi Province, locted in northwest Chin. Mterils nd methods Subjects Cses (n = 105) Controls (n = 109) Age (yers) 58±11 56±10 Sex Mle Femle Smoking Yes No Histology Adenocrcinom 44 Squmous 32 Smll Cell Crcinom 29 The smoking history is identified by the definition of WHO of 1997: consecutive or ccumultive smoking time exceeds 6 months. All subjects in this study were unrelted Hn Chinese from northwest Chin. The cses were ptients with histopthologiclly confirmed primry lung cncer in the Respirtory Deprtment of the Xijing Hospitl (the First Affilited Hospitl of the Fourth Militry Medicl University, Shnnxi Province) from 2011 to The dignosis of lung cncer ws bsed on surgicl nd pthologicl reports s well s rdiogrphy results. Subjects were excluded if they hd histories of mlignncies, utoimmune disorders, respirtory disorders, or hereditry diseses. Ptients who hd infections within 4 weeks before the study were lso excluded. Personl informtion ws obtined from medicl files. In the control group, 110 helthy controls were recruited rndomly from volunteers t The Medicl Exmintion Center of Xijing Hospitl. All helthy controls were frequency-mtched (1:1) to ptients bsed on ge, gender nd smoking sttus. During the sttisticl nlysis, we removed the smples filed in DNA genotyping. Ultimtely, this study included 105 lung cncer ptients nd 109 helthy controls. This study ws pproved by the Institutionl Review Bord of the Fourth Militry Medicl University nd informed consent ws obtined from ech prticipnt. DNA extrction nd genotyping Genomic DNA ws extrcted from blood smples using TIANmp Genomic DNA Kits (Beijing, Chin) ccording to mnufcturer protocols. The CD40-1C/T SNP ws identified by polymerse chin rection-restriction frgment length polymorphism (PCR-RFLP) ssys. Primer sequences were designed by Primer Premier softwre nd synthesized by Sngon Biotech (Shnghi, Chin). The primer sequences were: Sense, 5 -ACACAGCAAGATGCGTCCCTAAACT-3 (Tm = 65.6 C); Anti-sense, 5 -TCCTTCTCATT- CCCCACTCCCAACT-3 (Tm = 68.2 C). The length of the PCR product ws 334 bp. PCR ws performed in totl volume of 50 μl contining: 200 ng genomic DNA, 25 μl Premix Tq (TKR TqTM Version 2.0 plus dye) (TKR, Dlin, Chin), 1 μl of ech primer (20 μm), ddh 2 O were dded to bring smples to finl volume of 50 μl. The cycling conditions were consisted of: 5 min t 94 C; 35 cycles of denturtion for 30 s t 94 C, nneling for 30 s t 55 C, nd extension for 45 s t 72 C. After the 35 cycles, there is finl extension step for 7 min t 72 C. After purifiction with TIANmp PCR Purifiction Kits (Beijing, Chin), PCR products were digested by the restriction enzyme Ncol I t 37 C for 6 h in volume of 20 μl contining: 1 μl of Ncol I (2 U); 2 μl of 10 K Buffer; 2 μl of 0.1% bovine serum lbumin (BSA); 10 μl of PCR product; nd 5 μl of ddh 2 O. The restriction digest products were nlyzed by electrophoresis on 2.0% grose gel. The -1CC homozygotes showed 2 frgments (106 bp nd 228 bp), the -1TT homozygotes showed only 1 frgment (334 bp), nd the -1CT hetero Int J Clin Exp Pthol 2015;8(11):

