GENETIC TESTING AND MOLECULAR BIOMARKERS Volume 19, Number 3, 2015 ª Mary Ann Liebert, Inc. Pp. 133–137 DOI: 10.1089/gtmb.2014.0259
VEGF Gene Polymorphisms and Susceptibility to Colorectal Cancer ¨ zhan,1 Hakan Teoman Yanar,2 and Buket Alpertunga1 Aysxe Tarbın Jannuzzi,1 Gu¨l O
Aims: Colorectal cancer (CRC) is the third most common cancer in the world and its etiology involves the interaction of genetic and environmental factors. New blood vessels form through a process called angiogenesis and have an essential role in tumor growth, progression, and metastasis of malignant tumors. The vascular endothelial growth factor (VEGF), one of the most important angiogenic factors, is a specific mitogen for vascular endothelial cells. In the present case–control study, we carried out the study to evaluate whether the VEGF single-nucleotide polymorphisms play a role in modulating susceptibility to CRC. Methods: We evaluated the VEGF - 2578A > C, + 936C > T, and - 460C > T genotypes obtained from 103 patients with CRC and 129 healthy controls by using the polymerase chain reaction–restriction fragment length polymorphism (PCRRFLP) assay. Also, haplotype analysis was determined. Odds ratios (ORs) and 95% confidence intervals (CI) were estimated. Results: - 2578A > C was significantly associated with CRC risk (OR 1.81; 95% CI 0.94–3.47; p = 0.0495), while distribution of + 936C > T and - 460C > T genotypes in cases and controls did not significantly differ. The VEGF A2578-T936-T460 haplotype might be associated with the development of CRC (OR 8.77; 95% CI 1.05–73.36; p = 0.0434). There was significant haplotype effect for all eight haplotypes ( p = 0.02). Conclusions: These results suggest that the VEGF polymorphisms might play a role in the development of CRC. Therefore, the VEGF polymorphisms might be further investigated to use in the determination of risk factors for CRC and to have a predictive value for anti-VEGF-targeted cancer therapies.
Introduction
C
olorectal cancer (CRC) is one of the most frequent tumors and the main reason for the high mortality ratio among different types of cancer sufferers in developed countries (Parkin et al., 2005; Ferlay et al., 2013; American Cancer Society, 2014). During recent years, there have been many studies to define the biological profile of CRC to improve early diagnosis and the prognostic stratification, and eventually find a cure (Pokorny et al., 2000; Crawford et al., 2003). However, the biological factors involved in the development of CRC are not yet clear (Petrova et al., 2008; Campa et al., 2012). Angiogenesis, the formation of a new blood vessel from endothelial precursors, is a prerequisite for the development, growth, and progression of solid malignancies (Takahashi et al., 1995; Yancopoulos et al., 2000). Indeed, it is generally supposed that microvessel formation around tumors is stimulated by various angiogenic factors secreted by the tumor cells (Hanahan and Folkman, 1996; Yancopoulos et al., 2000). The vascular endothelial growth factor (VEGF) is considered one of the strongest promoters of tumor angiogenesis 1 2
and has become a potential target for cancer therapy (Takahashi et al., 1995; Carmeliet and Jain, 2000). There are some studies related to the role of VEGF in tumor angiogenesis. For instance, Bevacizumab (Avastin), a neutralizing monoclonal antibody to VEGF, was the first antiangiogenic agent that was approved for the treatment of CRC (Roy et al., 2006; Loupakis et al., 2013; Morita et al., 2013). The VEGF gene is located on chromosome 6p21.3 and consists of eight exons that exhibit alternative splicing to form a family of proteins (Vincenti et al., 1996; Watson et al., 2000). Several polymorphisms have been associated with variations in VEGF protein production and reported to be involved in the development of several malignancies, including breast (Toi et al., 1994), gastrointestinal (Brown et al., 1993a), urinary tract (Brown et al., 1993b), and ovarian (Olson et al., 1994) tumors, and autoimmune diseases (Han et al., 2004; Salvarani et al., 2004). The VEGF + 936C > T (rs3025039) in the 3¢ untranslated region and the VEGF - 2578A > C (rs699947) and - 460C > T (rs833061) in the promoter region have been associated with altered levels of VEGF protein. Three VEGF variants are very common and functional single-nucleotide polymorphisms (SNPs). The
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey. Department of General Surgery, Faculty of Medicine, Istanbul University, Istanbul, Turkey.
