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Carcinogenesis Advance Access originally published online on November 2, 2005
Carcinogenesis 2006 27(4):798-802; doi:10.1093/carcin/bgi258
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Identification of a novel polymorphism Arg290Gln of esophageal cancer related gene 1 (ECRG1) and its related risk to esophageal squamous cell carcinoma

Yuanyuan Li, Xuemei Zhang, Ge Huang, Xiaoping Miao, Liping Guo, Dongxin Lin and Shih-Hsin Lu *

Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China

* To whom correspondence should be addressed. Fax: 86 10 67712368; Email: shlu{at}public.bta.net.cn


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
We previously cloned and identified the esophageal cancer related gene 1 (ECRG1), a novel candidate tumor suppressor gene, from human esophageal cells. A single nucleotide polymorphism (Arg290Gln) was identified in the coding region of ECRG1 and might play a role in susceptibility to esophageal squamous cell carcinoma (ESCC). To examine this hypothesis, we analyzed 998 ESCC patients and 1252 controls in a hospital-based, case–control study in a Chinese population for this polymorphism. We observed a statistically significantly increased risk of ESCC associated with the ECRG1 290Arg/Gln and 290Gln/Gln genotypes compared with the 290Arg/Arg [odds ratio (OR) = 1.23, 95% confidence interval (CI) =1.03–1.46; P < 0.05]. A greater than multiplicative joint effect between the ECRG1 polymorphism and tobacco smoking exposure was also observed (OR = 1.95, 95% CI = 1.48–2.56; P < 0.001). Furthermore, the elevated risk of ESCC associated with the ECRG1 polymorphism was increased consistently with cumulative smoking dose. ORs (95% CI) for 290Arg/Gln and 290Gln/Gln genotypes among non-smokers and smokers who smoked ≤27 and >27 pack-years were 1.03 (0.78–1.35), 1.91 (1.36–2.67) and 2.08 (1.48–2.92), respectively (P trend test < 0.001). Taken together, our results indicate that the ECRG1 290Gln variant allele might be a genetic susceptibility factor for developing ESCC, especially in the smoking population.

Abbreviations: CI, confidence interval; ECRG1, esophageal cancer related gene 1; ESCC, esophageal squamous cell carcinoma; Miz-1, Myc-interacting Zn2+ finger protein-1; RFLP, restriction fragment length; OR, odds ratio; SSCP, single-strand conformational polymorphism; SNP, single nucleotide polymorphism


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Esophageal cancer ranks among the 10 most frequent cancers in the world, with a marked regional variation in incidence and mortality worldwide (1). In northern China, esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancers with a 5 year survival rate of <10%, and annually, there are ~250 000 newly diagnosed ESCC cases and 157 000 deaths (2). Epidemiological and ecological studies of ESCC have revealed that the incidence of ESCC is associated with several environmental risk factors, such as tobacco smoking, heavy alcohol drinking, micronutrient deficiency and dietary carcinogen exposure, all of which may be involved in the etiology of ESCC (3–6). However, studies in high-risk areas have also demonstrated a strong tendency towards familial aggregation or clustering of cases within families, suggesting that genetic susceptibility factors may also play an important role in the etiology of ESCC.

Although genetic abnormalities in several oncogenes and tumor suppressor genes frequently occur in ESCC and esophageal cancer cell lines (7–9), the events underlying the transformation of normal esophageal epithelia to malignant tumor cells are poorly understood. In recent years, many studies on this cancer have been focused on cloning and identifying novel esophageal cancer related genes, which might play important roles in the initiation and development of ESCC (10–11). The esophageal cancer related gene 1, ECRG1 (GenBank accession no. AF071882 [GenBank] .1), was cloned by the effective mRNA differential display technique through comparing the differential gene expression between normal esophageal epithelium and esophageal cancer cells from high incidence families of ESCC in our laboratory (12). We obtained a 1376 bp full-length cDNA of the ECRG1 gene by SMARTTM RACE technique (13) and found that the gene contains 10 exons, spanning ~54 014 bp on chromosome 4q13.2 and has a 1254 bp open reading frame encoding a 418 amino acid polypeptide. Furthermore, bioinformatics analysis indicates that the product of ECRG1 is a member of the membrane-anchored serine protease family (14), which contains three conserved tandem serine protease domains (His, Asp and Ser) that play a key role in proteolytic activity.

