Forkhead Box M1 positively regulates UBE2C and protects glioma cells from autophagic death.
ABSTRACT: Ubiquitin-conjugating enzyme E2C (UBE2C) is characterized as a crucial molecule in cancer cell growth that plays an essential role in the development of gliomas, but the detailed mechanisms have not been fully elucidated. In this study, we found that Forkhead box transcription factor M1 (FoxM1) overexpression increased UBE2C expression, whereas FoxM1 suppression inhibited UBE2C expression in glioma cells. In addition, high FoxM1/UBE2C expression was significantly correlated with poor prognosis in glioma. We subsequently demonstrated that UBE2C was a direct transcriptional target of FoxM1, and site-directed mutations markedly down-regulated UBE2C promoter activity. Moreover, UBE2C siRNA (si-UBE2C) significantly induced glioma cell autophagy and increased both mCherry-LC3 punctate fluorescence and LC3B-II/LC3-I expression. Notably, the si-UBE2C-induced decrease in cell viability was markedly inhibited by the autophagy inhibitor bafilomycin A1. The silencing of UBE2C resulted in a distinct inhibition of the PI3K-Akt-mTOR pathway, which functions in the negative modulation of autophagy. Collectively, our findings provide clinical and molecular evidence that FoxM1 promotes glioma progression by enhancing UBE2C transcription and that the inhibition of UBE2C partially induces autophagic glioma cell death. Thus, targeting the FoxM1-UBE2C axis has therapeutic potential in the treatment of gliomas.
Project description:FOXM1 (forkhead box protein M1) is a transcription factor that participates in all stages of tumor development, mainly through the control of cell cycle and proliferation, regulating the expression of genes involved in G1/S and G2/M transition and M phase progression. The ubiquitin conjugating enzyme E2 (UBE2C) is a member of the anaphase promoting complex/cyclosome, promoting the degradation of several target proteins along cell cycle progression, during metaphase/anaphase transition. FOXM1 and UBE2C have been found overexpressed in a wide range of different solid tumors. Therefore, the aim of this study was to investigate whether UBE2C is a transcriptional target of FOXM1, using esophageal squamous cell carcinoma (ESCC) as a model, in addition to several cancer-deposited data. Our results show that FOXM1 and UBE2C expression present a positive correlation in normal tissues and in 25 distinct tumor types, including ESCC, where these genes are overexpressed. Moreover, FOXM1 binds to UBE2C promoter region in ESCC cell line and transcriptionally activates it, leading to UBE2C upregulation. In conclusion, this study provides evidences that FOXM1 transcriptionally regulates UBE2C expression in ESCC and their deregulation may be a general phenomenon in human neoplasias.
Project description:Ubiquitination is one of the main post-translational modification of proteins. It plays key roles in a broad range of cellular functions, including protein degradation, protein interactions, and subcellular location. In the ubiquitination system, different proteins are involved and their dysregulation can lead to various human diseases, including cancers. By using data available from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases, we here show that the ubiquitin conjugating enzyme, E2C (UBE2C), is overexpressed in all 27 cancers we investigated. UBE2C expression is significantly higher in late-stage tumors, which might indicate its involvement in tumor progression and invasion. This study also revealed that patients with higher UBE2C levels showed a shorter overall survival (OS) time and worse OS prognosis. Moreover, our data show that UBE2C higher-expression leads to worse disease-free survival prognosis (DFS), indicating that UBE2C overexpression correlates with poor clinical outcomes. We also identified genes with positive correlations with UBE2C in several cancers. We found a number of poorly studied genes (family with sequence similarity 72-member D, FAM72D; meiotic nuclear divisions 1, MND1; mitochondrial fission regulator 2, MTFR2; and POC1 centriolar protein A, POC1A) whose expression correlates with UBE2C. These genes might be considered as new targets for cancers therapies since they showed overexpression in several cancers and correlate with worse OS prognosis.
