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. Author manuscript; available in PMC: 2021 Jun 15.
Published in final edited form as: Oncogene. 2020 Dec 15;40(6):1077–1090. doi: 10.1038/s41388-020-01583-7

Induction of APOBEC3B expression by chemotherapy drugs is mediated by DNA-PK directed activation of NF-κB

Manikandan Periyasamy 1, Anup K Singh 1, Carolina Gemma 1, Raed Farzan 1,6, Rebecca C Allsopp 2, Jacqueline A Shaw 2, Sara Charmsaz 3, Leonie S Young 3, Paula Cunnea 1, R Charles Coombes 1, Balázs Győrffy 4,5, Lakjaya Buluwela 1, Simak Ali 1,*
PMCID: PMC7116738  EMSID: EMS114702  PMID: 33323971

Abstract

The mutagenic APOBEC3B (A3B) cytosine deaminase is frequently over-expressed in cancer and promotes tumour heterogeneity and therapy resistance. Hence, understanding the mechanisms that underlie A3B over-expression is important, especially for developing therapeutic approaches to reducing A3B levels, and consequently limiting cancer mutagenesis. We previously demonstrated that A3B is repressed by p53 and p53 mutation increases A3B expression. Here, we investigate A3B expression upon treatment with chemotherapeutic drugs that activate p53, including 5-fluorouracil, etoposide and cisplatin. Contrary to expectation, these drugs induced A3B expression and concomitant cellular cytosine deaminase activity. A3B induction was p53-independent, as chemotherapy drugs stimulated A3B expression in p53 mutant cells. These drugs commonly activate ATM, ATR and DNA-PKcs. Using specific inhibitors and gene knockdowns, we show that activation of DNA-PKcs and ATM by chemotherapeutic drugs promotes NF-kB activity, with consequent recruitment of NF-kB to the A3B gene promoter to drive A3B expression. Further, we find that A3B knockdown re-sensitises resistant cells to cisplatin, and A3B knockout enhances sensitivity to chemotherapy drugs. Our data highlight a role for A3B in resistance to chemotherapy and indicate that stimulation of A3B expression by activation of DNA repair and NF-kB pathways could promote cancer mutations and expedite chemoresistance.

Keywords: Cancer, cancer mutations, cytosine deaminases, APOBEC3, chemotherapy, NF-κB

Introduction

Apolipoprotein B mRNA editing catalytic polypeptides (APOBECs) form a family of zinc-dependent cytidine deaminases that, in humans, include activation-induced deaminase (AID), APOBEC1, APOBEC2, APOBEC4 and seven APOBEC3 genes (A3A, A3B, A3C, A3D, A3F, A3G and A3H) [1]. Members of the APOBEC3 subfamily are capable of deaminating cytidine-to-uridine (C-to-U) on single stranded DNA (ssDNA), to generate potential cytidine-to-thymine (C-to-T) transition and cytidine-to-guanine (C-to-G) transversion mutations in 5’-TC motifs [2]. As part of the innate immune system, APOBEC3 genes are implicated in defence against viral pathogens such as retroviruses, papillomaviruses, hepatitis virus, and in the restriction of endogenous retroelements. Numerous studies have revealed the capacity of APOBEC3 proteins to cause genomic DNA mutation [3–5]. Concomitant with these reports has been the understanding, in the wake of cancer genome sequencing, that APOBEC3 cytidine deaminase activity drives somatic mutations in many cancers, including breast, ovarian, head and neck, and lung cancer [5–7]. The most compelling evidence links A3B to these cancer mutations, [2, 5, 7, 8], with A3B-mediated mutagenesis implicated in localised clustering of mutations (kataegis) [4], generation of cancer driver mutations in PIK3CA and TP53 [8–10] and triggering of tumour heterogeneity [9], which may drive tumour evolution [11] and resistance to cancer therapies [12, 13].

The induction of APOBEC3 expression following the interferon response upon viral infection is well-established [14]. Less clear are the mechanisms by which APOBEC3 overexpression, and particularly A3B, are elevated in cancer. We previously showed that p53 is an important repressor of A3B expression in diverse cancer types [15]. Surprisingly, chemotherapy agents stimulated, rather than inhibited, A3B expression, despite their activation of p53. Indeed, induction of A3B expression by these drugs was p53-independent. Using chemical inhibitors, siRNA and CRISPR-Cas9 knockout cells, together with expression analysis and chromatin immunoprecipitations (ChIP), we now show that chemotherapy induction of A3B expression and activity, as well as A3B-directed activation of base excision repair (BER) is directed by DNA-PKcs/ATM/AKT activation of NF-κB. We provide evidence for elevated levels of activated ATM, DNA-PKcs, NF-κB and associated higher A3B expression in ovarian cancer cell lines derived from patients following progression on cisplatin, suggesting that activation of this pathway may underlie continued mutational activity in the generation of chemotherapy resistance and tumour evolution. Importantly, through CRISPR mediated gene knockout, and siRNA studies, we also show that sensitivity to chemotherapy agents is modulated by A3B.

