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

Davuluri G, Augoff K, Schiemann WP, Plow EF, Sossey-Alaoui K - PLoS ONE (2014)

Bottom Line: 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.Our results identify a novel function of WAVE3 in NFκB signaling, where its activity is essential for the regulation of invadopodia and ECM degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cardiology, Cleveland Clinic Lerner Institute, Cleveland, Ohio, United States of America.

ABSTRACT
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.

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Over-expression of WAVE3 activates NFκB signaling.(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)
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pone-0110627-g002: Over-expression of WAVE3 activates NFκB signaling.(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)

Mentions: To better understand how WAVE3 modulates NFκB signaling, we applied a combination of genetic and pharmacologic means to manipulate effector molecules in the NFκB pathway. First, we examined whether overexpression of exogenous WAVE3 can affect NFκB activity. To do so, we overexpressed either GFP alone or GFP-WAVE3 fusion protein in MDA-MB-231 cells and assessed for NFκB activity. Using the luciferase-based NFκB reporter assay described in Figure 1A, we found overexpression of WAVE3 (Fig. 2A) stimulated NFκB activity by 3-fold increase (Fig. 2B). Consistent with this observation, WAVE3-overexpresion also stimulated (>3-fold increase) p65 phosphorylation without affecting total p65 expression levels (Fig. 2C). Conversely, treatment with a specific inhibitor of IKKβ (IKKβ-I), the upstream activator of NFκB signaling, blunted the TNFα-mediated stimulation of phospho-p65 in the WAVE3-overexpressing MDA-MB-231 cells (0.9-fold change in the TNFα and IKKβ-I-treated cells vs. 3.2-fold change in TNFα only treated cells (Fig. 2D)). Thus, these data further support the role of WAVE3 in the regulation of NFκB signaling. Next, we monitored p65 protein phosphorylation and turnover in the GFP and WAVE3-overexpressing MDA-MB-231 cells after treatment with cyclohexamide (CHX), a potent inhibitor of de novo protein synthesis. While phosphorylation levels of p65 were increased (∼3- to 4-fold increase) in the WAVE3-overexpressing cells, treatment with CHX did not affect the expression levels of total p65 (Fig. 2E). Likewise, treatment with the MG-132, a compound that inhibits the proteasome-mediated protein degradation, did not affect the WAVE3-mediated increase of phosphorylated p65 in the WAVE3 overexpressing cells (Fig. 2F) while the levels of total p65 remained unchanged (Fig. 2F). Thus, these data demonstrate that the WAVE3-mediated modulation of NFκB signaling does not require the involvement of WAVE3 in protein neo-synthesis (the CHX data, Fig. 2E) or t proteasome-mediated protein degradation (the MG-132 data, Fig. 2F).


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

Davuluri G, Augoff K, Schiemann WP, Plow EF, Sossey-Alaoui K - PLoS ONE (2014)

Over-expression of WAVE3 activates NFκB signaling.(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)
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199728&req=5

pone-0110627-g002: Over-expression of WAVE3 activates NFκB signaling.(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)
Mentions: To better understand how WAVE3 modulates NFκB signaling, we applied a combination of genetic and pharmacologic means to manipulate effector molecules in the NFκB pathway. First, we examined whether overexpression of exogenous WAVE3 can affect NFκB activity. To do so, we overexpressed either GFP alone or GFP-WAVE3 fusion protein in MDA-MB-231 cells and assessed for NFκB activity. Using the luciferase-based NFκB reporter assay described in Figure 1A, we found overexpression of WAVE3 (Fig. 2A) stimulated NFκB activity by 3-fold increase (Fig. 2B). Consistent with this observation, WAVE3-overexpresion also stimulated (>3-fold increase) p65 phosphorylation without affecting total p65 expression levels (Fig. 2C). Conversely, treatment with a specific inhibitor of IKKβ (IKKβ-I), the upstream activator of NFκB signaling, blunted the TNFα-mediated stimulation of phospho-p65 in the WAVE3-overexpressing MDA-MB-231 cells (0.9-fold change in the TNFα and IKKβ-I-treated cells vs. 3.2-fold change in TNFα only treated cells (Fig. 2D)). Thus, these data further support the role of WAVE3 in the regulation of NFκB signaling. Next, we monitored p65 protein phosphorylation and turnover in the GFP and WAVE3-overexpressing MDA-MB-231 cells after treatment with cyclohexamide (CHX), a potent inhibitor of de novo protein synthesis. While phosphorylation levels of p65 were increased (∼3- to 4-fold increase) in the WAVE3-overexpressing cells, treatment with CHX did not affect the expression levels of total p65 (Fig. 2E). Likewise, treatment with the MG-132, a compound that inhibits the proteasome-mediated protein degradation, did not affect the WAVE3-mediated increase of phosphorylated p65 in the WAVE3 overexpressing cells (Fig. 2F) while the levels of total p65 remained unchanged (Fig. 2F). Thus, these data demonstrate that the WAVE3-mediated modulation of NFκB signaling does not require the involvement of WAVE3 in protein neo-synthesis (the CHX data, Fig. 2E) or t proteasome-mediated protein degradation (the MG-132 data, Fig. 2F).

Bottom Line: 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.Our results identify a novel function of WAVE3 in NFκB signaling, where its activity is essential for the regulation of invadopodia and ECM degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cardiology, Cleveland Clinic Lerner Institute, Cleveland, Ohio, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus