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Association between Gαi2 and ELMO1/Dock180 connects chemokine signalling with Rac activation and metastasis.

Li H, Yang L, Fu H, Yan J, Wang Y, Guo H, Hao X, Xu X, Jin T, Zhang N - Nat Commun (2013)

Bottom Line: Binding of CXCL12 to CXCR4 triggers activation of heterotrimeric Gi proteins that regulate actin polymerization and migration.CXCL12 triggers a Gαi2-dependent membrane translocation of ELMO1, which associates with Dock180 to activate small G-proteins Rac1 and Rac2.In vivo, ELMO1 expression is associated with lymph node and distant metastasis, and knocking down ELMO1 impairs metastasis to the lung.

View Article: PubMed Central - PubMed

Affiliation: Tianjin Medical University Cancer Institute and Hospital and Research Center of Basic Medical Sciences, He Xi District, Tianjin 300060, China.

ABSTRACT
The chemokine CXCL12 and its G-protein-coupled receptor CXCR4 control the migration, invasiveness and metastasis of breast cancer cells. Binding of CXCL12 to CXCR4 triggers activation of heterotrimeric Gi proteins that regulate actin polymerization and migration. However, the pathways linking chemokine G-protein-coupled receptor/Gi signalling to actin polymerization and cancer cell migration are not known. Here we show that CXCL12 stimulation promotes interaction between Gαi2 and ELMO1. Gi signalling and ELMO1 are both required for CXCL12-mediated actin polymerization, migration and invasion of breast cancer cells. CXCL12 triggers a Gαi2-dependent membrane translocation of ELMO1, which associates with Dock180 to activate small G-proteins Rac1 and Rac2. In vivo, ELMO1 expression is associated with lymph node and distant metastasis, and knocking down ELMO1 impairs metastasis to the lung. Our findings indicate that a chemokine-controlled pathway, consisting of Gαi2, ELMO1/Dock180, Rac1 and Rac2, regulates the actin cytoskeleton during breast cancer metastasis.

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ELMO1 functions in breast cancer chemotaxis and invasion.(a) Western blotting analysis of ELMOs endogenous expression in MDA-MB-231, BT549, ZR-75-30, T47D, MCF-7, Bcap-37, SKBR3, MCF10A and HBL100 cells. Actin was used as control. (b) Chemotaxis analysis of ELMO1 knockdown cells. Western blotting analysis of ELMO1 expression in MDA-MB-231, T47D and MCF-7 breast cancer cells. ELMO2 was used as a loading control (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way analysis of variance (ANOVA), ***P<0.0001). (c) Scratch assay of siELMO1 cells. MDA-MB-231 cells were plated in six-well plates and formed a fluent monolayer. The medium was replaced by a RPMI medium supplied with 1% BSA. One of ELMO1 siRNA wells was pretreated with 0.1 μg ml−1 PTX in medium for 2 h. The gap distance was measured at 0, 3, 6, 9, 12 and 24 h (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001). (d) The reduction of ELMO1 impaired invasion of MDA-MB-231 cells, especially under 0.1 μg ml−1 PTX treatment for transfected ELMO1 siRNA cells (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001). (e) Actin polymerization in siELMO1 cells was decreased. Time course of relative F-actin content in normal, control, siELMO1 and siELMO1+0.1 μg ml−1 PTX cells followed by CXCL12 stimulation. The value was measured at 0, 4, 8, 15, 30, 60, 120 and 300 s (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001). (f) MTT assay: cell proliferation activity was not inhibited in siELMO1 cells (points=mean of three independent experiments; scale bars=s.e.m.; n=6; one-way ANOVA, P=0.8725>0.05).
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f1: ELMO1 functions in breast cancer chemotaxis and invasion.(a) Western blotting analysis of ELMOs endogenous expression in MDA-MB-231, BT549, ZR-75-30, T47D, MCF-7, Bcap-37, SKBR3, MCF10A and HBL100 cells. Actin was used as control. (b) Chemotaxis analysis of ELMO1 knockdown cells. Western blotting analysis of ELMO1 expression in MDA-MB-231, T47D and MCF-7 breast cancer cells. ELMO2 was used as a loading control (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way analysis of variance (ANOVA), ***P<0.0001). (c) Scratch assay of siELMO1 cells. MDA-MB-231 cells were plated in six-well plates and formed a fluent monolayer. The medium was replaced by a RPMI medium supplied with 1% BSA. One of ELMO1 siRNA wells was pretreated with 0.1 μg ml−1 PTX in medium for 2 h. The gap distance was measured at 0, 3, 6, 9, 12 and 24 h (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001). (d) The reduction of ELMO1 impaired invasion of MDA-MB-231 cells, especially under 0.1 μg ml−1 PTX treatment for transfected ELMO1 siRNA cells (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001). (e) Actin polymerization in siELMO1 cells was decreased. Time course of relative F-actin content in normal, control, siELMO1 and siELMO1+0.1 μg ml−1 PTX cells followed by CXCL12 stimulation. The value was measured at 0, 4, 8, 15, 30, 60, 120 and 300 s (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001). (f) MTT assay: cell proliferation activity was not inhibited in siELMO1 cells (points=mean of three independent experiments; scale bars=s.e.m.; n=6; one-way ANOVA, P=0.8725>0.05).

