Life-span inhalation exposure to mainstream cigarette smoke induces lung cancer in B6C3F1 mice through genetic and epigenetic pathways (original) (raw)
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Oncogene, 2005
A/J mice with genetic alterations in K-ras, p53, or Ink4a/ Arf were employed to investigate whether mice carrying these germline mutations would be susceptible to tobacco smoke-induced lung tumorigenesis. Transgenic mice of both genders and their wild-type littermates were exposed to environmental cigarette smoke for 6 months, followed by recovery in air for 5 months. A significant increase of lung tumor multiplicity was observed in K-ras, p53, or Ink4a/Arf mutant mice when compared with wild-type mice. Furthermore, an additive effect was observed between the mice with a mutant p53 transgene and an Ink4A/Arf deletion during tobacco smoke-induced lung tumorigenesis. Sequence analysis of the K-ras gene indicated that the mutations had occurred at either codon 12/13 or 61 in both spontaneously occurring (air control) and tobacco smoke-induced lung tumors. K-ras mutations were found in 62% of the tumors from air-control animals and 83% in those exposed to tobacco smoke. The mutation spectrum found in tumors from mice exposed to tobacco smoke is somewhat similar to that in tumors from air-control mice. In addition, we identified three novel mutations at codon 12: GGT (Gly)-TTT (Phe), ATT (Ile), and CTT (Leu). These findings provide evidence that K-ras, p53, and Ink4a/Arf mutations play a role in tobacco smoke-related lung carcinogenesis. The similarity of the mutation spectra in the K-ras oncogene observed in tobacco smoke-induced tumors, as compared to spontaneous tumors, suggests that tobacco smoke enhances lung tumorigenesis primarily through promoting spontaneously occurring K-ras mutations.
Carcinogenesis, 2003
The retinoic acid receptor beta (RAR-b) gene encodes one of the primary receptors for retinoic acid, an important signaling molecule in lung growth, differentiation and carcinogenesis. RAR-b has been shown to be down-regulated by methylation in human lung cancer. We have used previously lung tumors induced in mice to evaluate the timing and effect of specific carcinogen exposures on targeting genes altered in human lung cancer. These studies were extended to characterize the role of methylation of the RAR-b gene in murine lung cancers. After treatment with the demethylating agent 5-aza-2 H -deoxycytidine (DAC), RAR-b was re-expressed in silenced cell lines or expressed at a higher rate than without DAC, supporting methylation as the inactivating mechanism. Bisulfite sequencing detected dense methylation in the area of the CpG island that contained the 5 H untranslated region and the first translated exon in non-expressing cell lines, compared with minimal and heterogeneous methylation in normal mouse lung. Methylation-specific PCR revealed that this gene is targeted differentially by carcinogen exposures with the detection of methylated alleles in virtually all primary tumors associated with cigarette smoke or 4-methylnitrosamino-1-(3-pyridyl)-butanone (NNK) in contrast to half of tumors induced by methylene chloride or vinyl carbamate. RAR-b methylation was also detected in 54% of preneoplastic hyperplasias induced by treatment with NNK. Bisulfite sequencing of both premalignant and malignant lesions detected dense methylation in the same area observed in cell lines, substantiating that this gene is functionally inactivated at the earliest histologic stage of adenocarcinoma development. These studies demonstrate that aberrant methylation of RAR-b is an early and common alteration in murine lung tumors induced by several environmentally relevant exposures.
Predominant K-ras Codon 12 G → A Transition in Chemically Induced Lung Neoplasms in B6C3F1 Mice
Toxicologic Pathology, 2004
Based on long-term toxicity and carcinogencity studies in B6C3F1 mice conducted by the National Toxicology Program, 2,2-Bis(bromomethyl)-1,3-propanediol (BMP) and tetranitromethane (TNM) have been identified as carcinogens. Following 2 yr of exposure to 312, 625, or 1,250 ppm BMP in feed, or exposure to 0.5 or 2 ppm TNM by inhalation, increased incidences of lung neoplasms were observed in B6C3F1 mice at all exposure concentrations compared to unexposed mice. The present study characterizes genetic alterations in the K-ras protooncogene in BMP-and TNMinduced lung neoplasms, respectively, and compares the findings to spontaneous lung neoplasms from corresponding control mice. The frequencies of the K-ras mutations were 57% (29/51) in BMP-induced lung neoplasms compared to 15% (3/20) in lung neoplasms from dosed feed control mice, and 54% (14/26) in TNM-induced lung neoplasms compared to 60% (3/5) in lung neoplasms from inhalation control mice. G → A transitions at the second base of the K-ras codon 12 (GGT → GAT) were the most frequent pattern of K-ras mutations identified in BMP-induced (20/29) and TNM-induced lung neoplasms (13/14), which differed from the mutational patterns identified in the lung neoplasms from unexposed control mice. These results indicate that mutations in the K-ras gene are involved in B6C3F1 lung carcinogenesis following BMP-and TNM-exposure, and the high frequency and specificity of the ras mutation profile in lung neoplasms (G → A transition) may be due to in vivo genotoxicity by the parent compounds or their metabolites.
