The highly invasive human prostate cancer PC3 cell line was found to express the αvβ3 integrin; in contrast, the noninvasive LNCaP prostate cancer cell line did not express αvβ3. PC3 cells adhered to and migrated on vitronectin (VN), an αvβ3 ligand expressed in mature bone where prostate cancer cells preferentially metastasize. In contrast, LNCaP cells did not adhere to or migrate on VN. Analysis of primary human prostate cancer cells isolated from 16 surgical specimens, showed that these cells expressed αvβ3, whereas normal prostate epithelial cells did not. In addition, only primary prostate cancer cells adhered to and migrated on VN. The role of αvβ3 in mediating prostate epithelial cell migration was confirmed using LNCaP cell transfectants expressing β33-LNCaP). Exogenous expression of αvβ3 induced LNCaP cells to adhere to and migrate on VN. In response to αvβ3 engagement, increased tyrosine phosphorylation of focal adhesion kinase (FAK), a signaling molecule activated by integrins and able to modulate cell migration, was detected. Transfection of FAK-related nonkinase, known to compete with FAK for its correct localization and phosphorylation, caused inhibition of β3-LNCaP cell migration, specifically on VN. These data indicate that de novo expression of αvβ3 integrin in prostate cancer cells generates a migratory phenotype that is modulated by a FAK signaling pathway. This study points to αvβ3 as potential target in prostate cancer cell invasion and metastasis.

Prostatic carcinoma has been estimated to be the second leading cause of death due to cancer among men in the United States (1). Several studies have indicated that prostate cancer cell lines (2, 3, 4, 5), as well as human cancer and benign prostatic tissues (6, 7, 8, 9), express different members of the integrin family that are known to mediate interactions between cells and ECM4 proteins. Integrins are heterodimeric cell surface receptors that consist of noncovalently associated α and β subunits; these receptors have been shown to play a role in cell migration, proliferation, and gene transcription and can affect cancer cell invasion and growth (10, 11, 12, 13, 14, 15). Alterations of integrin expression in cancer cells correlate with tumor growth and progression, increased invasiveness, and metastatic potential (16).

Integrins provide a direct link between ECM and cytoskeleton, thus, controlling cell motility and, therefore, cancer cell invasion. The αvβ3 integrin, specifically, mediates adhesion and migration of several cell types on VN-coated substrates, although its stimulation can result in invasion through basement membrane matrices (17, 18, 19). Several receptors for VN have been described; specifically, in epithelial cells, αvβ5 integrin can replace the function of αvβ3(20, 21, 22, 23). Signaling from the αvβ3 can be synergized by growth factor receptors (24, 25).

Several signaling molecules, specifically FAK, Cas, and members of the MAP kinase family, play a role in modulating integrin-mediated cell migration. FAK is a nonreceptor tyrosine kinase localized in focal contacts that becomes tyrosine phosphorylated and subsequently activated on integrin-mediated cell adhesion to several matrix proteins, including VN (26, 27). FAK has been shown in vitro to bind β integrin cytoplasmic domain mimetic peptides (28). Domains within the amino- and COOH-terminal regions of the β3 integrin cytoplasmic tail, including the highly conserved NPXY motif, are required for stimulation of FAK tyrosine phosphorylation (29). In all instances, with the exception of a Cas-binding mutant (FAK P712/715A), FAK is tyrosine phosphorylated in migratory cells (30, 31, 32, 33, 34), although in some cell types, FAK tyrosine phosphorylation correlates with reduced migration (35). The COOH-terminal domain of FAK or pp41/43FRNK contains binding sites for a number of signaling molecules, including Cas, as well as a focal adhesion targeting sequence that is sufficient to recruit FAK into focal contacts (36). FRNK acts as a negative regulator of FAK and has been shown, when overexpressed, to prevent tyrosine phosphorylation of FAK and paxillin (37). FRNK has also been shown to delay formation of focal adhesions and cell spreading on FN in chicken embryo cells (37), as well as motility and proliferation of human endothelial cells (32). Cas is a cytoplasmic protein that does not have an intrinsic catalytic activity and that, in response to integrin-mediated cell adhesion, becomes tyrosine phosphorylated and serves as a docking protein for downstream signaling molecules, including FAK (38, 39, 40). Recently, increased tyrosine phosphorylation of Cas has been shown to correlate with increased integrin-mediated CHO or COS cell migration (34, 41). Integrin engagement has also been shown to stimulate activation of two members of the MAP kinase family, extracellular-regulated kinase-1 and -2 (14), which are involved in Ras-mediated control of gene expression in response to extracellular stimuli. Additionally, integrin-mediated cell migration of FG pancreatic carcinoma cells, macrophages expressing α6Aβ1, and human umbilical vein endothelial cells has been shown to occur via a mechanism that requires activation of the MAP kinase signaling cascade (42, 43).

In this study, we show that highly invasive human prostate cancer PC3 epithelial cells express the αvβ3 integrin and migrate on VN, an αvβ3 ligand expressed in mature bone where prostate cancer cells preferentially metastasize. In contrast, noninvasive LNCaP cells do not adhere to or migrate on VN. Primary prostate epithelial cells obtained from prostatic adenocarcinoma, but not cells obtained from normal prostate tissue, express the αvβ3 integrin, and only cancer cells adhere to and migrate on VN. Forced expression of αvβ3 in noninvasive LNCaP cells generates a migratory phenotype on VN-coated substrates, that correlates with a specific increase in tyrosine phosphorylation of FAK. Cotransfection of β3 and FRNK prevents cell migration on VN, suggesting a dominant role for FAK in this cellular function. These results describe a novel pathway mediated by the αvβ3 integrin that regulates migration of human prostate cancer cells and is relevant to the understanding of the mechanisms that control metastatic spread of these cells.

Cell Lines and Transfections.

