The Deubiquitylase USP52 Regulates Cell Proliferation and Ferroptosis through Stabilizing SLC7A11/xCT in Bladder Cancer

Ferroptosis, a recently coined non-apoptotic form of cell death, is triggered by lethal accumulation of iron-dependent lipid peroxidation. SLC7A11/xCT, acts as a central hub in ferroptosis, is closely related to the development of multiple human diseases. However, the specific functions and potential regulatory mechanisms of ferroptosis, especially xCT protein, in bladder cancer (BLCA) remain poorly understood. Herein, through an unbiased siRNA screen targeting 96 deubiquitinases (DUBs), we identified USP52/PAN2 as a top regulator essential for xCT protein translational activity and ferroptosis process. Functionally, loss of USP52 inhibits glutathione (GSH) synthesis through xCT degradation, leading to elevated lipid peroxidation and ferroptosis, thereby suppresses BLCA progression. Mechanistically, USP52 physically interacts with xCT through its WD40 domain, biochemically hydrolyzes the K48-conjugated ubiquitin chains of xCT at K4 and K12, thereby promotes its protein stability. Importantly, USP52 expression demonstrates a positive correlation with xCT expression in clinical BLCA samples, and high USP52 expression is linked to a worse progression and prognosis. Additionally, USP52 depletion and IKE synergistically restrained the progression of BLCA by triggering ferroptosis in vivo. Collectively, our findings reveal a molecular mechanism of USP52-xCT axis in ferroptosis, and uncover a previously unappreciated role of USP52 in BLCA tumorigenesis. To investigate the downstream mechanism of USP52 regulation of bladder cancer, we performed RNA sequencing on three repeated siNC and siUSP52-treated T24 cells.

铁死亡（Ferroptosis）是近年发现的一种非凋亡性细胞死亡形式，由铁依赖性脂质过氧化的致死性蓄积所触发。SLC7A11/xCT作为铁死亡通路的核心枢纽，与多种人类疾病的发生发展密切相关。然而，铁死亡尤其是xCT蛋白在膀胱癌（BLCA）中的具体功能及潜在调控机制仍有待阐明。

本研究通过针对96种去泛素化酶（deubiquitinases, DUBs）的无偏倚siRNA筛选，鉴定出USP52/PAN2是调控xCT蛋白翻译活性与铁死亡进程的关键调节因子。功能实验显示，敲低USP52可通过促进xCT蛋白降解抑制谷胱甘肽（glutathione, GSH）合成，进而升高脂质过氧化水平并诱发铁死亡，最终抑制膀胱癌的进展。

机制层面上，USP52可通过其WD40结构域与xCT发生物理相互作用，并在赖氨酸4（K4）与赖氨酸12（K12）位点水解xCT的K48位连接泛素链，从而增强xCT的蛋白稳定性。值得注意的是，临床膀胱癌样本中USP52的表达与xCT的表达呈正相关，且高表达USP52与患者较差的疾病进展及不良预后密切相关。

此外，体内实验表明，联合敲低USP52与IKE处理可通过诱发铁死亡协同抑制膀胱癌的进展。综上，本研究揭示了USP52-xCT轴在铁死亡中的分子调控机制，并阐明了USP52在膀胱癌发生发展中此前未被认知的重要作用。为探究USP52调控膀胱癌的下游分子机制，本研究对3个生物学重复的、经阴性对照siRNA（siNC）与靶向USP52的siRNA（siUSP52）处理的T24细胞开展了RNA测序（RNA sequencing）。