Integrin {alpha}vß3 mediates K1735 murine melanoma cell motility in vivo and in vitro

Xiaowu Li1, Joseph Regezi1, F. Patrick Ross3, Scott Blystone4, Dusko Ilic1, Stanley P. L. Leong2 and Daniel M. Ramos1,*

1 Department of Stomatology, University of California San Francisco, San Francisco, CA 94143, USA
2 Department of Surgery, University of California San Francisco, San Francisco, CA 94143, USA
3 Department of Pathology, Washington University School of Medicine, St Louis, MO 63110, USA
4 Departments of Anatomy and Cell Biology, SUNY Health Science Center, Syracuse, NY 13210, USA
* Author for correspondence (e-mail: dramos{at}itsa.ucsf.edu )


    SUMMARY
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 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The integrin {alpha}vß3 has been shown to be tightly linked to progression of human melanoma. In this study, using two clones from the K1735 murine melanoma system, we investigated the role of {alpha}vß3 in metastasis. The highly metastatic K1735M2 cells express the {alpha}vß3 integrin, whereas the poorly metastatic K1735C23 cells do not. When transduced with the ß3 integrin subunit cDNA, the K1735C23 cells produced lung lesions and, in two animals, cardiac metastases, whereas the parental C23 cells did not. By contrast, transduction of the full-length ß3 integrin antisense DNA into the K1735M2 cells suppressed metastatic colonization. To specifically investigate the activation of ß3 integrin-mediated pathways, the ß3-positive and the ß3-negative K1735 cells were plated onto vitronectin, a major matrix molecule of both primary and metastatic melanomas. Tyr397 of FAK was phosphorylated several times higher in ß3-expressing K1735 melanoma cells than in ß3-negative cells. To determine whether phosphorylation of FAK was associated with K1735 melanoma motility, we expressed the FAK-related non-kinase (FRNK) in the highly metastatic K1735M2 cells. Exogenous expression of FRNK suppressed phosphorylation of FAK at Tyr397 and decreased the invasive ability of these cells. In addition, expression of a constitutively active mutant Src in poorly metastatic K1735C23 cells increased invasion in vitro; whereas expression of a kinase-inactive Src mutant suppressed invasion. Our results suggest that signals initiated by {alpha}vß3 promote metastasis in K1735 melanoma cells through the phosphorylation of FAK and activation of Src.

Key words: ß3 integrins, Melanoma, Focal adhesion kinase, Src, Motility


    INTRODUCTION
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 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interactions between tumor cells and the surrounding extracellular matrix (ECM) activate a variety of intracellular signaling cascades. These signals, through cytoskeletal reorganization and regulation of gene expression, can modulate cell migration and cell proliferation (Ruoslahti, 1999Go; Seftor et al., 1992Go; Curran and Murray, 1999Go).

During invasion, tumor cells adhere to and penetrate the ECM. These processes are mediated by the integrin family of adhesion receptors. Integrins are heterodimeric cell surface receptors consisting of an {alpha} and a ß subunit (Hynes, 1992Go). Several studies have linked the expression of the {alpha}vß3 integrin with invasion of various types of malignant tumors (Zheng et al., 1999Go; Seftor et al., 1992Go). {alpha}vß3 has also been shown to be neo-expressed in melanomas when they switch from being a superficially spreading to a vertically invasive phenotype (Albelda et al., 1990Go; McGregor et al., 1989Go). Additionally, it was shown that overexpression of {alpha}vß3 in radial growth phase primary melanoma cells initiates invasive growth in three dimensional skin reconstructs and increases tumor growth in vivo (Hsu et al., 1998Go). We have also shown previously that expression of {alpha}vß3 is associated with the invasive phenotype in the K1735 melanoma system (Li et al., 1998Go).

