Inactivation of Rho GTPases by p190 RhoGAP reduces human pancreatic cancer cell invasion and metastasis

Abstract

A number of small GTPases are involved in cancer cell proliferation, migration and invasion, acting as molecular switches that cycle between GTP- and GDP-bound states. GTPase-activating proteins (GAPs) have been established as a major class of negative regulators of Rho GTPase signaling. To investigate the biological function of p190 RhoGAP toward RhoA in cancer cell invasion and metastasis, we generated a chimera made of the RhoGAP domain of p190 and the C-terminus of RhoA (p190-RhoA chimera), and transfected it into human pancreatic cancer cells, AsPC-1. Epidermal growth factor (EGF)-induced activation of RhoA, as well as RhoB and RhoC, to a lesser extent, was significantly inhibited in p190-RhoA chimera-transfected AsPC-1 cells compared with that of control cells (mock-infected), when assessed by pull-down assay for GTP-bound RhoA, RhoB, and RhoC, respectively. EGF-induced invasion of p190-RhoA chimera transfectants was significantly inhibited compared with that of mock-infected cells in a modified Boyden chamber assay. Furthermore, the mice injected intrasplenically with AsPC-1 cells that overexpressed the p190-RhoA chimera had a marked reduction in the number and size of metastatic nodules in the liver. These data suggest that the inhibitory action of p190 RhoGAP toward RhoA offers a novel approach to the treatment of invasion and metastasis of cancer cells.

Figure 1

Effect of blocking the Rho signal transduction pathway on AsPC‐1 cell invasion. (a) The invasive capacity of AsPC‐1 cells are depend on Rho signaling. Cells were incubated in medium containing 10% FBS and digitonin (25 µM) with the indicated concentration of C3 for 24 h. After the cells were washed twice with PBS, cells were resuspended in serum‐free medium containing 0.1% BSA and subjected to Matrigel invasion assay (left panel). To examine the effect of Y‐27632 on EGF‐induced AsPC‐1 cell invasion, cells were resuspended in serum‐free medium containing 0.1% BSA with the indicated concentration of Y27632 and subjected to Matrigel invasion assay (right panel). Data are expressed as mean ± SEM of three separate experiments in triplicate wells. *P < 0.05, **P < 0.01 versus control cells with EGF stimulation. (b) The p190‐RhoA chimera was expressed in AsPC‐1 cells by retroviral transfection. The Flag‐p190‐RhoA chimera proteins were detected by anti‐Flag antibody. Cells were incubated for 24 h and photographed by phase‐contrast microscopy. (c)(d) Inhibitory effect of the p190‐RhoA chimera on human pancreatic cancer cell invasion. Mock‐transfected or p190‐RhoA transfected AsPC‐1 cells (c) or PANC‐1 cells (d) were treated with or without EGF and were subjected to Matrigel invasion assay. Data are expressed as mean ± SEM of three separate experiments in triplicate wells. *P < 0.05 versus control (mock‐infected).

Figure 2

Alteration of Rho signaling by p190‐RhoA chimera transfection. (a) The specificity of the antibodies against RhoA, B, and C. The lysate from myc‐RhoA‐transfected HEK293 cells was subjected to Western blotting with antibodies against myc‐tag (myc), RhoA (A), RhoB (B), and RhoC (C), respectively. The location of each band detected by corresponding antibodies is shown by lines as indicated. (b) Effect of the p190‐RhoA chimera on EGF‐induced RhoA, B, and C activation. Mock‐transfected or p190‐RhoA transfected AsPC‐1 cells were incubated in the absence or presence of 1 nM EGF. The active form of Rho proteins which bound to the RBD were shown on upper panels. The total Rho proteins were shown on lower panels. Representative data from three experiments are shown. Relative intensity values of the blot are shown above the corresponding panel.

Figure 3

Effect of the p190‐RhoA chimera on EGF‐stimulated phosphorylation of p44/42 MAP kinase and proliferation in AsPC‐1 cells. (a) The top panel represents MAPK phosphorylation detected using an antibody specific for the phosphorylated form of MAPK. The bottom panel represents a Western blot probed with an anti‐MAPK antibody to demonstrate the equal loading. (b) Effect of the p190‐RhoA chimera on EGF‐induced proliferation of AsPC‐1 cells. The MTT assay results shown are from a representative of four experiments. The mean ± SEM of quadruplicate are shown.

Figure 4

Antimetastatic effect of the p190‐RhoA chimera in vivo. Nude mice were injected intrasplenically with 1 × 10⁶ AsPC‐1 control cells (mock‐infected) (a) or p190‐RhoA chimera transfectants (b). Gross appearance of tumors in spleen (lower row) and liver (upper row), 40 days after injection was shown.