Hyaluronan-CD44 Interaction with Neural Wiskott-Aldrich Syndrome Protein (N-WASP) Promotes Actin Polymerization and ErbB2 Activation Leading to β-Catenin Nuclear Translocation, Transcriptional Up-regulation, and Cell Migration in Ovarian Tumor Cells*

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 formation and ovarian tumor cell migration. In addition, HA binding promotes CD44-N-WASP association with ErbB2 and activates ErbB2 kinase activity that 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. Because 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. Transfection of SK-OV-3.ipl cells with N-WASP-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 assembly and tumor cell migration. Overexpression of the N-WASP-VCA domain 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 and ovarian cancer progression.

The Wiskott-Aldrich syndrome protein (WASP) family of proteins includes two types of hematopoietic WASP proteins, the ubiquitously expressed neural WASP (N-WASP) (25,26) and three WASP family verproline-homology proteins (WAVEs) (27,28). These proteins integrate upstream signaling events with changes in the actin cytoskeleton (29,30). WASP family proteins such as N-WASP contain a number of functional domains and motifs known to interact with both the cytoskeleton and various signaling complexes. These domains include a verprolin homology, a cofilin homology, and an acidic domain (VCA) at the C terminus; a proline-rich region in the center of the molecule, a GTPase binding domain/CRIB motif, a WASP-homology 1 (WH1) domain, and a basic region at the N terminus (25,26,28). Some of these motifs are involved in N-WASP interaction with specific proteins, such as Cdc42 (32), WASP-interacting protein (33), Src (34), Nck (35), Grb2/Ash (34), phospholipase C␥ (34), and phosphatidylinositol 3-kinase (34). In particular, the verprolin homology (V) binds to actin directly, whereas the cofilin homology-acidic (CA) region interacts with an actin-related protein (Arp) 2/3 complex (a 7-component protein complex), initiating the nucleation reaction of actin filaments in vitro and inducing actin polymerization/filamentous actin (F-actin) formation in vivo (26 -28, 36). Thus, these findings suggest that N-WASP not only serves as a scaffolding protein (mediating multiprotein Arp2/3 complex aggregation) but also directly participates in F-actin assembly and cytoskeleton reorganization. The VCA domain also interacts with the middle region of the N-WASP, generating an autoinhibitory configuration and preventing Arp2/3 association. Subsequently, this VCA-mediated autoinhibitory process blocks actin polymerization (37,38). However, a number of signaling regulators (e.g. Cdc42, phosphatidylinositol bisphosphate (39), Grb2/Ash (40), Nck (41), WISH (42), and Src family tyrosine kinases (43)) can promote the ability of Arp2/3 to nucleate actin filaments by releasing the autoinhibitory conformation of inactive N-WASP. Thus, the C-terminal VCA sequence of N-WASP is considered as one of the important regulatory domains of N-WASP in regulating F-actin formation and cytoskeleton function required for a variety of cellular functions, including filopodia/lamellipodia formation (27,37) and vesicle movement (44 -47).
The HER2 oncogene (also called ErbB2 or neu) encodes a 185-kDa membrane protein (p185 HER2 ) that contains a single transmembrane spanning region and a tyrosine kinase-associated cytoplasmic domain (48,49). Overexpression or amplification of HER2 oncogenes appears to correlate with poor survival rates of many known cancers, including ovarian cancer (50,51). However, the cellular and molecular mechanisms by which ErbB2 enhances the growth and survival of ovarian cancer cells are not completely understood. Previously, we have determined that CD44 and ErbB2 are physically linked to each other via interchain disulfide bonds (11) or signaling linker molecules (19) in human ovarian tumor cells (SK-OV-3.ipl cell line). The binding of HA to a CD44-asociated ErbB2 complex stimulates ErbB2 kinase activity and promotes Ras-mediated stimulation of a downstream kinase cascade, which includes the Raf-1/MEK/MAPK(ERK) pathway leading to tumor cell growth and migration (11,19).
The cytoskeletal protein ␤-catenin is a multifunctional protein known to play a key role in cell-cell adhesion and the Wnt signaling pathway (52)(53)(54)(55). In normal cells, ␤-catenin is associated with E-cadherin and mediates cell-cell adhesion (52)(53)(54)(55). To maintain homeostasis, the ␤-catenin/E-cadherin association is regulated in part by serine/tyrosine phosphorylation on specific residues. Serine phosphorylation in the N terminus of ␤-catenin targets it for proteosomal degradation, whereas tyrosine phosphorylation in the C terminus of ␤-catenin influences its interaction with E-cadherin (52)(53)(54)(55). In the Wnt signaling pathway, glycogen synthase kinase-3␤ (GSK-3␤, a kinase that normally phosphorylates excess ␤-catenin and causes its ubiquitination and degradation) is inactivated when soluble Wnt protein binds to the frizzled receptor (52)(53)(54)(55). ␤-Catenin then accumulates in the cytoplasm and translocates to the nucleus where it can interact with members of the TCF/LEF-enhancing factor family as well as other transcription factors (56,57).
␤-Catenin also contributes to oncogenesis of some tumors (52,55). In cancer cells, an uncomplexed phosphorylated form of ␤-catenin also accumulates in the cytoplasm prior to translocation into the nucleus where it binds to the TCF/LEF family of transcription factors and activates transcription of downstream genes such as cyclin D1 and c-myc (58,75). ␤-Catenin has been shown to be linked to ErbB2 tyrosine kinase or other receptor tyrosine kinases (59 -63). In particular, ␤-catenin phosphorylation by ErbB2 tyrosine kinase destabilizes the E-cadherin-catenin complexes leading to a decrease of cell adhesion and a shifting of ␤-catenin into the oncogenic pathway (59 -64). Until the present time, the mechanism by which HA/CD44 activation of ErbB2 modulates ␤-catenin-mediated signal transduction and oncogenesis in ovarian tumor cells has not been addressed.
In this study, we have discovered a new mechanistic role for HA/CD44-mediated N-WASP signaling and ErbB2 activation that is centered on F-actin formation and ␤-catenin phosphorylation/nuclear translocation. These events then promote cell migration and transcriptional up-regulation in ovarian tumor cells.

