Study Of The Function And Underlying Mechanism Of E3 Ligase HUWE1 In Glioblastoma Progression

Among various types of tumors,malignant brain tumor is the most daunting one,not only because of its poor prognosis,but also because it seriously affects quality of patient's life and cognitive function.Epidemiological studies found that a total of 138,054 patients in the United States were diagnosed with primary malignant brain tumor in 2010,among which malignant glioma accounted for the majority(about 80%).Globally,the annual incidence of glioblastoma(GBM)is 5.25 per 100,000,and approximately 17,000 people are diagnosed with GBM each year.Despite the combined treatment of surgery,radiotherapy and chemotherapy for GBM treatment in recent years,the median survival of patients has not improved significantly.Currently,temozolomide(TMZ)is used as a first-line chemotherapy drug to treat GBM in clinical treatment,However,due to the presence of blood-brain barrier,low methylation rate of gene promoter of 06-methylguanine DNA methyltransferase and other factors,the therapeutic effect of GBM with temozolomide is unsatisfactory,so it is urgent to find new targets for GBM treatment.Ubiquitin proteasome system(USP)plays an important role in eukaryocytes and it is widely involved in all aspects of cell function.The dysregulation of ubiquitination process will lead to serious consequences,including inflammation,metabolic disorders and even tumors.The imbalance between "oncoprotein" and "tumor suppressor protein" caused by the abnormal regulation of USP may promote the occurrence and development of GBM.E3 ligases play a key role in the USP process,specifically identifying substrates and mediating ubiquitin delivery leading the degradation or functional regulation of substrates.Therefore,it is particularly important to focus attention on E3 ligases and identify them as the novel therapeutic targets in treating GBM.HUWE1 is a member of the HECT E3 ubiquitin ligase with a huge molecular weight of 482 KD.Since the full length of HUWE1 gene was cloned in 2005,it enables us do a comprehensive and in-depth study of the function of it.HUWE1 contains the DUF908 and DUF913 domains,ubiquitin-associated domain(UBA)and the BH3 domain which is similar to the structure of the Bcl-2 family.The WWE domain on HUWE1 is involved in regulating ubiquitin-dependent protein degradation.Although the polyubiquitin chains synthesized by E3 ligases with HECT domain are mostly ubiquitin-site biased,HUWE1 can mediate the formation of polyubiquitin chains connected by K48,K63 and K6 sites,and also mediate mono-ubiquitination and Multi-sites ubiquitination of substrates to regulate cellular functions in a non-protein degradation pattern.HUWE1 can mediate the degradation of Mcl-1?P53?C-Myc?Cdc6?Dvl?Atoh1?HDAC2 and so on in different cell contexts.The role of HUWE1 in tumor progression is controversial due to the fact that its substrate contains both "oncoprotein" and "tumor suppressor protein".On the other hand,HUWE1 is highly expressed in colorectal cancer,gastric cancer,prostate cancer,breast cancer and ovarian cancer,suggesting that it may promote the occurrence and development of these cancers.However,HUWE1 is significantly lowly expressed in renal clear cell carcinoma,teratoma and head/neck tumors,suggesting that it may inhibit tumor progression in these types of cancer.Nevertheless,more and more research on HUWE1 does not make the function of it properly explained,but makes it more complicated.Therefore,the elaboration of the role of HUWE1 in specific types of tumors will have important significance for the interpretation of the occurrence and development of this tumor.However,there is no information about the functional and clinical value of HUWE1 in GBM.Therefore,it's necessary to explore the role of HUWE1 in the progression of GBM.In this work,we utilized bioinformatics method to identify HUWE1 through the conjoint analysis of TCGA,Rembrandt and Gravendeel databases in GBM.The techniques of Transwell invasion and migration assays,RNAi,PCR,Western blot,immunohistochemical(IHC),immunofluorescence(IF),Chromatin immunoprecipitation(CHIP),RNA-seq,Duo-link assay,co-immunoprecipitation(CO-IP),CRISPR synergistic activation mediator(SAM),dual luciferase reporter gene,in vivo xenograft experiments were used to explore the roles of HUWE1 on GBM cells and the underlying mechanisms.This study was divided into four parts.Firstly,we screened and identified E3 ligase HUWE1 and detected its expression in GBM cells and patients samples and then we predicted the role of HUWE1 in GBM by bioinformatics methods.Secondly,we testified and verified the role of HUWE1 in GBM cells in vitro and in vivo.Thirdly,we explored the underlying mechanism of HUWE1 in GBM cells.At last,we utilized SAM mediated by dCas9 to force expression of HUWE1 and demonstrated that it markedly suppressed GBM progression in vitro and in vivo.The main results and conclusions are as the followings:1.Cconjointly analyzing the gene expression profile of GBM patients with Rembrandt and Gravendeel databases and identifies E3 ligase HUWE1.It was found HUWE1 was significantly decreased in the tissue samples and paraffin sections of GBM patients and patients with high expression of HUWE1 had better prognosis.2.By utilizing GSEA and GO Function Enrichment of TCGA,Rembrandt,Gravendeel database and RNA-seq gene profiles,we found that HUWE1 is related to the migration,invasion,proliferation of GBM.3.Knockdown of HUWE1 significantly increased the migration,invasion,proliferation and intracranial tumorigenesis of GBM cells confirming by in vitro cell experiments and in situ tumor transplantation in GBM intracranial tumor experiments.4.HUWE1 mediates the ubiquitination degradation of N-Myc and N-Myc attached polyubiquitin chain formation is dependent on ubiquitin K6 and K48 sites.N-myc can directly bind and transcriptionally activate the promoter region of DLL1 for transcriptional activation and increased DLL1 activates the downstream Notch1 signaling pathway.5.The endogenous overexpression of HUWE1 was successfully achieved by using dCas9-SAM and it was confirmed that overexpression of HUWE1 can significantly suppress the migration,invasion,proliferation of GBM cells.In summary,the main conclusions of this study are as the followings:1.E3 ligase HUWE1 significantly decreases in GBM and has important prognostic value for the survival outcome of patients with GBM.2.HUWE1 can significantly suppress the migration,invasion and proliferation of GBM cells.3.HUWE1-N-Myc-DLL1-Notch1 axis mediates the suppression effects of HUWE1 on GBM.4.Overexpression of HUWE1 by using dCas9-SAM can significantly suppress the migration,invasion,proliferation of GBM cells.The above results will provide theoretical and experimental basis for elucidation of the mechanism of HUWE1 in the occurrence,progression of GBM,as well as the the role and molecular mechanism of ubiquitin proteasome system in the occurrence and development of GBM.For the first time,HUWE1 was overexpresses by dCas9-SAM and the antitumor effect of HUWE1 on GBM was confirmed,providing a new target for gene therapy strategies against GBM.