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The Drosophila insulin receptor (dInR) regulates cell growth and proliferation through the dPI3K/dAkt pathway, which is conserved in metazoan organisms. Here we report the identification and functional characterization of the Drosophila forkhead-related transcription factor dFOXO, a key component of the insulin signaling cascade. dFOXO is phosphorylated by dAkt upon insulin treatment, leading to cytoplasmic retention and inhibition of its transcriptional activity. Mutant dFOXO lacking dAkt phosphorylation sites no longer responds to insulin inhibition, remains in the nucleus, and is constitutively active. dFOXO activation in S2 cells induces growth arrest and activates two key players of the dInR/dPI3K/dAkt pathway: the translational regulator d4EBP and the dInR itself. Induction of d4EBP likely leads to growth inhibition by dFOXO, whereas activation of dInR provides a novel transcriptionally induced feedback control mechanism. Targeted expression of dFOXO in fly tissues regulates organ size by specifying cell number with no effect on cell size. Our results establish dFOXO as a key transcriptional regulator of the insulin pathway that modulates growth and proliferation. During the development of multicellular organisms, growth is tightly regulated by controlling cell number and cell size so that each organ reaches its appropriate dimensions in relation to the size of the organism. Many studies indicate that growth and proliferation are coordinated but distinct processes and that cells progress through the cell cycle only when sufficient mass, size, and macromolecular biosynthesis have been reached. (Hartwell et al. 1974;Johnston et al. 1977;Weigmann et al. 1997). Organism growth is controlled by coordinating both cell cycle progression and survival, which is modulated by nutrient availability, growth factors, and temperature. Growth factors can stimulate cell division and survival by activating the insulin receptor, which in turn acts through two main signal transduction cascades: the Ras/MAP kinase (Lee and McCubrey 2002) and the PI3K/ Akt kinase pathways (Cantley 2002). Insulin-mediated activation of PI3K increases production of 3Ј-phosphorylated phosphoinositide lipids (PIP 3 ) that serve as second messengers to recruit Akt to the plasma membrane (Datta et al. 1999). Once properly localized in the membrane, Akt becomes activated by phosphorylation and in turn phosphorylates a number of downstream targets that ultimately regulate cell growth. For example, Akt stimulates protein synthesis through activation of the target of rapamycin (TOR) kinase, which subsequently phosphorylates and inactivates the translational repressor eukaryotic initiation factor 4E-binding protein (4EBP; Gingras et al. 2001).In addition to modulating translation, Akt regulates transcription through the forkhead-related FOXO family of transcription factors FOXO1, FOXO3a, and FOXO4 (Burgering and Kops 2002) by phosphorylating these proteins at three conserved serine/threonine residues. This leads to retention of FOXO transcription factor...

Summary Background Alectinib, a highly selective, central nervous system (CNS)-active anaplastic lymphoma kinase (ALK) inhibitor, demonstrated promising clinical activity in crizotinib-naïve and crizotinib-resistant ALK-positive non-small-cell lung cancer (NSCLC). This phase 2 study evaluated the safety and efficacy of alectinib in ALK-positive NSCLC patients who progressed on previous crizotinib. Methods This ongoing North American study (NCT01871805) enrolled patients with stage IIIB/IV ALK-positive NSCLC, who had progressed following crizotinib. Patients were treated with oral alectinib 600 mg twice daily until progression, death or withdrawal. Primary endpoint was overall response rate (ORR) by independent review committee (IRC) using RECIST v1.1. Secondary endpoints included progression-free survival (PFS), duration of response (DOR), intracranial ORR and DOR, safety, and patient-reported outcomes. The intent-to-treat population was used for efficacy and safety analyses, with the response evaluable population used for response endpoints. Findings A total of 87 patients were enrolled in the intent-to-treat population. All patients had received prior crizotinib therapy, and 64 patients (74%) had also received prior chemotherapy. Fifty-two patients (60%) had baseline CNS metastases, of whom 18 (35%) had received no prior brain radiation therapy. At the time of primary analysis (median follow-up 4.8 months), ORR by IRC was 48% (95% CI 36–60). Adverse events were predominantly grade 1 or 2, most commonly constipation, fatigue, myalgia and peripheral edema. The most common grade ≥3 AEs were changes in laboratory values, including increased blood creatine phosphokinase (in 8%, n=7), increased alanine aminotransferase (in 6% n=5), and increased aspartate aminotransferase (in 5% n=4). Interpretation Alectinib demonstrated clinical efficacy and was well tolerated in patients with ALK-positive NSCLC who had progressed on crizotinib. Alectinib was active in the CNS, as demonstrated by durable responses in the majority of crizotinib-resistant patients with CNS disease. Therefore, alectinib could be a suitable treatment for patients with ALK-positive disease who have progressed on crizotinib.

Despite the high prevalence of nonalcoholic fatty liver disease (NAFLD), little is known of its pathogenesis based on study of human liver samples. By the use of Affymetrix GeneChips (17,601 genes), we investigated gene expression in the human liver of subjects with extreme steatosis due to NAFLD without histological signs of inflammation (liver fat 66.0 +/- 6.8%) and in subjects with low liver fat content (6.4 +/- 2.7%). The data were analyzed by using sequence-based reannotation of Affymetrix probes and a robust model-based normalization method. We identified genes involved in hepatic glucose and lipid metabolism, insulin signaling, inflammation, coagulation, and cell adhesion to be significantly associated with liver fat content. In addition, genes involved in ceramide signaling (MAP2K4) and metabolism (UGCG) were found to be positively associated with liver fat content. Genes involved in lipid metabolism (PLIN, ACADM), fatty acid transport (FABP4, CD36), amino acid catabolism (BCAT1), and inflammation (CCL2) were validated by real-time PCR and were found to be upregulated in subjects with high liver fat content. The data show that multiple changes in gene expression characterize simple steatosis.

We report here that the apoptosis-promoting protein TIA-1 regulates alternative pre-mRNA splicing of the Drosophila melanogaster gene male-specific-lethal 2 and of the human apoptotic gene Fas. TIA-1 associates selectively with pre-mRNAs that contain 5' splice sites followed by U-rich sequences. TIA-1 binding to the U-rich stretches facilitates 5' splice site recognition by U1 snRNP. This activity is critical for activation of the weak 5' splice site of msl-2 and for modulating the choice of splice site partner in Fas. Structural and functional similarities with the Saccharomyces cerevisiae splicing factor Nam8 suggest striking evolutionary conservation of a mechanism of pre-mRNA splicing regulation that controls biological processes as diverse as meiosis in yeast, dosage compensation in fruit flies, or programmed cell death in humans.