טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLange Tali
SubjectThe Involvement of Semaphorin 3C in Tumor Progression
and Angiogenesis
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Gera Neufeld
Full Thesis textFull thesis text - English Version


Abstract

 Class-3 semaphorins such as semaphorin-3A (sema3A) and semaphorin-3F (sema3F) that activate signal transduction by binding to the neuropilin-1 (np1) or neuropilin-2 (np2) receptors have been found to inhibit angiogenesis and tumor progression. In contrast, semaphorin-3C (sema3C) expression was found to be up-regulated in several types of malignant cells. We have expressed recombinant sema3C in several cell types and have found that even though it was highly expressed, sema3C was not secreted from these cells. The signal sequence of sema3C was active since it replaced successfully the signal sequence of sema3F, but the opposite was not true since the signal sequence of sema3F was unable to induce secretion of sema3C from cells. Nevertheless, the development of tumors from sema3C expressing HEK293 cells was strongly inhibited compared to empty vector transfected controls. The proliferation rate of these cells in cell culture was not affected by sema3C expression and the cells did not repel endothelial cells. In contrast, a fusion protein in which the sema3C was fused to the C-terminal of alkaline-phosphatase (AP-sema3C) was secreted efficiently, repelled endothelial cells, and induced apoptosis of endothelial cells. Furthermore, AP-sema3C inhibited VEGF induced phosphorylation of ERK-1/2 in endothelial cells. SiRNA inhibition experiments indicated that the effects of sema3C on endothelial cells are mediated by both neuropilins as suggested previously by binding experiments. AP-sema3C bound to np2 and induced cell contraction of cells co-expressing np2 and plexin-A4 but not of cells co-expressing np2 and plexin-A1.

Sema3C expressing HEK293 cells inhibited significantly bFGF induced angiogenesis in matrigel plug assays, suggesting that sema3C has anti-angiogenic properties. Sema3C also seemed to inhibit the maturation of new blood vessels invading the matrigel plugs because the average area of blood vessels was significantly smaller in plugs containing sema3C producing cells. Both the secreted AP-sema3C as well as the non-secreted sema3C inhibited bFGF induced angiogenesis in this assay, suggesting that sema3C may be secreted from the transfected cells in-vivo.