Mentions: To determine which ELMO proteins were involved in the migration of breast cancer cells, we first examined the expression of ELMOs in various breast cancer cells. Expression of ELMO1 and ELMO2 was examined in nine human cell lines, including MDA-MB-231, BT549, ZR-75-30, T47D, MCF-7, Bcap-37, SKBR3, MCF10A and HBL100, by western blotting analyses (Fig. 1a). Our result indicated that ELMO1 and ELMO2 were expressed in these nine cell lines, and protein levels of ELMO1 and ELMO2 were relatively high in metastatic breast cancer cells, such as MDA-MB-231 and BT549.


Association between Gαi2 and ELMO1/Dock180 connects chemokine signalling with Rac activation and metastasis.

Li H, Yang L, Fu H, Yan J, Wang Y, Guo H, Hao X, Xu X, Jin T, Zhang N - Nat Commun (2013)

ELMO1 functions in breast cancer chemotaxis and invasion.(a) Western blotting analysis of ELMOs endogenous expression in MDA-MB-231, BT549, ZR-75-30, T47D, MCF-7, Bcap-37, SKBR3, MCF10A and HBL100 cells. Actin was used as control. (b) Chemotaxis analysis of ELMO1 knockdown cells. Western blotting analysis of ELMO1 expression in MDA-MB-231, T47D and MCF-7 breast cancer cells. ELMO2 was used as a loading control (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way analysis of variance (ANOVA), ***P<0.0001). (c) Scratch assay of siELMO1 cells. MDA-MB-231 cells were plated in six-well plates and formed a fluent monolayer. The medium was replaced by a RPMI medium supplied with 1% BSA. One of ELMO1 siRNA wells was pretreated with 0.1 μg ml−1 PTX in medium for 2 h. The gap distance was measured at 0, 3, 6, 9, 12 and 24 h (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001). (d) The reduction of ELMO1 impaired invasion of MDA-MB-231 cells, especially under 0.1 μg ml−1 PTX treatment for transfected ELMO1 siRNA cells (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001). (e) Actin polymerization in siELMO1 cells was decreased. Time course of relative F-actin content in normal, control, siELMO1 and siELMO1+0.1 μg ml−1 PTX cells followed by CXCL12 stimulation. The value was measured at 0, 4, 8, 15, 30, 60, 120 and 300 s (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001). (f) MTT assay: cell proliferation activity was not inhibited in siELMO1 cells (points=mean of three independent experiments; scale bars=s.e.m.; n=6; one-way ANOVA, P=0.8725>0.05).
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f1: ELMO1 functions in breast cancer chemotaxis and invasion.(a) Western blotting analysis of ELMOs endogenous expression in MDA-MB-231, BT549, ZR-75-30, T47D, MCF-7, Bcap-37, SKBR3, MCF10A and HBL100 cells. Actin was used as control. (b) Chemotaxis analysis of ELMO1 knockdown cells. Western blotting analysis of ELMO1 expression in MDA-MB-231, T47D and MCF-7 breast cancer cells. ELMO2 was used as a loading control (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way analysis of variance (ANOVA), ***P<0.0001). (c) Scratch assay of siELMO1 cells. MDA-MB-231 cells were plated in six-well plates and formed a fluent monolayer. The medium was replaced by a RPMI medium supplied with 1% BSA. One of ELMO1 siRNA wells was pretreated