APOBEC3B is an enzymatic source of mutation in breast cancer

Abstract

Several mutations are required for cancer development, and genome sequencing has revealed that many cancers, including breast cancer, have somatic mutation spectra dominated by C-to-T transitions. Most of these mutations occur at hydrolytically disfavoured non-methylated cytosines throughout the genome, and are sometimes clustered. Here we show that the DNA cytosine deaminase APOBEC3B is a probable source of these mutations. APOBEC3B messenger RNA is upregulated in most primary breast tumours and breast cancer cell lines. Tumours that express high levels of APOBEC3B have twice as many mutations as those that express low levels and are more likely to have mutations in TP53. Endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell-line extracts. Knockdown experiments show that endogenous APOBEC3B correlates with increased levels of genomic uracil, increased mutation frequencies, and C-to-T transitions. Furthermore, induced APOBEC3B overexpression causes cell cycle deviations, cell death, DNA fragmentation, γ-H2AX accumulation and C-to-T mutations. Our data suggest a model in which APOBEC3B-catalysed deamination provides a chronic source of DNA damage in breast cancers that could select TP53 inactivation and explain how some tumours evolve rapidly and manifest heterogeneity.

Figure 1. A3B up-regulation and activity in breast cancer cell lines

a, A3B levels in indicated cell lines. Each point represents the mean of 3 reactions presented relative to TBP (s.d. shown unless smaller than symbol). b, A3B-eGFP or A3F-eGFP localization in MDA-MB-453 (nuclei are blue). c, Nuclear DNA C-to-U activity in extracts from MDA-MB-453 transduced with shControl or shA3B lentiviruses (n=3; s.d. shown unless smaller than symbol). d, Intrinsic dinucleotide DNA deamination preference of endogenous A3B in extracts from MDA-MB-453 (n=3; s.d. smaller than symbols).

Figure 2. A3B-dependent uracil lesions and mutations in breast cancer genomic DNA

a, Workflow for genomic uracil quantification by UPLC-MS. b, Average uracil loads in the indicated cell lines (n=3; errors, s.d.). c, Dot plots representing TK mutant frequencies of HCC1569 subclones expressing shControl or shA3B. Each dot corresponds to one subclone. Medians are labeled. d, Agarose gel and mutation analysis of TP53 3D-PCR amplicons from HCC1569 cells expressing shControl or shA3B (n≥35 sequences per condition). See Fig. S10 for additional data.

Figure 3. Cancer phenotypes triggered by inducing A3B over-expression

a, Cell viability at indicated times post-induction (mean and s.d. for n=3 per condition). b & c, Representative fields of cells imaged for γ-H2AX and A3A-eGFP (1 day) or A3B-eGFP (3 days) post-induction, and γ-H2AX quantification. Abnormal, multinuclear clusters are typical of induced A3B-eGFP (white arrows). d, Representative images of A3-induced DNA comets. e, C-to-T mutations in TP53 detected by sequencing 3D-PCR products 4 days post-induction (n>12 sequences per condition).

Figure 4. A3B up-regulation and mutation in breast tumors

a, A3B and A3G mRNA levels in the indicated tissues. Each symbol represents the mean mRNA level of three RTqPCR reactions presented relative to TBP (s.d. shown unless smaller than symbol; off-scale values are indicated numerically). b, RNAseq data for A3B and A3G in the indicated samples (off-scale values are indicated numerically). c, Local sequence contexts for all genomic cytosines (expected), cytosines deaminated by recombinant A3B (Fig. S13), and observed C-to-T transitions in the indicated cancers. Font size is proportional to each nucleotide frequency. d, Percent C-to-T mutations in the indicated tumors. e & f, C-to-T and total mutation counts for tumors in (b) grouped into lower, middle, and upper thirds based on A3B levels (medians are labeled; p values from Mann Whitney U test; off-scale values are indicated numerically). g, Relationship between A3B level and TP53 status for tumors in (b) (p values from Mann Whitney U test; off-scale values are indicated numerically).