Project description:An increased expression of UBE2C (Ubiquitin-conjugating enzyme E2C) has been associated with high tumor grade and cancer progression. It is an essential indicator of the mitotic destruction events. Our microarray study on cervical cancers showed UBE2C to be over expressed in cervical cancer. Subsequent studies from our laboratory, showed that inhibition of UBE2C can enhance radiation and chemosensitivity. Therefore it can be an appropriate target for drug development to identify potential and specific inhibitor of cancer. To identify small molecule inhibitors, a computational approach was used to model UBE2C and further docking studies were carried out. Different ligand subsets such as ChemBank, PDB, KEGG, Drug-likeness NCI, Not annotated NCI of ligand library ligands were downloaded and docked with UBE2C. Schrodinger tools were used for identifying active sites and docking studies of ligands with UBE2C. Based on glide score, the potential ligands were screened and its interaction with UBE2C was identified. We also analyzed the drug like properties such as absorption, distribution, metabolism, excretion and toxicity (ADME/T) of docked compounds. Our results suggest that 2,4-diimino-1-methyl-1,3,5-triazepan-6-one, sulfuric acid compound with 5,6-diamino-2,4-pyrimidinediol (1:1) and 7-alpha-d-ribofuranosyl-2-aminopurine-5'-phosphate may act as best inhibitors and further in vitro studies, may lead to development of novel and best inhibitor of UBE2C.
Project description:The roles of aberrantly regulated autophagy in human malignancy and the mechanisms that initiate and sustain the repression of autophagy in carcinogenesis are less well defined. Activation of the oncogene UBE2C and repression of autophagy are concurrently underlying the initiation, progression, and metastasis of lung cancer and exploration of essential association of UBE2C with autophagy will confer more options in searching novel molecular therapeutic targets in lung cancer. Here we report that aberrant activation of UBE2C in lung tumors from patients associates with adverse prognosis and enhances cell proliferation, clonogenicity, and invasive growth of NSCLC. UBE2C selectively represses autophagy in NSCLC and disruption of UBE2C-mediated autophagy repression attenuates cell proliferation, clonogenicity, and invasive growth of NSCLC. Autophagy repression is essentially involved in UBE2C-induced cell proliferation, clonogenicity, and invasive growth of NSCLC. Interference of UBE2C-autophagy repression axis by Norcantharidin arrests NSCLC progression. UBE2C is repressed post-transcriptionally via tumor suppressor miR-381 and epitranscriptionally stabilized with maintenance of lower m<sup>6</sup>A level within its mature RNAs due to the upregulation of m<sup>6</sup>A demethylase ALKBH5 in NSCLC. Collectively, our results indicated that deregulated UBE2C-autophagy repression axis drives NSCLC progression which renders varieties of potential molecular targets in cancer therapy of NSCLC.
Project description:Substantial evidence implicates the ubiquitin-conjugating enzyme E2C (UBE2C) gene, in several human cancers, including colorectal carcinoma (CRC). We therefore investigated the prognostic value of UBE2C alterations in CRC and UBE2C signaling in CRC cell lines. UBE2C protein expression and UBE2C gene copy number were evaluated on clinical samples by immunohistochemistry and fluorescence in situ hybridization in a TMA format. The effect of the proteasome inhibitor bortezomib and small-interfering RNA knockdown was assessed by apoptotic assays and immunoblotting. UBE2C dysregulation was associated with proliferative marker Ki-67, accumulation of cyclin A and B1, and a poor overall survival. UBE2C expression was an independent prognostic marker in early-stage (I and II) CRC. UBE2C depletion resulted in suppression of cellular growth and accumulation of cyclin A and B1. In vitro, bortezomib treatment of CRC cells caused inhibition of cell viability via down-regulation of UBE2C. UBE2C knockdown by bortezomib or transfection with specific small-interfering RNA against UBE2C also caused cells to be arrested at the G2/M level, leading to accumulation of cyclin A and cyclin B1. In vivo, a significant reduction in tumor volume and weight was noted in mice treated with a combination of subtoxic doses of oxaliplatin and bortezomib compared with treatment with oxaliplatin or bortezomib alone. Altogether, our results suggest that UBE2C and the ubiquitin-proteasome pathway may be potential targets for therapeutic intervention in CRC.
