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USP21 positively correlates with FOXD1 protein levels and is associated with the poor prognosis of MES GBM patients. a Representative IHC images of USP21 and FOXD1 expression in MES GBM specimens. b Correlation between USP21 and FOXD1 proteins in 91 MES GBM IHC results. c Kaplan-Meier plots of overall survival and progression-free survival of 91 MES GBM patients stratified by protein expression of USP21 showing that USP21 hi MES GBM patients displayed significantly shorter overall survival and progression-free survival than USP21 low MES GBM patients. **P < 0.01. d Schematic illustration of USP21-mediated FOXD1 stabilization, promoting self-renewal and tumorigenicity of MES GSCs.
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Recent studies suggest that Forkhead box D1 (FOXD1) plays an indispensable role in maintaining the mesenchymal (MES) properties of glioblastoma (GBM) stem cells (GSCs). Thus, understanding the mechanisms that control FOXD1 protein expression is critical for guiding GBM treatment, particularly in patients with therapy-resistant MES subtypes. In this...
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... To determine the possible clinical relevance between USP21 and FOXD1, we examined the expression levels of USP21 and FOXD1 in four clinical MES GBM tissues. In case 1 and 2, a high expression of USP21 correlated with a high expression of FOXD1. Conversely, in case 3 and 4, a low expression of USP21 correlated with a low expression of FOXD1 (Fig. 6a). In addition, the other 91 IHC results supported the positive correlation between USP21 and FOXD1 (Fig. 6b). Moreover, Kaplan-Meier survival curve analysis of the 91 GBM tissues showed that the survival rate of patients with high USP21 expression was significantly lower than that of patients with low USP21 expression (Fig. 6c). In ...
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... of USP21 and FOXD1 in four clinical MES GBM tissues. In case 1 and 2, a high expression of USP21 correlated with a high expression of FOXD1. Conversely, in case 3 and 4, a low expression of USP21 correlated with a low expression of FOXD1 (Fig. 6a). In addition, the other 91 IHC results supported the positive correlation between USP21 and FOXD1 (Fig. 6b). Moreover, Kaplan-Meier survival curve analysis of the 91 GBM tissues showed that the survival rate of patients with high USP21 expression was significantly lower than that of patients with low USP21 expression (Fig. 6c). In addition, similar results were obtained from the CGGA database ( Supplementary Fig. 6a, b). Overall, our results ...
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... expression of FOXD1 (Fig. 6a). In addition, the other 91 IHC results supported the positive correlation between USP21 and FOXD1 (Fig. 6b). Moreover, Kaplan-Meier survival curve analysis of the 91 GBM tissues showed that the survival rate of patients with high USP21 expression was significantly lower than that of patients with low USP21 expression (Fig. 6c). In addition, similar results were obtained from the CGGA database ( Supplementary Fig. 6a, b). Overall, our results suggest that there is a strong positive clinical relevance between the levels of USP21 and FOXD1 and that high USP21 expression in patients indicates a poor ...
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... Kaplan-Meier survival curve analysis of the 91 GBM tissues showed that the survival rate of patients with high USP21 expression was significantly lower than that of patients with low USP21 expression (Fig. 6c). In addition, similar results were obtained from the CGGA database ( Supplementary Fig. 6a, b). Overall, our results suggest that there is a strong positive clinical relevance between the levels of USP21 and FOXD1 and that high USP21 expression in patients indicates a poor prognosis. ...
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... unclear. In this study, by adopting the unbiased DUB-focused siRNA screen, we identified USP21 as a novel DUB that governs FOXD1 stability. We found that USP21 interacted with FOXD1 and stabilized its protein levels by reducing its K48-linked polyubiquitination, thereby sustaining high FOXD1 expression and activating its downstream signaling (Fig. 6d). Recent studies have shown that USP21 is frequently dysregulated in multiple types of cancer and plays a critical role in cancer development and progression. Indeed, our clinical data showed a strong positive correlation between the expression levels of USP21 and FOXD1 in GBM patients and demonstrated that elevated USP21 expression was ...
Similar publications
Pancreatic ductal adenocarcinoma (PDAC), as an intractable malignancy, still causes an extremely high mortality worldwide. The ubiquitin-specific protease (USP) family constitutes the major part of deubiquitinating enzymes (DUBs) which has been reported to be involved in initiation and progression of various malignancies via the function of deubiqu...
