Hyaluronan-CD44 interaction with neural wiskott-aldrich syndrome protein (N-WASP) promotes actin polymerization and ErbB2 activation leading to β-catenin nuclear translocation, transcriptional upregulation and cell migration in ovarian tumor cells

2527 In this study we have investigated the interaction of hyaluronan (HA) and CD44 with the Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) in regulating actin polymerization and ErbB2/β-catenin signaling in human ovarian tumor cells (SK-OV-3.ipl cells). Biochemical and immunological analyses indicate that N-WASP is expressed in SK-OV-3.ipl cells and that the binding of HA stimulates N-WASP association with CD44 and Arp2/Arp3 leading to filamentous actin (F-actin) formation and ovarian tumor cell migration. In addition, HA binding promotes CD44-N-WASP association with ErbB2 and activates ErbB2 kinase activity which, in turn, increases phosphorylation of the cytoskeletal protein, β-catenin. Subsequently, phosphorylated β-catenin is transported into the nucleus leading to β-catenin-mediated TCF/LEF-transcriptional co-activation. Since HA-induced β-catenin phosphorylation, nuclear translocation and TCF/LEF transcriptional activation is effectively blocked by the ErbB2 inhibitor, AG825, we conclude that HA/CD44-N-WASP-associated ErbB2 activation is required for β-catenin-mediated signaling events. In assessing the regulatory role of N-WASP in HA-CD44 signaling and ErbB2 activation, we have determined that overexpression of a dominant-negative N-WASP mutant protein [by transfecting cells with N-WASP’s VCA (verpolin homology, cofilin homology, and acidic domain) fragment cDNA] not only blocks HA/CD44-induced N-WASP-Arp2/3 complex formation, but also inhibits actin polymerization/F-actin formation and tumor cell migration. Transfection of SK-OV-3.ipl cells with N-WASP-VCAcDNA also significantly reduces HA-induced ErbB2 recruitment to CD44, diminishes β-catenin phosphorylation/nuclear translocation and abrogates TCF/LEF-specific transcriptional co-activation by β-catenin. Taken together, our findings strongly suggest that N-WASP plays a pivotal role in regulating HA-mediated CD44-ErbB2 interaction, β-catenin signaling and actin cytoskeleton functions that are required for tumor-specific behaviors (e.g. transcriptional upregulation and tumor cell migration) and ovarian cancer progression.