Abstract 1524: Heparan sulfate and Sulf-2 expression in neuroblastoma.

Abstract Heparan sulfate (HS) present on the surface of almost every cell and in the extracellular matrix binds a diversity of growth factors and ligands and thus can regulate cell signaling. Recent evidence suggests its role within the tumour microenvironment. The large diversity of HS is based on several chain modifications by numerous biosynthetic enzymes. The sulfation of the HS chain is an essential structural determinant in HS function as it influences signaling events. Particularly the removal of 6-O-sulfate groups from internal glucosamine residues by the sulfatases (Sulfs) in highly sulfated HS domains affects protein ligand binding and has recently been shown to play a role in cancer progression and development. However very little is known about HS in neuroblastoma (NBL). Here we have examined the expression of 14 HS biosynthetic enzymes in 8 NBL cell lines and 8 other cancer and normal cell lines. We first demonstrate that in contrast to many other cancers Sulf-2 is significantly up regulated in NBL cell (three-fold at mRNA level and 5-fold by western blot). In particular Sulf-2 is strongly increased in MYCN amplified NBL cell lines when compared to MYCN-non amplified (MYCN-NA) NBL and other cancerous cell lines (quantitative RT-PCR and western blotting). As HS has a low immunogenicity we used a panel of phage display antibodies to localize HS in NBL cells. The staining showed that HS is localized at the cell surface and more abundant in MYCN amplified (MYCN-A) NBL cell lines. To monitor the action of Sulf-2 on cell surface we used phage display antibodies that recognize highly sulfated N- and O- saccharide domains of HS (RB4CD12 and HS3A8V) and found that in MYCN-A NBL cell lines there are high Sulf-2 levels associated with a decrease in the expression of highly sulfate HS. Immunohistochemistry (IHC) with an anti-Sulf-2 monoclonal antibody was then used to assess expression in primary human NBL tumors (n=12). A computerized color deconvolution method was employed to quantify the staining intensity of Sulf-2 IHC by means of optical density (OD). In primary human NBL tissues samples Sulf-2 was expressed predominantly on NBL cell surface and in their surrounding matrix. The analysis revealed a significant increase of Sulf-2 expression (measured as OD) in tumors with an unfavorable histology and MYCN-A when compared to samples with MYCN-NA with favorable or unfavorable histology, (OD: 0.3±0.05 vs 0.12±0.03 and 0.14±0.04; respectively); (ANOVA; p<.05). Altogether, we characterized for the first time the expression of HS and Sulf-2 in NBL. The data suggest that a decrease in highly sulfated HS in association with high Sulf-2 may contribute to the malignant behavior of MYCN-A NBL. This hypothesis is currently being tested by examining the effects of Sulf-2 modulation in NBL cell line behavior. Citation Format: Yves A. DeClerck. Heparan sulfate and Sulf-2 expression in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1524. doi:10.1158/1538-7445.AM2013-1524