Inhibition of the prenylation of K-Ras, but not H- or N-Ras, is highly resistant to CAAX peptidomimetics and requires both a farnesyltransferase and a geranylgeranyltransferase I inhibitor in human tumor cell lines

Abstract

The farnesyltransferase (FTase) inhibitor FTI-277 is highly e ective at blocking oncogenic H-Ras but not KRas4B processing and signaling. While inhibition of processing and signaling of oncogenic K-Ras4B is more sensitive to the geranylgeranyltransferase I (GGTase I) inhibitor GGTI-286 than it is to FTI-277 in K-Ras4B-transformed NIH3T3 cells, the sensitivity of K-Ras as well as H-and N-Ras to the CAAX peptidomimetics in human tumor cell lines is not known. Here, we report that a panel of ®ve human carcinoma cell lines from pancreatic, pulmonary, and bladder origins all express H-, N-, and K-Ras, and their respective prenylation sensitivities to the FTase and GGTase I inhibitors is variable. In all of the cell lines investigated, the prenylation of N-Ras was highly sensitive to FTI-277, and in two of the cell lines, N-Ras showed slight sensitivity to GGTI-298, an analog of GGTI-286. Although the prenylation of H-Ras was also sensitive to FTI-277, complete inhibition of H-Ras processing even at high concentrations of FTI-277 and/or GGTI-298 was never achieved. The prenylation of K-Ras, on the other hand, was highly resistant to FTI-277 and GGTI-298. Most signi®cantly, treatment of human tumor cell lines with both inhibitors was required for inhibition of K-Ras prenylation. In one cell line, the human lung adenocarcinoma A-549, prenylation of KRas was highly resistant even when co-treated with both inhibitors. Furthermore, soft agar experiments demonstrated that in all the human tumor cell lines tested inhibition of K-Ras prenylation was not necessary for inhibition of anchorage-independent growth. In addition, although GGTI-298 had very little e ect on soft agar growth, the combination of FTI-277 and GGTI-298 resulted in signi®cant growth inhibition. Therefore, the results demonstrate that while FTI-277 inhibits N-Ras and H-Ras processing in the human tumor cell lines evaluated, inhibition of K-Ras processing requires both an FTase inhibitor as well as a GGTase I inhibitor, and that inhibition of human tumor growth in soft agar does not require inhibition of oncogenic K-Ras processing.

Both farnesyltransferase and geranylgeranyltransferase I inhibitors are required for inhibition of oncogenic K-Ras prenylation but each alone is sufficient to suppress human tumor growth in nude mouse xenografts

“…Although FTI-276 was unable to inhibit K-Ras prenylation, it was very e cacious at suppressing tumor growth of both A-549 and Calu-1 cells in the nude mouse xenograft model. Furthermore, FTI-277 (50 mM) inhibited by 70% and 90% the anchorageindependent growth in soft agar of A-549 and Calu-1 cells, respectively (Lerner et al, 1997). Thus, in two models of transformation (nude mouse xenografts and soft agar), inhibition of growth did not require inhibition of oncogenic K-Ras processing.…”

Depletion of K-Ras promotes proteasome degradation of survivin

“…Human Pancreatic Nestin Expressing (HPNE) cells were the kind gift of Dr Channing Der and Dr Paul Campbell (University of North Carolina) and were maintained in media containing 3:1 mixture of DMEM and M3F base media (INCELL). NIH-3T3 cells that stably express mutant K-Ras and C7 cells that stably express mutant K-Ras were grown as described by us, 19,20 respectively.…”

Farnesyltransferase and geranylgeranyltransferase I inhibitors upregulate RhoB expression by HDAC1 dissociation, HAT association and histone acetylation of the RhoB promoter

“…In addition, in vitro and in vivo experiments have shown that K-Ras was geranylgeranylated in cells treated with FTIs (Rowell et al, 1997;Whyte et al, 1997;Sun et al, 1998). Thus, both FTase and GGTase I need to be inhibited to fully block K-Ras isoprenylation (Lerner et al, 1997). Whether inhibition of RhoB farnesylation is involved in the mechanism of action of FTIs is unclear at this time (Lebowitz and Prendergast, 1998;Prendergast, 2001;Sebti and Der, 2003).…”