Guoqing Zhang, Long Qiu, Kaiyuan Li, Jia Zhao, Xiangnan Li
Ubiquitin-specific proteases (USPs) function in multiple types of tumor progression by controlling protein turnover. However, the regulatory mechanism of ubiquitin carboxyl-terminal hydrolase 44 (USP44) in esophageal squamous cell carcinoma (ESCC) remains incompletely understood. Here, we show that USP44 expression correlates with tumor size, tumor stage, and TNM stage in ESCC patients. In vitro gain- and loss-of-function experiments validate that USP44 acts as a tumor suppressor in ESCC. Xenograft assays further demonstrate that USP44 overexpression suppresses the growth of ESCC xenografts, as well as lymph node and lung metastasis. Analysis of collected ESCC tumor samples reveals downregulated USP44 expression and upregulated USP44 pan-succinylation, along with two opposing correlations: a negative correlation with Carnitine O-palmitoyltransferase 1 (CPT1A) and a positive correlation with CDK5 regulatory subunit-associated protein 3 (CDK5RAP3). Mechanistic studies identify CPT1A as a lysine succinyltransferase that specifically succinylates USP44 at lysine 543 (K543). Such CPT1A-mediated K543 succinylation triggers ubiquitination-dependent degradation of USP44, thus explaining the downregulation of USP44 observed in clinical ESCC samples. Moreover, ubiquitination proteomics combined with survival analysis identifies CDK5RAP3 as a key deubiquitination substrate of USP44. USP44 directly interacts with CDK5RAP3 and stabilizes CDK5RAP3 by removing its ubiquitin chains at lysine 166 (K166), in a manner dependent on the catalytic cysteine 282 (C282) within its USP domain. In rescue experiments, CDK5RAP3 overexpression reverses the promotion of malignant behavior in ESCC cells and the activation of the Wnt/β-catenin signaling pathway caused by USP44 silencing. Collectively, USP44 may serve as a promising therapeutic target for ESCC.