thyroid cancer is a common endocrine malignancy with a rapidly rising incidence in recent decades and at least 458,403 patients with thyroid cancer are currently living in the united states ( howlader et al . , 2011 ) . the most common histological type of this cancer is papillary thyroid cancer ( ptc ) , which accounts for > 80% of all thyroid malignancies ( hundahl et al . , 1998 ; ptc can be further divided into several subtypes , including mainly , in the order of decreasing prevalences , conventional , follicular variant and tall - cell ptc . among the molecular derangements that drive thyroid tumorigenesis gene methylation is the covalent addition of a methyl group to the fifth carbon position of the cytosine residue in a cpg dinucleotide , which is usually associated with gene silencing and is a common mechanism for functional loss of key genes in human cancers ( esteller , 2008 ) . aberrant gene methylation in thyroid tumorigenesis often involves major tumor suppressor genes , such as those for the tissue inhibitor of metalloproteinase-3 ( timp3 ) , slc5a8 , death - associated protein kinase ( dapk ) , and retinoic acid receptor 2 ( rar2 ) , particularly in ptc ( hoque et al . , 2005 ; hu et al . , the tumor suppressor functions of these genes have been well characterized and they are frequently methylated in cancers . for example , the anti - tumor role of the rar2 gene , which is often silenced by aberrant methylation in cancers , is mediated through inhibition of cancer cell proliferation and metastasis and induction of apoptosis ( raffo et al . conversely , re - expression of rar2 is associated with significant reduction of cell growth in thyroid cancer cells ( miasaki et al . , 2008 ) . ionizing radiation exposure , the best - established risk factor for thyroid cancer , can induce ret / ptc rearrangements and consequent development of ptc , particularly in the pediatric population ( nikiforov , 2006 ) . smoking has also been widely shown to be a significant risk factor for the development of thyroid nodule / goiter , particularly in women ( lio et al . , 1989 ; ericsson and lindgarde , 1991 ; galanti et al . , 2005 ) . thyroid nodule / goiter is , in turn , a strong risk factor for thyroid cancer ( preston - martin et al . , 1993 ; although some studies did not show an increased risk associated with smoking for thyroid cancer ( ron et al . , 1987 ; kreiger and parkes , 2000 ; bandurska - stankiewicz et al . , 2011 ) , others did show an association of smoking with occurrence of thyroid cancer ( sokic et al . , 1994 ) . interestingly , smoking has been found to be associated with aberrant gene methylation in some cancers , such as the rar2 gene methylation in human lung cancer ( tomizawa et al . , 2004 ) and animal lung cancer models ( vuillemenot et al . , 2004 ) . the effect of smoking on gene methylation has not been explored in thyroid cancer . in the present study , we investigated the relationship of smoking with methylation of several major tumor suppressor genes in ptc . with institutional review board approval and , where required , informed patient consent , we included 138 patients ( 99 female and 39 male , age 1585  years ) , who were operated for ptc between 1990 and 2006 and had recorded history of smoking status , in this study . the smoking history of a patient was obtained through a retrospective review of the patient s records . smoking histories of the ever smokers varied from 2 to 35 pack years of cigarette smoking . a pack - year is defined as equivalent to smoking one pack of cigarettes daily for a period of 1  year . paraffin - embedded ptc tumors were microdissected and processed and genomic dna was isolated as described previously ( hu et al . , 2006 ) . bisulfite - treated dna was subjected to methylation analysis for the promoters of the tumor suppressor genes timp3 , slc5a8 , dapk , and rar2 using real - time quantitative methylation - specific pcr . the primers for these genes and pcr reaction conditions were as described previously ( hu et al . , 2006 ) . any detectable level of methylation for the indicated genes defined as a positive methylation result and zero value on the current detection system was defined as a negative methylation result . categorical data were summarized with frequencies and percentages , and continuous data with medians and ranges . age and gender distribution were analyzed utilizing mann  whitney rank sum test and fisher s exact test , respectively . smoking status groups were compared using fisher s exact test for categorical data , and the non - parametric wilcoxon rank sum test for continuous measures . all reported p values are two - sided . a p - value of < 0.05 was considered to be statistically significant . analysis was performed using sas version 9.1.3 software ( sas institute inc . , cary , nc , usa ) . with institutional review board approval and , where required , informed patient consent , we included 138 patients ( 99 female and 39 male , age 1585  years ) , who were operated for ptc between 1990 and 2006 and had recorded history of smoking status , in this study . the smoking history of a patient was obtained through a retrospective review of the patient s records . smoking histories of the ever smokers varied from 2 to 35 pack years of cigarette smoking . a pack - year is defined as equivalent to smoking one pack of cigarettes daily for a period of 1  year . paraffin - embedded ptc tumors were microdissected and