Aryl hydrocarbon receptor (AhR) is a key transcription factor that modulates the differentiation of T helper 17 (Th17) cells. How AhR is regulated at the post-translational level in Th17 cells remains largely unclear. Here, we identify USP21 as a newly defined deubiquitinase of AhR. We demonstrate that USP21 interacts with and stabilizes AhR by removing the K48-linked polyubiquitin chains from AhR. Interestingly, USP21 inhibits the transcriptional activity of AhR in a deubiquitinating-dependent manner. USP21 deubiquitinates AhR at the K432 residue, and the maintenance of ubiquitination on this site is required for the intact transcriptional activity of AhR. Moreover, the deficiency of USP21 promotes the differentiation of Th17 cells both in vitro and in vivo. Consistently, adoptive transfer of USP21-deficient naïve CD4+ T cells elicits more severe colitis in Rag1-/- recipients. Therefore, our study reveals a novel mechanism in which USP21 deubiquitinates AhR and negatively regulates the differentiation of Th17 cells.

Disulfiram (DSF), primarily applied in the therapy for alcohol addiction, has been demonstrated to possess the promising capability of anti-tumor in many human cancers, including esophageal squamous cell carcinoma (ESCC). To date, almost all studies about DSF in ESCC are focusing on investigating either drug combinations or nanoparticle-based delivery systems. However, the exact molecular mechanisms mediating the response to DSF in ESCC are totally unknown. An increasing number of studies reported that aberrant expression of acetylation-related genes is closely involved in regulating the response of cancer cells to anti-tumor drugs. Here, we defined DSF-sensitive and -resistant cells by measuring the half-maximal inhibitory concentration (IC50) of DSF in four ESCC cell lines, followed by detecting the protein expression of nine dysregulated histone acetyltransferase (HAT) genes in ESCC. Our results demonstrate that MOF is responsible for the sensitivity to DSF in ESCC cells. Consistently, DSF treatment markedly abolished MOF-driving ESCC progression and Wnt/β-Catenin signaling activation. Interestingly, DSF decreased MOF protein expression via the ubiquitin-proteasome system. Further exploration verified the essential role of USP21, among three candidates (USP2, USP21, and USP10), in DSF-mediated MOF protein levels. Mechanistically, USP21 binds to MOF protein and decreases the ubiquitination of its K257 site, while DSF notably impedes MOF-mediated ESCC malignant progression and Wnt/β-Catenin signaling activation by blocking USP21-governed MOF-K257 deubiquitination. In conclusion, our study elucidates the USP21/MOF-K257 axis regulating the response to DSF in ESCC, which provides novel and key evidence for the clinical application of DSF in individualized therapy for ESCC patients.

Deubiquitinases play essential roles in hepatocellular carcinoma (HCC) progression, however, the role of ubiquitin-specific peptidase 21 (USP21) in HCC development remains unclear. The present work aims to analyze the effect of USP21 on tumor property of HCC cells and the underlying mechanism. mRNA expression levels of USP21 and H2BFS were analyzed by quantitative real-time polymerase chain reaction. Protein expression of USP21, E-cadherin, N-cadherin, Vimentin, H2BFS and methyltransferase 3 (METTL3) was assessed by western blotting assay or immunohistochemistry assay. Clonogenicity assay was used to analyze cell proliferation. Flow cytometry assay was performed to quantify apoptotic rate of cells. Wound-healing assay and transwell assay were conducted to analyze cell migration and invasion, respectively. Xenograft mouse model assay was performed to determine the effect of USP21 knockdown on tumor formation. m6A RNA immunoprecipitation assay (MeRIP) was used to analyze the effect of METTL3 silencing on methylated level of USP21. USP21 expression was upregulated in HCC tissues and cells when compared with control groups. USP21 silencing inhibited proliferation, migration and invasion and induced apoptosis of HCC cells, accompanied by the increased E-cadherin protein expression and decreased N-cadherin and Vimentin protein expression. Moreover, USP21 knockdown delayed tumor formation in vivo. USP21 stabilized H2BFS by deubiquitination, and H2BFS overexpression attenuated USP21 silencing-induced effects in HCC cells. Further, METTL3-mediated m6A methylation of USP21. METTL3-mediated m6A methylation of USP21 promoted HCC progression by stabilizing H2BFS through deubiquitination.

Pancreatic cancer, the 12th-most common cancer, globally, is highly challenging to treat due to its complex epigenetic, metabolic, and genomic characteristics. In pancreatic ductal adenocarcinoma, USP21 acts as an oncogene by stabilizing the long isoform of Transcription Factor 7, thereby activating the Wnt signaling pathway. This study aims to inhibit activation of this pathway through computer-aided drug discovery. Accordingly, four libraries of compounds were designed to target the USP21's catalytic domain (Cys221, His518, Asp534), responsible for its deubiquitinating activity.

Utilizing an array of computer-aided drug design methodologies, such as molecular docking, virtual screening, principal component analysis, molecular dynamics simulation, and dynamic cross-correlation matrix, the structural and functional characteristics of the USP21-inhibitor complex were examined. Following the evaluation of the binding affinities, 20 potential ligands were selected, and the best ligand was subjected to additional molecular dynamics simulation study.

The results indicated that the ligand-bound USP21 exhibited reduced structural fluctuations compared to the unbound form, as evident from RMSD, RMSF, Rg, and SASA graphs. ADMET analysis of the top ligand showed promising pharmacokinetic and pharmacodynamic profiles, good bioavailability, and low toxicity. The stable conformations of the proposed drug when bound to their target cavities indicate a robust binding affinity of -9.3 kcal/mol. The drug exhibits an elevated pKi value of 6.82, a noteworthy pIC50 value of 5.972, and a pKd value of 6.023 proving its high affinity and inhibitory potential towards the target.

