FilGAP, a GAP protein for Rac, regulates front‐rear polarity and tumor cell migration through the ECM

Abstract

Malignant tumor cells acquire abnormal motility and invade through various extracellular matrix (ECM) components with distinct composition and architecture, including basement membrane and interstitial collagen networks, and leave a blood or lymphatic vessel and invade the surrounding tissue parenchyma. 1,2 Actin cytoskeletal dynamics is essential for cell polarization and migration. 3-6 Migrating cells are characterized by a sustained front-rear asymmetry, with a front enriched in filamentous actin (F-actin) and a rear made of actomyosin filaments. Rho small GTPase (Rho GTPase) Rac1 induces membrane protrusion at the front of migrating cells through actin polymerization, whereas the activity of another Rho GTPase RhoA is required for tail retraction at the rear through activation of actomyosin contraction. 3,7-10 This acquisition of front-rear polarity is important for effective motility of various cell types including tumor cells.

FilGAP, a GAP for Rac1, down-regulates invadopodia formation in breast cancer cells

“…The human Rac1 coding sequence was polymerase chain reaction (PCR)-amplified using a Rac1 construct, and the PCR products digested with EcoRI and XhoI were inserted into the pEGFP-c1 vector using the EcoRI and SalI sites and sequenced. The pcDNA3 vector (Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA) encoding mCherry-tagged cortactin was generated as described previously (Saito et al, 2021). The human cortactin cording sequence was PCR-amplified using a cortactin construct, and the PCR products digested with BamHI and EcoRI were inserted into the pcDNA3-mCherry plasmid using the BamHI and EcoRI sites and sequenced.…”

“…were cultured as previously described (Donnelly et al, 2017;Saito et al, 2021). Cells were transfected with plasmid DNA for 24 hours or siRNA for 48 hours using Lipofectamine 2000 (Invitrogen-Thermo Fisher Scientific) according to the manufacturer's instructions.…”

“…To check the efficiency of FilGAP depletion by siRNAs and the expression level of FilGAP in stable cell lines, the cells were solubilized with 1% sodium dodecyl sulfate (SDS) in phosphate-buffered saline (PBS) containing protease inhibitor cocktail (Sigma-Merck) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysates were separated by SDS-PAGE and immunoblotting was performed as previously described (Saito et al, 2021).…”

“…FilGAP (also called ARHGAP24) is a Rac-specific GAP that regulates cell morphology and cell spreading (Morishita et al, 2015;Nakamura, 2013;Ohta et al, 2006;Yamada et al, 2016). In breast cancer cells, FilGAP controls cell shape changes during migration and contributes to cell invasion through the ECM in vitro (Saito et al, 2021;Saito et al, 2012;Uehara et al, 2017). In in vivo study, the depletion of endogenous FliGAP in breast cancer cells reduced tumor cell extravasation (Saito et al, 2012).…”

Smurf1 regulates ameloblast polarization by ubiquitination‐mediated degradation of RhoA

“…Cell polarity refers to the asymmetric distribution of cytoskeleton, organelles and biomacromolecules due to the cooperation or rejection of polarity protein complexes, 3 so that different regions of the cell can perform different functions, which plays an important role in embryonic development, 4 cell differentiation, 5 cell migration, 6 asymmetric cell division (ACD) 7 and tumour development. 8 According to the distribution characteristics of polarity protein complexes and cell morphology, cell polarity can be divided into four categories: Apical‐basal polarity (ABP), 9 , 10 Planar cell polarity (PCP), 11 , 12 Front‐rear polarity (FRP) 13 , 14 , 15 and ACD. 16 , 17 , 18 ABP is the one category of cell polarity which closely relates to the differentiation and maturation of AB and the formation of enamel.…”