Molecular Cancer Therapeutics 1289 Inhibition of angiogenesis by growth factor receptor bound protein 2-Src homology 2 domain binding antagonists Jesus V. Soriano,1 Ningfei Liu,4 Yang Gao,2 characterized role of Grb2 in signaling cell cycle progres- Zhu-Jun Yao,2 Toshio Ishibashi,3 sion together with our present findings suggests that Charles Underhill,4 Terrence R. Burke, Jr.,2 Grb2-SH2 domain binding antagonists have the potential and Donald P. Bottaro1 to act as anticancer drugs that target both tumor and vascular cell compartments. [Mol Cancer Ther 2004;3(10): 1 2 Laboratories of Cellular and Molecular Biology and Medicinal 1289–99] Chemistry, National Cancer Institute, NIH, Bethesda, Maryland; 3Department of Otolaryngology, Social Insurance Central General Hospital, Tokyo, Japan; and 4Department of Cell Biology, Georgetown University, Washington, District of Columbia Introduction Angiogenesis, the sprouting of new capillaries from existing blood vessels, consists of a complex cascade of events in- Abstract cluding endothelial cell–mediated degradation and inva- Growth factor receptor bound protein 2 (Grb2) is an sion of the extracellular matrix, endothelial cell migration, intracellular adaptor protein that participates in the signal proliferation, differentiation, basement membrane deposi- transduction cascades of several angiogenic factors, tion, and the organization of endothelial cords into capil- including hepatocyte growth factor, basic fibroblast lary structures. This sequence of events is regulated by growth factor, and vascular endothelial growth factor. soluble angiogenic growth factors such as fibroblast growth We described previously the potent blockade of hepato- factor (FGF)-2, vascular endothelial growth factor (VEGF), cyte growth factor–stimulated cell motility, matrix inva- and hepatocyte growth factor (HGF) operating through an sion, and epithelial tubulogenesis by synthetic Grb2-Src intricate network of tyrosine kinase receptor–mediated homology 2 (SH2) domain binding antagonists. Here, we signaling pathways (1–5). The intracellular signal trans- show that these binding antagonists block basic morpho- duction pathways activated by various receptor tyrosine genetic events required for angiogenesis, including hep- kinases share a subset of common events and mediators. atocyte growth factor–, vascular endothelial growth On ligand binding, receptor kinases undergo autophos- factor–, and basic fibroblast growth factor–stimulated phorylation on multiple cytoplasmic tyrosine residues. endothelial cell proliferation and migration, as well as Phosphorylated tyrosine residues on receptors or receptor phorbol 12-myristate 13-acetate–stimulated endothelial substrates then become binding sites for Src homology 2 cell migration and matrix invasion. The Grb2-SH2 domain (SH2) domain and/or phosphotyrosine binding domain– binding antagonists also impair angiogenesis in vitro,as containing effector proteins. Growth factor receptor bound shown by the inhibition of cord formation by macrovas- protein 2 (Grb2; refs. 6–8) is an adaptor protein that binds cular endothelial cells on Matrigel. We further show that a phosphotyrosyl proteins through its SH2 domain and representative compound inhibits angiogenesis in vivo as interacts through its two SH3 domains with proteins measured using a chick chorioallantoic membrane assay. containing proline-rich motifs (9–11). These results suggest that Grb2 is an important mediator Grb2 was first found to link growth factor receptor ty- of key proangiogenic events, with potential application rosine kinases to the Ras signal transduction pathway to pathologic conditions where neovascularization con- (6, 12) as well as to other intracellular signaling proteins tributes to disease progression. In particular, the well- including Gab family members and phosphatidylinositol 3 (P1-3)-kinase, leading ultimately to cell cycle progression. In the epidermal growth factor (EGF) signaling pathway, receptor autophosphorylation allows Grb2 binding via its SH2 domain, bringing SH3 domain–bound SOS-1 into the Received 5/19/04; revised 7/14/04; accepted 8/11/04. EGF receptor (EGFR) signaling complex. SOS-1 then fa- Grant support: Swiss National Research Foundation Fellowship for Advanced Scientists (J.V. Soriano). cilitates guanine nucleotide exchange and activation of The costs of publication of this article were defrayed in part by the Ras and subsequently a cascade of kinase activation steps payment of page charges. This article must therefore be hereby marked involving Raf and mitogen-activated protein kinases advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (MAPKs), which in turn stimulate the transcription of key Note: J.V. Soriano is presently