Project description:The esophageal squamous cell carcinoma (ESCC) is widely known as a highly lethal and poor understood cancer, then requiring the search for novel molecular markers to improve its management and patients survival. Recently, ubiquitin-conjugating enzyme E2C (UBE2C) has been figuring as a prominent tumor biomarker candidate, once it has been recognized as a key player in cell cycle progression. In this way, the aim of this study was to evaluate the expression profile of UBE2C gene and protein in ESCC samples, as well as its diagnostic and prognostic marker potential, and its contribution to ESSC genesis and/or progression by performing in vitro functional assays. The analysis of UBE2C gene expression in 52 paired ESCC samples (tumor and respective histologically normal surrounding tissue), by qRT-PCR, revealed that this gene is overexpressed in 73% of ESCC samples. Subsequently, immunohistochemical analysis confirmed that UBE2C protein expression was upregulated in all ESCC cases, but absent in the histologically normal tumor surrounding tissues. Moreover, we showed that UBE2C mRNA expression was able to accurately discriminate ESCC tissue from both healthy esophageal and histologically normal tumor surrounding tissues, pointing out its role as a diagnostic marker for this cancer. Finally, we report that UBE2C affects proliferation rates and cell cycle profile of ESCC cell lines, by directly interfering with cyclin B1 protein levels, suggesting its involvement in crucial steps of ESCC carcinogenesis.
Project description:BACKGROUND:Gliomas are the most common primary tumors in central nervous system. Despite advances in diagnosis and therapy, the prognosis of glioma remains gloomy. Autophagy is a cellular catabolic process that degrades proteins and damaged organelles, which is implicated in tumorigenesis and tumor progression. Autophagy related 4C cysteine peptidase (ATG4C) is an autophagy regulator responsible for cleaving of pro-LC3 and delipidation of LC3 II. This study was designed to investigate the role of ATG4C in glioma progression and temozolomide (TMZ) chemosensitivity. METHODS:The association between ATG4C mRNA expression and prognosis of gliomas patients was analyzed using the TCGA datasets. The role of ATG4C in proliferation, apoptosis, autophagy, and TMZ chemosensitivity were investigated by silencing ATG4C in vivo. Ectopic xenograft nude mice model was established to investigate the effects of ATG4C on glioma growth in vivo. RESULTS:The median overall survival (OS) time of patients with higher ATG4C expression was significantly reduced (HR: 1.48, p = 9.91 × 10- 7). ATG4C mRNA expression was evidently increased with the rising of glioma grade (p = 2.97 × 10- 8). Knockdown ATG4C suppressed glioma cells proliferation by inducing cell cycle arrest at G1 phase. ATG4C depletion suppressed autophagy and triggered apoptosis through ROS accumulation. Depletion of ATG4C suppressed TMZ-activated autophagy and promoted sensitivity of glioma cells to TMZ. Additionally, ATG4C knockdown suppressed the growth of glioma remarkably in nude mice. CONCLUSION:ATG4C is a potential prognostic predictor for glioma patient. Targeting ATG4C may provide promising therapy strategies for gliomas treatment.
Project description:Although early stage ovarian cancer is in most cases a curable disease, some patients relapse even with appropriate adjuvant treatment. Therefore, the identification of patient and tumor characteristics to better stratify risk and guide rational drug development is desirable. Using transcriptomic functional annotation followed by protein-protein interacting (PPI) network analyses, we identified functions that were upregulated and associated with detrimental outcome in patients with early stage ovarian cancer. Some of the identified functions included cell cycle, cell division, signal transduction/protein modification, cellular response to extracellular stimuli or transcription regulation, among others. Genes within these functions included AURKA, AURKB, CDK1, BIRC5, or CHEK1 among others. Of note, the histone-lysine N-methyltransferase (EZH2) and the ubiquitin-conjugating enzyme E2C (UBE2C) genes were found to be upregulated and amplified in 10% and 6% of tumors, respectively. Of note, EZH2 and UBE2C were identified as principal interacting proteins of druggable networks. In conclusion, we describe a set of genes overexpressed in ovarian cancer with potential for therapeutic intervention including EZH2 and UBE2C.
