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B7–H4 is an immune checkpoint crucial for inhibiting CD8+ T-cell activity. A clinical trial is underway to investigate B7–H4 as a potential immunotherapeutic agent. However, the regulatory mechanism of B7–H4 degradation via the ubiquitin-proteasome pathway (UPP) remains poorly understood. In this study, we discovered that proteasome inhibitors effectively increased B7–H4 expression, while EGFR-activating mutants promoted B7–H4 expression through the UPP. We screened B7–H4 binding proteins by co-immunoprecipitation and mass spectrometry and found that USP2a acted as a deubiquitinase of B7–H4 by removing K48- and K63-linked ubiquitin chains from B7–H4, leading to a reduction in B7–H4 degradation. EGFR mutants enhanced B7–H4 stability by upregulating USP2a expression. We further investigated the role of USP2a in tumor growth in vivo. Depletion of USP2a in L858R/LLC cells inhibited tumor cell proliferation, consequently suppressing tumor growth in immune-deficient nude mice by destabilizing downstream molecules such as Cyclin D1. In an immune-competent C57BL/6 mouse tumor model, USP2a abrogation facilitated infiltration of CD95+CD8+ effector T cells and hindered infiltration of Tim-3+CD8+ and LAG-3+CD8+ exhausted T cells by destabilizing B7–H4. Clinical lung adenocarcinoma samples showed a significant correlation between B7–H4 abundance and USP2a expression, indicating the contribution of the EGFR/USP2a/B7–H4 axis to tumor immunosuppression. In summary, this study elucidates the dual effects of USP2a in tumor growth by stabilizing Cyclin D1, promoting tumor cell proliferation, and stabilizing B7–H4, contributing to tumor immunosuppression. Therefore, USP2a represents a potential target for tumor therapy. • EGFR activating mutants promote B7–H4 stability through ubiquitin-proteosome pathway. • Deubiquitinase USP2a interacts with B7–H4 and mediates EGFR MT-inducing B7–H4 stability. • USP2a is upregulated in lung adenocarcinoma harboring EGFR mutants. • EGFR/USP2a/B7–H4 axis contributes to tumor immunosuppression. [ABSTRACT FROM AUTHOR]

Mesenchymal glioma stem cells (MES GSCs) are a subpopulation of cells in glioblastoma (GBM) that contribute to a worse prognosis owing to their highly aggressive nature and resistance to radiation therapy. Here, OCT4 is characterized as a critical factor in sustaining the stemness phenotype of MES GSC. We find that OCT4 is expressed intensively in MES GSC and is intimately associated with poor prognosis, moreover, OCT4 depletion leads to diminished invasive capacity and impairment of the stem phenotype in MES GSC. Subsequently, we demonstrated that USP5 is a deubiquitinating enzyme which directly interacts with OCT4 and preserves OCT4 stability through its deubiquitination. USP5 was additionally proven to be aberrantly over-expressed in MES GSCs, and its depletion resulted in a noticeable diminution of OCT4 and consequently a reduced self-renewal and tumorigenic capacity of MES GSCs, which can be substantially restored by ectopic expression of OCT4. In addition, we detected the dominant molecule that regulates USP5 transcription, E2F1, with dual luciferase reporter gene analysis. In combination, targeting the E2F1-USP5-OCT4 axis is a potentially emerging strategy for the therapy of GBM. • OCT4 is overexpressed in MES GSCs and high OCT4 predicts poor survival in GBM patients. • OCT4 promotes tumorigenesis and metastasis in an immunodeficient mouse model. • USP5 deubiquitinated OCT4, and promotes the proliferation, invasion, and tumorigenicity of MES GSC. • Transcription factor E2F1 promotes USP5 expression in MES GSCs. [ABSTRACT FROM AUTHOR]