MATERIALS AND METHODS
Cell Culture-The SK-OV-3.ipl cell line was established from ascites that developed in a nu/nu mouse given an intraperitoneal injection of SK-OV-3 human ovarian carcinoma cell line (obtained from the American Type Culture Collection) as described previously (11,13,19,22). Cells were grown in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 10% fetal bovine serum. Cells were routinely serum-starved (and therefore deprived of serum HA) before adding HA.
DNA Constructs-A construct for expression of the C-terminal part of rat N-WASP containing the verprolin homology, cofilin, and acidic domains (VCA, amino acids 391-501, N-WASP-VCA) in mammalian cells (kindly provided by Britta Qualmann and Regis B. Kelly, University of California, San Francisco) was generated by PCR with primer 5Ј-CCGCTCG-AGGGTGACCATCAAGTTCCAG-3Ј and 5Ј-CGGAATTCA-GTCTTCCCACTCATCATC-3Ј using rat N-WASPcDNA as a template. The PCR product was cloned into the XhoI-EcoRI sites of a derivative of the pEGFP-C1 vector (Clontech), in which green fluorescent protein was replaced by the hemagglutinin tag.
Cell Transfection-To establish a transient expression system, SK-OV-3.ipl.cells were transfected with various plasmid DNAs (e.g. hemagglutinin-tagged N-WASP-VCA or vector alone) using Lipofectamine 2000 methods (Invitrogen). Briefly, cells were plated at a density of 2 ϫ 10 6 cells per 100-mm dish and transfected with 25 g/dish plasmid cDNA using Lipofectamine 2000. Transfected cells were grown in the culture medium for at least 24 -48 h. Various transfectants were then analyzed for their protein expression and functional properties as described above.
RNA Oligonucleotides-The siRNA sequence targeting human ␤-catenin (from mRNA sequence, GenBank TM accession number AJ251595) corresponds to the coding region relative to the first nucleotide of the start codon. Target sequences were selected using the software developed by Ambion Inc., UK. As recommended by Ambion, ␤-catenin-specific targeted regions were selected beginning 50 -100 nucleotides downstream from the start codon. Sequences close to 50% G/C content were chosen. Specifically, the ␤-catenin target sequence (5Ј-AGCUGAUAUUGAUGGACAG-3Ј) and scrambled sequences (5Ј-AAGUGCGUGAAGUAUUCGG-3Ј) were used. ␤-Catenin-specific target sequences were then aligned to the human genome data base in a BLAST search to eliminate sequences with significant homology to other genes. Sense and antisense oligonucleotides were provided by the Biomolecular Research Unit, University of California, San Francisco. For construction of the siRNA, a transcription-based kit from Ambion was used (Silencer TM siRNA construction kit). SK-OV-3.ipl cells were then transfected with siRNA using siPORT Lipid as transfection reagent (Silencer TM siRNA transfection kit; Ambion, TX) according to the protocol provided by Ambion. Cells were incubated with 50 pmol of ␤-catenin siRNA or 50 pmol of siRNA containing scrambled sequences or no siRNA for at least 48 h before biochemical experiments and/or functional assays were conducted as described below.
Preparations of Cell Lysate, Cytoplasmic and Nucleus Fractions-SK-OV-3.ipl cells (untransfected or transfected with N-WASP-VCAcDNA or vector alone) were serum-starved for 24 h followed by incubation with 50 g/ml HA (or pretreated with anti-CD44 antibody or 25 M AG825 for 1 h followed by HA treatment or no HA addition) for various time intervals (e.g. 0, 5, or 30 min) at 37°C. Cells were then lysed in a lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl 2 , 0.5% Nonidet P-40, 0.2 mM Na 3 VO 4 , 0.2 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 5 g/ml aprotinin). The lysate was then homogenized by 30 strokes in a tight fitting Dounce homogenizer. Both cytoplasmic and nuclear fractions were prepared using the extraction kit from Active Motif (Carlsbad, CA) with some modifications. Briefly, the cell homogenate was centrifuged at 1,500 ϫ g for 5 min to sediment the nuclei. The supernatant was then recentrifuged at 15,000 ϫ g for 5 min, and the resulting supernatants form "the cytosolic fraction." The nuclear pellet was washed three times and resuspended in the same buffer containing 0.5 M NaCl to extract nuclear proteins. The extracted material was centrifuged at 15,000 ϫ g for 10 min, and the resulting supernatant was termed "the nuclear fraction" (65).
Immunoblotting and Immunoprecipitation Techniques-The cell lysate or the cytosolic fraction isolated from SK-OV-3.ipl cells ((untransfected or transfected with N-WASP-VCAcDNA or vector alone in the presence of 50 g/ml HA (or pretreated with anti-CD44 antibody or 25 M AG825 for 1 h followed by HA treatment or no HA addition) for various time intervals (e.g. 0, 5,or 30 min) at 37°C) was immunoblotted using various immunoreagents (e.g. mouse anti-N-WASP antibody (5 g/ml) or rabbit anti-ErbB2 (5 g/ml) or rabbit anti-phospho-ErbB2 (5 g/ml)).
In some experiments, the cytosolic fraction isolated from SK-OV-3.ipl cells ((untransfected or transfected with N-WASP-VCAcDNA or vector alone in the presence of 50 g/ml HA (or pretreated with anti-CD44 antibody or 25 M AG825 for 1 h followed by HA treatment or no HA addition) for various time intervals (e.g. 0, 5, or 30 min) at 37°C) was immunoprecipitated with mouse anti-␤-catenin antibody-conjugated beads followed by immunoblotting with mouse anti-phosphotyrosine antibody or mouse anti-␤-catenin antibody. The cytosolic fraction of cells (transfected with N-WASP-VCAcDNA or vector alone) was also subjected to immunoprecipitation using mouse anti-N-WASP antibody-conjugated beads followed by immunoblotting with goat anti-Arp2 antibody or rabbit anti-Arp3 antibody. These samples were then incubated with horseradish peroxidase-conjugated secondary antibodies (e.g. rabbit anti-goat IgG, goat anti-rabbit IgG, or goat anti-mouse IgG (1:10,000 dilution)) at room temperature for 1 h. The blots were then developed using ECL reagent (Amersham Biosciences) according to the manufacturer's protocols.
