Abstract

Triple-negative breast cancer (TNBC) poses a formidable clinical challenge due to its aggressive behavior and limited therapeutic avenues. Here, we delineate a novel pathway wherein BPHL governs R-loop homeostasis to sustain cancer stem cell (CSC) stemness in TNBC. Through single-cell RNA sequencing (scRNA-seq) of 26 breast cancer samples, we identified low R-loop scores as a defining feature of TNBC CSCs, marked by elevated chromosomal instability and stemness signatures. Extensive cohort analysis further confirmed that this low R-loop profile significantly correlates with poor patient prognosis and advanced tumor staging. Bioinformatics interrogation pinpointed BPHL as the pivotal regulator, a finding corroborated by the strong positive correlation between BPHL expression and lactylation markers in clinical specimens. Functional studies across multiple TNBC models revealed that BPHL overexpression expanded the CSC compartment, bolstered sphere formation, and accelerated tumor growth in vitro and in vivo. Consistent with the low R-loop phenotype, genes enriched in low R-loop TNBC cells and CSCs were linked to cell cycle progression, DNA replication/checkpoint control, and p53-associated pathways.Mechanistically, BPHL engages POLR2A, curtailing its lactylation while augmenting BARD1-mediated ubiquitination to resolve R-loops and enable β-catenin nuclear translocation, thereby perpetuating Wnt signaling. Beyond intrinsic tumor programs, low R-loop states were also associated with enhanced intercellular communication (notably MIF/MK signaling) and elevated immune checkpoint gene expression (e.g., PDCD1, CTLA4, TIGIT, LAG3). Intriguingly, in vivo BPHL depletion curbed tumorigenesis—an effect partially mitigated by RNase H overexpression but reinstated by β-catenin blockade. These insights position BPHL as a compelling target for eradicating TNBC CSCs.