Mark Miller Lab
Molecular Pathogenesis of Lung Cancer, Transplacental Carcinogenesis
Lung cancer is the leading cause of cancer-related deaths in the U.S. Despite decades of research and the recent development of novel therapeutic approaches, the 5 year survival rate of lung cancer patients has remained an abysmal 10-15%. Thus, a better understanding of the molecular events that drive lung tumor progression would aid in the development of novel chemopreventive strategies and chemotherapeutic agents aimed at reducing the mortality and morbidity resulting from this deadly disease. Research in my laboratory is examining lung cancer from 3 different directions: 1) determining environmental/genetic interactions that affect an organism's susceptibility to lung cancer formation, particularly as it relates to the effects of environmental chemicals on the developing fetus; 2) the development of novel chemopreventive agents to prevent lung cancer formation in high risk individuals; and 3) use of imaging techniques to assess early lesion formation before the tumors become fully malignant.
The first major focus area of my laboratory has been to determine the effects of in utero exposures to environmental toxicants on lung cancer incidence. As fetal tissues are more sensitive to the effects of chemical and physical carcinogens than are adult tissues, the embryos and fetuses of pregnant women are at a greater risk of developing cancers from environmental exposures than is the adult population. My laboratory uses a variety of techniques to study this problem, starting with in vivo bioassays in rodents to assess the carcinogenicity of environmental chemicals found in the diet and as byproducts of fuel combustion and cigarette smoking. The tumors generated in these bioassays are then assayed at the histological, biochemical, and molecular levels. Studies from my laboratory have demonstrated the unique sensitivity of the fetus to environmental chemicals. As a result of the dynamic nature of fetal growth, the fetus displays a constantly changing picture of metabolism in response to environmental exposures as various gene systems are turned on and off during development. Thus, because of the differential expression of drug metabolic enzymes in the fetus relative to the adult, the fetus is unable to detoxify chemical carcinogens and is rendered more sensitive to the carcinogenic effects of these agents. In addition, our more recent studies have shown that different strains of fetal mice exhibit different sensitivities to chemical carcinogens. Thus, the interaction of genetic background and environmental exposure influences the relative sensitivity to carcinogen exposure. Identification of these genetic factors will aid in identifying individuals who may be particularly sensitive to exposure to toxic chemicals, especially during the vulnerable fetal period of growth.
Studies from my laboratory have shown that the type of mutations induced in the Ki-ras gene are associated with the histological stage of the lung lesions, suggesting that different Ki-ras mutations may have different oncogenic potential. This led to the development of a novel, “humanized” bitransgenic mouse model that conditionally expresses the mutant human Ki-rasG12C allele in a lung-specific fashion. This is achieved by placing the mutant human Ki-rasG12C allele downstream of a tetracycline-inducible promoter as shown in the figure below.
Figure Legend – Bitransgenic mouse model. The Tet-On system uses a reverse tet trans-activator (rtTA) protein that requires the presence of the ligand, doxycycline (DOX), in order for the rtTA gene product (consisting of the mutant tet repressor linked to the VP16 activation domain) to recognize the tetO sequence and thus stimulate gene transcription. In this approach, the cDNA of Ki-rasG12C is cloned into the tetO7-CMV plasmid, placing the transgene downstream of a tet-inducible promoter. Founder mice established with this construct are unable to express the Ki-ras transgene because they lack the rtTA protein. These mice are then crossed with a second trangenic mouse line that contains the rtTA protein linked to either the surfactant protein C (SP-C) or Clara cell secretory protein (CCSP) promoters, directing lung-specific expression of the rtTA protein. In the absence of DOX, the rtTA gene product is unable to recognize the tetO sequence and is thus unable to stimulate transcription. Treatment of the bitransgenic mice with DOX allows binding of the rtTA protein to the tetO enhancer, resulting in activation of the CMV promoter and transcription of the Ki-ras gene specifically in the lung.