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- 2578A > C and - 460C > T genotypes appear to be associated with a higher VEGF expression, whereas the 936T allele is correlated to a lower VEGF expression. They are also located in close proximity region in the VEGF gene, in which a high degree of linkage disequilibrium exists and haplotype effects might therefore be a possibility (Renner et al., 2000; Krippl et al., 2003; Hofmann et al., 2008; Dassoulas et al., 2009; Hansen et al., 2010a; Antonacopoulou et al., 2012). Although it has been indicated that VEGF could be a good marker for determining the risk of developing CRC and could be used as a therapeutic target for new molecular anticancer drugs (Hanrahan et al., 2003; Fernando and Hurwitz, 2004; Dassoulas et al., 2009; Vidaurreta et al., 2010), the data remain conflicting if VEGF gene polymorphisms are associated with the prognosis of CRC (Yamamori et al., 2004; Park et al., 2007; Bae et al., 2008; Maltese et al., 2009; Wu et al., 2009; Hansen et al., 2010b; Antonacopoulou et al., 2012; Jang et al., 2013b). Therefore, to test this hypothesis, we investigated possible associations between genetic variations at the - 2578A > C, + 936C > T, and - 460C > T polymorphic site in the VEGF gene, in patients who have had CRC compared to healthy individuals. Materials and Methods
We carried out a case–control study to examine the association between the VEGF genotypes and the susceptibility to CRC in the Turkish population. Genomic DNA samples were obtained from 103 patients with CRC and 129 ethnic-, gender-, and age-matched healthy volunteers at the Hospital of Istanbul University and Bagcilar Training and Research Hospital between 2011 and 2013. The criteria for diagnosis of CRC were as follows: a clinical history consistent with the disease, positive colonoscopic results, and routine laboratory analysis parameters. The histopathological examinations were evaluated according to the established clinical criteria (Compton and Gleene, 2004). Controls were hospital patients with various diagnoses (eye diseases, pulmonary diseases, cardiovascular diseases, neurological disorders, etc.) who have never had cancer. Written informed consent was obtained from all participants, and the study was approved by the Ethics Committee of Istanbul University (2011/87-555). For all subjects, we collected data about their ethnicity, smoking status, gender, age, and body–mass index (BMI, kg/ cm2) by in-person interview. The smoking status was categorized into two groups: nonsmokers (never) and smokers (former and current). A person who had smoked at least once
a day for > 1 year in his or her lifetime was regarded as a smoker. Patients showed no significant different gender (47 women and 56 men vs. 81 women and 48 men) and age (mean age 52.66 – 15.65 years vs. mean age 43.88 – 16.43 years) and BMI (26.64 – 5.24 kg/cm2 vs. 26.64 – 4.86 kg/cm2) compared with the control subjects. As smoking status, 49% of cases and 56% control subjects were smokers. Genotyping of - 2578A > C, + 936C > T, and - 460C > T variants was performed by polymerase chain reaction– restriction fragment length polymorphism (PCR-RFLP) methods (Table 1). The temperature was controlled by a programable heat block (Applied Biosystems Gene Amp PCR System 9700). Restriction enzymes were obtained from New England Biolabs and Fermentas. All other molecular biology chemicals were obtained from Fermentas and SigmaAldrich. For each polymorphism, 10% of the samples were randomly chosen and genotyped twice to assure the results and the concordance was 100%. The Hardy–Weinberg equilibrium analysis was performed to compare the observed and expected genotype frequencies among patients and control subjects by using the w2 test. To evaluate the association between the VEGF genotype frequencies and CRC, odds ratios (ORs) and 95% confidence interval (95% CI) were estimated. The haplotypes and their frequencies were estimated by PHASE (Version 2) (Stephens and Donnelly, 2003). All statistical analyses were performed using SPSS software (Version 17). The distribution of haplotypes in the cases and controls was compared by the v2 test. A two-sided p-value < 0.05 was considered to be statistically significant. Results
In the study, there were no significant differences between patients with CRC and healthy volunteers for age and sex distribution, and this suggested that the matching based on these two variables was adequate. There was no interaction between VEGF polymorphisms and smoking status in relation to CRC risk (results not shown). The genotype distributions did not significantly deviate from the Hardy–Weinberg equilibrium in cases and controls for any of the examined SNPs (Table 2). In the distribution of genotypes, the VEGF - 2578C, + 936T, and - 460C recessive allele frequencies were 0.379, 0.131, and 0.413 in cases compared with 0.488, 0.155, and 0.442 in controls, respectively. The - 2578AC genotype was statistically significantly associated with risk of developing CRC. The association was
Table 1. Details of Methodology Used in Genotyping
VEGF polymorphisms - 2578A > C (rs699947) + 936C > T (rs3025039) - 460T > C (rs833061)
Primer sequences 5¢-ggCCTTAggACACCATACC-3¢ 5¢-CACAgCTTCTCCCCTATCC-3¢ 5¢-AggAAgAgggACTCTgCgCAgAgC-3¢ 5¢-TAAATgTATgTATgTgggTgggTgTg TCTACAgg-3¢ 5¢-TgTgCgTgTggggTTgAgCg-3¢ 5¢-TACgTgCggACAgggCCTgA-3¢
bp, base pair; VEGF, vascular endothelial growth factor.
Restriction enzymes/fragment lengths (bp)
Annealing temperatures (C)
BstYI/455, 206, 249
52.1
NlaIII/208, 122, 86
64.5
BstUI/174, 154, 20
60.7
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Table 2. Allele Frequencies of Vascular Endothelial Growth Factor Polymorphisms in Patients with Colorectal Cancer and Healthy Subjects Frequencies VEGF polymorphisms
Genotypes
Cases (n = 103)
Controls (n = 129)
AA AC CC CC CT TT TT TC CC
17 44 42 78 23 2 19 83 1
34 64 31 93 32 4 15 114 0
- 2578A > C (rs699947) + 936C > T (rs3025039) - 460T > C (rs833061)
OR (95% CI)
p-Value
AA vs. any C 1.81 (0.94–3.47)
0.0495
CC vs. T 0.83 (0.46–1.50)
0.3183
TT vs. C 0.58 (0.28–1.21)
0.1019
CT, confidence interval; OR, odds ratio.