processed and genomic dna was isolated as described previously ( hu et al . , 2006 ) . bisulfite - treated dna was subjected to methylation analysis for the promoters of the tumor suppressor genes timp3 , slc5a8 , dapk , and rar2 using real - time quantitative methylation - specific pcr . the primers for these genes and pcr reaction conditions were as described previously ( hu et al . , 2006 ) . any detectable level of methylation for the indicated genes defined as a positive methylation result and zero value on the current detection system was defined as a negative methylation result . categorical data were summarized with frequencies and percentages , and continuous data with medians and ranges . age and gender distribution were analyzed utilizing mann  whitney rank sum test and fisher s exact test , respectively . smoking status groups were compared using fisher s exact test for categorical data , and the non - parametric wilcoxon rank sum test for continuous measures . analysis was performed using sas version 9.1.3 software ( sas institute inc . , cary , nc , usa ) . we analyzed 138 ptc for the methylation status of the tumor suppressor genes timp3 , slc5a8 , dapk , and rar2 . these genes were chosen for analysis because they are important human tumor suppressor genes and their methylation was frequently found in thyroid cancer ( hoque et al . , 2005 ; hu et al . , 2006 ) . overall , methylation of these genes were detected in 67 ( 48.5% ) , 36 ( 26.1% ) , 40 ( 29.0% ) , and 23 ( 16.7% ) of the 138 cases , respectively . as shown in table 1 , except for the age of the dapk methylation - positive patients that was significantly older in the ever - smoker group than the never - smoker group , there was no significant difference in the age of the methylation - positive patients for the remaining three genes between the ever - smoker and never - smoker groups . similarly , as shown in table 2 , there was no significant difference in gender distribution of methylation - positive patients for the four genes examined in the present study . also , smoking did not affect the relationship of gene methylation with tumor size and other tumor characteristics ( data not shown ) . comparison of mean ages of methylation - positive patients for the indicated genes between the ever - smoker and never - smoker groups . gender distribution of methylation - positive patients for the indicated genes [ n ( % ) ] we next analyzed the relationship of gene methylation in ptc with cigarette smoking history in 138 patients . as summarized in table 3 and figure 1 , among the four tumor suppressor genes analyzed , methylation of the rar2 gene was significantly associated with a history of smoking . specifically , 13/42 ( 31.0% ) ever smokers vs. 10/96 ( 10.4% ) never smokers harbored methylation in the promoter of the rar2 gene ( p  =  0.003 ) . although there was no statistically significant difference in the methylation of the timp3 , slc5a8 , and dapk genes between the never and ever smokers , there was a tendency of higher prevalences of methylation in these genes in the smokers ( figure 1 ; table 3 ) . relationship of methylation of the rar2 , dapk , timp3 , and slc5a78 genes in papillary thyroid cancer ( ptc ) with smoking of patients [ n ( % ) ] . aberrant gene methylation in papillary thyroid cancer in ever and never smokers . shown on y - axis is the methylation rate percentage of the methylation - positive cases ( % )  for each of the four genes indicated on the x - axis . * the methylation rate of the rar2 gene is significantly higher ( p  =  0.003 ) in the ever smoker group than the never smoker group . the methylation rate for the remaining three genes shows a higher trend for ever smokers but does not reach statistical significance . the 138 cases of ptc included 72 conventional ptc , 51 follicular variant ptc , and 15 tall - cell ptc . we also analyzed the relationship of smoking with gene methylation in each of the subtype groups of ptc . as shown in table 3 , similar to the results on the overall analysis of all the cases of ptc , rar2 methylation was significantly associated with smoking [ 8/17 ( 47% ) of ever smokers vs. 7/55 ( 13% ) of never smokers , p  =  0.005 ] in conventional ptc . in the smaller number of cases of follicular variant ptc , marginally significant association of smoking with rar2 methylation was demonstrated ( p  =  0.06 ) . this relationship was not seen in patients with tall - cell ptc , probably due to the small number of cases . a trend of association of smoking with the timp3 , slc5a8 , and dapk methylation was observed in these subtypes of ptc . the average methylation levels for each gene in the ever and never smokers were not significantly different ( table 4 ) . however , this result was likely limited by the inability of the methylation pcr approach used in the present study to accurately provide quantitative measurements . comparison of methylation levels for the indicated genes in papillary thyroid cancer between ever and never smokers . tobacco smoking has long been known to be a significant risk factor for human cancers . this has prompted numerous studies on its role in molecular derangements in cancers , including genetic and epigenetic alterations , such as aberrant gene methylation . smoking has also been shown to be associated with the development of thyroid nodules in many studies ( lio et al . , 1989 ; ericsson and lindgarde , 1991 ; galanti et al . , 2005 ) , a strong