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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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ELMO2 has a role in breast cancer chemotactic responses.(a) Coimmunoprecipitation assay of ELMO2-YFP, Gαi2, Dock180, Rac1 and Rac2. The immunoprecipitation was performed by using the μMACS GFP isolation kit. The eluted proteins were separated by SDS–PAGE and probed with antibodies. Input Gαi2 was used as control. (b) Overexpression of ELMO2-YFP activated Rac1 and Rac2. Rac activation assay was performed by Rac Activation Assay Biochem Kit. Cleared lysates were obtained by centrifugation and incubated with PAK–PBD beads with rotation at 4 °C for 1 h. (c) Actin polymerization in siELMO2 cells was decreased. 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 analysis of variance (ANOVA), ***P<0.0001). (d) Reduction of ELMO2 also inhibited chemotaxis of MDA-MB-231 cells, especially, when ELMO1 and ELMO2 were knocked down together (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, *** P<0.0001). (e) Western blotting analysis: lane 1 was loaded with MDA-MB-231 cells, lane 2 with ELMO2-YFP/MDA-MB-231 cells, lane 3 with siELMO2/MDA-MB-231 cells and lane 4 with siELMO2+ELMO2-YFP (expressing ELMO2-YFP in siELMO2 cells). Overexpression of ELMO2 promoted chemotaxis (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001), and overexpression of ELMO2 rescued the defects of siELMO1 (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001) and siELMO2 cells (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001).
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f4: ELMO2 has a role in breast cancer chemotactic responses.(a) Coimmunoprecipitation assay of ELMO2-YFP, Gαi2, Dock180, Rac1 and Rac2. The immunoprecipitation was performed by using the μMACS GFP isolation kit. The eluted proteins were separated by SDS–PAGE and probed with antibodies. Input Gαi2 was used as control. (b) Overexpression of ELMO2-YFP activated Rac1 and Rac2. Rac activation assay was performed by Rac Activation Assay Biochem Kit. Cleared lysates were obtained by centrifugation and incubated with PAK–PBD beads with rotation at 4 °C for 1 h. (c) Actin polymerization in siELMO2 cells was decreased. 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 analysis of variance (ANOVA), ***P<0.0001). (d) Reduction of ELMO2 also inhibited chemotaxis of MDA-MB-231 cells, especially, when ELMO1 and ELMO2 were knocked down together (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, *** P<0.0001). (e) Western blotting analysis: lane 1 was loaded with MDA-MB-231 cells, lane 2 with ELMO2-YFP/MDA-MB-231 cells, lane 3 with siELMO2/MDA-MB-231 cells and lane 4 with siELMO2+ELMO2-YFP (expressing ELMO2-YFP in siELMO2 cells). Overexpression of ELMO2 promoted chemotaxis (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001), and overexpression of ELMO2 rescued the defects of siELMO1 (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001) and siELMO2 cells (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001).

Mentions: ELMO2 shares 76% identity in amino acid sequence with ELMO1 and is also expressed in breast cancer cells (Fig. 1a), and thus, we investigated the role of ELMO2 in chemotaxis of MDA-MB-231 cells (Fig. 4). As shown in Fig. 4a, ELMO2-YFP pulled down Dock180, Gαi2, Rac1 and Rac2. Moreover, although CXCL12 stimulation induced activation of both Rac1 and Rac2, it failed to do so when Gαi signalling was blocked by PTX or when ELMO2 was knocked down (siELMO2; Fig. 4b). CXCL12-triggered actin polymerization and chemotaxis were also reduced in ELMO2 knockdown (siELMO2) cells (Fig. 4c and d). Our results suggest that ELMO1 and ELMO2 have similar roles in CXCL12-mediated signal transduction, leading to chemotaxis in MDA-MB-231 cells. Our observations that knockdown of either ELMO1 or ELMO2 in the cells results in defective chemotactic responses suggest that sufficient levels of ELMO1 and/or ELMO2 are required for robust CXCL12-triggered chemotaxis of the breast cancer cells. Consistent with this notion, we found that knockdown of both ELMO1 and ELMO2 further inhibited chemotaxis (Fig. 4d). In addition, expressing ELMO1-YFP in siELMO1 cells (Supplementary Fig. S6A), ELMO2-YFP in siELMO2 cells (Fig. 4e) or ELMO2-YFP in siELMO1 cells (Fig. 4e) could partially rescue chemotaxis defects in these cells.


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)

ELMO2 has a role in breast cancer chemotactic responses.(a) Coimmunoprecipitation assay of ELMO2-YFP, Gαi2, Dock180, Rac1 and Rac2. The immunoprecipitation was performed by using the μMACS GFP isolation kit. The eluted proteins were separated by SDS–PAGE and probed with antibodies. Input Gαi2 was used as control. (b) Overexpression of ELMO2-YFP activated Rac1 and Rac2. Rac activation assay was performed by Rac Activation Assay Biochem Kit. Cleared lysates were obtained by centrifugation and incubated with PAK–PBD beads with rotation at 4 °C for 1 h. (c) Actin polymerization in siELMO2 cells was decreased. 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 analysis of variance (ANOVA), ***P<0.0001). (d) Reduction of ELMO2 also inhibited chemotaxis of MDA-MB-231 cells, especially, when ELMO1 and ELMO2 were knocked down together (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, *** P<0.0001). (e) Western blotting analysis: lane 1 was loaded with MDA-MB-231 cells, lane 2 with ELMO2-YFP/MDA-MB-231 cells, lane 3 with siELMO2/MDA-MB-231 cells and lane 4 with siELMO2+ELMO2-YFP (expressing ELMO2-YFP in siELMO2 cells). Overexpression of ELMO2 promoted chemotaxis (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001), and overexpression of ELMO2 rescued the defects of siELMO1 (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001) and siELMO2 cells (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001).
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f4: ELMO2 has a role in breast cancer chemotactic responses.(a) Coimmunoprecipitation assay of ELMO2-YFP, Gαi2, Dock180, Rac1 and Rac2. The immunoprecipitation was performed by using the μMACS GFP isolation kit. The eluted proteins were separated by SDS–PAGE and probed with antibodies. Input Gαi2 was used as control. (b) Overexpression of ELMO2-YFP activated Rac1 and Rac2. Rac activation assay was performed by Rac Activation Assay Biochem Kit. Cleared lysates were obtained by centrifugation and incubated with PAK–PBD beads with rotation at 4 °C for 1 h. (c) Actin polymerization in siELMO2 cells was decreased. 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 analysis of variance (ANOVA), ***P<0.0001). (d) Reduction of ELMO2 also inhibited chemotaxis of MDA-MB-231 cells, especially, when ELMO1 and ELMO2 were knocked down together (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, *** P<0.0001). (e) Western blotting analysis: lane 1 was loaded with MDA-MB-231 cells, lane 2 with ELMO2-YFP/MDA-MB-231 cells, lane 3 with siELMO2/MDA-MB-231 cells and lane 4 with siELMO2+ELMO2-YFP (expressing ELMO2-YFP in siELMO2 cells). Overexpression of ELMO2 promoted chemotaxis (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001), and overexpression of ELMO2 rescued the defects of siELMO1 (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001) and siELMO2 cells (points=mean of three independent experiments; scale bars=s.e.m.; n=6; two-way ANOVA, ***P<0.0001).
Mentions: ELMO2 shares 76% identity in amino acid sequence with ELMO1 and is also expressed in breast cancer cells (Fig. 1a), and thus, we investigated the role of ELMO2 in chemotaxis of MDA-MB-231 cells (Fig. 4). As shown in Fig. 4a, ELMO2-YFP pulled down Dock180, Gαi2, Rac1 and Rac2. Moreover, although CXCL12 stimulation induced activation of both Rac1 and Rac2, it failed to do so when Gαi signalling was blocked by PTX or when ELMO2 was knocked down (siELMO2; Fig. 4b). CXCL12-triggered actin polymerization and chemotaxis were also reduced in ELMO2 knockdown (siELMO2) cells (Fig. 4c and d). Our results suggest that ELMO1 and ELMO2 have similar roles in CXCL12-mediated signal transduction, leading to chemotaxis in MDA-MB-231 cells. Our observations that knockdown of either ELMO1 or ELMO2 in the cells results in defective chemotactic responses suggest that sufficient levels of ELMO1 and/or ELMO2 are required for robust CXCL12-triggered chemotaxis of the breast cancer cells. Consistent with this notion, we found that knockdown of both ELMO1 and ELMO2 further inhibited chemotaxis (Fig. 4d). In addition, expressing ELMO1-YFP in siELMO1 cells (Supplementary Fig. S6A), ELMO2-YFP in siELMO2 cells (Fig. 4e) or ELMO2-YFP in siELMO1 cells (Fig. 4e) could partially rescue chemotaxis defects in these cells.

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