Recent evidence from the field of integrin biology has elucidated some of the signaling mechanisms involved in the regulation of cell motility and proliferation by ECM (Schwartz and Shattil, 2000Go; Giancotti and Ruoslahti, 1999Go). Two non-receptor protein tyrosine kinases (NRPTKs), focal adhesion kinase (FAK) and Src, were found to modulate integrin-mediated cell migration. FAK is a novel type of NRPTK that becomes tyrosine phosphorylated and subsequently activated when integrins adhere to various matrix proteins, including vitronectin (VN). Although intensively studied, the exact sequence of events leading to FAK activation is still not well defined. FAK can be phosphorylated in vivo at six or more tyrosines - two within the N-terminal domain (Tyr397 and Tyr407), two within the activation loop (Tyr576 and Tyr577) and two within the C-terminal region (Tyr861 and Tyr925) - and at several serines (Richardson et al., 1997Go; Calalb et al., 1996Go; Schlaepfer et al., 1999Go). It has been suggested that (auto)phosphorylation of FAK at Tyr397 recruits the Src-family members, Src or Fyn (Schaller et al., 1994Go). Binding of Src or Fyn to Tyr397 further augments FAK tyrosine kinase activity (Schaller et al., 1999Go). Src binding to FAK at Tyr397 is also required for phosphorylation of Tyr925, which recruits the adapter molecule Grb2, linking integrins to the Ras pathway (Schlaepfer and Hunter, 1996Go). Integrin activation of gene transcription and cytoskeletal reorganization may not always involve the formation of FAK/Src complexes. A FAK-independent pathway, regulated by another Src-family member, Fyn, has also been proposed (Giancotti and Ruoslahti, 1999Go; Wary et al., 1998Go).

Direct evidence linking integrin-ECM interactions with the phosphorylation of FAK and tumor cell motility has been described by others. Specifically, binding of {alpha}vß3 to VN results in an increase in FAK tyrosine phosphorylation during prostatic cell migration (Zheng et al., 1999Go). Activation of Src has also been observed following melanoma cell-osteopontin interactions; an event mediated by the {alpha}vß3 integrin (Duong et al., 1998Go). Tumor cell invasion requires ECM remodeling, which occurs through the action of various matrix metalloproteinases (MMPs). Both MMP-2 and MMP-9 are highly expressed in many different types of tumors, and their expression has been correlated with increased invasion (Kurschat et al., 1999Go; Pyke et al., 1992Go).

In the present study we show that overexpression of {alpha}vß3 in poorly invasive, ß3-negative K1735 melanoma cells enhances their metastatic behavior in vivo. We also show that phosphorylation of FAK and Src occurs as a result of post-{alpha}vß3-ligand interactions and is important for K1735 cell motility.


    MATERIALS AND METHODS
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and reagents
Hamster anti-mouse mAbs to {alpha}v (CD51) and ß3 (CD61) were purchased from Pharmingen (San Diego, CA). Rabbit polyclonal antibodies to ß1 were a generous gift of Richard Hynes (Massachusetts Institute of Technology, Cambridge, MA). Phosphotyrosine-specific polyclonal antibodies, which react with the phosphorylated tyrosine 397 (pTyr397) of FAK were obtained from Biosource/QCB (Hopkington, MA). Polyclonal antibodies to Src (N-16), N-terminal region of FAK (A-17), C-terminal region of FAK (C-20), and Fyn (Fyn3) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies to paxillin and GFP were purchased from Zymed (South San Francisco, CA). Clone 28, a monoclonal antibody that specifically recognizes unphosphorylated Tyr530 of Src kinase when in an activated form, was generously provided by H. Kawakatsu (University of California, San Francisco) (Kawakatsu et al., 1996Go; Kovacic et al., 1998Go, Sieg et al., 1998). All secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA).

Cell culture
The mouse K1735 melanoma cell lines, the highly metastatic K1735M2 (hereafter M2) and the poorly metastatic K1735C23 (hereafter C23) lines, were a kind gift from I. J. Fidler (University of Texas M.D. Anderson Hospital and Tumor Institute, Houston, TX). Their isolation and metastatic activity have previously been described (Fidler et al., 1981Go). All cell lines used in this study were generated from these two K1735 melanoma lines and listed in Table 1. Cells were routinely cultured in 10% fetal bovine serum in Dulbecco's minimal essential medium (DMEM) and passaged in log growth phase by a brief treatment with 0.25% trypsin/2 mM EDTA. To minimize phenotypic drift, cells in culture were replenished after 8-10 passages from frozen stocks.


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Table 1.
 

