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USP36 expression is positively correlated with YAP expression and interacts with YAP in ESCC cells. A Immunostaining demonstrated the colocalization of USP36 (green) and YAP (red) in ECA109 cells. Blue denoted cell nuclei. Scale bar, 10 µm. B Representative immunoblots to show the interaction between USP36 and YAP as assessed by immunoprecipitation (IP) for USP36 or YAP, compared to isotype control (IgG). C Control or USP36-slienced ECA109 cells were treated with MG132 (10 μM) for 24 h. YAP and USP36 protein were measured by immunoblotting. Depletion of USP36 decreased YAP protein levels, and this effect could be attenuated by MG132. D USP36 depletion decreased the YAP protein half-life in ECA109 cells. The cells were treated with 100 µmol/L CHX for the indicated time before being collected for western blot assays. Quantitative analysis of halftime of YAP and TAZ protein in (D). E Representative immunoblots to show the interaction between USP36 and WT or truncated YAP as indicated assessed by immunoprecipitation (IP) with USP36 (Flag). YAP binded to USP36 at its WW domain. (Top panel) Schematic diagram of vectors expressing Myc-tagged wild-type or serial deletion mutants of YAP. (Bottom panel) The WW domain of YAP interacted with USP36. F Representative immunoblots to show the interaction between YAP and WT or truncated USP36 as indicated assessed by immunoprecipitation (IP) with YAP (anti-Myc). USP36 binded to YAP at its UPS domain. (Top panel) Schematic diagram of vectors expressing Flag-tagged wild-type or serial deletion mutants of USP. (Bottom panel) The USP domain of USP36 interacted with YAP. G, H ECA109 cells were transfected with vector, WT, or truncated USP36 and then subjected to immunoblotting to examine YAP protein level or qPCR to examine YAP target gene expression. The USP domain was essential for USP36 to maintain YAP protein levels and YAP target gene expression. I, J ECA109 cells were transfected with vector, WT, or USP36 mutant(C131A) and then subjected to immunoblotting to examine YAP protein level or qPCR to examine YAP target gene expression. The C131A mutation of USP36 impaired its ubiquitination activity and reduced the ability of USP36 to maintain YAP protein levels and YAP target gene expression in ECA109 cells. **P < 0.01, ***P < 0.001.
Source publication
Esophageal squamous carcinoma (ESCC) is the major subtype of esophageal cancer in China, accounting for 90% of cases. Recent studies revealed that abnormalities in the Hippo/YAP axis are pervasive in ESCC and are recognized as the important driver of ESCC progression. Since the activity of Hippo signaling is controlled by phosphorylation cascade, i...
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... by inhibiting the K48-linked polyubiquitination of YAP Since USP36 modulates the Hippo/YAP axis and ESCC progression, we further investigated the potential mechanism. Immunostaining indicated that both the YAP protein and USP36 protein were enriched in the nucleus, while USP36 depletion significantly reduced YAP staining signals in the nucleus (Fig. 5A), this conclusion was observed by the western blot of the distribution of YAP in nucleo-cytoplasmic extracts. (Fig. S3N). The endogenous immunoprecipitation assay indicated that USP36 could combine with YAP in ESCC cells (Fig. 5B). Since YAP degradation relies on the effect of the proteasome, we utilized the proteasome inhibitor MG132. ...
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... protein were enriched in the nucleus, while USP36 depletion significantly reduced YAP staining signals in the nucleus (Fig. 5A), this conclusion was observed by the western blot of the distribution of YAP in nucleo-cytoplasmic extracts. (Fig. S3N). The endogenous immunoprecipitation assay indicated that USP36 could combine with YAP in ESCC cells (Fig. 5B). Since YAP degradation relies on the effect of the proteasome, we utilized the proteasome inhibitor MG132. USP36 depletion decreased YAP protein levels, while MG132 treatment diminished this decrease in YAP protein levels (Fig. 5C). The protein half-life assay demonstrated that USP36 reduction impaired YAP protein stability in ESCC ...
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... (Fig. S3N). The endogenous immunoprecipitation assay indicated that USP36 could combine with YAP in ESCC cells (Fig. 5B). Since YAP degradation relies on the effect of the proteasome, we utilized the proteasome inhibitor MG132. USP36 depletion decreased YAP protein levels, while MG132 treatment diminished this decrease in YAP protein levels (Fig. 5C). The protein half-life assay demonstrated that USP36 reduction impaired YAP protein stability in ESCC cells, which wasn't observed in TAZ protein (Fig. 5D) (Fig. 5E, F). We made deletion constructs targeting these domains for interaction analysis, which revealed that the WW domain was necessary for the binding of YAP to USP36, while ...