In-vitro testing of the top compound (MOLHYB-0436) could lead to its use as a potential treatment for pancreatic cancer.

Ubiquitin-specific proteases (USPs) are the main members of deubiquitinases (DUBs) that catalyze removing ubiquitin chains from target proteins, thereby modulating their half-life and function. Enzymatic activity of USP21 regulates protein degradation which is critical for maintaining cell homeostasis. USP21 determines the stability of oncogenic proteins and therefore is implicated in carcinogenesis. In this study, we investigated the effect of USP21 deletion on cancer cell metabolism. Transcriptomic and proteomic analysis of USP21 KO HAP-1 cells revealed that endogenous USP21 is critical for the expression of genes and proteins involved in mitochondrial function. Additionally, we have found that the deletion of USP21 reduced STAT3 activation and STAT3-dependent gene and protein expression in cancer cells. Genetic deletion of USP21 impaired mitochondrial respiration and disturbed ATP production. This resulted in cellular consequences such as inhibition of cell proliferation and migration. Presented results provide new insights into the biology of USP21, suggesting novel mechanisms for controlling STAT3 activity and mitochondrial function in tumor cells. Taken together, our findings indicate that targeting USP21 dysregulates the energy status of cancer cells offering new perspectives for anticancer therapy.

Increasing evidence has suggested that ubiquitin-specific protease 10 (USP10), a deubiquitinating enzyme, plays an essential role in targeted protein degradation and participates in cancer progression. However, the relationship between USP10 and pancreatic ductal adenocarcinoma (PDAC) is poorly understood. Here, we developed a USP-targeting siRNA library, combining a loss-of-function experimental screen in patient-derived PDAC cells. This approach identified USP10 as a master regulator of PDAC cell migration. High USP10 expression levels were observed in PDAC patient tissues, which were associated with poor prognosis. Furthermore, knockdown of USP10 expression inhibited PDAC cell proliferation and migration in vivo and in vitro. Mechanistically, USP10 increased FOXC1 protein stability via deubiquitination. The phosphorylation of FOXC1 at S272A was dependent on USP10-mediated deubiquitination of FOXC1. Additionally, USP10 promoted FOXC1 protein localization in the nucleus. Interestingly, FOXC1 could increase USP10 mRNA expression levels by transcriptional activation. Our data suggest that a positive feedback loop exists between USP10 and FOXC1 that can activate WNT signaling, thus facilitating PDAC malignant progression. Therefore, USP10 represents an exciting therapeutic target that could support new strategies for treating PDAC.

Bladder cancer is one of the most common malignancies of the urinary tract and a prevalent cancer worldwide, still requiring efficient therapeutic agents and approaches. 20-Hydroxyecdysone (20-HE), a steroid hormone, can be found in insects and few plants and mediate numerous biological events to control the progression of varying diseases; however, its impacts on bladder cancer remain unclear. In the study, we found that 20-HE treatments effectively inhibited the viability and proliferation of bladder cancer cells and induced apoptosis by activating Caspase-3. The migratory and invasive potential of bladder cancer cells was markedly repressed by 20-HE in a dose-dependent manner. The inhibitory effects of 20-HE on bladder cancer were confirmed in an established xenograft mouse model, as indicated by the markedly reduced tumor growth rates and limited lung and lymph node metastasis. High-throughput RNA sequencing was performed to explore dysregulated genes in bladder cancer cells after 20-HE treatment. We identified ubiquitin-specific protease 21 (USP21) as a key deubiquitinating enzyme for bladder cancer progression and a positive correlation between USP21 and nuclear factor-κB (NF-κB)/p65 in patients. Furthermore, 20-HE treatments markedly reduced USP21 expression, NF-κB/p65 mRNA, stability and phosphorylated NF-κB/p65 expression levels in bladder cancer cells, which were validated in animal tumor tissues. Mechanistic studies showed that USP21 directly interacted with and stabilized p65 by deubiquitinating its K48-linked polyubiquitination in bladder cancer cells, which could be abolished by 20-HE treatment, contributing to p65 degradation. Finally, we found that USP21 overexpression could not only facilitate the proliferation, migration, and invasion of bladder cancer cells, but also significantly eliminated the suppressive effects of 20-HE on bladder cancer. Notably, 20-HE could still perform its anti-tumor role in bladder cancer when USP21 was knocked down with decreased NF-κB/p65 expression and activation, revealing that USP21 suppression might not be the only way for 20-HE during bladder cancer treatment. Collectively, all our results clearly demonstrated that 20-HE may function as a promising therapeutic strategy for bladder cancer treatment mainly through reducing USP21/p65 signaling expression.

Lacking effective therapeutic targets heavily restricts the improvement of clinical prognosis for patients diagnosed with esophageal squamous cell carcinoma (ESCC). Ubiquitin Specific Peptidase 21 (USP21) is dysregulated in plenty of human cancers, however, its potential function and relevant molecular mechanisms in ESCC malignant progression as well as its value in clinical translation remain largely unknown. Here, in vitro and in vivo experiments revealed that aberrant upregulation of USP21 accelerated the proliferation and metastasis of ESCC in a deubiquitinase-dependent manner. Mechanistically, we found that USP21 binds to, deubiquitinates, and stabilizes the G3BP Stress Granule Assembly Factor 1 (G3BP1) protein, which is required for USP21-mediated ESCC progression. Further molecular studies demonstrated that the USP21/G3BP1 axis played a tumor-promoting role in ESCC progression by activating the Wnt/β-Catenin signaling pathway. Additionally, disulfiram (DSF), an inhibitor against USP21 deubiquitylation activity, markedly abolished the USP21-mediated stability of G3BP1 protein and significantly displayed an anti-tumor effect on USP21-driving ESCC progression. Finally, the regulatory axis of USP21/G3BP1 was demonstrated to be aberrantly activated in ESCC tumor tissues and closely associated with advanced clinical stages and unfavorable prognoses, which provides a promising therapeutic strategy targeting USP21/G3BP1 axis for ESCC patients.

