Ubiquitin-conjugating Enzyme E2T Research Articles

Backbone 1H, 15N and 13C resonance assignments for an E2 ubiquitin conjugating enzyme-UBE2T.

Ubiquitin-conjugating enzyme E2 T (UBE2T) plays important roles in ubiquitination of proteins through participation in transferring ubiquitin to its substrate. Due to its importance in protein modifications, UBE2T associates with diverse diseases and serves as an important target for drug discovery and development. The crystal structure of UBE2T has been determined and the structure reveals the lack of a druggable pocket for binding to small molecules for clinical applications. Despite the challenge, effort has been made to develop UBE2T inhibitors. We obtained UBE2T constructs with and without the C-terminal region which is flexible in solution. Herein, we report the backbone resonance assignments for human UBE2T without the C-terminal region. The backbone dynamics of UBE2T was also explored. The available assignments will be helpful for hit identification, determining ligand binding site and understanding the mechanism of action of UBE2T inhibitors.

UBE2T Promotes Temozolomide Resistance of Glioblastoma Through Regulating the Wnt/β-Catenin Signaling Pathway.

Patients with glioblastoma (GBM) have poor prognosis and limited therapeutic options, largely because of chemoresistance to temozolomide (TMZ) treatment. Ubiquitin conjugating enzyme E2 T (UBE2T) plays a key role in regulating the malignancy of various tumors, including GBM; however, its role in TMZ resistance of GBM has not been elucidated. The purpose of this study was to clarify the role of UBE2T in mediating TMZ resistance and investigate the specific underlying mechanism. Western blotting was used to detect the protein levels of UBE2T and Wnt/β-catenin-related factors. CCK-8, flow cytometry, and colony formation assays were used to examine the effect of UBE2T on TMZ resistance. Wnt/β-catenin signaling pathway activation was inhibited using XAV-939, and a xenograft mouse model was generated to clarify the function of TMZ in vivo. UBE2T knockdown sensitized GBM cells to TMZ treatment, whereas UBE2T overexpression promoted TMZ resistance. The specific UBE2T inhibitor, M435-1279, increased the sensitivity of GBM cells to TMZ. Mechanistically, our results demonstrated that UBE2T induces β-catenin nuclear translocation and increases the protein levels of downstream molecules, including survivin and c-Myc. Inhibition of Wnt/β-catenin signaling using XAV-939 blocked TMZ resistance due to UBE2T overexpression in GBM cells. In addition, UBE2T was shown to facilitate TMZ resistance by inducing Wnt/β-catenin signaling pathway activation in a mouse xenograft model. Combined treatment with TMZ and UBE2T inhibitor achieved superior tumor growth suppression relative to TMZ treatment alone. Our data reveal a novel role of UBE2T in mediating TMZ resistance of GBM cells via regulating Wnt/β-catenin signaling. These findings indicate that targeting UBE2T has promising potential to overcome TMZ resistance of GBM.

Targeting UBE2T Potentiates Gemcitabine Efficacy in Pancreatic Cancer by Regulating Pyrimidine Metabolism and Replication Stress

Although small patient subsets benefit from current targeted strategies or immunotherapy, gemcitabine remains the first-line drug for pancreatic cancer (PC) treatment. However, gemcitabine resistance is widespread and compromises long-term survival. Here, we identified ubiquitin-conjugating enzyme E2T (UBE2T) as a potential therapeutic target to combat gemcitabine resistance in PC. Proteomics and metabolomics were combined to examine the effect of UBE2T on pyrimidine metabolism remodeling. Spontaneous PC mice (LSL-KrasG12D/+, LSL-Trp53R172H/+, Pdx1-Cre; KPC) with Ube2t-conditional knockout, organoids, and large-scale clinical samples were used to determine the effect of UBE2T on gemcitabine efficacy. Organoids, patient-derived xenografts (PDX), and KPC mice were used to examine the efficacy of the combination of a UBE2T inhibitor and gemcitabine. Spontaneous PC mice with Ube2t deletion had a marked survival advantage after gemcitabine treatment, and UBE2T levels were positively correlated with gemcitabine resistance in clinical patients. Mechanistically, UBE2T catalyzes really interesting new gene 1 (RING1)-mediated ubiquitination of p53 and relieves the transcriptional repression of ribonucleotide reductase subunits M1 and M2, resulting in unrestrained pyrimidine biosynthesis and alleviation of replication stress. Additionally, high-throughput compound library screening using organoids identified pentagalloylglucose (PGG) as a potent UBE2T inhibitor and gemcitabine sensitizer. The combination of gemcitabine and PGG diminished tumor growth in PDX models and prolonged long-term survival in spontaneous PC mice. Collectively, UBE2T-mediated p53 degradation confers PC gemcitabine resistance by promoting pyrimidine biosynthesis and alleviating replication stress. This study offers an opportunity to improve PC survival by targeting UBE2T and develop a promising gemcitabine sensitizer in clinical translation setting.

