Association between Cigarette Smoking and RASSF 1 A Gene Promoter Hypermethylation in Lung Cancer Patients : a Meta-analysis

Lung cancer is one of the most frequent cause of cancer-related mortality in the world (Jemal et al., 2011). Primary lung cancer can be divided into two categories according to histopathology, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC, characterized by a long asymptomatic latency and poor prognosis, accounts for about 80% of primary lung cancers . NSCLC includes denocarcinoma (AC), squamous cell carcinoma (SCC), large cell carcinoma and others. Despite advances in both detection and treatment of lung cancer, most lung cancers are detected at a late stage, the overall 5-year survival rate is less than 15% (Jemal et al., 2011). Lung cancer was expected to accout for 26% of all female cancer deaths in 2013 (Siegel et al., 2013). Thus, it is necessary to identify biomarkers for effective early diagnosis of lung cancer. Cigarette smoking has been definitely established as the major cause of lung cancer. Smoking is thought to be responsible for 17.2% of NSCLC cases in men and 11.6% of cases in women, which is much higher than nonsmokers with 1.3% in men and 1.4% in women (Villeneuve


Introduction
Lung cancer is one of the most frequent cause of cancer-related mortality in the world (Jemal et al., 2011).Primary lung cancer can be divided into two categories according to histopathology, non-small cell lung cancer

Association between Cigarette Smoking and RASSF1A Gene Promoter Hypermethylation in Lung Cancer Patients: a Metaanalysis
Xiao-Ming Wu, Yu Chen, Yang Shao, Xiao-Long Zhou, Wen-Ru Tang* and Mao, 1994).However, the molecular mechanism responsible for the increased risk of lung cancer in young smokers remains unclear.
The genomic organization at 3p21.3, RASSF1A (Rasassociation domain family 1 isoform A), a major isoform of RASSF1 (the Ras-association domain family 1), has a RAS association (RA) domain and a cysteine-rich diacylglycerol-binding domain (C1 domain) in the amino terminal region (Dammann et al., 2000;Burbee et al., 2001).RASSF1A is a cell cycle-related tumor suppressor protein that that decreases colony formation, suppresses anchorage-independent growth, and dramatically reduces tumorgenicity in vivo.Previous studies have reported the involvement of RASSF1A promoter methylation in several cancers, including prostate (Ge et al., 2014), Ovarian (Vo et al., 2013), lung cancer (Liu et al., 2013).It has been reported that the RASSF1A gene is frequently inactivated in primary lung cancers by the de novo methylation of CpG islands in the promoter region (Zochbauer-Muller et al., 2001;Tomizawa et al., 2002).RASSF1A methylation has been identified in 30-60% of NSCLC and has been associated with poorer patient prognosis and earlier recurrence of disease (Dammann et al., 2000;Burbee et al., 2001;Tomizawa et al., 2002;Endoh et al., 2003;Kim et al., 2003).Despite the significance of RASSF1A gene promoter methylation in predicting incidence or prognosis and in epigenetic therapy of lung cancer, the manner in which these epigenetic lesions accumulate during carcinogenesis is not completely understood.
Molecular epidemiological studies have begun to link specific environmental carcinogens, including those in tobacco smoke, with DNA methylation changes in lung cancer progression.However, the results are inconsistent and inconclusive.The present study mainly focused on RASSF1A gene , which is transcriptionally silenced predominantly through aberrant promoter hypermethylation.Here, we performed a meta-analysis to quantitatively analyze the correlation between cigarette smoking and RASSF1A gene promoter methylation in lung cancer patients.

Publication Search
We searched the PubMed and Chinese biomedicine databases for all articles on the association between cigarette smoking and RASSF1A gene methylation in lung cancer (last search update, November 21, 2013).The following key words were used: 'smoking', ' Cigarette smoking', 'RASSF1A', 'methylation '. 'hypermethylation', Case-control studies containing frequencies of RASSF1A gene methylation in smoker and unsmoker patients were chosen.Of the studies with overlapping data published by the same author, only the most recent or complete study was included in this metaanalysis.

Statistical analysis
The strength of association was accessed by calculating crude odds ratios (ORs) and 95% confidence intervals (CIs).Heterogeneity assumption was evaluated by a chisquare based Q-test.A P-value of >0.05 for the Q-test indicated a lack of heterogeneity among the studies, the summary OR estimate of each study was calculated by the fixed effects model (Mantel and Haenszel, 1959;DerSimonian and Laird, 1986).The potential for publication bias was examined by a Begg's test (funnel plot method) and Egger's linear regression test (P<0.05considered representative of statistical significance) (Egger et al., 1997).All analyses were performed using Stata software (version 8.2; Stata Corporation, College Station, TX).

Eligible studies
In this meta-analysis, we identified 19 studies with 2287 lung cancer patients on the association between cigarette smoking and RASSF1A gene methylation, including 1449/838 smokers /unsmokers (Table 1).The following information was extracted from study: first author, publication year, country, ethnicity, histology of lung cancer, method, number of participants RASSF1A methylation frequencies in smokers and nonsmokers.In studies defining nonsmoker, there were three different definitions of nonsmoker: (1) less than 20 pack-years; (2) less than 100 cigarettes in entire lifetime; (3) daily cigarette consumption *years of smoking = 0.In our meta-analysis, we combined nonsmokers according to their original group in each individual study.

