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

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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 ring finger protein 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. CONCLUSIONS: 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.

UBE2T promotes stage I lung adenocarcinoma progression through PBX1 ubiquitination and PBX1/RORA regulation.

BACKGROUND: Post-translational modification pathway of protein ubiquitination is intricately associated with tumorigenesis. We previously reported elevated ubiquitin-conjugating enzyme 2T (UBE2T) as an independent risk factor in stage I lung adenocarcinoma and promoting cellular proliferation. However, its underlying mechanisms needed further investigation. METHODS: Immunohistochemistry was used to assess the expression of UBE2T and retinoic acid receptor-related orphan receptor α (RORA) in stage I LUAD. Cell proliferation, migration, and invasion of LUAD cell lines were measured by Cell Counting Kit-8 assay (CCK-8), Colony-forming assay and Transwell assay, respectively. Western blot analysis was performed to determine the expression of epithelial-mesenchymal transition (EMT) markers. A xenograft model was established to evaluate the proliferative capacity of UBE2T and its interaction with RORA in promoting LUAD. Mechanistic insights into the promotion of early-stage LUAD by UBE2T were obtained through luciferase reporter assay, chromatin immunoprecipitation and co-immunoprecipitation. RESULTS: UBE2T and RORA expression was significantly up- and down-regulated in early-stage LUAD patients which's proved to be associated with unfavorable outcomes, strengthened cell proliferation, migration, EMT and invasion through its interaction with RORA both in vivo and in vitro. The growth NSCLC xenografts was reduced by down-expression of UBE2T but was suppressed by RORA knockout. Mechanistically, UBE2T mediated the ubiquitination of the intermediate transcription factor PBX1, which played a transcriptional role in downstream regulation of RORA. CONCLUSION: The oncogenic role of UBE2T and the UBE2T-PBX1-RORA axis in driving malignant progression in Stage I LUAD had been established. UBE2T might be a novel and promising therapeutic target for LUAD treatment.

UBE2T mediates SORBS3 ubiquitination to enhance IL-6/STAT3 signaling and promote lung adenocarcinoma progression.

UBE2T is an oncogene in varying tumors, including lung adenocarcinoma (LUAD). SORBS3 is an important signaling regulatory protein that plays a crucial role in many cancers. This study aimed to investigate whether UBE2T promoted LUAD development by mediating the ubiquitination of SORBS3 and further explore its mechanism. Bioinformatics analysis was conducted to examine the expression of SORBS3 in LUAD tissues. Cell Counting Kit-8, Transwell, and flow cytometry were employed to analyze the cellular functions of SORBS3. Co-immunoprecipitation and ubiquitination analysis were employed to observe the correlation between UBE2T and SORBS3. In vitro and in vivo experiments verified the role of UBE2T in mediating SORBS3 ubiquitination to enhance interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling and promote LUAD development. We observed significant downregulation of SORBS3 in LUAD tissues and cells. Furthermore, SORBS3 inhibited the proliferation, migration, and invasion of LUAD cells, while facilitating apoptosis in vitro. UBE2T enhanced IL-6/STAT3 signaling by mediating ubiquitination and degradation of SORBS3, thereby promoting LUAD progression. Additionally, this mechanism was further validated in the xenograft animal model in vivo. This study confirmed that UBE2T-mediated SORBS3 ubiquitination enhanced IL-6/STAT3 signaling and promoted LUAD progression, providing a novel therapeutic target for LUAD.

UBE2T promotes glioblastoma malignancy through ubiquitination-mediated degradation of RPL6.

