From tobacco smoking to cancer mutational signature: a mediation analysis strategy to explore the role of epigenetic changes

Abstract

Background: Tobacco smoking is associated with a unique mutational signature in the human cancer genome. It is unclear whether tobacco smoking-altered DNA methylations and gene expressions affect smoking-related mutational signature.

Methods: We systematically analyzed the smoking-related DNA methylation sites reported from five previous casecontrol studies in peripheral blood cells to identify possible target genes. Using the mediation analysis approach, we evaluated whether the association of tobacco smoking with mutational signature is mediated through altered DNA methylation and expression of these target genes in lung adenocarcinoma tumor tissues.

Results: Based on data obtained from 21,108 blood samples, we identified 374 smoking-related DNA methylation sites, annotated to 248 target genes. Using data from DNA methylations, gene expressions and smoking-related mutational signature generated from ~ 7700 tumor tissue samples across 26 cancer types from The Cancer Genome Atlas (TCGA), we found 11 of the 248 target genes whose expressions were associated with smoking-related mutational signature at a Bonferroni-correction P < 0.001. This included four for head and neck cancer, and seven for lung adenocarcinoma. In lung adenocarcinoma, our results showed that smoking increased the expression of three genes, AHRR, GPR15, and HDGF, and decreased the expression of two genes, CAPN8, and RPS6KA1, which were consequently associated with increased smoking-related mutational signature. Additional evidence showed that the elevated expression of AHRR and GPR15 were associated with smoking-altered hypomethylations at cg14817490 and cg19859270, respectively, in lung adenocarcinoma tumor tissues. Lastly, we showed that decreased expression of RPS6KA1, were associated with poor survival of lung cancer patients.

Conclusions: Our findings provide novel insight into the contributions of tobacco smoking to carcinogenesis through the underlying mechanisms of the elevated mutational signature by altered DNA methylations and gene expressions.

Keywords: Gene expression; Mediation analysis; Methylation; Mutational signature; Tobacco smoking.

Fig. 1

Identification of genes and their associations with smoking-related mutational signature. a A flow chart to illustrate the identification of candidate smoking-related DNA methylations from the previously reported blood-based methylations in five EWAS. “N” represents the sample size for each study. b Smoking-related mutational signature displayed according to the 96 substitution classifications characterized by six substitution types, together with a flanking base pair to the mutated base (Alexandrov et al. 2013). c A scatter plot indicating tobacco smoking correlated with known smoking-related mutational signature in lung adenocarcinoma. The dotted line refers to association coefficient. Each point represents one sample. The x axis represents the number of packs per year for each sample, the y axis represents the contribution of smoking-related mutational signature to overall mutation burden for each sample. The color from red to green refers to a higher to lower density of samples (this note applies to all other figure legends). d Box plots of the enrichment score of smoking-related mutational signature across 26 cancer types. e Bar plots indicating the P value of associations between the candidate genes and smoking-related mutational signature in six cancer types. Only genes with a P value of less than 1 × 10^− 4 were presented. The dashed dot box highlights the genes with significant associations at a Bonferroni-correction P < 0.001. f Scatter plots for each gene with significant associations at a Bonferroni-correction P < 0.001. From the left to the right panel, four genes in head and neck and seven genes in lung adenocarcinoma are presented

Fig. 2

Mediation analysis illustrating the effect of the expression of five genes that would be altered by smoking on smoking-related mutational signature in lung adenocarcinoma. a Scatter plots indicating the statistical significance between five candidate genes and tobacco smoking in lung adenocarcinoma. b A diagram to illustrate a mediation analysis framework, where gene expression can be a mediator to affect smoking-related mutational signature. c Five candidate genes are presented with significant mediation effect (via gene expression on smoking-related mutational signature), at P < 0.05

Fig. 3

Mediation analysis illustrating the effect of tobacco smoking-altered methylation on gene expression in lung adenocarcinoma. a Scatter plots indicating the statistical significance of associations between methylations at three candidate CpG sites and tobacco smoking in lung adenocarcinoma. b Scatter plots indicating negative correlations between DNA methylation at three candidate CpG sites and gene expression in lung adenocarcinoma.c A diagram to illustrate a mediation analysis framework, where DNA methylation can be a mediator to affect the expression of tobacco smoking-altered genes. d Two candidate CpG sites are presented with significant mediation effects on gene expression, at P < 0.05. “ACME” refers to the average causal mediation effects via DNA methylation on gene expression