Neuropilin and Semaphorin Function in Development and Tumor Angiogenesis

Boston Children'S Hospital, Boston MA

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Abstract

Neuropilins (NRP1 and NRP2) are receptors that bind two disparate families of ligands, the semaphorins (e.g. SEMA3A and SEMA3F), that repel axons and collapse growth cones and the VEGF family of potent angiogenesis factors. NRPs are required for normal angiogenesis. They promote VEGF interactions with VEGF receptor-2. NRP1 silencing in zebrafish embryos results in loss of angiogenic sprouts. NRP1 knockouts in mice result in severely impaired blood vessel development. Soluble NRP (sNRP) inhibits tumor progression. SEMA3A and SEMA3F are anti-angiogenic. They inhibit endothelial cell (EC) adhesion and migration in culture. SEMA3F in vivo is a potent inhibitor of malignant melanoma tumor angiogenesis and metastasis. It is proposed to expand these studies to other tumors, for example carcinomas. Since VEGF promotes tumor angiogenesis, there has been strong interest in developing VEGF antagonists, for example, anti-VEGF neutralizing antibodies which are now in the clinic. We find that these anti-VEGF antibodies inhibit VEGF binding to VEGFR-2 but not to NRP. Thus, it is proposed that anti-VEGF antibodies in combination with NRP antagonists such as SEMA3A, SEMA3F and sNRP might enhance the anti-VEGF antibody effects. The mechanisms by which SEMAs act need to be clarified. SEMAs and VEGF are competitive inhibitors for NRP binding. A novel anti-angiogenesis mechanism is that SEMA3F repels EC in culture mimicking axon repulsion in the nervous system. Another mechanism being explored is that SEMAs act directly on EC and tumor cells by collapsing the cytoskeleton. SEMA3F depolymerizes F-actin by a pathway that includes inactivation of RhoA and activation of cofilin, a direct depolymerization factor. It is proposed to continue our zebrafish NRP1 studies, for example by analyzing the relative importance of heparin- and NRP-binding for optimal VEGF165 activity, and developing the exon 7 domain of VEGF (which is responsible for NRP binding) as a VEGF antagonist in zebrafish in vivo. The overall goal is to develop SEMA3A, SEMA3F, sNRP and exon7 peptide as inhibitors of tumor angiogenesis, growth and metastasis and to identify the mechanisms involved, Health-relatedness of the project: Our focus on the NRPs and SEMAs in tumor growth and metastasis, are clearly relevant to health, especially cancer. These studies will be useful in developing anti-cancer drugs that act directly on blood vessels and tumor cells, and indirectly by antagonizing VEGF. Our Specific aims are: 1.To analyze semaphorin-induced inhibition of tumor progression and metastasis;2 To analyze mechanisms of Semaphorin action in endothelial and tumor cells;3. To analyze the molecular basis of neuropilin function in Zebrafish embryos.