Rotenone Inhibited Osteosarcoma Cell Growth Through USP47-Induced Decreases in FEN1 Stability and DNA Integrity

Zhen Li,1 Xiang Ma,2 Hengwei Ma,3 Bao Chen,3 Kun Jiang,3 Ziqiang Zhu,3 Jianqiang Wang,3 Baoqing Wang,1 Yunqing Wang,3 Suwei Dong3 1Department of Medical Oncology, The Second Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221000, People’s Republic of China; 2Department of Orthopaedics, The Third Affiliated Hospital of Kunming Medical University, Kunming, 650118, People’s Republic of China; 3Department of Orthopaedics, The Second Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221000, People’s Republic of ChinaCorrespondence: Suwei Dong, Email [email protected]: Osteosarcoma (OS) is the most prevalent primary malignant bone tumor in children. We previously showed that rotenone suppressed OS cell metastasis. However, its effects on OS cell growth and the underlying mechanisms remain unclear. The purpose of this study was to investigate the role of rotenone in OS and identify its direct target.Methods: Molecular dockingand Biacore assay were used to confirm the interaction between rotenone and USP47. Co-immunoprecipitation, immunofluorescence, and cycloheximide assays were used to verify the relationship betweenUSP47 and FEN1. Cell cycle and apoptosis were examined by flow cytometry. Comet analyses were used to determine DNA damage. Deubiquination and Ub-VME assays were carried out to assess ubiquitination status and properties. Immunohistochemistry and a xenograft mouse model were utilized to validate the effects of various proteins and rotenone in vivo.Results: Knockdown of FEN1 and USP47 in OS cell lines induced cell cycle arrest and apoptosis through the induction of DNA damage. FEN1 exhibited a direct interaction with USP47. Mechanistically, wild-type USP47 regulated FEN1 protein stability through deubiquitination modification, whereas mutated Cys109Ser USP47 did not. Furthermore, rotenone modulated USP47 protein expression and the combined quantity of Ub-USP47 conjugation through physical interaction. Xenograft studies further confirmed the anti-OS activity of rotenone in vivo.Conclusion: Rotenone is a potential therapeutic agent for OS due to its direct targeting of USP47 and resultant decrease in FEN1 stability and DNA integrity.Keywords: osteosarcoma, rotenone, USP47, FEN1, DNA damage