PC3, LNCaP (ATCC, Rockville, MD), and BPH-1 (provided by Simon W. Hayward, University of California San Francisco, San Francisco, CA; Ref. 44) human prostate epithelial cells were cultured in RPMI 1640 supplemented with 10% (or 2.5% for BPH-1) FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.292 mg/ml l-glutamine (all from Gemini Bio-Products, Inc., Calabasas, CA), 0.1 mm nonessential amino acids, and 1 mm sodium pyruvate (Life Technologies, Inc., Gaithersburg, MD). CHO cells (ATCC) were cultured as described (45). To generate stable cell lines, LNCaP cells were transfected with either the full-length human β3A integrin cDNA (designated here as β3; provided by Tim O’Toole, The Scripps Research Institute, La Jolla, CA) or the full-length human ICAM-1 cDNA in pRc/CMV (designated here as ICAM; provided by Dario C. Altieri, Yale University, New Haven, CT). Chicken FRNK cDNA in the pCMV-c-Myc expression vector, downstream of a c-myc epitope tag, was provided by J. Thomas Parsons and Wen-Cheng Xiong (University of Virginia, Charlottesville, VA). The full-length human β3 cDNA was excised from CD3a (46) using XbaI, subcloned in pRc/CMV mammalian expression vector (Invitrogen, Carlsbad, CA). All transfections were performed using Lipofectin (Life Technologies, Inc.) according to the manufacturer’s instructions. pRc/CMV vector alone was used to generate mock-LNCaP-transfected cells. Stably transfected populations were obtained by growing the cells in growth media supplemented with 1 mg/ml geneticin (Life Technologies, Inc.). To obtain a population of cells that uniformly expressed the transfected surface protein, cells were sorted by FACS using either LM609 (1:500; provided by David A. Cheresh, The Scripps Research Institute) against human αvβ3 integrin or 2D5 (25 μg/ml) against human ICAM-1. β3- and FRNK-β3 (FRNK-β3-3 and FRNK-β3-4) LNCaP cell transfectants were also analyzed by immunoblotting for FRNK expression using 0.1 μg/ml C-20, polyclonal antibody to FAK (Santa Cruz Biotechnology, Santa Cruz, CA) that cross-reacts with human and chicken FAK and FRNK. After cell sorting, the stable transfectants were maintained in growth medium supplemented with 0.1 mg/ml geneticin. The levels of expression of β3-LNCaP in either β3- or FRNK-β3 LNCaP cells were comparable, as evaluated by FACS analysis performed using LM609 (data not shown).

Primary Cultures of Epithelial Cells from Human Prostate.

Primary cultures of human prostate epithelial cells were prepared as described previously (44). Human prostate tissue specimens were obtained under review board-approved protocols from 16 radical prostatectomies performed for prostatic adenocarcinoma at Yale-New Haven Hospital. Tissue samples of ∼0.5 cm3 were taken from the peripheral zone of the resected prostate in areas grossly suspicious for involvement by carcinoma. The prostate tissue was minced into small pieces (0.1 × 0.1 × 0.1 cm), ∼10% of which was fixed in formalin and embedded in paraffin for histological examination and used as a control. The remaining 90% of the prostate tissue was processed for epithelial cell isolation. Only those samples in which tumor cells represented more than 80% of the total after microscopic examination of the formalin-fixed paraffin-embedded controls were further analyzed. Pathological examination of tissue sections taken from areas immediately adjacent to the 0.5 cm3 sample obtained for the study further confirmed the presence of carcinoma in all cases. Surgical specimens were collected only from patients that had a localized tumor and lacked metastatic lesions. This study is limited to the use of two normal prostate samples for primary cell isolation, due to obvious difficulties in obtaining fresh normal prostate; autopsy specimens could not be used successfully for primary cell isolation because the specimens were available only several hours after death. For prostate epithelial cell isolation, tissue fragments were dissociated using 200 units/ml Collagenase type I (Sigma Chemical Co., St. Louis, MO) and 100 μg/ml DNase I (Sigma Chemical Co.) in PBS at 37°C for 16 h with gentle stirring. The following day, epithelial cells were separated from stromal cells by repeated unit gravity sedimentation. The primary epithelial cells were cultured in RPMI 1640 (Life Technologies, Inc.) supplemented with 2.5% dextran-coated charcoal-stripped heat-inactivated FBS, 1 μg/ml insulin (Sigma Chemical Co.), 10 ng/ml hydrocortisone (Sigma Chemical Co.), 5 μg/ml transferrin (Life Technologies, Inc.), 1 μg/ml sodium folate (Sigma Chemical Co.), 50 ng/ml phosphorylethanolamine (Sigma Chemical Co.), 5 μg/ml ascorbic acid (Sigma Chemical Co.), 5 ng/ml recombinant human epidermal growth factor (R&D Systems, Inc., Minneapolis, MN), and 50 ng/ml cholera toxin (Sigma Chemical Co.) at 37°C in a humidified 7.5% CO2 incubator. Primary cell characterization was performed using CKs 8 and 18, markers of epithelial cells (47). Indirect immunofluorescence of monolayers of primary cells using monoclonal antibodies to CKs 8 and 18 was performed as follows. Cells isolated by unit gravity sedimentation were seeded onto glass coverslips coated using 3 μg/ml human VN, fixed using acetone (J. T. Baker Inc., Phillipsburg, NJ), blocked using 50 μg/ml BSA (Sigma Chemical Co.) in PBS (pH 7.4; 137 mm NaCl, 2.7 mm KCl, 1 mm Na2HPO4, and 1.8 mm KH2PO4), incubated with a monoclonal antibody against either human CK 8 (1:300; Boehringer Mannheim, Mannheim, Germany) or CK 18 (1:300; Sigma Chemical Co.), and finally incubated with FITC-conjugated goat antimouse IgG (40 μg/ml; Cappel, Durham, NC) at 37°C for 1 h. Coverslips were washed and mounted on slides with Fluoromount-G (Southern Biotechnology, Birmingham, AL).

Flow Cytometric Analysis.

Two-color flow cytometric analysis was performed using the monoclonal antibody against human CK 18 (1:250) and a rabbit serum against the cytoplasmic domain of β3 integrin (1:200), provided by Erkki Ruoslahti (The Burnham Institute, La Jolla, CA). A monoclonal antibody against a vascular endothelial surface protein, 1C10 (1:250; Life Technologies, Inc.) and nonimmune rabbit serum (1:200) were used as negative controls. Prostate epithelial cell suspensions, either immediately after unit gravity sedimentation or in primary culture, were permeabilized using 0.3% Triton X-100 (Acros, Pittsburgh, PA) for 3 min at room temperature, then blocked using 2% normal horse serum in PBS at 4°C for 15 min. After washing with PBS, the cells were subsequently incubated with antibody to β3, followed by FITC-conjugated goat antirabbit IgG (40 μg/ml; Jackson, West Grove, PA) and then by antibody to CK 18, followed by PE-coupled goat antimouse IgG (40 μg/ml; DAKO Corp., Carpinteria, CA). The FACS analysis was performed using FACS Vantage (Becton Dickinson, San Jose, CA). One-color FACS analysis was performed using nonpermeabilized epithelial cells with one of the following monoclonal antibodies to human integrins: VNR147 (Life Technologies, Inc.) and L230 (ATCC) to αv; P1F6 (Life Technologies, Inc.) to αvβ5; 9G6B2 (provided by Robert Pytela, University of California San Francisco) to β6; TS2/16 (ATCC) or P4C10 (Life Technologies, Inc.) to β1; 1C10; X653, a negative control supernatant; LM609 to αvβ3 or 2D5 to ICAM-1; 14E11, a nonbinding antibody used as a negative control; P1E6 (Chemicon, Temecula, CA) to α2; 9F10 (PharMingen, San Diego, CA) to α4; P1D6 (Life Technologies, Inc.) to α5; CLB-701 (Chemicon) to α6; and P1B5 (provided by Elizabeth Wayner; The Fred Hutchinson Cancer Research Center, Seattle, WA) to α3. The cells were incubated with goat antimouse FITC-conjugated secondary antibody (40 μg/ml; Cappel) at 4°C for 30 min. FACS analysis and sorting were performed using a FACSort (Becton Dickinson).

