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

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.

Figure 1. 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).

Figure 2. N-terminal portion of ELMO1 interacts with Gαi2 subunit.

(a) Image of SDS–PAGE gel stained by silver stain. Lysates of MDA-MB-231 cells expressing ELMO1-YFP or YFP (as a control) were incubated with beads coupled with anti-GFP antibodies and stained with silver stain. (b) Coimmunoprecipitation assay of ELMO1-YFP, Gαi2 and Dock180. The immunoprecipitation was performed by using the μMACS GFP isolation kit. The eluted proteins were separated by SDS–PAGE and were probed with antibodies. Input Gαi2 was used as control. (c) N terminus of ELMO1 was required for the interaction with Gαi2. YFP served as a negative control and ELMO1-YFP was a positive control. (d) CXCL12-induced ELMO1 colocalization with Gαi2 on the plasma membrane by confocal microscopy analysis. After stimulation with 100 ng ml⁻¹ CXCL12 for 1 h at 37 °C, cells were fixed, permeabilized and blocked in 3% BSA. MDA-MB-231 cells were stained with anti-ELMO1, anti-Gαi2 antibodies and probed with an Alexa Fluor 488-conjugated or 546-conjugated secondary antibody. Colocalization efficiency was calculated through Image J software. (e) Knockdown of Gαi2 impaired CXCL12-induced membrane translocation of ELMO1. After stimulation with 100 ng ml⁻¹ CXCL12 for 1 h at 37 °C, cells were fixed, permeabilized and blocked in 3% BSA. Twenty-five images were analysed by ImageJ software. Western blotting analysis of biochemical fractionation showed a clear enrichment of ELMO1 upon CXCL12 stimulation.

Figure 3. CXCL12 induces membrane translocation and activation of Rac.

(a) Coimmunoprecipitation assay of ELMO1-YFP, 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. (b) Knockdown of ELMO1 impaired CXCL12-induced membrane translocation of Rac1/2. After stimulation with 100 ng ml⁻¹ CXCL12 for 1 h at 37 °C, cells were fixed, permeabilized and blocked in 3% BSA. Twenty-five images were analysed by Image J software. Western blotting of biochemical fractionation showed that a clear enrichment of Rac1/2 upon CXCL12 stimulation and knockdown ELMO1 impaired CXCL12-induced Rac1/2 translocation. (c) Overexpression of ELMO1-YFP pulled down activated Rac1 and Rac2. Rac activation assay was performed by Rac Activation Assay Biochem Kit (Cytoskeleton, Inc.). Cleared lysates were obtained by centrifugation and incubated with PAK–PBD beads with rotation at 4 °C for 1 h. (d) siELMO1 had no effect in membrane translocation of AKT and PDK1. Western blotting showed a clear enrichment of AKT and PDK1 upon CXCL12 stimulation, and knockdown ELMO1 did not impair CXCL12-induced AKT and PDK1 translocation. (e) Western blotting analysis of phosphorylation of AKT^{308 or 473}, ERK1/2 and PDK1. Control/MDA-MB-231 cells were stimulated by 10 ng ml⁻¹ EGF or 100 ng ml⁻¹ CXCL12 for 1 h. siELMO1/MDA-MB-231 cells were stimulated by 100 ng ml⁻¹ CXCL12 for 1 h. Total AKT, ERK1/2 and PDK1 served as control.

Figure 4. 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).

Figure 5. ELMO1 has a role in breast cancer metastasis in vivo.

(a) Immunohistochemical analysis of ELMO1 expression in invasive ductal breast carcinoma tissues and normal breast tissues. The entire sample (81 invasive ductal breast carcinoma tissues and 7 normal breast tissues) was blocked for 1 h. The antibodies and the dilution factors were as follows: ELMO1 (1:100), Polink-2 plus Polymer HRP Detection System for Goat Primary Antibody. (b) Immunohistochemical analysis of ELMO2 expression in invasive ductal carcinoma tissues and normal breast tissues. The entire sample (81 invasive ductal breast carcinoma tissues and 7 normal breast tissues) was blocked for 1 h. The antibodies and the dilution factors were as follows: ELMO2 (1:50), Polink-2 plus Polymer HRP Detection System for Goat Primary Antibody. (c) Western blotting analysis for four stable siELMO1 clones and comparison of tumour size in SCID mice. (d) Comparison of spontaneous lung metastasis and images of representative lung metastasis. (e) Human tumour foci on mouse lungs were visualized by haematoxylin and eosin staining. (f) The number of lung metastases was counted and plotted (n=10). (g) A model for ELMO1 regulated the migration and chemotaxis of breast cancer cells by associating with Dock180, Gαi2, Rac1 and Rac2.