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SDF-1alpha concentration dependent modulation of RhoA and Rac1 modifies breast cancer and stromal cells interaction.

Pasquier J, Abu-Kaoud N, Abdesselem H, Madani A, Hoarau-Véchot J, Thawadi HA, Vidal F, Couderc B, Favre G, Rafii A - BMC Cancer (2015)

Bottom Line: This interaction increases migration from primary sites as well as homing at distant sites.Increased migration was obtained at 50 and 100 ng/ml of SDF-1α; however migration was reduced at 200 ng/ml.The adhesion between breast cancer cells and BMHC was significantly increased by SDF-1α treatment at 200 ng/ml and reduced using a blocking monoclonal antibody against CXCR4.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Education City, Qatar Foundation, Doha, Qatar. jep2026@qatar-med.cornell.edu.

ABSTRACT

Background: The interaction of SDF-1alpha with its receptor CXCR4 plays a role in the occurrence of distant metastasis in many solid tumors. This interaction increases migration from primary sites as well as homing at distant sites.

Methods: Here we investigated how SDF-1α could modulate both migration and adhesion of cancer cells through the modulation of RhoGTPases.

Results: We show that different concentrations of SDF-1α modulate the balance of adhesion and migration in cancer cells. Increased migration was obtained at 50 and 100 ng/ml of SDF-1α; however migration was reduced at 200 ng/ml. The adhesion between breast cancer cells and BMHC was significantly increased by SDF-1α treatment at 200 ng/ml and reduced using a blocking monoclonal antibody against CXCR4. We showed that at low SDF-1α concentration, RhoA was activated and overexpressed, while at high concentration Rac1 was promoting SDF-1α mediating-cell adhesion.

Conclusion: We conclude that SDF-1α concentration modulates migration and adhesion of breast cancer cells, by controlling expression and activation of RhoGTPases.

No MeSH data available.


Related in: MedlinePlus

Functional consequences of inhibition of RhoA. a F-actin polymerisation in RhoA siRNA transfected MDA-MB231. Two days after si-RNA transfection, MDA-MB-231 were grown on glass bottom slides and actin cytosqueletton was revealed by a phalloïdin-fluorescein (1 μg/mL) labelling. Pictures present fluorescence microscope series of adherent MDA-MB-231 transfected with non-specific siRNA (ns-SiRNA), RhoA-specific (RhoA si-RNA) unstimulated or stimulated with SDF-1α (100 ng/mL). RhoA inhibition reverted the increased of stress fiber in the treated sample. b RhoA specific si-RNA transfected MDA-MB-231 plasticity on Matrigel. Two days after transfection, with non-specific si-RNA (ns-SiRNA) or RhoA specific (RhoA si-RNA) MDA-MB231 were seeded on a 96-wells plate, coated with Matrigel. Microscopic pictures of cellular networks after SDF-1α stimulation (100 ng/ml) were taken after 18 h of culture. Quantitative evaluation of the cellular interconnection is presented. The evaluation was made by counting the number of cellular interconnections on 10 different fields. RhoA inhibition reversed the interconnection number increase in the treated sample. c Wound Closure assay. Two days after transfection, with non-specific si-RNA (ns-SiRNA) or RhoA specific (RhoA si-RNA) MDA-MB231 migration ability was tested after a scratch with or without SDF-1α (100 ng/ml). RhoA inhibition supressed the effect of SDF-1α on MDA-MB231 motility. d Cell cycle analysis. Two days after transfection, with non-specific si-RNA (ns-SiRNA) or RhoA specific (RhoA si-RNA) MDA-MB231 were treated with or without SDF-1α (50 ng/ml) for 48 h and position in cell cycle were evaluated with NIM-DAPI by flow cytometry. The inhibition of RhoA doesn’t have any effect on the position of the cell cycle position of MDA-MB231. The results presents in this figure are representative of three different experiments. e Adherence of MDA-MB231 RhoA specific si-RNA transfected cells to BMHC. Stable eGFP-MDA-MB231 cells were seeded on the plate and allow to adhere for one hour. As displayed Si-RhoA transfected cancer cells displayed significantly increased adhesion compared to controls
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Fig5: Functional consequences of inhibition of RhoA. a F-actin polymerisation in RhoA siRNA transfected MDA-MB231. Two days after si-RNA transfection, MDA-MB-231 were grown on glass bottom slides and actin cytosqueletton was revealed by a phalloïdin-fluorescein (1 μg/mL) labelling. Pictures present fluorescence microscope series of adherent MDA-MB-231 transfected with non-specific siRNA (ns-SiRNA), RhoA-specific (RhoA si-RNA) unstimulated or stimulated with SDF-1α (100 ng/mL). RhoA inhibition reverted the increased of stress fiber in the treated sample. b RhoA specific si-RNA transfected MDA-MB-231 plasticity on Matrigel. Two days after transfection, with non-specific si-RNA (ns-SiRNA) or RhoA specific (RhoA si-RNA) MDA-MB231 were seeded on a 96-wells plate, coated with Matrigel. Microscopic pictures of cellular networks after SDF-1α stimulation (100 ng/ml) were taken after 18 h of culture. Quantitative evaluation of the cellular interconnection is presented. The evaluation was made by counting the number of cellular interconnections on 10 different fields. RhoA inhibition reversed the interconnection number increase in the treated sample. c Wound Closure assay. Two days after transfection, with non-specific si-RNA (ns-SiRNA) or RhoA specific (RhoA si-RNA) MDA-MB231 migration ability was tested after a scratch with or without SDF-1α (100 ng/ml). RhoA inhibition supressed the effect of SDF-1α on MDA-MB231 motility. d Cell cycle analysis. Two days after transfection, with non-specific si-RNA (ns-SiRNA) or RhoA specific (RhoA si-RNA) MDA-MB231 were treated with or without SDF-1α (50 ng/ml) for 48 h and position in cell cycle were evaluated with NIM-DAPI by flow cytometry. The inhibition of RhoA doesn’t have any effect on the position of the cell cycle position of MDA-MB231. The results presents in this figure are representative of three different experiments. e Adherence of MDA-MB231 RhoA specific si-RNA transfected cells to BMHC. Stable eGFP-MDA-MB231 cells were seeded on the plate and allow to adhere for one hour. As displayed Si-RhoA transfected cancer cells displayed significantly increased adhesion compared to controls

