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Originally published In Press as doi:10.1074/jbc.M204691200 on October 21, 2002

J. Biol. Chem., Vol. 277, Issue 52, 50373-50379, December 27, 2002
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Synergistic Effect of Focal Adhesion Kinase Overexpression and Hepatocyte Growth Factor Stimulation on Cell Transformation*

Po-Chao ChanDagger, Chun-Chi LiangDagger, Kuo-Ching Yu§, Ming-Chen Chang§, William L. Ho§, Bor-Huah Chen, and Hong-Chen Chen||

From the Department of Life Sciences and the  Graduate Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan and the § Department of Pathology, Taichung Veterans General Hospital, Taichung 40705, Taiwan

Received for publication, May 14, 2002, and in revised form, October 18, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although an elevated level of focal adhesion kinase (FAK) has been observed in a variety of invasive human tumors, forced expression of FAK alone in cultured cells does not cause them to exhibit transformed phenotypes. Therefore, the role of FAK in oncogenic transformation remains unclear. In this study, we have demonstrated that FAK overexpression in Madin-Darby canine kidney epithelial cells rendered them susceptible to transformation by hepatocyte growth factor (HGF). Using various FAK mutants, we found that the simultaneous bindings of Src and p130cas were required for FAK to potentiate cell transformation. Expression of FAK-related nonkinase, kinase-deficient Src, or the Src homology 3 domain of p130cas, which respectively serve as dominant negative versions of FAK, Src, and p130cas, apparently reversed the transformed phenotypes of FAK-overexpressed cells upon HGF stimulation. Moreover, FAK overexpression was able to enhance HGF-elicited signals, leading to sustained activation of ERK, JNK, and AKT, which could be prevented by the expression of the Src homology 3 domain of p130cas. Taken together, our results indicate that the synergistic effect of FAK overexpression and HGF stimulation leads to cell transformation and implicate a critical role of p130cas in this process.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Focal adhesion kinase (FAK),1 a 125-kDa cytoplasmic protein-tyrosine kinase localized in focal contacts, has been implicated to play a crucial role in the control of integrin-mediated cellular functions including cell spreading (1, 2), cell migration (3, 4), cell cycle progression (5, 6), and cell survival (7-10). The ability of FAK to transduce signals to the downstream depends on its ability to interact with several intracellular signaling molecules, including Src family kinases (11, 12), phosphatidylinositol 3-kinase (PI3K) (13, 14), phospholipase C-gamma 1 (15), Grb2 (16, 17), and p130cas (18). We have previously shown that the simultaneous bindings of PI3K and p130cas are required for FAK to promote cell migration (19) and survival (8). Almeida et al. (7) showed that the FAK-p130cas complex transduces matrix survival signals via c-Jun NH2-terminal kinase (JNK). This FAK-p130cas-JNK signaling pathway was also shown to be required for FAK to promote cell cycle progression (5). The FAK-Grb2 complex has been proposed to trigger downstream signaling pathways, leading to activation of extracellular signal-regulated kinases (ERKs) (17, 20), which has recently been shown to contribute partially to FAK-promoted cell migration (21).

Appropriate cell behavior requires coordinate signals from both cell adhesion and growth factors. Evidence has suggested that FAK may be a point of convergence of integrin and growth factor signaling pathways. In addition to cell adhesion, the tyrosine phosphorylation of FAK is also stimulated by growth factors (22-25) as a result of FAK association with growth factor receptor (26) and/or Src activation by growth factor receptor (22). The tyrosine phosphorylation of FAK stimulated by growth factors enhances its association with effectors, leading to amplification of downstream signals (22, 27). More recently, FAK was shown to integrate signals from growth factor receptors and integrins to facilitate cell migration (21, 26).

Hepatocyte growth factor (HGF), also known as scatter factor, is a multifunctional growth factor that elicits mitogenic, motogenic, and morphogenic activities in various cell types (28-31). The diverse biological effects of HGF are transmitted through activation of its transmembrane receptor encoded by the c-met proto-oncogene (32, 33). Inappropriate activation of HGF/Met signaling has been implicated in the etiology of a number of human tumors and has been shown to confer invasive and metastatic properties to neoplastic cells (34-38). Similarly, an increased level of FAK has been found in a variety of invasive human tumors and has been implicated to play a role in tumor progression to an invasive phenotype (39-41). We have previously shown that FAK overexpression in Madin-Darby canine kidney (MDCK) cells significantly enhances their migration in response to HGF stimulation (21), indicating that the synergy between FAK overexpression and HGF stimulation facilitates cell migration. In this study, we further demonstrate this synergy leads to cell transformation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Recombinant human HGF was purchased from R&D System, Inc. Fetal bovine serum and LipofectAMINE were purchased from Life Technologies, Inc. G418 sulfate and hygromycin B were purchased from Calbiochem. Matrigel was purchased from Collaborative Biomedical Products (Bedford, MA). The 24-well transwell chamber for invasion assay was purchased from Costar (Cambridge, MA). The rabbit polyclonal anti-FAK was described previously (12). The monoclonal anti-FAK (clone 77) and anti-phosphotyrosine (PY20) were purchased from Transduction Laboratories (Lexington, KY). The monoclonal anti-hemagglutinin (HA) epitope was purchased from Roche Molecular Biochemicals. The polyclonal anti-phospho-Met (Tyr1234/Tyr1235) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The monoclonal anti-Src (clone 327) was purchased from Calbiochem. The rabbit polyclonal anti-p130cas (C-20), anti-Grb2 (C-23), anti-ERK (K-23), and anti-JNK (C-17) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The rabbit polyclonal anti-phospho-ERK (Thr202/Tyr204), anti-phospho-JNK (Thr183/Tyr185), anti-AKT, and anti-phospho-AKT (Ser473) were purchased from New England Biolabs, Inc. (Beverly, MA). The pKH3 expression plasmid encoding HA epitope-tagged FAK-related nonkinase (FRNK) was described previously (21). The plasmids encoding kinase-deficient (kd) Src was kindly provided by Dr. David Shalloway (Cornell University, Ithaca, NY).

