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Toll-like receptors (TLR) play a pivotal role in sensing a wide range of pathogens, including bacteria, fungi and viruses. A dysregulation of TLR signaling may increase the risk of developing chronic inflammatory diseases and cancers. The aim of this study was to investigate the association of TLR2 R753Q,TLR4 D299G, and T399I polymorphisms with nasopharyngeal carcinoma (NPC) and to explore the effects of these polymorphisms on cytokine and chemokine expression in NPC biopsies. The genotypes of the three loci among 236 patients with NPC and 287 healthy controls were determined by PCR-RFLP. Cytokines and chemokines mRNA and protein in NPC biopsies were measured by real-time quantitative PCR and ELISA, respectively. Results showed that the combined CT/TT genotype of T399I was associated with increased NPC risk, with an odds ratio of 1.853 (95% confidence interval: 1.184–2.961). Also, individuals with the T allele of T399I showed a 1.842-fold increase in NPC risk compared to those with the T399I C allele (95% confidence interval: 1.213–3.015). Messenger RNA levels of interleukin (IL)-1α, tumor necrosis factor-α and IL-10 were significantly elevated in patients with T399I combined CT/TT genotype; IL-1α and IL-10 protein concentration significantly increased in NPC patients with T399I combined CT/TT genotype compared to those with the T399I CC genotype. Our data suggest that TLR4 T399I modify cytokines and chemokines patterns and play a role in the development of NPC. (Cancer Sci 2012; 103: 653–658)
A highly invasive and metastatic malignant tumor, NPC arises from the epithelial cells lining of the nasopharynx. It shows a distinct geographical distribution with the highest incidence in South-East Asia; NPC is rare in Europe and North America. Microscopically, NPC are characterized by a heavy infiltration of nonmalignant lymphocytes. The etiology of NPC is poorly understood, although various lines of evidence suggest that infections with EBV, alcohol consumption, consumption of salted fish, and genetic susceptibility, such as chromosomal aberrations and SNP, may play a role in the development of NPC.[2-4]
Inflammatory processes are involved in cancer incidence and progression, and accumulating evidence implicates inflammation in NPC. Inflammatory cytokines and chemokines are associated with the risk of cancer; in particular, IL-1, IL-2, IL-8, IL-10, IL-12 and TNF-α have shown to be associated with a higher risk for NPC,[6-9] and increased blood levels of IL-1 and IL-12 have been demonstrated in NPC patients.[7, 8]
The TLR family of receptors plays a pivotal role in sensing a wide range of pathogens, including bacteria, fungi and viruses. These receptors have been evolutionarily conserved to recognize pathogen-associated molecular patterns. Once TLR are initiated, the downstream transcription factors of TLR signaling, such as nuclear factor kappa-light-chain-enhancer of activated B cells, are activated and promote pro-inflammatory cytokines and chemokines production as well as innate, adaptive immune responses targeting invading pathogens. A dysregulation of TLR signaling may contribute to a higher risk of developing chronic inflammatory diseases and cancers.[11-13] A total of 10 functional human TLR have been identified, with TLR4 and TLR2 being the principal receptors involved in recognizing bacterial cell wall components and capsid ligands from several viruses.[14, 15]
The ability to respond properly to TLR ligands may be impaired by SNP within the TLR family of genes, which results in an altered susceptibility to inflammatory diseases and cancers. Polymorphisms in TLR4 and TLR2 have already been studied. Recently, most studies have focused on two SNP of the human TLR4 gene: an A/G substitution at 896 bp and a C/T substitution at 1196 pb. The A/G substitution at 896 bp results in an aspartic acid to glycine replacement in amino acid sequence 299 (D299G, SNP ID: rs 4986790); the C/T substitution at 1196 pb results in a theronine to isoleucine exchange in amino acid sequence 399 (T399I, SNP ID: rs 4986791). In the TLR2 gene, the G/A substitution at 2258 pb results in an arginine to glutamine substitution in amino acid sequence 753 (R753Q, SNP ID: rs5743708). Toll-like receptor 4 SNP may influence the expression of inflammatory cytokines and chemokines, such as IL-6, IL-8, TNF-α, IL-10, monocyte chemotactic protein 1 (MCP-1) and human macrophage inflammatory protein-1α (MIP-1α), and are associated with susceptibility to gastric cancer and lymphoma,[16-18] while TLR2 variants may influence susceptibility to marginal zone lymphoma.
Gene polymorphisms in the TLR pathway could alter response to infection and downstream inflammatory effects, influencing disease susceptibility and progression. EBV plays a pivotal role in the development of NPC, but only a few people develop the disease in areas where NPC is endemic even though most individuals have been exposed to EBV infections, which suggests that genetic differences may contribute to NPC.
To further explore whether SNP in TLR2 and TLR4 influence NPC pathogenesis and whether these polymorphisms are associated with modified inflammatory cytokine and chemokine responses in NPC biopsies, we conducted an investigation of TLR4 and TLR2 gene polymorphisms in a case-control study.
