Novel Inhibitor of Geranylgeranyltransferase-I Figure 7. Stable expression of farnesylated RhoA promotes partial resistance to the anti-proliferative effect of P61-E7. (A) Stable expression of farnesylated RhoA (3xHA-RhoA-F) partially prevents P61-E7 inhibition of proliferation in Panc-1 cells in a manner that depends on the expression levels of 3xHA-RhoA-F. Panc-1 cells and Panc-1 stable cell lines expressing either wild-type 3xHA- RhoA-GG (clone no. 9) or RhoA-F mutant (clone no. 1, 4, or 8) were treated with DMSO or various concentrations of P61-E7 for 72 h. Cell number was determined as described in ‘‘Materials and Methods.’’ Cell proliferation relative to the DMSO control (100% value) is plotted. Data shown are averages 6 S.D. of three independent experiments. (B) Protein levels of 3xHA-RhoA-GG (clone no. 9) and 3xHA-RhoA-F (clone no. 1, 4, or 8) in different clones of Panc-1 stable cell lines as analyzed by immunoblotting using antibodies against the HA tag or actin (loading control). doi:10.1371/journal.pone.0026135.g007 A6. In addition, P61-E7 showed higher level of p21CIP1/WAF1 induction in comparison to P61-A6. Furthermore, P61-E7 treatment resulted in a more pronounced inhibition of cellular proliferation under low serum condition when compared to P61- A6. The excellent correlation we observed between cellular effects and increased potency to inhibit protein geranylgeranylation supports the idea that the consequences of geranylgeranylation inhibition include inhibition of cell cycle progression, induction of p21CIP1/WAF1 and inhibition of proliferation. We also examined the mechanism of action of P61-E7 in this paper, and our work suggests that RhoA is a major player in GGTI-mediated effects on pancreatic cancer cells. We have shown that P61-E7 induces p27Kip1 level, suggesting that the treatment with GGTI results in the increases of both p21CIP1/WAF1 and p27Kip1 in Panc-1 cells. The mechanism of induction, however, appears to differ between the two Cdk inhibitors. In the case of p21CIP1/WAF1, it appears that the induction is regulated at the transcriptional level, as we have shown that GGTI induces expression of p21-reporter construct [20]. On the other hand, GGTI effect on p27Kip1is associated with the inhibition of phosphorylation of Thr187. Phosphorylation of p27Kip1 (T187) results in the association of p27Kip1 with SCF ubiquitin ligase, targeting p27Kip1 for degradation [36]. Because P61-E7 inhibits phosphorylation of p27Kip1 (T187), it is possible that P61-E7 prevents degradation of p27Kip1, causing accumulation of p27Kip1 in the nucleus in Panc-1 cells. The above results on the effects of GGTI on Cdk inhibitors raise the possibility that the GGTI effects are mediated by RhoA. First, it is known that RhoA acts as a negative regulator of p21CIP1/WAF1 expression [38,39,41]. Second, RhoA is shown to be essential for the degradation of p27Kip1 [42–44] and that RhoA stimulates p27Kip1 degradation through its regulation of cyclin E/CDK2 activity [43]. Further support for the idea that many of the GGTI effects we observed with Panc-1 cells are due to the inhibition of RhoA function comes from additional effects of P61-E7 we observed. We found that P61-E7 treatment results in cell rounding, actin cytoskeleton disorganization and inhibition of focal adhesion formation. To evaluate the possibility that GGTI targets RhoA functions, we have established stable cell lines of Panc-1 expressing a mutated form of RhoA (RhoA-F). RhoA-F has the CAAL (CLVL) box changed to CAAX (CLVS) so that the mutant protein is farnesylated instead of being geranylgeranylated. Characterization of these stable cell lines showed that the expression of RhoA-F reversed the ability of GGTI to induce p21CIP1/WAF1 and p27Kip1. Stable expression of RhoA-F rescued cells from cell rounding and focal adhesion inhibition caused by P61-E7. Finally, we observed partial rescue of GGTI-induced proliferation inhibition and this rescue correlated with the expression levels of RhoA-F. These results suggest that many of the GGTI effects are due to the inhibition of RhoA functions. Our results on the rescue of proliferation inhibition by the expression of RhoA-F is consistent with the previous report that the expression of farnesylatable RhoA and Cdc42 restores proliferation of GGTase-I deficient fibroblasts expressing oncogenic K-Ras [7]. There are other proteins implicated in the GGTI effects. One type of proteins is Ral suggested to be involved in the GGTI effects of MiaPaca-2 pancreatic cancer cells [4]. It is possible that the Ral proteins are involved in other aspects of GGTI effects such as tumor growth. In addition, it has been suggested that Rac1and Rac3 proteins are PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e26135