Previous analyses using reverse transcriptase-PCR and northern blot showed that the ECRG1 gene is expressed in normal esophagus, liver, colon and lung, but the expression is downregulated in tumors, especially in ESCC, and their adjacent tissues (13). In vitro and in vivo assays have also indicated that overexpression of ECRG1 protein inhibits tumor cell proliferation (15). Furthermore, it has been shown that ECRG1 protein is able to induce G1/S cell cycle arrest through upregulation of P15INK4b expression by specifically interacting with Myc-interacting Zn2+ finger protein-1 (Miz-1) in esophageal cell line 9706 (16). Together, these findings indicate that ECRG1 might play a role in the development of ESCC.

Mutations and genetic polymorphisms in coding sequences of a gene may cause functional alternation of the gene product, which in turn may be associated with certain disease phenotypes. The goal of this study was to investigate the potential relationship between single nucleotide polymorphisms (SNPs) in ECRG1 and the risk of developing ESCC. We screened mutations and SNPs in the coding region of ECRG1 in DNA samples from 80 individuals. Three SNPs were identified, which are located in exons 3, 8 and 9, respectively. Particularly, the SNP in exon 8 (869G->A) located in the Asp conserved region of ECRG1 serine protease catalytic domain results in 290Arg->Gln amino acid change. We hypothesized that the 290Arg->Gln polymorphism might be associated with increased risk of ESCC because this SNP might affect the function of ECRG1 protein (17–18). In this paper, we report a case–control study examining this hypothesis.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Study subjects
There were two populations in this study. The DNA samples of 80 patients with ESCC coming from Linxian, a high-risk area for esophageal cancer in northern China, were used to screen SNPs in the coding region of ECRG1. The case–control study consisted of 998 patients with ESCC and 1252 population control subjects, who were unrelated ethnic Han Chinese and residents of Beijing and the surrounding regions. Patients were recruited between July 20, 1999 and December 20, 2001, at the Cancer Hospital of the Chinese Academy of Medical Sciences (Beijing, China). All patients with histologically confirmed ESCC were enrolled, yielding a 94% response rate. There was no sex and age restriction. Of all the 998 patients, 791 (79.3%) underwent surgical resection and had detailed histological differentiation data. Control subjects were cancer-free individuals who were randomly selected from a nutritional survey database conducted in Beijing and the surrounding regions between July 28, 1999 and December 12, 2001. The response rate for control subjects was 89%. The selection criteria for the control subjects included no individual history of cancer, and control subjects were frequency matched to case patients on the basis of sex and age (±5 years). At recruitment, informed consent was obtained from each subject, and personal data from each participant regarding demographic characteristics, such as sex, age and related risk factors including tobacco smoking were collected via questionnaire. This study was approved by the Institutional Review Board of the Chinese Academy of Medical Sciences Cancer Institute.

Identification of SNPs in ECRG1 coding region
The genomic DNA samples of 80 patients with ESCC from Linxian were isolated using standard methods (19) from surgically resected tumors, normal tissues adjacent to the tumors and peripheral blood lymphocytes. PCR primers corresponding to the sequences of ECRG1 exon 1–10 were designed by software Primer 3. The PCR products were diluted 2–25-fold depending on the band intensity on 1.5% agarose gel and analyzed by single-strand conformation polymorphism (SSCP) method. For the SSCP analysis, 10 µl of PCR product was mixed with 10 µl of loading buffer (95% formamide, 20 mM EDTA, 0.05% xylene gyanole and 0.05% bromphenol blue). This mixture was denatured at 98°C for 5 min and then immediately put on ice and loaded on the 10% non-denaturing polyacrylamide gel (29:1, acrylamide:biasacrylamide). The gel was electrophoresed in Tris–borate–EDTA (TBE) buffer at 30 W for ~8–12 h at 4°C and subsequently stained with silver and photographed. Samples with band shifts were examined by direct sequencing to determine the patterns of nucleotide changes.

Genotype analysis in cases and controls
Genomic DNA was isolated from peripheral blood of the patients with ESCC and controls. The genotypes of exon 8 at the Arg290Gln site in ECRG1 were analyzed by PCR-restriction fragment length polymorphism (RFLP) assays as described below, which were performed in a blinded manner (i.e. the case–control status of participants was unknown to those performing this test). The PCR amplification was accomplished with a 25 µl reaction mixture consisting of 50 ng template DNA, 0.4 µM each primer, 0.2 mM each deoxynucleotidetriphosphate (dNTP), 2.0 mM MgCl2 and 1.0 U Taq DNA polymerase with 1x reaction buffer (Takara, Japan). PCR primers for amplifying DNA fragment containing the Arg290Gln polymorphism were 5-CAGGGCTTAGCGCTCTGTTA-3 and 5-GCTCATATACTTTGGGCAGCTT-3, which produce a 354 bp fragment. The reaction conditions consisted of an initial melting step of 2 min at 94°C; followed by 35 cycles of 30 s at 94°C, 30 s at 58°C and 30 s at 72°C; and a final elongation step of 7 min at 72°C.

The gain of a MspI (New England BioLabs, USA) restriction site occurs in the polymorphic allele, so the restriction enzyme MspI digestion was used to distinguish the Arg290Gln polymorphism. The 290Gln/Gln genotype has a single band representing the entire 354 bp fragment, the variant 290Arg/Arg genotype results in two fragments of 232 and 122 bp, and the heterozygous 290Arg/Gln genotype has all of three fragments of 354, 232 and 122 bp. The restricted product was analyzed by electrophoresis in a 2% agarose gel stained with ethidium bromide (Figure 1A.). Three genotypes revealed by RFLP with MspI digestion were confirmed by DNA sequencing (Figure 1B). To ensure quality control, >20% random DNA samples were genotyped twice by different persons, and the results were concordant for all masked duplicate sets.


Figure 1
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  		Fig. 1. Analysis of the ECRG1 Arg290Gln polymorphism. (A) Representative gel picture showing PCR–RFLP analysis of the ECRG1 genotypes in genomic DNAs of study subjects with the restriction enzyme MspI. M, DNA size markers; Subjects 1, 3 and 7, Arg/Arg genotype; Subjects 2 and 6, Arg/Gln genotype; Subjects 4 and 5, Gln/Gln genotype. (B) Partial DNA sequence of three different allelic PCR products analyzed directly with an ABI PRISM 377 automatic sequencer showing a G-to-A transition at the nucleotide location at which the arrow points.

 
Statistical analysis
The {chi}2-test was used to examine differences in demographic variables, smoking and distribution of genotypes between cases and controls. The associations between genotype and risk of ESCC were estimated by calculating odds ratios (ORs) and their 95% confidence intervals (95% CIs) with unconditional logistic regression models. The ORs were adjusted for age, sex and pack-years smoked. Smokers were considered current smokers if they smoked up to 1 year before the date of cancer diagnosis or if they smoked up to 1 year before the date of the interview for control subjects. Information was collected on the number of cigarettes smoked per day, the age at which the subjects started smoking and the age at which the smokers stopped smoking. Subjects who never smoked or smoked <1 year before the date of cancer diagnosis for case patients or the date of interview for control subjects were defined as non-smokers. The light and heavy smokers were categorized by using the 50 percentile pack-year [(cigarettes per day/20) x (years smoked)] values of the controls as the cut-off points (i.e. ≤27 and >27 pack-years). A P-value of <0.05 was used as the criterion of statistical significance, and all statistical tests were two-sided. All analyses were done with the computer programs of Statistical Analysis System (version 6.12; SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The 80 DNA samples isolated from tumors, normal esophageal tissues adjacent to the tumors and peripheral blood lymphocytes of patients from Linxian were screened to identify mutations and SNPs in the ECRG1 coding region. Although we did not find any somatic mutations in this region, three SNPs located in exons 3, 8 and 9 were identified (Table I). All these SNPs were presented in the ECRG1 gene in DNAs isolated from tumor, normal adjacent tissue and blood of the same subject. The SNP in exon 8 (869G->A) results in Arg->Gln amino acid change at codon 290, yielding three type variations, i.e. 290Arg/Arg, 290Arg/Gln and 290Gln/Gln (Figure 1), which was subsequently used for the molecular epidemiological study in a case–control set.


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  		Table I. The characterization of three new SNPs in the coding region of ECRG1

 
The demographic data of case–control study subjects are summarized in Table II. No statistically significant differences were found between the cases and controls in terms of age and sex distributions, suggesting that the frequency matching was adequate. Although there were more smokers in the case group (61.2%) than in the control group (53.7%; P < 0.001), no significant difference was found between cases and controls in value of pack-years (P = 0.21). Among the 791 case patients with detailed histological differentiation background, 120 (15.2%) had primary and well-differentiated tumors (Grade 0–I), 250 (31.6%) had moderate-differentiated tumors (Grade II), 385 (48.6%) had low-differentiated tumors (Grade III) and 36 (4.6%) had undifferentiated tumors (Grade IV).