risk factor for thyroid cancer ( preston - martin et al . , 1993 ; franceschi et al . , 1999 ) , and occurrence of thyroid cancer in some studies ( sokic et al . , 1994 ) . however , little is known about molecular derangements associated with smoking in thyroid cancer . in the present study , we for the first time investigated the relationship between patient smoking and aberrant gene methylation in ptc , the most common type of thyroid cancer . we found a significant association of smoking with methylation of the rar2 gene in ptc , both in overall ptc and in its major subtypes . this association represented a specific epigenetic event in ptc as the association of smoking with the methylation of other genes ( timp3 , slc5a8 , and dapk ) was not significant . some of the relationship patterns of smoking with methylation of tumor suppressor genes in ptc observed in the present study were similar to the findings in other cancers . for example , as in the present study which failed to show a significant association of dapk methylation with smoking in ptc , such an association was also not found in non - small lung cancer patients ( liu et al . , 2007 ) . similar to our findings in ptc , previous studies demonstrated a significant association of smoking with rar2 methylation in lung cancers in humans ( tomizawa et al . , 2004 ) and in animal models ( vuillemenot et al . , 2004 ) . interestingly , even in normal non - cancerous epithelium of upper aerodigestive tract , methylation of the rar2 gene and other genes was more frequently seen in smokers ( zochbauer - muller et al . , 2003 ) , supporting a direct effect of smoking on gene aberrant methylation . frequent methylation of the rar2 gene has also been recently detected in laryngeal squamous cell carcinomas ( paluszczak et al . , 2011 ) , a malignancy often associated with smoking . we previously demonstrated that methylation of the tumor suppressor gene rar2 was associated with its silencing and , conversely , un - methylation was associated with expression of this gene in thyroid cancer cells ( hu et al . , 2006 ) . re - expression of rar2 using demethylating agent 5-aza-2-deoxycytidine caused a significant inhibition of thyroid cancer cell growth ( miasaki et al . the association of smoking with rar2 methylation in ptc suggests that this gene may more commonly loose function in ptc of smokers . rar2 is a major type of retinoic acid ( ra ) receptors , which are transcription factors that bind with the biologically active metabolites of vitamin a to regulate cell growth and proliferation ( pfahl and chytil , 1996 ) . therapeutic potential of vitamin a and retinoic acid has been demonstrated in various human cancers . ra has been shown to be able to re - differentiate thyroid cancer cell lines and increase radioiodine uptake in some patients who lost radioiodine avidity ( haugen et al . , 2004 ; coelho et al . , 2005 ) . however , the overall treatment efficacy is poor . one cause for the failure of ra therapy for thyroid cancer is the loss of ra receptor expression in this cancer . in vitro studies showed that ra could only inhibit the growth of thyroid cancer cell lines that expressed rar and some other ra receptors ( haugen et al . expression of ra receptors is often decreased or lost in thyroid cancers , particularly in ptc in which expression of the rar gene was most frequently decreased or silenced ( tang et al . , 2003 ; elisei et al . , 2005 ) . our previous demonstration of aberrant rar2 gene methylation in thyroid cancer provided a molecular explanation ( hoque et al . , 2005 ; given these data and the results in the present study , it is clear that aberrant methylation of ra receptor genes is an important molecular event in ptc associated with smoking . how this aberrant methylation of rar2 gene specifically contributes to the tumorigenesis of ptc in smokers warrants further studies to elucidate . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
aberrant gene methylation is often seen in thyroid cancer , a common endocrine malignancy . 
 tobacco smoking has been shown to be associated with aberrant gene methylation in several cancers , but its relationship with gene methylation in thyroid cancer has not been examined . in the present study , we investigated the relationship between smoking of patients and aberrant methylation of tumor suppressor genes for timp3 , slc5a8 , death - associated protein kinase , and retinoic acid receptor 2 ( rar2 ) in papillary thyroid cancer ( ptc ) , the most common type of thyroid cancer . 
 the promoter methylation status of these genes was analyzed using quantitative real - time methylation - specific pcr on bisulfite - treated genomic dna isolated from tumor tissues and correlated with smoking history of the patients . among the four genes , 
 methylation of the rar2 gene was significantly associated with smoking and other three genes showed a trend of association . 
 specifically , among the 138 patients investigated , 13/42 ( 31.0% ) ever smokers vs. 10/96 ( 10.4% ) never smokers harbored methylation of the rar2 gene ( p  =  0.003 ) . 
 this association was highly significant also in the subset of conventional variant ptc ( p  =  0.005 ) and marginally significant in follicular variant ptc ( p  =  0.06 ) . 
 the results demonstrate that smoking - associated aberrant methylation of the rar2 gene is a specific molecular event that may represent an important mechanism in thyroid tumorigenesis in smokers .