Synergistic Effect of Focal Adhesion Kinase Overexpression and Hepatocyte Growth Factor Stimulation on Cell Transformation
2002; Elsevier BV; Volume: 277; Issue: 52 Linguagem: Inglês
10.1074/jbc.m204691200
ISSN1083-351X
AutoresPo-Chao Chan, Chun-Chi Liang, Kuo-Ching Yu, Ming-Chen Chang, William L. Ho, Bor-Huah Chen, Hong‐Chen Chen,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoAlthough 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 p130 cas were required for FAK to potentiate cell transformation. Expression of FAK-related nonkinase, kinase-deficient Src, or the Src homology 3 domain of p130 cas , which respectively serve as dominant negative versions of FAK, Src, and p130 cas , 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 p130 cas . 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 p130 cas in this process. 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 p130 cas were required for FAK to potentiate cell transformation. Expression of FAK-related nonkinase, kinase-deficient Src, or the Src homology 3 domain of p130 cas , which respectively serve as dominant negative versions of FAK, Src, and p130 cas , 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 p130 cas . 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 p130 cas in this process. focal adhesion kinase hepatocyte growth factor phosphatidylinositol 3-kinase c-Jun NH2-terminal kinase extracellular signal-regulated kinase Madin-Darby canine kidney hemagglutinin FAK-related nonkinase wild type kinase-deficient Src homology 3 the SH3 domain of p130 cas 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 (1Richardson A. Malik R.K. Hildebrand J.D. Parsons J.T. Mol. Cell. Biol. 1997; 17: 6906-6914Crossref PubMed Scopus (290) Google Scholar, 2Richardson A. Parsons J.T. Nature. 1996; 380: 538-540Crossref PubMed Scopus (454) Google Scholar), cell migration (3Cary L.A. Chang J.F. Guan J.-L. J. 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Chem. 1999; 274: 12361-12366Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) and survival (8Chan P.-C. Lai J.-F. Cheng C.-H. Tang M.-J. Chiu C.-C. Chen H.-C. J. Biol. Chem. 1999; 274: 26901-26906Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Almeida et al. (7Almeida E.A.C. Ilic D. Han Q. Hauck C.R. Jin F. Kawakatsu H. Schlaepfer D.D. Damsky C.H. J. Cell Biol. 2000; 149: 741-754Crossref PubMed Scopus (336) Google Scholar) showed that the FAK-p130 cas complex transduces matrix survival signals via c-Jun NH2-terminal kinase (JNK). This FAK-p130 cas -JNK signaling pathway was also shown to be required for FAK to promote cell cycle progression (5Oktay M. Wary K.K. Dans M. Rirge R.B. Giancotti F.G. J. Cell Biol. 1999; 145: 1461-1469Crossref PubMed Scopus (250) Google Scholar). The FAK-Grb2 complex has been proposed to trigger downstream signaling pathways, leading to activation of extracellular signal-regulated kinases (ERKs) (17Schlaepfer D.D. 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Chem. 1998; 273: 25777-25782Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 27Chen H.-C. Guan J.-L. J. Biol. Chem. 1994; 269: 31229-31233Abstract Full Text PDF PubMed Google Scholar). More recently, FAK was shown to integrate signals from growth factor receptors and integrins to facilitate cell migration (21Lai J.-F. Kao S.-C. Jiang S.-T. Tang M.-J. Chan P.-C. Chen H.-C. J. Biol. Chem. 2000; 275: 7474-7480Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 26Sieg D.J. Hauck C.R. Ilic D. Klingbell C.K. Schaefer E. Damsky C.H. Schlaepfer D.D. Nat. Cell Biol. 2000; 2: 249-256Crossref PubMed Scopus (1080) Google Scholar). 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 (28Montesano R. Matsumoto K. Nakamura T. Orci L. Cell. 1991; 67: 901-908Abstract Full Text PDF PubMed Scopus (1113) Google Scholar, 29Montesano R. 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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 (39Agochiya M. Brunton V.G. Owens D.W. Parkinson E.K. Paraskeva C. Keith W.N. Frame M.C. Oncogene. 2000; 18: 5646-5653Crossref Scopus (215) Google Scholar, 40Owens L.V., Xu, L. Craven R.J. Dent G.A. Weiner T.M. Knornberg L. Liu E.T. Cance W.G. Cancer Res. 1995; 55: 2752-2755PubMed Google Scholar, 41Weiner T.M. Liu E.T. Craven R.J. Cance W.G. Lancet. 1993; 342: 1024-1025Abstract PubMed Scopus (316) Google Scholar). We have previously shown that FAK overexpression in Madin-Darby canine kidney (MDCK) cells significantly enhances their migration in response to HGF stimulation (21Lai J.-F. Kao S.-C. Jiang S.-T. Tang M.-J. Chan P.-C. Chen H.-C. J. Biol. Chem. 2000; 275: 7474-7480Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), 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. 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 (12Xing Z. Chen H.-C. Nowlen J.K. Taylor S. Shalloway D. Guan J.-L. Mol. Biol. Cell. 1994; 5: 413-421Crossref PubMed Scopus (292) Google Scholar). 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-p130 cas (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 (21Lai J.-F. Kao S.-C. Jiang S.-T. Tang M.-J. Chan P.-C. Chen H.