APOBEC3 enzymes mediate efficacy of cisplatin and are epistatic with base excision repair and mismatch repair in platinum response

Abstract

Identifying the mechanisms mediating cisplatin response is essential for improving patient response. Previous research has identified base excision repair (BER) and mismatch repair (MMR) activity in sensitizing cells to cisplatin. Cisplatin forms DNA adducts including interstrand cross-links (ICLs) that distort the DNA helix, forcing adjacent cytosines to become extrahelical. These extrahelical cytosines provide a substrate for cytosine deaminases. Herein, we show that APOBEC3 (A3) enzymes are capable of deaminating the extrahelical cytosines to uracils and sensitizing breast cancer cells to cisplatin. Knockdown of A3s results in resistance to cisplatin and induction of A3 expression in cells with low A3 expression increases sensitivity to cisplatin. We show that the actions of A3s are epistatic with BER and MMR. We propose that A3-induced cytosine deamination to uracil at cisplatin ICLs results in repair of uracils by BER, which blocks ICL DNA repair and enhances cisplatin efficacy and improves breast cancer outcomes.

Figure 1.

A3 mediation of cisplatin and carboplatin response. MDA-MB-231 colony survival assay with shControl or shA3B treated for 2 h with (A) cisplatin, (B) carboplatin and (C) oxaliplatin. P-values determined using unpaired two-sided t-test. (D) MDA-MB-231 A3 expression determined by RT-PCR for shControl or shA3B. Unpaired two-sided t-test was used to determine P-values. (E) Modified alkaline comet assay in MDA-MB-231 with shControl or shA3B treated for 2 h with 10 μM cisplatin. Error bars are standard deviation. n.s. represents not significant, *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 2.

Specific A3 knockdown using siRNA to evaluate cisplatin response. (A) MDA-MB-231 colony survival assay with siControl or siA3B with 2-h cisplatin treatment. MDA-MB-231 colony survival assay with siControl or siA3D treated for 2 h with (B) cisplatin or (C) oxaliplatin. P-values determined using unpaired two-sided t-test. (D) In vitro deamination and incision assay. Undamaged DNA, lanes 1–4; ICL DNA, lanes 5–8 and 10–12. A3B-CTD, UDG, APE1 and A3C are signified with + if in the reaction. A 20mer marker was loaded in lane 9. Substrate depictions and possible reaction products are included next to the gel.

Figure 3.

Epistatic relationship of A3 enzymes, BER and MMR to mediate cisplatin response. (A) MDA-MB-231 colony survival assay with 2-h cisplatin treatment with shControl, shA3B, shA3B and shMSH6, shA3B and shPolβ, shMSH6 and shPolβ. (B) Modified alkaline comet assay with the cells from (A) treated for 2 h with 10 μM cisplatin. Colony survival assay with SK-BR-3 shControl, shUNG, shMSH6, shA3B and shPolβ with 2-h (C) cisplatin or (D) carboplatin treatment. Colony survival assays in SK-BR-3 cells with cisplatin treatment following overexpression of (E) A3C, (F) A3D or (G) A3G in SK-BR-3 cells compared to control constructs. Colony survival P-values determined using unpaired two-sided t-test. n.s. represents not significant, *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 4.

Induction of A3s with IFNα-2b and sensitization to cisplatin treatment. (A) A3 expression in SK-BR-3 determined by RT-PCR. P-values determined using unpaired two-sided t-test. SK-BR-3 colony survival assay with control or IFNα-2b treatment with 2-h (B) cisplatin or (C) carboplatin treatment. (D) Modified alkaline comet assay with SK-BR-3 control or IFNα-2b-treated cells with 2-h 5 μM cisplatin treatment. (E) SK-BR-3 colony survival assay with shControl or shA3B with or without IFNα-2b with 2-h cisplatin treatment. P-values determined using one-way ANOVA with Tukey’s post-hoc analysis. (F) Modified alkaline comet assay with the cells from (E) after 2-h 5 μM cisplatin treatment. (G) SK-BR-3 colony survival assay with shControl, shMSH6 and shPolβ with or without IFNα-2b with 2-h cisplatin treatment. P-values determined using one-way ANOVA with Tukey’s post-hoc analysis. (H) Modified alkaline comet assay with cells from (G) after 2-h 5 μM cisplatin treatment. n.s. represents not significant, *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 5.

Model of A3 activation of BER and MMR. Our data are consistent with A3 family members deaminating the extrahelical cytosine at cisplatin and carboplatin ICLs. The subsequent uracil induced by the A3s activates the BER pathway, specifically via UNG recruitment followed by APE1 incision at the abasic site. Polβ synthesizes DNA downstream of the ICL and preferentially misincorporates nucleotides, which leads to recruitment of the MMR proteins. The subsequent futile cycle of processing the flanking DNA at the ICLs results in blocking productive ICL DNA repair and leads to enhanced sensitivity to cisplatin and carboplatin.