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• W1506256987 abstract "Hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets during development, homeostasis and tissue regeneration. Inappropriate HGF signaling occurs in several human cancers, and the ability of HGF to initiate a program of protease production, cell dissociation, and motility has been shown to promote cellular invasion and is strongly linked to tumor metastasis. Upon HGF binding, several tyrosines within the intracellular domain of its receptor, c-Met, become phosphorylated and mediate the binding of effector proteins, such as Grb2. Grb2 binding through its SH2 domain is thought to link c-Met with downstream mediators of cell proliferation, shape change, and motility. We analyzed the effects of Grb2 SH2 domain antagonists on HGF signaling and observed potent blockade of cell motility, matrix invasion, and branching morphogenesis, with ED50 values of 30 nm or less, but only modest inhibition of mitogenesis. These compounds are 1000–10,000-fold more potent anti-motility agents than any previously characterized Grb2 SH2 domain antagonists. Our results suggest that SH2 domain-mediated c-Met-Grb2 interaction contributes primarily to the motogenic and morphogenic responses to HGF, and that these compounds may have therapeutic application as anti-metastatic agents for tumors where the HGF signaling pathway is active. Hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets during development, homeostasis and tissue regeneration. Inappropriate HGF signaling occurs in several human cancers, and the ability of HGF to initiate a program of protease production, cell dissociation, and motility has been shown to promote cellular invasion and is strongly linked to tumor metastasis. Upon HGF binding, several tyrosines within the intracellular domain of its receptor, c-Met, become phosphorylated and mediate the binding of effector proteins, such as Grb2. Grb2 binding through its SH2 domain is thought to link c-Met with downstream mediators of cell proliferation, shape change, and motility. We analyzed the effects of Grb2 SH2 domain antagonists on HGF signaling and observed potent blockade of cell motility, matrix invasion, and branching morphogenesis, with ED50 values of 30 nm or less, but only modest inhibition of mitogenesis. These compounds are 1000–10,000-fold more potent anti-motility agents than any previously characterized Grb2 SH2 domain antagonists. Our results suggest that SH2 domain-mediated c-Met-Grb2 interaction contributes primarily to the motogenic and morphogenic responses to HGF, and that these compounds may have therapeutic application as anti-metastatic agents for tumors where the HGF signaling pathway is active. Hepatocyte growth factor (HGF)1 stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets including epithelial and endothelial cells, hematopoietic cells, neurons, melanocytes, as well as hepatocytes (reviewed in Refs.1Michalopoulos G.K. DeFrances M.C. Science. 1997; 276: 60-66Crossref PubMed Scopus (2905) Google Scholar, 2Rubin J.S. Bottaro D.P. Aaronson S.A. Biochim. Biophys. Acta. 1993; 1155: 357-371Crossref PubMed Scopus (288) Google Scholar, 3Zarnegar R. Michalopoulos G.K. J. Cell Biol. 1995; 129: 1177-1180Crossref PubMed Scopus (553) Google Scholar). These pleiotropic effects play fundamentally important roles during development, organogenesis, and tissue regeneration. For example, HGF is essential for the normal development of both liver and placenta (reviewed in Ref. 4Birchmeier C. Gherardi E. Trends Cell Biol. 1998; 8: 404-410Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar), contributes to neural development (reviewed in Ref. 5Streit A.C. Stern C.D. Ciba Found. Symp. 1997; 212: 155-165PubMed Google Scholar) and branching morphogenesis in various organs (reviewed in Ref. 6Birchmeier W. Brinkmann V. Niemann C. Meiners S. DiCesare S. Naundorf H. Sachs M. Ciba Found. Symp. 1997; 212: 230-240PubMed Google Scholar), and promotes kidney and lung regeneration (7Yanagita K. Matsumoto K. Sekiguchi K. Ishibashi H. Niho Y. Nakamura T. J. Biol. Chem. 1993; 268: 21212-21217Abstract Full Text PDF PubMed Google Scholar,8Balkovetz D.F. Lipschutz J.H. Int. Rev. Cytol. 