Retroviral transduction experiments
Constitutively active Src (CA-Src), kinase dead Src (KD-Src), FAK-related non-kinase (FRNK) or vector alone were stably expressed in either M2 or C23 melanoma cell lines using the Retro-X system (Clontech, Palo Alto, CA) (Li et al., 1998Go; Kovacic et al., 1998Go). Cells with stable expression of the sense or the antisense ß3 integrin subunit were generated as described (Li et al., 1998Go).

Briefly, the mutant Src and FRNK cDNAs were inserted in the sense orientation into the multiple cloning site of the retroviral vector pLXSN. The retroviral vector was transfected into the PT67 packaging cell line by the lipofectin transfection method. Conditioned growth medium containing secreted retrovirus was collected and endpoint titers were determined on NIH3T3 cells. After G418 selection, the supernatant was supplemented with 8 µg/ml polybrene, sterile filtered, and incubated with the M2 cells (KD-Src and FRNK) or the C23 cells (CA-Src) for 24 hours. Transfected cells expressing the retrovirus-encoded cDNA were selected with G418 growth medium. Pooled cultures of G418-resistant cells were used for the described studies.

In vivo tumor growth and metastasis assays
To evaluate primary tumor growth, C3H/HeN syngeneic mice were anesthetized with Avertin (0.015 ml of a 2.5% solution per gram of body weight). 2x105 tumor cells in 50 µl of DMEM were injected transorally into the floor of the mouth. After 4 weeks, animals were sacrificed by cervical dislocation and the extent of tumor formation was evaluated. For metastasis assays, 2x105 cells were injected into the hind flank of C3H/HeN mice. The animals were sacrificed after 12 weeks and analyzed for the presence of tumors in the lungs or other sites.

Immunohistochemistry
Metastatic cardiac lesions were isolated from two syngeneic mice and directly embedded in OCT (optimal cutting temperature compound; Miles Laboratories, Elkhart, IN) embedding medium and snap frozen, or first immersed in 10% sucrose at 4°C for 4 hours and then embedded and frozen. Briefly, 4-µm cryostat sections of tissue samples were air dried for 30 minutes, fixed in -20°C acetone for 5 minutes, and air dried again. Endogenous peroxidase activity was blocked with Peroxoblock Solution (Zymed Laboratories) for 45 seconds at room temperature. After rinsing in PBS for 15 minutes to remove the OCT, sections were incubated for 30 minutes at room temperature with 0.5% casein/0.05% thimerosal/PBS and then incubated for 2 hours at room temperature with primary antibody (anti-ß3 integrin subunit) or without primary antibody as a control. The sections were further processed using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Some sections were counterstained with hematoxylin and eosin (H and E) (Sigma, St Louis, MO). Sections were mounted with Permount (Fisher Scientific, Pittsburg, PA) and analyzed by microscopy.

Western blotting
Cells were seeded at 70% confluence and deprived of serum for 16 hours. The cells were then detached and replated on VN-coated dishes (20 µg/ml) for 30 minutes at 37°C. Cells were lysed in modified RIPA buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, 1% Nonidet P-40) with freshly added proteinase (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin) and phosphatase (1 mM Na3VO4, 50 mM NaF) inhibitors.

Next, the lysates were spun down at 15,000 g for 15 minutes and precleared for 1 hour at 4°C with protein A-agarose. Protein concentrations were determined by BCA protein assay kit (Pierce, Rockford, IL). For immunoprecipitations, 300 µg of cell lysate was incubated at 4°C for 1 hour with the primary antibody. Immune complexes were captured with Protein A-Sepharose and washed with lysis buffer followed by wash buffer (10 mM Tris, pH 7.4, 0.01% sodium dodecyl sulfate (SDS), 1% glycerol, 0.1% ß-mercaptoethanol, 1 mM Na3VO4, 1 mM EDTA) and boiled in the presence of sample buffer. The samples were then separated by SDS-polyacrylamide gel electrophoresis (PAGE) followed by western blotting using commercially available anti-FAK, anti-pTyr397 FAK, anti-Fyn and anti-Src antibodies.

