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

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

Loss of WAVE3 attenuates the aggressiveness of the BC cells of Triple-negative subtype.

<p>(A) Quantitative Real-time RT-PCR of WAVE3 in the indicated BC cell lines after transient transfection with either a control siRNA or WAVE3-specific siRNA. (B) Western blot analysis of protein lysates following the same treatment as in (A). Actin was used a loading control and WAVE2 was used a negative control. (C and D) Matrigel invasion assay after the indicated treatments. Representative micrographs are shown in (C), and the quantitation is shown in (D). The results are shown as the mean±s. d. of at least 3 independent assays.</p

Increased Expression Levels of WAVE3 Are Associated with the Progression and Metastasis of Triple Negative Breast Cancer

<div><h3>Background</h3><p>Breast Cancer (BC) is a heterogeneous disease comprised of at least five genetically distinct subtypes, which together form the second leading cause of cancer death in women in the United States. Within BC subtypes, those classified as Triple Negative BCs (TNBCs) exhibit dismal survival rates due to their propensity to develop distant metastases. We have identified the WAVE3 protein, which is a critical regulator of actin cytoskeleton dynamics that are required for the motility and invasion of cancer cells through its activation of the Arp2/3 complex, as a key regulator of the different steps of the invasion-metastasis cascade in BC, especially in the more aggressive TNBCs. Our published studies have also shown that elevated expression levels of WAVE3 in the TNBC cell lines directly contribute to their increased invasion and metastasis potentials both <em>in vitro</em> and <em>in vivo</em> in murine models of BC metastasis.</p> <h3>Methodology/Principal Findings</h3><p>Herein, we utilized both immunohistochemistry (IHC) of primary human BC tumors as well as quantitative real-time RT-PCR of WAVE3 in the peripheral blood of BC patients to clearly establish that WAVE3 is a predictive marker of overall BC patients’ survival. High levels of WAVE3 were predictive for reduced distant recurrence-free survival as well as for decreased disease-specific mortality. Our analysis of WAVE3 expression levels in the peripheral blood of BC patients showed that WAVE3 is highly expressed in the blood of patients who developed metastatic breast cancer compared to those who did not. WAVE3 expression was also highly upregulated in the blood of BC patients with the more aggressive TNBC subtype.</p> <h3>Conclusions</h3><p>Together, these findings establish WAVE3 as a novel marker for increased risk of breast-cancer-specific mortality and for the metastatic potential of the TNBCs, and also identify WAVE3 as an attractive therapeutic target for the treatment of metastatic BC.</p> </div

WAVE3 expression levels positively correlate with the aggressiveness of breast cancer cell lines.

<p>(A) Western bolt analysis of protein lysates isolated from the indicated BC cell lines using WAVE3 antibody. Actin was used as a loading control. (B) Representative micrographs of Matrigel invasion of the indicated BC cell lines and quantification of the Matrigel invasion (C). The results are shown as the mean±s. d. of at least 3 independent assays.</p

Comparison of WAVE3 score by clinical outcome.

a<p>Measured as a continuous variable, Mann-Whitney test.</p