Neuropilin and semaphorin, anti-angiogenesis activities and mechanisms

Boston Children'S Hospital, Boston MA

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Abstract

Abstract Cancer is a major cause of death in the U.S. A novel therapeutic approach has been to identify drugs that inhibit tumor angiogenesis, the process by which tumors attract blood vessels that bring in the nutrients needed for tumor growth. Recently, there has been a surge in the development of angiogenesis inhibitors, some of which are already FDA approved, e.g., Avastin (Bevacizumab), an antibody that inhibits tumor- derived VEGF, a potent angiogenesis factor. This proposal focuses on identifying potentially novel angiogenesis inhibitors based on neuropilin (NRP) and semaphorin (SEMA) structure and function. NRPs are transmembrane receptors for class 3 semaphorins (SEMA3 A-G) and for VEGF. SEMA3F was initially shown to repel axons and collapse growth cones but it also induces morphological changes in endothelial cells (EC) and tumor cells characterized by inactivation of RhoA, loss of stress fibers, collapse of the F-actin cytoskeleton and inhibition of migration. SEMA3F inhibits tumor angiogenesis and metastasis, whereas NRPs, on the other hand, promote VEGF-dependent angiogenesis. A focus of this proposal is to design new angiogenesis inhibitors and elucidate their mechanisms. The first specific aim explores the mechanism of SEMA3F-induced anti-angiogenesis activity. It appears that SEMA3F induces changes in blood vessel morphology rather than blood vessel number. SEMA3E is an exception among the SEMA3 family in that it is NRP-independent and acts directly via plexin D1. However, there are conflicting results in which SEMA3E has been reported to be both pro- and anti-angiogenic. RhoA kinase (ROCK) inhibitors (e.g., Fasudil) mimic the morphological alterations induced by SEMA3F and therefore may be angiogenesis inhibitors. SEMA3F, SEMA3E and ROCK inhibitor will be tested for the ability to block tumor angiogenesis and growth. The second specific aim will be to develop novel NRP antagonists. NRPs enhance the angiogenic activity of VEGF. A goal is to block VEGF-NRP interactions. VEGF, via its exons 7 and 8 (ex7/8), binds to the NRP B domain. Alteration of the NRP2 B domain structure by site-directed mutagenesis improved the affinity of the B-NRP2 mutants to VEGF by almost one log but had no effect on SEMA3F affinity. The soluble mutated NRP2 B domain appears to be a strong inhibitor of tumor growth, possibly by sequestering VEGF efficiently. Excess VEGF ex7/8 protein inhibits VEGF binding to endothelial cells (EC), presumably by competition with binding to the B domain. VEGF ex7/8 will be tested for efficacy as an angiogenesis inhibitor. The third specific aim is to discern how NRP2 and SEMA3F, important proteins involved reciprocally in tumor growth, are regulated. Both promoters have been isolated. NRP2 is upregulated in EC by GATA2, a transcriptional regulator of the hematopoietic lineage. NRP2 Lacz+/- mice show strong expression of NRP2 in bladder smooth muscle cells (BSMC). SEMA3F alters the morphology of these SMC, a new SEMA3F function. SEMA3F expression is induced by E47, an HLH E-protein transcription factor. Inhibitors of differentiation (Ids) are dominant/negative inhibitors of E47 binding to DNA and suppress E47-induced SEMA3F expression. Thus, Id pro-angiogenic activity in vivo might stem in part from suppression of SEMA3F expression. Ids are upregulated in tumor EC isolated from the mouse TRAMP model of prostate carcinoma, suggesting that Ids are pro-angiogenic in vivo in spontaneous tumors. The Specific Aims are as follows: 1) To investigate signaling mechanisms by which semaphorins inhibit tumor angiogenesis;2) To develop NRP2 B domain mutants and VEGF exon 7/8 peptides as VEGF antagonists and inhibitors of angiogenesis;3) To discover novel functions of NRP2 and its ligand, SEMA3F.