Heregulin Regulates Cytoskeletal Reorganization and CellMigration through the p21-activated Kinase-1 viaPhosphatidylinositol-3 Kinase*

(Received for publication, May 27, 1998, and in revised form, July 14, 1998)

Liana Adam‡, Ratna Vadlamudi‡, Sudhir Babu Kondapaka, Jonathan Chernoff§,John Mendelsohn, and Rakesh Kumar¶

From the Cell Growth Regulation Laboratory, the University of Texas M. D. Anderson Cancer Center,Houston, Texas 77030 and the §Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

The mechanisms through which heregulin (HRG) reg-ulates the activities of breast cancer cells are currentlyunknown. We demonstrate that HRG stimulation of non-invasive breast cancer cells enhanced the conversion ofglobular to filamentous actin and the formation of mem-brane ruffles, stress fibers, filopodia, and lamellipodiaand accompanied by increased cell migration. In addi-tion, HRG triggered a rapid stimulation of p21-activatedkinase1 (PAK1) activity and its redistribution into theleading edges of motile cells. The HRG-induced stimula-tion of PAK1 kinase activity followed phosphatidylinosi-tol-3 kinase (PI-3 kinase) activation. Inhibition of PI-3kinase activity blocked the activation of PAK1 kinaseand also blocked cell migration in response to HRG.Furthermore, direct inhibition of PAK1 functions by thedominant-negative mutant suppressed the capacity ofHRG to reorganize actin cytoskeleon structures. We alsodemonstrated that HRG stimulation promoted physicalinteractions between PAK1, actin, and human epider-mal growth factor receptor 2 (HER2) receptors, andthese interactions were dependent on the activation ofPI-3 kinase. The blockade of HER2 receptor by an anti-HER2 monoclonal antibody resulted in the inhibition ofHRG-mediated stimulation of PI-3 kinase/PAK pathwayand also the formation of motile actin cytoskeletonstructures but not extracellular signal-regulated ki-nases. These findings suggest a role of PI-3 kinase/PAK1-dependent reorganization of the cortical actin cytoskel-eton in HRG-mediated increased cell migration, andthese changes may have significant consequences lead-ing to enhanced invasion by breast cancer cells.

Proto-oncogenes are a group of normal genes that play im-portant roles in the regulation of cell proliferation, differenti-ation, and viability. Abnormalities in the expression, structure,or activity of proto-oncogene products contribute to the devel-opment and maintenance of the malignant phenotype. For ex-ample, HER21 (also known as c-erbB2 or c-neu) encodes a

185-kDa transmembrane glycoprotein with intrinsic tyrosinekinase activity (1) that has been shown to be overexpressed,amplified, or both, in a number of human malignancies, includ-ing breast cancer (2). Overexpression of the HER2 receptor isassociated with increased progression and metastasis, an ag-gressive clinical course, and decreased disease-free survival inhuman breast cancer (3, 4). Recently, two additional members,HER3 and HER4, have been added to the human EGF receptor(HER) family. All of these receptors share sequence homologywith the tyrosine kinase domain of HER1 (5). Overexpressionof some growth factor receptors has been shown to inducetransformed properties in recipient cells (6), possibly because ofexcessive activation of signal transduction pathways. The reg-ulation of HER family members is complex, as they can betransactivated by heterodimeric interaction between two HERmembers and thus can utilize multiple pathways to executetheir biological functions (7, 8). For example, HER3 and HER4receptors bind to more than a dozen isoforms of the heregulins(HRGs) or neu differentiation factors (9, 10), and they canactivate the HER2 receptor as a result of HER2/HER3 orHER2/HER4 heterodimeric interactions (7, 8). A ligand thatinteracts with HER2 in the absence of other HER family mem-bers has yet to be identified.

Although the significance of HER2 in breast cancer is wellestablished, the mechanism involved remains poorly under-stood. It has been proposed that this may involve constitutiveactivation of the intrinsic tyrosine kinase activity due to eithermutations in the HER2 gene, overexpression, and/or transac-tivation via receptor-dimerization with other HER members.HER3 is unique among HER family members, as it has animpaired tyrosine kinase domain due to substitution of threeamino acids in the kinase domain (11). Despite the kinase deadnature of HER3, HRG binding to HER3 leads to increasedHER3 tyrosine phosphorylation, probably due to formation of ahigh-affinity co-receptor complex through heterodimeric inter-action with HER2 (12). It is believed that among the HERfamily, the HER2/HER3 dimer complex elicits the most potentmitogenic signal (7). This may be related to the fact that theCOOH-terminal phosphorylation domain of HER3 containsseveral consensus sites for the binding of signal-transducingproteins implicated in mitogenic signaling, including phos-phatidylinositol 3-kinase (PI 3-kinase). HRG stimulation ofbreast cancer cells enhances activation of PI 3-kinase andMAP-kinases (13, 14). Despite the widely acknowledged role ofthe HER2/heregulin pathway in breast cancer progression, thesignaling pathway(s) involved remains elusive.

* This study was supported in part by the Breast and Ovarian re-search programs of the University of Texas M. D. Anderson CancerCenter (to R. K.) and by National Institutes of Health Grants CA65746(to R. K.) and GM54168 (to J. C.). The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

‡ Contributed equally as first author.¶ To whom correspondence should be addressed: Cell Growth Regu-

lation Section (Box 36), the University of Texas M. D. Anderson CancerCenter, 1515 Holcombe Blvd., Houston, TX 77030. E-mail: rkumar@notes.mdacc.tmc.edu.

