ECT2 and RASAL2 Mediate Mesenchymal-Amoeboid Transition In Human Astrocytoma Cells

Malignant astrocytomas are highly invasive brain tumors. The Rho family of cytoskeletal GTPases are key regulators of astrocytoma migration and invasion; expression of the guanine nucleotide exchange factor ECT2 is elevated in primary astrocytomas and predicts both survival and malignancy. Mice bearing orthotopically implanted astrocytoma cells with diminished ECT2 levels following ECT2 knockdown exhibit longer survivaL Although ECT2 is normally expressed in the nucleus, we show that ECT2 is aberrantly localized to the cytoplasm in both astrocytoma cell lines and primary human astrocytomas, and colocalizes with RAC1 and CDC42 at the leading edge of migrating astrocytoma cells. Inhibition of ECT2 expression by RNA interference resulted in decreased RAC1 and CDC42 activity, but no change in RHO activity, suggesting that ECT2 is capable of activating these pro-migratory Rho family members. ECT2 overexpression in astrocytotna cells resulted in a transition to an amoeboid phenotype that was abolished with the ROCK inhibitor, Y-27632. Cytoplasmic fractionation of astrocytotua cells followed by ECT2 immunoprecipitation and mass spectrometry were used to identify protein-binding partners that modulate the activity of ECT2 toward RAC1 and RHO/ROCK_ We identified RASAL2 as an ECT2-interacting protein that regulates RHO activity in astrocytoma cells. RASAL2 knockdown leads to a conversion to an amoeboid phenotype. Our studies reveal that ECT2 has a novel role in mesenchymal-amoeboid transition in human astrocytoma cells. (Am J Pathol 2012, 181:662-674; http://dx.doi. org/10.1016/j.ajpath.2012.04.011)