The influence of genetic variability on the risk of developing malignant mesothelioma

Malignant mesothelioma (MM) is a rare and aggressive disease with poor survival. It has been associated with occupational and/or environmental exposure to asbestos in more than 86% of patients with this disease. 1,2 Malignant mesothelioma most commonly arises from pleura (65%–70%), peritoneum (30%) and very rarely other serous surfaces (1%). 3,4 Global incidence is expected to peak 30 to 40 years after the peak of asbestos usage that occurred in the 1960s and 1970s.3,5 However, recent studies still show a rise in incidence. 6

The implication of asbestos exposure in MM has been validated, but the mechanism of carcinogenesis is not yet completely understood. Asbestos fibre components, specifically iron, are hypothesized to contribute to reactive oxygen species (ROS) production. Iron catalyses both Fenton and Haber-Weiss reactions which produce hydroxyl radical (HO) from peroxide (H₂O₂). 7 Furthermore, all types of asbestos may cause frustrated phagocytosis in the macrofages, which produces ROS, reactive nitrogen species (RNS), cytokines, chemokines, proteases and growth factors. 8,9 This may lead to DNA damage, genomic instability and a malignant transformation of mesothelial cells.9 A number of studies show that ROS and RNS and inflammation could have a central role in asbestos fibre toxicity. 10,11,12,13

On the other hand, antioxidative enzymes such as catalase (CAT), superoxide dismutases (SOD-s), and NAD(P)H quinone dehydrogenase 1 (NQO1) participate in the enzymatic defence against ROS and RNS. 10 When the activity of these enzymes is decreased or changed, ROS concentrations increase and DNA damage may occur. One of the most important repair enzymes for oxidative DNA damage repair is human 8-oxoguanine glycosylase 1 (hOGG1). Functional polymorphisms that influence the expression level or activity have been reported in the genes coding for all these enzymes. CAT helps to maintain the oxidative balance by catalysing H₂O₂ to H₂O and O₂14,15 Numerous polymorphisms of CAT gene (CAT) have been described, rs1001179 being the most commonly studied one. It causes cytosine (C) to thymine (T) change at position -262 in the promoter region (c.262C>T).14 Our previous study investigating the association of this polymorphism with asbestosis found a slight increase in the risk of the TT genotype.16 Superoxide dismutases (SOD) convert superoxide into H₂O₂ and O₂. SOD2 is found in mitochondria, where the amount of ROS is very high. The most common polymorphism is rs4880, resulting in C to T substitution at position 201 (c.201C>T), which causes the change of alanine to valine at position 16 (p.Ala16Val). Several studies associate the 6q25 chromosome deletion with certain forms of cancer, which is why some authors consider SOD2 to be a tumour suppressor gene.17,18 HOGG1 catalyses the repair of 8-oxoguanine that may result from ROS damage to the DNA. Functional polymorphisms of the hOGG gene may impact DNA repair. In rs1052133 polymorphism, C replaces G in exon 7, causing the substitution of serine with cysteine in codon 326 (p.Ser326Cys). Although a changed structure of the polymorphic enzyme has not been proved, several studies have shown the association between hOGG1 Ser326Cys polymorphism and lung cancer risk. 19,20 NQO1 catalyses the reduction of quinones to hydroquinones, preventing the formation of free radicals. The most frequently studied single nucleotide polymorphism (SNP), rs1800566, results in C to T change (c.609C>T), which causes proline to serine substitution (p.Pro187Ser). 21 Some studies found this polymorphism to be associated with an increased risk of several malignant diseases: lung cancer, colorectal cancer, breast cancer and bladder cancer. 22,23

Only few studies have investigated the interplay between asbestos exposure and genetic variability in antioxidant defence system in MM so far. 24,25,26 Nevertheless, the interaction between asbestos exposure and genetic susceptibility due to genetic polymorphism of antioxidant enzymes has been shown for asbestosis. 27 We have previously described the association between SOD2 Ala/Ala genotype and asbestosis28 as well as association between CAT -262 TT genotype and asbestosis. 16 Landi et al. reported on the association between SOD2 and the risk of MM25, but according to our knowledge and available literature, the impact of NQO1, CAT and hOGG1 polymorphisms on the risk of developing MM has not been studied so far.