In some cases, the nuclear fractions of SK-OV-3.ipl cells (untransfected, transfected with N-WASP-VCAcDNA, or vector alone and incubated with 50 g/ml HA (or no HA) for various time intervals (e.g. 0, 5, or 30 min) at 37°C) were processed for immunoprecipitation using mouse anti-␤-catenin antibody-conjugated beads followed by immunoblotting with mouse anti-phosphotyrosine antibody or mouse anti-␤-catenin antibody, respectively. In some experiments, anti-␤-cateninmediated immunoblot of cell lysates (isolated from ␤-catenin siRNA-scrambled sequence-treated cells or ␤-catenin siRNAtreated cells) was also carried out as described above.
FACS Quantitation of F-actin-For measuring total F-actin levels, FACS analysis was used to quantitate FITC-phalloidin staining in fixed cell populations (67). SK-OV-3.ipl cells ((untransfected or transfected with N-WASP-VCAcDNA or vector alone were grown in 6-cm plates in the presence of 50 g/ml HA (or pretreated with anti-CD44 antibody or 20 g/ml cytochalasin D for 1 h followed by HA treatment or no HA addition) for various time intervals (e.g. 0, 5, or 30 min) at 37°C). First, cells were trypsinized, washed sequentially with Dulbecco's modified Eagle's medium, 1% horse serum/PBS, and then resuspended in 0.5 ml of PBS. Cells were then fixed by the addition of 0.5 ml of 8% paraformaldehyde in PBS for 10 min at room temperature, washed in PBS, permeabilized in 1 ml of 0.3% Triton X-100 in PBS. Subsequently, permeabilized cells were labeled with 33 nM FITC-phalloidin to measure F-actin content in 5% fetal calf serum in PBS for 45 min at 37°C. Finally, FITC-phalloidin-labeled cells were analyzed using a FACSCalibur TM . Mean F-actin content was evaluated and expressed as a percentage of that of untransfected cells (with no HA treatment) or vector-transfected cells (with no HA treatment) from the same experiment.
Immunofluorescence Staining-SK-OV-3.ipl cells (transfected with N-WASP-VCAcDNA or vector alone) were incubated with HA (50 g/ml) at 37°C for various time intervals (e.g. 0, 10, 30, or 60 min) (or pretreated with various agents (e.g. anti-CD44 antibody or 25 M AG825 or 20 g/ml cytochalasin D) followed by adding HA (50 g/ml) or no HA). These cells were then fixed with 2% paraformaldehyde. Subsequently, these cells were rendered permeable by ethanol treatment followed by incubating with FITC-conjugated anti-␤-catenin antibody followed by a monomeric cyanine nucleic acid staining, Topro-3 (a marker for nucleus) or stained with Texas Redconjugated phalloidin alone (to locate F-actin). To detect nonspecific antibody binding, Topro-3-labeled cells were incubated with FITC-conjugated normal IgG, respectively. No labeling was observed in such control samples. These fluorescence-labeled samples were then examined with a confocal laser scanning microscope.
Luciferase Reporter Assays-Transactivation assays were conducted with SK-OV-3.ipl cells (untreated or pretreated with anti-CD44 antibody, 25 M AG825, or 20 g/ml cytochalasin D or transfected with N-WASP-VCAcDNA (or vector alone) or treated with 50 pmol of scrambled siRNA or 50 pmol of ␤-catenin siRNA). Following 24 h of HA (50 g/ml) treatment (or no HA treatment), these cells (or transfectants) grown in 35-mm diameter dishes were transfected with 1.0 g of a plasmid containing a multimeric TCF/LEF-1 consensus-binding sequence driving the luciferase reporter gene (pTop-flash) or a mutant inactive form (pFop-flash) (kindly provided by Robert Nissenson, University of California and Veterans Affairs Medical Center, San Francisco). pTop-flash, but not pFop-flash, is responsive to co-activation of TCF/LEF by ␤-catenin (68). Therefore, relative luciferase units were expressed as the amount of pTopflash-derived luciferase activity divided by the amount from control pFop-flash. A plasmid encoding ␤-galactosidase (1.0 g) was also co-transfected to enable normalization for transfection efficiency. After 24 h, expression of the reporter (luciferase) and the control (␤-galactosidase) genes was determined using enzyme assay systems from Promega as per the manufacturer's instructions.
Tumor Cell Migration Assays-Twenty four transwell units were used for monitoring in vitro cell migration as described previously (12, 13, 18 -24). Specifically, the 5-m porosity polycarbonate filters (CoStar Corp., Cambridge, MA) were used for the cell migration assay. SK-OV-3.ipl cells (untransfected, transfected with N-WASP-VCAcDNA, or vector alone in the presence or absence of rat anti-CD44 antibody (5 g/ml), 20 g/ml cytochalasin D, or 50 pmol of scrambled siRNA or 50 pmol of ␤-catenin siRNA) were placed in the upper chamber of the transwell unit. The medium containing 50 g/ml HA or no HA was placed in the lower chamber of the transwell unit. After 18 h of incubation at 37°C in a humidified 95% air, 5% CO 2 atmosphere, cells on the upper side of the filter were removed by wiping with a cotton swab. Cell migration processes were determined by measuring the cells that migrated to the lower side of the polycarbonate filters by standard cell number counting methods. The CD44-specific cell migration was determined by subtracting nonspecific cell migration (i.e. cells migrate to the lower chamber in the presence of anti-CD44 antibody treatment). Each assay was performed in triplicate and repeated at least five times. The number of ovarian tumor cell migration in untreated SK-OV-3.ipl cells (control) or in SK-OV-3.ipl cells treated with no HA (or vector-transfected cells treated with no HA) is designated as 100%. All data were analyzed statistically by Student's t test, and statistical significance was set at p Ͻ 0.01.

HA/CD44-activated N-WASP Signaling and F-actin Formation in Ovarian Tumor Cells
The basic cellular and molecular processes underlying ovarian tumor cell invasion and metastasis are poorly understood at the present time. It is quite clear, however, that transmembrane interactions between receptor(s) and actin cytoskeleton are involved in tumor cell motility, invasion of surrounding tissue, and metastasis (69,70). The details of these interactions in ovarian tumor cell movement and infiltration of surrounding tissue remain largely unknown.