Results

Chemotherapeutic drugs stimulate A3B expression in cancer cells

To determine if A3B expression was correlated with response to chemotherapy treatments, we analysed two large, independent, breast cancer patient public gene expression data sets. Chemotherapy-treated breast cancer patients with higher A3B expression, progressed more rapidly than those with lower A3B expression in both data sets (Figure 1A-B). High A3B expression has been associated with poor outcome in ER+ breast cancer [16]. Dividing patients in this ER+ cohort into chemotherapy responders and non-responders, showed that non-responder groups were characterised by significantly higher A3B expression than responder groups (Mann-Whitney U test in the GEO series p=6.97x10-6 and in the METABRIC p=0.00036, respectively) (Figure 1C-D). Analysis of ER+ patients who received chemotherapy, but who were not treated with hormonal therapy, further confirms a greater incidence of recurrence with high A3B expression (HR = 1.8 (1.21-2.67), logrank p=0.003) (Supplementary Figure 1).

Figure 1. APOBEC3B expression is associated with poor patient response to chemotherapy in breast cancer.

Figure 1

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(A-B) KM plot of breast cancer patients from microarray data sets (A; GEO series) and METABRIC (B), only including patients who were reported to have been treated with chemotherapy. (C-D) A3B expression is lower in estrogen receptor-positive breast cancer patients who responded to chemotherapy than in patients who did not respond. P-values were generated using the Mann-Whitney U test. The GEO series includes sample with follow-up data from the gene expression microarray data sets GSE1456, GSE16391, GSE16446, GSE16716, GSE17907, GSE19615, GSE20271, GSE21653, GSE31519, GSE3494, GSE37946, GSE45255, GSE4611, GSE5327 and GSE69031. (E) Clonogenic assays were performed for MCF7 and MCF7-A3B-/- cells in the presence of the drugs at seven concentrations, ranging between 0.001 - 10 μM (cisplatin, etoposide, 5-FU), or 0.01 - 100 nM (doxorubicin, SN-38). The graphs plot IC50 values for 4 independent experiments. (F) Immunoblotting was performed using protein lysates prepared 24 hours following addition of Cisplatin (20 μM), Etoposide (10 μM), 5-FU (50 μM), doxorubicin (100 nM), or SN-38 (50 nM) to MCF7 and MCF7-A3B-/- cells.

To better understand response to chemotherapy in vitro, we deleted the A3B gene in the ER+ MCF7 breast cancer cell line. Interestingly, A3B knockout increased the sensitivity of MCF7 cells to chemotherapy agents (Figure 1E). Additionally, all treatments stimulated A3B expression in p53-WT MCF7 cells (Figure 1F), a response that was confirmed in the p53-WT HCT116 cells (Figure 2A, 2B). As we reported previously that p53 suppress A3B expression, we anticipated that the chemotherapy agents would also repress A3B expression. Contrary to our expectations, all agents increased A3B expression in a p53-independent manner; A3B expression was stimulated by chemotherapy agents in p53-WT and p53-null HCT116 and other p53-WT and -mutant breast cancer cells (Supplementary Figure 2-B). Expression of other APOBEC3 genes, namely A3C and A3H, was also induced by chemotherapy drugs, but this increase was strictly p53-dependent, consistent with direct p53 regulation of their expression, as reported previously [17]. ChIP-qPCR showed that Nutlin-3A, an activator of p53 [18], as well as etoposide and cisplatin, promote p53 recruitment to the A3C and A3H gene promoters (Figure 2C). This was confirmed by analysis of published p53 ChIP-seq data (results of one ChIP-seq data set [19] are shown (Figure 2D). However, p53 was not recruited to the A3B promoter (Figure 2C).

Figure 2. Cancer chemotherapy drugs promote APOBEC3B expression in a p53-independent manner.

Figure 2

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(A, B) RNA and protein lysates were prepared 24 hours following drug addition to HCT116 and p53-null HCT116 cells. RT-qPCR is shown relative to GAPDH levels (n=3). Significantly (p<0.05) higher (*) or lower (#) gene expression, relative to the vehicle control, are indicated. (C) p53 ChIP following addition of Nutlin3, etoposide or cisplatin for 24 hours to HCT116 cells (n=3). The NEU3 promoter region was identified as a negative control for p53 binding from analysis of p53 ChIP-seq data [19]. (D) Genome browser snapshot of human chromosome 22 region encoding APOBEC3 genes, with p53 ChIP-seq data for HCT116 cells treated for 12 hours with 10 μM Nutlin3, downloaded from NCBI GEO (GSE86222) [19].

A3B expression was also stimulated in a p53-independent manner by hydroxyurea (HU; DNA synthesis inhibitor), SN38 (topoisomerase I inhibitor) and by aphidicolin (DNA polymerase inhibitor), whereas expression of A3C, A3D and A3H by these agents required p53 (Supplementary Figure 2C-D). Stimulation of A3B expression, independent of p53 status, by the chemotherapeutic drugs was also confirmed in MCF7 cells in which p53 has been inactivated using CRISPR-Cas9 genome editing [15] (Supplementary Figure 2D, 2E). Taken together, these results show that a mechanistically broad variety of chemotherapy agents promote A3B expression independent of p53.

Stimulation of A3B expression by chemotherapy agents promotes APOBEC3 activity in cancer cells

The above results demonstrate that chemotherapy drugs stimulate the expression of several APOBEC3 genes, so treatment with these agents should increase cytidine deaminase (CD) activity. To test this, CD activity was assessed with a single-stranded oligonucleotide containing the sequence TCA, which is the optimal substrate for A3B [20] and is an efficient substrate for the other APOBEC3 genes [21, 22]. Consistent with increased levels of A3B and other APOBEC3 genes, CD activity was boosted by etoposide, cisplatin and by 5-FU (Figure 3A-C). However, CD activity was also increased in p53-null cells, implying that the augmentation of CD activity is due to A3B, as expression of other APOBEC3 genes remained low and was unchanged by the drugs in cell lines with mutant p53. Furthermore, CRISPR-Cas9 knockout of A3B expression in MCF7 and HCT116 yielded cells with low CD activity, regardless of drug treatment (Figure 3B-D, Supplementary Figure 2E-F), confirming that CD activity in HCT116 and MCF7 cells is mostly attributable to A3B. We note that expression of other APOBEC3 genes was similar to, or slightly higher in A3B-/- MCF7 cells compared to the parental MCF7 cells and expression increases following Nutlin-3A or chemotherapy treatments were similar to, or a little greater, than those observed in the parental MCF7 cells (Supplementary Figure 2G).