Glioblastoma (GBM) is the most frequent and aggressive malignant glioma. Due to patients' poor prognosis, it is of great clinical significance to determine new targets that may improve GBM treatment. In the present study, we showed that ubiquitin (Ub)-conjugating enzyme E2T (UBE2T) was significantly overexpressed in GBM and could promote proliferation, invasion, and inhibit apoptosis of GBM cells. Mechanistically, UBE2T functioned as the Ub enzyme of ribosomal protein L6 (RPL6) and induced the ubiquitination and degradation of RPL6 in an E3 ligase-independent manner through direct modification by K48-linked polyubiquitination, thus contributing to the malignant progression of GBM cells. Furthermore, inhibiting the expression of RPL6 by UBE2T could not only reduce the expression of wild-type p53, but also enhance the gain-of-function of mutant p53. Moreover, knockdown of UBE2T in LN229 cells obviously suppressed tumor growth in LN229 xenograft mouse models. Collectively, our study demonstrated that UBE2T promotes GBM malignancy through ubiquitination-mediated degradation of RPL6 regardless of the p53 mutation status. It will provide new candidates for molecular biomarkers and therapeutic targets for clinical application in GBM.

Identification and characterization of inhibitors covalently modifying catalytic cysteine of UBE2T and blocking ubiquitin transfer.

UBE2T is an E2 ubiquitin ligase critical for ubiquitination of substrate and plays important roles in many diseases. Despite the important function, UBE2T is considered as an undruggable target due to lack of a pocket for binding to small molecules with satisfied properties for clinical applications. To develop potent and specific UBE2T inhibitors, we adopted a high-throughput screening assay and two compounds-ETC-6152 and ETC-9004 containing a sulfone tetrazole scaffold were identified. Solution NMR study demonstrated the direct interactions between UBE2T and compounds in solution. Further co-crystal structures reveal the binding modes of these compounds. Both compound hydrolysation and formation of a hydrogen bond with the thiol group of the catalytic cysteine were observed. The formation of covalent complex was confirmed with mass spectrometry. As these two compounds inhibit ubiquitin transfer, our study provides a strategy to develop potent inhibitors of UBE2T.

Effects of the major formaldehyde catalyzer ADH5 on phenotypes of fanconi anemia zebrafish model.

BACKGROUND: Fanconi anemia (FA) is a devastating hereditary disorder for which we desperately need a novel therapeutic strategy. It is caused by mutations in one of at least 22 genes in the FA pathway and is characterized by developmental abnormalities, bone marrow failure, and cancer predisposition. The FA pathway is required for the efficient repair of damaged DNA, including interstrand cross-links (ICL). Recent studies indicate formaldehyde as an ultimate endogenous cause of DNA damage in FA pathophysiology. Formaldehyde can form DNA adducts as well as ICLs by inducing covalent linkages between opposite strands of double-stranded DNA. METHODS AND RESULTS: In this study, we generated a disease model of FA in zebrafish by disrupting the ube2t or fancd2 gene, which resulted in a striking phenotype of female-to-male sex reversal. Since formaldehyde is detoxified from the body by alcohol dehydrogenase 5 (ADH5), we generated fancd2-/-/adh5-/- zebrafish. We observed a body size reduction and a lower number of mature spermatozoa than wild-type or single knockout zebrafish. To evaluate if increased activity in ADH5 can affect the FA phenotype, we overexpressed human ADH5 in fancd2-/- zebrafish. The progress of spermatogenesis seemed to be partially recovered due to ADH5 overexpression. CONCLUSIONS: Our results suggest potential utility of an ADH5 enzyme activator as a therapeutic measure for the clearance of formaldehyde and treatment of FA.

Hsa_circ_0092887 targeting miR-490-5p/UBE2T promotes paclitaxel resistance in non-small cell lung cancer.