Diverse roles of UBE2T in cancer (Review).

As a leading cause of mortalities worldwide, cancer results from accumulation of both genetic and epigenetic alterations. Disruption of epigenetic regulation in cancer, particularly aberrant ubiquitination, has drawn increasing interest in recent years. The present study aimed to review the roles of ubiquitin‑conjugating enzyme E2 T (UBE2T) and its associated pathways in the pathogenesis of pan‑cancer, and the development of small‑molecule modulators to regulate ubiquitination for treatment strategies. The current study comprehensively investigated the expression landscape and functional significance of UBE2T, as well as its correlation with cancer cell sensitivity to chemotherapy/radiotherapy. Multiple levels of evidence suggested that aberrant UBE2T played important roles in pan‑cancer. Information was collected from 16 clinical trials on ubiquitin enzymes, and it was found that these molecules had an important role in the ubiquitin‑proteasome system. Further studies are necessary to explore their feasibility and effectiveness as diagnostic and prognostic biomarkers, or as up/down‑stream and therapeutic targets for cancer treatment.

UBE2T promotes breast cancer tumor growth by suppressing DNA replication stress.

Breast cancer is a leading cause of cancer-related deaths among women, and current therapies benefit only a subset of these patients. Here, we show that ubiquitin-conjugating enzyme E2T (UBE2T) is overexpressed in patient-derived breast cancer samples, and UBE2T overexpression predicts poor prognosis. We demonstrate that the transcription factor AP-2 alpha (TFAP2A) is necessary for the overexpression of UBE2T in breast cancer cells, and UBE2T inhibition suppresses breast cancer tumor growth in cell culture and in mice. RNA sequencing analysis identified interferon alpha-inducible protein 6 (IFI6) as a key downstream mediator of UBE2T function in breast cancer cells. Consistently, UBE2T inhibition downregulated IFI6 expression, promoting DNA replication stress, cell cycle arrest, and apoptosis and suppressing breast cancer cell growth. Breast cancer cells with IFI6 inhibition displayed similar phenotypes as those with UBE2T inhibition, and ectopic IFI6 expression in UBE2T-knockdown breast cancer cells prevented DNA replication stress and apoptosis and partly restored breast cancer cell growth. Furthermore, UBE2T inhibition enhanced the growth-suppressive effects of DNA replication stress inducers. Taken together, our study identifies UBE2T as a facilitator of breast cancer tumor growth and provide a rationale for targeting UBE2T for breast cancer therapies.

Prognostic Value of UBE2T and Its Correlation with Immune Infiltrates in Lung Adenocarcinoma.

Non-small cell lung cancer has a subtype with a high morbidity and mortality rate called lung adenocarcinoma (LUAD). It is critical to locate reliable prognostic biomarkers for LUAD at this time. Ubiquitin-conjugating enzyme E2T (UBE2T) has been found in numerous malignancies; however, its expression level and potential functions in LUAD are not completely understood at this time. A differentially expressed gene (DEG) screening method was used to identify genes that were expressed differently in 516 samples from LUAD and 59 samples from TCGA datasets. Clinicopathological markers were correlated with UBE2T expression. Using the Kaplan–Meier plotter database, UBE2T was evaluated for its prognostic value in the context of LUAD. In order to examine the importance of independent prognostic factors, both univariable and multivariable Cox regression models were applied. TIMER and CIBERSORT were utilized in order to investigate the connection that exists between UBE2T expression and tumor-infiltrating immune cells. This study collected 578 DEGs in total, as follows: 171 genes were significantly increased, while 408 genes were significantly decreased. We identified 9 survival-related DEGs in LUAD, including ASF1B, CA9, CCNB2, CCNE1, RRM2, SAPCD2, TCN1, TPX2, and UBE2T. Our attention focused on UBE2T, which was highly expressed in LUAD. A correlation was also found between high UBE2T expression and gender, age, advanced clinical stage, and decreased overall survival. In addition, multivariate analysis demonstrated UBE2T expression to be a significant independent diagnostic factor for patients suffering from LUAD. UBE2T was positively correlated with resting T cell CD4+ memory, myeloid dendritic cell resting, mast cell activated, macrophage M2, and B cell plasma, whereas it was negatively correlated with resting T cell CD4+ memory, MDC resting, MDC activated, macrophage M2, and B cell plasma. Overall, high expression levels of UBE2T correlated with poor overall survival in patients with LUAD, and UBE2T was an independent predictor involved in immune infiltration of LUAD. These findings offer fresh perspectives that contribute to our comprehension of the evolution of LUAD.

