Inhibition of Rho-Kinase Affects Astrocytoma Morphology, Motility, and Invasion through Activation of Rac1

Abstract

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 Mmol/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 twodimensional 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 pulldown 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.

Manipulation of prostate cancer metastasis by locus-specific modification of the CRMP4 promoter region using chimeric TALE DNA methyltransferase and demethylase

“…The metastasis-suppressing role of this novel signaling pathway identified for CRMP4 in this study (Figure 5h) is in agreement with reports implicating that Akt inhibits Rac1 activity by direct phosphorylation of Rac1 [34,35]. It is known that Rac1 enhances prostate cancer invasion through activation of Rho GTPases and MMPs [30,32,33]. It appears that CRMP4-mediated suppression of meta stasis involves multiple signaling pathways.…”

The regulation of Rhoa in focal adhesions by starD13 is essential for astrocytoma cell motility. (c2011)

“…These results are in accordance with a previous study that showed that the inhibition of the RhoA signaling pathway leads to a significant inhibition of glioblastoma cell migration (Manning, et al, 2000). However, contradictory results have also been reported, where the inhibition of ROCK, a downstream effector of Rho, lead to an increase in astrocytoma cell migration and invasion (Salhia, et al, 2005).…”

The Pivotal Roles of GSK3β in Glioma Biology

“…This also causes GBM cells to acquire a relatively round shape without extending cell processes (Chuang et al, 2004). In contrast, collapse of actin stress fiber formation by the inhibition of RhoA promotes migration (Salhia et al, 2005). As LiCl treatment is associated with a marked change in GBM cell morphology, with cells retracting their long extensions at their leading edge and losing lamellipodia formation (Nowicki et al, 2008), GSK3 signaling may involve small GTPases such as Rac1 and RhoA.…”