Cell Lines and Transfections-- MDCK II 3B5 cells overexpressing HA epitope-tagged wild type (WT) FAK, FAK mutants (D395A, Y397F, P712A/P715A, and Y925F), or FRNK have been described previously (8, 21) and were maintained in Dulbecco's modified Eagle's medium supplemented with 10% serum and 0.5 mg/ml G418. MDCK cells stably co-expressing HA epitope-tagged FAK and the SH3 domain p130cas have been described previously (8) and were maintained in growth medium containing 0.5 mg/ml G418 and 100 units/ml hygromycin.

To generate MDCK cells stably co-expressing HA epitope-tagged FAK and FRNK, MDCK cells that had already overexpressed FAK were grown on 60-mm dishes and co-transfected with 2 µg of pKH3-FRNK and 0.2 µg of pREP3 using 10 µl of LipofectAMINE following the manufacturer's instructions. Clones were selected in growth medium containing 0.5 mg/ml G418 and 100 units/ml Hygromycin and screened for FAK and FRNK expression by immunoblotting with anti-HA. To generate MDCK cells stably co-expressing HA epitope-tagged FAK and kd Src, MDCK cells that had already overexpressed FAK were co-transfected with 2 µg of pEVX-Src kd and 0.2 µg of pREP3. Clones were selected in growth medium containing 0.5 mg/ml G418 and 100 units/ml hygromycin and screened by in vitro Src activity assay.

Biological Assays-- For soft agar colony formation assay, 5 × 103 cells were suspended in 2 ml of Dulbecco's modified Eagle's medium containing 0.3% agar and 10% serum with or without 20 ng/ml HGF and added onto a layer of medium containing 0.5% agar and 10% serum in a 60-mm dish. 2 ml of medium containing 0.3% agar and 10% serum with or without 20 ng/ml HGF was added to the dish every other day. Each experiment was performed in duplicate. After 18 days, the number of colonies was measured.

For the Matrigel invasion assay, MDCK cells were pretreated with 10 ng/ml HGF in 5% serum for 12 h and then collected by trypsinization. 5 × 104 cells in 250 µl of serum-free medium were added to an inner cup of the 24-well transwell chamber that had been coated with 150 µl of Matrigel (1:10 dilution in serum-free medium). 750 µl of medium supplemented with 10% serum was added to the outer cup. After 24 h, cells that had migrated through Matrigel and filter membrane with 8-µm pores were fixed, stained, and counted under a light microscope. Each experiment was performed in triplicate.

Immunoprecipitations, Immunoblotting, and in Vitro Kinase Assays-- Cells were lysed in 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, and 1 mM Na3VO4) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.2 trypsin inhibitory units/ml aprotinin, and 20 µg/ml leupeptin). The lysates were centrifuged for 10 min at 4 °C to remove debris, and the protein concentrations were determined using the Bio-Rad protein assay. For immunoprecipitation, aliquots of lysates were incubated with 3 µl of various polyclonal antibodies or 6 µl of monoclonal anti-HA or anti-Src for 1.5 h at 4 °C. Immunocomplexes were collected on protein A-Sepharose beads. For monoclonal antibodies, Protein A-Sepharose beads were coupled with rabbit anti-mouse IgG before use. The beads were washed three times with 1% Nonidet P-40 lysis buffer, boiled for 3 min in SDS sample buffer, subjected to SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose (Schleicher and Schuell). Immunoblotting was performed with appropriate antibodies using the Amersham Biosciences enhanced chemiluminescence system for detection.

To measure Src activity, anti-Src immunoprecipitates were washed three times with 1% Nonidet P-40 lysis buffer and once in 20 mM Tris buffer. In vitro kinase reactions were carried out in 40 µl of kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MnCl2) containing 5 µg of acid-denatured enolase (Sigma) and 10 µCi of [r-32P]ATP (3000 Ci mmol-1; PerkinElmer Life Sciences) for 20 min at 25 °C. Reactions were terminated by the addition of SDS sample buffer, and proteins were resolved by SDS-polyacrylamide gel electrophoresis. To measure the PI3K activity associated with ectopically expressed FAK proteins in MDCK cells, epitope-tagged FAK proteins were immunoprecipitated with anti-HA from cell lysates and subjected to an in vitro PI3K assay as described previously (13).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Synergy between FAK Overexpression and HGF Stimulation Leads to Anchorage-independent Cell Growth and Matrigel Invasion-- To examine whether increased expression of FAK and concomitant exposure to HGF lead to cell transformation, stable MDCK cell lines overexpressing HA epitope-tagged FAK were established and subjected to the soft agar colony formation assay and the Matrigel invasion assay in the presence or absence of HGF. The expression of ectopic FAK in MDCK cells led to a ~3-fold increase in total FAK proteins, and the phosphorylation of FAK was increased upon HGF stimulation (Fig. 1A). The phosphorylation of Met was increased to a similar level in Neo control cells and FAK-overexpressed cells upon HGF stimulation (Fig. 1A). Because of no available antibody capable of recognizing canine Met in MDCK cells, Northern hybridization instead of immunoblotting was applied to analyze the expression of c-met. We found that the amount of Met transcripts was not increased by FAK overexpression in the presence or absence of HGF (data not shown). Therefore, it is less likely that FAK overexpression enhances the activation of Met upon HGF stimulation.


