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RalB regulates contractility-driven cancer dissemination upon TGFβ stimulation via the RhoGEF GEF-H1.

Biondini M, Duclos G, Meyer-Schaller N, Silberzan P, Camonis J, Parrini MC - Sci Rep (2015)

Bottom Line: RalA and RalB proteins are key mediators of oncogenic Ras signaling in human oncogenesis.RalB, but not RalA, is required for matrix deformation and cell dissemination acting via the RhoGEF GEF-H1, which associates with the Exocyst complex, a major Ral effector.These results provide a novel molecular mechanism underlying the control of cell invasion by RalB via a cross-talk with the Rho pathway.

View Article: PubMed Central - PubMed

Affiliation: 1] Institut Curie, Centre de Recherche, Paris Sciences et Lettres University, 75248 Paris, France [2] ART group, Inserm U830.

ABSTRACT
RalA and RalB proteins are key mediators of oncogenic Ras signaling in human oncogenesis. Herein we investigated the mechanistic contribution of Ral proteins to invasion of lung cancer A549 cells after induction of epithelial-mesenchymal transition (EMT) with TGFβ. We show that TGFβ-induced EMT promotes dissemination of A549 cells in a 2/3D assay, independently of proteolysis, by activating the Rho/ROCK pathway which generates actomyosin-dependent contractility forces that actively remodel the extracellular matrix, as assessed by Traction Force microscopy. RalB, but not RalA, is required for matrix deformation and cell dissemination acting via the RhoGEF GEF-H1, which associates with the Exocyst complex, a major Ral effector. Indeed, uncoupling of the Exocyst subunit Sec5 from GEF-H1 impairs RhoA activation, generation of traction forces and cell dissemination. These results provide a novel molecular mechanism underlying the control of cell invasion by RalB via a cross-talk with the Rho pathway.

No MeSH data available.


Related in: MedlinePlus

Dissemination of TGFβ-treated A549 cells in 2/3D is proteolysis independent.(A) Depiction of the 2/3D Circular Invasion Assay (CIA). Note that we kept the original name of the assay, but that the actual shape of the stopper we used is not circular but square. (B) EMT promotes cell dissemination. A549 cells were treated with 2 ng/mL TGFβ for 7 days, submitted to CIA and compared to untreated cells. Selected time points from a representative experiment are shown. See Supplementary Movie S1 for entire video sequence. Scale bar, 100 μm. (C) Quantification of cell dissemination. Individual cells were tracked using the ImageJ software. Number of cells n = 40 for untreated and n = 163 for TGFβ-treated conditions from at least four experiments per condition. (D) TGFβ greatly stimulates invadopodia formation. Cells were stimulated with TGFβ for 7 days and invadopodia were visualized as dots positive for matrix degradation (black), for cortactin staining (green) and for F-actin staining (red). Scale bar, 20 μm.(E) Quantification of cells positive for at least one invadopodia. Counting was performed on three independent experiments (n≥100 cells/condition per experiment). (F) TGFβ induces strong secretion of MMP2 and MMP9 metalloproteinases. Conditioned media from untreated and TGFβ-treated cells were collected and processed for gelatin zymogram assay. A representative zymogram image is shown. (G) MMP-dependent proteolysis is dispensable for TGFβ-induced dissemination in 2/3D. TGFβ-treated cells were incubated with 25 μM GM6001 MMPs inhibitor (2 hrs before CIA and during CIA) or depleted of MMP2 and MMP9, and submitted to CIA. Number of cells n>30 per condition from three experiments with GM6001 and one experiment with siRNAs. Error bars represent SEM. p values come from two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
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f1: Dissemination of TGFβ-treated A549 cells in 2/3D is proteolysis independent.(A) Depiction of the 2/3D Circular Invasion Assay (CIA). Note that we kept the original name of the assay, but that the actual shape of the stopper we used is not circular but square. (B) EMT promotes cell dissemination. A549 cells were treated with 2 ng/mL TGFβ for 7 days, submitted to CIA and compared to untreated cells. Selected time points from a representative experiment are shown. See Supplementary Movie S1 for entire video sequence. Scale bar, 100 μm. (C) Quantification of cell dissemination. Individual cells were tracked using the ImageJ software. Number of cells n = 40 for untreated and n = 163 for TGFβ-treated conditions from at least four experiments per condition. (D) TGFβ greatly stimulates invadopodia formation. Cells were stimulated with TGFβ for 7 days and invadopodia were visualized as dots positive for matrix degradation (black), for cortactin staining (green) and for F-actin staining (red). Scale bar, 20 μm.(E) Quantification of cells positive for at least one invadopodia. Counting was performed on three independent experiments (n≥100 cells/condition per experiment). (F) TGFβ induces strong secretion of MMP2 and MMP9 metalloproteinases. Conditioned media from untreated and TGFβ-treated cells were collected and processed for gelatin zymogram assay. A representative zymogram image is shown. (G) MMP-dependent proteolysis is dispensable for TGFβ-induced dissemination in 2/3D. TGFβ-treated cells were incubated with 25 μM GM6001 MMPs inhibitor (2 hrs before CIA and during CIA) or depleted of MMP2 and MMP9, and submitted to CIA. Number of cells n>30 per condition from three experiments with GM6001 and one experiment with siRNAs. Error bars represent SEM. p values come from two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.