at American Type Culture Collection, cell cycle regulators (13). SH2 domain–mediated recogni- P.O. Box 1549, Manassas, VA 20108. tion by Grb2 is specific for the phosphopeptide motif Requests for reprints: Donald P. Bottaro, Urologic Oncology Branch, pYXNX (where pY is phosphotyrosine, N is aspara- Center for Cancer Research, National Cancer Institute, Building 10, gine, and X is any residue), which is found in several Room 2B47, 9000 Rockville Pike, Bethesda, MD 20892-1501. Phone: 301-402-6499; Fax: 301-402-0922. tyrosine kinases and their substrates. Thus, ligand-inde- E-mail: [email protected] pendent EGFR activation, such as growth hormone– Copyright C 2004 American Association for Cancer Research. induced EGFR tyrosine phosphorylation by JAK2, also Mol Cancer Ther 2004;3(10). October 2004 Downloaded from mct.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. 1290 Inhibition of Angiogenesis Mediated by Grb2 leads to Grb2-mediated MAPK pathway activation and c- fos expression (14, 15). Similarly, mechanical stress leading to increased angiotensin II production and transactivation of EGFR and other intracellular kinases implicates Grb2 recruitment in cardiac hypertrophy and myocardial remod- eling (16, 17). Many of the signaling pathways in which Grb2 functions are critical for vasculogenesis, angiogenesis, and lymphan- giogenesis. The VEGF receptors VEGFR2 and VEGFR3 bind Grb2 directly, and Grb2 is also recruited to receptors through its interaction with Shc (18–21). Similarly, Grb2 participates in the signaling pathways of FGF-2–, angio- poietin-1-, and HGF-stimulated vasculogenesis and angio- genesis via direct association with activated receptors and/ or through intermediaries such as FGF receptor substrate 2, Gab1, and Shc (22–25). As a mediator of signals down- stream of potent epithelial and endothelial growth and motility factors, it is not surprising that Grb2 has been implicated in tumor progression and metastasis. For example, Grb2 is critical in metastatic signaling down- stream of ErbB-2/Neu in a mouse model of mammary tumorigenesis (26), and the critical role of Grb2 in linking Figure 1. Chemical structures of Grb2-SH2 domain binding antagonists. HGF-stimulated c-Met activation with Ras and Rac- X, position of phosphotyrosine (pY) in the basic peptidomimetic structure regulated cell migration in normal development seems to recognized by the Grb2-SH2 domain, which is replaced with phosphono- methyl phenylalanine in compound C126 or with two carboxylic acid extend to c-Met-driven cell transformation and tumor moieties in C90, as shown below the backbone structure. Both moieties metastasis as well (27–30). confer resistance to cellular phosphatases. While all of these properties make Grb2 an excellent target for the design of therapeutic antitumor agents, Grand Rapids, MI). The HGF isoform HGF/NK1, which evaluating Grb2-SH2 domain binding antagonist phospho- has the same biological properties as full-length HGF, was peptides using preclinical models has been hampered by produced in a bacterial expression system and then their relative inability to cross the cell membrane, resulting purified and refolded as described previously (38). The in poor bioavailability (31, 32). The binding antagonists Grb2-SH2 domain binding antagonists designated C126 used in this study (33, 34) are potent tripeptide mimetic and C90 were synthesized and purified as described inhibitors that seem to have significantly improved po- (33, 34). Human recombinant basic FGF (bFGF) and human tential for cell membrane penetration (33); their chemical recombinant VEGF were from R&D Systems (Minneapolis, structures are shown in Fig. 1. Replacement of phosphotyr- MN). Phorbol 12-myristate 13-acetate (PMA) was from osine at position X (Fig. 1) with phosphonomethyl phe- Sigma Chemical Co. (St. Louis, MO). nylalanine (compound C126) or related structures protects Cultured Cell Lines this moiety from phosphatases (35). In a further evolution Human dermal microvascular (HMEC-1) endothelial of this concept, compound C90 has two carboxylic acid cells (39) were grown in RPMI 1640 (Biofluids, Rockville, moieties as a phosphomimetic group. These moieties confer MD) containing 10% fetal bovine serum (Biofluids) and resistance to cellular phosphatases and are the most potent 2 mmol/L glutamine (Sigma Chemical). Human micro- non-phosphorous-containing phosphotyrosine mimetics vascular endothelial cells (HMVEC) from neonatal dermis yet reported for the Grb2-SH2 domain (36, 37). We have were purchased from Cascade Biologicals (Portland, OR) reported previously that these synthetic inhibitors of the and cultured in medium 131 containing MVGS (media Grb2-SH2 domain potently block HGF-stimulated cell supplement)