Influence of CMTM8 polymorphisms on lung cancer susceptibility in the Chinese Han population

Supplemental Digital Content is available in the text. Background Lung cancer is the leading cause of cancer-related mortality worldwide and CMTM8 is a potential tumor suppressor gene, which is down-regulated in lung cancer. The objective of this research was to assess the association of CMTM8 genetic polymorphisms with lung cancer risk. Methods To evaluate the correlation between CMTM8 polymorphisms and lung cancer risk, Agena MassArray platform was used for genotype determination among 509 lung cancer patients and 506 controls. Multiple genetic models, stratification analysis and Haploview analysis were used by calculating odds ratio (OR) and 95% confidence intervals (CIs). Results Significant associations were detected between CMTM8 rs6771238 and an increased lung cancer risk in codominant (adjusted OR = 1.57, 95% CI: 1.01–2.42, P = 0.044) and dominant (adjusted OR = 1.54, 95% CI: 1.01–2.36, P = 0.047) models. After sex stratification analysis, we observed that rs6771238 was related to an increased risk of lung squamous cell carcinoma, while rs6771238 was associated with an increased risk of lung adenocarcinoma. Rs9835916 was linked to increased risk of lymph node metastasis in lung cancer patients. Conclusion Our study first reported that CMTM8 polymorphisms were a risk factor for lung cancer, which suggested the potential roles of CMTM8 in the development of lung cancer.


Background
Lung cancer is the leading cause of cancer-related mortality worldwide. Lung cancer is also a major health problem in China, where 605 946 new cases of lung cancer (416 333 men and 189 613 women) and 486 555 lung cancer-related deaths were reported in 2010. Despite the advance of therapeutic strategies, the prognosis of lung cancer patients remains poor, and the 5-year survival rate remains less than 10% in most parts of the world [1,2]. The reason for this lack of improvement may be due to the high invasion and recurrence rate of lung cancer. Lung cancer development appears to result from a complex interaction between environmental exposures and genetic factors. And genetic factors may play a fundamental role in the development of lung cancer [3]. Therefore, novel biomarkers for predicting the tumor progression of lung cancer are urgently needed.
CMTM8 (CKLF-like MARVEL transmembrane domain containing 8), also known as CKLFSF8, belongs to the chemokine-like factor gene superfamily, a novel family that was first cloned by the Peking University Human Disease Genomics Research Center in 2003 [4,5]. The encoding product of this family gene has a special structure, which is between chemokines and four transmembrane proteins. Studies have shown that CMTM8 is a potential tumor suppressor that can participate in various signal transduction pathways to control tumor occurrence and development, thereby affecting tumor formation, development and metastasis [6]. CMTM8 is widely expressed in many normal human tissues and is often downregulated or absent in multiple solid tumors, including the liver, lung, colon, rectum, esophagus and stomach [7]. The over-expression of CMTM8 can inhibit the proliferation, migration, and invasion of carcinoma cells [8]. Hence, CMTM8 is a potential marker of early tumor detection in many cancers, including lung cancer.
Single nucleotide polymorphism (SNP), as a natural sequence variation, may affect the expression level of CMTM8. As for genetic variation, many SNPs sites affecting the occurrence of lung squamous cell carcinoma have been reported in GWAS studies in Chinese Han population [9,10]. However, the risk of CMTM8 polymorphisms on lung cancer has been unexplored to date. Therefore, we performed a case-control study to determine whether CMTM8 SNPs impact susceptibility to lung cancer in Chinese Han population.

Study participants
The present hospital-based case-control study included a total of 509 lung cancer cases and 506 healthy controls. Lung cancer subjects were recruited from the Tumor Hospital of Shaanxi province, China. Controls were taken from the people who visited the hospital for routine checkup with no history of cancer and any diseases associated with vital organs. All lung cancer cases were newly diagnosed and histopathologic confirmed.

Clinical data and demographic information
We use a standardized epidemiologic questionnaire including residential region, age, sex, smoking status, alcohol use, ethnicity, education status and family history of cancer to collect personal data in an in-person interview. Venous blood sample of 5 ml was drawn from each subject and used for DNA extraction and genotyping. All volunteers signed an informed consent form explaining the research purpose of the blood withdrawal.

