[CANCER RESEARCH 60, 2680 –2688, May 15, 2000] Mouse Mammary Tumor Virus-Ki-rasB Transgenic Mice Develop Mammary Carcinomas That Can Be Growth-inhibited by a Farnesyl:Protein Transferase Inhibitor Charles A. Omer, 1 Zunxuan Chen, 2 Ronald E. Diehl, Michael W. Conner, 3 Howard Y. Chen, Myrna E. Trumbauer, Shobhna Gopal-Truter, Gina Seeburger, Hema Bhimnathwala, Marc T. Abrams, Joseph P. Davide, Michelle S. Ellis, Jackson B. Gibbs, Ian Greenberg, 4 Kelly Hamilton, Kenneth S. Koblan, Astrid M. Kral, Dongming Liu, Robert B. Lobell, Patricia J. Miller, Scott D. Mosser, Timothy J. O’Neill, Elaine Rands, Michael D. Schaber, Edith T. Senderak, Allen Oliff, 5 and Nancy E. Kohl 6 Departments of Cancer Research [C. A. O., Z. C., R. E. D., H. B., M. T. A., J. P. D., M. S. E., J. B. G., I. G., K. H., K. S. K., A. M. K., D. L., R. B. L., P. J. M., S. D. M., T. J. O., E. R., M. D. S., A. O., N. E. K.], Safety Assessment [M. W. C.], and Biometrics Research [E. T. S.], Merck Research Laboratories, West Point, Pennsylvania 19486, and Departments of Metabolic Disorders [H. Y. C., M. E. T.] and Laboratory Animal Resources [S. G-T., G. S.], Merck Research Laboratories, Rahway, New Jersey 07065 ABSTRACT For Ras oncoproteins to transform mammalian cells, they must be posttranslationally modified with a farnesyl group in a reaction catalyzed by the enzyme farnesyl:protein transferase (FPTase). Inhibitors of FPTase have therefore been developed as potential anticancer agents. These com- pounds reverse many of the malignant phenotypes of Ras-transformed cells in culture and inhibit the growth of tumor xenografts in nude mice. Furthermore, the FPTase inhibitor (FTI) L-744,832 causes tumor regres- sion in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice and tumor stasis in MMTV-N-ras mice. Although these data support the further development of FTIs, it should be noted that Ki-ras is the ras gene most frequently mutated in human cancers. Moreover, Ki-RasB binds more tightly to FPTase than either Ha- or N-Ras, and thus higher con- centrations of FTIs that are competitive with the protein substrate may be required to inhibit Ki-Ras processing. Given the unique biochemical and biological features of Ki-RasB, it is important to evaluate the efficacy of FTIs or any other modulator of oncogenic Ras function in model systems expressing this Ras oncoprotein. We have developed strains of transgenic mice carrying the human Ki-rasB cDNA with an activating mutation (G12V) under the control of the MMTV enhancer/promoter. The predom- inant pathological feature that develops in these mice is the stochastic appearance of mammary adenocarcinomas. High levels of the Ki-rasB transgene RNA are detected in these tumors. Treatment of MMTV-Ki- rasB mice with L-744,832 caused inhibition of tumor growth in the absence of systemic toxicity. Although FPTase activity was inhibited in tumors from the treated mice, unprocessed Ki-RasB was not detected. These results demonstrate the utility of the MMTV-Ki-rasB transgenic mice for testing potential anticancer agents. Additionally, the data suggest that although the FTI L-744,832 can inhibit tumor growth in this model, Ki-Ras may not be the sole mediator of the biological effects of the FTI. INTRODUCTION Among the dominant-acting oncogenes, the ras genes are the most commonly mutated in human cancers and as such have been the focus for the development of new cancer chemotherapeutics (1). The three ras genes, Harvey (Ha)-, Kirsten (Ki)-, and N-ras, encode four highly homologous, 21-kDa GTP-binding proteins, Ha-Ras, Ki4A-Ras, and Ki4B-Ras (encoded by splicing variants of the Ki-ras gene), and N-Ras, which function in the transduction of growth promoting sig- nals from the membrane to the nucleus (2). Mutated forms of the ras genes, which encode constitutively active proteins, are found in 20% of all human cancers, including 90% of pancreatic tumors and 50% of colon tumors (3, 4). The biological activity of the Ras proteins is dependent upon localization to the inner surface of the plasma membrane. This local- ization is achieved after a series of posttranslational modifications, which increase the hydrophobicity of the protein (5). The first and obligatory step in this cascade is the addition of the 15-carbon farnesyl isoprenoid to the cysteine located four residues from the COOH- terminus of the Ras proteins. This cysteine residue is part of the COOH-terminal tetrapeptide referred to as a CA 1 A 2 X motif, in which C is cysteine, A is usually an aliphatic amino acid, and X is usually serine or methionine. Genetic experiments demonstrating that farne- sylation is essential for the transforming activity of the Ras oncopro- teins (6 –9) suggested that inhibitors of the enzyme that catalyzes the farnesylation reaction, FPTase, 7 would be useful in the treatment of Ras-dependent tumors. Recent studies have established that prenylation of the different Ras proteins is more complex than originally realized. Eukaryotic cells contain a related prenyl:protein transferase, GGPTase-I. This enzyme transfers the 20-carbon isoprenoid geranylgeranyl to the COOH- terminal cysteine of CA 1 A 2 X-containing proteins, which terminate in leucine or to a lesser extent phenylalanine or methionine. In vitro, all of the Ras proteins are normally substrates for FPTase. However, Ki- and N-Ras (which terminate in methionine), but not Ha-Ras (which terminates in serine), can also be modified by GGPTase-I. The effi- ciency of the Ras geranylgeranylation reaction is lower than the corresponding farnesylation reaction (10). Thus, in vivo, the Ras proteins are normally farnesylated, but when FPTase activity is ab- lated, as upon treatment of cells with a FTI, Ki- and N-Ras, but not Ha-Ras, become geranylgeranylated (11, 12). The geranylgeranylated forms of Ki- and N-Ras remain associated with the cellular membrane fraction. Furthermore, forms of oncogenic Ha-Ras and Ki-RasB en- gineered to be a substrates for GGPTase-I by modification of the CA 1 A 2 X motif retain the ability to transform rodent fibroblasts (9, 13). Potent inhibitors of FPTase that are selective versus GGPTase-I have been identified from screening of chemical collections and natural products as well as from rational design based on the protein and isoprenoid substrates of the reaction. In cell culture models, cell Received 9/24/99; accepted 3/21/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Present address: Department of Cancer Research, Parke-Davis, Ann Arbor, MI 48105. 2 Present address: Department of Renal Pharmacology, SmithKline Beecham Pharma- ceuticals, King of Prussia, PA 19406. 3 Present address: Advanced Medicine Inc., South San Francisco, CA 94080. 4 Present address: Eli Lilly & Co., Indianapolis, IN 46285. 5 Present address: Dupont Pharmaceuticals, Wilmington, DE 19880. 6 To whom requests for reprints should be addressed, at Department of Cancer Research, Merck Research Laboratories, WP16 –3, West Point, PA 19486. Phone: (215) 652-5646; Fax: (215) 652-7320; E-mail: nancy kohl@merck.com. 7 The abbreviations used are: FPTase, farnesyl:protein transferase; GGPTase-I, gera- nylgeranyl:protein transferase type I; FTI, FPTase inhibitor; MMTV, mouse mammary tumor virus; LTR, long terminal repeat; HaSV, Harvey sarcoma virus; MGR, mean growth rate. 2680 Research. on November 20, 2015. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from