Figure 3. Chemotherapeutic drugs stimulate A3B cytidine deaminase activity in cancer cells.

Figure 3

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(A-D) Protein lysates prepared from cell lines treated with drugs for 24 hours, were used in the cytidine deaminase assay. Positions of the substrate (probe) and the deamination product, are labelled. CAL51 are WT for p53, T47D and MDA-MB-453 express mutant p53. Levels of the deaminase product were quantified using densitometric analysis using Image J [58]. The results of three independent experiments are shown in the bar charts, including the individual values (circles). Asterisks represent statistically significant (p<0.05; n=3) differences in deaminase products for drug-treated samples, relative to vehicle controls. (E, F, G) AP sites in genomic DNA from indicated cell lines treated with drugs for 24 hours, were quantified by AP site conversion with a biotinylated aldehyde reactive probe and quantification of biotinylated DNA. Asterisks show statistically significant (p<0.05; n=3) differences in AP sites for drug-treated samples, relative to vehicle controls.

Cytidine deamination by APOBEC3 proteins generates U:G mismatches in genomic DNA, activating the base excision repair (BER) pathway in which uracil DNA glycosylases excise U:G mismatches to generate apurinic/apyrimidinic (AP) sites that are processed by AP endonuclease and which can be labelled and quantified [23]. All drugs increased global genomic AP sites in p53-WT (CAL51) and in p53 mutant (MDA-MB-453) cells (Figure 3E). Drug treatments similarly increased AP sites in MCF7 cells. AP sites were approximately 2-fold higher in p53-null MCF7 cells and drug treatments further increased AP sites (Figure 3F), consistent with the higher A3B expression in p53-null cells. Since there is low expression of the other APOBEC3 genes following drug treatments in p53-null cells, the increase in AP sites is likely to be due to A3B, as well as BER activation by direct DNA damage by these agents. While a key mechanism of action of 5-FU is through inhibition of thymidylate synthase, 5-FU and its metabolites can be incorporated into DNA, thus activating BER [24]. Intrastrand crosslinks (ICLs) induced by cisplatin and DNA damage induced by topoisomerase inhibitors, such as etoposide, also activate BER, as well as other repair pathways [25, 26]. A3B deletion impaired the stimulation of AP site generation by 5-FU in MCF7 cells by 46% (± SD = 3.9%, p=0.0036) (Figure 3F), further evidence that stimulation of A3B expression represents a mechanism for 5-FU-mediated DNA damage. The number of genomic AP sites were similarly lower in A3B-null MCF7 cells treated with etoposide (mean = 38%, SD = 1.08%, p=0.0004), cisplatin (mean = 28%, SD = 0.66, p=0.0003), hydroxyurea (mean = 48%, SD = 2.8, p=0.0018) and SN38 (mean = 38%, SD = 3.04, p=0.0033). Genomic AP sites were also reduced in A3B-null HCT116 cells by between 19% (SD = 0.77, p=0.0008) in the case of HU and 33% (SD = 3.6, p=0.0061) for cisplatin (Figure 3G). Since there is no reduction in expression of the other APOBEC3 genes in A3B-null cells, these results support the idea that A3B activity is an important driver for BER activation following treatment with diverse DNA damaging chemotherapeutic drugs.

The DREAM complex is not involved in stimulation of A3B expression by chemotherapy

Our previous work showed that repression of A3B expression by p53 involves a pathway in which p53 activation induces p21 expression, which inhibits CDK2/4/6 and so promotes recruitment of the transcriptional repressive E2F4/p107/p130-containing DREAM complex and concomitant displacement of the B-MYB transcriptional activation complex at the A3B gene [15]. As expected, and like Nutlin-3A, both etoposide and cisplatin stimulated p53 recruitment to the p21 and MDM2 gene promoters in ChIP assays (Supplementary Figure 3A), consistent with the activation of p53 by these drugs. By contrast, Nutlin-3A did not support p53 recruitment to the A3B gene, nor was p53 recruited to the survivin gene (Supplementary Figure 3A), another p53-regulated gene that is repressed by the DREAM complex (see ref [15]). Nutlin-3A stimulated DREAM complex recruitment to the A3B gene, as shown with ChIP for the DREAM complex subunits E2F4 and p130, as well as the E2F4-associated KDM5a/JARID1a H3K4 demethylase [27] (Figure 4A, Supplementary Figure 3A). This was accompanied by reduced B-MYB, p300, CBP and RNA polymerase II (PolII) at the A3B gene promoter. By contrast, DREAM complex recruitment was not stimulated by etoposide or cisplatin. Moreover, these treatments resulted in increased binding of the histone acetyltransferases CBP and p300, H3K27 acetylation (H3K27ac) and PolII recruitment, indicative of DREAM complex-independent induction of A3B expression by these drugs. Moreover, and consonant with A3B expression, the DREAM complex was not recruited to the A3B promoter in p53-null HCT116 cells, while CBP, p300, PolII and H3K27ac were enriched by etoposide and cisplatin (but not by Nutlin-3A) in p53-null HCT116 cells. In summary, despite the lack of DREAM complex recruitment, chemotherapy treatments did not stimulate B-MYB binding at the A3B promoter, indicating that B-MYB does not mediate the chemotherapy induction of A3B expression.