Migration assay
Cell migration was assayed in 24-well, 6.6-mm-internal-diameter Transwell cluster plates (8.0 µm pore size; Costar, Cambridge, MA) as described (Ramos et al., 1997Go; Ramos et al., 1998Go). Five random fields of each Transwell filter were counted at x200 magnification, and the cell numbers were calculated as total migrated cells per filter. Briefly, the lower surfaces of the filters were coated with VN (10 µg/ml) overnight at 4°C. Then 5x104 cells were placed onto the upper surfaces of the filters, and the chambers were incubated at 37°C for 2 hours. The upper surfaces of the filters were wiped clean, the filter was fixed and stained, and the number of cells that had migrated to the underside were counted in triplicate.

Invasion of a reconstituted basement membrane
A thin layer of a reconstituted basement membrane (RBM) (Collaborative Research, Bedford, MA) was overlaid onto a Transwell filter (8 µm pore size) and allowed to polymerize at 37°C for 1 hour. Then 5x104 cells were seeded onto the RBM-coated filter and allowed to invade for 16 hours. After the incubation period, the cells were processed as in the migration assay.

Kinase assay
In vitro autophosphorylation assays were performed as described (Kanazawa et al., 1996Go). Briefly, Src, Fyn or FAK immunoprecipitates were washed once with kinase reaction buffer (20 mM 1,4-piperazine bis-(ethane sulfonic acid) (PIPES), pH 7.4, 10 mM MnCl2) and labeled by addition of 0.5 ml [{gamma}-32P]ATP (3000 Ci/mmol, 10 mCi/ml) in kinase buffer (total volume 10 µl) for 30 minutes at 37°C. The reaction was terminated by the addition of sample buffer. The labeled protein, separated by SDS-PAGE, was transferred to a nylon membrane. The amount of Src protein on the membrane was visualized with alkaline phosphatase-conjugated secondary antibodies and BCIP/NBT substrate (Gibco BRL, Grand Island, NY). The membrane was then dried and exposed to X-ray film to detect incorporated radioactivity.


    RESULTS
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 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
K1735 cell primary tumor formation and eventual metastasis require ß3 integrins
Cell lines used in this study (listed in Table 1 were derived from the highly invasive, ß3-positive M2 cells or the poorly invasive, ß3-negative C23 cells. As previously described, the M2-Tß3 cell line was derived by transduction of the M2 cells with the full-length ß3 integrin cDNA in the antisense direction (Li et al., 1998Go). The resulting M2-Tß3 cells express significantly lower levels of the ß3-integrin than the parental M2 cell line (Li et al., 1998Go). The C23-mß3 cell line, which expresses high levels of ß3, was generated by introduction of the full-length ß3 integrin cDNA into the C23 cells (Li et al., 1998Go).

Tumor growth was subsequently evaluated in vivo. The highly invasive, ß3-positive M2 cells and the descendant M2-Tß3 cells were injected into the floor of the mouth of C3H/HeN syngeneic mice (12 mice per cell line). After 4 weeks, large primary tumors were formed in mice injected with the M2 cells (Fig. 1A, left, arrow), whereas tumors formed by the M2-Tß3 cells were extremely small (Fig. 1A, right), suggesting that expression of ß3 enhances K1735 tumor formation in vivo. The average weight of the tumors produced by the M2 cells (black histogram) was 5.0 g (±1 g), whereas the tumors formed by the M2-Tß3 cells averaged approximately 1.2 g (±0.4 g) (Fig. 1B).



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Fig. 1. K1735 primary tumor formation requires ß3 expression. 2x105 ß3-positive M2 cells or 2x105 ß3-antisense-expressing M2-Tß3 cells were injected transorally into the floor of the mouth of syngeneic mice. After 4 weeks the mice were sacrificed and evaluated for tumor formation. Note that the mice injected with the M2 cells developed large primary tumors (A, left), whereas those injected with the M2-Tß3 cells were considerably smaller (A, right). (B) The average weight of tumors formed by M2 cells was 5.0 g (±1 g), whereas tumors formed by the M2-Tß3 cells averaged 1.2 g (±0.4 g). 12 mice were injected with each cell line.

