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 Edwina C Lerner 1,3 , Ting-Ting Zhang 1 , David B. Knowles 2 , Yimin Qian 2 , Andrew D Hamilton 2 and SaõÈd M Sebti 1,3 Departments of 1 Pharmacology and 2 Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA The farnesyltransferase (FTase) inhibitor FTI-277 is highly eective at blocking oncogenic H-Ras but not K- Ras4B 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 K- Ras 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 proces- sing 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. Keywords: Ras; prenylation; human tumors; FTase inhibitors Introduction The Ras family of proteins are small guanine nucleotide binding proteins that cycle between their GTP (active) and GDP-bound (inactive) forms to transduce growth and dierentiation signals from receptor tyrosine kinases to the nucleus (McCormick et al., 1993). Mutations have been identi®ed in all three types of Ras (H-, K-, and N-Ras) proteins in a variety of human tumors, including approximately 90% of pancreatic adenocarcinomas, 50% of colon carcino- mas, 20% of lung carcinomas, and 40% of myeloid disorders, with K-Ras mutations occurring most frequently (Barbacid, 1987). In order for Ras to transduce its normal or oncogenic signal it must be anchored to the plasma membrane, which is accomp- plished by a series of post-translational modi®cations that increase its hydrophobicity (reviewed in Zhang et al., 1996). A key step in this process is catalyzed by farnesyltransferase (FTase), an enzyme which transfers farnesyl to the cysteine of the carboxyl-terminal CAAX of Ras, where A is an aliphatic amino acid, and X is serine, methionine, glutamine, or cysteine (Zhang et al., 1996). A closely related enzyme, geranylgeranyltrans- ferase I (GGTase I), shares the a-subunit of FTase, and attaches the lipid geranylgeranyl to the cysteine of the CAAX box of proteins where X is leucine (Casey et al., 1992; Moomaw et al., 1992). Because membrane association of Ras is required for its biological function, we and others have designed inhibitors of Ras prenylation as novel anticancer agents (reviewed in Sebti and Hamilton, 1997; Sattler and Tamanoi, 1996; Buss and Marsters, 1996). We have designed CAAM peptidomimetics such as FTI-276 that are potent and selective inhibitors of FTase over GGTase I in vitro, and that selectively block the processing of farnesylated but not geranylgeranylated proteins in whole cells (Lerner et al., 1995a,b). We have also designed CAAL peptidomimetics, such as GGTI-287 and GGTI-297, that are selective for GGTase I over FTase (Lerner et al., 1995a; McGuire et al., 1996; Vogt et al., 1996). Furthermore, we have shown that while H-Ras processing was highly sensitive to FTI-277 and resistant to GGTI-286, KB-Ras was more sensitive to GGTI-286 than FTI-277 (Lerner et al., 1995a). These results suggested that H-Ras is only farnesylated, while KB-Ras may be both farnesylated and geranylgerany- lated in whole cells (Lerner et al., 1995a). This was consistent with in vitro data that showed that KB-Ras is a substrate for both FTase and GGTase I (James et al., 1995). Our studies on the relative sensitivities of H- Ras and KB-Ras processing to the FTase and GGTase I inhibitors were carried out in NIH3T3 cells Correspondence: SM Sebti and/or AD Hamilton 3 Present address: H Lee Mot Cancer Center, Drug Discovery Program, Department of Biochemistry and Molecular Biology, University of South Florida, 12902 Magnolia Drive, Tampa, Florida 33612, USA Received 28 February 1997; revised 20 May 1997; accepted 20 May 1997 Oncogene (1997) 15, 1283 ± 1288  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00