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USP39 increases proliferation and migration of HCC through the wnt signaling pathway. A The expression of USP39 mRNA was analyzed by RT-PCR in PLC/PRF/5 and SK-hep-1 cells. B Western blot confirmed the expression of USP39 after cells were knocked down and overexpressed C The effects of USP39 on the proliferation of HCC cells (PLC/PRF/5 and SK-hep-1 cells) were examined by MTT assay in the presence or absence of ICG-001. D Colony formation assays were performed in HCC PLC/PRF/5 and SK-hep-1 cells (The colony numbers were normalized to controls and expressed as a percentage). E Wound-healing assays were performed to determine the effect of USP39 on HCC cells (PLC/PRF/5 and SK-hep-1 cells) migration in the presence or absence of ICG-001. All the data are representative of at least three independent experiments and presented as the means ± SD. (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 v.s. control by one-way ANOVA /two-way ANOVA or Student's t-test).
Source publication
Ubiquitin-specific protease 39(USP39) plays an important role in modulating pre-mRNA splicing and ubiquitin-proteasome dependent proteolysis as a member of conserved deubiquitylation family. Accumulating evidences prove that USP39 participates in the development of hepatocellular carcinoma (HCC). However, little is known about the mechanism especia...
Contexts in source publication
Context 1
... whether USP39 regulate β-catenin expression, cell proliferation, cell cycle, and tumor growth in HCC cells, knockdown and overexpression of USP39, β-catenin inhibitor ICG-001 were performed in HCC cell lines (SK-hep-1 and PLC/PRF/5). The efficiency of knockdown and complementation of USP39 was confirmed by quantitative real-time PCR (qRT-PCR) (Fig. 1A) and western blotting (WB) (Fig. 1B) respectively. HCC cell proliferation ability was assessed by MTT and colony formation assays. MTT results indicated that knockdown of USP39 reduced the proliferation of HCC cells compared with the control group, while replenishment of USP39 restored the proliferation of HCC cells. Nevertheless, USP39 ...
Context 2
... expression, cell proliferation, cell cycle, and tumor growth in HCC cells, knockdown and overexpression of USP39, β-catenin inhibitor ICG-001 were performed in HCC cell lines (SK-hep-1 and PLC/PRF/5). The efficiency of knockdown and complementation of USP39 was confirmed by quantitative real-time PCR (qRT-PCR) (Fig. 1A) and western blotting (WB) (Fig. 1B) respectively. HCC cell proliferation ability was assessed by MTT and colony formation assays. MTT results indicated that knockdown of USP39 reduced the proliferation of HCC cells compared with the control group, while replenishment of USP39 restored the proliferation of HCC cells. Nevertheless, USP39 replenishment failed to restore HCC ...
Context 3
... and colony formation assays. MTT results indicated that knockdown of USP39 reduced the proliferation of HCC cells compared with the control group, while replenishment of USP39 restored the proliferation of HCC cells. Nevertheless, USP39 replenishment failed to restore HCC cell proliferation when the wnt/β-catenin pathway was blocked by ICG-001 (Fig. 1C). Colony formation assays demonstrated that USP39 knockdown remarkably restrained colony formation ability of HCC cells, and this effect was recovered by replenishment of USP39. However, colony formation ability was inhibited again when treated with ICG-001 ( Fig. 1D). In addition, wound-healing assays showed that silencing USP39 ...
Context 4
... HCC cell proliferation when the wnt/β-catenin pathway was blocked by ICG-001 (Fig. 1C). Colony formation assays demonstrated that USP39 knockdown remarkably restrained colony formation ability of HCC cells, and this effect was recovered by replenishment of USP39. However, colony formation ability was inhibited again when treated with ICG-001 ( Fig. 1D). In addition, wound-healing assays showed that silencing USP39 significantly decreased the migration of HCC cells, replenishing USP39 rescued the migration ability impaired by silencing USP39, and the Wnt pathway inhibitor ICG-001 reverted this phenomenon. (Fig. 1E). These data demonstrated that USP39 increases the proliferation and ...
Context 5
... However, colony formation ability was inhibited again when treated with ICG-001 ( Fig. 1D). In addition, wound-healing assays showed that silencing USP39 significantly decreased the migration of HCC cells, replenishing USP39 rescued the migration ability impaired by silencing USP39, and the Wnt pathway inhibitor ICG-001 reverted this phenomenon. (Fig. 1E). These data demonstrated that USP39 increases the proliferation and migration of HCC cells through the Wnt/β-catenin ...
Context 6
... proteins separated from cytoplasmic and nuclear by western blotting showed that, knockdown of USP39 induced a reduction of nuclear β-catenin (Fig. 2F), whereas USP39 overexpression increased β-catenin level in the nucleus (Fig. 2G). Moreover, USP39 exerted its oncogenic properties by activating the relative genes of Wnt signals, including Axin, GSK3β, c-Myc and Cyclin D1 (Supplementary Fig. 1). These data suggested that USP39 affects the activity of the Wnt/β-catenin pathway. ...
Citations
... For instance, USP5 has been identified as a DUB for LSH, and its activity is linked to the ferroptosis suppression, thereby facilitating the tumorigenesis of HCC [11]. USP39 promotes HCC cell proliferation and migration by deubiquitinating β-catenin, activating WNT/β-catenin signaling pathway [12]. These findings underscore the complex role of deubiquitination in HCC, with various DUBs contributing to the progression of the disease by stabilizing oncoproteins or inhibiting cell death pathways. ...
STAM Binding Protein Like 1 (STAMBPL1), functions as a deubiquitinase (DUB) and plays a significant role in various types of cancers. However, its effect as a DUB participating in the HCC tumorigenesis and progression still unknown. In the study, the upregulation and strong prognosis value of STAMBPL1 were identified in HCC patients. Functionally, STAMBPL1 significantly promoted HCC cells proliferation and metastasis, and it interacts with TRAF2 and stabilize it via the deubiquitination at the K63 residue. The TRAF2 upregulation stabilized by STAMBPL1 overexpression transfers of P65 protein into the nucleus and activates the WNT/PI3K/ NF-kb signaling pathway. The 251–436 sites of STAMBPL1 particularly interact with the 294–496 sites of TRAF2, thereby exerting the function of DUB and removing the ubiquitin molecules attached to TRAF2. Our research unveiled a new function of STAMBPL1 in mediating TRAF2 deubiquitination and stabilization, thereby activating the WNT/PI3K/NF-kb signaling pathway, suggesting its potential as a novel biomarker and therapeutic target for HCC.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13062-024-00460-7.
... Twenty pairs of HCC and corresponding adjacent tissues were obtained from the Department of Pathology of Zhejiang Provincial People's Hospital. IHC procedures were carried out as previously reported [20]. Two experienced pathologists scored the staining intensity and percentage Fig. 1 USP40 is elevated in HCC and predicts poor prognosis. ...
Background
Hepatocellular carcinoma (HCC) is a prevalent malignant tumor that poses a major threat to people’s lives and health. Previous studies have found that multiple deubiquitinating enzymes are involved in the pathogenesis of HCC. The purpose of this work was to elucidate the function and mechanism of the deubiquitinating enzyme USP40 in HCC progression.
Methods
The expression of USP40 in human HCC tissues and HCC cell lines was investigated using RT-qPCR, western blotting and immunohistochemistry (IHC). Both in vitro and in vivo experiments were conducted to determine the crucial role of USP40 in HCC progression. The interaction between USP40 and Claudin1 was identified by immunofluorescence, co-immunoprecipitation and ubiquitination assays.
Results
We discovered that USP40 is elevated in HCC tissues and predicts poor prognosis in HCC patients. USP40 knockdown inhibits HCC cell proliferation, migration and stemness, whereas USP40 overexpression shows the opposite impact. Furthermore, we confirmed that Claudin1 is a downstream gene of USP40. Mechanistically, USP40 interacts with Claudin1 and inhibits its polyubiquitination to stabilize Claudin1 protein.
Conclusions
Our study reveals that USP40 enhances HCC malignant development by deubiquitinating and stabilizing Claudin1, suggesting that targeting USP40 may be a novel approach for HCC therapy.