The NADPH oxidase (Nox) family of enzymes is solely dedicated in the generation of reactive oxygen species (ROS). ROS generated by Nox are involved in multiple signaling cascades and a myriad of pathophysiological conditions including cancer. As such, ROS seem to have both detrimental and beneficial roles in a number of cellular functions, including cell signaling, growth, apoptosis and proliferation. Regulatory mechanisms are required to control the activity of Nox enzymes in order to maintain ROS balance within the cell. Here, we performed genome-wide screening for deubiquitinating enzymes (DUBs) regulating Nox organizer 1 (NoxO1) protein expression using a CRISPR/Cas9-mediated DUB-knockout library. We identified cylindromatosis (CYLD) as a binding partner regulating NoxO1 protein expression. We demonstrated that the overexpression of CYLD promotes ubiquitination of NoxO1 protein and reduces the NoxO1 protein half-life. The destabilization of NoxO1 protein by CYLD suppressed excessive ROS generation. Additionally, CRISPR/Cas9-mediated knockout of CYLD in PC-3 cells promoted cell proliferation, migration, colony formation and invasion in vitro. In xenografted mice, injection of CYLD-depleted cells consistently led to tumor development with increased weight and volume. Taken together, these results indicate that CYLD acts as a destabilizer of NoxO1 protein and could be a potential tumor suppressor target for cancer therapeutics. [ABSTRACT FROM AUTHOR]

Recently, many studies have shown that deubiquitination modification of proteins is of great significance in major physiological processes such as cell proliferation, apoptosis, and differentiation. The ubiquitin-specific peptidase (USP) family is one of the most numerous and structurally diverse of the deubiquitinates known to date. USP22, an important member of the USP family, has been found to be closely associated with tumor cell cycle regulation, stemness maintenance, invasion and metastasis, chemoresistance, and immune regulation. We focus on recent advances regarding USP22's function in cancer and discuss the prospect of USP22 in this review. [ABSTRACT FROM AUTHOR]

Immunotherapy based on PD-1/PD-L1 antagonists has been demonstrated to be efficacious in inducing tumor remission in patients with triple-negative breast cancer (TNBC). However, tumor immune evasion caused by the PD-1/PD-L1 pathway inhibits the immunotherapeutic effect of PD-1/PD-L1 inhibitors against TNBC. Therefore, identifying potential targets for blocking the PD-1/PD-L1 pathway is a compelling strategy for TNBC treatment. Here, we discovered that VGLL4 could inhibit PD-L1 transcription by suppressing STAT3 activation, thereby enhancing the efficacy of anti-PD-1 antibody immunotherapy in TNBC. Low expression of USP15, a deubiquitinating enzyme of VGLL4, was associated with reduced CD8+ T cell infiltration and poor prognosis in TNBC patients. USP15 was found to inhibit PD-L1 transcription, leading to increased CD8+ T cell infiltration and thus enhancing the efficacy of TNBC immunotherapy. Furthermore, SART3 regulated VGLL4 stability and PD-L1 transcription by influencing the nuclear translocation of USP15. In conclusion, our study provides new insights into the biological regulation of PD-L1, identifies a previously unrecognized regulator of this critical immune checkpoint, and highlights potential therapeutic targets for overcoming immune evasion in TNBC. • VGLL4 enhances the efficacy of anti-PD-1 antibody immunotherapy for TNBC by inhibiting PD-L1 transcription. • The C-terminal of USP15 is responsible for its interaction with TDU domains in VGLL4. • USP15 removes the K48-linked ubiquitination of VGLL4 and stabilizes VGLL4. • Low expression of USP15 is accompanied by reduced CD8+ T cell infiltration and poor prognosis in TNBC patients. • USP15 enhances the efficacy of anti-PD-1 antibody immunotherapy for TNBC. [ABSTRACT FROM AUTHOR]

