Abstract
Aim: Tissue inhibitors of metalloproteinases (TIMPs) are a family of multifunctional proteins whose expression has been shown to be up-regulated in various types of cancer. However, the contribution of TIMPs to lung cancer is not known. The present study aimed to evaluate the contribution of TIMP1 rs4898, rs6609533 and rs2070584 genetic polymorphisms to the risk of lung cancer. Materials and Methods: The contributions of these TIMP1 genotypes to lung cancer risk were investigated in 358 patients with lung cancer patients and 716 age- and gender-matched healthy controls. Results: The results showed that the percentages of TT, CT and CC for TIMP-1 rs4898 genotypes were 28.5%, 33.2% and 38.3% in the patient group and 34.5%, 41.2% and 24.3% in the non-cancer control group, respectively (p for trend=1.21×10−5). The CC genotype carriers were at higher risk for lung cancer (odds ratio=1.91, 95% confidence interval=1.38-2.63, p=0.0001) than the TT genotype carriers. We also analyzed the allelic frequency distributions and the results showed that the C allele of TIMP1 rs4898 increased lung cancer susceptibility (p=1.26×10−5). On the contrary, there was no difference in the distribution of genotypic or allelic frequencies among patients and the controls for TIMP1 rs6609533 and rs2070584. Conclusion: The CC genotype of TIMP1 rs4898 compared to the TT wild-type genotype may increase lung cancer risk in Taiwan and may serve as a marker for early detective and predictive purposes.
Statistically, lung cancer has been the leading cause of cancer mortality world wide for years (1, 2). Even though new antitumor therapies are being developed, the prognosis of patients with lung cancer remains poor, with a 5-year survival rate of less than 20% (3). The most well-known factor implicated in lung cancer etiology is the individual long-term habit of tobacco consumption, which is also useful for prognosis prediction (4, 5). Although cigarettes contain various kinds of carcinogens that may increase reactive oxygen species, DNA adducts and strand breaks in lung cells, however, there are also epidemiological reports showing that only 10 to 15% of all smokers actually develop lung cancer during their lifetime, suggesting that individual susceptibility to carcinogens in cigarette smoke is unpredictable (6, 7). In recent years, mounting case–control studies have shown that specific genotypes are associated with higher lung cancer risk for cigarette smokers than non-smokers (8-15) and vice versa (16-19). These population studies elucidating the gene–lifestyle interactions on lung cancer risk, especially for smokers and non-smokers, may provide predictive systems for revealing the personalized etiology of lung cancer and personalized therapy and genomic pharmacology.
The dysfunction of the extracellular matrix (ECM) contributes to the initial phase for microenvironmental remodeling during physiological processes of morphogenesis, angiogenesis, inflammation, wound healing and tumorigenesis (20). Among the various kinds of ECM component molecules, the matrix metalloproteinases (MMPs) are a family of zinc and calcium-dependent endopeptidases that play a key role in ECM remodeling in lung tissue (21). MMPs have the capacity to degrade the components of connective tissue matrices (20, 21), and are closely related to the regulation of lung cancer invasion and metastasis (22-24). The homeostasis of each MMP is also under the control of a complex network at several levels, including their interactions with specific inhibitors, e.g. the tissue inhibitors of metalloproteinases (TIMPs) (21). For instance, MMP1 and MMP2 catalyze degradation of connective tissue fibrillary collagen (type I collagen) and type IV globular basement membrane collagen, respectively (20). The expressions of MMP1 and MMP2 are under the control of their specific inhibitors, TIMP1 and TIMP2, respectively (21). In the interstitial space and beneath epithelial and endothelial cells, the balance between MMPs and TIMPs is very important since relatively higher expressions of MMPs would stimulate the degradation of collagen and lead to acute injury (25). In an animal model, exposure to side-stream cigarette smoke 5 days per week for one month induced an increase in MMP1 and TIMP1 mRNA levels and a decrease in TIMP2 and type collagen mRNA levels in the lung of male Wistar rats (26). Since smoking is a main factor for lung cancer etiology, it is possible that the balance between MMPs and TIMPs may play a role in the carcinogenesis of smoking-related lung cancer.
Distributions of selected demographic data of the 358 patients with lung cancer and the 716 matched controls.