A number of studies have been aimed at identifying the specific molecules expressed by tumor cells that correlate with actin-based tumor motility and metastatic behavior. One possible candidate in this area is N-WASP, which has been shown to play an important role in signal transduction and F-actin formation . Using a specific anti-N-WASP-mediated immunoblot technique, we have found that significant amounts of N-WASP (molecular mass ϳ55 kDa) are expressed in SK-OV-3.ipl cells (Fig. 1A, lane 2). We believe that the N-WASP protein detected by anti-N-WASP-mediated immunoblot is specific because no protein is detected in those cells incubated with preimmune rabbit IgG (Fig. 1A, lane 1).
In addition, we have addressed the question of whether there is a physical linkage between CD44 and N-WASP in ovarian tumor cells (SK-OV-3.ipl cell line). To this end we first carried out anti-CD44-mediated immunoprecipitation followed by anti-N-WASP immunoblot ( In response to external signals, the N-WASP often interacts with Arp2/3 complex and G-actin to stimulate actin polymerization in cells (26 -28, 36). In this study we have demonstrated that HA induces recruitment of Arp2 ( Using a cell-free system and pyrene-labeled actin (a fluorescent derivative of actin known to display higher fluorescence intensity when actin is assembled into filaments), we have observed that N-WASP (isolated from SK-OV-3ipl cells) markedly activates Arp2/3 complex-induced actin polymerization (Fig. 1C, line a). In contrast, the level of Arp2/3-mediated actin polymerization is relatively low in these samples treated with N-WASP alone (or Arp2/3 alone or the reaction buffer only) (Fig. 1C, lines b and c). These findings suggest that N-WASP is involved in Arp2/3-mediated actin assembly. In addition, our flow cytometry analyses of FITC-phalloidin-labeled SK-OV-3.ipl cells (an assay that selectively detect the expression of polymerized F-actin) indicate that HA-mediated actin polymerization is enhanced by ϳ295% in SK-OV-3.ipl cells (Table 1). In contrast, F-actin formation is greatly reduced in cells pretreated with anti-CD44 antibody followed by HA treatment (Table 1) or with no HA treatment ( Table 1). Treatment of cells with cytochalasin D (an inhibitor known to impair F-actin polymerization) also blocks HA-mediated F-actin formation ( Table 1). These findings suggest that HA-CD44 interaction promotes de novo actin polymerization in SK-OV-3.ipl cells.
Furthermore, using in vitro migration assays, we have observed that SK-OV-3.ipl cells undergo active cell migration (enhanced by ϳ285-290%) during HA treatment (Table 2). However, pretreatment of SK-OV-3.ipl cells with certain reagents, such as anti-CD44 antibody ( Table 2, part A) and cytochalasin D (an inhibitor known to impair F-actin polymerization) ( Table 2, part B), causes a significant inhibition of HA/CD44-mediated tumor cell migration (Table 2). Together, these findings indicate that HA/CD44-induced F-actin formation is required for ovarian tumor cell migration.

HA-mediated CD44-N-WASP Interaction with ErbB2 in Ovarian Tumor Cells
Previous studies have shown that CD44 and ErbB2 are both structurally and functionally linked in ovarian tumor cells (11,19). To examine the possible relationship between the CD44-N-WASP complex and ErbB2 in SK-OV-3.ipl cells, we have analyzed the anti-CD44-mediated immunoprecipitates from cell lysates by immunoblotting with anti-ErbB2 ( Fig. 2A, panel  b, lane 1) or anti-N-WASP ( Fig. 2A, panel a, lane 1) antibody, respectively. Our results demonstrate that ErbB2 ( Fig. 2A,  panel b, lane 1) is complexed with both CD44 (Fig. 2A, panel c, lane 1) and N-WASP ( Fig. 2A, panel a, lane 1). Furthermore, we have observed that HA treatment of SK-OV-3.ipl cells stimulates a significant increase in the amount of ErbB2 ( Fig. 2A, panel b, lane 2) and N-WASP ( Fig. 2A, panel a, lane 2), recruited into the CD44-associated ( Fig. 2A, panel c, lane 2) signaling complex. Using the supernatant fraction following anti-CD44 immunoprecipitation, we have observed that N-WASP (immunoprecipitated with anti-N-WASP antibody) does not interact with ErbB2 (analyzed by anti-ErbB2-mediated immunoblotting) (Fig. 2B, panels a and b, lane 2) in HA-treated cells. ErbB2 (immunoprecipitated with anti-ErbB2 antibody) also fails to form a complex with N-WASP in SK-OV-3.ipl cells treated with HA (Fig. 2C, panels a and b, lane 2). These findings suggest that N-WASP becomes associated with ErbB2 when a CD44-containing multimolecular complex is formed during HA signaling (Fig. 2A, panels a-c, lane 2).
In addition, we have confirmed that HA treatment of SK-OV-3.ipl cells stimulates ErbB2 tyrosine kinase activity (Fig.  3A, panels a and b, lanes 1 and 2). Pretreatment of cells with anti-CD44 antibody (Fig. 3A, panels a and b, lane 3) or an ErbB2 inhibitor, AG825 (Fig. 3A, panels a and b, lane 4), readily inhibits HA-mediated ErbB2 kinase activity. These results suggest that HA-mediated ErbB2 activation in SK-OV-3.ipl cells is CD44-dependent and ErbB2 tyrosine kinase-sensitive. The cytoskeletal protein, ␤-catenin is known to serve as a substrate for ErbB2 tyrosine kinase (59 -63). Here we have found that a 5-min HA treatment stimulates ␤-catenin tyrosine phosphorylation in the cytosol (Fig. 3B-(I), panels a and b, lanes  1 and 2). Phosphorylated ␤-catenin then becomes translocated from the cytosol into the nuclear fraction ( Fig. 3B-(II), panels a and b, lane 2) after a 30-min HA treatment of SK-OV-3.ipl cells. When cells were pretreated with anti-CD44 or AG825 (an ErbB2 inhibitor), both HA-mediated ␤-catenin phosphorylation ( Fig. 3B-(I), panels a and b, lanes 3 and 4) and nuclear translocation ( Fig. 3B-(II), panels a and b, lanes 3 and 4) are greatly reduced. Therefore, it is likely that ␤-catenin phosphorylation and nuclear translocation are closely linked to HA/CD44-N-WASP-associated ErbB2 signaling.