Smad3 is the key mediator of TGF-β1-triggered signal transduction and the related biological responses, promoting cell invasion and metastasis in various cancers, including lung cancer. However, the deubiquitinase stabilizing Smad3 remains unknown. In this study, we present a paradigm in which POH1 is identified as a novel deubiquitinase of Smad3 that plays a tumor-promoting role in lung adenocarcinoma (LUAD) by regulating Smad3 stability. POH1 markedly increased Smad3 protein levels and prolonged its half-life. POH1 directly interacted and colocalized with Smad3, leading to the removal of poly-deubiquitination of Smad3. Functionally, POH1 facilitated cell proliferation, migration, and invasion by stabilizing Smad3. Importantly, POH1 also promoted liver metastasis of lung cancer cells. The protein levels of both POH1 and Smad3 were raised in the tumor tissues of patients with LUAD, which predicts poor prognosis. Collectively, we demonstrate that POH1 acts as an oncoprotein by enhancing TGF-β1/Smad3 signaling and TGF-β1-mediated metastasis of lung cancer. • POH1 enhances TGF-β1-mediated signaling by increasing the stability of Smad3. • POH1 abolishes polyubiquitination of Smad3 via K63 of ubiquitin or a K48- branched chain based on K63-chain. • POH1 facilitates cell proliferation, migration, invasion, and metastasis of lung cancer cells by stabilizing Smad3. • Elevated expression of both POH1 and Smad3 are associated with poor survival of LUAD patients. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) has emerged as one of the most common malignancies worldwide. It is associated with a high mortality rate, as evident from its increasing incidence and extremely poor prognosis. The deubiquitinating enzyme 26S proteasome non-ATPase regulatory subunit 14 (PSMD14) has been reported to act as an oncogene in several human cancers. The present study aimed to reveal the functional significance of PSMD14 in HCC progression and the underlying mechanisms. We found that PSMD14 was significantly upregulated in HCC tissues. Overexpression of PSMD14 correlated with vascular invasion, tumor number, tumor recurrence, and poor tumor-free and overall survival of patients with HCC. Knockdown and overexpression experiments demonstrated that PSMD14 promoted proliferation, migration, and invasion in HCC cells in vitro, and facilitated tumor growth and metastasis in vivo. Mechanistically, we identified PSMD14 as a novel post-translational regulator of GRB2. PSMD14 inhibits degradation of GRB2 via deubiquitinating this oncoprotein in HCC cells. Furthermore, pharmacological inhibition of PSMD14 with O-phenanthroline (OPA) suppressed the malignant behavior of HCC cells in vitro and in vivo. In conclusion, our findings suggest that PSMD14 could serve as a novel promising therapeutic candidate for HCC. [ABSTRACT FROM AUTHOR]

Hippo pathway plays a crucial role in the progression of hepatocellular carcinoma (HCC), and yes-associated protein (YAP) is one of the major factors of the Hippo pathway. However, the mechanism of abnormal YAP activation in HCC has not been well elucidated. Here, we screened a Deubiquitinating enzymes' (DUB) siRNA library targeting DUBs, and identified Ubiquitin Specific Peptidase 19 (USP19) as a specific deubiquitinating enzyme of YAP in HCC, which could stabilize YAP at K76 and K90 sites via removing the K48- and K11-linked ubiquitin chains. USP19 knockdown decreased the expression of YAP protein and its target gene (CTGF, CYR61, ANKRD1) expression. Through substantial in vivo and in vitro experiments, we prove that USP19 facilities the proliferation and migration of HCC. More importantly, we found that USP19 was upregulated in HCC tissues and associated with poor prognosis. In general, our research revealed a novel post-translational mechanism between USP19 and YAP in HCC, suggesting that USP19 may be a pivotal therapeutic target for HCC treatment. • USP19 is a novel deubiquitinating enzyme for YAP. • The USP domain of USP19 interacted with the WW domain of YAP. • USP19 stabilizes YAP at K76 and K90 sites via removing the K48- and K11-linked ubiquitin chains. • USP19 promotes hepatocellular carcinoma progression and metastasis. • USP19 are elevated in hepatocellular carcinoma and correlate with poor prognosis. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) is extremely malignant with limited treatment options. Deubiquitinases (DUBs), which cleave ubiquitin on substrates, can regulate tumor progression and are appealing therapeutic targets, but there are few related studies in PDAC. In our study, we screened the expression levels and prognostic value of USP family members based on published databases and selected USP10 as the potential interventional target in PDAC. IHC staining of the PDAC microarray revealed that USP10 expression was an adverse clinical feature of PDAC. USP10 promoted tumor growth both in vivo and in vitro in PDAC. Co-IP experiments revealed that USP10 directly interacts with PABPC1. Deubiquitination assays revealed that USP10 decreased the K27/29-linked ubiquitination level of the RRM2 domain of PABPC1. Deubiquitinated PABPC1 was able to couple more CLK2 mRNA and eIF4G1, which increased the translation efficiency. Replacing PABPC1 with a mutant that could not be ubiquitinated impaired USP10 knock-down-mediated tumor suppression in PDAC. Targeting USP10 significantly delayed the growth of cell-derived xenograft and patient-derived xenograft tumors. Collectively, our study first identified USP10 as the DUB of PABPC1 and provided a rationale for potential therapeutic options for PDAC with high USP10 expression. • USP10 expression is an adverse clinical feature in patients with PDAC and supports the malignant phenotype of PDAC. • The N-terminal domain of USP10 physically interacts with the C-terminal domain of PABPC1. • USP10 removes the K27/29-linked ubiquitination on the RRM2 domain of PABPC1. • Deubiquitinated PABPC1 binds more CLK2 mRNA and eIF4G1, which increases the translation efficiency of CLK2 mRNA. • Targeting USP10 suppresses the growth of CDX and PDX tumors of PDAC. [ABSTRACT FROM AUTHOR]