Ubiquitin-specific proteases family is crucial to host immunity against pathogens. However, the correlations between USP21 and immunosurveillance and immunotherapy for colorectal cancer (CRC) have not been reported.

The differential expression of USP21 between CRC tissues and normal tissues was analyzed using multiple public databases. Validation was carried out in clinical samples through qRT-PCR and IHC. The correlation between USP21 and the prognosis, as well as clinical pathological characteristics of CRC patients, was investigated. Moreover, cell models were established to assess the influence of USP21 on CRC growth and progression, employing CCK-8 assays, colony formation assays, and wound-healing assays. Subsequently, gene set variation analysis (GSVA) was used to explore the potential biological functions of USP21 in CRC. The study also examined the impact of USP21 on cytokine levels and immune cell infiltration in the tumor microenvironment (TME). Finally, the effect of USP21 on the response to immunotherapy and chemotherapy in CRC was analyzed.

The expression of USP21 was significantly upregulated in CRC. High USP21 is correlated with poor prognosis in CRC patients and facilitates the proliferation and migration capacities of CRC cells. GSVA indicated an association between low USP21 and immune activation. Moreover, low USP21 was linked to an immune-activated TME, characterized by high immune cell infiltration. Importantly, CRC with low USP21 exhibited higher tumor mutational burden, high PD-L1 expression, and better responsiveness to immunotherapy and chemotherapeutic drugs.

This study revealed the role of USP21 in TME, response to therapy, and clinical prognosis in CRC, which provided novel insights for the therapeutic application in CRC.

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 reviewcomprehensively 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.

Ubiquitin-specific protease (USP), an enzyme catalyzing protein deubiquitination, is involved in biological processes related to metabolic disorders and cancer proliferation. We focused on constructing predictive models tailored to unveil compounds boasting USP21 inhibitory attributes. Six models, Extra Trees Classifier, Random Forest Classifier, LightGBM Classifier, XGBoost Classifier, Bagging Classifier, and a convolutional neural network harnessed from empirical data were selected for the screening process. These models guided our selection of 26 compounds from the FDA-approved drug library for further evaluation. Notably, nifuroxazide emerged as the most potent inhibitor, with a half-maximal inhibitory concentration of 14.9 ± 1.63 μM. The stability of protein-ligand complexes was confirmed using molecular modeling. Furthermore, nifuroxazide treatment of HepG2 cells not only inhibited USP21 and its established substrate ACLY but also elevated p-AMPKα, a downstream functional target of USP21. Intriguingly, we unveiled the previously unknown capacity of nifuroxazide to increase the levels of miR-4458, which was identified as downregulating USP21. This discovery was substantiated by manipulating miR-4458 levels in HepG2 cells, resulting in corresponding changes in USP21 protein levels in line with its predicted interaction with ACLY. Lastly, we confirmed the in vivo efficacy of nifuroxazide in inhibiting USP21 in mice livers, observing concurrent alterations in ACLY and p-AMPKα levels. Collectively, our study establishes nifuroxazide as a promising USP21 inhibitor with potential implications for addressing metabolic disorders and cancer proliferation. This multidimensional investigation sheds light on the intricate regulatory mechanisms involving USP21 and its downstream effects, paving the way for further exploration and therapeutic development.

The gastrointestinal tract can be affected by a number of diseases that pancreatic cancer (PC) is a malignant manifestation of them. The prognosis of PC patients is unfavorable and because of their diagnosis at advanced stage, the treatment of this tumor is problematic. Owing to low survival rate, there is much interest towards understanding the molecular profile of PC in an attempt in developing more effective therapeutics. The conventional therapeutics for PC include surgery, chemotherapy and radiotherapy as well as emerging immunotherapy. However, PC is still incurable and more effort should be performed. The molecular landscape of PC is an underlying factor involved in increase in progression of tumor cells. In the presence review, the newest advances in understanding the molecular and biological events in PC are discussed. The dysregulation of molecular pathways including AMPK, MAPK, STAT3, Wnt/β-catenin and non-coding RNA transcripts has been suggested as a factor in development of tumorigenesis in PC. Moreover, cell death mechanisms such as apoptosis, autophagy, ferroptosis and necroptosis demonstrate abnormal levels. The EMT and glycolysis in PC cells enhance to ensure their metastasis and proliferation. Furthermore, such abnormal changes have been used to develop corresponding pharmacological and nanotechnological therapeutics for PC.

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.

To investigate the relationship between USP21 and ZEB1 in CRC progression.

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.

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.

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.

Bladder cancer (BC) is a prevalent and deadly disease. circCD2AP was suggested to be highly expressed in BC. However, the exact mechanism needs further investigation. In this study, circCD2AP was observed to be upregulated in BC and linked to poor prognosis in individuals. Functionally, circCD2AP or USP21 knockdown inhibited BC cell EMT and stemness both in vitro and in vivo. Mechanistically, circCD2AP interacted with ELAVL1 to enhance the stability of USP21 mRNA, which, in turn, inhibited the ubiquitination degradation of FOXQ1. Through rescue assay, USP21 or FOXQ1 knockdown was found to abolish the promoting effects of circCD2AP or USP21 overexpression on BC cell EMT and stemness. Overall, this study has unveiled the role of circCD2AP/ELAVL1/USP21/FOXQ1 axis in BC EMT and stemness regulation, offering insights into the mechanisms underlying BC progression, with potential implications for therapeutic strategies.

USP54, a ubiquitin-specific protease in the deubiquitinase (DUB) family, facilitates the malignant progression of several types of cancer. However, the role of USP54 in prostate cancer (PCa), especially castration-resistant prostate cancer (CRPC), remains unknown.

