Mdm2 Induces Mono-Ubiquitination of FOXO4

Background: The Forkhead box O (FOXO) class of transcription factors are involved in the regulation of several cellular responses including cell cycle progression and apoptosis. Furthermore, in model organisms FOXOs act as tumor suppressors and affect aging. Previously, we noted that FOXOs and p53 are remarkably similar within their spectrum of regulatory proteins [1]. For example, the de-ubiquitinating enzyme USP7 removes ubiquitin from both FOXO and p53. However, Skp2 has been identified as E3 ligase for FOXO1, whereas Mdm2 is the prime E3 ligase for p53. Principal Findings/Methodology: Here we provide evidence that Mdm2 acts as an E3 ligase for FOXO as well. In vitro incubation of Mdm2 and FOXO results in ATP-dependent (multi)mono-ubiquitination of FOXO similar to p53. Furthermore, in vivo co-expression of Mdm2 and FOXO induces FOXO mono-ubiquitination and consistent with this result, siRNAmediated depletion of Mdm2 inhibits mono-ubiquitination of FOXO induced by hydrogen peroxide. Regulation of FOXO ubiquitination by Mdm2 is likely to be direct since Mdm2 and FOXO co-immunoprecipitate. In addition, Mdm2-mediated ubiquitination regulates FOXO transcriptional activity. Conclusions/Significance: These data identify Mdm2 as a novel E3 ligase for FOXOs and extend the analogous mode o

Aberrant over-expression of a forkhead family member, FOXO1A, in a brain tumor cell line

<p>Abstract</p> <p>Background</p> <p>The mammalian FOXO (forkhead box, O subclass) proteins are a family of pleiotropic transcription factors involved in the regulation of a broad range of cellular processes critical for survival. Despite the essential and diverse roles of the FOXO family members in human cells and their involvement in tumor pathogenesis, the regulation of <it>FOXO </it>expression remains poorly understood. We have addressed the mechanisms underlying the high level of expression of the <it>FOXO1A </it>gene in a cell line, PER-453, derived from a primitive neuroectodermal tumor of the central nervous system (CNS-PNET).</p> <p>Methods</p> <p>The status of the <it>FOXO1A </it>locus in the PER-453 CNS-PNET cell line was investigated by Southern blotting and DNA sequence analysis of the proximal promoter, 5'-UTR, open reading frame and 3'-UTR. FOXO1A expression was assessed by conventional and quantitative RT-PCR, Northern and Western blotting.</p> <p>Results</p> <p>Quantitative real-time RT-PCR (qRT-PCR) data indicated that after normalization to <it>ACTB </it>mRNA levels, canonical <it>FOXO1A </it>mRNA expression in the PER-453 cell line was 124-fold higher than the average level of five other CNS-PNET cell lines tested, 24-fold higher than the level in whole fetal brain, and 3.5-fold higher than the level in fetal brain germinal matrix cells. No mutations within the <it>FOXO1A </it>open reading frame or gross rearrangements of the <it>FOXO1A </it>locus were detected. However, a single nucleotide change within the proximal promoter and several nucleotide changes within the 3'-UTR were identified. In addition, two novel <it>FOXO1A </it>transcripts were isolated that differ from the canonical transcript by alternative splicing within the 3'-UTR.</p> <p>Conclusion</p> <p>The CNS-PNET cell line, PER-453, expresses <it>FOXO1A </it>at very high levels relative to most normal and cancer cells from a broad range of tissues. The <it>FOXO1A </it>open reading frame is wild type in the PER-453 cell line and the abnormally high <it>FOXO1A </it>mRNA expression is not due to mutations affecting the 5'-UTR or proximal promoter. Over expression of <it>FOXO1A </it>may be the result of PER-453 specific epimutations or imbalances in regulatory factors acting at the promoter and/or 3'-UTR.</p

A novel germline mutation of PTEN associated with brain tumours of multiple lineages

We have identified a novel germline mutation in the PTEN tumour suppressor gene. The mutation was identified in a patient with a glioma, and turned out to be a heterozygous germline mutation of PTEN (Arg234Gln), without loss of heterozygosity in tumour DNA. The biological consequences of this germline mutation were investigated by means of transfection studies of the mutant PTEN molecule compared to wild-type PTEN. In contrast to the wild-type molecule, the mutant PTEN protein is not capable of inducing apoptosis, induces increased cell proliferation and leads to high constitutive PKB/Akt activation, which cannot be increased anymore by stimulation with insulin. The reported patient, in addition to glioma, had suffered from benign meningioma in the past but did not show any clinical signs of Cowden disease or other hereditary diseases typically associated with PTEN germline mutations. The functional consequences of the mutation in transfection studies are consistent with high proliferative activity. Together, these findings suggest that the Arg234Gln missense mutation in PTEN has oncogenic properties and predisposes to brain tumours of multiple lineages

Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

BACKGROUND: Hypoxia-induced mitogenic factor (HIMF), a lung-specific growth factor, promotes vascular tubule formation in a matrigel plug model. We initially found that HIMF enhances vascular endothelial growth factor (VEGF) expression in lung epithelial cells. In present work, we tested whether HIMF modulates expression of fetal liver kinase-1 (Flk-1) in endothelial cells, and dissected the possible signaling pathways that link HIMF to Flk-1 upregulation. METHODS: Recombinant HIMF protein was intratracheally instilled into adult mouse lungs, Flk-1 expression was examined by immunohistochemistry and Western blot. The promoter-luciferase reporter assay and real-time RT-PCR were performed to examine the effects of HIMF on Flk-1 expression in mouse endothelial cell line SVEC 4–10. The activation of NF-kappa B (NF-κB) and phosphorylation of Akt, IKK, and IκBα were examined by luciferase assay and Western blot, respectively. RESULTS: Intratracheal instillation of HIMF protein resulted in a significant increase of Flk-1 production in lung tissues. Stimulation of SVEC 4–10 cells by HIMF resulted in increased phosphorylation of IKK and IκBα, leading to activation of NF-κB. Blocking NF-κB signaling pathway by dominant-negative mutants of IKK and IκBα suppressed HIMF-induced Flk-1 upregulation. Mutation or deletion of NF-κB binding site within Flk-1 promoter also abolished HIMF-induced Flk-1 expression in SVEC 4–10 cells. Furthermore, HIMF strongly induced phosphorylation of Akt. A dominant-negative mutant of PI-3K, Δp85, as well as PI-3K inhibitor LY294002, blocked HIMF-induced NF-κB activation and attenuated Flk-1 production. CONCLUSION: These results suggest that HIMF upregulates Flk-1 expression in endothelial cells in a PI-3K/Akt-NF-κB signaling pathway-dependent manner, and may play critical roles in pulmonary angiogenesis

A Role for Drosophila dFoxO and dFoxO 5′UTR Internal Ribosomal Entry Sites during Fasting

One way animals may cope with nutrient deprivation is to broadly repress translation by inhibiting 5′-cap initiation. However, under these conditions specific proteins remain essential to survival during fasting. Such peptides may be translated through initiation at 5′UTR Internal Ribosome Entry Sites (IRES). Here we show that the Drosophila melanogaster Forkhead box type O (dFoxO) transcription factor is required for adult survival during fasting, and that the 5′UTR of dfoxO has the ability to initiate IRES-mediated translation in cell culture. Previous work has shown that insulin negatively regulates dFoxO through AKT-mediated phosphorylation while dFoxO itself induces transcription of the insulin receptor dInR, which also harbors IRES. Here we report that IRES-mediated translation of both dFoxO and dInR is activated in fasted Drosophila S2 cells at a time when cap-dependent translation is reduced. IRES mediated translation of dFoxO and dInR may be essential to ensure function and sensitivity of the insulin signaling pathway during fasting

Curcumin induces the apoptosis of human monocytic leukemia THP-1 cells via the activation of JNK/ERK Pathways

<p>Abstract</p> <p>Background</p> <p>Curcumin is a principal compound of turmeric, commonly used to treat tumors and other diseases. However, its anti-cancer activity in human acute monocytic leukemia THP-1 cells is not clear. This study aimed to study the anti-cancer effect and action of curcumin on THP-1 cells.</p> <p>Methods</p> <p>THP-1 parental cells and PMA-treated THP-1 cells, were used as <it>in vitro </it>models to evaluate the anti-cancer effect and mechanism of curcumin. Apoptosis and its mechanism were evaluated by WST-1, flow cytometry and Western blotting. MAPK inhibitors were used to further confirm the molecular mechanism of curcumin-induced THP-1 cell apoptosis.</p> <p>Results</p> <p>Curcumin induced cell apoptosis of THP-1 cells as shown by cell viability, cell cycle analysis and caspase activity. Curcumin significantly increased the phosphorylation of ERK, JNK and their downstream molecules (c-Jun and Jun B). Inhibitor of JNK and ERK reduced the pro-apoptotic effect of curcumin on THP-1 cells as evidenced by caspase activity and the activation of ERK/JNK/Jun cascades. On the contrary, the pro-apoptotic effect of curcumin was abolished in the differentiated THP-1 cells mediated by PMA.</p> <p>Conclusions</p> <p>This study demonstrates that curcumin can induce the THP-1 cell apoptosis through the activation of JNK/ERK/AP1 pathways. Besides, our data suggest its novel use as an anti-tumor agent in acute monocytic leukemia.</p

