(339c) Sprouty2 Regulation of Glioblastoma Adhesion and Invasion

Glioblastoma multiforme (GBM) is a devastating form of brain cancer where patients typically succumb within two years of diagnosis. Most tumors will recur even after surgical resection due to the presence of residual tumor cells beyond the margin of the resected tumor mass, highlighting the importance of identifying the key regulators of GBM cell invasion. Previous work from our group identified the protein Sprouty2 (SPRY2) as a possible tumor promoter in GBM. Specifically, SPRY2 knockdown reduced proliferation of human glioblastoma cells and antagonized the growth of subcutaneous tumor xenografts in mice. New observations from our lab suggest that SPRY2 may also regulate GBM cell adhesion and migration phenotypes. In 3D cultures of tumor cell spheroids, GBM cells demonstrated extensive invasion into the matrix as revealed by live-cell microscopy studies. This phenotype was largely abrogated by SPRY2 knockdown. In 2D cultures, SPRY2-replete cells tended to grow as monolayers, with most cells clearly adhered to tissue culture plastic. In contrast, similarly dense SPRY2-deficient cells displayed a tendency to grow on top of each other, suggesting a preference for cell-cell adhesions over spreading on tissue culture plastic. Based on these findings, we hypothesized that SPRY2 may play a role in the invasion of GBM cells away from the main tumor body. Further, this role of SPRY2 may contribute to the ineffectiveness of initial surgical resections in resolving GBM. Quantitative immunofluorescence measurements of phosphorylated paxillin (p-paxillin, Y118) indicate that SPRY2 knockdown antagonizes formation of p-paxillin containing focal adhesions. Based on this finding, we hypothesized that SPRY2 plays a role in the regulation of p-paxillin containing focal adhesions in a manner that allows GBM cells to more efficiently invade their surrounding matrix. Focal adhesion kinase (FAK) is reported to be an upstream regulator of paxillin phosphorylation, and thus we hypothesized that FAK may drive cell spreading. Surprisingly, treatment with a FAK inhibitor increased cell spread area and antagonized the formation of cell-cell adhesions. Ongoing studies will aim to elucidate a mechanistic model that unites these apparently conflicting findings. Ultimately, these studies are relevant to the identification of targeted therapeutics that may limit putative SPRY2-mediated GBM invasion, and thus has relevance to the improvement of patient outcomes.