We established the CRPC LNCaP-AI cell line from the hormone-sensitive prostate cancer (HSPC) LNCaP cell line. RNA-Seq was utilized to explore DUB expression levels in LNCaP and LNCaP-AI. USP54 was knocked down, and its effects on cell growth were evaluated in vitro and in vivo. Bioinformatics analyses were conducted to explore signaling pathways affected by USP54 in PCa. Quantitative polymerase chain reaction was used to confirm key signaling pathways involved.

USP54 was the most strongly upregulated DUB in LNCaP-AI cells compared with LNCaP cells. USP54 levels were higher in PCa than in normal tissues. USP54 silencing suppressed the proliferation of PCa cell lines, both in vitro and in vivo. USP54 expression was positively correlated with the androgen receptor (AR) signaling level in PCa samples, and USP54 knockdown inhibited AR signaling in PCa cells.

USP54 was upregulated during HSPC progression to CRPC. USP54 depletion suppressed CRPC cell proliferation both in vitro and in vivo. USP54 may facilitate PCa progression by regulating AR signaling.

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.

In recent years, the ubiquitin-proteasome system (UPS) has become a hot spot in medical research in cervical cancer (CC) and has received extensive attention. Among them, ubiquitin-specific protease 14 (USP14) is involved in a wide variety of typical cell signaling pathways and is recognized to be involved in the progression of most known tumors. However, the expression and significance of USP14 in CC have not been directly studied. Through database analysis, we found that USP14 was overexpressed in CC, which influenced the FIGO stage and prognosis of CC patients, and it was positively correlated with the expression level of β-catenin. In this study, USP14 promoted the G1-S phase transition of Hela and Siha cells and inhibited cell apoptosis, thereby promoting the proliferation, migration, and invasion of CC cells. In addition, USP14 also significantly promoted the growth of subcutaneous tumor in nude mice. We also found that overexpression of USP14 significantly upregulated β-catenin expression and increased the activity of Wnt/β-catenin signaling pathway. While knockdown of USP14 resulted in the opposite. These results suggest that USP14 may promote the proliferation of CC by up-regulating the expression of β-catenin, contributing to a deeper understanding of the mechanisms of CC and providing a potential therapeutic target.

Ovarian cancer is the most aggressive and lethal of all gynecologic malignancies. Although the overexpression (OE) of ubiquitin-specific peptidase 21 (USP21) has been observed in multiple cancers, its expression profile and biological function in ovarian cancer remain unknown. The expression levels of USP21 in ovarian cancer cells and tissues as well as adjacent normal tissues were assessed by qRT-PCR or Western blot assay. The biological function of USP21 in ovarian cancer cells was assessed by cell growth assay in vitro and a tumor growth model in vivo. Our study revealed that USP21 was markedly elevated in ovarian carcinoma tissues compared with adjacent normal tissues. Downregulation of USP21 attenuated the expression levels of MEK2 and p-ERK1/2. Depletion of USP21 resulted in suppressed cell growth of ovarian cancers in vitro and inhibited tumor growth in vivo. Conversely, OE of USP21 promoted the cell proliferation of ovarian cancers and conferred resistance to BAY 11-7082. These findings provide evidences supporting the notion of USP21 as a promising therapeutic target for the treatment of ovarian cancer.

The ubiquitin-proteasome system (UPS) is a fundamental regulatory mechanism in cells, vital for maintaining cellular homeostasis, compiling signaling transduction, and determining cell fates. These biological processes require the coordinated signal cascades of UPS members, including ubiquitin ligases, ubiquitin-conjugating enzymes, deubiquitinases, and proteasomes, to ubiquitination and de-ubiquitination on substrates. Recent studies indicate that ubiquitination code rewriting is particularly prominent in pancreatic cancer. High frequency mutation or aberrant hyperexpression of UPS members dysregulates ferroptosis, tumor microenvironment, and metabolic rewiring processes and contribute to tumor growth, metastasis, immune evasion, and acquired drug resistance. We conduct an in-depth overview of ubiquitination process in pancreatic cancer, highlighting the role of ubiquitin code in tumor-promoting and tumor-suppressor pathways. Furthermore, we review current UPS modulators and analyze the potential of UPS modulators as cancer therapy.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignant tumor of the digestive system, characterized by rapid progression and being prone to metastasis. Few effective treatment options are available for PDAC, and its 5-year survival rate is less than 9%. Many cell biological and signaling events are involved in the development of PDAC, among which protein post-translational modifications (PTMs), such as ubiquitination, play crucial roles. Catalyzed mostly by a three-enzyme cascade, ubiquitination induces changes in protein activity mainly by altering their stability in PDAC. Due to their role in substrate recognition, E3 ubiquitin ligases (E3s) dictate the outcome of the modification. Ubiquitination can be reversed by deubiquitylases (DUBs), which, in return, modified proteins to their native form. Dysregulation of E3s or DUBs that disrupt protein homeostasis is involved in PDAC. Moreover, the ubiquitination system has been exploited to develop therapeutic strategies, such as proteolysis-targeting chimeras (PROTACs). In this review, we summarize recent progress in our understanding of the role of ubiquitination in the development of PDAC and offer perspectives in the design of new therapies against this highly challenging disease.

Colon adenocarcinoma (COAD) represents a type of common malignant tumor originating in the digestive tract. Long non-coding RNAs (lncRNAs) have been identified to engage in regulating the initiation and development of COAD. LncRNA LINC02253 has been reported abnormal expressed in COAD, but the underlying mechanism has not been discussed so far. This study aimed to determine the role and the molecular biology mechanism of LINC02253 in COAD progression and unearthed its specific molecular mechanism.

