title: Ubiquitin-specific protease 8 links the PTEN-Akt-AIP4 pathway to the control of FLIPS stability and TRAIL sensitivity in glioblastoma multiforme.
abstract: The antiapoptotic protein FLIP(S) 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 FLIP(S) 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 FLIP(S) 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 FLIP(S) ubiquitination, decreased FLIP(S) half-life, decreased FLIP(S) 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 FLIP(S) ubiquitination, USP8 seemed to control FLIP(S) ubiquitination through an intermediate target. Consistent with this idea, overexpression of wild-type USP8 decreased the ubiquitination of the FLIP(S) E3 ubiquitin ligase AIP4, an event previously shown to increase AIP4-FLIP(S) interaction, whereas siRNA-mediated suppression of USP8 increased AIP4 ubiquitination. Furthermore, the suppression of FLIP(S) 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 FLIP(S) stability and TRAIL sensitivity.
authors.affiliation: Brain Tumor Research Center, Department of Neurological Surgery, University of California San Francisco, San Francisco, California, USA.
authors.forename: Amith
authors.initials: A
authors.lastname: Panner
authors.forename: Courtney A
authors.initials: CA
authors.lastname: Crane
authors.forename: Changjiang
authors.initials: C
authors.lastname: Weng
authors.forename: Alberto
authors.initials: A
authors.lastname: Feletti
authors.forename: Shanna
authors.initials: S
authors.lastname: Fang
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Parsa
authors.forename: Russell O
authors.initials: RO
authors.lastname: Pieper
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D056827:Endosomal Sorting Complexes Required for Transport; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C432966:ITCH protein, human; D044767:Ubiquitin-Protein Ligases; D051057:Proto-Oncogene Proteins c-akt; D051059:PTEN Phosphohydrolase; C487757:Pten protein, mouse; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase; C541536:Usp8 protein, mouse