Immunoblotting and Immunoprecipitation.

Cells from primary cultures were lysed using the following lysis buffer: 50 mm Tris (pH 7.5; American Bioanalytical), 1% NP40 (Calbiochem), 2.5 mg/ml sodium deoxycholate, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 1 mm sodium orthovanadate, 20 mm NaF, 0.2 mm EGTA, and 1 mm EDTA (pH 8; all from Sigma Chemical Co.). Antibodies were rabbit sera (1:500), against the cytoplasmic domain of human β3 or αv (provided by Erkki Ruoslahti), and nonimmune serum (1:500). Protein concentrations were determined using the BCA protein assay reagent (Pierce Chemical Co., Rockford, IL), and 100 μg of lysate/lane were resolved by 10% SDS-PAGE under reducing conditions. Proteins were transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) and immunoblotted. Quantitative analysis was conducted using a computing densitometer (Molecular Dynamics, Sunnyvale, CA). To control for protein loading, membranes were stripped and blotted using rabbit antibodies against SOS-1 (4 μg/ml; Upstate Biotechnology Inc., Lake Placid, NY).

For detection of tyrosine phosphorylated forms of FAK and Cas, cells were detached using 0.05% trypsin and 0.53 mm EDTA, washed once with 0.5 mg/ml soybean trypsin inhibitor and washed twice with RPMI 1640, resuspended in serum-free RPMI 1640, and incubated at 37°C with agitation for 30 min. Cells were then plated on 60-mm dishes coated with either human VN, or human FN, or BSA, as described below, and allowed to adhere at 37°C for 45 min. Cells were lysed in the 1% NP40 lysis buffer described above. The protein concentration of each lysate was determined using BCA protein assay reagent (Pierce Chemical Co.). Precleared lysates were then incubated using either 0.5 μg of C-20, 4 μg of polyclonal antibody to p130Cas (Upstate Biotechnology Inc.), or an equivalent amount of nonimmune rabbit IgG (Sigma Chemical Co.). Western blotting analysis was performed using 1 μg/ml antiphosphotyrosine monoclonal antibody, PY20 (ICN, Costa Mesa, CA), as described previously (48). To detect immunoprecipitated proteins, membranes were stripped and stained using an antibody against FAK, C-20 (0.1 μg/ml), or a monoclonal antibody against Cas ( 0.25 μg/μl; Transduction Laboratories, Lexington, KY). Experiments were repeated three times.

Adhesion Assay.

Adhesion assays were performed as described previously (19). VN and FN were purified as described (49, 50). BSA, L230, or protein-free hybridoma medium (used as a negative control) were from Life Technologies, Inc. For antibody coating, wells were precoated with 10 μg/ml goat antimouse IgG (Cappel). Cell adhesion was quantitated by measuring the absorbance at 630 nm. Inhibition assays were performed by incubating cells in the presence of either LM609 or 1C10 or affinity purified antibodies to the VN receptor (19), or GRGESP or GRGDSPK (1 mg/ml; Life Technologies, Inc). Duplicate observations were performed in each of the above experiments. In some experiments, adhesion was quantitated using cells that had been labeled with [51Cr]sodium chromate (Amersham Corp., Arlington Heights, IL), as described previously (51). Each condition was performed in triplicate.

Migration Assay.

Cells (5–8 × 105) were resuspended in RPMI 1640 containing 1 mg/ml BSA and 0.5% FBS and plated in transwell migration chambers (12-mm pores from Corning Costar Corporation, Cambridge, MA), as described (19). For antibody coating, the inserts were incubated with RPMI 1640 containing 1 mg/ml BSA and 0.5% FBS at room temperature for 30 min. Cells were allowed to migrate for the indicated times at 37°C in the presence of 5% CO2. Cells were fixed using 3% paraformaldehyde and subsequently stained with 5 mg/ml crystal violet at room temperature. Cells that had not migrated were removed by wiping the top of the membrane with a cotton swab. The stained cells in 10 randomly chosen fields/filter were counted by microscopic examination. The numbers of migrated cells/mm2 are shown. In some experiments, cells were incubated on ice for 15 min before plating in the presence of 1:500 dilutions of either LM609 or 1C10, or 1 mg/ml peptides (GRGDSPK or GRGESP).

Statistical Analysis.

Statistical analysis was performed using the Student’s t test or one-way ANOVA, Sigma Stat (Jandel Scientific, San Rafael, CA).

Expression of αvβ3 in Prostate Cells Supports Migration on VN.

Two prostate epithelial cell lines, PC3 and LNCaP, have been shown to have high and low metastatic potentials, respectively. PC3 cells form i.p. tumors and extravasate from skeletal tissue to form metastatic lesions in nude mice. LNCaP cells do not form i.p. tumors, however, on injection into the medulla of the femur, they do form tumors that are not capable of metastasizing (2, 52). The αvβ3 integrin, a receptor for VN and other ligands, was found differentially expressed in these cell lines: specifically, PC3 but not LNCaP cells expressed αvβ3 (Fig. 1,A), as evaluated by monoclonal antibodies to the αvβ3 complex (LM609) and to αv (VNR147). Another VN receptor, αvβ5, as well as α(s)β1, were expressed at comparable levels in both cell types. A previous study (2) similarly showed lack of expression of αvβ3 by LNCaP cells, although αv-containing complexes were immunoprecipitated by a polyclonal antibody to αvβ3. The differential expression of αvβ3 in PC3 and LNCaP cells correlated with a different ability of these cells to adhere and migrate on VN (Fig. 1, B and C). PC3 cells adhered to VN and FN, whereas LNCaP cells adhered only to FN (Fig. 1,B). Both LNCaP and PC3 cells expressed an endogenous alternative VN receptor on their cell surface, αvβ5 (Fig. 1,A); however, these receptor levels were not able to mediate LNCaP cell adhesion to VN (Fig. 1,B). PC3 cells migrated on VN and FN, whereas LNCaP cells migrated only on FN (Fig. 1 C).

