Jing-zhe Sun, Xue-xi Yang*, Ni-ya Hu, Xin Li, Fen-xia Li, Ming Li,

1School of Biotechnology, Southern Medical University, Guangzhou 510515, China

2Department of Clinical Laboratory, the First Affiliated Hospital, Nanchang University, Nanchang 330006, China

3Da An Gene Co., Ltd. of Sun Yat-sen University, Guangzhou 510665, China

Genetic Variants in MMP9 and TCF2 Contribute to Susceptibility to Lung Cancer

Jing-zhe Sun1, Xue-xi Yang1*, Ni-ya Hu2, Xin Li1, Fen-xia Li1, Ming Li1,3**

1School of Biotechnology, Southern Medical University, Guangzhou 510515, China

2Department of Clinical Laboratory, the First Affiliated Hospital, Nanchang University, Nanchang 330006, China

3Da An Gene Co., Ltd. of Sun Yat-sen University, Guangzhou 510665, China

Objective: The Wnt signaling pathway is crucial for pulmonary development and differentiation; dysregulation of the Wnt signaling pathway may impair lung function. Indeed, single nucleotide polymorphisms (SNPs) of Wnt pathway-related genes have been suggested as risk factors for certain types of cancers. In this study, we aimed to evaluate the influence of SNPs in Wnt-related genes (TCF2,MMP9) on susceptibility to lung cancer.

Methods: Polymorphisms ofTCF2rs4430796,MMP9rs2250889, andMMP9 rs17576 were studied in Han Chinese subjects, including 135 patients with lung cancer and 176 controls, using the Sequenom MassARRAY platform. The association of genotypes with susceptibility to lung cancer was analyzed using odds ratio (OR), with 95% confidence interval (95% CI) andχ2.

Results: The three SNPs (rs4430796, rs2250889, and rs17576) were found to be significantly associated with an increased risk of lung cancer. The AA genotype and AG+AA genotype of rs4430796 showed a significantly increased susceptibility to lung cancer compared with the GG genotype (adjusted OR=6.03, 95% CI: 1.30-28.09,P=0.022; 5.55, 95% CI: 1.20-25.58,P=0.028). Compared with the rs17576 GG genotype, the AG and AG+AA genotypes were also associated with a significant risk (adjusted OR=2.65, 95% CI: 1.60-4.37,P≤0.001; 2.57, 95% CI: 1.59-4.19,P≤0.001) whereas the rs2250889 CG and CG+GG genotypes had 2.97-fold (95% CI: 1.81-4.85;P≤0.001) and 2.80-fold increased associations with lung cancer (95% CI: 1.73-4.54;P≤0.001), respectively, compared with the rs2250889 CC genotype. Furthermore, the association of rs4430796 with lung cancer became insignificant (P＞0.05) after adjusting for gender and rs2250889.

Conclusion: The three SNPs may play a role in the predisposition of members of the Han Chinese population to lung cancer.

Single nucleotide polymorphisms (SNPs);TCF2;MMP9; Susceptibility; Lung cancer

INTRODUCTION

Lung cancer is one of the most commonly diagnosed cancers worldwide and causes the highest rate of morbidity and mortality of all cancers[1]. Despite the advances in both diagnosis and treatment in the last few decades, the prognosis for someone diagnosed with lung cancer remains poor[1]. The development of lung cancer is considered as a multifaceted and polygenic event. Although smoking is the most prominent etiological factor for the development of lung cancer[2], single nucleotide polymorphisms (SNPs) have recently been identified as important factors for tumorigenesis. An increasing number of SNPs associated 111with susceptibility to lung cancer have been discovered, suggesting that SNP is an important mechanism underlying lung cancer development[3-6].

The Wnt signaling pathway is involved in regulating cell function and tumorigenesis. It also plays a critical role in pulmonary development and differentiation[7-10], and its activation is dysregulated in lung adenocarcinoma cells[11,12]. Recently, SNPs of Wnt signaling genes have been reported to correlate with the risk of cancer[13,14]. We therefore hypothesized that SNPs of the Wnt pathway-related genesTCF2andMMP9may be associated with an increased risk for the development of lung cancer.

