Anti-UBE2T antibody: A novel biomarker of progressive-fibrosing interstitial lung disease.

BACKGROUND: Anti-fibrotic therapy has demonstrated efficacy against progressive-fibrosing interstitial lung disease (PF-ILD); therefore, identifying disease behavior before progression has become a priority. As autoimmunity is implicated in the pathogenesis of various ILDs, this study explored circulating biomarkers that could predict the chronic progressive behavior of ILDs. METHODS: A single-center retrospective cohort study was conducted. Circulating autoantibodies in patients with ILD were screened using microarray analysis to identify candidate biomarkers. An enzyme-linked immunosorbent assay was performed with a larger sample set for the quantification of antibodies. After 2 years of follow-up, ILDs were reclassified as PF or non-PF. The relationship between the participants' autoantibody levels measured at enrolment and final diagnosis of PF-ILD was determined. RESULTS: In total, 61 healthy participants and 66 patients with ILDs were enrolled. Anti-ubiquitin-conjugating enzyme E2T (UBE2T) antibody was detected as a candidate biomarker. Anti-UBE2T antibody levels were elevated in patients with idiopathic pulmonary fibrosis (IPF). After following up on the study participants for 2 years, anti-UBE2T levels measured at enrolment significantly correlated with the new PF-ILD diagnosis. Immunohistochemical staining of normal lung tissues revealed sparsely located UBE2T in the bronchiole epithelium and macrophages, whereas IPF lung tissues showed robust expression in the epithelial lining of honeycomb structures. CONCLUSION: To our knowledge, this is the first report to describe an anti-UBE2T antibody, a new biomarker that is significantly elevated in patients with ILD who present future disease progression.

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 ubiquitin­conjugating enzyme E2 T (UBE2T) and its associated pathways in the pathogenesis of pan­cancer, and the development of small­molecule 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 pan­cancer. Information was collected from 16 clinical trials on ubiquitin enzymes, and it was found that these molecules had an important role in the ubiquitin­proteasome system. Further studies are necessary to explore their feasibility and effectiveness as diagnostic and prognostic biomarkers, or as up/down­stream and therapeutic targets for cancer treatment.

UBE2T is upregulated, predicts poor prognosis, and promotes cell proliferation and invasion by promoting epithelial-mesenchymal transition via inhibiting autophagy in an AKT/mTOR dependent manner in ovarian cancer.

Aberrant upregulation and oncogenic roles of UBE2T are revealed in several cancers. However, the expression, clinical significance, and functions of UBE2T have not been explored in ovarian cancer (OC). In this study, the expression of UBE2T and its relation with clinicopathological features and prognosis of OC patients were explored by analyzing online data and experimental data. Besides, the functions of UBE2T in OC cells were investigated by in vitro experiments, including CCK-8, plate clone formation, and Transwell assays. Finally, the underlying mechanism of UBE2T associated functions in OC was analyzed. The results indicated that UBE2T was significantly upregulated in OC tissues. UBE2T expression was notably correlated with clinical features, such as primary T stage and FIGO stage in OC patients. UBE2T, acting as an independent prognostic indicator, was inversely associated with the prognosis of OC patients. The UBE2T knockdown remarkably suppressed the growth, proliferation, and invasion of OC cells, indicated by impaired cell viability, fewer cell clones, and invasive cells. Mechanistically, UBE2T depletion suppressed epithelial-mesenchymal transition (EMT), which was caused by autophagy activation due to inactivation of AKT/mTOR in OC cells with UBE2T knockdown. Collectively, our findings confirm that UBE2T upregulation predicts poor prognosis and promotes malignant progression in OC. UBE2T upregulation suppresses autophagy and subsequently boosts EMT via activating the AKT/mTOR axis, which accounts for the underlying mechanism of oncogenic roles of UBE2T in OC.

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 non­small cell lung cancer (NSCLC). To quantify the expression levels of FANCI and ubiquitin­conjugating enzyme E2T (UBE2T) in NSCLC tissues, reverse­transcription quantitative PCR and western blotting were employed. Cell Counting Kit­8, 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 co­immunoprecipitation assay. Epithelial­to­mesenchymal 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.

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.

