Pyrazolo–pyrimidine-derived c-Src inhibitor reduces angiogenesis and survival of squamous carcinoma cells by suppressing vascular endothelial growth factor production and signaling Sandra Donnini 1 , Martina Monti 1 , Cinzia Castagnini 1 , Raffaella Solito 1 , Maurizio Botta 2 , Silvia Schenone 3 , Antonio Giachetti 1 and Marina Ziche 1 * 1 Dipartimento di Biologia Molecolare, Universita` degli Studi di Siena, Via Aldo Moro, 2, 53100, Siena, Italy 2 Dipartimento Farmaco Chimico Tecnologico, Universita` degli Studi di Siena, Via Aldo Moro, 2, 53100, Siena, Italy 3 Dipartimento Scienze Farmaceutiche, Universita` degli Studi di Genova, Viale Benedetto XV, 16132 Genova, Italy Src tyrosine kinase family cooperates with activated growth factor receptors to regulate growth, invasion and metastasis. The authors examined the influence of a novel c-Src inhibitor, 1l, derived from 4-amino-substituted-pyrazolo–pyrimidines, on tumor angiogenesis and on the angiogenic output of squamous carcinoma cells, A431 and SCC-4. The effect of 1l was assessed on growth and microvessel density in A431 tumors and its effect compared with the established c-Src inhibitor PP-1. The effects of c-Src inhibition were investi- gated on vascular endothelial growth factor (VEGF) expression and activity in tumor cells grown in vivo and in vitro, as well as on VEGF mediated signaling and on endothelial cell functions. Nano- molar concentrations of 1l decreased tumor volume promoted by A431 implanted in nude mice, without affecting in vitro cell tumor survival. This effect was related to 1l inhibition of VEGF produc- tion, and secondary to an effect on tumor microvessel density. The rabbit cornea assay confirmed that 1l markedly decreased neovessel growth induced by VEGF. In cultured endothelial cells, 1l inhibited the VEGF-induced phosphorylation on tyr416 of c-Src, resulting in a reduced cell proliferation and invasion. Consistently, 1l dowregu- lated endothelial nitric oxide synthase, MAPK-extracellular recep- tor kinase 1–2 (ERK1-2) activity and matrix metalloproteinases (MMP-2/MMP-9), while the tissue inhibitors of metalloproteinases (TIMP2/TIMP-1) were upregulated. These results demonstrate that nM concentrations of c-Src kinase inhibitors (1l and PP-1), by reducing the production of VEGF released by tumor cell and its en- dothelial cell responses, have a highly selective antiangiogenesis effect, which might be useful in combination therapies. ' 2006 Wiley-Liss, Inc. Key words: c-Src kinase inhibitors; tumor angiogenesis; vascular endothelial growth factor; squamous cell carcinoma Angiogenesis, the formation of new capillaries, is associated with the progression of tumor growth and metastasis. 1 The formation of new capillaries is a process that requires secretion of proteases, en- dothelial cell invasion, migration, proliferation and differentiation, which are cellular processes in part regulated by Src family of tyro- sine kinases (SFKs). 2 The SFKs consist of 8 members. Src, Yes and Fyn are ubiquitously expressed, while Lck, Hck, Fgr, Lyn and Blk have more tissue-restricted expression. 3 SFKs are activated in response to stimulation of a variety of cell surface receptors such as tyrosine kinase receptors, integrin receptors, G-protein coupled receptors and by cellular stress. 2 Recent studies have shown that c- Src, a nonreceptor tyrosine kinase, exhibits elevated protein levels and activity in numerous types of human cancers. 2–4 Specifically, Src activity was found to be elevated in breast, pancreatic, ovarian, oesophageal, lung, gastric, colon and head and neck cancers. 4–8 The frequency with which elevated expression and/or activity of Src occurs in epithelial cancers strongly suggests its implication in facili- tating malignant progression. Src activity has been found to be a crit- ical component of multiple signaling pathways that regulate prolifer- ation, survival, metastasis and angiogenesis. 2,9 However, the mecha- nism by which Src activity contributes to cancer progression is still not well understood. Recently, numerous papers focus on Src effects on tumor metastasis and angiogenesis. 9–12 Because of its important role in oncogenic processes, it represents a therapeutic target ripe for exploitation. Vascular endothelial growth factor (VEGF) has been identified as one of the most important factors mediating angiogenesis in physiological and pathological conditions. The key mechanism by which VEGF promotes angiogenesis and permeability is c-Src activation. 11 Indeed, the tyrosine kinase c-Src coordinates both the VEGF-induced Ras-extracellular signal-related kinase (ERK) cas- cade, which mediates endothelial cell proliferation, survival, inva- sion and gene expression, 13 and the VEGF-induced focal adhesion kinase-avb5 integrin cascade, which regulates vascular perme- ability. 14 Activation of Src following VEGF interaction with its receptor VEGFR-2 has also been reported to mediate endothelial nitric oxide (eNOS) activity, 15,16 which is known to control both VEGF-mediated vascular permeability and the angiogenesis pro- cess. 10,17 The finding that expression of a dominant negative c-Src mutant inhibits VEGF induced angiogenesis in vivo suggests that c-Src might be a suitable pharmacological target for inhibition of angiogenesis. 18,19 Computer modeling of the pyrazolo–pyrimidine class, namely, 4-amino-substituted-1-(2-chloro-2-phenylethyl)-1H-pyrazolo[3,4- d]pyrimidines, predicted that the 1l compound would dock in the ATP pocket of the c-Src tyrosine kinase. 20 Biochemical studies in tumor cells revealed that 1l potently inhibited the activity of c-Src and subsequently downregulated phosphorylation of many down- stream signaling proteins at the cellular level such as mitogen acti- vated protein kinases, MAPKs. 20 Here we report that lM con- centration of 1l or PP-1, a known c-Src inhibitor, significantly reduced squamous carcinoma cell A431 and SCC-4 survival and invasion. At low concentration, in the nanomolar range, c-Src in- hibition markedly reduced VEGF-induced endothelial cell growth and invasion in vitro, and neovessel growth in vivo. This ulti- mately leads to reduction in tumor growth in immunodeficient mice. The molecular mechanisms involved the ability of c-Src to regulate both VEGF production in tumors and VEGF-mediated functions in endothelial cells. Materials and methods Reagents and chemicals Bovine calf serum (BCS) was from Hyclone (Celbio, Milan, Italy), Dulbecco’s modified Eagle’s medium (DMEM), trypsin, gelatine and analytical grade chemicals were purchased from This publication reflects only the authors’ views. The Commission is not liable for any use that may be made of information herein. Grant sponsor: Italian Ministry for Research; Grant number: 2004065317_003; Grant sponsor: EU project EICOSANOX FP6 funding; Grant number: LSHM-CT-2004-005033; Grant sponsor: NuGO; Grant number: FOOD-CT-2004-506360. *Correspondence to: Department of Molecular Biology, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy. Fax 139-0577-234343. E-mail: ziche@unisi.it Received 23 May 2006; Accepted after revision 14 September 2006 DOI 10.1002/ijc.22410 Published online 27 November 2006 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 120, 995–1004 (2006) ' 2006 Wiley-Liss, Inc. Publication of the International Union Against Cancer