TCF2, also known asHNF1β, is a downstream transcription activator of the Wnt signaling pathway that is widely expressed in a variety of tissues, and it is crucial in embryonic development[15,16]. It also regulates the expression of many other tissue-specific genes by binding the proximal region of the promoter sequences of genes such as alpha 1-antitrypsin[17,18], which, when overexpressed, is associated with lung cancer development[19]. Interestingly, the SNP ofTCF2is associated with renal cancer and prostatecancer[20,21], but the association with lung cancer remains unknown. Therefore, a better understanding of the polymorphism ofTCF2, particularly its contribution to lung cancer development, is needed.

As a transcriptional target of Wnt signaling through tandem LEF/TCF binding sites,MMP9, plays an important role in regulating tumor cell behaviors, including growth, differentiation, apoptosis, migration, invasion, tumor angiogenesis, and immune surveillance[22-25].MMP9overexpression is seen in lung cancer samples[26], suggesting that it may contribute to cancer development. SNPs ofMMP9have been reported to correlate with susceptibility to lung cancer[26,27], but the data are inconsistent.

The aim of the present study was to investigate whether genetic variations ofMMP9andTCF2contribute to increased risk of lung cancer development. We focused on three polymorphisms,TCF2rs4430796,MMP9rs2250889, andMMP9rs17576, to evaluate the associations between the genotypes and the risk of lung cancer development.

MATERIALS AND METHODS

Subjects

The subjects were 135 unrelated lung cancer patients and 176 controls. All the subjects were genetically Han Chinese and were recruited from patients at the First Affiliated Hospital of Nanchang University, Nanchang, China. All lung cancer patients were confirmed by pathology tests based on clinical examination, and the control group was defined by having no history of cancer. The median ages of the patients and control group were 58.3 and 59.0 years old, respectively.

Genotyping

Genomic DNA was extracted from a 200 μl peripheral blood sample using a Genomic DNA Purification Kit (Tianamp Biotech, Beijing, China) according to the manufacturer’s instructions and stored at -70°C until use. Three SNPs, rs4430796, rs17576, and rs2250889, were genotyped using SEQUENOM MassARRAY matrix-assisted laser desorption ionization-time of flight mass spectrometry platform (Sequenom, USA). Primers were designed using a semiautomated method (Assay Design3.1, Sequenom). The call rate for each assay was set at ＞90%.

Statistical Analysis

Hardy–Weinberg equilibrium (HWE) was examined using Haploview4.1. The characteristics of the subjects and the odds ratio (OR) were assessed using SPSS, version 13.0. The characteristics of the patients and the controls were compared using a two-samplet-test for continuous variables and Chi-square for categorical variable, with 95% confidence interval (95% CI). The associations between polymorphisms and lung cancer risk were estimated by computing the OR and 95% CI from binary logistic regression analyses with adjustment for age and gender. All the tests were two-sided, andP＜0.05 was considered statistically significant.

RESULTS

The demographics of the patients and controls enrolled in this study are shown in Table 1. No significant difference in age distribution was observed between the case and control groups (P=0.619). However, gender was significantly different between the two groups (P=0.011). So, age and gender adjustment were carried in the subsequent statistical analyses. The observed genotype frequencies for rs17576, rs2250889, and rs4430796 were in Hardy-Weinberg equilibrium in the control group, and thePvalues were 0.878, 0.959, and 0.656, respectively. The frequency distribution of the alleles and genotypes for the three polymorphisms is presented in Tables 2 and 3.

