Effect of farnesyltransferase inhibitor FTI-276 on established lung adenomas from A/J mice induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Laura E. Lantry, Zhongqiu Zhang, Rusheng Yao, Keith A. Crist1, Yian Wang, Junko Ohkanda2, Andrew D. Hamilton2, Said M. Sebti3, Ronald A. lubet4 and Ming You5

Department of Pathology and
1 Department of Surgery, Medical College of Ohio, Toledo, OH 43699,
2 Department of Chemistry, Yale University, New Haven, CT 06511,
3 H.Lee Moffitt Comprehensive Cancer Center and Research Institute, Department of Biochemistry and Molecular Biology, University of South Florida, Tampa, FL 33647 and
4 Chemoprevention Branch, National Cancer Institute, Rockville, MD 20892, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Ras protein undergoes a series of post-translational modifications at the C-terminal CAAX motif, which culminates with the anchoring of p21 Ras to the plasma membrane where it relays growth regulatory signals from receptor tyrosine kinases to various pathways of cell signal transduction. FTI-276 is a CAAX peptidomimetic of the carboxyl terminal of Ras proteins. Pharmacokinetic analysis of FTI-276 in A/J mice with a time-release pellet system showed a dose of 50 mg/kg body wt achieved an average serum level of 1.68 µg/ml for up to 30 days following implantation. In the present study, 4 week old A/J mice were initiated with a single dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (100 mg/kg), and monitored for 18 weeks. Mice were grouped for daily delivery (time-release pellet) of 50 mg/kg of FTI-276 for 30 days (n = 12) and the control group (n = 12). Analysis of tumors from time-release pellet treated animals showed a 60% reduction in tumor multiplicity and a 42% reduction in tumor incidence. Moreover, FTI-276 treatment resulted in a significant reduction in tumor volume (~58%). Mutation analysis of the lung tumors from both treatment groups revealed that most of the tumors harbored mutations in the codon 12 of K-ras and there is no significant difference in the incidence and types of mutations between tumors from the treated and control animals. This is the first demonstration of chemotherapeutic efficacy of a synthetic CAAX peptidomimetic farnesyltransferase inhibitor in a primary lung tumor model.

Abbreviations: DTT, dithiothreitol; FPP, farnesyl pyrophosphate; FTase, farnesyltransferase; FTIs, FTase inhibitors; GGTase I, geranylgeranyltransferase I; MAP, mitogen-activated pathway; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; RFLP, restriction fragment length polymorphism; SSCP, single-strand conformation polymorphism.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Ras family of small GTP/GDP-binding proteins has gained attention in the search for mechanism-based anticancer rational drug discovery (1). In the mammalian genome, four species of Ras proteins have been identified and found to be expressed in a tissue- and cell-dependent manner, including H-Ras, N-Ras, K-Ras4A and K-Ras4B (2). Normally, these 21 kDa proteins function in cell communication pathways by cycling between a GDP- to a GTP-bound state, conveying signals between cell-surface tyrosine kinase receptors and various intracellular second messengers e.g. mitogen-activated pathway (MAP) kinase pathway to effect changes in growth, differentiation and organization of the cytoskeleton (3,4). Activation of ras proto-oncogenes, generally resulting from a point mutation at codons 12, 13 or 61, has been identified in several types of cancers in humans, with K-ras4B found predominately in lesions of colon, pancreas and lung (2,3,5,6).

All nascent Ras proteins undergo a series of post-translational modifications in order to associate with the inner plasma membrane, and thus attain biological activity (79). The first of these is catalyzed by the enzyme farnesyltransferase (FTase) which transfers a 15 carbon, farnesyl moiety from farnesyl pyrophosphate (FPP) to the cysteine thiol of the carboxyl terminus of the newly synthesized protein (10). This enzyme recognizes tetrapeptides at the C-terminus, referred to as the CAAX motif. The farnesylation step alone has been shown to be sufficient for Ras-dependent transformation in some systems (11). These findings have led to the development of FTase inhibitors (FTIs) designed to prevent the isoprenylation and therefore the translocation of oncogenic Ras to the plasma membrane, thus inhibiting its constitutive activity (1214). This fundamental knowledge of the enzyme recognition site, the CAAX motif, has enabled the rational design of peptide mimetics, as well as non-peptide inhibitors of FTase (1517).