There is a compelling evidence that transcriptional co-activation by ␤-catenin occurs through the binding to TCF/LEF transcription factors (56,57). Consequently, we examined the potential impact of HA/CD44-ErbB2 activation on ␤-catenin signaling (␤-catenin tyrosine phosphorylation and nuclear translocation)-mediated co-activation of TCF/LEF transcription using luciferase reporter assays. Specifically, we utilized firefly luciferase reporter plasmids containing either pTopflash (a wild-type containing TCF/LEF-binding sites for ␤-catenin) or pFop-flash (a mutant lacking TCF/LEF-binding sites for ␤-catenin), which are transiently transfected into SK-OV-3.ipl cells. With this technique HA/CD44-ErbB2 signaling-related and ␤-catenin-mediated TCF/LEF-transcriptional co-activation can be measured by the ratio of pTop-flash to pFop-flash luciferase units. Our results indicate that the level of ␤-cateninmediated TCF/LEF-transcriptional co-activation is low in cells treated with no HA (Fig. 4A, bar a) or pretreated with anti-CD44 followed by HA treatment (Fig. 4A, bar b). However, TCF/LEF transcriptional co-activation by ␤-catenin is greatly enhanced in SK-OV-3.ipl cells treated with HA (Fig. 4A, bar c). The level of ␤-catenin-mediated TCF/LEF transcriptional coactivation is greatly reduced if these cells were pretreated with AG825 followed by no HA or with HA addition (Fig. 4A, bars d  and e). These findings clearly indicate that TCF/LEF-transcriptional co-activation by ␤-catenin requires HA/CD44-activated ErbB2 tyrosine kinase in SK-OV-3.ipl cells.
Although cytochalasin D treatment significantly blocks HAmediated F-actin formation (Table 1), it does not cause a noticeable inhibition of HA-mediated phosphorylation of ␤-catenin (Fig. 3B-(I), panels a and b, lane 5). Thus, F-actin assembly appears not to be involved in the initiation of HA-CD44 interaction (so-called "inside-out signaling") in SK-OV-3.ipl cells. The fact that cytochalasin D treatment significantly abrogates HA/CD44-mediated ␤-catenin nuclear translocation ( Fig. 3B-(II), panels a and b, lane 5, and Fig. 8B-(iii), panels a-c) and ␤-catenin-TCF/LEF transcriptional co-activation (Fig. 4A,  bars f and g) suggests that the F-actin cytoskeleton (regulated  by N-WASP and Arp2/Arp3) is actively participating in HA/CD44-mediated "outside-in" signaling cascades in ovarian tumor cells.

Effects of N-WASP-VCA Overexpression on HA/CD44-N-WASP Signaling and ErbB2 Activation in SK-OV-3.ipl Cells
N-WASP Signaling-The VCA domain of N-WASP is an important region for Arp2/3 binding and actin polymerization (26 -28, 36). Overexpression of the VCA domain of N-WASP in cells (by transfecting cells with N-WASP-VCAcDNA) has been shown to inhibit N-WASP-Arp2/3 signaling and impair actin cytoskeleton-mediated cellular functions (47). In this study we have used a VCA fragment construct that was cloned into a hemagglutinin-tagged expression vector (Fig. 5A) to examine the possible involvement of N-WASP-VCA in HA/ CD44-mediated signaling. Using a cell-free system, we have confirmed that the hemagglutinin-tagged recombinant VCA fusion protein of N-WASP can promote a significant up-regulation of Arp2/3-induced actin polymerization (Fig. 5B, line a) as compared with these samples treated with VCA alone (or Arp2/3 alone or reaction buffer alone) (Fig. 5B, lines b-d). Moreover, we have demonstrated that HA is capable of promoting the recruitment of endogenous Arp2 (Fig. 5C, panel a, lanes 1 and 2) together with Arp3 (Fig. 5C, panel b, lanes 1 and 2) into a complex with N-WASP (Fig. 5C, panel c, lanes 1 and 2) in vector-transfected cells. In contrast, transfection of SK-OV-3.ipl cells with N-WASP-VCAcDNA causes a significant reduction in HA-mediated endogenous Arp2/3 (Fig. 5C, panels a and b,  lanes 3 and 4) association with N-WASP (Fig. 5C, panel c, lanes  3 and 4). These findings indicate that overexpression of the VCA domain acts as a potent competitive inhibitor for endogenous Arp2/3 binding to N-WASP in SK-OV-3.ipl cells.
Furthermore, we have examined both F-actin formation and tumor cell migration in vector-transfected or N-WASP-VCAcDNA-transfected cells treated with HA or without HA ( Staining of SK-OV-3.ipl cells with fluorescent phalloidin reveals that the assembly of both cortical actin fibrils and cell body actin fibrils occurs in vector-transfected cells following HA treatment (Fig. 6, B and C). In the majority of those cells treated with HA for 10 min, the actin filaments were present in numerous stress fibers and in a thick layer immediately beneath the plasma membrane (Fig. 6B). After 60 min of HA treatment, radiating actin filaments start to disassemble and become reorganized/aggregated at the cell margins as well as the membranous projections (Fig.  6C). It is possible that the disassembly of cell body actin fibrils and reorganization/thickening of cortical fibril during 60 min of HA treatment is involved in membrane motility and cell migration. In contrast, we have found that a small amount of F-actin fragments was randomly distributed in the cytosol and located around the cell periphery in vector-transfected cells treated with no HA (Fig. 6A) or pretreated with anti-CD44 antibody plus HA (Fig. 6, B, inset i, and C, inset i). These findings suggest thatHA-inducedF-actinformationandreorganizationareCD44dependent. Furthermore, we have noted that stress fibers were no longer apparent, and the total amount of actin was greatly reduced, and the small amount of remaining actin was primarily located at the cell periphery in N-WASP-VCAcDNA-transfected cells either treated with (Fig. 6, E and F) or untreated with HA (Fig. 6D). These observations support the notion that disruption of the CD44/N-WASP interaction affects the assembly and reorganization of both cortical actin fibrils and cell body actin fibers. Consequently, F-actin-based cytoskeletal function appears to be impaired during HA-mediated CD44 signaling.
