The MAP3K1/c-JUN Signaling Axis Regulates The Invasion Of Glioblastoma Stem Cells Mechanism Research

Glioblastoma(GBM)is the most frequent primary malignant brain tumor in adults,and despite aggressive treatment,including surgical resection,chemotherapy,and radiation therapy,the prognosis for patients is very poor,with an average overall survival time of just 12-18 months.The main cause of lethality is the diffuse infiltration of GBM tumor cells into the surrounding healthy brain parenchyma,preventing complete resection without interfering with normal brain function.In addition,numerous findings at the molecular level suggest that GBM stem cells(GSC)are the key cells responsible for resistance to anticancer drug radiotherapy,invasion and spread.However,the mechanism of GSC invasion is still not fully understood and is in its infancy.To identify the driving signaling pathway and genes that may play pivotal roles in GBM invasion,we performed a deeper analysis of single-cell transcriptome sequencing using surgically isolated tumor core compared to surrounding peripheral tissue in GBM patients and discovered that the mitogen-activated protein kinase(MAPK)signaling pathway is one of the three signaling pathways that are highly activated in the tumor periphery.Because of its kinase capacity and targeting convenience,we selected MAPK signaling pathway as a potential regulatory pathway for GBM invasion for further analysis.Among the 28 genes involved in the MAPK signaling pathway,MAP3K1 showed the highest ranking(glioma versus non-tumor)in the REMBRANDT m RNA database.Furthermore,we found that the high transcript levels of MAP3K1 markedly correlated with poor prognosis in GBM patients.Therefore,we identified MAP3K1 as a potential key target gene for regulating GBM invasion.To define how MAP3K1 regulates GSC invasion and tumorigenicity,we selectively knocked down MAP3K1 in GSCs using two different interfering short hairpin RNAs(sh RNAs)and detected the tumorsphere-forming ability,GSC proliferation,invasion,as well as in vivo tumor formation ability.According to limiting dilution assay,which is a widely used method for detecting the self-renewal capability of GSCs,knocking down of MAP3K1 significantly suppressed the tumorsphere-forming ability of GSCs.Knockdown of MAP3K1 also significantly repressed GSC growth and invasion ability.To explore the tumorigenicity and infiltration of MAP3K1 in vivo,xenograft mouse models were established using X01 GSCs infected with a sh MAP3K1-expressing lentiviral or sh Ctrl constructs.The tumor area from the whole brain tissue sections revealed that knocking down of MAP3K1 dramatically delayed GBM progression.As expected,knockdown of MAP3K1 also reduced the infiltration field along the corpus callosum and significantly prolonged mice survival.Collectively,these findings suggest that MAP3K1 is capable of regulating the stemness and invasion of GSCs and the tumorigenicity and infiltration of GBM.To further demonstrate the regulatory mechanism of MAP3K1 in GSC invasion,we analyzed phosphoproteome data from the CPTAC(clinical proteomic tumor analysis consortium)GBM database for c-JUN,since MAP3K1 regulates epithelial migration in a c-JUN-dependent manner has been reported.The protein abundances of MAP3K1 and c-JUN were notably higher in GBM than in normal tissues.The phospho-JUN-S63 abundance was remarkably higher in GBM than normal tissues.We further analyzed the Pearson’s correlation coefficient between c-JUN and phospho-JUN-S63 and found that they showed a significantly positive correlation.Moreover,GBM patients with high protein abundance of phospho-JUNS63 showed much worse prognosis.Furthermore,we validated MAP3K1 regulates GSC properties and invasion through c-JUN-S63 phosphorylation in MAP3K1 knockdown GSCs as well as employing a c-JUN inhibitor,JNK-IN-8.In conclusion,this thesis mainly reported that silencing MAP3K1 gene had significant effects on proliferation,stemness and invasiveness of GSC,and secondly,MAP3K1 gene was found to regulate its proliferation,stemness and invasive ability through c-JUN signaling pathway in GSC cells,which also suggests that MAP3K1 may become an important target for the treatment of infiltrative GBM.