Mentions: To confirm the essential role of both RhoA and Rac1 we used an inhibition strategy. Using a Si-RhoA (Additional file 1: Figure S5C), we were able to show reduced actin polymerization when MDA-MB231RhoA- were treated with SDF-1α (Fig. 5a). The number of cellular extension was also decreased by the inhibition of RhoA (data not shown). SDF-1α mediated increase of intercellular connection was reversed in Si-RhoA transfected cells (Fig. 5b). The inhibition of RhoA has a drastic negative effect on the migration and proliferation of the MDA-MB231 (Fig. 5c and d). However adhesion to BMHC was increased in Si-RhoA transfected cells (Fig. 5e) suggesting that activation of RhoA has a negative effect on the MDA-MB231 binding to the BMHC. As a decrease in RhoA expression was leading to increased adhesion, we hypothesized that the balance between RhoA and Rac1 could be a mediator of the SDF-1α effect. We used the cell-sorting gate set-up in Fig. 2a to separate MDA-MB231 after a co-culture of 2 or 5 days with BMHC. The sorted cells displayed an increase of Rac1 and Cdc42, but a decrease of Rock2 and RhoA (Fig. 6a). Using NSC23766, a widely used inhibitor of Rac1 activation, we were able to demonstrate a decrease of MDA-MB231 adhesion to both plastic and BMHC despite SDF-1α treatment (Fig. 6b). We then generated a knock-down of Rac1 through ShRNA (Additional file 1: Figure S5C). We previously demonstrated an up-regulation of αV, β1 and β3 protein after 4 h of stimulation with 200 ng/ml of SDF-1α. Interestingly, when Rac1 was silenced in MDA-MB231, a 200 ng/ml of SDF-1α treatment didn’t lead to increased integrin expression confirming the major role of Rac1 in MDA-MB231 adhesion through integrin αV, β1 and β3 (Fig. 6c). When MDA-MB231 ShRac1 cells were co-cultured for 6 days with BMHC, we noticed a decrease in the number of cancer cells present on BMHC (Fig. 6d top panels). Moreover, MDA-MB231 ShRac1 cells co-cultured with BMHC in serum free cytokine free media didn’t display any proliferative advantage as compared to MDA-MB231 Mock (Fig. 6d bottom panel). Finally, we confirmed that Rac1 inhibition reduced the number of proliferating cells using a cell cycle analysis in presence of SDF-1α (Fig. 6e).Fig. 5


SDF-1alpha concentration dependent modulation of RhoA and Rac1 modifies breast cancer and stromal cells interaction.