BACKGROUND: Chemoresistance is a major contributing factor to cancer treatment failure. Emerging research reveals that circular RNA (circRNA) dysregulation is implicated in chemoresistance. Our current study aimed to investigate the involvement of hsa_circ_0092887 in paclitaxel (PTX) resistance in non-small cell lung cancer (NSCLC). METHODS: RT-qPCR as well as western blotting were used for the analysis of hsa_circ_0092887, miR-490-5p and UBE2T expression in PTX-resistant NSCLC tumor tissues and cells. CCK-8 assay was done to determine the IC50 value of PTX. CCK-8 assay, wound healing assay, analysis of apoptosis related proteins (Bax and Bcl-2), and xenograft mouse models were utilized to investigate the role of hsa_circ_0092887 in PTX-resistance in NSCLC. The binding sites of miR-490-5p to hsa_circ_0092887 or UBE2T were predicted by bioinformatics tools and were verified by RIP and dual-luciferase assays. RESULTS: Expression of hsa_Circ_0092887 was upregulated in NSCLC tumor samples/cell lines, and its expression was also higher in PTX-resistant tumor samples/cell lines when compared with their respective controls. Silencing of hsa_circ_0092887 in PTX-treated NSCLC cells inhibited cell proliferation and migration, induced apoptosis, and suppressed tumor growth in xenograft mouse models in vivo. MiR-490-5p was a direct target of hsa_circ_0092887, and UBE2T was a functional downstream target of hsa_circ_0092887/miR-490-5p axis. Hsa_circ_0092887 depletion-induced anti-cancer effects in PTX-treated NSCLC cells were reversed by miR-490-5p inhibitor. Furthermore, inhibition of miR-490-5p strengthened UBE2T expression, thereby attenuating the anti-cancer effects caused by UBE2T knockdown. CONCLUSION: Hsa_circ_0092887 depletion alleviated PTX-resistance in NSCLC cells via modulating the miR-490-5p/UBE2T axis, and the targeted management of hsa_circ_0092887-mediated signaling axis might contribute to PTX-resistance intervention in NSCLC.

UBE2T is a prognostic biomarker and correlated with Th2 cell infiltrates in retinoblastoma.

OBJECTIVE: This study aimed to screen anaplasia-related genes that influence the progression of retinoblastoma (RB) and to identify immune cells associated with the poor prognosis. METHODS: Differentially expressed genes (DEGs) between retina and RB samples were acquired from gene expression omnibus (GEO) database. Candidate hub genes were screened by taking intersections among the co-expressed genes, the hub nodes, and DEGs of the validation set. The hub genes were identified by receiver operating characteristic (ROC) and quantitative real-time PCR (qPCR). Immune infiltration levels of RB tissues were estimated using single-sample gene set enrichment analysis (ssGSEA). The functions of RB cells were detected by CCK8, EDU and flow cytometry assays. RESULTS: 665 DEGs involved in the genesis and progression of RB were acquired from GEO database. 29 candidate hub genes were screened by examining 43 co-expressed genes and 63 hub nodes. 9 hub genes (CHEK1, EXO1, FANCI, GTSE1, MELK, MKI67, NCAPH, PRC1, and UBE2T) strongly related to the anaplastic grades were validated by ROC curve analysis (AUC >0.8). Based on the ssGSEA scores, the immune infiltration levels of Th2 cells were positively associated with anaplastic grade. qPCR assay showed that 9 hub genes were upregulated in RB cells, and UBE2T expressed remarkably high. CCK 8, EDU, and flow cytometry assays revealed that UBE2T silencing inhibited the proliferation of RB cells and incited apoptosis. CONCLUSIONS: The increased infiltration of Th2 cells and upregulated expression of 9 hub genes predict a poor prognosis of RB. UBE2T can be a therapeutic target for RB treatment.

UBE2T promotes proliferation, invasion and glycolysis of breast cancer cells by regualting the PI3K/AKT signaling pathway.

PURPOSE: Breast cancer (BCa) is one of the most common gynecological malignancies. Ubiquitin-coupled enzyme E2T (UBE2T) has been demonstrated to play crucial roles in various tumors. METHODS: UBE2T levels were detected using quantitative real time PCR and western blot. CCK-8 and colony formation assays were used to evaluate cell proliferation. A xenograft model was used to evaluate the effects of UBE2T on tumor growth in mice, and immunohistochemistry (IHC) assay was performed to detect the expression of UBE2T and Ki-67. Transwell assay was performed to determine cell migration and invasion. The ATP level, glucose consumption and lactate production were measured using the corresponding commercial kits. Western blot assay was used to detect the levels of epithelial-mesenchymal transformation (EMT), glycolytic and the PI3K/AKT pathway related proteins regulated by UBE2T. RESULTS: Upregulation of UBE2T expression in human BCa tissues was found in human clinical BCa tissues and The Cancer Genome Atlas (TCGA) dataset. The expression of UBE2T was confirmed to be up-regulated in BCa cells compared to normal breast epithelial cell line (MCF-10A). Overexpression of UBE2T promoted proliferation, migration, invasion and glycolysis in BCa cells, while UBE2T knockdown showed the opposite results. Moreover, UBE2T knockdown suppressed tumor growth in mice. Further mechanism analysis shows that UBE2T participated in the regulation of BCa progression through affecting the PI3K/AKT signaling pathway. CONCLUSION: UBE2T promoted proliferation, invasion and glycolysis through modulating PI3K/AKT signaling pathway in BCa, implying that UBE2T may provide a promising therapeutic target for the therapy of BCa.

