Secretory Pathway Kinase FAM20C Promotes Malignant Progression And Immunosuppression Of Glioma

Objective: Glioma is a common primary tumor of the central nervous system,which has a high recurrence rate and mortality,especially glioblastoma(GBM).Although the existing surgical treatment and concurrent chemo-radiotherapy can temporarily relieve the pain of patients,it still does not change the prognosis significantly,so new treatment strategies are urgently needed.Traditional clinical studies of glioma usually focus on the tumor cells but ignore the interaction between tumor cells and glioma microenvironment.In fact,tumor cells,stromal cells and immune cells can interact with each other.Our previous research found that some kinases have important regulatory effects on tumor development,such as AXL,is the key regulators of stem cell maintenance and tumor propagation in mesenchymal glioblastoma stem cells,and the purity of glioma can be used as an important indicator to predict the survival of glioma patients,indicate that the important kinases can modify the microenvironment of glioma to inhibit the growth of glioma cells.The secretory pathway kinase or kinaselike proteins(SPKKPs)have been shown to mediate multiple physiological functions by phosphorylating extracellular proteins and proteoglycans.However,their role in cancer,especially GBM,remains unclear.FAM20 C is one of the core members of SPKKPs.Exploring its function in glioma and its effect on tumor microenvironment can provide a new strategy for the treatment of GBM.Methods: 1,Organize and analyze four glioma related public databases,including The Cancer Genome Atlas(TCGA),Chinese Glioma Genome Atlas(CGGA),Gene Expression Omnibus(GSE16011)and Clinical Proteomic Tumor Analysis Consortium(CPTAC).The expression of all SPKKPs related genes reported in the previous literature were sorted out in the public database.The least absolute shrinkage and selection operator(LASSO)regression was employed for establishing the SPKKPs signature for IDH wild type(wt)GBM prognosis.The correlation between the risk score and the survival was verified by the survival curve,and the correlation between each member of the model and the clinical information of glioma patients was analyzed by heatmap and Pearson correlation.2,The interaction between SPKKPs was analyzed by the web protein interaction analysis tool STRING.In TCGA and CGGA databases,Spearman correlation and univariate Cox analysis were used to find the core member of SPKKPs.The Oncomine online database was used to analyze the differential expression of FAM20 C in various cancers.3,The expression of FAM20 C in different grades of gliomas was detected by immunohistochemistry(IHC).The results were then verified by analyzing TCGA,CGGA and CPTAC databases.Ivy GAP RNA-seq data set and IHC were used to explore the localization of FAM20 C in glioma tissues.The receiver operator characteristic(ROC)curves were further used for evaluating its association with progressive malignancy in glioma and GBM patients' survival.4,Gene set enrichment analysis(GSEA)and Kyoto encyclopedia of genes and genomes(KEGG)were used to interpret its functions in GBM.The humanderived glioma cell line LN229 was cultured in vitro,the small interference sequence was customized in Sangon Biotech and transfected into LN229 cells to further explore the function of FAM20 C in glioma.RT-q PCR and Western Blot were used to detect the knockdown efficiency of FAM20 C and the expression of MMP2 and MMP9;Transwell assay was used to detect the migration and invasion ability of glioma cells after knockdown of FAM20C;Clone formation assay was used to detect the change of tumor cell clone formation ability;The proliferation of tumor cells was measured by MTS method;Apoptosis assay was used to detect the changes of apoptosis ability of tumor cells after knockdown of FAM20 C.5,The potential phosphorylation substrates of FAM20 C reported in the previous literature were sorted out,the correlation between the substrates and FAM20 C was analyzed by protein interaction analysis tool STRING.The univariate Cox analysis and Spearman correlation analysis showed that FN1 was the most relevant substrate of FAM20 C in GBM.Furthermore,the binding between FAM20 C and FN1 was simulated by molecular docking method,and the binding mode between FAM20 C and FN1 was shown by Py MOL 2.3.2 and Ligplus 2.1.Moreover,the combination was verified by immunoprecipitation.6,Estimate R package was used to calculate the tumor purity,immune score and stroma score of TCGA and CGGA data samples.The correlation between FAM20 C and tumor purity,immune score and stroma score was shown by heatmap and Spearman correlation.x Cell and EPIC tools were used to calculate the content of immune cells in TCGA and CGGA samples.The expression of FAM20 C in different cells was analyzed through single cell database.Further,the effect of FAM20 C on tumor microenvironment was determined by the recombinant protein.Results: 1.FAM20 C is the core member of SPKKPs in glioblastoma and affects the biological function of tumor cells.A SPKKPs classifier was constructed with 3 genes of this family.This signature could effectively distinguish IDH wt GBM survival.Family with sequence similarity 20C(FAM20C)was further identified as the core member of this family in glioma.Elevated FAM20 C expression was not only closely correlated with glioma malignancy progression and the mesenchymal subtype of GBM,but also indicated unfavorable survival of GBM patients.Cell function experiment in vitro showed that the deletion of FAM20 C could affect the migration,invasion and clone formation ability of glioma cells.2.FAM20 C phosphorylates FN1 and promotes immune cell migration in glioblastoma.FN1 was identified as the core phosphorylation substrate of FAM20 C in glioma.Previous reports suggest that FN1 is essential for the migration and invasion of glioma cells.Further molecular docking analysis showed that amino acids(Pro200,Asn185,Trp202,Ala219 and Glu217)in FAM20 C protein bound to amino acids(Arg550,Gly551 and Arg552)in FN1 protein through hydrogen bonding interaction.At the same time,FAM20 C was also found to be associated with the destruction of immune response in GBM microenvironment.Tumor cells secreted FAM20 C enhanced the migration ability of immune cells.Conclusions: The prognosis of patients with high expression of FAM20 C is worse in GBM.FAM20 C promote the development of glioma by binding FN1 and enhancing immune cell migration.These data indicate that the potential of FAM20 C serving as a predictive molecule and a therapeutic target for GBM.