Osteopontin promoter polymorphisms associated with
susceptibility of gastric cancer
Short Title: OPN polymorphisms in gastric cancer
Teng-Yu Lee, Jaw-Town Lin4, 吳 誠 中 ， 余 政 展 ， Ming-Shiang Wu4, Tso-Ching Lee5,
Hsiao-Ping Chen1,6, and Chun-Ying Wu1,2,3
1Division of Gastroenterology, Taichung Veterans General Hospital, Taichung, Taiwan; 2College of Public Health, China Medical University, Taichung, Taiwan;
3Department of Internal Medicine, National Yang-Ming University, Taipei, Taiwan; 4Division of Gastroenterology, National Taiwan University Hospital, Taipei, Taiwan; 5Molecular Genetic Laboratory, Taichung Veterans General Hospital, Taichung, Taiwan; 6Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan 7Department of Dermatology, Taichung Veterans General Hospital, Taichung, Taiwan; 8College of Public Health, National Taiwan University, Taipei, Taiwan;
* Corresponding author:
Dr. Chun-Ying Wu, Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan. 155, Sec. 2, Linong Street, Taipei 112, Taiwan, Tel: +886-4-23592525 # 3304, Fax: +886-4-23741331, E-mail: [email protected]
Acknowledgement: this work was supported by the National Science Council, Executive Yuan, Taipei, Taiwan (Grant number: NSC-96-2314-B-075A-007).
ABSTRACT
Aims: The expression of osteopontin (OPN) has been correlated with development, invasiveness, metastasis and survival of gastric cancer, but the role of polymorphisms in the OPN promoter is not investigated. We aim to evaluate the significance of OPN genotypes in the susceptibility of gastric cancer.
Methods: We enrolled 146 gastric cancer patients and 128 controls. DNA was extracted from peripheral blood leucocytes. Single-nucleotide polymorphisms (SNPs) in the OPN promoter (-66, -156, -443, -616, -1748, and -1776) were analyzed by PCR-direct sequencing method. Results: SNP -443 C/C and -616 T/T of the OPN promoter were significantly associated with gastric cancer (OR=2.88 (95% CI 1.16 – 7.12) and 1.95 (95% CI 1.35 – 2.82), respectively). Checking the combination effect of OPN promoter SNPs, combination of SNP -443 T/C, -443 C/C, -616 T/T and -616 T/G had the most significant association with gastric cancer (OR=3.95; 95% CI 1.58 – 9.90).
Conclusion: Our results suggest polymorphisms in the OPN promoter play an important role in the development of gastric cancer, and the combination of SNP -433 (T/C+C/C) and -616 (T/T+T/G) most significantly increases susceptibility of gastric cancer.
INTRODUCTION
Gastric cancer remains a leading cause of cancer mortality, and as high as 80% of gastric cancer patients have advanced cancer on diagnosis with poor prognosis.1 Even some
risk factors of gastric cancer, such as Helicobacter pylori (HP) infection, intestinal metaplasia, salty diet, smoking, alcohol, family history, genetics and epigenetic factors, have been identified, only detection for early-stage gastric cancer can improve outcomes of due to poor responses to advanced gastric cancer.2 Aggressive endoscopic screening strategy for
persons in high risk of gastric cancer development may early detect gastric cancer and improve outcomes3, so finding useful indicators for identifying high risk population,
especially who have multiple risk factors, is important.
Osteopontin (OPN), also known as early T cell activation gene 1 (Eta-1) or secreted phosphoprotein 1 (SPP1), is a secreted protein involved in wide variety of different functions such as stress response, inflammation, wound healing, and immunoregulatory responses.4
OPN has played an important role for cancer progression and prognosis in multiple tumor types5, such as lung cancer6, breast cancer7, colorectal cancer8, and hepatocellular carcinoma9.
The expression of OPN in tissues has been correlated with development, invasiveness, metastasis and survival of gastric cancer10-12, and we also found plasma OPN level might have
potential usefulness as a diagnostic and prognostic factor for gastric cancer13. However, the
role of OPN gene polymorphisms in gastric cancer is still not investigated, so we aim to evaluate the significance of OPN genotypes in the susceptibility of gastric cancer.
OPN gene polymorphism may affect binding affinity of transcriptional factors and gene expression, and it has been associated with disease development, phenotypes and severity in various diseases such as asthma, type 1 diabetes mellitus, Lewy body disease, systemic lupus erythematosus, systemic sclerosis and urinary calcium stones.14-19 Moreover, polymorphisms
have been reported to play an important role in the pathogenesis of some diseases such as diastolic dysfunction in hypertensive heart22, Duchenne muscular dystrophy23, urolithiasis24,25,
chronic hepatitis C26, oral squamous cell carcinoma27, melanoma28, and atherosclerosis29. The
relation between polymorphisms in the OPN promoter and susceptibility of gastric cancer has not been studied, so we evaluate their association in the present study.
