title: Mechanism of p27 upregulation induced by downregulation of cathepsin B and uPAR in glioma.
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. p27(Kip1) (p27) is one of the major cyclin-CDK regulators in the G1 phase. uPAR and cathepsin B downregulation was recently shown to induce p27 expression through PI3K/Akt/FOXO3a signaling. Since uPAR and cathepsin B knockdown also decreased phosphorylation of ERK, we hypothesized that ERK also has a role to play in p27 induction. As induction of p27 is due to an increase in gene transcription, we investigated the roles of c-Myc and E2F1 transcription factors which have been shown to potently affect p27 promoter activity. In the present study, shRNA against cathepsin B and uPAR as well as specific inhibitors, Wortmannin (10 μM) and U0126 (10 μM), were used to determine the roles of AKT and ERK signaling on p27 expression. Immunoblot analysis demonstrated that downregulation of both p-ERK and p-AKT downstream of EGFR and β1 integrin are involved in the p27 upregulation. Cathepsin B and uPAR downregulation induced E2F1 and decreased phosphorylaion of pocket proteins and c-Myc expression. CHIP analysis and luciferase expression studies confirmed the functional association of transcription factor E2F1 to the p27 promoter. Further, c-Myc-Max interaction inhibitor studies showed an inverse pattern of c-Myc and p27 expression. Also, cathepsin B and uPAR downregulation reduced tumor growth and increased p27 nuclear expression in vivo. In summary, cathepsin B and uPAR downregulation reduced p-ERK levels and c-Myc expression, increased expression of E2F1 and FOXO3a, decreased phosphorylation of pocket proteins and thus upregulated p27 expression in glioma cells.
authors.affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656, USA.
authors.forename: Sreelatha
authors.initials: S
authors.lastname: Gopinath
authors.forename: Kiranmai
authors.initials: K
authors.lastname: Alapati
authors.forename: Rama Rao
authors.initials: RR
authors.lastname: Malla
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Gondi
authors.forename: Sanjeeva
authors.initials: S
authors.lastname: Mohanam
authors.forename: Dzung H
authors.initials: DH
authors.lastname: Dinh
authors.forename: Jasti S
authors.initials: JS
authors.lastname: Rao
mesh_terms: D000818:Animals; D002401:Cathepsin B / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D066246:ErbB Receptors / Q000378:metabolism; D016193:G1 Phase; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D005910:Glioma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D039081:Integrin alpha5beta1 / Q000378:metabolism; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism*; D016192:Resting Phase, Cell Cycle; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: D039081:Integrin alpha5beta1; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D055293:Receptors, Urokinase Plasminogen Activator; D050760:Cyclin-Dependent Kinase Inhibitor p27; D066246:ErbB Receptors; D002401:Cathepsin B