ABSTRACT: Background: Resistance to chemotherapy remains an obstacle for triple-negative breast cancer (TNBC) patients. The core components of the ubiquitin-proteasome system have been demonstrated to regulate chemoresistance of TNBC. As a novel DUB, ubiquitin-specific peptidase 51 (USP51) plays a pivotal role in chemotherapeutic resistance in multiple malignancies. Herein, we sought to better understand how USP51 performs a cell-intrinsic role in mediating chemotherapeutic resistance in TNBC. Methods: Western blotting, RNA-sequencing and CCK8 assay were used to determine the important DUB for chemoresistance of breast cancer. Coimmunoprecipitation and pulldown analyses, protein deubiquitination assay, immunohistochemistry and molecular biology methods were then used to determine the significance of USP51 in TNBC chemoresistance and associated signaling pathways in several different cell lines, mouse models and patient tissue samples. Ubiquitin-7-amido-4-methylcoumarin (Ub-AMC)-based deubiquitinase activity, cellular thermal shift and surface plasmon resonance (SPR) analyses were performed to investigate the activity of USP51 inhibitors. Results: To identify the critical DUB involved in breast cancer chemoresistance, we established doxorubicin-resistant Cal51 and MDA-MB-231 by multiple dose exposure. We found ectopic USP51 in doxorubicin-resistant cells compared with parental cell. Moreover, USP51-interfered chemoresistant tumor cells exhibited impaired cell viability as well as increasing DNA damage and apoptosis after treated with doxorubicin. Inversely, ectopic USP51 not inactive mutant USP51C372S enhanced cell viability and decreased DNA damage and apoptosis after treated with doxorubicin. At the molecular level, GRP78 was identified as a bona fide substrate of USP51. Importantly, knockdown USP51 increased doxorubicin-induced DNA damage and apoptosis, which was recovered by overexpressing GRP78 in vitro and in vivo. In addition, the activity of ABCB1, the main efflux pump of doxorubicin, was enhanced by GRP78. Consistently, the expression of USP51, GRP78 and ABCB1 were correlated with doxorubicin-resistance in TNBC patients. Finally, we explored a small molecular inhibitor of USP51, WCY-4-1, which confered chemosensitization in TNBC. Conclusion: These findings collectively indicated that USP51 enhances the activity of ABCB1 by deubiquitinating and stabilizing GRP78, which leads to decreased accumulation of doxorubicin as well as decreased DNA damage and apoptosis in TNBC. Our results also revealed that inhibition of USP51 impairs doxorubicin-resistance in TNBC.
