Osteopontin induces AP-1-mediated secretion of urokinase-type plasminogen activator through c-Src-dependent epidermal growth factor receptor transactivation in breast cancer cells.

We have recently reported that osteopontin (OPN) stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase-type plasminogen activator (uPA) through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells (Das, R., Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278, 28593-28606). However, the role(s) of OPN on AP-1-mediated uPA secretion and cell motility and the involvement of c-Src/epidermal growth factor receptor (EGFR) in these processes in breast cancer cells are not well defined. In this study we report that OPN induces alpha(v)beta(3) integrin-mediated c-Src kinase activity in both highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. Ligation of OPN with alpha(v)beta(3) integrin induces kinase activity and tyrosine phosphorylation of EGFR in MDA-MB-231 and wild type EGFR-transfected MCF-7 cells, and this was inhibited by the dominant negative form of c-Src (dn c-Src) indicating that c-Src kinase plays a crucial role in this process. OPN induces association between alpha(v)beta(3) integrin and EGFR on the cell membrane in a macromolecular form with c-Src. Furthermore, OPN induces alpha(v)beta(3) integrin/EGFR-mediated ERK1/2 phosphorylation and AP-1 activation. Moreover, dn c-Src also suppressed the OPN-induced phosphatidylinositol (PI) 3-kinase activity in these cells indicating that c-Src acts as master switch in regulating MEK/ERK1/2 and phosphatidylinositol 3-kinase/Akt signaling pathways. OPN-induced ERK phosphorylation, AP-1 activation, uPA secretion, and cell motility were suppressed when cells were transfected with dn c-Src or pretreated with alpha(v)beta(3) integrin antibody, c-Src kinase inhibitor (pp2), EGFR tyrosine kinase inhibitor (PD153035), and MEK-1 inhibitor (PD98059). To our knowledge, this is the first report that OPN induces alpha(v)beta(3) integrin-mediated AP-1 activity and uPA secretion by activating c-Src/EGFR/ERK signaling pathways and further demonstrates a functional molecular link between OPN-induced integrin/c-Src-dependent EGFR phosphorylation and ERK/AP-1-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells.

Osteopontin (OPN) 1 a non-collagenous, sialic acid-rich, and glycosylated phosphoprotein is a member of the extracellular matrix (ECM) protein family (1,2). OPN acts both as chemokine and cytokine. It is produced by osteoclast, macrophages, T cells, hematopoietic cells, and vascular smooth muscle cells (3). It has an N-terminal signal sequence, a highly acidic region consisting of nine consecutive aspartic acid residues, a GRGDS cell adhesion sequence predicted to be flanked by the ␤-sheet structure (4). This protein has a functional thrombin cleavage site and is a substrate for tissue transglutaminase (2). It binds with several integrins and CD44 variants in an RGD sequencedependent and -independent manner (5,6). This protein is involved in normal tissue-remodeling process such as bone resorption, angiogenesis, wound healing, and tissue injury as well as certain diseases such as restenosis, atherosclerosis, tumorigenesis, and autoimmune diseases (6 -8). OPN expression is up-regulated in several cancers and is reported to associate with tumor progression and metastasis (9 -11). The level of OPN was high in the plasma of patients with breast cancer and has been shown to correlate with poor prognosis of breast cancer (10,12). OPN regulates cell adhesion, cell migration, ECM invasion, and cell proliferation by interacting with its receptor ␣ v ␤ 3 integrin in various cell types (6). Previous data indicated that OPN induces pro-matrix metalloproteinase-2 (pro-MMP-2) activation and urokinase-type plasminogen activator (uPA) secretion, cell motility, ECM invasion, and tumor growth (13)(14)(15).
Integrins are non-covalently associated, heterodimeric, cellsurface glycoproteins with ␣and ␤-subunits. The various combinations of the ␣and ␤-subunits form integrin dimers with diverse ligand specificity and biological activities. The interaction of cell-surface integrin with ECM proteins can lead to the regulation of cell growth, differentiation, adhesion, and migration. Integrin-dependent signaling includes Ca 2ϩ influx, cytoplasmic alkalinization, potassium channel activation, activation of lipid mediators, tyrosine phosphorylation of cytoplasmic proteins, and activation of mitogen-activated protein kinases (MAPK), ERK1 and ERK2 (16 -20). Integrin transduces intracellular signaling by interacting with FAK and Src (21,22). After activation by integrins, p125 FAK is phosphorylated at Tyr-397 which serves as a binding site for SH2 domain of c-Src and other Src family kinases (23). The Src kinase then phosphorylates Tyr-925 of FAK. This generates binding site of the Grb2-SOS complex that leads to the activation of MAPK cascades. Src family kinase could also activate MAPK pathway independent of FAK (24).
Epidermal growth factor receptor (EGFR) belongs to the ErbB growth factor receptor family. The overexpression of EGFR has been correlated with tumor progression and poor metastasis in breast and colon cancers (25,26). The oncogenic effects of EGFR include enhanced DNA synthesis, cell growth, invasion, and metastasis. In fibroblasts, c-Src synergistically increases the oncogenic activity of EGFR suggesting that these two kinases may cooperate in the progression of a malignant phenotype (27). c-Src activity has been shown to be required for both the mitogenic effect of EGF in fibroblasts (28) and the migratory effects of EGF in epithelial cells (29). Earlier reports have indicated that OPN induces migration of human mammary epithelial cells through activation of EGFR (30). It has been described that c-Src catalytic activity is required for ␣ v ␤ 3 integrin-EGFR macromolecular complex formation followed by EGFR phosphorylation (31). EGFR has been shown to be instrumental in the activation of the MAPK pathway (32). Inhibition of ERK1 and ERK2 by the MEK inhibitor, PD98059, resulted in the significant inhibition of the basal cell migration (33). MEK-1 is a serine/threonine kinase that modulates the ERK1/2 pathway (34). ERK1/2 plays key role in regulation of the transcription factor, AP-1, as its activation leads to the induction of c-Fos, which associates with c-Jun to form an AP-1 heterodimeric complex that can promote targeted gene expression (35). However, the molecular mechanism by which OPN regulates c-Src-dependent EGFR transactivation and whether this leads to the activation of AP-1 through MAPK signaling pathway is not clearly understood. uPA is a member of the serine protease family that interacts with the uPA receptor and facilitates the conversion of inert zymogen plasminogen into widely acting serine protease plasmin (36). Plasmin regulates cell invasion by degrading matrix proteins such as fibronectin, type IV collagen, and laminin or indirectly by activating matrix metalloproteinases (MMPs) (37,38). uPA is also involved in cell adhesion, chemotaxis, tumor growth, and metastasis (39 -41). uPA is regulated at the transcriptional level by multiple transcription factors. We have demonstrated recently (15) that OPN regulates NFB-mediated uPA secretion in breast cancer cells. AP-1 transcription factor duplex also plays a major role in regulation of uPA expression through binding to its promoter (42). The molecular mechanism by which OPN controls AP-1-mediated uPA expression and cell motility in breast cancer cells is not well defined.
