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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.

SUBMITTER: Gopinath S

PROVIDER: S-EPMC3183416 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMeningiomas are the most commonly occurring intracranial tumors and account for approximately 15-20% of central nervous system tumors. Surgery and radiation therapy is a common treatment for brain tumors, however, patients whose tumors recur after such treatments have limited therapeutic options. Earlier studies have reported important roles of uPA, uPAR and cathepsin B in tumor progression.Methodology/principal findingsIn the present study, we examined the therapeutic significance of RNAi-mediated simultaneous down regulation of these proteolytic networks using two bicistronic siRNA constructs, pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) either alone or in combination with radiation in two different meningioma cell lines. Transfection of meningioma cells with pUC and pU2 significantly reduced angiogenesis as compared to control treatment both in vitro and in vivo nude mice model. This effect is mediated by inhibiting angiogenic molecules (Ang-1, Ang-2 and VEGF). Expression of focal adhesion kinase (FAK) is elevated in malignant meningioma, yet the role of intrinsic FAK activity in promoting tumor progression remains undefined. We found that pUC treatment reduced FAK phosphorylation at Y925 more efficiently compared to pU2 treatment. In immunoprecipitation assay, we found pronounced reduction of FAK (Y925) interaction with Grb2 in meningioma cells transfected with pUC with and without irradiation. Transient over-expression of uPAR and cathepsin B by full length uPAR/cathepsin B (FLpU/C) in pUC transfected meningioma cells promoted vascular phenotype, rescued expression of Ang-1, Ang-2, VEGF, FAK (Y925) and Grb2 both in vitro and in vivo mice model.Conclusion/significanceThese studies provide the first direct proof that bicistronic siRNA construct for uPAR and cathepsin B (pUC) reduces Y925-FAK activity and this inhibition is rescued by overexpression of both uPAR and cathepsin B which clearly demonstrates that pUC could thus be a potential therapeutic approach as an anti-angiogenic agent in meningioma.

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Mechanism of p27 upregulation induced by downregulation of cathepsin B and uPAR in glioma.

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