Expression of ubiquitin-conjugating enzyme E2T in colorectal cancers and clinical implications.

Ubiquitin-conjugating enzyme E2T (UBE2T) plays a significant role in carcinogenesis. Previous studies have demonstrated that UBE2T promotes the development and progression of numerous types of cancer. However, the association between UBE2T expression and colorectal cancer (CRC) remains unclear. In the present study, UBE2T protein expression was examined in the tissues of patients with CRC via immunohistochemistry. In addition, UBE2T expression data and corresponding clinical information were obtained from The Cancer Genome Atlas (TCGA). In the clinical samples, the associations between UBE2T expression and clinicopathological factors were evaluated by the χ2 or Fisher's exact tests. In TCGA data, associations between UBE2T expression and clinical characteristics were evaluated using a logistic regression model. Overall survival was analyzed using Kaplan-Meier and Cox regression analyses. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting assays were used to examine UBE2T expression in normal and CRC cell lines. Gene set enrichment analysis (GSEA) was performed on the dataset from TCGA. UBE2T protein was highly expressed in the cytoplasm of tumor cells in 29/50 clinical samples, whereas in the adjacent normal tissues, it was only highly expressed in 2/50 samples. Furthermore, UBE2T expression was associated with the N classification (P<0.001), clinical TNM stage (P<0.001) and histological grade of tumors (P=0.010). Survival analysis showed an association between high UBE2T expression and poor survival rate in patients with CRC (P=0.002). Cox regression analysis also revealed that UBE2T expression was an independent prognostic factor for these patients (P=0.006). RT-qPCR and western blotting showed that UBE2T was expressed in CRC cell lines at higher levels than that in a normal colon cell line. Analysis of TCGA data revealed that UBE2T was highly expressed in tumor samples compared with normal samples, but was not associated with prognosis. GSEA showed that high expression of UBE2T was associated with the Kyoto Encyclopedia of Genes and Genomes pathways 'cell cycle', 'oxidative phosphorylation', 'DNA replication', 'p53 signaling pathway', 'ubiquitin mediated proteolysis' and 'pentose phosphate pathway'. These results indicate that UBE2T may play an important role in the progression of CRC and serve as a potential prognostic factor during the treatment of cancer.

Role of ubiquitin-conjugating enzyme E2T in the carcinogenesis and progression of pancreatic cancer.

Ubiquitin-conjugating enzyme E2T (UBE2T) is a recently discovered oncogenic protein. Numerous studies reported that UBE2T is highly expressed in various types of human cancer; however, its role in the carcinogenesis and progression of pancreatic cancer remains unknown. The aim of the present study was to investigate the role of UBE2T in pancreatic cancer progression through in vitro experiments in pancreatic cancer tissues and cell lines. The results obtained in the present study demonstrated that UBE2T served an important role in the initiation and progression of pancreatic cancer. Furthermore, increased expression of UBE2T in human pancreatic cancer tissues and pancreatic cancer cells was observed compared with normal tissues and cells. The effect of upregulating and downregulating UBE2T in pancreatic cancer cell lines was investigated using the MTT, wound-healing and migration and invasion assays. The results demonstrated that overexpression of UBE2T significantly promoted pancreatic cancer cell proliferation, migration and invasion compared with controls. However, UBE2T downregulation resulted in the inhibition of pancreatic cancer cell proliferation, migration and invasion. In addition, the results demonstrated that UBE2T may promote the epithelial mesenchymal transition in pancreatic cancer cells.

UBE2T-regulated H2AX monoubiquitination induces hepatocellular carcinoma radioresistance by facilitating CHK1 activation.