Single nucleotide polymorphisms selection and genotyping
We screened the SNPs of CMTM8 with minor allele frequencies greater than 5% in 1000 genome project (http://www.internationalgenome.org/). The distribution of SNPs genotypes in control group was in accordance with Hardy-Weinberg equilibrium (HWE) (P > 0.05). We used Agena MassARRAY RS1000 to genotype and we retained the SNPs with call rate over 95.0%. Six SNPs were genotyped on the MassARRAY iPLEX (Agena Bioscience, San Diego, California, USA) platform using matrix-assisted laser desorption ionization time-of-flight. Then, the Haploview software package (version 4.2) was used to estimate pairwise linkage disequilibrium (LD) at CMTM8 polymorphism loci. When r 2 (the measure value of LD) > 0.8, the SNP can represent all the polymorphisms in a block. According to the above selection criteria, we selected the six SNPs (rs9853415, rs6796318, rs6771238, rs9835916, rs1077868 and rs6802418) in CMTM8 as the gene variation to study. Genomic DNA was isolated from peripheral whole blood employing the Gold Mag -Mini Whole Blood Genomic DNA Purification Kit (Gold Mag Co. Ltd., Xi'an, China) following the manufacturer's instructions and quantified by Nano Drop spectrophotometer 2000C (Thermo Scientific, Waltham, Massachusetts, USA). Primers in this study were listed in Supplementary Table S1, Supplemental digital content 1, http://links.lww.com/FPC/B387. SNPs genotyping analysis was performed on Agena MassARRAY RS1000 instrument (Shanghai, China) system according to the standard scheme recommended by the manufacturer, and data were managed and analyzed by Agena Typer 4.0 software [11,12].

Statistical analyses
Allele frequencies and genotype frequencies for each SNP of case and control subjects were compared using the Chi-squared test. HWE of each SNP in control group was tested by Fisher s exact test [5]. Odds ratios (ORs) and 95% confidence intervals (CIs) were determined using unconditional logistic regression analysis with adjustments for age, sex, smoking and drinking [13][14][15]. Associations between genotypes and lung cancer risk were tested in different genetic models (co-dominant, dominant, recessive, and log-additive) by SNPStats website software (http://bioinfo.iconcologia.net/snpstats/start.htm) [16,17]. All statistical analysis was performed using SPSS statistical package, version 19.0 (SPSS Inc., Chicago, Illinois, USA). Haploview software version 4.2 was used to analyze the association between haplotypes and the lung cancer [18]. The power of the significant difference was calculated by Power and Sample Size Calculation software (http:// biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize) [19]. All P values in this study were two-sided, and a P-value of less than 0.05 as the cutoff value for statistical significance.

General characteristics
Essential characteristics of the case and control groups were revealed in Table 1. This study involved 1015 subjects, including 509 patients (354 males and 155 females; age at diagnosis: 58.53 ± 10.12 years) and 506 healthy controls (355 males and 151 females; age: 61.43 ± 9.47 years). There were no significant differences in terms of   age, sex or smoking status between lung cancer patients and healthy controls, but there were significant differences in alcohol consumption.

Hardy-Weinberg equilibrium and single nucleotide polymorphisms alleles
The minor allele frequency distribution of selected six SNPs among all subjects was summarized in Table 2.
The allele frequency of each SNP in controls was consistent with the CHB population (Han Chinese in Beijing, China) in the 1000 genome project. Furthermore, all six SNP locus in the control subjects conformed to HWE (P > 0.05).

Association of single nucleotide polymorphisms with lung cancer risk
Four genetic analysis models (co-dominant, dominant, recessive and log-additive) were applied to analyze and assess the association between each variant and lung cancer risks. In Table 3   Stratified subgroup in the case of lymph node metastasis, rs9835916 was found to be associated with lymph node metastasis risk in patients with lung cancer.  Table 6).

Association of haplotypes with lung cancer risk
A haplotype-based association study was performed to show the association between CMTM8 haplotype and risk of lung cancer. Among the subpopulation (staging), two SNPs (rs1077868 and rs6802418) form an LD block (Fig. 1). The frequencies' distribution of haplotypes in case and control group is presented in Table 7. The haplotypes 'GG' and 'AG' was found to prominently increase the risk of lung cancer staging (OR = 1.71; 95% CI: 1.02-2.88; P = 0.043).

Single nucleotide polymorphism functional evaluation
In order to evaluate the possible function of the six selected variants in the CMTM8 gene, we performed a bioinformatics analysis using the HaploReg v4.1 database. The results showed that all the variants were predicted to be regulatory SNPs with different biologic functions (Supplementary Table S2, Supplemental digital content 1, http://links.lww.com/FPC/B387).