3 Tble 2. Genotypes nd llele frequencies of CD40-1C/T in lung cncer ptients nd controls Anlysis model Allele or Genotype Allele Cses (n = 105) No. (%) Controls (n = 109) No. (%) Crude OR (95% CI) Crude P Adjusted OR (95% CI) Adjusted P C 122 (58.10) 152 (69.72) 1.00 (Ref) 1.00 (Ref) T 88 (41.90) 66 (30.28) 1.66 ( ) ( ) Genetic model Genotype Co-Dominnt CC 36 (34.29) 53 (48.62) 1.00 (Ref) 1.00 (Ref) CT 50 (47.62) 46 (42.20) 1.60 ( ) ( ) TT 19 (18.10) 10 (9.17) 2.80 ( ) ( ) Additive 1.65 ( ) ( ) Dominnt CC 36 (34.29) 53 (48.62) 1.00 (Ref) 1.00 (Ref) CT/TT 69 (65.71) 56 (51.38) 1.81 ( ) ( ) Recessive CT/CC 86 (81.90) 99 (90.83) 1.00 (Ref) 1.00 (Ref) TT 19 (18.10) 10 (9.17) 2.19 ( ) ( ) Adjusted for ge, smoking sttus nd gender. zygotes showed 3 frgments (334 bp, 106 bp, nd 228 bp). To ensure the ccurcy of genotyping for this PCR-RFLP nlyse, 10% of the ptient nd control smples were rndomly selected for vlidtion by direct sequencing. Sttisticl nlyses The cses nd controls were compred using Student s t-tests for continuous vribles nd χ 2 tests for ctegoricl vribles. The Hrdy- Weinberg equilibrium ws tested in the control group with the expected genotype frequencies. The cncer risk ssocited with the genotypes ws estimted s odds rtios (OR) with 95% confidence intervls (CI) using logistic regression. Crude ORs nd ORs djusted for possible confounders (gender, ge nd smoking sttus) were clculted. Multivrite logistic regression nlyses were performed to nlyze the ssocition between genotypes nd lung cncer risk fter strtifying subjects ccording to ge (medin yers), gender, smoking sttus. P vlues <0.05 were considered significnt for ll of nlyses. Sttisticl nlyses were performed using SPSS Results Bsic chrcteristics of the lung cncer ptients nd controls re summrized in Tble 1. No sttisticlly significnt differences in gender, ge, or smoking sttus were observed between the lung cncer ptients nd controls (P>0.05), which suggested dequte mtching bsed on those vribles. The genotype nd llele frequencies of the CD40-1C/T polymorphism in 105 lung cncer ptients nd 109 control subjects re shown in Tble 2. The genotype distributions of the polymorphism mong the controls were within the Hrdy-Weinberg equilibrium (χ 2 = ; P>0.05). A significnt difference regrding the frequency of llele T ws found between the lung cncer ptients (41.9%) nd controls (30.3%) (djusted P = 0.008; OR = 1.72; 95% CI = ). Significntly incresed lung cncer risks were suggested to be ssocited with the TT genotype of CD40-1C/T compred with the CC genotype (djusted P = 0.017; OR = 2.94; 95% CI = ). In dominnt model, compred to the CC genotype, the TT/CT genotype ws ssocited with significntly incresed risk of lung cncer (djusted P = 0.020; OR = 1.95; 95% CI = ). When the cses were ctegorized by tumor histology, we found the incidence of the CD40-1C/T polymorphisms in squmous cell crcinom cses differed significntly from tht of the controls. The CC genotype ws observed in 31.3% ptients versus 48.6% in controls. The CT genotype ws observed in 42.2% of ptients versus 37.5% of controls nd the TT genotype ws observed in 31.3% of ptients versus 9.2% of controls (djusted P in dditive model = 0.004). Compred to the CC genotype, the TT genotype significntly incresed the risk of squmous cell crcinom (djusted OR = 6.53; 95% CI = ; P = 0.002). The ssocition between the CD40-1C/T polymorphism nd the risk of lung cncer ws further exmined by grouping the subjects ccording to gender, ge nd smoking sttus (Tble Int J Clin Exp Pthol 2015;8(11):