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  		Table II. Distributions of select characteristics of case–control status

 
Allelic frequencies and genotype distributions of the ECRG1 Arg290Gln polymorphism in cases and controls are shown in Table III. The allelic frequencies for ECRG1 290Arg and 290Gln were 82.2 and 17.8% among controls, and 79.6 and 20.4% among ESCC cases, respectively, with the 290Gln allele being significantly more prevalent in cases than in the controls, suggesting that it might be a risk allele for ESCC. Because the 290Gln/Gln genotype was relatively infrequent, it was combined with the 290Arg/Gln genotype as a group for analysis. The distributions of ECRG1 genotypes in cases and controls were consistent with Hardy–Weinberg equilibrium (P = 0.65 and 0.80, respectively). The frequencies for ECRG1 290Arg/Gln and 290Gln/Gln genotypes among cases were significantly higher than those among controls, suggesting that subjects carrying at least one 290Gln allele had an increased risk for the development of ESCC compared with subjects carrying the 290Arg/Arg genotype (OR = 1.23, 95% CI = 1.03–1.46, P < 0.05).


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  		Table III. Genotypic and allelic frequencies of ECRG1 among 998 patients and 1252 controls and their associations with the risk of ESCC

 
The effects of the ECRG1 genotypes (Arg/Gln and Gln/Gln versus Arg/Arg) were further examined by stratifying for potential variables such as age, sex, smoking status, smoking level and pathologic characteristics (Table IV). Because smoking is a risk factor for ESCC and affects the expression and function of many tumor suppressor genes (4,20–21), we first investigated whether an interaction existed between the ECRG1 290 Arg->Gln polymorphism and smoking status. Among the non-smokers, the variant genotypes of ECRG1 290Arg/Gln and 290Gln/Gln were not associated with elevated risk of ESCC (OR = 1.03; 95% CI = 0.78–1.35; P = 0.831). However, among smokers, the combined 290Arg/Gln and 290Gln/Gln genotypes were associated with a significantly increased risk of ESCC (OR = 1.95; 95% CI = 1.48–2.56), which was greater than that for the 290Arg/Arg genotype (OR = 1.35; 95% CI = 1.07–1.71; P < 0.004). Because the OR for the presence of both smoking and 290Arg/Gln or 290Gln/Gln genotype was greater than the product of OR for smoking and OR for the genotype (P < 0.001), these data clearly suggested a multiplicative joint effect between smoking and the ECRG1 polymorphism. Moreover, when the risk associated with the ECRG1 polymorphism was further evaluated with smoking levels (≤27 and >27 pack-years smoked), it increased consistently with cumulative smoking dose among smokers in which a multiplicative joint effect between the susceptible genotypes and categories of pack-years smoked was also observed. ORs (95% CI) of 290Arg/Gln and 290Gln/Gln genotypes for non-smokers and smokers who smoked (27 and >27 pack-years were 1.03 (0.78–1.35), 1.91 (1.36–2.67) and 2.08 (1.48–2.92), respectively (P < 0.001 for trend test). Thus, there appears to be an interaction between the ECRG1 290 Arg->Gln polymorphism and tobacco smoking that contributes to the risk for ESCC. In addition, effect of the ECRG1 290Arg/Gln and 290Gln/Gln genotypes on risk of ESCC seemed to be more pronounced in male subjects (OR = 1.33; 95% CI = 1.09–1.62; P < 0.005) than in female subjects. No association was found between ECRG1 genotypes and histological differentiation of ESCC (data not shown).


