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Tumor Biology |
vß3Integrin Is Modulated by a Focal Adhesion Kinase Pathway1
Departments of Pathology [D-Q. Z., M. F., G. T., L. R. L.] and Biology [A. S. W.], Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 integrin; in contrast, the noninvasive LNCaP prostate cancer cell line did not express vß3. PC3 cells adhered to and migrated on vitronectin (VN), an vß3 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 vß3, whereas normal prostate epithelial cells did not. In addition, only primary prostate cancer cells adhered to and migrated on VN. The role of vß3 in mediating prostate epithelial cell migration was confirmed using LNCaP cell transfectants expressing ß3 (ß3-LNCaP). Exogenous expression of vß3 induced LNCaP cells to adhere to and migrate on VN. In response to vß3 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 ß3-LNCaP cell migration, specifically on VN. These data indicate that de novo expression of vß3 integrin in prostate cancer cells generates a migratory phenotype that is modulated by a FAK signaling pathway. This study points to vß3 as potential target in prostate cancer cell invasion and metastasis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß 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 vß3 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, vß5 integrin can replace the function of vß3 (20, 21, 22, 23)
. Signaling from the vß3 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 ß integrin cytoplasmic domain mimetic peptides (28)
. Domains within the amino- and COOH-terminal regions of the ß3 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 6Aß1, 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 vß3 integrin and migrate on VN, an vß3 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 vß3 integrin, and only cancer cells adhere to and migrate on VN. Forced expression of vß3 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 ß3 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 vß3 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.
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MATERIALS AND METHODS |
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vß3 integrin or 2D5 (25 µg/ml) against human ICAM-1. ß3- and FRNK-ß3 (FRNK-ß3-3 and FRNK-ß3-4) 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 ß3-LNCaP in either ß3- or FRNK-ß3 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 x 0.1 x 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 ß3 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 ß3, 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 v; P1F6 (Life Technologies, Inc.) to vß5; 9G6B2 (provided by Robert Pytela, University of California San Francisco) to ß6; TS2/16 (ATCC) or P4C10 (Life Technologies, Inc.) to ß1; 1C10; X653, a negative control supernatant; LM609 to vß3 or 2D5 to ICAM-1; 14E11, a nonbinding antibody used as a negative control; P1E6 (Chemicon, Temecula, CA) to 2; 9F10 (PharMingen, San Diego, CA) to 4; P1D6 (Life Technologies, Inc.) to 5; CLB-701 (Chemicon) to 6; and P1B5 (provided by Elizabeth Wayner; The Fred Hutchinson Cancer Research Center, Seattle, WA) to 3. 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 ß3 or v (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 x 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).
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RESULTS |
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vß3 in Prostate Cells Supports Migration on VN.vß3 integrin, a receptor for VN and other ligands, was found differentially expressed in these cell lines: specifically, PC3 but not LNCaP cells expressed vß3 (Fig. 1A)
, as evaluated by monoclonal antibodies to the vß3 complex (LM609) and to v (VNR147). Another VN receptor, vß5, as well as (s)ß1, were expressed at comparable levels in both cell types. A previous study (2)
similarly showed lack of expression of vß3 by LNCaP cells, although v-containing complexes were immunoprecipitated by a polyclonal antibody to vß3. The differential expression of vß3 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. 1B)
. Both LNCaP and PC3 cells expressed an endogenous alternative VN receptor on their cell surface, vß5 (Fig. 1A)
; however, these receptor levels were not able to mediate LNCaP cell adhesion to VN (Fig. 1B)
. PC3 cells migrated on VN and FN, whereas LNCaP cells migrated only on FN (Fig. 1C)
.
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vß3 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 ß3 integrin cDNA and obtained vß3 stable transfectants. The transfected ß3 integrin associated with the endogenously expressed v, as shown using LM609, an vß3 complex-specific and function-blocking monoclonal antibody (Fig. 2A)
. As a control, we also generated stable LNCaP cell transfectants that either expressed a distinct surface receptor, the human ICAM-1 (Fig. 2D)
, which is not expressed in LNCaP cells (Fig. 2E)
and is constitutively expressed in PC3 cells (Fig. 2F)
, or were mock-transfected (Fig. 2E)
. We examined the expression levels of endogenous integrins in mock-transfected LNCaP and in ß3-LNCaP cells to control that potential changes in cell behavior were specifically due to surface expression of vß3 integrin. We found that exogenous expression of ß3 in LNCaP cells did not significantly alter surface expression levels of the following integrins, which are known to be expressed in epithelial cells (23)
: 2, 3, 5, 6, v, ß1, and ß5; neither 4 nor ß6 were expressed on the surface of LNCaP cells (data not shown and Fig. 1A
).