In this paper, we investigate the involvement of OPN in ␣ v ␤ 3 integrin/c-Src/EGFR-dependent activation of AP-1 and uPA secretion in breast cancer cells. Taken together, we demonstrate that OPN induces ␣ v ␤ 3 integrin-mediated c-Src kinase activity that ultimately phosphorylates ERK1/2 through EGFRdependent and -independent pathways. This leads to the induction of uPA secretion, cell motility, and ECM invasion through activation of AP-1, which is a prerequisite for proteolytic degradation, invasion, and distant metastasis.

EXPERIMENTAL PROCEDURES
Materials-The rabbit polyclonal anti-EGF, anti-EGFR, anti-ERK1/2, anti-c-Src, anti-c-Fos, anti-c-Jun, anti-actin, mouse monoclonal anti-phosphotyrosine, anti-phospho-ERK1/2, anti-␣ v ␤ 3 integrin, and goat polyclonal anti-TGF-␣ antibodies were purchased from Santa Cruz Biotechnology. Mouse monoclonal anti-uPA antibody was obtained from Oncogene. Mouse monoclonal anti-human ␣ v ␤ 3 integrin antibody was from Chemicon International. PD153035, pp2, and PD98059 were obtained from Calbiochem. Rabbit muscle enolase was from ICN. LipofectAMINE Plus and GRGDSP and GRGESP peptides were purchased from Invitrogen. The anti-OPN antibody was purchased from R & D Systems. The dual luciferase reporter assay system and AP-1 consensus oligonucleotide were from Promega. Boyden-type cell migration chambers were obtained from Corning Glass, and Bio-Coat Matrigel TM invasion chambers were from Collaborative Biomedical. [␥-32 P]ATP was purchased from the Board of Radiation and Isotope Technology (Hyderabad, India). All other chemicals were analytical grade.
Cell Culture-The MDA-MB-231 and MCF-7 cells were purchased from the ATCC (Manassas, VA). Both MDA-MB-231 and MCF-7 cells were cultured in Dulbecco's modified Eagle's medium. The medium was supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM glutamine in a humidified atmosphere of 5% CO 2 and 95% air at 37°C.
OPN Purification-OPN was purified from human milk as described previously (13). Briefly, the cleared milk sample was loaded onto a DEAE-Sephadex column. The fraction containing partially purified OPN was purified further on an FPLC Resource-Q column and rechromatographed on the same Resource-Q column. The purity of OPN was checked by SDS-PAGE followed by Coomassie Blue or Silver staining. The purified OPN was characterized by Western blot using anti-OPN antibody. Because EGF and TGF-␣ are also present in milk, we therefore have checked whether trace amounts of EGF or TGF-␣ are present in the purified OPN preparation. Accordingly, purified OPN was resolved by SDS-PAGE and analyzed by Western blot using either a mixture of anti-OPN and anti-EGF or anti-TGF-␣ antibody. This purified OPN was used throughout these studies.
Plasmids and DNA Transfection-Wild type EGFR (EGFR-WT) and kinase-inactive mutant EGFR (EGFR-K Ϫ ) cDNA constructs were generous gifts from Dr. A. N. Habib (Beth Israel Deaconess Medial Center and Harvard Medical School, Boston). Both MCF-7 and MDA-MB-231 cells were transiently transfected with cDNA using LipofectAMINE Plus according to the manufacturer's instructions (Invitrogen). Briefly, cDNA (8 g) was mixed with Plus reagent, and then cDNA reagent Plus was incubated with LipofectAMINE. The LipofectAMINE Plus cDNA complex was added to the cells and incubated further at 37°C for 12 h. The control cells received LipofectAMINE Plus alone. The cell viability was detected by a trypan blue dye exclusion test. After incubation, the medium was removed, and the cells were refed with fresh medium and maintained for an additional 12 h. In other experiments, these cells were individually transfected with the dominant negative form of c-Src cDNA (K296R/Y528F, dn c-Src) in pUSEamp (Upstate Biotechnology) under the same conditions as described above. These transfected cells were used for EGFR phosphorylation, ERK1/2 phosphorylation, uPA expression by Western blot analysis, AP-1 activity by luciferase reporter gene assay, cell migration, and ECM invasion assays.
Western Blot Analysis-To delineate the role of OPN in regulation of ERK1/2 phosphorylation, both MDA-MB-231 and MCF-7 cells were treated with 5 M OPN at 37°C for 0 -60 min. In another experiment, both MDA-MB-231 and MCF-7 cells were individually pretreated with anti-␣ v ␤ 3 integrin blocking antibody (20 g/ml), GRGDSP (10 M), GRGESP (10 M), EGFR tyrosine kinase inhibitor (25 pM PD153035), c-Src kinase domain inhibitor (2 nM pp2), or MEK-1 inhibitor (50 M PD98059) for 1 h and then treated with OPN as described above. In separate experiments, MCF-7 cells were transiently transfected with wild type EGFR (EGFR-WT) or kinase-inactive mutant of EGFR (EGFR-K Ϫ ) cDNA, or both these cells were transfected with dn c-Src cDNA in the presence of LipofectAMINE Plus and then stimulated with OPN. Cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM iodoacetamide, 2 mM phenylmethylsulfonyl fluoride, 20 g/ml leupeptin, and 2 mM EDTA) containing 25 mM NaF and 2 mM Na 3 VO 4 . The cleared lysates were collected by centrifugation at 12,000 ϫ g for 15 min at 4°C. The protein concentration in the lysate was measured by Bio-Rad protein assay. The lysates containing equal amounts of total proteins were resolved by SDS-PAGE. The proteins were electrotransferred from gel to nitrocellulose membrane. The membrane was incubated with mouse monoclonal anti-phospho-ERK1/2 antibody and incubated further with anti-mouse horseradish peroxidaseconjugated IgG. The membrane was washed and detected by the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences) according to the manufacturer's instructions. The membrane was reprobed with rabbit polyclonal anti-ERK1/2 antibody to ensure equal protein loading.