UBE2T regulates FANCI monoubiquitination to promote NSCLC progression by activating EMT.

Fanconi anemia complementation group I (FANCI) is a critical protein for maintaining DNA stability. However, the exact role of FANCI in tumors remains to be elucidated. The present study aimed to explore the role and potential mechanism of action of FANCI in non-small cell lung cancer (NSCLC). To quantify the expression levels of FANCI and ubiquitin-conjugating enzyme E2T (UBE2T) in NSCLC tissues, reverse-transcription quantitative PCR and western blotting were employed. Cell Counting Kit-8, wound healing and Transwell assays along with flow cytometry analysis and tumor xenograft were used to investigate the biological effects of FANCI in NSCLC in vitro and in vivo. The binding of FANCI with UBE2T was confirmed using a co-immunoprecipitation assay. Epithelial-to-mesenchymal transition (EMT) protein markers were quantified via western blotting. The results showed that FANCI expression level was higher in NSCLC tumor tissues, compared with adjacent tissues. In A549 and H1299 cells, knockdown of FANCI inhibited cell proliferation, migration, invasion, cell cycle and EMT in vitro. Tumor growth was repressed in vitro, upon downregulation of FANCI expression. UBE2T was observed to directly bind to FANCI and regulate its monoubiquitination. Overexpression of UBE2T reversed the effects induced by FANCI knockdown in NSCLC cells. Furthermore, it was noted that FANCI interacted with WD repeat domain 48 (WDR48). Overexpression of WDR48 reversed the effects of FANCI on cell proliferation, migration and EMT. In conclusion, FANCI was identified to be a putative oncogene in NSCLC, wherein FANCI was monouniubiquitinated by UBE2T to regulate cell growth, migration and EMT through WDR48. The findings suggested that FANCI could be used as a prognostic biomarker and therapeutic target for NSCLC.

Circ_0000291 contributes to hepatocellular carcinoma tumorigenesis by binding to miR-1322 to up-regulate UBE2T

Introduction and objectivesCircular RNAs (circRNAs) are identified to show important regulatory functions in cancer biology. We attempted to analyze the role of circ_0000291 in hepatocellular carcinoma (HCC) progression and its related mechanism. MethodsThe circular characteristic of circ_0000291 was tested using exonuclease RNase R. Cell proliferation was analyzed by 5-Ethynyl-2’-deoxyuridine (EdU) incorporation and colony formation assays. Cell apoptosis was measured by flow cytometry and a caspase 3 activity assay kit. Transwell assays were performed to analyze cell migration and invasion abilities. Sphere formation assay was conducted to analyze cell stemness. Dual-luciferase reporter and RNA-pull down assays were conducted to verify the interaction between microRNA-1322 (miR-1322) and circ_0000291 or ubiquitin conjugating enzyme E2 T (UBE2T). ResultsCirc_0000291 was markedly up-regulated in HCC tissues and cell lines. HCC patients with high expression of circ_0000291 displayed a low survival rate. Circ_0000291 knockdown restrained the proliferation, migration, invasion, and stemness and induced the apoptosis of HCC cells. Circ_0000291 directly interacted with miR-1322 and negatively regulated miR-1322 expression. Circ_0000291 knockdown-mediated anti-tumor impacts in HCC cells were largely overturned by the interference of miR-1322. miR-1322 directly paired with the 3’ untranslated region (3’UTR) of UBE2T, and UBE2T was negatively regulated by miR-1322. UBE2T overexpression largely reversed circ_0000291 silencing-induced effects in HCC cells. Circ_0000291 positively regulated UBE2T expression by absorbing miR-1322 in HCC cells. Circ_0000291 silencing notably reduced the tumorigenic potential in vivo. ConclusionCirc_0000291 facilitated HCC progression by targeting miR-1322/UBE2T axis, which provided novel potential biomarkers and targets for HCC patients.