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... on the effect of the proteasome, we utilized the proteasome inhibitor MG132. USP36 depletion decreased YAP protein levels, while MG132 treatment diminished this decrease in YAP protein levels (Fig. 5C). The protein half-life assay demonstrated that USP36 reduction impaired YAP protein stability in ESCC cells, which wasn't observed in TAZ protein (Fig. 5D) (Fig. 5E, F). We made deletion constructs targeting these domains for interaction analysis, which revealed that the WW domain was necessary for the binding of YAP to USP36, while the USP domain was required for the association of USP36 with YAP (Fig. 5E, F). We further overexpressed the deletion Fig. 3 USP36 depletion inhibited ESCC ...
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... of the proteasome, we utilized the proteasome inhibitor MG132. USP36 depletion decreased YAP protein levels, while MG132 treatment diminished this decrease in YAP protein levels (Fig. 5C). The protein half-life assay demonstrated that USP36 reduction impaired YAP protein stability in ESCC cells, which wasn't observed in TAZ protein (Fig. 5D) (Fig. 5E, F). We made deletion constructs targeting these domains for interaction analysis, which revealed that the WW domain was necessary for the binding of YAP to USP36, while the USP domain was required for the association of USP36 with YAP (Fig. 5E, F). We further overexpressed the deletion Fig. 3 USP36 depletion inhibited ESCC cell ...
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... reduction impaired YAP protein stability in ESCC cells, which wasn't observed in TAZ protein (Fig. 5D) (Fig. 5E, F). We made deletion constructs targeting these domains for interaction analysis, which revealed that the WW domain was necessary for the binding of YAP to USP36, while the USP domain was required for the association of USP36 with YAP (Fig. 5E, F). We further overexpressed the deletion Fig. 3 USP36 depletion inhibited ESCC cell progression via Hippo/YAP signaling. A Cell viability was determined by CCK8 assay in ESCC cell lines transfected with siControl or two independent USP36 siRNA. CCK-8 assay showed that USP36 depletion inhibited the proliferation of ECA109 and KYSE150 ...
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... of USP36 to determine their impact on the Hippo/YAP axis. The western blotting data showed that full-length USP36 and the USP domain could stabilize the YAP protein (Fig. 5G). The qPCR data showed that the USP domain was necessary and sufficient to increase endogenous Hippo target gene expression (Fig. 5H). Since previous studies indicated that cysteine 131 is critical for USP36 to exert its deubiquitinase function [29], we made a mutation variant and transfected it into HEK293 cells. The western blotting ...
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... of USP36 to determine their impact on the Hippo/YAP axis. The western blotting data showed that full-length USP36 and the USP domain could stabilize the YAP protein (Fig. 5G). The qPCR data showed that the USP domain was necessary and sufficient to increase endogenous Hippo target gene expression (Fig. 5H). Since previous studies indicated that cysteine 131 is critical for USP36 to exert its deubiquitinase function [29], we made a mutation variant and transfected it into HEK293 cells. The western blotting data indicated that the C131 site of USP36 was required for its regulation of YAP protein expression (Fig. 5I). The qPCR data ...
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... Hippo target gene expression (Fig. 5H). Since previous studies indicated that cysteine 131 is critical for USP36 to exert its deubiquitinase function [29], we made a mutation variant and transfected it into HEK293 cells. The western blotting data indicated that the C131 site of USP36 was required for its regulation of YAP protein expression (Fig. 5I). The qPCR data indicated that the C131A mutant form abolished the effect of USP36 on Hippo target gene expression (Fig. 5J). Since USP36 belongs to the deubiquitinase family, we subsequently assessed the role of USP36 in YAP ubiquitination. The ubiquitination assay indicated that USP36 depletion could significantly increase the total ...
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... its deubiquitinase function [29], we made a mutation variant and transfected it into HEK293 cells. The western blotting data indicated that the C131 site of USP36 was required for its regulation of YAP protein expression (Fig. 5I). The qPCR data indicated that the C131A mutant form abolished the effect of USP36 on Hippo target gene expression (Fig. 5J). Since USP36 belongs to the deubiquitinase family, we subsequently assessed the role of USP36 in YAP ubiquitination. The ubiquitination assay indicated that USP36 depletion could significantly increase the total YAP ubiquitination level, but USP36 overexpression could decrease the total YAP ubiquitination level (Fig. 6A, B). In ...
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Citations
... Imbalanced ubiquitination induced by dysregulated E3 or DUBs impairs many cellular physiological processes, such as DNA damage repair, cell cycle, gene expression, and signaling transduction, which are closely associated with the initiation and progression of human cancers [10][11][12]. Our previous study clarified that ubiquitinspecific peptidase 36 (USP36) accelerates ESCC proliferation and metastasis through stabilizing YAP protein [13]. However, further studies are still needed to further elucidate the ambiguous mechanisms underlying dysregulated ubiquitination in ESCC progression. ...