Up until now, the genomic contribution of TIMP1 to cancer has not been elucidated. For lung cancer, TIMP1 in serum was found to be higher in patients with lung cancer than in controls, and high TIMP1 (>300 ng/ml) correlated to poor cumulative survival in patients with lung cancer, especially those with squamous cell cancer and in advanced non-small cell lung cancer (NSCLC) (27-29). In 2007, Safranek and colleagues investigated the mRNA expression levels of TIMP1 in 20 patients with NSCLC, finding that the expression of TIMP1 mRNA was higher in the non-tumorous surrounding lung tissue, and in adenocarcinoma than in the epidermoid form of NSCLC (30). In 2009, the same group extended their investigation to a larger NSCLC population with 91 patients, finding that significantly higher expression of TIMP1 mRNA was demonstrated in NSCLC tissue in comparison to normal lung tissue from the same patients (31). To date, as far as we are aware of, there is no investigation on the contribution of TIMP1 to lung cancer susceptibility at the genomic level. In the current study, we aimed to investigate the contribution of TIMP1 genotypes at rs4898, rs6609533 and rs2070584 single nucleotide polymorphic (SNP) sites to the risk of lung cancer in Taiwan.
Materials and Methods
Investigated population. Three hundred and fifty-eight patients diagnosed with lung cancer were recruited at the Outpatient Clinics of General Surgery at the China Medical University Hospital during 2005-2008. The clinical characteristics of patients, including histological details, were all graded and defined by expert surgeons. Patients with history of any other cancer and pulmonary diseases such as chronic obstructive pulmonary disease, pneumothorax and asthma were excluded from the databank. All participants voluntarily completed a self-administered questionnaire and provided 5 ml of their peripheral blood samples. Twice as many non-lung cancer healthy volunteers as controls were selected by matching for age, gender and smoking behavior after initial random sampling from the Health Examination Cohort of the hospital. The exclusion criteria of controls included previous malignancy, metastasized cancer from other or unknown origin, and any genetic or familial diseases. The characteristics of patients in this study are summarized in Table I. The genotyping study was approved by the Institutional Review Board of the China Medical University Hospital (DMR100-IRB-284) and written-informed consent was obtained from all participants.
The summary of primer sequences, polymerase chain reaction-based sequence and polymerase chain reaction-restriction fragment length polymorphisms for rs4898, rs6609533 and rs2070584 polymorphic sites.
Distribution of tissue inhibitor of metalloproteinase-1 (TIMP1) genotypes among the 358 patients with lung cancer and the 716 matched controls.
Genotyping conditions. Genomic DNA from the peripheral blood leucocytes of each patient and controls was prepared using the QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan) and further stored and processed as our previous articles (10, 11, 32). The polymerase chain reaction (PCR) cycling conditions were: one cycle at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. The sequences of forward and reverse primers and the restriction enzymes for each SNP were designed by our laboratory and are summarized in Table II. The genotypic process was performed by two researchers independently and blindly. Five percent of the samples for rs6609533 were randomly selected for direct sequencing and the results from PCR–restriction fragment length polymorphisms and direct sequencing were 100% concordant. The results from direct sequencing for rs4898 and rs2070584 were also 100% concordant between the results using forward and reverse primers.
Statistical analyses. All of the 716 of the controls and 358 cases with genotypic and clinical data were analyzed. To ensure that the controls used were representative of the general population and to exclude the possibility of genotyping error, the deviation of the genotype frequencies of TIMP1 SNPs in the controls from those expected under the Hardy–Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson's Chi-square test was used to compare the distribution of the TIMP1 genotypes between the cases and the controls. The associations between the TIMP1 genotypes and lung cancer risk were estimated by computing odds ratios (ORs) and their 95% confidence intervals (CIs) from logistic regression analysis. A value of p<0.05 was considered statistically significant, and all statistical tests were two-sided.
Results
The frequency distributions of selected characteristics such as age, gender, and smoking status for the 358 patients with lung cancer and 716 non-cancer controls are summarized and compared in Table I. The average age of the patients was 64.0 years and the ratio of male versus female patients was about 7:3 (Table I). Since we applied frequency matching to recruit the non-cancer healthy controls, there was no difference in the distributions of age and gender between the control and case groups (Table I).
The distributions of the TIMP1 genotypes at rs4898, rs6609533 and rs2070584 among the non-cancer controls and the patients with lung cancer are presented and statistically analyzed in Table III. The genotypes of TIMP1 rs4898 were differently distributed between lung cancer and non-cancer control groups (p for trend=1.21×10−6) (Table III). In detail, TIMP1 rs4898 heterozygous CT was not associated with lung cancer risk (OR=0.98, 95% CI=0.71-1.34, p=0.9362), while the homozygous CC genotype seemed to be associated with increased lung cancer risk (OR=1.91, 95% CI=1.38-2.63) and only the latter was statistically significant (Table III). On the contrary, there was no association between the genotypes for rs6609533 nor rs2070584 and lung cancer risk (Table III).