seen with VEGF - 2578A > C ( p = 0.0495); specifically, patients carrying the A allele in comparison to patients carrying the C allele had a significantly higher risk of disease (OR 1.81; 95% CI 0.94–3.47) (Table 2). To confirm the genotyping results for - 2578A > C, which has association with CRC, the selected PCR-amplified DNA samples (n = 6, for each genotype in both cases and controls) were examined by DNA sequencing, and the results were also 100% concordant. On the other hand, the other SNPs were not associated with the development of CRC, at least in the Turkish population (Table 2). In developing the risk of CRC, the results of haplotypebased analysis are shown in Table 3. There was a significant haplotype effect for all of haplotypes ( p = 0.02). Eight haplotypes were found, with the VEGF haplotype C2578-C936T460 being the most common. VEGF haplotype A2578-T936T460 might be associated with the development of CRC (OR 8.77; 95% CI 1.05–73.36; p = 0.0434). Discussion
In the present study, the VEGF - 2578A > C SNP and A2578-T936-T460 haplotype were associated with the risk of CRC. However, the distributions of VEGF + 936C > T and - 460C > T genotypes in cases and controls did not significantly differ as was also true for the other VEGF haplotypes. The present findings are consistent with the results from
some previous studies (Chae et al., 2008; Hoffmann et al., 2008; Kim et al., 2008; Dassoulas et al., 2009; Maltese et al., 2009; Wu et al., 2009; Antonacopoulou et al., 2012; Jang et al., 2013a). Some studies concluded that - 2578A > C gene polymorphism was not associated with individual susceptibility to CRC (Hoffmann et al., 2008; Dassoulas et al., 2009), whereas Maltese et al. (2009) found a reduced risk of CRC in individuals carrying the - 2578AC and CC genotypes (OR 0.34; 95% CI 0.16–0.68 and OR 0.38; 95% CI 0.18–0.78, respectively), similar to our results. They reported that AA homozygotes of SNP - 2578 had increased susceptibility to CRC in the Italian population. Similarly, Antonacopoulou et al. (2012) reported that the - 2578A allele was more frequently detected in CRC patients compared to healthy controls. Conversely, Park et al. (2007) indicated that the - 2578AC + AA genotype was associated with reduced risk in patients with proximal colon cancer (OR 0.55; 95% CI 0.31– 0.97; p = 0.049). However, they reported that the VEGF - 2578C > A polymorphisms could be a genetic determinant of colon cancer risk in Koreans. Regarding + 936C > T gene polymorphism, the majority of studies report no association with CRC in Caucasians (Hoffmann et al., 2008; Dassoulas et al., 2009; Wu et al., 2009). However, the + 936T allele increases the risk for CRC in Korean and Chinese populations (Bae et al., 2008; Wu et al., 2009). Similarly, Jang et al. (2013a, 2013b) found that
Table 3. Haplotype Frequencies Constructed by PHASE Algorithm and the Association with Colorectal Cancer Risk VEGF haplotypes - 2578A > C
Frequencies
+ 936C > T
- 460T > C
Cases n = 103
Controls n = 129
OR (95% CI)
p-Value
C C C C T T T T
T C C T T T C C
0.442 0.189 0.141 0.097 0.010 0.029 0.019 0
0.279 0.256 0.178 0.132 0.124 0.031 0 0.008
2.15 (1.01–4.59) 1 (reference) — — 8.77 (1.05–73.36) — ND ND
0.0574 — — — 0.0434 —
C A C A A C A C ND, not determinated.
—
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the + 936T allele was associated with an increased susceptibility to CRC. In gastric cancer, Tzanakis et al. (2006) found a strong association between the - 2578AA, + 936CT, and + 936TT genotypes and a larger tumor size, and the - 2578AA genotype was strongly correlated to poor differentiation. However, some researchers indicated that the results did not support an association with tumor size, histological grading, tumor stage, lymph node metastasis, and age at diagnosis in CRC cases and the contribution of each genotype to tumor characteristics might be closely related to the type of organ that carries the malignancy (Hofmann et al., 2008; Dassoulas et al., 2009). As for the VEGF - 460C > T SNP, the C allele was associated with increased VEGF gene expression in CRC tissue and with increased protein concentration of VEGF in normal colorectal tissue (Yamamori et al., 2004; Hansen et al., 2010b) in contrast with our results. In the present study, there was no association for the VEGF + 936C > T and - 460C > T variants in the risk of CRC (Table 2). Haplotype analysis has been thought to be an effective method for the identification of functional genetic variations in complex diseases, including CRC. Even though VEGF SNPs produce conflicting results in CRC development and progression, haplotype analysis can provide genetic information relative to the analysis of SNPs. In some studies (Stevens et al., 2003; Tzanakis et al., 2006; Chae et al., 2008; Kim et al., 2008; Maltese et al., 2009; Antonacopoulou et al., 2012; Jang et al., 2013a), the effect of VEGF locus haplotypes on cancer risk has been assessed. However, no metaanalysis and comparison could be performed to date because the haplotypes considered by different authors are heterogeneous. According to Antonacopoulou et al. (2012), the haplotype C2578-T1498 was less frequent in CRC patients, while the A2578-C1498 haplotype was significantly more frequent in patients compared to healthy controls. Maltese et al. (2009) identified a protective haplotype A2578-T460-G405 (OR 0.04; 95% CI 0.01–0.19) and two different high-risk haplotypes A2578-C460-G405 (OR 1.90; 95% CI 1.31–2.27) and C2578-C460-C405 (OR 9.62; 95% CI 1.3–70.87). According to Kim et al. (2008), the A2578-G634-T936 haplotype exhibited a significantly worse survival when compared with the wild C2578-G634-C936 haplotype (overall survival [OS]:hazard ratio [HR] 3.9; p < 0.001). Jang et al. (2013a) found that the A2578-G634-C936-G1154 haplotype was significantly associated with a decreased OS rate in Korean CRC patients (HR 2.5; 95% CI 1.34–4.78; p = 0.004). Also, they found that the VEGF - 2578CA genotype was associated with a significantly poorer prognosis for rectal cancers compared to the CC genotype (HR 2.2; 95% CI 1.09–4.27; p = 0.028). Chae et al. (2008) found that the C634-C936 and G634-T936 haplotypes were associated with a decreased susceptibility to CRC (OR 0.53 and 0.56, respectively; p < 0.001). In the present study, a protective A2578-T936-T460 haplotype was identified in CRC development in Turkish populations. If the - 2578C and - 460T variants were disease-causing alleles, all haplotypes containing the alleles would be expected to be positively associated with CRC and should therefore be over-represented in cases. In our population, the frequencies of the C2578-T936-T460 and C2578-C936-T460 haplotypes were 0.442 and 0.029 in cases and 0.279 and 0.031 in controls, respectively (Table 3). There was no significant association between their frequencies and CRC risk. On the contrary, the
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haplotypes containing the + 936T allele, recessively protective allele, were over-represented in controls compared to cases even if we found no relationship between the + 936C > T variant and CRC risk (Table 2 and 3). In conclusion, the present study is the first to provide evidence of the risk association of VEGF polymorphisms with CRC in the Turkish population. Further large population studies are needed to understand the role of the VEGF polymorphisms, especially the - 2578A > C variant, and haplotypes in the development and progression of CRC. Additional studies will be also warranted to clarify the ethnic differences because the studies highlight the possibility of ethnic-specific susceptibility to CRC. Acknowledgment
This work was supported by the Research Fund of Istanbul University ONAP-24985. Author Disclosure Statement
All authors declare that they have no financial conflicts of interest. References
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Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J, et al. (2013) Cancer incidence and mortality patterns in Europe: Estimates for 40 countries in 2012. Eur J Cancer 49:1374–1403. Fernando NH, Hurwitz HI (2004) Targeted therapy of colorectal cancer: clinical experience with bevacizumab. Oncologist 9:11–18. Han SW, Kim GW, Seo JS, et al. (2004) VEGF gene polymorphisms and susceptibility to rheumatoid arthritis. Rheumatology 43:1173–1177. Hanahan D, Folkman J (1996) Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86:353–364. Hanrahan V, Currie MJ, Gunningham SP, et al. (2003) The angiogenic switch for vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, and VEGF-D in the adenoma–carcinoma sequence during colorectal cancer progression. J Pathol 200:183–194. Hansen TF, Spindler KLG, Andersen RF, et al. (2010a) The prognostic value of haplotypes in the vascular endothelial growth factor A gene in colorectal cancer. Cancers 2:1405–1418. Hansen TF, Spindler KL, Lorentzen KA, et al. (2010b) The importance of - 460 C/T and + 405 G/C single nucleotide polymorphisms to the function of vascular endothelial growth factor A in colorectal cancer. J Cancer Res Clin Oncol 136:751–758. Hoffmann G, Langsenlehner U, Renner W, et al. (2008) Common single nucleotide polymorphisms in the vascular endothelial growth factor gene and colorectal cancer risk. J Cancer Res Clin Oncol 134:591–595. Jang MJ, Kim JW, Jeon YJ, et al. (2013a) Prognostic significance of vascular endothelial growth factor gene polymorphisms in patients with colorectal cancer. Int J Clin Oncol 18:1032–1041. Jang MJ, Jeon YJ, Kim JW, et al. (2013b) Association of VEGF and KDR single nucleotide polymorphisms with colorectal cancer susceptibility in Koreans. Mol Carcinog 52(S1):60–69. Kim JG, Chae YS, Sohn SK, et al. (2008) Vascular endothelial growth factor gene polymorphisms cancer associated with prognosis for patients with colorectal. Clin Cancer Res 14: 62–66. Krippl P, Langsenlehner U, Renner W, et al. (2003) A common 936C/T gene polymorphism of vascular endothelial growth factor is associated with decreased breast cancer risk. Int J Cancer 106:468–471. Loupakis F, Cremolini C, Yang D, et al. (2013) Prospective validation of candidate SNPs of VEGF/VEGFR pathway in metastatic colorectal cancer patients treated with first-line FOLFIRI plus Bevacizumab. PLoS One 8:e66774. Maltese P, Canestrari E, Ruzzo A, et al. (2009) VEGF gene polymorphisms and susceptibility to colorectal cancer disease in Italian population. Int J Colorectal Dis 24:165–170. Morita S, Uehara K, Nakayama G, et al. (2013) Association between bevacizumab-related hypertension and vascular endothelial growth factor (VEGF) gene polymorphisms in Japanese patients with metastatic colorectal cancer. Cancer Chemother Pharmacol 71:405–411. Olson TA, Mohanraj D, Carson LF, et al. (1994) Vascular permeability factor gene expression in normal and neoplastic human ovaries. Cancer Res 54:276–280. Park HM, Hong SH, Kim JW, et al. (2007) Gender-specific association of the VEGF–2578C > A polymorphism in Korean patients with colon cancer. Anticancer Res 27:2535–2540. Parkin DM, Bray F, Ferlay J, et al. (2005) Global cancer statistics, 2002. CA Cancer J Clin 55:74–108. Petrova DT, Nedeva P, Maslyankov S, et al. (2008) No association between MDR1 (ABCB1) 2677G > T and 3435C > T