The zinc metalloprotease STAM-binding protein-like 1 (STAMBPL1) has been identified as a deubiquitinase by specifically cleaving Lys-63-linked polyubiquitin chains, but its cellular function remains unclear. Here we described the potential role of STAMBPL1 in suppression of the intrinsic apoptosis. We observed substantially high amounts of STAMBPL1 proteins in androgen-independent prostate cancer PC3 and DU145 cell lines. STAMBPL1 RNAi depletion triggered caspase-3/-7-dependent apoptosis in PC3 and DU145 cells. STAMBPL1 knockdown-induced apoptosis was accompanied by accumulation of cellular ROS and a decrease in endogenous caspase inhibitor XIAP protein content. Treatment cells with antioxidant NAC delayed STAMBPL1 silencing-induced apoptosis, whereas ectopic expression of XIAP almost completely abrogated apoptosis. We further elucidated that STAMBPL1 knockdown diverted XIAP protein to lysosomal degradation pathway. Taken together, these studies show that STAMBPL1 depletion induces apoptosis by promoting XIAP lysosomal degradation, and suggest that targeting deubiquitinase STAMBPL1 might offer promising therapeutic strategy for prostate cancer. [ABSTRACT FROM AUTHOR]

The serine/threonine kinase, CHK2 (checkpoint kinase 2), is a key mediator in DNA damage response and a tumor suppressor, which is implicated in promoting cell cycle arrest, apoptosis and DNA repair. Accumulating evidence suggests that these functions are primarily exerted through phosphorylation downstream factors such as p53 and BRCA1. Recent studies have shown that ubiquitination is an important mode of regulation of CHK2. However, it remains largely unclear whether deubiquitinases participate in regulation of CHK2. Here, we report that a deubiquitinase, USP39, is a new regulator of CHK2. Mechanistically, USP39 deubiquitinates and stabilizes CHK2, which in turn enhances CHK2 stability. Short hairpin RNA (shRNA) mediated knockdown of USP39 led to deregulate CHK2, which resulted in compromising the DNA damage-induced G2/M checkpoint, decreasing apoptosis, and conferring cancer cells resistance to chemotherapy drugs and radiation treatment. Collectively, we identify USP39 as a novel regulator of CHK2 in the DNA damage response. [ABSTRACT FROM AUTHOR]

