[CANCER RESEARCH 59, 5307–5314, October 15, 1999] Urokinase Receptor Interacts with  v  5 Vitronectin Receptor, Promoting Urokinase-dependent Cell Migration in Breast Cancer 1 Maria V. Carriero, 2 Silvana Del Vecchio, Monica Capozzoli, Paola Franco, Laura Fontana, Antonella Zannetti, Gerardo Botti, Giuseppe D’Aiuto, Marco Salvatore, and M. Patrizia Stoppelli National Cancer Institute [M. V. C., M. C., P. F., A. Z., G. B., G. D.], Nuclear Medicine Center (C. N. R.), University Federico II [S. D. V., M. S.], and International Institute of Genetics and Biophysics [L. F., M. P. S.], 80131 Naples, Italy ABSTRACT Perturbation of adhesive interactions at cell-substratum and cell-cell contact sites is a critical event in the multistep process of cancer invasion. Recent studies indicate that the urokinase receptor (uPAR) is associated in large molecular complexes with other molecules, such as integrins. To test the possibility that uPAR may physically and functionally interact with vitronectin (Vn) receptors, we determined the expression level of uPAR,  v  3 , and  v  5 Vn receptors in 10 human breast carcinomas. Here, we show the ability of uPAR to physically associate with  v  5 in the breast carcinomas examined. The functional effects of this interaction were studied using HT1080 human fibrosarcoma and MCF-7 human breast carcinoma cell lines, both exhibiting a urokinase-dependent physical as- sociation between uPAR and  v  5 . Both cell lines respond to urokinase or to its noncatalytic amino-terminal fragment by exhibiting remarkable cytoskeletal rearrangements that are mediated by  v  5 and require pro- tein kinase C activity. On the contrary, binding of Vn to  v  5 results in the protein kinase C-independent formation of F-actin containing microspike- type structures. Furthermore,  v  5 is required for urokinase-directed, receptor-dependent MCF-7 and HT1080 cell migration. These data show that uPAR association with  v  5 leads to a functional interaction of these receptors and suggest that uPAR directs cytoskeletal rearrangements and cell migration by altering  v  5 signaling specificity. INTRODUCTION The invasive ability of malignant cells requires a complex interplay of various cell surface-associated components participating to the proteolytic disruption of ECM 3 and the modification of cell adhesion properties (1). A large body of evidence assigns to urokinase [urokinase-type plasminogen activator (uPA)] a key role in tumor progression and invasion, by virtue of its ability to activate plasminogen, which degrades many ECM components, such as fibronectin, laminin, and proteoglycans, and activates latent collagenases (2). The inoculation of metastatic Lewis lung carcinoma cells into plasminogen-deficient mice results in the formation of smaller and less hemorrhagic tumors than in control wild type mice (3). Furthermore, the absence of uPA negatively affects the progression of chemically induced melanocytic neoplasms in mice (4). In addition to its proteolytic role, uPA can regulate cell mobiliza- tion, adhesion, proliferation, and transcription of specific genes through a catalytic-independent mechanism (5–7). A key player in this process is the specific cell surface uPAR, which binds with high affinity the ATF of uPA (8). uPAR is a highly glycosylated 55,000 – 60,000 M r protein that includes the N-terminal uPA binding domain, designated D1, a connecting domain (D2), and the COOH-terminal domain bearing a glycosylphosphatidylinositol anchor (D3; Ref. 2). D2 and D3 domains have the property to recognize the matrix-like form of Vn (9). The multiple molecular events following uPAR ligation with uPA include diacylglycerol formation in endothelial cells, activation of PKC, and phosphorylation of cytokeratins 8 and 18 in human epithelial cells (10, 11). A transient modification of the Src-family kinase p56/59 hck activation state has been also reported in mielomonocytic cells (12, 13). All of these cell responses raise a question concerning the modality of uPAR signaling transmission, as this is restricted to the outer leaflet of the membranous bilayer and therefore requires a transmembrane “adaptor.” Coimmunoprecipita- tion studies show that uPAR is associated in large molecular com- plexes with integrins, caveolin, and Src kinases (14 –16). The revers- ible association with other receptors is supported by the uPAR lateral mobility in the plasma membranous bilayer and its focal redistribution upon interaction with uPA (17). The relevance of uPAR lateral mo- bility to signaling is further sustained by the finding that a nonsignal- ing uPA variant is also unable to mobilize the receptor (18). Integrin receptors are composed of  and  subunits that heterodimerize to produce more than 20 different receptors, capable of mediating a variety of cell responses, such as spreading and migration, control of gene expression, growth, and differentiation (19 –21). Changes in integrin structure and/or expression are frequently associated with malignant transformation and tumor progression (22). Overexpression of  v integrins occurring in human mammary carcinomas is associ- ated to a widespread deregulated expression of other integrins, such as  2 and  6  4 (23). uPAR and integrins share the ability to activate members of the Src family, as well as pp125FAK, further supporting the possibility that uPAR impinges on cell function via integrins (24). Following engage- ment with uPA, uPAR colocalizes or physically associates with  1 ,  2 ,  3 , or  5 integrins both in vivo and in vitro (15, 25, 26). It is presently known that uPAR association suppresses the normal adhe- sive function of different integrins, suggesting that they may acquire new functional properties (16). A shared ligand between integrins and uPAR is Vn that binds and stabilizes plasminogen activator inhibitor PAI-1 (27). Both, uPAR and PAI-1 bind to the somatomedin-like domain of Vn, and PAI-1 pre- vents Vn binding to the VnR, therefore stimulating cell detachment (28, 29). The physical linkage between uPAR and VnR is supported by the colocalization of uPAR and  v  5 at focal contacts of human keratinocytes (30). Furthermore, Vn-dependent migration of human pancreatic carcinoma cells is inhibited by anti-uPAR or by anti- v  5 antibody, suggesting a functional coupling between these two recep- tors (31). The two known VnRs exhibit different functional properties, as they regulate two distinct pathways in angiogenesis (32, 33). Unlike  v  3 ,  v  5 can direct cell migration, as well as redistribution of talin, vinculin, and -actinin only in the presence of PKC activators (31, 34). We have previously shown the ability of membrane-associated Received 4/12/99; accepted 8/19/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Associazione Italiana per la Ricerca sul Cancro. A. Z. is a recipient of a fellowship from Fondazione Italiana per la Ricerca sul Cancro. 2 To whom requests for reprints should be addressed, at National Cancer Institute, Via M. Semmola, 80131 Naples, Italy. Phone: 39-081-5903569; Fax: 39-081-5461688; E- mail: stoppelli@iigbna.iigb.na.cnr.it. 3 The abbreviations used are: ECM, extracellular matrix; uPA, urokinase-type plas- minogen activator; uPAR, urokinase receptor; Vn, vitronectin; VnR, vitronectin receptor; ATF, amino-terminal fragment of urokinase; PKC, protein kinase C; mAb, monoclonal antibody. 5307 Research. on February 6, 2015. © 1999 American Association for Cancer cancerres.aacrjournals.org Downloaded from