טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBarash Uri
SubjectA Novel Human Heparanase Splice Variant; T5; Endowed with
Pro-Tumorigenic Characteristics
DepartmentDepartment of Medicine
Supervisor Professor Israel Vlodavsky
Full Thesis textFull thesis text - English Version


Abstract

Heparanase is an endo-β-D-glucuronidase capable of cleaving heparan sulfate (HS) side chains. Heparanase activity is implicated in neovascularization, inflammation, and autoimmunity, involving migration of vascular endothelial cells and activated cells of the immune system.

In silico analysis, has predicted three novel splice variants of human heparanase. The expression of one, termed T5, has been confirmed in various tumor-derived cell lines and tumor biopsies. In this splice variant, 144 bp of intron 5 are jointed with exon 4, resulting in a truncated, 169 amino acid protein that lack enzymatic activity typical of heparanase. T5 appears to get secreted and facilitate Src phosphorylation. Over expression of T5 by pharynx (FaDu), myeloma (CAG) and embryonic kidney (293) cells resulted in enhanced proliferation and larger colony formation in soft agar, which was attenuated by Src inhibitors. Likewise, T5 gene silencing was associated by reduced cell proliferation, indicating that endogenous levels of T5 govern tumor cells proliferation. Moreover, the development of tumor xenografts produced by heparanase- and T5-infected cells was markedly enhanced compared with xenografts generated by control cells. In addition, tumors xenografts developed by T5 expressing cells exhibited higher vessel density, which were decorated with more smooth muscle actin (SMA)-positive cells (pericytes), indication of vessel maturation. Furthermore, we established an inducible system by which the expression of heparanase variants is controlled by addition of doxycycline (Dox), to the cell culture medium or drinking water of mice. Colony formation in soft agar was markedly enhanced following heparanase, heparanase C-terminal domain (8C), or T5 induction by Dox. Moreover, xenografts development was profoundly increased in mice inoculated with CAG cells infected with inducible heparanase, 8C, and T5 constructs following supplementation of Dox in their drinking water. These results emphasize that signaling capacity of heparanase by the 8C domain is sufficient to facilitate tumor progression in this model system. The clinical relevance of T5 critically emerged from analysis of renal cell carcinoma biopsies, where T5 and heparanase expression appeared to be induced in 75% of the cases. In addition , we generated a T5-specific monoclonal antibody (mAb), 9c9 that preferentially recognizes T5 vs. heparanase by immunoblotting. mAb 9c9 is suitable for immunohistochemical analysis and clearly revealed the occurrence of T5 in myeloma and renal cell carcinoma tumor biopsies, thus enabling prospective analysis of tumor specimens and correlating T5 expression with clinical and molecular determinants. We concluded, therefore, that T5 is an abundant functional splice variant of human heparanase.