Abstract 1974: EphB2 signaling activity plays a vital role in medulloblastoma cell adhesion and invasion
Authors: Sikkema Arend H. , den Dunnen Wilfred F.A. , Diks Sander H. , Kampen Kim R. , Scherpen Frank J.G. , van Vuurden Dannis G. , Hulleman Esther , Kamps Willem A. , Peppelenbosch Maikel P. , de Bont Eveline S.J.M. 
Published: August 2014

Eph/ephrin signaling has been implicated in various types of cancer, enhancing key processes like migration, proliferation and angiogenesis. In medulloblastoma, invading tumor cells characteristically lead to early recurrence and a decreased prognosis. Based on kinase activity profiling data published recently we hypothesized on a key function of the Eph/ephrin signaling system in medulloblastoma invasion.

In a panel of medulloblastoma cell lines a limited subset of Eph receptors was found to be expressed of which EphB2 was the most predominant. Furthermore, a significantly higher expression of EphB2 and its ligands ephrin-B1 and ephrin-B3 was observed in medulloblastoma patient tissue compared to normal cerebellum. To determine the relative importance of the EphB and EphA receptor families, 3 medulloblastoma cell lines (Daoy, Uw-402, Res-256) were stimulated with either ephrin-A1 or ephrin-B1 followed by assessment of the migration and adhesion capacity of the cells. Upon stimulation with ephrin-A1 no phenotype could be observed. However, stimulation with ephrin-B1 resulted in a marked increase in migration capacity of Daoy and, to a lesser extent, Uw-402 in concentrations as low as 0.2 ug/mL. The adhesion capacity of these cell lines upon stimulation with ephrin-B1 was determined employing collagen and laminin coated culture dishes. Again, no effects could be observed on Res-256 whereas the adhesive capacity of Daoy and Uw-402 decreased significantly. In addition, stable transfection of Daoy medulloblastoma cells with shRNA against EphB2 significantly reduced the ephrin-B1 induced effects on tumor cell adhesion.

The observed differences in the effects of EphB receptor stimulation correspond with the expression levels of EphB2 in the cell lines. Furthermore, a substantial increase of EphB2 and EphB4 phosphorylation could be observed for Daoy and to a lesser extent Uw-402. Res-256 had a low mRNA expression of EphB2 and B4 and also showed no increase in phosphorylation of these receptors upon stimulation. Interestingly, the increase in phosphorylated EphB2 and EphB4 is accompanied by a sharp decrease in EGFR phosphorylation. Possibly this plays a role in the ephrin-B1 induced effects, as has been reported recently for ephrin-A5 in glioblastoma.

Recent studies report an epigenetic regulation by hypermethylation of Eph/ephrin family gene expression in acute lymphoblastic leukemia. To assess the presence of epigenetic regulation of EphB receptor expression in medulloblastoma we treated the medulloblastoma cell lines with the demethylating agent 5-aza-2′-deoxycytidine. This resulted in a substantial increase in expression of all EphB type receptors in Daoy.

In conclusion, our results indicate a selective Eph expression profile in favor of medulloblastoma invasion. Furthermore, the expression of Eph receptors is regulated, at least in part, through DNA methylation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1974. doi:10.1158/1538-7445.AM2011-1974