BACKGROUND: Radioresistance is the major obstacle in radiation therapy (RT) for hepatocellular carcinoma (HCC). Dysregulation of DNA damage response (DDR), which includes DNA repair and cell cycle checkpoints activation, leads to radioresistance and limits radiotherapy efficacy in HCC patients. However, the underlying mechanism have not been clearly understood. METHODS: We obtained 7 pairs of HCC tissues and corresponding non-tumor tissues, and UBE2T was identified as one of the most upregulated genes. The radioresistant role of UBE2T was examined by colony formation assays in vitro and xenograft tumor models in vivo. Comet assay, cell cycle flow cytometry and γH2AX foci measurement were used to investigate the mechanism by which UBE2T mediating DDR. Chromatin fractionation and immunofluorescence staining were used to assess cell cycle checkpoint kinase 1(CHK1) activation. Finally, we analyzed clinical data from HCC patients to verify the function of UBE2T. RESULTS: Here, we found that ubiquitin-conjugating enzyme E2T (UBE2T) was upregulated in HCC tissues, and the HCC patients with higher UBE2T levels exhibited poorer outcomes. Functional studies indicated that UBE2T increased HCC radioresistance in vitro and in vivo. Mechanistically, UBE2T-RNF8, was identified as the E2-E3 pair, physically bonded with and monoubiquitinated histone variant H2AX/γH2AX upon radiation exposure. UBE2T-regulated H2AX/γH2AX monoubiquitination facilitated phosphorylation of CHK1 for activation and CHK1 release from the chromatin to cytosol for degradation. The interruption of UBE2T-mediated monoubiquitination on H2AX/γH2AX, including E2-enzyme-deficient mutation (C86A) of UBE2T and monoubiquitination-site-deficient mutation (K119/120R) of H2AX, cannot effectively activate CHK1. Moreover, genetical and pharmacological inhibition of CHK1 impaired the radioresistant role of UBE2T in HCC. Furthermore, clinical data suggested that the HCC patients with higher UBE2T levels exhibited worse response to radiotherapy. CONCLUSION: Our results revealed a novel role of UBE2T-mediated H2AX/γH2AX monoubiquitination on facilitating cell cycle arrest activation to provide sufficient time for radiation-induced DNA repair, thus conferring HCC radioresistance. This study indicated that disrupting UBE2T-H2AX-CHK1 pathway maybe a promising potential strategy to overcome HCC radioresistance.

UBE2T promotes glioblastoma invasion and migration via stabilizing GRP78 and regulating EMT.

Glioblastoma (GBM) generally has a dismal prognosis, and it is associated with a poor quality of life as the disease progresses. However, the development of effective therapies for GBM has been deficient. Ubiquitin-conjugating enzyme E2T (UBE2T) is a member of the E2 family in the ubiquitin-proteasome pathway and a vital regulator of tumour progression, but its role in GBM is unclear. In this study, we aimed to clarify the role of UBE2T in GBM. Bioinformatics analysis identified UBE2T as an independent risk factor for gliomas. Immunohistochemistry was used to measure UBE2T expression in GBM and normal tissue samples obtained from patients with GBM. The effects of UBE2T on GBM cell invasion and migration were analysed using the Transwell assay. BALB/c nude mice were used for the in vivo assays. Immunoblotting and immunoprecipitation were performed to determine the molecular mechanisms. UBE2T was highly expressed in GBM tissues, and its expression was linked to a poor prognosis. In vitro, depletion of UBE2T significantly suppressed cell invasion and migration. Moreover, UBE2T depletion suppressed the growth of GBM subcutaneous tumours in vivo. Further experiments revealed that UBE2T suppressed invasion and migration by regulating epithelial- mesenchymal transition (EMT) via stabilising GRP78 in GBM cells. We uncovered a novel UBE2T/GRP78/EMT regulatory axis that modulates the malignant progression and recurrence of GBM, indicating that the axis might be a valuable therapeutic target.

Identification of UBE2T as an independent prognostic biomarker for gallbladder cancer.

