|Ph.D Student||Shteingauz Anna|
|Subject||Interaction of Heparanase with Heparan Sulfate|
and its Cellular and Molecular Consequences
|Department||Department of Medicine||Supervisor||Professor Israel Vlodavsky|
|Full Thesis text|
Heparanase is the only functional endoglycosidase capable of cleaving heparan sulfate (HS) in mammals, activity that is highly implicated in cell dissemination associated with tumor metastasis, inflammation and angiogenesis. Heparanase uptake is considered a pre-requisite for the delivery of latent 65 kDa heparanase to lysosomes and its subsequent proteolytic processing and activation into 8 and 50 kDa protein subunits by cathepsin L. Efficient uptake of heparanase was evident also by GPI-deficient cells (i.e., lack cell surface glypicans), suggesting preferential involvement of syndecans in this process. The molecular mechanism underlying heparanase uptake is still largely obscure. Here, we examined the necessity of syndecan 1 cytoplasmic domain for heparanase internalization and processing. To this end, we transfected cells with full length mouse syndecan 1 or deletion constructs lacking the entire cytoplasmic domain (delta), the conserved (C1 or C2) or variable (V) regions. Heparanase binding, internalization and processing were then evaluated by immunofluorescent staining and immunoblotting. Heparanase uptake was markedly increased following syndecan 1 over expression, thus challenging the notion that HS coat of the cell membrane is at saturation and does not limit ligand binding. In contrast, heparanase was retained at the cell membrane and its processing was impaired in cells over expressing syndecan 1 deleted for the entire cytoplasmic tail. We have next identified that conserved domain 2 (C2) and variable (V) regions of syndecan 1 cytoplasmic tail mediate heparanase internalization and processing. Furthermore, we found that syntenin, known to interact with syndecan C2 domain and α actinin, mediates heparanase uptake. These results further illustrate the tight regulation of heparanase uptake and bioavailability.
The role of heparanase under normal conditions has not been revealed yet. Residing for relatively long period of time in the lysosome led us to hypothesize that heparanase may have a role in autophagy machinery. Here, we provide evidence that heparanase resides within autophagosomes. Moreover, autophagy extent was decreased in heparanase knockout mice and cells, and was augmented significantly by heparanase over expression in transgenic mice and cancer-derived cells, evident by immunofluorescent staining, immunoblotting and electron microscopy. Furthermore, we show that the pro-tumorigenic properties of heparanase are mediated, at least in part, by its pro-autophagic function. This is demonstrated by decreased cell survival, colony number and size, and tumor xenografts growth by inhibitors of the lysosome (chloroquine) and heparanase (PG545) alone and in combination. We also show that heparanase over expressing cells are more resistant to stress and chemotherapy, resistance that also results from increased autophagy and was reversed by chloroquine. Collectively, the results indicate, for the first time, a role for endogenous heparanase in modulating autophagy that endows cancer cells with growth advantage and chemo resistance.