eNOS Genetic Polymorphisms and Cancer Risk: A Meta-Analysis and a Case-Control Study of Breast Cancer.

The association between endothelial nitric oxide synthase (eNOS) polymorphisms (intron 4a/b, -786T>C and 894G>T) and cancer risk remains elusive. In addition, no studies focused on their associations with the risk of breast cancer in Chinese Han population. Thus, a meta-analysis was conducted to determine the relationship between eNOS polymorphisms and cancer risk, and then a case-control study in Chinese Han population was performed to assess their associations with breast cancer susceptibility.Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association. The pooled analysis indicated that eNOS intron 4a/b and -786T>C polymorphisms were significantly associated with an increased risk of overall cancer. In subgroup analyses based on cancer type, the significant association was found between eNOS intron 4a/b polymorphism and prostate cancer risk, eNOS -786T>C polymorphism and risk of prostate, bladder and breast cancers, and eNOS 894G>T polymorphism and breast cancer risk. In subgroup analyses based on ethnicity, eNOS intron 4a/b and -786T>C polymorphisms were associated with an increased risk of cancer in Caucasians. In consistent with our meta-analysis results, a case-control study in Chinese Han population showed significant associations of eNOS -786T>C and 894G>T polymorphisms with the increased risk of breast cancer. In addition, stratified analyses based on pathological type showed that eNOS 894G>T polymorphism was only associated with the risk of infiltrative ductal carcinoma. Stratified analyses by tumor stage showed that eNOS -786T>C polymorphism was only associated with the risk of tumor stage III and IV.In conclusion, our meta-analysis and case-control study suggest that eNOS -786T>C and 894G>T polymorphisms are associated with the increased risk of breast cancer.

INTRODUCTION

Nitric oxide (NO) is a short-lived and small molecule, which is closely related to inflammatory status and regarded as a key inflammation mediator. Overproduction of NO can cause DNA damage and inhibit DNA repair.[1] In addition, NO also promotes tumor angiogenesis and metastasis.[2] Therefore, NO plays a significant role in the development of cancer. NO is produced from L-arginine by nitric oxide synthases (NOSs), which have 3 different isoforms and are divided into 2 functional classes. Constitutive class includes endothelial nitric oxide synthase (eNOS) and neuronal-NOS (nNOS) while the other class contains inducible form of NOS (iNOS).[3] eNOS is a Ca2+ dependent enzyme and firstly defined in the vascular endothelial cells. Increased expression of eNOS has been noted in the vasculature of various tumor tissues, including bladder, colon, and pancreatic cancers.[4-6] Previous studies have shown that eNOS can modulate cancer-related events, such as angiogenesis, invasion, and metastasis.[7-9] eNOS is a central mediator of several endothelium growth stimulators, such as vascular endothelial growth factor (VEGF) and prostaglandin E2. The former (VEGF) can increase angiogenesis in both iNOS+/+ and iNOS−/− mice but not in eNOS−/− mice, suggesting a predominant role of eNOS in VEGF-induced angiogenesis.[7] In addition, an in vivo study has indicated that high eNOS expression is correlated to trophoblast cancer cell vascular invasion.[8] Tumor cells in lung metastatic sites are always strongly eNOS-positive, suggesting that eNOS expression facilitates metastasis.[9]

The gene-encoding eNOS is located on chromosome 7q35 and has more than 168 polymorphisms.[10] Among these polymorphisms, intron 4a/b, -786T>C (rs2070744), and 894G>T (rs1799983) polymorphisms seem to be functional and have been widely investigated for their associations with cancer risk.[10-42] However, results were inconsistent. Therefore, we performed a comprehensive meta-analysis to derive a more precise estimation of the relationship between eNOS intron 4a/b, -786T>C, and 894G>T polymorphisms and cancer risk. Additionally, considering that no studies focused on the association of eNOS intron 4a/b, -786T>C, and 894G>T polymorphisms with the risk of breast cancer in Chinese Han population, we performed a case–control study to assess the association.