To investigate whether the differential expression of αvβ3 in PC3 and LNCaP cells could have a causal role in modulating the differing abilities of these two cell types to adhere to and migrate on VN (2, 52), we transfected LNCaP cells using human β3 integrin cDNA and obtained αvβ3 stable transfectants. The transfected β3 integrin associated with the endogenously expressed αv, as shown using LM609, an αvβ3 complex-specific and function-blocking monoclonal antibody (Fig. 2,A). As a control, we also generated stable LNCaP cell transfectants that either expressed a distinct surface receptor, the human ICAM-1 (Fig. 2,D), which is not expressed in LNCaP cells (Fig. 2,E) and is constitutively expressed in PC3 cells (Fig. 2,F), or were mock-transfected (Fig. 2,E). We examined the expression levels of endogenous integrins in mock-transfected LNCaP and in β3-LNCaP cells to control that potential changes in cell behavior were specifically due to surface expression of αvβ3 integrin. We found that exogenous expression of β3 in LNCaP cells did not significantly alter surface expression levels of the following integrins, which are known to be expressed in epithelial cells (23): α2, α3, α5, α6, αv, β1, and β5; neither α4 nor β6 were expressed on the surface of LNCaP cells (data not shown and Fig. 1 A).

β3-LNCaP cells adhered to VN-coated surfaces (Fig. 2,G) in a concentration-dependent manner (data not shown), whereas ICAM-LNCaP cells did not (Fig. 2,G). The two cell lines attached comparably well to L230, a monoclonal antibody to αv (Fig. 2,G). To confirm that adhesion of the β3-LNCaP cells to VN occurred in an αvβ3-dependent manner, assays were conducted in the presence of LM609. This antibody completely inhibited β3-LNCaP cell adhesion to VN, but it did not affect attachment to FN (Fig. 2,H). Similarly, LM609 or an affinity purified antibody to the VN receptor completely inhibited primary prostate cancer cell adhesion to VN (Fig. 2 I); this inhibitory effect by LM609 was consistently observed using four independent cancer cell populations (data not shown). To further confirm that adhesion to VN occurred via αvβ3 and to exclude the role of non-RGD binding receptors for VN, such as the urokinase receptor (53), we tested the ability of a RGD-containing peptide to inhibit attachment. “β3-LNCaP” cell adhesion to VN was blocked by a RGD peptide, not by a RGE-containing peptide; similarly, inhibition by RGD was consistently observed using three prostate cancer cell populations (data not shown).

To examine whether expression of αvβ3 integrin in LNCaP cells correlated with a migratory phenotype, we performed migration assays using a modified Boyden chamber system. Expression of αvβ3, but not of ICAM, in LNCaP cells resulted in migration on VN-coated surfaces, confirming that the observed effect was αvβ3-dependent (Fig. 3,A). Both ICAM-LNCaP and β3-LNCaP cells migrated equally well on TS2/16, an antibody to human β1 integrin (Fig. 3 B).

Differential Migratory Properties of Epithelial Cells from Adenocarcinoma and Normal Tissue.

We examined the role of the αvβ3 integrin in modulating migration and adhesion of epithelial cells isolated from either prostate carcinoma or from normal prostate tissue. Epithelial cells from prostate carcinoma showed a strong migratory response on VN (Fig. 3,C) and FN (not shown), whereas epithelial cells from normal prostate tissue did not (Fig. 3,D). Two cell populations obtained from independent normal tissue specimens showed similar results (Fig. 3,D and data not shown). The differences in cell migration and adhesion on VN between epithelial cells from prostatic adenocarcinoma and normal tissue were statistically significant (P < 0.0001 and P < 0.05, respectively). Epithelial cells from prostate carcinoma strongly adhered to VN and FN (Fig. 3,E), whereas epithelial cells from normal prostate tissue adhered only to FN (Fig. 3,F). All epithelial cell populations adhered and migrated equally well on FN (data not shown); therefore, FN was used as 100% control in Fig. 3, E–G. A migratory response on VN, which was similar or slightly reduced compared with FN-mediated migration, was observed using primary cells obtained from 10 independent prostate carcinoma tissues (Fig. 3 G); in contrast, the adhesive response to VN of these cell populations was variable. Epithelial cell populations isolated from six additional specimens and analyzed only for their migratory response, consistently migrated on VN (data not shown). Thus, in this regard, PC3 and LNCaP cells seem to have a phenotype similar to cells derived from cancer and normal tissue specimens, respectively.

Primary cancer cells showed a typical epithelial morphology and were stained by CK 8 and CK18 antibodies (data not shown). These cells expressed high levels of the αvβ3 integrin; a striking differential expression in cancer and normal cells (Figs. 4 and 5) was found, whereas subtle or no differences were observed in the expression of αv, β5, and β1 integrins (Fig. 5,A). To confirm that β3 expression was not due to a contaminant cell population or to culture conditions, we performed two-color flow cytometric analysis (Fig. 4, A–H) that is a more sensitive analysis than immunohistochemical staining. Fig. 4, C and G, shows that ∼36% of the cells directly isolated from the tissue specimen, and ∼89% of the cells in primary cultures were epithelial because they expressed CK 18. Fig. 4, D and H, shows that ∼46% (57% minus 11%, due to nonspecific staining) of the cells directly isolated from prostate tissue and 66% of the CK 18-positive primary cancer cells expressed β3.

Lysates of epithelial cells, isolated from either adenocarcinoma or normal tissue, were analyzed by immunoblotting using an antibody against the β3 cytoplasmic domain (Fig. 5). The results show that epithelial cells isolated from adenocarcinoma expressed β3, whereas epithelial cells from normal tissue did not (Fig. 5,C); equal protein amounts were loaded in both lanes, as evaluated using a control antibody to SOS-1 (Fig. 5,B). As expected, the β3 integrin formed a heterodimer with the αv subunit as demonstrated by immunoprecipitation using an antibody to the αv cytoplasmic domain, followed by immunoblotting using an antibody to the β3 cytoplasmic domain (Fig. 5,E). PC3 and BPH-1 prostate epithelial cells are shown as positive and negative controls for β3 expression (Fig. 5,E). The β5, αv, and β1 integrin subunits showed a similar expression pattern in cells isolated from either adenocarcinoma or normal tissues (Fig. 5 A); specifically, the αvβ5 integrin, an alternative VN receptor, was found to be poorly expressed. In conclusion, epithelial cells, isolated from fresh adenocarcinoma tissues, express β3 integrin, and this is not a consequence of de novo synthesis of the αvβ3 subunit due to cell culture conditions. On the basis of these results, we focused on studying the role of the αvβ3 integrin in prostate cancer cell adhesion and migration.

Engagement of αvβ3 in LNCaP Cells by VN Increases FAK Phosphorylation.

