Ailanthone induces triple-negative breast cancer cells death involving the inhibition of OTUB1-mediated ERRα deubiquitylation

INTRODUCTION: Triple-negative breast cancer (TNBC) remains the most aggressive subtype of breast cancer, and effective therapeutic strategies are needed. Estrogen-related receptor alpha (ERRα) is considered a promising target for managing TNBC. OBJECTIVES: Here, we aimed to screen natural products to find downregulator of ERRα and elucidate its mechanism of action. METHODS: TNBC cells (MDA-MB-231, MDA-MB-468, MDA-MB-453, and BT-549) were used for in vitro studies, and a subcutaneous MDA-MB-231 tumor model was created for in vivo studies. Immunofluorescence assessed protein distribution, while competitive activity-based protein profiling identified potential target proteins. Co-immunoprecipitation detected protein interactions and modifications, and a luciferase reporter assay evaluated ERRα transcriptional activity. RESULTS: The natural product Ailanthone (AIL) effectively induced cell death in TNBC cells by reducing the protein level of ERRα. The mechanism of action involved AIL promoting the degradation of ERRα through the ubiquitin-proteasome system, consequently reducing its transcriptional activity. The competitive-ABPP method mapped the profile of target proteins for AIL, and OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) was identified as a pivotal target of AIL in regulating ERRα protein levels. OTUB1 was validated as a novel deubiquitinating enzyme for ERRα, with its C91 residue being crucial for this deubiquitination process. AIL was found to inhibit the enzyme activity of OTUB1 by interacting with the C91 residue and disrupt the interaction between OTUB1 and ERRα, ultimately leading to the inhibition of ERRα. CONCLUSION: AIL is a promising downregulator of ERRα, and the mechanism of this downregulation has been elucidated. Additionally, a new regulatory relationship between ERRα and OTUB1 is identified. The research presented in this article is anticipated to yield potential lead compounds for ERRα regulatory agents and to stimulate the development of novel therapeutic strategies designed to modulate ERRα activity for the treatment of TNBC.