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.
... It stabilizes YAP proteins by inhibiting the polyubiquitination of the K48 chain of YAP proteins ( Figure 2(A)). Thus, targeting Hippo/YAP signaling may be a viable therapeutic approach for patients with ESCC [31]. ...
... By modulating USP36 levels or activity, it may be possible to influence Hippo/YAP signaling and potentially impede the progression of ESCC. However, further research is imperative in order to fully comprehend the complex molecular mechanisms underlying this interaction and to determine the feasibility and effectiveness of targeting USP36 as a therapeutic strategy for ESCC [31]. ...
... In humans, USP36 was found to be highly expressed in the brain, endocrine tissues, liver, gallbladder, and female tissues ( Figure 4). USP36 is upregulated in multiple cancers including esophageal carcinoma [31], glioblastoma [37], HCC [73], colorectal cancer [74], breast cancer [75], and T cell lymphoma [76]. Although the analysis of Kaplan-Meier survival rate and log-rank test revealed a correlation between USP36 levels and overall survival (OS) in liver hepatocellular carcinoma (LIHC) (Figure 5), there are lots of studies verifying that USP36 is implicated in the progression and drug resistance of many other cancers. ...
The balance between ubiquitination and deubiquitination is instrumental in the regulation of protein stability and maintenance of cellular homeostasis. The deubiquitinating enzyme, ubiquitin-specific protease 36 (USP36), a member of the USP family, plays a crucial role in this dynamic equilibrium by hydrolyzing and removing ubiquitin chains from target proteins and facilitating their proteasome-dependent degradation. The multifaceted functions of USP36 have been implicated in various disease processes, including cancer, infections, and inflammation, via the modulation of numerous cellular events, including gene transcription regulation, cell cycle regulation, immune responses, signal transduction, tumor growth, and inflammatory processes. The objective of this review is to provide a comprehensive summary of the current state of research on the roles of USP36 in different pathological conditions. By synthesizing the findings from previous studies, we have aimed to increase our understanding of the mechanisms underlying these diseases and identify potential therapeutic targets for their treatment.
... Recent findings have revealed the significance of ubiquitin modification in modulating YAP function and Hippo kinase activity [17]. Specifically, a group of E3 ubiquitin ligases and deubiquitinases (DUBs) delicately control the protein stability of YAP, with selective inhibition of certain DUBs having the potential to reverse this balance and facilitate YAP degradation [18][19][20][21]. Our study seeks to identify the critical deubiquitinase that targets YAP stability. ...
... It has been demonstrated that some deubiquitinases can modulate the stability of YAP and promote the progression of gastric cancer in several recent studies. Nevertheless, these studies are based on the siRNA screening in HEK293 cells [18,21]. In our study, we did the siRNA screening from one typical gastric cancer cell line AGS cells, and identify crucial deubiquitinases that affect the progression of gastric cancer and are potentially significant for treatment. ...
The dysregulation of Hippo signaling is a crucial factor driving the progression of gastric cancer, making the targeting of the Hippo pathway a promising therapeutic strategy. However, effective drugs targeting the Hippo/YAP axis remain unavailable. Thus, identifying potential therapeutic targets and mechanisms that inhibit the activity of the Hippo/YAP axis in gastric cancer is of paramount importance. The ubiquitination modification of the Hippo/YAP pathway plays a significant role in signaling transduction and cancer progression. In an effort to shed light on effective therapeutic targets, we conducted a screening using a deubiquitinase small interfering RNA library, leading to the identification of USP12 as an important deubiquitinase in the context of Hippo/YAP axis and the progression of gastric cancer. Our bioinformatic analysis further demonstrated a correlation between USP12 and poor survival, as well as a positive association with classical YAP target genes in gastric cancer samples. Notably, USP12 depletion was found to inhibit gastric cancer progression via the Hippo/YAP axis, whereas USP12 overexpression exhibited the opposite effect, promoting gastric cancer growth and enhancing YAP activity. Further studies through immuno-staining and immuno-precipitation assays indicated the nuclear localization of USP12 and its association with YAP to enhance YAP stability. Specifically, our findings revealed that USP12 could inhibit K48-linked poly-ubiquitination of YAP, predominantly at the K315 site. As a result, we have identified a novel regulatory mechanism involving USP12 and Hippo signaling in the progression of gastric cancer, with the potential for blockade of USP12 to materialize as a promising strategy for combating gastric cancer.