 

To evaluate a possible link between expression of the ß3 integrin and metastatic potential in these two melanoma cell lines, we performed in vivo metastasis assays. M2 or M2-Tß3 cells were injected into the hind flank of C3H/HeN syngeneic mice (12 animals/cell line). After 12 weeks, the mice were sacrificed and analyzed for the formation of metastases. On average, 100 lung lesions (±20 colonies) were formed by the M2, whereas none were formed by the M2-Tß3 cells (Fig. 2A and B, respectively). The ß3-negative C23 and the C23-mß3 cells, stably expressing the ß3 subunit, were also evaluated by spontaneous metastasis assays. The C23-mß3 cells formed an average of 40 lung lesions per animal (±10 colonies) (Fig. 2D), whereas the C23 cells did not metastasize (Fig. 2C). These results indicate that expression of {alpha}vß3 potentiates metastatic colonization. Quantitation of experiment is included (Fig. 2E).



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Fig. 2. K1735 melanoma metastasis requires expression of ß3 integrin. 2x105 M2 (A), M2-Tß3 (B), C23 (C), or C23-mß3 (D) cells were injected subcutaneously into the hind flank of C3H/HeN mice. After 12 weeks the mice were sacrificed and analyzed for metastases. On average 100 colonies (±20) were formed by M2 (A, arrows) and 40 lesions (±10) by the C23-mß3 cells (D, arrows). By contrast, M2-Tß3 (B) and C23 cells (C) did not form pulmonary metastases. Quantitation of the experiment is shown in E.

 

Metastatic lesions formed by the C23-mß3 cells retain expression of ß3 integrin
C23-mß3 cells produced not only lung metastases but also cardiac muscle lesions in two of the 12 animals (Fig. 3A, arrows). Immunostaining of frozen tumor tissue sections with anti-ß3 antibodies localized the integrin to the tumor cell membrane (Fig. 3B, arrow). These results indicate that the C23-mß3 cells continue to express the ß3 integrin in vivo.



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Fig. 3. Metastatic lesions formed by C23-mß3 cells retain ß3 integrin expression. (A) In addition to pulmonary lesions, two mice injected with the C23-mß3 cells developed cardiac metastases. H and E staining of the lesions showed an infiltration by the tumor cells into the cardiac muscle. (B) Sections of cardiac lesions were incubated with anti-ß3 antibodies (without counterstaining) to localize the expression of this integrin. Note the intense membrane staining (arrows) indicating continued expression of ß3. The inset shows the area marked by the arrows at high magnification.

 

ß3 ligand-binding induces phosphorylation of FAK and activation of Src
Integrin engagement triggers a variety of intracellular signaling cascades. One of the foremost events is the phosphorylation of FAK on Tyr397 (Schlaepfer et al., 1999Go). The K1735 cell lines (M2, M2-Tß3, C23, C23-mß3) were plated on VN at 70% confluence. Serum was withdrawn for 16 hours to avoid the confounding effects of exogenous growth factors. The cells were detached and then replated on VN for an additional 30 minutes in the absence of serum. FAK was immunoprecipitated from the cell lysates. Tyr397 phosphorylation was examined by western blotting of the FAK immunoprecipitates with phospho-specific anti-pTyr397 antibodies (Fig. 4A, top). Levels of pTyr397 in the M2 and the C23 cells were comparable; however, higher levels of pTyr397 were detected in the M2 and the C23-mß3 cell lysates when compared directly with the M2-Tß3 or C23 lysates, respectively (Fig. 4A, top). The membrane was stripped and reblotted with anti-FAK antibodies to confirm that our results were not due to unequal amounts of immunoprecipitated protein (Fig. 4A, bottom). Results were further confirmed by comparison of {gamma}-32P incorporated into FAK in FAK (auto)phosphorylation assays (data not shown). Phosphorylation of FAK on Tyr397 appeared in part to be associated with expression of the ß3 integrin.