Figure 4. NF-κB mediates induction of APOBEC3B gene expression by chemotherapy drugs.

Figure 4

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(A-C) Bar graphs show ChIP-qPCR enrichment for transcription factors or the H3K27ac mark, for ChIP lysates prepared 24 hours following addition of drugs, as fold enrichment relative to vehicle-treated cells. Asterisks show significant (p<0.05) differences in factor enrichment relative to the vehicle control for each of the two cell lines. (D) ChIP was performed following addition of drugs ± NF-κB inhibitors BAY 11-7208 (BAY) or TPCA-1 (n=3; * = p<0.05). (E, F) RNA and protein lysates prepared from T47D cells, 24 hours following drug addition, were used for RT-qPCR and immunoblotting, respectively. RT-qPCR results were normalised for GAPDH expression (n=3). Statistically significant reductions in A3B expression, for NF-κB inhibitor-treated samples, compared to the expression for each chemotherapy drug, are marked by asterisks (p<0.05).

Chemotherapy drugs promote NF-κB recruitment to the A3B gene promoter to stimulate its expression

Stimulation of A3B expression, through PKC-directed recruitment of the NF-κB RelB subunit, but not of RelA/p65, has recently been reported [28]. However, evidence for regulation of A3B expression by RelA/p65 has also been forthcoming [29]. In agreement with the latter, analysis of reported ChIP-seq data (GSE24518, GSE102796, GSE67295) showed that NF-κB activation induces RelA/p65 recruitment to the A3B gene promoter (Supplementary Figure 3B), with additional binding to a region approximately 10 kb 5’ to the A3A gene and a region upstream of the A3G gene. Consistent with this, RelA/p65, p50, RelB and p52 were recruited to the A3B gene upon addition of etoposide and cisplatin in HCT116, p53-null HCT116 and in p53-mutant T47D cells (Figure 4B-C); the results were suggestive of greater recruitment of RelA/p65 and p50 than of RelB or p52. Etoposide and cisplatin similarly promoted NF-κB recruitment to well-characterised NF-κB target genes (ICAM1, IL8, CXCL1), generally with greater enrichment of p65 and p50 than of RelB and p52 (Supplementary Figure 3C-D). NF-κB recruitment following etoposide or cisplatin treatment occurred irrespective of p53 status and NF-κB recruitment was not influenced by Nutlin-3A. The NF-κB inhibitors, BAY 11-7082 [30] and TPCA-1 [31] reduced NF-κB recruitment to the A3B gene promoter (Figure 4D, Supplementary Figure 4A) and blocked stimulation of A3B expression by cisplatin, etoposide or HU (Figure 4E-F, Supplementary Figure 4B-C). Consistent with NF-κB recruitment primarily at the A3B promoter (Supplementary Figure 3B), NF-κB inhibitors largely did not affect the chemotherapy-stimulated expression of other APOBEC3 genes (Supplementary Figure 4C). Finally, siRNA-mediated p65/p50 knockdown also blunted the stimulation of A3B expression by cisplatin and etoposide (Supplementary Figure 4D). Thus, DNA damage by cytotoxic drugs activates NF-κB, which, in turn, drives A3B expression.

DNA-PKcs and ATM mediate NF-κB-directed expression of the APOBEC3B gene

The DNA damage response activated upon genotoxic stress is mediated by three related kinases, ATM, ATR and DNA-PKcs, with ATM and DNA-PKcs being central to the response to DNA double strand breaks (DSB) formation of the type engendered by cytotoxic chemotherapies [32]. Moreover, ATM activates NF-κB through a mechanism in which interaction of ATM with the the regulatory subunit of IκB kinase (IKKγ, also known as the NF-κB essential modulator, NEMO), promotes its export to the cytoplasm, facilitating IκB phosphorylation and consequent degradation [33] (Figure 5A). DNA-PKcs can also activate NF-κB, by directly phosphorylating IκB [34] and via activation of AKT [35], a key mediator of IκB kinase [36]. Therefore, we determined if the chemotherapy induced expression of A3B results from activation of NF-κB signaling by these DNA damage response (DDR) kinases [37].

Figure 5. Stimulation of APOBEC3B expression is promoted by DNA-PK directed NF-κB activation.

Figure 5

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(A) Pathway from DNA damaging chemotherapy drugs to NF-κB stimulation of A3B expression. (B) RT-qPCR using RNA prepared from T47D cells treated with increasing concentrations of the DNA-PKcs inhibitor, NU7441, for 24 hours. Asterisks highlight significant (p<0.05; n=3) differences relative to vehicle treatment. (C) T47D cells were treated with cisplatin or etoposide ± 5 μM NU7441, for 24 hours. Asterisks denote statistically significant differences in A3B expression, when comparing the NU7441-treated samples with corresponding samples not treated with NU7441 (p<0.05; n=3). (D) Immunoblotting of T47D cell lysates following treatment with cisplatin or etoposide ± 5 μM NU7441. (E) T47D cells were transfected with siRNA for DNA-PKcs (siDNA-PK) or a control siRNA (siControl). Cisplatin or etoposide were added 24 hours following transfection and protein lysates prepared after a further 24 hours. (F, G) Protein lysates from T47D cells treated with cisplatin or etoposide for ± DNA-PKcs (NU7441), ATM (KU55933), AKT (MK2206) or NF-κB (BAY-11-7082, TPCA1) inhibitors for 24 hours, were used in the cytidine deaminase assay. Densitometric quantification of deaminase products using Image J (n=3 independent experiments), is shown in the bar charts. Asterisks show statistically significant (p<0.05) differences in deaminase products for inhibitor-treated samples, relative to the cisplatin- or etoposide-treated samples. The circles show values for each sample and error bars = standard errors of the mean.

