Abstract LB-115: EphA2/ephrinA1 system may regulate invadopodia formation of glioblastoma multiforme (GBM) cells

Abstract

Abstract Background. Grade IV astrocytoma, or glioblastoma multiforme (GBM), is the most common, most malignant primary brain tumor in humans. With a median survival of approximately14 months, GBMs are characterized for their aggressive and highly invasive nature. We have previously explored the function of the EphA2 receptor and its ligand ephrinA1 in GBM. EphA2 was found to be over-expressed in various GBM cells, but not normal brain. EphrinA1, a naturally occurring GPI-linked protein originally found on epithelial cells, plays a tumor suppressive function by binding to and down-regulating cell surface expression of EphA2, leading to a subsequent decrease in cell migration and invasion. Invadopodia are electron-dense, actin-rich protrusions that bind to and proteolytically degrade extracellular matrix (ECM) proteins, potentially allowing for increased invasion. We have thus begun exploring the role of the EphA2/ephrinA1 complex in invadopodia formation and matrix degradation. Methods. Invadopodia formation and matrix degradation were assessed by an in situ gelatin zymography assay combined with fluorescence microscopy. Expression of the EphA2 receptor was examined by immunoblot using the following GBM cell lines: parental, vector control and ephrinA1-expressing U-251 cells, A-172, G48a, and Snb-19. Invadopodia-mediated degradation of the gelatin matrix was examined by fluorescence microscopy at various time points for each cell line. Src-transformed NIH3T3 (Src3T3) cells were used as a positive control. Results. The Snb-19 cells showed the most degradation of gelatin matrix among the GBM cell lines tested, closely mimicking control Src3T3 cells. This punctate pattern of gelatin degradation started to form as early as 2 hr after cell plating and continued until at least 72 hr. Other GBM cell lines degraded the matrix in the following order: Snb-19 ≫ A-127 > U-251 ephrinA1 > U-251 vector > U-251 parental > G-48a. Unexpectedly, immunoblot analysis showed low levels of EphA2 among the cells that degraded the most ECM. In preliminary experiments, diminishing levels of EphA2, either by knockdown or ligand activation, evoked more prominent gelatin-degrading invadopodia activity. Conclusions. GBM cells form invadopodia-like structures that degrade the ECM, with the Snb-19 cell line exhibiting the greatest degradation activity among the tested cell lines. Our preliminary results suggest a significant involvement of the EphA2/ephrinA1 system in invadopodia function based on an inverse correlation between matrix degradation and EphA2 expression. These findings have potentially important implications concerning the role of the EphA2/ephrinA1 complex in brain tumor migration and invasion. 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 LB-115. doi:10.1158/1538-7445.AM2011-LB-115