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Cellular Characterization of a Novel Focal Adhesion Kinase Inhibitor

2007; Elsevier BV; Volume: 282; Issue: 20 Linguagem: Inglês

10.1074/jbc.m606695200

1083-351X

Jill K. Slack‐Davis, Karen H. Martin, Robert W. Tilghman, Marcin Iwanicki, Ethan Ung, Christopher Autry, Michael J. Luzzio, Beth Cooper, John C. Kath, W. Gregory Roberts, J. Thomas Parsons,

Platelet Disorders and Treatments

Focal adhesion kinase (FAK) is a member of a family of non-receptor protein-tyrosine kinases that regulates integrin and growth factor signaling pathways involved in cell migration, proliferation, and survival. FAK expression is increased in many cancers, including breast and prostate cancer. Here we describe perturbation of adhesion-mediated signaling with a FAK inhibitor, PF-573,228. In vitro, this compound inhibited purified recombinant catalytic fragment of FAK with an IC50 of 4 nm. In cultured cells, PF-573,228 inhibited FAK phosphorylation on Tyr397 with an IC50 of 30–100 nm. Treatment of cells with concentrations of PF-573,228 that significantly decreased FAK Tyr397 phosphorylation failed to inhibit cell growth or induce apoptosis. In contrast, treatment with PF-573,228 inhibited both chemotactic and haptotactic migration concomitant with the inhibition of focal adhesion turnover. These studies show that PF-573,228 serves as a useful tool to dissect the functions of FAK in integrin-dependent signaling pathways in normal and cancer cells and forms the basis for the generation of compounds amenable for preclinical and patient trials. Focal adhesion kinase (FAK) is a member of a family of non-receptor protein-tyrosine kinases that regulates integrin and growth factor signaling pathways involved in cell migration, proliferation, and survival. FAK expression is increased in many cancers, including breast and prostate cancer. Here we describe perturbation of adhesion-mediated signaling with a FAK inhibitor, PF-573,228. In vitro, this compound inhibited purified recombinant catalytic fragment of FAK with an IC50 of 4 nm. In cultured cells, PF-573,228 inhibited FAK phosphorylation on Tyr397 with an IC50 of 30–100 nm. Treatment of cells with concentrations of PF-573,228 that significantly decreased FAK Tyr397 phosphorylation failed to inhibit cell growth or induce apoptosis. In contrast, treatment with PF-573,228 inhibited both chemotactic and haptotactic migration concomitant with the inhibition of focal adhesion turnover. These studies show that PF-573,228 serves as a useful tool to dissect the functions of FAK in integrin-dependent signaling pathways in normal and cancer cells and forms the basis for the generation of compounds amenable for preclinical and patient trials. The ability of cells to respond appropriately to environmental cues is critical to maintaining cellular, tissue, and organism homeostasis. One such environmental cue is derived from cellular adhesion to the extracellular matrix. The loss of adhesion-dependent cellular regulation can lead to increased cellular proliferation, decreased cell death, changes in cellular differentiation status, and altered cellular migratory capacity, all of which are critical components of cell carcinogenesis and metastatic progression. The FAK 4The abbreviations used are: FAK, focal adhesion kinase; ELISA, enzyme-linked immunosorbant assay; FN, fibronectin; PBS, phosphate-buffered saline; TIRF, total interference reflection fluorescence; MDCK, Madin-Darby canine kidney. 