Expression of AMAP1, an ArfGAP, provides novel targets to inhibit breast cancer invasive activities

Abstract

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.

Regulation of actin cytoskeleton dynamics by Arf-family GTPases

“…Interestingly, formation of these structures requires the presence of ASAP1 but not its GAP activity [61], suggesting that it functions primarily as a scaffold in this context. Moreover, disruption of the ASAP-paxillin-cortactin complex inhibits the metastasis of murine breast tumor cells in an animal model [62]. These findings highlight the fact that ASAPs have both GAP-dependent and GAP-independent functions.…”

“…Paxillin and cortactin are key components of invadopodia, and ASAP1 (AMAP1) binds both proteins in a tripartite complex. Knockdown of ASAP1 (AMAP1) or disruption of the ASAPpaxillin-cortactin complex prevents the formation of invadopodia or related structures called podosomes [61,62]. Interestingly, formation of these structures requires the presence of ASAP1 but not its GAP activity [61], suggesting that it functions primarily as a scaffold in this context.…”

Autonomous regulation of osteosarcoma cell invasiveness by Wnt5a/Ror2 signaling

“…Invasive tumor cells form actin-rich protrusions, called invadopodia, that degrade and extend into ECMs (Bowden et al, 1999;Onodera et al, 2005;Weaver, 2006;Cortesio et al, 2008). To evaluate the ability of invadopodia formation during in vitro invasion of SaOS-2 cells, Alexa-labeled gelatin (red) was utilized as ECM (see Materials and Methods).…”

“…Fluorescent images were obtained using a laser scanning confocal imaging system (LSM510, Carl Zeiss, Go¨ttingen, Germany). For ECM degradation assay, glass-bottom dishes were coated with gelatin (Type A with 300 Bloom; Sigma) conjugated with Alexa 594 (Invitrogen) (Alexa-gelatin) and then treated with 0.5% glutaraldehyde, as described earlier (Bowden et al, 1999;Onodera et al, 2005). Cells were cultured on these glassbottom dishes in DMEM, fixed and stained with anti-cortactin antibody or Alexa 488-phalloidin.…”

Arf GTPase-activating Protein ASAP1 Interacts with Rab11 Effector FIP3 and Regulates Pericentrosomal Localization of Transferrin Receptor–positive Recycling Endosome

“…Moreover, Nuf, the Drosophila orthologue of mammalian FIP3 and FIP4, associates with the pericentrosomal recycling endosomes during cellularization, and a nuf mutant disrupts recruitment of actin to the cortical cleavage furrow . ASAP1 is cytoplasmic and in cell adhesion sites including focal adhesions, invadopodia, and podosomes in fibroblasts and invasive epithelial tumor cells Onodera et al, 2005;Bharti et al, 2007). The interaction of ASAP1 with FIP3 described above led us to consider the possibility that the two proteins operate together in the endocytic recycling compartment or cell adhesion sites.…”