Abstract 5852: Doxorubicin increases the level of a BRCA2 exon 3-skipping mRNA isoform in MCF7

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. It is therefore critical to distinguish pathogenic mutations from sequence variants of unknown clinical significance (VUSs). Among the VUSs that require clinical classification are those that could potentially disrupt normal splicing patterns. The challenge in determining the spliceogenic nature of a VUS is the ubiquity of naturally occurring alternative splicing variants. BRCA2 exon 3, which encodes a binding site for the interacting proteins EMSY and PALB2, is frequently skipped during alternative splicing events to different levels in different cell types under different conditions. It is not yet known whether BRCA2 exon 3-skipping is a regulated event contributing to the spectrum of BRCA2 functions or is a stochastic occurrence. A genomic deletion that results in complete allelic loss of exon 3 has been shown to be pathogenic, yet VUSs 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. Alternative splicing patterns have also been observed in response to DNA damaging agents, potentially linking some therapies to risks from genome-wide aberrant splicing. 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. Methods: MCF7 cells were treated with doxorubicin according to 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 (Δ3). RT-PCR products were visualized using agarose gel electrophoresis. Results and Conclusion: Preliminary results suggest that doxorubicin increases the level of exon 3-skipping in MCF7. These results suggest doxorubicin, used to treat breast and other cancers, may compromise the repair functions of BRCA2 in addition to other transcriptome-wide effects from aberrant splicing patterns. However, it is not known whether this difference in isoform accumulation between treated and untreated cells is the result of increased frequency of alternative splicing events or changes in relative stabilities of the full-length and Δ3 isoforms, so it cannot yet be determined whether any potential physiological defects associated with alternative splicing of BRCA2 exon 3 result from insufficiency of the full-length isoform. Citation Format: Zoha Hameedi, Talia Ishfaq, Zaain Ahmad, James D. Fackenthal. Doxorubicin increases the level of a BRCA2 exon 3-skipping mRNA isoform in MCF7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5852.