4 Tble 3. The genotype frequencies of CD40-1C/T in different histopthology type of lung cncer Histopthology type Genotype, No. (%) Adjusted OR (95% CI) CC CT TT CC CT TT Controls 53 (48.60) 46 (42.20) 10 (9.20) Squmous 10 (31.30) 12 (37.50) 10 (31.30) (0.61~4.16) 6.53 (1.97~21.61) b Adenocrcinom 17 (38.60) 22 (50.00) 5 (11.40) (0.72~3.34) 1.72 (0.50~5.85) Smll Cell Crcinom 9 (31.03) 16 (55.20) 4 (13.79) (0.81~5.14) 2.39 (0.60~9.50) Adjusted for ge, gender nd smoking sttus. b P = ). The effect of the TT genotype on the risk of lung cncer ws sttisticlly significnt in mles (djusted OR = 4.14; 95% CI = ; P = 0.015), wheres it hd no significnt effect in femles (djusted OR = 1.39; 95% CI = ; P = 0.672) (Tble 4). When strtified ccording to medin ge, the TT genotype ws ssocited with significntly incresed risk of lung cncer in older individuls (djusted OR = 4.53; 95% CI = ; P = 0.034), nd the CT genotype hd significnt effect in younger individuls (djusted OR = 2.70; 95% CI = ; P = 0.024). However, in n dditive model, the three genotypes were similr in younger individuls (djusted OR = 1.76; 95% CI = ; P = 0.064) nd older individuls (djusted OR = 1.77; 95% CI = ; P = 0.056). With regrds to smoking, the occurrence of the TT genotype ws significntly different between the lung cncer ptients nd controls (djusted OR = 3.62; 95% CI = ; P = 0.047). Discussion Most previous studies tht exmined links between the CD40-1C>T gene polymorphism nd cncers suggested tht the T-contining genotypes could significntly increse the risk of tumors. The ssocition we observed between the polymorphism in CD40-1C>T gene nd the susceptibility to lung cncer hs not been previously reported. In this cse-control study, we found significntly different distribution of polymorphisms between the lung cncer ptients nd the controls. Notbly, the frequencies of the T-contining genotypes (TT nd TT/CT) in lung cncer ptients were much higher thn tht in the controls, indicting tht T-contining muttions my increse the risk of lung cncer, which ws consistent with previous studies. Although the mechnism underlying this ssocition remins to be elucidted, one possible explntion is tht the CD40-1C/T polymorphism chnge from C llele to T llele could cuse mjor ltertions in the initition of gene trnsltion. The -1C>T SNP plces t the 5 -untrnslted region within the Kozk consensus sequence, which is in most eukryotic mrnas nd fcilittes the binding of mrna to the smll subunit of the ribosome nd then promotes the initition of trnsltion [13, 18]. Accordingly, this SNP might ffect the trnsltion ctivity of CD40 gene, nd subsequent cell rectivity to the stimultion of cytokines nd T cells [19]. A previous study showed tht the TT genotype ws relted to lower circulting levels of soluble CD40 nd reduced CD40 expression levels on the surfces of monocyte-derived ctivted dendritic cells nd B cells [20]. Moreover, the T-contining genotypes could influence the stbility of mrna-ribosome complex, thus down-regulting CD40 expression nd incresing susceptibility to brest cncer [14]. Interestingly the CC genotype enhnced CD40 trnsltion nd hs been shown to induce the development of utoimmune diseses, such s Grve s disese [20]. In this study, we found tht the TT genotype ws ssocited with significntly incresed risk of squmous cell crcinom, suggesting tht different histopthologicl types my hve different etiologies, not only in reltion to environmentl risk fctors but genetic susceptibility. However, the smll smple size in this cse group did not llow exmintion of gene-environment interctions, nd therefore lrger studies re needed to explore this. After djusting for possible confounders (ge nd smoking sttus), we observed tht men with the TT vrint genotype hd higher risk of lung cncer, but surprisingly, we found no dditionl risk in women, even with the sme level of tobcco exposure. We lso found tht the ssocition between the CD40-1C>T vrint nd lung cncer risk ws more pronounced Int J Clin Exp Pthol 2015;8(11):