Meta-analysis
In the meta-analysis, 2287 lung cancer patients including 1449 smokers and 838 nonsmokers were included in pooling the overall correlation estimation.
The results of the association between the cigarette smoking and the frequency of RASSF1A methylation and the heterogeneity test are shown in smoker with lung cancer had a 1.297-fold higher risk for methylation (Figure 1).Stratified analysis also revealed that the associations varied among the subjects from different regions.The association between cigarette smoking and RASSF1A methylation tended to be stronger in 15 Asian studies (OR =1.481, 95% CI: 1.179~1.861,p=0.001, p=0.186) compared to the other studies (Caucasian: OR=1.187, 95%CI =0.558-2.525,p=0.657;Mix: OR=0.769, 95%CI =0.483-1.224,p=0.268) (Figure 1).

Publication bias
Funnel plot and Egger's test were performed to quantitatively evaluate the publication bias of literatures on RASSF1A methylation.The results of Begg's test provided statistical evidence for funnel plot symmetry (p=0.970) in overall results, suggesting the absence of publication bias.

Discussion
In this study, we investigated that cigarette smoking is positively related to RASSF1A hypermethylation in tumor tissues from lung cancer patients.The frequency of RASSF1A hypermethylation was 1.297 times higher in smoker patients than that in nonsmoker patients.The association appeared to be stronger in Asian patients.We speculated that RASSF1A hypermethylation might be an early marker for cancer diagnosis, particularly in cigarette smoking patients.
Epidemiological studies indicate that some characteristics of lung cancer among smokers significantly differ from those of unmokers.Kras mutations appear more often in smoker lung cancers, while EGFR is activated by gene mutations in unsmoker lung cancers.The biological consequences of Kras and EGFR mutations share similaries in regulation of cell proliferation.It is well known that RASSF1A could also lead to cell cycle arrest by participating in the Rb family cell cycle checkpoint (Hesson et al., 2007).Inhibition of cyclin D1 by RASSF1A occurs post-transcriptionally and may be at the level of translational control (Shivakumar et al., 2002).Moreover, RASSF1A serve as Ras effector, whose function analysis has revealed an involvement in apoptotic signaling (Khokhlatchev et al., 2002), microtubule stabilization (Liu et al., 2003) and mitotic progression (Tomizawa et al., 2002;Mathe, 2004).It has been reported that the RASSF1A gene is frequently inactivated in lung cancer by the de novo methylation of CpG islands in the promoter region (Zochbauer-Muller et al., 2001;Honorio et al., 2003;Grote et al., 2006).RASSF1A promoter methylation might play a pivotal role in the development of Lung cancer (Liu et al., 2013).
Links among cigarette smoking, RASSF1A hypermethylation, and lung cancer remain unclear.DNA methylation is mediated by the members of the DNA methyltransferase (DNMT) family, conventionally classified as de novo (DNMT3a and DNMT3b) and maintenance (DNMT1).It is estimated that DNMT1 overexpression was found in many types of cancers including lung cancers, particularly in patients who were smokers (Lin et al., 2007;Lin et al., 2010).DNMT1 accumulation and subsequent hypermethylation of the promoter of RASSF1A may lead to tumorigenesis and poor prognosis and provide an important link between tobacco smoking and lung cancer.Moreover, expression ofΔDNMT3B variants, a subfamily of DNMT3B, was detected in 80% tumors but in only 18% of the corresponding normal lungs (Wang et al., 2008).DNMT3B may play an important role in the development of promoter methylation.Demethylation by 5'-aza-2'deoxycytidine treatment resulted in the restoration of RASSF1A gene expression and the transfection and expression of RASSF1A in lung cancer cells resulted in the suppression of colony formation, anchorageindependent soft agar growth and tumorigenesis in nude mice (Burbee et al., 2001).These findings indicated that tobaccoinduced DNMTs overexpression might be responsible for maintaining the hypermethylation status of RASSF1A gene.
There are some limitations that are present in this analysis, which may affect the objectivity of the conclusion of this meta-analysis.It is well known that the risk for developing lung cancer in smokers is 8 to 13 times higher than that in nonsmokers, while the risk of RASSF1A methylation in lung cancer patients with smoking habits was only 1.297 times.It is possible that differences in RASSF1A hypermethylation between smokers and unsmokers are influenced by other factors, such as gender, age, state of lung cancer, histological tumour type.Therefore, a meta-analysis including more high-quality designed epidemiology studie is necessary in the future in this field.
This meta-analysis with sufficient individual data to be stratify results by ethnicity.This analysis supports conclusions that the cigarette smoking had increased risk of RASSF1A methylation (OR =1.297, 95% CI: 1.066~1.580,p=0.010, p=0.087) in lung cancer patients, especially in the case of Asian (OR =1.481, 95% CI: 1.179~1.861,p=0.001, p=0.186) , which highlight the

Figure 1 .
Figure 1.Forest Plot of ORs of RASSF1A Methylation in Smoker Compared to Nonsmoker Lung Cancer Patients.The squares and horizontal lines correspond to the study-specific OR and 95% CI.The area of the squares reflects the study-specific weight.The diamond represents the pooled OR and 95% CI