PD01-05 MIR-182-5P INHIBITS THE TUMORIGENESIS OF CLEAR CELL RENAL CELL CARCINOMA BY REPRESSING UBE2T

You have accessJournal of UrologyCME1 May 2022PD01-05 MIR-182-5P INHIBITS THE TUMORIGENESIS OF CLEAR CELL RENAL CELL CARCINOMA BY REPRESSING UBE2T Wu Yucai, Gong Yanqing, Li Xuesong, and Zhou Liqun Wu YucaiWu Yucai More articles by this author , Gong YanqingGong Yanqing More articles by this author , Li XuesongLi Xuesong More articles by this author , and Zhou LiqunZhou Liqun More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002516.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Ubiquitin-conjugating enzyme E2T (UBE2T), a member of the E2 family, has been reported to be overexpressed in certain tumor types and to have an important role in the Fanconi anemia pathway. However, the role of UBE2T in clear cell renal cell carcinoma (ccRCC) has not been clarified. MicroRNAs (miRNAs) participate in tumorigenesis by binding to genes and proteins that regulate cell proliferation or cell apoptosis. The aim of this study was to determine the role of UBE2T and the relationship between miR-182-5p and UBE2T in ccRCC. METHODS: UBE2T expression levels were detected by real-time quantitative PCR (RT-qPCR) and western blot in ccRCC cell lines and tissues. Protein expression of UBE2T was assessed in a total of 93 ccRCC patients from Peking University First Hospital (PKU) by immunohistochemistry (IHC). The effects of UBE2T knockdown/overexpression on the proliferation, invasion and metastasis of ccRCC cells were assessed by MTS assays, wound healing assays, transwell invasion assays and flow cytometry. The effects of in vivo treatment were evaluated through xenograft experiments. The relationship between miR-182-5p and UBE2T was verified by dual luciferase reporter gene assay. RESULTS: UBE2T was highly expressed in ccRCC cells and tissues. High expression of UBE2T was positively correlated with advanced pathological stage, histological grade, maximum diameter and distant metastasis of the tumor. Multivariate analysis revealed that the UBE2T expression was an independent risk factor for overall survival (OS) and recurrence-free survival (RFS) in patients with ccRCC. Knockdown of UBE2T significantly suppressed proliferation, migration and invasion of RCC. Flow cytometry analysis showed that knocking down UBE2T could promote RCC cell cycle arrest at G2/M phase and increase cell apoptosis. A xenograft model also confirmed that suppression of UBE2T significantly delayed tumor formation and growth in vivo. In addition, miR-182-5p inhibited the expression of UBE2T protein through targeting UBE2T mRNA, and then inhibited the proliferation, migration and invasion of ccRCC. CONCLUSIONS: Our research reveals that miR-182-5p and UBE2T probably play a critical role in ccRCC progression, and they may be the potential therapeutic targets for ccRCC. Source of Funding: The present study was funded by the National Key R&D Program of China (grant no. 2019YFA09006001) and the National Natural Science Foundation of China (grant nos. 81772703 and 81972380) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e31 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Wu Yucai More articles by this author Gong Yanqing More articles by this author Li Xuesong More articles by this author Zhou Liqun More articles by this author Expand All Advertisement PDF DownloadLoading ...

UBE2T promotes β-catenin nuclear translocation in hepatocellular carcinoma through MAPK/ERK-dependent activation.

Ubiquitin‐conjugating enzyme E2T (UBE2T) has been implicated in many types of cancer including hepatocellular carcinoma (HCC). Epithelial–mesenchymal transition (EMT) process plays a fundamental role during tumor metastasis and progression. However, the molecular mechanisms underlying EMT in HCC in accordance with UBE2T still remain unknown. In this study, we showed that UBE2T overexpression augmented the oncogenic properties and specifically EMT in HCC cell lines, while its silencing attenuated them. UBE2T affected the activation of EMT‐associated signaling pathways: MAPK/ERK, AKT/mTOR, and Wnt/β‐catenin. In addition, we revealed that the epithelial protein complex of E‐cadherin/β‐catenin, a vital regulator of signal transduction in tumor initiation and progression, was totally disrupted at the cell membrane. In particular, we observed that UBE2T overexpression led to E‐cadherin loss accompanied by a simultaneous elevation of both cytoplasmic and nuclear β‐catenin, while its silencing resulted in a strong E‐cadherin turnover at the cell membrane. Interestingly, chemical inhibition of the MAPK/ERK, AKT/mTOR, and Wnt/β‐catenin signaling pathways demonstrated that the nuclear translocation of β‐catenin and subsequent EMT was enhanced mainly by MAPK/ERK. Collectively, our findings demonstrate the UBE2T/MAPK‐ERK/β‐catenin axis as a critical regulator of cell state transition and EMT in HCC.

