Abstract 2129: Translational regulation of Id1 expression in glioma cells by the PI-3K pathway involves PPM1G-mediated dephosphorylation of 4E-BP1

Abstract

Abstract Increasing evidence suggests that Id1, a helix-loop-helix transcriptional modulator that inhibits differentiation and is elevated in stem cells, is involved in the initiation and aggressive behavior of malignant glial neoplasms. Our own previous study also found that overexpression of Id1 increases the transforming potential of human glioma cells (Xu et al., Oncotarget 5:1241-52, 2014). Since a high percentage of glioblastomas show overactivity of the PI-3K pathway and a link between this signaling pathway and Id1 expression has not previously been established, we sought to determine whether PI-3K signaling regulates Id1 expression. We explored this question using multiple established glioma cell lines (including SF767, LN229, U87, U251, U118, SF539). We initially find higher basal Id1 expression in glioma cell lines with dysregulated PI-3K signaling. To determine the role of PI-3K signaling on Id1 expression, glioma cell lines with increased flux through this signaling pathway were further assessed. In each case, forced expression of wild-type PTEN or treatment with either the PI-3K inhibitor LY294002 or the AKT inhibitor MK2206 leads to lower Id1 expression at the protein but not mRNA level. Pulse-chase assay and polyribosome profile analysis of Id1 mRNA confirm that inhibition of the PI-3K pathway reduces Id1 translation but not protein stability. The PI-3K pathway is known to regulate protein translation through activation of TORC1 leading to hyper-phosphorylation of 4E-BP1, a form of this protein that can no longer bind and inhibit the translation initiation factor eIF4E. Interestingly, while inhibition of PI-3K and AKT reduces 4E-BP1 phosphorylation and expression of Id1 in all cases, inhibition of TORC1 with rapamycin did not consistently have a similar effect. These results suggest a PI-3K/AKT-dependent, mTOR-independent mechanism for increasing Id1 translation. In fact, we identify a potential mechanism for this regulation of Id1 protein expression involving the serine-threonine phosphatase PPM1G. PPM1G and 4E-BP1 are associated by co-immunoprecipitation indicating interactions in the cell. In addition, knockdown of PPM1G expression by siRNA increases both 4E-BP1 phosphorylation and Id1 expression. Furthermore, PPM1G is not only phosphorylated but this phosphorylation is regulated by PI-3K activity. Finally, we find that PI-3K-dependent phosphorylation of PPM1G reduces its activity using an in vitro phosphatase assay and speculate that this regulation of PPM1G directly increases 4E-BP1 phosphorylation and activation of Id1 translation. In conclusion, our findings provide the first evidence that the PI-3K/AKT signaling pathway translationally regulates Id1 expression through modulation of PPM1G activity, possibly by phosphorylation, altering the balance between hyper- and hypo-phosphorylated 4E-BP1. Citation Format: Kaiming Xu, Lanfang Wang, Hui-Kuo G. Shu. Translational regulation of Id1 expression in glioma cells by the PI-3K pathway involves PPM1G-mediated dephosphorylation of 4E-BP1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2129. doi:10.1158/1538-7445.AM2015-2129