Regarding HRASG12V, check FAQ it is evident that Rac1 plays an important role in EMT properties of Caco H cells, since inhibition of this GTPase with specific inhibitor, resulted in decreased capacity of the cells to migrate and invade in vitro. It is worth mentioning that inhibition of Rac1 was also attempted using specific siRNA, but downregu lation of Rac1 was not significant. Although activation of Rac1 in Caco H cells is moder ate, as compared to Caco 2, activity of RhoA is reduced, potentially due to antagonistic action of RhoA and Rac1 in actin cytoskeleton organization. Inhibitors,Modulators,Libraries Regulation of Rho GTPases pathway differs in each case of oncogene transformation a. BRAFV600E and RhoA In our system, cross talk between BRAFV600E and RhoA is mainly mediated through MEK ERK pathway, as indi cated by cell treatment with a MEK inhibitor.

Additional Inhibitors,Modulators,Libraries data which link BRAFV600E to Rho signalling were recently derived from microarray analysis preformed with Caco BR cells in our lab. Global gene expression analysis revealed that RhoA spe cific guanine nucleotide exchange factors, like GEF11 and GEF18 were upregulated in Caco BR cells. This indicates that mutant BRAF can positively regulate RhoA activity by modulating the expression of its regulatory factors. Inhibitors,Modulators,Libraries Remarkably, as presented in a recent study, ERK can pro mote Rho dependent Inhibitors,Modulators,Libraries focal adhesion formation by sup pressing p190A RhoGAP. Nevertheless, in our system RhoA ROCK axis does not appear to play crucial role in the enhanced cell migration and invasion proper ties, since inhibition of ROCK does not alter the capacity of Caco Inhibitors,Modulators,Libraries BR cells to migrate and invade in vitro.

In agree ment with this data, previous studies have shown that treatment of human endometrial stromal cells and NIH 3T3 mouse fibroblasts with ROCK inhibitor Y 27632 resulted in enhanced selleckbio cell motility. A possi ble explanation may be the fact that RhoA has alternative effectors, such as Dia1 which was shown to be involved in RhoA dependent cytoskeletal properties. In human colon cancer cells Dia1 can act downstream of RhoA to regulate the actin network. Previous studies using HeLa or breast cancer cells showed that active RhoA is required for the induction of membrane ruffles in migrat ing cells also mediated by Dia1 and not ROCK. Here, active RhoA may potentially act mainly through Dia1 and not ROCK to induce migration and invasion in Caco BR cells and for that reason downregulation of ROCK may not affect these cell properties. Notably, cross talk analysis of small GTPases by means of selective siRNA revealed that RhoA may have an antagonistic function with Cdc42 in Caco BR13 cells. This can be achieved though competition for common regulatory molecules, like Rho guanine nucleotide dissociation inhibitors.