טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGross-Cohen Miriam
SubjectElucidating the Biological Features of Heparanase
Homolog, Heparanase 2, as a Tumor Suppressor
DepartmentDepartment of Medicine
Supervisor Professor Israel Vlodavsky
Full Thesis textFull thesis text - English Version


Abstract

Heparanase is an endoglycosidase that specifically cleaves heparan sulfate (HS) side chains of proteoglycans, activity that is highly implicated in tumor metastasis and angiogenesis. Heparanase 2 (Hpa2) is a close homolog of heparanase that lacks intrinsic HS-degrading activity but retains the capacity to bind HS with high affinity. In head and neck cancer patients, Hpa2 expression was markedly elevated, correlating with prolonged time to disease recurrence and inversely correlating with tumor cell dissemination to regional lymph nodes, suggesting that Hpa2 functions as a tumor suppressor. The molecular mechanism associated with favorable prognosis following Hpa2 induction is unclear. Here, we demonstrate that Hpa2 overexpression in head and neck cancer markedly reduces tumor vascularity and growth, likely due to reduced Id1 expression, a transcription factor implicated in VEGF-A and VEGF-C gene regulation. Notably, reduced tumor growth in response to Hpa2 occurred in heparanase-, and HS-independent manner.

Subsequent studies revealed that Hpa2 is expressed by normal bladder, breast, and oral epithelium and its levels are decreased substantially in the corresponding carcinomas, expression pattern typical of a tumor suppressor. Notably, bladder and breast tumors that retain high levels of Hpa2 were diagnosed as low grade. Moreover,overexpression of Hpa2 in 5637 bladder carcinoma cells resulted in smaller tumors diagnosed as low grade. Overexpression of Hpa2 was associated with a marked increase in lysyl oxidase (LOX) in head and neck and bladder tumor models and in bladder cancer patients.

Moreover, in normal epithelium of the breast, Hpa2 was localized predominantly to the cell nucleus. In striking contrast, Hpa2 levels were lower and appeared diffused in the cytoplasm of breast cancer cells, suggesting that both the expression levels and cellular localization of Hpa2 play a role in tumorigenesis. This notion is critically supported by ZR-75-1 breast cancer cells engineered to overexpress nuclear-targeted Hpa2 variant. Notably, targeting Hpa2 to the cell nucleus attenuated tumor growth. Repression (i.e., VEGF-A, VEGF-C) and induction (i.e., cytokeratin-13 & 15, LOX) of selected genes by Hpa2 imply that nuclear Hpa2 plays a transcriptional function required to maintain a normal differentiated epithelial phenotype.

In other studies, we found that Hpa2 enhances cell adhesion.  Briefly, cells adhered to Hpa2/heparanase pre-coated plates in a HS-dependent and independent manner, indicating that cell adhesion is mediated by cell membrane HSPGs and another yet unknown cell surface molecule(s). Hpa2 was also found to affect cell migration and wound closure. This was exemplified by inhibition of wound closure by endothelial and tumor-derived cells upon exogenous addition of Hpa2, further supporting the notion that Hpa2 restrains pro-tumorigenic properties of cancer cells.

Taken together, our results support the notion that Hpa2 functions as a tumor suppressor in bladder, head & neck and breast carcinomas. We further show that Hpa2 function is not restricted to modulation of heparanase activity or interaction with HSPGs but is apparently involved in regulation of selected genes that affect tumor vascularity (Id1, VEGF-A,  VEGF-C), tumor fibrosis (LOX), and cell differentiation (cytokeratins 13 & 15), thus expanding significantly the repertoire of Hpa2 functions and its mode of action.