1 The abbreviations used are: HRG, heregulin-b1; PAK, p21-activated

kinase; MBP, myelin basic protein; PI-3 kinase, phosphatidylinositol-3kinase, HA, hemagglutinin epitope; Ab, antibody; mAb, monoclonal Ab;EGF, epidermal growth factor; HER, human EGF receptor; MMP, ma-trix metalloproteinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 43, Issue of October 23, pp. 28238–28246, 1998© 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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The exposure of cells to growth factors has been shown tocause cytoskeleton reorganization, formation of lamellipodia,membrane ruffling, and altered cell morphology, and accord-ingly, it has been implicated in stimulating cell migration andinvasion (15, 16). Most eukaryotic cells possess the capacity tomove over or through a substrate, and cell migration plays akey role in both normal physiology and diseases, includinginvasion and metastasis (17). In many tissues, the motilityfunction of cells is normally repressed but can be activated byappropriate stimuli and/or oncogenic transformation. In fact,one of the earliest response of cells to many extracellulargrowth factors is rapid reorganization of their cytoskeleton andcell shape. The leading edge of a motile cell is composed of thinprotrusions of membrane that continuously extend and retract,mediating the initial stage of cell movement and determiningthe direction of advance. The underlying cytoskeleton of aleading edge is composed of actin-filament bundles (in filo-podia) or meshworks (in lamellipodia) oriented toward themembrane (16, 17). In addition, the cell motility also involvesthe formation of long bundles of actin stress fibers that end infocal adhesions, points of attachment of the plasma membranesto the substratum.

The small GTPases, including Cdc42, Rac1, and RhoA, havebeen implicated in the regulation of morphology; the formationof filopodia/lamellopodia, membrane ruffles, and stress fibers;and motility of mammalian cells (15). The small GTPase shut-tles between the active GTP-bound form and the inactive GDP-bound form and act as molecular switches in intracellularsignaling. There is also evidence to suggest that the smallGTPases are effectors of PI-3 kinase in the signal transductionpathway leading to growth factor-induced actin reorganization,membrane ruffling, and chemotaxis (18–20). Although thesmall GTPase family members have been shown to be neces-sary for morphological changes in response to growth factors,the mechanisms by which they initiate and regulate the forma-tion of cytoskeletal structures are not completely understood.Recently, a family of serine/threonine kinases known as p21-activated kinases (PAKs) have been identified as a direct targetof activated GTPases (21, 22). Activation of PAK1 has beenshown to result in phenotypic changes reminiscent of thoseproduced by GTPases. The activation of PAK1 either by over-expression of dominant active PAK1 mutants or by stimulationof cells with growth factors causes the accumulation of F-actinand the formation of membrane ruffles, lamellipodia, and filo-podia. Furthermore, activated PAK1 has been also shown to beco-localized with F-actin at the leading edge of cells (21, 22).

Growth factors play an essential role in the regulation ofproliferation of mammary epithelial cells. HRG has been shownto be involved in morphogenesis and ductal migration in mam-mary epithelium (23, 24). In addition to HER2 overexpression,accumulating evidence suggests that the HRG pathway may beinvolved in the progression of breast cancer cells to a moreinvasive phenotype. In the present study, we investigated themechanism(s) by which HRG-b1 signaling may participate inthe acquisition of invasive phenotype in breast cancer. Ourresults demonstrate that HRG stimulation of a noninvasivehuman breast cancer cell leads to the activation of PAK1 ki-nase via PI-3 kinase; an increased physical association betweenthe activated PAK1, actin, and HER2 receptor; and a redistri-bution of PAK1 at the leading edges. These interactions weredependent on the activation of PI-3 kinase by HRG. In addition,these molecular changes were accompanied by the develop-ment of lamellipodia/filopodia and stress fibers, and they alsoincreased the migration of breast cancer cells through a porousmembrane.

MATERIALS AND METHODS

Cell Cultures and Reagent—MCF-7 human breast cancer and HER2-overexpressing MCF-7 cells (25, 26) were maintained in Dulbecco’smodified Eagle’s medium-F12 (1:1) supplemented with 10% fetal calfserum. Antibodies against EGF receptor (catalog no. MS320-P1), HER2(catalog no. MS-301-P1), and HER3 (catalog no. MS262-P) and recom-binant heregulin- b1 were obtained from Neomarkers Inc. Antibodiesagainst HER2 and EGF receptors have been described previously (27,28).

Northern Hybridization, Cell Extracts, Immunoblotting, and Immu-noprecipitation—Total cytoplasmic RNA was analyzed by Northernhybridization using a complementary actin probe for human actinmRNA as described (29). Cell extracts were prepared as described (30)and resolved on a 10% SDS-polyacrylamide gel, transferred to nitrocel-lulose, and probed with the appropriate antibodies, using an enhancedchemiluminescence method or alkaline phosphatase-based color reac-tion method (30).

PI-3 Kinase and PAK-1 Kinase Assays and Transfections—PI-3 ki-nase assays were performed according to Royal and Park (31). Briefly,cell lysates (1 mg) were immunoprecipitated with anti-phosphotyrosinemAb PY20 (Neomarkers) and subjected to in vitro kinase reaction in 50ml of kinase buffer containing 0.2 mg/ml phosphatidylinositol (Sigma)and 20 mCi of [g-32P]ATP and 20 mM MgCl2. The reaction products wereseparated on thin layer chromatography plates using chloroform:meth-anol:ammonium hydroxide:water (86:76:10:14). For PAK kinase assays,200 mg of lysate was immunoprecipitated with anti PAK-1, and kinaseassays were performed in kinase buffer using myelin basic protein(MBP) (Sigma) as substrate. For transfection, we used hemagglutinin(HA)-tagged dominant-negative PI-3 kinase (the p85 subunit that lacksthe binding site for P110) (32) and a mutant Pak1 (pCMV6 M-Pak1H83L/H86L) (21).

Analysis of F-actin Distribution and Cell Morphology—For F-actinstaining, cells that had been cultured on glass coverslips or in chamberslides (Falcon) were fixed with 3.7% paraformaldehyde followed by ashort incubation with acetone at –20 °C as indicated by the manufac-turer. In some cases, coverslips were coated with a thin layer of Matri-gel before plating the cells. Rhodamine phalloidin has been added for 30min at ambient temperature to stain the filamentous actin. Slides weremounted using the Slow Fade Antifade kit (Molecular Probes).

Other Assays—We have performed the cell migration assays as de-scribed, with some modifications (33). Subconfluent cells weretrypsinized. Results are expressed as the percentage of migrating cellscompared with the total number of cells (cells present in all Z-sections)and represent the means 6 S.E. of quadruplicate wells from three ormore experiments. Immunofluorescence and laser scanning confocalmicroscopy was essentially performed as described (30).

