(Chest. 2004;125:144S-147S.)
© 2004
American College of Chest Physicians
Chemoprevention of Lung Cancer in Transgenic Mice*
Ronald A. Lubet, PhD;
Zhongqiu Zhang, MD, PhD;
Yian Wang, MD, PhD and
Ming You, MD, PhD
* From Chemoprevention Branch (Dr. Lubet), National Cancer Institute, Bethesda, MD; and Department of Surgery and The Alvin J. Siteman Cancer Center (Drs. Zhang, Wang, and You), Campus Box 8109, Washington University School of Medicine, St. Louis, MO.
Correspondence to: Ronald A. Lubet, PhD, Chemoprevention Branch, National Cancer Institute, Bethesda, MD 20892; e-mail: lubetr{at}mail.nih.gov
Key Words: animal models • chemopreventive agents • chemotherapeutic agents • lung cancer • organ tropic carcinogens • transgenic mice • tumor suppressor genes
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Introduction
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Lung cancer is the leading cause of cancer deaths in men and women in the United States.1 Epidemiologic and laboratory animal model studies2345 have demonstrated that smoking and environmental exposure to carcinogens are closely linked to increased lung cancer risk. Tobacco exposure has been implicated in 90% of lung carcinomas, and smokers have a 20-fold greater risk of acquiring lung cancer compared with persons who have never smoked.6 Although approximately one half of all people who had ever smoked are now former smokers, many people are unable or unwilling to stop smoking. For these reasons, chemoprevention is a potentially important approach to reduce the large number of tobacco-caused cancer deaths, especially for former smokers. Chemoprevention is the use of pharmacologic or natural agents to inhibit the development of cancer. A primary mode of chemoprevention action includes reversing the progression of premalignant cells by stimulation of the cell to repair DNA or other cell damage that initiates carcinogenesis. Numerous studies have found chemoprevention methods can prevent or improve the outcome of a wide variety of cancer.7 This approach is especially useful in targeting persons at high risk for cancer, such as patients who have a genetic predisposition to cancer, or patients who are at high risk for secondary primary tumors after surgical removal of a tumor.7 The targets for pharmacologic intervention are the various stages of tumor development, including hyperplasia and dysplasia. There are two major classes of cancer chemopreventive agents: blocking agents and suppressing agents.8910 Blocking agents prevent metabolic activation of carcinogens to reduce the likelihood of DNA damage. Suppressing agents can block expansion of carcinogen-initiated cells by suppressing cell replication or by causing apoptosis of precancerous or cancerous cells.
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Use of A/J Mice in Chemoprevention Studies
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The A/J mouse lung model of chemical carcinogenesis has been the most frequently employed murine model both for testing for potential chemical carcinogens and to screen for agents that prevent carcinogenesis (chemopreventive agents). The model has been shown to respond to a wide variety of potential chemical carcinogens, including the tobacco-related carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo(a)pyrene, yielding multiple peripheral adenomas.11 Among the agents tested in A/J mice, several groups of chemicals have shown significant efficacy against mouse lung tumor development. For example, glucocorticoids, green tea, nonsteroidal anti-inflammatory drugs, isothiocyanates, and farnesyltransferase inhibitors (FTIs) are among the most effective compounds.12 Table 1
summarizes the efficacy of glucocorticoids and FTIs that have been tested in A/J mice and transgenic mice. Glucocorticoids were found to be a strong inhibitor of carcinogenesis in skin, forestomach, and lung in rodents.13141516 Wattenberg and Estensen16 reported that dexamethasone, a synthetic glucocorticoid, inhibits lung tumorigenesis by 56%, and by 86% when dexamethasone was administered with myo-inositol.16 In order to minimize the systemic toxic effects, glucocorticoids such as budesonide can be successfully delivered by aerosol, and by this method inhibit lung tumor development by > 90%.1718 Recently, we have found that FTI is chemopreventive on both the established lung adenomas model and the complete carcinogenesis model from A/J mice induced by NNK.1920 FTI-276 was