1999; 186: 225-260Crossref PubMed Google Scholar). The biological responses to HGF are mediated by its cell surface receptor, c-Met, a transmembrane tyrosine kinase. Upon HGF binding, several tyrosines residues within the c-Met intracellular domain are phosphorylated, some of which are essential for catalytic activity, and some of which mediate the binding of signaling proteins such as the p85 subunit of phosphoinositide 3-kinase (PI3K), phospholipase C-γ, Shc, Gab1, and Grb2 (reviewed in Ref. 9Bardelli A. Comoglio P.M. Ciba Found. Symp. 1997; 212: 133-144PubMed Google Scholar). In epithelial cells, PI3K activity is required for both HGF-stimulated scatter and mitogenesis (10Royal I. Park M. J. Biol. Chem. 1995; 270: 27780-27787Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 11Rahimi N. Tremblay E. Elliott B. J. Biol. Chem. 1996; 271: 24850-24855Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), Gab1 is sufficient for tubulogenesis (12Weidner K.M. Di Cesare S. Sachs M. Brinkmann V. Behrens J. Birchmeier W. Nature. 1996; 384: 173-176Crossref PubMed Scopus (505) Google Scholar), and Grb2 binding appears to be required for HGF-stimulated cell motility and branching tubulogenesis (13Ponzetto C. Bardelli A. Zhen Z. Maina F. dalla Zonca P. Giordano S. Graziani A. Panayotou G. Comoglio P.M. Cell. 1994; 77: 261-271Abstract Full Text PDF PubMed Scopus (893) Google Scholar, 14Zhu H. Naujokas M.A. Fixman E.D. Torossian K. Park M. J. Biol. Chem. 1994; 269: 29943-29948Abstract Full Text PDF PubMed Google Scholar, 15Fournier T.M. Kamikura D. Teng K. Park M. J. Biol. Chem. 1996; 271: 22211-22217Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The small GTP-binding proteins Ras, Rho, and Rac are required for HGF-stimulated cytoskeletal rearrangements and subsequent cell motility (16Hartmann G. Weidner K.M. Schwarz H. Birchmeier W. J. Biol. Chem. 1994; 269: 21936-21939Abstract Full Text PDF PubMed Google Scholar, 17Ridley A.J. Comoglio P.M. Hall A. Mol. Cell. Biol. 1995; 15: 1110-1122Crossref PubMed Google Scholar). In addition to its critical roles in normal development and adult homeostasis, HGF signaling is strongly linked to cancer, including colon, breast, lung, thyroid, and renal carcinomas; melanoma; and several sarcomas; as well as glioblastoma (reviewed in Ref. 18Vande Woude G.F. Jeffers M. Cortner J. Alvord G. Tsarfaty I. Resau J. Ciba Found. Symp. 1997; 212: 119-130PubMed Google Scholar). The inappropriate expression of c-Met in certain mesenchymal cells can lead to a carcinogenic transformation in which the tumor cells express both mesenchymal and epithelial markers (19Tsarfaty I. Rong S. Resau J.H. Rulong S. da Silva P.P. Vande Woude G.F. Science. 1994; 263: 98-101Crossref PubMed Scopus (189) Google Scholar). Inherited mutations in c-Met that result in constitutive activation of the HGF signaling pathway are associated with human renal papillary carcinoma (20Schmidt L. Duh F.M. Chen F. Kishida T. Glenn G. Choyke P. Scherer S.W. Zhuang Z. Lubensky I. Dean M. Allikmets R. Chidambaram A. Bergerheim U.R. Feltis J.T. Casadevall C. Zamarron A. Bernues M. Richard S. Lips C.J. Walther M.M. Tsui L.C. Geil L. Orcutt M.L. Stackhouse T. Zbar B. et al.Nat. Genet. 