Table 1. Characteristics of the study population

Table 2. Summary results forTCF2SNP showing a promising association with lung cancer risk

Table 3. Summary results forMMP9SNPs showing a promising association with lung cancer risk

TCF2Polymorphism and Lung Cancer

The genotype frequencies of rs4430796 were 1.6% (GG), 38.9% (AG), and 59.5% (AA) in lung cancer patients, which were significantly different from those observed in the control group. Compared with the GG genotype, the AA and AG genotypes were associated with lung cancer (adjusted OR=6.03, 95% CI: 1.30–28.09; 4.85, 95% CI: 1.02–23.01, respectively). Under the dominant model of inheritance, the AG+AA genotype had an adjusted OR (95% CI) of 5.55 (1.20–25.58;P=0.028) as compared with the homozygous GG genotype (Table 2). The A allele frequency of rs4430796 was higher in patients with lung cancer than in controls (OR=1.50; 95% CI: 1.02–2.20), as shown in Table 2.

MMP9Polymorphism and Lung Cancer

The genotype distributions ofMMP9rs17576 and rs2250889 among lung cancer patients and healthy controls are listed in Table 3. Significant increases in the frequency of the AG genotype and A allele of rs17576 were observed in patients with lung cancer compared with controls (OR=2.65; 95% CI: 1.60-4.37; OR=1.86; 95% CI: 1.28-2.70, respectively). Under the dominant model of inheritance, we observed a significant increase in the frequency of the AG+AA genotype of rs17576 (OR=2.57; 95% CI: 1.59-4.19) in patients with lung cancer. For rs2250889, significantly increased frequencies of the CG genotype (OR=2.97; 95% CI: 1.81–4.85) and of the G allele (OR=2.01; 95% CI: 1.35–2.99) were observed in the patients with lung cancer. Under the dominant model of inheritance, the CG+GG genotype correlated with an increased risk for lung cancer (OR=2.80; 95% CI: 1.73-4.54).

Linkage Disequilibrium and Interactions Analysis inTCF2andMMP9Gene Polymorphisms

We used Haploview 4.1 to calculate the pairwiser2between SNPs. TheMMP9rs2250889 polymorphism was in linkage disequilibrium (LD) with rs17576 (r2=0.59). TheTCF2rs4430796 was not in LD withMMP9rs2250889 (r2=0). Statistical differences in SNP–SNP interactions betweenTCF2andMMP9were examined (Table 4). The relationship between rs4430796 and the risk of lung cancer became insignificant when rs2250889 was considered as a covariable.

Table 4. Multivariate logistic regression analysis for significant SNPs

DISCUSSION

SNPs are the most common inherited genetic markers, accounting for 90% of heterogeneity among individuals[28]. Recent reports have emphasized that some SNPs of Wnt signaling genes are related to the risk of cancer[13,14]. In thisstudy, we investigated the effects of three SNPs in two Wnt signaling genes on the development of lung cancer, including one inTCF2(rs4430796) and two inMMP9(rs2250889 and rs17576). The results indicated that rs4430796, rs2250889, and rs17576 were significantly associated with the occurrence of lung cancer.

TCF2rs4430796 lies in intron 2 of theTCF2gene; an A-to-G substitution in this region had no obvious effect on the expression of protein[21]. Recently, some papers have reported that rs4430796 is associated with prostate cancer[21,29]and type 2 diabetes[30]. In the present study, we show an association between rs4430796 and susceptibility to lung cancer. The location of rs17576 is in the catalytic domain ofMMP9, particularly in the fibronectin type II domain, which plays important roles in substrate binding[31]. For the homozygous genotype (A/A) of rs17576, Wu, et al. found a significantly increased risk for esophageal squamous cell carcinoma (ESCC) (OR=1.70, 95% CI: 0.62–4.66)[32]; Jin, et al. reported a higher risk of death for non-small-cell lung cancer(NSCLC) (adjusted HR=1.62, 95% CI: 1.08–2.44)[27]. Our results also showed that A, as a risk allele of rs17576, increased the risk for lung cancer. The finding that the SNP rs17576 has a potential effect on the activity of this enzyme suggests a possible mechanism for the development or predisposition to lung cancer[26]. The rs2250889 is located in the haemopexin-like domain, which is an important site for binding with a metalloproteinase inhibitor[33]. The effect of the SNP rs2250889 in these functional domains has not been fully determined. Wu, et al. found that the rs2250889 GG genotype was associated with a significantly increased risk for ESCC (OR=4.08, 95% CI: 1.58–10.52)[32]. Our study indicated that G, as a risk allele of rs2250889, increased the risk for lung cancer. However, Hu, et al. found that rs17576 G and rs2250889 C as the risk alleles significantly increased the risk of lung cancer[26]. The differences in outcomes between studies may be due to differences in the ethnicity of the patients studied.

