The Roles And Molecular Mechanisms Of CUL4B In Regulating Therapeutic Effect Of Temozolomide On Glioma

Glioma is the most common type of primary tumor of the central nervous system.According to the criteria of the World Health Organization(WHO),the majority of gliomas can be classified into four grades of malignancy(?-?).Grade IV gliomas are the most aggressive,also known as Glioblastoma Multiforme(GBM).Adult GBM accounts for 56.6%of all gliomas and 48%of all central nervous system malignancies.As one of the most lethal malignancies with a 5-year survival rate of just under 5%,GBM represents a great clinical challenge.Current standard care for GBM includes maximal safe surgical resection followed by radiotherapy and chemotherapy.The initial treatment for GBM is surgical resection.However,complete removal of the tumor is very difficult due to the diffuse and infiltrative growth pattern of the tumor.Following surgery,patients receive radiotherapy and temozolomide(TMZ)chemotherapy,concomitant and later as adjuvant chemotherapy,which improved the median,2-year,and 5-year survival rates in newly diagnosed GBM patients.However,the overall clinical efficacy was disappointing due to the inherent or therapy-induced resistance of tumor cells to TMZ.The recurrence of glioblastoma was almost inevitable.Chemoresistance to TMZ is a vital factor of glioma recurrence.Several studies had shown that TMZ resistance was not mediated by a single molecule or pathway,but by the involvement of multiple molecules or signaling pathways.CUL4B acts a scaffold protein of the Cullin 4B-RING E3 ubiquitin ligases(CRL4Bs)complex,belonging to a member of the Cullin gene family.The main roles of CRL4B are to ubiquitinate specific substrates to promote their degradation,and to act as epigenetic regulators by ubiquitinating histones to regulate histone methylation,acetylation,and DNA methylation.CRL4B participates in a broad variety of physiological processes,including cell cycle,cell signal transduction,gene transcription and embryonic development.The expression of CUL4B is abnormal in a wide variety of diseases.On the one hand,the mutations in the human CUL4B gene causes intellectual disability syndrome.On the other hand,CUL4B is overexpressed in a variety of solid tumors and plays an oncogenic role by transcriptionally inhibiting the expression of a group of tumor suppressor genes.It is worth pointing out that CUL4B acts as a tumor suppressor through limiting the accumulation and activation of MDSCs,an immunosuppressive component in the tumor microenvironment.CUL4B plays a very important role in the development of tumors.Though CUL4B was reported to promote the proliferation and invasion of glioma cells,the relationship between CUL4B expression and clinical prognosis was still unclear.In particular,its regulatory effect and mechanisms on TMZ resistance have not been elucidated.The aim of this study was to decipher the role and mechanism of CUL4B in TMZ therapeutic effect on gliomas.The main finding was as the following:Part?CUL4B was overexpressed in gliomas and correlated with poor prognosisFirst,after analyzing the expression of CUL4B in 29 pairs of glioma tumor tissues and adjacent non-tumor tissue samples by Western blot,we found that CUL4B expression was significantly upregulated in gliomas,and the expression of CUL4B was significantly increased in high-grade gliomas compared with low-grade gliomas.Through further analysis of mRNA expression of CUL4B in public databases such as TCGA,Rembrandt,CGGA and GEO,we validated the findings that the expression level of CUL4B was significantly increased in glioma compared with non-tumor tissues,that CUL4B increased with tumor malignancy,and that the overexpression of CUL4B was associated with poor prognosis.Through bioinformatic analysis,we found that in the GBM patients with TMZ treatment,the overall survival of patients with high CUL4B expression was significantly lower than that of patients with low CUL4B expression.Compared with primary gliomas,the expression of CUL4B in gliomas was significantly increased in recurrent gliomas.Through the analysis of the molecular subtypes of GBMs,the expression of CUL4B in mesenchymal