1997; 16: 68-73Crossref PubMed Scopus (1322) Google Scholar, 21Schmidt L. Junker K. Nakaigawa N. Kinjerski T. Weirich G. Miller M. Lubensky I. Neumann H.P. Brauch H. Decker J. Vocke C. Brown J.A. Jenkins R. Richard S. Bergerheim U. Gerrard B. Dean M. Linehan W.M. Zbar B. Oncogene. 1999; 18: 2343-2350Crossref PubMed Scopus (434) Google Scholar, 22Jeffers M. Schmidt L. Nakaigawa N. Webb C.P. Weirich G. Kishida T. Zbar B. Vande Woude G.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11445-11450Crossref PubMed Scopus (387) Google Scholar). Importantly, in addition to its mitogenic activity, the ability of HGF to initiate a program of cell dissociation and increased cell motility coupled with increased protease production has been shown to promote cellular invasion through extracellular matrix substrates, and is correlated with tumor metastasis in vivo (reviewed in Refs. 6Birchmeier W. Brinkmann V. Niemann C. Meiners S. DiCesare S. Naundorf H. Sachs M. Ciba Found. Symp. 1997; 212: 230-240PubMed Google Scholar, 9Bardelli A. Comoglio P.M. Ciba Found. Symp. 1997; 212: 133-144PubMed Google Scholar, and18Vande Woude G.F. Jeffers M. Cortner J. Alvord G. Tsarfaty I. Resau J. Ciba Found. Symp. 1997; 212: 119-130PubMed Google Scholar). Increased extracellular matrix proteolysis, cell dissociation, and increased cell motility are essential for tumor metastasis, and HGF may be unique among extracellular signaling molecules in its ability to initiate all of these events. Artificial inhibitors of protein and lipid kinases, phosphatases, and protein-protein interactions have been used extensively to examine the roles of individual intracellular effector molecules in specific HGF-stimulated activities, and to explore their potential as anticancer drugs. Many intracellular effectors interact with receptor tyrosine kinases through conserved amino acid sequence modules, such as the Src homology 2 (SH2) domain (reviewed in Refs. 23Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1900) Google Scholar and 24Pawson T. Saxton T.M. Cell. 1999; 97: 675-678Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). SH2 domains directly recognize phosphotyrosine (Tyr(P)) (25Matsuda M. Mayer B.J. Fukui Y. Hanafusa H. Science. 1990; 248: 1537-1539Crossref PubMed Scopus (285) Google Scholar), with additional secondary binding interactions within two or three amino acids C-proximal to the Tyr(P) residue introducing differential affinity toward SH2 domain subfamilies (26Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2186) Google Scholar, 27Zhou S. Cantley L.C. Trends Biochem. Sci. 1995; 20: 470-475Abstract Full Text PDF PubMed Scopus (330) Google Scholar). These and other observations have led to the development of potent tripeptide inhibitors of SH2 domain interactions. Modification of the Tyr(P) residue to phosphonomethyl phenylalanine, or related structures, protects this moiety from phosphatases (28Marseigne I. Roques B.P. J. Org. Chem. 1988; 53: 3621-3624Crossref Scopus (83) Google Scholar, 29Burke Jr., T.R. Smyth M. Nomizu M. Otaka A. Roller P. J. Org. Chem. 1993; 58: 1336-1340Crossref Scopus (174) Google Scholar), whereas other modifications have been identified that increase inhibitor affinity and the potential for cell membrane penetration (30Yao Z.