RESULTS

HRG Enhances the Expression of F-actin and Cell Migrationof the Breast Cancer MCF-7 Cell—During an investigation intothe mechanism of HRG action, we noticed that HRG rapidlyenhances the expression of actin mRNA (Fig. 1a) and also thesynthesis of 35S-labeled actin (data not shown). Because mod-ulation of cytoskeleton proteins has been linked with cellgrowth and invasion (17), we proceeded to investigate the roleof cytoskeletal proteins in the action of HRG. The results shownin Fig. 1b demonstrate that HRG-induced actin exists predom-inantly as filamentous form (F-actin). The HRG-mediated en-hanced expression of F-actin was accompanied by the forma-tion of filopodias and membrane ruffles and also stress fibers(Fig. 1b, top right and bottom panels). Filamentous actin inserum-starved cells forms is mainly seen in the more periph-eral regions, corresponding to the cortical actin. Stress fibers,as well as filopodia-like structures, which provide attachmentand movement of cytoplasmic components that are responsiblefor cell migration, were seen only in HRG-treated cells. Toinvestigate the biological significance of the formation of F-actin structures in HRG-treated cells, we explored whetherHRG could influence the migration of breast cancer cells.MCF-7 cells were plated in the upper part of Boyden chamberscontaining an 8-mm porous membrane coated with Matrigel onthe lower face and were cultured with or without HRG for 16 h.Cell migration was quantitated by determining the number of

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cells trans-migrating to the lower side of the membrane (facingthe lower chamber) and expressed as percentage of the totalcells. The results shown in Fig. 1c demonstrate that HRG is avery potent cell migratory growth factor.

Because overexpression of HER2 has been shown to be fre-quently associated with an aggressive clinical course in humanbreast cancer (4), we also examined the effect of HER2 overex-pression on the cell migration and reorganization of actin-containing structures, using well characterized HER2-overex-pressing (MCF-7/HER2) cells (25, 26). Results demonstratedthat HER2-overexpressing MCF-7 cells were more motile andhave more constitutively polymerized actin at the free edges ascompared with MCF-7 cells but responded to HRG by furtherenhanced actin polymerization and cell migration (data notshown).

HRG Stimulates PAK1 Activation and Cell Migration—Be-cause activation of PAK1, the downstream target of GTPases,has been shown to result in the phenotypic changes, includinga formation of lamellipodia and filopodia (21, 22), we exploredthe potential involvement of PAK1 in the action of HRG. Toevaluate this possibility, we first investigated whether PAK1 isactivated by HRG in MCF-7 cells. Cell lysates from control andHRG-treated (30 min) MCF-7 cells were immunoprecipitatedwith anti-PAK1 Ab, and precipitated PAK1 was assayed for itskinase activity using exogenous MBP as substrate. The resultsshown in Fig. 2a demonstrated that HRG rapidly increasesPAK1 kinase activity, and there was autophosphorylation ofPAK1. In addition to activating PAK1 kinase, the activation ofPI-3 kinase has been also shown to induce actin reorganizationand membrane ruffling (18), possibly through guanine ex-change factors. Because HRG is known to activate PI-3 kinasein breast cancer cells, we investigated the potential role of PI-3kinase in HRG-mediated alterations in cytoskeletal structures.To this end, we first examined the kinetic relationship betweenthe stimulation of HER2 and PI-3 kinase and PAK1 activities.As shown in Fig. 2, b–d, activation of HER2 receptors bybinding of HRG to its receptors resulted in rapid increase in theactivities of all three kinases. The PI-3 kinase activity wasinduced to a maximum 13.3-fold compared with control cellswithin 5 min of HRG treatment and persisted up to 5 h post-

treatment. It was noteworthy that HRG-induced activation ofPAK1 followed the kinetics of PI-3 kinase activation with amaximum 7-fold activation by 30 min of treatment, and PAK-1kinase remained activated over the period of 5 h compared tothe levels in control untreated cells.

Because HRG-induced activation of PAK1 followed the ki-netics of PI-3 kinase, we investigated whether PAK1 activationis a downstream event of HRG-induced activation of PI-3 ki-nase. The results shown in Fig. 2, e–g, demonstrate that inhi-bition of PI-3 kinase by a specific inhibitor, wortmannin, wasaccompanied by concurrent inhibition of HRG-induced activa-tion of PAK1 in MCF-7 cells (Fig. 2, e and f). Interestingly,treatment of MCF-7 cells with wortmannin was also accompa-nied by suppression of HRG-mediated stimulation of cell mi-gration through a porous membrane (Fig. 2g). The resultsshown in Fig. 2e also demonstrate that although wortmanninpartially inhibited the production of phosphatidylinositol3-phosphate, it completely blocked both PAK1 activity and cellmigration in HRG-treated cells. It is possible that a phospho-tyrosine immunoprecipitation that was used to measure PI-3kinase activity also contains other PI kinases that are notsensitive to wortmannin.

The results in Fig. 3 show the redistribution of PAK1 at themotile free edges in the polarized, migrating HRG-treated cells.In HRG-activated cells, PAK-1 was clearly colocalized to char-acteristic peripheral complexes situated internal from the cellborder but in the proximity of the area that resembles theleading edge of a cell that becomes polarized due to cell move-ment. A cytoplasmic punctate distribution could also be seenaround the cell nucleus. Control cells consistently displayeddifferent distribution of PAK-1, where it colocalized mainlywith cytokeratin 19. Different subcellular localizations, such asfocal adhesion complexes or pinocytic vesicles of exogenouslyintroduced PAK-1, and not its endogenous activation as wedescribe here, have been stated before (22). Indeed, PAK-1subcellular localization have been shown to depend on its ex-pression level and on its interplay with GTPases (21, 22).

