Antagonism of Sphingosine-1-Phosphate Receptors by FTY720 Inhibits Angiogenesis and Tumor Vascularization Kenneth LaMontagne, 1 Amanda Littlewood-Evans, 2 Christian Schnell, 2 Terence O’Reilly, 2 Lorenza Wyder, 2 Teresa Sanchez, 3 Beatrice Probst, 2 Jeannene Butler, 1 Alexander Wood, 4 Gene Liau, 4 Eric Billy, 2 Andreas Theuer, 2 Timothy Hla, 3 and Jeanette Wood 2 1 Novartis Institutes for BioMedical Research, East Hanover, New Jersey; 2 Novartis Institutes for BioMedical Research, Basel, Switzerland; 3 University of Connecticut Health Center, Farmington, Connecticut; and 4 Novartis Institutes for BioMedical Research, Inc., Cambridge, Massachusetts Abstract FTY720, a potent immunomodulator, becomes phosphorylated in vivo (FTY-P) and interacts with sphingosine-1-phosphate (S1P) receptors. Recent studies showed that FTY-P affects vascular endothelial growth factor (VEGF)–induced vascular permeability, an important aspect of angiogenesis. We show here that FTY720 has antiangiogenic activity, potently abro- gating VEGF- and S1P-induced angiogenesis in vivo in growth factor implant and corneal models. FTY720 administration tended to inhibit primary and significantly inhibited meta- static tumor growth in a mouse model of melanoma growth. In combination with a VEGFR tyrosine kinase inhibitor PTK787/ ZK222584, FTY720 showed some additional benefit. FTY720 markedly inhibited tumor-associated angiogenesis, and this was accompanied by decreased tumor cell proliferation and increased apoptosis. In transfected HEK293 cells, FTY-P internalized S1P 1 receptors, inhibited their recycling to the cell surface, and desensitized S1P receptor function. Both FTY720 and FTY-P apparently failed to impede VEGF-produced increases in mitogen-activated protein kinase activity in human umbilical vascular endothelial cells (HUVEC), and unlike its activity in causing S1PR internalization, FTY-P did not result in a decrease of surface VEGFR2 levels in HUVEC cells. Pretreatment with FTY720 or FTY-P prevented S1P- induced Ca 2+ mobilization and migration in vascular endo- thelial cells. These data show that functional antagonism of vascular S1P receptors by FTY720 potently inhibits angiogen- esis; therefore, this may provide a novel therapeutic approach for pathologic conditions with dysregulated angiogenesis. (Cancer Res 2006; 66(1): 221-31) Introduction Angiogenesis, the formation of new blood vessels from preexist- ing vessels, is a normal aspect of the physiologic remodeling processes that occurs in wound healing and during the female reproductive cycle. However, in pathologic situations, such as rheumatoid arthritis, diabetic retinopathy, and tumor development, abnormally enhanced neovascularization is a major contributory factor for disease progression (1, 2). The initiation of pathology- associated angiogenesis involves vascular permeability changes, driven by angiogenic factors, such as vascular endothelial growth factor (VEGF; ref. 3). This leads to fibrin deposition, plasmin activation, basement membrane degradation, and ultimately endothelial cell migration and proliferation, recruitment of mural cells, and vessel maturation (4). Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metab- olite secreted by platelets upon activation, is a potent proangio- genic molecule, which acts by binding various members of the G-protein–coupled receptor (GPCR) family of S1P receptors (S1P-R; refs. 5, 6). A novel immunosuppressant agent currently in clinical trials for renal transplant rejection (FTY720) and its metabolite of cellular kinase(s) FTY720 phosphate (FTY-P; ref. 7) bear structural similarity to sphingosine and S1P, respectively. FTY-P binds at low nanomolar concentrations to four of five S1P-Rs, S1P 1 , S1P 3 , S1P 4 , and S1P 5 (8). Recently, we have shown that FTY-P can act in a similar manner to S1P, stimulating endothelial cell signaling, migration, survival, and differentiation (9). By recruiting adherens junction proteins to the endothelial cell-cell junctions (10), FTY720 has also been shown to antagonize VEGF-induced permeability of blood vessels (10, 11). Tumor-associated blood vessels are permeable and elicit tissue extracellular fluid extravasation; therefore, this prompted us to investigate whether FTY720 exerts any antiangiogenic and antitumor activity in vivo by affecting vessel permeability. In this report, we show that FTY720 at clinically relevant doses, inhibits both S1P- and VEGF-induced angiogenesis, and impedes primary and metastatic tumor growth in a murine model of melanoma. Additionally, combination of FTY720 with the VEGFR tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) further reduces the growth of the tumors and metastases. These findings suggest that targeting S1P receptors may provide a novel ther- apeutic approach in cancer treatment. Materials and Methods Materials. S1P was purchased from BioMol Research Labs, Inc. (Plymouth Meeting, PA). FTY720 and all other related compounds mentioned herein were generously provided by the Novartis Transplanta- tion Group (Basel, Switzerland) and prepared as described previously (9). Human umbilical vein endothelial cells (HUVEC), from Vec Technologies, Inc. (Rensselaer, NY), were maintained in MCDB 131 Complete media (Vec Technologies) and were used from passages 4 to 7. Female C57/Bl6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) or IFFA Credo (L’Arbresle, France). Female mice (MAG and NIH/Tif), weighing 18 to 20 g (6-8 weeks old), were obtained from the Novartis animal breeding facility. All animal experiments done in Switzer- land were done in strict adherence to the Swiss law for animal protection, Note: K. LaMontagne and A. Littlewood-Evans contributed equally to this work. K. LaMontagne and J. Butler are currently at J&J PRD, Raritan, NJ (klamontagne@ prdus.jnj.com). PTK787/ZK222584 is a co-development compound by Novartis AG and Schering AG. Requests for reprints: Amanda Littlewood-Evans, Novartis NIBR AG, K125.1.20, Klybeck Strasse, Basel, CH4002, Switzerland. Phone: 41-61-696-1023; Fax: 41-61-696- 6242; E-mail: amanda.littlewood-evans@novartis.com. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-2001 www.aacrjournals.org 221 Cancer Res 2006; 66: (1). 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