p53 controls expression of the DNA deaminase APOBEC3B to limit its potential mutagenic activity in cancer cells

Abstract

Cancer genome sequencing has implicated the cytosine deaminase activity of apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) genes as an important source of mutations in diverse cancers, with APOBEC3B (A3B) expression especially correlated with such cancer mutations. To better understand the processes directing A3B over-expression in cancer, and possible therapeutic avenues for targeting A3B, we have investigated the regulation of A3B gene expression. Here, we show that A3B expression is inversely related to p53 status in different cancer types and demonstrate that this is due to a direct and pivotal role for p53 in repressing A3B expression. This occurs through the induction of p21 (CDKN1A) and the recruitment of the repressive DREAM complex to the A3B gene promoter, such that loss of p53 through mutation, or human papilloma virus-mediated inhibition, prevents recruitment of the complex, thereby causing elevated A3B expression and cytosine deaminase activity in cancer cells. As p53 is frequently mutated in cancer, our findings provide a mechanism by which p53 loss can promote cancer mutagenesis.

Figure 1.

APOBEC3B expression is elevated in breast cancer with mutated p53. (A) Analysis of A3B expression in METABRIC and TCGA breast cancer samples. For METABRIC, p53 mutational status was available for 820 samples (n = 99 (mutant p53) and 721 (WT p53)). The TCGA dataset comprised 297 samples with mutant and 802 samples with wild-type (WT) p53. (B) A3B expression analysis for the TCGA gene expression datasets for lung and endometrial cancer according to p53 mutational status. (C) RT-qPCR shows that A3B expression is higher in p53 mutant breast cancers than in p53 WT tumours. A3B expression is shown relative to GAPDH levels. (D) RT-qPCR of 50 breast cancer cell lines shows that A3B expression is elevated in cell lines with p53 mutations. A3B expression is shown relative to expression of GAPDH. Asterisks show cell lines (SkBr3, HCC38) that encode the A3A-A3B variant and so do not express A3B.

Figure 2.

p53 represses APOBEC3B expression in cancer cells. Nutlin (10 μM) was added for 24 h in all experiments. (A) RT-qPCR of WT and mutant p53 breast cancer cell lines, treated with Nutlin (n = 3). A3B expression is shown relative to GAPDH. Asterisks show significant (P < 0.05) differences between vehicle and Nutlin-treated cells. (B) Immunoblotting of cell lysates following Nutlin treatment. The filled triangle shows position of Cyclin B1. (C) RT-qPCR of MCF7 cells in which exon 3 of the TP53 gene was targeted using CRISPR-Cas9 (MCF7-Δp53). Expression of all examined genes was significantly (P < 0.05) different between parental and Δp53 MCF7 cells. (D) Immunoblotting of protein lysates from MCF7 and MCF7-Δp53 cells. (E) Protein lysates from HCT116 and p53-null HCT116 (HCT116-p53^−/−) cells ± Nutlin. (F) Twenty-four hours following transfection of HCT116 cells with siRNA for p53, Nutlin was added. RT-qPCR was performed using RNA prepared 48 h following addition of Nutlin. (G) Immunoblotting of HCT116 cells transfected with si-p53.

Figure 3.

p53 regulation of APOBEC3B expression is mediated by p21 acting through the E2F4 DREAM transcriptional complex. (A) Shown is a model depicting the mechanism by which the DREAM complex regulates gene expression following p53 activation. (B) RT-qPCR for p21-null HCT116 cells treated with Nutlin for 24 h. (C) RT-qPCR of HCT116 cells transfected with p21 siRNA. Nutlin was added for 24 h. Significant (P < 0.05; n = 3) differences between vehicle and Nutlin-treated cells are highlighted by asterisks. (D) Immunoblotting of HCT116 cell lysates following p21 knockdown. (E and F) RT-qPCR and immunoblotting of HCT116 cells transfected with siRNA for E2F4.

Figure 4.

ChIP analysis of DREAM complex enrichment at the APOBEC3B gene promoter. (A–H) ZR-75–1 cells were treated with Nutlin (10 μM, 24 h), followed by ChIP for transcription factors in the DREAM complex and for histone marks associated with active genes. Asterisks identify significant (P < 0.05; n = 3) differences in transcription factor recruitment and histone marks for the Nutlin-treated cells, relative to vehicle controls. (I) ChIP-qPCR for Nutlin-treated samples are shown, as fold enrichment relative to vehicle. (J) ChIP-qPCR for the A3B gene in HCT116 cells, in p21-null or in p53-null HCT116 cells. The heat map shows Nutlin-promoted changes in factor recruitment to the A3B gene, relative to vehicle controls. The full ChIP-qPCR data are shown in Supplementary Figure S5.

Figure 5.

Inhibition of APOBEC3B expression by p53 controls mutagenesis in cancer cells. (AandB) Protein lysates prepared from Nutlin-treated cells were used in an in vitro cytidine deaminase assay. Positions of the substrate (probe) and the deamination product, are labelled. (C) Cytidine deamination with lysates prepared from ZR-75–1 cells, following transfection with two independent A3B siRNAs. (D–F) AP or abasic sites in genomic DNA were biotin labelled by conversion with an aldehyde reactive probe (ARP) and quantification of biotinylated DNA. Asterisks show significant (P < 0.05; n = 3) differences in AP sites for Nutlin-treated samples, relative to vehicle controls. (G) Analysis of A3B mutational signature for WT and mutant p53 in breast, lung and endometrial cancer from TCGA. Values on the y-axis represent the number of A3B mutational signatures 2 and 13 in each cancer. P-values were calculated using Mann–Whitney–Wilcoxon statistical test.

Figure 6.

HPV16 E6 and E7 viral oncogenes promote APOBEC3B expression and activity by inhibiting the action of the E2F4/p107/p130 DREAM transcriptional repression complex. (A) HCT116 and p53-null HCT116 cells were transfected with HPV16 E6 or the E6-SAT mutant that does not interact with p53. RT-qPCR was performed using RNA prepared 24 h following addition of Nutlin (10 μM). Asterisks show significant (P < 0.05; n = 3) differences in mRNA expression. (B) Cells treated as above, were processed for immunoblotting. (C) Normal immortalized human keratinocyte line (NIKS) stably transduced with E6, the E6-SAT mutant, E7, E6/E7, E7/mutant E6 or vector only, were treated with Nutlin (10 μM) for 24 h, followed by preparation protein lysates for immunoblotting. (D) NIKS were treated as in C, followed by preparation of total RNA and RT-qPCR. Asterisks show significant (P < 0.05) reduction in mRNA expression for Nutlin-treated samples relative to the vehicle treated NIKS for each transduced line. Cross-hatches show significant (P < 0.05) difference in mRNA expression relative to the vehicle treated and vector transduced NIKS. Results for three experiments are shown. (E) The in vitro cytidine deaminase assay was used to assess A3B activity in protein lysates prepared 24 h following addition of 10 μM Nutlin to the E6 and E7 transduced NIKS cells. (F) Quantification of biotin-labelled ARP conversion of abasic sites in genomic DNA from E6 and E7 transduced NIKS cells. Asterisks show significant (P < 0.05) differences in AP sites compared to vehicle-treated vector transduced NIKS; cross-hatches denote statistically significant reduction in AP sites by Nutlin treatment for each transduction. (G) ChIP-qPCR for vector, E6 or E7 transduced NIKS cells is shown as fold change in transcription factor/histone mark enrichment at the A3B gene promoter, relative to vehicle controls. The actual factor enrichment relative to input is shown in Supplementary Figure S7B.