MiR-182-5p inhibits the tumorigenesis of clear cell renal cell carcinoma by repressing UBE2T.

Ubiquitin-conjugating enzyme E2T (UBE2T), a member of the E2 family, has been reported to be overexpressed in certain tumor types and to have an important role in the Fanconi anemia pathway. However, the role of UBE2T in clear cell renal cell carcinoma (ccRCC) has not been clarified. MicroRNAs (miRNAs) participate in tumorigenesis by binding to genes and proteins that regulate cell proliferation or cell apoptosis. The aim of this study was to determine the role of UBE2T and the relationship between miR-182-5p and UBE2T in ccRCC. In the present study, UBE2T expression levels in ccRCC tissues and cells were assessed using real-time quantitative PCR (RT-qPCR) and western blotting. UBE2T protein expression was assessed in a total of 93 ccRCC patients from Peking University First Hospital (PKU) via immunohistochemistry (IHC). The effects of UBE2T knockdown on ccRCC cells were assessed with MTS assays, wound healing assays, Transwell invasion assays and flow cytometry. The effects of in vivo treatment were evaluated through xenograft experiments. The relationship between miR-182-5p and UBE2T was verified with a dual-luciferase reporter gene assay. We found that UBE2T was highly expressed in ccRCC cells and tissues. High UBE2T expression was positively correlated with advanced pathological stage, histological grade, maximum tumor diameter and distant metastasis. Multivariate analysis revealed that UBE2T expression was an independent risk factor for overall survival (OS) and recurrence-free survival (RFS) in patients with ccRCC. Knockdown of UBE2T significantly suppressed RCC cell proliferation, migration and invasion. Flow cytometry analysis showed that UBE2T knockdown promoted RCC cell cycle arrest at G2/M phase and increased cell apoptosis. The xenograft model confirmed that suppression of UBE2T significantly delayed tumor formation and growth in vivo. In addition, miR-182-5p inhibited UBE2T protein expression by targeting UBE2T mRNA and then inhibited the proliferation, migration and invasion of ccRCC cell. Our research reveals that UBE2T likely plays a critical role in ccRCC progression and may be a potential therapeutic target for ccRCC.

UBE2T-mediated Akt ubiquitination and Akt/\u03b2-catenin activation promotes hepatocellular carcinoma development by increasing pyrimidine metabolism

The oncogene protein ubiquitin-conjugating enzyme E2T (UBE2T) is reported to be upregulated in hepatocellular carcinoma (HCC) and correlated with poor clinical outcomes of HCC patients. However, the underlying mechanism by which UBE2T exerts its oncogenic function in HCC remains largely unexplored. In this study, in vitro and in vivo experiments suggested that UBE2T promoted HCC development including proliferation and metastasis. GSEA analysis indicated that UBE2T was positively correlated with pyrimidine metabolism, and LC/MS-MS metabolomics profiling revealed that the key products of pyrimidine metabolism were significantly increased in UBE2T-overexpressing cells. UBE2T overexpression led to the upregulation of several key enzymes catalyzing de novo pyrimidine synthesis, including CAD, DHODH, and UMPS. Moreover, the utilization of leflunomide, a clinically approved DHODH inhibitor, blocked the effect of UBE2T in promoting HCC progression. Mechanistically, UBE2T increased Akt K63-mediated ubiquitination and Akt/β-catenin signaling pathway activation. The disruption of UBE2T-mediated ubiquitination on Akt, including E2-enzyme-deficient mutation (C86A) of UBE2T and ubiquitination-site-deficient mutation (K8/14 R) of Akt impaired UBE2T’s effect in upregulating CAD, DHODH, and UMPS. Importantly, we demonstrated that UBE2T was positively correlated with p-Akt, β-catenin, CAD, DHODH, and UMPS in HCC tumor tissues. In summary, our study indicates that UBE2T increases pyrimidine metabolism by promoting Akt K63-linked ubiquitination, thus contributing to HCC development. This work provides a novel insight into HCC development and a potential therapeutic strategy for HCC patients.