We examined the phosphorylation state of FAK in β3-LNCaP, PC3, and primary epithelial cells on αvβ3 integrin engagement by VN. It has been reported previously that LNCaP cells, harvested from their own ECM, have a reduced tyrosine phosphorylation of FAK compared with PC3 cells (54, 55). In agreement with this finding, we observed a very low level of FAK tyrosine phosphorylation in LNCaP cells on engagement of α5β1 (data not shown). Fig. 6,A shows a 7.3-fold increase in FAK tyrosine phosphorylation in β3-LNCaP cells plated on VN (Fig. 6,A, Lane 1) compared with cells held in suspension (Fig. 6,A, Lane 2); five experiments were performed, and an average of 6.5-fold increase was observed. FAK tyrosine phosphorylation was also increased in PC3 cells plated on VN (Fig. 6,A, Lane 4) compared with cells held in suspension (Fig. 6,A, Lane 5). In primary cells, FAK phosphorylation increased in response to adhesion to VN (Fig. 6 B, Lane 1) compared with cells held in suspension (Lane 2). Thus, FAK tyrosine phosphorylation is stimulated by engagement of αvβ3 integrin in human prostate cancer cells. Conversely, Cas tyrosine phosphorylation was not increased in β3-LNCaP or PC3 cells plated on VN (data not shown) compared with cells held in suspension. CHO cells plated on FN served as a positive control to show that Cas phosphorylation could be detected using the same experimental conditions (data not shown).

Modulation of LNCaP Cell Migration on VN by the FAK-Pathway.

To investigate whether FAK signaling would have a causal role in prostate epithelial cell migration, we cotransfected LNCaP cells using FRNK and β3 cDNAs. LNCaP that, in addition to β3, expressed FRNK (FRNK-β3-3 and FRNK-β3-4; Fig. 7,A and data not shown) or failed to express FRNK (β3-9; Fig. 7,A and data not shown) were analyzed in cell adhesion and cell migration assays (Fig. 7, BE). FRNK expression did not alter LNCaP cell transfectant adhesion to VN (Fig. 7,D), whereas it did inhibit cell migration on VN (Fig. 7,B). The effect seemed to be specific for VN because cell motility on FN remained unaffected (Fig. 7, B and C). Therefore, we conclude that engagement of αvβ3 integrin by VN in β3-LNCaP cells is accompanied by a specific tyrosine phosphorylation of FAK and that the FAK signaling pathway plays a causal role in the migration of these cells.

In this study, we show that the highly invasive PC3 cells express the αvβ3 integrin and migrate on VN. Tumor-derived human prostate epithelial cells isolated from surgical specimens, but not normal cells, also express αvβ3 and migrate on VN. Furthermore, we show that forced expression of the αvβ3 integrin induces noninvasive prostate LNCaP cells to migrate on VN. Finally, we demonstrate that the FAK-signaling pathway modulates prostate epithelial cell migration on VN.

The αvβ3 integrin, although not frequently found in epithelial cells, is very abundant in bone-residing breast cancer metastases and in malignant ovarian carcinomas (56, 57, 58); it is also abundant in metastatic melanomas both in vivo(59) and in vitro(60). Furthermore, expression of αvβ3 causes increased in vivo tumorigenicity and metastatic potential of human melanoma cells (61) and predicts subsequent metastatic progression in patients with primary cutaneous melanoma (62). Although the involvement of αvβ3 in mediating an invasive phenotype of human prostate cancer cells has not been analyzed due to the obvious difficulties in obtaining suitable samples from patients with metastatic prostate cancer, it can be speculated, on the basis of these observations, that the increased αvβ3-mediated migration of prostate cancer cells is likely to generate a metastatic phenotype in vivo. It should be stressed that a strong correlation between in vivo metastatic spread by β3-melanoma cell transfectants and in vitro β3-mediated melanoma cell migration, as evaluated using Boyden chamber assays, has been shown (63). Further support to the hypothesis that this integrin plays a role in prostate cancer cell metastatic spread derives from the observation that VN, the best characterized ligand of αvβ3, is found in mature bone tissue where these cells preferentially metastasize (64, 65). In addition to VN, another bone matrix protein, osteopontin, binds αvβ3; however, it should be pointed out that the role of osteopontin seems to be predominantly in regulating prostate epithelial cell proliferation (66).

De novo expression of the αvβ3 integrin and its engagement by VN in prostate cancer cells generate a migratory phenotype that correlates with a specific increase in FAK tyrosine phosphorylation. A correlation between FAK tyrosine phosphorylation and metastatic lesions of prostatic adenocarcinoma has been shown (54). Our results strongly suggest for the first time a causal role for FAK-signaling pathways in prostate epithelial cell migration on VN since FRNK, a negative regulator of FAK, blocks migration of these cells (37). Thus, it is conceivable that activation of FAK will modulate in vivo migration and invasion of prostate cancer cells via αvβ3. The mechanism that allows inhibition of cell migration by FRNK in a substrate-specific manner (i.e., on VN and not on FN) and the potential ability of FRNK to block parallel pathways, remain to be investigated. A specific role for the β3 cytodomain in FAK phosphorylation and cell migration has been described by two independent studies, showing that the NPXY motif in the β3 cytodomain is required to support FAK phosphorylation in fibroblasts (29) as well as in melanoma cell migration and metastatic spread in vivo(63). It remains to be established whether prostate cancer cells also use this motif for their VN-mediated cell migration and FAK phosphorylation.

Cas and PI 3-kinase, both of which form complexes with FAK, are believed to act as downstream effectors of FAK and to control cell migration (39, 40, 67). In our system, although FAK is autophosphorylated and is known to phosphorylate Cas (34), the latter did not seem to be involved in αvβ3 signaling in LNCaP cells because its tyrosine phosphorylation remains undetectable in response to VN attachment (data not shown). A role for PI 3-kinase, a signaling molecule that has been shown to play a role in integrin-mediated epithelial cell motility (68, 69), as a potential downstream mediator of αvβ3 and FAK-activated pathways, remains to be investigated. Other downstream effectors of FAK are members of the MAP kinase family. The role of MAP kinase in prostate cell migration does not seem to be predominant because a specific inhibitor of MEK-1, PD98059 (70), did not affect cell migration of β3-LNCaP cells on VN, whereas it did inhibit endothelial cell migration, as described previously (43).5 Similarly, Cary et al.(34) have shown that PD98059 had no effect on FAK/Cas-dependent CHO cell migration, indicating a cell type-dependent activity of the MAP kinase pathway on migration.

Studies performed using tissue sections or cell lines have shown changes in integrin expression between cancer and benign prostate epithelial cells; specifically, redistribution of α6β1(7) as well as β1C down-regulation in prostate cancer tissues have been described (9); furthermore, α6β4 has been shown to be up-regulated in metastatic prostate cancer cell lines (3). For the first time, in this study, an analysis of integrin expression using prostate cells isolated from fresh tissue samples has been performed. The data show that: (a) αvβ3 is expressed only by tumor-derived primary cells, but not by normal, prostate epithelial cells; and (b) the expression of αvβ3 is not induced by culture conditions, but is found constitutively in freshly isolated epithelial cells. Although it is conceivable that ultimately the altered cancerous phenotype will be contributed to by several surface receptors, our study provides, for the first time, evidence that the αvβ3 integrin is up-regulated in freshly isolated prostate cancer cells and is a predominant player in the control of migration of these cells. LNCaP cells express αvβ5, an alternative receptor for VN (20), that can mediate cell migration of epithelial and melanoma cells on growth factor stimulation (71); however, this integrin that binds VN in several cell types (20, 71, 72) does not participate in β3-LNCaP binding to VN because a complex-specific antibody to αvβ3 completely inhibited β3-LNCaP cell adhesion to VN.

