USP10 Deubiquitinates RUNX1 And Promotes Proneural-to-mesenchymal Transition In Glioblastoma

Objective:To clarify the deubiquitination of ubiquitin-specific protease 10(USP10)and its specific mechanism on RUNX1 in glioblastoma(GBM),and to explore the mechanism by which the upregulated RUNX1 by USP10 induces proneural(PN)-to-mesenchymal(MES)transition in GBM and maintains the characteristics of MES subtypes.The expression relationship between USP10 and RUNX1 in clinical tissue samples of GBM was analyzed to determine the prognostic value of USP10/RUNX1 axis in patients with GBM.Methods:1.A DUB si RNA library was screened to identify Deubiquitinases(DUBs)capable of inhibiting the protein levels of RUNX1,and Co-immunoprecipitation(Co-IP)was used to identify DUBs that could bind to RUNX1 protein.The screened DUB,wild-type USP10(USP10-WT)and inactivated USP10(USP10-C424A)were overexpressed in HEK293 T cells in a dose-dependent manner,and their effects on the protein level of RUNX1 were detected.Quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the m RNA expression levels of USP10,RUNX1,PN and MES related marker genes in different subtypes(PN,classical [CL] and MES)of GBM tissues.The protein expression levels of USP10 in normal brain tissue and different GBM subtypes(PN,CL and MES)was detected by immunohistochemistry and Western Blot.USP10 protein levels in normal astrocytes,4 GBM cell lines and 2 primary GBM cells were detected by Western Blot.USP10 was knocked down in LN229 and GBM2 cells,and RUNX1 protein levels were detected by Western Blot after transfection with USP10-WT or USP10-C424 A plasmids.2.The levels of USP10,RUNX1 and PN subtype marker proteins(Olig2 and PDGFRα)and MES marker proteins(YKL-40,MET and COL5A1)were detected by Western Blot in U251 cell line with PN isotype and primary GBM1 cells that expressed Vector/USP10 or sh Ctrl/sh RUNX1.Transwell assay was performed to determine the effects of Vector/USP10 or sh Ctrl/sh RUNX1 overexpression on the invasion and migration ability of U251 and GBM1 cells.The effects of Vector/USP10 or sh Ctrl/sh RUNX1 overexpression on the proliferation ability of U251 and GBM1 cells were evaluated by CCK-8 assay.The effects of Vector/USP10 or sh Ctrl/sh RUNX1 overexpression in U251 and GBM1 cells on tumor growth and survival in nude mice were investigate by intracranial tumorigenesis.3.Transfection of sh Ctrl/sh USP10 or Vector/RUNX1 into LN229 cell lines and GBM2 primary cells with the characteristics of MES subtype were performed.The levels of USP10,RUNX1 and PN subtype marker proteins(Olig2 and PDGFRα),and MES marker proteins(YKL-40,MET and COL5A1)were detected by Western Blot.The effects of sh Ctrl/sh USP10 or Vector/RUNX1 transfection on the invasion and migration abilities of LN229 and GBM2 cells were assessed by Transwell assay.The effect of sh Ctrl/sh USP10 or Vector/RUNX1 on the proliferation of LN229 and GBM2 cells was evaluated by CCK-8 assay.Furthermore,the effect of LN229 and GBM2 cells transfected with sh Ctrl/sh USP10 or Vector/RUNX1 on tumor growth and survival in nude mice was investigated by intracranial tumor formation in situ.4.Co-IP and GST pull-down tests in HEK293 T cells were conducted to determine whether there was a direct interaction between WT-USP10 or USP10-C424 A and RUNX1.The confocal immunofluorescence was used to determine whether there was colocalization of USP10 and RUNX1 expression in LN229 and GBM2 cells,and Co-IP was carried out to verify whether there was a direct interaction between USP10 and RUNX1 in LN229 and GBM2 cells.Different truncated mutants of USP10 and RUNX1 were constructed,and Co-IP experiments were performed in HEK293 T cells to determine the amino acid regions where USP10 protein interacts with RUNX1 protein.5.Western Blot was performed to examine the expression levels of RUNX1 protein in LN229 and GBM2 cells with knockdown of USP10 and treatment of the proteasome inhibitor MG132.Besides,western Blot was also employed to determine the effect of USP10 knockdown on GBM cells with the characteristics of MES subtype,and the effect of overexpression of USp10-WT or USP10-C424 A on the stability of RUNX1 protein in GBM cells with the characteristics of PN subtype.In addition,the effects of USP10 knockdown in GBM cells with the characteristics of MES subtype and USp10-WT or USP10-C424 A overexpression on the ubiquitination levels of RUNX1 protein in GBM cells with the characteristics of PN subtype were detected by Western Blot as well.In vitro deubiquitination experiments were conducted to investigate whether USP10 could directly deubiquitinate RUNX1 protein by directly cutting the polyubiquitin chain of RUXN1 protein through its DUB activity.In vitro deubiquitination experiments were also conducted to determine whether the deubiquitination modification of RUNX1 by USP10 was mediated by lysine 48-linked polyubiquitin chain or lysine 63-linked polyubiquitin chain.Finally,the effect of USP10 knockdown on RUNX1 protein expression with overexpression of lysine 48 mutation was detected by Western Blot.6.Western Blot was used to detect the effects of USP10 small molecule inhibitor Spautin-1 on the expression and stability of RUNX1 protein in LN229 and GBM2 cells;Western Blot was also performed to determine the effect of Spautin-1treatment on the ubiquitination level of RUNX1 in LN229 and GBM2 cells,as well as in U251 and GBM1 cells overexpressing USP10.Furthermore,western Blot was used to detect the expression levels of PN subtype marker proteins(Olig2 and PDGFRα)and MES marker proteins(YKL-40,MET and COL5A1)in LN229 and GBM2 cells transfected with Vector or RUNX1 after Spautin-1 treatment,as well as in U251 and GBM1 cells overexpressing USP10 and transfected with Vector or RUNX1 after Spautin-1 treatment.In vivo and in vitro experiments were performed to evaluate the effects of Spautin-1 treated LN229 and GBM2 cells transfected with Vector or RUNX1 on tumor cell invasion,migration,proliferation,tumor growth and survival in nude mice.7.Immunohistochemistry and Western Blot were used to compare the expression of USP10 and RUNX1 in tumor tissue samples from GBM patients with primary PN subtype and recurrent MES subtype before and after two surgeries.The correlation between the expression levels of USP10 and RUNX1 in GBM tissue samples and the relationship between the expression level of USP10 and patient survival were also analyzed.Results:1.Knockdown of USP10 can inhibit RUNX1 protein levels,and USP10 interacts with RUNX1 protein.The expression levels of RUNX1 protein gradually increased with the increasing levels of USP10-WT overexpression,while overexpression of USP10-C424 A had no effect on RUNX1 protein expression.The m RNA expression levels of USP10,RUNX1 and MES related marker genes in MES subtype of GBM tissue were higher than those in PN subtype,and the m RNA expression levels of PN marker genes were lower than those in PN subtype.USP10 expression in normal brain tissue was lower than that in GBM tissue,but it was higher in MES subtype of GBM tissue than that in PN subtype.USP10 expression was lower in astrocytes than GBM cells,but higher in GBM cells with the characteristics of MES subtype than those in GBM cells with the characteristics of PN subtype.Overexpression of USp10-WT reversed the effect of USP10 sh RNA transfection on USP10 protein expression,while overexpression of USP10-C424 A did not reverse the effect of USP10 sh RNA transfection on USP10 protein expression.2.Overexpression of USP10 in U251 and GBM1 cells resulted in an increase in MES labeled proteins(YKL-40,MET and COL5A1)and a decrease in PN subtype labeled proteins(Olig2 and PDGFRα).Knockdown of RUNX1 could