The Mechanism Of ZDHHC4-mediated GSK3β Palmitoylation In Glioblastoma Chemoresistance

Glioblastoma(GBM)is one of the most common primary tumors in the adult central nervous system,accounting for about 12-15%of all intracranial tumors.The main modalities for GBM treatment are surgery,radiotherapy and chemotherapy.Due to the blood-brain barrier,temozolomide(TMZ)has become the most commonly been used chemotherapy drug in clinical practice.Glioblastoma stem cell(GSC)is an important factor causing GBM chemotherapy resistance.Many literatures have reported that GSC is involved in the regulation mechanism of GBM resistance to TMZ.Protein post-translational modification participates in various biological processes by affecting the conformation,activity and function of proteins.Palmitoylation is one of the post-translational modification mediated by palmitoyltransferase.The changes in protein function caused by abnormal palmitoylation are closely related to diseases such as tumors,but its function in GBM occurrence,progression,and chemoradiotherapy resistance remains unclear.In this study,we found that palmitoyltransferase ZDHHC4 was highly expressed in glioma tissues through data analysis combined with clinical sample validation.The expression of ZDHHC4 was positively correlated with the degree of malignancy of glioma patients,and the survival time of patients with high ZDHHC4 expression was relatively shorter.To explore the specific regulatory mechanism of ZDHHC4 in glioma progression,we used co-immunoprecipitation to identify and prove that GSK3β was a specific palmitoylation substrate of ZDHHC4.GSK3β is a serine protein kinase.The kinase activity of GSK3β is mainly regulated by phosphorylation at the Tyr216 site of the kinase domain,and its substrate-mediated cascade transmission is modified by phosphorylation,resulting in changes in biological functions.Post-translational modification of GSK3β protein is an important mode of regulation of kinase activity.We identified for the first time that the cysteine residue at position 14(Cys14)of GSK3β was palmitoylated by ZDHHC4.Moreover,ZDHHC4 enhanced the phosphorylation of p-Tyr216 by inhibiting the modification of p-Ser9 of GSK3β by competitively binding with modified kinases AKT1 and p70S6K upstream of GSK3β.This showed that ZDHHC4-mediated palmitoylation modification altered the kinase activity of GSK3β and caused the malignant biological phenotype of GBM cells.At the cytological level,by enhancing or inhibiting the expression of ZDHHC4,the results showed that high levels of ZDHHC4 increased the resistance of GBM cells to TMZ treatment.Meanwhile,silencing of ZDHHC4 or overexpression of palmitoylation-inactive GSK3β(C14A)mutants contributed to increased TMZ sensitivity in GBM cells.Screening of TMZ-resistant GBM cell lines,compared with normal cells,it was found that the expression of ZDHHC4 in TMZ-resistant cells was up-regulated.and the GSK3β palmitoylation enhanced its p-Tyr216 phosphorylation activation,which resulted in promotion of GBM cell proliferation,migration,and GSC self-renewal ability,which in turn promotes the up-regulation of the TMZ resistance of GBM cells.In order to further explore the ZDHHC4-mediated palmitoylation of GSK3β and the molecular mechanism of TMZ drug resistance in the downstream signaling axis regulating GBM.Using protein chip analysis,it was found that the downstream STAT3 signaling pathway was positively correlated with the expression of ZDHHC4.The ZDHHC4-GSK3β-STAT3 pathway was persistently activated in TMZ-resistant cells.Palmitoylated GSK3β could bind to intracellular STAT3 and increase its phosphorylation at Tyr705.Mainly GSK3β changed the methylation level of Lys180 of STAT3 by promoting the phosphorylation modification of Ser21 of EZH2.In addition,the enhanced activity of the transcription factor STAT3 could also enhance the expression of ZDHHC4 by binding to the promoter sequence of ZDHHC4,so that a positive feedback regulatory loop is formed between ZDHHC4-GSK3β-STAT3.Taken together,these data suggest that ZDHHC4-mediated palmitoylation of GSK3βincreases TMZ resistance in GBM cells by activating the EZH2/STAT3 signaling axis.To verify signaling axis transmission,GBM cells were treated with Niclosamide,a specific small-molecule inhibitor targeting GSK3β.In vitro,Niclosamide-treated GBM-resistant cells significantly reduced the activity of STAT3,effectively inhibited the spheroidization ability of GSC stem cells,and promoted apoptosis.Cytological experiments confirmed that inhibition of ZDHHC4 expression,inhibition of palmitoyl modification of GSK3β,and Niclosamide treatment of drug-resistant GBM cells significantly inhibited tumor growth.At the same time,by establishing a mouse intracranial xenograft model,reducing the expression of ZDHHC4,or combined with GSK3β small molecule inhibition,could inhibit the GSK3β/STAT3 signaling axis,thereby enhancing the tumor-killing effect of TMZ.These data suggest that ZDHHC4 can be used as a potential target,and GSK3β/STAT3 could be regarded as an intervention target for TMZ-resistant GBM cells,which provided theoretical support for potential clinical applications.Taken together,this study revealed that ZDHHC4-mediated palmitoylation of GSK3β enhanced GSC self-renewal capacity and TMZ resistance in GBM cells by activating the EZH2-STAT3 signaling axis.This helps to further understand the mechanism of drug resistance and recurrence after GBM treatment,and also provides a new theoretical basis for the regulatory mechanism of protein palmitoylation modification in the malignant progression of GBM.