In T47D cells, the DNA-PKcs inhibitor, NU7441, inhibited A3B expression in a dose-dependent manner, whereas ATM (KU55933) and ATR (VE-821) inhibitors had only small effects on A3B mRNA levels (Figure 5B, Supplementary Figure 5A). Similar results were obtained in HCT116 cells, except that the highest dose of VE-821 inhibited expression of all A3 genes (Supplementary Figure 5B). Although VE-821 reduced basal A3B expression, it only modestly suppressed the cisplatin and etoposide induction of A3B expression, by 22% (SEM ± 5.01, p=0.0001) and 28% (SEM ± 1.04, p=0.0001), respectively (Supplementary Figure 5C). This compares with 55% (SEM ± 1.1, p=0.0001) and 50% (SEM ± 1.3, p=0.0001) inhibition of the cisplatin and etoposide mediated increase in A3B expression by KU55933 (Supplementary Figure 5C). NU7441 reduced the cisplatin and etoposide induction of A3B by 68% (SEM ± 0.7, p=0.0001) and 66% (SEM ± 1.9, p=0.0001) (Figure 5C).

Knockdown of each DDR kinase confirmed the importance of DNA-PKcs and ATM in directing chemotherapy-induced A3B expression. Whereas ATR knockdown had no effect on A3B expression (Supplementary Figure 5D), DNA-PK and ATM siRNA significantly reduced the impact of cisplatin and etoposide on A3B expression. Immunoblotting showed that DNA-PKcs and ATM inhibitors lessened A3B induction by cisplatin/etoposide (Figure 5D, Supplementary Figure 5E), confirming the RT-qPCR results. These findings demonstrate the importance of ATM and DNA-PKcs activities for inducing A3B expression by chemotherapy drugs.

DNA-PKcs activation of NF-κB in promoting A3B expression is mediated via ATM and AKT

NU7441 inhibited the action of cisplatin/etoposide in all cell lines, regardless of p53 status (Supplementary Figure 6A-F). Previous reports show that lack of DNA-PKcs causes ATM down-regulation [38], providing an explanation for the reduction in ATM mRNA and protein levels observed for NU7441 addition or following DNA-PKcs knockdown (Supplementary Figure 5D, Supplementary Figure 7A-B). Chemical inhibition or knockdown of DNA-PKcs also reduced AKT phosphorylation at the activating serine-473 (S473) (Supplementary Figure 7A, B), in agreement with previous reports [36]. Taken together, these results demonstrate the key role of DNA-PKcs in regulating the induction of A3B expression by DNA damaging drugs.

When activated, IκB kinase (IKKα/ß/γ) phosphorylates IκB, which promotes its ubiquitination and subsequent proteasomal degradation, allowing nuclear translocation of NF-κB and consequent activation of NF-κB target genes [36] (Figure 5A). Cisplatin/etoposide increased IKKα/ß and IκBα phosphorylation (Fig 4F), which was prevented by chemical inhibition or siRNA-knockdown of DNA-PKcs (Figure 5D, 5E). Consequently, NU7441 inhibited NF-κB recruitment to the A3B gene promoter tiggered by cisplatin, etoposide and hydroxyurea (Supplementary Figure 7C-E).

KU55933 also reduced A3B levels and strongly inhibited IKKα/β, IκBα phosphorylation, but had only a small effect on DNA-PKcs phosphorylation (Supplementary Figure 5E). Finally, the AKT inhibitor MK2206, inhibited phosphorylation of IKKα/ß, without affecting ATM or DNA-PKcs phosphorylation induced by cisplatin or etoposide (Supplementary Figure 7F). Taken together, these results reveal that NF-κB induces A3B expression in response to DNA damaging drugs and that its activation is mediated by ATM, AKT and DNA-PKcs, with DNA-PKcs having a pivotal role in regulating the chemotherapy-induced activation of NF-κB, via ATM and AKT. Stimulation of A3B expression by this mechanism is consistent with the cytidine deaminase activity in cancer cells, since inhibiting DNA-PKcs, ATM, AKT or NF-κB robustly reduced cisplatin and etoposide stimulation of cytidine deamination activity in lysates from T47D cells (Figure 5F,G).

Finally, we determined if this pathway also stimulated A3B expression in the non-tumourigenic breast epithelial MCF10A cell line. As in all cancer cell lines tested, chemotherapy drugs stimulated A3B expression (Supplementary Figure 8A). A3B expression was also induced in primary metastatic breast cancer cells (PEOO3), obtained from a pleural effusion [39] (Supplementary Figure 8B). In MCF10A, as well as PEOO3 and in primary normal human mammary epithelial cells (HMEC), DNA-PK, ATM, AKT or NF-κB inhibitors reduced cisplatin-induced A3B expression and cellular cytidine deaminase activity (Supplementary Figure 8C-F). Together, these results demonstrate that this mechanism for A3B induction is not restricted to established cancer cell lines.