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  		Fig. 1.   The synergistic effect of FAK overexpression and HGF stimulation leads to anchorage-independent cell growth and Matrigel invasion. A, MDCK cells stably overexpressing HA epitope-tagged FAK or control cells (Neo) were treated with (+) or without (-) 10 ng/ml HGF for 15 min before lysis. FAK was immunoprecipitated (IP) by polyclonal anti-FAK and analyzed by immunoblotting (IB) with anti-phosphotyrosine (PY) or monoclonal anti-FAK. Whole cell lysates (WCL) were analyzed by immunoblotting with anti-HA to detect ectopically expressed FAK or anti-phospho-Met to measure the phosphorylation of Met. B, FAK-overexpressed cells and control cells (Neo) were subjected to a soft agar colony formation assay in the presence or absence of 20 ng/ml HGF, as described under "Experimental Procedures." Colonies were counted after 18 days of incubation. Representative photographs of the experiments are shown. C, stable MDCK cell clones were treated with or without 10 ng/ml HGF in the medium containing 5% serum for 12 h. Cells were trypsinized and subjected to a Matrigel invasion assay, as described under "Experimental Procedures." 24 h later, invaded cells were fixed, stained, and counted using a light microscope. Values (means ± S.E.) are from nine data points from three independent experiments using three different clones for each experimental group.

The soft agar colony formation assay has been used to measure anchorage-independent cell growth, a hallmark of cell transformation. As shown in Fig. 1B, FAK-overexpressed cells failed to grow in soft agar without the supplement of HGF, indicating that elevated expression of FAK by itself is not sufficient to confer an anchorage-independent phenotype to MDCK cells. Likewise, HGF stimulation alone was not sufficient to support the growth of control cells in soft agar either. However, HGF stimulation allowed FAK-overexpressed cells to grow in soft agar and finally form discrete cell colonies, indicating that the synergistic effect of FAK overexpression and HGF stimulation leads to anchorage-independent cell growth.

Enhanced invasiveness is another characteristic of transformed cells. To examine whether FAK-overexpressed cells acquire invasive potential in response to HGF stimulation, stable MDCK cell lines overexpressing FAK were pretreated with HGF for 12 h and then subjected to an in vitro invasion assay (Fig. 1C). This assay measures the capability of cells to migrate through a reconstituted basal membrane (Matrigel) and mimics aspects of tumor cell invasion during metastatic process. In the absence of HGF stimulation, MDCK cells did not exhibit any invasive properties, no matter whether FAK was overexpressed (Fig. 1C). Although HGF has been shown to stimulate the invasiveness of various tumor cell lines (24, 42, 43), it failed to promote control MDCK cells to invade Matrigel under our experimental conditions. Importantly, HGF stimulation dramatically enhanced the invasive property of MDCK cells overexpressing FAK (Fig. 1C). In addition to MDCK cells, the synergism between FAK overexpression and HGF stimulation was examined in Chang liver cells. Similarly, FAK overexpression in Chang liver cells renders them susceptible to transformation upon HGF stimulation, judging from the two in vitro transformation assays above (data not shown).

To further confirm the effect of FAK overexpression on promoting HGF-dependent cell transformation, FRNK, a dominant negative construct of FAK, was expressed in MDCK cells that had already expressed WT FAK (Fig. 2A). Although FRNK has been reported to be cytotoxic in some cell types (44), it is not the case for MDCK cells, where FRNK only caused a decreased growth rate but not cell death (data not shown). Our result showed that the expression of FRNK significantly (~80%) inhibited FAK-overexpressed cells to grow in soft agar and invade Matrigel upon HGF stimulation (Fig. 2B), supporting the specific role of FAK overexpression in HGF-dependent cell transformation.


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  		Fig. 2.   Inhibitory effect of FRNK on FAK promotion of HGF-dependent transformation. A, cell lysates from control cells (Neo) and stable MDCK cells expressing HA epitope-tagged FRNK or FAK alone and in combination (FAK/FRNK) were analyzed by immunoblotting with anti-HA. B, MDCK cells as described for A were subjected to the soft agar colony formation assay and the Matrigel invasion assay in the presence of HGF. Values (mean ± S.E.) are from nine data points from three independent experiments using three independent clones for each experimental group. Relative colony formation and Matrigel invasion were calculated based on the level of cells overexpressing FAK alone.