... As a result, the deubiquitinase-independent functions of USP39, particularly its role as a splicing factor in pre-mRNA splicing, have been extensively studied [27,[33][34][35][36][37]. However, several studies, including our own, have provided evidence that USP39 is fully active and can deubiquitinate and stabilize its substrate proteins, such as β-catenin, SP1, IkBα (a negative regulator of NF-κB), CHK2, STAT1, NAT10, and cyclin B1, regulating various biological functions [38][39][40][41][42][43][44]. These findings suggest that the cysteine residue responsible for enzymatic activity may not be located at position 234 but at another site within the USP domain. ...
ETS2 is a member of the ETS family of transcription factors and has been implicated in the regulation of cell proliferation, differentiation, apoptosis, and tumorigenesis. The aberrant activation of ETS2 is associated with various human cancers, highlighting its importance as a therapeutic target. Understanding the regulatory mechanisms and interacting partners of ETS2 is crucial for elucidating its precise role in cellular processes and developing novel strategies to modulate its activity. In this study, we conducted binding assays using a human deubiquitinase (DUB) library and identified USP39 as a novel ETS2-binding DUB. USP39 interacts with ETS2 through their respective amino-terminal regions, and the zinc finger and PNT domains are not required for this binding. USP39 deubiquitinates ETS2 without affecting its protein stability. Interestingly, however, USP39 significantly suppresses the transcriptional activity of ETS2. Furthermore, we demonstrated that USP39 leads to a reduction in the nuclear localization of ETS2. Our findings provide valuable insights into the intricate regulatory mechanisms governing ETS2 function. Understanding the interplay between USP39 and ETS2 may have implications for therapeutic interventions targeting ETS2-related diseases, including cancer, where the dysregulation of ETS2 is frequently observed.
... Aberrant functioning of signal transduction, regulatory and metabolic cascades significantly contribute to malignant transformation [1], therefore, many ways of pharmacological correction of molecular components of cascades are being under development [2][3][4]. In recent years, significant progress has been made in the identification of proteins, which are directly involved in oncogenic cascades and modulate them by means of protein-protein interactions (PPIs) and post-transcriptional regulation [5][6][7][8]. Thus, the exploration of a spectrum of novel cancer-associated proteins is a promising strategy in anticancer drug discovery. ...
Citation: Ershov, P.; Yablokov, E.; Mezentsev, Y.; Ivanov, A. Uncharacterized Proteins CxORFx: Subinteractome Analysis and Abstract: Functions of about 10% of all the proteins and their associations with diseases are poorly annotated or not annotated at all. Among these proteins, there is a group of uncharacterized chromosome-specific open-reading frame genes (CxORFx) from the 'Tdark' category. The aim of the work was to reveal associations of CxORFx gene expression and ORF proteins' subinteractomes with cancer-driven cellular processes and molecular pathways. We performed systems biology and bioinformatic analysis of 219 differentially expressed CxORFx genes in cancers, an estimation of prognostic significance of novel transcriptomic signatures and analysis of subinteractome composition using several web servers (GEPIA2, KMplotter, ROC-plotter, TIMER, cBioPortal, DepMap, EnrichR, PepPSy, cProSite, WebGestalt, CancerGeneNet, PathwAX II and FunCoup). The subinteractome of each ORF protein was revealed using ten different data sources on physical protein-protein interactions (PPIs) to obtain representative datasets for the exploration of possible cellular functions of ORF proteins through a spectrum of neighboring annotated protein partners. A total of 42 out of 219 presumably cancer-associated ORF proteins and 30 cancer-dependent binary PPIs were found. Additionally, a bibliometric analysis of 204 publications allowed us to retrieve biomedical terms related to ORF genes. In spite of recent progress in functional studies of ORF genes, the current investigations aim at finding out the prognostic value of CxORFx expression patterns in cancers. The results obtained expand the understanding of the possible functions of the poorly annotated CxORFx in the cancer context.