-J. King C.R. Cao T. Kelley J. Milne G.W.A. Voigt J.H. Burke Jr., T.R. J. Med. Chem. 1999; 42: 25-35Crossref PubMed Scopus (94) Google Scholar). In this study we describe the effects of several artificial tripeptide-based inhibitors of the Grb2 SH2 domain on HGF-stimulated mitogenesis, motogenesis, and extracellular matrix invasion in epithelial and hematopoietic target cell models. Grb2 is thought to link HGF-stimulated c-Met activation with the activation of Rho, Ras, and Rac (17Ridley A.J. Comoglio P.M. Hall A. Mol. Cell. Biol. 1995; 15: 1110-1122Crossref PubMed Google Scholar), and to regulate critical steps in early embryonic development, as well as in malignant transformation (31Cheng A.M. Saxton T.M. Sakai R. Kulkarni S. Mbamalu G. Vogel W. Tortorice C.G. Cardiff R.D. Cross J.C. Muller W.J. Pawson T. Cell. 1998; 95: 793-803Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). We show that these compounds potently block HGF-stimulated cell motility, matrix invasion, and branching morphogenesis, but not HGF-stimulated mitogenesis, with ED50 values of 1–30 nm. The anti-motility and anti-invasion effects were characterized using four different assay systems, with no evidence of toxicity, or loss of contractility required for cellular functions other than locomotion. The active compounds did not alter HGF-stimulated c-Met tyrosine kinase activation, and their potency for inhibiting HGF-stimulated motility correlated well with their affinity for binding to Grb2 in vitro. Overall, our results suggest that c-Met-Grb2 interaction contributes primarily to the motogenic and morphogenic cellular responses to HGF, and that these compounds may have therapeutic application as anti-metastatic drugs in tumors where the HGF signaling pathway is active. Human HGF protein and the cDNA for human c-Met in the pMOG vector were gifts from Dr. G. Vande Woude. The truncated HGF isoform HGF/NK1 was produced in a bacterial expression system, purified, and refolded as previously described (32Stahl S.J. Wingfield P.T. Kaufman J.D. Pannell L.K. Cioce V. Sakata H. Taylor W.G. Rubin J.S. Bottaro D.P. Biochem. J. 1997; 326: 763-772Crossref PubMed Scopus (48) Google Scholar). HGF/NK1 produced by this method stimulates all of the major biological responses of full-length HGF (33Montesano R. Soriano J.V. Malinda K.M. Ponce L.M. Kleinman H.K. Bafico A. Bottaro D.P. Aaronson S.A. Cell Growth Differ. 1998; 9: 355-365PubMed Google Scholar). The Grb2 SH2 domain antagonists designated 1–4 (Fig. 1) were synthesized and purified as described (30Yao Z.-J. King C.R. Cao T. Kelley J. Milne G.W.A. Voigt J.H. Burke Jr., T.R. J. Med. Chem. 1999; 42: 25-35Crossref PubMed Scopus (94) Google Scholar, 34Gao Y. Luo J. Yao Z.-J. Guo R. Zou H. Kelley J. Voigt J.H. Yang D. Burke Jr., T.R. J. Med. Chem. 2000; 43: 911-920Crossref PubMed Scopus (67) Google Scholar). The human mammary epithelial cell line 184B5 (35Rubin J.S. Chan A.M.L. Bottaro D.P. Burgess W.H. Taylor W.G. Cech A.C. Hirshfield D.W. Wong J. Miki T. Finch P.W. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 415-419Crossref PubMed Scopus (471) Google Scholar) was maintained in RPMI 1640 + 10% fetal bovine serum (FBS) and 5 ng/ml epidermal growth factor (Becton Dickinson). Balb/MK keratinocytes (35Rubin J.S. Chan A.M.L. Bottaro D.P. Burgess W.H. Taylor W.G. Cech A.C. Hirshfield D.W. Wong J. Miki T. Finch P.W. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 415-419Crossref PubMed Scopus (471) Google Scholar) were maintained in low calcium Eagle's minimal essential medium + 10% dialyzed FBS and 5 ng/ml mouse epidermal growth factor (R&D Systems). The human gastric carcinoma cell line Okajima (36Motoyama T. Hojo H. Suzuki T. Oboshi S. Acta Med. Biol. 1979; 27: 49-63Google Scholar) was maintained in RPMI + 15% FBS. The murine interleukin-3-dependent cell line 32D (37Day R.M. Cioce V. Breckenridge D. Castagnino P. Bottaro D.P. Oncogene. 1999; 18: 3399-3406Crossref PubMed Scopus (79) Google Scholar) was cultured in RPMI 1640 + 15% FBS and 5% WEHI-3B conditioned medium. 