Dissection of non-conventional pathways of BRCA1-driven tumorigenicity

BRCA1 is a tumor suppressor gene encoding a protein with critical roles in DNA repair, transcriptional regulation, RNA processing, and replication fork protection. Carriers of BRCA1 pathogenic variants are at increased risk of developing breast and ovarian cancers. While BRCA1’s fundamental role in maintaining genomic integrity through DNA repair and cell cycle regulation is well-established, the specific molecular and cellular mechanisms driving tissue-specific tumors in the case of BRCA1 deficiency are not fully understood. In particular, whether BRCA1 insufficiency alters RNA splicing programs or how it compromises replication fork stability before full gene loss are open questions. The importance of these studies lies in the fact that changes in RNA splicing contribute to tumorigenesis by generating cancer-specific mRNA isoforms that drive uncontrolled cell proliferation, decreased apoptosis, elevated migration, and invasive capacity. In addition, replication fork instability leads to tumorigenesis by creating a high level of genomic instability. This instability is caused by stalled or collapsed replication forks due to replication stress and DNA damage. Replication stress and splicing are interconnected, as replication stress can induce alternative mRNA splicing, generating isoforms with distinct properties that may regulate cellular functions during replication stress. Based on prior studies, we first investigated whether BRCA1 influences the expression of target gene isoforms through changing alternative splicing decisions. RNA sequencing in BRCA1+/- FT282 cells, combined with LeafCutter splicing analysis, identified LAMA3 as a strong candidate for BRCA1-dependent splicing regulation, with evidence of an isoform switch from LAMA3 variant 1 (V1) to variant 2 (V2) in FT282 BRCA1+/- cells. However, droplet digital PCR (ddPCR) validation in breast BRCA1 LOF models (HBL100, MCF7, and MDAMB231) suggested global LAMA3 upregulation rather than variant-specific control, highlighting context- and cell-type-dependent regulation of splicing. Second, we explored replication stress signatures of BRCA1 haploinsufficiency, regarding replication fork protection. Western blot and immunofluorescent analysis revealed attenuated phosphorylation of RPA2 in BRCA1+/- FT282 and MCF10A cells after hydroxyurea (HU) treatment, suggesting the lower levels of single-stranded DNA (ssDNA) in these cells, attributed to the decrease of BRCA1, which protects ssDNA against nuclease degradation. DNA fiber assays confirmed increased fork degradation in BRCA1+/- MCF10A cells, partially rescued by MRE11 inhibitor, mirin, indicating that BRCA1 haploinsufficiency compromises fork stability through nucleolytic degradation. Taken together, these findings suggest that BRCA1 haploinsufficiency impacts replication fork protection and might alter RNA processing, thereby creating an environment permissive to genome instability and transcriptional dysregulation even before complete BRCA1 loss. Future work could focus on the context and tissue-specific mechanisms that BRCA1 applies to regulate RNA processing, as well as exploring if there are replication stress-dependent splicing changes in BRCA1+/- cells. Uncovering the connection between replication stress and splicing regulation in those cells not only advances mechanistic understanding but also suggests possible therapeutic approaches.