Journal: The Journal of Biological Chemistry

Article Title: Integrin αvβ3–associated DAAM1 is essential for collagen-induced invadopodia extension and cell haptotaxis in breast cancer cells

doi: 10.1074/jbc.RA117.000327

Figure Lengend Snippet: DAAM1 activation is required for collagen-induced cell haptotaxis. A–D, type IV collagen triggered haptotaxis of MDA-MB-231 and MDA-MB-453 breast cancer cells. MDA-MB-231, MDA-MB-453, and MCF10A cells were examined for cell haptotaxis for 8 h in 8.0-μm porous Boyden chamber membranes coated with vehicle (Uncoated) or type IV collagen on upper sides (Upper), both sides (Both), or lower sides (Lower). Migratory cells on the lower side of the membrane were counted per field of the microscope. Bar, 50 μm. Objective lens, magnification, ×20; numerical aperture, 0.75. E–G, DAAM1 activation was significantly elevated by type IV collagen coated on the lower sides of Boyden chamber membranes. MDA-MB-231 or MDA-MB-453 cells were seeded on Boyden chamber membranes coated with vehicle (Uncoated) or type IV collagen on upper sides (Upper), both sides (Both), or lower sides (Lower). MDA-MB-231 or MDA-MB-453 cells were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for the active DAAM1 by a pulldown assay using a GST-RHOA as a bait. H, efficiency of gene knockdown was analyzed by immunoblotting for DAAM1. MDA-MB-231 and MDA-MB-453 cells were transfected with scrambled or DAAM1 shRNAs (#1 and #2). Total protein extracts from MDA-MB-231 and MDA-MB-453 cells transfected with scrambled or DAAM1 shRNAs were analyzed by immunoblotting for DAAM1. I and J, knockdown of DAAM1 significantly inhibited cell haptotaxis induced by collagen coated on the lower sides of Boyden chamber membranes. MDA-MB-231 and MDA-MB-453 cells transfected with DAAM1 shRNA or scrambled shRNAs were allowed to migrate for 8 h, and the migration rate was determined by Boyden chamber assays. K and P, the expression of empty vector and HA-tagged N-DAAM1 (N terminus of DAAM1) was verified using total protein from cells and immunoblotted using anti-HA antibody. The expression level of DAAM1 was tested using immunoblotting and proved the efficiency of N-DAAM1 expression. L and M, the expression of N-DAAM1 significantly inhibited cell haptotaxis induced by collagen coated on the lower sides of Boyden chamber membranes. MDA-MB-231 and MDA-MB-453 cells transfected with N-DAAM1 were allowed to migrate for 8 h, and the number of migratory cells were counted per field of the microscope. N and P, the expression of empty vector and HA-tagged C-DAAM1 (C terminus of DAAM1) was verified using total protein from cells and immunoblotted using anti-HA antibody. The expression level of DAAM1 was tested using immunoblotting and proved the efficiency of C-DAAM1 expression. O, MDA-MB-231 cells transfected with C-DAAM1 were seeded on Boyden chamber membranes and allowed to migrate for 8 h, and the number of migratory cells were counted per field of the microscope. Error bars, S.D.

Article Snippet: Equal amounts of total cellular protein were incubated with GTPγS-incubated GST-RHOA beads captured on MagneGST GSH particles (Promega, Madison, WI) at 4 °C with constant rotation for 90 min.

Techniques: Activation Assay, Microscopy, Western Blot, Transfection, shRNA, Migration, Expressing, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Integrin αvβ3–associated DAAM1 is essential for collagen-induced invadopodia extension and cell haptotaxis in breast cancer cells

