Bin Zhang,Ruowen Sun,Min Gu,Zehui Jiang,Ye Wang,Linlin Zhang,Xiaoyang Liu,Zuofei Chi
Acute T-lymphocyte leukemia (T-ALL) is a malignant tumor disease. RNA-binding protein neotumor ventral antigen-1 (NOVA1) is highly expressed in bone marrow mononuclear cells of T-ALL patients, while the role of NOVA1 in T-ALL progression remains unknown. The gain- and loss-of-function studies for NOVA1 were performed in Jurkat and CCRF-CEM cells. NOVA1 overexpression promoted cell proliferation and cell cycle progression. NOVA1 knockdown increased the apoptosis rate of T-ALL cells. Ubiquitin-specific protease 44 (USP44), a nuclear protein with deubiquitinase catalytic activity, has been reported to play an oncogene role in human T-cell leukemia. USP44 expression was positively associated with NOVA1, and RNA immunoprecipitation assay verified the binding of NOVA1 to the mRNA of USP44. USP44 knockdown partially abolished NOVA1-induced cell proliferation and inhibition of apoptosis. The in vivo xenograft experiment was performed by injection of T-ALL tumor cells into the tail vein of NOD/SCID mice. The knockdown of NOVA1 had lower tumorigenicity. NOVA1 knockdown alleviated pathological changes in lung and spleen tissues, and increased the overall survival period and the weight of T-ALL mice. Thus, NOVA1 acts as an accelerator in T-ALL, and its function might be achieved by binding to and stabilizing USP44 mRNA.
T-cell acute lymphoblastic leukemia (T-ALL) is a common hematologic malignancy. Based on the data from {"type":"entrez-geo","attrs":{"text":"GSE66638","term_id":"66638"}} GSE66638 and {"type":"entrez-geo","attrs":{"text":"GSE141140","term_id":"141140"}} GSE141140 , T-ALL patients depicted a higher USP44 level. However, its role in T-ALL is still unclear. In the present study, we investigated the role of USP44 in T-ALL growth. USP44 overexpression elevated the proliferation of CCRF-CEM cells, while USP44 knockdown suppressed the proliferation of Jurkat and MOLT-4 cells. In addition, USP44 accelerated the cell cycle progression, with boosted cyclinD and PCNA levels. However, USP44 knockdown induced apoptosis in Jurkat and MOLT-4 cells, with an upheaval among cleaved caspase-3 and PARP levels. Mechanistically, USP44 co-localized and interacted with WDR5, leading to the repression of its ubiquitination and degradation. Interestingly, WDR5 overexpression abolished the apoptosis induced by USP44 knockdown. Consistently, the in vivo study revealed that USP44 knockdown restricted the leukemic engraftments in the bone marrow and spleens and reduced the infiltration of T-ALL cells in the livers and lungs. In conclusion, this study indicated that USP44 enhanced the growth of T-ALL through interacting with WDR5 and repressing its ubiquitination. This study highlights the potential use of USP44 as a therapeutic target of T-ALL.
Guoqing Zhang,Long Qiu,Kaiyuan Li,Jia Zhao,Xiangnan Li
ESCC samples show higher USP44 succinylation and lower expression vs adjacent non-tumor tissues. • CPT1A binds USP44 and specifically mediates its K543 succinylation, reducing USP44 stability. • K543 succinylation triggers USP44's ubiquitination-mediated degradation in ESCC tissues. • USP44 stabilizes CDK5RAP3 by K166 deubiquitylation, dependent on its catalytic C282 residue. • USP44 inhibits ESCC xenograft growth, lung and lymphatic metastasis by stabilizing CDK5RAP3. 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.
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