subtypes was significantly increased.The analysis of GBM sub-anatomical location showed that CUL4B was highly expressed in GBM microvascular proliferation zone.After further analysis of several factors affecting TMZ resistance of gliomas,we found that the expression of CUL4B was independent of the methylation status of the O6-methylguanine-DNA methyltransferase(MGMT)promoter.Part?CUL4B attenuated TMZ sensitivity in glioma cellsWe further experimentally verified the relationship between CUL4B expression and TMZ resistance in gliomas in vivo and in vitro.After TMZ treatment,the expression of CUL4B was upregulated in glioma cells.Analysis of the expression of CUL4B protein in 6 types of glioma cells showed that CUL4B was low in TMZ-sensitive glioma cells,while CUL4B was highly expressed in TMZ-resistant glioma cells.The expression of CUL4B was negatively correlated with TMZ sensitivity.Bioinformatics analysis in pubic databases showed that the expression of CUL4B in 17 types of glioma cells confirmed the above results.For further validation the effect of CUL4B expression on TMZ sensitivity,using the lentivirus transfection,we constructed glioma cells with CUL4B knockdown or overexpression.We found that CUL4B knockdown enhanced TMZ sensitivity,and TMZ sensitivity in CUL4B-overexpressing glioma decreased.Compared to xenograft of control cells,xenograft with reduced CUL4B exhibited higher sensitivity to TMZ.Part ?The mechanism of CUL4B in regulating TMZ sensitivity in gliomaWe explored the molecular mechanism of CUL4B in regulating TMZ sensitivity in glioma.Through senescence-related ?-Gal staining,we found that TMZ induced senescence of glioma cells.Reduced expression of CUL4B enhanced the TMZ-induced cellular senescence.The cell cycle analyses demonstrated that compared to control cells,more cells with reduced CUL4B were arrested in S phase upon TMZ treatment.Overexpression of CUL4B attenuated TMZ-induced cellular senescence.Cell cycle assays showed that the cell cycle of CUL4B-overexpressing gliomas did not change significantly.These results suggested that CUL4B promoted TMZ resistance of glioma cells by inhibiting senescence.After TMZ treatment,the expression of p21 protein in glioma cells increased,and the level of p21 protein in CUL4B knocking down cells increased more significantly than that in control cells.The results of immunohistochemical experiments also showed that after TMZ treatment,the expression of p21 significantly increased in glioma cells with low expression of CUL4B.Transfection of p21 expression plasmid into glioma cells overexpressing CUL4B effectively restored TMZ-induced cell senescence.These results suggested that CUL4B inhibit TMZ induced senescence of glioma cells by negatively regulating the expression of p21 protein in glioma cells.Real-time quantitative PCR(qRT-PCR)confirmed that reduction of CUL4B up-regulated the expression of p21 at the transcriptional level.After TMZ treatment,the up-regulation of p21 mRNA in glioma cells with low expression of CUL4B was more significant.The ChIP experiment demonstrated that CUL4B and HD AC 1/3 bound to the promoter of CDKN1A.The qRT-PCR results showed that similar to inhibiting the expression of CUL4B,HDAC inhibitors also promoted the transcription of CDKV1A.These results suggest that CUL4B and HDAC1/3 coacted together to inhibit the transcription of CDKN1A.These results collectively suggested that coordinated with HADC1/3,CUL4B directly bound to the promoter of CDKN1A to repress its transcriptional activity to inhibit senescence.In summary,we found that CUL4B was highly expressed in gliomas,positively related to the malignancy,poor prognosis of the tumor and TMZ chemoresistance.CUL4B promoted TMZ resistance of glioma cells,inhibited TMZ-induced cell senescence,and inhibited S phase arrest of the cell cycle.Together with HD AC,CUL4B bound to the CDKN1A promoter,and inhibited the transcription of CDKN1A through epigenetic regulation.This study will greatly enrich our understanding of the role of CUL4B in the regulation of TMZ therapeutic effect in gliomas,and provide a new therapeutically beneficial strategy to discover novel drug targets for the glioma treatment.