ErbB2 Activation and ␤-Catenin Function-To assess the effects of the N-WASP-VCA domain in regulating HA/CD44-mediated ErbB2 and ␤-catenin signaling, we have transfected SK-OV-3.ipl cells with N-WASP-VCAcDNA. Our results show that these transfectants exhibit a marked inhibition of HA-mediated recruitment of N-WASP (Fig. 7A, panel a, lanes 3 and 4) and ErbB2 (Fig. 7A, panel b, lanes 3 and 4) to CD44 (Fig. 7A,  panel c, lanes 3 and 4). In contrast, we have demonstrated that HA is capable of promoting the recruitment of endogenous N-WASP (Fig. 7A, panel a, lanes 1 and 2) together with ErbB2 (Fig. 7A, panel b, lanes 1 and 2) into a complex with CD44 (Fig.  7A, panel c, lanes 1 and 2) in vector-transfected cells. These findings suggest that the N-WASP containing the VCA domain functions as a strong dominant-negative mutant for blocking N-WASP and ErbB2 accumulation into HA-induced CD44 signaling complexes.
Using immunofluorescence staining and confocal analyses, we have confirmed that ␤-catenin is primarily distributed in the cytosol of vector-transfected cells in the absence of HA (Fig.  8A). Very low levels of this molecule are detected in the nucleus (as indicated by Topro-3 nuclear staining) in these vectortransfected cells without any HA treatment (Fig. 8A). However, 30 min after HA treatment, ␤-catenin is translocated into the nucleus (Fig. 8B, inset i). We have also noted that overexpression of N-WASP-VCA by transfecting SK-OV-3.ipl with N-WASP-VCAcDNA (Fig. 8, C and D) significantly inhibits HA-mediated ␤-catenin nuclear translocation in these transfectants. The fact that treatment of vector-transfected cells with AG825 (an ErbB2 inhibitor) (Fig. 8B, inset ii) or cytochalasin D (a blocker for actin polymerization) (Fig. 8B, inset iii) effectively blocks HA-induced ␤-catenin nuclear localization further confirms the direct involvement of both ErbB2 tyrosine kinase and actin cytoskeleton in HA-mediated ␤-catenin nuclear localization.
In addition, our results show that ␤-catenin-associated TCF/ LEF transcriptional co-activation is greatly stimulated in vector-transfected cells treated with HA (Table 3) as compared with vector-transfected cells not treated with HA ( Table 3). Transfection of SK-OV-3.ipl cells with N-WASP-VCAcDNA significantly inhibits HA-activated ␤-catenin-associated TCF/ LEF-transcriptional co-activation (Table 3). Together, these findings demonstrate that the VCA domain of N-WASP is closely involved in HA/CD44-mediated recruitment/activation of ErbB2 kinase and ␤-catenin signaling-related nuclear translocation leading to TCF/LEF transcriptional co-activation in ovarian tumor cells.
To address whether ␤-catenin-mediated TCF/LEF transcriptional co-activation plays a role in regulating cell migration, we have used a ␤-catenin siRNA technique to down-regulate ␤-catenin expression (Fig. 4B-(II), panels a and b, lanes 1  and 2) and inhibit ␤-catenin-mediated TCF/LEF transcriptional co-activation in cells treated with HA ( Fig. 4B-(I), bars a-d). Our results indicate that reduction of ␤-catenin expression and ␤-catenin-mediated TCF/LEF transcriptional co-activation abrogates HA-mediated ovarian tumor cell migration ( Table 2, part D). These findings suggest that ␤-catenin signaling is closely coupled with HA-mediated cell migration. Identification of the ␤-catenin-mediated TCF/LEF targets genes encoding cytoskeletal proteins is under investigation in our laboratory.

DISCUSSION
Ovarian cancer has the highest mortality rate among all gynecological malignancies. HA, a glycosaminoglycan, is present as a loose pericellular layer that coats the mesothelium required for both protection and lubrication within this body cavity (3,4). Overexpression of HA has been found to be associated with ovarian tumor progression (3,4). HA binds to specific tumor cell-surface receptors such as CD44, which is present in at least 94% of ovarian tumor cells (6,7). HA-CD44 binding stimulates oncogenic signaling and ovarian tumor cellspecific properties (3, 6 -9). Thus, it has been suggested that the interaction between the HA pericellular coat of mesothelial cells and CD44 on the surface of tumor cells is one of the important steps in the peritoneal spread of ovarian cancer.
In the search for HA/CD44-linked cytoskeletal regulators that correlate with metastatic behavior, a prime candidate (named N-WASP), has been identified. N-WASP contains a number of functional domains known to interact with various signaling regulators and actin cytoskeleton (25,26,28). These include the WH1 domain, IQ region, a highly basic region, the GBD/CRIB motifs, a proline-rich region, and the VCA domain (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). Specifically, the WH1 domain binds to WASP-interacting protein family proteins that are important for N-WASP localization (25,26,28). Both the basic region and the GBD/CRIB motif contribute to N-WASP activation through phosphatidylinositol 4,5-bisphosphate/ Cdc42 binding (32,39). The proline-rich domain regulates the activation of N-WASP by interacting with Grb2/Ash (34,40), Nck (35,41), and WISH (42). The VCA domain contains a conserved tryptophan residue that is essential for triggering actin nucleation and polymerization by binding to the Arp2/3 complexes (25,26,28). Consequently, N-WASP-mediated actin assembly can be regulated by many different mechanisms and is apparently involved in the formation of membrane projections, vesicular trafficking, cell migration, and gene regulation (27, 37, 44 -47). In this study we have focused on the role of N-WASP (in particular, the VCA domain) in regulating HA/CD44-mediated actin assembly and ErbB2-regulated ␤-catenin signaling as well as specific ovarian tumor cell behaviors.
Our initial results demonstrate that N-WASP is expressed in ovarian tumor cells (SK-OV-3.ipl cells) (Fig. 1). We have also presented evidence for an HA-induced physical interaction between N-WASP and the transmembrane glycoprotein, CD44, in SK-OV-3.ipl cells (Fig. 1). Treatment of cells with HA also promotes an association of the CD44-N-WASP complex with the actin-related protein (Arp) 2/3 complex (Fig. 1) leading to the nucleation of new actin filaments in a cell-free system (Fig. 1) and in SK-OV-3.ipl cells (Table 1; Fig. 6). Inhibition of actin polymerization by cytochalasin D effectively reduces HA/CD44-mediated F-actin formation (Table 1) and tumor cell migration (Table 2). These studies indicate that HA-CD44 interaction serves as an important signal for N-WASP/Arp2/3regulated F-actin polymerization and ovarian tumor cell migration.