doi: 10.1074/jbc.RA117.000327

Figure Lengend Snippet: DAAM1 is associated with integrin β3 and acts as a downstream target of the integrin receptor. A, integrin inhibitors blocked type IV collagen–induced haptotaxis of breast cancer cells. MDA-MB-231 and/or MDA-MB-453 cells, treated with cilengitide trifluoroacetate (an integrin inhibitor for αvβ3 receptor and αvβ5 receptor, 10 nmol/liter) or cyclo(-RGDfK) (an αvβ3 integrin inhibitor, 20 nmol/liter), were examined for cell haptotaxis for 8 h in 8.0-μm porous Boyden chamber membranes coated with type IV collagen on the lower sides. CT, cilengitide trifluoroacetate. Cy, cyclo(-RGDfK). B and C, collagen-induced DAAM1 activation was blocked by αvβ3 integrin inhibitor treatment. MDA-MB-231 or MDA-MB-453 cells were seeded on Boyden chamber membranes coated with collagen type IV on both sides (Both) or lower sides (Lower) and treated with cyclo(-RGDfK) (20 nmol/liter). MDA-MB-231 or MDA-MB-453 cells were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for the active DAAM1 by a pulldown assay using a GST-RHOA as a bait. Cy, cyclo(-RGDfK). D and E, the lysates of MDA-MB-231 or MDA-MB-453 cells were subjected to immunoprecipitation (IP) with antibody to DAAM1, followed by immunoblotting with antibody to integrin β3 and DAAM1. F, HA-tagged DAAM1 constructs were expressed in HEK-293T cells. Cell lysates were harvested and cleared, and integrin was immunoprecipitated with anti-HA antibodies. Immune precipitates were analyzed by immunoblotting for anti-HA or anti-integrin β3. Whole-cell lysates were used as a control. G, purified GST or GST-fused C terminus (amino acids 716–762) of integrin β3 was incubated with purified HA-tagged full-length DAAM1. The amounts of HA-DAAM1 co-purified with GST or GST-integrin (pulldown) were analyzed by immunoblotting. Error bars, S.D.

Article Snippet: Equal amounts of total cellular protein were incubated with GTPγS-incubated GST-RHOA beads captured on MagneGST GSH particles (Promega, Madison, WI) at 4 °C with constant rotation for 90 min.

Techniques: Activation Assay, Immunoprecipitation, Western Blot, Construct, Purification, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Integrin αvβ3–associated DAAM1 is essential for collagen-induced invadopodia extension and cell haptotaxis in breast cancer cells

doi: 10.1074/jbc.RA117.000327

Figure Lengend Snippet: RHOA is a downstream target of DAAM1, and its activation is required for collagen-induced cell haptotaxis. A–C, RHOA, RAC1, and CDC42 activations were elevated by type IV collagen treatment. MDA-MB-231 cells were seeded on Boyden chamber membranes coated with vehicle (Uncoated) or type IV collagen on upper sides (Upper), both sides (Both), or lower sides (Lower). Cells were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for RHOA, RAC1, and CDC42 activation assays. D, collagen-induced RHOA activation was inhibited by DAAM1 silence. Stable DAAM1 knockdown MDA-MB-231 cells (DAAM1 shRNA #1 and #2) and control cells were seeded on Boyden chamber membranes coated with collagen type IV on the lower sides. Cells were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for RHOA, RAC1, and CDC42 activation assays. E, collagen-induced RHOA activation was inhibited by cyclo(-RGDfK) treatment. MDA-MB-231 cells were seeded on Boyden chamber membranes coated with collagen type IV on the lower sides. Cells treated with cyclo(-RGDfK) (20 nmol/liter) were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for RHOA activation assays. Cy, cyclo(-RGDfK). F and G, CCG-1423 (RHOA-specific inhibitor) or RHOA-N19 (dominant-negative RHOA) inhibited collagen-induced haptotaxis of MDA-MB-231 cells. MDA-MB-231 cells treated with CCG-1423 (1 μmol/liter) or RHOA-N19–overexpressing stable cells were seeded in Boyden chamber membranes coated with type IV collagen on both sides (Both) or lower sides (Lower). Cells were allowed to migrate toward collagen for 8 h. H, CCG-1423 or RHOA-N19 inhibited collagen-induced haptotaxis of MDA-MB-453 cells. MDA-MB-453 cells treated with CCG-1423 (1 μmol/liter) or transfected with RHOA-N19 were seeded in Boyden chamber membranes coated with type IV collagen on both sides (Both) or lower sides (Lower). Cells were allowed to migrate toward collagen for 8 h. I and J, DAAM1 activation was not altered by RHOA-N19 overexpression. RHOA-N19–overexpressing stable cells were seeded on Boyden chamber membranes coated with type IV collagen on both sides (Both) or on lower sides (Lower). Cells were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for the active DAAM1 by a pulldown assay using a GST-RHOA as a bait. K, RHOA-V14 rescued the haptotaxis of N-DAAM1–expressed MDA-MB-231 cells. MDA-MB-231 cells transfected with N-DAAM1 and/or RHOA-V14 were seeded in Boyden chamber membranes coated with type IV collagen on lower sides. Cells were allowed to migrate toward collagen for 8 h. Error bars, S.D.