Wu, et al. reported thatMMP9is a transcriptional target of the Wnt pathway and that the β-catenin/LEF/TCF transcription factor complex can regulate the expression ofMMP9by binding its proximal promoter sequences[22]. From multivariate logistic regression analyses, we found that the effect ofTCF2rs4430796 became insignificant for the risk for lung cancer whenMMP9rs2250889 was considered as a covariate. LD was not found betweenMMP9rs2250889 andTCF2rs4430796; however, we did find an association between the two SNPs. The association between the genes caused by polymorphism may be related to the complex regulation of the signaling pathway, but the exact mechanism needs to be studied further.

In conclusion, the current study provides evidence thatTCF2rs4430796,MMP9rs2250889, andMMP9 rs17576 may be associated with susceptibility to lung cancer, and we report an association between the rs4430796 SNP and susceptibility to lung cancer. Our findings are an important addition to previously published work on the association between the Wnt pathway and lung cancer susceptibility. Further validation with meta-analysis, functional studies of genetic variants, and mechanical studies of the association between rs4430796 and rs2250889 are necessary.

REFERENCES

1. Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002. CA Cancer J Clin 2005; 55:74-108.

2. Alberg AJ, Samet JM. Epidemiology of lung cancer. Chest 2003; 123(1 Suppl):21S-49S.

3. Grimminger PP, St?hlmacher J, Vallb?hmer D, et al. Prognostic Significance and Clinicopathological Associations of COX-2 SNP in Patients with Nonsmall Cell Lung Cancer. J Oncol 2009; 2009:139590.

4. Crosbie PA, McGown G, Thorncroft MR, et al. Association between lung cancer risk and single nucleotide polymorphisms in the first intron and codon 178 of the DNA repair gene, O6-alkylguanine-DNA alkyltransferase. Int J Cancer 2008; 122:791-5.

5. Yin Z, Su M, Li X, et al. ERCC2, ERCC1 polymorphisms and haplotypes, cooking oil fume and lung adenocarcinoma risk in Chinese non-smoking females. J Exp Clin Cancer Res 2009; 28:153.

6. Kiyohara C, Horiuchi T, Takayama K, et al. IL1B rs1143634 polymorphism, cigarette smoking, alcohol use, and lung cancer risk in a Japanese population. J Thorac Oncol 2010; 5:299-304.

7. Daniel VC, Peacock CD, Watkins DN. Developmental signaling pathways in lung cancer. Respirology 2006; 11:234-40.

8. Reynolds SD, Zemke AC, Giangreco A, et al. Conditional stabilization of beta-catenin expands the pool of lung stem cells. Stem Cells 2008; 26: 1337-46.

9. Niehrs C. Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene 2006; 25:7469-81.

10. Pishvaian MJ, Byers SW. Biomarkers of Wnt signaling. Cancer Biomark 2007; 3:263-74.

11. Nguyen DX, Chiang AC, Zhang XH, et al. WNT/TCF signaling through LEF1 and HOXB9 mediates lung adenocarcinoma metastasis. Cell 2009; 138:51-62.

12. Akiri G, Cherian MM, Vijayakumar S, et al. Wnt pathway aberrations including autocrine Wnt activation occur at high frequency in human non-small-cell lung carcinoma. Oncogene 2009; 28:2163-72.

13. Pan KF, Liu WG, Zhang L, et al. Mutations in components of the Wnt signaling pathway in gastric cancer. World J Gastroenterol 2008; 14: 1570-4.

14. Hirata H, Hinoda Y, Nakajima K, et al. Wnt antagonist gene polymorphisms and renal cancer. Cancer 2009; 115:4488-503.

15. Roose J, Clevers H. TCF transcription factors: molecular switches in carcinogenesis. Biochim Biophys Acta 1999; 1424:M23-M37.