This study aimed to investigate whether functional polymorphisms in NQO1, CAT, SOD2 and hOGG1 genes influence the risk of MM, to investigate the interactions between genetic variability in antioxidative and DNA repair mechanisms and to investigate the interactions between asbestos exposure and the investigated polymorphisms in MM patients.

The clinical characteristics of MM patients and controls are presented in Table 1. There was no statistical difference in gender between the cases and controls (p = 0.315), but MM patients were notably older (p < 0.001) and a much higher number of the patients were smokers (p < 0.001). All the controls (122) and 126 (83.4%) MM patients had been exposed to asbestos. For all the controls and 64 (50.8%) MM patients, asbestos exposure could be categorized into the groups. Among the subjects with known asbestos exposure, the MM patients had a significantly higher asbestos exposure compared to asbestos exposed subjects without any asbestos related disease (p < 0.001, Table 1).

Table 1

Clinical characteristics of MM patients and controls

Univariate regression logistic analysis has shown that the risk of MM was influenced by smoking, age and asbestos exposure, but not by gender. The risk of MM was increased in smokers (OR = 9.30; 95% CI = 4.83–17.98; p < 0.001) and older patients (OR = 1.10; 95% CI = 1.08–1.14; p < 0.001). Compared to a low exposure to asbestos, medium and high asbestos exposures increased the risk of MM 7-fold (OR = 7.05; 95% CI = 3.59–13.83; p < 0.001). Gender did not influence MM risk (OR = 0.76; 95% CI = 0.44–1.30; p = 0.316).

Genotype frequencies for controls and MM patients are presented in Table 2. Minor allele frequencies were 13.9% for NQO1 rs1800566, 22.4% for CAT rs1001179, 52.5% for SOD2 rs4880 and 18.8% for hOGG1 rs1052133. In controls, all SNPs were in Hardy-Weinberg equilibrium (all p > 0.05). The association between MM and the investigated polymorphisms was tested with univariate logistic regression using a dominant genetic model. The carriers of at least one polymorphic NQO1 allele (CT and TT genotypes) had an increased risk of MM compared to those with CC genotype (OR = 1.73; 95% CI = 1.02–2.96; p = 0.043). No association was observed between MM and other genetic polymorphisms (Table 2).

Table 2

The distribution of antioxidative and repair gene polymorphisms in MM patients and controls and risk of MM

Multivariate analysis was used to determine the combined effect of genetic determinants and clinical variables such as smoking, age and asbestos exposure. The association between NQO1 and MM risk remained significant after adjustment for smoking, but the risk was slightly lower when adjusted by age or asbestos exposure. The association between other investigated polymorphisms and MM risk remained nonsignificant also after taking into account the effect of age, smoking and asbestos exposure (Table 2).

Next, gene-gene interactions between the investigated NQO, CAT, SOD2 and hOGG1 genotypes and the interactions between genotypes and asbestos exposure were calculated. The interaction between CAT rs1001179 and hOGG1 rs1052133 had a protective effect on the risk of MM (OR_int = 0.27; 95% CI = 0.10–0.77; p = 0.014). On the other hand, no gene-gene interactions were observed between other investigated polymorphisms (Table 3).

Table 3

Gene-gene interactions between rs1800566 NAD(P)H quinone dehydrogenase 1 (NQO1), rs1001179 catalase (CAT), rs4880 superoxide dismutase 2 (SOD2), and rs1052133 human 8-oxoguanine glycosylase 1 (hOGG1)

Finally, we investigated the influence of interactions between polymorphisms and asbestos exposure on the risk of MM, but no interaction was found (Table 4).

Table 4

The influence of interactions between the investigated polymorphisms and asbestos exposure on risk of malignant mesothelioma

The association between asbestos exposure and MM has been clearly proved, but not much has been known about the influence of genetic polymorphisms that may modify the risk of developing this aggressive cancer. Our present study investigated the effect of genetic polymorphisms of some of the most important enzymes involved in removal of ROS and RNS (NQO1, CAT, SOD2) and DNA damage repair (hOGG1) on the risk of MM, as well as the impact of interactions between the observed genetic polymorphisms and between genetic polymorphisms and asbestos exposure on the risk of developing this cancer.