MATERIALS AND METHODS

Meta-Analysis

A comprehensive literature search was performed by PubMed and EMBASE databases with the following key words “endothelial nitric oxide synthase or eNOS or NOS3,” “polymorphism or variation,” and “cancer or tumor” (up to October 1, 2014). In addition, references of retrieved articles were also screened. The inclusion criteria were as follows: evaluation of the association between eNOS polymorphisms and cancer risk; case–control studies; detailed genotype data for estimating of odds ratios (ORs) and 95% confidence intervals (CIs); and no deviation from Hardy-Weinberg equilibrium (HWE) among the controls. As described previously, data were independently extracted from all eligible studies by 2 investigators, and any disagreement was resolved by discussion.[43] The following information was collected from each study: first author, publication year, ethnicity, cancer type, total number of cases and controls, and number of different genotypes in cases and controls.

Case–Control Study

All recruited subjects were ethnically homogenous Han Chinese. A total of 873 patients (age 50.62 ± 10.20) with histopathologically diagnosed breast cancer and 1034 age-matched healthy women (age 51.02 ± 10.79) were consecutively recruited between October 2013 and May 2014 at the Affiliated Hospital of Bengbu Medical College. Clinicopathologic information were collected from medical records and pathology reports (Table 1). Written informed consent was obtained from all participants. The research protocol was approved by the ethics committee of the Affiliated Hospital of Bengbu Medical College. Genotyping method and material of eNOS polymorphisms, including primer sequences, PCR program, selected restriction enzymes, and fragment sizes, were presented in Table 2. PCR-RFLP assay was performed for genotyping of -786T>C and 894G>T loci. PCR products were digested by restriction enzyme MspI for -786T>C and BanII for 894G>T overnight according to the manufacture's protocols. The digestion products of -786T>C and 894G>T as well as the amplifying product of intron 4a/b locus were analyzed by electrophoresis on a 3% agarose gel.

TABLE 1

Distribution of Clinicopathologic Features Among Breast Cancer Cases

TABLE 2

Genotyping Method and Material of eNOS Polymorphisms

Statistical Analysis

Crude ORs with 95% CIs were calculated by the Stata version 12.0 software. Heterogeneity among studies was evaluated using the χ2-based Cochran Q statistic test. The random effect model was used to estimate a pooled OR when there was heterogeneity between studies (PH < 0.05); otherwise, the fixed effect model was adopted. HWE among the controls was verified using a goodness-of-fit χ2 test. The association between eNOS polymorphisms and cancer risk was examined under allele contrast (4a vs 4b, C vs T, and T vs G), recessive model (4a/4a vs 4a/4b + 4b/4b, CC vs CT + TT, and TT vs TG + GG), dominant model (4a/4a + 4a/4b vs 4b/4b, CC + CT vs TT, and TT + TG vs GG), homozygote contrast (4a/4a vs 4b/4b, CC vs TT, and TT vs GG), and heterozygote contrast (4a/4b vs 4b/4b, CT vs TT, and TG vs GG). Additionally, sensitivity analysis was performed by removing each individual study in turn from the total and reanalyzing the remainder. Finally, the Begg funnel plot and Egger test were employed to investigate the potential publication bias. In case–control study, logistic regression was used to analyze the association between eNOS polymorphisms and the risk of breast cancer. These statistical analyses were implemented in Statistic Analysis System software 8.0. All P < 0.05 was used as the criterion of statistical significance.