Ras Effector Mutant Expression Suggest a Negative Regulator Inhibits Lung Tumor Formation
PLoS ONE, 2014
Lung cancer is currently the most deadly malignancy in industrialized countries and accounts for 18% of all cancer-related deaths worldwide. Over 70% of patients with non-small cell lung cancer (NSCLC) are diagnosed at a late stage, with a 5-year survival below 10%. KRAS and the EGFR are frequently mutated in NSCLC and while targeted therapies for patients with EGFR mutations exist, oncogenic KRAS is thus far not druggable. KRAS activates multiple signalling pathways, including the PI3K/Akt pathway, the Raf-Mek-Erk pathway and the RalGDS/Ral pathway. Lung-specific expression of BrafV600E, the most prevalent BRAF mutation found in human tumors, results in Raf-Mek-Erk pathway activation and in the formation of benign adenomas that undergo widespread senescence in a Cre-activated Braf mouse model (Braf CA). However, oncogenic KRAS expression in mice induces adenocarcinomas, suggesting additional KRAS-activated pathways cooperate with sustained RAF-MEK-ERK signalling to bypass the oncogene-induced senescence proliferation arrest. To determine which KRAS effectors were responsible for tumor progression, we created four effector domain mutants (S35, G37, E38 and C40) in G12V-activated KRAS and expressed these alone or with BrafV600E in mouse lungs… The S35 and E38 mutants bind to Raf proteins but not PI3K or RalGDS; the G37 mutant binds to RalGDS and not Raf or PI3K and the C40 mutant is specific to PI3K. We designed lentiviral vectors to code for Cre recombinase along with KRAS mutants (V12, V12/S35, V12/G37, V12/E38 or V12/C40) or EGFP as a negative control.. These lentiviruses were used to infect Braf CA and wild-type mice. Surprisingly there was a significant decrease in tumor number and penetrance with each KRAS effector domain mutant relative to controls, suggesting that KRAS directly activates effectors with tumor suppressive functions.
Requirement for Rac1 in a K-ras Induced Lung Cancer in the Mouse
Cancer Research, 2007
Given the prevalence of Ras mutations in human cancer, it is critical to understand the effector pathways downstream of oncogenic Ras leading to transformation. To directly assess the requirement for Rac1 in K-ras-induced tumorigenesis, we employed a model of lung cancer in which an oncogenic allele of K-ras could be activated by Cre-mediated recombination in the presence or absence of conditional deletion of Rac1. We show that Rac1 function is required for tumorigenesis in this model. Furthermore, although Rac1 deletion alone was compatible with cell viability and proliferation, when combined with K-ras activation in primary epithelial cells, loss of Rac1 caused a profound reduction in proliferation. These data show a specific requirement for Rac1 function in cells expressing oncogenic K-ras. [Cancer Res 2007;67(17):8089-94]
American Journal of Respiratory Cell and Molecular Biology, 2009
Lung cancer is the leading cause of cancer deaths in the United States. In addition to genetic abnormalities induced by cigarette smoke, several epidemiologic studies have found that smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lungs, have an increased risk of lung cancer (1.3-to 4.9fold) compared to smokers without COPD. This suggests a link between chronic airway inflammation and lung carcinogenesis, independent of tobacco smoke exposure. We studied this association by assaying the inflammatory impact of products of nontypeable Haemophilus influenzae, which colonizes the airways of patients with COPD, on lung cancer promotion in mice with an activated Kras mutation in their airway epithelium. Two new mouse models of lung cancer were generated by crossing mice harboring the LSL-Kras G12D allele with mice containing Cre recombinase inserted into the Clara cell secretory protein (CCSP) locus, with or without the neomycin cassette excised (CCSP Cre and CCSP Cre-Neo , respectively). Lung lesions in CCSP Cre-Neo /LSL-K-ras G12D and CCSP Cre /LSL-Kras G12D mice appeared at 4 and 1 month of age, respectively, and were classified as epithelial hyperplasia of the bronchioles, adenoma, and adenocarcinoma. Weekly exposure of CCSP Cre /LSL-Kras G12D mice to aerosolized nontypeable Haemophilus influenzae lysate from age 6-14 weeks resulted in neutrophil/macrophage/ CD8 T-cell-associated COPD-like airway inflammation, a 3.2-fold increase in lung surface tumor number (156 6 9 versus 45 6 7), and an increase in total lung tumor burden. We conclude that COPD-like airway inflammation promotes lung carcinogenesis in a background of a G12D-activated K-ras allele in airway secretory cells.
