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 astrocytoma cells resulted in a transition to an amoeboid phenotype that was abolished with the ROCK inhibitor, Y-27632. Cytoplasmic fractionation of astrocytoma 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.
Division of Neurosurgery, Department of Surgery, Arthur and Sonia Labatt Brain Tumour Research Centre, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada.
Adrienne
A
Weeks
Nadia
N
Okolowsky
Brian
B
Golbourn
Stacey
S
Ivanchuk
Christian
C
Smith
James T
JT
Rutka
D000293:Adolescent; D000328:Adult; D000368:Aged; D000708:Anaplasia; D000818:Animals; D001253:Astrocytes / Q000378:metabolism / Q000473:pathology; D001254:Astrocytoma / Q000378:metabolism* / Q000473:pathology*; D002352:Carrier Proteins / Q000378:metabolism*; D002465:Cell Movement; D002648:Child; D002675:Child, Preschool; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D020690:GTPase-Activating Proteins; D006801:Humans; D008297:Male; D008648:Mesoderm / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008875:Middle Aged; D008954:Models, Biological; D060787:Neoplasm Grading; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011554:Pseudopodia / Q000378:metabolism; D055815:Young Adult; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D002352:Carrier Proteins; C489850:ECT2 protein, human; D020690:GTPase-Activating Proteins; D011518:Proto-Oncogene Proteins; C107953:RAC1 protein, human; C576391:RASAL2 protein, human; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein