Limits...
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.

Show MeSH

Related in: MedlinePlus

Down-regulation of WAVE3 sensitizes MDA-MB-231 cells to TNFα induced apoptosis through Akt signaling.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).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4199728&req=5

pone-0110627-g008: Down-regulation of WAVE3 sensitizes MDA-MB-231 cells to TNFα induced apoptosis through Akt signaling.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).

Mentions: It is well established that the resistance of many cancer cells to apoptosis and cell death is dependent on activation of the NFκB signaling pathway. Additionally, most cancer cells, including MBA-MB-231 BC cells, exhibit constitutively activated NFκB signaling and thereby can escape cell death [17]. We therefore investigated the role of WAVE3-mediated modulation of NFκB signaling in apoptosis and cell death in cancer cells. WAVE3-knockdown or non-targeting shRNA MDA-MB-231 cells were treated with the TNFα for 2 h, and apoptosis was assessed using flow cytometry analysis of Annexin V (apoptosis) and propidium iodide (cell death). Under these conditions, treatment of the control (Ctrl-sh) cells with TNFα induced little apoptosis (only 5% of the total cell population showed staining for Annexin V). In sharp contrast, the percent of apoptotic cells in WAVE3-knockdown population increased to 37% (Fig. 8A, (p<0.05)). Likewise, cell death, as measured by propidium iodide staining, was increased by more than 3.5-fold (p<0.05) in the WAVE3-knockdown population (Fig. 8B). Thus, WAVE3 is critical for resistance to apoptosis and cell death in cancer cells. This observation was further confirmed when TNFα-treated WAVE3-knockdown cells were assessed for apoptosis using immunocytofluorescence staining for Annexin V and Caspase 3. In the non-targeting shRNA-transfected cells (Ctrlsh), TNFα-treatment did not have a significant effect on apoptosis as measured by either Annexin V staining (Fig. 8C and 8D) or Caspase 3 staining (Fig. 8C and 8E). However, the WAVE3-knockdown cells were significantly sensitized (p<0.05) to apoptosis both in the untreated (Fig. 8C –TNFα panels, and Fig. 8D-E) and the TNFα-treated conditions (Fig. 8C +TNFα panels, and Fig. 8D-E).


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)

Down-regulation of WAVE3 sensitizes MDA-MB-231 cells to TNFα induced apoptosis through Akt signaling.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).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110627-g008: Down-regulation of WAVE3 sensitizes MDA-MB-231 cells to TNFα induced apoptosis through Akt signaling.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).
Mentions: It is well established that the resistance of many cancer cells to apoptosis and cell death is dependent on activation of the NFκB signaling pathway. Additionally, most cancer cells, including MBA-MB-231 BC cells, exhibit constitutively activated NFκB signaling and thereby can escape cell death [17]. We therefore investigated the role of WAVE3-mediated modulation of NFκB signaling in apoptosis and cell death in cancer cells. WAVE3-knockdown or non-targeting shRNA MDA-MB-231 cells were treated with the TNFα for 2 h, and apoptosis was assessed using flow cytometry analysis of Annexin V (apoptosis) and propidium iodide (cell death). Under these conditions, treatment of the control (Ctrl-sh) cells with TNFα induced little apoptosis (only 5% of the total cell population showed staining for Annexin V). In sharp contrast, the percent of apoptotic cells in WAVE3-knockdown population increased to 37% (Fig. 8A, (p<0.05)). Likewise, cell death, as measured by propidium iodide staining, was increased by more than 3.5-fold (p<0.05) in the WAVE3-knockdown population (Fig. 8B). Thus, WAVE3 is critical for resistance to apoptosis and cell death in cancer cells. This observation was further confirmed when TNFα-treated WAVE3-knockdown cells were assessed for apoptosis using immunocytofluorescence staining for Annexin V and Caspase 3. In the non-targeting shRNA-transfected cells (Ctrlsh), TNFα-treatment did not have a significant effect on apoptosis as measured by either Annexin V staining (Fig. 8C and 8D) or Caspase 3 staining (Fig. 8C and 8E). However, the WAVE3-knockdown cells were significantly sensitized (p<0.05) to apoptosis both in the untreated (Fig. 8C –TNFα panels, and Fig. 8D-E) and the TNFα-treated conditions (Fig. 8C +TNFα panels, and Fig. 8D-E).

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