Project description:Forkhead box M1 (FoxM1) is a member of the forkhead transcription factor family and is overexpression in malignant gliomas. However, the molecular mechanisms by which FoxM1lead to glioma carcinogenesis and progression are still not well known. In the present study, we show that Anxa1 was overexpression in gliomas and predicted the poor outcome. Furthermore, Anxa1 closely related to the FoxM1 expression and was a direct transcriptional target of FoxM1. Overexpression of FoxM1 up-regulated Anxa1 expression, whereas suppression of FoxM1 expression down-regulated Anxa1 expression in glioma cells. Finally, FoxM1 enhanced the proliferation, migration, and angiogenesis in Anxa1-dependent manner both in vitro and in vivo. Our findings provide both clinical and mechanistic evidences that FoxM1 contributes to glioma development by directly up-regulating Anxa1 expression.
Project description:Background: Recent evidence indicates that UBE2C participates in carcinogenesis by regulating the cell cycle, apoptosis, metastasis, and transcriptional processes. Additionally, miR-548e-5p dysregulation plays a vital role in tumor progression. However, the molecular mechanism via which UBE2C is directly targeted by miR-548-5p, resulting in increase in cellular growth and invasiveness of cancer cells, and its interactions with the epithelial-mesenchymal transition (EMT) marker protein ZEB1/2 in non-small cell lung cancer (NSCLC) is not understood. Methods: Expression of UBE2C and miR-548e-5p was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The protein level of UBE2C and ZEB1/2 was analyzed using western blotting and immunofluorescence staining. Cellular proliferation was detected using the cell counting kit 8 (CCK8) and 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell migration, invasion, and growth were analyzed using the wound healing and transwell assay. Promoter activity and transcription was analyzed using the luciferase reporter assay. Chromatin immunoprecipitation was used to detect binding of UBE2C to 5'UTR-ZEB1/2. Results: We observed that 4,5-ubiquitin-conjugating enzyme E2C (UBE2C) expression was higher in NSCLC tissue than in the adjacent normal tissue and was associated with increased cell proliferation and invasion. UBE2C enhanced NSCLC progression and metastasis by affecting the cell cycle and inhibiting apoptosis. We also observed that miR-548e-5p was significantly downregulated in lung cancer tissue specimens, which decreased the expression of its direct substrate, UBE2C. Moreover, miR-548e-5p overexpression and UBE2C under-expression significantly suppressed lung cancer cell proliferation, migration, and invasion. Luciferase reporter and chromatin immunoprecipitation assays indicated that miR-548e-5p directly binds to the 3'-UTR of UBE2C and decreases UBE2C mRNA expression. Furthermore, UBE2C knockdown downregulated the mesenchymal marker vimentin and upregulated the epithelial marker E-cadherin. Bioinformatics assays, coupled with western blotting and luciferase assays, revealed that UBE2C directly binds to the 5'-untranslated region (UTR) of the transcript of the E-cadherin repressor ZEB1/2 and promotes EMT in lung cancer cells. Conclusion: miR-548e-5p directly binds to the 3'-UTR of UBE2C and decreases UBE2C mRNA expression. UBE2C is an oncogene that promotes EMT in lung cancer cells by directly targeting the 5'-UTR of the transcript encoding the E-cadherin repressor ZEB1/2. miR-548e-5p, UBE2C, and ZEB1/2 constitute the miR-548e-5p-UBE2C-ZEB1/2 signal axis, which enhances cancer cell invasiveness by directly interacting with e EMT marker proteins. We believe that the miR-548e-5p-UBE2C-ZEB1/2 signal axis may be a suitable diagnostic marker and a potential target for lung cancer therapy.