Hepatic RACK1 deficiency protects against fulminant hepatitis through myeloid-derived suppressor cells

Fulminant hepatitis (FH) is a life-threatening disease with partially understood pathogenesis. It has been demonstrated that myeloid-derived suppressor cells (MDSCs) are recruited into the liver during this process, and their augmented accumulation by various strategies protects against liver injury. However, the underlying mechanism(s) remain elusive. Receptor for activated C kinase 1 (RACK1), a multi-functional scaffold protein, is highly expressed in normal liver and has been implicated in liver physiology and diseases, but the in vivo role of hepatic RACK1 in FH remains unknown.Methods: Survival curves and liver damage were monitored to investigate the in vivo role of hepatic RACK1 in FH. The liver microenvironment was explored by microarray-based transcriptome analysis, flow cytometry, immunoblotting, and immunohistochemistry. MDSCs were identified with phenotypic and functional characteristics. Functional antibodies were used to target MDSCs. Co-culture techniques were used to study the underlying mechanism(s) of protection. The interaction of RACK1 with histone deacetylase 1 (HDAC1) and the consequent effects on HDAC1 ubiquitination were analyzed. Ectopic expression of HDAC1 with recombinant adeno-associated virus serotype 8 was conducted to confirm the role of HDAC1 in the protective effects of hepatic RACK1 deficiency against FH. Post-translational modifications of RACK1 were also investigated during the induction of FH.Results: Liver-specific RACK1 deficiency rendered mice resistant to FH. RACK1-deficient livers exhibited high basal levels of chemokine (C-X-C motif) ligand 1 (CXCL1) and S100 calcium-binding protein A9 (S100A9), associated with MDSC accumulation under steady-state conditions. Targeting MDSCs with an antibody against either Gr1 or DR5 abrogated the protective effects of liver-specific RACK1 deficiency. Accumulated MDSCs inhibited inflammatory cytokine production from macrophages and enhanced IκB kinase (IKK)/NF-κB pathway activation in hepatocytes. Further investigation revealed that RACK1 maintained HDAC1 protein level in hepatocytes by direct binding, thereby controlling histone H3K9 and H3K27 acetylation at the Cxcl1 and S100a9 promoters. Ectopic expression of HDAC1 in livers with RACK1 deficiency partially reversed the augmented Cxcl1/S100a9 → MDSCs → IKK/NF-κB axis. During FH induction, RACK1 was phosphorylated at serine 110, enhancing its binding to ubiquitin-conjugating enzyme E2T and promoting its ubiquitination and degradation.Conclusion: Liver-specific RACK1 deficiency protects against FH through accelerated HDAC1 degradation and the consequent CXCL1/S100A9 upregulation and MDSC accumulation.

MiR-543 impairs breast cancer cell phenotypes by targeting and suppressing ubiquitin-conjugating enzyme E2T (UBE2T)

ABSTRACT Breast cancer, with high morbidity worldwide, is a threat to the life of women. MiR-543 was identified as playing an active part in the development of breast cancer involving multiple molecules. The goal of this study was to explore the molecular mechanisms of the involvement of miR-543 in the development of breast cancer. Quantitative real-time PCR (qRT-PCR) or Western blotting was used to detect mRNA or protein expression. Cell counting kit-8 (CCK-8), and the 5-bromo-2ʹ-deoxyuridine (BrdU), wound healing, and Transwell assays were the main experimental procedures. Furthermore, subcutaneous tumor formation experiments were conducted to detect the function of miR-543 in breast cancer development in vivo. The match of miR-543 and ubiquitin-conjugating enzyme E2T (UBE2T) was detected through a dual-luciferase reporter experiment and RNA pull-down assay. Based on these results, miR-543 exhibited reduced expression in breast cancer tissues and cell lines, whereas UBE2T exhibited high levels. Furthermore, miR-543 directly targeted UBE2T, and a negative correlation between miR-543 and UBE2T was also observed in breast cancer tissues. Moreover, miR-543 overexpression led to inhibition of viability, proliferation, migration, and invasion of breast cancer. Furthermore, miR-543 overexpression undermined the UBE2T promotional effect by inhibiting ERK/MAPK pathway activity in breast cancer cells. Our study revealed that miR-543 impaired breast cancer progression by targeting UBE2T and downregulating UBE2T expression through the ERK/MAPK pathway, which suggested that miR-543 and UBE2T might serve as promising therapeutic gene targets for breast cancer in clinical application.