MATERIALS AND METHODS
Study Subjects
In this nested case-control study, blood samples were prospectively collected from individuals participating in 2 national projects to investigate the risk factors of gastric cancer in Taiwan between January 1989 and January 1995. All participants consented to join on a voluntary basis after a full verbal explanation of the study. All patient-derived specimens were collected and archived under protocols approved and supported by the National Science Council, Taiwan. Newly diagnosed patients with gastric cancer undergoing gastrectomy in the inpatient unit and outpatient cancer clinics of three major medical centers in Taiwan were recruited. Inclusion and exclusion criteria were detailed previously.30,31 Gastric
adenocarcinoma was histopathologically confirmed by surgical specimens. No patients in this study had been prescribed chemotherapy or radiotherapy prior to surgery, and there was no evidence of any other malignancy. Patients who died of surgical complications in the first 30 days were excluded. In total, we studied 146 consecutive patients with gastric cancer, for whom complete clinical data and a peripheral blood leukocytes DNA sample were available. All patients were Han Chinese, and none had a family history of gastric malignancy.
Control blood samples were obtained from 128 individuals who visited health examination clinics with minimal gastritis or normal appearance of the gastric mucosa on gastroscopic examination. The controls were matched by age ( 3 years) and date of blood
collection ( 3 months).
Laboratory analysis
Genomic DNA from study subjects were isolated from cryopreserved white blood cells by standard proteinase K digestion and phenol-chloroform method. Polymorphism analyses for OPN (-66T/G, -156del G/G, -443T/C, -616T/G, -1748A/G, -1776T/C) were performed in duplicate by pyrosequencing. The forward, reverse and pyrosequencing primers were summarized in Table 1. PCR began with at 5-min incubation at 95C for initial denaturing, followed by 45 cycles at 30s for 95C, 30s at 60C, and 30s at 72C. and at 72C for 10 minute. The amplification was verified on an agarose gel (1%) followed directly by pyrosequencing and sequencing with an automatic sequencer using fluorescent DNA capillary electrophoresis. The forward primers were used for the sequencing primer. All laboratory assays were conducted and interpreted blindly without the knowledge of the case or control status. H. pylori status was determined by serum samples using standard enzyme-linked immunosorbent assay.32
Statistical analysis
The demographic characteristics of patients and controls were compared using the chi-square test and Student’s t test. Logistic regression analyses were used to evaluate the associations between promoter polymorphisms and development of gastric cancer. Odds ratios (ORs) were presented with 95% confidence intervals (95% CIs) and adjusted for age and sex. Genotype combination effect of OPN promoter SNPs in gastric cancer patients and controls were also analyzed using logistic regression analysis. All p values were two-tailed with statistical significance indicated by a value of p < 0.05. All data were analyzed via the SPSS program for Windows 11.0 (SPSS Inc. Chicago, Illinois, USA).
RESULTS
Demographic data
Totally, we recruited 146 gastric cancer patients and 128 controls, and their demographic data are summarized in Tables 2. There was no difference in the distributions of age and gender. Gastric cancer patients had higher prevalence of H. pylori infection when compared with control subjects (61.0% vs. 48.4%, p = 0.04).
Polymorphisms in the OPN promoter
Prevalence of Single-nucleotide polymorphisms (SNPs) in the promoter region of OPN gene in gastric cancer patients and controls are summarized in Tables 3. There was no statistically significant difference in the prevalence of promoter SNP -66 T/G, -66 T/T, -56 delG/delG, -56 delG/G, -56 G/G, -443 T/T, -443 T/C, -616 G/G, -616 T/G, -1748 A/A, -1748 A/G, -1748 G/G, -1776 T/T, and -1776 T/C. However, gastric cancer patients had higher prevalence of SNP -443 C/C when compared with control subjects (14.4% vs. 6.3%, p = 0.022), and SNP -443 C/C had significantly higher risk of developing gastric cancer when compared with -443 T/T (OR 2.88 ; 95% CI: 1.16 – 7.12). In addition, gastric cancer patients had higher prevalence of SNP -616 T/T when compared with control subjects (28.1% vs. 21.5%, p < 0.001), and SNP -616 T/T had significantly higher risk of developing gastric cancer when compared with -616 G/G (OR 1.95; 95% CI: 1.35 – 2.82).
Combination effect of different genotypes
We also analyzed the combined effect of different promoter genotypes, which including various haplotypes of SNP -443 and -616, on the risk of gastric cancer (Table 4). No statistically significant combination effect was found when various haplotypes combined with homozygous SNP -443 T/T or -616 G/G, but gastric cancer patients had higher prevalence of SNP -443 T/C, -443 C/C, -616 T/T and -616 T/G combination when compared with control
subjects (43.2% vs. 24.2%, p < 0.01). The combination of SNP -443 T/C, -443 C/C, -616 T/T and -616 T/G had the most significant association with risk of gastric cancer development (OR=3.95; 95% CI 1.58 – 9.90).
