Ubiquitin-Specific Protease 8 Links the PTEN-Akt-AIP4 Pathway to the Control of FLIPS Stability and TRAIL Sensitivity in Glioblastoma Multiforme

The antiapoptotic protein FLIPS is a key suppressor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human glioblastoma multiforme (GBM) cells. We previously reported that a novel phosphatase and tensin homologue (PTEN)-Akt-atrophin-interacting protein 4 (AIP4) pathway regulates FLIPS ubiquitination and stability, although the means by which PTEN and Akt were linked to AIP4 activity were unclear. Here, we report that a second regulator of ubiquitin metabolism, the ubiquitin-specific protease 8 (USP8), is a downstream target of Akt, and that USP8 links Akt to AIP4 and the regulation of FLIPS stability and TRAIL resistance. In human GBM xenografts, levels of USP8 correlated inversely with pAkt levels, and genetic or pharmacologic manipulation of Akt regulated USP8 levels in an inverse manner. Overexpression of wild-type USP8, but not catalytically inactive USP8, increased FLIPS ubiquitination, decreased FLIPS half-life, decreased FLIPS steady-state levels, and decreased TRAIL resistance, whereas short interfering RNA (siRNA)mediated suppression of USP8 levels had the opposite effect. Because high levels of the USP8 deubiquitinase correlated with high levels of FLIPS ubiquitination, USP8 seemed to control FLIPS ubiquitination through an intermediate target. Consistent with this idea, overexpression of wild-type USP8 decreased the ubiquitination of the FLIPS E3 ubiquitin ligase AIP4, an event previously shown to increase AIP4-FLIPS interaction, whereas siRNA-mediated suppression of USP8 increased AIP4 ubiquitination. Furthermore, the suppression of FLIPS levels by USP8 overexpression was reversed by the introduction of siRNA targeting AIP4. These results show that USP8, a downstream target of Akt, regulates the ability of AIP4 to control FLIPS stability and TRAIL sensitivity. Cancer Res; 70(12); 5046-53. (C) 2010 AACR.