Citations
... Several DUBs have been directly implicated in GSC PMT marker deubiquitination. USP21 promotes selfrenewal and tumorigenicity of mesenchymal glioblastoma stem cells by deubiquitinating and stabilizing FOXD1 [49]. We identified USP9X as a bona fide deubiquitinase of ALDH1A3 in MES GSCs [6]. ...
Background
The high degree of intratumoral genomic heterogeneity is a major obstacle for glioblastoma (GBM) tumors, one of the most lethal human malignancies, and is thought to influence conventional therapeutic outcomes negatively. The proneural-to-mesenchymal transition (PMT) of glioma stem cells (GSCs) confers resistance to radiation therapy in glioblastoma patients. POLD4 is associated with cancer progression, while the mechanisms underlying PMT and tumor radiation resistance have remained elusive.
Method
Expression and prognosis of the POLD family were analyzed in TCGA, the Chinese Glioma Genome Atlas (CGGA) and GEO datasets. Tumorsphere formation and in vitro limiting dilution assay were performed to investigate the effect of UCHL3-POLD4 on GSC self-renewal. Apoptosis, TUNEL, cell cycle phase distribution, modification of the Single Cell Gel Electrophoresis (Comet), γ-H2AX immunofluorescence, and colony formation assays were conducted to evaluate the influence of UCHL3-POLD4 on GSC in ionizing radiation. Coimmunoprecipitation and GST pull-down assays were performed to identify POLD4 protein interactors. In vivo, intracranial xenograft mouse models were used to investigate the molecular effect of UCHL3, POLD4 or TCID on GCS.
Result
We determined that POLD4 was considerably upregulated in MES-GSCs and was associated with a meagre prognosis. Ubiquitin carboxyl terminal hydrolase L3 (UCHL3), a DUB enzyme in the UCH protease family, is a bona fide deubiquitinase of POLD4 in GSCs. UCHL3 interacted with, depolyubiquitinated, and stabilized POLD4. Both in vitro and in vivo assays indicated that targeted depletion of the UCHL3-POLD4 axis reduced GSC self-renewal and tumorigenic capacity and resistance to IR treatment by impairing homologous recombination (HR) and nonhomologous end joining (NHEJ). Additionally, we proved that the UCHL3 inhibitor TCID induced POLD4 degradation and can significantly enhance the therapeutic effect of IR in a gsc-derived in situ xenograft model.
Conclusion
These findings reveal a new signaling axis for GSC PMT regulation and highlight UCHL3-POLD4 as a potential therapeutic target in GBM. TCID, targeted for reducing the deubiquitinase activity of UCHL3, exhibited significant synergy against MES GSCs in combination with radiation.
... These enzymes are also associated with GBM malignancy and poor prognosis. The USP9X inhibitor WP1130 and the USP21 inhibitor disulfiram have shown good therapeutic efficacy in MES-GSC-derived GBM xenograft models [77][78][79][80] . Furthermore, PRL1 promotes GBM progression by activating the USP36-mediated deubiquitination of Snail2 81 . ...
Among central nervous system-associated malignancies, glioblastoma (GBM) is the most common and has the highest mortality rate. The high heterogeneity of GBM cell types and the complex tumor microenvironment frequently lead to tumor recurrence and sudden relapse in patients treated with temozolomide. In precision medicine, research on GBM treatment is increasingly focusing on molecular subtyping to precisely characterize the cellular and molecular heterogeneity, as well as the refractory nature of GBM toward therapy. Deep understanding of the different molecular expression patterns of GBM subtypes is critical. Researchers have recently proposed tetra fractional or tripartite methods for detecting GBM molecular subtypes. The various molecular subtypes of GBM show significant differences in gene expression patterns and biological behaviors. These subtypes also exhibit high plasticity in their regulatory pathways, oncogene expression, tumor microenvironment alterations, and differential responses to standard therapy. Herein, we summarize the current molecular typing scheme of GBM and the major molecular/genetic characteristics of each subtype. Furthermore, we review the mesenchymal transition mechanisms of GBM under various regulators.
