Activation of ERβ hijacks the splicing machinery to trigger R-loop formation in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with aggressive behavior and poor prognosis. Current therapeutic options available for TNBC patients are primarily chemotherapy. With our evolving understanding of this disease, novel targeted therapies, including poly ADP-ribose polymerase (PARP) inhibitors, antibody-drug conjugates, and immune-checkpoint inhibitors, have been developed for clinical use. Previous reports have demonstrated the essential role of estrogen receptor β (ERβ) in TNBC, but the detailed molecular mechanisms downstream ERβ activation in TNBC are still far from elucidated. In this study, we demonstrated that a specific ERβ agonist, LY500307, potently induces R-loop formation and DNA damage in TNBC cells. Subsequent interactome experiments indicated that the residues 151 to 165 of U2 small nuclear RNA auxiliary factor 1 (U2AF1) and the Trp 439 and Lys 443 of ERβ were critical for the binding between U2AF1 and ERβ. Combined RNA sequencing and ribosome sequencing analysis demonstrated that U2AF1-regulated downstream RNA splicing of 5-oxoprolinase ( OPLAH ) could affect its enzymatic activity and is essential for ERβ-induced R-loop formation and DNA damage. In clinical samples including 115 patients from The Cancer Genome Atlas (TCGA) and 32 patients from an in-house cohort, we found a close correlation in the expression of ESR2 and U2AF1 in TNBC patients. Collectively, our study has unraveled the molecular mechanisms that explain the therapeutic effects of ERβ activation in TNBC, which provides rationale for ERβ activation-based single or combined therapy for patients with TNBC.