Abstract 2938: Inhibition of PI3K/AKT and MEK/ERK pathways act independently on p27 upregulation induced by downregulation of cathepsin B and uPAR in glioma

Abstract

Abstract Cathepsin B and urokinase plasminogen activator receptor (uPAR) are overexpressed in gliomas. Deregulation of the G1 phase cell cycle machinery is a common feature of cancers. The abundance of cyclin-dependent kinase inhibitor p27Kip1 (p27) during the cell cycle determines whether cells will proliferate or become quiescent. In the present study, we carried out transfections using shRNA against cathepsin B and uPAR. We assessed cell cycle arrest using FACS analysis and used Western blot analysis to detect expression of ERK, AKT, p27, cyclin E, cyclin D, CDK4, CDK2, EGFR, β1 integrins, and the pocket proteins (p-Rb, p107 and p130). Specific inhibitors like Wortmanin (10 µM) and U0126 (10 µM) were used to further determine the roles of the AKT and ERK pathways. We used immunoprecipitation analysis to detect the respective interactions between EGFR and β1, p27 and CDK4, and p27 and CDK2. CHIP analysis was used to check for the recruitment of transcription factors FOXO3a, E2F1 and AP-1 to the p27 promoter. The results of the present study show that shRNA treatment efficiently downregulated expression of cathepsin B and uPAR and induced G0/G1 arrest through upregulation of p27. Downregulation of both p-ERK and p-AKT downstream of EGFR and β1 are involved, but independently, in p27 upregulation. Multiple transcription factors (namely, AP-1, E2F1 and FOXO3a) were involved in upregulation of p27 as determined by luciferase expression under the influence of promoter regions to which these transcription factors bind individually. Also, cathepsin B and uPAR downregulation reduced tumor growth and increased p27 nuclear expression in vivo. In summary, the reduction of p-ERK and AP-1, the increased expression of E2F1 and FOXO3a, and the reduced association of cyclin D-CDK4 and cyclin E-CDK2 complexes contributed to p27 upregulation as well as G0/G1 arrest induced by downregulation of cathepsin B and uPAR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2938. doi:10.1158/1538-7445.AM2011-2938