To investigate the role of Immunoprecipitation-To examine the effect of OPN in regulation of EGFR phosphorylation, both MDA-MB-231 and MCF-7 cells were treated with 5 M OPN at 37°C for 0 -60 min. In separate experiments, MCF-7 cells were transfected with EGFR-WT or EGFR-K Ϫ and then treated with 5 M OPN for 0 -60 min as described above. In other experiments, MDA-MB-231 cells or EGFR-WT transfected MCF-7 cells were either pretreated with anti-␣ v ␤ 3 integrin blocking antibody (20 g/ml), GRGDSP (10 M), GRGESP (10 M), pp2 (2 nM), or transfected with dn c-Src and then treated with 5 M OPN for 30 min. Cell lysates containing equal amounts of total proteins were immunoprecipitated with rabbit polyclonal anti-EGFR antibody. The immunocomplexes were analyzed by Western blot using mouse monoclonal anti-phosphotyrosine antibody. The same blots were reprobed with rabbit polyclonal anti-EGFR antibody as loading controls.
To delineate whether OPN regulates any direct interactions between ␣ v ␤ 3 integrin and EGFR or c-Src in breast cancer cells, non-transfected MDA-MB-231 cells or EGFR-WT-transfected MCF-7 cells were individually treated with OPN (5 M) for 0 -30 min. Cell lysates were immunoprecipitated with mouse monoclonal anti-␣ v ␤ 3 integrin antibody. Half of the immunocomplex was immunoblotted with anti-EGFR antibody, and the other half was immunoblotted with anti-c-Src antibody.
c-Src and PI 3-Kinase Assays-To check the role of OPN in regulation of c-Src kinase activity, both MDA-MB-231 and MCF-7 cells were treated with 5 M OPN at 37°C for 0 -30 min. In separate experiments, cells were pretreated with anti-␣ v ␤ 3 integrin antibody (20 g/ml), GRGDSP (10 M), or GRGESP (10 M) for 1 h and then treated with 5 M OPN at 37°C for 5 min. Cells were lysed in Src lysis buffer (150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, 2 mM orthovanadate, 10 mg/ml pepstatin, 2.5 mM EDTA), and the lysates were used for c-Src kinase assay as described (43). Briefly, cell lysates were immunoprecipitated with rabbit polyclonal anti-c-Src antibody. Half of the immunocomplex was incubated with kinase assay buffer (20 mM Hepes (pH 7.4), 10 mM MnCl 2 ) containing 0.2 g of acid-denatured enolase, 10 Ci of [␥-32 P]ATP, and 10 M ATP and incubated at 30°C for 30 min. The samples were resolved by SDS-PAGE and autoradiographed. The remaining half of the immunoprecipitated samples were analyzed by Western blot using anti-c-Src antibody.
In separate experiments, both these cells were either pretreated with pp2 (2 nM) or transfected with dn c-Src and then treated with OPN. These treated or transfected cells were used for PI 3-kinase assay as described previously (15).
EGFR Kinase Assay-In vitro EGFR kinase assays were performed as described by Tuck et al. (30). The MDA-MB-231 cells were treated with 5 M OPN for 0 -60 min at 37°C. Cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.5), containing 150 mM NaCl, 1% Nonidet P-40, 1 mM Na 3 VO 4 , 50 mM NaF, 2 mM EGTA, 2 g/ml aprotinin, 2 g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride), and the cell lysates were immunoprecipitated with anti-EGFR antibody. The immunoprecipitated samples were incubated with 5 Ci of [␥-32 P]ATP in kinase assay buffer at 30°C for 10 min and resolved by SDS-PAGE and autoradiographed. The remaining half of the immunoprecipitated samples were analyzed by Western blot using anti-EGFR antibody.
Nuclear Extracts and Western Blot-To check the level of c-Fos expression in the nucleus, both MDA-MB-231 and MCF-7 cells were treated with 5 M OPN for 0 -4 h at 37°C. The nuclear extracts were prepared as described (15,44). Briefly, cells were incubated in hypotonic buffer (10 mM Hepes (pH 7.9), 1.5 mM MgCl 2 , 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol) and allowed to swell on ice for 10 min. Cells were homogenized in a Dounce homogenizer. The nuclei were separated by spinning at 3300 ϫ g for 5 min at 4°C. The nuclear pellet was extracted in nuclear extraction buffer (20 mM Hepes (pH 7.9), 0.4 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 25% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol) and centrifuged at 12,000 ϫ g for 30 min. The supernatant was used as nuclear extract. The protein concentration was measured by the Bio-Rad protein assay. The nuclear extracts were resolved by SDS-PAGE, and the level of c-Fos was detected by Western blot analysis using rabbit anti-c-Fos antibody.
EMSA-Both MDA-MB-231 and MCF-7 cells were treated with 5 M OPN for 0 -4 h at 37°C. The nuclear extracts were prepared as described above and incubated with 16 fmol of 32 P-labeled doublestranded AP-1 oligonucleotide (5Ј-CGC TTG ATG ACT CAG CCG GAA-3Ј) in binding buffer (25 mM Hepes (pH 7.9), 0.5 mM EDTA, 0.5 mM dithiothreitol, 1% Nonidet P-40, 5% glycerol, and 50 mM NaCl) contain-ing 2 g of poly(dI-dC). The DNA-protein complex was resolved on a native polyacrylamide gel and analyzed by autoradiography. For supershift assay, the OPN-treated nuclear extracts from MDA-MB-231 cells were individually incubated with anti-c-Fos or anti-c-Jun antibody for 30 min at room temperature and analyzed by EMSA.