Dominant-negative PI-3 Kinase Mutant and a PAK1 MutantPrevent HRG-mediated Reorganization of Actin-containingStructures—Actin reorganization is the central molecular

FIG. 1. Effect of HRG on the expres-sion and organization of actin inMCF-7 cells. a, cells were cultured withor without HRG (20 ng/ml) for 6 or 24 h,and expression of actin mRNA was deter-mined by Northern analysis. b, cells werecultured with or without HRG (20 ng/ml)for 5 h, and the reorganization of filamen-tous actin was determined with fluores-cein isothiocyanate-phalloidin staining byimmunofluorescence microscopy. Bottompanel is a 3 63 magnification of HRG-treated MCF-7 cells. Note that the fila-mentous actin in starved cells forms ring-like structures, as well as some rare fibersat the border of some cells. The appear-ance of F-actin and stress fibers, as wellas filopodia-like structures, was seen onlyin HRG-treated cells. Of interest were thetypical lamellipodia, which contained across-section disposition of F-actin. c, ef-fect of HRG (20 ng/ml for 16 h) on cellmigration, using Boyden chambers.

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event essential for the development of motile cytoskeletalstructures in response to an appropriate stimulus. In order toestablish that inhibition of cell migration by wortmannin wasmediated by suppression of PI-3 kinase, we also inhibited PI-3kinase function by the direct expression of a HA-tagged domi-nant-negative human mutant p85a (DNp85), which lacks abinding site for p110 (32), and determined the effect on HRG-mediated reorganization of actin in MCF-7 cells. As demon-strated in Fig. 4, a–c, inhibition of PI-3 kinase in transfected

HA-positive cells (green color, arrow) resulted in suppression ofHRG-mediated actin polymerization. In contrast, active actinpolymerization (stress fibers) was observed in cells that did notexpressed the mutant PI-3 kinase (negative cells, arrowhead).The inhibitory effect of DNp85 on actin structures was observedin more than 50% of the transfected cells. The observed sup-pression of actin polymerization by DNp85 was HRG-depend-ent, as there was no effect of overexpression of DNp85 alone(data not shown).

FIG. 2. Regulation of signalingpathways and cell migration by HRG.a, activation of PAK-1 by HRG. Cell ly-sates from control and HRG-treated (20ng/ml for 30 min) MCF-7 cells were im-munoprecipitated (IP) with PAK-1 Ab,and subjected to an in vitro kinase reac-tion using MBP as a substrate. b, MCF-7cells were cultured with HRG (20 ng/ml)for the indicated times. Cell lysates wereimmunoprecipitated with an anti-HER2mAb and blotted with anti-phosphoty-rosine mAb PY-69. Bottom arrow, celllysates were blotted with a nonspecificcyclin A Ab. c, cell lysates were immuno-precipitated with anti-phosphotyrosinemAb PY-20 and subjected to a PI-3 kinaseassay. d, cell lysates were immunoprecipi-tated with anti-PAK-1 Ab and subjectedto an in vitro kinase assay using MBP asa substrate and then immunoblotted withanti-PAK Ab to demonstrate potentialvariations in immunoprecipitation effi-ciencies between different samples. e–g,effect of the PI-3 kinase inhibitor wort-mannin on PI3 kinase activity (e), PAK-1kinase activity (f), and cell migration (g).MCF-7 cells were pretreated with wort-mannin (20 mM) for 30 min and then stim-ulated with HRG (20 ng/ml) for 5 min.HRG-mediated stimulation of PAK1 ki-nase activity was observed in five inde-pendent experiments. Ori, origin; PIP,phosphatidylinositol 3-phosphate.

FIG. 3. Distribution of PAK-1 andcytokeratin-19. Cells were culturedwith or without HRG (20 ng/ml) for 5 h,followed by co-staining with affinity-puri-fied anti-PAK-1 Ab (red) and mousemonoclonal anti-CK-19 Ab (green). Ar-rows indicate areas where PAK-1 has dis-tinct localization (around the nucleus andat the leading edges) as compared withcontrol cells.

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In order to demonstrate that PAK-1 is downstream of PI-3kinase in HRG signaling and is involved in mediating theeffects of HRG on cytoskeletal system, we examined the effectof direct expression of a Myc-tagged PAK-1 mutant on HRG-induced alterations in the reorganization of actin in MCF-7cells. A mutated PAK1 molecule (DH83L/H86L) chosen forthese studies contains mutations within the p21 (Rac/Cdc42)binding site, rendering it unable to associate with Rac andCdc42 (21). Although active as a protein kinase, this form ofPak1 may sequester downstream signaling elements, thus po-tentially interfering with Rac and/or Cdc42 signals. The resultsshown in Fig. 4, d–f, demonstrate that inhibition of PAK1function in cells transfected with the Myc-tagged PAK1 mutant(green color, arrow) resulted in complete blockade of HRG-mediated actin polymerization. In transfected cells, F-actinwas weakly seen at cell-cell contact sites, but it was not highlypolymerized. In contrast, actin was highly polymerized in theuntransfected cells (arrowhead). The inhibitory effect of domi-nant-negative PAK1 on actin structures was observed in morethan 60% of the transfected cells. There was no inhibitory effectof the transfection of Myc-tagged control vector on HRG-in-duced reorganization of actin polymerization or cell attachment

(Fig. 4, g–i). In brief, these results suggested that the HRG-initiated signals leading to actin reorganization could beblocked by inhibiting PI-3 kinase and/or PAK1 kinase.

HRG Enhances Physical Interaction between HER2, PAK1,and Actin—Because HRG triggered relocalization of PAK1 intothe leading edge of cells, we explored the potential interactionsbetween the activated HER2 receptor and the PAK1 and actinby immunoblotting PAK1 immunoprecipitates with an anti-HER2 mAb. A modest but significant amount of HER2 receptorwas associated with PAK1/actin complexes only in HRG-stim-ulated MCF-7 cells (Fig. 5a, lanes 1 and 2). As a control, celllysates from HRG-treated or control MCF-7 cells were immu-noprecipitated with normal rabbit serum, and there was noco-immunoprecipitation of actin or HER2 or PAK1 (Fig. 5a,lanes 3 and 4). The observed interactions among HER2, PAK1,and actin were dependent on the activation of PI-3 kinase byHRG, as pretreatment of MCF-7 cells with the PI-3 kinaseinhibitor wortmannin disrupted the interaction between PAK1and HER2 receptor and actin (Fig. 5b). The results shown inFig. 5b also demonstrate that there was detectable interactionbetween the HER2 receptor and PAK1 in HER2-overexpress-ing MCF-7 cells, and HRG stimulation could further enhance