Simultaneous Bindings of Src and p130cas, but Not PI3K, Are Essential for FAK to Potentiate HGF-dependent Cell Transformation-- To investigate the signals downstream of FAK required for HGF-dependent cell transformation, stable MDCK cell lines overexpressing HA epitope-tagged FAK mutants including D395A, Y397F, P712A/P715A, and Y925F, deficient in binding to PI3K, Src, p130cas, and Grb2, respectively, were established. We first demonstrated that the FAK mutant stably expressed in MDCK cells was indeed specific to the intended defect in the FAK signaling (Fig. 3A). Next, those cells were subjected to the soft agar colony formation assay and the Matrigel invasion assay. We found that the cells expressing FAK Y397F or P712A/P715A mutant failed to grow in HGF-containing soft agar and invade Matrigel upon HGF stimulation (Fig. 3, B and C), suggesting that simultaneous bindings of Src and p130cas are required for FAK to potentiate HGF-dependent cell transformation. The potentials of cells expressing FAK D395A mutant to grow in HGF-containing soft agar and to invade Matrigel upon HGF stimulation were similar to those of cells expressing WT FAK, suggesting that the PI3K binding may be dispensable for FAK to induce cell transformation upon HGF stimulation. Interestingly, although cells expressing the FAK Y925F mutant displayed poor invasive properties after HGF stimulation, their colony forming potential in HGF-containing soft agar remained ~60% of that of cells expressing WT FAK, suggesting that the interaction of FAK and Grb2 may differentially contribute to anchorage-independent cell growth and Matrigel invasion.


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  		Fig. 3.   Src and p130cas are required for FAK to potentiate HGF-dependent cell transformation. A, FAK mutants stably overexpressed in MDCK cells exhibits intended defects in binding to other cellular proteins. Cell lysates prepared from control cells (Neo) and cells stably expressing HA epitope-tagged FAK (WT) or its mutants including D395A, Y397F, P712A/P715A, and Y925F were subjected to immunoprecipitation (IP) with antibodies as indicated. The washed immunoprecipitates were subsequently analyzed by immunoblotting (IB) with antibodies as indicated. To detect the PI3K activity associated with ectopically expressed FAK, the anti-HA immunoprecipitates were subjected to an in vitro kinase assay for PI3K activity as described under "Experimental Procedures." B, stable MDCK cell clones were subjected to the soft agar colony formation assay in the presence of 20 ng/ml HGF. Values (means ± S.E.) are from nine data points from three independent experiments using three independent clones for each experimental group. Relative colony formation was calculated based on the level of cells overexpressing WT FAK. C, stable MDCK cell clones were treated with 10 ng/ml HGF in the medium containing 5% serum for 12 h. Cells were trypsinized and subjected to the Matrigel invasion assay. 24 h later, invaded cells were fixed, stained, and counted using a light microscope. Data (means ± S.E.) are from 12 data points from four independent experiments using three independent clones for each experimental group.

Inhibitory Effects of Kinase-deficient Src and the SH3 Domain of p130cas on FAK Promotion of HGF-dependent Cell Transformation-- As demonstrated in Fig. 3, the Src/p130cas signaling pathway is likely to be most critical for FAK to potentiate the HGF-dependent cell transformation. To examine the significance of Src in this model, kd Src, which functions as a dominant negative version of Src, was stably expressed in MDCK cells that had already overexpressed FAK (Fig. 4). Although the expression of Src kd mutant did not apparently increase the total amount of Src proteins, it significantly suppressed the total Src activity and the tyrosine phosphorylation of FAK stimulated by HGF (Fig. 4A), supporting an essential role for Src in HGF-stimulated FAK phosphorylation, as previously suggested by our laboratory (22). Moreover, the expression of Src kd mutant inhibited the potentials of FAK-overexpressed cells to grow in soft agar and invade Matrigel upon HGF stimulation by ~70% (Fig. 4B). Consistently, the selective Src family kinase inhibitor PP1 was found to completely block the transformed phenotypes of FAK-overexpressed cells upon HGF stimulation (Fig. 6B). To examine the significance of p130cas in this transformation model, the SH3 domain of p130cas (CasSH3) was expressed in MDCK cells that had already expressed WT FAK (Fig. 5A). We have previously demonstrated that the SH3 domain of p130cas competes with endogenous p130cas for FAK binding (8, 19). In this study, we showed that the expression of the SH3 domain of p130cas in FAK-overexpressed MDCK cells significantly (~90%) inhibited their abilities to grow in soft agar and to invade Matrigel upon HGF stimulation (Fig. 5B). Together, these results support the significance of the Src/p130cas signaling cascade in cell transformation.


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  		Fig. 4.   Inhibitory effect of kinase-deficient Src on FAK promotion of HGF-dependent cell transformation. A, MDCK cells overexpressing FAK alone (FAK) or both FAK and Src kd (FAK/Src kd) were treated with (+) or without (-) 10 ng/ml HGF for 15 min before lysis. Src proteins were immunoprecipitated by monoclonal anti-Src and subjected to immunoblotting with anti-Src or an in vitro kinase assay for Src activity. To analyze the phosphorylation of FAK, FAK proteins were immunoprecipitated and subjected to immunoblotting with anti-phosphotyrosine or anti-FAK. B, MDCK cells as described in A were subjected to the soft agar-colony formation assay and the Matrigel invasion assay in the presence of HGF. Data (mean ± S.E.) are from nine data points from three independent experiments using three independent clones for each experimental group. Relative colony formation and Matrigel invasion were calculated based on the level of cells overexpressing FAK alone.


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  		Fig. 5.   Inhibitory effect of the SH3 domain of p130cas on FAK promotion of HGF-dependent cell transformation. A, cell lysates from control cells (Neo) and stable MDCK cells expressing HA epitope-tagged FAK, CasSH3, or both FAK and CasSH3 (FAK/CasSH3) were analyzed by immunoblotting with anti-HA. B, MDCK cells as described for A were subjected to the soft agar colony formation assay and the Matrigel invasion assay in the presence of HGF. Data (mean ± S.E.) are from nine data points from three independent experiments using three independent clones for each experimental group. Relative colony formation and Matrigel invasion were calculated based on the level of cells overexpressing FAK alone.