In conclusion, our study suggests that the αvβ3 integrin and the signaling molecules downstream to αvβ3 are potential targets to prevent prostate cancer invasion and metastatic spread.

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

Supported by NIH Grants CA-71870 and DK-52670, Donaghue Medical Research Foundation Grant 95-006, and Army PCRP Grant DAMD17-98-1-8506 (to L. R. L). M. F. is recipient of the Donaghue Medical Research Foundation Fellowship Award.

                  
4

The abbreviations used are: ECM, extracellular matrix; FAK, focal adhesion kinase; FRNK, FAK-related nonkinase; MAP, mitogen-activated protein; PE, Phycoerythrin; VN, vitronectin; FN, fibronectin; FACS, fluorescence-activated cell sorter; FBS, fetal bovine serum; ATCC, American Type Culture Collection; Cas, Crk-associated substrate; CK, cytokeratin.

      
5

A. S. Woodard and L. R. Languino, unpublished results.

Fig. 1.

PC3 and LNCaP cell differential expression of αvβ3 integrin and migration on VN. A, expression of integrins in PC3 and LNCaP cells by FACS analysis is shown. Monoclonal antibodies to αv VNR147 (1:500), αvβ3 LM609 (1:500), β5 P1F5 (1:500), β6 9G6B2 (1:10), and β1 P4C10 (1:500) were used. BPH-1 cells were used as positive control for the β6 integrin. B, PC3 or LNCaP cells (2.5 × 104) were labeled using [51Cr]sodium chromate and allowed to attach to VN (▪, 30 μg/ml), FN (▨, 3 μg/ml), or BSA (□, 10 μg/ml) at 37°C for 2 h. C, PC3 or LNCaP cells (8 × 105) were allowed to migrate on VN (5 μg/ml), FN (0.5 μg/ml), or BSA (10 μg/ml) at 37°C for 16 h. BSA was used as negative control. In B and C, migration and adhesion experiments were repeated at least three times with consistent results. Bars, mean ± SE (n = 3).

Fig. 1.

PC3 and LNCaP cell differential expression of αvβ3 integrin and migration on VN. A, expression of integrins in PC3 and LNCaP cells by FACS analysis is shown. Monoclonal antibodies to αv VNR147 (1:500), αvβ3 LM609 (1:500), β5 P1F5 (1:500), β6 9G6B2 (1:10), and β1 P4C10 (1:500) were used. BPH-1 cells were used as positive control for the β6 integrin. B, PC3 or LNCaP cells (2.5 × 104) were labeled using [51Cr]sodium chromate and allowed to attach to VN (▪, 30 μg/ml), FN (▨, 3 μg/ml), or BSA (□, 10 μg/ml) at 37°C for 2 h. C, PC3 or LNCaP cells (8 × 105) were allowed to migrate on VN (5 μg/ml), FN (0.5 μg/ml), or BSA (10 μg/ml) at 37°C for 16 h. BSA was used as negative control. In B and C, migration and adhesion experiments were repeated at least three times with consistent results. Bars, mean ± SE (n = 3).

Close modal
Fig. 2.

β3-LNCaP and primary cancer cells adhere to VN in an αvβ3-dependent manner. AF, surface expression of αvβ3 is shown by FACS analysis: β3-LNCaP (A), mock-LNCaP (B and E), PC3 (C and F), or ICAM-LNCaP (D). AC, the bold line shows cells labeled using LM609, monoclonal antibody to αvβ3, and the thin line shows cells labeled using 1C10, negative control monoclonal antibody. DF, the bold line shows cells labeled using 2D5, monoclonal antibody to ICAM-1, and the thin line shows cells labeled using 14E11, negative control monoclonal antibody. G–I, β3-LNCaP and primary cancer cell adhesion to VN. G, β3-LNCaP or ICAM-LNCaP cells (1 × 105) were allowed to attach to VN (▪) or BSA (□; both at 3 μg/ml), L230 (), or negative control (▨) hybridoma medium (coating concentrations, 1:500) for 1 h. H, β3-LNCaP cells (1 × 105) were allowed to attach to VN or FN (both at 3 μg/ml) in the presence of LM609 or 1C10 (both at 1:1000) for 105 min. H, the results are shown as the percentage of attachment to VN or FN in the presence of the negative control antibody 1C10. The differences between 1C10 and LM609 effects on VN attachment were statistically significant (*, P < 0.03; , 1C10; □, LM609). I, effect of LM609 on primary prostate cancer cell adhesion to VN. Primary cancer cells (2.5 × 104) labeled by [51Cr]sodium chromate were allowed to attach to VN in the presence of 1C10 (), LM609 (□), control IgG (▨), or rabbit antibody () to the VN receptor (all at 1:500), or BSA (; 3 μg/ml) for 120 min. The differences in cell adhesion to VN between LM609 and 1C10 or between antibody to the VN receptor and control IgG, indicated by asterisks, were statistically significant (P < 0.05). In all of the above, consistent results were obtained from at least two different experiments. Representative experiments are shown. GI, bars are the mean ± SE (n = 3).

Fig. 2.

β3-LNCaP and primary cancer cells adhere to VN in an αvβ3-dependent manner. AF, surface expression of αvβ3 is shown by FACS analysis: β3-LNCaP (A), mock-LNCaP (B and E), PC3 (C and F), or ICAM-LNCaP (D). AC, the bold line shows cells labeled using LM609, monoclonal antibody to αvβ3, and the thin line shows cells labeled using 1C10, negative control monoclonal antibody. DF, the bold line shows cells labeled using 2D5, monoclonal antibody to ICAM-1, and the thin line shows cells labeled using 14E11, negative control monoclonal antibody. G–I, β3-LNCaP and primary cancer cell adhesion to VN. G, β3-LNCaP or ICAM-LNCaP cells (1 × 105) were allowed to attach to VN (▪) or BSA (□; both at 3 μg/ml), L230 (), or negative control (▨) hybridoma medium (coating concentrations, 1:500) for 1 h. H, β3-LNCaP cells (1 × 105) were allowed to attach to VN or FN (both at 3 μg/ml) in the presence of LM609 or 1C10 (both at 1:1000) for 105 min. H, the results are shown as the percentage of attachment to VN or FN in the presence of the negative control antibody 1C10. The differences between 1C10 and LM609 effects on VN attachment were statistically significant (*, P < 0.03; , 1C10; □, LM609). I, effect of LM609 on primary prostate cancer cell adhesion to VN. Primary cancer cells (2.5 × 104) labeled by [51Cr]sodium chromate were allowed to attach to VN in the presence of 1C10 (), LM609 (□), control IgG (▨), or rabbit antibody () to the VN receptor (all at 1:500), or BSA (; 3 μg/ml) for 120 min. The differences in cell adhesion to VN between LM609 and 1C10 or between antibody to the VN receptor and control IgG, indicated by asterisks, were statistically significant (P < 0.05). In all of the above, consistent results were obtained from at least two different experiments. Representative experiments are shown. GI, bars are the mean ± SE (n = 3).

