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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

Exocyst/GEF-H1 interaction is required for dissemination, RhoA activity and generation of traction forces of TGFβ-treated cells.(A) Exocyst/GEF-H1 uncoupling impairs TGFβ-induced dissemination in CIA. TGFβ-treated cells stably expressing Cherry (control) or Cherry-GEF-H1aa119–236 (the peptide aa119–236 competes for the binding of endogenous GEF-H1 to Sec5) were submitted to CIA. Dissemination speeds were measured by manual tracking: Cherry n = 60, Cherry-GEF-H1aa119–236 n = 45, from two experiments. (B) Exocyst/GEF-H1 uncoupling perturbs RhoA activity. TGFβ-treated cells stably expressing Cherry or Cherry-GEF-H1aa119–236 were transfected with a FRET biosensor to visualize RhoA activity. Graphic shows mean whole-cell FRET values: Cherry n = 54, Cherry-GEF-H1aa119–236 n = 39, from two experiments. (C) Exocyst/GEF-H1 uncoupling impairs generation of traction forces. The ability of Cherry or Cherry-GEF-H1aa119–236 stably expressing cells to generate forces was investigated by Traction Force Microscopy (TFM). Phase contrast images and corresponding traction force maps are shown. Color scale bar denotes traction stress (Pa). Scale bar, 50 μm. (D) Quantification of the strain energy. Cherry-GEF-H1aa119–236 cells generate significantly less strain energies compared to Cherry. Number of Cherry cells n = 72, Cherry-GEF-H1aa119–236 cells n = 51, from two experiments. Error bars represent SEM. p values come from two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.
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f5: Exocyst/GEF-H1 interaction is required for dissemination, RhoA activity and generation of traction forces of TGFβ-treated cells.(A) Exocyst/GEF-H1 uncoupling impairs TGFβ-induced dissemination in CIA. TGFβ-treated cells stably expressing Cherry (control) or Cherry-GEF-H1aa119–236 (the peptide aa119–236 competes for the binding of endogenous GEF-H1 to Sec5) were submitted to CIA. Dissemination speeds were measured by manual tracking: Cherry n = 60, Cherry-GEF-H1aa119–236 n = 45, from two experiments. (B) Exocyst/GEF-H1 uncoupling perturbs RhoA activity. TGFβ-treated cells stably expressing Cherry or Cherry-GEF-H1aa119–236 were transfected with a FRET biosensor to visualize RhoA activity. Graphic shows mean whole-cell FRET values: Cherry n = 54, Cherry-GEF-H1aa119–236 n = 39, from two experiments. (C) Exocyst/GEF-H1 uncoupling impairs generation of traction forces. The ability of Cherry or Cherry-GEF-H1aa119–236 stably expressing cells to generate forces was investigated by Traction Force Microscopy (TFM). Phase contrast images and corresponding traction force maps are shown. Color scale bar denotes traction stress (Pa). Scale bar, 50 μm. (D) Quantification of the strain energy. Cherry-GEF-H1aa119–236 cells generate significantly less strain energies compared to Cherry. Number of Cherry cells n = 72, Cherry-GEF-H1aa119–236 cells n = 51, from two experiments. Error bars represent SEM. p values come from two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.

Mentions: Second, we perturbed the endogenous binding between the Sec5 Exocyst subunit and GEF-H1 by expressing a Cherry-fused GEF-H1aa119–236 competing peptide which corresponds to the minimal GEF-H1 domain interacting with Sec526. Previously, this strategy was successfully used to uncouple Sec5 from GEF-H1 and to show that the Exocyst/GEF-H1 interaction modulates vesicle trafficking26. By lentiviral infection, we stably expressed the Cherry-fused GEF-H1aa119–236 competing peptide and the Cherry control in TGFβ-treated A549 cells. Comparing to the control, the expression of GEF-H1aa119–236 led to decreased dissemination in CIA (Fig. 5A), to decreased RhoA activity (Fig. 5B) and to decreased capacity to generate traction forces (Fig. 5C,D), without impacting on cell proliferation (Supplementary Figure S3C). These results indicate that the binding of GEF-H1 to Exocyst is necessary to activate RhoA and to promote force-driven dissemination of TGFβ-treated cells.


