Abstract 4556: USP51-GRP78-ABCB1 axis promotes chemoresistance of triple negative breast cancer by decreasing the accumulation of doxorubicin in cells

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 deubiquitinase (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 assays were used to identify the DUBs for chemoresistance of breast cancer. Coimmunoprecipitation, GST-pulldown, protein deubiquitination and immunohistochemistry assays were then used to determine the biological phenotypes of ectopic 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 DUBs involved in breast cancer chemoresistance, we established doxorubicin-resistant Cal51 and MDA-MB-231 TNBC cell lines by multiple dose exposure. We found ectopic USP51 in doxorubicin-resistant cells compared with their parental cells. Moreover, USP51-interfered chemoresistant tumor cells exhibited impaired cell viability as well as increased DNA damage and cell apoptosis upon treatment with doxorubicin. On the contrary, overexpression of USP51 performed the opposite effects to enhance cell viability but decrease DNA damage and apoptosis in response to doxorubicin; however, these outcomes were not shown for its catalytically inactive mutant USP51C372S. 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 rescued by overexpression of 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 chemoresistant phenotypes in TNBC patients. Of note, we also explored a small molecular inhibitor of USP51, WCY-4-1, which conferred chemosensitization in TNBC. Conclusion: These findings collectively indicated that ectopic 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 cell apoptosis in TNBC. Our results also revealed that specific inhibition of USP51 significantly impairs doxorubicin-resistance in TNBC. Citation Format: Yang Ou, Kun Zhang, Qiuying Shuai, Chenyang Wang, Huayu Hu, Lixia Cao, Chunchun Qi, Min Guo, Zhaoxian Li, Jie Shi, Yuxin Liu, Siyu Zuo, Xiao Chen, Yanjing Wang, Mengdan Feng, Hang Wang, Yi Shi, Guang Yang, Shuang Yang. USP51-GRP78-ABCB1 axis promotes chemoresistance of triple negative breast cancer by decreasing the accumulation of doxorubicin in cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 4556.