Abstract 44: EGFR and Dock180 activate MLK3 to drive invasion of glioblastoma cells

Abstract

Abstract Glioblastoma (GBM) is the most common and deadly form of brain tumor in adults, with an average post-diagnosis survival time of 15 months. While GBMs rarely metastasize to distant organs, they readily invade into surrounding brain tissue, leading to incomplete surgical resection and subsequent tumor recurrence. Epidermal Growth Factor Receptor (EGFR) signaling is aberrantly activated in a majority of GBM tumors, and is clearly implicated in multiple malignant phenotypes, including migration and invasion. However, direct targeting of EGFR has been largely unsuccessful, for multiple reasons, including compensatory upregulation of other RTKs. Thus, identifying and targeting key signaling nodes that are downstream of multiple RTKs is of therapeutic importance. Several guanine nucleotide exchange factors (GEFs), including Dock180, a Rac1 GEF, are implicated in GBM invasion through activation of small GTPases, Rac and Cdc42. The Mixed Lineage Kinases (MLKs) are a family of cytosolic MAP3Ks that activate multiple MAPK pathways, including the c-Jun N-terminal kinase (JNK), ERK, and p38 pathways. MLK3 is activated through the binding of activated, GTP-bound Rac and/or Cdc42 to induce dimerization and activation. Herein, the role of MLK signaling on GBM cell migration is investigated using an ATP-competitive pan-MLK inhibitor (previously in phase II/III clinical trials for Parkinson's disease), as well as siRNA targeting MLK3. Data from this study demonstrate that both MLK inhibition and silencing of MLK3 dramatically reduce the migration and invasion of GBM cells. EGF-induced activation of JNK in multiple GBM cell lines is blocked by a pan-MLK inhibitor, as well as an MLK3-specific siRNA. Furthermore, the MLK inhibitor blocks EGF-induced migration in a 2D wound healing assay. Mutant EGFRvIII-induced JNK activation is blocked by MLK inhibition, and invasion of primary GBM6 cells (express EGFRvIII) is blocked in a 3D invasion assay. Dock180, a Rac GEF has been shown to be critical for multiple malignant phenotypes in GBM cells, including migration and 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 MLK3. Our data demonstrate that Dock180 complexes with MLK3, and that ectopic expression of Dock180 activates MLK3 activation in a Rac-dependent manner. Furthermore, siRNA-mediated silencing of Dock 180 and of MLK3 reduce EGF-induced JNK activation to similar extents, consistent with the idea that they act in the same signaling axis leading to 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. Citation Format: Sean A. Misek, Jian Chen, Kathleen A. Gallo. EGFR and Dock180 activate MLK3 to drive invasion of glioblastoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 44. doi:10.1158/1538-7445.AM2015-44