Enhancement of HGF-elicited Signal Transduction Pathways by FAK Overexpression-- To identify the signal transduction pathways involved in FAK promotion of HGF-dependent cell transformation, control cells and stable MDCK cell lines overexpressing FAK with or without the expression of the SH3 domain of p130cas were serum-starved and exposed to HGF for a short (15 min) or long period (6 h) of time before harvest. The activation of intracellular signaling molecules that have been reported to be involved in HGF signaling was then analyzed by immunoblotting with phospho-specific antibodies (Fig. 6A). HGF stimulation led to sustained activation of ERK, JNK, and AKT in FAK-overexpressed cells but transient activation of these in control cells. Although STAT3 and phospholipase Cgamma were reported to be activated by HGF (45, 46), their phosphorylation levels were not altered in our experiments (data not shown). The expression of the SH3 domain of p130cas was capable of preventing the sustained activation of ERK, JNK, and AKT by both FAK overexpression and HGF stimulation. Taken together, although we cannot exclude the possibility that signaling molecules other than those examined in this study may participate in FAK promotion of HGF-dependent cell transformation, the sustained activations of ERK, JNK, and AKT are likely to account at least partially for the acquisition of transformed phenotypes. Accordingly, we found that the specific ERK kinase (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) inhibitor PD98059 efficiently blocked HGF-dependent transformation of FAK-overexpressed cells (Fig. 6B), supporting the importance of the ERK pathway in this transformation model. In addition, the translation inhibitor cycloheximide could completely inhibit the cell transformation (Fig. 6B), suggesting that de novo protein synthesis is required for cell transformation induced by the synergy between FAK overexpression and HGF stimulation.


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  		Fig. 6.   Enhancement of HGF-elicited signalings by FAK overexpression. A, control cells (Neo) and stable MDCK cells overexpressing FAK or both FAK and CasSH3 (FAK/CasSH3) were serum-starved for 24 h and then incubated with 10 ng/ml HGF for 15 min or 6 h before lysis. An equal amount of cell lysates was analyzed by immunoblotting with antibodies as indicated. A representative result of three experiments is shown. B, MDCK cells overexpressing FAK were subjected to the soft agar-colony formation assay and the Matrigel invasion assay in the presence of HGF and an inhibitor. 100 µM PD98059, 10 µM PP1, or 10 µg/ml cycloheximide was used in both assays. The solvent Me2SO was used as a control. Data (means ± S.E.) are from three independent experiments. Relative colony formation and Matrigel invasion were calculated based on the level of cells in the presence of HGF and Me2SO.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Since FAK has been implicated to play a critical role in regulating integrin-mediated cellular functions including cell migration, cell cycle progression, and cell survival, it is generally believed that an increased level of FAK may contribute to a more aggressive phenotype in tumors. However, the precise role of FAK in tumorigenesis has not been clarified. In this study, we have established an in vitro model to examine the effect of increased expression of FAK on cell transformation. The results described in this report demonstrate that a synergism between FAK overexpression and HGF stimulation leads to MDCK cell transformation, judging from two in vitro characteristics, anchorage-independent cell growth and Matrigel invasion. Notably, neither FAK overexpression nor HGF stimulation alone is capable of transforming MDCK cells. Therefore, our results suggest that an elevated expression of FAK may sensitize cells to HGF stimulation, leading to aberrant amplification of certain intracellular signals important for the control of fundamental cellular functions. It is possible that FAK may play roles in two crucial stages during tumorigenesis by its capability to confer an anchorage-independent growth and to promote invasiveness of tumor cells upon growth factor stimulation. It has previously been shown that the expression of the constitutively activated form of FAK (CD2-FAK) by anchoring it to the plasma membrane protected MDCK cells against apoptosis upon detachment and led to an anchorage-independent growth in soft agar and tumor formation in nude mice (9). Because the activity and phosphorylation of FAK can be activated by HGF stimulation in a manner independent of integrin-mediated cell adhesion (22), it is likely that signals activated by the synergy between FAK overexpression and HGF stimulation may be analogous to those transduced by CD2-FAK.

Because integrins and growth factor receptors share many common elements in their signaling pathways, it is clear that there are many opportunities for integrin signals to modulate growth factor signals and vice versa. For example, integrins can induce various growth factor receptors including Met to undergo ligand-independent activation, at least to the extent of enhanced tyrosine phosphorylation after cell adhesion (47). Because FAK is known to be activated by cell adhesion, it was thought to be involved in this process. However, FAK overexpression seems not to enhance the phosphorylation of Met in the presence or absence of HGF stimulation (Fig. 1A), suggesting that the transformed cell phenotype observed in this study is not simply the result of an enhanced activation of Met by FAK overexpression. In fact, FAK has been demonstrated not to be involved in cell adhesion-elicited activation of Met or epidermal growth factor receptor (47, 48). Nevertheless, our results suggest that FAK is more likely to serve as a functional amplifier to potentiate HGF-triggered activation of signaling pathways. Similarly, in human embryonic kidney 293 cells (22), the expression of Src kd mutant or the addition of a selective Src inhibitor prevents HGF-induced FAK phosphorylation in MDCK cells (Fig. 4). We propose that following Met activation, Src in turn is activated to phosphorylate FAK independent of integrin activation, thereby generating an additional signaling platform to potentiate HGF-elicited signals. Alternatively, HGF may cause the tyrosine phosphorylation of certain integrins such as alpha 6beta 4 (49), which then induces FAK to undergo autophosphorylation. Recently, FAK has been shown to be activated by integrin beta 4 (50).