... In pancreatic cancer, USP21 activation promotes tumor growth via Wnt pathway activation [8]. In addition, USP21 has also been reported to promote tumorigenicity in mesenchymal glioblastoma stem cells by deubiquitinating FOXD1 [9]. Although it has been reported that USP21 expression is upregulated in cholangiocarcinoma [10], the mechanism of USP21 in the progression of CCA has not been studied. ...
Deubiquitylating enzymes (DUBs) play an essential role in targeted protein degradation and represent an emerging therapeutic paradigm in cancer. However, their therapeutic potential in cholangiocarcinoma (CCA) has not been explored. Herein, based on The Cancer Genome Atlas (TCGA) and The Gene Expression Omnibus (GEO) databases, we found that ubiquitin-specific protease 21 (USP21) was upregulated in CCA, high USP21 level was associated with poor prognosis. In vivo and in vitro, we identified USP21 as a master regulator of CCA growth and maintenance, which directly interacted with deubiquitinates and stabilized the heat shock protein 90 (HSP90) through K48-linked deubiquitination, and in turn, this stabilization increased HIF1A expression, thus upregulating key glycolytic enzyme genes ENO2, ENO3, ALDOC, ACSS2, and then promoted aerobic glycolysis, which provided energy for CCA cell proliferation. In addition, USP21 could directly stabilize alpha-Enolase 1 (ENO1) to promote aerobic glycolysis. Furthermore, increased USP21 level enhanced chemotherapy resistance to the gemcitabine-based regimen. Taken together, we identify a USP21-regulated aerobic glycolysis mechanism that involves the USP21/HSP90/HIF1A axis and USP21/ENO1 axis in CCA tumorigenesis, which could serve as a potential target for the treatment of CCA.
... The study of Li et al. revealed that USP21 was upregulated in nasopharyngeal carcinoma tissues and cell lines, and it was found to promote cell migration and inhibit invasion by regulating FOXM1 (Li and Li 2023). Similarly, USP21 exerted a pro-oncogenic effect in cholangiocarcinoma and mesenchymal glioblastoma stem cells (Zhang et al. 2022b;Zhou et al. 2021). The function of USP21 in endometriosis has yet to be reported. ...
The mechanism involved in the pathogenesis of endometriosis is poorly understood. The purpose of this study is to identify key deubiquitinating enzymes (DUBs) for endometriosis diagnosis and elucidate the possible mechanism, offering novel insights for noninvasive early diagnosis and treatment. Four gene expression datasets were employed from the Gene Expression Omnibus to identify differentially expressed genes (DEGs) between endometriosis and normal controls. GO and KEGG pathways were performed for enrichment analysis. Calibration curves, ROC, DCA, and clinical impact curves verified the clinical usefulness of the nomogram model. In addition, the ssGSEA method was conducted to estimate 23 types of immune cells. A specific DUB gene signature was constructed with Lasso regression, univariate logistic regression, and SVM analysis. RT-qPCR validated the expression of biomarkers. A total of 85 endometriosis-related DUBs were identified in the eutopic endometrium. Among them, 20 DUBs were found to be correlated with the severity of endometriosis. A diagnostic risk model based on five DUB-related genes (USP21, USP48, ZRANB1, COPS5, and EIF3F) was developed using lasso-cox regression analysis. The nomogram model exhibited a strong predictive ability to diagnose endometriosis. KEGG analysis revealed that ubiquitin-mediated proteolysis was activated in patients suffering from severe symptoms. Analysis of immune cell infiltration revealed a positive correlation between USP21 and multiple immune cells in the eutopic endometrium. However, EIF3F showed an opposite relationship. Dysregulation of DUBs was related to the immune microenvironment in endometriosis. Results from RT-qPCR confirmed the expression of DEGs in clinical samples. In summary, the diagnostic model for endometriosis constructed using five differentially expressed DUB genes demonstrates strong diagnostic capability, suggesting that these genes could serve as potential candidate biomarkers and therapeutic targets.
... Recent reports have indicated that the abnormal overexpression of FOXD1 is related to tumorigenesis and the progression of several types of cancers [28][29][30]. The regulation of FOXD1 stability was uncharacterized until Zhang et al. reported that USP21 is a critical deubiquitinase of FOXD1 that removes its K48-linked polyubiquitin chains and stabilizes its protein to maintain the mesenchymal properties of glioblastoma stem cells [31]. However, the E3 ligases that can mediate the K48-linked polyubiquitination of FOXD1 are still unknown. ...