RT-qPCR and Western blot assays were conducted to detect gene expression. Function assays were performed to evaluate the effect of gene expression on COAD cell phenotype. Mechanism analyses were done to verify the association among genes after bioinformatics analysis. The obtained data revealed that LINC02253 demonstrated a high expression in COAD tissues and cells. This gene served as an oncogene, permitting to stimulate proliferation and suppress apoptosis of COAD cells. Mechanically, it was found that LINC02253 recruited FUS to stabilize WWP1 mRNA and WWP1 could mediate SMAD3 ubiquitination, thereby promoting the malignant phenotype formation of COAD cells.

LINC02253 was uncovered to exert an oncogenic role, enhancing the proliferation of COAD cells and repressing the cell apoptosis by recruiting FUS and encouraging WWP1-mediated SMAD3 ubiquitination.

Canonical Wnt signaling is essential for a plethora of biological processes ranging from early embryogenesis to aging. Malfunctions of this crucial signaling pathway are associated with various developmental defects and diseases, including cancer. Although TCF/LEF transcription factors (TCF/LEFs) are known to be essential for this pathway, the regulation of their intracellular levels is not completely understood. Here, we show that the lysine demethylase KDM2A promotes the proteasomal destabilization of TCF/LEFs independently of its demethylase domain. We found that the KDM2A-mediated destabilization of TCF/LEFs is dependent on the KDM2A zinc finger CXXC domain. Furthermore, we identified the C-terminal region of TCF7L2 and the CXXC domain of KDM2A as the domains responsible for the interaction between the two proteins. Our study is also the first to show that endogenous TCF/LEF proteins undergo KDM2A-mediated proteasomal degradation in a neddylation-dependent manner. Here, we reveal a completely new mechanism that affects canonical Wnt signaling by regulating the levels of TCF/LEF transcription factors through their KDM2A-promoted proteasomal degradation.

Colorectal cancer (CRC) is one of the most common human malignancies due to its invasiveness and metastasis. Recent studies revealed the pivotal roles of long noncoding RNAs (lncRNAs) in tumorigenesis and progressions of various tumors. However, the biological roles and molecular mechanisms of long intergenic noncoding RNA 00174 (LINC00174) in human CRC remain unclear. Here, we report that LINC00174 expression was higher in human CRC tissues and cell lines than in adjacent normal tissues and a colon epithelial cell line (FHC). High expression of LINC00174 was positively correlated with poor overall and disease-free survival in patients with CRC. Loss- and gain-of-function of LINC00174 demonstrated its critical roles in promoting cell proliferation, apoptosis resistance, migration, and invasion of CRC cells in vitro. Moreover, overexpression of LINC00174 enhanced tumor growth in vivo. Mechanistic experiments revealed that LINC00174 could bind to microRNA (miR)-2467-3p and augment the expression and function of ubiquitin-specific peptidase 21 (USP21). Rescue assays found that miR-2467-3p inhibition can offset the actions of LINC00174 or USP21 knockdown in CRC cells. Additionally, transcriptional factor c-JUN transcriptionally activated LINC00174 expression and mediated LINC00174-induced malignant phenotypes of CRC cell lines. Totally, our findings shed light on a new therapeutic strategy in modulating LINC00174/miR-2467-3p, which may interfere with the expression of USP21, and revealed that LINC00174 could be a new therapeutic target or prognostic marker in CRC.

Cancer stem cells (CSCs), initially identified in leukemia in 1994, constitute a distinct subset of tumor cells characterized by surface markers such as CD133, CD44, and ALDH. Their behavior is regulated through a complex interplay of networks, including transcriptional, post-transcriptional, epigenetic, tumor microenvironment (TME), and epithelial-mesenchymal transition (EMT) factors. Numerous signaling pathways were found to be involved in the regulatory network of CSCs. The maintenance of CSC characteristics plays a pivotal role in driving CSC-associated tumor metastasis and conferring resistance to therapy. Consequently, CSCs have emerged as promising targets in cancer treatment. To date, researchers have developed several anticancer agents tailored to specifically target CSCs, with some of these treatment strategies currently undergoing preclinical or clinical trials. In this review, we outline the origin and biological characteristics of CSCs, explore the regulatory networks governing CSCs, discuss the signaling pathways implicated in these networks, and investigate the influential factors contributing to therapy resistance in CSCs. Finally, we offer insights into preclinical and clinical agents designed to eliminate CSCs.

Diffuse large B cell lymphoma (DLBCL) is a type of cancer that originates from abnormal B cells in the lymph nodes or other lymphoid tissues. Dysfunction of deubiquitinases is frequently implicated in malignant progression. This study planned to uncover the biological roles of deubiquitinase USP25 during DLBCL tumorigenesis. In this study we identified USP25 as a novel oncogene which is frequently upregulated in DLBCL and associated with dismal prognosis of patients. Moreover, USP25 silencing was found to inhibit DLBCL growth, migration, while induced an obvious increase in apoptosis in vitro. Meanwhile, USP25 could promote DLBCL tumour growth and lung metastasis in vivo. Mechanistically, the co-immunoprecipitation test provided a mechanistic explanation, showing that USP25 directly interacted with murine double minute 2 (MDM2) and MDM2 protein stability was maintained by USP25 mediated deubiquitination. In addition, overexpression of USP25 with C178A mutation failed to decrease its modification on MDM2 stability. Further mechanism-of-action studies demonstrated that USP25 promoted DLBCL progression via stabilizing MDM2 and consequently decreasing p53 expression. In addition, further analysis showed that the oncogenic effect of USP25 was relied on MDM2-p53 signaling pathway-mediated cell-cycle accelerating. Collective, USP25 was shown to be an important upstream regulator of the MDM2-p53 signaling pathway in DLBCL, and it has the potential to be employed as a novel target gene in the development of new therapeutic applications.