4The abbreviations used are: FAK, focal adhesion kinase; ELISA, enzyme-linked immunosorbant assay; FN, fibronectin; PBS, phosphate-buffered saline; TIRF, total interference reflection fluorescence; MDCK, Madin-Darby canine kidney. family kinases (which include FAK and Pyk2) regulate cell adhesion, migration, and proliferation in a variety of cell types (for review see Refs. 1Parsons J.T. J. Cell Sci. 2003; 116: 1409-1416Crossref PubMed Scopus (1119) Google Scholar, 2Mitra S.K. Hanson D.A. Schlaepfer D.D. Nat. Rev. Mol. Cell Biol. 2005; 6: 56-68Crossref PubMed Scopus (1902) Google Scholar, 3Abbi S. Guan J.L. Histol. Histopathol. 2002; 17: 1163-1171PubMed Google Scholar). Adhesion of cells to the extracellular matrix is mediated by heterodimeric transmembrane integrin receptors located within sites of close opposition to the underlying matrix called focal adhesions. Integrin engagement and clustering stimulates FAK phosphorylation on Tyr397, creating a high affinity binding site for Src and Src family kinases. The FAK·Src complex phosphorylates many components of the focal adhesion, resulting in changes in adhesion dynamics and the initiation of signaling cascades. In addition to FAK catalytic activity, FAK also functions as a scaffold to organize structural and signaling proteins within focal adhesions. The importance of FAK as a regulator of normal cellular function is underscored by the number of cancers reported to have alterations in FAK expression and/or activity, including colon, breast, thyroid, prostate, cervical, ovarian, head and neck, oral, liver, stomach, sarcoma, glioblastoma, and melanoma (4Gabarra-Niecko V. Schaller M.D. Dunty J.M. Cancer Metastasis Rev. 2003; 22: 359-374Crossref PubMed Scopus (296) Google Scholar, 5McLean G.W. Carragher N.O. Avizienyte E. Evans J. Brunton V.G. Frame M.C. Nat. Rev. Cancer. 2005; 5: 505-515Crossref PubMed Scopus (839) Google Scholar). Additionally, alterations in FAK expression and/or activity have been associated with tumorigenesis and increased metastatic potential (4Gabarra-Niecko V. Schaller M.D. Dunty J.M. Cancer Metastasis Rev. 2003; 22: 359-374Crossref PubMed Scopus (296) Google Scholar, 5McLean G.W. Carragher N.O. Avizienyte E. Evans J. Brunton V.G. Frame M.C. Nat. Rev. Cancer. 2005; 5: 505-515Crossref PubMed Scopus (839) Google Scholar). Currently, it is unclear how the catalytic and/or scaffolding function of FAK contributes to tumor progression. To date studies of FAK function have relied on the expression of dominant interfering mutants or elimination of FAK expression by genetic knock-out, antisense oligonucleotide expression, or small interfering RNA. Herein, we report the biochemical and cellular characterization of a novel small molecule inhibitor, PF-573,228 (here after referred to as PF-228), that targets FAK catalytic activity. The inhibitor interacts with FAK in the ATP-binding pocket and effectively blocks the catalytic activity of recombinant FAK protein or endogenous FAK expressed in a variety of normal and cancer cell lines. Treatment of cells with PF-228 blocked FAK phosphorylation on Tyr397 and concomitantly reduced the tyrosine phosphorylation of paxillin, a recognized downstream effector of FAK signaling. Drug treatment of normal and cancer cells resulted in decreased cell migration and inhibited adhesion turnover, biological activities previously ascribed to FAK. Interestingly, inhibition of FAK activity had little effect on normal or cancer cell growth or apoptosis in culture. PF-573,228 provides an appropriate tool to dissect the role of FAK in regulation of cell adhesion signaling and the regulation of adhesion dynamics. Chemical Synthesis—PF-573,228 was identified through a combination of high throughput screening, structure based drug design, and conventional medicinal chemistry approaches (38Luzzio M.J. Autry C. Berliner M. Coleman K. Cooper B. Emerson E. Griffor M. Hulford C. Jani J. Kath J. LaGreca S. Lin J. Lorenzen M. Matt E. Martinez-Alsina L. Patel N. Richter D. Schmitt E. Ung E. Vajdos F. Wessell M. Whalen P. Yao L. Roberts W.G. Proc. Annu. Meet. Am. Assoc. Cancer Res. 2007; 48: 1283Google Scholar). PF-573,228 was prepared according to the procedures described in a patent (35Kath J.C. Luzzio M.J. U. S. Patent Application WO 2004056786. 