32D/c-Met cells were generated by co-transfection of 32D cells with pMOG/c-Met and pCEV27 encoding neomycin-resistance as described (37Day R.M. Cioce V. Breckenridge D. Castagnino P. Bottaro D.P. Oncogene. 1999; 18: 3399-3406Crossref PubMed Scopus (79) Google Scholar). The human leiomyosarcoma cell line SK-LMS-1 and Madin-Darby canine kidney (MDCK) cells were maintained in DMEM + 10% FBS. TAC-2 (38Soriano J.V. Pepper M.S. Nakamura T. Orci L. Montesano R. J. Cell Sci. 1995; 108: 413-430Crossref PubMed Google Scholar), a normal mammary gland epithelial cell line, was cultured in high glucose DMEM (Life Technologies, Inc.) supplemented with 10% FBS. c-Met autophosphorylation was analyzed by immunoprecipitation and immunoblotting as described (32Stahl S.J. Wingfield P.T. Kaufman J.D. Pannell L.K. Cioce V. Sakata H. Taylor W.G. Rubin J.S. Bottaro D.P. Biochem. J. 1997; 326: 763-772Crossref PubMed Scopus (48) Google Scholar). c-Met-Grb2 interaction was analyzed by co-immunoprecipitation of c-Met with agarose-conjugated antibodies against Grb2 (Santa Cruz Biotechnology), followed by immunoblot detection using antibodies against c-Met (Santa Cruz Biotechnology), phosphotyrosine (Tyr(P); Upstate Biotechnology), and Grb2 (Upstate Biotechnology). Briefly, intact cells were serum-deprived for 16 h in the presence or absence of Grb2 antagonists. Cells were then growth factor-treated for 10 min as indicated, and lysed in cold buffer containing non-ionic detergents and protease and phosphatase inhibitors. After immunoprecipitation for 2 h on ice, immunocomplexes were washed, eluted with SDS sample buffer, resolved by SDS-polyacrylamide gel electrophoresis, transferred to Immobilon P (Millipore), and detected by ECL (Amersham Pharmacia Biotech). HGF/NK1-stimulated PI3K activation in intact cells after 16 h of incubation in the presence or absence of Grb2 antagonists was analyzed by co-immunoprecipitation of the p85 subunit of PI3K with agarose-conjugated anti-Tyr(P), followed by immunoblot detection using antibodies against PI3K (Upstate Biotechnology). Cells were treated similarly to assess Akt and MAP kinase activation, except that SDS-polyacrylamide gel electrophoresis-resolved whole cell lysates were immunoblotted using anti-phospho-Akt and anti-Akt, or anti-phospho-MAP kinase (New England Biolabs), respectively. Mitogenic assays were performed using 184B5 cells or 32D/c-Met cells as described (35Rubin J.S. Chan A.M.L. Bottaro D.P. Burgess W.H. Taylor W.G. Cech A.C. Hirshfield D.W. Wong J. Miki T. Finch P.W. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 415-419Crossref PubMed Scopus (471) Google Scholar, 37Day R.M. Cioce V. Breckenridge D. Castagnino P. Bottaro D.P. Oncogene. 1999; 18: 3399-3406Crossref PubMed Scopus (79) Google Scholar). Briefly, cells were serum-deprived for 24 h (184B5) or 4 h (32D/c-Met) before the addition of growth factors and/or antagonists for 16 h (184B5) or 36 h (32D/c-Met). Cells were incubated with [3H]thymidine for 6 h, and DNA synthesis was measured by liquid scintillation counting. The migration of Okajima, 184B5, and SK-LMS-1 cells in modified Boyden chambers was measured using Biocoat Cell Environment control inserts (8-μm pore size; Becton Dickinson). Lower chambers contained DMEM + 0.1% bovine serum albumin, to which HGF (50 ng/ml) and/or Grb2 inhibitors were added. Cells were trypsinized, washed in DMEM + 0.1% bovine serum albumin, added to upper chambers (2 × 105 cells/ml) with growth factors and/or inhibitors, and incubated for 16 h at 37 °C. Cells on the upper surface of each filter were removed with a cotton swab, whereas cells that had traversed to the bottom surface of the filter were fixed and stained using Diff-Quik (Dade Diagnostics), and counted using brightfield microscopy. Mean values from 10 randomly selected unit areas were calculated for each of triplicate wells. The ratio of growth factor-treated to control migrating cells is designated on they axis as “migration (-fold increase).” 