Ubiquitination is one of the most significant post-translational modifications that regulate almost all physiological processes like cell proliferation, autophagy, apoptosis, and cell cycle progression. Contrary to ubiquitination, deubiquitination removes ubiquitin from targeted protein to maintain its stability and thus regulate cellular homeostasis. Ubiquitin-Specific Protease 12 (USP12) belongs to the biggest family of deubiquitinases named ubiquitin-specific proteases and has been reported to be correlated with various pathophysiological processes. In this review, we initially introduce the structure and biological functions of USP12 briefly and summarize multiple substrates of USP12 as well as the underlying mechanisms. Moreover, we discuss the influence of USP12 on tumorigenesis, tumor immune microenvironment (TME), disease, and related signaling pathways. This study also provides updated information on the roles and functions of USP12 in different types of cancers and other diseases, including prostate cancer, breast cancer, lung cancer, liver cancer, cardiac hypertrophy, multiple myeloma, and Huntington's disease. Generally, this review sums up the research advances of USP12 and discusses its potential clinical application value which deserves more exploration in the future.

As the ubiquitin-proteasome system (UPS) regulates almost every biological process, the dysregulation or aberrant expression of the UPS components causes many pathological disorders, including cancers. To find a novel target for anticancer therapy, the UPS has been an active area of research since the FDA's first approval of a proteasome inhibitor bortezomib in 2003 for treating multiple myeloma (MM). Here, we summarize newly described UPS components, including E3 ubiquitin ligases, deubiquitinases (DUBs), and immunoproteasome, whose malfunction leads to tumorigenesis and whose inhibitors have been investigated in clinical trials as anticancer therapy since 2020. We explain the mechanism and effects of several inhibitors in depth to better comprehend the advantages of targeting UPS components for cancer treatment. In addition, we describe attempts to overcome resistance and limited efficacy of some launched proteasome inhibitors, as well as an emerging PROTAC-based tool targeting UPS components for anticancer therapy.

Pancreatic ductal adenocarcinoma (PDAC) is still one of the deadliest cancers in oncology because of its increasing incidence and poor survival rate. More than 90% of PDAC patients are KRAS mutated (KRASmu), with KRASG12D and KRASG12V being the most common mutations. Despite this critical role, its characteristics have made direct targeting of the RAS protein extremely difficult. KRAS regulates development, cell growth, epigenetically dysregulated differentiation, and survival in PDAC through activation of key downstream pathways, such as MAPK-ERK and PI3K-AKT-mammalian target of rapamycin (mTOR) signaling, in a KRAS-dependent manner. KRASmu induces the occurrence of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) and leads to an immunosuppressive tumor microenvironment (TME). In this context, the oncogenic mutation of KRAS induces an epigenetic program that leads to the initiation of PDAC. Several studies have identified multiple direct and indirect inhibitors of KRAS signaling. Therefore, KRAS dependency is so essential in KRASmu PDAC that cancer cells have secured several compensatory escape mechanisms to counteract the efficacy of KRAS inhibitors, such as activation of MEK/ERK signaling or YAP1 upregulation. This review will provide insights into KRAS dependency in PDAC and analyze recent data on inhibitors of KRAS signaling, focusing on how cancer cells establish compensatory escape mechanisms.

Pancreatic cancer (PC) is one of the most aggressive malignancies worldwide. Increasing evidence suggests that the capacity for self-renewal, proliferation, and differentiation of pancreatic cancer stem cells (PCSCs) contribute to major challenges with current PC therapies, causing metastasis and therapeutic resistance, leading to recurrence and death in patients. The concept that PCSCs are characterized by their high plasticity and self-renewal capacities is central to this review. We focused specifically on the regulation of PCSCs, such as stemness-related signaling pathways, stimuli in tumor cells and the tumor microenvironment (TME), as well as the development of innovative stemness-targeted therapies. Understanding the biological behavior of PCSCs with plasticity and the molecular mechanisms regulating PC stemness will help to identify new treatment strategies to treat this horrible disease.

Ubiquitin is a small protein that can be added onto target protein for inducing target degradation, thereby modulating the activity and stability of protein. Relatively, deubiquitinases (DUBs), a class catalase that can remove ubiquitin from substrate protein, provide a positive regulation of the protein amount at transcription level, post-translational modification, protein interaction, etc. The reversible and dynamic ubiquitination-deubiquitination process plays an essential role in maintaining protein homeostasis, which is critical to almost all the biological processes. Therefore, the metabolic dysregulation of deubiquitinases often lead to serious consequences, including the growth and metastasis of tumors. Accordingly, deubiquitinases can be served as key drug targets for the treatment of tumors. The small molecule inhibitors targeting deubiquitinases has become one of the hot spots of anti-tumor drug research areas. This review concentrated on the function and mechanism of deubiquitinase system in the proliferation, apoptosis, metastasis and autophagy of tumor cells. The research status of small molecule inhibitors of specific deubiquitinases in tumor treatment is introduced, aiming to provide reference for the development of clinical targeted drugs.

The Ubiquitin-proteasome system (UPS) performs a crucial role in immune activation and tumorigenesis. Nevertheless, the comprehensive role of the ubiquitin-proteasome system in the low-grade glioma (LGG) tumor microenvironment (TME) remains unknown. Ubiquitination modification patterns in LGG patients and corresponding characteristics of tumor immune traits, CSC stemness, and cellular senescence were evaluated via a comprehensive analysis of 20 ubiquitination modification regulators. For quantification of the ubiquitination modification status of individual patients, the UM-score was constructed and associated with TME characteristics, clinical features, cancer stem cell stemness, cellular senescence, prognosis, and immunotherapy efficacy. We identified that alterations in multiple ubiquitination regulators are linked to patient survival and the shaping of the tumor microenvironment. We found two different styles of ubiquitination modification in patients with low-grade glioma (immune-inflamed differentiation and immune-exclude dedifferentiation), characterized by high and low UM-score, and the two regulatory patterns of ubiquitination modification on immunity, stemness feature, and cellular senescence. We demonstrate that the UM-score could forecast the subtype of LGG, the immunologic infiltration traits, the biological process, the stemness feature, and the cellular senescence trait. Notably, the UM-score was related to immunotherapeutic efficacy, implying that modifying ubiquitination modification patterns by targeting ubiquitination modification regulators or ubiquitination modification pattern signature genes to reverse unfavorable TME properties will provide new insights into cancer immunotherapy. This research indicated that the ubiquitin-proteasome system is crucial in the formation of TME complexity and multiformity. The UM-score can determine ubiquitination modification status in individual patients, bringing about more personalized and effective immunotherapeutic tactics.

