Chinese-German Journal of Clinical Oncology

May 2009, Vol. 8, No. 5, P269–P272

DOI 10.1007/s10330-009-0054-2

Vascular endothelial growth factor polymorphisms and risk of lung cancer* Jing Liang, Xinshuang Yu, Xiaolin Liu, Dianshui Sun, Hairong Liu, Wei Hu, Aizhong Qu, Yan Li Cancer Diagnosis and Therapy Center, Qianfoshan Hospital in Shandong, Jinan 250014, China Received: 24 March 2009 / Revised: 3 April 2009 / Accepted: 17 April 2009 Abstract Objective: We investigated the potential association between vascular endothelial growth factor (VEGF) polymorphisms and the risk of lung cancer. Methods: In the case-control study, we used PCR-RFLP technique to determine two VEGF genotypes –2578C/A and 936C/T in 171 lung cancer patients and 172 healthy controls for conformation, and constructed haplotypes of the two gene sites by PHASE1.0 software. Unconditional logistic regression model was used to analyze the statistical association of genontypes or haplotypes in the two groups adjusted by gender and age. Results: Compared with at least one –2578A allele, individuals with –2578CC genotype found associated with a significantly decreased risk of lung cancer [P=0.001; adjusted odds ratio (OR), 0.391; 95% confidence interval (95% CI), 0.226–0.686]. Analyses stratified by gender showed that the combined –2578 CA and AA genotype were also associated with a significantly decreased risk of lung cancer. (P = 0.016; OR = 0.303; 95% CI = 0.153–0.601 and P = 0.018; OR = 0.547; 95% CI = 0.331–0.903, respectively). The distribution of the two haplotypes (936C/–2578C and 936C/–2578A) were significantly different between case-and -control groups (P = 0.016, OR = 0.317, 95% CI = 0.124–0.809 and P = 0.018, OR = 0.547, 95% CI = 0.331–0.903). Analyses categorized by tumor histology showed that Haplotype C-C was associated with a significantly decreased risk of adenocarcinoma compared with the reference haplotypes. (P = 0.004; OR = 0.237; 95% CI = 0.090–0.627). Conclusion: These results suggest that the VEGF polymorphisms may be a critical factor for the risk of lung cancer. Key words

lung neoplasms; vascular endothelial growth factor; risk factor; genetic polymorphism

There is substantial experimental evidence that angiogenesis is an essential process in the development, growth, and metastasis of malignant tumors, so is lymphatic metastasis of tumor. Many researches have confirmed that vascular endothelial growth factor (VEGF) is tightly associated with angiogenesis and lymphatic metastasis of tumors. The gene encoding VEGF is located on chromosome 6p21.3 and comprises a 28 kb region with 7 introns and 8 exons. Recently, several studies have investigated the impact of VEGF polymorphisms (–460T > C, –405C > G, –936C > T) on risk of lung cancer [1]. Rare reports have been made about the association of VEGF polymorphisms and risk of lung cancer in China. In this report, we described a case-control study of lung cancer in a Chinese population to explore the empact of genetic polymorphisms of the gene on host susceptibility to lung cancer, and provide theoretical basis or lung cancer prevention.

Correspondence to: Xinshuang Yu. Email: [email protected] * Supported by a grant from the Shandong Provincal Natural Sciences Foundation (No. 2005ZX04).

Materials and methods Sample source We used strict histopathologically case-controlled study method. All incident patients were consecutively recruited from Shandong Qianfoshan Hospital, Qilu Hospital and Shandong Tumor Hospital from 2005 to 2006. The diagnosis of PLC was all determined by histopathology analysis. The average age of all 171 cases was 57.5 ± 11.0 years, included 121 males and 50 females. Controls were randomly selected from a pool of healthy subjects who visited the general health check-up center during the same period. The average age of all 171 controls was 56.9 ± 13.8 years, included 108 males and 64 females. There were no differences by age and gender between case and control groups. Two mL peripheral blood was extracted from cases and controls and stored at –20 ć after EDTA anticoagulant. DNA extraction Genomic DNA was extracted from peripheral blood lymphocytes of all 343 people by proteinase K digestion and phenol–chloroform extraction. The extracted DNA was purified by Glass milk precipitation method.

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Polymorphic sites selection and primers designing We searched the whole gene codes of VEGF from Gene Bank (Gene Bank accession number AF437895). PCR primers, designed by Primer 5.0 software, were based on the Gene Bank reference sequence (Gene Bank accession number AF437895). Primers were synthetize by Shennengbocai Biological Technological Limited Corporation of Shanghai (China). PCR reaction system and conditions process The PCR reaction conditions were determined as follows: 40.5 μL ddH2O, 5 μL 10*PCR buffer (contains 1.5 mmol/L Mg2+), 10 mmol/L dNTPs, 0.5 μL (5 U/μL) of Taq polymerase, 1 μL of Upstream and downstream primers separately, 1 μL DNA template (500 ng). PCR cycle were 30 cycles of amplification reactions, which were consisted of an initial denaturation step at 94 ć for 2 min, followed by of annealing for 40 s at 64.2 ć for –2578 site, denaturation 40 s at 64.5ć for 936 site, and extension at 72 ć for 40 s, and a final elongation at 72 ć for 10 min.