used, which is a CAAX peptidomimetic of the carboxyl terminal of Ras proteins. FTI-276 inhibited lung tumor size by 58% in an established mouse lung tumor model,19 and by 79% in a complete chemoprevention model.20 These results demonstrated that FTI-276 is chemopreventive in both the established lung adenomas model and the complete carcinogenesis model. These studies support the importance of using animal models in testing and characterization of cancer chemopreventive agents. More recently, a specific farnesyltransferase inhibitor, R115777, was tested for its efficacy against lung tumor development in A/J mice using NNK, and was found to exhibit 58% inhibition of lung tumor multiplicity.21
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Transgenic Mouse Model for Lung Adenocarcinomas
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Although the A/J mouse lung adenoma model is valuable because of similar histology to a subtype of human adenocarcinoma,25 there are some features yet to be desired in order to make this model better. For example, the progression of lung adenomas to adenocarcinomas is rare in A/J mice.11 The most common genetic alteration associated with these tumors being carcinogen-specific mutations is K-ras.26 The limited number of large adenocarcinomas produced in this model has been shown to produce characteristic LOH changes.27 However, even most of these larger adenocarcinomas do not show mutations or loss of the p53 tumor suppressor gene or lost expression of the cyclin D1/cyclin D kinase inhibitor p16 gene, although these or closely related genes are routinely altered in human lung cancer.25 In order to incorporate these specific alterations into the A/J tumor model, we placed two different transgenes onto the A/J background.2024 We employed a transgene that has a mouse p53 gene, with a missense mutation, at position 135 (Arg to Val), under the control of its own endogenous p53 promoter.28 The resulting mice and tumors have three copies of the mutated p53 gene, as well as two copies of the wild-type p53 alleles.23 However, the mutated protein is considered to be dominant over the wild-type p53 protein. To alter p16, we employed a knockout of the INK 4A locus that includes the genes for both p16 as well as the gene for ARF.29 Finally mice with alterations of both p53 and INK 4A on the A/J background were produced.
In a recent study, p53 transgenic mice, Ink4a/Arf heterozygous deficiency and transgenic mice with both defects were employed to determine the role of p53 and Ink4a/Arf on benzo(a)pyrene-induced lung tumorigenesis and progression.2024 Six-week-old A/J, p53+/+Ink4a/Arf+/-, p53+/-Ink4a/Arf+/+, and p53+/-Ink4a/Arf+/- mice were randomized into eight groups according to the p53 and Ink4a/Arf genotypes and treatments. One group of mice was administered vehicle controls, and another group of mice was administered benzo(a)pyrene. Animals were killed 40 weeks after exposure to benzo(a)pyrene. The incidence of lung tumors in all four groups of treated mice was 100%. p53+/-Ink4a/Arf+/+, and p53+/-Ink4a/Arf+/- mice carrying a mutant p53 transgene (Val135) with or without Ink4a/Arf heterozygous deletion had a higher number of lung tumors (an average of 25 tumors per mouse) after treatment with benzo(a)pyrene than wild-type and p53+/+Ink4a/Arf+/- mice (an average of 12.0 tumors per mouse). More interestingly, mice with p53-dominant negative mutation and Ink4A/Arf heterozygous deficiency (p53+/-Ink4a/Arf+/-) exhibited a striking increase in tumor volume (approximately 23-fold) compared to a ninefold increase in tumor volume in mice with only the p53-dominant negative mutation (p53+/-Ink4a/Arf+/+). There was also an approximate 50% and an approximate fourfold increase in tumor volume in Ink4a/Arf heterozygous-deficient mice (p53+/+Ink4a/Arf+/-), indicating that the effect of Ink4A/Arf heterozygous deficiency is mostly on late-stage lung tumor progression. In addition, most of the lung tumors (approximately 60%) from mice with a p53 mutation and deletion of Ink4A/Arf (p53+/-Ink4a/Arf+/-) were lung adenocarcinomas. In contrast, lung adenocarcinomas were seen in < 10% of the lung tumors from the wild-type mice, and approximately 50% of the lung tumors from either p53 transgenic mice or Ink4a/Arf heterozygous-deficient mice. These results clearly indicate a significant synergistic interaction between the presence of a mutant p53 transgene and the Ink4A/Arf deletion during lung tumor progression.
