This study aimed to investigate the association between SHS exposure and DNA methylation of a large number of CpG loci bladder cancer cases who had never smoked. We tested the hypothesis that epigenetic changes in bladder tumors are associated with SHS exposure. Our finding of specific CpG loci methylation being associated with either adulthood, childhood, occupational or total SHS exposure, provides considerable additional evidence for the biological plausibility for the association between SHS exposure and bladder cancer risk, and may aid in explaining the incidence of bladder cancer amongst non-primary smoking individuals. CpG loci with statistically significant associations between their methylation status and SHS exposures demonstrated exclusively greater extents of methylation suggesting that SHS exposure may induce and/or select for specific hypermethylation events in bladder cancer.
Previous work has shown associations between methylation of the promoter region of several cancer-related genes in bladder tumors and primary tobacco smoking, particularly identifying associations between current and recent former smoking and hypermethylation of the CDKN2A
). A propensity for hypermethylation of a panel of tumor suppressor genes was also significantly more pronounced in current smokers (23
). Our results suggest that SHS exposure may also lead to hypermethylation of key tumor suppressor genes. Interestingly, we did not identify those loci whose hypermethylation was previously found to be associated with primary smoking exposures, including CDKN2A
, MDM2, or the SFRP
). This was confirmed when we ran individualized generalized linear models between the methylation beta value for each locus and former and current smoking status, where we found no overlap with any of the specific loci that were identified with the strongest associations (FDR Q<0.05) between methylation and any type of SHS exposure. We did find that two specific CpG loci that were found to be associated at the p<0.05 level with former smoking status were also associated with Childhood and Adulthood exposure to SHS respectively, and that one specific CpG loci that was found to be associated at the p<0.05 level with current smoking status was also associated with Adulthood exposure to SHS. This may suggest that the types of exposure encountered by the bladders of SHS-exposed individuals are different than those of primary smoking individuals, and thus contribute different selective pressures resulting in differential methylation. In addition, the fact that the genes whose CpGs were significantly associated with either adulthood, childhood or occupational SHS exposure were all distinct from one another, suggest that the specific type or timing of SHS exposure is important epigenetically. Indeed, there may be variability in the way the different SHS exposures were experienced; this could lead to stochastic epigenetic changes due to the less consistent way individuals may experience these individual types of SHS exposures. These SHS exposures are themselves likely to be less intense and less consistent than those experienced by primary smokers. We also checked the correlation between our SHS exposures and key bladder cancer risk factors (namely age, gender, tumor grade and stage) and found that these covariates largely are not associated with the change in methylation observed between exposed and unexposed individuals (reflected by low correlation coefficients).
A number of CpG loci associated with genes in various cellular receptor kinase families were found to have significantly greater methylation associated with individual types of SHS exposure. The insulin-like growth factor-I receptor (IGF1R
) gene and the insulin-like growth factor-II receptor (IGF2R
) gene have opposite roles in oncogenesis with IGF1R
acting as a mediator in tumor cell growth (as shown in pancreatic cancer) by inhibiting tumor apoptosis, while IGF2R
acts as a tumor suppressor decreasing cellular proliferation and increasing apoptosis (35
has been found to be hypermethylated in ovarian and breast tumors showing that both IGF1R
could potentially be good targets for alterations by the components of SHS exposure (36
). Other receptor kinases found to be hypermethylated with exposure to SHS were the fms-related tyrosine kinase 1 (FLT1
), the EPH receptor B2 (EPHB2
) and the ephrin-A1 (EFNA1
) gene (37
). All three have been shown to have aberrant methylation in tumors, with FLT1
showing CpG island methylator phenotype (CIMP)-associated DNA hypermethylation in colorectal tumors, EPHB2
showing altered methylation of its promoter regulatory sequences in colorectal tumors, and EFNA1
being hypermethylated in leukemia cells (37
). Also of particular interest was the hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT
) associated with adulthood SHS exposure. MGMT
is involved in DNA repair and has been found to be aberrantly hypermethylated in tumors such as breast cancer (40
). Its methylation state in glioma serves as a clinically useful marker of susceptibility to the chemotherapy temozolamide (41
). Finally, the connective tissue growth factor (CTGF
) was found to be hypermethylated after exposure to both occupational and total SHS. CTGF
is a secreted protein belonging to the CCN family shown to be hypermethylated in ovarian cancer; it may therefore be a factor in the carcinogenesis of ovarian cancer (42
Of the pathways found to be significantly enriched amongst genes associated with any type of exposure to second hand smoking in our canonical pathways analysis, the JAK/Stat, IL-3 IL-4, CTLA4, T-cell receptor, and erythropoietin signaling pathways were all found to be significantly associated with adulthood and total exposure to SHS. The pathways are of interest, both in that they have been linked to tumorigenesis, and are critical regulators of the immune response (43
). The role of the immune system in bladder cancer is of great interest as patients with localized disease are often treated with Bacillus Calmette-Guérin therapy, which is thought to illicit a local inflammatory response against the bladder tumor. Epigenetic alteration of key regulators of these immune responses may help to explain differential response to this treatment.
The extracellular-signal-regulated kinase 5 (ERK5
) and the renin-angiotensin signaling pathways were also enriched amongst genes whose methylation status was associated with SHS exposure (49
). This suggests that these exposures may be influencing vascular remodeling and angiogenic pathways with may be critical in sustaining or enhancing tumor growth (49
Strengths of this study included the population-based nature of the study, as well as the use of the Illumina GoldenGate Methylation Bead Array for methylation profiling. Limitations of this study include the small sample size and the use of a questionnaire to ascertain second hand smoking exposure of study participants, as their responses may have been subject to recall bias. Future studies with larger sample sizes and adequately detailed SHS data are warranted.
In summary, this study demonstrates that passive smoking, like active smoking is associated with DNA methylation across numerous CpG loci in bladder cancer, but appears to be targeting different genes than those that have been associated with active smoking. The novelty of these results lies in the use of array-based methodologies to examine methylation of a large number of CpG loci in relation to SHS to help further clarify the potential association and biological plausibility of the risk of bladder cancer and SHS exposure. These findings indicate that SHS exposure has effects on methylation levels of genes and in turn affect important biological pathways which may have impacts on overall bladder cancer risk. However, due to our small sample size, we recognize the necessity to replicate and extend these results in a larger sample in the future.