Inhibition of Rho-kinase affects astrocytoma morphology, motility, and invasion through activation of Rac1.
Malignant astrocytomas are highly invasive neoplasms infiltrating diffusely into regions of normal brain. Whereas the molecular and cellular mechanisms governing astrocytoma invasion remain poorly understood, evidence in other cell systems has implicated a role for the Rho-GTPases in cell motility and invasion. Here, we examine how the inhibition or activation of Rho-kinase (ROCK) affects astrocytoma morphology, motility, and invasion. ROCK was inhibited in astrocytoma cells by using 5 to 100 mumol/L of Y27632 or by expressing the dominant-negative ROCK mutant, RB/PH TT. ROCK activation was achieved by expressing a constitutively active mutant, CAT. ROCK inhibition led to morphologic and cytoskeletal alterations characterized by an increase in the number and length of cell processes, increased membrane ruffling, and collapse of actin stress fibers. Using two-dimensional radial migration and Boyden chamber assays, we show that astrocytoma migration and invasion were increased at least 2-fold by ROCK inhibition. On the contrary, ROCK activation significantly inhibited migration and invasion of astrocytoma cells. Furthermore, using a Rac-GTP pull-down assay, we show that Rac1 is activated as a consequence of ROCK inhibition. Finally, we show that treatment of astrocytoma cells with small interfering RNA duplexes specific for Rac1-reversed stellation, prevented membrane ruffling formation and abrogated the increased motility observed following treatment with Y27632. Our data show that Rac1 plays a major role in astrocytoma morphology, motility, and invasion. These findings warrant further investigation to determine precisely how the modulation of Rac1 and ROCK can be exploited to inhibit glioma invasion.
Salhia
Bodour
B
The Arthur and Sonia Labatt Brain Tumour Research Center and Division of Neurosurgery, The Hospital for Sick Children, The University of Toronto, Toronto, Ontario, Canada.
Rutten
Frederieke
F
Nakada
Mitsutoshi
M
Beaudry
Christian
C
Berens
Michael
M
Kwan
Allison
A
Rutka
James T
JT
D000199:Actins / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D001254:Astrocytoma / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids / Q000494:pharmacology; D009154:Mutation; D009361:Neoplasm Invasiveness; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D054460:rho-Associated Kinases
D000199:Actins; D000577:Amides; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C107953:RAC1 protein, human; C108830:Y 27632; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020830:rac1 GTP-Binding Protein; C032881:lysophosphatidic acid