... The regulation of FOXD1 protein was recently described. Zhang et al. reported that USP21, a deubiquitinase of FOXD1, removes K48-linked polyubiquitin chains from FOXD1 and maintains its stability [31]. However, the E3 ligases of FOXD1 are still unknown. ...
Chronic hyperglycaemia is a devastating factor that causes diabetes-induced damage to the retina and kidney. However, the precise mechanism by which hyperglycaemia drives apoptotic cell death is incompletely known. Herein, we found that FOXD1, a FOX family transcription factor specifically expressed in the retina and kidney, regulated the transcription of BCL-2, a master regulator of cell survival. Intriguingly, the protein level of FOXD1, which responded negatively to hyperglycaemic conditions, was controlled by the TRIM21-mediated K48-linked polyubiquitination and subsequent proteasomal degradation. The TRIM21-FOXD1-BCL-2 signalling axis was notably active during diabetes-induced damage to murine retinal and renal tissues. Furthermore, we found that tartary buckwheat flavonoids effectively reversed the downregulation of FOXD1 protein expression and thus restored BCL-2 expression and facilitated the survival of retinal and renal tissues. In summary, we identified a transcription factor responsible for BCL-2 expression, a signalling axis (TRM21-FOXD1-BCL-2) underlying hyperglycaemia-triggered apoptosis, and a potential treatment for deleterious diabetic complications.
... Due to their critical role in tumor growth, GSCs have emerged as a key focus in glioma research. GSCs are a subpopulation of glioma cells, which closely resemble neural precursor cells and have self-renewal activity and multilineage differentiation potential [25,26]. These cells are believed to be closely associated with various malignant behaviors of glioma, including rapid proliferation, invasion, drug resistance, and recurrence [27]. ...
... GSCs share similarities with neural precursor cells, exhibiting self-renewal capabilities and multipotent differentiation potential. GSCs are believed to be one of the sources contributing to various malignant behaviors observed in gliomas, including rapid proliferation, invasion, drug resistance, and recurrence [26]. Indeed, GSCs are currently one of the hottest topics in glioma research. ...
Background
Glioblastoma, the most common primary malignant tumor of the brain, is associated with poor prognosis. Glioblastoma cells exhibit high proliferative and invasive properties, and glioblastoma stem cells (GSCs) have been shown to play a crucial role in the malignant behavior of glioblastoma cells. This study aims to investigate the molecular mechanisms involved in GSCs maintenance and malignant progression.
Methods
Bioinformatics analysis was performed based on data from public databases to explore the expression profile of Mitotic arrest deficient 2 like 2 (MAD2L2) and its potential function in glioma. The impact of MAD2L2 on glioblastoma cell behaviors was assessed through cell viability assays (CCK8), colony formation assays, 5-Ethynyl-2ʹ-deoxyuridine (EDU) incorporation assays, scratch assays, and transwell migration/invasion assays. The findings from in vitro experiments were further validated in vivo using xenograft tumor model. GSCs were isolated from the U87 and LN229 cell lines through flow cytometry and the stemness characteristics were verified by immunofluorescence staining. The sphere-forming ability of GSCs was examined using the stem cell sphere formation assay. Bioinformatics methods were conducted to identified the potential downstream target genes of MAD2L2, followed by in vitro experimental validation. Furthermore, potential upstream transcription factors that regulate MAD2L2 expression were confirmed through chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays.
Results
The MAD2L2 exhibited high expression in glioblastoma samples and showed significant correlation with patient prognosis. In vitro and in vivo experiments confirmed that silencing of MAD2L2 led to decreased proliferation, invasion, and migration capabilities of glioblastoma cells, while decreasing stemness characteristics of glioblastoma stem cells. Conversely, overexpression of MAD2L2 enhanced these malignant behaviors. Further investigation revealed that MYC proto-oncogene (c-MYC) mediated the functional role of MAD2L2 in glioblastoma, which was further validated through a rescue experiment. Moreover, using dual-luciferase reporter gene assays and ChIP assays determined that the upstream transcription factor E2F-1 regulated the expression of MAD2L2.
Conclusion
Our study elucidated the role of MAD2L2 in maintaining glioblastoma stemness and promoting malignant behaviors through the regulation of c-MYC, suggesting its potential as a therapeutic target.
... FOXD1 is located on chromosome 5q12, and it plays a major role in transcriptional regulation in embryogenesis. Recent studies have proved that FOXD1 is associated with tumorigenesis and cancer progression, serving as a prognostic indicator and a prospective target in some sorts of cancers [9][10][11][12][13]. In our research, we demonstrated that FOXD1 was over expressed in HNSCC tissues and related to bad overall survival (OS) and disease-free survival (DFS) according to The Cancer Genome Atlas (TCGA) database. ...
Forkhead box D1 (FOXD1) belongs to the FOX protein family, which has been found to function as a oncogene in multiple cancer types, but its role in head and neck squamous cell carcinoma (HNSCC) requires further investigation. Our research aimed to investigate the function of FOXD1 in HNSCC. Bioinformatics analysis indicated that mRNA level of FOXD1 was highly expressed in HNSCC tissues, and over-expressed FOXD1 was related to poor prognosis. Moreover, FOXD1 knockdown increased the ratio of senescent cells but decreased the proliferation ability, while FOXD1 overexpression obtained the opposite results. In vitro experiments revealed that FOXD1 bound to the p21 promoter and inhibited its transcription, which blocked the cyclin dependent kinase 2 (CDK2)/retinoblastoma (Rb) signaling pathway, thus preventing senescence and accelerating proliferation of tumor cells. CDK2 inhibitor could reverse the process to some extent. Further research has shown that miR-3oe-5p serves as a tumor suppressant by repressing the translation of FOXD1 through combining with the 3’-untranslated region (UTR). Thus, FOXD1 resists cellular senescence and facilitates HNSCC cell proliferation by affecting the expression of p21/CDK2/Rb signaling, suggesting that FOXD1 may be a potential curative target for HNSCC.
Although certain members of the Ubiquitin-specific peptidases (USPs) have been recognized as promising therapeutic targets for various diseases, research progress regarding USP21 has been relatively sluggish in its early stages. USP21 is a crucial member of the USPs subfamily, involved in diverse cellular processes such as apoptosis, DNA repair, and signal transduction. Research findings from the past decade demonstrate that USP21 mediates the deubiquitination of multiple well-known target proteins associated with critical cellular processes relevant to both disease and homeostasis, particularly in various cancers. This review comprehensively summarizes the structure and biological functions of USP21 with an emphasis on its role in tumorigenesis, and elucidates the advances on the discovery of tens of small-molecule inhibitors targeting USP21, which suggests that targeting USP21 may represent a potential strategy for cancer therapy.
BACKGROUND
Colorectal cancer (CRC) is one very usual tumor together with higher death rate. Ubiquitin-specific protease 21 (USP21) has been confirmed to take part into the regulation of CRC progression through serving as a facilitator. Interestingly, the promotive function of USP21 has also discovered in the progression of CRC. ZEB1 has illustrated to be modulated by USP7, USP22 and USP51 in cancers. However, the regulatory functions of USP21 on ZEB1 in CRC progression need more investigations.
AIM
To investigate the relationship between USP21 and ZEB1 in CRC progression.
METHODS
The mRNA and protein expressions were assessed through RT-qPCR, western blot and IHC assay. The interaction between USP21 and ZEB1 was evaluated through Co-IP and GST pull down assays. The cell proliferation was detected through colony formation assay. The cell migration and invasion abilities were determined through Transwell assay. The stemness was tested through sphere formation assay. The tumor growth was evaluated through in vivo mice assay.
RESULTS
In this work, USP21 and ZEB1 exhibited higher expression in CRC, and resulted into poor prognosis. Moreover, the interaction between USP21 and ZEB1 was further investigated. It was demonstrated that USP21 contributed to the stability of ZEB1 through modulating ubiquitination level. In addition, USP21 strengthened cell proliferation, migration and stemness through regulating ZEB1. At last, through in vivo assays, it was illustrated that USP21/ZEB1 axis aggravated tumor growth.
CONCLUSION
For the first time, these above findings manifested that USP21 promoted tumorigenicity and stemness of CRC by deubiquitinating and stabilizing ZEB1. This discovery suggested that USP21/ZEB1 axis may provide novel sights for the treatment of CRC.