with 0.1 μg ml−1 PTX in medium for 2 h. The gap distance was measured at 0, 3, 6, 9, 12 and 24 h (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001). (d) The reduction of ELMO1 impaired invasion of MDA-MB-231 cells, especially under 0.1 μg ml−1 PTX treatment for transfected ELMO1 siRNA cells (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001). (e) Actin polymerization in siELMO1 cells was decreased. Time course of relative F-actin content in normal, control, siELMO1 and siELMO1+0.1 μg ml−1 PTX cells followed by CXCL12 stimulation. The value was measured at 0, 4, 8, 15, 30, 60, 120 and 300 s (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001). (f) MTT assay: cell proliferation activity was not inhibited in siELMO1 cells (points=mean of three independent experiments; scale bars=s.e.m.; n=6; one-way ANOVA, P=0.8725>0.05).
Mentions: To determine which ELMO proteins were involved in the migration of breast cancer cells, we first examined the expression of ELMOs in various breast cancer cells. Expression of ELMO1 and ELMO2 was examined in nine human cell lines, including MDA-MB-231, BT549, ZR-75-30, T47D, MCF-7, Bcap-37, SKBR3, MCF10A and HBL100, by western blotting analyses (Fig. 1a). Our result indicated that ELMO1 and ELMO2 were expressed in these nine cell lines, and protein levels of ELMO1 and ELMO2 were relatively high in metastatic breast cancer cells, such as MDA-MB-231 and BT549.

Bottom Line: Binding of CXCL12 to CXCR4 triggers activation of heterotrimeric Gi proteins that regulate actin polymerization and migration.CXCL12 triggers a Gαi2-dependent membrane translocation of ELMO1, which associates with Dock180 to activate small G-proteins Rac1 and Rac2.In vivo, ELMO1 expression is associated with lymph node and distant metastasis, and knocking down ELMO1 impairs metastasis to the lung.

View Article: PubMed Central - PubMed

Affiliation: Tianjin Medical University Cancer Institute and Hospital and Research Center of Basic Medical Sciences, He Xi District, Tianjin 300060, China.

ABSTRACT
The chemokine CXCL12 and its G-protein-coupled receptor CXCR4 control the migration, invasiveness and metastasis of breast cancer cells. Binding of CXCL12 to CXCR4 triggers activation of heterotrimeric Gi proteins that regulate actin polymerization and migration. However, the pathways linking chemokine G-protein-coupled receptor/Gi signalling to actin polymerization and cancer cell migration are not known. Here we show that CXCL12 stimulation promotes interaction between Gαi2 and ELMO1. Gi signalling and ELMO1 are both required for CXCL12-mediated actin polymerization, migration and invasion of breast cancer cells. CXCL12 triggers a Gαi2-dependent membrane translocation of ELMO1, which associates with Dock180 to activate small G-proteins Rac1 and Rac2. In vivo, ELMO1 expression is associated with lymph node and distant metastasis, and knocking down ELMO1 impairs metastasis to the lung. Our findings indicate that a chemokine-controlled pathway, consisting of Gαi2, ELMO1/Dock180, Rac1 and Rac2, regulates the actin cytoskeleton during breast cancer metastasis.

Show MeSH
Related in: MedlinePlus