APOBEC3B (A3B), a DNA cytosine deaminase, contributes to mutagenesis and genomic instability in prostate cancer (PCa). Despite its known association with therapy resistance, the pathways regulating A3B expression and its functional effects in PCa remain unclear. Here we investigated the functional consequences of A3B overexpression in androgen-driven prostate cancer, and its regulation by ATF4, a transcription factor and effector of the integrated stress response.

The potential for A3B regulation by androgen was assessed by treating LNCaP cells (cultured in charcoal-stripped media) with synthetic androgen (R1881), followed by qPCR and WB for A3B expression. The role of TRIB3 as a negative regulator of the ATF4/A3B pathway was investigated using TRIB3 siRNA in DU145 doxycycline (Dox)-inducible ATF4-HA cells. A Dox-inducible A3B-HA expression system was established in DU145 and LNCaP prostate cancer cell lines to assess A3B-induced DNA damage via Western blotting (WB) and immunofluorescence. To explore A3B-driven molecular changes, bulk RNA sequencing (RNA-seq) was performed on LNCaP cells expressing Doxycycline-inducible A3B-HA, followed by differential expression analysis.

R1881 treatment significantly upregulated A3B and ATF4 expression (2-fold, p<0.05), confirming androgen-mediated regulation. Similarly, ATF4 induction led to a 2-fold increase in A3B expression (p<0.05). TRIB3 knockdown further enhanced A3B expression following ATF4 induction, suggesting that TRIB3 is a negative feedback regulator within the ATF4/A3B axis. Additionally, A3B overexpression significantly increased γH2AX levels in DU145 and LNCaP cells (2-fold, p<0.05), confirming its role in DNA damage induction. RNA-seq analysis of A3B-overexpressing LNCaP cells identified 1,087 differentially expressed genes (fold change ≥ 1.0; adjusted p<0.05), including the upregulation of DNA repair pathway genes such as BRCA2, SETD2, and XRCC2, likely in response to A3B-driven DNA damage.

Our findings demonstrate that both androgen signalling and ATF4 induction upregulate A3B, with TRIB3 acting as a negative feedback regulator. Additionally, A3B overexpression induces DNA damage and alters transcriptomic profiles linked to genomic instability in prostate cancer. These findings highlight potential therapeutic targets for mitigating A3B-driven mutagenesis and therapy resistance in PCa.