Snail is an important transcription factor of epithelial-mesenchymal transition (EMT) and related to poor prognosis and distant metastasis of tumor patients. Snail is a liable protein and degraded by ubiquitin-proteasome system. There are various E3 ligases mediating its degradation, but the deubiquitinating enzyme reversed Snail degradation is not fully understood. In this study, we screened a DUB library and found USP26 is a potent deubiquitinase mediating Snail stabilization. We also identified that USP26 is a booster of esophageal squamous cell carcinoma (ESCC) cell migration and invasion, and it is highly expressed in ESCC samples. Our observation demonstrates that USP26 is a novel deubiquitinating enzyme of Snail and it provides a potential target for the therapy of esophageal cancer metastasis. [ABSTRACT FROM AUTHOR]

Continuous estrogen signaling is thought to be the main mechanism causing endometrial cancer (EC). Studies have demonstrated that CD163+ macrophages could promote the development of estrogen-dependent EC, but the mechanisms involved remain unclear. We found that CD163+ macrophages were the dominant macrophages in atypical endometrial hyperplasia and cancer, and their infiltration was positively associated with ERα expression. CD163+ macrophages mainly increased ERα protein levels but with little upregulatory effect on ESR1 (ERα coding gene) transcripts. The ubiquitin-editing enzyme A20, screened from the endometrial microarray obtained from mice receiving a high-fat diet and sustained estrogen-intervened, was highly expressed in endometrial lesions rich with CD163+ macrophages, and positively correlated with ERα expression. Similarly, A20 and ERα were both upregulated by CD163+ macrophages via cytokines such as IL1α, IL17A and TNFα. Mechanistically, A20 overexpression in EC cells prolonged ERα protein half-life without affecting ESR1 transcripts. A20 increased functional ERα protein levels and enhanced estrogen-driven EC cell proliferation through preventing ERα protein degradation by its deubiquitinase activity. Our study revealed that A20-mediated deubiquitination of ERα might be an important mechanism by which CD163+ macrophages sensitize EC cells to estrogen. [ABSTRACT FROM AUTHOR]

Dysregulation of deubiquitination pathway is associated with poor prognosis in cancers such as hepatocellular carcinoma (HCC). The mammalian target of rapamycin, mTOR, has become an attractive cancer therapeutic target in HCC. However, whether and how aberrant expression of deubiquitination pathway regulates mTOR pathway has remained elusive. Here we report that ubiquitin-specific protease 10 (USP10) functions as a tumor suppressor which inhibits mTOR pathway by stabilizing PTEN and AMPKα in HCC cells. Mechanistically, USP10 interacts and stabilizes PTEN and AMPKα by inhibiting their polyubiquitylation. This stabilization in turn inhibits AKT phosphorylation and mTOR Complex1 (mTORC1) activation. In human liver cancer, USP10 expression is downregulated in HCC tumor tissues across three independent HCC cohorts, and lower-expression of USP10 will generate poor prognosis outcome. Collectively, our results uncover an undescribed mechanism where USP10, as a tumor suppressor, negatively regulates mTORC1 activation and AKT phosphorylation by stabilizing AMPKα and PTEN in HCC cells. This study sheds light on the theoretical basis of mTOR signaling pathway-oriented targeting treatment in clinic. [ABSTRACT FROM AUTHOR]

As a newly discovered deubiquitinating enzyme, ubiquitin-specific protease 22 (USP22) is predictive of therapeutic outcomes in individual cancer patients. However, its clinical effects on malignancy and its roles in conferring resistance to EGFR-TKIs (epidermal growth factor receptor-tyrosine kinase inhibitors) in lung adenocarcinoma (ADC) remain largely unknown. Here, we showed that USP22 promotes cell proliferation, migration and invasion, and contributes to resistance to EGFR-TKIs in EGFR mutant lung ADC cells. Mechanistically, USP22 deubiquitinates EGFR localized on late endosomes, prevents ubiquitination mediated EGFR degradation and enhances recycling of EGFR after EGF stimulation. Additionally, USP22 sustained the activation of multiple EGFR downstream signaling pathways, including STAT3, AKT/mTOR and MEK/ERK pathways, in lung ADC cell lines H1975 and PC9. Furthermore, USP22 stabilizes EGFR protein expression, which correlates with USP22 expression in EGFR-mutant lung ADC patient samples. We are the first to demonstrate that silencing USP22 counteracts EGFR-TKIs resistance both in vitro and in vivo. We propose USP22 as a potential therapeutic target for EGFR-TKIs-resistant lung ADC. [ABSTRACT FROM AUTHOR]