To confirm the findings in Table III, the analysis of allelic frequency distribution for the three TIMP1 SNPs was also conducted and the results are summarized in Table IV. Supporting the findings that the homozygous CC genotype of IL-4 rs4898 was associated with increased lung cancer risk, 54.9% in the patient group, the C allele was significantly more frequent higher than in controls (44.9%; p=1.26×10−6). Again, there was no significant difference in the allelic frequencies of rs6609533 or rs2070584 between the control and patient groups (Table IV).
Discussion
In the current case–control association study, the contribution of three TIMP1 SNPs, rs4898, rs6609533 and rs2070584, with lung cancer risk was evaluated, to our knowledge for the first time. For rs4898, we found that the CC genotype of TIMP1 rs4898 was significantly associated with an increased risk of lung cancer (Table III). However, the heterogeneous CT genotype was not associated at all (Table III). Combining the CT and CC genotypes to compare with the wild-type TT genotype, the OR is 1.32 and 95% CI is 1.00-1.74 (p=0.0529; data not shown). For the other two SNPs, no obvious differential distribution in the genotypes of TIMP1 rs6609533 or TIMP1 rs2070584 was found (Table III). The allelic frequency analysis supports our finding that the C allele of TIMP1 rs4898 was associated with increased lung cancer risk (IV).
Distribution of tissue inhibitor of metalloproteinase-1 (TIMP1) allelic frequencies among the 358 patients with lung cancer and the 716 matched controls.
The MMP members are involved in the degradation of the basement membrane and ECM. Under normal conditions, MMPs are expressed at a relative low level and the TIMP1 protein can bind with MMP1 to suppress its activity (33, 34). The dynamic balance between MMPs and TIMPs plays a pivotal role for the maintenance of normal physiological conditions of cells. In 2012, Liu and colleagues performed a meta-analysis exploring the association between MMP1 promoter -1607 1G/2G polymorphism and risk of several types of cancer, and the results showed that an elevated cancer risk was found regarding breast, colorectal, genitourinary neoplasm but not lung cancer (35). A combined contribution of MMP1 and TIMP1 genotypes may provide further evidence for the contribution of genotypes for these genes.
Lung cancer is a gender-related cancer. In the National Health Insurance Research Database of Taiwan investigating 33,919 patients with lung cancer recorded from 2002 to 2008, about two-thirds of the patients were male (36), and the ratio is very similar to the gender ratio in this study. During recent years, there has been an increasing trend for the female patients with lung cancer in Taiwan and the prevalence and mortality rates of females with NSCLC adenocarcinoma are very high in Taiwan. Therefore, we were interested in whether the genotype of TIMP1 rs4898 contributes to the gender difference in lung cancer susceptibility. After stratification by gender, it was found that the genotypes of TIMP1 rs4898 were not differently distributed among males or females (data not shown).
Lung cancer is also a smoking-related cancer. Cigarette smoke may enhance remodeling in the developing human airway smooth muscle through hyperplasia and ECM production, thus contributing to development of neonatal and pediatric airway disease (37). Therefore, the interaction of the genotype of TIMP1 rs4898 and the cigarette smoking status of the participants was also analyzed. However, the results showed that the genotypic distribution of the variant genotypes of IL-4 rs2243250 was not significantly different between lung cancer and control groups who were ever smokers or those who were non-smokers (data not shown). In 2005, Rozynska and colleagues found that smoking behavior increased the level of MMP1 in the serum from patients with chronic obstructive pulmonary disease, increasing with pack-years of cigarette consumed (38). There is still no direct evidence to show that an altered expression of MMP1 or TIMP1 is essential in lung cancer carcinogenesis in any cell culture or animal models. Moreover, the detailed investigations of the mechanism of the contribution of TIMP1 rs4898 genotype to smoking-related or smoking-non-related lung cancer development are needed.
In conclusion, our pilot study provides evidence that the C allele of TIMP1 rs4898 is associated with an increased lung cancer risk, that should be confirmed in multicenter and multi-population studies.
Acknowledgements
The Authors declare no conflicts of interest. We appreciate Tissue-bank of China Medical University Hospital for their excellent technical assistance. This study was supported partially by research grant from Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW104-TDU-B-212-113002).
Footnotes
↵* These Authors contributed equally to this study.
- Received October 10, 2015.
- Revision received November 2, 2015.
- Accepted November 5, 2015.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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