Epidermal growth factor (EGF) receptor (EGFR) signalling is implicated in tumour invasion and metastasis. However, whether there are EGFR signalling pathways specifically used for tumour invasion still remains elusive. Overexpression of Arf6 and its effector, AMAP1, correlates with and is crucial for the invasive phenotypes of different breast cancer cells. Here we identify the mechanism by which Arf6 is activated to induce tumour invasion. We found that GEP100/BRAG2, a guanine nucleotide exchanging factor (GEF) for Arf6, is responsible for the invasive activity of MDA-MB-231 breast cancer cells, whereas the other ArfGEFs are not. GEP100, through its pleckstrin homology domain, bound directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6. Overexpression of GEP100, together with Arf6, caused non-invasive MCF7 cells to become invasive, which was dependent on EGF stimulation. Moreover, GEP100 knockdown blocked tumour metastasis. GEP100 was expressed in 70% of primary breast ductal carcinomas, and was preferentially co-expressed with EGFR in the malignant cases. Our results indicate that GEP100 links EGFR signalling to Arf6 activation to induce invasive activities of some breast cancer cells, and hence may contribute to their metastasis and malignancy.

Identification of the molecular machinery employed in cancer invasion, but not in normal adult cells, will greatly contribute to cancer therapeutics. Here we found that an ArfGAP, AMAP1/PAG2, is expressed at high levels in highly invasive breast cancer cells, but at very low levels in noninvasive breast cancer cells and normal mammary epithelial cells. siRNA-mediated silencing of AMAP1 effectively blocked the invasive activities. AMAP1 expression in human breast primary tumors also indicated its potential correlation with malignancy. Paxillin and cortactin have been shown to colocalize at invadopodia and play a pivotal role in breast cancer invasion. We found that AMAP1 is also localized at invadopodia, and acts to bridge paxillin and cortactin. This AMAP1-mediated trimeric protein complex was detected only in invasive cancer cells, and blocking this complex formation effectively inhibited their invasive activities in vitro and metastasis in mice. Our results indicate that AMAP1 is a component involved in invasive activities of different breast cancers, and provide new information regarding the possible therapeutic targets for prevention of breast cancer invasion and metastasis.

Integrins can intercommunicate with cadherins. Here, we examined their possible relationship by use of small interfering RNA–mediated protein knockdown in HeLa cells. We found that a subset of integrin signaling molecules, namely Fak and paxillin, but not p130 Crk-associated substrate or proline-rich tyrosine kinase 2, participate in processes regulating N-cadherin–based cell–cell adhesion. Paxillin was found to be required primarily for the recruitment of Fak to robust focal adhesions. Our results suggest that at least some signals involving Fak are linked to a mechanism down-regulating Rac1 activity at the cell periphery, which appears to be important for the formation of N-cadherin–based adhesions in motile cells. Our analyses simultaneously exemplified the essential role of Fak in the maintenance of cell–cell adhesions in collective cell migration, a type of migration occurring in embryonic development and carcinoma invasion.

RhoA activity is transiently inhibited at the initial phase of integrin engagement, when Cdc42- and/or Rac1-mediated membrane spreading and ruffling predominantly occur. Paxillin, an integrin-assembly protein, has four major tyrosine phosphorylation sites, and the phosphorylation of Tyr31 and Tyr118 correlates with cell adhesion and migration. We found that mutation of Tyr31/118 caused enhanced activation of RhoA and premature formation of stress fibers with substantial loss of efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells. These phenotypes were similar to those induced by RhoA(G14V) in parental cells, and could be abolished by expression of RhoA(T19N), Rac1(G12V), or p190RhoGAP in the mutant-expressing cells. Phosphorylated Tyr31/118 was found to bind to two src homology (SH)2 domains of p120RasGAP, with coprecipitation of endogenous paxillin with p120RasGAP. p190RhoGAP is known to be a major intracellular binding partner for the p120RasGAP SH2 domains. We found that Tyr31/118-phosphorylated paxillin competes with p190RhoGAP for binding to p120RasGAP, and provides evidence that p190RhoGAP freed from p120RasGAP efficiently suppresses RhoA activity during cell adhesion. We conclude that Tyr31/118-phosphorylated paxillin serves as a template for the localized suppression of RhoA activity and is necessary for efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells.

Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. Retrotransposon insertion was found to produce an N-terminally truncated form (Δγ1) of the B56γ1 regulatory subunit isoform of protein phosphatase (PP) 2A in BL6 cells, but not in F10 cells. We found an interaction of paxillin with PP2A C and B56γ subunits by co-immunoprecipitation. B56γ1 co-localized with paxillin at focal adhesions, suggesting a role for this isoform in targeting PP2A to paxillin. In this regard, Δγ1 behaved similarly to B56γ1. However, the Δγ1-containing PP2A heterotrimer was insufficient for the dephosphorylation of paxillin. Transfection with Δγ1 enhanced paxillin phosphorylation on serine residues and recruitment into focal adhesions, and cell spreading with an actin network. In addition, Δγ1 rendered F10 cells as highly metastatic as BL6 cells. These results suggest that mutations in PP2A regulatory subunits may cause malignant progression.