CYT387,as An IKBKE Inhibitor,Suppresses Human Glioblastoma Malignant Progression By Activating The Hippo Pathway

Recent studies showed IKBKE(inhibitor of nuclear factor kappa-B kinase subunit epsilon)was overexpressed in several kinds of cancers and IKBKE-knockdown inhibited tumour progression.In this article,we firstly certified U87-MG and LN229 was sensitive to CYT387 via measuring half maximal inhibitory concentration(IC50)by CCK-8 assay and testified that CYT387,as a potent IKBKE inhibitor,suppressed glioblastoma cells ability of proliferation,migration as well as invasion.Meanwhile CYT387 also induced cell apoptosis and arrested cell cycle at G2 phase in vitro.So we showed CYT387 didn’t simply inhibit IKBKE activity but also decrease IKBKE expression on protein level rather than mRNA level.For more detailed mechanism,we certified that IKBKE interacted with YAP1 and TEAD2 to influence Hippo pathway using Co-immunoprecipitation(co-IP)and silencing of IKBKE could inhibit YAP1 and TEAD2 translocation to nucleus to induce associated genes transcription.And the following experiments in vivo,we found out that CYT387 inhibited subcutaneous nude mice tumours growth but few effects on intracranial orthotopic xenografts probably due to its difficulty to penetrate blood brain barrier(BBB).These results suggested that CYT387 potentially became a new anti-glioblastoma drug but need to seek out the approach to pass through blood brain barrier(BBB).Methods We measured CYT387 half maximal inhibitory concentration(IC50)of glioblastoma cell lines U87-MG and LN229 at different times using CCK-8 assay.We certified the proliferative ability of glioblastoma cells treated with CYT387 compared to blank control and DMSO groups using CCK-8 assay and clone formation test.Then the migrative and invasive capacities of glioblastoma cells treatment with CYT387 were measured by wound healing test and transwell assay.Next we test the cell cycle and cell apoptosis of glioblastoma cells treated with CYT387 using flow cytometry,besides,the cell cycle and cell apoptosis associated markers such as cyclinD1,cyclinA2,cdk1,cdc25 c,cyclinB1 and Bax,Bcl-2,caspase-3,caspase-9 were measured by western blot.Furthermore,we measured the protein expression ofIKBKE,the core Hippo pathway markers LATS2,YAP1,TEAD2,p-YAP1(s127)and its downstream factors Axl,c-myc using western blot after silencing of IKBKE using IKBKE-shRNA and then detected these markers after overexpressing IKBKE using recombinant plasmid of Flag-IKBKE and next measured these markers after cells treated with CYT387 in dose-and time-dependent manners using western blot.For more detailed mechanism,we testified that IKBKE,as a serine/threonine kinase,interacted with YAP1,TEAD2 using Co-immunoprecipitation(co-IP)in vivo and in vitro.And we used western blot and immunofluorescence(IF)to certify that suppression of IKBKE could inhibit YAP1 and TEAD2 translocation to nucleus.We also measured mRNA of YAP1 and TEAD2 after knocking down IKBKE using real-time RT-PCR.Additionally,we constructed subcutaneous tumor nude mice model using U87-MG.The experimental group were fed with CYT387(100mg/kg/day)and negative control group were fed with same concentration solvent.Measure the subcutaneous tumors’ volume and weight after mice were sacrificed for30 days.Detect associated markers using immunohistochemical(IHC)staining.Then we established intracranial orthotopic tumor model using U87-MG transfected with luciferase virus.Monitor the mice weight and survival rate.Stain the markers using IHC.Results 1,Glioblastoma cell lines U87-MG and LN229 are sensitive to CYT387 and CYT387 remarkably inhibits glioblastoma cell proliferation in vitro.2,CYT387 dramatically inhibits migration and invasion ability of glioblastoma cell lines in vitro.3,CYT387 accelerates glioblastoma cells apoptosis.4,CYT387 arrests glioblastoma cell cycle on G2 phase.5,CYT387 suppresses IKBKE protein expression rather than m RNA of IKBKE expression.6,CYT387 enhances Hippo pathway via inhibiting IKBKE expression to suppress glioblastoma cell growth.7,IKBKE interacts with YAP1 and TEAD2 to influence on the Hippo pathway and inhibition of IKBKE could suppress YAP1 and TEAD2 translocation to nucleus.8,CYT387 inhibited tumour growth in subcutaneous nude mice model but few effects on intracranial orthotopic model.Conclusion 1,CYT387 remarkably inhibits glioblastoma cells growth and accelerates glioblastoma cells apoptosis in vitro.2,CYT387 accelerates the Hippo pathway induced by IKBKE activity and quantity.3,IKBKE interacts with YAP1 and TEAD2 to influence on the Hippo pathway,promoting YAP1 and TEAD2 translocation to nucleus.4,CYT387 inhibited tumour growth in subcutaneous nude mice model but few effects on intracranial orthotopic model probably due to its difficulty to penetrate the blood brain barrier(BBB).