The present study was designed to test the chemotherapeutic effect of FTI-276, a tetrapeptide mimetic of the carboxyl terminus of K-Ras4B, K-Ras4A and N-Ras, first described by Lerner et al. (18). FTI-276 is highly selective in vitro, inhibiting FTase at 100 times lower concentration than is needed to inhibit the related enzyme, geranylgeranyltransferase I (GGTase I) (19). In addition, FTI-276 has been reported to selectively inhibit K-Ras-dependent tumor growth in a nude mouse model involving subcutaneous growth of a xenograft (20, 21). Unlike previous reports, the current study examines the chemotherapeutic effects of FTI-276 on primary lung tumorigenesis in an immunocompetent mouse. Mouse lung tumors induced with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in this model are well documented to harbor the codon 12 mutation in K-Ras4B at an early stage of tumorigenesis (22). We demonstrate that FTI-276 reduces the tumor burden in a primary mouse lung tumor model.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Reagents
NNK was purchased from Chemsyn Science Laboratories, Lenexa, KS. The stock solution was prepared in warm PBS, and the solution tested for purity (99%) by HPLC by Dr Mark Morse at the Ohio State University. FTI-276 (95% pure) was synthesized as described previously (18). Custom-made time-release pellets of FTI-276 were produced by Innovative Research of America (Tampa, FL).

Lung tumor bioassay
Four-week-old, male A/J mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed four per cage in plastic cages with hardwood bedding and dust covers, in a HEPA filtered, environmentally controlled room (24 ± 1°C, 12/12 h light/dark cycle). The mice were maintained on Rodent Lab Chow, #5001 (Purina) and water ad libitum. After a 1 week quarantine, they were given one i.p. injection of NNK, at 100 mg/kg body weight in PBS, and monitored for 18 weeks.

On the first day of FTI-276 treatment (18 weeks after NNK administration), the animals were randomized into treatment groups as follows: group 1, FTI-276 time-release pellet (n = 12); group 2, pellet matrix control (n = 12). The custom-made time-release pellets contained the equivalent concentration of FTI-276 to deliver 1.25 mg/day for 30 days, based on 50 mg/kg/day to a 25 g mouse (group 1), or matrix alone (group 2). The pellets were inserted under sterile conditions, one per animal (s.c.), at the right dorsal base of the neck.

At the end of the fourth week of treatment with FTI-276, the mice were killed by CO2 asphyxiation. The lungs were harvested, and the fresh tumors were sized and counted, and then frozen for K-ras mutation analysis. The remaining lungs were then fixed for 3 days in Tellyesniczky's [90% ethanol (70% v/v), 5% glacial acetic acid, 5% formalin (10% v/v buffered formalin)]. Each lung was examined independently by two investigators under a dissecting microscope to obtain the size of individual tumors (only >=0.5 mm were counted), and the surface tumor count was added to the harvested tumor number for a total tumor count. Tumor volume (V) was calculated by V = (4/3){pi}r3, where r is the radius of the lung tumor.

DNA isolation
DNA was extracted from frozen tumors by standard methods. After homogenization, lung tumors were incubated overnight at 37°C in lysis solution [pronase 0.4 mg/ml, 10% SDS (w/v), 10 mM Tris, 400 mM NaCl and 2 mM EDTA] followed by phenol–chloroform extraction and precipitation with ice-cold alcohol.

Polymerase chain reaction (PCR)
The region containing K-ras codons 12, 13 and 61 was amplified by PCR using primers that have been published previously (22). The reaction mixture for PCR contained ~100 ng genomic DNA, 10 mM Tris–HCl pH 8.5, 50 mM KCl, 2.5 mM MgCl2, 100 µM of each deoxyribonucleoside triphosphate (dATP, dCTP, dGTP, dTTP), 1.0 U of Taq DNA polymerase (Promega, Madison, WI) and 40 pmol of each primer. This mixture was overlaid with one drop of sterile mineral oil and subjected to 35 cycles of PCR amplification. Each cycle consisted of 1 min each at 94, 60 and 72°C.