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  		Table IV. Risk of ESCC associated with ECRG1 genotypes by sex, smoking status and pack-year value

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this study, a new Arg290Gln variant allele in exon 8 of ECRG1, resulting in the expression of either Arginine (Arg) or Glutamine (Gln) at codon 290, which locates in the Asp conserved region of ECRG1 serine protease catalytic domain, was identified by PCR-based SSCP and DNA sequencing analysis. Because the substitution of amino acid located in the protein conserved domain always contributed to decreasing function of the protein (17–18), we hypothesized that this polymorphism might influence proteolytic activity of ECRG1 protein. A case–control study of ESCC in a Chinese population was carried out to examine this hypothesis and a significant association between the Arg290Gln polymorphism and susceptibility to ESCC was subsequently uncovered. Individuals who carried at least one 290Gln allele (290Gln/Gln or 290Arg/Gln genotype) were at increased risk for the development of ESCC. More importantly, we found a significant increased risk associated with the ECRG1 variant genotype among smokers, in which a multiplicative joint effect between the ECRG1 290Gln/Gln or 290Arg/Gln genotype and cigarette smoking was observed. Furthermore, the elevated risk of ESCC associated with the ECRG1 polymorphism was >2-fold among heavy smokers who carried at least one 290Gln allele compared with non-smokers who carried the 290Arg/Arg allele. These results are consistent with our hypothesis and warrants further functional studies on this polymorphism in ECRG1.

ECRG1 is a novel candidate of tumor suppressor gene identified in our laboratory (12). Previous studies have shown that the expression of ECRG1 is gradually downregulated from normal esophageal epithelium, adjacent tissue to esophageal cancer (13). The in vitro and in vivo assays also indicated that overexpression of ECRG1 protein inhibits tumor cell proliferation (15). In addition, ECRG1 has been shown to induce G1/S cell cycle arrest through upregulation of P15INK4b expression in esophageal cell line 9706 by specifically interacting with Miz-1 (16). Thus, ECRG1, which acts as a multifunctional protein associated with regulation of cell proliferation and cell cycle arrest in esophageal cancer cells, may play a role in the development of ESCC.

Bioinformatic analysis suggests that the ECRG1 protein is a member of the membrane-anchored serine protease family (14), which participates in proteolytic reactions that are essential to a diverse range of physiological and pathological processes (22). Regulation of such processes involves cell surface proteolysis, which is important not only for matrix remodeling but also for the regulation of growth and differentiation through activation and/or release of functionally diverse effector molecules, including cytokines, growth factors and cell surface receptors. Furthermore, the involvement of several serine proteases like ECRG1 during the stages of tumor suppression has been documented (23–24). The enzymatic properties of serine proteinases are dependent on a catalytic triad of His, Asp and Ser amino acids (25), which are presented in motifs that are highly conserved among family members. In the present study, individuals who carried at least one 290Gln allele had 1.23-fold higher risk for developing ESCC than those carrying homozygous 290Arg/Arg genotype. We, therefore, propose that the Arg-to-Gln substitution located in the conserved catalytic domain might reduce the ECRG1 catalytic capacity by impacting protein three-dimensional conformation and thereby cause genetic susceptibility to ESCC.

Importantly, a 2-fold increased ESCC risk associated with the ECRG1 290Gln variants among smokers but not non-smokers is an example for gene–environment interaction that may have played a role in the etiology of ESCC in this Chinese population. Higher risk of ESCC among smokers with the variant ECRG1 genotype may be attributed to the normal esophageal cells or initiated esophageal cancer cells resulting from exposure to tobacco carcinogen such as the nitrosonornicotine (NNK) (26), which in turn increases the possibility to become malignant under the condition of lower ECRG1 function. ESCC is a complex disease that may be attributed to the integrated outcome of exposure to endogenous and/or exogenous carcinogens (27). Therefore, interindividual differences in metabolic activation (28), detoxification process (29), DNA repair capacity (30) and tumor suppresser gene (31) reflecting the acquired and inherent host status may influence risk of developing this cancer. Our observation may be consistent with the fact that the genetic effect can be overwhelmed by the environmental effect. So our findings make a fine example for gene–environment interaction that plays a role in the etiology of ESCC in this Chinese population. Consequently, smoking and carrying at least one ECRG1 290Gln allele increase the risk of developing ESCC.

In conclusion, our study demonstrates a significant association between the ECRG1 genetic polymorphism and ESCC in a Chinese population. The association between the ECRG1 Arg290Gln polymorphism and risk of ESCC displayed a gene–environment interaction with tobacco smoking. These molecular epidemiological findings provide additional evidence that ECRG1 as a candidate tumor suppressor gene is an important determinant factor in susceptibility to ESCC.


   Acknowledgments
 
We thank professor Wei Jiang of the Buruham Institute Cancer Center for critical reading of the manuscript prior to publication. This study was supported by the State Key Basic Research Program (grant no.2004CB518701).

Conflict of Interest Statement: None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 5, 2005; revised October 18, 2005; accepted October 26, 2005.


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