Abstract 407: RNA:DNA hybrid/R-loop determinants variation in distinct basal-like breast cancer cells
Authors: Kapali Ojashpi Singh , Kuznetsov Vladimir 
Published: March 2024

Abstract
Introduction: Despite progress in treatment methods and diagnosis, breast cancer remains one of the world's leading cancers causes of death. Among the various subtypes, basal-like breast cancer (BLBC) is highly diverse, low differentiated, most aggressive form with poor prognosis. Co-translational R-loops are created by combining G-rich RNA with complementary C-rich DNA (RNA: DNA hybrid), which creates a three-stranded nucleic acid structure. R-loops are often associated with genome instability and aggressiveness of cancers. However, their role in the diversity of BLBC remains controversial and poorly understood. This study aims to characterize RNA: DNA hybrid variations in BLBC from a cellular, genetic, and biochemical perspective.
Methods: This study investigates the variation of RNA: DNA hybrid formation in stable BLBC cells. We utilize a panel of stable basal-like cell lines (MDAMB231, MDAMB436, SUM149PT) and proliferative non-cancer cells (MCF10A). They represent different genetic and cell lineages in BLBC development which allows us to study R-loop distribution in different basal lineage cells. Luminal breast cancer cells (MCF7) were used in comparative analysis. To detect RNA: DNA hybrids, we optimized Immunofluorescence (IF) specificity using RNase III and H enzymes. Predictions from Quantitative Model of R-loop Forming Sequences (QmRLFS) identify RLFS in specific gene regulatory regions. This design allows us to define R-loop position and boundaries, and optimize primers for the DRIP-qPCR which quantifies RNA: DNA hybrid enrichment in transcription regulatory sites for several breast cancer-associated genes such as BRCA1, TP53, PTEN, and AURKA. The expression of these genes was detected using RTqPCR.
Results: Our IF and computational image analyses demonstrated that RNA: DNA hybrids are present not only luminal cells (as found by others), but also in the studied BLBC cells. The non-cancer proliferative cells (MCF10A) show low RNA: DNA hybrid signals bolstering our experimental technique. Variation of the RNA: DNA hybrid signals were distinct in various BLBC cells which was supported by the DRIP-qPCR and RTqPCR findings. The R-loop formation data variations was referred to the cell line contexts and the studied genes responsible for tumor suppression, DNA damage/repair signaling and impair apoptosis in breast cancer.
Conclusion: Our study suggests that RNA: DNA hybrids are formed and distributed heterogeneously in the genomes and gene regions of stable BLBC cell lines, while in basal proliferative (non-cancer) cells hybrid signals are weak. A subset of the RNA: DNA hybrids might be particularly significant at certain gene loci contributing to tumor progression, indicating their significance in BLBC pathogenesis. This study provides insights into the cellular and molecular basis of the BLBC heterogeneity and implicates the RNA:DNA hybrid as a potential diagnostic and therapeutic markers.
Citation Format: Ojashpi Singh Kapali, Vladimir Kuznetsov. RNA:DNA hybrid/R-loop determinants variation in distinct basal-like breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 407.