Mentions: To study invasion we took advantage of the Circular Invasion Assay (CIA)20 because it allows detailed time-lapse microscopic analysis during the invasion process (Fig. 1A). In this assay the cells are in contact with the matrix (Matrigel) both at the dorsal and frontal sides but they ventrally adhere to the bottom-glass dish; therefore it is a 2-3D situation which mimics a topography that is often observed during cancer invasion in vivo21. We compared the invasive phenotype of untreated versus TGFβ-treated cells. Untreated cells invaded through the matrix in a collective-like manner, keeping cell-cell coordination and with a rather compact front, but they were never able to invade deeply during the 2-day observation time (Fig. 1B and Supplementary Movie S1). On the contrary, TGFβ-treated cells displayed more chaotic dynamics inside the monolayer; some cells were detaching from the group and disseminating through the matrix till distant sites (Fig. 1B and Supplementary Movie S1). By single-cell tracking analysis we measured a “dissemination speed”, defined as the velocity of the cell movements in this 2-3D setting. The dissemination speed was almost 2-fold higher in TGFβ-treated cells (12.74 μm/hr ± 0.51 SEM) as compared to untreated cells (7.09 μm/hr ± 0.56 SEM) (Fig. 1C). We concluded that, in 2-3D Matrigel environment, TGFβ-induced EMT increases A549 cell invasion and promotes a shift from collective to single-cell dissemination.


RalB regulates contractility-driven cancer dissemination upon TGFβ stimulation via the RhoGEF GEF-H1.

Biondini M, Duclos G, Meyer-Schaller N, Silberzan P, Camonis J, Parrini MC - Sci Rep (2015)

Dissemination of TGFβ-treated A549 cells in 2/3D is proteolysis independent.(A) Depiction of the 2/3D Circular Invasion Assay (CIA). Note that we kept the original name of the assay, but that the actual shape of the stopper we used is not circular but square. (B) EMT promotes cell dissemination. A549 cells were treated with 2 ng/mL TGFβ for 7 days, submitted to CIA and compared to untreated cells. Selected time points from a representative experiment are shown. See Supplementary Movie S1 for entire video sequence. Scale bar, 100 μm. (C) Quantification of cell dissemination. Individual cells were tracked using the ImageJ software. Number of cells n = 40 for untreated and n = 163 for TGFβ-treated conditions from at least four experiments per condition. (D) TGFβ greatly stimulates invadopodia formation. Cells were stimulated with TGFβ for 7 days and invadopodia were visualized as dots positive for matrix degradation (black), for cortactin staining (green) and for F-actin staining (red). Scale bar, 20 μm.(E) Quantification of cells positive for at least one invadopodia. Counting was performed on three independent experiments (n≥100 cells/condition per experiment). (F) TGFβ induces strong secretion of MMP2 and MMP9 metalloproteinases. Conditioned media from untreated and TGFβ-treated cells were collected and processed for gelatin zymogram assay. A representative zymogram image is shown. (G) MMP-dependent proteolysis is dispensable for TGFβ-induced dissemination in 2/3D. TGFβ-treated cells were incubated with 25 μM GM6001 MMPs inhibitor (2 hrs before CIA and during CIA) or depleted of MMP2 and MMP9, and submitted to CIA. Number of cells n>30 per condition from three experiments with GM6001 and one experiment with siRNAs. Error bars represent SEM. p values come from two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
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Related In: Results  -  Collection