Pasquier J, Abu-Kaoud N, Abdesselem H, Madani A, Hoarau-Véchot J, Thawadi HA, Vidal F, Couderc B, Favre G, Rafii A - BMC Cancer (2015)

Functional consequences of inhibition of RhoA. a F-actin polymerisation in RhoA siRNA transfected MDA-MB231. Two days after si-RNA transfection, MDA-MB-231 were grown on glass bottom slides and actin cytosqueletton was revealed by a phalloïdin-fluorescein (1 μg/mL) labelling. Pictures present fluorescence microscope series of adherent MDA-MB-231 transfected with non-specific siRNA (ns-SiRNA), RhoA-specific (RhoA si-RNA) unstimulated or stimulated with SDF-1α (100 ng/mL). RhoA inhibition reverted the increased of stress fiber in the treated sample. b RhoA specific si-RNA transfected MDA-MB-231 plasticity on Matrigel. Two days after transfection, with non-specific si-RNA (ns-SiRNA) or RhoA specific (RhoA si-RNA) MDA-MB231 were seeded on a 96-wells plate, coated with Matrigel. Microscopic pictures of cellular networks after SDF-1α stimulation (100 ng/ml) were taken after 18 h of culture. Quantitative evaluation of the cellular interconnection is presented. The evaluation was made by counting the number of cellular interconnections on 10 different fields. RhoA inhibition reversed the interconnection number increase in the treated sample. c Wound Closure assay. Two days after transfection, with non-specific si-RNA (ns-SiRNA) or RhoA specific (RhoA si-RNA) MDA-MB231 migration ability was tested after a scratch with or without SDF-1α (100 ng/ml). RhoA inhibition supressed the effect of SDF-1α on MDA-MB231 motility. d Cell cycle analysis. Two days after transfection, with non-specific si-RNA (ns-SiRNA) or RhoA specific (RhoA si-RNA) MDA-MB231 were treated with or without SDF-1α (50 ng/ml) for 48 h and position in cell cycle were evaluated with NIM-DAPI by flow cytometry. The inhibition of RhoA doesn’t have any effect on the position of the cell cycle position of MDA-MB231. The results presents in this figure are representative of three different experiments. e Adherence of MDA-MB231 RhoA specific si-RNA transfected cells to BMHC. Stable eGFP-MDA-MB231 cells were seeded on the plate and allow to adhere for one hour. As displayed Si-RhoA transfected cancer cells displayed significantly increased adhesion compared to controls
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Fig5: Functional consequences of inhibition of RhoA. a F-actin polymerisation in RhoA siRNA transfected MDA-MB231. Two days after si-RNA transfection, MDA-MB-231 were grown on glass bottom slides and actin cytosqueletton was revealed by a phalloïdin-fluorescein (1 μg/mL) labelling. Pictures present fluorescence microscope series of adherent MDA-MB-231 transfected with non-specific siRNA (ns-SiRNA), RhoA-specific (RhoA si-RNA) unstimulated or stimulated with SDF-1α (100 ng/mL). RhoA inhibition reverted the increased of stress fiber in the treated sample. b RhoA specific si-RNA transfected MDA-MB-231 plasticity on Matrigel. Two days after transfection, with non-specific si-RNA (ns-SiRNA) or RhoA specific (RhoA si-RNA) MDA-MB231 were seeded on a 96-wells plate, coated with Matrigel. Microscopic pictures of cellular networks after SDF-1α stimulation (100 ng/ml) were taken after 18 h of culture. Quantitative evaluation of the cellular interconnection is presented. The evaluation was made by counting the number of cellular interconnections on 10 different fields. RhoA inhibition reversed the interconnection number increase in the treated sample. c Wound Closure assay. Two days after transfection, with non-specific si-RNA (ns-SiRNA) or RhoA specific (RhoA si-RNA) MDA-MB231 migration ability was tested after a scratch with or without SDF-1α (100 ng/ml). RhoA inhibition supressed the effect of SDF-1α on MDA-MB231 motility. d Cell cycle analysis. Two days after transfection, with non-specific si-RNA (ns-SiRNA) or RhoA specific (RhoA si-RNA) MDA-MB231 were treated with or without SDF-1α (50 ng/ml) for 48 h and position in cell cycle were evaluated with NIM-DAPI by flow cytometry. The inhibition of RhoA doesn’t have any effect on the position of the cell cycle position of MDA-MB231. The results presents in this figure are representative of three different experiments. e Adherence of MDA-MB231 RhoA specific si-RNA transfected cells to BMHC. Stable eGFP-MDA-MB231 cells were seeded on the plate and allow to adhere for one hour. As displayed Si-RhoA transfected cancer cells displayed significantly increased adhesion compared to controls
Mentions: To confirm the essential role of both RhoA and Rac1 we used an inhibition strategy. Using a Si-RhoA (Additional file 1: Figure S5C), we were able to show reduced actin polymerization when MDA-MB231RhoA- were treated with SDF-1α (Fig. 5a). The number of cellular extension was also decreased by the inhibition of RhoA (data not shown). SDF-1α mediated increase of intercellular connection was reversed in Si-RhoA transfected cells (Fig. 5b). The inhibition of RhoA has a drastic negative effect on the migration and proliferation of the MDA-MB231 (Fig. 5c and d). However adhesion to BMHC was increased in Si-RhoA transfected cells (Fig. 5e) suggesting that activation of RhoA has a negative effect on the MDA-MB231 binding to the BMHC. As a decrease in RhoA expression was leading to increased adhesion, we hypothesized that the balance between RhoA and Rac1 could be a mediator of the SDF-1α effect. We used the cell-sorting gate set-up in Fig. 2a to separate MDA-MB231 after a co-culture of 2 or 5 days with BMHC. The sorted cells displayed an increase of Rac1 and Cdc42, but a decrease of Rock2 and RhoA (Fig. 6a). Using NSC23766, a widely used inhibitor of Rac1 activation, we were able to demonstrate a decrease of MDA-MB231 adhesion to both plastic and BMHC despite SDF-1α treatment (Fig. 6b). We then generated a knock-down of Rac1 through ShRNA (Additional file 1: Figure S5C). We previously demonstrated an up-regulation of αV, β1 and β3 protein after 4 h of stimulation with 200 ng/ml of SDF-1α. Interestingly, when Rac1 was silenced in MDA-MB231, a 200 ng/ml of SDF-1α treatment didn’t lead to increased integrin expression confirming the major role of Rac1 in MDA-MB231 adhesion through integrin αV, β1 and β3 (Fig. 6c). When MDA-MB231 ShRac1 cells were co-cultured for 6 days with BMHC, we noticed a decrease in the number of cancer cells present on BMHC (Fig. 6d top panels). Moreover, MDA-MB231 ShRac1 cells co-cultured with BMHC in serum free cytokine free media didn’t display any proliferative advantage as compared to MDA-MB231 Mock (Fig. 6d bottom panel). Finally, we confirmed that Rac1 inhibition reduced the number of proliferating cells using a cell cycle analysis in presence of SDF-1α (Fig. 6e).Fig. 5

