Roles of the Rac1 and Rac3 GTPases in human tumor cell invasion

Abstract

Members of the Rho family of small GTPases have been shown to be involved in tumorigenesis and metastasis. Currently, most of the available information on the function of Rho proteins in malignant transformation is based on the use of dominant-negative mutants of these GTPases. The specificity of these dominant-negative mutants is limited however. In this study, we used small interfering RNA directed against either Rac1 or Rac3 to reduce their expression specifically. In line with observations using dominant-negative Rac1 in other cell types, we show that RNA interference-mediated depletion of Rac1 strongly inhibits lamellipodia formation, cell migration and invasion in SNB19 glioblastoma cells. Surprisingly however, Rac1 depletion has a much smaller inhibitory effect on SNB19 cell proliferation and survival. Interestingly, whereas depletion of Rac3 strongly inhibits SNB19 cell invasion, it does not affect lamellipodia formation and has only minor effects on cell migration and proliferation. Similar results were obtained in BT549 breast carcinoma cells. Thus, functional analysis of Rac1 and Rac3 using RNA interference reveals a critical role for these GTPases in the invasive behavior of glioma and breast carcinoma cells.

Involvement of Rac and Rho signaling in cancer cell motility in 3D substrates

“…Rac3 has been reported to be involved in the invasion of glioblastoma cells, but its function remains unknown (Chan et al, 2005). The involvement of Rac3 in cellsubstrate adhesion during cell invasion is a novel finding in this study.…”

“…Inhibition of Rac signaling by the expression of Rac1 T17N in U87MG cells changed the morphology of the cells from elongated to round (Figures 3e and f). It has been reported that Rac1 and Rac3 play pivotal roles in the invasive process of glioblastoma cells (Chan et al, 2005); thus, the expression of Rac1 and Rac3 was repressed by RNAi in U87MG cells (Figure 3a). As we did not have an antibody specific for Rac3, the effect of Rac3-directed siRNA (Rac3-RNAi) on protein expression was confirmed using an antibody that recognizes both Rac1 and Rac3.…”

“…This suggested that Rac proteins other than Rac1 could compensate for Rac1 deficiency in U87MG cells. Therefore, in U87MG cells, Rac signaling was inhibited by the expression of a dominant-negative mutant of Rac1 (Rac1 T17N), which has been thought to inhibit the function of all Rac proteins (Chan et al, 2005). Inhibition of Rac signaling by the expression of Rac1 T17N in U87MG cells changed the morphology of the cells from elongated to round (Figures 3e and f).…”

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

“…The Cdc42 siRNA oligo was: 5′-AAAGACTCCTTTCTTGCTTGT-3′. MTLn3 cells were transfected with 100nM Rac or Cdc42 siRNA or with control siRNA sequences targeting GL2 luciferase [28] (Ambion) for 48 hours prior to the day of the experiment using Oligofectamine (Invitrogen) as described by the manufacturer. Rac and Cdc42 suppression of protein levels was analyzed by Western blotting using antibodies against total Rac and Cdc42 respectively.…”

“…The time interval between successive frames was 1 min. Motility parameters were calculated using DIAS software [28], and the data are the mean ± SEM from 15-40 cells per condition.…”