The DNA-PKcs/AKT/ATM/NF-κB axis is constitutively active and A3B expression is upregulated in platinum-resistant high-grade serous ovarian carcinoma cells

High-grade serous ovarian carcinoma (HGSOC) shows a high degree of genomic instability, copy number alterations, p53 mutation and chemoresistance. Platinum based chemotherapy is used as first-line therapy for the treatment of HGSOC [40], but most patients progress with platinum-resistant disease. Increased DNA-PKcs activity and DNA-PKcs-directed activation of AKT has also been proposed as a mechanism for driving resistance to platinum in ovarian cancer cells [41]. Therefore, we wondered if the increased DNA-PKcs and AKT activity in platinum-resistant cancer cells, is accompanied by NF-κB activation and higher A3B expression. To this end, we used the isogenic HGSOC paired cell lines PEA1, an HGSOC cell line generated from a patient prior to cisplatin treatment and PEA2, obtained from the same patient following relapse after cisplatin treatment [42].

A3B expression was considerably higher in the cisplatin-resistant PEA2 cells, compared with PEA1 cells (Figure 6A-B). Expression of other NF-κB target genes was also elevated in PEA2 cells than in PEA1 cells, indicative of higher NF-κB activity in the cisplatin-resistant cells. Immunoblotting confirmed higher activities of DNA-PKcs, AKT, ATM and NF-κB, as defined by the phosphorylation levels of DNA-PKcs, ATM, AKT, IKK and IκB in PEA2 cells (Figure 6B). Together with higher A3B expression and cytidine deaminase activity in PEA2 over PEA1 cells (Figure 6C) and the greater enrichment of p65/p50 NF-κB subunits at the A3B gene promoter (Figure 6D), these results are consistent with the data linking ATM/NF-κB and DNA-PKcs/AKT/NF-κB in regulating A3B expression and cytidine deaminase activity in response to cisplatin. Inhibiting ATM, DNA-PKcs, AKT or NF-κB reduced expression of A3B in the absence of chemotherapy drugs in the cisplatin-resistant PEA2 cells (Figure 6E), which implies that these pathways are constitutively active in the cisplatin-resistant HGSOC cells and are responsible for A3B expression. Inhibiting NF-κB increased the sensitivity of PEA2 cells to cisplatin, whereas cisplatin-sensitive PEA1 cells were not much affected by TPCA1, consistent with the elevated NF-κB activity PEA2 (Supplementary Figure 9). In keeping with the increased DNA-PK, ATM and AKT activities in PEA2, compared with PEA1 cells, these inhibitors were also more effective in promoting cisplatin-directed growth inhibition in PEA2 cells.

Figure 6. The DNA-PKcs/AKT/NF-κB axis is constitutively active and APOBEC3B expression is increased in cisplatin-resistant ovarian cancer cells.

Figure 6

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(A) RT-qPCR using RNA prepared from PEA1 and PEA2 ovarian cancer cells for A3B and NF-κB target genes. (B) Immunoblotting of PEA1 and PEA2 protein lysates. (C) Cytidine deaminase assay performed with lysates prepared from PEA1 and PEA2 cells. Protein lysate was omitted in the no lysate control. The bar chart shows the results of three independent experiments. Cytidine deaminase activity was significantly different between PEA1 and PEA2 cells (* = p<0.05). (D) ChIP-qPCR enrichment for NF-κB proteins in PEA1 and PEA2 cells. (E) Protein lysates prepared 24 hours following drug treatment of PEA2 cells were used for immunoblotting. (F) PEA1 and PEA2 cells were transfected with A3B or control siRNA. Cisplatin was added 24 hours following transfection at concentrations ranging from 0.8 nM to 50 μM (4-fold serial dilutions). Growth was determined using the SRB assay after a further 48 hours. IC50 values for 4 independent experiments are plotted. The graph on the right shows A3B expression relative to expression of GAPDH. (G, H) Cisplatin (20 μM) was added as in (F) and cell lysates were assessed for caspase 3/7 activity (n=3) or immunoblotted, as shown.

We also determined if the increased expression of A3B in the cisplatin-resistant ovarian cancer cells is associated with the resistance to cisplatin. Interestingly, A3B knockdown re-sensitised the cisplatin-resistant PEA2 cells to cisplatin (Figure 6F), reducing the IC50 from 20.5 μM to 6.8 μM (difference between means ± SEM = 13.7 ± 1.0, p<0.0001); similar to the IC50 values for cisplatin in the cisplatin-sensitive PEA1 cells (5.3 μM) (the difference between means of ± SEM for siA3B-PEA2 and siControl-PEA1 = 1.5 ± 0.5, p=0.02). Moreover, A3B knockdown increased cisplatin-induced apoptosis, as judged by the increase in caspase activity, as well as levels of cleaved PARP and γH2AX (Figure 6G, H). A3B knockdown also increased sensitivity of PEA1 cells to cisplatin, albeit, the reduction in IC50 values was much more modest (difference between means ± SEM = 0.4 ± 1.0, p=0.003) (Figure 6F).

