Determining the mutagenic factors that underlie the mix of mutations within tumors is important for a general understanding of carcinogenesis. However, this analysis is daunting as it often requires the testing of numerous poorly defined hypotheses. Here, we have developed a single detailed hypothesis, that APOBEC cytidine deaminases are a significant source of mutagenesis in human cancer genomes. This hypothesis is based on knowledge of the sequence- and single strand-specificity of APOBEC enzymes, their capacity to generate strand-coordinated mutation clusters in model systems and the impressive correlation between experimentally determined APOBEC mutagenesis patterns and the pattern of mutations in strand-coordinated clusters found in cancers. While formally, we cannot exclude that another mutagenic factor may closely mimic both the motif and mutagenic specificities of the APOBEC mutation pattern, there is yet no indication that such a factor exists. Furthermore, our observed correlation between the APOBEC mutagenesis pattern and APOBEC expression in cancer samples provides strong support for this hypothesis. Additional support could be sought in correlations with the germline genotype of patients as soon as such information would be available.
Our TCGA-based analysis indicates a widespread APOBEC mutagenesis pattern and suggests that this pattern is associated with biological mechanisms underlying carcinogenesis. With our approach, we establish a resource for identifying this pattern in the rapidly growing TCGA database as well as in other databases of genome- or exome-wide human mutations. In addition, the predominance of APOBEC-signature mutations across tumors of multiple cancer types sets the next round of questions to be resolved including the identification of the specific APOBEC proteins responsible for mutagenesis, the presence of this mutagenesis in other types and subtypes of cancers, identifying the stage(s) of cancer development that are most prone to APOBEC mutagenesis, and evaluation of the relative impact of this mutagenesis on genome changes that lead to cancer.
Multiple mechanisms could facilitate APOBEC mutagenesis. Environmental and physiological factors may trigger and/or support mutagenesis by (i) affecting the cellular abundance or activity of APOBEC proteins, (ii) altering access to nuclear DNA, and (iii) increasing the amount and/or persistence of ssDNA substrates for APOBEC cytidine deamination. Ours and previous analyses suggest that the level of APOBEC3B
transcription impacts APOBEC mutagenesis. How increased 3B
transcription levels are established remains unclear. Among the factors that could increase the amount of APOBEC(s) are the presence of viral and retrotransposable elements that these enzymes restrict6,33
. Such factors can stimulate APOBEC expression through a complex network of innate immunity signaling including components like Toll-like receptors, interferons, interleukins and even the “usual suspect” in carcinogenesis, the p53 protein34–37
. Infection with several viruses38
as well as retrotransposition39
are associated with carcinogenesis; however the mechanisms of this association are far from clear. A potential relationship between APOBEC mutagenesis and viral infection is appealing as cervical, bladder, and head and neck cancer, which are highly associated with HPV infection, display a strong enrichment in APOBEC mutagenesis.
Despite a positive correlation between APOBEC3B
expression and APOBEC mutagenesis, the extent of the association is relatively small (Spearman r = 0.30). Thus other factors likely contribute more prominently to APOBEC mutagenesis. Factors which could increase the abundance and persistence of ssDNA include DNA damaging agents40,41
as well as defects in DNA transactions that impede break repair42,43
and replication integrity44,45
. Our work in yeast demonstrated that proliferation in the presence of an alkylation agent leads to the formation of ssDNA at DSB sites and dysfunctional forks and subsequently to mutation clusters13
. Importantly, a high level of APOBEC deamination may itself lead to DNA breakage27
, which could generate a ssDNA substrate for APOBEC hyper-mutation. It is generally acknowledged that carcinogenesis requires the accumulation of multiple genetic changes46
. As discussed in13
, simultaneous mutations in scattered stretches of ssDNA formed at DSBs, replication forks and other cell contexts would be excellent substrates for APOBEC mutagenesis, which in turn may produce multiple changes without excessive genome-wide mutation and provide a means to accumulate multiple carcinogenic mutations in a single or a few generations.