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in a concentration-dependent manner (data not shown), whereas ICAM-LNCaP cells did not (Fig. 2G)
. The two cell lines attached comparably well to L230, a monoclonal antibody to v (Fig. 2G)
. To confirm that adhesion of the ß3-LNCaP cells to VN occurred in an vß3-dependent manner, assays were conducted in the presence of LM609. This antibody completely inhibited ß3-LNCaP cell adhesion to VN, but it did not affect attachment to FN (Fig. 2H)
. Similarly, LM609 or an affinity purified antibody to the VN receptor completely inhibited primary prostate cancer cell adhesion to VN (Fig. 2I)
; 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 vß3 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. "ß3-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 vß3 integrin in LNCaP cells correlated with a migratory phenotype, we performed migration assays using a modified Boyden chamber system. Expression of vß3, but not of ICAM, in LNCaP cells resulted in migration on VN-coated surfaces, confirming that the observed effect was vß3-dependent (Fig. 3A)
. Both ICAM-LNCaP and ß3-LNCaP cells migrated equally well on TS2/16, an antibody to human ß1 integrin (Fig. 3B)
.
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vß3 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. 3C)
and FN (not shown), whereas epithelial cells from normal prostate tissue did not (Fig. 3D)
. Two cell populations obtained from independent normal tissue specimens showed similar results (Fig. 3D
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. 3E)
, whereas epithelial cells from normal prostate tissue adhered only to FN (Fig. 3F)
. 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. 3G)
; 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 vß3 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 v, ß5, and ß1 integrins (Fig. 5A)
. To confirm that ß3 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 ß3.
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. The results show that epithelial cells isolated from adenocarcinoma expressed ß3, whereas epithelial cells from normal tissue did not (Fig. 5C)
; equal protein amounts were loaded in both lanes, as evaluated using a control antibody to SOS-1 (Fig. 5B)
. As expected, the ß3 integrin formed a heterodimer with the v subunit as demonstrated by immunoprecipitation using an antibody to the v cytoplasmic domain, followed by immunoblotting using an antibody to the ß3 cytoplasmic domain (Fig. 5E)
. PC3 and BPH-1 prostate epithelial cells are shown as positive and negative controls for ß3 expression (Fig. 5E)
. The ß5, v, and ß1 integrin subunits showed a similar expression pattern in cells isolated from either adenocarcinoma or normal tissues (Fig. 5A)
; specifically, the vß5 integrin, an alternative VN receptor, was found to be poorly expressed. In conclusion, epithelial cells, isolated from fresh adenocarcinoma tissues, express ß3 integrin, and this is not a consequence of de novo synthesis of the vß3 subunit due to cell culture conditions. On the basis of these results, we focused on studying the role of the vß3 integrin in prostate cancer cell adhesion and migration.
Engagement of vß3 in LNCaP Cells by VN Increases FAK Phosphorylation.
We examined the phosphorylation state of FAK in ß3-LNCaP, PC3, and primary epithelial cells on vß3 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 5ß1 (data not shown). Fig. 6A
shows a 7.3-fold increase in FAK tyrosine phosphorylation in ß3-LNCaP cells plated on VN (Fig. 6A
, Lane 1) compared with cells held in suspension (Fig. 6A
, 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. 6A
, Lane 4) compared with cells held in suspension (Fig. 6A
, Lane 5). In primary cells, FAK phosphorylation increased in response to adhesion to VN (Fig. 6B
, Lane 1) compared with cells held in suspension (Lane 2). Thus, FAK tyrosine phosphorylation is stimulated by engagement of vß3 integrin in human prostate cancer cells. Conversely, Cas tyrosine phosphorylation was not increased in ß3-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).
