Activation Of PI3-kinase Signaling Research Articles

Dilp-2-mediated PI3-kinase activation coordinates reactivation of quiescent neuroblasts with growth of their glial stem cell niche.

Dietary nutrients provide macromolecules necessary for organism growth and development. In response to animal feeding, evolutionarily conserved growth signaling pathways are activated, leading to increased rates of cell proliferation and tissue growth. It remains unclear how different cell types within developing tissues coordinate growth in response to dietary nutrients and whether coordinated growth of different cell types is necessary for proper tissue function. Using the early Drosophila larval brain, we asked whether nutrient-dependent growth of neural stem cells (neuroblasts), glia, and trachea is coordinated and whether coordinated growth among these major brain cell types is required for neural development. It is known that in response to dietary nutrients and PI3-kinase activation, brain and ventral nerve cord neuroblasts reactivate from quiescence and ventral nerve cord glia expand their membranes. Here, we assay growth in a cell-type specific manner at short time intervals in the brain and determine that growth is coordinated among different cell types and that coordinated growth is mediated in part through activation of PI3-kinase signaling. Of the 7 Drosophila insulin-like peptides (Dilps), we find that Dilp-2 is required for PI3-kinase activation and growth coordination between neuroblasts and glia in the brain. Dilp-2 induces brain cortex glia to initiate membrane growth and make first contact with quiescent neuroblasts. Once reactivated, neuroblasts promote cortex glia growth to ultimately form a selective membrane barrier. Our results highlight the importance of bidirectional growth signaling between neural stem cells and surrounding cell types in the brain in response to nutrition and demonstrate how coordinated growth among different cell types drives tissue morphogenesis and function.

Loss of Dnmt3a Dysregulates Hematopoietic Homeostasis and Myeloid Skewing Via the PI3Kinase Pathway: PI3Kα/β Inhibition Corrects Extramedullary Hematopoiesis and Myeloid Skewing Due to Loss of Dnmt3a

Mutations in DNA methyltransferase type 3A (DNMT3A) are frequently associated with myeloid malignancies including myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and myeloproliferative neoplasms (MPN) including systemic mastocytosis (SM). Presence of this mutation is associated with poor prognosis and reduced overall patient survival. Earlier studies have shown that hematopoietic specific loss of Dnmt3a in mice results in lethal, fully penetrant MPN with myelodysplasia (MDS/MPN) characterized by extramedullary hematopoiesis, peripheral cytopenias, splenomegaly and hepatomegaly. However, it's unclear how DNMT3A mutations contribute to myeloid skewing, and induce myeloid malignancies. Further, the mechanism(s) by which loss of DNMT3A impairs various mast cell functions leading to mastocytosis are also not defined. We show that loss of Dnmt3a in vitro results in accelerated and enhanced differentiation of mast cells from its bone marrow (BM) precursors and in vivo results in increased number of mast cells. Genome wide transcriptome analysis revealed elevated expression of the transcription factor MITF and a profound repression in the expression of C/EBPα in BM precursors derived from Dnmt3a-/- mice. Importantly, restoring the expression of Dnmt3a in Dnmt3a deficient BM cells, completely restored enhanced differentiation, along with correction in levels of MITF and C/EBPα, suggesting that early loss of C/EBPα in Dnmt3a-/- cells contributes to enhanced maturation of mast cells from its precursors. Furthermore, biochemical analysis revealed greater PI3Kinase activation in Dnmt3a-/- cells, and these findings were supported by genome wide transcriptome analysis. Importantly, mast cells derived from Dnmt3a-/- mice lacking the expression of p85α regulatory subunit of PI3Kinase or pharmacologic inhibition of PI3Kinase completely corrected cytokine induced hyperproliferation in these cells to near normal levels. Insights from mast cell studies led us to hypothesize that the aberrant hematopoietic regulation in Dnmt3a-/- mice described above may be due to hyperactivation of the PI3Kinase signaling in hematopoietic stem and progenitors (HSCPs). We investigated the role of PI3Kinase signaling in Dnmt3a loss induced myeloid malignancy using pharmacological inhibitor, GDC0941 (PI3Kα/δ inhibitor) and BAY1082439 (PI3Kα/β inhibitor). PI3Kα/β inhibitor treatment of Dnmt3a-/- mice markedly reduced monocytosis, neutrophilia, enhanced WBC counts, and improved RBCs, hematocrits and platelet numbers compared to control mice. In contrast, PI3Kα/δ inhibition moderately corrected the monocytosis and WBC aberrancy, however correction in erythroid dysregulation was not observed. Inhibiting PI3Kα/β signaling dramatically reduced splenomegaly and hepatomegaly in Dnmt3a-/- mice. In contrast, PI3Kα/δ inhibition moderately reduced the spleen size, however the correction in hepatomegaly was insignificant. Importantly, clonal hematopoiesis due to loss of Dnmt3a in a competitive transplant setting was rescued by inhibiting the PI3Kinase pathway. In the BM, PI3Kα/β inhibition moderately decreased LSK cells, however a marked decrease in GMP, and significant increase in MEP, and CLP was observed compared to controls. Flow cytometry analysis revealed that PI3Kα/β inhibition reduced Dnmt3a-/- BM cell migration to liver, and completely corrected the extramedullary hematopoiesis in the spleen compared to controls. Further, Dnmt3a loss induced alteration in erythroid development in the BM, spleen and PB was corrected upon PI3Kα/β blockade compared to controls. Genome wide transcriptome analysis revealed that PI3Kα/β inhibition markedly reduced the expression of GMP related genes, fetal liver hematopoiesis transcription program, and expression levels of inflammatory cytokines, growth factors, and chemokines in Dnmt3a depleted malignant mice. However PI3Kα/β inhibition increased the expression of genes involved in erythroid development in Dnmt3a-/- mice compared to controls. These results demonstrate that Dnmt3a ablation induces liver specific expansion of hematopoietic cells, and extramedullary hematopoiesis in spleen via aberrant activation of PI3Kinase signaling in HSCPs. Consistent with this notion, PI3K inhibitor treatment of malignant Dnmt3a-/- bearing mice showed significantly improved overall survival compared to controls. Disclosures No relevant conflicts of interest to declare.

