Abstract P3-13-03: Heterozygous BRCA1 mutation influences alternative splicing in human benign mammary organoids
Authors: Lee Oukseub , Choi Mi-Ran , Cottone Gannon , Patel Priyam , Clare Susan E , Khan Seema A. 
Published: March 2022

Abstract
Background: Women carrying a germline BRCA1 mutation, have 60% risk of developing breast cancer during their lifetime and frequently develop triple-negative, basal-like, aggressive breast tumors. Humans produce around 150,000 different proteins from their 25,000-30,000 genes. This is accomplished by alternative splicing (AS). We hypothesized that AS in BRCA1 mutation carriers is different from those women with wild type BRCA1, therefore, we examined AS, expression of genes encoding splicing factors, and enrichment of binding motifs for RNA-binding proteins in BRCA1 carriers (BRCA1mut/+) and controls (BRCA1 normal). Methods: Prophylactic mastectomy (BRCA1mut/+) and reduction mammoplasty (BRCA1 normal) specimens were collected at Prentice Women’s Hospital of Northwestern Medicine under approval by Northwestern’s Institutional Review Board (NU15B07). We isolated organoids from these tissues, and cryopreserved them for future use. We selected organoids of premenopausal women, age-matched (28-46) without oral contraceptive usage within 3 years: BRCA1mut/+ (n=12) and BRCA1 normal (n=4). Organoids were maintained in complete MammoCult medium in ultra-low attachment plates at 37 °C, 5% CO2 for 72 hrs, and harvested for RNA extraction (NucleoSpin RNA Plus, TaKaRa). 100 ng of total RNA (RIN 7+) were used for RNA sequencing assay with the KAPA mRNA Hyper Prep Kit (Roche Corporate) and NovaSeq 6000 for 100b paired-end sequencing (Illumina, Inc). The preprocessed reads were aligned to the human genome (hg38) using STAR. Differential expression analysis (DESeq2, nominal p-value &lt; 0.05) and pathway analysis (GSEA, size ≥30, NES &gt;2, FDR q-value &lt;0.05, and enriched genes ≥ 5) were performed to compare BRCA1mut/+ with BRCA1 normal. We used rMATS- turbo v4.1.0 to perform multivariate analysis of transcript splicing and chose an FDR &lt; 0. 05, to identify significant differential alternative splicing events. To determine which RNA binding proteins and splicing factors were employed in BRCA1mut/+ tissue, we examined the cis-regulatory motifs adjacent to splice sites using rMAPs. Results: BRCA1mut/+ organoids displayed 155 upregulated genes compared to BRCA1 normal organoids (P &lt;0.05). Among top 20 enriched pathways, we identified that expression of mRNA spliceosome (POLR2L, U2AF2, NUP188, SNRPF, CSTF3, HNRNPM, SRSF2), and, in addition, cell cycle, oxidative phosphorylation, HIV infection, and protein metabolism genes were significantly upregulated in BRCA1mut/+ organoids. We identified significant differential AS events (FDR &lt;0.05) between BRCA1mut/+ and BRCA1 normal organoids: 2159 genes (skipped exon), 433 genes (mutually exclusive exon), 325 genes (retained intron), 304 genes (alternative 3’ splice sites), 168 genes (alternative 5’ splice sites). RNA splicing analysis indicated that highly expressed genes (SRSF2, ALG3, AP1B1 and PMF1) in BRCA1mut/+ organoids were also alternatively spliced. In addition, many other serine/arginine-rich splicing factors (SRSF) themselves (SRSF1, 2, 3, 5, 6, 7) were alternatively spliced (skipped exon events). An examination of binding motifs of RNA-binding proteins with significantly increased expression in BRCA1 carriers revealed multiple significantly enhanced and silenced regions flanking exons involved in AS. Conclusions: Our data suggest that AS of BRCA1mut/+ mammary tissue significantly differ from BRCA1 normal tissue, and may alter RNA spliceosome activity, and consequently play a role in malignant transformation and warrant further study.
Citation Format: Oukseub Lee, Mi-Ran Choi, Gannon Cottone, Priyam Patel, Susan E Clare, Seema A. Khan. Heterozygous BRCA1 mutation influences alternative splicing in human benign mammary organoids [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-13-03.