Mutational signatures associated with tobacco smoking in human cancer

Abstract

Tobacco smoking increases the risk of at least 17 classes of human cancer. We analyzed somatic mutations and DNA methylation in 5243 cancers of types for which tobacco smoking confers an elevated risk. Smoking is associated with increased mutation burdens of multiple distinct mutational signatures, which contribute to different extents in different cancers. One of these signatures, mainly found in cancers derived from tissues directly exposed to tobacco smoke, is attributable to misreplication of DNA damage caused by tobacco carcinogens. Others likely reflect indirect activation of DNA editing by APOBEC cytidine deaminases and of an endogenous clocklike mutational process. Smoking is associated with limited differences in methylation. The results are consistent with the proposition that smoking increases cancer risk by increasing the somatic mutation load, although direct evidence for this mechanism is lacking in some smoking-related cancer types.

Fig. 1. Comparison between tobacco smokers and lifelong non-smokers.

Bars are used to display average values for numbers of somatic substitutions per megabase, numbers of indels per megabase, numbers of dinucleotide mutations per megabase, numbers of breakpoints per megabase, fraction of the genome that shows copy number changes and numbers of mutations per megabase attributed to mutational signatures found in multiple cancer types associated with tobacco smoking. Light gray bars are non-smokers, while dark gray bars are smokers. Comparisons between smokers and non-smokers for all features, including mutational signatures specific for a cancer type and overall DNA methylation are provided in Table S2. Error bars correspond to 95% confidence intervals for each feature. Each q-value is based on a two-sample Kolmogorov-Smirnov test corrected for multiple hypothesis testing for all features in a cancer type. Cancer types are ordered based on their age adjusted odds ratios for smoking as provided in Table 1. Data for numbers of breakpoints per megabase and fraction of the genome that shows copy number changes were not available for liver cancer and small cell lung cancer. Adeno stands for Adenocarcinoma; Esophag. stands for Esophagus. Note that the presented data include only a few cases (<10) of nonsmokers for lung small cell, lung squamous and cancer of the larynx.

Fig. 2. Mutational signatures associated with tobacco smoking.

(A) Each panel contains 25 randomly selected cancer genomes (represented by individual bars) from either smokers or non-smokers in a given cancer type. The y-axes reflect numbers of somatic mutations per megabase. Each bar is colored proportionately to the numbers of mutations per megabase attributed to the mutational signatures found in that sample. Naming of mutational signatures is consistent with previous reports (–18). (B) Each panel contains the pattern of a mutational signature associated with tobacco smoking. Signatures are depicted using a 96 substitution classification defined by the substitution type and sequence context immediately 5’ and 3’ to the mutated base. Different colors are used to display different types of substitutions. The percentages of mutations attributed to specific substitution types are on the vertical axes, while the horizontal axes display different types of substitutions. Mutational signatures are depicted based on the trinucleotide frequency of the whole human genome. Signatures 2, 4, 5, 13 and 16 are extracted from cancers associated with tobacco smoking. The signature of benzo[a]pyrene is based on in vitro experimental data (19). Numerical values for these mutational signatures are provided in Table S6.

Fig. 3. Differentially methylated individual CpGs in tobacco smokers across cancers associated with tobacco smoking.

Each dot represents an individual CpG. The horizontal axes reflect differences in methylation between lifelong non-smokers and smokers, where positive values correspond to hyper-methylation and negative values to hypo-methylation. The vertical axes depicted levels of statistical significance. Results satisfying a Bonferroni threshold of 10^-7 (above the red line) are considered statistically significant.