TLR4 promoter rs1927914 variant contributes to the susceptibility of esophageal squamous cell carcinoma in the Chinese population

Introduction

Esophageal cancer, as the sixth leading cause of cancer death, is one of the most common malignant tumors worldwide (Bray et al., 2018). Esophageal cancer contains two common histological types: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC). There are clear differences between EAC and ESCC that affect their distribution and incidence in the world (Domper Arnal, Ferrández Arenas & Lanas Arbeloa, 2015; Yang, Chen & Tu, 2016). In China, most of the cases of esophageal cancer are squamous cell cancer (Lin et al., 2013). ESCC is caused by environmental and genetic factors. Epidemiological studies have reported that tobacco smoking, alcohol drinking, ingesting hot substances and so on played a role in the development of ESCC (Yu et al., 2018a). However, not all individuals who have been exposed to these hazards eventually get ESCC. In recent years, genetic polymorphisms have been reported to impact the development of esophageal cancer (Hiyama et al., 2007; Yue et al., 2017).

Single nucleotide polymorphism (SNP) is one of the most common genetic variants in the genome. Over the past decade, large-scale SNP analyses, known as genome-wide association studies (GWAS), have provided a new way to identify genetic loci which might be associated with the cancer susceptibility, survival prognosis or drug response (Wu et al., 2013; Yu et al., 2018b; Zhang et al., 2020). The SNPs located in specific genes, which involved in cancer-related pathway, may modulate gene expression or protein activity and further involved in cancer initiation and development. For example, the functional genetic variants in cyclooxygenase-2 and 12-lipoxygenase have been reported to be associated with the risk of esophageal cancer (Guo et al., 2007; Zhang et al., 2005). The mutations in Flap endonuclease 1 (Fen1), which is one of key components in long-patch DNA base-excision repair, resulted in autoimmunity, chronic inflammation and various cancers (Zheng et al., 2007).

The interaction between the immune system and malignant cells has an impact on tumorigenicity (Terme & Tanchot, 2017). On one hand, the immune system kills or clears malignant transformed cells; on the other hand, malignant cells struggle to escape immune surveillance (De Visser, Eichten & Coussens, 2006; Schreiber, Old & Smyth, 2011). As the most studied pattern recognition receptor, Toll-like receptors (TLRs) can enhance the innate immune response and stimulate antigen-derived cells such as dendritic cells, and then activate the tumor-specific T cells immune which involving in the development of tumors (Kaczanowska, Joseph & Davila, 2013; Pham et al., 2010). TLR4 can not only recognize extracellular antigens, but also respond to intracellular injury related factors (Jacobsen, Aasenden & Hensten-Pettersen, 1991; Rocha et al., 2016). A study showed that TLR4 induced by LPS promoted the secretion of immunosuppressive cytokines which promoted the proliferation of lung cancer and ESCC cells (He et al., 2007; Zu et al., 2017). TLR4 also involved in the antitumor T-cell immune response by induced by danger-associated molecular patterns (DAMPs) (Fang et al., 2014). Studies have shown that TLR4 is overexpressed in a variety of malignant tumors and associated with poor prognosis in cancer patients (Li et al., 2017; Pandey, Chauhan & Jain, 2018; Sheyhidin et al., 2011; Wang et al., 2017; Zhao et al., 2019). TLR4 has been identified as a potential drug target for the immuno-therapeutics in various cancers (Shetab Boushehri & Lamprecht, 2018).

In view of the important role of TLR4 in tumors, we screened out the potential functional SNPs in TLR4 using bioinformatic methods and then performed a case-control study in Chinese population to determine whether they were correlated with the occurrence of ESCC.

Materials and Methods

TLR4 SNPs selection

In this study, we predicted the possible functional SNPs in the regulatory region of TLR4. All included SNPs located in the promoter region or the 3′ untranslated region with MAF ≥0.05. For SNPs in the promoter region of TLR4, transcription factor binding capability was predicted by TRANSFAC program (Wingender et al., 1996). For the SNPs located in the 3′ untranslated region, microRNA binding ability was predicted using SNPinfo Web Server (Xu & Taylor, 2009). Finally, TLR4 rs1927914 in the promoter region and rs11536891 and rs7873784 in the 3′ untranslated region were selected for further genotyping (Fig. 1A).

Results

Discussion

Because the symptoms of esophageal cancer are not obvious in the early stage, most of the patients are diagnosed in the middle and late stages and often accompanied by malnutrition. A multicenter study, which investigated the potential epidemiological and clinical risk factors affecting the survival of esophageal cancer patients in China, demonstrated that the overall 5-year survival rate is around 39% (He et al., 2020). Multiple large clinical studies have shown that concurrent chemoradiotherapy (CCRT) can significantly improve the local control rate and the overall survival rate of esophageal cancer (Kang et al., 2018; Takeda et al., 2018). Therefore, CCRT is still the standard therapy for patients with locally advanced esophageal cancer who cannot receive or refuse surgical treatment. However, CCRT is not tolerated in patients with advanced age, severe cardiopulmonary complications or malnutrition. In the past decade, targeted therapy has brought cancer treatment into the era of precision therapy with its low toxic side effects and high therapeutic efficiency. The discovery of EGFR, ALK and other driving genes in lung cancer provides an example for targeted therapy of malignant tumors. Therefore, it is still necessary to look for potential molecular targets to guide the clinical treatment of esophageal cancer.