AP-1 Luciferase Reporter Gene Assay-The semiconfluent cells (MDA-MB-231 and MCF-7) grown in 24-well plates were transiently transfected with a luciferase reporter construct (pAP-1-Luc) containing seven tandem repeats of the AP-1-binding site (Stratagene) using LipofectAMINE Plus reagent (Invitrogen). The transfection efficiency was normalized by cotransfecting the cells with pRL vector (Promega) containing a full-length Renilla luciferase gene under the control of a constitutive promoter. After 24 h of transfection, the cells were treated with OPN (5 M) for 6 h or pretreated with anti-␣ v ␤ 3 integrin antibody (20 g/ml), GRGDSP (10 M), GRGESP (10 M), PD153035 (25 pM) (MDA-MB-231 alone), pp2 (2 nM), or PD98059 (50 M) for 1 h and then treated with OPN (5 M) for an additional 6 h at 37°C. In other experiments, both these cells were individually cotransfected with EGFR-WT, EGFR-K Ϫ , or dn c-Src with pAP-1-Luc and then treated with OPN (5 M) for 6 h. Cells were harvested in passive lysis buffer (Promega). The luciferase activities were measured by a luminometer (Lab Systems) using the dual luciferase assay system according to the manufacturer's instructions (Promega). Changes in luciferase activity with respect to control were calculated.
Cell Migration Assay-The migration assay was conducted using a Transwell cell culture chamber according to the standard procedure as described previously (15,41). Both MDA-MB-231 and MCF-7 cells were individually pretreated with anti-␣ v ␤ 3 integrin antibody (20 g/ml), GRGDSP (10 M), GRGESP (10 M), PD153035 (25 pM) (MDA-MB-231 alone), pp2 (2 nM), and PD98059 (50 M) for 3 h. In separate experiments, both these cells were individually transfected with EGFR-WT, EGFR-K Ϫ , or dn c-Src and used for migration assay. The cells were harvested with trypsin-EDTA and centrifuged at 800 ϫ g for 10 min. The cell suspension (5 ϫ 10 5 cells/well) was added to the upper chamber of the prehydrated polycarbonate membrane filter. The lower chamber was filled with fibroblast condition medium, which acted as chemoattractant. Purified human OPN (5 M) was added to the upper chamber. After treatment, these cells were incubated in a humidified incubator in 5% CO 2 and 95% air at 37°C for 16 h. The non-migrated cells on the upper side of the filter were scraped, and the filter was washed. The migrated cells in the reverse side of the filter were fixed with methanol and stained with Giemsa. The migrated cells on the filter were counted under an inverted microscope (Olympus). The experiments were repeated in triplicate. Preimmune IgG served as a nonspecific control.
Chemoinvasion Assay-The chemoinvasion assay was performed using Matrigel TM -coated invasion chamber as described (14,15). MDA-MB-231 or MCF-7 cell suspension (5 ϫ 10 5 cells/well) was added to the upper portion of the prehydrated Matrigel TM -coated chamber. The lower chamber was filled with fibroblast conditioned medium, which acted as chemoattractant. Purified OPN (5 M) was added to the upper chamber. In separate experiments, these cells were individually pretreated with anti-␣ v ␤ 3 integrin antibody (20 g/ml), GRGDSP (10 M), GRGESP (10 M), PD153035 (25 pM) (MDA-MB-231 alone), pp2 (2 nM), and PD98059 (50 M) for 3 h. In other experiments, both these cells were individually transfected with EGFR-WT, EGFR-K Ϫ , or dn c-Src and used for invasion assay. The cells were incubated at 37°C for 16 h. The non-migrating cells and Matrigel TM from the upper side of the filter were scraped and removed using a moist cotton swab. The invaded cells in the lower side of the filter were stained with Giemsa and washed with phosphate-buffered saline (pH 7.6). The invaded cells were then counted under the inverted microscope (Olympus). The experiments were repeated in triplicate. Preimmune IgG served as a nonspecific control.

OPN Purification and Western Blot Analysis-
The OPN was partially purified from human milk by DEAE-Sephadex chromatography followed by final purification on an FPLC Resource-Q column. The final purity of OPN was checked by SDS-PAGE, followed by Coomassie Blue (Fig. 1, panel A, lanes  1-4) and Silver (panel B, lanes [1][2][3][4] staining. This was characterized by Western blot analysis using established goat anti-OPN antibody (panel C, lanes [1][2][3]. To confirm further whether any trace amount of EGFR ligands (e.g. EGF or TGF-␣) are present in the purified OPN preparation, this was resolved by SDS-PAGE and analyzed by Western blot using a mixture of anti-OPN and anti-EGF or anti-TGF-␣ antibody. The results showed that EGF or TGF-␣ was absent in purified OPN preparations although these proteins were present in whole milk (panels D and E, lanes [1][2][3]. OPN Induces ␣ v ␤ 3 Integrin-mediated Autophosphorylation of c-Src and Its Kinase Activity-To investigate the role of OPN on c-Src kinase activity and to demonstrate the involvement of ␣ v ␤ 3 integrin in this activation process, both MDA-MB-231 and MCF-7 cells were treated with 5 M OPN for 0 -30 min or pretreated with ␣ v ␤ 3 integrin antibody or RGD peptide and then treated with OPN for 5 min. Cell lysates were immunoprecipitated with anti-c-Src antibody. Half of the immunoprecipitated samples were used for c-Src kinase assay using enolase as a substrate. The maximum c-Src kinase activity and autophosphorylation were observed at 5 min of OPN stimulation in both MDA-MB-231 and MCF-7 cells (Fig. 2, upper panels A and B, lanes [1][2][3][4][5]. Pretreatment of cells with anti-␣ v ␤ 3 integrin antibody or GRGDSP but not with GRGESP peptide suppressed the OPN-induced c-Src autophosphorylation and kinase activity in both these cells (upper panels A and B, lanes 6 -8), suggesting that OPN induces c-Src kinase activity through ␣ v ␤ 3 integrin-mediated pathway. The other half of the immunoprecipitated samples was analyzed by Western blot using anti-c-Src antibody, and the level of c-Src was unchanged in these cells (lower panels A and B, lanes [1][2][3][4][5][6][7][8]. The bands were analyzed densitometrically (Kodak Digital Science), and the fold changes were calculated.