DISCUSSION
In the present study, the association of OPN promoter polymorphisms with gastric cancer is firstly reported, and we found that SNP -443 C/C and -616 T/T of the OPN promoter were significantly associated with risk of gastric cancer development. It has been proven that different haplotypes related to variants in the promoter of OPN gene may influence the individual degree of gene expression, and the levels of OPN protein expression are therefore different.20,21 Moreover, it has also been reported that polymorphisms in the
OPN promoter increased binding affinity of transcriptional factors, and promoter activity could be marked enhanced.21 The expression of OPN has been correlated with carcinogenesis
of gastric cancer10-12, and it is reasonable that polymorphisms of the OPN promoter are
significantly associated with risk of gastric cancer development.
Although polymorphisms in the OPN promoter may affect its transcriptional activity20,21,
we found that some promoter SNPs, such as -66, -156, -1748, and -1776, are not associated with gastric cancer. Variability in sequences may be targets of different factors triggered by stimulations such as inflammation, necrosis, growth factors, and hormones, and the functional properties of individual variability can be different in various clinical conditions.20
Data regarding polymorphisms of the OPN promoter in gastric cancer is lacking, so our findings cannot be directly elucidated by data coming from conditions other than gastric cancer. Moreover, OPN is a multifunctional protein, and other levels of regulation should also be considered. Even OPN activity affected by polymorphisms in the OPN promoter have been reported in healthy individuals or specific disease conditions20-22,27,28, only specific
cancer development in the present study.
The SNP -443 has been reported that might have differential binding with a putative binding site for the MYT1 zinc finger transcription factor20, which may involve in the
regulation of 213Bi-induced death in gastric cancer cells33. In addition, the -443 T/C can
influence OPN gene expression in melanoma cells via binding of c-Myb transcription factor28, which may involve in the regulation of prostaglandin E2-induced thioredoxin-related
protein-1 in gastric cancer cells34. In a study of oral squamous cell carcinoma, polymorphic
genotypes -443 C/C and -443 T/C were found to be 2.6 and 1.35 folds higher than those in controls, and the linkage disequilibrium of the -156 and -443 SNPs was also found.27
Previous findings suggested that the -443 SNP may involve in carcinogenesis, and further investigation focusing on the cascades of SNP -443-related gene expressions in gastric cancer is needed.
In studies of chronic hepatitis C26,35, SNPs in the promoter region of -443, -616 and
-1748 with linkage disequilibrium were found to be useful as a marker to predict the therapeutic efficacy of interferon therapy, especially in patients with genotype 1b and a high viral load, and the SNPs may be crucial in provoking diverse Th1 immune reactions against viruses through the regulation of OPN expression in the liver. Helicobacter pylori infection is a major risk factor for gastric cancer, and attention to the role of Th1 pathway in H. pylori-related gastric diseases is increasing.36 In a recent study, Helicobacter pylori infection
increased gastric OPN expression, and OPN expression was correlate with severity of gastric inflammation and intestinal metaplasia.37 Moreover, they found OPN predominantly
originates from inflammatory cells. The involvement of OPN in immune response has been proven, and immunoregulatory responses of OPN involved in gastric cancer development need further studies. Although studies on the cascades of SNP -616-related gene expressions are sparse, our findings provide a useful clue for further investigations.
One of the interesting findings of this study was that combination of SNP -443 T/C, -443 C/C, -616 T/T and -616 T/G in the OPN promoter had the most significant association with gastric cancer, and above mentioned OPN genotype -443 C/C and -616 T/T, which significantly associated with gastric cancer, were included. In recent years, there has been an increasing recognition that combination of indicators may have better diagnostic or prognostic value than individual one.5,27 The synergistic effects of different OPN SNPs had
been observed, and for example, all the patients who had SNP -1748 G/G or G/A and -443 T/T obtained a successful virological response after interferon-based therapies for chronic hepatitis C35. The mechanisms of combination effect may be due to influence on
transcriptional activity, epigenetic modification, binding with transcriptional factors or post-transcriptional modification, and further studies are needed.
There are several limitations to our study. First, selection bias could not be completely excluded in this hospital-based case-control study, although we tried to enroll cases from
three different hospitals in different regions of Taiwan. Future studies based on community populations will be helpful in excluding selection bias. Second, the sample size of the present study is relatively small for detecting the moderately increased risk of gastric cancer associated with the promoter polymorphisms. For example, the statistical significance of OPN promoter SNP -616 T/G in gastric cancer risk were only borderline (p = 0.052). Further studies with larger sample size may help to clarify the impact of SNPs in borderline statistical significance.
In conclusion, our study provides the first evidence that polymorphisms in the OPN promoter play an important role in the carcinogenesis of gastric cancer, and the combination of OPN SNPs -433 (T/C+C/C) and -616 (T/T+T/G) most significantly increases susceptibility of gastric cancer. Additional studies, especially among different ethnic populations, are necessary to confirm these observations.
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