2004; Google Scholar). The structure and inhibitory activity are shown in Fig. 1. Recombinant Kinase Assay—Purified activated FAK kinase domain (amino acids 410–689) was reacted with 50 μm ATP, and 10 μg/well of a random peptide polymer of Glu and Tyr (molar ratio of 4:1), poly(Glu/Tyr) in kinase buffer (50 mm HEPES, pH 7.5, 125 mm NaCl, 48 mm MgCl2) for 15 min. Phosphorylation of poly(Glu/Tyr) was challenged with serially diluted compounds at ½-Log concentrations starting at a top concentration of 1 μm. Each concentration was run in triplicate. Phosphorylation of poly(Glu/Tyr) was detected with a general anti-phospho-tyrosine (PY20) antibody, followed by horseradish peroxidase-conjugated goat anti-mouse IgG antibody. The standard horseradish peroxidase substrate 3, 3′, 5, 5′-tetramethylbenzidine was added, and Optical Density readings at 450 nm were obtained following the addition of stop solution (2 m H2SO4). The IC50 values were determined using the Hill slope model. Broad kinase selectivity profiling was performed using the KinaseProfiler™ selectivity screening service available through Upstate Biotechnology, Inc. For more information please see: www.upstate.com/discovery/services/kp_overview.q. Cellular Kinase Assays—Using the GeneSwitch inducible system from Invitrogen, stable A431 epithelial carcinoma clones were generated to express either wild type V5-tagged FAK protein or mutant FAK Y397F V5-tagged protein under the inducible regulation of mifepristone (36Ung E.J. Whalen P.M. Roberts W.G. Cell Assay for Inhibitors of Focal Adhesion Kinase (FAK). 2002; (Patent Cooperation Treaty application number W004027018)Google Scholar). Stable clones were grown in Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, 750 μg/ml Zeocin, and 50 μg/ml Hygromycin. One day prior to running the FAK cell ELISA, A431·FAKwt cells were seeded at 1.2 × 106 cells/ml in growth medium in 96-well U-bottom plates. After 4–6 h at 37 °C, 5% CO2, FAK expression was induced with 0.1 nm miferpristone. Uninduced controls were included. Goat anti-mouse or anti-rabbit plates were subsequently coated with either anti-V5 or anti-FAK (1.0 μg/ml) or an irrelevant antibody control in Superblock Tris-buffered saline buffer. Anti-V5- or anti-FAK-coated plates were blocked in 3% bovine serum albumin, 0.5% Tween for 1 h at room temperature. The cells were treated with ½-Log serial dilutions starting at a top concentration of 1 μm for 30 min at 37 °C, 5% CO2. Lysates from cells treated with indicated concentrations of compound were prepared in P-lysis buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm Na3VO4, 1 mm NaF, and protease inhibitors) and transferred to the anti-V5- or anti-FAK-coated plates to capture total induced or total FAK protein. Anti-phosphospecific FAK[Y397] was used to detect autophosphorylated FAK Tyr397, followed by secondary reporter antibody. Horseradish peroxidase substrate was added, and plates were read at 450 nm. The IC50 values were determined using the Hill slope model. For Western blot analysis, REF52 cells were treated with the indicated concentrations of inhibitor for the indicated periods of time prior to lysis in CH buffer (50 mm HEPES, 0.15 m NaCl, 2 mm EDTA, 1% Nonidet P-40, and 0.5% sodium deoxycholate, pH 7.2) containing 1 mm phenylmethylsulfonyl fluoride, 100 mm leupeptin, and 0.05 TIU/ml aprotinin, 1 mm Na3VO4, 40 mm NaF, and 10 mm Na4P2O7. For suspension/replating