University of Dundee p 53 controls expression of the DNA deaminase APOBEC 3 B to limit its potential mutagenic activity in cancer cells

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 overexpression 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. INTRODUCTION The APOBEC3 family of cytosine deaminases are mediators of intrinsic immunity to retroviruses and endogenous retrotransposons, which act by causing cytosine-touracil (C-to-U) deamination in single-stranded DNA that is generated during reverse transcription (1,2), to promote deleterious mutations. The seven members of the APOBEC3 gene family (A3A/B/C/D/F/G/H), are related to the APOBEC1, APOBEC2, APOBEC4 and activationinduced deaminase (AID). APOBEC1 functions in RNA editing and DNA editing by AID is required for classswitch recombination and somatic hypermutation of immunoglobulin genes in B cells to augment antibody diversity (3). Additionally, apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) genes, in particular AID, have been implicated in the epigenetic regulation of gene expression by directing the deamination of 5-hydroxymethylcytosine generated by TET enzyme conversion of 5-methylcytosine (for reviews see refs. (4–6)). Here, deamination by AID facilitates base excision repair, resulting in cytosine demethylation. Moreover, we recently reported a DNA methylation-independent role for A3Bmediated cytidine deamination and repair as a mechanism for chromatin remodelling that facilitates estrogen receptor (ER) target gene expression in breast cancer cells (7). The mutational capacity of APOBECs has led to the proposal that inappropriate and/or upregulated expression of these genes could promote mutations in genomic DNA, a *To whom correspondence should be addressed. Tel: +44 20 7594 2811; Email: simak.ali@imperial.ac. uk C © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup. com/nar/article-abstract/doi/10.1093/nar/gkx721/4082858/p53-controls-expression-of-the-DNA-deaminase by University Library user on 10 October 2017 2 Nucleic Acids Research, 2017 possibility bolstered by the demonstration that AID can cause chromosomal mutations and rearrangements (8–10) and AID, as well as APOBEC1 promote tumourigenesis in transgenic mouse models (11–13). Ectopic expression studies in yeast and mammalian cells have shown that APOBEC3 enzymes can also promote mutations in genomic DNA (14–17). Importantly, sequencing of cancer genomes reveals that a large proportion of somatic mutations in diverse cancer types, including breast, ovarian, cervical, bladder, head and neck and lung cancer, are attributable to APOBEC activity (17–23). A3B is the only one of the 11 APOBEC genes that is consistently expressed at high levels in these cancer types and A3B expression correlates with the number of C-to-T and overall mutational load in cancer genomes (17,21,24). Genome sequencing of yeast cells expressing A3B identify kataegic mutational patterns similar to those that are observed in breast cancer genomes (25) and the incidence of C-to-T mutations in breast cancer cells is reduced by A3B knockdown (17). Together, these studies provide a compelling case for A3B as a driver of the mutational landscape and tumour evolution in many common cancers. Gene expression analysis shows that A3B levels are low in normal tissues, but are elevated in many cancer types (7,17). Understanding the mechanisms that regulate A3B expression will provide important insights into the processes driving acquisition of cancer mutations and tumour evolution. Originally cloned on the basis of its induction by phorbol ester treatment of normal keratinocytes, A3B expression is stimulated by NFB activation by protein kinase C (26,27). Interestingly, mutational signatures associated with A3B activity are especially strong in cervical and head/neck cancers, in which human papillomaviruses (HPV) are important causative agents (20,21,28). Recently, the E6 and E7 viral oncogenes in high-risk HPVs were shown to promote A3B expression (29–32), highlighting a potential explanation for the A3B-associated mutator phenotype in HPVpositive cervical and head/neck cancer. As inhibition of p53 tumour suppressor activity/levels is a key function of E6 (33), we reasoned that p53 might be a direct regulator of A3B expression. Here, we show that p53 represses A3B expression and cytosine deaminase activity in cancer cells, through a p21-dependent mechanism and that loss of p53 activity through its mutation or HPV-16 E6/E7-mediated downregulation, causes A3B upregulation. Further, by assessing cellular cytosine deaminase activity and abasic site generation in genomic DNA, we show that loss of p53 activity through mutation or HPV-directed downregulation can promote increased mutagenic capacity of normal and cancer cells. MATERIALS AND METHODS