Single-strand conformation polymorphism (SSCP) analysis
SSCP used to screen for K-ras activating mutations in exons 1 and 2, and 3 was performed essentially as described (24). After PCR, the amplified DNAs were separated by agarose gel electrophoresis (1.2%), and purified using Qiagen affinity beads. T4 polynucleotide kinase (Pharmacia, Piscataway, NJ) was used to label ~5 ng of purified PCR product with [{gamma}-32P]ATP in buffer containing 10 mM Tris–acetate and 50 mM potassium acetate, at 37°C for 45 min, followed by 65°C for 10 min. The reaction was stopped by 10 µl of stop buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol FF). The mixture was heated to 95°C for 5 min, chilled on ice for 1 min, followed by electrophoresis on an 18% non-denaturing polyacrylamide gel (4°C, 18–24 h, 30 W). The gels were exposed to X-ray film for 3–24 h at –80°C.

Sequence analysis
A representative number of shifted bands were confirmed for ras mutation by sequence analysis as described by Tindall and Stankowski (25). Briefly, primers were end-labeled as above, and then annealed to 20 ng of amplified PCR DNA by heat denaturation at 95°C for 5 min, followed by chilling on ice for 5 min. The sample was divided between four tubes, each containing 1.5 U of Sequenase (US Biochemical), 3 µl of 80 mM deoxyribonucleotide triphosphates and 8 mM dideoxyribonucleotide triphosphates. The reaction was terminated after 5 min at 37°C, by the addition of formamide dye mix. The samples were heated to 94°C for 5 min, followed by electrophoresis on an 8% polyacrylamide gel. The gel was dried and exposed to X-ray film for 16–24 h.

Statistical analysis
Tumor incidence, multiplicity and volume were evaluated for significance by Student's t-test, comparing the compound group with the control group.


    Results
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 Materials and methods
 Results
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In order to determine whether treatment with FTI-276 would result in the inhibition of lung tumor growth, we initiated lung tumorigenesis in A/J mice with a single injection of NNK (100 mg/kg body weight), and monitored for 18 weeks. The dependability of the pellet treatment was assessed in a pharmacokinetic study, in which mice treated with time-release pellets showed a cumulative average serum level of 1.68 µg/ml over a 30 day period (day 7 average 1.44 µg/ml; day 14 average 4.15 µg/ml; day 21 average 0.60 µg/ml; day 30 average 1.07 µg/ml) (S.M.Sebti, unpublished data). As shown in Table IGo, FTI-276 delivered by time-release pellet reduced the percent of mice with lung tumors by 42% (P < 0.01), the multiplicity by 60% (P < 0.01), and the volume by 58% (P < 0.05).


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Table I. Chemotherapeutic effect of FTI-276 in the A/J mouse lung tumor modela
 
As shown in Table IIGo, mutation analysis demonstrated that eight out of nine tumors from tumor-bearing animals treated with FTI-276, and all of the tumors (7/7) from the control group were positive for K-ras mutations in exon 1 of the K-ras gene. The frequency and type of mutations in the K-ras gene was similar in the group treated with FTI-276 to those in the control group.


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Table II. Analysis of K-ras mutations in lung tumors from FTI-276 treated A/J mice
 

    Discussion
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 Abstract
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 Materials and methods
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This study was designed to test the chemotherapeutic efficacy of the CAAX peptidomimetic, FTI-276. The present study shows that the CAAX peptidomimetic FTI-276 has a strong chemotherapeutic efficacy in a chemically induced model of lung cancer in mice when given via time-release pellet. This specific model results in lung tumors with two of the most common genetic alterations observed in human lung cancer, i.e. mutations in K-ras and decreased expression of p16INK4a (2,26,27). Treatment with FTI-276 began 18 weeks after initiation with the potent tobacco-related lung carcinogen, NNK. Powdered FTI-276 was embedded within a stable time-release matrix which was designed to deliver 1.25 mg/day (50 mg/kg body wt) of FTI-276 at a constant rate over 30 days. In a pellet-delivery study we found that over a 30 day period serum levels averaged ~1.7 µg/ml. Use of this method resulted in a >60% decrease in tumor multiplicity and an ~58% decrease in tumor volume. All of the target tumor tissues for treatment with the FTI-276 contained a K-ras mutation as demonstrated in the control group (7/7), suggesting that the observed chemotherapeutic efficacy of FTI-276 might be in part mediated through its inhibition of post-translational modifications of the K-Ras.

