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.

The serine/threonine kinase HPK1 is a member of the germinal center kinase (GCK) family that has been implicated in the regulation of MAP kinase pathways. Here, we demonstrate the involvement of HPK1 in antigen receptor signaling. Engagement of the TCR or the BCR resulted in a marked induction of HPK1 catalytic activity. Subsequent analysis revealed that Src and Syk/ZAP-70 tyrosine kinases and the adaptor proteins LAT, SLP-76, BLNK, Grb2, and Grap are involved in HPK1 activation. Overexpression of HPK1 inhibited TCR activation of AP-1 and ERK2, whereas the kinase-inactive mutant of HPK1 potentiated these responses. Neither form of HPK1 affected PMA or v-Ras activation of AP-1 and ERK2. Thus, HPK1 is a negative regulator of the TCR-induced AP-1 response pathway.

SummaryMitogen-activated protein (MAP) kinase family members, including extracellular signal-regulated kinase (ERK), c-Jun NH 2 -terminal kinase ( JNK), and p38 MAP kinase, have been implicated in coupling the B cell antigen receptor (BCR) to transcriptional responses. However, the mechanisms that lead to the activation of these MAP kinase family members have been poorly elucidated. Here we demonstrate that the BCR-induced ERK activation is reduced by loss of Grb2 or expression of a dominant-negative form of Ras, RasN17, whereas this response is not affected by loss of Shc. The inhibition of the ERK response was also observed in phospholipase C (PLC)-␥ 2-deficient DT40 B cells, and expression of RasN17 in the PLC-␥ 2-deficient cells completely abrogated the ERK activation. The PLC-␥ 2 dependency of ERK activation was most likely due to protein kinase C (PKC) activation rather than calcium mobilization, since loss of inositol 1,4,5-trisphosphate receptors did not affect ERK activation. Similar to cooperation of Ras with PKC activation in ERK response, both PLC-␥ 2-dependent signal and GTPase are required for BCR-induced JNK and p38 responses. JNK response is dependent on Rac1 and calcium mobilization, whereas p38 response requires Rac1 and PKC activation.