GEPIA database analysis on gene expression
Furthermore, GEPIA database analyzed the expression of CMTM8 gene in lung cancer and found that the expression level of CMTM8 gene in lung adenocarcinoma was lower than that in normal tissues, which indicates that

Discussion
In recent years, a growing number of studies have found that the CMTM8 gene plays an important role in the tumor s formation, development and metastasis, and the expression of CMTM8 is down-regulated in lung cancer. In our study, we genotyped six polymorphisms of CMTM8 and evaluated their correlations with the risk of lung cancer. Our results firstly showed that rs6771238 was associated with an increased lung cancer susceptibility in Chinese Han Population. Stratified analysis showed that rs6771238 was related to an increased the risk of lung squamous cell carcinoma, rs6771238 was associated with an increased the risk of lung adenocarcinoma, rs9835916 and rs1077868 were correlated with lung cancer staging, and rs9835916 was correlated with an increased the risk of lymph node metastasis in lung cancer patients. Haplotype analysis illuminated that 'GG' and 'AG' were closely correlated with lung cancer staging, and 'AG' was correlated with an increased lung cancer risk among individuals older than 50 years. To our knowledge, this is the first study to explore the association between CMTM8 gene polymorphism and lung cancer risk in Chinese Han Population.
Human CMTM8 localizes to chromosome 3p22.3, where other known tumor suppressor genes that are frequently deleted or methylated in tumors reside [20,21]. CMTM8 may be silenced or down-regulated in a similar manner during tumorigenesis. Previous studies demonstrated that CMTM8 induces caspase-dependent and caspase-independent apoptosis in multiple tumor cell lines [4]. Downregulation of CMTM8 in epithelial cells induces epithelial-mesenchymal transformation through MEK-ERK signaling [6]. Overexpression of CMTM8 can accelerate the rate of epidermal growth factor receptor internalization, attenuates epidermal growth factor receptor-mediated signaling, and inhibits tumor cell growth [5]. At present, indications for tumor suppressive function of CMTM8 gene products have been found in several tumor types. In osteosarcoma, it was confirmed that CMTM8 was identified as a candidate tumor suppressor gene, which inhibited the EGFR signaling pathway and affected the occurrence of osteosarcoma [5,22]. CMTM8 underexpression may result in upregulation of EGFR signaling. In bladder cancer, CMTM8 is also an important tumor suppressor gene and a useful prognostic indicator for patients with bladder cancer [8,23]. It is inferred that CMTM8 overexpression blocks c-MET signaling in-vivo model of bladder cancer. Research has also demonstrated that the downregulation of CMTM8 induced epithelial-to-mesenchymal transition-like changes via c-MET/ extracellular signal-regulated kinase signaling in HepG2 hepatocellular carcinoma cells, thereby affecting the cancer process [6]. In addition, CMTM8 was negatively correlated with the tumorigenesis and development of clear-cell renal cell carcinoma, and the location and intensity of expression were significantly correlated with prognosis. Reduced CMTM8 expression is associated with lower survival rates in lung squamous cell carcinoma progression [24].
However, the expression of CMTM8 in lung cancer is only known to be down-regulated, and other relevant reports are relatively few. Our results suggested that rs6771238 was associated with an increased lung cancer. Stratified analysis showed that rs6771238 was associated with the risk of lung squamous cell carcinoma, rs6771238 was associated with an increased the risk of lung adenocarcinoma, rs9835916 and rs1077868 were associated with lung cancer stage, and rs9835916 was associated with lymph node risk in lung cancer patients. In addition, it should be noted that the incidence of lung cancer significantly different according to the different stratification. Considering the potential function of the selected SNPs in our study, we speculated that SNPs may affect the carcinogenic process by changing the protein expression and this process may be influenced by individual background, thus leading to different outcomes on lung cancer risk.
Finally, I need to explain the deficiencies of our experiment. Because the examinee come from the same hospital, inherent selection bias and information bias are inevitable problems. Our current research is only at the gene level, which needs to be verified and explored in many aspects. We just provide the theoretical basis for the follow-up scientific research.

Conclusion
Our study revealed a novel association between CMTM8 polymorphisms and the risk of lung cancer among Chinese Han population. These results may help elucidate the underlying mechanisms for CMTM8 polymorphisms in lung cancer. Larger sample sizes and mechanism studies are necessary to further explore and confirm the role of these variants in increasing lung cancer risk, which will help in better understanding the genetic heterogeneity in complex diseases like lung cancer.