ErbB2 (p185 HER2 ) belongs to the EGFR tyrosine kinase subfamily (48,49). Lacking a ligand, its intrinsic kinase domain is activated upon heterodimerization with another activated member of the EGFR family resulting in the phosphorylation of specific tyrosine residues within the cytoplasmic tail (48,49). These phosphorylated residues can serve as docking sites for the recruitment of a range of proteins resulting in the activation of certain intracellular signaling pathways. ErbB2 is implicated in important biological events such as proliferation, migration, and differentiation and has been shown to be overexpressed in a number of human cancers, including ovarian cancer. This overexpression often correlates with a more aggressive disease and a poor prognosis for the patient (50,51).
Hyaluronan has been shown to constitutively regulate ErbB2 activity and to influence ErbB2 interaction with phosphatidylinositol 3-kinase/AKT signaling in tumor cells (71). Previously, we have determined that CD44 and p185 HER2 are physically linked to each other via interchain disulfide bonds in human ovarian tumor cells (SK-OV-3.ipl cell line) (11). Most importantly, HA binding to a CD44-associated p185 HER2 complex activates the p185 HER2 tyrosine kinase activity and promotes ovarian carcinoma cell growth (2). We believe that direct crosstalk between the two surface molecules, CD44 and the p185 HER2 , may be one of the most important signaling events in human ovarian carcinoma development. HA-mediated CD44 association with p185 HER2 signaling complexes is also mediated by molecular scaffolds and adaptors such as Vav2 (a Rac-specific GEF) and Grb2 (19) in ovarian tumor cells. Specifically, endogenous Vav2 and Grb2 are associated with CD44 and p185 HER2 in a signaling complex, and HA treatment induces recruitment of both Vav2 and Grb2 into CD44v3-p185 HER2containing multimolecular complexes leading to the co-activation of Rac1 and Ras signaling and ovarian tumor cell growth and migration (19).
In this study we have demonstrated that the CD44-N-WASP complex interacts with ErbB2 (Fig. 5). HA treatment of SK-OV-3.ipl cells causes a significant increase in the amount of ErbB2 recruited into the CD44-N-WASP complex and stimulates ErbB2 tyrosine kinase activity (Fig. 3) leading to ␤-catenin phosphorylation (Fig. 3) and nuclear translocation (Fig. 3). The cell-cell adhesion regulator, ␤-catenin, has been found to be a critical downstream mediator of Wnt signaling (52,53). In the normal ovary ␤-catenin degradation through the ubiquitin pathway is facilitated by GSK-3␤-mediated serine/threonine phosphorylation in connection with APC (the tumor suppressor gene product adenomatous polyposis coli) and Axin (72). Overexpression of ␤-catenin has been shown to be closely associated with ovarian cancer progression (52, 59 -64, 73, 74). Misregulated Wnt signaling in tumor cells often causes ␤-catenin accumulation in the cytoplasm and nuclear translocation (74). ␤-Catenin also binds to the transcription factor TCF/LEF in the nucleus. This is followed by transcriptional activation of target genes such as c-myc, E-cadherin, and cyclin D1 (75). A number of studies also indicate that ErbB2 kinase is capable of inducing tyrosine phosphorylation of ␤-catenin, which can shift ␤-catenin from a normal cell-cell adhesion state into the oncogenic pathways (59 -64, 76). Our results indicate that HAmediated and CD44/N-WASP-associated ErbB2 kinase can induce ␤-catenin phosphorylation and nuclear translocation ( Fig. 3 and Fig. 8) resulting in ␤-catenin-mediated TCF/LEFspecific transcription co-activation (Fig. 4) in ovarian tumor cells. The fact that ErbB2 kinase inhibitor (AG825) can effectively block HA-mediated ␤-catenin phosphorylation, nuclear translocation, and TCF/LEF-transcriptional co-activation (Figs. 3, 4, and 7) strongly suggests that HA interaction with CD44-N-WASP complex-associated ErbB2 kinase plays a critical role in ␤-catenin signaling and oncogenic events during SK-OV-3.ipl cell activation. In human hepatocellular carcinoma, the extracellular signal-regulated kinase (ERK) has been shown to be involved in the inactivation of GSK-3␤ and the up-regulation of ␤-catenin (77). A previous study showed that the binding of HA to CD44 promotes the association of ERK with the IQGAP1 molecule that stimulates both ERK phosphorylation and kinase activity leading to Elk-1 and estrogen-responsive element-specific transcriptional activation in ovarian tumor cells (22). The question of whether HA/CD44-mediated ERK activation is also involved in GSK-3␤ inactivation and ␤-catenin-mediated oncogenic signaling in ovarian tumor cells awaits further investigation.
The VCA domain of N-WASP is known to interact with Arp2/3 complexes and to promote actin polymerization (26 -28, 36). In this study we have confirmed that the VCA domain of N-WASP is responsible for the Arp2/3-mediated actin polymerization in a cell-free system (Fig. 5). To further assess the role of the N-WASP-VCA domain in regulating CD44-and ErbB2-mediated cytoskeleton function and signaling events in human ovarian tumor cells (SK-OV-3.ipl cells), we have transfected SK-OV-3.ipl cells with N-WASP-VCAcDNA (Fig. 5) or vector alone. Our results indicate that transfection of SK-OV-3.ipl cells with N-WASP-VCAcDNA effectively blocks HA-induced endogenous Arp2/3 association with N-WASP (Fig. 5). These findings further support our conclusion that the N-WASP-VCA domain acts as a potent competitive inhibitor that is capable of interfering with endogenous Arp2/3 interaction with N-WASP. Subsequently, HAmediated actin polymerization (Table 1) and tumor cell migration ( Table 2) in these transfectants are inhibited. These observations strongly indicate the importance of the VCA domain of N-WASP in regulating HA/CD44-mediated F-actin formation and ovarian tumor cell migration.
