Mutation of the Zebrafish Nucleoporin elys Sensitizes Tissue Progenitors to Replication Stress

The recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress

WAVE3-NFκB interplay is essential for the survival and invasion of cancer cells.

The WAVE3 cytoskeletal protein promotes cancer invasion and metastasis. We have shown that the WAVE3-mediated activation of cancer cell invasion is due, in part, to its regulation of expression and activity of key metalloproteinases (MMPs), including MMP9, which is centrally involved in invadopodia-mediated degradation of the extracellular matrix (ECM). MMP9 is also a major NFκB target gene, suggesting a potential linkage of WAVE3 to this pathway, which we sought to investigate. Mechanistically, we found that loss of WAVE3 in cancer cells leads to inhibition of NFκB signaling as a result of a decrease in the nuclear translocation of NFκB and therefore loss of activation of NFκB target genes. Conversely, overexpression of WAVE3 was sufficient to enhance NFκB activity. Both pharmacologic and genetic manipulations of NFκB effector molecules show that the biological consequence of loss of WAVE3 function in the NFκB pathway result the inhibition of invadopodia formation and ECM degradation by cancer cells, and these changes are a consequence of decreased MMP9 expression and activity. Loss of WAVE3 also sensitized cancer cells to apoptosis and cell death driven by TNFα, through the inhibition of the AKT pro-survival pathway. Our results identify a novel function of WAVE3 in NFκB signaling, where its activity is essential for the regulation of invadopodia and ECM degradation. Therefore, targeted therapeutic inhibition of WAVE3 will sensitize cancer cells to apoptosis and cell death, and suppress cancer invasion and metastasis

Smooth muscle tension induces invasive remodeling of the zebrafish intestine.

The signals that initiate cell invasion are not well understood, but there is increasing evidence that extracellular physical signals play an important role. Here we show that epithelial cell invasion in the intestine of zebrafish meltdown (mlt) mutants arises in response to unregulated contractile tone in the surrounding smooth muscle cell layer. Physical signaling in mlt drives formation of membrane protrusions within the epithelium that resemble invadopodia, matrix-degrading protrusions present in invasive cancer cells. Knockdown of Tks5, a Src substrate that is required for invadopodia formation in mammalian cells blocked formation of the protrusions and rescued invasion in mlt. Activation of Src-signaling induced invadopodia-like protrusions in wild type epithelial cells, however the cells did not migrate into the tissue stroma, thus indicating that the protrusions were required but not sufficient for invasion in this in vivo model. Transcriptional profiling experiments showed that genes responsive to reactive oxygen species (ROS) were upregulated in mlt larvae. ROS generators induced invadopodia-like protrusions and invasion in heterozygous mlt larvae but had no effect in wild type larvae. Co-activation of oncogenic Ras and Wnt signaling enhanced the responsiveness of mlt heterozygotes to the ROS generators. These findings present the first direct evidence that invadopodia play a role in tissue cell invasion in vivo. In addition, they identify an inducible physical signaling pathway sensitive to redox and oncogenic signaling that can drive this process

L-leucine Supplementation Reverses Hyperammonemia Induced Skeletal Muscle Mitochondrial Dysfunction

Background. Skeletal muscle hyperammonemia occurs in cirrhosis and contributes to sarcopenia or loss of skeletal muscle mass. Skeletal muscle ammonia disposal occurs in the mitochondria by cataplerosis (loss of tricarboxylic acid- TCA- cycle intermediates) of a-ketoglutarate to generate glutamate and glutamine. Skeletal muscle hyperammonemia also results a unique cellular stress response with partitioning of L-leucine into the mitochondria to assist ammonia disposal, potentially promoting anaple- rosis (entry of TCA cycle intermediates) either directly or indirectly. We have previously reported that skeletal muscle hyperammonemia decreases cellular oxygen consumption, lowers tricarboxylic acid cycle intermediates and increases mitochondrial reactive oxygen species in the muscle. We therefore hypothesized that ammonia induced skeletal muscle mitochondrial dysfunction will be reversed by L-leucine supplementation that is sufficient to satisfy the metabolic demand in the mitochondria. Methods. Differentiated C2C12 murine myotubes were treated with 10mM ammonium acetate or vehicle alone and the response to L leucine 5mM was quantified. Cellular oxygen consumption in the basal state and the response to substrates for components of the electron transport chain (pyruvate and malate for complex I; succinate for complex II) were measured using high sensitivity respirometry in intact and permeabilized myotubes. Concentration of TCA cycle intermediates in cell lysates were quantified using isotope ratio mass spectrometry respectively. Mitochondrial reactive oxygen species were measured by flow cytometry using the fluoroprobe, MitoSox®. ATP content was measured by a fluorometric assay. Complementary studies were performed in myotubes grown in leucine deficient medium that were treated with L-leu- cine. All experiments were done in at least 4 biological replicates. Results. Hyperammonemia decreased intact cell and substrate dependent oxygen consumption in myotubes that were reversed by 5mM L-leucine. Increased reactive oxygen species during hyperammonemia was also decreased by L-Leucine Hyperammonemia resulted in a reduction in TCA cycle intermediates and the intermediate ratios at 6 and 24 h that was reversed by L-leucine. Low cellular ATP content and ATP synthesis were also reversed by L-leucine supplementation. L-leucine deficient medium resulted in a depletion of TCA cycle intermediates that was aggravated by hyperammonemia and reversed by L leucine. Conclusions. We demonstrate for the first time that ammonia induced skeletal muscle mitochondrial dysfunction is reversed by L leucine supplementation and restores the anaplerosis/cataplerosis balance

Down-regulation of WAVE3 sensitizes MDA-MB-231 cells to TNFα induced apoptosis through Akt signaling.