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Fig. 4. ß3 integrin mediates autophosphorylation of FAK and the activity of Src. The cells were plated on VN without serum for 30 minutes on all panels. (A) Cells were extracted and immunoprecipitated with anti-FAK antibodies followed by western blotting with anti-pTyr397 antibodies (top). The membrane was stripped and reprobed with anti-FAK antibodies (bottom). (B) The cell lysates were immunoprecipitated with anti-Src antibodies. The immune complexes were analyzed for the incorporation of {gamma}-32P by kinase assay (top). To ensure equivalent amounts of immunoprecipitated protein, the lysates were immunoprecipitated followed by western blotting with anti-Src antibodies (bottom). Autophosphorylation of Fyn was analyzed by kinase assay (top). The relative amount of immunoprecipitated protein was analyzed by an immunoprecipitation followed by western blotting with anti-Fyn antibodies (bottom). (C) To detect putative complex formation, samples were immunoprecipitated with anti-Src antibodies followed by western blotting with antibodies to FAK (top). Relative amounts of immunoprecipitated protein were determined by an immunoprecipitation followed by western blotting with anti-Src antibodies (bottom). To detect FAK/Fyn complex formation, samples were immunoprecipitated with anti-FAK antibodies followed by western blotting with anti-Fyn antibodies (top). Relative amounts of Fyn were identified by immunoprecipitation followed by western blotting with anti-Fyn antibodies (bottom).

 

The predominant partners of FAK in integrin-mediated signaling are Src and Fyn (Schlaepfer et al., 1999Go). Both Src and Fyn are known to bind to FAK and augment its kinase activity. We assessed both Src and Fyn kinase activity in our system to attempt to make a physiological distinction between Src and Fyn activity and metastatic potential (Fig. 4B). We detected Src but not Fyn kinase activity in the cell lysate (Fig. 4B). Greater incorporation of {gamma}-32P into Src corresponded to samples that also showed higher levels of pTyr397 FAK (M2 and C23-mß3) (Fig. 4B, top). Results of Src and Fyn autophosphorylation assays were confirmed by blots with clone 28, an antibody that specifically recognizes the active form of Src and Fyn (Kawakatsu et al., 1996Go) (data not shown). These data suggest that Src, rather than Fyn, may be involved in the ß3-mediated invasive phenotype of K1735 cells.

Expression of ß3 integrin correlates with an increase in FAK/Src complex formation in K1735 melanoma cells
We wished to determine if FAK formed a complex with Src or Fyn in these cell lines (M2 and C23-mß3). Src and Fyn were immunoprecipitated from the lysates of cells plated on VN for 30 minutes and prepared as in the previous experiments. The immune complexes were resolved by SDS-PAGE and probed with anti-FAK antibodies. A complex containing both FAK and Src was found to be several times higher in the M2 and C23-mß3 cells than in the M2-Tß3 and the C23 cells (Fig. 4C). A complex containing FAK and Fyn was not detected in any of the four cell lines (Fig. 4C). Reblotting with antibodies to Src or Fyn indicated that our observations were not due to differences in overall protein level (Fig. 4C; bottom). These results indicate that, in K1735 cells, a FAK/Src complex is formed that may be important for ß3-integrin-mediated invasion. This finding represents one of the first insights into phenotypic differences that may be a result of association between FAK/Src and not FAK/Fyn.

Phosphorylation of FAK on Tyr397 is required for K1735 cell motility
The independently expressed C-terminal region of FAK, termed FAK-related non-kinase (FRNK), can serve as a dominant-negative regulator of FAK function by competing with full-length FAK for a site in focal adhesions (Richardson and Parsons, 1996Go). We examined whether phosphorylation of FAK on Tyr397 is required for the transduction of signals initiated by the binding of the ß3 integrin to VN and subsequent motility. We introduced FRNK fused with green fluorescent protein (GFP) into the K1735M2 cell line using a retroviral vector. The resulting cell line was termed M2GFP-FRNK. The expression level of GFP-FRNK was verified by western blotting with antibodies to GFP (data not shown). Expression of GFP-FRNK was also verified by western blotting using antibodies to the C-terminus of FAK (Fig. 5A). A decrease in phosphorylation of FAK at Tyr397 was detected in the M2GFP-FRNK cell lysate when compared with the M2 cell lysate (Fig. 5A, top). No change in overall FAK protein expression was detected (Fig. 5A, middle). Western blot of whole cell lysate with antibodies to the C-terminus of FAK clearly detected the FRNK construct in the M2GFP-FRNK cell line.