TLRs are important components of inflammatory response by effecting on innate immune response. So far, 10 members (TLR1-TLR10) have been identified in TLR family which involved in multiple biological processes, such as inflammatory response, immune response, apoptosis and angiogenesis and further contributed to the development of various cancers (Belmont et al., 2014; Dajon, Iribarren & Cremer, 2017; Garcia et al., 2016; Paone et al., 2010; Vijay, 2018). TLR4 locates in chromosome 9q32-33. TLR4 mRNA can be polyadenylated at 3′ UTR to produce 5432nt and 12853nt transcripts that both encode the same 839aa protein. Kutikhin et al. found that the high expression of TLR4 in cancer tissues can promote the metastasis and invasion of tumor cells, and it is not suppressed by the immune system (Davoodi, Hashemi & Seow, 2013; Kutikhin et al., 2014). The overexpression of TLR4 in ESCC tissues was also associated with the poor prognosis (Li et al., 2018; Sato et al., 2020).

So far, several studies have found that TLR4 polymorphisms influence cancer susceptibility, such as gastric cancer, myeloma and hepatocellular carcinoma (Bagratuni et al., 2016; He et al., 2018; Huang et al., 2017). In Chinese population, Huang et al. found that there is a significantly decreased risk of gastric cancer in individuals carrying of the allele C for the rs10116253 and allele T for the rs1927911 in TLR4 (Huang et al., 2014). Similar results were found in hepatocellular carcinoma (Minmin et al., 2011). Song et al. (2009) found that both TLR4 rs1927911 and rs11536858 polymorphism increased the susceptibility of prostate cancer in Korean Men.

In this study, the online databases of TRANSFAC and SNPinfo Web Server were used to predict the SNPs that may affect the expression of TLR4. The prediction results showed that rs1927914 in the promoter TLR4 affected the binding capability of the organic cation transporter 1 (Oct-1) which is a member of the POU homeodomain family of transcription factors (Verrijzer & Van der Vliet, 1993). The main feature of this family is its highly conserved original POU domain composed of 150 amino acids, which has a high affinity for the octamer binding sequence 5′-ATGCAAAT-3′ (Verrijzer et al., 1992). Studies have showed that Oct-1 was abnormally expressed in a variety of cancers and the overexpression of Oct-1 was associated with the poor prognosis in well-differentiated gastric adenocarcinoma patients (Jeong et al., 2014; Rhodes et al., 2007). In this study, our results demonstrated that TLR4 rs1927914 A>G genetic polymorphism contributed to a reduced risk of esophageal cancer. This finding was further supported by luciferase reporter assay which showed that TLR4 rs1927914 G-containing constructure displayed higher luciferase activity than rs1927914A-containing constructure. We also found that the oligonucleotide probe with TLR4 rs1927914G could bind with the nuclear extract from esophageal cancer cells using EMSA; however, that with rs1927914A allele couldn’t. There were several studies reported the association of rs1927914 polymorphism with the risk of other cancer types. For example, Shi and Minmin et al. reported that TLR4 rs1927914 genetic variations are correlated with the hepatocellular carcinoma susceptibility (Minmin et al., 2011; Shi et al., 2017). However, researchers didn’t find the correlation between TLR4 rs1927914 and the risk of lung or gastric cancer (Huang et al., 2010; Wu et al., 2020). Therefore, it is suggested that TLR4 rs1927914 may be associated with the occurrence of certain cancer type. For rs11536891 polymorphisms, we predicted that it affected the binding capability of hsa-miR-519a/hsa-miR-519b-3p; however, our study didn’t show this SNP on the risk of esophageal cancer. At present, there are few studies on the correlation between TLR4 rs11536891 polymorphism and cancer susceptibility. Researchers didn’t find that TLR4 rs11536891 was associated with the risk of prostate cancer and lung cancer (Song et al., 2009; Wu et al., 2020). Tsilidis et al. reported that this SNP was contributed to the colorectal cancer risk (Tsilidis et al., 2009). These findings suggested that TLR4 might promote esophageal cancer cell proliferation through different pathways.

In addition to genetic factors, epidemiological evidence also proved that cigarette smoking strongly elevated the susceptibility to ESCC (Abnet, Arnold & Wei, 2018; Chen et al., 2010; Dong & Thrift, 2017). Thus, we performed stratification analysis by smoking status and found TLR4 rs1927914 GG genotype carriers had decreased risk of ESCC among non-smokers, but not among smokers. Meanwhile, we found that GG genotype is a protective factor for older subjects. These results suggest that the risk of ESCC is mainly caused by the combination of environmental and genetic factors.

Conclusion

In summary, we found that rs1927914 A>G polymorphism in the promoter of TLR4 could affect the transcriptional activity of TLR4 and contributed to the susceptibility to ESCC. These data further supported the hypothesis that naturally occurring variants in innate immune genes conferred individual’s susceptibility to esophageal cancer. The TLR4 polymorphism might serve as a biomarker for evaluation of esophageal cancer risk.

Supplemental Information