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f1: Dissemination of TGFβ-treated A549 cells in 2/3D is proteolysis independent.(A) Depiction of the 2/3D Circular Invasion Assay (CIA). Note that we kept the original name of the assay, but that the actual shape of the stopper we used is not circular but square. (B) EMT promotes cell dissemination. A549 cells were treated with 2 ng/mL TGFβ for 7 days, submitted to CIA and compared to untreated cells. Selected time points from a representative experiment are shown. See Supplementary Movie S1 for entire video sequence. Scale bar, 100 μm. (C) Quantification of cell dissemination. Individual cells were tracked using the ImageJ software. Number of cells n = 40 for untreated and n = 163 for TGFβ-treated conditions from at least four experiments per condition. (D) TGFβ greatly stimulates invadopodia formation. Cells were stimulated with TGFβ for 7 days and invadopodia were visualized as dots positive for matrix degradation (black), for cortactin staining (green) and for F-actin staining (red). Scale bar, 20 μm.(E) Quantification of cells positive for at least one invadopodia. Counting was performed on three independent experiments (n≥100 cells/condition per experiment). (F) TGFβ induces strong secretion of MMP2 and MMP9 metalloproteinases. Conditioned media from untreated and TGFβ-treated cells were collected and processed for gelatin zymogram assay. A representative zymogram image is shown. (G) MMP-dependent proteolysis is dispensable for TGFβ-induced dissemination in 2/3D. TGFβ-treated cells were incubated with 25 μM GM6001 MMPs inhibitor (2 hrs before CIA and during CIA) or depleted of MMP2 and MMP9, and submitted to CIA. Number of cells n>30 per condition from three experiments with GM6001 and one experiment with siRNAs. Error bars represent SEM. p values come from two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
Mentions: To study invasion we took advantage of the Circular Invasion Assay (CIA)20 because it allows detailed time-lapse microscopic analysis during the invasion process (Fig. 1A). In this assay the cells are in contact with the matrix (Matrigel) both at the dorsal and frontal sides but they ventrally adhere to the bottom-glass dish; therefore it is a 2-3D situation which mimics a topography that is often observed during cancer invasion in vivo21. We compared the invasive phenotype of untreated versus TGFβ-treated cells. Untreated cells invaded through the matrix in a collective-like manner, keeping cell-cell coordination and with a rather compact front, but they were never able to invade deeply during the 2-day observation time (Fig. 1B and Supplementary Movie S1). On the contrary, TGFβ-treated cells displayed more chaotic dynamics inside the monolayer; some cells were detaching from the group and disseminating through the matrix till distant sites (Fig. 1B and Supplementary Movie S1). By single-cell tracking analysis we measured a “dissemination speed”, defined as the velocity of the cell movements in this 2-3D setting. The dissemination speed was almost 2-fold higher in TGFβ-treated cells (12.74 μm/hr ± 0.51 SEM) as compared to untreated cells (7.09 μm/hr ± 0.56 SEM) (Fig. 1C). We concluded that, in 2-3D Matrigel environment, TGFβ-induced EMT increases A549 cell invasion and promotes a shift from collective to single-cell dissemination.

Bottom Line: RalA and RalB proteins are key mediators of oncogenic Ras signaling in human oncogenesis.RalB, but not RalA, is required for matrix deformation and cell dissemination acting via the RhoGEF GEF-H1, which associates with the Exocyst complex, a major Ral effector.These results provide a novel molecular mechanism underlying the control of cell invasion by RalB via a cross-talk with the Rho pathway.

View Article: PubMed Central - PubMed

Affiliation: 1] Institut Curie, Centre de Recherche, Paris Sciences et Lettres University, 75248 Paris, France [2] ART group, Inserm U830.

ABSTRACT
RalA and RalB proteins are key mediators of oncogenic Ras signaling in human oncogenesis. Herein we investigated the mechanistic contribution of Ral proteins to invasion of lung cancer A549 cells after induction of epithelial-mesenchymal transition (EMT) with TGFβ. We show that TGFβ-induced EMT promotes dissemination of A549 cells in a 2/3D assay, independently of proteolysis, by activating the Rho/ROCK pathway which generates actomyosin-dependent contractility forces that actively remodel the extracellular matrix, as assessed by Traction Force microscopy. RalB, but not RalA, is required for matrix deformation and cell dissemination acting via the RhoGEF GEF-H1, which associates with the Exocyst complex, a major Ral effector. Indeed, uncoupling of the Exocyst subunit Sec5 from GEF-H1 impairs RhoA activation, generation of traction forces and cell dissemination. These results provide a novel molecular mechanism underlying the control of cell invasion by RalB via a cross-talk with the Rho pathway.

No MeSH data available.


Related in: MedlinePlus