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polymorphism and sporadic colorectal cancer among Bulgarian patients. J Cancer Res Clin Oncol 134:317–322. Pokorny RM, Hunt L, Galandiuk S (2000) What’s new with tumor markers for colorectal cancer? Dig Surg 17:209–215. Renner W, Kotschan S, Hoffmann C, et al. (2000) A common 936C/T mutation in the gene for vascular endothelial growth factor is associated with vascular endothelial growth factor plasma levels. J Vasc Res 37:443–448. Roy H, Bhardwaj S, Yla-Herttuala S (2006) Biology of vascular endothelial growth factors. FEBS Lett 580:2879–2887. Salvarani C, Boiardi L, Casali B, et al. (2004) Vascular endothelial growth factor gene polymorphisms in Behcet’s disease. J Rheumatol 31:1785–1789. Stephens M, Donnelly PA (2003) A comparison of Bayesian methods for haplotype reconstruction from population genotype data. Am J Hum Genet 73:1162–1169. Stevens A, Soden J, Brenchley PE, et al. (2003) Haplotype analysis of the polymorphic human vascular endothelial growth factor gene promoter. Cancer Res 63:812–816. Takahashi Y, Kitadai Y, Bucana CD, et al. (1995) Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res 55:3964–3968. Toi M, Hoshina S, Takayanagi T, et al. (1994) Association of vascular endothelial growth factor expression with tumor angiogenesis and with early relapse in primary breast cancer. Jpn J Cancer Res 85:1045–1049. Tzanakis N, Gazouli M, Rallis G, et al. (2006) Vascular endothelial growth factor polymorphisms in gastric cancer development, prognosis, and survival. J Surg Oncol 2006; 94:624–630. Vidaurreta M, Sanchez-Munoz R, Veganzones S, et al. (2010) Vascular endothelial growth factor gene polymorphisms in patients with colorectal cancer. Rev Esp Enferm Dig 102: 20–31. Vincenti V, Cassano C, Rocchi M, et al. (1996) Assignment of the vascular endothelial growth factor gene to human chromosome 6p21.3. Circulation 93:1493–1495. Watson CJ, Webb NJ, Bottomley MJ, et al. (2000) Identification of polymorphisms within the vascular endothelial growth factor (VEGF) gene: correlation with variation in VEGF protein production. Cytokine 12:1232–1235. Wu GY, Hasenberg T, Magdeburg R, et al. (2009) Association between EGF, TGF-beta1, VEGF gene polymorphism and colorectal cancer. World J Surg 33:124–129. Yamamori M, Sakaeda T, Nakamura T, et al. (2004) Association of VEGF genotype with mRNA level in colorectal adenocarcinomas. Biochem Biophys Res Commun 325:144–150. Yancopoulos GD, Davis S, Gale NW, et al. (2000) Vascularspecific growth factors and blood vessel formation. Nature 407:242–248.
Address correspondence to: ¨ zhan, PhD Gu¨l O Department of Pharmaceutical Toxicology Faculty of Pharmacy Istanbul University Beyazit Istanbul 34116 Turkey E-mail: [email protected]
VEGF Gene Polymorphisms and Susceptibility to Colorectal Cancer ¨ zhan,1 Hakan Teoman Yanar,2 and Buket Alpertunga1 Aysxe Tarbın Jannuzzi,1 Gu¨l O
Aims: Colorectal cancer (CRC) is the third most common cancer in the world and its etiology involves the interaction of genetic and environmental factors. New blood vessels form through a process called angiogenesis and have an essential role in tumor growth, progression, and metastasis of malignant tumors. The vascular endothelial growth factor (VEGF), one of the most important angiogenic factors, is a specific mitogen for vascular endothelial cells. In the present case–control study, we carried out the study to evaluate whether the VEGF single-nucleotide polymorphisms play a role in modulating susceptibility to CRC. Methods: We evaluated the VEGF - 2578A > C, + 936C > T, and - 460C > T genotypes obtained from 103 patients with CRC and 129 healthy controls by using the polymerase chain reaction–restriction fragment length polymorphism (PCRRFLP) assay. Also, haplotype analysis was determined. Odds ratios (ORs) and 95% confidence intervals (CI) were estimated. Results: - 2578A > C was significantly associated with CRC risk (OR 1.81; 95% CI 0.94–3.47; p = 0.0495), while distribution of + 936C > T and - 460C > T genotypes in cases and controls did not significantly differ. The VEGF A2578-T936-T460 haplotype might be associated with the development of CRC (OR 8.77; 95% CI 1.05–73.36; p = 0.0434). There was significant haplotype effect for all eight haplotypes ( p = 0.02). Conclusions: These results suggest that the VEGF polymorphisms might play a role in the development of CRC. Therefore, the VEGF polymorphisms might be further investigated to use in the determination of risk factors for CRC and to have a predictive value for anti-VEGF-targeted cancer therapies.