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To our knowledge, this is the first study to investigate whether TLR4 and TLR2 gene polymorphisms are related to the occurrence of NPC in a Chinese population and whether these gene polymorphisms correlate to mRNA and protein expression of inflammation-related cytokines and chemokines in NPC biopsies. In this study, we have shown that the C/T polymorphism at 1196 pb of TLR4, an asparagine (T) to lysine (I) substitution in amino acid sequence 399 (T399I), was significantly associated with the risk of NPC. The combined CT/TT genotype of T399I was associated with increased NPC risk compared to the CC genotype (OR = 1.853, 95% CI: 1.184–2.961, P = 0.007). Moreover, individuals with the T allele of T399I showed a 1.842-fold increase in NPC risk compared with the T399I C allele (95% CI: 1.213–3.015, P = 0.004). We also found that mRNA expression of IL-1α, IFN-α and IL-10 and protein concentrations of IL-1α and IL-10 were significantly increased in patients with the combined CT/TT genotype of T399I polymorphism. These findings suggest that the TLR4 T399I polymorphism could be a contributing factor in NPC risk, but it could be used as a genetic susceptibility marker for NPC.
In most parts of the world, NPC is rare, but it occurs at a high frequency in South-East Asia and China and at a somewhat lower but still elevated rate in North and East Africa. Microscopically, one striking feature of NPC is a heavy infiltration of nonmalignant lymphocytes, and most of these lymphocytes have been shown to be T cells. Another important feature of NPC is its association with the EBV infection. Studies have shown that the expression of EBV gene products is involved in the latent and lytic cycles in NPC specimens, and some of these viral gene products might have the capacity to induce or influence inflammatory cytokine production, such as IL-10, IL-6 and TGF-β.[23, 24] The contribution of inflammation and inflammatory cells to the process of tumor development and progression has been increasingly recognized.[25, 26] Elevated expression of cytokines is a common phenomenon of tumor cell lines derived from many cancers, such as melanomas, leukemias, and gastric and ovarian carcinoma.[27-30] Evidence has shown that a substantial proportion of malignant tumors worldwide arise from infection and chronic inflammation. Both inflammatory and tumor cells produce an assorted array of cytokines and chemokines, which mediate all aspects of inflammation and profoundly affect the development and progression of cancer.[32, 33] Previous studies have shown that NPC is associated with overexpression of numerous cytokines in NPC biopsies. EBV infection is ubiquitous in the world, but NPC incidence differs according to geographic region, and the reasons for this selective susceptibility are not fully understood. However, it is clear that environmental, viral and host factors play a role.
As a family of receptors, TLR play a pivotal role in sensing a wide range of pathogens, including bacteria, fungi and viruses. Recent works have documented that TLR may be an important pattern recognition receptor in the immune response directed against EBV infection, which may evade the immune system by modulation of the TLR signaling pathway.[34, 35] TLR signaling initiated by various pathogens triggers a cascade of signaling events that stimulate the production of pro-inflammatory cytokines and chemokines, initiating the inflammatory response. As a consequence, a dysregulation of TLR signaling may contribute to an imbalance between pro- and anti-inflammatory cytokines and lead to a higher risk of developing chronic inflammatory diseases and cancer. Several SNP within individual TLR have been identified. The ability to respond properly to TLR ligands may be impaired by SNP within TLR genes, causing an altered susceptibility to the outcome of infectious or inflammatory diseases and cancers.[16, 11] A number of molecular epidemiologic studies on the TLR4 and TLR2 genotypes and cancer susceptibility have been reported. Zheng et al. showed that the 11381G/C variant was positively associated with prostate cancer in a Swedish population, but Chen et al. demonstrated that the 11381G/C variant was not associated with prostate cancer. Cheng et al. suggested that rs10759932 was associated with a four-fold increased risk of prostate cancer. Garza-Gonzalez et al. and Trejo-de la O et al. found no significant difference in frequency of TLR4D299G and TLR4T399I genotypes among gastric cancer patients and controls.[13, 16] However, Trejo-de la O et al. showed that TLR4 polymorphisms could influence the expression of cytokine and chemokine in the gastric mucosa. Santini et al. demonstrated that TLR4Thr399Ile polymorphism is linked with an increased susceptibility to gastric cancer. Nischalke et al. suggested that the TLR2 −196 to −174del allele affects hepatitis C virus loads and increases the risk for hepatocellular carcinoma in hepatitis C virus genotype 1-infected patients.
The mechanisms by which TLR polymorphisms affect cancer risk remain unknown, but evidence shows that chronic inflammation pathogenesis is the underlying pathological event in these malignancies. NPC is an epithelial cancer that is causally associated with EBV infection; studies have shown that cytokines synthesis might contribute to lymphocyte infiltration and/or tumor growth during NPC development. Therefore, it is plausible that the various phenotypes of TLR may result in individuals with various inflammatory response and NPC risk.
Our study shows that TLR4 polymorphisms probably play a major role in susceptibility to NPC. Genotypes of TLR4 T399I TT and CT are associated with increased NPC susceptibility and IL-10 expression in NPC biopsies and are a risk factor for NPC development. A possible mechanism for TLR4 polymorphism in modulating NPC susceptibility and tumor development is through the regulation of the expression of cytokines. TLR signaling initiated by various pathogens, such as EBV, triggers a cascade of signaling events that stimulate the production of pro-inflammatory cytokines and chemokines. Of them, TNF-α stimulates tumor cell growth, affects stromal cells and enhances both metastasis and angiogenesis; IL-10 plays a major role in the development of cancer. We speculated that the ability to respond properly to TLR ligands may be impaired by SNP within TLR genes, and a dysregulation of TLR signaling may contribute to an imbalance between pro- and anti-inflammatory cytokines, causing an altered susceptibility to the outcome of cancers. Although the precise mechanisms by which TLR4 gene polymorphisms are associated with NPC remains undetermined, additional functional studies could provide valuable characterization of the molecular mechanisms by which TLR are involved in susceptibility to NPC. Continued study of the role of TLR polymorphisms to NPC susceptibility from other ethnic populations would also be of great value.