32D/c-Met cell migration was assayed using a modified Boyden chamber with 5-μm pore size Nucleopore filters (Corning). HGF/NK1 (300 ng/ml) and/or Grb2 inhibitors were added to both chambers, and cells were applied to the upper chamber at a final density of 2 × 106 cells/ml. After incubation for 8 h at 37 °C, cells that migrated to the lower chamber were counted with an automated cell counter (Coulter, Inc.), and migration was expressed as described for Okajima cells. SK-LMS-1 cell invasion was analyzed using Matrigel Invasion Chambers (Becton Dickinson) and quantitated essentially as described for Okajima cell migration across uncoated membranes. Briefly, SK-LMS-1 cells were seeded at 1 × 104 cells/chamber in DMEM + 5% FBS. Chambers were placed into 24-well culture plates containing DMEM + 5% FBS alone, or with HGF (10 ng/ml) and/or compounds 1–4 as indicated for 40 h at 37 °C. The ratio of growth factor-treated to control invading cells is designated on the y axis as “invasion (-fold increase).” MDCK cell invasion into three-dimensional collagen gels was analyzed as described (39Montesano R. Schaller G. Orci L. Cell. 1995; 66: 697-711Abstract Full Text PDF Scopus (436) Google Scholar). Briefly, type I collagen (1.5 mg/ml; Cohesion Technologies) was mixed with 10× minimal essential medium and sodium bicarbonate (11.76 mg/ml) at a ratio of 8:1:1 (v/v/v) on ice, and 0.4-ml aliquots were dispensed into 16-mm tissue culture wells and allowed to gel at 37 °C for 20 min. Cells were seeded onto gels (1 × 104 cells/well) in 0.4 ml of growth medium containing HGF and/or compounds 1 or 4 as indicated. After 5 days, cells were fixed in situ, and cells that had invaded the gel below the surface monolayer in 10 randomly selected fields (1 × 1.4 mm) were counted microscopically using a 20× phase contrast objective. Depth of cellular invasion into the collagen gel was quantitated in the same 10 fields per treatment group using a calibrated fine focusing micrometer. Values shown in Table I are the mean number of invading cells/field or mean invasion depth/cell in micrometers ± S.E.Table IMDCK cell invasion into collagen matricesTreatment groupMean invading cells/fieldMean invasion depth/cellμmControl00HGF29.7 ± 2.635.6 ± 2.7HGF + compound 112.6 ± 1.917.9 ± 1.9HGF + compound 434.3 ± 2.730.8 ± 2.9MDCK cells were left untreated (Control), treated with HGF (10 ng/ml), or HGF + compound 1 or 4 (100 nM), and invasion into collagen matrices was quantitated microscopically as described under “Experimental Procedures.” Values are the mean of 10 or more randomly selected fields ± S.E. Open table in a new tab MDCK cells were left untreated (Control), treated with HGF (10 ng/ml), or HGF + compound 1 or 4 (100 nM), and invasion into collagen matrices was quantitated microscopically as described under “Experimental Procedures.” Values are the mean of 10 or more randomly selected fields ± S.E. MDCK cell scatter, observed as the dispersion of single cells from tightly grouped colonies, was assayed as described (32Stahl S.J. Wingfield P.T. Kaufman J.D. Pannell L.K. Cioce V. Sakata H. Taylor W.G. Rubin J.S. Bottaro D.P. Biochem. J. 1997; 326: 763-772Crossref PubMed Scopus (48) Google Scholar). Briefly, MDCK cells were seeded into 24-well plates (2 × 10 4 cells/well) in DMEM containing various concentrations of inhibitors and/or HGF (30 ng/ml). Cells were incubated for 16 h at 37 °C. The scatter of fixed and stained cells was observed by brightfield microscopy; images were captured at 