In this study, we have found that FAK mutants deficient in Src or p130cas binding failed to transform cells even with HGF stimulation, indicating that simultaneous binding of both Src and p130cas are required for FAK to exert its transforming function. In addition, the finding that the expression of the SH3 domain of p130cas resulted in reversion of HGF-dependent transformed phenotypes of FAK-overexpressed cells further demonstrates the significance of p130cas in this transformation model. p130cas has previously been shown to be essential for FAK to promote cell survival (7, 8) and migration (21, 51). Thus, it is likely that p130cas may contribute to cell transformation by its function at least in promoting cell survival and migration, both of which are relevant to anchorage-independent growth and invasion, respectively. In fact, p130cas was originally identified as a major tyrosine-phosphorylated protein in cells transformed by v-src (52) or v-crk (53). Its phosphorylation and subsequent association with adaptor protein Crk has been shown to be critical for its function (54). Thus, although Src may contribute to cell transformation by pathways independent of p130cas, its necessity in FAK promotion of HGF-dependent transformation may be the result of its function, at least, in phosphorylating p130cas.

We have previously shown that unlike FAK WT, the D395A mutant failed to protect MDCK cells from UV-induced apoptosis (8) and to promote cell migration on matrix proteins (21). In this study, however, we found that the FAK D395A mutant, similar to WT, was able to confer a transformed phenotype to MDCK cells upon HGF stimulation (Fig. 3, B and C), indicating that the direct binding of PI3K is dispensable for FAK to exert its transforming activity upon HGF stimulation. Nevertheless, our results did not exclude the necessity of PI3K activity in this transformation model. It is known that HGF treatment substantially activates PI3K in MDCK cells (55, 56); therefore, it is possible that the extent of PI3K activation by HGF stimulation may have already reached its threshold for cell transformation, regardless of the fraction of PI3K activated by FAK. Consistent with this, we found that the specific PI3K inhibitor LY294002 completely inhibited FAK-overexpressed cells from growing in HGF-containing soft agar and to invade Matrigel upon HGF stimulation (data not shown).

To identify the downstream signals responsible for cell transformation by the synergistic effect of FAK overexpression and HGF stimulation, the HGF-elicited activation of intracellular signaling molecules was compared between FAK-overexpressed cells and control cells. Among the molecules examined in this study, the duration of ERK, JNK, and AKT activation elicited by HGF stimulation was prolonged by FAK overexpression, rendering it likely that the sustained activation of these signaling molecules may at least partially account for cell transformation in this model. In addition, because of the inhibitory effect of the SH3 domain of p130cas on the sustained activation of ERK, JNK, and AKT, it is possible that the signals downstream of p130cas may target to these molecules. In fact, the signals transmitted by p130cas have been shown to activate ERK (57) and JNK (5, 7); however, the possibility for p130cas to activate AKT in this model remains to be tested.

The HGF receptor is the product encoded by the c-met proto-oncogene (32, 33). Inappropriate activation of the HGF/Met pathway has been implicated in the etiology of a number of human tumors and has been shown to confer invasive and metastatic properties to cancer cells (34-38). Similarly, an elevated level of FAK has been implicated to play a role in tumor progression to an invasive phenotype (39-41). In this study, we provide evidence that an increased level of FAK may render cells susceptible to transformation by HGF at a concentration that does not induce cell transformation, suggesting a synergism between HGF/Met signaling and FAK signaling. In addition to HGF, several growth factors have been shown to activate FAK (22-25). Accordingly, epidermal growth factor also induced a transformed phenotype in MDCK cells overexpressing FAK, although to a lesser extent (data not shown). Therefore, our results implicate that increased expression of FAK may generally accompany growth factors in tumorigenesis and support the use of FAK expression level as a marker for occult invasion in premalignant conditions.

    ACKNOWLEDGEMENT

We thank Dr. David Shalloway (Cornell University, Ithaca, NY) for providing pEVX-Src kd.

    FOOTNOTES

* This work was supported by National Health Research Institutes, Taiwan, Grant NHRI-EX91-8919SC, National Science Council, Taiwan, Grant NSC-91-2311-B-005-044, and Taichung Veterans General Hospital, Taiwan, Grants TCVGH905801A and TCVGH915802A.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors contributed equally to this work.

|| To whom correspondence should be addressed. Tel.: 886-4-22854922; Fax: 886-4-22851797; E-mail: hcchen@nchu.edu.tw.