Pan-cancer analysis of UBE2T with a focus on prognostic and immunological roles in lung adenocarcinoma.

BACKGROUND: Ubiquitin-conjugating enzyme E2 T (UBE2T) is a potential oncogene. However, Pan-cancer analyses of the functional, prognostic and predictive implications of this gene are lacking. METHODS: We first analyzed UBE2T across 33 tumor types in The Cancer Genome Atlas (TCGA) project. We investigated the expression level of UBE2T and its effect on prognosis using the TCGA database. The correlation between UBE2T and cell cycle in pan-cancer was investigated using the single-cell sequencing data in Cancer Single-cell State Atlas (CancerSEA) database. The Weighted Gene Co-expression Network analysis (WGCNA), Univariate Cox and Least absolute shrinkage and selection operator (LASSO) Cox regression models, and receiver operating characteristic (ROC) were applied to assess the prognostic impact of UBE2T-related cell cycle genes (UrCCGs). Furthermore, the consensus clustering (CC) method was adopted to divide TCGA-lung adenocarcinoma (LUAD) patients into subgroups based on UrCCGs. Prognosis, molecular characteristics, and the immune panorama of subgroups were analyzed using Single-sample Gene Set Enrichment Analysis (ssGSEA). Results derived from TCGA-LUAD patients were validated in International Cancer Genome Consortium (ICGC)-LUAD data. RESULTS: UBE2T is highly expressed and is a prognostic risk factor in most tumors. CancerSEA database analysis revealed that UBE2T was positively associated with the cell cycle in various cancers(r > 0.60, p < 0.001). The risk signature of UrCCGs can reliably predict the prognosis of LUAD (AUC1 year = 0.720, AUC3 year = 0.700, AUC5 year = 0.630). The CC method classified the TCGA-LUAD cohort into 4 UrCCG subtypes (G1-G4). Kaplan-Meier survival analysis demonstrated that G2 and G4 subtypes had worse survival than G3 (Log-rank test PTCGA training set < 0.001, PICGC validation set < 0.001). A comprehensive analysis of immune infiltrates, immune checkpoints, and immunogenic cell death modulators unveiled different immune landscapes for the four subtypes. High immunophenoscore in G3 and G4 tumors suggested that these two subtypes were immunologically "hot," tending to respond to immunotherapy compared to G2 subtypes (p < 0.001). CONCLUSIONS: UBE2T is a critical oncogene in many cancers. Moreover, UrCCG classified the LUAD cohort into four subgroups with significantly different survival, molecular features, immune infiltrates, and immunotherapy responses. UBE2T may be a therapeutic target and predictor of prognosis and immunotherapy sensitivity.

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

INTRODUCTION AND OBJECTIVES: Circular 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. METHODS: The 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). RESULTS: Circ_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. CONCLUSION: Circ_0000291 facilitated HCC progression by targeting miR-1322/UBE2T axis, which provided novel potential biomarkers and targets for HCC patients.

UBE2T promotes autophagy via the p53/AMPK/mTOR signaling pathway in lung adenocarcinoma.