... Wnt/β-catenin target genes regulate cell proliferation and apoptosis, thereby mediating initiation and progression in many cancers [38] . Emerging studies have shown that Wnt/β-catenin signaling pathway plays an important role in EMT and tumor progression and β-catenin accumulation is closely associated with HCC progression and poor prognosis [39] . β-catenin destruction complex and β-catenin degradation regulated by the ubiquitin-proteasome pathway are the core mechanisms regulating intracellular βcatenin levels [40] . ...
Endometrial cancer (EC) is a common malignant tumor of female reproductive system. It seriously affects women's health. The mechanical characteristics of EC have not been paid noticed. However, the mechanical characteristics of different types of EC are diverse. These characteristics will greatly influence the development and prognosis of cancer cells. In this study, we explored traction force in primary EC cells and EC cell lines for the first time. It was found that the traction force of primary EC cells or cell lines with metastatic capability was lower. Various clinicopathological features of EC patients are significantly associated with traction force. In addition, we also confirmed that SLC8A1 is the main molecule which had an effect on traction force. The stiffness of extracellular matrix (ECM) had an influence on its downstream cytoskeleton expression through SLC8A1, thus changing the traction force and metastatic capacity of EC cells. The mechanism behind the SLC8A1 may be related to Wnt-β- catenin pathway and ion-channel activity. SLC8A1 can significantly regulate the downstream cytoskeleton F-actin and promote EC cell metastasis. Therefore, in this study, we found that SLC8A1 is an important molecule that affects the mechanical characteristics of endometrial cancer. The molecular mechanism is through changing the cytoskeleton by regulating Wnt- β- catenin pathway.
Proteomics, together with genomics and other large-scale omics-based approaches, provides extensive data that can explain the molecular mechanisms of cancer. One particular area, cancer biomarkers, is an everlasting “hot topic” in proteomics studies. Several papers in this Special Issue (SI) deal with the study of cancer biomarkers. Recently, scientists have started to pay more attention not only to quantitative changes in protein levels, but also to monitoring variation at the level of specific protein forms—proteoforms. Different methods, including mass spectrometry and immunology, can be applied to achieve this aim. The other papers of the SI, not directly involved in cancer biomarkers, offer insight into general aspects of the cellular proteome organization and protein-protein interactions.
Stanislav Naryzhny
Editor
Signaling pathways in hepatocellular carcinoma are primarily mediated by the phosphorylation and ubiquitination of post-translational proteins. In mammalian cells, ubiquitin-specific proteases (USPs) account for the majority of protein deubiquitination activities. In addition to transcriptional and post-translational regulation, ubiquitination plays an important role in the regulation of key proteins. There is a possibility that altered biological processes may lead to serious human diseases, including cancer. Recent studies have revealed the role of USPs in hepatocellular carcinoma tumorigenesis. The purpose of this review is to summarize the involvement of this class of enzymes in the regulation of cell signaling in hepatocellular carcinoma and the therapeutic development of inhibitors that target USPs, which may lead to novel therapies to treat hepatocellular carcinoma.
Recent studies have reported a correlation between ubiquitination or deubiquitination and cancer development. But mechanisms underlying the roles of genes associated with E3 ubiquitin ligases and deubiquitinating enzymes (DUB) in liver cancer remain to be explored. We analyzed and screened differentially expressed genes related to E3 ubiquitin ligases and DUB in liver cancer on the basis of public databases. Cluster analysis was utilized to classify liver cancer samples into different subtypes. Survival analysis, immune analysis, and pathway enrichment analysis were performed on the subtypes. We constructed a protein-protein interaction network using STRING to screen hub genes. Finally, we used the Connectivity Map (CMap) database to predict targeted small molecules. The results show that a total of 139 differentially expressed E3/DUB genes in liver cancer were screened. Then, liver cancer was classified into two subtypes, cluster 1 and cluster 2, based on E3-related and DUB-related genes. Patients in cluster 1 had higher survival rates and immune levels than those in cluster 2. Four hub genes (RPSA, RPS5, RPL30, and RPL8) significantly affecting the survival of the two subtypes of liver cancer patients were identified based on cluster 1 and cluster 2. Finally, the CMap database predicted that small-molecule drugs including probenecid, dexamethasone, and etomidate may improve the prognosis of liver cancer patients. These findings may offer a reference for risk stratification studies and drug development in liver cancer.
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