FIG. 4. Inhibition of formation of actin-containing structures by dominant-negative PI-3 kinase or PAK-1 mutants. Cells weretransfected with HA-tagged dominant-negative PI-3K mutant (a–c), Myc-tagged PAK-1 mutant (d–f), or Myc-tagged control vector (g–i). MCF-7cells were double stained for F-actin using rhodamine phalloidin (b, e, and h) and with fluorescein isothiocyanate-coupled anti-HA mAb (a) oranti-c-Myc mAb followed by fluorescein isothiocyanate-coupled goat anti-mouse secondary antibody (d and g). After transfection, the cells weretreated with 20 ng/ml HRG for 4 h except for cells transfected with the Myc-tagged dominant active PAK-1 mutant (j–l). Small arrows show cellswith strong c-Myc or HA protein immunoreactivity (transfected cells), arrowheads indicate the nontransfected cells, and thick arrows show arounded cell with HA immunoreactivity. Note the absence of actin polymerization after HRG treatment in cells in which the function of PI-3K orof PAK1 was impaired (b and e, respectively). The vector alone had no effect on F-actin (h).

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the interactions among HER2 receptor, PAK1, and actin. Theseresults are consistent with the existence of motile cytoskeletonstructures in MCF-7/HER2 cells. Results of other experimentsindicated that HER2-overexpressing MCF-7 cells have 2–3times more baseline PAK1 activity than MCF-7 cells (data notshown). These results suggest that HRG stimulates physicalinteractions among HER2, PAK1, and actin. It remains to beinvestigated whether these interactions are direct or mediatedthrough other protein(s).

An Essential Role of HER2 Receptor in HRG-mediated ActinReorganization and Increased Cell Migration—To investigatethe possible contribution of HER2 receptors in HRG-inducedmotile responses in breast cancer cells, we examined the effectof an anti-HER2 mAb, 4D5 (27), on the development of motilestructures by HRG in an extracellular matrix, Matrigel. Theresults shown in Fig. 6a demonstrate that treatment of MCF-7cells with mAb 4D5 was accompanied by complete suppressionof HRG-mediated development of F-actin containing cytoskel-etal structures. This inhibitory effect of anti-HER2 mAb 4D5was specific, as there was no significant inhibitory effect ofanti-EGF receptor mAb 225 on the development of actin-con-taining structures by HRG. The ability of anti-HER2 mAb 4D5to suppress the cell migratory function of breast cancer cellswas also verified by the Boyden chamber assay. The resultsshown in Fig. 6b demonstrate that co-treatment of MCF-7 cellswith mAb 4D5 and HRG resulted in the suppression of HRG-induced cell migration. Because one of the most importantsteps during the process of migration and/or invasion is the

destruction of extracellular matrix by matrix metalloprotein-ases (MMPs) (34), we examined the potential regulation ofMMPs by HRG and its blockage by mAb 4D5. The results inFig. 6c show that stimulation of MCF-7 cells with HRG leads toa significant increase in the expression of MMP-9 protein, aswell as its gelatinolytic activity in the conditioned medium, andthese changes could be prevented by co-treatment with mAb4D5.

Anti-HER2 mAb 4D5 Blocks HRG-induced Signaling Path-ways and Interaction between HER2 and HER3 Receptors—Tofurther delineate the mechanism of inhibitory effects of mAb4D5 on HRG-mediated functions in MCF-7 cells, we examinedthe effect of blocking HER2 receptor by mAb 4D5 on HRG-activated signaling pathways. MCF-7 cells were pretreatedwith or without mAb 4D5 or an unrelated anti-EGF receptormAb 225 for 30 min, followed by stimulation with HRG for 30min, and cell lysates were assayed for the activation of HER2,PI-3k, PAK1, JNK, and ERK kinases. As shown in Fig. 7a,co-treatment of cells with mAb 4D5 failed to block the activa-tion of HER2 receptor by HRG and, in contrast, resulted in theactivation of HER2 receptor (top panel, lane 3). However, mAb4D5 treatment was accompanied by 67% suppression in thelevels of HRG-induced PI-3 kinase activity. Treatment withmAb 225 blocked the HRG-mediated activation of PI-3 kinaseby 25.7%. Treatment with mAb 4D5 also resulted in the inhi-bition of HRG-induced activation of PAK1 kinase and JNKkinase activities to a significant extent (Fig. 7b). In contrast, itwas interesting to note that mAb 4D5 failed to block the acti-vation of ERK kinase by HRG (Fig. 7b, bottom panel). Theresults shown in Fig. 7a also demonstrate that mAb 4D5-induced the activation of HER2 receptor was not accompaniedby activation of any signaling kinases analyzed in this study,thus suggesting the possibility of a nonproductive nature ofHER2 activation by mAb 4D5 in MCF-7 cells. In the past, mAb4D5 has been shown to induce the activation of HER2 pathwayin breast cancer cells overexpressing HER2 receptors (35), butnot in cells with a normal level of HER2 receptors, such asMCF-7 cells, as shown in the present study. Taken together,the above findings suggested that mAb 4D5 may selectivelyinhibit the activation of PI-3 kinase-dependent pathway lead-ing to actin reorganization, but not the signals leading to ERK2activation.

To further understand the biochemical basis of inhibitoryeffects of mAb 4D5 in MCF-7 cells, we explored the possibilityof an interfering effect of pretreatment of mAb 4D5 on HRG-induced interactions between HER2 and HER3 receptors. Theresults shown in Fig. 7c demonstrate that pretreatment of cellsmAb 4D5 for 30 min blocked interactions between HER2 andHER3 receptors in HRG-treated cells, because there was noco-immunoprecipitation of HER3 receptor with HER2 mAb incells cotreated with mAb 4D5 and HRG (lane 4) compared withthe cells treated with HRG alone (lane 2) or mAb 225 and HRGtogether (lane 6). As shown in Fig. 7c, there was no down-regulation of HER2 receptor by 1 h of treatment with mAb 4D5.

