Abstract 2241: Differential regulation of Gli1 transcriptional activity by MAPK and PI3K pathways in neuroblastoma
Authors: Pritha Paul, Jingbo Qiao, Natasha S. Volny, Sora Lee, Dai H. Chung
Journal: Cancer Research (2012)

Abstract Activation of Hedgehog (Hh) pathway has been reported in multitude of cancers including neuroblastoma. Activation of Gli1, the nuclear effector of Hh pathway, is associated with differentiation and indicates good prognosis in neuroblastoma patients. However, the mechanism by which Gli1 protein is regulated in neuroblastoma remains unknown. MAPK pathway, but not PI3K/AKT pathway, synergizes with Hh signaling and positively regulates Gli1 activity in gastric and pancreatic cancers. Interestingly, activation of PI3K/AKT is associated with poor outcomes in neuroblastoma. Hence, the aim of this study was to examine the regulation of Gli1 transcriptional activity by RAF-MEK-ERK and PI3K-AKT signaling pathways. METHODS. NIH3T3, HEK 293T and the human neuroblastoma cell line, BE(2)-C, were used for our study. Gli1 transcriptional activity was measured by a Dual-Glo Luciferase Assay using the reporter 8X3′GLI-BS-Luc or its mutant form. Chemical inhibition and genetic manipulations were performed to modulate specific genes of the MAPK and PI3K signaling pathways. Transfection was achieved using Lipofectamine 2000 and cycloheximide chase assay was employed to study Gli1 protein degradation. RESULTS. Gli1 luciferase activity in neuroblastoma cells was decreased by specific MEK1/2 inhibitor U0126, but enhanced by PI3K inhibitor, LY294002. Activation of MAPK pathway by overexpression of its components (BRAF, MEK or ERK) enhanced Gli1 activity. Inhibiting the PI3K signaling by using deletion mutant of the PI3K subunit p85 (p≤85), a dominant negative form of AKT (dnAKT) or PTEN overexpression potentiated Gli1 transcriptional activity in neuroblastoma cells, whereas, constitutively active PI3K (p110α) decreased Gli1 luciferase activity. Interestingly, targeted silencing of AKT1 isoform inhibited Gli1 transcriptional activity, whereas, silencing the AKT2 isoform enhanced Gli1 signaling in neuroblastoma cells. Alternatively, using constitutively activated AKT1 or AKT2 (myr-AKT1 or myr-AKT2), we found opposing regulation of Gli1 transcriptional activity by these isoforms in neuroblastoma cells. Stable knockdown of AKT2 using shRNA further confirmed the increase in Gli1 transcriptional activity by stabilization of Gli1 protein. CONCLUSION. Here, we report that MAPK pathway positively regulates Gli1 transcriptional activity, whereas, PI3K pathway inhibits it. AKT isoforms 1 and 2 have opposing effect on the activity of Gli1. AKT2 appears to enhance Gli1 protein degradation in neuroblastoma cells. Hence, AKT2 activation in neuroblastoma might indicate a shift to a more aggressive phenotype and require the suppression of Gli1, which is pro-differentiation. The biological significance of this negative regulation in the progression of neuroblastoma remains to be determined. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2241. doi:1538-7445.AM2012-2241