Published, JBC Papers in Press, October 21, 2002, DOI 10.1074/jbc.M204691200

    ABBREVIATIONS

The abbreviations used are: FAK, focal adhesion kinase; HGF, hepatocyte growth factor; PI3K, phosphatidylinositol 3-kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; MDCK, Madin-Darby canine kidney; HA, hemagglutinin; FRNK, FAK-related nonkinase; WT, wild type; kd, kinase-deficient; SH3, Src homology 3; CasSH3, the SH3 domain of p130cas.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Richardson, A., Malik, R. K., Hildebrand, J. D., and Parsons, J. T. (1997) Mol. Cell. Biol. 17, 6906-6914[Abstract]
2. Richardson, A., and Parsons, J. T. (1996) Nature 380, 538-540[CrossRef][Medline] [Order article via Infotrieve]
3. Cary, L. A., Chang, J. F., and Guan, J.-L. (1996) J. Cell Sci. 109, 1787-1794[Abstract]
4. Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M., Yamamoto, T., and Aizawa, S. (1995) Nature 377, 539-544[CrossRef][Medline] [Order article via Infotrieve]
5. Oktay, M., Wary, K. K., Dans, M., Rirge, R. B., and Giancotti, F. G. (1999) J. Cell Biol. 145, 1461-1469[Abstract/Free Full Text]
6. Zhao, J.-H., Reiske, H., and Guan, J.-L. (1998) J. Cell Biol. 143, 1997-2008[Abstract/Free Full Text]
7. Almeida, E. A. C., Ilic, D., Han, Q., Hauck, C. R., Jin, F., Kawakatsu, H., Schlaepfer, D. D., and Damsky, C. H. (2000) J. Cell Biol. 149, 741-754[Abstract/Free Full Text]
8. Chan, P.-C., Lai, J.-F., Cheng, C.-H., Tang, M.-J., Chiu, C.-C., and Chen, H.-C. (1999) J. Biol. Chem. 274, 26901-26906[Abstract/Free Full Text]
9. Frisch, S. M., Vuori, K., Ruoslahti, E., and Chan-Hui, P.-Y. (1996) J. Cell Biol. 134, 793-799[Abstract/Free Full Text]
10. Ilic, D., Almeida, E. A. C., Schlaepfer, D. D., Dazin, P., Aizawa, S., and Damsky, C. H. (1998) J. Cell Biol. 143, 547-560[Abstract/Free Full Text]
11. Cobb, B. S., Schaller, M. D., Leu, T.-H., and Parsons, J. T. (1994) Mol. Cell. Biol. 14, 147-155[Abstract/Free Full Text]
12. Xing, Z., Chen, H.-C., Nowlen, J. K., Taylor, S., Shalloway, D., and Guan, J.-L. (1994) Mol. Biol. Cell 5, 413-421[Abstract]
13. Chen, H.-C., and Guan, J.-L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10148-10152[Abstract/Free Full Text]
14. Chen, H.-C., Appeddu, P. A., Isoda, H., and Guan, J.-L. (1996) J. Biol. Chem. 271, 26329-26334[Abstract/Free Full Text]
15. Zhang, X., Chattopadhyay, A., Ji, Q.-S., Owen, J. D., Ruest, P. J., Carpenter, G., and Hanks, S. T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9021-9026[Abstract/Free Full Text]
16. Schlaepfer, D. D., Hanks, S. K., Hunter, T., and van der Geer, P. (1994) Nature 372, 786-791[Medline] [Order article via Infotrieve]
17. Schlaepfer, D. D., and Hunter, T. (1996) Mol. Cell. Biol. 16, 5623-5633[Abstract]
18. Polte, T. R., and Hanks, S. K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10678-10682[Abstract/Free Full Text]
19. Reiske, H. R., Kao, S.-C., Cary, L. A., Guan, J.-L., Lai, J.-F., and Chen, H.-C. (1999) J. Biol. Chem. 274, 12361-12366[Abstract/Free Full Text]
20. Schlaepfer, D. D., and Hunter, T. (1997) J. Biol. Chem. 272, 13189-13195[Abstract/Free Full Text]
21. Lai, J.-F., Kao, S.-C., Jiang, S.-T., Tang, M.-J., Chan, P.-C., and Chen, H.-C. (2000) J. Biol. Chem. 275, 7474-7480[Abstract/Free Full Text]
22. Chen, H.-C., Chan, P.-C., Tang, M.-J., Cheng, C.-H., and Chang, T.-J. (1998) J. Biol. Chem. 273, 25777-25782[Abstract/Free Full Text]
23. Hatai, M., Hashi, H., Mogi, A., Soga, H., Yokota, J., and Yaoi, Y. (1994) FEBS Lett. 350, 113-116[CrossRef][Medline] [Order article via Infotrieve]
24. Matsumoto, K., Matsumoto, K., Nakamura, T., and Kramer, R. H. (1994) J. Biol. Chem. 269, 31807-31813[Abstract/Free Full Text]
25. Rankin, S., and Rozengurt, E. (1994) J. Biol. Chem. 269, 704-710[Abstract/Free Full Text]
26. Sieg, D. J., Hauck, C. R., Ilic, D., Klingbell, C. K., Schaefer, E., Damsky, C. H., and Schlaepfer, D. D. (2000) Nat. Cell Biol. 2, 249-256[CrossRef][Medline] [Order article via Infotrieve]
27. Chen, H.-C., and Guan, J.-L. (1994) J. Biol. Chem. 269, 31229-31233[Abstract/Free Full Text]
28. Montesano, R., Matsumoto, K., Nakamura, T., and Orci, L. (1991) Cell 67, 901-908[CrossRef][Medline] [Order article via Infotrieve]
29. Montesano, R., Schaller, G., and Orci, L. (1991) Cell 66, 697-711[CrossRef][Medline] [Order article via Infotrieve]
