FOXO3A-short is a novel regulator of non-oxidative glucose metabolism associated with human longevity.

Intronic single-nucleotide polymorphisms (SNPs) in FOXO3A are associated with human longevity. Currently, it is unclear how these SNPs alter FOXO3A functionality and human physiology, thereby influencing lifespan. Here, we identify a primate-specific FOXO3A transcriptional isoform, FOXO3A-Short (FOXO3A-S), encoding a major longevity-associated SNP, rs9400239 (C or T), within its 5' untranslated region. The FOXO3A-S mRNA is highly expressed in the skeletal muscle and has very limited expression in other tissues. We find that the rs9400239 variant influences the stability and functionality of the primarily nuclear protein(s) encoded by the FOXO3A-S mRNA. Assessment of the relationship between the FOXO3A-S polymorphism and peripheral glucose clearance during insulin infusion (Rd clamp) in a cohort of Danish twins revealed that longevity T-allele carriers have markedly faster peripheral glucose clearance rates than normal lifespan C-allele carriers. In vitro experiments in human myotube cultures utilizing overexpression of each allele showed that the C-allele represses glycolysis independently of PI3K signaling, while overexpression of the T-allele represses glycolysis only in a PI3K-inactive background. Supporting this finding inducible knockdown of the FOXO3A-S C-allele in cultured myotubes increases the glycolytic rate. We conclude that the rs9400239 polymorphism acts as a molecular switch which changes the identity of the FOXO3A-S-derived protein(s), which in turn alters the relationship between FOXO3A-S and insulin/PI3K signaling and glycolytic flux in the skeletal muscle. This critical difference endows carriers of the FOXO3A-S T-allele with consistently higher insulin-stimulated peripheral glucose clearance rates, which may contribute to their longer and healthier lifespans.

Mesenchymal-Type Neuroblastoma Cells Escape ALK Inhibitors.

Cancer therapy frequently fails due to the emergence of resistance. Many tumors include phenotypically immature tumor cells, which have been implicated in therapy resistance. Neuroblastoma cells can adopt a lineage-committed adrenergic (ADRN) or an immature mesenchymal (MES) state. They differ in epigenetic landscape and transcription factors, and MES cells are more resistant to chemotherapy. Here we analyzed the response of MES cells to targeted drugs. Activating anaplastic lymphoma kinase (ALK) mutations are frequently found in neuroblastoma and ALK inhibitors (ALKi) are in clinical trials. ALKi treatment of ADRN neuroblastoma cells with a tumor-driving ALK mutation induced cell death. Conversely, MES cells did not express either mutant or wild-type ALK and were resistant to ALKi, and MES cells formed tumors that progressed under ALKi therapy. In assessing the role of MES cells in relapse development, TRAIL was identified to specifically induce apoptosis in MES cells and to suppress MES tumor growth. Addition of TRAIL to ALKi treatment of neuroblastoma xenografts delayed relapses in a subset of the animals, suggesting a role for MES cells in relapse formation. While ADRN cells resembled normal embryonal neuroblasts, MES cells resembled immature precursor cells, which also lacked ALK expression. Resistance to targeted drugs can therefore be an intrinsic property of immature cancer cells based on their resemblance to developmental precursors. SIGNIFICANCE: In neuroblastoma, mesenchymal tumor cells lack expression of the tumor-driving ALK oncogene and are resistant to ALKi, but dual treatment with ALKi and mesenchymal cell-targeting TRAIL delays tumor relapse.

A NOTCH feed-forward loop drives reprogramming from adrenergic to mesenchymal state in neuroblastoma.

Transition between differentiation states in development occurs swift but the mechanisms leading to epigenetic and transcriptional reprogramming are poorly understood. The pediatric cancer neuroblastoma includes adrenergic (ADRN) and mesenchymal (MES) tumor cell types, which differ in phenotype, super-enhancers (SEs) and core regulatory circuitries. These cell types can spontaneously interconvert, but the mechanism remains largely unknown. Here, we unravel how a NOTCH3 intracellular domain reprogrammed the ADRN transcriptional landscape towards a MES state. A transcriptional feed-forward circuitry of NOTCH-family transcription factors amplifies the NOTCH signaling levels, explaining the swift transition between two semi-stable cellular states. This transition induces genome-wide remodeling of the H3K27ac landscape and a switch from ADRN SEs to MES SEs. Once established, the NOTCH feed-forward loop maintains the induced MES state. In vivo reprogramming of ADRN cells shows that MES and ADRN cells are equally oncogenic. Our results elucidate a swift transdifferentiation between two semi-stable epigenetic cellular states.

FOXO3a is a major target of inactivation by PI3K/AKT signaling in aggressive neuroblastoma.

Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system with a highly variable prognosis. Activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway in neuroblastoma is correlated with poor patient prognosis, but the precise downstream effectors mediating this effect have not been determined. Here we identify the forkhead transcription factor FOXO3a as a key target of the PI3K/AKT pathway in neuroblastoma. FOXO3a expression was elevated in low-stage neuroblastoma tumors and normal embryonal neuroblasts, but reduced in late-stage neuroblastoma. Inactivation of FOXO3a by AKT was essential for neuroblastoma cell survival. Treatment of neuroblastoma cells with the dual PI3K/mTOR inhibitor PI-103 activated FOXO3a and triggered apoptosis. This effect was rescued by FOXO3a silencing. Conversely, apoptosis induced by PI-103 or the AKT inhibitor MK-2206 was potentiated by FOXO3a overexpression. Furthermore, levels of total or phosphorylated FOXO3a correlated closely with apoptotic sensitivity to MK-2206. In clinical specimens, there was an inverse relationship between gene expression signatures regulated by PI3K signaling and FOXO3a transcriptional activity. Moreover, high PI3K activity and low FOXO3a activity were each associated with an extremely poor prognosis. Our work indicates that expression of FOXO3a and its targets offer useful prognostic markers as well as biomarkers for PI3K/AKT inhibitor efficacy in neuroblastoma.

Sequencing of neuroblastoma identifies chromothripsis and defects in neuritogenesis genes.

Neuroblastoma is a childhood tumour of the peripheral sympathetic nervous system. The pathogenesis has for a long time been quite enigmatic, as only very few gene defects were identified in this often lethal tumour. Frequently detected gene alterations are limited to MYCN amplification (20%) and ALK activations (7%). Here we present a whole-genome sequence analysis of 87 neuroblastoma of all stages. Few recurrent amino-acid-changing mutations were found. In contrast, analysis of structural defects identified a local shredding of chromosomes, known as chromothripsis, in 18% of high-stage neuroblastoma. These tumours are associated with a poor outcome. Structural alterations recurrently affected ODZ3, PTPRD and CSMD1, which are involved in neuronal growth cone stabilization. In addition, ATRX, TIAM1 and a series of regulators of the Rac/Rho pathway were mutated, further implicating defects in neuritogenesis in neuroblastoma. Most tumours with defects in these genes were aggressive high-stage neuroblastomas, but did not carry MYCN amplifications. The genomic landscape of neuroblastoma therefore reveals two novel molecular defects, chromothripsis and neuritogenesis gene alterations, which frequently occur in high-risk tumours.

Integrin Cytoplasmic domain-Associated Protein-1 (ICAP-1) promotes migration of myoblasts and affects focal adhesions.

Integrin Cytoplasmic domain-Associated Protein-1 (ICAP-1) binds specifically to the beta1 integrin subunit cytoplasmic domain. We observed that RNAi-induced knockdown of ICAP-1 reduced migration of C2C12 myoblasts on the beta1 integrin ligand laminin and that overexpression of ICAP-1 increased this migration. In contrast, migration on the beta3 integrin ligand vitronectin was not affected. ICAP-1 knockdown also greatly diminished migration of microvascular endothelial cells on collagen. The number of central focal adhesions in C2C12 cells on laminin was reduced by ICAP-1 knockdown and increased by ICAP-1 overexpression. Previously, we demonstrated that ICAP-1 binds to the ROCK-I kinase and translocates ROCK-I to the plasma membrane. We show here that the ROCK kinase inhibitor Y27362 reduces migration on laminin and causes a loss of central focal adhesions, similarly as ICAP-1 knockdown. ICAP-1 and ROCK were co-immune-precipitated from C2C12 cells, and in cells that overexpressed ICAP-1, YFP-ROCK was translocated to membrane ruffles. These results indicate that ICAP-1 regulates beta1 integrin-dependent cell migration by affecting the pattern of focal adhesion formation. This is likely due to ICAP-1-induced translocation of ROCK to beta1 integrin attachment sites.

Integrin cytoplasmic domain-associated protein-1 (ICAP-1) interacts with the ROCK-I kinase at the plasma membrane.

The integrin cytoplasmic domain-associated protein-1 (ICAP-1) binds via its C-terminal PTB (phosphotyrosine-binding) domain to the cytoplasmic tails of beta1 but not other integrins. Using the yeast two-hybrid assay, we found that ICAP-1 binds the ROCK-I kinase, an effector of the RhoA GTPase. By coimmunoprecipitation we show that ICAP-1 and ROCK form complexes in cells and that ICAP-1 contains two binding sites for ROCK. In cells transfected with both ICAP-1 and ROCK, the proteins colocalized at the cell membrane predominantly in lamellipodia and membrane ruffles, but also in retraction fibers. ROCK was not found at these sites when ICAP-1 was not co-transfected, indicating that ICAP-1 translocated ROCK. In lamellipodia ICAP-1 and ROCK colocalized with endogenous beta1 integrins and this colocalization was also observed with the isolated ICAP-1 PTB domain. The plasma membrane localization of ROCK did not depend on beta1 integrin ligation or ROCK kinase activity, and in truncated ROCK proteins it required the presence of the ICAP-1-binding domain. To show that the interaction was direct, we measured fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) fused to ICAP-1 and yellow fluorescent protein (YFP) fused to ROCK. FRET was observed in lamellipodia in cells that were induced to spread. These results indicate that ICAP-1-mediated binding of ROCK to beta1 integrin serves to localize the ROCK-I kinase to both the leading edge and the trailing edge where ROCK affects cell migration.