Role of cytokine gene (interferon‐γ, transforming growth factor‐β1, tumor necrosis factor‐α, interleukin‐6, and interleukin‐10) polymorphisms in the risk of oral precancerous lesions in Taiwanese

Introduction

Oral squamous cell carcinoma is the eighth most common cancer in the world, with approximately 300,000 new cases diagnosed annually. In Taiwan, this disease is the fourth leading cause of cancer‐related deaths in males. Oral squamous cell carcinoma can be preceded by some benign oral lesions with malignant potential, such as leukoplakia, erythroplakia, oral lichen planus, and oral submucous fibrosis. There are chronic inflammatory and immunologic processes. Although the most common oral premalignant lesions appear to be related to the habit of chewing betel quid, tobacco smoking, and heavy alcohol consumption, the shift in the balance between pro‐ and anti‐inflammatory cytokines may affect the inflammation‐mediated oncogenesis.

Cytokines are important mediators of inflammatory responses. They may either be involved in the antitumor effector immune mechanisms or may enhance malignant transformation and tumor growth. Th1 type cytokines such as interferon (IFN)‐γ are required for antitumor immunity, whereas Th2 type and several inflammatory cytokines such as tumor necrosis factor (TNF)‐α and transforming growth factor (TGF)‐β1 favor tumor development [1]. Interleukin (IL)‐6, another cytokine of Th2 type, plays an important role in different physiologic and pathophysiologic processes such as inflammation, bone metabolism, and carcinogenesis [2]. In a previous study, we found that there were different releasing of cytokines using arecoline cultured peripheral blood mononuclear cells from patients with oral mucous diseases and controls [3].

Single nucleotide polymorphisms (SNPs) in genes encoding for susceptibility factors may influence gene expression, protein function, and disease predisposition. An increasing number of recent reports suggested that SNPs in cytokine genes, such as TNF‐α (−308), TGF‐β1 (codons 10 and 25), IL‐10 (−1082, −819, and −592), IL‐6 (−174), and IFN‐γ (+874), have been correlated with an increased risk for developing oral malignancies [[4], [5], [6]]. However, reports discussing about the association of cytokine polymorphism and oral precancerous lesion are limited. The aim of this study was to determine any possible gene polymorphisms of TNF‐α (−308), TGF‐β1 (codons 10 and 25), IL‐10 (−1082, −819, and −592), IL‐6 (−174), and IFN‐γ (+874) that may influence any susceptibility to oral precancerous lesions.

Materials and methods

Results

Table 1 presents clinical characteristics of the study participants. Although there were differences in the age group, sex distribution, betel quid chewing habit, drinking habit, and smoking habit between the cases and controls, these were taken into consideration during the computation of ORs to compare between genotype. The age range of the patients with OPL was 26–79 years, with a median age of 50.83 years and a 95% CI of 36.69–63.97 years. The respective age range of the healthy controls was 23–83 years, with a median age of 59.20 years and a 95% CI of 46.29–72.11 years. Table 2 presents the histological diagnosis of OPLs. We included the patient with OPL among leukoplakia, erythroplakia, oral lichen planus, and oral submucous fibrosis. Genotype distribution of the controls satisfied the Hardy–Weinberg equilibrium.

Discussion

In the present study, we found that functional polymorphisms at TNF‐α −308 G > A, TGF‐β1 codon 25 G > C, and IL‐6 −174 G > C have significant effects on the risk of OPL development. The female patients were more than males in the study because the blood examination doesn't be involved in the routine protocol of treatment of the oral precancerous lesions and female patients were more willing to join the study. We assume that there is no difference between females and males regarding the distribution of polymorphisms of those cytokines.

Expressions of these cytokines in different polymorphisms are shown in Table 5. A G‐to‐A transition in the promoter region (−308) of the TNF‐α gene results in higher expression of TNF‐α in vitro and in vivo [7]. TNF‐α high‐expression allele increases the risk of oral cancer [7]. TNF‐α is a proinflammatory multifunctional cytokine produced by macrophages. It plays an important role in the regulation of immune response, because its increase after traumatic injury generates a cytokine cascade resulting in activation, proliferation, and hypertrophy of mononuclear and phagocytic cells. TNF‐α‐initiated signaling pathways activate proinflammatory gene expression via the transcription factor nuclear factor‐kappa B. TNF‐α has been implicated in the pathogenesis and progression of various malignancies. In comparison to healthy individuals, increased serum levels of TNF‐α have been observed in European patients with oral cancer [8]. Significantly higher levels of salivary IL‐6 and TNF‐α were found in patients with oral leukoplakia when compared with healthy controls [9]. SNPs in inflammation genes have been shown to alter their expression and functions. TNF‐α and IFN‐γ polymorphisms contribute to susceptibility to oral lichen planus [10]. A high‐producing G/A genotype of TNF‐α at position −308 was more commonly found among the OPL patients of this study.