RESULTS

Meta-Analysis of eNOS Polymorphisms and Cancer Risk

A total of 33 articles met the inclusion criteria and were included in the meta-analysis (Figure 1 and Table 3). For eNOS intron 4a/b polymorphism, 16 studies with 3850 cases and 4180 controls met the inclusion criteria. Among these studies, there were 4 studies of Asians and 12 studies of Caucasians. For eNOS -786T>C polymorphism, 10 studies with 4593 cases and 4355 controls were included in the meta-analysis. Four studies were carried out in Asians and 6 studies in Caucasians. For eNOS 894G>T polymorphism, there were 25 studies met the inclusion criteria with 9199 cases and 9726 controls. Among these studies, there were 2 studies of Asians, 21 studies of Caucasians, 1 study of African-American population, and 1 study of mixed population.

FIGURE 1

A flow chart of the study selection procedure.

TABLE 3

The Main Characteristics of Studies Included in the Meta-Analysis

As shown in Table 4  , meta-analysis for eNOS intron 4a/b polymorphism showed significant associations in overall cancer. In the subgroup analysis based on cancer type, significant associations were found in prostate cancer. In the subgroup analysis based on ethnicity, significant associations were observed in Caucasians. For eNOS -786T>C polymorphism, significant associations were also observed in overall cancer. Subsequently, subgroup analysis by cancer type showed statistically significant associations in breast, prostate, and bladder cancers. Subgroup analysis by ethnicity showed significant associations among Caucasians. For eNOS 894G>T polymorphism, no significant associations were found in overall cancer, but stratified analysis by cancer type revealed that eNOS 894G>T polymorphism was associated with the risk of breast cancer.

TABLE 4

Results of Meta-Analysis Between eNOS Polymorphisms and Cancer Risk

The sensitivity analysis was performed to assess the influence of an individual study on the overall OR. For eNOS -786T>C polymorphism, the omission of Lu J study slightly affected the overall OR under recessive model (CC vs CT + TT: OR = 1.22, 95%CI: 0.99–1.50), and so did Jang MJ study under heterozygote contrast (CT vs TT: OR = 1.10, 95%CI: 0.99–1.23).

Both Begg funnel plot and Egger test were conducted to assess the publication bias. The shape of the funnel plot for the overall results seemed symmetrical. Similarly, Egger test showed no evidence of publication bias in the eNOS intron 4a/b, -786T>C, and 894G>T polymorphisms (Table 4  ).

eNOS Polymorphisms and Breast Cancer Risk in Chinese Han Population

As summarized in Table 5, the genotype distributions for eNOS intron 4a/b, -786T>C, and 894G>T polymorphisms did not deviate from HWE in the controls (PHWE = 0.19, 0.41, and 0.18, respectively). No statistical association was found between the eNOS intron 4a/b polymorphism and breast cancer risk. However, eNOS -786T>C and 894G>T polymorphisms were associated with breast cancer risk (For eNOS -786T>C polymorphism: C vs T, OR = 1.32, 95%CI: 1.02–1.70, P = 0.04; CC vs TT, OR = 2.19, 95%CI: 1.01–4.76, P = 0.05. For eNOS 894G>T polymorphism: TT vs GG, OR = 1.69, 95%CI: 1.19–2.42, P = 0.00; TT vs TG + GG, OR = 1.69, 95%CI: 1.18–2.41, P = 0.00).

TABLE 4 (Continued)