-C. J. Biol. Chem. 2000; 275: 7474-7480Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The plasmids encoding kinase-deficient (kd) Src was kindly provided by Dr. David Shalloway (Cornell University, Ithaca, NY). 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 (8Chan P.-C. Lai J.-F. Cheng C.-H. Tang M.-J. Chiu C.-C. Chen H.-C. J. Biol. Chem. 1999; 274: 26901-26906Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 21Lai J.-F. Kao S.-C. Jiang S.-T. Tang M.-J. Chan P.-C. Chen H.-C. J. Biol. Chem. 2000; 275: 7474-7480Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) 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 p130 cas have been described previously (8Chan P.-C. Lai J.-F. Cheng C.-H. Tang M.-J. Chiu C.-C. Chen H.-C. J. Biol. Chem. 1999; 274: 26901-26906Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) 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. 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. Cells were lysed in 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mm Tris-HCl, pH 8.0, 137 mmNaCl, 10% glycerol, and 1 mmNa3VO4) 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 mmTris 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 anin vitro PI3K assay as described previously (13Chen H.-C. Guan J.-L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10148-10152Crossref PubMed Scopus (486) Google Scholar). 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. 1 A). The phosphorylation of Met was increased to a similar level in Neo control cells and FAK-overexpressed cells upon HGF stimulation (Fig. 1 A). 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. The soft agar colony formation assay has been used to measure anchorage-independent cell growth, a hallmark of cell transformation. As shown in Fig. 1 B, 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 anin vitro invasion assay (Fig. 1 C). 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. 1 C). Although HGF has been shown to stimulate the invasiveness of various tumor cell lines (24Matsumoto K. Matsumoto K. Nakamura T. Kramer R.H. J. Biol. Chem. 1994; 269: 31807-31813Abstract Full Text PDF PubMed Google Scholar, 42Jeffers M. Rong S. Vande Woude G.F. Mol. Cell. Biol. 1996; 16: 1115-1125Crossref PubMed Scopus (298) Google Scholar, 43Nakamura T. Matsumoto K. Kiritoshi A. Tano Y. Nakamura T. Cancer Res. 1997; 57: 3305-3313PubMed Google Scholar), 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. 1 C). 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. 2 A). Although FRNK has been reported to be cytotoxic in some cell types (44Xu L.-H. Yang X. Carven R.J. Cance W.G. Cell Growth Differ. 1998; 9: 999-1005PubMed Google Scholar), 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. 2 B), supporting the specific role of FAK overexpression in 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, p130 cas , 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. 3 A). 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 andC), suggesting that simultaneous bindings of Src and p130 cas 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. As demonstrated in Fig. 3, the Src/p130 cas 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. 4 A), supporting an essential role for Src in HGF-stimulated FAK phosphorylation, as previously suggested by our laboratory (22Chen H.-C. Chan P.-C. Tang M.-J. Cheng C.-H. Chang T.-J. J. Biol. Chem. 1998; 273: 25777-25782Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). 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. 4 B). Consistently, the selective Src family kinase inhibitor PP1 was found to completely block the transformed phenotypes of FAK-overexpressed cells upon HGF stimulation (Fig. 6 B). To examine the significance of p130 cas in this transformation model, the SH3 domain of p130 cas (CasSH3) was expressed in MDCK cells that had already expressed WT FAK (Fig. 5 A). We have previously demonstrated that the SH3 domain of p130 cas competes with endogenous p130 cas for FAK binding (8Chan P.-C. Lai J.-F. Cheng C.-H. Tang M.-J. Chiu C.-C. Chen H.-C. J. Biol. Chem. 1999; 274: 26901-26906Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 19Reiske H.R. Kao S.-C. Cary L.A. Guan J.-L. Lai J.-F. Chen H.-C. J. Biol. Chem. 1999; 274: 12361-12366Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). In this study, we showed that the expression of the SH3 domain of p130 cas in FAK-overexpressed MDCK cells significantly (∼90%) inhibited their abilities to grow in soft agar and to invade Matrigel upon HGF stimulation (Fig. 5 B). Together, these results support the significance of the Src/p130 cas signaling cascade in cell transformation.Figure 6Enhancement 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 μmPP1, 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Inhibitory effect of the SH3 domain of p130 cas 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 forA 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 p130 cas 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. 6 A). HGF stimulation led to sustai
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