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

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

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

Over-expression of WAVE3 activates NFκB signaling.

<p>(A) Western blot analysis of WAVE3-GFP protein levels in GFP and GFP-W3-expressing cells. (B) Luciferase-based NFκB reporter assay in GFP and GFP-WAVE3 expressing cells (*, p<0.05). (C & D) Western blot analysis with the indicated antibodies of cell lysates from the GFP and WAVE3-GFP expressing cells. The numbers below the GFP panel indicate the fold change p-p65 levels with respect to the GFP cells. (E & F) Western blot analysis with the indicated antibodies of cell lysates from the GFP and WAVE3-GFP expressing cells after treatment with cyclohexamide (CHX, E) and the proteasome inhibitor MG132 (F). The numbers below the GFP panel indicate the fold change p-p65 levels with respect to the GFP cells. β-Actin was used a loading control. All data are representative of 3 independent experiments, or are the mean ± SD (n = 3; *, p <0.05; Student's t-test)</p

WAVE3 is involved in invadopodia formation and ECM degradation.

<p>(A) Confocal microscopic micrographs of MDA-MB-231 cells grown on FITC-labeled gelatin and stained for (a) F-actin filaments (red). The white arrow-heads point to invadopodia structures (white spots). (b) Areas of ECM degradation (black arrow-head) are shown as black spots. The invadopodia structures coincide with the areas of ECM degradation in the merged image (c). (d) In the 3-dimensional reconstructed image the black arrows point to invadopodia (red) infiltrating the gelatin bed (green). (B) Confocal microscopic micrographs of MDA-MB-231 cells grown on collagen-coated coverslips, treated with TNFα (50 ng/μl) for 15 min, and stained for WAVE3 (left panel) and Cortactin (middle panel). The arrow-heads point to areas with invadopodia where both Cortactin and WAVE3 colocalize in the merged image in the right panel (yellow spots).</p

Src induces formation of invadopodia-like protrusions in the intestine of wild type zebrafish larvae.

<p>(A–D) Sagittal confocal scans through the intestine of 74 hpf wild type and <i>mlt</i> larvae that express Src-mCherry (red) and Lifeact-GFP (green) in the intestinal epithelium. (A) In WT, Src (red) is localized at the apical (ap) and lateral epithelial cell membrane. (B) In <i>mlt</i>, Src also localizes to sites of actin-rich (green) invadopodia-like protrusions (arrowheads B) arising from the basal epithelial cell membrane (ba). (C) Constitutively active Src (caSrc; red) localizes to invadopodia-like protrusions (green) in <i>mlt</i> (arrowheads). (D) caSrc induces formation of the protrusions in WT (arrowheads). (E) Histological cross-sections through the intestine of a 74 hpf wild type larva showing caSrc-rich protrusions (green) protruding through small degraded regions of the basal lamina (laminin immunostain, red). Additional examples are shown in high power images (E, E′, and E″).</p

Oxidative stress induces invasive remodeling in <i>mlt</i> heterozygous larvae.

<p>(A–C) Lateral images of live WT (A), <i>mlt</i> homozygous (B), and <i>mlt</i> heterozygous larvae (C). The WT and <i>mlt</i> heterozygous larvae received 3 h of treatment with Menadione beginning at 73 hpf. Menadione treated heterozygote (C) larvae have an intestinal phenotype (arrowheads) resembling the untreated <i>mlt</i> homozygous larvae (B). (D–F) Corresponding histological cross-sections (representative of larvae in A, C) with intestinal epithelial cells labeled red (anti-keratin immunostain) and basement membrane in green (anti-laminin immunostain). Menadione causes epithelial cell invasion (arrows) and stratification (asterisks) in <i>mlt</i> heterozygous larvae (E, F) but does not affect epithelial architecture in the WT intestines (D). (G–I) Sagittal confocal scans through the intestine of WT and <i>mlt</i> heterozygotes treated with menadione. Both larvae express LifeAct-GFP in a subset of intestinal epithelial cells. Actin-rich invadopodia-like protrusions (green) are seen arising from the basal epithelial cell membrane of menadione treated heterozygous larvae (arrowheads, H, I). Actin is located nearly exclusively in the apical brush border of WT epithelial cells (G): Red -membrane mCherry; ba, basal epithelial cell border; ap, apical epithelial cell border.</p

Src inhibition rescues invasion but not formation of invadopodia-like protrusions in <i>mlt</i>.

<p>(A–C) Lateral views of live 5 dpf WT (A) and <i>mlt</i> (B, C) larvae. Treatment with the Src-I1 inhibitor rescues invasion in <i>mlt</i> (C). The size of the intestinal epithelium in the treated <i>mlt</i> larva (C) is reduced compared with the untreated <i>mlt</i> larva (B). (D–G) Histological cross-sections of 4 dpf WT (D) and <i>mlt</i> (E–G) <i>Tg(miR194:Lifeact-GFP)</i> larvae immunostained with antibodies against laminin (red) and GFP (green). Nuclei stained blue with DAPI. Arrows point to invadopodia-like protrusions arising from the basal epithelial cell membrane of Src-I1 treated <i>mlt</i> larvae (F, G). Pronounced invasion with distortion of intestinal architecture is evident in the untreated <i>mlt</i> larva (E). Note that invasion is markedly reduced in the Src-I1 treated <i>mlt</i> larvae despite the presence of the invadopodia-like protrusions (white arrows in F, G).</p

Intestinal epithelial invasion in <i>mlt</i> larvae.

<p>(A–C) Live images of wild type (WT) and <i>mlt</i> larvae. In WT (A) the posterior intestine forms a smooth cylindrical tube (box), whereas in <i>mlt</i> at 74 hpf the intestinal contour is irregular (B). Cystic expansion of the intestine is evident in 86 hpf <i>mlt</i> larvae (C). (D–H) Histological cross-sections through the posterior intestine of larvae immunostained for laminin (green) and cytokeratin (red). The WT intestine is comprised of a simple epithelial sheet consisting of a single layer of cells, whereas in <i>mlt</i> epithelial stratification (asterisks) and invasive cells that have breached the basement membrane are evident (E–G arrowheads). The initial invasive behavior is followed by expansive growth and loss of epithelial architecture (H).</p