Close modal
Fig. 3.

β3-LNCaP and primary prostate cancer cells migrate on VN. A, β3-LNCaP or ICAM-LNCaP cells (4 × 105) were allowed to migrate on VN or BSA (both at 3 μg/ml) in each insert at 37°C for 4 h. B, β3-LNCaP or ICAM-LNCaP cells (4 × 105) were allowed to migrate on TS2/16 (coating concentrations, 1:500) or BSA (3 μg/ml) in each insert at 37°C for 4 h. CG, epithelial cells from prostatic adenocarcinoma or normal prostate tissue differentially adhere to and migrate on VN-coated substrates. Epithelial cells (2 × 105) from prostate carcinoma (C) or normal (D) prostate tissue were allowed to migrate at 37°C for 16 h in Boyden chambers that were coated using VN (3 μg/ml) or BSA (10 μg/ml). The differences between cancer and normal epithelial cell migration on VN were statistically significant (P < 0.0001). Epithelial cells (E; 2.5 × 104) from prostate carcinoma or normal prostate (F) tissue were labeled using [51Cr]sodium chromate and allowed to attach to VN (3 μg/ml), BSA (10 μg/ml), or FN (3 μg/ml) at 37°C for 2 h. E–G, the results are expressed as the percentage of control; either migration on or adhesion to FN for each cell population was used as 100% control. G, BSA values were subtracted in migration and adhesion assays. The differences between cancer and normal epithelial cell adhesion to VN were statistically significant (P < 0.05). Cell migration and adhesion assays were performed using 10 prostatic adenocarcinoma tissue specimens. Each symbol (▴, migrated cells; •, attached cells) represents one independent case of prostatic adenocarcinoma. AF, bars are the mean ± SE (n = 3).

Fig. 3.

β3-LNCaP and primary prostate cancer cells migrate on VN. A, β3-LNCaP or ICAM-LNCaP cells (4 × 105) were allowed to migrate on VN or BSA (both at 3 μg/ml) in each insert at 37°C for 4 h. B, β3-LNCaP or ICAM-LNCaP cells (4 × 105) were allowed to migrate on TS2/16 (coating concentrations, 1:500) or BSA (3 μg/ml) in each insert at 37°C for 4 h. CG, epithelial cells from prostatic adenocarcinoma or normal prostate tissue differentially adhere to and migrate on VN-coated substrates. Epithelial cells (2 × 105) from prostate carcinoma (C) or normal (D) prostate tissue were allowed to migrate at 37°C for 16 h in Boyden chambers that were coated using VN (3 μg/ml) or BSA (10 μg/ml). The differences between cancer and normal epithelial cell migration on VN were statistically significant (P < 0.0001). Epithelial cells (E; 2.5 × 104) from prostate carcinoma or normal prostate (F) tissue were labeled using [51Cr]sodium chromate and allowed to attach to VN (3 μg/ml), BSA (10 μg/ml), or FN (3 μg/ml) at 37°C for 2 h. E–G, the results are expressed as the percentage of control; either migration on or adhesion to FN for each cell population was used as 100% control. G, BSA values were subtracted in migration and adhesion assays. The differences between cancer and normal epithelial cell adhesion to VN were statistically significant (P < 0.05). Cell migration and adhesion assays were performed using 10 prostatic adenocarcinoma tissue specimens. Each symbol (▴, migrated cells; •, attached cells) represents one independent case of prostatic adenocarcinoma. AF, bars are the mean ± SE (n = 3).

Close modal
Fig. 4.

β3 integrin and CK 18 coexpression in human prostate cancer cells. Freshly isolated (A–D) or primary (E–H) cultures of prostate cancer cells were incubated with serum against the cytoplasmic domain of the β3 integrin and labeled using FITC-conjugated antibody to rabbit IgG (B and F) or with a monoclonal antibody antihuman CK 18 and labeled using PE-conjugated antibody to murine IgG (C and G). D and H, cells were double stained with antibodies to β3 integrin and to CK 18, followed by FITC- and PE-conjugated antibodies. As negative control, double-staining was performed using nonimmune rabbit serum and 1C10 monoclonal antibody (A and E), followed by FITC- and PE-conjugated antibodies. Fluorescence intensity is expressed in arbitrary units. The experiment was repeated using either three or two independently obtained prostate cell populations with consistent results. A–H, a representative dot plot showing staining for β3 on the horizontal axis and for CK 18 on the vertical axis is shown; the percentage of cells expressing the single epitope is shown. D and H, CK 18-positive cells express β3 (top right). G, a typical cell population in primary culture was composed of 89% epithelial cells (top left).

Fig. 4.

β3 integrin and CK 18 coexpression in human prostate cancer cells. Freshly isolated (A–D) or primary (E–H) cultures of prostate cancer cells were incubated with serum against the cytoplasmic domain of the β3 integrin and labeled using FITC-conjugated antibody to rabbit IgG (B and F) or with a monoclonal antibody antihuman CK 18 and labeled using PE-conjugated antibody to murine IgG (C and G). D and H, cells were double stained with antibodies to β3 integrin and to CK 18, followed by FITC- and PE-conjugated antibodies. As negative control, double-staining was performed using nonimmune rabbit serum and 1C10 monoclonal antibody (A and E), followed by FITC- and PE-conjugated antibodies. Fluorescence intensity is expressed in arbitrary units. The experiment was repeated using either three or two independently obtained prostate cell populations with consistent results. A–H, a representative dot plot showing staining for β3 on the horizontal axis and for CK 18 on the vertical axis is shown; the percentage of cells expressing the single epitope is shown. D and H, CK 18-positive cells express β3 (top right). G, a typical cell population in primary culture was composed of 89% epithelial cells (top left).

Close modal
Fig. 5.

Differential expression of β3 integrin in epithelial cells derived from human adenocarcinoma or normal prostate. A, FACS analysis of epithelial cells obtained from adenocarcinoma or normal tissue was performed using monoclonal antibodies against human αv or β5 or β1 integrins. BPH-1 cells were used as positive control for the antibody against β5. Primary epithelial cell lysates from either tumor (B–D, Lane 1) or normal (B–D, Lane 2) tissue specimens were separated using a 7.5% SDS-PAGE, and immunoblotting was performed using serum against the β3 integrin (C), nonimmune serum (D), or an antibody against SOS-1 (B); the last was used as control for protein loading. E, association of αv and β3 integrin subunits was shown by immunoprecipitation using serum against αv (Lanes 4 and 6), followed by immunoblotting using serum against β3. Nonimmune rabbit serum was used in immunoprecipitations as a negative control (Lanes 3 and 5). Two independent primary epithelial cell populations (Lanes 3 and 4 and Lanes5 and 6, respectively) isolated from tumor specimens are shown. Total lysates from PC3 (Lane 1) or BPH-1 (Lane 2) cells were used as positive and negative controls for β3 integrin expression, respectively.

