The Role And Mechanism Of ZYXIN In The Invasion Of Glioblastoma

Glioblastoma multiforme(GBM)is characterized with highly invasive growth,which leads to extensive infiltration and difficulty to complete removal.Incomplete surgical resection pivotally contributes to frequent disease recurrence and poor prognosis of GBM patients.Therefore,identifying reliable biomarkers for invasibility of GBM is crucial to predict patient prognosis and finding novel therapeutic targets.Cytoskeleton proteins are tightly related with cell spreading,migration,and invasion.Through analysis on public GBM databases,we identified an actin-interacted protein,ZYXIN,as a promising candidate involved in GBM invasion.ZYXIN is one of the key focal adhesion proteins and involved in fundamental cellular activities,such as cell adhesion,migration and proliferation by interacting with a variety of cellular proteins.Previous studies have confirmed that ZYXIN plays an important role in the invasion of many malignant tumors,but the role of ZYXIN in the progression of GBM remains unclear.In the present study,through analysis on our own glioma cohort as well as The Cancer Genome Atlas(TCGA),Rembrandt,Gravendeel databases,we noticed that increased ZYXIN expression was significantly correlated with tumor progression and poor prognosis of glioma patients.In vitro and in vivo experiments further confirmed the oncogenic roles of ZYXIN and proved the involvement of ZYXIN in GBM invasive growth.Moreover,RNA-seq and Mass-Spectrum data using ZYXIN-knockdown LN229 and its control cells revealed STMN1 was a potential target of ZYXIN.Indeed,STMN1 at both m RNA and protein levels was positively regulated by ZYXIN.Functionally,STMN1 not only promoted invasion of GBM cells but also rescued the invasion repression caused by ZYXIN loss.Taken together,our results indicated that high ZYXIN expression was associated with worse prognosis and highlighted that the ZYXIN-STMN1 axis might be considered as a potential therapeutic target for GBM.The main results and conclusions are as the followings:1.ZYXIN was screened out and its expression in GBM and its relationship with patient prognosis was investigated1.1 ZYXIN was potentially responsible for mobility and invasibility of GBM cells.To investigate possible candidate genes responsible for cell migration and invasion in GBM,we first analyzed three invasion-related genesets,i.e.KEGG FOCAL ADHESION,GO CELL SUBSTRATE ADHESION,and GO CELL SUBSTRATE JUNCTION.Genes in the three genesets play critical roles in tumor invasion through regulation on interactions between cell and extracellular matrix.Venn diagram revealed that 26 genes were involved in all three genesets.To further evaluate the potential involvements of the 26 genes in GBM invasion,we analyzed their expression levels in two widely used databases of GBM,TCGA＿GBM and Rembrandt.Interestingly,the result showed that ZYXIN was the most increased gene in both databases,which implied a critical implication of ZYXIN in mobility of GBM cells.Then,we examined the expression of ZYXIN proteins in a panel of GBM cell lines with a normal glia line as control.The data showed that ZYXIN protein level in GBM cell lines was higher than that in control cells.Furthermore,we also collected 6 freshly resected tumor tissues and their adjacent non-tumor tissues.Supporting cell line result,ZYXIN protein in tumor tissues were higher than that in paired non-tumor tissues.Therefore,ZYXIN was a GBM-related gene and could be a candidate to be used for invasion marker.1.2 Increased ZYXIN was correlated with disease progression and poor prognosis of glioma.To comprehensively explore the pathological relevance of ZYXIN in gliomas,we analyzed ZYXIN expression in clinical glioma samples containing grade II,III,and IV(GBM).In a cohort of 84 cases of glioma,immunohistochemistry results indicated that ZYXIN protein in GBM was much higher than that in grade II and III gliomas.In 12 cases of fresh human glioma samples,we also observed higher ZYXIN protein level in GBM than grade II and III gliomas measured by western blotting.The analyses on three public glioma databases,i.e.TCGA＿GBMLGG,Rembrandt,and Gravendeel,consistently revealed that the ZYXIN mRNA in GBM were significantly higher than that in grade II and III gliomas.Accordingly,ZYXIN was positively correlated with glioma progression.Moreover Kaplan-Meier survival analysis on the three databases suggested that the patients with high ZYXIN level showed shorter overall survival than those with low ZYXIN level in both all gliomas and GBM only,which further supported the oncogenic roles of ZYXIN in gliomas.In classical histological classification,the expression of ZYXIN m RNA in GBM with the worst prognosis was significantly higher than that in other types by analyzing three databases of Rembrandt,Gravendeel and TCGA.Similar results were obtained in the latest molecular typing.Therefore,ZYXIN might act as a marker for disease progression and poor prognosis of glioma patients.2.Using RNA-Seq and databases bioinformatics to investigate the roles of ZYXIN in glioblastoma.Since ZYXIN plays pivotal roles in cell-extracellular substrate interaction,we speculated that ZYXIN might lead to glioma progression through promoting invasion of tumor cells.To examine this hypothesis,we stably knocked down ZYXIN