The present study, based on the accumulated evidences from 19 cross-sectional studies, indicates that cigarette smoking is positively related to p16INK4α hypermethylation in tumor tissues from NSCLC patients. The frequency of p16INK4α methylation in smoker lung cancer patients was 2.25 times higher than that in nonsmoker patients. The association appeared to be stronger in Asian patients and in studies with a larger number of subjects, but without a histology (AC or SCC) specificity. However, this positive correlation did not exist in adjacent noncancerous tissues from NSCLC patients and in biological specimens of ‘healthy’ subjects without cancer. Given the results that cigarette smoking leading to p16INK4α hypermethylation was related to the stage progression of tumorigenesis, we speculated that p16INK4α hypermethylation might be an early marker for cancer diagnosis, particularly in cigarette smoking patients.
Recent epidemiological studies have revealed that molecular mechanisms underlying the development of lung cancers differed between nonsmokers and smokers. For instance, EGFR
pathway is frequently activated by gene mutations in nonsmoker lung cancers, while mutations of KRAS
often occur in smoker lung cancer. However, the mutations in either gene in lung adenocarcinomas are rarely seen although the biological consequences of KRAS and EGFR mutations share similarities in regulation of cell proliferation, survival and apoptosis 
. In the present study, we demonstrate that the frequency of p16INK4α
hypermethylation is slightly but significantly higher in smoker patients than that in nonsmoker patients. It is well known that p16INK4α
plays an essential role in the development of most human cancers for the reason that the p16/cyclinD1/CDK4/RB signaling pathway controls the cell cycle at the G1/S transition 
. Hypophosphorylated RB inhibits G1/S transition by binding to E2F1 transcription factor and exerts its tumor-suppressor function. Once hyperphosphorylated by the cyclinD1/CDK4 complex, RB releases E2F1, which results in transition from G1 to S phase. p16 prevents RB from phosphorylation by inhibition of CDK4, leading to a cell cycle arrest. Suppression of p16 expression allows unregulated phosphorylation of the RB protein and leads to uncontrolled cell cycle progression and cell division 
has a low frequency of mutations in lung cancer 
. Its inactivation is mainly through gene promoter hypermethylation 
. For example, Nakata et al. found that in tumors with p16INK4α
hypermethylation, 63.3% showed reduced expression; whereas, in tumors without p16INK4α
hypermethylation, only 33.7% showed reduced expression (P
. The positive correlation between cigarette smoking and p16INK4α
hypermethylation demonstrates that cigarette smoking plays an important role in determining the molecular signatures involved in lung cancer development.
The mechanism for cigarette smoking inducing gene-specific hypermethylation, e.g. p16INK4α
, remains unclear. De novo
methylation uses S-adenosyl-methionine as a methyl donor and adds a methyl group to the cytosine ring to form methyl cytosine, which is catalyzed by DNA methyltransferases (DNMT) 1, 3a, or 3b 
. It is estimated that DNMT1 is responsible for about 90% of methyltransferase activity in mammalian cells 
. DNMT1 overexpression was found in many types of cancers including lung cancers, particularly in patients who were smokers 
. A recent study found that DNMT1 was highly expressed in tumor tissues in a dose response manner compared with the non neoplastic stroma tissues, not only in tobacco-specific carcinogen nicotine-derived nitrosamine ketone (NNK)-induced mouse lung cancer but also in human lung cancer associated with cigarette smoking 
. Moreover, it was demonstrated that NNK increased DNMT1 expression and activity by blocking its degradation related to ubiquitin-proteasome 
. AKT/GSK3β/βTrCP signaling is implicated in the accumulation of nuclear DNMT1, which leads to hypermethylation of p16INK4α
, fragile histidine triad gene (FHIT
) and retinoic acid receptor β (RARB
), and ultimately leads to tumorigenesis and poor prognosis 
. Although the direct interaction of DNMT1 to the p16INK4α
gene promoter is not yet characterized 
, these findings indicated that tobacco-induced DNMT1 overexpression might be responsible for maintaining the hypermethylation status of p16INK4α
Lung cancer in nonsmokers is now a prominent public health concern. However, the major causes of them have yet not been identified. Environmental tobacco smoke (ETS), for example, second-hand smoke, has been recognized as a high risk factor 
. According to the report from International Agency for Research on Cancer (IARC), the risk for developing lung cancer from ETS exposure might reach 35% in men and 25% in women 
. Given that cigarette smoking has a cause-effect on p16INK4α
hypermethylation, ETS exposure may explain, at least partly, the variable percentage of p16INK4α
hypermethylation in nonsmoker patients. Other factors such as exposed to asbestos, chromium, arsenic, cadmium, silica, or nickel, or outdoor air pollutants, previous lung disease, and dietary factors have also been implicated in non smoking-related risk 
. But so far there is still a missing link between environmental factors, p16INK4α
hypermethylation, and lung cancer, which limits the use of gene-specific hypermethylation as a biomarker to detect lung cancer in early stage. Thus, the molecular mechanism underlying lung cancer, irrespective of tobacco-association, should be further elucidated.
In this study, we observed that the frequency of p16INK4α hypermethylation in NSCLC patients varied among different studies. The combined frequency in the present meta-analysis was less than 35%. The discrepancy and the relative low frequency might be due to the method used for detection of methylation, the variation in defining cigarette smokers, and insufficient information of clinical outcome. Although MSP is sufficiently sensitive, the conditions of PCR may affect the results to a large extent. The results seemed to be a little artificial particularly when PCR reaction was performed using both methylated and unmethylated primers. As for definition of cigarette smoker or nonsmoker, it lacked a consistent criterion followed by each investigation. In addition, current smokers and former smokers were not clearly distinguished, and the quantity of smoking was not calculated in the meta-analysis due to limited data. Moreover, insufficient clinical information such as the stage of NSCLC made it difficult to predict the prognosis based on the results provided.
In conclusion, cigarette smoking is suggested to be positively related to p16INK4α methylation in human NSCLC, highlighting the potential importance of p16INK4α promoter methylation in early cancer diagnosis. Furthermore, 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 p16INK4α hypermethylation in lung cancer patients with smoking habits was only 2.2 times increased than that in nonsmoker patients, we speculate that many other aberrant epigenetic modifications, together with the genetic damage are involved in lung cancer development, which needs to be addressed in further investigation.