The epithelial-mesenchymal transition (EMT) transcription factor SNAIL is associated with distant metastasis and poor prognosis of esophageal squamous cell carcinoma (ESCC) patients. The proteolysis of SNAIL is mediated by the ubiquitin-proteasome system. Several E3 ligases have been characterized to promote SNAIL ubiquitination and degradation. However, the reverse process - deubiquitination of SNAIL remains largely unknown. In this study, we performed a mass spectrometry to examine the interaction between SNAIL and deubiquitinating enzyme(s). Subsequently, the deubiquitinating enzyme PSMD14 was identified to target SNAIL for deubiquitination and stabilization. Furthermore, knockdown of PSMD14 significantly blocks SNAIL-induced EMT and then suppresses tumor cell migration and invasion in vitro and tumor metastasis in vivo. In addition, the high expression level of PSMD14 predicts poor prognosis for esophageal cancer patients. These findings suggest PSMD14 as a bona fide deubiquitinating enzyme to regulate SNAIL at the post-translational level and provide a promising therapeutic strategy against tumor metastasis of esophageal cancer. [ABSTRACT FROM AUTHOR]

ID1 is a labile protein implicated in the development and progression of several malignant tumors, but the mechanisms regulating ID1 stability and function in human esophageal squamous cell carcinoma (ESCC) are largely unclear. In this study, we performed an immunoprecipitation assay to screen for deubiquitinases that may interact with ID1 and identified USP8 as a novel deubiquitinase for ID1. USP8 interacts with ID1, and increases the protein level and stability of ID1 by reducing its ubiquitination. Ectopic expression of USP8 promotes ESCC tumorigenesis by suppressing ID1 degradation, whereas knockdown of USP8 results in the opposite phenotypes in vitro and in vivo. Moreover, we found that TXNIP is a novel downstream target of ID1, and USP8 promotes ESCC tumorigenesis by activating the ID1-TXNIP pathway. Increased expression of USP8 and ID1 positively correlates with reduced TXNIP expression in ESCC tissues and predicts an advanced tumor stage. Overall, our data indicate that USP8 is a novel deubiquitinase for ID1 and show the importance of the USP8-ID1-TXNIP axis in promoting ESCC tumorigenesis. [ABSTRACT FROM AUTHOR]

Continuous estrogen signaling is thought to be the main mechanism causing endometrial cancer (EC). Studies have demonstrated that CD163 + macrophages could promote the development of estrogen-dependent EC, but the mechanisms involved remain unclear. We found that CD163 + macrophages were the dominant macrophages in atypical endometrial hyperplasia and cancer, and their infiltration was positively associated with ERα expression. CD163 + macrophages mainly increased ERα protein levels but with little upregulatory effect on ESR1 (ERα coding gene) transcripts. The ubiquitin-editing enzyme A20, screened from the endometrial microarray obtained from mice receiving a high-fat diet and sustained estrogen-intervened, was highly expressed in endometrial lesions rich with CD163 + macrophages, and positively correlated with ERα expression. Similarly, A20 and ERα were both upregulated by CD163 + macrophages via cytokines such as IL1α, IL17A and TNFα. Mechanistically, A20 overexpression in EC cells prolonged ERα protein half-life without affecting ESR1 transcripts. A20 increased functional ERα protein levels and enhanced estrogen-driven EC cell proliferation through preventing ERα protein degradation by its deubiquitinase activity. Our study revealed that A20-mediated deubiquitination of ERα might be an important mechanism by which CD163 +  macrophages sensitize EC cells to estrogen., (Copyright © 2018 Elsevier B.V. All rights reserved.)