5 Tble 4. Strtifiction nlysis of the CD40-1C/T genotype frequencies in lung cncer cses nd controls Genotype, No. (%) Adjusted OR (95% CI) Vribles Cses (n = 105) Controls (n = 109) Co-dominnt model Additive model CC CT TT CC CT TT CC CT TT Gender Femle 10 (29.41) 19 (55.88) 5 (14.71) 14 (35.90) 20 (51.28) 5 (12.82) (0.48~3.93) 1.39 (0.30~6.42) 1.22 (0.59~2.54) Mle 26 (36.62) 31 (43.66) 14 (19.72) 39 (55.71) 26 (37.14) 5 (7.14) (0.89~3.84) 4.14 (1.32~13.00),e 1.97 (1.19~3.27),e Age (yer) d (26.92) 31 (59.62) 7 (13.46) 28 (46.67) 25 (41.67) 7 (11.67) (1.14~6.37) b,e 2.28 (0.64~8.11) b 1.76 (0.97~3.21) b >57 22 (41.51) 19 (35.85) 12 (22.64) 25 (51.02) 21 (42.86) 3 (6.12) (0.48~2.78) b 4.53 (1.12~18.28) b,e 1.77 (0.99~3.16) b Smoking sttus Non-smoking 13 (30.95) 21 (50.00) 8 (19.05) 21 (42.86) 22 (44.90) 6 (12.24) (0.60~4.10) c 2.20 (0.61~7.94) c 1.50 (0.80~2.79) c Smoking 23 (36.51) 29 (46.03) 11 (17.46) 32 (53.33) 24 (40.00) 4 (6.67) (0.82~3.83) c 3.62 (1.01~12.94) c,e 1.85 (1.07~3.22) c,e Adjusted for ge nd smoking sttus. b Adjusted for gender nd smoking sttus. c Adjusted for gender nd ge. d The medin of ge is 57. e P< Int J Clin Exp Pthol 2015;8(11):

6 in smokers. This finding is consistent with previous results tht the effect of two other CD40 SNPs (rs nd rs ) on nonsmll cell lung cncer (NSCLC) ws only significnt for smokers [21], indicting tht genetic susceptibility of CD40 vrints is often ssocited with smoking sttus. In conclusion, our study provides evidence tht links CD40-1C/T polymorphisms nd lung cncer susceptibility in Chinese popultion. Additionl studies re needed to better understnd the different pthwys nd fctors tht contribute to these ssocitions. With dvnces in clinicl moleculr biology, genetic informtion cn be redily utilized in clinicl decision mking. Acknowledgements This study ws supported by grnt from the Ntionl Science nd Technology Mjor Project of the Ministry of Science nd Technology of Chin (2013ZX ). Disclosure of conflict of interest None. Address correspondence to: Dr. Lihu Chen, Deprtment of Immunology, Fourth Militry Medicl University, Xi n, Chin. Tel: ; E-mil: Dr. Feng Zho, Deprtment of Respirtory Medicine, Xijing Hospitl, Fourth Militry Medicl University, Xi n, Chin. E-mil: References [1] Chen W, Zheng R, Zhng S, Zho P, Zeng H, Zou X, He J. Annul report on sttus of cncer in Chin, Chin J Cncer Res 2014; 26: [2] Rosell R, Wei J. Single nucleotide polymorphisms (SNPs) in non-smll cell lung cncer (NSCLC) ptients. Oncologist 2012; 17: [3] Elguet R, Benson MJ, de Vries VC, Wsiuk A, Guo Y, Noelle RJ. Moleculr mechnism nd function of CD40/CD40L enggement in the immune system. Immunol Rev 2009; 229: [4] Kroczek R, Hmelmnn E. T-cell costimultory molecules: optiml trgets for the tretment of llergic irwy disese with monoclonl ntibodies. J Allergy Clin Immunol 2005; 116: [5] Murugiyn G, Mrtin S, Sh B. CD40-induced countercurrent conduits for tumor escpe or elimintion? Trends