BACKGROUND: Ubiquitin-conjugating enzyme E2T (UBE2T) acts as an oncogene in various types of cancer. However, the mechanisms behind its oncogenic role remain unclear in lung cancer. This study aims to explore the function and clinical relevance of UBE2T in lung cancer. METHODS: Lentiviral vectors were used to mediate UBE2T depletion or overexpress UBE2T in lung cancer cells. CCK8 analysis and western blotting were performed to investigate the effects of UBE2T on proliferation, autophagy, and relevant signaling pathways. To exploit the clinical significance of UBE2T, we performed immunohistochemistry staining with an anti-UBE2T antibody on 131 NSCLC samples. Moreover, we downloaded the human lung adenocarcinoma (LUAD) dataset from The Cancer Atlas Project (TCGA). Lasso Cox regression model was adopted to establish a prognostic model with UBE2T-correlated autophagy genes. RESULTS: We found that UBE2T stimulated proliferation and autophagy, and silencing this gene abolished autophagy in lung cancer cells. As suggested by Gene set enrichment analysis, we observed that UBE2T downregulated p53 levels in A549 cells and vice versa. Blockade of p53 counteracted the inhibitory effects of UBE2T depletion on autophagy. Meanwhile, the AMPK/mTOR signaling pathway was activated during UBE2T-mediated autophagy, suggesting that UBE2T promotes autophagy via the p53/AMPK/mTOR pathway. Interestingly, UBE2T overexpression increased cisplatin-trigged autophagy and led to cisplatin resistance of A549 cells, whereas inhibiting autophagy reversed drug resistance. However, no association was observed between UEB2T and overall survival in a population of 131 resectable NSCLC patients. Therefore, we developed and validated a multiple gene signature by considering UBE2T and its relevance in autophagy in lung cancer. The risk score derived from the prognostic signature significantly stratified LUAD patients into low- and high-risk groups with different overall survival. The risk score might independently predict prognosis. Interestingly, nomogram and decision curve analysis demonstrated that the signature's prognostic accuracy culminated while combined with clinical features. Finally, the risk score showed great potential in predicting clinical chemosensitivity. CONCLUSIONS: We found that UBE2T upregulates autophagy in NSCLC cells by activating the p53/AMPK/mTOR signaling pathway. The clinical predicting ability of UBE2T in LUAD can be improved by considering the autophagy-regulatory role of UBE2T.

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

BACKGROUND: A 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. METHODS: The 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. RESULTS: Bioinformatic 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. CONCLUSIONS: Collectively, 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.

UBE2T promotes hepatocellular carcinoma cell growth via ubiquitination of p53.

Deregulation of Ubiquitin-conjugating enzyme E2T (UBE2T) contributes to the progression of human cancers. However, its clinical significance and role in hepatocellular carcinoma (HCC) remain unclear. Here, we show that UBE2T is up-regulated in HCC and exerts oncogenic activities via ubiquitination of p53. High UBE2T expression was correlated with higher pathological grade, advanced TNM stage, tumor vascular invasion, and poor overall and disease-free survivals in two independent cohorts containing 827 patients with HCC. UBE2T was further identified as an independent factor for overall survival by multivariate analyses. Luciferase reporter assays confirmed that UBE2T was directly targeted by miR-543 which was down-regulated in HCC. In vitro experiments demonstrated that UBE2T overexpression promoted, whereas UBE2T knockdown inhibited HCC cell growth. Ectopic expression of UBE2T resulted in the decreases of p53, p21 and Noxa. Further studies revealed that UBE2T facilitated the degradation of p53 protein via enhancing its ubiquitination. Collectively, our findings suggest UBE2T serves as a promising prognostic factor for HCC and functions as an oncogene. The newly identified miR-543/UBE2T/p53 axis may represent a new potential therapeutic target for HCC intervention.

Mechanism and disease association of E2-conjugating enzymes: lessons from UBE2T and UBE2L3.

Ubiquitin signalling is a fundamental eukaryotic regulatory system, controlling diverse cellular functions. A cascade of E1, E2, and E3 enzymes is required for assembly of distinct signals, whereas an array of deubiquitinases and ubiquitin-binding modules edit, remove, and translate the signals. In the centre of this cascade sits the E2-conjugating enzyme, relaying activated ubiquitin from the E1 activating enzyme to the substrate, usually via an E3 ubiquitin ligase. Many disease states are associated with dysfunction of ubiquitin signalling, with the E3s being a particular focus. However, recent evidence demonstrates that mutations or impairment of the E2s can lead to severe disease states, including chromosome instability syndromes, cancer predisposition, and immunological disorders. Given their relevance to diseases, E2s may represent an important class of therapeutic targets. In the present study, we review the current understanding of the mechanism of this important family of enzymes, and the role of selected E2s in disease.

