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Background We have previously shown that the zinc finger transcription repressor SNAI2 (SLUG) represses tumor suppressor BRCA2-expression in non-dividing cells by binding to the E2-box upstream of the transcription start site. However, it is unclear how proliferating breast cancer (BC) cells that has higher oxidation state, overcome this repression. In this study, we provide insight into the mechanism of de-silencing of BRCA2 gene expression by PRDX5A, which is the longest member of the peroxiredoxin5 family, in proliferating breast cancer cells. Methods We used cell synchronization and DNA affinity pulldown to analyze PRDX5A binding to the BRCA2 silencer. We used oxidative stress and microRNA (miRNA) treatments to study nuclear localization of PRDX5A and its impact on BRCA2-expression. We validated our findings using mutational, reporter assay, and immunofluorescence analyses. Results Under oxidative stress, proliferating BC cells express PRDX5 isoform A (PRDX5A). In the nucleus, PRDX5A binds to the BRCA2 silencer near the E2-box, displacing SLUG and enhancing BRCA2-expression. Nuclear PRDX5A is translated from the second AUG codon in frame to the first AUG codon in the PRDX5A transcript that retains all exons. Mutation of the first AUG increases nuclear localization of PRDX5A in MDA-MB-231 cells, but mutation of the second AUG decreases it. Increased mitronic hsa-miRNA-6855-3p levels under oxidative stress renders translation from the second AUG preferable. Mutational analysis using reporter assay uncovered a miR-6855-3p binding site between the first and second AUG codon in the PRDX5A transcript. miR-6855-3p mimic increases accumulation of nuclear PRDX5A and inhibits reporter gene translation. Conclusion Oxidative stress increases miR-6855-3p expression and binding to the inter-AUG sequence of the PRDX5A transcript, promoting translation of nuclear PRDX5A. Nuclear PRDX5A relieves SLUG-mediated BRCA2 silencing, resulting in increased BRCA2-expression. Graphical abstract

The precursor of the human microRNA hsa-miR-6855-3p is a 5-tailed mitronic pre-miRNA that is coded by intron #13 of the deubiquitinase USP20 gene. Our preliminary data suggest that the biosynthesis of this miRNA is upregulated by oxidative stress in the SLUG-high metastatic breast cancer cells, promoting DNA double-strand break repair (DSBR) through the indirect enhancement of BRCA2 gene expression. We found that miR-6855-3p favors BRCA2 biosynthesis through the enhancement of the level of an alternately translated form of the BRCA2 gene enhancer protein SPRDX5. We hypothesize that BRCA2 gene silencer acts as a scaffolding to co-recruit SLUG and SPRDX5 and promote SPRDX5-mediated oxidative inactivation of the BRCA2 negative regulator protein SLUG. Here, we present evidence that (a) the expression of miR-6855-3p is essential for the expression of BRCA2 in the SLUG-high metastatic breast cancer cells; (b) knockdown of miR-6855-3p affects DSBR in these cells; and (c) the miR-6855-3p knocked-down cells are susceptible to the synthetic lethality by the PARP inhibitors. Our study reveals a novel mechanism of regulation of BRCA2 gene expression in the aggressive breast cancer cells, offering an alternative strategy to chemically intervene in the progression of hard-to-cure, highly aggressive SLUG-high breast cancer. We propose that the inhibition of miR-6855-3p will induce BRCA2 deficiency in the BLBC cells, thus increasing their susceptibility to standard chemotherapy as well as to PARP inhibitors as has been described for other BRCA2-deficient cells. Supported in part by DOD-CDMRP IDEA Expansion Grant #BC103645 and NIH/NCI grant 1R21CA181920-01 to GC and 1U54RR026140 to SM. Note: This abstract was not presented at the conference. Citation Format: Marshall JD Ellison, Smita Misra, Gautam Chaudhuri. Characterization of the mitronic tsmiR miR6855-3p as a DNA damage repair regulator in the SLUG-high metastatic breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A12.