experiments, REF52 cells were suspended in serum-free medium in the presence or absence of the indicated concentrations of inhibitor and were allowed to reattach to plates coated with 5 μg/ml FN for 30 min in the continued presence or absence of inhibitor. The cells were lysed in CH-buffer, and Western blot analysis of whole cell lysates was performed using 25–50 μg of protein. Cell Growth and Apoptosis—Growth assays were performed by seeding 1 × 104 REF52 or PC3 cells/well of a 24-well plate in triplicate 24 h prior to daily treatment with the indicated concentrations of each inhibitor for 3 days. Subsequently, the cells were harvested and counted. Apoptosis assays were performed using a cell death detection ELISA (Roche Applied Science) according to the manufacturer’s instructions. Briefly, REF52, PC3 or MDCK cells were treated for 24 h (16 h for MDCK) with the indicated concentrations of each inhibitor prior to lysis. Cells suspended for 16–24 h in serum-free medium served as a positive control. The cell lysates were incubated in duplicate in the ELISA system. The data represent the means ± standard deviation of one of three experiments performed in duplicate. Cell Migration Assays—Cell migration was assessed using modified Boyden chambers (tissue culture-treated, 6.5-mm diameter, 10-mm thickness, 8-mm pores; BioCoat; Fisher) as described previously (6Slack J.K. Catling A.D. Eblen S.T. Weber M.J. Parsons J.T. J. Biol. Chem. 1999; 274: 27177-27184Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Chemokinesis was determined to be the number of cells migrating to the lower side of the chamber in serum-free medium. For growth factor-mediated chemotaxis, 10% fetal bovine serum was included in the lower chamber. To assess FN-stimulated haptotaxis, the underside of the membrane was coated with 1.5 μg/ml FN suspended in PBS in the lower chamber, and PBS was placed in the upper chamber. After incubating the chambers overnight at 4 °C, the FN solutions were removed, and Dulbecco’s modified Eagle’s medium without serum was added to each chamber. For inhibitor studies, 1 × 105 REF52 cells were pretreated with each inhibitor for 30 min prior to harvest and addition to the transwell. The inhibitor remained in both chambers of each well for the duration of the migration assay. The cells were allowed to migrate for 6 h at 37 °C. Nonmigrating cells on the upper side of the membrane were removed with a cotton swab. The cells that had migrated to the underside of the membrane were washed twice in PBS, fixed with 4% paraformaldehyde at room temperature for 20 min, washed twice with PBS, and stained with crystal violet. Migration was assessed by counting four 320× fields using a Zeiss Axiovert 135TV inverted fluorescence microscope. The data presented represent the means ± standard deviation of one of three experiments performed in triplicate normalized to the untreated control. Each experimental group was analyzed using single-factor analysis of variance. If the global F test for differences among any one of the groups was significant at the 5% level, we proceeded to investigate which treated sample(s) differed from untreated controls using Student’s t tests assuming unequal variance. Statistical significance was defined as p ≤ 0.05. Wound Healing Assay—Confluent REF52 monolayers on 35-mm Bioptechs delta-T dishes (Fisher) were wounded with a 10-μl pipette tip, and cells migrating into the wound were filmed for 9 h at 37 °C by time lapse microscopy. PF-573,228 was added at the time of wounding, and filming was initiated 1 h post-wounding. Time lapse microscopy was performed using a Nikon TE200 inverted microscope with a 20× differential interference contrast objective and a Bioptechs heated stage. The images