The Stability Of FOXM1and Mechanism Of Enhancing Transcriptional Activity Of Wnt/β-Catenin Pathway

Glioblastoma represents the most common primary intrinsic malignant brain tumor. The clinical hallmarks of glioblastoma are its aggressive growth and inexorable recurrence despite multimodal therapy with surgery followed by radiation and temozolomide therapy. Although more basic and clinical research achievements are being accumulated, challeges in GBM prevention and therapy still remain. Therefore, it is necessary to explore the rules of glioma tumorigenesis and development. Previous studies indicate that FoxM1is overexpressed in glioma, and associated with higher gade of glioma and critical for malignant proliferation, angiogenesis and invasion. Recently, we also find that FoxMl can mediate P-catenin nuclear translocation and enhance the transcription of Wnt signaling target genes to maintain self-renewal of glioma stem cells and tumorigenesis. However, it is unclear how Wnt signaling regulates FoxMl stability and how FoxMl enhances interaction of P-catcnin/TCF-4.Objective (1) To uncover the mechanism of FoxM1stability regulated by Wnt signaling;(2) To clarify the mechanism of FoxM1enhancing the transcriptional activity of P-catenin/TCF-4.Methods (1) To analyze the effects of Wnt3a and its inhibitor DK.K.1on FoxMl stability using western blotting (WB).(2) To analyze the effects of GSK-3β expression and activity on F’oxMl stability using western blotting (WB).(3) To analyze interaction of GSK-3P and FoxMl using Co-IP.(4) To analyze FoxMl phosphorylation mediated by GSK-3β using in vitro kinase assay.(5) To analyze the stability and degradation of F’oxMl mutants (putative GSK.-3β motif) using site-directed mutagenesis kit and Co-IP.(6) To analyze interaction among F’oxMl. ICAT and P-catenin using Co-IP.(7) To analyze the relative activity of TOPfiash changes induced by interaction among FoxMl. ICAT and β-catenin using dual luciferase assay.(8) To analyze the ability of TCF-4binding to axinl promoter using ChIP assay and determine the expression levels of Wnt signaling target genes by WB.(9) To analyze the effect of constitutive stable FoxMl mutant on cell cycle by How eytometry (FCM). Results (1) FoxM1protein levels were increased in dose-(0,10,20,40,80ng/ml) and time-(0,2,4,6h) dependent manner in U87MG and LN229treated with Wnt3a, wheras DKK1, Wnt inhibitor, markedly reduced FoxM1levels, which indicated that Wnt signaling regulated FoxM1turnover.(2) GSK-3β inhibitor LiC1(20mM) can stabilize FoxM1protein level in time-dependent manner in LN229and U87MG cells. Knocking down GSK-3β can increase FoxM1expression in LN229cells. GSK-3βKD inhibited FoxM1degradation, wheras GSK-3βCA promoted FoxM1degradation. Furthermore, FoxM1was dramatically stable not in MEF-WT cells, but in MEF-KO cells. These results indicated that Wnt signaling played an important role in FoxM1turnover.(3) FoxM1can directly interact with GSK-3β in LN229and U87MG cells. Moreover, FoxM1C-terminal (CT) had higher affinity with GSK-3β. Also, FoxM1was phosphorylated by GSK-3β kinase using in vitro kinase assay.(4) Using kinase-specific phosphorylation site prediction software (GPS2.1) and online tool (scansite.mit.edu) to predict probable sites, we proposed that FoxM1S474probably might be GSK-3β target site, and S478be the priming site of S474. Thus, FoxM1site-directed mutants S474A, S478A, S228A and T309A were conducted using site-directed mutatgenensis kit.We found that S474A and S478A were obviously resistant to degradation among FoxM1mutants, and S474A also was resistant to ubiquitination and had lower affinity to GSK-3β.(5)Ck1inhibitor D4476reduced FoxM1turnover in dose-and time-dependent manner. We also found that FoxM1could bind to CK1δ and CK1δ knockdown increased FoxM1protein level. It was suggested that CK1δ was a potential kinase for S478priming site.(6) Nuclear FoxM1was increased in time-dependent manner under Wnt3a treatment.(7) FoxM1competed with ICAT binding to β-catenin, and thus affected the transcriptional activity of TCF-4, which was confirmed by dual luciferase assay. FoxM1enhanced the ability of TCF-4binding to axin2promoter by ChIP assay, wheras ICAT competitively inhibited this effect. Expression of Wnt signaling target genes were consistan with the above results.(8) S474A significantly promoted G2/M transition by cell cycle analysis.Conclusion (1) FoxM1stability is regulated by Wnt signaling via GSK-3β and CK1δ dual kinase.(2) Wnt signaling promotes FoxM1nuclear translocation, and FoxM1competes with ICAT binding to β-catenin, and enhances the ability of β-catenin/TCF-4complex binding to promoters of target genes, and finally promotes G2/M transition.