OPN Enhances ␣ v ␤ 3 Integrin and c-Src-mediated EGFR Kinase Activity and Tyrosine Phosphorylation-Previous studies (31) have demonstrated that ligand binding to integrins could lead to transactivation of receptor tyrosine kinase. Therefore, we sought to determine the role of OPN on EGFR transactivation in breast cancer cells. Accordingly, MDA-MB-231 cells were treated with 5 M OPN for 0 -60 min, and cell lysates were immunoprecipitated with anti-EGFR antibody. The immunocomplex was used for kinase assay. The maximum EGFR kinase activity was detected at 30 min of OPN stimulation (Fig.  3, upper panel A, lanes [1][2][3][4][5][6]. The level of non-phospho-EGFR was detected by Western blot analysis, and it remained the same (lower panel A, lanes [1][2][3][4][5][6]. To examine the role of OPN in regulating tyrosine phosphorylation of EGFR, both MDA-MB-231 and MCF-7 cells were treated with 5 M OPN for 0 -60 min. Cell lysates were immunoprecipitated with anti-EGFR antibody. The immunoprecipitated samples were analyzed by Western blot using anti-phosphotyrosine antibody, and the blots were reprobed with anti-EGFR antibody. The results indicated that maximum OPNinduced tyrosine phosphorylation of EGFR was observed at 30 min in MDA-MB-231 cells (Fig. 3, upper panel B, lanes 1-6). However, no induction of EGFR tyrosine phosphorylation was detected in MCF-7 cells upon OPN stimulation (upper panel C, lanes [1][2][3][4][5][6]. This could be because of less expression of EGFR in MCF-7 cells (45). To examine further the status of OPN-induced EGFR phosphorylation in MCF-7 cells overexpressing EGFR, these cells were transfected with EGFR-WT or EGFR-K Ϫ and then treated with OPN for 0 -60 min.  A and B, lanes 1-8). The remaining half of the immunoprecipitated samples was analyzed by Western blot using anti-c-Src antibody (lower panels A and B, lanes 1-8). The arrows indicate the specific bands of 32 P-c-Src, 32 P-enolase and c-Src. All these bands were quantified by densitometric analysis, and the fold changes were calculated. The results shown here represent three experiments exhibiting similar effects.  , lanes 1-6), and the remaining half was used for Western blot using anti-EGFR antibody (lower panel, lanes 1-6). Panels B and C, EGFR tyrosine phosphorylation. Both MDA-MB-231 (panel B) and MCF-7 (panel C) cells were treated with 5 M OPN for 0 -60 min. Cell lysates were immunoprecipitated with anti-EGFR antibody, and the immunocomplexes were analyzed by Western blot with antiphosphotyrosine antibody (lanes 1-6). Panels D and E, MCF-7 cells were transfected with wild type EGFR (EGFR-WT) (panel D) or with the kinase-inactive mutant of EGFR (EGFR-K Ϫ ) (panel E) in the presence of LipofectAMINE Plus and then treated with 5 M OPN for 0 -60 min. Cell lysates were immunoprecipitated with anti-EGFR antibody and immunoblotted with anti-phosphotyrosine antibody (lanes 1-6). These blots were reprobed with anti-EGFR antibody (panels B-E, lower panels). Note that maximum EGFR phosphorylation was observed at 30 min in MDA-MB-231 and EGFR-WT-transfected MCF-7 cells. All these bands were quantified by densitometric analysis, and the fold changes were calculated. The results shown here represent three experiments exhibiting similar effects. 231 and EGFR-WT-transfected MCF-7 cells were further transfected with dn c-Src and then treated with 5 M OPN for 30 min. Cell lysates were immunoprecipitated with anti-EGFR antibody, and the immunoprecipitated samples were immunoblotted with anti-phosphotyrosine antibody. The same blots were reprobed with anti-EGFR antibody. The results showed that OPN-induced tyrosine phosphorylation of EGFR was inhibited by ␣ v ␤ 3 integrin antibody and GRGDSP but not by GRGESP peptide in MDA-MB-231 (Fig. 4, upper panel A, lanes  1-5) and MCF-7 (upper panel B, lanes 1-5) C and D, lanes 1-3) suppressed the OPN-induced EGFR phosphorylation, indicating that the kinase domain of c-Src played an important role in OPN-induced ␣ v ␤ 3 integrin-dependent EGFR phosphorylation. The level of non-phospho-EGFR remained identical (lower panels A-D). The bands were analyzed densitometrically, and the fold changes were calculated.

EGFR Interacts with ␣ v ␤ 3 Integrin and c-Src in the Presence of
OPN-It is reported that ␣ v ␤ 3 integrin associates with c-Src and EGFR on the cell membrane in a macromolecular complex form, which leads to the phosphorylation of EGFR (31). Because we have shown that OPN induces EGFR phosphorylation, we therefore sought to determine whether OPN could regulate this macromolecular complex formation in MDA-MB-231 and MCF-7 cells. Accordingly, MDA-MB-231 and EGFR-WT transfected MCF-7 cells were stimulated with 5 M OPN for 0 -30 min. Cell lysates were immunoprecipitated with anti-␣ v ␤ 3 integrin antibody, and half of the immunoprecipitated samples were used for Western blot analysis by using anti-EGFR antibody and the other half of the samples were immunoblotted by anti-c-Src antibody. The results revealed that ␣ v ␤ 3 integrin interacts with EGFR and c-Src in the presence of OPN in MDA-MB-231 (Fig. 4,  upper and lower panels E, lanes 1-4) and in EGFR-WT-transfected MCF-7 (upper and lower panels F, lanes 1-4) cells. These interactions were transient, and maximum interactions were observed at 5 min (lane 2) in both these cells.  E and F, lanes 1-4). All these bands were quantified by densitometric analysis, and the fold changes were calculated. The results shown here represent three experiments exhibiting similar effects.  A and C, lanes 1-6). However, the level of phosphorylation in EGFR-K Ϫ -transfected MCF-7 cells (upper panel D, lanes 1-6) was almost identical as non-transfected MCF-7 cells (upper panel B, lanes 1-6), indicating that the kinase domain of EGFR is crucial in this process. The expression of non-phospho-ERK remained the same (lower panels A-D).
