CS-22 * MIXED LINEAGE KINASE SIGNALING IS REQUIRED FOR GLIOBLASTOMA CELL MIGRATION AND INVASION

Abstract

Glioblastoma multiforme (GBM) tumors are the most deadly of brain tumors due to their diffuse infiltrative nature and tremendous heterogeneity. EGFR signaling is aberrantly activated in most GBM tumors, and clearly contributes to malignancy. However, targeting EGFR directly has been largely unsuccessful, for multiple reasons, including compensatory upregulation of other receptor tyrosine kinases (RTKs). Identifying and targeting key common signaling nodes downstream of multiple RTKs that drive invasion may be an effective therapeutic strategy. The Mixed Lineage Kinases (MLKs) are a family of MAP3Ks that activate multiple MAPK pathways, including c-Jun N-terminal kinase (JNK). Binding of GTP-bound Rac/Cdc42 activates MLK3. We investigated the role of MLK signaling on GBM cell migration and 3D spheroid invasion using an ATP-competitive pan-MLK inhibitor, as well as siRNA-mediated silencing of MLK3. Our data demonstrate that both MLK inhibition and MLK3 silencing dramatically reduce GBM migration and 3D invasion. EGF-induced GBM cell migration, as well as JNK activation, is blocked by a pan-MLK inhibitor and by MLK3 silencing. The Rac GEF, Dock180, is critical for GBM invasion. Based upon Drosophila genetics, the Dock180 homolog (MBC), a Rac GEF, acts upstream of Rac, the MLK homolog (slipper) and JNK. Therefore we hypothesized that Dock180 might be an upstream activator of MLKs. Our data show that Dock180 complexes with MLK3; and that ectopic expression of Dock180 activates both MLK3 and JNK in a Rac-dependent manner. Furthermore, silencing of either Dock180 or MLK3 leads to a similar reduction in EGF-induced JNK activation. Finally, ectopic expression of MLK3 (which leads to its auto-activation) is sufficient to rescue the defect in migration due to Dock180 silencing. Based upon these data, we propose that an EGFR-Dock180-Rac-MLK-JNK signaling axis drives GBM migration and 3D invasion, and that targeting MLKs may be a useful therapeutic approach for treating patients with GBM.