One of the hallmarks of cancer is the ability of tumor cells to invade surrounding tissues and metastasize. During metastasis, cancer cells degrade the extracellular matrix, which acts as a physical barrier, by developing a specialized actin-rich membrane protrusion structure called invadopodia. The formation of invadopodia is regulated by Rho GTPases, a family of proteins that regulates the actin cytoskeleton. Here, we describe a novel role for RhoG in the regulation of invadopodia disassembly in human breast cancer cells. Our results show that RhoG and Rac1 have independent and opposite roles in the regulation of invadopodia dynamics. We also show that SGEF is the exchange factor responsible for the activation of RhoG during invadopodia disassembly. When the expression of either RhoG or SGEF is silenced, invadopodia are more stable and have a longer lifetime than in control cells. Our findings also demonstrate that RhoG and SGEF modulate the phosphorylation of paxillin, which plays a key role during invadopodia disassembly. In summary, we have identified a novel signaling pathway involving SGEF, RhoG, and paxillin phosphorylation, which functions in the regulation of invadopodia disassembly in breast cancer cells.
Supplementary Movie 1. Invadopodia formation in CTRL SUM159 cells in response to PDBu.
SUM159 cells expressing a CTRL shRNA were infected with mCherry-cortactin adenovirus and plated on glass-bottom coverslips 24 h after infection. Cells were imaged at 37°C in a CO2 and humidity controlled environment. At indicated times cells were stimulated to form invadopodia with PDBu. Images were captured at 15 sec intervals for the duration of the movie.
Supp Movie 1-non targeting 360x480_15fps.mov
Supplementary Movie 2. Invadopodia formation in Rac1 KO cells.
Rac1 KO SUM159 cells were infected with adenovirus encoding mCherry-cortactin as in Supp Movie 1. At indicated times cells were stimulated to form invadopodia with PDBu. Images were captured at 15 sec intervals for the duration of the movie.
Supp Movie 2-Rac KO-360x480.mov
Supplementary Movie 3. Invadopodia formation in Rac1 KO/rescue cells.
Rac1 KO SUM159 cells were co-infected with adenovirus encoding mCherry-cortactin and mycRac1. At indicated times cells were stimulated to form invadopodia with PDBu. Images were captured at 15 sec intervals for the duration of the movie.
Supp Movie 3-Rac KO- Rescue_360x480.mov
Supplementary Movie 4. Invadopodia in RhoG KD SUM159 cells.
SUM159 cells stably expressing a RhoG targeting shRNA were infected with mCherry-cortactin adenovirus as in Supp Movie 1. At indicated times cells were stimulated to form invadopodia with PDBu. Images were captured at 15 sec intervals for the duration of the movie.
Supp Movie 4_RhoG KD_480x360 37min.mov
Supplementary Movie 5. Invadopodia formation in SGEF KD cells.
SUM159 cells stably expressing SGEF targeting shRNA were infected with mCherry-cortactin adenovirus as in Supp Movie 1. At indicated times cells were stimulated to form invadopodia with PDBu. Images were captured at 15 sec intervals for the duration of the movie.
Supp Movie 5 -SGEF-KD-360x480.mov
Supplementary Movie 6. Invadopodia formation in RhoG KD/rescue cells.
RhoG KD SUM159 cells were co-infected with adenoviruses encoding mCherry-cortactin and shRNA resistant mycRhoG. At indicated times cells were stimulated to form invadopodia with PDBu. Images were captured at 15 sec intervals for the duration of the movie.
Supp Movie 6-RhoG-rescue_480x360.mov
Supplementary Movie 7. Invadopodia formation in SGEF KD/rescue cells.
SGEF KD SUM159 cells were co-infected with adenovirus encoding mCherry-cortactin and shRNA resistant mycSGEF. At indicated times cells were stimulated to form invadopodia with PDBu. Images were captured at 15 sec intervals for the duration of the movie.
Supp Movie 7_SGEF-rescue 480x360.mov