<p>Representative histograms using flow cytometry of control shRNA (ctrl-sh, green)- or sh-WAVE3-expressing (red) MDA-MB-231 cells after TNFα treatment stained by Annexin V for apoptosis (A) and by Propidium Iodide for cell death (B). (C) Representative confocal images of Ctrl-sh and sh-W3 MDA-MB-231 cells stained Annexin V (Green) and cleaved caspase3 (Red) before and after TNFα treatment (50 ng/μl for 15 min). The bright field images in the right panels indicate healthy cells. High resolution enlarged images are shown in the insets. (D & E) Quantification of Annexin V staining levels (D) and Caspase 3 staining levels. (F) Western blot analysis with the indicated antibodies of cell lysates form the Ctrl-sh and sh-W3 cells after treatment with TNFα at the indicated times. The numbers below the p-AKT and the p-p38 panels indicate their respective fold change with respect to the untreated Ctrl-sh cells. All data are representative of 3 independent experiments, or are the mean (±SE; n = 3; *, p <0.05; Student's t-test).</p

Loss of WAVE3 inhibits the NFκB-mediated stimulation of MMP9 expression and activity.

<p>(A) Western blot analysis with the indicated antibodies of cell lysates of MDA-MB-231 cells transfected with non-targeting shRNA (Ctrl-sh), and two different sh-WAVE3 clones (sh-W3-1 and sh-W3-2). The numbers below the WAVE3 and MMP9 panels indicate the fold change WAVE3 and MMP9 levels with respect to the untreated Ctrl-sh cells. β-Actin was used as a loading control. (B) Gelatin zymography of activated MMP9 and MMP2 in conditioned media of the Ctrl-sh two sh-W3 clones. C) Gelatin zymography of activated MMP9 before and after treatment with TNFα in the conditioned media of the Ctrl-sh and two sh-W3 clones. In both (B) and (C), the Red-Ponceau-stained gels are shown as loading controls. The numbers below the zymography panels indicate the fold change of MMP9 levels with respect to the untreated Ctrl-sh cells.</p

WAVE3 is required for NFκB activation.

<p>(A) Luciferase-based NFκB reporter assay in MDA-MB-231 cells with stable transfection of a non-targeting shRNA (Ctrl-sh) or the WAVE3-trageting shRNA (sh-W3). (Inset) Western blot analysis of protein lysates of cells described in (A) with anti-WAVE3 antibody. β-Actin was used as a loading control. (*, p<0.05). (B) Western blot analysis with the indicated antibodies of protein lysates from Ctrl-sh MDA-MB-231 and two different shWAVE3-derived clones (sh-W3-1 and sh-W3-2), before and after TNFα treatment (50 ng/μl for 15 min). The numbers below the p-p65 and WAVE3 panels indicate the fold change of p-p65 and WAVE3 levels, respectively, as compared to the untreated Ctrl-sh cells. (C) Quantification of p-p65 levels in the indicated conditions. (D) Western blot analysis with p65 antibody of the nuclear fraction lysates from the Ctrl-sh and the sh-W3 MDA-MB-231 cells, with or without TNFα treatment. H2b was used as a loading control for the nuclear fraction. The numbers below the H2b panel indicate the fold change p65 levels with respect to the untreated Ctrl-sh cells. (E) Immuno-staining for nuclear translocation (white arrows) of p65 protein (Red) in Ctrl-sh and shWAVE3 MDA-MB-231 cells. Cells nuclei are counter-stained with DAPI (Blue). (F) Quantification of p65 nuclear staining. All data are representative of 3 independent experiments, or are the mean ± SD (n = 3; *, p <0.05; Student's t-test)</p

The WAVE3:NFκB interplay involves Akt signaling to regulate invadopodia and ECM degradation in cancer cells.

<p>(A) Western blot analysis with the indicated antibodies of cell lysates of untreated MDA-MB-231 cells or treated with TNFα, MK-2206 or both. β-Actin was used a loading control. (B) Confocal microscopy micrographs of MDA-MB-231 cells grown on FITC-labeled gelatin, treated as indicated and stained for F-actin filaments (left panels). The white arrow-heads point to invadopodia structures (white spots). Areas of ECM degradation (black arrow-heads) are shown as black spots (middle panels). The invadopodia structures coincide with the areas of ECM degradation in the merged image (right panels). (C) Quantification of number of invadopodia per cell in the control and treated cells. (D) Quantification of area of gelatin degradation per cell in the control and treated cells. All data are representative of 3 independent experiments, or are the means ± SD.</p