were captured with a Hamamatsu Orca camera and compiled using Improvision Openlab software (Improvision, Lexington, MA). Following image capture, the nuclei of single cells were tracked over time as they migrated into the wound, and the cell speed was calculated by dividing the length of the cell track by the total time of the movie. Immunofluorescence and Cell Spreading—Adherent REF52 cells were washed and treated with increasing concentrations of PF-573,228 for 1 h in the absence of serum. For replating experiments REF52 cells were held in suspension for 30 min without serum and in the presence of increasing amounts of the FAK inhibitors. The cells were plated onto FN-coated (5 μg/ml) coverslips (for immunofluorescence) or delta-T dishes (for differential interference contrast time lapse movies) for 60 min in the continued presence of inhibitors. The cells were fixed for 20 min with fresh 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 2 min and blocked in 20% goat serum, 2% bovine serum albumin for 30 min. The cells were then stained with antibodies to paxillin (Transduction Laboratories, Franklin Lakes, NJ) or FAK Tyr(P)397 (BioSource International, Camarillo, CA). Secondary antibodies were labeled with Alexa Fluor 488 or 594 dyes (Molecular Probes, Eugene, OR). Images were acquired using a Nikon Eclipse E600 microscope with a 60× oil objective, a Hamamatsu digital camera, and the Openlab imaging software package (Improvision). TIRF Microscopy and Adhesion Turnover Analysis—NIH-3T3 cells were stably transfected with green fluorescent protein-paxillin using the FLP-In system (Invitrogen). Confluent monolayers on delta-T dishes were treated with PF-573,228 and wounded with a pipette tip as described above. Fluorescent adhesions were visualized at 37 °C using a Nikon TE2000-E Eclipse inverted TIRF microscope equipped with a 60 × 1.45 N.A. TIRF objective, a Retiga digital camera (QImaging) and QCapture Pro acquisition software. The images were acquired every 5 min for 60–90 min. Image analysis was performed using Openlab software. The intensity of each adhesion was measured over time, and the peak (maximal) intensity for each adhesion was determined. Adhesion lifetime was calculated as the sum of the time required for the adhesion to reach the peak intensity from its half-peak intensity and the time required for the adhesion to return to its half-peak intensity from its peak intensity. At least seven adhesions were analyzed per cell. Inhibition of FAK Kinase Activity in Vitro and in Vivo—PF-573,228, (6-[(4-((3-(methanesulfonyl)benzyl)amino)-5-trifluoromethylpyrimidin-2-yl)amino]-3,4-dihydro-1H-quinolin-2-one) is a competitive inhibitor of ATP with specificity for FAK family protein-tyrosine kinases (Fig. 1A). PF-573,228 was one of several compounds evaluated for biochemical and cellular activities against FAK and was chosen for additional studies based on its specificity for FAK, minimal activity against other kinases tested, and utility for in vitro evaluation. Using a catalytically active fragment of FAK, the inhibitory properties of PF-228 were assessed initially by measuring the phosphorylation of poly(Glu/Tyr) by enzymatically active recombinant FAK protein. PF-228 inhibited catalytic activity in this assay with an IC50 of 4 nm (Fig. 1B and Table 1). In a parallel assay, PF-228 inhibited Pyk2 with an IC50 of 1 μm (Table 1). PF-228 was screened against a commercially available panel of recombinant enzymes (Upstate Biotechnology, Inc.; (7Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3910) Google Scholar)) at 1 μm (Table 1). In this assay, 1 μm PF-228 (250-fold the IC50 for FAK) inhibited the cyclin-dependent kinases CDK1/cyclin B and CDK7/cyclin H 77% and 86%, respectively, and