In the studies of mice with the dominant negative p53 mutant transgene on an A/J background, either from control mice or mice treated with lung carcinogens, we have observed a low incidence of lymphomas (< 15%) or sarcomas (< 5%).2021 242627 This may reflect both the A/J background as well as the fact that mice were kept for 
10 months. The p16-deficient mice have routinely had rapidly growing sarcomas, in agreement with other studies. Furthermore, mice with both p53 and p16 alterations acquired sarcomas in a relatively high percentage of mice. This may pose some limitations for routine studies with these doubly transgenic mice. This problem might be overcome with a lung-specific knockout of p16.
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Use of Transgenic Mouse Model in Chemoprevention Studies
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We examined the effects of two potential agents on the development of adenomas/adenocarcinomas in these mice.2024 The two classes of agents examined were budesonide2224 and the FTI inhibitor L778,123.20 The former is a glucocorticoid that presumably interacts with the glucocorticoid receptor, and has been shown to be a profoundly effective agent in the A/J mouse model. L778,123 inhibits the farnesyltransferase enzyme that transfers farnesyl onto proteins and allows their transport to and activation in the cell membrane. FTI inhibitors were initially produced because they should block the farnesylation of the mutant Ras oncogenes (H-ras, N-ras, and K-ras). Although they effectively block H-ras and N-ras farnesylation and activation, they do not block farnesylation of K-ras due apparently to the high affinity of this protein for the farnesyltransferase enzyme. We examined the abilities of these agents to inhibit tumor formation either in a prevention setting in which budesonide24 was administered beginning prior to benzo(a)pyrene and throughout the study or in a delayed experiment, which we feel may be closer to that achieved in former or current smokers. As shown in Table 2
, budesonide or the FTI20 were administered beginning 12 to 20 weeks after benzo(a)pyrene when small adenoma exist, and were administered for a period of 10 to 18 weeks. Budesonide administration was initiated early, and continually resulted in striking decreases in both tumor multiplicity (65 to 77%) and tumor volume (77 to 98%) in all mice. When budesonide was administered for 18 weeks (weeks 12 to 30) a moderate decrease in tumor multiplicity (36 to 53%) was observed, while the effects on tumor volume were more variable. Tumor volume was decreased 64 to 82% in wild-type or INK 4A-deficient mice, while volume was decreased only 33 to 45% in mice with a p53 mutation or a double-mutant animal. Thus, much of the efficacy of delayed treatment with budesonide was lost if the tumors had a mutation in p53. When FTI was administered for 10 weeks (weeks 20 to 30), a moderate decrease in tumor multiplicity was observed (43 to 47%), while the effects on tumor volume were more variable. Tumor volume was decreased roughly 50% in wild-type or INK 4A-deficient mice, while volume was decreased 84% and 72% in mice with a p53 mutation or a double mutant animal, respectively. Interestingly, the efficacy of delayed administration of FTI were even more striking in tumors with a p53 mutation in contrast to delayed administration of budesonide.
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Table 2.. Effects of Budesonide or the FTI Inhibitor (L-778,123) on Lung Tumor Multiplicity and Volume in B(a)P-Treated A/J Mice*
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Conclusion
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We feel that these models may be more relevant in studying both preventive and therapeutic agents than the standard A/J model. The late intervention, as employed with both budesonide and FTI, appears to be closer to the proposed experiments in humans where individuals, either former or current smokers, with known dysplasia and multiple genetic changes are employed. Furthermore, the model should prove useful for testing chemotherapeutic agents in a model in which tumors contain certain known relevant genetic alterations and grow and progress in situ. This in situ development of tumors has certain advantages over xenograft models, in which transplantable tumors grow subcutaneously and appear to be profoundly susceptible to agents that affect angiogenesis.
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Footnotes
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Abbreviations: FTI = farnesyltransferase inhibitor; NNK = 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
This research was supported in part by National Institutes of Health grants and contract R01CA96103, R01CA58554, and N01CN25104.
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References
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Introduction
Use of A/J Mice...