Discussion

We have shown that DNA damaging chemotherapy drugs stimulate A3B expression, in a p53-independent manner, suggesting that these agents promote A3B expression by a mechanism that is distinct from the DREAM transcription repression complex, previously reported as the mediator of p53-directed A3B inhibition [15]. Compatible with this proposition, treatment with etoposide or cisplatin did not, promote DREAM complex recruitment to the A3B gene promoter. Unlike the p53 activator, Nutlin-3A, these agents also stimulated recruitment of RNA PolII and CBP/p300 histone acetyltransferases, together with increased H3K27 acetylation at the A3B gene promoter. The action of these drugs acting independently of p53 was confirmed in cell lines expressing mutant p53, or cells in which p53 had been ablated by gene knockout.

As has been shown previously for the DNA damaging agent doxorubicin [17], expression of A3C, A3F, A3D and A3H was stimulated by etoposide, cisplatin and hydroxyurea, but as we also show, stimulation of their expression is dependent on the presence of functional p53. ChIP-qPCR confirmed direct recruitment of p53 to their gene promoters upon addition of Nutlin-3A, or chemotherapy drugs. Although interrogation of public p53 ChIP-seq data did not identify p53 binding sites at the A3D and A3F gene promoters, p53 binding to the upstream A3C gene may be sufficient to provide p53 regulation of A3D and A3F expression. Importantly, the results presented here collectively reveal that the increased cellular cytidine deaminase activity caused by the chemotherapeutic agents is due to A3B, with little or no involvement of the other APOBEC3 genes.

Our results using specific inhibitors of each kinase and siRNA-mediated knockdown, show that DNA-PKcs and ATM are necessary for A3B expression in response to etoposide and cisplatin. ATR, by contrast, did not greatly impact on the etoposide/cisplatin stimulation of A3B expression, despite the fact that ATR is activated by genotoxic stresses [32]. In addition, RNAi and inhibitor data demonstrate clearly the importance of DNA-PKcs and ATM for chemotherapy drug induced expression of A3B.

A3B expression was recently shown to be stimulated by PMA-directed activation of NF-κB [28, 29]. Analysis of published ChIP-seq data showed that RelA (p65) is recruited to the A3B gene promoter following TNFα and/or IL1ß treatments. Our results show that cisplatin and etoposide also promote recruitment of RelA/p50, and to a lesser extent RelB/p52, to the A3B gene promoter and highlight the particular importance of the canonical NF-κB subunits RelA/p50 in mediating the chemotherapy induction of A3B expression. Our results demonstrate that induction of DNA-PKcs and ATM caused by DNA damaging chemotherapy drugs activates NF-κB, which in turn stimulates expression of A3B and promotes A3B-directed cytidine deamination. These findings emphasize the particular importance of DNA-PKcs in regulating A3B expression by relaying signals from chemotherapy-induced DNA damage to ATM and AKT, for activation of NF-κB and are consistent with previous reports of NF-κB activation upon DNA-PKcs activation following DNA damage by genotoxic agents, including ionising radiation (IR), etoposide and doxorubicin [43–47].

To further understand the role of A3B in chemotherapy sensitivity, we developed MCF7 cells, in which the A3B gene was deleted by CRISPR-Cas9 gene editing and have found that these knockout cells displayed greater sensitivity to genotoxic drugs. RNAi-directed A3B knockdown similarly increased sensitivity of ovarian cancer cells to cisplatin and re-sensitised cisplatin-resistant ovarian cancer cells. Genomic mutations caused by A3B are proposed as a mechanism for promoting tumour evolution and facilitating mutations in genes that cause resistance to cancer therapies [6, 12, 48, 49]. However, our results imply that A3B itself, also plays an important role in protecting cancer cells from the growth inhibitory effects of genotoxic drugs. Several potential mechanisms can be envisaged. Firstly, up-regulation of A3B expression by genotoxic drugs may activate BER pathways, to stimulate DNA damage repair and thus reduce the effectiveness of chemotherapeutic drugs. A3B also plays an intriguing role as a transcriptional co-activator for estrogen receptor-α, to promote growth of breast cancer cells [16]. Another role for A3B in gene regulation is highlighted by the observed interaction of A3B with several miRNA precursors [50]. The importance of these transcriptional functions of A3B in the cellular response to genotoxic drugs needs further exploration.

In conclusion, we present evidence that A3B expression is induced by chemotherapy in a p53-independent manner, via DNA-PKcs and ATM mediated activation of the NF-κB pathway, resulting in elevated A3B expression, with possible consequences for the generation of somatic mutations that drive tumour evolution. Offsetting this, our results also highlight a converse role for A3B in protecting cells from genotoxic stress, perhaps through activation of DNA repair pathways. A potential chemoprotective role of A3B may also underlie the increased incidence of breast cancer in women with a germline copy number polymorphism that results in deletion of the A3B gene [51] and the higher APOBEC mutational signature that has been described for patients with the A3B deletion polymorphism, compared with patients lacking the A3B deletion [51]. While a role for A3B in protecting the genome from DNA damage may seem to be at odds with repression of its expression by p53, it has previously been demonstrated that p53 reduces BER, by inhibiting expression of the AP endonuclease, APE1 [52], which processes the apurinic sites that would be generated by DNA glycosylase action on A3B-mediated cytidine deamination. While our study demonstrates an important role for A3B in reducing sensitivity to chemotherapy drugs, segregation of tumours on the basis of A3B levels did not show an association with patient relapse in ER-negative breast cancer [16]. In addition, a trend towards better progression-free survival has been previously noted for clear cell ovarian cancer patients with high A3B expression [53]. In both cases, other factors such as altered DNA repair capacities, for example due to BRCA1/2 mutations and the additional involvement of expression of APOBEC3 proteins in tumour infiltrating cells [54], are all likely to contribute to, and further complicate responses to chemotherapy mediated by DNA damage. Notwithstanding, inhibition of NF-κB activation, with ATM, DNA-PKcs, AKT or with NF-κB inhibitors, does identify a means of limiting chemoresistance and tumour evolution by A3B.