NEDD4L-induced ubiquitination mediating UBE2T degradation inhibits progression of lung adenocarcinoma via PI3K-AKT signaling

BackgroundA number of studies have indicated that Ubiquitin-conjugating enzyme E2T (UBE2T), as an oncogene, promotes progression and metastasis of lung cancer, including lung adenocarcinoma (LUAD), but it is completely unknown whether and how UBE2T is ubiquitylated and degraded, and by which E3 ligase. NEDD4L plays a critical role in the regulation of cellular processes of various cancers, most of which is attributed to its E3 ubiquitin ligase function. However, the relationship between NEDD4L and UBE2T in LUAD has not been elucidated.MethodsThe relationship between NEDD4L and UBE2T in LUAD tissues and cells was found by bioinformatic analyses and immunoblotting. Cell counting kit-8, colony formation assay, half-life analysis and the in vivo ubiquitylation assay, generation of xenograft model were performed to determine how NEDD4L regulates UBE2T and its downstream signaling pathway in vitro and in vivo.ResultsBioinformatic analyses found that NEDD4L, as a potential correlation E3 ligase of UBE2T, was negatively correlated with UBE2T in LUAD. Consistently, UBE2T protein half-life was shortened or extended by NEDD4L overexpression or depletion, respectively. NEDD4L inhibited LUAD cell progression in vitro and in vivo via inducing the ubiquitination-mediated UBE2T degradation, which repressed PI3K-AKT signaling. Similarly, NEDD4L predicted a better patient survival, whereas UBE2T predicted a worse survival.ConclusionsCollectively, our results reveal that NEDD4L is a novel E3 ligase of UBE2T, which can inhibit PI3K-AKT signaling by targeting for UBE2T ubiquitination and degradation, resulting in repression of LUAD cell progression.

LncRNA CASC11 Promotes Hepatocellular Carcinoma Progression via Upregulation of UBE2T in a m6A-Dependent Manner.

Hepatocellular carcinoma (HCC) is one of the most frequent malignancies and the third leading cause of cancer-related deaths worldwide. Besides, it has been revealed that long non-coding RNA (LncRNA) cancer susceptibility candidate 11 (CASC11) is involved in cancer progression. However, the functional role and underlying mechanism of CASC11 in HCC remains largely unknown. In this context, here, it was found that CASC11 was upregulated in HCC tissues and associated with tumor grades, metastasis, and prognosis of HCC patients. Functionally, CASC11 facilitated HCC cell proliferation, migration, and invasion in vitro, and enhanced tumor growth and metastasis in vivo. Mechanistically, CASC11 associated with and stabilized Ubiquitin-conjugating enzyme E2T (UBE2T) mRNA. To be specific, it decreased UBE2T N6-methyladenosine (m6A) level via recruiting ALKBH5. Moreover, CASC11 inhibited the association between UBE2T mRNA and m6A reader protein YTHDF2. Taken together, our findings demonstrate the epigenetic mechanism of CASC11 in the regulation of UBE2T expression and possibly provide a novel therapeutic target for HCC treatment.

The expression of circ_0090049 in hepatocellular carcinoma and the molecular regulation mechanism of other biological functions.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in liver cancer. Circular RNA_0090049 (circ_0090049) has been shown to be involved in the advance of HCC. However, the interaction between circ_0090049 and microRNA (miRNA) in HCC has not been studied. Quantitative real-time PCR was used to detect the expression of related genes. Through detection of cell proliferation, migration, invasion, and rate of tumor sphere formation, the capping experiment was carried out to verify the regulatory relationship between miRNA and circ_0090049 or circ_0090049 and ubiquitin-conjugating enzyme E2 T (UBE2T). The expression of related proteins was detected by Western blotting. The interaction of miRNA with circ_0090049 or UBE2T was notarized by Dual-luciferase reporter assay. Xenotransplantation experiments confirmed the function of circ_0090049 in vivo. Circ_0090049 and UBE2T were upregulated in liver cancer. Silencing circ_0090049 reduced the proliferation, migration, invasion, and tumor spheroid formation rate of Huh7 and HCCLM3 cells. MiR-605-5p and miR-548c-3p were identified as targets of circ_0090049, and UBE2T was the target of miR-605-5p and miR-548c-3p. Anti-miR-605-5p, anti-miR-548c-3p or UBE2T overexpression restored the inhibitory effect of circ_0090049 knockdown on HCC cells. Animal experiments confirmed the antitumor effect of silence circ_0090049. Circ_0090049 regulates the expression of UBE2T by regulating miR-605-5p or miR-548c-3p, thereby promoting the development of HCC cells.

Ubiquitin-conjugating enzyme E2T promotes tumor stem cell characteristics and migration of cervical cancer cells by regulating the GRP78/FAK pathway.

Ubiquitin-conjugating enzyme E2T (UBE2T) functions as an E2 ubiquitin-conjugating enzyme in the ubiquitin-proteasome degradation system and mediates cellular processes, such as cell cycle, proliferation, and differentiation. UBE2T has been considered to be an oncogene in a variety of tumors. However, the oncogenic role of UBE2T in cervical cancer remains unclear. In this study, our results first showed that the expression of UBE2T was higher in both of cervical cancer tissues and cells than that in the normal tissues and cells. Knockdown of UBE2T reduced cervical cancer cell viability and suppressed the proliferation, invasion, and migration. However, overexpression of UBE2T contributed to cervical cancer cell growth and metastasis. Moreover, UBE2T overexpression cervical cancer cells demonstrated enhanced self-renewal capacity with upregulation of SOX2, Oct-4, and Nanog protein. Silencing of UBE2T downregulated protein expression of SOX2, Oct-4, and Nanog in cervical cancer cells reduced self-renewal capacity. Furthermore, ectopic UBE2T expression promoted protein expression of glucose-regulated protein 78 (GRP78) and focal adhesion kinase (FAK) phosphorylation in cervical cancer cells. The knockdown of UBE2T reduced protein expression of GRP78 and FAK phosphorylation. Collectively, UBE2T promoted cervical cancer stem cell traits and exerted an oncogenic role through activation of the GRP78/FAK pathway.

Comprehensive analysis of differentially expressed genes reveals the promotive effects of UBE2T on colorectal cancer cell proliferation.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Via analysis using The Cancer Genome Atlas database, the present study identified 1,835 genes that were differentially expressed in CRC, including 811 upregulated and 1,024 downregulated genes. Enrichment analyses using the Database for Annotation, Visualization and Integrated Discovery tool revealed that these differentially expressed genes were associated with the regulation of CRC progression by modulating multiple pathways, such as ‘Cell Cycle, Mitotic’, ‘DNA Replication’, ‘Mitotic M-M/G1 phases’ and ‘ATM pathway’. To identify the key genes in CRC, protein-protein interaction (PPI) network analysis was performed and the hub modules in upregulated and downregulated PPI networks were identified. Ubiquitin-conjugating enzyme E2 T (UBE2T), a member of the E2 family, was identified to be a key regulator in CRC. To the best of our knowledge, the present study was the first to demonstrate that UBE2T expression was upregulated in CRC samples compared with normal tissues. Kaplan-Meier analysis revealed that higher expression levels of UBE2T were associated with worse prognosis compared with lower UBE2T expression levels in CRC. Additionally, the present study demonstrated that knockdown of UBE2T inhibited CRC cell proliferation. Flow cytometry assays revealed that UBE2T knockdown induced cell cycle arrest at G1 phase and apoptosis in vitro. These results suggested that UBE2T may be a novel potential biomarker for CRC.

DEPDC1B regulates the progression of human chordoma through UBE2T-mediated ubiquitination of BIRC5

Chordoma is a rare bone malignancy with a high rate of local recurrence and distant metastasis. Although DEP domain-containing protein 1B (DEPDC1B) is implicated in a variety of malignancies, its relationship with chordoma is unclear. In this study, the biological role and molecular mechanism of DEPDC1B in chordoma were explored. The function of DEPDC1B in chordoma cells was clarified through loss-of-function assays in vitro and in vivo. Furthermore, molecular mechanism of DEPDC1B in chordoma cells was recognized by RNA sequencing and Co-Immunoprecipitation (Co-IP) assay. The malignant behaviors of DEPDC1B knockdown chordoma cells was significantly inhibited, which was characterized by reduced proliferation, enhanced apoptosis, and hindered migration. Consistently, decreased expression of DEPDC1B suppressed tumor growth in xenograft mice. Mechanically, DEPDC1B affected the ubiquitination of baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) through ubiquitin-conjugating enzyme E2T (UBE2T). Simultaneous downregulation of BIRC5 and DEPDC1B may exacerbate the inhibitory effects of chordoma. Moreover, BIRC5 overexpression reduced the inhibitory effects of DEPDC1B knockdown in chordoma cells. In conclusion, DEPDC1B regulates the progression of human chordoma through UBE2T-mediated ubiquitination of BIRC5, suggesting that it may be a promising candidate target with potential therapeutic value.