APOBEC3B Upregulation and Genomic Mutation Patterns in Serous Ovarian Carcinoma

Endogenous APOBEC3B activity in ovarian cancer cell lines. A, a schematic of the fluorescence-based DNA cytosine deamination assay. The single-stranded DNA substrate has a target cytosine, 5′ fluorescent group (F) and 3′ fluorescence-quenching group (Q). Deamination and uracil excision create an abasic site, hydroxide breaks the DNA backbone, and the fluorescent group escapes quenching. B, APOBEC3B qRT-PCR data from the indicated ovarian cancer cell lines expressing control shRNA (Con) or one of two shRNAs specific to APOBEC3B (1 or 2; n = 3; mean and SD shown for each condition). Fractionation is confirmed by immunoblots of the cytoplasmic (C) and nuclear (N) protein fractions from each condition (TUB, anti-tubulin; H3, anti-histone H3). C, DNA C-to-U deaminase activity elicited by cytoplasmic (top) and nuclear (bottom) protein extracts from the indicated cell lines. These experiments used a single-stranded DNA substrate with a single 5′-TC deamination target. Symbol colors match the knockdown bar colors in B.

Intrinsic DNA deamination preferences of recombinant APOBEC3B. A, representative gel images of APOBEC3B catalytic domain DNA deamination products accumulating over the indicated reaction times for the 5′-TCA (most preferred) and 5′-GCA (least preferred) trinucleotide contexts. Complete deamination by APOBEC3A is shown as a positive control (Con). B, APOBEC3B catalytic domain deamination kinetics using 5′-TCN, CCN, GCN, and ACN single-stranded DNA substrates (n = 16 reaction conditions done each in triplicate; mean values are shown with SD smaller than symbols in all but one instance). Reactions with 5′-RCN substrates had indistinguishably low activity (R = A or G).

APOBEC3B expression in ovarian tumors. A, APOBEC3B levels in the indicated normal (green triangles; n = 21) and unmatched cancerous (red circles; n = 66) ovarian tissues. Cancer history is indicated by open (no history) or filled green symbols (some history; see Supplementary Tables S2 and S4 for additional patient information). Tumor stage is indicated by open (early-stage) or closed (late-stage) red symbols. Data points in each category are arranged from lowest to highest APOBEC3B expression level. Each point reports the mean APOBEC3B level of 3 independent qRT-PCR reactions presented relative to mRNA levels of the constitutive housekeeping gene TBP (error bars = 1 SD). Asterisks indicate samples that are also in TCGA data sets with the alternative identifiers listed in Supplementary Table S5. B, dot plot showing APOBEC3B expression in matched normal and tumor specimens (n = 11 unrelated to specimens in A). Lines connect matched specimens. P values were calculated using the Signed rank test. C and D, dot plots showing the relationship between APOBEC3B levels (as in A) and tumor stage (early vs. late) or tumor grade (low vs. high). P values were calculated using the Wilcoxon rank sum test. E, relative microarray APOBEC3 expression levels based on data from the indicated probe sets. A false-positive APOBEC3A signal is expected due to high nucleotide identity with APOBEC3B and cross-hybridizing probe sets (see supplement to ref. 7). F, APOBEC3B quantification by RNA sequencing of 188 TCGA ovarian tumors. APOBEC3B mRNA levels are presented relative to those of the housekeeping gene TBP and plotted from lowest to highest. No normal tissues were available for comparison. G, a two-dimensional plot comparing qRT-PCR and RNA sequencing data for tumor samples common to each analysis (n = 32). P values calculated using the Spearman correlation.

Ovarian cancer genomic mutation patterns. A, correlation between APOBEC3B expression and total mutation loads in whole-genome sequences of 16 early-stage serous ovarian carcinomas (Supplementary Table S4) assessed using the Spearman correlation. B, grouped analysis of whole-genome mutation types in all 16 cancers. C, correlation between APOBEC3B expression levels and mutation type at C/G base pairs in whole-genome sequences assessed using the Spearman correlation. D, trinucleotide context of the mutated C for transversions (top) and transitions (bottom) in whole-genome sequences (16,986 transversions and 13,232 transitions). E–H, as above for A–D, except these analyses were done using RNAseq-confirmed mutations from the same 16 early-stage serous ovarian carcinomas (1,468 transversions and 1,198 transitions).