reverse the effect of USP10 overexpression on tumor cell-related protein expression.Overexpression of USP10 also enhanced the invasion,migration and proliferation ability of GBM cells,as well as the tumor growth ability,but shortened the survival time of tumor-bearing nude mice.Knockdown of RUNX1 could reverse the effects of USP10 overexpression on the invasion,migration and proliferation ability of GBM cells,as well as the tumor growth and survival time of nude mice.3.Knockdown of USP10 in LN229 and GBM2 cells results in a decrease in the levels of MES marker proteins(YKL-40,MET and COL5A1)and an increase in the levels of PN subtype marker proteins(Olig2 and PDGFRα).Overexpression of RUNX1 could reverse the effects of USP10 knockdown on tumor cell-related protein expression.Knockdown of USP10 also weakened the invasion,migration and proliferation ability of GBM cells,as well as the tumor growth ability,and prolonged the survival time of tumor-bearing nude mice.Overexpression of RUNX1 could reverse the effects of USP10 knockdown on the invasion,migration and proliferation ability of GBM cells,as well as the tumor growth and survival time of nude mice.4.In HEK293 T cells,both WT-USP10 and USP10-C424 A had direct interaction with RUNX1.In GBM cells,the expression of USP10 and RUNX1 was colocalized and directly interacted with each other.The N terminus of USP10 and the N terminus of RUNX1 containing Runx domain represented the amino acid regions where protein-protein interactions took place.5.MG132 treatment in LN229 and GBM2 cells reversed the down-regulation of RUNX1 protein expression caused by USP10 knockdown;USP10 could promote the stability of RUNX1 protein and reduce its ubiquitination-mediated degradation level.USP10 directly deubiquitinated RUNX1 protein by cleaving off its lysine 48-linked polyubiquitin chain through its DUB activity.Knockdown of USP10 had no effect on the expression of RUNX1 protein after overexpression of lysine 48 mutant.6.Treatment of GBM cells with USP10 small molecule inhibitor Spautin-1resulted in decreased expression and stability of RUNX1 protein,as well as increased ubiquitination-mediated degradation level;Treatment of GBM cells with Spautin-1 led to increased expression of PN subtype marker proteins(Olig2 and PDGFR α)and decreased expression of MES marker proteins(YKL-40,MET and COL5A1).Overexpression of RUNX1 could reverse the effect of Spautin-1 on GBM cells.Treatment of LN229 and GBM2 cells with Spautin-1 resulted in reduced tumor cell invasion,migration and proliferation,and slower tumor growth in nude mice,leading to prolonged survival.Overexpression of RUNX1 could reverse the effect of Spautin-1on intracranial tumor growth and survival of nude mice with GBM cells.7.The expression levels of USP10 and RUNX1 in recurrent MES subtype tumor tissues of GBM patients were higher than those in primary PN subtype tumor tissues;There was a correlation between the expression levels of USP10 and RUNX1 in GBM tissue samples,and GBM patients with high expression levels of USP10 had shorter progression-free survival and overall survival.Conclusions:1.USP10 is a deubiquitination enzyme that regulates the stability of RUNX1 and is expressed at the highest levels in MES subtype of GBM.USP10 up-regulates RUNX1 to induce PN-to-MES transition in GBM,thereby maintaining the characteristics of MES subtype;2.USP10 directly interacts with RUNX1,promoting the deubiquitination of RUNX1 and stabilizing the expression of RUNX1 protein;3.The USP10 inhibitor Spautin-1 induces RUNX1 degradation and inhibits the characteristics of MES subtype in vitro and in vivo;4.The expression of USP10 is closely related to that of RUNX1 in clinical tissue samples of GBM and has predictive value for the prognosis of GBM patients.The USP10/RUNX1 axis may be a potential target for new GBM treatment.