CBX3 Accelerates Glioblastoma Progression By Stabilizing EGFR Expression

Glioblastoma(GBM)is a common neuroepithelial tumor in the adult central nervous system,by the world health organization(WHO)classified as Ⅳ grade glioma,is the most malignant primary brain tumor and the most deadly solid tumor.The prognosis of GBM patients is extremely poor,with a median survival of only 14 months after diagnosis.The cause of GBM is unknown in most patients,and the only known cause of GBM is ambient ionizing radiation.Although surgery,radiotherapy and chemotherapy have made great progress,they still cannot effectively improve the survival of patients.The 5-year survival rate of GBM patients is only 5.1%,so it is urgent to screen and identify effective biomarkers,explore the therapeutic targets of GBM and clarify the pathogenesis of GBM,so as to ensure the long-term survival of GBM patients.CBX3,also known as HP1γ,is a core member of the heterochromatin protein 1(HP1)family and a highly conserved multifunctional protein.It contains a CD domain(Chromodomain)that specifically recognizes the H3K9me2/3 site.Therefore,CBX3 is also known as a histone methylation reader and performs the function of transcriptional silencing.According to research,CBX3 has played an important role in the occurrence and development of many types of tumors,including colorectal cancer,pancreatic cancer,lung cancer,and hepatocellular carcinoma.However,the biological role and molecular regulatory mechanism of CBX3 in GBM have not been reported.In this paper,we will use molecular pathology and molecular biology and other experimental methods to focus on exploring the fine regulatory mechanisms of CBX3 in GBM cell proliferation,migration and invasion,and tumorigenesis,to provide new diagnostic ideas for targeted therapy of GBM.The main research results obtained are as follows:(1)High expression of CBX3 is associated with poor prognosis in patients with GBMTo determine the role of CBX3 in GBM,we analyzed the online R2 clinical database and found that when CBX3 is highly expressed,the patient had a low survival rate and a poor prognosis.And the relationship between CBX3 and the grade of malignancy of glioma was analyzed,and it was found that the higher the grade of malignancy of glioma,the higher the expression of CBX3.This suggested that CBX3 might play an oncogene role in GBM.We examined the expression of CBX3 mRNA and protein in normal astrocytes and GBM cell lines,and found that the expression of CBX3 in GBM was significantly higher than that of normal astrocytes.We further found that the expression of CBX3 in tumor tissue samples was significantly higher than that in normal tissue samples by immunohistochemical staining and Western blotting.In summary,CBX3 was indeed highly expressed in GBM and is closely related to the prognosis of the patient,which also implied that high expression of CBX3 could be used as an indicator of poor prognosis in patients with GBM.(2)CBX3 regulates the proliferation,metastasis and tumorigenesis of GBM cellsTo explore the effect of CBX3 on the proliferation of GBM,we interfered with the expression of CBX3 in LN229 and U-87 MG cells by lentiviral-mediated RNAi technology,and the cell proliferation was significantly slowed by MTT and BrdU experiments.Flow cytometry was used to detect the cell cycle after CBX3 interference,and it was found that the cells were arrested in the S phase after CBX3 interference.When we knocked down CBX3,the expression of related cyclins was detected.As a result,it was found that related protein to the S phase process were significantly changed.We found that the tumorigenic capacity of CBX3-knockdown cell were significantly inhibited by soft agar,Subcutaneous and in situ neoplasia assay.To study the effect of CBX3 on the migration and invasion of GBM,we knocked down CBX3 in LN229 and U-87 MG cells and found that the cell migration ability was significantly weakened by Transwell and wound healing experiment.And we detected the expression of related cell mesenchymal markers(N-cadherin,β-catenin,Slug,MMP13).When we knocked down CBX3,the expression of these proteins were significantly down-regulated.To confirm that knockdown of CBX3 inhibited the proliferation and metastasis of GBM cells was not caused by off-target effect,we restored the expression of CBX3 in CBX3-knockdown LN229 and U-87 MG cells,and we found that restored the expression of CBX3 could rescue the inhibitory effect of CBX3 on GBM cells proliferation,metastasis and tumor formation.The above results indicated that CBX3 played an important role in the proliferation,metastasis and tumorigenicity of GBM cells.(3)CBX3 regulates EGFR signaling pathwayEGFR was highly expressed in GBM,and the downstream signaling pathway mediated by EGFR played an important role in tumor cell proliferation and migration.In order to further explore whether CBX3 had a regulatory effect on EGFR,we first detected that EGFR expression was significantly down-regulated in CBX3-knockdown LN229 and U-87 MG cells,but its mRNA level did not change.When overexpressed CBX3 in GBM cells,and we treated these cells and control cells with erlotinib(100 μM),an EGFR tyrosine kinase inhibitor.We found that erlotinib can significantly decreased the promotion effect of CBX3 on the proliferation,migration and invasion of GBM cells,and found that overexpression of CBX3 colud cause activation of EGFR and downstream pathways,but the addition of erlotinib could effectively inhibit the activation of EGFR signaling pathways.Since the mRNA level of EGFR did not change significantly in CBX3-knockdown GBM cells,we speculated that CBX3 regulated EGFR expression from the post-translational level.We treated cells with protease inhibitor(MG132)and protein synthesis inhibitor cycloheximide(CHX)and in vitro ubiquitination experiments was performed,the results revealed that CBX3 maintained EGFR stability by regulated the ubiquitination of its.We next analyzed the online R2 database to obtain a negative correlation between PARK2 、STUB1 and CBX3,with correlation indexes of-0.467 and-0.336,respectively.We tested whether CBX3 regulated the E3 ligase of EGFR(PARK2 and STUB1)at the transcription level.A chromatin immunoprecipitation(Ch IP)was performed.The ChIP results showed that CBX3 was enriched at the 33 to 261 and-452 to-211 on the PARK2 and STUB1 proximal promoter regions,respectively.Next,a co-IP was performed and found that CBX3 interacted with PARK2,but not bound with STUB1.Then we examined the effect of the interaction between CBX3 and PARK2 on PARK2.We treated GBM cells with protein synthesis inhibitor cycloheximide(CHX)and in vitro ubiquitination was performed and found that the interaction between CBX3 and PARK2 regulated the level of ubiquitination degradation of PARK2 protein.We down-regulated the expression of PARK2 or STUB1 in CBX3-knockdown LN229 and U-87 MG cells and revealed that restored the inhibitory effect of CBX3-knockdown on the development of GBM in vivo and vitro.These data verified that PARK2 and STUB1 are downstream effectors of CBX3.Therefore,the experimental results showed that CBX3 stabilized the activation of EGFR-mediated signaling pathways by regulating the expression of STUB1 and PARK2.Through our above experiments,we have found the precise regulation mechanism of CBX3 to regulate the process of glioblastoma.CBX3 not only transcriptionally inhibit the expression of E3 ubiquitin ligase PARK2 and STUB1 to protect the stability of EGFR,but also can regulate PARK2 ubiquitin degradation by interacting with PARK2 to further protect the stability of EGFR.In summary,CBX3 promotes the protection of the stability of EGFR,and then EGFR accelerates important cell processes through its downstream signaling pathways,such as cell proliferation,migration and invasion.