Introduction
C
olorectal cancer (CRC) is one of the most frequent tumors and the main reason for the high mortality ratio among different types of cancer sufferers in developed countries (Parkin et al., 2005; Ferlay et al., 2013; American Cancer Society, 2014). During recent years, there have been many studies to define the biological profile of CRC to improve early diagnosis and the prognostic stratification, and eventually find a cure (Pokorny et al., 2000; Crawford et al., 2003). However, the biological factors involved in the development of CRC are not yet clear (Petrova et al., 2008; Campa et al., 2012). Angiogenesis, the formation of a new blood vessel from endothelial precursors, is a prerequisite for the development, growth, and progression of solid malignancies (Takahashi et al., 1995; Yancopoulos et al., 2000). Indeed, it is generally supposed that microvessel formation around tumors is stimulated by various angiogenic factors secreted by the tumor cells (Hanahan and Folkman, 1996; Yancopoulos et al., 2000). The vascular endothelial growth factor (VEGF) is considered one of the strongest promoters of tumor angiogenesis 1 2
and has become a potential target for cancer therapy (Takahashi et al., 1995; Carmeliet and Jain, 2000). There are some studies related to the role of VEGF in tumor angiogenesis. For instance, Bevacizumab (Avastin), a neutralizing monoclonal antibody to VEGF, was the first antiangiogenic agent that was approved for the treatment of CRC (Roy et al., 2006; Loupakis et al., 2013; Morita et al., 2013). The VEGF gene is located on chromosome 6p21.3 and consists of eight exons that exhibit alternative splicing to form a family of proteins (Vincenti et al., 1996; Watson et al., 2000). Several polymorphisms have been associated with variations in VEGF protein production and reported to be involved in the development of several malignancies, including breast (Toi et al., 1994), gastrointestinal (Brown et al., 1993a), urinary tract (Brown et al., 1993b), and ovarian (Olson et al., 1994) tumors, and autoimmune diseases (Han et al., 2004; Salvarani et al., 2004). The VEGF + 936C > T (rs3025039) in the 3¢ untranslated region and the VEGF - 2578A > C (rs699947) and - 460C > T (rs833061) in the promoter region have been associated with altered levels of VEGF protein. Three VEGF variants are very common and functional single-nucleotide polymorphisms (SNPs). The
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey. Department of General Surgery, Faculty of Medicine, Istanbul University, Istanbul, Turkey.
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- 2578A > C and - 460C > T genotypes appear to be associated with a higher VEGF expression, whereas the 936T allele is correlated to a lower VEGF expression. They are also located in close proximity region in the VEGF gene, in which a high degree of linkage disequilibrium exists and haplotype effects might therefore be a possibility (Renner et al., 2000; Krippl et al., 2003; Hofmann et al., 2008; Dassoulas et al., 2009; Hansen et al., 2010a; Antonacopoulou et al., 2012). Although it has been indicated that VEGF could be a good marker for determining the risk of developing CRC and could be used as a therapeutic target for new molecular anticancer drugs (Hanrahan et al., 2003; Fernando and Hurwitz, 2004; Dassoulas et al., 2009; Vidaurreta et al., 2010), the data remain conflicting if VEGF gene polymorphisms are associated with the prognosis of CRC (Yamamori et al., 2004; Park et al., 2007; Bae et al., 2008; Maltese et al., 2009; Wu et al., 2009; Hansen et al., 2010b; Antonacopoulou et al., 2012; Jang et al., 2013b). Therefore, to test this hypothesis, we investigated possible associations between genetic variations at the - 2578A > C, + 936C > T, and - 460C > T polymorphic site in the VEGF gene, in patients who have had CRC compared to healthy individuals. Materials and Methods
We carried out a case–control study to examine the association between the VEGF genotypes and the susceptibility to CRC in the Turkish population. Genomic DNA samples were obtained from 103 patients with CRC and 129 ethnic-, gender-, and age-matched healthy volunteers at the Hospital of Istanbul University and Bagcilar Training and Research Hospital between 2011 and 2013. The criteria for diagnosis of CRC were as follows: a clinical history consistent with the disease, positive colonoscopic results, and routine laboratory analysis parameters. The histopathological examinations were evaluated according to the established clinical criteria (Compton and Gleene, 2004). Controls were hospital patients with various diagnoses (eye diseases, pulmonary diseases, cardiovascular diseases, neurological disorders, etc.) who have never had cancer. Written informed consent was obtained from all participants, and the study was approved by the Ethics Committee of Istanbul University (2011/87-555). For all subjects, we collected data about their ethnicity, smoking status, gender, age, and body–mass index (BMI, kg/ cm2) by in-person interview. The smoking status was categorized into two groups: nonsmokers (never) and smokers (former and current). A person who had smoked at least once
