The Role And Mechanism Of TRIM59 In EGFR/EGFRvⅢ Regulated Glioblastoma Tumorigenesis And Progression

Section Ⅰ: TRIM59 Promotes Glioblastoma Tumorigenesis and Progression by Inhibiting TC45 Dephosphorylation of STAT3Objective: The pathogenesis factors and mechanisms of EGFR/EGFRvⅢ glioblastoma are unknown.This study was to investigate the role and mechanism of TRIM59 in the tumorigenesis and development of EGFR/EGFRvⅢ glioblastoma.Methods: Immunohistochemical staining was used to detect the expression level of TRIM59 in glioblastoma tissues and its relationship with prognosis of glioblastoma patients.Cell proliferation assay,soft agar colony formation assay and intracranial orthotopic transplantation tumor model to observe the effect of knockdown of TRIM59 on the biological behavior of EGFR/EGFRvⅢ glioblastoma.To detect the regulation of TRIM59 expression by EGFR/EGFRvⅢ through Western blot,real-time quantitative PCR and dual luciferase reporter gene assay.RNA-Seq and Gene enrichment analysis were used to detect the effect of knockdown of TRIM59 on the downstream signaling of EGFR/EGFRvⅢ.The molecular mechanism of TRIM59-mediated EGFR/EGFRvⅢ regulation of STAT3 pathway was explored by Western blot and co-immunoprecipitation.Results: The expression of TRIM59 in glioblastoma is significantly higher than that in normal and low-grade glioma tissues.The prognosis of glioblastoma patients with highexpression of TRIM59 was poor.Knockdown of TRIM59 significantly inhibited EGFR/EGFRvⅢ-promoting glioblastoma cells proliferation,clonal formation in vitro and tumor growth in vivo.EGFR/EGFRvⅢ transcriptionally up-regulated TRIM59 expression via SOX9.Knockdown of TRIM59 significantly inhibited STAT3 phosphorylation and expression of its target genes.EGFR activation promoted binding of TRIM59 to STAT3 in the nucleus,and the TRIM59-STAT3 association was critical for EGFR promoted glioblastoma cell proliferation,clonal formation in vitro and tumor growth in vivo.The association of TRIM59 and STAT3 competitively inhibited TC45 binding to STAT3,leading to inhibiting TC45 dephosphorylation of STAT3 and maintain STAT3 hyperphosphorylation level and activation of STAT3 pathway.Finally,the prognosis of patients with high expression of p-EGFR/TRIM59 or TRIM59/p-STAT3 was poor.Conclusion: TRIM59 could be used as a clinical marker for the prognosis of glioblastoma.TRIM59 competitively inhibits TC45 dephosphorylation of STAT3 and activates the STAT3 pathway,which is important for EGFR/EGFRvⅢ to promote glioblastoma tumorigenesis.Section Ⅱ: CDK5-dependent phosphorylation and nuclear translocation of TRIM59 promotes the tumorigenesis and development of EGFR/EGFRvⅢ glioblastomaObjective: To explore the mechanism of TRIM59 phosphorylation and nuclear translocation and to investigate the molecular mechanism of nuclear TRIM59 involved in the regulation of EGFR/EGFRvⅢ glioblastoma.Methods: Immunofluorescence assay and nucleoplasmic separation assay were used to detect the effect of EGFR/EGFRvⅢ on the nuclear translocation of TRIM59.Small molecular kinase inhibitors and nucleoplasmic isolation assays were used to screen for kinases that mediate EGFR activation-induced TRIM59 nuclear translocation.Immunofluorescence experiment and nucleoplasmic separation experiment were performed to detect the effect of CDK5 on the nuclear translocation of TRIM59.The binding of TRIM59 to CDK5 was detected by co-immunoprecipitation and GST pull-down.The phosphorylation of TRIM59 by CDK5 was detected via in vitro kinase assay.Western blot,co-immunoprecipitation and Immunofluorescence assay for the role of PIN1/importin α5 in the nuclear translocation of TRIM59.Co-immunoprecipitation and protein mass spectrometry were used to detect proteins binding to nuclear TRIM59.Western blot and protein ubiquitination assay for the role of TRIM59 on stability and ubiquitination of macro H2A1.Chromatin immunoprecipitation and real-time quantitative PCR to detect the effect of macro H2A1 degradation by TRIM59 on STAT3 target genes expression.Cell proliferation,colony formation and intracranial orthotopic transplantation model to detect effect of TRIM59 nuclear translocation on biological behavior of glioblastoma.Immunohistochemical staining for detection of clinical significance of the EGFR/CDK5/TRIM59/STAT3 signal axis.Results: EGFR activation promoted TRIM59 nuclear translocation,and EGFR tyrosine kinase inhibitor erlotinib treatment inhibited TRIM59 nuclear translocation.CDK5 inhibitor Roscovitine inhibited EGFR activation-promoted TRIM59 nuclear translocation.Knockout of CDK5 significantly inhibited TRIM59 nuclear translocation,and TRIM59 nuclear translocation was dependent on CDK5 kinase activity.EGFR activation promoted the association of CDK5 and TRIM59.CDK5 phosphorylated TRIM59 at S308.TRIM59 nuclear translocation was dependent on TRIM59 phosphorylation.Phosphorylation of TRIM59 recruited PIN1,and PIN1 promotedTRIM59 binding to importin α5.TRIM59 nuclear translocation was dependent on itself nuclear localization signal sequence and importin α/β1 transport system.Nuclear TRIM59 bound to macro H2A1 and regulated macro H2A1 stability through ubiquitination.Degradation of macro H2A1 by TRIM59 promoted STAT3 target genes expression.Nuclear translocation of TRIM59 promoted proliferation and colony formation of glioblastoma cells and tumor growth in vivo.Co-expression of p-CDK5,p-TRIM59,and p-STAT3 was present in p-EGFR-positive glioblastoma tissues,and these patients had a poor prognosis.Conclusion: CDK5 phosphorylation of TRIM59 promotes TRIM59 nuclear translocation.Nuclear TRIM59 ubiquitination and degradation of macro H2A1 promotes STAT3 target genes expression,and EGFR/CDK5/TRIM59/STAT3 signaling axis is involved in the regulation of EGFR/EGFRvⅢ glioblastoma.