BRD4 Regulates Self-renewal Ability And Tumorigenicity Of Glioma Initiating Cells By Enrichment In The Notch1 Promoter Region

Background:Glioblastoma(GBM)is the most familiar and fatal primary tumor of the central nervous system.Based on maximal surgical resection,even as targeted therapy and immunotherapy have made progress,GBMs still have a poor prognosis.Due to the complexity caused by intratumoral molecular heterogeneity,targeted or customized therapies targeting specific subtypes or mutations mostly failed.Most tumors become resistant to treatment and quickly recur.Diverse studies have manifested the existence of a small subpopulation of immature,undifferentiated tumor cells with tumor-initiating capacity in many types of cancer,includes GBM.These cells called glioma initiating cells(GICs),also called glioma stem like cells,which commit self-renewal,perpetual proliferation,multi-directional differentiation,and vigorous tumorigenicity,sustain the peculiar genetic and epigenetic diversification in the GBM patients.Importantly,the resistance to chemotherapy and radiotherapy of GICs,which ultimately leads to tumor recurrence,recommends that GICs can be definitely targeted during therapy to diminish the jeopardy of tumor recurrence.Bromodomain and extraterminal domain(BET)family proteins are epigenetic readers that administer gene expression via recognizing acetylated lysine residues on histone and non histone chromatin factors.BRD4 is the most researched member of this family and is associated with multiple human cancers.In cancer stem cells,several studies mentioned that targeting BRD2 and BRD4 inhibited GIC proliferation and BRD4-depleted induced GIC apoptosis.However,it is not so clear that the molecular mechanism by which BET protein take part in GIC processes.Purpose:This study aimed to investigate the relationship between BET family proteins and GICs,investigate the effects of BET family proteins on the self-renewal and tumorigenicity of GICs and explore the molecular mechanisms involved,and provide a new theoretical basis for clinical translational therapy targeting GICs.Methods:1.Based on the bioinformatics analysis of glioma patients’ data in TCGA and CGGA databases,the relationship between the mRNA expression levels of BET family proteins BRD2,BRD3 and BRD4 and the prognosis of glioma patients was evaluated,and BRD4 expression in different WHO grades,pathological types and GBM molecular subtypes was analyzed.BRD4 expression in GBM tissues was detected by Western blot and immunohistochemistry.2.By CCK8,single cell neurosphere formation and limiting dilution assay,the effects of BET proteins inhibition on self-renewal and tumorigenicity of GICs were detected after interference of BET inhibitor I-BET151 and shBRD2/BRD3/BRD4.The expressions of stem cell related proteins Nestin,CD 133 and Sox2 were detected by Western blot and immunofluorescence staining.To evaluate the effect of I-BET151 on the tumorigenicity of GICs,the bioluminescence imaging,survival analysis,HE and immunohistochemistry.3.Western blot and immunofluorescence staining were used to detect the expression of Notch1,NICD and Hes1 after BRD2,BRD3 and BRD4 inhibition.The enrichment of BRD4 in Notch1 promoter region was detected by immunoprecipitation(CHIP)-qPCR.Notch 1 mRNA expression was detected by qPCR after BRD4 inhibition.After sequencing the whole genome of U251 GICs,CHIP-Seq data was processed by UCSC database visualization,the enrichment of BRD4 in Notch1 promoter region was observed.Based on CHIP-Seq data,GO(gene ontology)analysis and KEGG pathway analysis were used to predict the biological process of BRD4 in GICs.4.Based on establishment of GBM cells overexpressing BRD4,the effects of Notch1 pathway inhibition on the self-renewal and tumorigenicity of GICs were detected by CCK8,single cell Neurosphere formation and limiting dilution assay.Western blot was used to analyze the effects the expression of stem cell related protein and Notch1 pathway related protein.The correlation of BRD4 with stem cell related protein and Notch1 pathway related protein was analyzed by bioinformatics.Results:1.TCGA and CGGA data showed that BRD4 mRNA expression was closely related to the overall survival of glioma patients.The higher the BRD4 expression level,the worse the prognosis of glioma patients.BRD2 and BRD3 data did not support this view.BRD4 expression was the highest in WHO grade Ⅳ GBM patients,and also the highest in the GBM classical subtypes.Based on GBM and normal tissue samples,the expression of BRD4 in GBM was higher than that of normal tissue.2.Single cell neurosphere formation assay showed that I-BET151 could effectively block the formation of GIC spheroidization.The number and size of spheroidization in U87,U251 and primary cells were measured and quantified to prove the effectiveness of I-BET151 treatment.CCK8 test confirmed that I-BET151 reduced GIC proliferation.In limiting dilution assay,with the increase of I-BET151 concentration,the proportion of non spheroidization cells increased.I-BET151 inhibited the expression of stem cell related proteins;these results suggest that targeted BET protein inhibited the self-renewal ability of GICs.Further shRNA data suggest that BRD4 is responsible for the regulation of GIC self-renewal.In vivo bioluminescence imaging and HE staining showed that the tumor volume was smaller and the survival of mice was longer after treatment with I-BET151.Immunohistochemistry results showed that the expression of stem cell related protein and Notch1 pathway related protein decreased after treatment with I-BET151.These results indicate that I-BET151 inhibits the tumorigenicity of GICs.3.Western blot and immunofluorescence staining showed that the expression of Notch1 pathway related proteins decreased after BRD4 was inhibited,BRD2 and BRD3 data did not support this view.After inhibition of BRD4,the results of CHIP-qPCR showed that BRD4 protein was less enriched in Notch1 promoter region.qPCR results showed that BRD4 inhibition reduced Notch 1 mRNA expression.After CHIP-Seq data was processed by UCSC database visualization,it showed that BRD4 was significantly enriched in Notchl promoter region.Through GO and KEGG analysis of BRD4 significantly enriched genes,it was found that BRD4 was related to the metabolism process of GICs.4.In BRD4 overexpression cells,inhibition of Notchl pathway can effectively block the formation of GIC neurospheres.CCK8 assay confirmed that inhibition of Notchl pathway decreased the proliferation of GIC.In the limiting dilution assay,inhibition of Notchl pathway increased the proportion of non spheroids.Similarly,inhibition of Notchl pathway decreased the expression of stem cell-related proteins.These results indicated that Notchl pathway inhibition could inhibit the proliferation of GIC.It is suggested that targeting Notchl pathway can inhibit the self-renewal ability of GICs even if BRD4 is overexpressed.Conclusions:Our findings put forward a novel glioma treatment strategy.Our findings demonstrate that BRD4 is enriched at the promoter region of Notchl,and that BRD4 inhibition is capable of downregulating self renewal and tumorigenicity in GICs.This can be a promising strategy for beating GICs.Thus,studies of molecular targeting and/or signaling pathways that target BRD4 and modulate Notch1 promoter region transcription can be performed further on as they might facilitate the development of an appropriate approach for treating GBMs.