Inhibition of FOXO3 Tumor Suppressor Function by βTrCP1 through Ubiquitin-Mediated Degradation in a Tumor Mouse Model

The ubiquitin-proteasome system is the primary proteolysis machine for controlling protein stability of the majority of regulatory proteins including those that are critical for cancer development. The forkhead box transcription factor FOXO3 plays a key role in regulating tumor suppression; however, the control of FOXO3 protein stability remains to be established. It is crucial to elucidate the molecular mechanisms underlying the ubiquitin-mediated degradation of FOXO3 tumor suppressor.Here we show that betaTrCP1 oncogenic ubiquitin E3-ligase interacts with FOXO3 and induces its ubiquitin-dependent degradation in an IkappaB kinase-beta phosphorylation dependent manner. Silencing betaTrCP1 augments FOXO3 protein level, resulting in promoting cellular apoptosis in cancer cells. In animal models, increasing FOXO3 protein level by silencing betaTrCP1 suppresses tumorigenesis, whereas decreasing FOXO3 by over-expressing betaTrCP1 promotes tumorigenesis and tumor growth in vivo.This is a unique demonstration that the betaTrCP1-mediated FOXO3 degradation plays a crucial role in tumorigenesis. These findings significantly contribute to understanding of the control of FOXO3 stability in cancer cells and may provide opportunities for developing innovative anticancer therapeutic modalities

Evolution of the TOR Pathway

The TOR kinase is a major regulator of growth in eukaryotes. Many components of the TOR pathway are implicated in cancer and metabolic diseases in humans. Analysis of the evolution of TOR and its pathway may provide fundamental insight into the evolution of growth regulation in eukaryotes and provide a practical framework on which experimental evidence can be compared between species. Here we performed phylogenetic analyses on the components of the TOR pathway and determined their point of invention. We find that the two TOR complexes and a large part of the TOR pathway originated before the Last Eukaryotic Common Ancestor and form a core to which new inputs have been added during animal evolution. In addition, we provide insight into how duplications and sub-functionalization of the S6K, RSK, SGK and PKB kinases shaped the complexity of the TOR pathway. In yeast we identify novel AGC kinases that are orthologous to the S6 kinase. These results demonstrate how a vital signaling pathway can be both highly conserved and flexible in eukaryotes

NFV, an HIV-1 protease inhibitor, induces growth arrest, reduced Akt signalling, apoptosis and docetaxel sensitisation in NSCLC cell lines

HIV-1 protease inhibitor (PI), nelfinavir (NFV) induced growth arrest and apoptosis of NCI-H460 and -H520, A549, EBC-1 and ABC-1 non-small-cell lung cancer (NSCLC) cells in association with upregulation of p21waf1, p27 kip1 and p53, and downregulation of Bcl-2 and matrix metalloproteinase (MMP)-2 proteins. We found that NFV blocked Akt signalling in these cells as measured by Akt kinase assay with glycogen synthase kinase-3α/β (GSK-3α/β) as a substrate. To explore the role of Akt signalling in NFV-mediated growth inhibition of NSCLC cells, we blocked this signal pathway by transfection of Akt small interfering RNA (siRNA) in these cells; transient transfection of Akt siRNA in NCI-H460 cells decreased the level of Bcl-2 protein and slowed their proliferation compared to the nonspecific siRNA-transfected cells. Conversely, forced-expression of Akt partially reversed NFV-mediated growth inhibition of these cells, suggesting that Akt may be a molecular target of NFV in NSCLC cells. Also, we found that inhibition of Akt signalling by NFV enhanced the ability of docetaxel to inhibit the growth of NCI-H460 and -H520 cells, as measured by MTT assay. Importantly, NFV slowed the proliferation and induced apoptosis of NCI-H460 cells present as tumour xenografts in nude mice without adverse systemic effects. Taken together, this family of compounds might be useful for the treatment of individuals with NSCLC