Monoubiquitinated lysine 119 of histone H2A (H2AK119ub) is a highly abundant epigenetic mark, associated with gene repression and deposited on chromatin by the polycomb repressor complex 1 (PRC1), which is an essential regulator of diverse transcriptional programs in mammalian development and tissue homeostasis. While multiple deubiquitinases (DUBs) with catalytic activity for H2AK119ub (H2A-DUBs) have been identified, we lack systematic analyses of their roles and cross-talk in transcriptional regulation. Here, we address H2A-DUB functions in epigenetic regulation of mammalian development and tissue maintenance by conducting a meta-analysis of 248 genomics datasets from 32 independent studies, focusing on the mouse model and covering embryonic stem cells (ESCs), hematopoietic, and immune cell lineages. This covers all the publicly available datasets that map genomic H2A-DUB binding and H2AK119ub distributions (ChIP-Seq), and all datasets assessing dysregulation in gene expression in the relevant H2A-DUB knockout models (RNA-Seq). Many accessory datasets for PRC1-2 and DUB-interacting proteins are also analyzed and interpreted, as well as further data assessing chromatin accessibility (ATAC-Seq) and transcriptional activity (RNA-seq). We report co-localization in the binding of H2A-DUBs BAP1, USP16, and to a lesser extent others that is conserved across different cell-types, and also the enrichment of antagonistic PRC1-2 protein complexes at the same genomic locations. Such conserved sites enriched for the H2A-DUBs and PRC1-2 are proximal to transcriptionally active genes that engage in housekeeping cellular functions. Nevertheless, they exhibit H2AK119ub levels significantly above the genomic average that can undergo further increase with H2A-DUB knockout. This indicates a cooperation between H2A-DUBs and PRC1-2 in the modulation of housekeeping transcriptional programs, conserved across many cell types, likely operating through their antagonistic effects on H2AK119ub and the regulation of local H2AK119ub turnover. Our study further highlights existing knowledge gaps and discusses important directions for future work.

Laryngeal cancer is a usual malignant tumor of the head and neck. The role and mechanism of deubiquitinase USP21 in laryngeal cancer are still unclear. We aimed to explore whether USP21 affected laryngeal cancer progress through deubiquitinating AURKA. USP21 and AURKA levels were evaluated by qRT-PCR and Western blot. Kaplan-Meier analysis was conducted by survival package. MTT was performed to detect cell proliferation. The wound healing assay was applied to evaluate cell migration. Transwell was used to measure cell invasion. Co-IP and GST-pull down determined the interaction between USP21 and AURKA. In addition, AURKA ubiquitination levels were analyzed. USP21 was signally elevated in laryngeal cancer tissues and cells. USP21 level in clinical stages III-IV was higher than that in clinical stages I-II, and high levels of USP21 were highly correlated with poor prognosis in laryngeal cancer. USP21 inhibition suppressed AMC-HN-8 and TU686 cell proliferation, migration and invasion. Co-IP and GST-pull down confirmed the interaction between USP21 and AURKA. Knockdown of USP21 markedly increased the ubiquitination level of AURKA, and USP21 restored AURKA activity through deubiquitination. In addition, overexpression of AURKA reversed the effects of USP21 knockdown on cell growth, migration, and invasion. USP21 stabilized AURKA through deubiquitination to promote laryngeal cancer progression.

USP21 belongs to the ubiquitin-specific protease (USP) subfamily of deubiquitinating enzymes (DUBs). Due to its relevance in tumor development and growth, USP21 has been reported as a promising novel therapeutic target for cancer treatment. Herein, we present the discovery of the first highly potent and selective USP21 inhibitor. Following high-throughput screening and subsequent structure-based optimization, we identified BAY-805 to be a non-covalent inhibitor with low nanomolar affinity for USP21 and high selectivity over other DUB targets as well as kinases, proteases, and other common off-targets. Furthermore, surface plasmon resonance (SPR) and cellular thermal shift assays (CETSA) demonstrated high-affinity target engagement of BAY-805, resulting in strong NF-κB activation in a cell-based reporter assay. To the best of our knowledge, BAY-805 is the first potent and selective USP21 inhibitor and represents a valuable high-quality in vitro chemical probe to further explore the complex biology of USP21.

Laryngeal cancer (LC) is a rare and challenging clinical problem. Our aim was to investigate the mechanism of salt-like transcription factor 4 (SALL4) in LC. LC tissue and paracancerous tissue were collected. Relative mRNA or protein levels were measured by quantitative real-time polymerase chain reaction or Western blot. MTT, wound healing, and transwell assay were performed to evaluate cell proliferation, migration and invasion. The binding relationship between SALL4 and USP21 promoter was verified by dual-luciferase assay and ChIP. Co-IP and glutathione-S-transferase (GST)-pull down were performed to measure the protein interaction between USP21 and YY1. Additionally, YY1 ubiquitination level was analyzed. It was found that SALL4 mRNA and SALL4 protein levels were elevated in LC clinical tissues and various LC cells. Knockdown of SALL4 inhibited epithelial-mesenchymal transition (EMT) of LC cells. USP21 was transcriptionally activated by SALL4. Co-IP and GST-pull down confirmed USP21 interacted with YY1. USP21 protected YY1 from degradation through deubiquitination. Furthermore, overexpression of USP21 reversed the effect of knockdown of SALL4 on YY1 and EMT in LC cells. In general, SALL4 facilitated EMT of LC cells through modulating USP21/YY1 axis.

Necroptosis is a necrotic programmed cell death with potent immunogenicity. Due to the dual effects of necroptosis on tumor growth, metastasis and immunosuppression, we evaluated the prognostic value of necroptosis-related genes (NRGs) in hepatocellular carcinoma (HCC).

We first analyzed RNA sequencing and clinical HCC patient data obtained to develop an NRG prognostic signature based on the TCGA dataset. Differentially expressed NRGs were further evaluated by GO and KEGG pathway analyses. Next, we conducted univariate and multivariate Cox regression analyses to build a prognostic model. We also used the dataset obtained from the International Cancer Genome Consortium (ICGC) database to verify the signature. The Tumor Immune Dysfunction and Exclusion (TIDE) algorithm was used to investigate the immunotherapy response. Furthermore, we investigated the relationship between the prediction signature and chemotherapy treatment response in HCC.

We first identified 36 differentially expressed genes out of 159 NRGs in hepatocellular carcinoma. Enrichment analysis showed that they were mainly enriched in the necroptosis pathway. Four NRGs were screened by Cox regression analysis to establish a prognostic model. The survival analysis revealed that the overall survival of patients with high-risk scores was significantly shorter than that of patients with low-risk scores. The nomogram demonstrated satisfactory discrimination and calibration. The calibration curves validated a fine concordance between the nomogram prediction and actual observation. The efficacy of the necroptosis-related signature was also validated by an independent dataset and immunohistochemistry experiments. TIDE analysis revealed that patients in the high-risk group were possibly more susceptible to immunotherapy. Furthermore, high-risk patients were found to be more sensitive to conventional chemotherapeutic medicines such as bleomycin, bortezomib, and imatinib.

We identified 4 necroptosis-related genes and established a prognostic risk model that could potentially predict prognosis and response to chemotherapy and immunotherapy in HCC patients in the future.

The purpose of this work was to explore the molecular mechanisms by which USP21 regulates nasopharyngeal carcinoma tumor growth and cancer cell stemness. In this study, the USP21 transcript data was obtained from TCGA database. Then, qPCR and western blot tests revealed that, in contrast to normal tissue or normal nasopharyngeal epithelial cells, the expression of USP21 was greater in nasopharyngeal carcinoma tissues or cell lines, respectively. CCK-8 and EdU immunofluorescent staining assays revealed that USP21 promoted the proliferation of nasopharyngeal carcinoma cells. Meanwhile, scratch and transwell assays showed that USP21 facilitated migration and invasion of nasopharyngeal carcinoma cells. Sphere formation assay was performed on nasopharyngeal carcinoma cells after knockdown of USP21, which revealed that knockdown of USP21 inhibited the stemness profiles of nasopharyngeal carcinoma cells. Then, the western blot assays indicated that knockdown of USP21 in nasopharyngeal carcinoma cells would inhibit FOXM1 expression, and overexpression of FOXM1 could reverse the cell proliferation ability, cell migration and invasion ability, and cell stemness profiles. Finally, a nasopharyngeal xenograft model suggested that USP21 facilitated tumor growth in mice. These findings proved that USP21 promoted tumor growth and cancer cell stemness in nasopharyngeal carcinoma by regulating FOXM1.

Cancer stem cells (CSCs) play significant roles in cancer development, drug resistance and cancer recurrence. In cancer treatments based on the CSC characteristics and inducing factors, MYC is a promising target for therapeutic molecules. Although it has been regarded as an undrugable target, its stability tightly regulated by the ubiquitin-proteasome system offers a new direction for molecule targeting and cancer treatment. Herein we report our discoveries in this research area, and we have found that deubiquitinase USP45 can directly bind with MYC, resulting in its deubiquitination and stabilization. Further, USP45 overexpressing can upregulate MYC, and this overexpressing can significantly enhance cancer development, cancer cell stemness and drug resistance. Interestingly, without enhancing cancer development, MYC silencing with shRNA can only suppress USP45-induced stemness and drug resistance. Moreover, we have identified that USP45 can be specifically bound and inhibited by a natural small molecule (α-mangostin), in turn significantly suppressing USP45-induced stemness and drug resistance. Since USP45 is significantly expressed in cervical tumors, we have discovered that the combination of α-mangostin and doxorubicin can significantly inhibit USP45-induced cervical tumorigenesis in an animal model. In general, on the basis of our USP45 discoveries on its MYC deubiquitination and α-mangostin inhibition, suppressing USP45 has opened a new window for suppressing cancer development, stemness and drug resistance.

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 study, we identify the ubiquitin-specific peptidase 21 (USP21) as a critical FOXD1 deubiquitinase in MES GSCs. We find that USP21 directly interacts with and stabilizes FOXD1 by reverting its proteolytic ubiquitination. Silencing of USP21 enhances polyubiquitination of FOXD1, promotes its proteasomal degradation, and ultimately attenuates MES identity in GSCs, while these effects could be largely restored by reintroduction of FOXD1. Remarkably, we show that disulfiram, a repurposed drug that could block the enzymatic activities of USP21, suppresses GSC tumorigenicity in MES GSC-derived GBM xenograft model. Additionally, we demonstrate that USP21 is overexpressed and positively correlated with FOXD1 protein levels in GBM tissues, and its expression is inversely correlated with patient survival. Collectively, our work reveals that USP21 maintains MES identity by antagonizing FOXD1 ubiquitination and degradation, suggesting that USP21 is a potential therapeutic target for the MES subtype of GBM.