in LN229 cells(LN229/shZYXIN#1)and used scramble shRNA as control(LN229/Ctrl).LN229/sh ZYXIN#1 and LN229/Ctrl cells were subjected to RNA-seq in triplicate.The data showed that ZYXIN loss resulted in upregulation of 489 genes and downregulation of 219 genes(P < 0.05 and Fold > 2).Gene Ontology(GO)analysis revealed that the genes significantly altered by ZYXIN knockdown were enriched in several cell migration and invasion-related gene clusters under the context of biological process,such as cell adhesion,integrin-mediated signaling pathway,extracellular matrix organization,cell-matrix adhesion.Gene set enrichment assay(GSEA)further showed that ZYXIN was positively correlated with the levels of genes responsible for glioma invasion,which were defined in our previous work.The public databases were also analyzed and the result s consistently indicated that high expression of ZYXIN enriched genes involved in glioma invasion and cell migration(Wu＿Cell＿Migration).Thus,both our own data and public databases identified potential involvement of ZYXIN in glioma invasion.3.ZYXIN knockdown prevents migration and invasion of GBM cells in vivo and in vitroNext,we explored whether ZYXIN functionally affected the migration and invasion of GBM cells through Transwell assays.The results indicated ZYXIN knockdown significantly inhibited serum-induced migration and invasion of tumor cells.We also intracranially inoculated control GBM cells or sh ZYXIN#1-transfected GBM cells into NOD-SCID mice followed by pathological analysis.We observed that the margin of tumors formed by sh ZYXIN#1-transfected GBM cells was smooth and the tumor and non-tumor areas could be clearly distinguished,while the margin of tumors formed by control GBM cells had obvious protrudes into non-tumor area.IHC staining of ZYXIN indicated that ZYXIN was highly expressed in the invasive front of tumor formed by GBM1/Ctrl cells.Consistent with the attenuated invasiveness of sh ZYXIN#1-transfected GBM cells,the mice with ZYXIN-knockdown GBM showed significantly longer survival time than those with control GBM.Therefore,ZYXIN was indeed positively correlated with invasiveness of GBM cells.4.The mechanism of ZYXIN promoting GBM invasion was explored.4.1 ZYXIN regulated the transcription of STMN1 in GBM.To explore the underlying mechanism of ZYXIN-mediated invasion,we performed iTraq-based Mass-Spectrum using LN229/Ctrl and LN229/sh ZYXIN#1 cells to profile altered proteins related with ZYXIN loss.Through comparing with the data of RNAseq,we found that STMN1,IGTA4,SRM and ASNS were downregulated with ZYXIN knockdown in both RNA-seq and Mass-Spectrum.Among the four genes,we noticed that STMN1 has been well known to play critical roles in tumor cell invasion.Hence,we further studied STMN1 in GBM cells.Consistent with the omics data,both qRT-PCR and western blotting confirmed the downregulation of STMN1 by ZYXIN knockdown in LN229 and GBM1 cells.The Pearson correlation analysis of IHC scores of ZYXIN and STMN1 in a glioma cohort containing 61 cases showed that the protein expression of ZYXIN was indeed positively correlated with that of STMN1.In addition,immunofluorescence confirmed that knocked down expression of ZYXIN resulted in downregulation of STMN1 in sh ZYXIN#1-transfected GBM cells compared with control GBM cells.We also confirmed the upregulation of ZYXIN on the transcription of STMN1 in GBM cells by dual-luciferase reporter assay system.To further evaluate the relationship between ZYXIN and STMN1,we analyzed the mRNA expression of the two genes in GBM subtypes.Highest level of ZYXIN mRNA was observed in Classical subtype of GBM,but STMN1 mRNA expressed at the highest level in Proneural subtype of GBM.Although the distribution of ZYXIN and STMN1 seemed inconsistent,correlation analysis in individual subtype revealed that ZYXIN and STMN1 were significantly positively correlated in Proneural and Classical subtypes.In other two subtypes,however,no correlation was observed.Thus,the regulation of STMN1 by ZYXIN might be genetic background-dependent.Together,our results for the first time revealed that STMN1 might be a target of ZYXIN in GBM cells under specific genetic background.4.2.STMN1 possibly functions as a downstream target of ZYXIN in GBM cells.As ZYXIN regulated the transcription of STMN1 in GBM,we examined whether overexpression of STMN1 could reverse the tumor invasion inhibited by knockdown of ZYXIN.For this purpose,STMN1-overexpressing lentivirus was transfected into LN229 and GBM1 cells with or without ZYXIN knockdown.Migration assay using Transwell showed that STMN1 overexpression not only increased migration of GBM cells but also partially rescued the inhibitory effect of ZYXIN knockdown on migration.Invasion assay also supported that STMN1 promoted invasion and rescued ZYXIN knockdown-induced repression of invasion.Therefore,our results indicated that STMN1 could be a downstream mediator for ZYXIN functions in GBM cells.The following conclusions can be drawn:(1)ZYXIN is highly expressed in GBM and can act as a new prognostic biomarker for GBM patients.(2)ZYXIN is an important molecule to promote the invasion of GBM cells in vitro and in vivo,thereby involved in the progression of GBM.(3)ZYXIN transcriptionally regulates the expression of STMN1 to promote the migration and invasion of GBM.