16. Ulinski T, Bensman A, Lescure S. Abnormalities of hepatocyte nuclear factor (HNF)-1beta: biological mechanisms, phenotypes, and clinical consequences. Arch Pediatr 2009; 16:1049-56.

17. Terasawa K, Toyota M, Sagae S, et al. Epigenetic inactivation of TCF2 in ovarian cancer and various cancer cell lines. Br J Cancer 2006; 94: 914-21.

18. Hu C, Perlmutter DH. Cell-specific involvement of HNF-1beta in alpha (1)-antitrypsin gene expression in human respiratory epithelial cells. Am J Physiol Lung Cell Mol Physiol 2002; 282:L757-65.

19. Topic AS, Jelic-Ivanovic ZD, Spasojevic-Kalimanovska VV, Spasic SM. Association of moderate alpha-1 antitrypsin deficiency with lung cancer in the Serbian population. Arch Med Res 2006; 37:866-70.

20. Rebouissou S, Vasiliu V, Thomas C, et al. Germline hepatocyte nuclear factor 1alpha and 1beta mutations in renal cell carcinomas. Hum Mol Genet 2005; 14:603-14.

21. Levin AM, Machiela MJ, Zuhlke KA, et al. Chromosome 17q12 variants contribute to risk of early-onset prostate cancer. Cancer Res 2008; 68: 6492-5.

22. Wu B, Crampton SP, Hughes CC. Wnt signaling induces matrixmetalloproteinase expression and regulates T cell transmigration. Immunity 2007; 26:227-39.

23. Sheu BC, Hsu SM, Ho HN, et al. A novel role of metalloproteinase in cancer-mediated immunosuppression. Cancer Res 2001; 61:237-42.

24. Bergers G, Brekken R, McMahon G, et al. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol 2000; 2:737-44.

25. Yu Q, Stamenkovic I. Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Genes Dev 2000; 14:163-76.

26. Hu Z, Huo X, Lu D, et al. Functional polymorphisms of matrix metalloproteinase-9 are associated with risk of occurrence and metastasis of lung cancer. Clin Cancer Res 2005; 11:5433-9.

27. Jin G, Miao R, Hu Z, et al. Putative functional polymorphisms of MMP9 predict survival of NSCLC in a Chinese population. Int J Cancer 2009; 124:2172-8.

28. Tost J, Gut IG. Genotyping single nucleotide polymorphisms by MALDI mass spectrometry in clinical applications. Clin Biochem 2005; 38: 335-50.

29. Sun J, Zheng SL, Wiklund F, et al. Evidence for two independent prostate cancer risk-associated loci in the HNF1B gene at 17q12. Nat Genet 2008; 40:1153-5.

30. Wang C, Hu C, Zhang R, et al. Common variants of hepatocyte nuclear factor 1beta are associated with type 2 diabetes in a Chinese population. Diabetes 2009; 58:1023-7.

31. Zhang B, Henney A, Eriksson P, et al. Genetic variation at the matrix metalloproteinase-9 locus on chromosome 20q12.2-13.1. Hum Genet 1999; 105:418-23.

32. Wu J, Zhang L, Luo H, et al. Association of matrix metalloproteinases-9 gene polymorphisms with genetic susceptibility to esophageal squamous cell carcinoma. DNA Cell Biol 2008; 27:553-7.

33. Gohlke U, Gomis-Rüth FX, Crabbe T, et al. The C-terminal (haemopexin-like) domain structure of human gelatinase A (MMP2): structural implications for its function. FEBS Lett 1996; 378:126-30.

10.1007/s11670-011-0183-3

2011-02-19; Accepted 2011-05-17

This work was supported by the Key Programs for Science and Technology Development of Guangzhou (No. 2008A1-E4151), the National “863” High Technology Research and Development Program of China (No. 2006AA02A311).

*Contributed equally to this study.

**Corresponding author.

E-mail: mingli2006_2006@126.com

? Chinese Anti-Cancer Association and Springer-Verlag Berlin Heidelberg 2011