12.5× total magnification. TAC-2 cells were suspended in three-dimensional collagen gels as described (38Soriano J.V. Pepper M.S. Nakamura T. Orci L. Montesano R. J. Cell Sci. 1995; 108: 413-430Crossref PubMed Google Scholar) at 1 × 104 cells/ml in collagen and incubated in complete medium containing HGF and/or Grb2 inhibitors as indicated. After 3 days, the cultures were fixed with 2.5% glutaraldehyde in 0.1 mcacodylate buffer, and brightfield microscopic images of three or more randomly selected fields per experimental condition were digitally recorded in each of three separate experiments. The total length of the tubular structures (cord length) in each colony present in each optical field was measured using IPLab software (Scanalytics, Inc.). Cord length was considered “0” for spheroid colonies and for elongated colonies with length to diameter ratios less than 2. The mean values for each experimental condition were compared with controls using Student's unpaired t test. The structures of the Grb-2 SH2 domain binding antagonists used in this study are shown in Fig. 1. The affinity of these compounds for binding to Grb2 SH2 domainsin vitro have been described (30Yao Z.-J. King C.R. Cao T. Kelley J. Milne G.W.A. Voigt J.H. Burke Jr., T.R. J. Med. Chem. 1999; 42: 25-35Crossref PubMed Scopus (94) Google Scholar, 34Gao Y. Luo J. Yao Z.-J. Guo R. Zou H. Kelley J. Voigt J.H. Yang D. Burke Jr., T.R. J. Med. Chem. 2000; 43: 911-920Crossref PubMed Scopus (67) Google Scholar). Each compound contains a common backbone structure corresponding to the invariant Grb2 SH2 domain binding motif pY-X-N, with distinct modifications to the Tyr(P)-mimetic moiety that render each resistant to phosphatases, but maintain the net charge (−2) of this moiety. Compound 4 also has these features, but differs from the others in that it has greater than 100-fold lower affinity for binding to Grb2 SH2 domains in vitro (30Yao Z.-J. King C.R. Cao T. Kelley J. Milne G.W.A. Voigt J.H. Burke Jr., T.R. J. Med. Chem. 1999; 42: 25-35Crossref PubMed Scopus (94) Google Scholar). The similar charge and overall structure of compound 4 to compounds 1–3 thus made it an excellent negative control throughout the course of the biological characterization of the effects of these compounds on HGF signaling. To rule out the possibility that the Grb2 antagonists might interfere with HGF binding or receptor activation, we examined HGF-stimulated c-Met autophosphorylation in intact Okajima cells in the presence and absence of various concentrations of compound 1 (Fig.2). As shown in panel A, HGF/NK1-stimulated tyrosine phosphorylation of the 145-kDa c-Met β subunit was well above the level observed in control, untreated cells (compare left and right upper panels). The lower panels confirm that equal amounts of c-Met protein were present in each lane (Fig. 2 A). No differences in the level of HGF/NK1-stimulated c-Met autophosphorylation were observed in cells treated with up to 1 μm compound 1 (Fig. 2 A). Identical results were obtained for compound 2, as well as for both compounds on HGF-stimulated c-Met activation in intact 32D/c-Met cells (data not shown). Additional evidence that these compounds did not block HGF-c-Met interaction or subsequent c-Met activation is provided in the analysis of HGF-stimulated mitogenesis described below. Direct evidence that these compounds blocked c-Met-Grb2 interaction was obtained from co-immunoprecipitation/immunoblot analysis (Fig.2 B). Intact Okajima cells were treated with Grb2 inhibitors for 16 h, then briefly stimulated with HGF/NK1 before lysis, immunoprecipitation with anti-Grb2 antibodies, and immunoblotting with anti-c-Met, anti-Tyr(P), or anti-Grb2 (Fig. 2 