Abstract PS19-09: Alternative splicing events from progesterone exposure differ based on BRCA1 mutation status
Authors: Cottone Gannon , Davaadelger Batzaya , Yadav Shivangi , Kim J Julie , Khan Seema A. , Clare Susan 
Published: February 2021

Background: Progesterone (P) and progestin are instrumental in the breast cancer risk associated with reproductive hormones. Progesterone receptor (PR) signaling has been shown to be inhibited by the wild-type BRCA1 protein and it is hypothesized that if this inhibition is lost, due to gene mutation, potentially tumorigenic consequences may result. Our efforts are focused on understanding the role PR and P play in BRCA1 mutation carriers. The purpose of this study was to determine if the presence of a BRCA1 mutation effects alternative splicing (AS) events.

Methods: Mammary organoids from 12 patients, six with a BRCA1 mutation and six without (WT, control), were cultured in vitro. The organoids from each patient were divided into two groups and treated over the course of 28 days to mimic a normal menstrual cycle. One group was treated was estradiol and progesterone (EP) while the other was treated with EP and the PR antagonist telapristone acetate (TPA) to identify PR-mediated effects. After treatment, RNA was extracted from both groups and RNA-sequencing performed. A statistical model, “replicate Multivariate Analysis of Transcript Splicing” (rMATS) was employed to identify AS events. Output from rMATS was then entered into “RNA Map Analysis And Plotting Server” (rMAPS) to identify RNA-binding proteins (RBP) motifs in the region of the splices. Droplet digital PCR (ddPCR) from Bio-Rad was employed, with the use of Evagreen intercalating dye, to validate a subset of the AS events identified by the RNA-Seq data. RNA from eight of the EP treated samples was reverse transcribed and ddPCR performed. The data was analyzed using QuantaSoft Analysis Pro (Bio-Rad). The identified AS events were validated additionally using data generated from solid tissue normals (STN) from premenopausal, BRCA1mut and BRCA1WT patients, who developed breast cancer, which was available for download from The Cancer Genome Atlas (TCGA).

Results: Genes involved in the epithelial-mesenchymal-transition (EMT), progesterone metabolism, and cellular proliferation are significantly differentially spliced in the EP treated BRCA1mut tissue (p-value &lt; 0.01 and FDR &lt; 5%) and absent in those with TPA treatment, suggesting the AS is a PR-mediated effect. CD44, associated with cellular proliferation and migration, NCOR2 associated with tamoxifen resistance, and AKR1C2 associated with progesterone metabolism all showed significant skipped exon (SE) events. Exon 11 in CD44 and exon 45 in NCOR2 were skipped in both the BRCA1 organoids as well as in BRCA1 STN from TCGA. Skipping of Exon 4 in AKR1C2 was validated using the ddPCR: the BRCA1mut organoids displayed two isoforms, indicative of a skipped exon.

Conclusions: PR-mediated alternative splicing events differ in mammary organoids of BRCA1 carriers compared to non-carriers. We hypothesize that the skipping of Exon 4 of AKR1C2 in BRCA1mut results in a structural alteration that decreases protein activity, leading to increased concentrations of P4 and 5α-pregnane-3,20-dione. This may explain the previously reported high median luteal phase serum P levels (p=0.00034) in BRCA1mut/BRCA2mut (PMID: 24140203).

Citation Format: Gannon Cottone, Batzaya Davaadelger, Shivangi Yadav, J Julie Kim, Seema A. Khan, Susan Clare. Alternative splicing events from progesterone exposure differ based on BRCA1 mutation status [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-09.