Fig. 5.

Differential expression of β3 integrin in epithelial cells derived from human adenocarcinoma or normal prostate. A, FACS analysis of epithelial cells obtained from adenocarcinoma or normal tissue was performed using monoclonal antibodies against human αv or β5 or β1 integrins. BPH-1 cells were used as positive control for the antibody against β5. Primary epithelial cell lysates from either tumor (B–D, Lane 1) or normal (B–D, Lane 2) tissue specimens were separated using a 7.5% SDS-PAGE, and immunoblotting was performed using serum against the β3 integrin (C), nonimmune serum (D), or an antibody against SOS-1 (B); the last was used as control for protein loading. E, association of αv and β3 integrin subunits was shown by immunoprecipitation using serum against αv (Lanes 4 and 6), followed by immunoblotting using serum against β3. Nonimmune rabbit serum was used in immunoprecipitations as a negative control (Lanes 3 and 5). Two independent primary epithelial cell populations (Lanes 3 and 4 and Lanes5 and 6, respectively) isolated from tumor specimens are shown. Total lysates from PC3 (Lane 1) or BPH-1 (Lane 2) cells were used as positive and negative controls for β3 integrin expression, respectively.

Close modal
Fig. 6.

αvβ3 engagement by VN stimulates FAK phosphorylation. A, β3-LNCaP or PC3 cells were plated on VN (Lanes 1 and 4; 3 μg/ml) or held in suspension (Lanes 2, 3, 5, and 6) for 45 min and lysed; β3-LNCaP (500 μg) or 200 μg PC3 lysate/immunoprecipitation were used. B, primary epithelial cells from prostatic adenocarcinoma were plated on VN (Lane 1; 3 μg/ml) or held in suspension (Lane 2) for 70 min and lysed; lysate (300 μg)/immunoprecipitation were used. A and B, anti-FAK immunoprecipitates were separated by 7.5% SDS-PAGE under reducing conditions and immunoblotted using PY20, antiphosphotyrosine monoclonal (top), and C-20, anti-FAK polyclonal antibody (bottom).

Fig. 6.

αvβ3 engagement by VN stimulates FAK phosphorylation. A, β3-LNCaP or PC3 cells were plated on VN (Lanes 1 and 4; 3 μg/ml) or held in suspension (Lanes 2, 3, 5, and 6) for 45 min and lysed; β3-LNCaP (500 μg) or 200 μg PC3 lysate/immunoprecipitation were used. B, primary epithelial cells from prostatic adenocarcinoma were plated on VN (Lane 1; 3 μg/ml) or held in suspension (Lane 2) for 70 min and lysed; lysate (300 μg)/immunoprecipitation were used. A and B, anti-FAK immunoprecipitates were separated by 7.5% SDS-PAGE under reducing conditions and immunoblotted using PY20, antiphosphotyrosine monoclonal (top), and C-20, anti-FAK polyclonal antibody (bottom).

Close modal
Fig. 7.

FRNK expression inhibits β3-LNCaP cell migration on VN. A, LNCaP cells cotransfected using β3 and FRNK cDNAs are designated FRNK-β3-3, FRNK-β3-4, or β3-9. Each cell lysate (30 μg) was separated using a 7.5% SDS-PAGE and analyzed by immunoblotting using C-20, antibody against FAK (bottom), which also recognizes FRNK (top). Only FRNK-β3-3 and FRNK-β3-4 transfectants expressed detectable levels of FRNK. Computing densitometric analysis showed that FRNK-β3-3 had >2-fold expression of FRNK compared with FRNK-β3-4, whereas β3-9 did not express it. FRNK-β3-3, FRNK-β3-4, or β3-9 expressed comparable levels of endogenous FAK (1.08, 1.00, and 0.94, respectively). B and C, LNCaP cells (4 × 105) expressing β3 and FRNK (▪), or expressing β3 but not FRNK (▧), were allowed to migrate on VN (3 and 10 μg/ml), FN (3 μg/ml), or BSA (3 μg/ml) at 37°C for 4 h. D and E, FRNK-β3-3 or β3-9 cells (2.5 × 104)/well were allowed to attach to VN (3, 10, and 30 μg/ml), FN (3 μg/ml), or BSA (3 μg/ml) at 37°C for 2 h. B–E, bars are the mean ± SE (n = 3).

Fig. 7.

FRNK expression inhibits β3-LNCaP cell migration on VN. A, LNCaP cells cotransfected using β3 and FRNK cDNAs are designated FRNK-β3-3, FRNK-β3-4, or β3-9. Each cell lysate (30 μg) was separated using a 7.5% SDS-PAGE and analyzed by immunoblotting using C-20, antibody against FAK (bottom), which also recognizes FRNK (top). Only FRNK-β3-3 and FRNK-β3-4 transfectants expressed detectable levels of FRNK. Computing densitometric analysis showed that FRNK-β3-3 had >2-fold expression of FRNK compared with FRNK-β3-4, whereas β3-9 did not express it. FRNK-β3-3, FRNK-β3-4, or β3-9 expressed comparable levels of endogenous FAK (1.08, 1.00, and 0.94, respectively). B and C, LNCaP cells (4 × 105) expressing β3 and FRNK (▪), or expressing β3 but not FRNK (▧), were allowed to migrate on VN (3 and 10 μg/ml), FN (3 μg/ml), or BSA (3 μg/ml) at 37°C for 4 h. D and E, FRNK-β3-3 or β3-9 cells (2.5 × 104)/well were allowed to attach to VN (3, 10, and 30 μg/ml), FN (3 μg/ml), or BSA (3 μg/ml) at 37°C for 2 h. B–E, bars are the mean ± SE (n = 3).

Close modal

We acknowledge Drs. D. A. Cheresh, R. Pytela, E. Ruoslahti, and E. Wayner for generously providing antibodies and Dr. S. W. Hayward for providing BPH-1 cells. We are grateful to Drs. J. T. Parsons and W. Xiong and to Drs. T. O’Toole and K. Vuori for providing cDNA constructs. Special thanks to Drs. M. Centrella, F. Peracchia, and C. A. Steger for critical comments on the manuscript. In addition, we thank R. Carbone, Q. Cui, L. DeDios, and A. E. Slear for excellent technical assistance and N. Bennett for helping with preparation of the manuscript.

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