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.

In most human breast cancer cell lines, there is a direct correlation between their in vivo invasive phenotypes and in vitro invasion activities. Here, we found that ADP-ribosylation factor 6 (Arf6) is localized at the invadopodia of the cultured breast cancer cells MDA-MB-231, and its suppression by a small-interfering RNA duplex effectively blocks the invasive activities of the cells, such as invadopodia formation, localized matrix degradation and Matrigel transmigration but not the cell-adhesion activity. We also found that the GTP hydrolysis-defective mutant Arf6(Q67L) and the GTP-binding defective mutant Arf6(T27N) both blocked these invasive activities but not cell adhesion, suggesting the necessity of continued activation and cycling of the Arf6 GTPase cycle in invasion. Among the different human breast cancer cell lines that we examined, cell lines with high invasive activities expressed higher amounts of Arf6 protein than those in weakly invasive and noninvasive cell lines, although no notable correlation was found between Arf6 mRNA expression levels and invasive activities. Moreover, Matrigel-transmigration activity of all of these invasive cells was blocked effectively by an Arf6 small-interfering RNA duplex. Hence, Arf6 appears to be an integral component of breast cancer invasive activities, and we propose that Arf6 and the intracellular machinery regulating Arf6 during invasion should be considered as therapeutic targets for the prevention of breast cancer invasion.

Invasive potentials of carcinomas greatly contribute to their metastasis, which is a major threat in most cancers. We have recently shown that Arf6 plays a pivotal role in breast cancer invasive activities and identified AMAP1 as an effector of GTP-Arf6 in invasion. Expression of AMAP1 correlates well with invasive phenotypes of primary tumors of the human breast. We also have shown that AMAP1 functions by forming a trimeric protein complex with cortactin and paxillin. In this complex, AMAP1 binds to the src homology 3 (SH3) domain of cortactin via its proline-rich peptide, SKKRPPPPPPGHKRT. SH3 domains are known to bind generally to the proline-rich ligands with a one-to-one stoichiometry. We found that AMAP1͞cortactin binding is very atypical in its stoichiometry and interface structure, in which one AMAP1 proline-rich peptide binds to two cortactin SH3 domains simultaneously. We made a cell-permeable peptide derived from the AMAP1 peptide, and we show that this peptide specifically blocks AMAP1͞cortactin binding, but not other canonical SH3͞proline bindings, and effectively inhibits breast cancer invasion and metastasis. Moreover, this peptide was found to block invasion of other types of cancers, such as glioblastomas and lung carcinomas. We also found that a small-molecule compound, UCS15A, which was previously judged as a weak inhibitor against canonical SH3͞proline bindings, effectively inhibits AMAP1͞cortactin binding and breast cancer invasion and metastasis. Together with fine structural analysis, we propose that the AMAP1͞cortactin complex, which is not detected in normal mammary epithelial cells, is an excellent drug target for cancer therapeutics.cell-permeable peptide ͉ molecular target ͉ NMR ͉ small-molecule inhibitor ͉ x-ray structure

In neutrophils, superoxide anion production generally accompanies chemotaxis and functions in killing invading pathogens. The GIT2 GTPase-activating protein binds to the guanine nucleotide-exchange factor alphaPIX. Here we show that GIT2 was necessary for directional chemotaxis and for the suppression of superoxide production in G protein-coupled receptor-stimulated neutrophils. GIT2 was also necessary for the orientation of superoxide production toward chemoattractant sources. GIT2 suppressed the activity of ADP ribosylation factor 1 and was a component of the Gbetagamma subunit-mediated direction-sensing machinery 'downstream' of G protein-coupled receptor signaling. This study establishes a function for GIT2 in linking chemotaxis and superoxide production in neutrophils and shows that loss of GIT2 in vivo leads to an immunodeficient state.