To examine further whether c-Src and MEK-1 play important roles in OPN-induced ERK phosphorylation, both these cells were either transfected with dn c-Src or pretreated with pp2 or PD98059 and then treated with OPN. The data revealed that both genetic dnc-Src and pharmacological (pp2) inhibitors of c-Src and MEK-1 inhibitor (PD98059) suppressed OPN-induced ERK1/2 phosphorylation in both MDA-MB-231 (upper panel C, lanes 1-5) and MCF-7 (upper panel D, lanes 1-5) cells. These data suggested that OPN-induced ERK1/2 phosphorylation is regulated by both c-Src and MEK. The expression of non-phospho-ERK remained identical (lower panels A-D).
c-Src Regulates PI 3-Kinase Activity in the Presence of OPN-We have shown that c-Src kinase activity is required for OPN-induced ␣ v ␤ 3 integrin/EGFR-mediated ERK1/2 phosphorylation. Therefore, we have delineated whether c-Src plays any role in OPN-induced ␣ v ␤ 3 integrin-mediated PI 3-kinase activity. Accordingly, cells were either transfected with dn c-Src or pretreated with pp2 and then treated with OPN, and PI 3-kinase activity was measured. The data revealed that both genetic (dn c-Src) and pharmacological (pp2) inhibitors of c-Src suppressed OPN-induced PI 3-kinase activity in MDA-MB-231 (panel E, lanes 1-4) and MCF-7 ( panel F, lanes 1-4), cells suggesting that c-Src regulates OPN-induced PI 3-kinase activity.
OPN Induces c-Fos Expression, AP-1-DNA Binding, and AP-1 Transactivation-An earlier report (35) showed that activation of ERK could induce c-Fos expression and leads to AP-1 activation. Therefore, we first examined the level of c-Fos expression in MDA-MB-231 and MCF-7 cells. Accordingly, both these cells were treated with 5 M OPN for 0 -4 h, and nuclear extracts were prepared, and the level of c-Fos expression was detected by Western blot analysis using anti-c-Fos antibody. The results indicated that OPN induced c-Fos expression, and the level of c-Fos was higher at 1 h in MDA-MB-231 (Fig. 7,  panel A, lanes 1-5) and MCF-7 (panel B, lanes 1-5) cells.
To check whether OPN induces AP-1-DNA binding, both  1 activity (panels F and G). Similarly, dn c-Src inhibited and EGFR-WT enhanced the OPN-induced AP-1 activity in these cells (panels F and G). EGFR-K Ϫ has no effect on OPN-induced AP-1 transactivation (panels F and G). These data suggested that OPN induces AP-1 transactivation, and this activation was mediated by ␣ v ␤ 3 integrin, EGFR, c-Src, and ERK in these cells.
OPN Induces EGFR and ERK1/2-dependent uPA Secretion-Because the AP-1-binding sequences are present in the promoter with dn c-Src or pretreated with pp2 and PD98059 and then treated with OPN as described above. Cell lysates were analyzed by Western blot using anti-phospho-ERK1/2 antibody (upper panels A-D). All these blots were reprobed with anti-ERK1/2 antibody (lower panels A-D). Panels E and F, effect of c-Src on OPN-induced PI 3-kinase activity. MDA-MB-231 (panel E) and MCF-7 (panel F) cells were either treated with OPN alone or pretreated with pp2 or transfected with dn c-Src and then treated with OPN. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody, and the immunocomplexes were used for PI 3-kinase assay. All these bands were quantified by densitometric analysis, and the fold changes were calculated. The results shown here represent three experiments exhibiting similar effects. region of uPA, and we have shown in this paper that OPN induced AP-1-DNA binding and AP-1 transactivation, we therefore sought to determine whether ␣ v ␤ 3 integrin, c-Src, and ERK which regulate AP-1 activity are involved in OPN-induced uPA secretion in MDA-MB-231 and MCF-7 cells. Accordingly, both these cells were treated with 5 M OPN or pretreated with anti-␣ v ␤ 3 integrin blocking antibody (20 g/ml), RGD peptide (10 M), PD153035 (25 pM) (MDA-MB-231 alone), pp2 (2 nM), or PD98059 (50 M) and then treated with OPN (5 M). Cell lysates were analyzed by Western blot using mouse monoclonal anti-uPA antibody. The data indicated that OPN induces the uPA secretion, and OPN-induced uPA secretion was suppressed by ␣ v ␤ 3 integrin antibody, GRGDSP but not GRGESP peptide, PD153035, pp2, or PD98059 in MDA-MB-231 (Fig. 8, upper panel  A, lanes 1-9) and MCF-7 (upper panel B, lanes 1-7) cells.
In separate experiments, both these cells were individually transfected with wild type EGFR (EGFR-WT), EGFR-K Ϫ or dn c-Src in the presence of LipofectAMINE Plus and then treated with OPN (5 M) as described above. The results indicated that EGFR-WT (panels C and D, lane 3) enhanced and dn c-Src (lane 5) suppressed the OPN-induced uPA secretion compared with cells treated with OPN alone (lane 2). EGFR-K Ϫ had no effect on OPN-induced uPA secretion (lane 4). As loading controls, all these blots were reprobed with anti-actin antibody (lower panels A-D). In all these experiments, the uPA-specific bands were quantified by densitometric analysis, and the values of fold changes are indicated. These results demonstrated that OPN induces uPA secretion via ␣ v ␤ 3 integrin/c-Src/EGFR/ERK-mediated pathways and further suggested that AP-1 is involved in this process.
EGFR, c-Src, and ERK1/2 Play Crucial Roles in OPN-induced ␣ v ␤ 3 Integrin-mediated Cell Migration and Chemoinvasion-We have shown earlier that OPN regulates ␣ v ␤ 3 integrin-mediated c-Src and EGFR-dependent ERK1/2 phos- phorylation and uPA secretion in MDA-MB-231 and MCF-7 cells. Therefore, we have checked whether OPN-induced c-Src/ EGFR/ERK-dependent AP-1 activation and uPA secretion play any role in breast cancer cell migration and invasion. Accordingly, both these cells were individually treated with anti-␣ v ␤ 3 integrin antibody, RGD peptide, PD153035 (MDA-MB-231 alone), pp2, or PD98059, and treated cells were used for the migration assay. The results indicated that OPN induces the cell migration, and OPN-induced migration was suppressed by ␣ v ␤ 3 integrin antibody, GRGDSP but not GRGESP, PD153035, pp2, and PD98059 in MDA-MB-231 (Fig. 9, panel A) and MCF-7 (Fig. 9, panel B) cells. In other experiments, cells were transiently transfected with EGFR-WT, EGFR-K Ϫ , or dn c-Src in the presence of LipofectAMINE Plus and then used for migration assay. The data showed that dn c-Src inhibited and EGFR-WT but not EGFR-K Ϫ enhanced the OPN-induced cell migration in MDA-MB-231 (Fig. 9, panel C) and MCF-7 (Fig. 9, panel D) cells.
