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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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WAVE3 is required for invadopodia formation and ECM degradation through NFκB signaling.(A) Confocal microscopy micrographs of control shRNA- or sh-WAVE3-expressing MDA-MB-231 cells grown on FITC-labeled gelatin 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). (B) Quantification of number of invadopodia per cell in the control shRNA and shWAVE3 cells. (C) Quantification of area of gelatin degradation per cell in the shRNA and shWAVE3 cells. (D) Confocal microscopy micrographs of control shRNA- or sh-WAVE3-expressing MDA-MB-231 cells grown on FITC-labeled gelatin and stained for F-actin filaments with or without treatment with TNFα (50 ng/μl for 15 min). (E) Quantification of number of invadopodia formed in the shRNA and shWAVE3 cells with or without treatment with TNFα. All data are representative of 3 independent experiments, or are the mean (±SE; n = 3; *, **, p <0.05; Student's t-test).
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pone-0110627-g006: WAVE3 is required for invadopodia formation and ECM degradation through NFκB signaling.(A) Confocal microscopy micrographs of control shRNA- or sh-WAVE3-expressing MDA-MB-231 cells grown on FITC-labeled gelatin 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). (B) Quantification of number of invadopodia per cell in the control shRNA and shWAVE3 cells. (C) Quantification of area of gelatin degradation per cell in the shRNA and shWAVE3 cells. (D) Confocal microscopy micrographs of control shRNA- or sh-WAVE3-expressing MDA-MB-231 cells grown on FITC-labeled gelatin and stained for F-actin filaments with or without treatment with TNFα (50 ng/μl for 15 min). (E) Quantification of number of invadopodia formed in the shRNA and shWAVE3 cells with or without treatment with TNFα. All data are representative of 3 independent experiments, or are the mean (±SE; n = 3; *, **, p <0.05; Student's t-test).

Mentions: We used this gelatin-based invadopodia assay to assess the effect of WAVE3 on the formation of invadopodia and subsequently the degradation of the ECM by MDA-MB-231 cells. We found a significant reduction of both the number of invadopodia (Fig. 6A, left panel and Fig. 6B), as well as the total area of invadopodia-mediated degradation of gelatin in WAVE3-knockdown cells compared to the non-targeting shRNA control (Ctrl-sh) MDA-MB-231 cells (Figure 6A, middle panel and Fig. 6C). There was more than 3-fold reduction (p<0.05) in the number of invadopodia (Fig. 6B) and more than 10-fold reduction (p<0.05) in the area of ECM degradation in the WAVE3-knockdown cells compared to the control cells (Fig. 6C). While Our data show that loss of WAVE3 inhibits both invadopodia formation and degradation of ECM, this phenomenon seems to affect the majority of the cells observed under the microscope, and careful analysis of our data did not find a decrease in the number of cells that form invadopodia and degrade the ECM, rather, loss of WAVE3 seems to have a global effect. Treatment with TNFα, which resulted in a significant increase in the number of invadopodia in the control cells (p<0.01), was unable to rescue the inhibition of invadopodia caused by loss of WAVE3 (Fig. 6D & 6E). Conversely, over-expression of WAVE3 in the non-invasive MCF7 BC cells (Fig S7A in File S1), which we had previously shown to stimulate their invasiveness [30], resulted in a clear increase in both invadopodia formation and gelatin degradation by TNFα-stimulated cells (Fig. S7B in File S1).


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)

WAVE3 is required for invadopodia formation and ECM degradation through NFκB signaling.(A) Confocal microscopy micrographs of control shRNA- or sh-WAVE3-expressing MDA-MB-231 cells grown on FITC-labeled gelatin 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). (B) Quantification of number of invadopodia per cell in the control shRNA and shWAVE3 cells. (C) Quantification of area of gelatin degradation per cell in the shRNA and shWAVE3 cells. (D) Confocal microscopy micrographs of control shRNA- or sh-WAVE3-expressing MDA-MB-231 cells grown on FITC-labeled gelatin and stained for F-actin filaments with or without treatment with TNFα (50 ng/μl for 15 min). (E) Quantification of number of invadopodia formed in the shRNA and shWAVE3 cells with or without treatment with TNFα. All data are representative of 3 independent experiments, or are the mean (±SE; n = 3; *, **, p <0.05; Student's t-test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199728&req=5

pone-0110627-g006: WAVE3 is required for invadopodia formation and ECM degradation through NFκB signaling.(A) Confocal microscopy micrographs of control shRNA- or sh-WAVE3-expressing MDA-MB-231 cells grown on FITC-labeled gelatin 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). (B) Quantification of number of invadopodia per cell in the control shRNA and shWAVE3 cells. (C) Quantification of area of gelatin degradation per cell in the shRNA and shWAVE3 cells. (D) Confocal microscopy micrographs of control shRNA- or sh-WAVE3-expressing MDA-MB-231 cells grown on FITC-labeled gelatin and stained for F-actin filaments with or without treatment with TNFα (50 ng/μl for 15 min). (E) Quantification of number of invadopodia formed in the shRNA and shWAVE3 cells with or without treatment with TNFα. All data are representative of 3 independent experiments, or are the mean (±SE; n = 3; *, **, p <0.05; Student's t-test).
Mentions: We used this gelatin-based invadopodia assay to assess the effect of WAVE3 on the formation of invadopodia and subsequently the degradation of the ECM by MDA-MB-231 cells. We found a significant reduction of both the number of invadopodia (Fig. 6A, left panel and Fig. 6B), as well as the total area of invadopodia-mediated degradation of gelatin in WAVE3-knockdown cells compared to the non-targeting shRNA control (Ctrl-sh) MDA-MB-231 cells (Figure 6A, middle panel and Fig. 6C). There was more than 3-fold reduction (p<0.05) in the number of invadopodia (Fig. 6B) and more than 10-fold reduction (p<0.05) in the area of ECM degradation in the WAVE3-knockdown cells compared to the control cells (Fig. 6C). While Our data show that loss of WAVE3 inhibits both invadopodia formation and degradation of ECM, this phenomenon seems to affect the majority of the cells observed under the microscope, and careful analysis of our data did not find a decrease in the number of cells that form invadopodia and degrade the ECM, rather, loss of WAVE3 seems to have a global effect. Treatment with TNFα, which resulted in a significant increase in the number of invadopodia in the control cells (p<0.01), was unable to rescue the inhibition of invadopodia caused by loss of WAVE3 (Fig. 6D & 6E). Conversely, over-expression of WAVE3 in the non-invasive MCF7 BC cells (Fig S7A in File S1), which we had previously shown to stimulate their invasiveness [30], resulted in a clear increase in both invadopodia formation and gelatin degradation by TNFα-stimulated cells (Fig. S7B in File S1).

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