DISCUSSION

Abnormality in the action of the HER2 receptor and HRG,which activates HER2 receptor via its binding to HER3 orHER4 receptors, has been shown to be frequently associatedwith the progression of breast cancer cells to a more invasiveand aggressive phenotype. Because the process of progressionto a more malignant phenotype must involve physical locomo-tion of cancer cells, and because HRG has been shown toinfluence ductal migration in mouse mammary epithelium (23,24), we investigated the nature of HRG-generated signals thatmay be involved in the regulation of cell migration. The resultspresented here indicate that HRG treatment of noninvasive

FIG. 5. HRG induced physical interactions between PAK1, ac-tin, and HER2 receptor. a, MCF-7 cells were cultured with HRG (20ng/ml) for the indicated times. Cell lysates were immunoprecipitatedwith anti-PAK1 Ab (lanes 1 and 2) or normal rabbit serum (NRS) (lanes3 and 4), resolved by 10% SDS-polyacrylamide gel electrophoresis, andtransferred onto a nitrocellulose membrane. Direct cell lysate was usedas a positive control (lane 5). The membrane was cut into three partsand blotted with the indicated antibodies. b, MCF-7 and MCF-7/HER2cells were pretreated with wortmannin (20 mM) for 30 min and thenstimulated with HRG (20 ng/ml) for 30 min. Cell lysates were immu-noprecipitated with nonimmune serum (lanes 1 and 2) or PAK-1 Ab(lanes 3–8) and blotted with the indicated antibodies.

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MCF-7 breast cancer cells leads to a significant enhancement ofcell migration. Our conclusion that HRG is a very active cellmigratory factor is supported by the following lines of evidence:(i) HRG-mediated increased expression of F-actin was accom-panied by the development of motile structures, such as filop-odia and lamellipodia; (ii) HRG stimulated the formation ofstress fibers, the appearance of a leading edge, and membraneruffles at the leading edge; (iii) HRG stimulated the trans-migration of MCF-7 cells across an 8-mm porous membrane;and (iv) HRG also induced cell spreading and F-actin contain-

ing structures when cells were cultured on a reconstitutedbasal membrane, Matrigel. Taken together, these data provideevidence that HRG induces the formation of motile actin cy-toskeleton structures and is a very potent migratory factor forbreast cancer cells.

Among the various pathways leading to actin reorganization,the Rho family of the small GTPases, including cdc42, Rac1,and RhoA, has been implicated in the regulation of morphologyand motility of mammalian cells. Recent studies have identi-fied the p21-activated kinases (PAKs) as a direct target of

FIG. 6. Inhibition of the invasive behavior of HRG-stimulated cells by anti-HER2 mAb 4D5. a, MCF-7 cells were grown on Matrigel-coated coverslips and cultured with or without HRG (20 ng/ml) in the presence of either 3 nM mAb 4D5 or 3 nM mAb 225 for 16 h and then werestained with rhodamine-phalloidin. b, MCF-7 cells were cultured with or without HRG (20 ng/ml) in the presence or absence of 3 nM mAb 4D5 for16 h, and cell migration was assayed using Boyden chamber method. c, MCF-7 cells were cultured with or without HRG (20 ng/ml) in the presenceor absence of 3 nM mAb 4D5 for 16 h. Cell lysates were immunoblotted with MMP-9 (middle panel) and control TIMP-1 mAb (bottom panel), andconditioned medium was assayed for MMP-9 gelatinolytic activity (top panel).

FIG. 7. Inhibition of HRG-induced PI-3 kinase pathway but not ERK kinase by mAb 4D5. MCF-7 cells were pretreated without or with3 nM mAb 4D5 or mAb 225 for 30 min and incubated with or without HRG (20 ng/ml) for an additional 30 min. a, cell lysates wereimmunoprecipitated (IP) with an anti-HER2 mAb and blotted with anti-phosphotyrosine mAb PY-20 (top panel). Cell lysates were immunopre-cipitated with anti-phosphotyrosine mAb PY-20 and subjected to a PI-3 kinase assay (bottom panel). PIP, phosphatidylinositol 3-phosphate. b, celllysates were immunoprecipitated with PAK1 Ab (top panel), JNK2 Ab (middle panel), and ERK2 Ab (bottom panel) and subjected to immunecomplex kinase assays using the indicated substrates. Lanes 1–4 represent the samples from lanes 1–4 of panel a. Quantitation of signal ispresented as fold induction over control untreated cells. c, inhibition of HRG-induced interaction between HER2 and HER3 receptors by mAb 4D5but not mAb 225. MCF-7 cells were pretreated without or with 3 nM mAb 225 or mAb 225 for 30 min and incubated with or without HRG (20 ng/ml)for an additional 30 min. Cell lysates were immunoprecipitated with an anti-HER2 mAb, followed by sequential blotting with anti-phosphotyrosinemAb PY-20 (top panel), HER2 mAb (middle panel), and HER3 mAb (bottom panel).

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activated GTPases, because activation of PAK1 has been shownto result in the phenotypic changes reminiscent of those pro-duced by Rac and/or cdc42 (21, 22). Furthermore, a direct roleof PAK in promoting cell migration has been suggested by thephysical interaction between PAK1 and F-actin in response toupstream stimuli (21, 22). In this context, our observation thatHRG rapidly stimulated PAK1 kinase activity and also itsrelocalization to the leading edges and membrane ruffles of theactivated cells opens a new area of investigation linking, for thefirst time, HER2 activation to cytoskeleton signaling in breastcancer cells. The involvement of PAK1 activation in HRG sig-naling was evident from the following independent findings: (i)PAK1 activation followed the kinetic tyrosine phosphorylationof HER2 (Fig. 2); (ii) activated PAK1 activity has been shown tobe associated with receptor tyrosine kinases (36); and (iii)PAK1 physically associated with the HER2 receptors only inHRG-stimulated cells and not in control cells (Fig. 5). Thesignificance of PAK1 activation in the action of HRG upon cellmotility was deduced from the observations that HRG trig-gered a relocalization of PAK1 to the leading edges of activatedcells, the PAK1 physically associated with actin in HRG-stim-ulated cells, and the expression of a PAK1 mutant completelyprevented HRG-mediated actin polymerization required for thereorganization of actin-containing structures. These findingssuggest a role of PAK1 activation in HRG-mediated reorgani-zation of actin-containing motile structures in breast cancercells.

