The DNA cytosine deaminase APOBEC3B promotes tamoxifen resistance in ER-positive breast cancer

Abstract

Breast tumors often display extreme genetic heterogeneity characterized by hundreds of gross chromosomal aberrations and tens of thousands of somatic mutations. Tumor evolution is thought to be ongoing and driven by multiple mutagenic processes. A major outstanding question is whether primary tumors have preexisting mutations for therapy resistance or whether additional DNA damage and mutagenesis are necessary. Drug resistance is a key measure of tumor evolvability. If a resistance mutation preexists at the time of primary tumor presentation, then the intended therapy is likely to fail. However, if resistance does not preexist, then ongoing mutational processes still have the potential to undermine therapeutic efficacy. The antiviral enzyme APOBEC3B (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3B) preferentially deaminates DNA C-to-U, which results in signature C-to-T and C-to-G mutations commonly observed in breast tumors. We use clinical data and xenograft experiments to ask whether APOBEC3B contributes to ongoing breast tumor evolution and resistance to the selective estrogen receptor modulator, tamoxifen. First, APOBEC3B levels in primary estrogen receptor-positive (ER⁺) breast tumors inversely correlate with the clinical benefit of tamoxifen in the treatment of metastatic ER⁺ disease. Second, APOBEC3B depletion in an ER⁺ breast cancer cell line results in prolonged tamoxifen responses in murine xenograft experiments. Third, APOBEC3B overexpression accelerates the development of tamoxifen resistance in murine xenograft experiments by a mechanism that requires the enzyme's catalytic activity. These studies combine to indicate that APOBEC3B promotes drug resistance in breast cancer and that inhibiting APOBEC3B-dependent tumor evolvability may be an effective strategy to improve efficacies of targeted cancer therapies.

Keywords: APOBEC3B; DNA deamination; breast cancer; cancer mutagenesis; drug resistance; tumor evolution.

Fig. 1. High A3B levels in primary ER⁺ breast tumors predict poor response to tamoxifen therapy after tumor recurrence.

(A) Schematic of the clinical time course. Timeline breaks depict variable intervals between clinical milestones. (B) Relative A3B expression levels in each observation group [mean ± SD of n = 72 (quartiles 1 and 3), n = 70 (quartile 2), and n = 71 (quartile 4)]. (C) Kaplan-Meier curves showing the periods of PFS after initiating tamoxifen therapy for patients whose primary tumors expressed A3B at low (dark blue line), intermediate (light blue and orange lines), or high levels [red line; patient groups and color scheme match those in (B)].

Fig. 2. Endogenous A3B depletion does not alter MCF-7L ER⁺ breast cancer cells in culture.

(A) A3B mRNA levels in MCF-7L cells expressing shA3B or shCON constructs (TBP, TATA-binding protein mRNA; each bar represents the mean ± SD of three RT-qPCR assays). (B) A3B DNA cytosine deaminase activity in soluble whole-cell (W), cytoplasmic (C), and nuclear (N) extracts of MCF-7L cells expressing shA3B or shCON constructs. Vector (V) and A3B-transfected 293T cell lysates were used as controls (S, substrate; P, product). (C) Light microscopy images of shA3B and shCON expressing MCF-7L pools. (D and E) Growth kinetics and doubling times of cultured MCF-7L cells expressing shA3B versus shCON constructs (mean ± SD of n = 6 cultures per condition).

Fig. 3. A3B is required for the development of tamoxifen-resistant tumors in mice.

(A) Schematic of the A3B knockdown xenograft study design and time course (see text for details). (B) Growth kinetics of engrafted MCF-7L cells expressing shA3B or shCON in the absence or presence of tamoxifen (TAM) treatment. Tumor volumes were measured weekly (mean + SEM shown for clarity of data presentation). (C) A3B mRNA levels in xenografted tumors recovered from the experiment shown in (B) (TBP mRNA; each bar represents the mean ± SD of three RT-qPCR assays). (D) MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] data comparing tamoxifen susceptibility of input MCF-7L cells versus tamoxifen resistance of a representative MCF-7L shCON tumor [tamoxifen (10, 100, and 1000 ng/ml)].

Fig. 4. Novel lentivirus-based system for conditional A3B overexpression.

(A) Schematic of the lentiviral construct for conditional A3B overexpression (see text for details). LTR, long terminal repeat; RSV, Rous sarcoma virus; CTD, C-terminal domain; NTD, N-terminal domain; CMV, cytomegalovirus; SV40, simian virus 40. (B) A3B mRNA levels relative to TBP in MCF-7L cells expressing lentivirus-delivered A3B or a catalytic mutant derivative (E255Q) as well as endogenous A3B (mean ± SD of three RT-qPCR assays). (C) Doubling times of cultured MCF-7L cells overexpressing A3B or A3B-E255Q (mean ± SD of four replicates).

Fig. 5. Overexpression of catalytically active A3B accelerates the development of tamoxifen-resistant tumors in mice.

(A) Schematic of the A3B overexpression xenograft study design and time course (see text for details). (B) Growth kinetics of engrafted MCF-7L cells overexpressing A3B or A3B-E255Q in the absence or presence of tamoxifen treatment. The graph reports tumor volumes measured weekly (mean + SEM shown for clarity of data presentation). Average tumor volumes from the untreated control arms are shown by gray symbols, and overlapping error bars are omitted for clarity of presentation.