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and data not shown) or failed to express FRNK (ß3-9; Fig. 7A
and data not shown) were analyzed in cell adhesion and cell migration assays (Fig. 7, B–E)
. FRNK expression did not alter LNCaP cell transfectant adhesion to VN (Fig. 7D)
, whereas it did inhibit cell migration on VN (Fig. 7B)
. 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 vß3 integrin by VN in ß3-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.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 integrin and migrate on VN. Tumor-derived human prostate epithelial cells isolated from surgical specimens, but not normal cells, also express vß3 and migrate on VN. Furthermore, we show that forced expression of the vß3 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 vß3 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 vß3 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 vß3 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 vß3-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 ß3-melanoma cell transfectants and in vitro ß3-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 vß3, is found in mature bone tissue where these cells preferentially metastasize (64
, 65)
. In addition to VN, another bone matrix protein, osteopontin, binds vß3; 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 vß3 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 vß3. 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 ß3 cytodomain in FAK phosphorylation and cell migration has been described by two independent studies, showing that the NPXY motif in the ß3 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 vß3 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 vß3 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 ß3-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 6ß1 (7)
as well as ß1C down-regulation in prostate cancer tissues have been described (9)
; furthermore, 6ß4 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) vß3 is expressed only by tumor-derived primary cells, but not by normal, prostate epithelial cells; and (b) the expression of vß3 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 vß3 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 vß5, 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 ß3-LNCaP binding to VN because a complex-specific antibody to vß3 completely inhibited ß3-LNCaP cell adhesion to VN.
In conclusion, our study suggests that the vß3 integrin and the signaling molecules downstream to vß3 are potential targets to prevent prostate cancer invasion and metastatic spread.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. 
2 These authors contributed equally to this report.
3 To whom requests for reprints should be addressed, at Department of Pathology, Yale University School of Medicine, P. O. Box 208023, 310 Cedar Street, New Haven, CT 06520. Phone: (203) 737-1454; Fax: (203) 737-1455; E-mail: lucia.languino{at}Yale.Edu
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.
Received 11/13/98. Accepted 2/ 2/99.
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3ß1 and 6ß4 integrins in highly invasive and metastatic variants of human prostate carcinoma cells selected by in vitro invasion through reconstituted basement membrane. Clin. Exp. Metastasis, 11: 391-400, 1993.[Medline]
6 expression in human prostate carcinoma cells is associated with a migratory and invasive phenotype in vitro and in vivo. Clin. Exp. Metastasis, 13: 481-491, 1995.[Medline]
6 and 2 very late antigen integrins in the normal, hyperplastic, and neoplastic prostate: simultaneous demonstration of cell surface receptors and their extracellular ligands. Hum. Pathol., 24: 243-248, 1993.[Medline]
6-integrins in the normal and neoplastic prostate. Am. J. Pathol., 145: 167-174, 1994.[Abstract]
vß3 integrin in human melanoma cell invasion. Proc. Natl. Acad. Sci. USA, 89: 1557-1561, 1992.vß3 integrin and PDGF receptor increases cell migration. J. Cell Sci., 111: 469-478, 1998.[Abstract]
vßx) integrin is responsible for distinct adhesive properties of carcinoma cells. Cell, 57: 59-69, 1989.[Medline]
subunit (v) in a human ostesarcoma cell line and is a substrate for protein kinase C. EMBO J., 8: 2955-2965, 1989.[Medline]
vß8 interaction with vitronectin and functional divergence of the ß8 cytoplasmic domain. J. Biol. Chem., 269: 28708-28715, 1994.vß3 integrin associates with a 190-kDa protein that is phosphorylated on tyrosine in response to platelet-derived growth factor. J. Biol. Chem., 268: 17270-17276, 1993.6 integrin subunit. J. Biol. Chem., 273: 5903-5907, 1998.vß3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis. J. Cell Biol., 140: 1255-1263, 1998.vß3 integrins in ovarian cancer. Gynecol. Oncol., 58: 216-225, 1995.[Medline]
vß3 expression by bone-residing breast cancer metastases. Diagn. Mol. Pathol., 5: 127-135, 1996.[Medline]
vß3 integrin is less frequent in ovarian epithelial tumors of low malignant potential in contrast to ovarian carcinomas. Hum. Path., 28: 443-449, 1997.[Medline]
v gene expression in human melanoma tumorigenicity. J. Clin. Invest., 89: 2018-2022, 1992.
6ß4 integrin promotes carcinoma invasion. Cell, 91: 949-960, 1997.[Medline]
vß5-directed cell motility but not adhesion on vitronectin. J. Cell Biol., 127: 859-866, 1994.This article has been cited by other articles:
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, 30 µg/ml), FN (
, 3 µg/ml), or BSA (
, 10 µg/ml) at 37°C for 2 h. C, PC3 or LNCaP cells (8 x 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).
), or negative control (
, 1C10; 
) 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. G–I, bars are the mean ± SE (n = 3).
, migrated cells; •, attached cells) represents one independent case of prostatic adenocarcinoma. A–F, bars are the mean ± SE (n = 3).
), 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-ß3-3 or ß3-9 cells (2.5 x 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).