Inhalation Toxicology Laboratory; Lovelace Respiratory Research Institute
Lung cancer is the second most common cancer in men and women and is the leading cause of cancer death in both sexes. The major risk factor for lung cancer is cigarette smoking; however, exposure to radiation in the form of radon also increases the risk of lung cancer. Identifying the specific carcinogens within tobacco and the role of radiation in inducing the molecular mechanisms that underlie tumor development is critical for developing approaches to prevent and treat lung cancer. CpG island methylation, an epigenetic modification of DNA associated with silencing of gene transcription, is an alternative mechanism to mutation in inactivating tumor suppressor genes. For example, methylation of promoter CpG islands has been implicated as one mechanism for the inactivation of the p16INK4a (p16), estrogen receptor (ER), and O6-methylguanine methyltransferase (O6MGMT) genes. The purpose of the current studies was to define the role of these genes and the influence of known environmental exposures in the development of non-small cell lung cancer (NSCLC). Identifying epigenetic alterations during the development of human squamous cell carcinomas (SCCs) and adenocarcinomas induced in rodents was the focus of the study. Defined exposures indicative of the type of DNA damage induced by chemicals present within tobacco were used to induce the rodent tumors.
Studies in primary rat lung tumors induced by ionizing radiation and corresponding derived cell lines have revealed that CpG island methylation is the major mechanism for inactivating the p16 gene in these tumors and have substantiated the primary tumor as the origin for dysfunction of the p16 gene. These studies were extended to determine the timing for inactivation of p16 during the development of rat lung tumors induced by the tobacco-specific carcinogen NNK and in human SCCs. In the rat, 94% of adenocarcinomas induced by NNK were hypermethylated at the p16 gene promoter. Most importantly, this methylation change was frequently detected in precursor lesions to the tumors: adenomas, and hyperplastic lesions. The timing for p16 methylation was recapitulated in human SCCs where the p16 gene was coordinately methylated in 75% of carcinoma in situ lesions adjacent to SCCs harboring this change. Moreover, the frequency of this event increased during disease progression from basal cell hyperplasia to squamous metaplasia to carcinoma in situ lesions. These studies show for the first time that an epigenetic alteration, aberrant methylation of the p16 gene, can be an early event in lung cancer. Furthermore, this gene appears to be a target for inactivation by other types of environmental carcinogens (particulates) such as beryllium metal and diesel exhaust.
The high frequency for targeting p16 methylation irrespective of environmental exposure is contrasted by studies of ER methylation. Methylation of ER was examined in NSCLCs from smokers and never-smokers. Among the never-smokers, 37% of the tumors were positive for ER methylation, while only 20% of tumors were methylated among smokers. This potential heterogeneity for methylation of the ER gene in lung cancer was examined in tumors induced in rodents by several environmental exposures. In A/J mice, 83% of spontaneous tumors had significant methylation of the ER gene promoter region, while methylation was detected in only 22% of NNK-induced tumors in this mouse strain. Tumors induced by NNK in the rat had a similar low frequency of ER methylation. In contrast, 83% and 45% of tumors induced by plutonium oxide and X-rays, respectively, showed ER methylation. This is the first study to demonstrate that gene-specific promoter methylation can be modulated differentially depending on the carcinogen exposure.
The O6MGMT represents another class of genes that can be silenced in cancer through CpG island methylation. This gene is involved in the repair of methyl-adducts formed through the metabolism of nitrosamines such as NNK. Preliminary studies indicate that this gene is inactivated in approximately 25% of human SCCs associated with tobacco. Studies are in progress to determine the effect of radiation exposure (radon) on inactivating this gene in SCCs and its role in rodent lung tumor development. (Supported by DE-FC04-9AL76406 and NIH grant 5P50CA58184)