a day for > 1 year in his or her lifetime was regarded as a smoker. Patients showed no significant different gender (47 women and 56 men vs. 81 women and 48 men) and age (mean age 52.66 – 15.65 years vs. mean age 43.88 – 16.43 years) and BMI (26.64 – 5.24 kg/cm2 vs. 26.64 – 4.86 kg/cm2) compared with the control subjects. As smoking status, 49% of cases and 56% control subjects were smokers. Genotyping of - 2578A > C, + 936C > T, and - 460C > T variants was performed by polymerase chain reaction– restriction fragment length polymorphism (PCR-RFLP) methods (Table 1). The temperature was controlled by a programable heat block (Applied Biosystems Gene Amp PCR System 9700). Restriction enzymes were obtained from New England Biolabs and Fermentas. All other molecular biology chemicals were obtained from Fermentas and SigmaAldrich. For each polymorphism, 10% of the samples were randomly chosen and genotyped twice to assure the results and the concordance was 100%. The Hardy–Weinberg equilibrium analysis was performed to compare the observed and expected genotype frequencies among patients and control subjects by using the w2 test. To evaluate the association between the VEGF genotype frequencies and CRC, odds ratios (ORs) and 95% confidence interval (95% CI) were estimated. The haplotypes and their frequencies were estimated by PHASE (Version 2) (Stephens and Donnelly, 2003). All statistical analyses were performed using SPSS software (Version 17). The distribution of haplotypes in the cases and controls was compared by the v2 test. A two-sided p-value < 0.05 was considered to be statistically significant. Results
In the study, there were no significant differences between patients with CRC and healthy volunteers for age and sex distribution, and this suggested that the matching based on these two variables was adequate. There was no interaction between VEGF polymorphisms and smoking status in relation to CRC risk (results not shown). The genotype distributions did not significantly deviate from the Hardy–Weinberg equilibrium in cases and controls for any of the examined SNPs (Table 2). In the distribution of genotypes, the VEGF - 2578C, + 936T, and - 460C recessive allele frequencies were 0.379, 0.131, and 0.413 in cases compared with 0.488, 0.155, and 0.442 in controls, respectively. The - 2578AC genotype was statistically significantly associated with risk of developing CRC. The association was
Table 1. Details of Methodology Used in Genotyping
VEGF polymorphisms - 2578A > C (rs699947) + 936C > T (rs3025039) - 460T > C (rs833061)
Primer sequences 5¢-ggCCTTAggACACCATACC-3¢ 5¢-CACAgCTTCTCCCCTATCC-3¢ 5¢-AggAAgAgggACTCTgCgCAgAgC-3¢ 5¢-TAAATgTATgTATgTgggTgggTgTg TCTACAgg-3¢ 5¢-TgTgCgTgTggggTTgAgCg-3¢ 5¢-TACgTgCggACAgggCCTgA-3¢
bp, base pair; VEGF, vascular endothelial growth factor.
Restriction enzymes/fragment lengths (bp)
Annealing temperatures (C)
BstYI/455, 206, 249
52.1
NlaIII/208, 122, 86
64.5
BstUI/174, 154, 20
60.7
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Table 2. Allele Frequencies of Vascular Endothelial Growth Factor Polymorphisms in Patients with Colorectal Cancer and Healthy Subjects Frequencies VEGF polymorphisms
Genotypes
Cases (n = 103)
Controls (n = 129)
AA AC CC CC CT TT TT TC CC
17 44 42 78 23 2 19 83 1
34 64 31 93 32 4 15 114 0
- 2578A > C (rs699947) + 936C > T (rs3025039) - 460T > C (rs833061)
OR (95% CI)
p-Value
AA vs. any C 1.81 (0.94–3.47)
0.0495
CC vs. T 0.83 (0.46–1.50)
0.3183
TT vs. C 0.58 (0.28–1.21)
0.1019
CT, confidence interval; OR, odds ratio.
seen with VEGF - 2578A > C ( p = 0.0495); specifically, patients carrying the A allele in comparison to patients carrying the C allele had a significantly higher risk of disease (OR 1.81; 95% CI 0.94–3.47) (Table 2). To confirm the genotyping results for - 2578A > C, which has association with CRC, the selected PCR-amplified DNA samples (n = 6, for each genotype in both cases and controls) were examined by DNA sequencing, and the results were also 100% concordant. On the other hand, the other SNPs were not associated with the development of CRC, at least in the Turkish population (Table 2). In developing the risk of CRC, the results of haplotypebased analysis are shown in Table 3. There was a significant haplotype effect for all of haplotypes ( p = 0.02). Eight haplotypes were found, with the VEGF haplotype C2578-C936T460 being the most common. VEGF haplotype A2578-T936T460 might be associated with the development of CRC (OR 8.77; 95% CI 1.05–73.36; p = 0.0434). Discussion
In the present study, the VEGF - 2578A > C SNP and A2578-T936-T460 haplotype were associated with the risk of CRC. However, the distributions of VEGF + 936C > T and - 460C > T genotypes in cases and controls did not significantly differ as was also true for the other VEGF haplotypes. The present findings are consistent with the results from
some previous studies (Chae et al., 2008; Hoffmann et al., 2008; Kim et al., 2008; Dassoulas et al., 2009; Maltese et al., 2009; Wu et al., 2009; Antonacopoulou et al., 2012; Jang et al., 2013a). Some studies concluded that - 2578A > C gene polymorphism was not associated with individual susceptibility to CRC (Hoffmann et al., 2008; Dassoulas et al., 2009), whereas Maltese et al. (2009) found a reduced risk of CRC in individuals carrying the - 2578AC and CC genotypes (OR 0.34; 95% CI 0.16–0.68 and OR 0.38; 95% CI 0.18–0.78, respectively), similar to our results. They reported that AA homozygotes of SNP - 2578 had increased susceptibility to CRC in the Italian population. Similarly, Antonacopoulou et al. (2012) reported that the - 2578A allele was more frequently detected in CRC patients compared to healthy controls. Conversely, Park et al. (2007) indicated that the - 2578AC + AA genotype was associated with reduced risk in patients with proximal colon cancer (OR 0.55; 95% CI 0.31– 0.97; p = 0.049). However, they reported that the VEGF - 2578C > A polymorphisms could be a genetic determinant of colon cancer risk in Koreans. Regarding + 936C > T gene polymorphism, the majority of studies report no association with CRC in Caucasians (Hoffmann et al., 2008; Dassoulas et al., 2009; Wu et al., 2009). However, the + 936T allele increases the risk for CRC in Korean and Chinese populations (Bae et al., 2008; Wu et al., 2009). Similarly, Jang et al. (2013a, 2013b) found that
Table 3. Haplotype Frequencies Constructed by PHASE Algorithm and the Association with Colorectal Cancer Risk VEGF haplotypes - 2578A > C
Frequencies
+ 936C > T
- 460T > C
Cases n = 103
Controls n = 129
OR (95% CI)
p-Value
C C C C T T T T
T C C T T T C C
0.442 0.189 0.141 0.097 0.010 0.029 0.019 0
0.279 0.256 0.178 0.132 0.124 0.031 0 0.008
2.15 (1.01–4.59) 1 (reference) — — 8.77 (1.05–73.36) — ND ND
0.0574 — — — 0.0434 —
C A C A A C A C ND, not determinated.
—
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the + 936T allele was associated with an increased susceptibility to CRC. In gastric cancer, Tzanakis et al. (2006) found a strong association between the - 2578AA, + 936CT, and + 936TT genotypes and a larger tumor size, and the - 2578AA genotype was strongly correlated to poor differentiation. However, some researchers indicated that the results did not support an association with tumor size, histological grading, tumor stage, lymph node metastasis, and age at diagnosis in CRC cases and the contribution of each genotype to tumor characteristics might be closely related to the type of organ that carries the malignancy (Hofmann et al., 2008; Dassoulas et al., 2009). As for the VEGF - 460C > T SNP, the C allele was associated with increased VEGF gene expression in CRC tissue and with increased protein concentration of VEGF in normal colorectal tissue (Yamamori et al., 2004; Hansen et al., 2010b) in contrast with our results. In the present study, there was no association for the VEGF + 936C > T and - 460C > T variants in the risk of CRC (Table 2). Haplotype analysis has been thought to be an effective method for the identification of functional genetic variations in complex diseases, including CRC. Even though VEGF SNPs produce conflicting results in CRC development and progression, haplotype analysis can provide genetic information relative to the analysis of SNPs. In some studies (Stevens et al., 2003; Tzanakis et al., 2006; Chae et al., 2008; Kim et al., 2008; Maltese et al., 2009; Antonacopoulou et al., 2012; Jang et al., 2013a), the effect of VEGF locus haplotypes on cancer risk has been assessed. However, no metaanalysis and comparison could be performed to date because the haplotypes considered by different authors are heterogeneous. According to Antonacopoulou et al. (2012), the haplotype C2578-T1498 was less frequent in CRC patients, while the A2578-C1498 haplotype was significantly more frequent in patients compared to healthy controls. Maltese et al. (2009) identified a protective haplotype A2578-T460-G405 (OR 0.04; 95% CI 0.01–0.19) and two different high-risk haplotypes A2578-C460-G405 (OR 1.90; 95% CI 1.31–2.27) and C2578-C460-C405 (OR 9.62; 95% CI 1.3–70.87). According to Kim et al. (2008), the A2578-G634-T936 haplotype exhibited a significantly worse survival when compared with the wild C2578-G634-C936 haplotype (overall survival [OS]:hazard ratio [HR] 3.9; p < 0.001). Jang et al. (2013a) found that the A2578-G634-C936-G1154 haplotype was significantly associated with a decreased OS rate in Korean CRC patients (HR 2.5; 95% CI 1.34–4.78; p = 0.004). Also, they found that the VEGF - 2578CA genotype was associated with a significantly poorer prognosis for rectal cancers compared to the CC genotype (HR 2.2; 95% CI 1.09–4.27; p = 0.028). Chae et al. (2008) found that the C634-C936 and G634-T936 haplotypes were associated with a decreased susceptibility to CRC (OR 0.53 and 0.56, respectively; p < 0.001). In the present study, a protective A2578-T936-T460 haplotype was identified in CRC development in Turkish populations. If the - 2578C and - 460T variants were disease-causing alleles, all haplotypes containing the alleles would be expected to be positively associated with CRC and should therefore be over-represented in cases. In our population, the frequencies of the C2578-T936-T460 and C2578-C936-T460 haplotypes were 0.442 and 0.029 in cases and 0.279 and 0.031 in controls, respectively (Table 3). There was no significant association between their frequencies and CRC risk. On the contrary, the
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haplotypes containing the + 936T allele, recessively protective allele, were over-represented in controls compared to cases even if we found no relationship between the + 936C > T variant and CRC risk (Table 2 and 3). In conclusion, the present study is the first to provide evidence of the risk association of VEGF polymorphisms with CRC in the Turkish population. Further large population studies are needed to understand the role of the VEGF polymorphisms, especially the - 2578A > C variant, and haplotypes in the development and progression of CRC. Additional studies will be also warranted to clarify the ethnic differences because the studies highlight the possibility of ethnic-specific susceptibility to CRC. Acknowledgment
This work was supported by the Research Fund of Istanbul University ONAP-24985. Author Disclosure Statement
All authors declare that they have no financial conflicts of interest. References
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Address correspondence to: ¨ zhan, PhD Gu¨l O Department of Pharmaceutical Toxicology Faculty of Pharmacy Istanbul University Beyazit Istanbul 34116 Turkey E-mail: [email protected]