Similarly, both these cells were pretreated with anti-␣ v ␤ 3 integrin antibody, RGD peptide, PD153035 (MDA-MB-231 alone), pp2, and PD98059 and used for chemoinvasion assay. In separate experiments, cells were transfected with EGFR-WT, EGFR-K Ϫ , or dn c-Src and then used for invasion assay. The results showed that OPN-induced invasion was suppressed by ␣ v ␤ 3 integrin antibody, GRGDSP but not GRGESP, PD153035, pp2, and PD98059 in MDA-MB-231 (Fig. 10, panel A) and MCF-7 (Fig. 10, panel B) cells. Moreover, dn c-Src inhibited and EGFR-WT but not EGFR-K Ϫ enhanced the OPN-induced inva-sion in MDA-MB-231 (Fig. 10, panel C) and MCF-7 (Fig. 10, panel D) cells. These data demonstrated that OPN-induced ␣ v ␤ 3 -integrin-mediated cell migration, ECM invasion, and uPA secretion are regulated by c-Src and ERK1/2 in MDA-MB-231 and MCF-7 cells and further suggested that EGFR modulates these processes. DISCUSSION In a recent study (15), we have demonstrated that OPN stimulates cell motility and NFB-mediated secretion of uPA through PI 3-kinase/Akt signaling pathways in breast cancer cells. In this paper, we have delineated the molecular mechanisms by which OPN induces EGFR transactivation and AP-1-mediated uPA secretion in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. We have shown that OPN induced ␣ v ␤ 3 integrin-mediated c-Src kinase activity and EGFR phosphorylation. The data also revealed that c-Src kinase is required for OPN-induced EGFR phosphorylation. OPN induced transient interaction between ␣ v ␤ 3 integrin, c-Src, and EGFR which is prerequisite for EGFR transactivation. OPN binding to ␣ v ␤ 3 integrin also enhanced ERK1/2 phosphorylation and AP-1 activation, and these were regulated by c-Src and EGFR. OPN also enhanced uPA secretion, cell motility, and ECM invasion through c-Src/EGFR/ERK/AP-1-mediated pathways.
OPN plays significant roles in tissue remodeling processes such as bone resorption, angiogenesis, wound healing, and tissue injury as well as certain diseases such as restenosis, atherosclerosis, tumorigenesis, and autoimmune diseases (6 - 8). Integrins are cell-surface glycoproteins that bind to the extracellular matrix proteins. In fibroblast, Src family kinases are known to regulate integrin-mediated cell attachment, spreading, and cell migration (46). It has been reported that many primary breast tumors showed elevated levels of c-Src and are correlated with breast carcinomas (47). These results prompted us to investigate whether ligation of OPN with ␣ v ␤ 3 integrin regulates c-Src kinase activity in breast cancer cells. In this study, we have demonstrated that OPN induces ␣ v ␤ 3 integrin-mediated c-Src kinase activity in both MDA-MB-231 and MCF-7 cells.
Transactivation of growth factor receptors contributes to integrin-mediated activation of the Ras-ERK pathways (48). Recently Tuck et al. (30) reported that OPN activates EGFR and regulates the migration of breast epithelial cells. However, the molecular mechanism(s) by which OPN regulates EGFR transactivation which leads to breast cancer cell migration is not clearly understood. We have demonstrated that OPN stimulated kinase activity and tyrosine phosphorylation of EGFR in MDA-MB-231 cells, whereas it was almost absent in normal MCF-7 cells. This could be due to the low level of expression of EGFR in MCF-7 cells. Accordingly, the level of EGFR was enhanced in MCF-7 cells by transfecting with wild type EGFR (EGFR-WT) and then treated with OPN. These data revealed that OPN enhanced the kinase activity (data not shown) and tyrosine phosphorylation of EGFR in these transfected cells. Furthermore, we have shown that OPN-induced tyrosine phosphorylation of EGFR was suppressed by anti-␣ v ␤ 3 integrin blocking antibody and RGD peptide indicating that EGFR transactivation is mediated by ␣ v ␤ 3 integrin rather than through direct binding of EGFR with OPN. Moreover, our data suggested that c-Src kinase activity is required for OPN-induced ␣ v ␤ 3 integrin-mediated EGFR phosphorylation. This was confirmed by the fact that both genetic and pharmacological inhibitors of c-Src kinase suppressed the OPN-induced EGFR phosphorylation. Our results also revealed that OPN stimulated the association between ␣ v ␤ 3 integrin and EGFR in MDA-MB-231 cells and wild type EGFR (EGFR-WT)-transfected MCF-7 cells. The association was transient and remained up to 5 min upon OPN stimulation. Moro et al. (31) have recently reported that ␣ v ␤ 3 integrin and EGFR form a complex with tyrosine kinase such as c-Src, p130 Cas, and Crk. Our data indicated that in addition to EGFR, c-Src also coimmunoprecipitated with the ␣ v ␤ 3 integrin antibody in cells stimulated with OPN, suggesting that OPN enhances macromolecular complex formation between ␣ v ␤ 3 integrin, EGFR and c-Src.
It is well established that MAPKs including ERKs, c-Jun N-terminal kinases, and p38 could be activated by integrin upon ligation with ECM proteins and triggered many cellular responses. For example, ␣ v integrin ligation with vitronectin activates p38 MAPK and increases uPA expression in invasive breast cancer cells (49). Fibronectin stimulates Src kinase activity, which further activates ERK1/2 and promotes cell migration through phosphorylation and enhanced activation of myosin light chain kinase (50). However, it is still not clear whether OPN ligation to integrin can activate MAPKs, especially ERK1/2, which may regulate uPA expression and cell migration in breast cancer cells. Our results indicated that OPN-induced ERK1/2 phosphorylation and the maximum phosphorylation was retained up to 30 min in MDA-MB-231 cells, while it was retained up to 5 min in MCF-7 cells. These results led us to hypothesize that EGFR, which was expressed and phosphorylated in high levels in MDA-MB-231 but not in MCF-7 cells, may be responsible for the retaining of long term ERK1/2 phosphorylation in MDA-MB-231 cells. Accordingly, MCF-7 cells were transfected with EGFR-WT and stimulated with OPN. The data showed that ERK1/2 phosphorylation was also retained up to 30 min in these transfected cells indicating the role of EGFR in OPN-induced ERK phosphorylation.