Data from the literature (18) and from these studies suggestthat the activation of PI-3 kinase is a common growth factorresponse that may be involved in actin reorganization andmembrane ruffling. The finding that the stimulation of PAK1kinase activity follows PI-3 kinase activation is important, as itimplies that PI-3 kinase may constitute an initial HRG-acti-vated signal for actin reorganization and cell migration. Thisview is further supported by the observations that (i) inhibitionof PI-3 kinase by the specific inhibitor wortmannin was accom-panied by concurrent inhibition of HRG-mediated stimulationof PAK1 activity and cell migration (Fig. 2, e–g); (ii) expressionof dominant-negative PI-3 kinase or PAK-1 mutant blockedHRG-mediated reorganization of actin-containing structures(Fig. 4); (iii) inhibition of PI-3 kinase activity by wortmannincompletely prevented physical interactions between PAK1 andHER2 receptor and actin in HRG-treated cells (Fig. 5b); and(iv) anti-HER2 mAb 4D5 inhibited both activation of PI-3 ki-nase/PAK pathway and HRG-induced cell migration (Fig. 7).These results suggest that PAK1 may be downstream of PI-3kinase in the HRG signaling pathway, leading to cytoskeletalreorganization and increased migration of breast cancer cells.

Another notable finding in this study was the essential roleof HER2 receptor in mediating the cell migratory function ofHRG. The evidence that a growth inhibitory anti-HER2-mAb4D5 has the capacity to inhibit the biological effects of HRG,including reorganization of actin cytoskeleton and migration ofbreast cancer cells, is of special interest, as it strongly suggeststhat HRG primarily utilizes the HER2 receptor to transduce itssignals in breast cancer cells. This hypothesis is further sup-ported by our findings that pretreatment of MCF-7 cells withanti-HER2 mAb 4D5, and not an unrelated anti-EGF receptormAb 225, completely blocked the development of motile actin-containing structures. At the moment, we do not know themechanism by which anti-HER2 mAb 4D5 executes its pro-found inhibitory effects on the functions of HRG. It is possiblethat binding of anti-HER2 mAb to HER2 receptors may lead toa conformational change in the HER2 receptor in a mannerthat interferes with its interaction with HER3 or HER4 recep-tors in response to HRG. Alternately, it is also possible that

mAb 4D5 binding to HER2 receptor may create steric hin-drance so that HER2 cannot efficiently interact with HER3receptor, and/or pretreatment with mAb 4D5 may reduce thelevels of HER2 monomers available for interaction with HER3in response to HRG. In either event, mAb 4D5 treatment mayresult in the possible blockade of signals, initiated by the for-mation of HER2/HER3 heterodimers in HRG-treated cells. Atthe moment, we do not understand why mAbs blocked theactivation of the PI3 kinase-PAK pathway but not that ofERK2. Because there is a PI-3 kinase binding motif in thecarboxyl-terminal domain of HER3 receptors (37), one simpleexplanation may include direct recruitment of PI-3 kinase byactivated HER3 receptor and ERK2 kinase may be activated byHER2. In this context, it is possible that the observed activa-tion of HER2 receptor by mAb 4D5 could be due to the forma-tion of HER2/HER2 homodimers. Studies are in progress toinvestigate these and other possibilities. Regardless of themechanism, our findings have clearly established that anti-HER2 mAb 4D5 inhibits the development of actin-based motilestructures as well as cell migration.

In the present study, we have not delineated the pathway(s)that connects the PI-3 kinase with PAK1 kinase in HRG-stim-ulated cells. Previous studies have shown that the small GT-Pases Rac1 and cdc42 are both activators of PAK1 kinase andeffectors of PI-3 kinase in the signaling pathways. Ongoingstudies are aimed at defining the roles of phosphorylated in-ositides and the small GTPases in HRG signaling. In thiscontext, it was interesting to note that the expression of adominant-negative PAK1 not only blocked the expected reor-ganization of actin at the leading edges but also blocked for-mation of stress fibers (Fig. 4, d–f). In the past, stress fiberformation has been shown to require RhoA activation andtyrosine phosphorylation (38). Therefore, it is possible thatRhoA may be in the pathway(s) leading to PAK1 activation inHRG-treated cells. Alternatively, RhoA may be downstream ordistantly regulated by PAK1 in HRG signaling. In either event,inhibition of PAK1 activity may lead to suppression of RhoA-dependent formation of stress fibers, therefore indicating a rolefor RhoA in HRG signaling. This is further supported by ourrecent finding that the inactivation of endogenous Rho by itsspecific ADP-ribosylation using botulium C3 exoenzyme blocksHRG-mediated formation of stress fibers.2 Taken together, ourpresent findings of regulation of actin reorganization and cellmigration functions by HRG and its complete suppression byan anti-HER2 mAb open a new avenue of investigation closelylinking HER2 receptor, cytoskeleton signaling, and breast can-cer cell activity. In addition, our present finding raises thepossibility of therapeutic effects of mAb 4D5 in inhibiting theinvasion and metastasis of breast cancer.

Acknowledgments—We thank Mahitosh Mandal for help with theactin Northern and protein, Masato Kasuga for providing the PI-3kinase constructs, and Genentech Inc. for mAb 4D5.

REFERENCES

1. Stern, D. F., Heffernan, P. A., and Weinberg, R. A. (1986) Mol. Cell. Biol. 6,1729–1740

2. Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A., andMcGuire, W. L. (1987) Science 235, 177–181

3. Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. E.,Levin, W. J., Stuart, S. G., Udove, J., and Ullrich, A. (1989) Science 244,707–712

4. Muss, H. B., Thor, A. D., Berry, D. A., Kute, T., Liu, E. T., Koerner, F.,Cirrincione, C. T., Budman, D. R., Wood, W. C., and Barcos, M. (1994) NewEng. J. Med. 330, 1260–1266

5. Plowman, G. D., Culouscou, J. M., Whitney, G. S., Green, J. M., Carlton, G. W.,Foy, L., Neubauer, M. G., and Shoyab, M. (1993) Proc. Natl. Acad. Sci.U. S. A. 90, 1746–1750

6. Heidaran, M. A., Fleming, T. P., Bottaro, D. P., Bell, G. I., DiFiore, P. P., andAaronson, S. A. (1990) Oncogene 5, 1265–1270