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Fig. 5. Expression of FRNK suppresses phosphorylation of FAK at Tyr397 and K1735 cell motility. M2 and M2GFP-FRNK cell lysates were immunoprecipitated with anti-FAK and then analyzed by western blotting with anti-pTyr397 antibodies (top). The membrane was stripped and reprobed with anti-FAK antibodies to ensure that amounts of protein were equivalent (middle). Detection of the FRNK construct was evaluated by a western blot of whole cell lysate with antibodies to the C-terminus of FAK (lane 2, bottom). (B) M2 and M2GFP-FRNK cells were seeded onto VN-coated filters and monitored for migration over a 2 hour period or plated onto RBM-coated filters and allowed to invade overnight, as described in Materials and Methods. The results (means±s.e.m.) are representative of three independent experiments each performed in triplicate. The average number of M2 cells migrating on VN was 238 (±11.5), whereas, the average number of migrating M2GFP-FRNK cells was 11.5 (±1). Similarly, invasion of an RBM was suppressed approximately 80% by expression of FRNK (B). The average number of M2 cells invading was 46 (±19), whereas only 8 (±1) M2GFP-FRNK cells invaded. These results suggest that phosphorylation of FAK on Tyr397 is required for maximum K1735 cell motility.

 

We next evaluated a possible role for FRNK in cell motility (Fig. 5B). We found that migration of GFP-FRNK cells was reduced by approximately 90% when compared with the M2 cells (Fig. 5B). On average, 238 (±11.5) M2 cells migrated on VN, whereas 11.5 (±1) M2GFP-FRNK cells migrated. Similarly, invasion of an RBM was suppressed approximately 80% by expression of FRNK (Fig. 5B). On average 46 (±19) M2 cells invaded, whereas only 8 (±1) M2-GFP-FRNK cells invaded. These results suggest that phosphorylation of FAK on Tyr397 is required for maximum K1735 cell motility.

Src activity affects K1735 melanoma cell motility
Having demonstrated increased activation of Src and increased FAK/Src complex formation in the ß3 positive cells, we next addressed the effect of Src activation on cell motility. We expressed a kinase dead Src in the highly invasive M2 cells and derived the M2KD-Src cell line. We also expressed a constitutively active Src in the non-invasive C23 cells to generate the C23CA-Src cell line (Fig. 6A). When examined by immunoprecipitation/western blot or western blotting of whole cell lysates, the M2KD-Src and C23CA-Src cells showed slightly higher expression of total Src levels (paxillin blot was used as a loading control). A Src autophosphorylation assay showed markedly higher incorporation of {gamma}-32P into Src immunoprecipitates from M2 vs M2KD-Src cells. The amount of {gamma}-32P incorporated into Src in C23CA-Src cells was 20-fold that seen in the original C23 cells (Fig. 6A).



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Fig. 6. K1735 cell migration requires active Src. (A) M2, M2KD-Src, C23, and C23CA-Src cells were analyzed for autophosphorylation of Src by kinase assay (top-left). Immunoprecipitation followed by western blotting using anti-Src antibodies was done to detect changes in expression as a result of transduction with the mutant constructs (bottom-left). Western blotting of whole cell lysate was done to detect changes in Src expression as a result of transduction with the Src constructs (top-right). Western blotting of whole cell lysate with antibodies to paxillin was used as protein loading control (bottom-right). (B) Migration of the four cell lines was evaluated on VN. The results (means±s.e.m.) are representative of three independent experiments each performed in triplicate.

 

We next evaluated the effect Src activation plays in K1735 cell migration. The C23CA-Src cells were four times as motile on VN as the original C23 cells. The expression of a KD-Src reduced M2 cell line migration by two thirds (Fig. 6B). These results suggest that activation of Src is required for K1735 cell migration.


    DISCUSSION
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Invasion and metastasis are complex processes requiring both cell-matrix and cell-cell interactions. Both processes generate a variety of intracellular signals promoting the assembly and disassembly of the actin cytoskeleton (Ruoslahti, 1999Go). Cytoskeletal reorganization results in changes to both cell shape and motility.

The heterogeneity of tumors is well documented (Nicolson, 1988Go). Within the primary tumor, there is a subset of cells that may become metastatic. These cells acquire specific characteristics that may promote the invasive phenotype. Acquisition of specific integrin receptors may be one such characteristic.

