A Cyp2a polymorphism predicts susceptibility to NNK-induced lung tumorigenesis in mice.

Lung tumors from smokers as well as lung tumors from mice exposed to tobacco carcinogens such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), often carry mutations in K-ras, which activates downstream-signaling pathways such as PI3K/AKT/mTOR pathway. Mice with genetic deletion of one of three isoforms of AKT were used to investigate the role of AKT in mutant K-ras-induced lung tumorigenesis in mice. Although deletion of Akt1 or Akt2 decreased NNK-induced lung tumor formation by 90%, deletion of Akt2 failed to decrease lung tumorigenesis in two other mouse models driven by mutant K-ras. Genetic mapping showed that Akt2 was tightly linked to the cytochrome P450 Cyp2a locus on chromosome 7. Consequently, targeted deletion of Akt2 created linkage to a strain-specific Cyp2a5 polymorphism that decreased activation of NNK in vitro. Mice with this Cyp2a5 polymorphism had decreased NNK-induced DNA adduct formation in vivo and decreased NNK-induced lung tumorigenesis. These studies support human epidemiological studies linking CYP2A polymorphisms with lung cancer risk in humans and highlight the need to confirm phenotypes of genetically engineered mice in multiple mouse strains.

Nicotine does not enhance tumorigenesis in mutant K-ras-driven mouse models of lung cancer.

Smoking is the leading cause of preventable cancer deaths in the United States. Nicotine replacement therapies (NRT) have been developed to aid in smoking cessation, which decreases lung cancer incidence. However, the safety of NRT is controversial because numerous preclinical studies have shown that nicotine enhances tumor cell growth in vitro and in vivo. We modeled NRT in mice to determine the effects of physiologic levels of nicotine on lung tumor formation, tumor growth, or metastasis. Nicotine administered in drinking water did not enhance lung tumorigenesis after treatment with the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Tumors that develop in this model have mutations in K-ras, which is commonly observed in smoking-related, human lung adenocarcinomas. In a transgenic model of mutant K-ras-driven lung cancer, nicotine did not increase tumor number or size and did not affect overall survival. Likewise, in a syngeneic model using lung cancer cell lines derived from NNK-treated mice, oral nicotine did not enhance tumor growth or metastasis. These data show that nicotine does not enhance lung tumorigenesis when given to achieve levels comparable with those of NRT, suggesting that nicotine has a dose threshold, below which it has no appreciable effect. These studies are consistent with epidemiologic data showing that NRT does not enhance lung cancer risk in former smokers.

Metformin prevents tobacco carcinogen--induced lung tumorigenesis.

Activation of the mammalian target of rapamycin (mTOR) pathway is an important and early event in tobacco carcinogen-induced lung tumorigenesis, and therapies that target mTOR could be effective in the prevention or treatment of lung cancer. The biguanide metformin, which is widely prescribed for the treatment of type II diabetes, might be a good candidate for lung cancer chemoprevention because it activates AMP-activated protein kinase (AMPK), which can inhibit the mTOR pathway. To test this, A/J mice were treated with oral metformin after exposure to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Metformin reduced lung tumor burden by up to 53% at steady-state plasma concentrations that are achievable in humans. mTOR was inhibited in lung tumors but only modestly. To test whether intraperitoneal administration of metformin might improve mTOR inhibition, we injected mice and assessed biomarkers in liver and lung tissues. Plasma levels of metformin were significantly higher after injection than oral administration. In liver tissue, metformin activated AMPK and inhibited mTOR. In lung tissue, metformin did not activate AMPK but inhibited phosphorylation of insulin-like growth factor-I receptor/insulin receptor (IGF-1R/IR), Akt, extracellular signal-regulated kinase (ERK), and mTOR. This suggested that metformin indirectly inhibited mTOR in lung tissue by decreasing activation of insulin-like growth factor-I receptor/insulin receptor and Akt upstream of mTOR. Based on these data, we repeated the NNK-induced lung tumorigenesis study using intraperitoneal administration of metformin. Metformin decreased tumor burden by 72%, which correlated with decreased cellular proliferation and marked inhibition of mTOR in tumors. These studies show that metformin prevents tobacco carcinogen-induced lung tumorigenesis and support clinical testing of metformin as a chemopreventive agent.