Amlexanox,a Selective Inhibitor Of IKBKE,Inhibits Human Glioblastoma By Disrupting The Hippo Pathway

IKBKE(inhibitor of nuclear factor kappa-B kinase subunit epsilon)is a non-canonical IKK family member that is important in inflammatory and immune diseases.Recently,IKBKE is believed to function as an oncogenic protein.Targeting IKBKE has become an avenue for the development of novel therapeutic interventions for tumors.As an inhibitor of IKBKE,several reports have demonstrated that amlexanox has anti-inflammatory,antiallergic,immunomodulation activity,and it been used for treating aphthous ulcer,allergic rhinitis,asthma and obesity in the clinic.In our article,amlexanox not only suppressed GBM cells proliferation,migration and invasion,but also induced G0/G1 phase arrest and cell apoptosis.Amlexanox has been shown to block IKBKE activity and reduce IKBKE protein level.IKBKE can bind to and regulate LATS1/2,which are the core kinase of the Hippo pathway.Silencing IKBKE affected the abundance of LATS1/2,phosphorylation status of YAP1 and the expression of downstream targets.In addition,amlexanox also suppresses GMB growth in subcutaneous and intracranial xenograft models by down-regulating IKBKE.These findings indicate that amlexanox is a promising chemotherapeutic compound for the treatment of GBM.Methods 1.Treatment with amlexanox inhibited cell viability in a dose-and time-dependent manner at different concentrations,the viability rate of U87 and U251 cells was assessed by CCK-8.Based on the IC50,different concentrations were selected to be used in the experiments.2.The effect of amlexanox on the cell cycle progression and apoptosis of U87 and U251 cells was evaluated by DNA content analysis.3.The effect of amlexanox on proliferation ability of U87 and U251 cells was evaluated by colony formation assay.The effect of amlexanox on migration and invasion of U87 and U251 cells were examined by the transwell assay.4.The effect of amlexanox on the protein expression of IKBKE,the core kinases of the Hippo pathway and the downstream targets was detected by western blot.5.The effect of IKBKE knockdown or over-expression on the core kinases of the Hippo pathway and the downstream targets was detected by WB.6.Immunofluorescence analysis and WB were used to assessed the expression of YAP1 in the cytoplasm and nucleus when IKBKE was silenced.7.The LATS1/2 mRNA expression in the control and shIKBKE groups was determined by qRT-PCR.The interaction between IKBKE and LATS1/2 was performed by Co-IP experiments.LATS2 ubiquitination levels in IKBKE knockdown U87 cells was assessed by WB.8.Subcutaneous glioma model was established by using U87 cells.DMSO or amlexanox(100mg/kg/day)were intraperitoneally injected every day for 21 days.The tumor volume was measured using a digital caliper.At the end of the experiment,all mice were sacrificed,and tumors were extracted and weighed.The tumors were fixed in 10% formalin,and embedded in paraffin for HE and IHC.9.To establish an intracranial model,U87 cells with a luciferase-encoding lentivirus were injected into the mice stereotactically.Intracranial tumor growth was detected by using the IVIS Spectrum Live Imaging System.Mice brains were extracted and embedded in paraffin for HE and IHC.Results 1.Amlexanox suppressed the proliferation and colony formation of GBM cells.2.Amlexanox inhibited the migration and invasion of GBM cells.3.Amlexanox induced GBM cell G0/G1 phase arrest and apoptosis.4.Amlexanox affected IKBKE at the post-transcriptional level,which increased the level of LATS1/2 and p-YAP1(Ser127).5.IKBKE directly bind to and down-regulate LATS1/2 by promoting their degradation,which activates the Hippo pathway.6.After treatment with the proteasome inhibitor MG132,the IKBKE-induced reduction of LATS2 expression was blocked.7.Amlexanox suppressed tumor growth in subcutaneous xenograft models.There was no significant differences in body weights between the control group and amlexanox treatment.IHC showed that the expression levels of IKBKE,the core kinase of the Hippo pathway and the downstream targets changed.8.Amlexanox suppressed intracranial tumor growth and prolonged the survival time of tumor bearing mice.The expression of IKBKE,the core kinase of the Hippo pathway and the downstream targets changed according to the IHC.Conclusion 1.Amlexanox could suppress GBM cells growth and inhibit the IKBKE protein and alter the abundance and phosphorylation status of the Hippo pathway proteins.2.IKBKE can directly bind to LATS1/2 and regulate LATS1/2 stability through promoting its polyubiquitin degradation.IKBKE knockdown activates the Hippo pathway and inhibits GBM growth by increasing the level of LATS1/2.3.Amlexanox suppresses glioblastoma growth in subcutaneous and intracranial xenograft models by down-regulating IKBKE.