Malignant mesotheliomas (MMs) are very aggressive tumors that respond poorly to standard chemotherapeutic approaches. The phosphatidylinositol 3-kinase (PI3K)/ AKT pathway has been implicated in tumor aggressiveness, in part by mediating cell survival and reducing sensitivity to chemotherapy. Using antibodies recognizing the phosphorylated/activated form of AKT kinases, we observed elevated phospho-AKT staining in 17 of 26 (65%) human MM specimens. In addition, AKT phosphorylation was consistently observed in MMs arising in asbestos-treated mice and in MM cell xenografts. Consistent with reports implicating hepatocyte growth factor (HGF)/Met receptor signaling in MM, all 14 human and murine MM cell lines had HGF-inducible AKT activity. One of nine human MM cell lines had elevated AKT activity under serum-starvation conditions, which was associated with a homozygous deletion of PTEN, the first reported in MM. Treatment of this cell line with the mTOR inhibitor rapamycin resulted in growth arrest in G1 phase. Treatment of MM cells with the PI3K inhibitor LY294002 in combination with cisplatin had greater efficacy in inhibiting cell proliferation and inducing apoptosis than either agent alone. Collectively, these data indicate that MMs frequently express elevated AKT activity, which may be targeted pharmacologically to enhance chemotherapeutic efficacy. These findings also suggest that mouse models of MM may be useful for future preclinical studies of pharmaceuticals targeting the PI3K/AKT pathway.

Younger children may require a longer isolation period and more aggressive treatment.

mice, we confirm genetically that Nef requires PACS-2 to localize to the paranuclear region and assemble the multikinase cascade. Moreover, genetic loss of PACS-2 or inhibition of class I PI3K prevents Nef-mediated MHC-I down-regulation, demonstrating that short interfering RNA knockdown of PACS-2 phenocopies the gene knock-out. This PACS-2-dependent targeting pathway is not restricted to Nef, because PACS-2 is also required for trafficking of an endocytosed cation-independent mannose 6-phosphate receptor reporter from early endosomes to the TGN. Together, these results demonstrate PACS-2 is required for Nef action and sorting of itinerant membrane cargo in the TGN/endosomal system. HIV-12 negative factor (Nef), a 27-kDa N-myristoylated protein, enhances viral replication and virion infectivity, and it is required for the onset of AIDS following HIV-1 infection (1, 2). Nef affects cells in many ways, including altering T-cell activation and maturation (3-5), subverting the apoptotic machinery, and down-regulating CD4 molecules and major histocompatibility complex class I (MHC-I) molecules encoded by the HLA-A and -B loci (2,6). But the precise mechanism of how Nef mediates these pathways has remained elusive.Nef diverts cell-surface MHC-I molecules to trans-Golgi network (TGN)-associated endosomal compartments by an endocytic pathway that is stimulated by class I phosphoinositide 3-kinase (PI3K) and dependent on ADP-ribosylation factor-6 (ARF6) (2,7,8). This MHC-I down-regulation requires the action of three motifs (1, 2) as follows: an N-proximal ␣-helical region (residues 7-26) (9) containing a critical methionine (Met 20 ) that promotes association of MHC-I with the heterotetrameric adaptor AP-1 (10, 11); an acidic cluster (EEEE 65 ) required for binding to phosphofurin acidic cluster sorting protein-1 (PACS-1) (12, 13); and an SH3-binding domain formed by a type II polyproline helix (PXXP 75 ) (9, 12) that promotes association of Nef with Src family tyrosine kinases (SFKs) (14 -17). The conservation of these three motifs in the pandemic M group HIV-1, which accounts for over 90% of all AIDS cases worldwide, suggests they control an essential pathway required for HIV-1 pathogenesis (18,19).The EEEE 65 and PXXP 75 sites act sequentially to recruit and stimulate class I PI3K by directing the assembly of an SFK-ZAP-70/Syk-PI3K cascade in primary CD4 ϩ T-cells (8). Assembly of this multikinase complex is initiated by the EEEE 65 -dependent targeting of Nef to the paranuclear region, which enables the PXXP 75 SH3 domain-binding motif to recruit a TGN-localized SFK. This Nef-activated SFK then stimulates tyrosine phosphorylation of ZAP-70/Syk, recruiting class I PI3K by an unknown mechanism to increase endocytosis of MHC-I through an ARF6-regulated pathway (7,8). MHC-I molecules internalized by this signaling pathway are then redistributed to paranuclear endosomal compartments by a process * This work was supported by National Institutes of Health National Research Service Awards DK076343 (to R. T. Y.), T32 NS007...

Summary TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis.