Heparanase is highly expressed in GH-adenomas and regulates proliferation of pituitary tumor cells
Hadara Rubinfeld1, Neta Ilan2, Dvora Nass3, Victoria Cohen-Kaplan2, Orit Pappo4, Moshe Hadani5, Israel Vlodavsky2 & Ilan Shimon1
Background: Pituitary adenomas are monoclonal neoplasms that may be either functional (hormone-secreting) or non-functional. The processes associated with pituitary tumor development and progression are mostly unknown. Remodeling of the extracellular matrix (ECM) and cell adhesion were suggested to be involved in pituitary tumorigenesis. Heparanase, an endoglycosidase capable of degrading heparan sulfate, the main polysaccharide constituent of the ECM, is implicated in diverse processes associated with ECM remodeling, such as development and morphogenesis, angiogenesis, and tumor invasion. Here, we examined the role of heparanase in pituitary tumorigenesis.
Methods: Human normal pituitaries and pituitary tumors were examined for heparanase mRNA and protein expression using real-time PCR and immunohistochemistry, respectively. Cell viability, assessed by XTT, and cell proliferation, assessed by colony formation assays, were tested following incubation of GH3 and MtT/S cells with recombinant heparanase. Cell viability and cell cycle progression, were evaluated following heparanase gene silencing.
Results: Higher heparanase mRNA expression was noted in GH-tumors as compared with normal pituitary. Non-functioning tumors usually presented low levels of heparanase. Normal pituitaries and GH-tumors showed heparanase protein expression, whereas it was absent in most non-functional tumors. Increased cell viability of GH3 and MtT/S cells was observed following addition of recombinant heparanase. Heparanase over-expression in these cells resulted in increased colonies number by 23 fold, compared with control cells. Cell viability decreased by 50%, following heparanase gene silencing, due to elevation of cells at S and subG1 phases. Notably, addition of heparanase to MtT/S cells enhanced Akt phosphorylation.
Conclusions: Our results suggest that heparanase enhances pituitary cell proliferation and may thus contribute to pituitary tumor development and progression. This effect is mediated, in part, by facilitating Akt phosphorylation, a well known survival factor.