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)

Exocyst/GEF-H1 interaction is required for dissemination, RhoA activity and generation of traction forces of TGFβ-treated cells.(A) Exocyst/GEF-H1 uncoupling impairs TGFβ-induced dissemination in CIA. TGFβ-treated cells stably expressing Cherry (control) or Cherry-GEF-H1aa119–236 (the peptide aa119–236 competes for the binding of endogenous GEF-H1 to Sec5) were submitted to CIA. Dissemination speeds were measured by manual tracking: Cherry n = 60, Cherry-GEF-H1aa119–236 n = 45, from two experiments. (B) Exocyst/GEF-H1 uncoupling perturbs RhoA activity. TGFβ-treated cells stably expressing Cherry or Cherry-GEF-H1aa119–236 were transfected with a FRET biosensor to visualize RhoA activity. Graphic shows mean whole-cell FRET values: Cherry n = 54, Cherry-GEF-H1aa119–236 n = 39, from two experiments. (C) Exocyst/GEF-H1 uncoupling impairs generation of traction forces. The ability of Cherry or Cherry-GEF-H1aa119–236 stably expressing cells to generate forces was investigated by Traction Force Microscopy (TFM). Phase contrast images and corresponding traction force maps are shown. Color scale bar denotes traction stress (Pa). Scale bar, 50 μm. (D) Quantification of the strain energy. Cherry-GEF-H1aa119–236 cells generate significantly less strain energies compared to Cherry. Number of Cherry cells n = 72, Cherry-GEF-H1aa119–236 cells n = 51, from two experiments. Error bars represent SEM. p values come from two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4495419&req=5

f5: Exocyst/GEF-H1 interaction is required for dissemination, RhoA activity and generation of traction forces of TGFβ-treated cells.(A) Exocyst/GEF-H1 uncoupling impairs TGFβ-induced dissemination in CIA. TGFβ-treated cells stably expressing Cherry (control) or Cherry-GEF-H1aa119–236 (the peptide aa119–236 competes for the binding of endogenous GEF-H1 to Sec5) were submitted to CIA. Dissemination speeds were measured by manual tracking: Cherry n = 60, Cherry-GEF-H1aa119–236 n = 45, from two experiments. (B) Exocyst/GEF-H1 uncoupling perturbs RhoA activity. TGFβ-treated cells stably expressing Cherry or Cherry-GEF-H1aa119–236 were transfected with a FRET biosensor to visualize RhoA activity. Graphic shows mean whole-cell FRET values: Cherry n = 54, Cherry-GEF-H1aa119–236 n = 39, from two experiments. (C) Exocyst/GEF-H1 uncoupling impairs generation of traction forces. The ability of Cherry or Cherry-GEF-H1aa119–236 stably expressing cells to generate forces was investigated by Traction Force Microscopy (TFM). Phase contrast images and corresponding traction force maps are shown. Color scale bar denotes traction stress (Pa). Scale bar, 50 μm. (D) Quantification of the strain energy. Cherry-GEF-H1aa119–236 cells generate significantly less strain energies compared to Cherry. Number of Cherry cells n = 72, Cherry-GEF-H1aa119–236 cells n = 51, from two experiments. Error bars represent SEM. p values come from two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.
Mentions: Second, we perturbed the endogenous binding between the Sec5 Exocyst subunit and GEF-H1 by expressing a Cherry-fused GEF-H1aa119–236 competing peptide which corresponds to the minimal GEF-H1 domain interacting with Sec526. Previously, this strategy was successfully used to uncouple Sec5 from GEF-H1 and to show that the Exocyst/GEF-H1 interaction modulates vesicle trafficking26. By lentiviral infection, we stably expressed the Cherry-fused GEF-H1aa119–236 competing peptide and the Cherry control in TGFβ-treated A549 cells. Comparing to the control, the expression of GEF-H1aa119–236 led to decreased dissemination in CIA (Fig. 5A), to decreased RhoA activity (Fig. 5B) and to decreased capacity to generate traction forces (Fig. 5C,D), without impacting on cell proliferation (Supplementary Figure S3C). These results indicate that the binding of GEF-H1 to Exocyst is necessary to activate RhoA and to promote force-driven dissemination of TGFβ-treated cells.

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