Abstract
Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B) to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80–90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.
Highlights
Cancer genome sequencing studies have defined approximately 30 distinct mutation signatures
A3B-eGFP clones were uniformly GFPnegative without Dox treatment, but eGFP only clones showed a low level of leaky expression possibly related to greater protein stability
A base substitution mutation was defined as a clear single nucleotide polymorphism (SNP) difference between each daughter clone and her respective granddaughter clone. These analyses revealed a wide range of SNP alterations among granddaughter clones, ranging from a low of
Summary
Cancer genome sequencing studies have defined approximately 30 distinct mutation signatures (reviewed by [1,2,3,4]). Some signatures are large-scale confirmations of established sources of PLOS ONE | DOI:10.1371/journal.pone.0155391. Breast Cancer Research Program (BC121347), the Jimmy V Foundation for Cancer Research, the Norwegian Centennial Chair Program, the Minnesota Partnership for Biotechnology and Medical Genomics, and the Randy Shaver Cancer Research and Community Fund. Salary support for GJS was provided by a National Science Foundation Graduate Research Fellowship (DGE 13488264). RSH is an Investigator of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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