טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentCohen Victoria
SubjectInvolvement of Heparanase in lymphangiogenesis and Signal
Transduction
DepartmentDepartment of Medicine
Supervisor Professor Israel Vlodavsky
Full Thesis textFull thesis text - English Version


Abstract

Heparanase is an endoglycosidase that specifically cleaves heparan sulfate side chains and strongly implicated in tumor metastasis attributed to remodeling of the basement membranes. Moreover, heparanase up-regulation was noted in an increasing number of primary human tumors.

We hypothesized that heparanase facilitates the formation of lymphatic vessels. We provide evidence that lymphatic vessel density (LVD) correlates with head and neck lymph node metastasis, and inversely correlates with tumor cell differentiation. Notably, heparanase staining correlated with LVD and, moreover, with VEGF C levels. We further demonstrate that heparanase over expression by human tumor-derived cell lines induces elevation in VEGF C expression in vitro, and facilitates tumor xenograft lymphangiogenesis in vivo, while heparanase gene silencing was associated with decreased VEGF C levels.

In addition, in the present study we substantiate the ability of heparanase to activate Src in a variety of human tumor-derived cell lines. We hypothesized that Src stimulation by heparanase will facilitate the phosphorylation and activation of Src substrates such as epidermal growth factor receptor (EGFR). We provide evidence that active and inactive heparanase proteins enhance the phosphorylation of EGFR in a Src-dependent manner. Activation of EGFR results in increased cell migration and colony formation, which were attenuated by Src inhibitors. Similarly, heparanase gene silencing by means of siRNA was associated with reduced Src and EGFR phosphorylation levels and decreased cell proliferation. Notably, heparanase expression correlated with increased phospho-EGFR levels and progression of head & neck carcinoma, thus providing a strong clinical support for EGFR modulation by heparanase.

We further characterized the signaling cascade downstream the EGFR and found that heparanase enhances the phosphorylation of signal transducer and activator of transcription (STAT)3 and STAT5b but not STAT5a. Enhanced STAT5b phosphorylation by heparanase was attenuated by selective inhibitors of Src and EGFR, but not a MEK inhibitor. Moreover, enhanced proliferation of heparanase transfected cells was attenuated by STAT3- and STAT5b-, but not STAT5a- or STAT1- siRNA. Clinically, STAT3 phosphorylation was associated with head & neck cancer progression and with EGFR phosphorylation and heparanase expression and cellular localization. Notably, cytoplasmic rather than nuclear phospho-STAT3 correlated with increased tumor size, number of infected neck lymph nodes, and reduced patients' survival.

Thus, heparanase appears to modulate two critical systems involved in tumor progression and metastasis, namely VEGF and VEGF C expression, and Src-EGFR-STAT activation. Neutralizing heparanase enzymatic and nonenzymatic functions is therefore expected to profoundly affect tumor growth, angiogenesis, and metastasis.