Ubiquitin-Conjugating Enzyme E2T is an Independent Prognostic Factor and Promotes Gastric Cancer Progression.

Ubiquitin-conjugating enzyme E2T (UBE2T) is a member of the E2 family that mediates the ubiquitin-proteasome system and regulates gene expression. It is a major oncogene in several cancers such as lung cancer and breast cancer, while the potential functions of UBE2T in gastric cancer (GC) remains largely unknown. Here, we identified the roles of UBE2T in GC progression and its potential to act as a prognostic marker of GC. Our data demonstrated that UBE2T was significantly upregulated in gastric cancer tissues, and the high expression of UBE2T was significantly correlated with poor differentiation, high T classification, and poor prognosis. In vitro experiments indicated that UBE2T promoted cell proliferation and inhibited cell cycle arrest. In addition, we observed that UBE2T modulated cell mobility by inducing epithelial-mesenchymal transition. Collectively, these findings suggest that UBE2T plays an important role in the tumorigenesis of gastric cancer and could act as a potential independent prognostic factor for cancer therapy.

Knockdown of Ubiquitin-Conjugating Enzyme E2 T Abolishes the Progression of Head and Neck Squamous Cell Carcinoma by Inhibiting NF-Κb Signaling and inducing Ferroptosis.

BACKGROUND: Ubiquitin-conjugating enzyme 2T (UBE2T) has been reported to be associated with uncontrolled cell growth and tumorigenesis in multiple cancer types. However, the understanding of its regulatory role in the carcinogenesis of Head And Neck Squamous Cell Carcinoma (HNSC) is limited. METHODS: UBE2T expression in HNSC patient samples and the correlation between its expression and patients' survival rates were evaluated using The Cancer Genome Atlas (TCGA) database. Cell survival and proliferation were investigated in UM-SCC1 and UM-SCC15 cells infected with control and shUBE2T lentivirus. The xenograft mouse model was established using UM-SCC15 cells to examine HNSC tumorigenesis with or without UBE2T. Western blot, qRT-PCR, and ferroptosis assays were carried out to disclose the interaction between UBE2T and NF-κB signaling and ferroptosis. RESULTS: The increased expression of UBE2T was noted in tumor tissues of patients with HNSC, correlating with a significantly reduced overall survival time in this patient cohort. Knockdown of UBE2T inhibited HNSC tumorigenesis and tumor growth. Mechanistically, inhibition of UBE2T suppressed NF-κB signaling and induced ferroptosis in HNSC. CONCLUSION: Our study underscores the multifaceted role of UBE2T in HNSC, illuminating its potential as a biomarker and therapeutic target.

Identification of small-molecule binding sites of a ubiquitin-conjugating enzyme-UBE2T through fragment-based screening.

UBE2T is an attractive target for drug development due to its linkage with several types of cancers. However, the druggability of ubiquitin-conjugating E2 (UBE2T) is low because of the lack of a deep and hydrophobic pocket capable of forming strong binding interactions with drug-like small molecules. Here, we performed fragment screening using 19 F-nuclear magnetic resonance (NMR) and validated the hits with 1 H-15 N-heteronuclear single quantum coherence (HSQC) experiment and X-ray crystallographic studies. The cocrystal structures obtained revealed the binding modes of the hit fragments and allowed for the characterization of the fragment-binding sites. Further screening of structural analogues resulted in the identification of a compound series with inhibitory effect on UBE2T activity. Our current study has identified two new binding pockets in UBE2T, which will be useful for the development of small molecules to regulate the function of this protein. In addition, the compounds identified in this study can serve as chemical starting points for the development of UBE2T modulators.