B). Consistent with previous studies describing the phosphorylation-dependent binding of Grb2 to Tyr-1356 in c-Met (14Zhu H. Naujokas M.A. Fixman E.D. Torossian K. Park M. J. Biol. Chem. 1994; 269: 29943-29948Abstract Full Text PDF PubMed Google Scholar), HGF/NK1 stimulated the co-immunoprecipitation of the 145 kDa c-Met β subunit with Grb2 (Fig. 2 B). When cells were pretreated for 16 h with 30 nm compound 1 prior to HGF/NK1 stimulation, the amount of HGF receptor that was co-immunoprecipitated with Grb2 was reduced by ∼50% (Fig.2 B, right lanes). Thus, the effective concentration of this compound for blocking c-Met/Grb2 interaction was almost identical to its relative affinity for Grb2 SH2 domain binding measured in vitro (34Gao Y. Luo J. Yao Z.-J. Guo R. Zou H. Kelley J. Voigt J.H. Yang D. Burke Jr., T.R. J. Med. Chem. 2000; 43: 911-920Crossref PubMed Scopus (67) Google Scholar). At 300 nm compound 1, Grb2-c-Met interaction was completely abolished (Fig. 2 B). The lower panel of Fig. 2 B confirms that equal amounts of Grb2 protein were present in each lane. Similar results were obtained using compound 2 (data not shown). Despite complete inhibition of Grb2-c-Met interaction by the active compounds in intact cells, other SH2 domain-mediated interactions such as HGF-stimulated PI3K activation were not affected (Fig.2 C). The ability of HGF/NK1 to stimulate anti-Tyr(P)-immunoprecipitability of the PI3K p85 subunit, as well as activation of the downstream effector Akt, was identical in 32D/c-Met cells treated with compound 1 at 300 nm for 16 h and control cells (Fig. 2 C). Consistent with their known dependence on PI3K, HGF-stimulated activation of the MAP kinases ERK1 and ERK2 was also completely unaffected by compound 2 after incubation of intact 32D/c-Met cells for 16 h with antagonist concentrations as high as 1 μm (Fig. 2 D). Together, our data demonstrate that these antagonists act selectively on Grb2 SH2 domain binding interactions in vivo at concentrations more than 100-fold over biologically effective doses. To characterize the mechanism by which the Grb2 SH2 domain antagonists entered intact cells, we analyzed HGF/NK1-stimulated Grb2-c-Met interaction by co-immunoprecipitation following treatment of cells with compound 1 for varying time periods (Fig. 2 E). Very short (10 min) exposure of cells to 300 nm compound 1 had no effect on Grb2-c-Met interaction (data not shown). With increasing time of exposure to this treatment, Grb2-c-Met association diminished gradually over 6 h; association was inhibited by more than 50% after 2–4 h (Fig. 2 E). Similar experiments using 10-fold lower concentrations of compound 1 also revealed a gradual increase in inhibition, reaching 50% inhibition after 8 h (data not shown). Although these experiments are not definitive, the simple time- and concentration-dependent inhibition of Grb2-c-Met interaction observed is consistent with passive diffusion of these compounds across the plasma membrane. Three well established cultured cell systems representing hematopoietic and epithelial HGF targets were used to evaluate the effects of the Grb2 SH2 domain antagonists on HGF-stimulated DNA synthesis: 32D/c-Met cells (37Day R.M. Cioce V. Breckenridge D. Castagnino P. Bottaro D.P. Oncogene. 1999; 18: 3399-3406Crossref PubMed Scopus (79) Google Scholar), 184B5 human mammary epithelial cells (35Rubin J.S. Chan A.M.L. Bottaro D.P. Burgess W.H. Taylor W.G. Cech A.C. Hirshfield D.W. Wong J. Miki T. Finch P.W. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 415-419Crossref PubMed Scopus (471) Google Scholar), and Balb/MK mous" @default.
• W1506256987 created "2016-06-24" @default.
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