GSK3β 64%. Therefore, the inhibitory effect of PF-228 on recombinant CDK1/cyyclin B, CDK7/cyclin H, and GSK3β kinase activity was determined. The IC50 values for PF-228 on all kinases were 50–250 times greater than the IC50 for FAK (Table 1).TABLE 1Kinase selectivity profile of PF-228KinasePF-228aPercentage of inhibition.1 μmIC50NmFAK4Pyk2>1000Abl(h)8ACK29AKT0ASK1BTK11c-RAF(h)0CaMKIV(h)0CDK1/cyclinB(h)77486CDK2/cyclinA(h)50CDK2/cyclinE(h)44CDK3/cyclinE(h)44CDK5/p35(h)0CDK6/cyclinD3(h)15CDK7/cyclinH/MAT1(h)86197CHK113cSRC(h)1EGFR(h)0EphA215EphB211Fyn28GSK3β(h)64>1000IR27IGF-1R21IKKβ(h)56JAK327JNK1α1(h)4c-kit25LCK0MAPK1(h)48MEK1(h)20MKK48MKK616PDGFR-β2P70S6K(h)31PI 3-Kγ(h)0PKA(h)0PKBα(h)13PKCα(h)12PKC-θ36ROCK-II(h)8a Percentage of inhibition. Open table in a new tab Two cell-based assays were used to assess the activity of PF-228. First, A431 epithelial carcinoma cells expressing epitope-tagged FAK under the inducible regulation of mifepristone were incubated with increasing concentrations of PF-228, and the phosphorylation of FAK Tyr397 was measured in a quantitative ELISA (see “Experimental Procedures”). PF-228 inhibited FAK phosphorylation in A431 cells with IC50 of 11 nm (Fig. 2A). To assess the ability of PF-228 to block endogenous FAK activity, the phosphorylation of FAK Tyr397 was measured in cultured REF52 cells (a nontransformed, immortalized rat fibroblast cell line). Treatment of cells with increasing concentrations of PF-228 for 60 min revealed half-maximal inhibition of Tyr397 phosphorylation of ∼100 nm. Greater than 75–80% inhibition was routinely achieved with PF-228 concentrations of 0.3–3 μm (Fig. 2B). PF-228 also blocked FAK Tyr397 phosphorylation in PC3 (prostate carcinoma), SKOV-3 (ovarian carcinoma), L3.6p1 and F-G (pancreatic carcinomas), and MDCK cells with IC50 of 30–500 nm (Fig. 2B, inset). FAK Tyr397 phosphorylation decreased within 15 min of treatment with 1 μm PF-228 and was accompanied by a decrease in the phosphorylation of a downstream substrate, paxillin Tyr31 (Fig. 2C). FAK localizes to and is activated within focal adhesions (1Parsons J.T. J. Cell Sci. 2003; 116: 1409-1416Crossref PubMed Scopus (1119) Google Scholar, 2Mitra S.K. Hanson D.A. Schlaepfer D.D. Nat. Rev. Mol. Cell Biol. 2005; 6: 56-68Crossref PubMed Scopus (1902) Google Scholar). Therefore, the subcellular localization of phosphorylated FAK was examined in adherent REF52 cells 1 h after treatment with increasing concentrations of the FAK inhibitors in the absence of serum. Paralleling the Western blot analysis, there was a dose-dependent decrease in FAK phosphorylation within paxillin-containing focal adhesions, particularly noticeable in the larger adhesions underlying the cell body (Fig. 2D). Effects of FAK Inhibition on Cell Growth and Apoptosis—Adhesion-dependent signaling events have been reported to regulate cell growth and apoptosis (8Playford M.P. Schaller M.D. Oncogene. 2004; 23: 7928-7946Crossref PubMed Scopus (416) Google Scholar, 9Cary L.A. Han D.C. Guan J.L. Histol. Histopathol. 1999; 14: 1001-1009PubMed Google Scholar). Because increased FAK expression has been correlated with increased tumorigenicity (reviewed in Refs. 4Gabarra-Niecko V. Schaller M.D. Dunty J.M. Cancer Metastasis Rev. 2003; 22: 359-374Crossref PubMed Scopus (296) Google Scholar and 5McLean G.W. Carragher N.O. Avizienyte E. Evans J. Brunton V.G. Frame M.C. Nat. Rev. Cancer. 2005; 5: 505-515Crossref PubMed Scopus (839) Google Scholar), the growth inhibitory effects of PF-228 on PC3 cells, a prostate adenocarcinoma with increased FAK expression (10Slack J.K. Adams R.B. Rovin J.D. Bissonette E.A. Stoker C.E. Parsons J.T. Oncogene. 