England BioLabs), PCR reactions were done in a 20 μL reaction volume containing 1 μg genomic DNA, 5 U Endonuclease, 2 μL restriction enzymes 10*Buffer, supplied with ddH2O to final 20 μL, all of which were in bain-marie at 37 ć for 3 min. At last, the products were separated on a 2% agarose gel to determine the genotypes by electrophoresis strip bp numbers. Statistical analysis The age distribution between cases and controls were compared using Mann-Whitney-test and the chi-square test for gender variables. The allele frequencies of VEGF were tested for the Hardy-Weinberg equilibrium Haplo-

PFLP method to determine VEGF genotypes in lung cancer group After PCR product was purified, the 2578, 936 bp of VEGF gene was digested with BglII and Nla III (New Table 1

Patient characteristics

Variable

Patients (%)

Controls (%)

Mean age Gender Male Female Smoking Yes No

57.5 ± 11.0

56.9 ± 13.8

121 (70.76) 50 (29.24)

108 (62.79) 64 (37.21)

83 (48.54) 88 (51.46)

37 (21.51) 135 (78.49)

Fig. 1 Product of the VEGF gene–2578 C/A polymorphism showed on agarose electrophoresis. M: marker, 100 bp ladder

Table 2 The association of VEGF 936C/T and –2578 C/A polymorphisms and lung cancer risk Genotype 936C/T CC CT TT CT+TT –2578 C/A CC CA AA CA + AA

Patients Controls

P*

OR (95%CI)

123 40 8 48

131 38 3 41

0.205 0.250 0.248

1.439 (0.819–2.526) 2.316 (0.552–9.720) 1.362 (0.807–2.298)

129 28 14 42

112 56 4 60

0.001 0.059 0.029

0.397 (0.226–0.698) 3.198 (0.958–10.675) 0.571 (0.345–0.944)

* Unconditional Logistic regression model adjusted by gender, age, smoking, the genotype C/C as the reference group

Fig. 2 Product of the VEGF gene 936 C/T polymorphism shown on agarose electrophoresis. M: marker, 100 bp ladder

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Table 3 The stratified analysis of lung cancer risk associated with VEGF genotypes 936 genotype –2578 genotype P * value P * value CC CT + TT OR (95%CI) CC CA + AA OR (95%CI) Smoking Yes 62/25** 21/12 0.834 (0.226–3.225) 0.816 65/23 18/14 0.356 (0.089–1.421) 0.144 No 61/106 27/29 1.579 (0.836–2.982) 0.159 64/89 24/46 0.799 (0.431–1.483) 0.478 Histological type Adenocarcinoma 61/131 26/41 1.330 (0.730–2.424) 0.351 64/112 23/60 0.659 (0.365–1.189) 0.166 SCC 41/131 19/41 2.111 (0.937–4.784) 0.073 44/112 16/60 0.532 (0.0.237–1.192) 0.125 17/131 1/41 0.226 (0.028–1.804) 0.160 15/112 3/60 0.454 (0.121–1.701) 0.242 SCLC# Gender Male 88/84 33/24 1.826 (0.904–3.689) 0.093 95/67 26/41 0.303 (0.153–0.601) 0.001 Female 35/47 15/17 0.956 (0.384–2.381) 0.922 34/45 16/19 1.098 (0.452–2.665) 0.836 * Unconditional Logistic regression model adjusted by gender, age, smoking, the genotype C/C as the reference group; ** Number of cases/number of controls; # Other 4 cases were too little to be considered Table 4

Distribution of VEGF 936C/T and –2578 C/A haplotypes among cases and controls Haplotype C-C C-A T-A N% OR (95%CI) N% OR (95%CI) N% OR (95%CI) 59/113 21/151 Controls 165/7# Cases 152/19 0.317 (0.124–0.809)* 38/133 0.547 (0.331–0.903)* 17/154 0.716 (0.352–1.456) SCC 56/4 0.601 (0.137–2.636) 14/46 0.560 (0.257–1.219) 10/50 1.325 (0.515–3.410) Adenoca73/14 0.237 (0.090–0.627)* 21/66 0.625 (0.344–1.136) 6/81 2.488 (0.185–1.283) rcinoma SCLC 17/1 0.622 (0.067–5.779) 3/15 0.336 (0.099–1.349) 1/17 0.378 (0.046–3.092)

T-C OR* (95%CI)

N% 41/131 42/129 15/45 25/62

1.050 (0.625–1.765) 1.243 (0.558–2.771) 1.180 (0.649–2.146)

0/18

–

* Unconditional Logistic regression model adjusted by gender, age, smoking; # Number of haplotype in the analysis/number of any other haplotypes

types and their frequencies were estimated using Phase 1.0 program. Unconditional logistic regression analysis was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs), with adjustment for possible confounders (gender, age, etc). All reported P values were based on two-sided tests. P < 0.05 were considered statistically significant. All analyses were performed using SPSS for Windows, version 10.0.

