Abstract P3-09-07: R-loop forming sequences determine early response genes and tumorigenic pathways driven in BRCA1-deficient carriers

Abstract

Abstract Transcriptional R-loops are triple-stranded RNA:DNA hybrid genome structures which are defined in more than 75% of genes of the human genome, functionally versatile in cells, and mechanistically involved in tumorigenesis. In cancer cells, excessive/aberrant R-loop structures promote mutagenesis and genome instability due to their key regulatory roles in transcription and genotoxic stress. Thus, a balance in the regulation of R-loop initiation, stabilization, and suppression is required for proper function. Recent studies have shown that R-loop formation increases in luminal but not in basal-like cells of the tumor-free mammary epithelium of BRCA1 mutation carriers. R-loop forming sequences (RLFSs) are strand-specific G-rich regions of ssDNA that initiate and stabilize the formation of R-loops. The roles of the RLFSs in functional R-loops associated with premalignant states of BRCA1-mc have not been studied. Using our QmRLFS model and R-loopDB (http://rloop.bii.a-star.edu.sg/), we identified RLFS-associated (RLFS(+)) and non-associated (RLFS(-)) RNA:DNA hybrids detected by published DRIP-seq data in gene regions in luminal precursors, mature luminal, and basal-like epithelial cell type populations, extracted from BRCA1 wild-type and mutated non-cancer breast tissue samples (GSE96672). We found that genome regions of RLFS(+) RNA:DNA hybrid signals are strongly associated with G4-quadruplex, ssDNA regions, histone markers (acetylation, demethylation), and other open chromatin markers while the RLFS(-) RNA:DNA hybrid signal regions were not significantly associated with the positions of these regulatory markers. Our statistical modeling and cell-type-specific gene analysis of normal and BRCA1-deficient non-cancer epithelial cells samples shown that RLFS(+) R-loop frequency in luminal cells was mostly increased; in contrast, RLFS(+) R-loops were essentially decreased in basal-like breast cells. Analyzing published gene expression profiles of mammary epithelial cells obtained from disease-free prophylactic mastectomy tissues of BRCA1-mutation carriers and reduction mammoplasty tissues from non-mutation carriers (GSE25835 & GSE19383), we found the differential expressed genes (DEGs) associated with RLFS(+) RNA-DNA hybrid (DRIP-seq) signals. Integrating these findings with our RLFS RNA-DNA hybrid characterizations, we dichotomized the DEGs referring to the luminal and basal-like cell types. According to pathway analysis, a majority of BRCA1-deficient altered transcripts referring to luminal cell’s genes are involved metabolism and cell cycle while basal-like cells involved immune functions, cell signaling, and epithelial-mesenchymal transition pathways. We also found that the genes associated with RLFS clusters present on the sense strand are more associated with nucleic acid binding and transcription regulation, while the genes with an RLFS present on the anti-sense strand are more associated with immune system functions depleted in BRCA1 deficient non-cancer carriers. Thus, for the first time, we demonstrate the roles of RLFS mediated R-looping in the luminal and basal-like breast cells from BRCA1-mutation carrier tissues that specify aberrantly transcribed genes involving in tumorigenesis and immune function pathways. This study suggests novel regulatory mechanics of BRCA1-driven pre-malignant state development and prospective biomarker discovery. Citation Format: Andre Grageda, Vladimir A Kuznetsov. R-loop forming sequences determine early response genes and tumorigenic pathways driven in BRCA1-deficient carriers [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 P3-09-07.