30. Ridley, A., Comoglio, P. M., and Hall, A. (1995) Mol. Cell. Biol. 15, 1110-1122[Abstract]
31. Zarnegar, R., and Michalopoulos, G. K. (1995) J. Cell Biol. 129, 1177-1180[Free Full Text]
32. Bottaro, D. P., Rubin, J. S., Faletto, D. L., Chan, A. M.-L., Kmiecik, T. E., Vande Woulde, G. F., and Aaronson, S. A. (1991) Science 251, 802-804[Abstract/Free Full Text]
33. Naldini, L., Vigna, E., Ferracini, R., Longati, P., Gandino, L., Prat, M., and Comoglio, P. M. (1991) Mol. Cell. Biol. 11, 1793-1803[Abstract/Free Full Text]
34. Beviglia, L., Matsumoto, K., Lin, C.-S., Ziober, B., and Kramer, R. H. (1997) Int. J. Cancer 74, 301-309[CrossRef][Medline] [Order article via Infotrieve]
35. Di Renzo, M. F., Olivero, M., Giacomini, A., Porte, H., Chastre, E., Mirossay, L., Nordlinger, B., Bretti, S., Bottardi, S., Giordano, S., Plebani, M., Gespach, C., and Comoglio, P. M. (1995) Clin. Cancer Res. 1, 147-154[Abstract]
36. Di Renzo, M. F., Olivero, M., Martone, T., Maffe, A., Maggiora, P., De, Stefani, A., Valente, G., Giordano, S., Cortesina, G., and Comoglio, P. M. (2000) Oncogene 19, 1547-1555[CrossRef][Medline] [Order article via Infotrieve]
37. Giordano, S., Bardelli, A., Zhen, Z., Menard, S., Ponzetto, C., and Comoglio, P. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13868-13872[Abstract/Free Full Text]
38. Jeffers, M., Rong, S., and Vande Woude, G. F. (1996) J. Mol. Med. 74, 505-513[CrossRef][Medline] [Order article via Infotrieve]
39. Agochiya, M., Brunton, V. G., Owens, D. W., Parkinson, E. K., Paraskeva, C., Keith, W. N., and Frame, M. C. (2000) Oncogene 18, 5646-5653
40. Owens, L. V., Xu, L., Craven, R. J., Dent, G. A., Weiner, T. M., Knornberg, L., Liu, E. T., and Cance, W. G. (1995) Cancer Res. 55, 2752-2755[Abstract/Free Full Text]
41. Weiner, T. M., Liu, E. T., Craven, R. J., and Cance, W. G. (1993) Lancet 342, 1024-1025[CrossRef][Medline] [Order article via Infotrieve]
42. Jeffers, M., Rong, S., and Vande Woude, G. F. (1996) Mol. Cell. Biol. 16, 1115-1125[Abstract]
43. Nakamura, T., Matsumoto, K., Kiritoshi, A., Tano, Y., and Nakamura, T. (1997) Cancer Res. 57, 3305-3313[Abstract/Free Full Text]
44. Xu, L.-H., Yang, X., Carven, R. J., and Cance, W. G. (1998) Cell Growth Differ. 9, 999-1005[Abstract]
45. Boccaccio, C., Ando, M., Tamagnone, L., Bardelli, A., Michieli, P., Battistini, C., and Comoglio, P. M. (1998) Nature 391, 285-288[CrossRef][Medline] [Order article via Infotrieve]
46. Ponzetto, C., Bardelli, A., Zhen, Z., Maina, F., dalla Zonca, P., Giordano, S., Graziani, A., Panayotou, G., and Comoglio, P. M. (1994) Cell 77, 261-271[CrossRef][Medline] [Order article via Infotrieve]
47. Wang, R., Kobayashi, R., and Bishop, J. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8425-8430[Abstract/Free Full Text]
48. Moro, L., Dolce, L., Cabodi, S., Bergatto, E., Erba, E. B., Smeriglio, M., Turco, E., Retta, S. F., Giuffrida, M. G., Venturino, M., Godovac-Zimmermann, J., Conti, A., Schaefer, E., Beguinot, L., Tacchetti, C., Gaggini, P., Silengo, L., Tarone, G., and Defilippi, P. (2002) J. Biol. Chem. 277, 9405-9414[Abstract/Free Full Text]
49. Trusolino, L., Bertotti, A., and Comoglio, P. M. (2001) Cell 107, 643-654[CrossRef][Medline] [Order article via Infotrieve]
50. Abdel-Ghany, M., Cheng, H.-C., Elble, R. C., and Pauli, B. U. (2002) J. Biol. Chem. 277, 34391-34400[Abstract/Free Full Text]
51. Cary, L. A., Han, D. C., Polte, T. R., Hanks, S. K., and Guan, J.-L. (1998) J. Cell Biol. 140, 211-221[Abstract/Free Full Text]
52. Reynolds, A. B., Kanner, S. B., Wang, H.-C. R., and Parsons, J. T. (1989) Mol. Cell. Biol. 9, 3951-3958[Abstract/Free Full Text]
53. Mayer, B. J., and Hanafusa, H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2638-2642[Abstract/Free Full Text]
54. Klemke, R. L., Leng, J., Molander, R., Brooks, P. C., Vuori, K., and Cheresh, D. A. (1998) J. Cell Biol. 140, 961-972[Abstract/Free Full Text]
55. Khwaja, A., Lehmann, K., Marte, B. M., and Downward, J. (1998) J. Biol. Chem. 273, 18793-18801[Abstract/Free Full Text]
56. Royal, I., and Park, M. (1995) J. Biol. Chem. 270, 27780-27787[Abstract/Free Full Text]
57. Hakak, Y., and Martin, S. (1999) Mol. Cell. Biol. 19, 6953-6962[Abstract/Free Full Text]

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