Mouse models of K-ras-initiated carcinogenesis
Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2005
Activating mutations of the oncogene K-ras are found in one third of all human cancers. Much of our knowledge on K-ras signal transduction and its influence on tumor initiation and progression comes from in vitro studies with cell lines. However, mouse models of human cancer allow a much more faithful recapitulation of the human disease, and the in vivo perspective is crucial for our understanding of neoplasia. In recent years, several new murine models for K-ras-induced tumorigenesis have been described. They allow new insights into the specific role that oncogenic K-ras proteins play in different solid tumors, and they permit the molecular dissection of the pathways that are initiated by somatic mutations in subsets of cells. Key advances have been made by the use of tissue-specific and inducible control of expression, which is achieved by the Cre/LoxP technology or the tetracycline system. from these sophisticated models, a common picture emerges: The effects of K-ras on tumor initiation depend strongly on the cellular context, and different tissues vary in their susceptibility to Kras transformation. D
Toxicologic Pathology, 2007
Ethylene oxide is a multisite carcinogen in rodents and classified as a human carcinogen by the National Toxicology Program. In 2-year mouse studies, ethylene oxide (EO) induced lung, Harderian gland (HG), and uterine neoplasms. We evaluated representative EO-induced and equivalent spontaneous neoplasms for K-ras mutations in codons 12, 13, and 61. K-ras mutations were identified in 100% (23/23) of the EO-induced lung neoplasms and 25% (27/108) of the spontaneous lung neoplasms. Codon 12 G to T transversions were common in EO-induced lung neoplasms (21/23) but infrequent in spontaneous lung neoplasms (1/108). K-ras mutations were found in 86% (18/21) of the EO-induced HG neoplasms and 7% (2/27) of the spontaneous HG neoplasms. Codon 13 G to C and codon 12 G to T transversions were predominant in the EO-induced HG neoplasms but absent in spontaneous HG neoplasms (0/27). K-ras mutations occurred in 83% (5/6) of the EO-induced uterine carcinomas and all were codon 13 C to T transitions. These data show a strong predilection for development of K-ras mutations in EO-induced lung, Harderian gland, and uterine neoplasms. This suggests that EO specifically targets the K-ras gene in multiple tissue types and that this event is a critical component of EO-induced tumorigenesis.
BMC Cancer, 2007
Epidemiological studies indicate that some characteristics of lung cancer among never-smokers significantly differ from those of smokers. Aberrant promoter methylation and mutations in some oncogenes and tumor suppressor genes are frequent in lung tumors from smokers but rare in those from never-smokers. In this study, we analyzed promoter methylation in the ras-association domain isoform A (RASSF1A) and the death-associated protein kinase (DAPK) genes in lung tumors from patients with primarily non-small cell lung cancer (NSCLC) from the Western Pennsylvania region. We compare the results with the smoking status of the patients and the mutation status of the K-ras, p53, and EGFR genes determined previously on these same lung tumors.
Molecular and Biological Basis of Lung Cancer-Part I
2020
Lung cancer (LC) consists the 5th leading cause of death worldwide, and an important cause of morbidity and mortality, nowadays, as the 5-year survival is extremely poor. LC molecular biology may lead to customized treatment based on targeting specific genes and signaling path-ways. The main signaling pathways in LC development concern growth promoting pathways such as EGFR/Ras/PI3K, growth inhibitory pathways such as p53/Rb/P14ARF, STK11, apoptotic pathways such as Bcl-2/Bax/Fas/FasL, and DNA repair and immortalization genes. Epigenetic alterations in LC are also responsible for cell transformation by modifying chromatin structures and the specific expression of genes and are involved in tumor suppressor genes silencing whereas enhancing oncogenes expression. In the present review, is presented the current state of knowledge regarding the cascade of events that are associated with LC development giving emphasis to oncogenes, tumor suppressor-genes and signaling pathways that are im...