IL‐6 is an anti‐inflammatory cytokine [11]. It has already been well discussed that cigarette smoking affects the immune system [12]. Cigarette smoking decreases bioactive IL‐6 secretion by alveolar macrophages. Betel quid component also influences inflammatory reaction. Areca nut extract induces IL‐6 production by gingival keratinocyte and also induces Cox‐2 and IL‐6 mRNA expression [13]. Betel quid chewing contributes to the pathogenesis of cancer and oral submucous fibrosis by impairing T cell activation and inducing IL‐6 production, which affect oral mucosal inflammation and growth of oral fibroblast and oral epithelial cells [14].

IL‐6 activates mainly the JAK1/STAT3 signal pathway and functions as a proinflammatory factor [15]. Expression rates of IL‐6R and IL‐6 mRNA transcripts in tumor specimens were much higher than those in nontumor mucosa [16]. A G/C SNP in the promoter region (−174) of IL‐6 was shown to affect transcription and alter plasma IL‐6 levels. As reported in the literature, the IL‐6 variant genotype (C/C) frequency varied dramatically among different ethnic groups; for example, it is very low in Asian populations [17]. Our control frequency, compared with other studies about Southern Chinese population [18], is slightly higher. The IL‐6 polymorphisms have been linked to various malignancies such as gastric, hepatocellular, lung, colorectal, cervical, breast [[19], [20], [21]], and bladder cancers [22]. Significantly higher levels of salivary IL‐6 were found in patients with oral leukoplakia when compared to healthy controls [9]. In this study, we found an association between the variant C allele of the −174 G > C SNP in the IL‐6 promoter region with an increasing OPL risk.

TGF‐β1 induces tumor regression in vivo by Smad4‐independent pathways that sensitize keratinocytes to mitochondrial‐mediated apoptosis [23]. TGF‐β1 gene is located on chromosome 19q13. SNPs in cytokine genes may influence the expression or function of cytokines via cytokine network, and many have been evaluated for their role in inflammatory diseases and cancer predisposition. Two polymorphisms of TGF‐β1 are located on codon 10 (869T > C::Leu/Pro) and codon 25 (915 G > C::Arg/Pro) of exon 1 that encodes leader sequence of the protein and have functional importance in modulating its transmembrane transport. TGF‐β1 low‐production genotypes (TGF‐β1 codon 10 CC) were associated with not only an increased risk of breast cancer [24], but also an increased odd of breast cancer relapse and reduced 5‐year disease‐free survival rate [25]. The present study revealed that the codon 10 genotype is not a critical factor in the development of OPL. Codon 25 of TGF‐β1 increased serum levels of TGF‐β1 in the presence of the G allele, which might contribute to long‐term suppression of epithelium proliferation, leading to a decrease in the risk of OPL. The C allele at codon 25 may contribute to weaker suppression of the epithelium proliferation, thereby increasing the risk of OPL. TGF‐β1 codon 25, not codon 10, is an important risk factor of OPL, as found in the study. TGF‐β1 is the major cytokine responsible for the regulation of fibroblast proliferation and differentiation [26]. The rough fibers present in betel nuts may cause multiple invisible tiny abrasive wounds during betel quid chewing. Wound healing was affected by the chemical component, which may modulate cytokine secretion.

Polymorphism analysis of IFN‐γ, an important cytokine in antitumor host defense, showed no genotype difference at position +874 in OPL patients and controls. However, the higher‐producer T/T genotype at position +874 was reported to be significantly higher in hepatocellular carcinoma in Brazil [27].

IL‐10 is a multifunctional cytokine with both immunosuppressive and antiangiogenic functions, and consequently has both tumor‐promoting and tumor‐inhibiting properties [28]. Raised levels of serum and peritumoral IL‐10 production have been reported in many malignancies, which have been interpreted in support of a role for IL‐10 in tumor escape from the immune response [29]. The IL‐10 SNP and haplotype have been reported to be associated with differential IL‐10 expression in vitro, with the −1082 A, −819 T, and −592 A haplotypes being associated with a decreased IL‐10 expression, compared with the −1082 G, −819 C, and −592 C haplotypes. SNPs at position −1082 A/G in the IL‐10 gene promoter are significantly associated with the risk of oral cancer [30]. However, it has not been found to be associated with OPL in our study.

Limitations of the present study need to be addressed. It has been suggested that the sample size is always an important issue. Statistical power of the analysis would generally increase with increasing size of sample. Because of the relatively small sample size, this study may not be powerful enough to test the interaction among genes with low frequencies.

This study provides evidence for the association between the variants IL‐6, TNF‐α (−308), and TGF‐β1 codon 10 genotype, and an overall increased OPL risk. While these findings need to be replicated in larger, prospective studies, they support the role of chronic inflammation as an etiologic factor in this population.