In this study we demonstrated that expression of the ß3 integrin subunit increased K1735 melanoma cell invasion and metastasis. We also show that phosphorylation of FAK at Tyr397, and Src activity are strongly correlated with enhanced K1735 cell invasion.

In vivo, K1735 cells expressing the ß3 integrin (M2 and C23-mß3) formed multiple metastatic foci in syngeneic mice; whereas ß3-negative cells did not. Therefore, we suggest a specific mechanism by which the K1735 melanoma cells become metastatic: expression of the {alpha}vß3 integrin.

Adhesion to the ECM `clusters' the integrin, generating intracellular signals, similar to those demonstrated by growth factors, resulting in cytoskeletal rearrangements (Juliano, 1996Go).

Two NRPTKs, FAK and Src, have been reported to act as signaling molecules linking the integrin to cytoskeletal reorganization and subsequent migration (Schlaepfer et al., 1999Go). In our model system, adhesion to VN resulted in the phosphorylation of FAK at Tyr397 and an increase in Src activity. This suggests that FAK and Src are responsive to clustering of the {alpha}vß3 integrin on VN. To explore this further, we perturbed Src function by expressing either a constitutively active or a kinase-inactive Src mutant. Expression of a constitutively active Src increased K1735 cell motility, whereas expression of a kinase-dead mutant Src significantly suppressed cell movement.

We expressed FRNK in the M2 cell line to evaluate the role of FAK in K1735 tumor cell motility. FRNK, a naturally occurring C-terminal domain of FAK, acts as a dominant-negative regulator of FAK function. The stable expression of FRNK in the highly metastatic M2 cells decreased their invasive behavior and the phosphorylation of FAK at Tyr397. These results indicate that phosphorylation of Tyr397 of FAK is a contributing factor to the invasive potential of K1735 melanoma cells. The suggestion that FAK is important for tumor progression is not novel. Several other groups have reported that the expression of FAK is upregulated in a variety of different cancer cells (Judson et al., 1999Go; Zheng et al., 1999Go; Tremblay et al., 1996Go; Owens et al., 1995Go; Owens et al., 1996Go; Weiner et al., 1993Go). However, its function in tumor cell invasion and metastasis is still unclear. Recently, data from three independent laboratories demonstrated that the phosphorylation of FAK may also be involved in cell proliferation, a characteristic central to tumor progression (Wang et al., 2000Go; Zhao et al., 1998Go; Oktay et al., 1999Go). In our system we also observed a slight decrease in K1735 cell proliferation when FAK phosphorylation was suppressed by expression of FRNK.

Phosphorylated Tyr397 of FAK serves as a binding site for the Src-family kinases, Src and Fyn. It has also been shown by several groups that formation of a FAK/Src or FAK/Fyn complex can lead to further phosphorylation of FAK by Src or Fyn, and thereby increase FAK kinase activity. However, to date, there is no strong evidence to indicate which biological events may be mediated through a FAK/Src vs a FAK/Fyn complex (Schlaepfer et al., 1999Go; Parsons and Parsons, 1997Go; Thomas and Brugge, 1997Go). Our data suggest that the specific complex of FAK with Src, but not with Fyn, will lead to increased cell migration, invasion and metastasis.

In summary, we detected in a single, well-characterized system that (1) the specific clustering of {alpha}vß3 results in phosphorylation of FAK at Tyr397 and activation of Src, not Fyn; and (2) expression of Src mutants affects K1735 cell motility. Therefore, we suggest that suppressing the function or expression of {alpha}vß3, or targeting one or more of its downstream signaling molecules, will have a significant impact on melanoma progression in a clinical setting.


    ACKNOWLEDGMENTS
 
This work was supported by grants 1 R29 DE11930-03A1 and R01 DE12856-01A2 from the NIDCR (DMR) and grants from the University of California Cancer Research Coordinating Committee and the University of California Cancer Center Clinical Investigator Research Program (D.M.R.). D.I. was supported by K01CA87652-01 (The Howard Temin Award) from the NCI and from an Individual Investigator Award from the UCSF Academic Senate. We wish to thank Evangeline Leash for editing the manuscript and Dean Sheppard for helpful discussions.


    REFERENCES
 Top
 SUMMARY
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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