Fig 2: Rhoa G17E induced Vav1 expression and contributed to the invasion of MKN74 cells. (A) Vav1 mRNA expression in MKN74 cells transduced with a vector containing a RhoA mutant (N = 5). Real-time PCR revealed that Vav1 mRNA expression was significantly increased in cells with the RhoA G17E mutant. (B) Vav1 protein expression of MKN74 cells transduced with a vector containing mutant RhoA. GAPDH was used as an internal control. Western blotting showed a significant elevation of Vav1 and phosphorylated Vav1 in cells with RhoA G17E and Y42C mutants. (C) and (D) Vav1 was retrovirally knocked down by Vav1 shRNA, and its expression was compared with that in cells transfected with scramble shRNA in MKN74 cells transduced with the RhoA G17E mutant by real-time PCR and western blotting. (E) Migration assays of cells containing the RhoA G17E mutant with Vav1 shRNA and scrambled control shRNA (N = 4). The increased migration of cells with RhoA G17E was reduced by Vav1 knockdown but not significantly. (F) Invasion assays of MKN74 cells were transduced with a vector containing the RhoA G17E mutant and treated with Vav1 shRNA and scrambled control shRNA (N = 4). Increased infiltration of cells transduced with the RhoA G17E mutant was significantly reduced by Vav1 knockdown. *P < 0.05.

Fig 3: Vav1 promoted the formation of peritoneal disseminated nodules in vivo. (A) Number of peritoneal disseminated nodules after intraperitoneal administration of RhoA WT (N = 4) and RhoA G17E mutant (N = 4). Arrows indicate peritoneal disseminated nodules. The number of intraperitoneal nodules was significantly higher in mice injected with RhoA G17E-containing vector-transduced cells than in cells transduced with a vector containing WT RhoA. (B) Number of peritoneal disseminated nodules after intraperitoneal administration of RhoA G17E mutant-containing vector-transduced cells with Vav1 shRNA or scrambled control shRNA. The number of peritoneal nodules in RhoA G17E mutant-containing vector-transduced cells with Vav1 knockdown (N = 4) was greatly reduced compared to that in RhoA G17E mutant-containing vector-transduced cells without Vav1 knockdown (scramble shRNA) (N = 4). *P < 0.05.

Fig 4: Rhoa G17E and active Vav1 interacted through direct binding and induced cancer invasion via matrix metalloproteinase-9 (MMP-9). (A) Binding of Vav1 to RhoA G17E detected by immunoprecipitation. Phosphorylated Vav1 was specifically immunoprecipitated with RhoA G17E but not with RhoA WT. (B) Expression of proteins related to the RhoA-ROCK pathway after treatment with Vav1 shRNA. The expression of ROCK and phosphorylated cofilin was unchanged after Vav1 knockdown. (C) Enzymatic activities of MMP-2 and MMP-9 were examined by gelatin zymography. Expression of MMP-9 was elevated by RhoA G17E gene transduction; this effect was abolished by knockdown of Vav1. MMP-2 expression showed no change. (D) Expression of proteins related to the MAPK pathway, which is thought to be downstream of Vav1, after treatment with Vav1 shRNA, was reduced in RhoA G17E mutant-containing cells compared to that in WT RhoA-containing cells; this reduction was unaffected by knockdown of Vav1.