If you can’t beat me, join me: collaborating oncogenes circumventing senescence and causing cancer

death. Signaling routes commonly altered in cancer include the RAS-MAP Kinase and PI3 Kinase (PI3K) pathways. Activation of the first can take place at many levels, most frequently through mutations in members of the RAS and RAF families, while also further upstream receptor tyrosine kinases can be activated by mutation or amplification. PI3K pathway activation occurs largely through mutations in its catalytic subunit PIK3CA or amplification of AKT family members. Additionally, the Phosphatase and Tensin Homolog (PTEN), a phosphatase negatively regulating the PI3K pathway by dephosphorylating the lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3), is inactivated in several tumor types by different mechanisms including mutation, deletion, and epigenetic silencing. Perhaps somewhat counter intuitively, although PI3K is an established downstream effector of RAS in oncogenic transformation, simultaneous mutations in RAS and in the PIK3CA ⁄ PTEN ⁄AKT pathway do occur in human cancer. Consistently, in several mouse models, they have been shown to cooperate in accelerating tumorigenesis. While it is reasonably well understood why mutant RAS and deficiencies in the pRB or p53 pathways collaborate in cancer, it is less clear what the mechanistic basis is for activating mutations at two points in a presumed single signaling pathway, like RAS and PI3K. Kennedy et al. recently reported on a molecular interplay model in which the second oncogene, acting downstream, counteracts the senescence-inducing effect of the first (Kennedy et al.,

OncomiRs: the discovery and progress of microRNAs in cancers

microRNAs (miRNAs) are evolutionarily conserved, endogenous, small, noncoding RNA molecules of about 22 nucleotides in length that function as posttranscriptional gene regulators. They are deemed to play a crucial role in the initiation and progression of human cancer, and those with a role in cancer are designated as oncogenic miRNAs (oncomiRs). For example, miR-15 and miR-16 induce apoptosis by targeting Bcl2. miRNAs from the miR-17-92 cluster modulate tumor formation and function as oncogenes by influencing the translation of E2F1 mRNA. miR-21 modulates gemcitabine-induced apoptosis by phosphatase and tensin homolog deleted on chromosome 10-dependent activation of PI 3-kinase signaling. miR-34a acts as a suppressor of neuroblastoma tumorigenesis by targeting the mRNA encoding E2F3 and reducing E2F3 protein levels. The chromosomal translocations associating with human tumors disrupt the repression of High mobility group A2 by let-7 miRNA. In addition, the oncomiRs expression profiling of human malignancies has also identified a number of diagnostic and prognostic cancer signatures. This article introduces the roles of oncomiRs in neoplasm development, progression, diagnosis, prognostication, as well as their mechanism of actions on target mRNAs and the functional outcomes of their actions on mRNAs. The paper ends with a brief perspective to the future of oncomiRs.

Involvement of Human Micro-RNA in Growth and Response to Chemotherapy in Human Cholangiocarcinoma Cell Lines

Micro-RNA (miRNA) are endogenous regulatory RNA molecules that modulate gene expression. Alterations in miRNA expression can contribute to tumor growth by modulating the functional expression of critical genes involved in tumor cell proliferation or survival. Our aims were to identify specific miRNA involved in the regulation of cholangiocarcinoma growth and response to chemotherapy. miRNA expression in malignant and nonmalignant human cholangiocytes was assessed using a microarray. Expression of selected miRNA and their precursors was evaluated by Northern blots and real-time polymerase chain reaction, respectively. The effect of selected miRNA on cell growth and response to chemotherapy was assessed using miRNA-specific antisense oligonucleotides to decrease miRNA expression or with precursor miRNA to increase cellular expression. miRNA expression was markedly different in malignant cholangiocytes, with decreased expression of many miRNA compared with nonmalignant cells. A cluster of miRNA, including miR-320, miR-200b, miR-21, miR-23a, miR-141, miR-27a, and miR-34a, were expressed in all cell lines. MiR-21, miR-141, and miR-200b were highly over-expressed in malignant cholangiocytes. Inhibition of miR-21 and miR-200b increased sensitivity to gemcitabine, whereas inhibition of miR-141 decreased cell growth. Treatment of tumor cell xenografts with systemic gemcitabine altered the expression of a significant number of miRNA. miR-21 modulates gemcitabine-induced apoptosis by phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-dependent activation of PI 3-kinase signaling. Potential target genes that were modulated by selected miRNA were identified. Alterations in miRNA expression contribute to tumor growth and response to chemotherapy. Aberrantly expressed miRNA or their targets will provide mechanistic insight and therapeutic targets for cholangiocarcinoma.