Results of Meta-Analysis Between eNOS Polymorphisms and Cancer Risk

Stratification Analysis of eNOS Polymorphisms With Breast Cancer Risk

As summarized in Tables 6 and 7, stratified analysis based on pathological type indicated that eNOS -786T>C polymorphism was associated with the risk of infiltrative ductal carcinoma (C vs T: OR = 1.34, 95%CI: 1.02–1.76, P = 0.03) and other carcinoma (CC vs TT: OR = 3.40, 95%CI: 1.36–8.46, P = 0.01; CC vs CT + TT: OR = 3.37, 95%CI: 1.36–8.37, P = 0.01). However, eNOS 894G>T polymorphism was only associated with the risk of infiltrative ductal carcinoma (T vs G: OR = 1.25, 95%CI: 1.02–1.52, P = 0.03; TT vs GG: OR = 1.73, 95%CI: 1.20–2.50, P = 0.00; TT vs TG + GG: OR = 1.71, 95%CI: 1.19–2.47, P = 0.00). Furthermore, stratified analysis by tumor stage suggested that eNOS -786T>C polymorphism was only associated with the risk of tumor stage III and IV (C vs T: OR = 1.99, 95%CI: 1.35–2.93, P = 0.00; CC vs TT: OR = 4.42, 95%CI: 1.97–9.89, P = 0.00; CC + CT vs TT: OR = 1.74, 95%CI: 1.12–2.68, P = 0.01; CC vs CT + TT: OR = 4.34, 95%CI: 1.94–9.71, P = 0.00). However, eNOS 894G>T polymorphism was associated not only with the risk of tumor stage I and II (TT vs GG, OR = 1.57, 95%CI: 1.07–2.29, P = 0.02; TT vs TG + GG, OR = 2.45, 95%CI: 1.15–5.23, P = 0.02), but also with tumor stage III and IV (TT vs GG, OR = 2.14, 95%CI: 1.34–3.40, P = 0.00; TT vs TG + GG, OR = 4.56, 95%CI: 1.81–11.52, P = 0.00).

TABLE 4 (Continued)

Results of Meta-Analysis Between eNOS Polymorphisms and Cancer Risk

TABLE 5

Association of eNOS Polymorphisms With Breast Cancer Risk Among Chinese Han Population

DISCUSSION

Multiple lines of evidence supported an important role for genetics in determining cancer risk, and understanding polymorphisms associated with cancer risk may be valuable for providing personalized diagnosis and therapy of certain cancers. Since the identification of eNOS intron 4a/b, -786T>C, and 894G>T polymorphisms, an increasing number of studies suggested that eNOS intron 4a/b, -786T>C, and 894G>T polymorphisms may play important roles in cancer risk. Epidemiological studies of eNOS intron 4a/b, -786T>C, and 894G>T polymorphisms, if large and unbiased, can provide insight into the association between the gene and cancer risk. However, previous results are inconclusive. To derive a more precise estimation of the association, we performed this meta-analysis. The eNOS intron 4a/b and -786T>C polymorphisms were significantly associated with overall cancer risk. In contrast, no association was observed between eNOS 894G>T polymorphism and overall cancer risk. In subgroup analyses based on cancer type, significant associations were found between eNOS intron 4a/b polymorphism and the risk of prostate cancer, eNOS -786T>C polymorphism and the risk of prostate, bladder and breast cancers, and eNOS 894G>T polymorphisms and the risk of breast cancer. In subgroup analyses based on ethnicity, eNOS intron 4a/b and -786T>C polymorphisms were associated with cancer risk in Caucasians.

In current case–control study of 873 patients with breast cancer and 1034 healthy women, we found that eNOS -786T>C and 894G>T polymorphisms were associated with breast cancer risk in Chinese Han population, which were consistent with our meta-analysis results. Furthermore, stratified analyses based on pathological type showed that eNOS 894G>T polymorphism was only associated with risk of infiltrative ductal carcinoma. In stratified analyses by tumor stage, we found that eNOS -786T>C polymorphism was only associated with the risk of tumor stage III and IV.

To a certain extent, our meta-analysis and case–control study still include some limitations, which should be interpreted and taken into consideration. For the present meta-analysis, we did not have original data for all studies to adjust estimates and perform a more precise analysis. For the case–control study, all participants were from hospital, which may result in inherent selection bias.

In conclusion, our meta-analysis and case–control study suggest that eNOS -786T>C and 894G>T polymorphisms are associated with the risk of breast cancer. However, our findings need to be further validated in well-designed studies.

TABLE 6

Genotype Distribution of eNOS Polymorphisms in Stratification Analysis

TABLE 7

Stratification Analysis of eNOS Polymorphisms With Breast Cancer Risk