These mice develop relatively benign tumor lesions that remain extremely small (#1 mm) and do not progress beyond the early adenoma stage. Thus, these mice represent the early stages of tumor development that would be seen in smokers and ex-smokers, in which the lung tissue contains a field of mutated cancer cells that have the potential to progress to malignant tumors in the presence of further toxic insults. This model thus offers a unique opportunity to determine the role of inflammation in lung tumor progression and assess the efficacy of chemopreventive agents during the development of lung cancer. To mimic the promotional phase of tumorigenesis, mice are treated with pro-inflammatory agents such as butylated hydroxytoluene to drive tumor progression and allow the development of higher grade lesions. We are utilizing this model to both examine the gene pathways that drive tumorigenesis, as well as to examine the efficacy of potential chemopreventive agents and determine their mechanisms of action. As we have identified genes that appear to play a role in lung cancer formation, current studies are focusing both on the development of chemopreventive strategies to inhibit lung tumorigenesis by targeting the early pathways that mediate tumor progression as well as the testing of novel, mechanism-based anti-neoplastic agents that will specifically target the genetic lesions that are found to be altered in the tumors.
Figure Legend – CCSP/Ki-ras lung morphology following DOX treatment. H&E stained lung tissue following A) no DOX treatment, 10X; B) 12 days of DOX treatment, 20X, focal hyperplasia; C) 3 mo of DOX treatment, 10X, focal hyperplasia; D) 3 mo of DOX treatment, 40X, focal hyperplasia; E) 6 mo of DOX treatment, 10X, focal hyperplasia with regular cuboidal cells lining alveolar septa; F) 6 mo of DOX treatment, 10X, solid adenoma; G) 9 mo of DOX treatment, 4X, pneumocyte hyperplasia; H) 9 mo of DOX treatment, 40X, focal hyperplasia; I) 9 mo of DOX treatment, 4X, solid adenoma; J) 9 mo of DOX treatment, 40X, solid adenoma with regular closely aligned pneumocytes arranged in a ribbon pattern; K) 9 mo of DOX treatment , 40X, solid adenocarcinoma exhibiting a solid sheet of atypical epithelial cells with pale pleomorphic nuclei, some with prominent nucleoli, and indistinct cytoplasmic borders; and L) 9 mo of DOX treatment followed by 1 month of withdrawal, 10X.
The final area of research focus has been to examine the effects of routine CT screening on lung tumor incidence in the high risk population of smokers and ex-smokers. The ability to detect early stage lesions in otherwise asymptomatic patients would increase the likelihood of survival. Annual low-dose CT has been studied to determine its value for screening asymptomatic individuals that are at high risk for lung cancer. However, the efficacy and long term effects of this annual CT lung screening are controversial. Presently, there are unknown risks associated with the CT screening for lung tumors, particularly in individuals with a significant smoking history, because the low energy ionizing radiation employed has the potential to initiate, promote and/or accelerate the growth of lung tumors. Our bitransgenic mouse model recapitulates the earliest stages of lung tumor formation and would be representative of asymptomatic light ex-smokers who harbor genetic damage in their lung epithelial cells and contain small, undetectable, and relatively benign early stage lesions. We are thus looking at the effect of clinically relevant CT screening procedures on radiation-induced carcinogenesis, and tracking tumor development in real time through the use of MRI imaging.
Figure Legend – Imaging of lung tumors from mice bitransgenic, DOX-treated mice. A DOX-treated mouse from our ongoing CT studies was analyzed by MRI 6 and 8 months after the initiation of DOX (to increase mutant Ki-ras gene expression). The attached images were acquired using a respiratory and ECG gated Fast Low Angle Shot (FLASH) gradient echo pulse sequence, with a repetition time (TR) = 50ms, an echo time (TE) = 3ms, a flip angle = 25 degrees, 8 averages, a field of view FOV = 3 cm, a matrix size of 256x256 (giving an in plane resolution of 137 um), and a slice thickness of 0.6 mm. The tumors' dimensions were measured on a Linux workstation running Bruker's ParaVision4.0. The imaging results demonstrate the ability to detect sub-millimeter sized tumors and monitor tumor growth over the course of the studies.
Students rotating through my laboratory will be taught the basic techniques of molecular biology and, depending on the length of the rotation and the student's interest, some of the more advanced methodologies for gene mutation analysis. Because of the diversity of models, the student will be exposed to a wide range of toxicological techniques, from treatment of animals in vivo to biochemical and histochemical analyses followed by molecular approaches to determine the mechanism(s) of tumor development.