Stimulation of Cytokine Expression in Human Airway Epithelial Cells by IL‐17A: Polarity of Receptor Distribution and Dependence on PI3 Kinase/NF‐κB Signaling Pathways

The pro-inflammatory effects of IL-17A on airway epithelial cells have been recognized. In order to elucidate the signaling of IL-17A in the airway, primary human tracheobronchial epithelial cells (TBE) and HBE1 cell line were cultured under an air-liquid biphasic condition. Differential gene expression was determined by Affymetrix gene chips. We have observed gene induction of IL-19, GRO-α, -β, -γ, CXCL-5 and GCP-2 by IL-17A in addition to those in previous studies, such as HBD2, CCL20, and mucin genes, which were further confirmed by Q-PCR. Polarity of the treatment and anti-IL-17 receptor A (IL-17AR) antibody staining consistently showed the localization preference of IL-17AR is at the basal-lateral side of the epithelium. Inhibitor studies demonstrated that the gene induction is sensitive to the inhibition of PI3K and NF-κB activation. This is further supported by protein analysis, nuclear translocation and HBD-2 promoter analysis. However, PI3K inhibitor (LY294002) has no effects on NF-κB translocation, suggesting an additional PI3K-independent event is involved in coordinating the NF-κB-based transcriptional regulation. Based on these results, IL-17A basal-laterally acts through the IL-17 AR of the polarized airway epithelium to exert the activation of PI3 kinase signaling and NF-κB translocation to activate the expression of various cytokines.

Membrane localization of all class I PI 3‐kinase isoforms suppresses c‐Myc‐induced apoptosis in Rat1 fibroblasts via Akt

Phosphoinositide 3'-kinases (PI3Ks) constitute a family of lipid kinases implicated in signal transduction through tyrosine kinase receptors and heterotrimeric G protein-linked receptors. PI3Ks are heterodimers made up of four different 110-kDa catalytic subunits (p110alpha, p110beta, p110gamma, and p110delta) and a smaller regulatory subunit. Despite a clear implication of PI3Ks in survival signaling, the contribution of the individual PI3K isoforms has not been elucidated. To address this issue, we generated Rat1 fibroblasts that co-express c-Myc and membrane targeted derivates of the different p110 isoforms. Here we present data for the first time showing that activation of PI3-kinase signaling through membrane localization of p110beta, p110gamma, and p110delta protects c-Myc overexpressing Rat1 fibroblasts from apoptosis caused by serum deprivation like it has been described for p110alpha. Expression of each p110 isoform reduces significantly caspase-3 like activity in this apoptosis model. Decreased caspase-3 activity correlates with the increase in Akt phosphorylation in cells that contain one of the myristoylated p110 isoforms. p110 isoform-mediated protection from cell death was abrogated upon expression of a kinase-negative version of Akt.

Visualizing PI3 Kinase-Mediated Cell-Cell Signaling during Dictyostelium Development

Background: Starving amoebae of Dictyostelium discoideum communicate by relaying extracellular cAMP signals, which direct chemotactic movement, resulting in the aggregation of thousands of cells into multicellular aggregates. Both cAMP relay and chemotaxis require the activation of PI3 kinase signaling. The spatiotemporal dynamics of PI3 kinase signaling can be followed in individual cells via the cAMP-induced membrane recruitment of a GFP-tagged PH domain-containing protein, CRAC, which is required for the activation of adenylylcyclase.Results: We show that polarized periodic CRAC-GFP translocation occurs during the aggregation and mound stages of development in response to periodic cAMP signals. The duration of CRAC translocation to the membrane is determined by the duration of the rising phase of the cAMP signal. The system shows rapid adaptation and responds to the rate of change of the extracellular cAMP concentration. When the cells are in close contact, it takes 10 s for the signal to propagate from one cell to the next. In slugs, all cells show a permanent polarized PI3 kinase signaling in their leading edge, which is dependent on cell-cell contact.Conclusions: Measuring the redistribution of GFP-tagged CRAC has enabled us to study the dynamics of PI3 kinase-mediated cell-cell communication at the individual cell level in the multicellular stages of Dictyostelium development. This approach should also be useful to study the interactions between cell-cell signaling, cell polarization, and movement in the development of other organisms.