FOXA1/UBE2T Inhibits CD8+T Cell Activity by Inducing Mediates Glycolysis in Lung Adenocarcinoma.

BACKGROUND: Immune escape is a key factor influencing survival rate of lung adenocarcinoma (LUAD) patients, but molecular mechanism of ubiquitin binding enzyme E2T (UBE2T) affecting immune escape of LUAD remains unclear. The objective was to probe role of UBE2T in LUAD. METHODS: Bioinformatics means were adopted for analyzing UBE2T and forkhead box A1 (FOXA1) expression in LUAD tissues, the gene binding sites, the pathway UBE2T regulates, and the correlation between UBE2T and glycolysis genes. Dual luciferase and chromatin immunoprecipitation (ChIP) assays were conducted for validating the binding relationship between the two genes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot were employed to evaluate UBE2T, FOXA1, and programmed death ligand 1 (PD-L1) levels in cancer cells. MTT assay was conducted for detecting cell viability. Cytotoxicity assay detected CD8+T cell toxicity. Cytokine expression was assayed by enzyme linked immunosorbent assay (ELISA). Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were assayed by extracellular flow analyzer. Glycolytic gene expression was analyzed by qRT-PCR, and glycolysis-related indicators were detected by ELISA. Immunohistochemistry (IHC) detected CD8+T cell infiltration in tumor tissues. RESULTS: FOXA1 and UBE2T were up-regulated in LUAD, and a binding site existed between UBE2T and FOXA1. Overexpressing UBE2T could increase PD-L1 expression and inhibit toxicity of CD8+T cells to LUAD cells. Overexpressing UBE2T repressed CD8+T cell activity in LUAD by activating the glycolysis pathway, and the addition of glycolysis inhibitor 2-deoxy-d-glucose (2-DG) reversed the above results. Mechanistically, FOXA1 promoted the immune escape of LUAD by up-regulating UBE2T and thus mediating glycolysis. In vivo experiments revealed that UBE2T knockdown hindered tumor growth, inhibited PD-L1 expression, and facilitated CD8+T cell infiltration. CONCLUSION: FOXA1 up-regulated the expression of UBE2T, which activated glycolysis, and thus inhibited activity of CD8+T cells, causing immune escape of LUAD.

Identification and validation of a novel ubiquitination-related gene UBE2T in Ewing's sarcoma.

Background: Ewing's sarcoma (ES) is one of the most prevalent malignant bone tumors worldwide. However, the molecular mechanisms of the genes and signaling pathways of ES are still not well sufficiently comprehended. To identify candidate genes involved in the development and progression of ES, the study screened for key genes and biological pathways related to ES using bioinformatics methods. Methods: The GSE45544 and GSE17618 microarray datasets were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified, and functional enrichment analysis was performed. A protein-protein interaction (PPI) network was built, and key module analysis was performed using STRING and Cytoscape. A core-gene was gained and was validated by the validation dataset GSE67886 and immunohistochemistry (IHC). The diagnostic value and prognosis evaluation of ES were executed using, respectively, the ROC approach and Cox Regression. Results: A total of 187 DEGs, consisting of 56 downregulated genes and 131 upregulated genes, were identified by comparing the tumor samples to normal samples. The enriched functions and pathways of the DEGs, including cell division, mitotic nuclear division, cell proliferation, cell cycle, oocyte meiosis, and progesterone-mediated oocyte maturation, were analyzed. There were 149 nodes and 1246 edges in the PPI network, and 15 hub genes were identified according to the degree levels. The core gene (UBE2T) showed high expression in ES, validated by using GSE67886 and IHC. The ROC analysis revealed UBE2T had outstanding diagnostic value in ES (AUC = 0.75 in the training set, AUC = 0.90 in the validation set). Kaplan-Meier (analysis of survival rate) and Cox Regression analyses indicated that UBE2T was a sign of adverse results for sufferers with ES. Conlusion: UBE2T was a significant value biomarker for diagnosis and treatment of ES, thereby presenting a novel potential therapeutic target for ES as well as a new perspective for assessing the effect of treatment and prognostic prediction.