In the study, we have found that smoking increased the risk of MM. It has been well proved that exposure to asbestos fibres results in an increased generation of ROS.8,9 Many studies have also investigated the association between ROS and carcinogenesis, caused by tobacco smoke.31 According to the free radical hypothesis of aging, ROS and RNS can drive the accumulation of cell and DNA damage32 leading to carcinogenesis and cancer.33,34,35,36 The combined effect of both asbestos and smoking may thus greatly increase the amount of ROS in the cells and may cause more DNA damage than smoking or asbestos exposure alone. That could explain the observed higher risk of MM among smokers exposed to asbestos compared to non-smokers.

Our study also showed a slight increase in MM susceptibility in older patients, which is in line with other studies in which MM is found predominantly as disease of the elderly.37 Mortality due to pleural MM increased between 75 and 89 years of age and in peritoneal MM between 65 and 84 years of age.37 This may be due to the long latency time, which is the period from the first exposure to the diagnosis of MM, and can range from 20 to over 50 years.38 There are many factors affecting the latency period, including dose response, age, gender and location of MM.39

An important finding of our study is that medium and high asbestos exposures increase the risk of MM by 7-fold compared to low asbestos exposure. This is in line with the results of some studies that have also reported that the MM risk is related to the amount of exposure.40,41,42,43 An Australian study reported an increased risk of MM with higher and longer occupational or environmental exposure to asbestos.40, 41 A Norwegian study also observed a correlation between the duration of occupational exposure and risk of MM42,43 However, in our previous study, low levels of asbestos exposure were reported in almost 36% of patients with MM.2

Another important finding of the current study indicates a higher risk of MM among subjects with the NQO1 rs1800566 T allele. According to the available literature, the association between NQO1 polymorphisms and MM has not been investigated yet. However, some studies have found an increased risk of lung cancer21, colorectal cancer44 and bladder cancer22 among the carriers of the polymorphic allele. On the other hand, a risk of MM was not statistically significantly increased for other investigated polymorphisms. Contrary to the findings of this study, the only other study investigating the SOD2 polymorphism in relation to MM risk25 showed an increased risk of pleural MM in SOD2 Ala/Ala genotype. Regarding other asbestos related diseases, we have previously reported an association between SOD2 Ala/Ala genotype and higher risk of asbestosis.28 Furthermore, a significantly higher risk of lung cancer was reported in carriers of both Ala/Val and Val/Val genotypes.45,46

Even though there was no association between CAT rs1001179 or hOGG1 rs1052133 alone and MM, one of the key findings of this study was that the interactions between CAT rs1001179 and hOGG1 rs1052133 have a protective effect on the risk of MM. This can be explained by the fact that CAT as an antioxidative enzyme constitutes a part of the primary defence system against ROS, while hOGG1 as a repair enzyme removes oxidized bases such as 8-oxoguanine. According to the above-mentioned mechanisms of defence against ROS, we can consider our observations as biologically plausible. We have also previously reported slightly increased risk of asbestosis among the carriers of the CAT rs1001179 TT genotype.16 Furthermore, Erculj et al. have observed an association between hOGG1 Ser326Cys polymorphism and higher DNA damage levels in healthy young population.47

A limitation of this study was that MM patients were significantly older than controls, however we accounted for that with adjustment for age in the statistical analysis. Furthermore, cumulative asbestos exposure could not be determined for all MM patients, as proper assessment is very difficult, especially for environmental or occasional exposure. Therefore, some of the analyses were only performed on the subgroup of MM patients. On the other hand, our study is one of the few that investigated gene-gene as well as gene-environment interactions in MM patients.48 Neri et al. analysed a different set of genes, including several glutathione S-transferases that also contribute to antioxidative defence mechanisms and also showed the presence of gene-gene interactions as well as geneenvironment interactions in the development of MM.48 Therefore, further studies including a larger number of subjects with well-defined asbestos exposure are needed to elucidate the role of gene-environment interactions in the development of MM. Considering that pathogenesis of MM is still not completely understood, polymorphisms of other enzymes that could affect the removal of ROS and RNS and other DNA repair mechanisms, also need to be investigated.

In conclusion, our study showed for the first time that NQO1 polymorphism influences the risk of MM both independently and after adjustment by smoking. Another key observation is the protective effect of the interaction between CAT rs1001179 and hOGG1 rs1052133 polymorphisms, indicating the importance of interaction between antioxidative and DNA repair mechanisms. The results of this and future studies will improve our understanding of MM pathogenesis and may consequently enable better preventive measures for the exposed populations, earlier diagnosis and new approaches to treatment of this aggressive malignant disease.