Bottom Line: This interaction increases migration from primary sites as well as homing at distant sites.Increased migration was obtained at 50 and 100 ng/ml of SDF-1α; however migration was reduced at 200 ng/ml.The adhesion between breast cancer cells and BMHC was significantly increased by SDF-1α treatment at 200 ng/ml and reduced using a blocking monoclonal antibody against CXCR4.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Education City, Qatar Foundation, Doha, Qatar. jep2026@qatar-med.cornell.edu.

ABSTRACT

Background: The interaction of SDF-1alpha with its receptor CXCR4 plays a role in the occurrence of distant metastasis in many solid tumors. This interaction increases migration from primary sites as well as homing at distant sites.

Methods: Here we investigated how SDF-1α could modulate both migration and adhesion of cancer cells through the modulation of RhoGTPases.

Results: We show that different concentrations of SDF-1α modulate the balance of adhesion and migration in cancer cells. Increased migration was obtained at 50 and 100 ng/ml of SDF-1α; however migration was reduced at 200 ng/ml. The adhesion between breast cancer cells and BMHC was significantly increased by SDF-1α treatment at 200 ng/ml and reduced using a blocking monoclonal antibody against CXCR4. We showed that at low SDF-1α concentration, RhoA was activated and overexpressed, while at high concentration Rac1 was promoting SDF-1α mediating-cell adhesion.

Conclusion: We conclude that SDF-1α concentration modulates migration and adhesion of breast cancer cells, by controlling expression and activation of RhoGTPases.

No MeSH data available.


Related in: MedlinePlus