2 L. Adam, R. Vadlamudi, and R. Kumar, manuscript in preparation.

Regulation of Actin Dynamics and Cell Migration by HRG 28245

by guest on May 17, 2016

http://ww

w.jbc.org/

Dow

nloaded from

7. Alroy, L., and Yarden, Y. (1997) FEBS Lett. 410, 83–868. Hynes, N. C., and Stern, D. F. (1994) Biochim. Biophys. Acta 1198, 165–1849. Peles, E., Bacus, S. S., Koski, R. A., Lu, H. S., Wen, D., Ogden, S. G., Levy,

R. B., and Yarden, Y. (1992) Cell 69, 205–21610. Wen, D., Peles, E., Cupples, R., Suggs, S. V., Bacus, S. S., Luo, Y., Trail, G., Hu,

S., Silbiger, S. M., and Levy, R. B. (1992) Cell 69, 559–57211. Guy, P. M., Platko, J. V., Cantley, L. C., Cerione, R. A., and Carraway, K. L.,

III (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8132–813612. Horan, T., Wen, J., Arakawa, T., Liu, N., Brankow, D., Hu, S., Ratzkin, B., and

Philo, J. S. (1995) J. Biol. Chem. 270, 24604–2460813. Marte, B. M., Meyers, T., Stabel, S., Standke, G. J., Jaken, S., Fabbro, D., and

Hynes, N. E. (1994) Cell Growth Differ. 5, 239–24714. Pinkas-Kramarski, R., Soussan, L., Waterman, H., Levkowitz, G., Alroy, I.,

Klapper, L., Lavi, S., Seger, R., Ratzkin, B. J., Sela, M., and Yarden, Y.(1997) EMBO J. 15, 2452–2467

15. Hall, A. (1998) Science 279, 509–51416. Mitchison, T. J., and Cramer, L. P. (1996) Cell 84, 371–37917. Lauffenburger, D. A., and Horwitz, A. H. (1996) Cell 84, 359–36918. Kotani, K., Yonezawa, K., Hara, K., Ueda, H., Kitamura, Y., Sakaue, H., Ando,

A., Chavanieu, A. Calas, B., and Grigorescu, F. (1994) EMBO J. 13,2313–2321

19. Hawkins, P. T., Eguinoa, A., Qiu, R. G., Stokoe, D., Cooke, F. T., Walters, R.,Wennstrom, S., Claesson-Welsh, L., Evans, T., and Symons, M. (1995) Curr.Biol. 5, 393–403

20. Hooshmand-Rad, R., Claesson-Welsh, L., Wennstrom, S., Yokote, K.,Siegbahn, A., and Heldin, C. H. (1997) Exp. Cell Res. 234, 434–441

21. Sells, M. A., Knaus, U. G., Bagrodia, S., Ambrose, D. M., Bokoch, G. M., andChernoff, J.(1997) Curr. Biol. 7, 202–210

22. Dharmawardhane, S., Sanders, L. C., Martin, S. S., Daniels, R. H., andBokoch, G. M. (1997) J. Cell Biol. 138, 1265–1278

23. Yang, Y., Spitzer, E., Meyer, D., Sach, M., Niemann, C., Hartmann, G.,Weidner, K. M., Birchmeier, C., and Birchmeier, W. (1995) J. Cell Biol. 131,

215–22624. Jones, F. E., Jerry, D. J., Guarino, B. C., Andrews, G. C., and Stern, D. F.

(1996) Cell Growth Differ. 7, 1031–103825. Benz, C. C., Scott, G. K., Sarup, J. C., Johnson, R. M., Tripathy, D., Coronado,

E., Shepard, H. M., and Osborne, CK. (1993) Breast Cancer Res. Treat. 24,85–95

26. Kumar, R., Mandal, M., Ratzkin, B. J., Liu, N., and Lipton, A. (1995) J. Cell.Biochem. 62, 102–112

27. Kumar, R., Shepard, H. M., and Mendelsohn, J. (1991) Mol. Cell. Biol. 11,979–986

28. Fan, Z., Mendelsohn, J., Masui, H., and Kumar, R. (1993) J. Biol. Chem. 268,21073–21079

29. Kumar, R., and Atlas, I. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6599–660330. Bandyopadhyay, D., Mandal, M., Adam, L., Mendelsohn, J., and Kumar, R.

(1998) J. Biol. Chem. 273, 1568–157331. Royal, I., and Park, M. (1995) J. Biol. Chem. 270, 27780–2778732. Hara, K., Yonezawa, K., Sakaue, H., Kotani, K., Kitamura, Y., Ueda, H.,

Stephens, L., Jackson, T. R. Hawkins, P. T., Dhand, R., Clark, A. E.,Holman, G. D., Waterfield, M. D., and Kasuga, M. (1994). Proc. Natl. Acad.Sci. U. S. A. 91, 7415–7419

33. Adam. L., Crepin, M., Lelong, J. C., Spanakis, E., and Israel, L. (1994) Int. JCancer 59, 262–268

34. Liotta, L. A., Steeg, P. S., and Stetler-Stevenson, W. G. (1991) Cell 64, 327–33635. Scott, G. K., Dodson, J. M., Montgomery, P. A., Johnson, R. M., Sarup, J. C.,

Wong, W. L., Ullrich, A., Shepard, H. M., and Benz, C. C. (1991) J. Biol.Chem. 266, 14300–14305

36. Galisteo, M. L., Chernoff, J., Su, Y.-C., Skolnik, E. Y., and Schlessinger, J.(1996) J. Biol. Chem. 271, 20997–21000

37. Kim, H., Sierke, S. L., and Koland, J. G. (1994) J. Biol. Chem. 269,24747–24755

38. Ridley, A. J., and Hall, A. (1994) EMBO J. 13, 2600–2610

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Mendelsohn and Rakesh KumarLiana Adam, Ratna Vadlamudi, Sudhir Babu Kondapaka, Jonathan Chernoff, John

p21-activated Kinase-1 via Phosphatidylinositol-3 KinaseHeregulin Regulates Cytoskeletal Reorganization and Cell Migration through the

doi: 10.1074/jbc.273.43.282381998, 273:28238-28246.J. Biol. Chem. 

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