The OPN-induced ERK phosphorylation was inhibited when both MDA-MB-231 and MCF-7 cells when pretreated with ␣ v ␤ 3 integrin-blocking antibody or RGD peptide indicating that OPN induces ERK phosphorylation through ␣ v ␤ 3 integrin-mediated pathways. Similarly, treatment of MDA-MB-231 cells with either EGFR inhibitor (PD153035) or in combination of PD153035 and ␣ v ␤ 3 integrin antibody blocked the OPN-induced ERK phosphorylation. In contrast, ␣ v ␤ 3 integrin antibody inhibited the OPN-induced ERK phosphorylation, whereas PD153035 failed to inhibit it in MCF-7 cells. These results clearly suggested that ␣ v ␤ 3 integrin and EGFR can activate the OPN-induced ERK1/2 phosphorylation independently; however, the presence of EGFR can enhanced this ␣ v ␤ 3 integrin-mediated phosphorylation process. Our data demon- AP-1, a family of transcription factors, consists of homodimers or heterodimers of Jun, Fos, or activating transcription factor protein (51). Previous reports (51) have demonstrated that AP-1 is involved in several cellular processes such as cell growth, apoptosis, and cell motility. In addition, AP-1 activity is elevated in several pathological conditions including cancers. The activation of ERK induces the level of c-Fos, which can associate with c-Jun comprising the transcription factor AP-1 (35). Because we have shown that OPN induced ERK1/2 phosphorylation and because the AP-1-binding site is present in the promoter region of the uPA gene, we therefore sought to determine the level of c-Fos expression upon OPN stimulation. OPN enhances the nuclear level of c-Fos in both MDA-MB-231 and MCF-7 cells. Our results also suggested that OPN induces AP-1-DNA binding activity. The OPN-induced c-Fos expression correlates with AP-1-DNA binding because maximum c-Fos expression and AP-1-DNA binding were observed at same time period in both these cells. OPN also enhanced AP-1 transcriptional activity in both these cells. Both pharmacological and genetic inhibitors of c-Src, ␣ v ␤ 3 integrin antibody and MEK kinase inhibitor suppressed the OPN-induced AP-1 transactivation in these cells. However, the level of AP-1 activity was significantly higher in MDA-MB-231 cells compared with MCF-7 cells. These results clearly suggested that OPN-induces AP-1 activity through ␣ v ␤ 3 integrin/c-Src/ERK-mediated pathways in breast cancer cells.
Several studies have indicated the correlation between uPA expression and metastatic potential and have suggested that uPA plays a major role in controlling cell migration and ECM invasion in various cancer cells (15,41). Here we have reported that OPN induces uPA secretion. However, the levels of uPA secretion, cell motility, and invasiveness were significantly higher in MDA-MB-231 cells compared with MCF-7 cells. Both pharmacological and genetic inhibitors of c-Src and ␣ v ␤ 3 integrin antibody and inhibitors of EGFR and MEK kinase suppressed the OPN-induced uPA secretion, cell motility, and invasion in these cells. Our data revealed that OPN-induced uPA secretion could be regulated by activations of the c-Src kinase, EGFR, and ERK1/2 which ultimately control the ␣ v ␤ 3 integrinmediated cell motility and invasiveness in these cells.
reports (52,53) indicated that c-Src could induce PI 3-kinase activity either by directly interacting with the Src homology 3 domain of the p85 subunit of PI 3-kinase or through the FAKmediated pathway. Here we have shown that the dominant negative form of c-Src (dn c-Src) suppressed the OPN-induced PI 3-kinase activity, suggesting that OPN induces PI 3-kinase activity via c-Src-dependent mechanisms. c-Src kinase activity is also required in OPN-induced ERK1/2 phosphorylation. Our data demonstrated that OPN-induced uPA secretion, cell motility, and ECM invasion were totally blocked by pp2, a c-Src kinase inhibitor. All these results suggested that OPN-induced uPA secretion can be regulated both by PI 3-kinase and MAPKmediated pathways, and c-Src can act as master switch in regulating both these pathways.
Previous reports (54,55) have indicated that nanomolar concentrations of OPN are used in regulation of NO production, NFB activation, Akt phosphorylation, cell adhesion, and migration in endothelial and other cell types. However, we and other groups (13-15, 30, 56 -58) have demonstrated that micromolar concentrations of OPN are required to regulate PI-3kinase activation, uPA production, pro-MMP-2 activation, cell migration, and ECM invasion in melanoma, breast cancer, and other cell types. Therefore, it is possible that various concentrations of OPN used to regulate these cellular functions may depend on types of cell lines used in these studies.
In summary, we have demonstrated for the first time that OPN induces AP-1 transactivation through phosphorylation of EGFR by inducing the c-Src kinase activity in breast cancer cells. Ligation of OPN with the ␣ v ␤ 3 integrin induces phosphorylation and kinase activity of EGFR, and this was blocked by dn c-Src indicating that c-Src kinase plays a crucial role in these processes. OPN induces ␣ v ␤ 3 integrin/EGFR-mediated ERK1/2 phosphorylation. OPN stimulates c-Fos expression, AP-1-DNA binding, AP-1 transactivation, uPA secretion, cell motility, and invasion in these cells. OPN-induced ERK phosphorylation, AP-1 activation, uPA secretion, and cell motility were suppressed when these cells were transfected with dn c-Src or pretreated with ␣ v ␤ 3 integrin antibody, c-Src kinase domain inhibitor, EGFR tyrosine kinase inhibitor, or MEK-1 inhibitor. We have delineated the mechanism by which OPN induces AP-1 activation and uPA secretion by activating c-Src kinase/ERK-mediated and EGFR-dependent or -independent signaling pathways (Fig. 11). These findings may be useful in designing novel therapeutic interventions that block the OPNregulated c-Src kinase-dependent EGFR phosphorylation and AP-1 activation resulting in reduction of uPA secretion and consequent blocking of cell motility, invasiveness, and metastatic spread of breast cancer.