Immunol 2007; 28: [6] Loskog AS, Eliopoulos AG. The Jnus fces of CD40 in cncer. Semin Immunol 2009; 21: [7] Selvrj S, Rundhl M, Ptidr A, Sh B. Anti-VEGF ntibody enhnces the ntitumor effect of CD40. Int J Cncer 2014; 135: [8] Krimi MH, Pourfthollh AA. CD40 nd tolernce induction. Irn J Allergy Asthm Immunol 2012; 11: [9] Jureck-Lubienieck B, Ploski R, Kul D, Krol A, Bednrczuk T, Kolosz Z, Tukiendorf A, Szpk-Ulczok S, Stnjek-Cichorck A, Polnsk J, Jrzb B. Assocition between ge t dignosis of Grves disese nd vrints in genes involved in immune response. PLoS One 2013; 8: e [10] Kim SH, Lee JE, Jee YK, Kim YK, Prk HS, Min KU, Prk HW. Allelic vrints of CD40 nd CD40L genes interct to promote ntibiotic-induced cutneous llergic rections. Clin Exp Allergy 2009; 39: [11] Skibol CF, Nieters A, Brcci PM, Curry JD, Agn L, Skibol DR, Hubbrd A, Becker N, Smith MT, Holly EA. A functionl TNFRSF5 gene vrint is ssocited with risk of lymphom. Blood 2008; 111: [12] Rychudhuri S, Remmers EF, Lee AT, Hckett R, Guiducci C, Burtt NP, Ginniny L, Kormn BD, Pdyukov L, Kurreemn FA, Chng M, Ctnese JJ, Ding B, Wong S, vn der Helm-vn Mil AH, Nele BM, Coblyn J, Cui J, Tk PP, Wolbink GJ, Crusius JB, vn der Horst-Bruinsm IE, Criswell LA, Amos CI, Seldin MF, Kstner DL, Ardlie KG, Alfredsson L, Costenbder KH, Altshuler D, Huizing TW, Shdick NA, Weinbltt ME, de Vries N, Worthington J, Seielstd M, Toes RE, Krlson EW, Begovich AB, Klreskog L, Gregersen PK, Dly MJ, Plenge RM. Common vrints t CD40 nd other loci confer risk of rheumtoid rthritis. Nt Genet 2008; 40: [13] Prk JH, Chng HS, Prk CS, Jng AS, Prk BL, Rhim TY, Uh ST, Kim YH, Chung IY, Shin HD. Assocition nlysis of CD40 polymorphisms with sthm nd the level of serum totl IgE. Am J Respir Crit Cre Med 2007; 175: [14] Shung C, Dlin L, Weigung Y, Zhenkun F, Fengyn X, D P, Li D. Assocition of CD40 gene polymorphisms with spordic brest cncer in Chinese Hn women of Northest Chin. PLoS One 2011; 6: e [15] Liu Y, Ling WB, Go LB, Wng YY, Zhng L. Assocition of CD40-1C/T polymorphism in the 5 -untrnslted region nd chronic obstructive Int J Clin Exp Pthol 2015;8(11):

7 pulmonry disese. Clin Chim Act 2009; 408: [16] Zhng B, Wu T, Song C, Chen M, Li H, Guo R. Assocition of CD40--1C/T polymorphism with cerebrl infrction susceptibility nd its effect on scd40l in Chinese popultion. Int Immunophrmcol 2013; 16: [17] M Y, Wng SX, Liu Y, Peng GG, Wn g XM, Zhng B, Wu BH, Yu JM. Single nucleotide polymorphism of CD40 in the 5 -untrnslted region is ssocited with ischemic stroke. Gene 2013; 529: [18] Tomer Y, Dvies TF, Greenberg DA. Wht is the contribution of Kozk SNP in the CD40 gene to Grves disese? Clin Endocrinol (Oxf) 2005; 62: 258. [19] Jcobson EM, Huber AK, Akeno N, Sivk M, Li CW, Concepcion E, Ho K, Tomer Y. A CD40 Kozk sequence polymorphism nd susceptibility to ntibody -medited utoimmune conditions: the role of CD40 tissue-specific expression. Genes Immun 2007; 8: [20] Jcobson EM, Concepcion E, Oshi T, Tomer Y. A Grves disese-ssocited Kozk sequence single-nucleotide polymorphism enhnces the efficiency of CD40 gene trnsltion: cse for trnsltionl pthophysiology. Endocrinology 2005; 146: [21] Pthk A, Wenzlff AS, Hylnd PL, Cote ML, Keele GR, Lnd S, Boulton ML, Schwrtz AG. Apoptosis-Relted Single Nucleotide Polymorphisms nd the Risk of Non-Smll Cell Lung Cncer in Women. J Cncer Ther Res 2014; Int J Clin Exp Pthol 2015;8(11):