Article Snippet: Equal amounts of total cellular protein were incubated with GTPγS-incubated GST-RHOA beads captured on MagneGST GSH particles (Promega, Madison, WI) at 4 °C with constant rotation for 90 min.

Techniques: Activation Assay, shRNA, Dominant Negative Mutation, Transfection, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Integrin αvβ3–associated DAAM1 is essential for collagen-induced invadopodia extension and cell haptotaxis in breast cancer cells

doi: 10.1074/jbc.RA117.000327

Figure Lengend Snippet: DAAM1 activation is essential for collagen-induced invadopodia extension. A and B, type IV collagen triggered invadopodia extension of MDA-MB-231 and MDA-MB-453 cells. MDA-MB-231 or MDA-MB-453 cells were examined for invadopodia extension for 6 h in 3.0-μm porous Boyden chamber membranes coated with vehicle (Uncoated) or type IV collagen on upper sides (Upper), both sides (Both), or lower sides (Lower). Invadopodia on the lower sides of the membrane were stained with cortactin antibodies and counted per field of the microscope. Bar, 10 μm. Objective lens, magnification, ×40; numerical aperture, 0.95. C, DAAM1 silence significantly inhibited collagen-induced invadopodia extension. Stable DAAM1 knockdown MDA-MB-231 cells (DAAM1 shRNA #1 and #2) or control cells were allowed to extend invadopodia for 6 h. The number of extended invadopodia was determined by 3.0-μm porous Boyden chamber assays and counted per field of the microscope. D, cyclo(-RGDfK) suppressed collagen-induced invadopodia extension. MDA-MB-231 cells were treated with 20 nmol/liter cyclo(-RGDfK) or vehicle and allowed to extend invadopodia for 6 h. The number of extended invadopodia was determined by 3.0-μm porous Boyden chamber assays and counted per field of the microscope. E, DAAM1 silence or cyclo(-RGDfK) significantly inhibited collagen-induced invadopodia extension. MDA-MB-453 cells transfected with DAAM1 shRNA or treated with cyclo(-RGDfK) were allowed to extend invadopodia for 6 h. The number of extended invadopodia was determined by 3.0-μm porous Boyden chamber assays and counted per field of the microscope. Bo, collagen coated on both sides. Lo, collagen coated on the lower sides. F–H, abundant active DAAM1 were located in invadopodia. MDA-MB-231 or MDA-MB-453 cells were seeded on 3.0-μm porous Boyden chamber membranes coated with collagen type IV on the lower sides. The invadopodia of MDA-MB-231 or MDA-MB-453 cells were allowed to extend toward collagen for 4 h. Cell bodies and invadopodia were separated as described under “Experimental procedures.” Cellular lysates were assayed for the active DAAM1 by a pulldown assay using a GST-RHOA as a bait. Error bars, S.D.

Article Snippet: Equal amounts of total cellular protein were incubated with GTPγS-incubated GST-RHOA beads captured on MagneGST GSH particles (Promega, Madison, WI) at 4 °C with constant rotation for 90 min.

Techniques: Activation Assay, Staining, Microscopy, shRNA, Transfection