Most studies have predicted that FTIs would be cytostatic rather than cytotoxic. Previous studies with FTI-276 employing the human lung adenocarcinoma cell lines CaLu-1 and A-549, which bear a mutant K-ras oncogene, demonstrated inhibition of growth of xenografts but failed to demonstrate regression of larger palpable masses of CaLu-1 cells (20,21). In contrast, the recent work of Kohl et al. (28) demonstrated tumor regression in mammary and salivary tumors in a transgenic mouse with a mutant H-ras oncogene when employing another FTI. We presume that the greater efficacy observed in mice exposed to FTI-276 via time-release pellet was related to the fact that this methodology was able to sustain the serum level of the inhibitor. Our data suggest that the inhibitory effect of the FTI-276 on established lung adenomas may be mediated through blocking the activity of mutated K-Ras proteins.

FTI-276 has been shown to selectively inhibit farnesylation of p21 Ras proteins in vitro but does not affect the geranylgeranylation of most proteins at low dose (19). The latter is a potentially significant issue when considering treatment in humans since numerous proteins are geranylgeranylated. Most studies employing specific FTIs, however, have been conducted on H-Ras systems, even though mutations in K-Ras are primarily associated with human cancer e.g., lung, colon and pancreas (2). The work of James et al. (29) and Lerner et al. (19,31) demonstrated that it is more difficult to inhibit the farnesylation of K-Ras than H-Ras because the binding motif for K-Ras has a higher affinity for FTase than does the CAAX motif found in H-Ras. Thus, it has been shown that it takes at least a 10-fold higher concentration of FTI-276 to block farnesylation of K-Ras4B than it does to inhibit farnesylation of H-Ras (19).

In the present study, the frequency and type of mutations in the K-ras gene in groups treated with FTI-276 were similar as those in control groups indicating that FTI-276 could limit the growth rate of an established tumor that expresses an activated K-ras gene. However, the question of the specific role of inhibition of K-Ras farnesylation by FTIs in their therapeutic effect is still somewhat problematic. Lerner et al. (19,31) has proposed that unlike H-Ras, K-Ras is actually post-translationally geranylgeranylated when FTase is inhibited by FTIs. More recent studies imply that although K-Ras may be farnesylated under normal circumstances, in the presence of inhibitors of FTase, K-Ras4B may be geranylgeranylated (19,31,32). However, the possibility remains that geranylgeranylated K-Ras proteins, although able to translocate to the inner plasma membrane, may be spatially and/or functionally distinct from membrane-bound farnesylated K-Ras. Furthermore, in a study on human tumor cell lines, L-744,832, an FTI peptidomimetic, inhibited the growth of anchorage-dependent and -independent cell lines, even in cell lines with no known ras mutations (17). The latter observation implies that this farnesyltransferase inhibitor may be affecting important cellular proteins other than Ras. In their recent review, Cox and Der proffer the explanation that this disparity between ras mutation status and ability to be inhibited by FTI may be a function of whether a tumor is Ras-dependent rather than simply its ras mutation status (33). Since there are other proteins that may be associated with tumorigenesis which appear to require farnesylation, e.g. Rho B, further studies are required to determine the role of specific proteins in the observed inhibition. Since virtually all of the tumors in this system harbor mutations in the K-ras oncogene, the present studies do not help to delineate any differences in sensitivity of K-ras mutant versus non-K-ras mutant tumors, nor do we directly address the mechanism by which FTI-276 accomplishes its biological effects.

The present studies constitute the first demonstration of the efficacy of farnesyltransferase inhibitors in a primary tumor model, which arises in situ and has mutations in K-ras, a potentially important substrate for FTase in lung cancer. In addition, the reduction in total tumor volume with treatment is the first demonstration of inhibition of tumor development by an FTI in a primary lung tumor model.


    Acknowledgments
 
We would like to thank Drs Gary D.Stoner, Herman A.J.Schut and Christopher R.Herzog for critical reading of this manuscript. We would also like to thank Dr Peter Kova from Abbott Laboratories for FTI-276 serum level determination and Dr Mark Morse for analysis of NNK. We acknowledge Rusheng Yao, Lisa Twinings, David Hu, Feng Gao, Ping Gu and Michelle Truesdale for excellent technical assistance, and Lynda Titterington from The Ohio State University for her assistance in statistical analysis. This study was supported by National Cancer Institute (CN-55184 and CA58554).


    Notes
 
5 To whom correspondence should be addressed Email: myou{at}mco.edu Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 13, 1999; revised July 16, 1999; accepted August 27, 1999.