... Esophageal cancer is the seventh most frequently diagnosed cancer and the sixth leading cause of cancer death in the world, with an estimated 604,000 new cases and 544,000 deaths in 2020, according to the GLOBOCAN estimates of cancer incidence and mortality produced by the International Agency for Research on Cancer [1]. Esophageal squamous cell carcinoma (ESCC) is the most common histologic subtype of esophageal cancer in China, accounting for 90% of cases [2]. Although considerable advances achieved in diagnosis and multimodality therapies, the patients with ESCC often have a very poor prognosis, with the 5-year overall survival rate less than 15%, mainly due to the high incidences of tumor metastasis [3]. ...
Background
Metastasis is still a major cause of poor pathological outcome and prognosis in esophageal squamous cell carcinoma (ESCC) patients. NUAK1 has been reported highly expressed in many human cancers and is associated with the poor prognosis of cancer patients. However, the role of NUAK1 and its underlying signaling mechanism in ESCC metastasis remain unclear.
Methods
Expression of NUAK1 in ESCC was detected by real-time quantitative RT-PCR (qRT-PCR), Western blotting and immunohistochemical staining. MTT, colony formation, wound-healing and transwell assays were used to determine the role NUAK1 in vitro. Metastasis was evaluated by use of an experimental pulmonary metastasis model in BALB/c-nu/nu mice. The mechanisms were assessed by using coimmunoprecipitation, immunofluorescence and dual-luciferase reporter gene experiments.
Results
NUAK1 was highly expressed in ESCC tissues compared with the adjacent normal esophageal epithelial tissues. Moreover, the elevated expression of NUAK1 positively correlated with tumor invasion depth, lymph node metastasis, pathological TNM stage, and poor survival in ESCC patients. Further experiments showed that NUAK1 overexpression did not change the cell viability and colony formation of ESCC cells, while remarkably promoted the migration and invasion in vitro and experimental pulmonary metastasis in vivo. Mechanistically, NUAK1 enhanced the transcription level of Slug, which enhanced the migratory and invasive capability of ESCC cells. Consistently, silencing Slug almost completely diminished the migration and invasion of NUAK1-overexpressing ESCC cells. Further studies demonstrated that NUAK1 upregulated the transcription activity of Slug through activating the JNK/c-Jun pathway.
Conclusion
These results demonstrated that NUAK1 promoted the metastasis of ESCC cells through activating JNK/c-Jun/Slug signaling, indicating NUAK1 is a promising therapeutic target for metastatic ESCC.
... Esophageal cancer represents a prevalent and lethal form of cancer, histologically classified into two subtypes: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC) (52). ...
... The Hippo/YAP signaling pathway plays an important role in HNSCC [103]. USP36 promotes proliferation and invasion of esophageal squamous cell carcinoma by deubiquitination of YAP [104]. USP36 may also play an important role in head and neck cancer. ...
Ubiquitination and deubiquitination are two popular ways for the post-translational modification of proteins. These two modifications affect intracellular localization, stability, and function of target proteins. The process of deubiquitination is involved in histone modification, cell cycle regulation, cell differentiation, apoptosis, endocytosis, autophagy, and DNA repair after damage. Moreover, it is involved in the processes of carcinogenesis and cancer development. In this review, we discuss these issues in understanding deubiquitinating enzyme (DUB) function in head and neck squamous cell carcinoma (HNSCC), and their potential therapeutic strategies for HNSCC patients are also discussed.
Yes-associated protein 1 (YAP1) mediated hippo pathway has attracted several research importance in various types of malignancies. The hampered Hippo-YAP1 axis in bladder cancer (BC) was identified as a major driver of BC progression and oncogenesis. The activity of Hippo pathway is controlled via regulation of phosphorylation cascade of MST1/2-LATS1/2-YAP1, while other modification as ubiquitination of hippo pathway protein also mediated the activity of Hippo pathway through the co-regulation of E3 ligases and deubiquitinases. In this study, we identified USP20 as a Hippo/YAP1 pathway activity related deubiquitinase through combined analysis of siRNA screening and deubiquitinase over-expression. Further analysis identified that USP20 directed regulated the YAP1 expression and target gene of YAP1, connective tissue growth factor (CTGF) as well as cysteine-rich angiogenic inducer 61 (CYR61). Tissue microarray assay confirmed that USP20 was elevated in tumor tissue and correlated with YAP1 expression. Mechanism study identified that USP20 directly interacted with YAP1 protein and promoted the stability of YAP1 protein through hampering the K48-linked poly-ubiquitination. Our findings in this study revealed that USP20 as a novel deubiquitinase in regulating the Hippo-YAP1 pathway in BC.