Moreover, we have observed that HA not only stimulates the recruitment of N-WASP and ErbB2 into a CD44 complex (Fig.  7) but also promotes ErbB2 signaling (e.g. ErbB2 kinase activation, ␤-catenin phosphorylation, nuclear translocation, and TCF/LEF-specific transcriptional co-activation) in vectortransfected cells (Fig. 7). In contrast, the failure of endogenous Arp2/3 to become associated with N-WASP in N-WASP-VCAcDNA-transfected cells (Fig. 5) almost completely abolishes HA-mediated N-WASP association with membrane proteins such as CD44 and ErbB2 leading to an  impairment of ErbB2-associated signaling events (e.g. ErbB2 kinase activity, ␤-catenin phosphorylation, nuclear translocation, and TCF/LEF-specific transcriptional co-activation) ( Fig.  7 and Fig. 8; Table 3). Thus, these results provide strong evidence that N-WASP (via the VCA domain) not only serves as an activator for Arp2/3-induced actin assembly but also provides a novel linker function to recruit ErbB2 into a CD44 signaling complex resulting in HA-induced ErbB2-␤-catenin signaling required for tumor-specific behaviors (e.g. tumor cell migration and transcriptional activation). Most importantly, we believe that successful identifications of specific linker molecule(s) between CD44 and tyrosine kinase receptors (e.g. ErbB2) could lead to the identification of potential new drug targets. For example, signaling perturbation procedures designed to overexpress a dominant-negative mutant protein, such as the VCA domain of N-WASP in ovarian tumor cells, could prevent the HA/CD44-induced activation of multiple signaling pathways from being initiated in the first place. Although the ovarian carcinomas cells are frequently detected in an HA-enriched environment, the biological effects or activities generated by the HA polysaccharides may vary significantly depending on the HA sizes and concentrations. Apparently, both HA synthase isozymes (e.g. HA synthase 1 (HAS1), HA synthase 2 (HAS2), and HA synthase 3 (HAS3)) and hyaluronidases (e.g. hyaluronidase 1 (Hyal-1), hyaluronidase 2 (Hyal-2), and PH20) are involved in regulating HA sizes and concentrations required for outside-in signaling events (e.g. HA-CD44 binding, ErbB2-␤-catenin signaling, and cytoskeleton organization). Therefore, it is possible that the enhanced ErbB2 signaling and ␤-catenin phosphorylation are triggered by locally accumulated HA at the tumor attachment site(s) when the required sizes and concentrations of HA become available. The question regarding how various HAS molecules and hyaluronidases are regulated in the extracellular matrix components during ovarian cancer progression will be addressed in our future studies. Preliminary data indicate that large size HA (Ͼ1-2 ϫ 10 6 daltons) has a minimal signaling activation capability. In contrast, HA fragments ranging from 500,000 to 100,000 daltons induce a strong stimulation of cellular signaling. The cellular and molecular mechanisms involved in these selective signaling responses induced by large HA fragments versus small HA fragments await further investigation in our laboratory.
Previously, HA-mediated CD44 interaction with cytoskeleton proteins (e.g. ankyrin and ERM) and various signaling molecules (e.g. Tiam1, Vav2, RhoGEF/RhoA-activated ROK, Cdc42-IQGAP1, and PKN) have been reported (18 -23). However, the regulatory mechanism involved in HA/CD44-mediated ErbB2 activation in ovarian tumor cells has not been well understood. In this study we have obtained new evidence indicating that N-WASP participates in a multimolecular complex formation containing CD44 and ErbB2. Most importantly, N-WASP regulates HA/CD44-mediated ErbB2 activation and ␤-catenin signaling (e.g. ␤-catenin phosphorylation/nuclear translocation and TCF/LEF transcriptional co-activation) needed for transcriptional activation and ovarian tumor cell growth. N-WASP is also involved in HA/CD44-mediated F-actin assembly required for cytoskeleton-dependent tumor-spe-cific behavior such as tumor cell migration. These findings strongly suggest that N-WASP not only mediates multiprotein complex assembly but also participates in cytoskeleton activation and CD44-ErbB2 signaling cross-talk processes.
As summarized in Fig. 9, we would like to propose that upon binding of HA, CD44 is tightly coupled with N-WASP and ErbB2 in a complex (step 1) that induces the formation of the N-WASP-Arp2/3 complex (step 2a) and ErbB2 tyrosine kinase-mediated ␤-catenin phosphorylation (step 2b). These N-WASP-Arp2/3 complexes then stimulate actin polymerization (Fig. 9, step 3a) and cytoskeleton activation resulting in tumor cell migration. At the same time, ␤-catenin phosphorylation (Fig. 9, step 3b), because of HA-mediated CD44-N-WASP-ErbB2 tyrosine kinase activation (together with cytoskeleton function), promotes ␤-catenin nuclear translocation (step 4) and TCF/LEF-specific transcriptional co-activation as well as tumor cell migration. ␤-Catenin-TCF/LEF complexes The binding of HA to SK-OV-3.ipl cells promotes CD44 association with N-WASP and ErbB2 (step 1), which induces N-WASP-Arp2/3 complex formation (step 2a) and ErbB2 tyrosine kinase-mediated ␤-catenin phosphorylation (step 2b). These N-WASP-Arp2/3 complexes then stimulate actin polymerization (step 3a) and cytoskeleton activation resulting in tumor cell migration. At the same time, ␤-catenin phosphorylation (step 3b) because of HA-mediated CD44-N-WASP-ErbB2 tyrosine kinase activation (together with cytoskeletal function) promotes ␤-catenin nuclear translocation (step 4) and TCF/LEF-specific transcriptional up-regulation as well as tumor cell migration. Taken together, these results indicate that CD44 interaction with N-WASP and ErbB2 plays a pivotal role in stimulating HA-dependent actin polymerization and ␤-catenin signaling leading to the concomitant stimulation of cell migration and transcriptional activation required for ovarian cancer progression. are known to be involved in transcription of specific target genes such as cyclin D1 and c-myc (58,75). Cyclin D1 is a major regulator of the progression of cells into the proliferative stage of the cell cycle (78). Overexpression of cyclin D1 is associated with poor survival in epithelial ovarian cancers (79,80). Protooncogene c-myc codes for several phosphoproteins that regulate cell cycle and cell proliferation (81). Amplification of c-myc also plays a critical role in the development of ovarian epithelial neoplasms (54,79). Consequently, ␤-catenin-mediated TCF/ LEF transcriptional co-activation of these target genes (e.g. cyclin D1 and c-myc) is a critical component in the tumorigenesis pathway by dysregulating cell cycle progression and cell growth. Taken together, these results clearly indicate that CD44 interaction with N-WASP and ErbB2 plays a pivotal role in stimulating HA-dependent actin polymerization and ␤-catenin signaling leading to the concomitant stimulation of cell migration and transcriptional activation required for ovarian cancer progression.