2001; 20: 1152-1163Crossref PubMed Scopus (186) Google Scholar), as well as a nontransformed cell line (REF52) were assessed. REF52 or PC3 cells were treated with PF-228 each day for 3 days, after which the total number of cells was determined and compared with the Me2SO-treated controls (Table 2). Treatment of cells with 1 μm PF-228 failed to inhibit cell growth, although this concentration of PF-228 was sufficient to inhibit FAK phosphorylation by greater than 80–90%. Growth of REF52 and PC3 cells was significantly inhibited at the higher concentration of 10 μm PF-228 (Table 2). To assess whether the inhibition of growth observed at higher concentrations was perhaps due to off target effects of PF-228, FAK-deficient fibroblasts (11Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1576) Google Scholar) were treated with increasing concentrations of PF-228. Inhibition of growth was observed using 10 μm PF-228 (data not shown), indicating that prolonged treatment of cells at high concentrations of drug may inhibit cell growth in a FAK-independent manner.TABLE 2Effects of PF-228 on cell growthPF-228Me2SO1 μm10 μmGrowthaPercentage of Me2SO control.REF52100141 ± 357 ± 5PC3100109 ± 617 ± 6a Percentage of Me2SO control. Open table in a new tab To examine whether inhibition of FAK catalytic activity induced apoptosis, REF52, PC3, or MDCK cells were treated with the indicated concentrations of PF-228 for 24 h, and apoptosis was assessed using an ELISA assay, which measures the presence of free histones in lysates (Fig. 3). As previously reported suspension of MDCK cells for 16 h readily induced apoptosis (Fig. 3) (12Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2735) Google Scholar). Similarly, suspending REF52 cells for 24 h also induced apoptosis, although to a lesser extent than the apoptosis induced in MDCK cells. PC3 cells did not undergo apoptosis in suspension. Treatment of attached REF52, PC3, or MDCK cells with PF-228 failed to induce histone accumulation at concentrations shown to significantly block FAK Tyr397 phosphorylation. Effects of FAK Inhibition on Cell Spreading and Migration—Cell adhesion to extracellular matrix components such as FN stimulates FAK activity and cell spreading (1Parsons J.T. J. Cell Sci. 2003; 116: 1409-1416Crossref PubMed Scopus (1119) Google Scholar, 2Mitra S.K. Hanson D.A. Schlaepfer D.D. Nat. Rev. Mol. Cell Biol. 2005; 6: 56-68Crossref PubMed Scopus (1902) Google Scholar). To assess the ability of PF-228 to block adhesion-dependent FAK activation, REF52 cells were suspended in serum-free medium containing increasing concentrations of PF-228 or vehicle control for 1 h and plated on FN-coated dishes for 30 min in the continued presence or absence of inhibitor (Fig. 4A). Treatment of cells with 1–3 μm PF-228 reduced FN-stimulated FAK Tyr397 phosphorylation by ∼65–85% (Fig. 4A). In addition, treatment of cells with PF-228 also reduced the tyrosine phosphorylation of paxillin on Tyr31 in a dose-dependent manner (data not shown). To examine the effects of PF-228 on the morphology of cell spreading, REF52 cells were suspended in the presence of the inhibitor and then plated on 5 μg/ml FN for 60 min in the continued presence of inhibitor. Immunostaining showed a maximal inhibition of FAK phosphorylation and a loss of paxillin organization in focal adhesions with 3 μm PF-228 (Fig. 4B). A role for FAK catalytic activity in cell migration was assessed in transwell assays using 10% serum or FN as chemoattractants. REF52 cells were pretreated with the indicated concentrations of PF-228 for 30 min and allowed to migrate toward 10% serum or FN for 6 h in the continued presence of inhibitor (Table 3). Treatment of cells with 1.0 μm PF-228 (a concentration that reduces FAK phosphorylation by 80%) had no significant effect on random