טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentShafat Itay
SubjectCharacterization of Mechanisms Involved in Secretion of
Active Heparanase by Cancer Cells
DepartmentDepartment of Medicine
Supervisor Professor Israel Vlodavsky
Full Thesis textFull thesis text - English Version


Abstract

Heparanase is an endo-β-D-glucuronidase involved in extracellular matrix remodeling and implicated in tumor metastasis, angiogenesis and inflammation. The enzyme is synthesized as a latent 65-kDa protein and is processed to an active 58-kDa heterodimer in the lysosomal compartment, where it is stored in a stable form. In contrast, its heparan sulfate substrate is localized extracellularly, suggesting the existence of mechanisms that trigger heparanase secretion.

In this work, we show that secretion of active heparanase is mediated by the protein kinase A and C pathways. Moreover, secretion of active heparanase was noted upon cell stimulation with physiological concentrations of adenosine, ADP, and ATP, secretion that was shown to be mediated by the purine receptors P2Y1 and P2Y2. The kinetics of heparanase secretion resembled the secretion of cathepsin D, indicating that the secreted heparanase is of a lysosomal origin. In addition, activation of P2Y receptors increased the ability of heparanase expressing cells to migrate.

Apart of nucleotides, the potent cancer-related growth factor IGF-I also induced heparanase secretion, correlating with cathepsin D secretion, elevated ECM-degrading ability and increased migratory capacity of heparanase expressing tumor cells.

We suggest that secretion of active heparanase is initiated by extracellular cues activating the protein kinase A and C signaling pathways, and possibly other secretory routs. The secreted enzyme then facilitates cell invasion associated with cancer metastasis and inflammation.

The results of the first part led us to seek a means to identify and quantify heparanase in biological samples. We developed a sensitive ELISA suitable for determining and quantifying human heparanase. The assay preferentially detects the 58-kDa active heparanase vs. the latent 65-kDa proenzyme. It detects heparanase at concentrations as low as 100 pg/ml and correlates with immunoblot analysis of heparanase-containing samples.

Utilizing the ELISA we conducted several clinical studies, demonstrating elevated levels of heparanase in the urine and plasma of patients with various cancers, correlating with the stage and grade of the patient's tumor. The diagnostic potential of the ELISA was revealed in a study on bladder cancer, where 60% of urinary-heparanase-positive samples were derived from cancer patients, while only 2.3% originated from healthy controls. Moreover, another study of pediatric patients with hematological cancers, plasma levels of heparanase predicted the patients' response to anti-tumor treatments for over 80% of the patients. These results highlight heparanase as a tumor marker, and the newly developed ELISA method as an appropriate tool to identify and quantify the heparanase protein.