Materials and Methods

Cell lines and culturing

Cell lines, as well as HMEC cells were obtained from ATCC (LGC Standards, UK) and cultured in DMEM containing 10% FCS, except for HMEC cells which were cultured according to supplier instructions. MCF7 p53-/- cells [15] and HCT116 p53-/- cells [55] have been described. The paired high grade serous ovarian carcinoma lines PEA1 and PEA2 [42] were cultured in RPMI containing 10% FCS. PE003 breast cancer cells have been described [39]. All cell lines were routinely monitored for mycoplasma negativity and were regularly genotyped. Details of drugs used in this study are provided in Supplementary table 1.

CRISPR-Cas9 mediated A3B knockout

Details of the scheme for generating the A3B knockout and guide RNA sequences are provided in supplemental information. pCAG-Cas9-GFP [56] was a gift from Kiran Musunuru (Addgene, http://n2t.net/addgene:44719).

siRNA transfections

Cells were transfected by reverse transfection using Lipofectamine RNAiMAX (Invitrogen, Thermofisher Scientific, UK), as described [16]. siRNA details are provided in Supplementary Table 2.

Proliferation and clonogenic assays

Cell growth was assessed using the sulphorhodamine B assay (SRB), as described previously [16]. For clonogenic assays, 200 cells/well were seeded in 96 well plates, prior to addition of the indicated drugs or vehicle. Cells were incubated with drugs continuously for 14 days, with changes in media ± drug every 4 days. After 14 days of treatment, cells were processed using the SRB method.

RT-qPCR and ChIP-qPCR

Total RNA extraction and RT-qPCR was undertaken using Taqman Gene Expression Assays (Applied Biosystems, UK) qPCR on an ABI 7900HT machine, as described [16]. Chromatin preparation and immunoprecipitation (ChIP) was performed also as described [16]. ChIP antibodies, Taqman assays and the primer sequences for qPCR are detailed in Supplementary tables 3, 4 and 5, respectively.

Immunoblotting and Cytosine Deaminase assays

Cell lysates were prepared in HEPES lysis buffer (25mM HEPES (pH 7.4), 10% glycerol, 150mM NaCl, 0.5% Triton X-100, 1mM EDTA, 1mM MgCl2 and 1mM ZnCl2), supplemented with protease and phosphatase inhibitors (Roche, UK), sonicated, and lysates were immunoblotted as described [15], using antibodies listed in Supplementary table 6. The cytosine deaminase assay has also been described [15].

Caspase 3/7 Assay

Cells were seeded in 96-well clear bottom white plates and treated for 24 hours with cisplatin or vehicle. The Caspase-Glo 3/7 Assay was performed according to manufacturer’s instructions (Promega, UK).

Apurinic/apyrimidinic (AP) site determination

Genomic DNA was prepared and assayed using the Oxiselect DNA damage ELISA kit (AP sites) (STA-324), according to manufacturer’s protocol (Cell Biolabs Inc. San Diego, CA, USA). Aldehyde reactive probe (ARP) DNA standards provided with the kit, facilitated estimation of the number of genomic AP sites.

Analysis of A3B expression in METABRIC breast cancers and GEO data sets

We utilized two independent patient cohorts to assess the correlation to survival and to chemotherapy response in breast cancer patients. Samples for the GEO dataset were identified by searching the GEO repository (https://www.ncbi.nlm.nih.gov/geo/) for chemotherapy-treated datasets with at least 30 samples (PMID: 31020993). Altogether, we have identified 15 datasets (GSE1456, GSE16391, GSE16446, GSE16716, GSE17907, GSE19615, GSE20271, GSE21653, GSE31519, GSE3494, GSE37946, GSE45255, GSE4611, GSE5327 and GSE69031). A second cohort of samples included chemotherapy-treated tumour samples from the METABRIC dataset [57]. Survival analysis was performed by dividing the patient samples into two cohorts using the expression of A3B and the two cohorts were compared by Cox proportional hazards regression. Response to therapy was assessed by dividing samples into two cohorts by their response status (responders vs. non-responders) and A3B expression was compared between these two groups by the Mann-Whitney U-test.

Statistical analyses

P-values were calculated using the two-tailed t-test in GraphPad Prism version 8.0, for results of at least three replicates.

Supplementary Material

1
2
3

Acknowledgements

We thank Dr B Vogelstein for p53-null HCT116 cells. We are grateful to Dr S Langdon for the PEA1 and PEA2 lines. Our thanks also go to Drs S Hong and L Magnani for their kind gift of PEOO3 cells. This study was supported by Cancer Research UK grant C37/A18784. We thank the LMS/NIHR Imperial Biomedical Research Centre Flow Cytometry Facility for support. Additional support was provided by the Imperial Experimental Cancer Medicine Centre, Imperial NIHR Biomedical Research Centre, and the Cancer Research UK Imperial Centre. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

Footnotes

The authors declare no conflict of interest

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Supplementary Materials

1
EMS114702-supplement-1.docx (1.1MB, docx)
2
EMS114702-supplement-2.docx (29.6KB, docx)
3
EMS114702-supplement-3.pdf (9.1MB, pdf)

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