migration of REF52 cells (Table 3). However, at this concentration of PF-228, both FN and serum-stimulated migration were significantly inhibited. Treatment of cells with 10 μm PF-228 blocked random migration and also efficiently blocked serum and FN-stimulated migration (Table 3).TABLE 3Effects of PF-228 on cell migrationAssayPF-228Me2SO1.0 μm10 μmTranswell assayaThe data represent fold increase over mock-treated, Me2SO control ± standard error.Mock1.01.2 ± 0.7cNot significantly different from Me2SO.0.4 ± 0.110% serum4.0 ± 2.01.0 ± 0.10.3 ± 0.02FN4.0 ± 1.82.2 ± 0.230.5 ± 0.04Wound healingbSee “Experimental Procedures.”10% serum300 nm/min220 nm/min (27%)120 nm/min (60%)a The data represent fold increase over mock-treated, Me2SO control ± standard error.b See “Experimental Procedures.”c Not significantly different from Me2SO. Open table in a new tab Inhibition of cell migration by PF-228 was also assessed in wound assays performed with REF52 cells (supplemental Fig S1). Treatment of cultures with 1 μm PF-228 significantly reduced the rate of movement of individual cells into the wound, supporting the results from the transwell assay (Table 3). Cell migration requires the coordinated formation and disassembly of focal adhesions, a phenomenon known as focal adhesion turnover (13Webb D.J. Donais K. Whitmore L.A. Thomas S.M. Turner C.E. Parsons J.T. Horwitz A.F. Nat. Cell Biol. 2004; 6: 154-161Crossref PubMed Scopus (1051) Google Scholar). The effects of PF-228 treatment on focal adhesion turnover were analyzed using a wound assay in NIH-3T3 cells stably expressing green fluorescent protein-paxillin. Focal adhesions in cells at the edge of the wound were analyzed by TIRF microscopy. The lifetime of the adhesions was measured by monitoring the fluorescence intensity of single adhesions over time. Untreated cells exhibited active adhesion assembly and disassembly (Fig. 5A, arrowheads), and the average adhesion lifetime was 21.9 ± 11.3 min (Fig. 5B). In contrast, all of the adhesions in the cells pretreated with PF-228 showed a decrease in the rate of disassembly (Fig. 5A, arrowheads) and exhibited significantly longer lifetimes than the adhesions in the untreated cells (Fig. 5B, average adhesion lifetime of 33.1 ± 12.9 and 42.0 ± 14.4 min for 1 and 10 μm PF-228, respectively). Treatment with PF-228 did not inhibit the assembly of new adhesions (Fig. 5A, arrow). Interestingly, whereas the adhesions in the cells treated with PF-228 showed a lack of turnover, they exhibited a pattern of movement toward the center of the cell (see supplemental Movies S1 and S2). In this study we characterize the biochemical and cellular properties of a novel and specific small molecule inhibitor of FAK, PF-228. The inhibitor effectively blocked recombinant FAK catalytic activity and FAK Tyr397 phosphorylation in cultured cells. Treatment of cultured cells with concentrations of the inhibitor that significantly inhibited FAK autophosphorylation of Tyr397 failed to inhibit growth or apoptosis. However, similar treatment of cells with PF-228 resulted in inhibition of serum or FN-directed migration and decreased focal adhesion turnover. Because increased FAK expression and activity are hallmarks of many cancers and the observations that increased cell migration often characterizes tumor cells with increased metastatic potential, FAK inhibitors may have therapeutic potential for the treatment of cancer. PF-228 inhibited recombinant FAK kinase activity with an IC50 of 4 nm, 50–250-fold lower than any other kinase tested. In cultured cells, PF-228 inhibited FAK phosphorylation to varying degrees. ELISA measurements of FAK phosphorylation in A431 cells engineered to overexpress FAK produced an IC50 of 11 nm compared with ∼1