Abstract 6095: A DNA damaging agent affects BRCA2 exon 3-skipping

Abstract

Abstract Introduction: BRCA2 is a tumor suppressor that participates in DNA repair. Inherited pathogenic mutations in BRCA2 are associated with a 38%-84% risk for breast cancer and a 16.5%-27% risk for ovarian cancer. BRCA2 exon 3, which encodes a binding site for the interacting proteins EMSY and PALB2, is frequently skipped during alternative splicing events, often due to spliceogenic mutations, but also for as yet uncharacterized environmental conditions. A genomic deletion that results in complete allelic loss of exon 3 has been shown to be pathogenic, yet sequence variants that result in increased frequency of exon 3-skipping have been shown to be non-pathogenic. Thus, the allele-specific ability of BRCA2 to contribute to DNA repair is affected by the level of exon 3-skipping. In this study we explore the effects of the topoisomerase inhibitor doxorubicin on the frequency of BRCA2 exon 3-skipping. Isoform-specific end-point RT-PCR shows that doxorubicin increases the frequency of BRCA2 exon 3-skipping in the breast cancer cell line MCF7 at concentrations below those required for promoting apoptosis. Methods: MCF7 cells were treated with doxorubicin according to standard protocols. IC50 levels of doxorubicin were determined using a crystal violet assay. Flow cytometry was performed using standard protocols. RNA was prepared using Qiagen reagents and amplified with Invitrogen One-Step RT-PCR reagents. Isoform-specific RT-PCR primers were designed to amplify the full-length region surrounding BRCA2 exon 3 independently from the region left by an exon-skipping event (∆3). RT-PCR products were visualized using agarose gel electrophoresis. Results and Conclusion: Results suggest that doxorubicin increases the level of BRCA2 exon 3-skipping in MCF7 at concentrations below those required for promoting apoptosis. This suggests doxorubicin may compromise the DNA repair functions of BRCA2 and other alternatively spliced mRNAs, which may contribute to the therapeutic benefits and/or toxicity of the drug. Citation Format: Jordan R. Werner, Hiba Siddiqui, Ethan Castillo, Mohammed Ansari, James D. Fackenthal. A DNA damaging agent affects BRCA2 exon 3-skipping [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6095.