2).

Results

Distribution of VEGF –2578 and 936 sites and the relationship with lung cancer risk As shown in Table 2, there was no significant difference of the –936 genotypes distribution between cases and controls. –2578 genotype polymorphism was separated into two groups for comparison: (1) C/C genotype, (2) C/C and C/A genotypes. The distributions of C/C and C/A were significantly different from C/A genotype. When the cases were categorized by smoking history or tumor histology, there was no significant difference of the two genotypes between two groups; Analyses stratified by gender showed that the combined –2578 CA and AA genotype were also associated with a significantly decreased risk of lung cancer (Table 3).

Mann-Whitney test The demographics of the cases and controls enrolled in this study were shown in Table 1. There were no significant differences in the mean age and gender distribution between cases and controls (P = 0.656; P = 0.117). VEGF gene sites electrophoresis –2578 gene site was observed of two alleles (C A) and three genotypes (CC CA AA). AA genotype has two bands (248 bp, 152 bp), CA have three bands (400 bp, 248 bp, 152 bp). CC genotype have only one band (400 bp) (Fig. 1). –936 gene site was observed of two alleles (C T) and three genotypes (CC CT TT). TT genotype has two bands (264 bp, 137 bp), CT have three bands (401 bp, 264 bp, 137 bp); TT genotype have only one band (401 bp; Fig.

Hardy-weinberg equilibrium test As the polymorphism sites of both 171 lung cancer patients and 172 subjects were clear, the haplotype distributions of either VEGF –2578 or 936 site among cases and controls were in Hardy--Weinberg equilibrium (P > 0.05).

Distribution of haplotypes of VEGF –2578 and

272

936 sites in cases and controls As shown in Table 4, there were four haplotypes (C-C, C-A, T-C, T-A respectively) of VEGF 936C/T and 2578C/ A polymorphisms. The distribution of the two haplotypes (936C/–2578C and 936C/–2578A) were significantly different between case-and -control groups (P = 0.016, OR = 0.317, 95% CI, 0.124–0.809 and P = 0.018, OR = 0.547, 95% CI 0.331–0.903). Analyses categorized by tumor histology showed that Haplotype C-C was associated with a significantly decreased risk of adenocarcinoma compared with the reference haplotypes (P = 0.004; OR, 0.237; 95% CI, 0.090–0.627).

Discussion Although cigarette smoking is the major cause of lung cancer, only a small fraction of smokers develop this disease, which suggests that genetic factors contribute to lung cancer risk. VEGF receptors are expressed on various kinds of cells such as megalokaryocytes, monocytes etc. Only those express on endothelium have the ability to response to VEGF [2, 3]. The main function of VEGF are as follows: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen. It promotes vascular permeability during many pathological processes, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis and psoriasis. When VEGF combine with its receptor, it can pathologic induce proliferation of endothelium and promote inappropriate angiogenesis, thus the establishment of vascular nets can furnish tumor growth. VEGF-C is a specific induction for angiogenesis [4] and lymphangiogenesis. VEGF can alter gene expression of endothelium, induce Interstitial Collagenase, plasminogen activator genesis for matrix deregulation, which can make ease vasogenesis more and help cancer cells dropped from tissues. The VEGF-induced alternation of coded protein activity and the production can affect tumor angiogenesis. Thus contribute to genetic variation to lung cancer among individuals [5]. Recently, many polymorphic gene sites was found consequently [5–7], some of these are associated with the ability to produceVEGF protein. But these are not consenous yet. Renner et al [7] have reported 936C/T allele was associated with low titer; of VEGF in the plasma, and also associated with low risk of breast cancer. Jacobs et al [8] suggested limited support for the hypothesis that the –2578C and –1154G VEGF alleles are associated with increased risk for invasive breast cancer in American postmenopausal women. Another research about breast cancer from Poland and Germany demonstrated that the –2578AA genotype and the –2578/–634 AG haplotype were significantly associated with low histologic grade

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tumors (P = 0.04) [9]. Some VEGF polymorphisms were significantly associated with low expression of VEGF and low angiogenesis, which assessed genetic variation is the major cause for degradation of VEGF [10]. Our investigation showed that The 936 genotypes were not associated with risk of lung cancer, Compared with at least one –2578A allele, Individuals with –2578CC genotype were associated with a significantly decreased risk of lung cancer. Lee et al (Korea) showed that the 936 CT genotype and the variable genotypes (936 CT and combined 936 CT and TT genotype) were also associated with a significantly decreased risk of PLC compared with the 936 CC genotype, and the reason was still unknown, which may related with the sample size we studied, the specifically of carcinogen contacted and genetic background varility between ethnic groups. Further studies are needed to clarify the association between the VEGF polymorphism and lung cancer in diverse ethnic populations.

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