Gallbladder cancer is the most common biliary tract malignant tumor, with unfavorable patient outcomes. The present study aimed to identify potential diagnostic or prognostic biomarkers for gallbladder cancer. To do so, differentially expressed genes in the gallbladder walls and tumor tissues of patients with gallbladder cancer were analyzed via microarray. Furthermore, a protein-protein interaction network was constructed and genes with a degree score >10 were selected as hub genes. As ubiquitin conjugating enzyme E2T (UBE2T) was considered to be a hub gene, its expression was assessed via reverse transcription-quantitative (RT-q)PCR and immunohistochemistry (IHC). In addition, the association between UBE2T expression and the clinicopathological characteristics of patients with gallbladder cancer was analyzed using the χ2 test. Furthermore, all patients were divided into high- and low groups based on UBE2T expression level and overall survival analysis was performed. Univariate and multivariate Cox regression analyses were performed to determine whether UBE2T may serve as an independent risk factor for gallbladder cancer. The results demonstrated that UBE2T expression was upregulated in the gallbladder walls and tumor tissues of patients with gallbladder cancer. Furthermore, UBE2T expression level was confirmed to be upregulated following RT-qPCR, and results from IHC demonstrated that UBE2T was predominantly expressed in the cytoplasm of gallbladder cancer cells. In addition, high UBE2T expression level was associated with clinical stage, T classification, N classification and M classification. The results from Univariate and multivariate analyses indicated that UBE2T expression level may be considered as an independent risk factor for gallbladder cancer. Taken together, the findings from this study suggested that high UBE2T expression level may contribute to the poor prognosis of patients with gallbladder cancer, and that UBE2T may act as an independent prognostic biomarker for these patients.

Ubiquitin-conjugating enzyme E2T knockdown suppresses hepatocellular tumorigenesis via inducing cell cycle arrest and apoptosis.

BACKGROUND: Hepatocellular carcinoma (HCC) is now the most common primary liver malignancy worldwide, and multiple risk factors attribute to the occurrence and development of HCC. Recently, increasing studies suggest that ubiquitin-conjugating enzyme E2T (UBE2T) serves as a promising prognostic factor in human cancers, although the molecular mechanism of UBE2T in HCC remains unclear. AIM: To investigate the clinical relevance and role of UBE2T in HCC development. METHODS: UBE2T expression in HCC tissues from the TCGA database and its association with patient survival were analyzed. A lentivirus-mediated strategy was used to knock down UBE2T in HCC cells. qRT-PCR and Western blot assays were performed to check the effect of UBE2T silencing in HCC cells. Cell growth in vitro and in vivo was analyzed by multiparametric high-content screening and the xenograft tumorigenicity assay, respectively. Cell cycle distribution and apoptosis were determined by flow cytometry. The genes regulated by UBE2T were profiled by microarray assay. RESULTS: UBE2T was overexpressed in HCC tissues compared with paired and non-paired normal tissues. High expression of UBE2T predicted a poor overall survival in HCC patients. In vitro, lentivirus-mediated UBE2T knockdown significantly reduced the viability of both SMMC-7721 and BEL-7404 cells. In vivo, the xenograft tumorigenesis of SMMC-7721 cells was largely attenuated by UBE2T silencing. The cell cycle was arrested at G1/S phase in SMMC-7721 and BEL-7404 cells with UBE2T knockdown. Furthermore, apoptosis was increased by UBE2T knockdown. At the molecular level, numerous genes were dysregulated after UBE2T silencing, including IL-1B, FOSL1, PTGS2, and BMP6. CONCLUSION: UBE2T plays an important role in cell cycle progression, apoptosis, and HCC development.

MicroRNA-1305 Inhibits the Stemness of LCSCs and Tumorigenesis by Repressing the UBE2T-Dependent Akt-Signaling Pathway.

MicroRNAs (miRNAs) are involved in the maintenance of the cancer stem cell (CSC) phenotype by binding to genes and proteins that modulate cell proliferation and/or cell apoptosis. In our study, we aimed to investigate the role of miR-1305 in the proliferation and self-renewal of liver CSCs (LCSCs) via the ubiquitin-conjugating enzyme E2T (UBE2T)-mediated Akt-signaling pathway. Differentially expressed genes in human hepatocellular carcinoma (HCC) were obtained by in silico analysis. The relationship between miR-1305 and UBE2T was verified by dual luciferase reporter gene assay. qRT-PCR and western blot analysis were performed to determine the expression of UBE2T, the Akt-signaling pathway, and stemness-related factors in LCSCs. In addition, miR-1305 disrupted the activation of the Akt-signaling pathway by targeting UBE2T, and, ultimately, it repressed the sphere formation, colony formation, and proliferation, as well as tumorigenicity of LCSCs. In summary, miR-1305 targeted UBE2T to inhibit the Akt-signaling pathway, thereby suppressing the self-renewal and tumorigenicity of LCSCs. Those findings may provide an enhanced understanding of miR-1305 as a therapeutic target to limit the progression of LCSCs.