Education

Fordham University, B.S. (1977), Columbia University, M.A. (1978), M.Phil. (1980), Ph.D. (1983)

Postdoctoral Fellow, Laboratory of Toxicology, Massachusetts Institute of Technology, Cambridge, MA (1983-1986)

Senior Staff Fellow, Perinatal Carcinogenesis Section, FCRDC, National Cancer Institute, Frederick, MD (1986-1990)

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 environmental/genetic interactions that affect an organism's susceptibility to lung cancer formation. My research focuses on both the effects of environmental chemicals on the developing fetus following in utero exposures, as well as determining the molecular and genetic factors that influ­ence individual susceptibility to lung carcinogenesis.

One major focus 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.

Figure Legend – Imaging of lung tumors from mice exposed in utero to 3-methylcholanthrene.

Pregnant Balb/c mice were injected with 3-methylcholanthrene on the 17^th day of gestation.  The offspring were housed for 14 months.  Mice were anesthesized with isoflurane and images of their lungs collected by CT on a Light Speed 16 pro CT system.  Mice were then euthanized, lung tumors counted, and the tumor diameter determined.  Arrow in lung slide and + symbols in CTs point to tumors in the dissected lungs that were successfully imaged by CT.  Technical parameters were optimized for high spatial resolution by using a small scan field of view, detailed reconstruction algorithm, high resolution 2i channel configuration, small focal spot and maximizing technique (120 KV and 300 mA0 for enhanced signal to noise ratio. Rapid image acquisition of 400 msec was used to futher reduce motion unsharpness. These settings result in submillimeter spatial resolution. In plane resolution is 195 microns x 195 microns and each volume element (or voxel) measures 195 microns x 195 microns x 625 microns. To compensate for the anisotropic voxel size (i.e. the slice thickness of 625 microns is 3 times the in slice resolution), each mouse was repositioned and scanned in the axial and sagittal planes.

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-ras^G12C allele in a lung-specific fashion. This is achieved by placing the mutant human Ki-ras^G12C 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-ras^G12C 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, future studies will focus 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.

A second area of interest that is being pursued in my laboratory has been to extend our rodent work into human tumors. We are assessing the effect of drug metabolic enzymes on the relative levels of gene mutations in human breast cancer patients. Human breast tissue was assayed for specific genotypes of cytochrome P450s and glutathione S-transferases, and the specific forms of these enzymes were correlated with the presence of gene mutations in the p53 gene, a tumor suppressor gene found to be mutated in approximately 30% of all human breast cancer patients. The results of these experiments have demonstrated that individuals with specific genotypes are more likely to harbor mutations in the p53 gene, and demonstrate the gene-smoking and gene-gene interactions that may impact the prevalence of p53 mutations in breast cancer.  Thus, individuals with certain metabolic genotypes may be at greater risk for breast cancer following exposure to cigarette smoke or certain dietary carcinogens. These studies may aid in the development of effective prevention strategies for individuals based on genetic risk factors.

Figure Legend – Identification of mutations in p53 in  human breast cancer tissue.

DNA was isolated from paraffin-embedded tissue obtained from breast cancer patients and amplified by PCR.  The PCR products were analyzed by single strand conformation polymorphism (SSCP) analysis to identify band shifts between the DNA obtained from breast cancer tissue and a blood samples from the same patient.  Lanes in the gel exhibiting band shifts were cut out and sequenced.  The arrows point to band shifts leading to a misssense mutation in codon 248 (CGG →CAG, resulting in a ARG →GLN substitution) and a silent mutation in the 3^rd base of codon 241 (TCC →TCT).

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 used (in vivo rodent bioassays and human tissues), 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.

Recent Publications

Miller, M.S.: Tumor suppressor genes in rodent lung carcinogenesis - mutation of p53 does not appear to be an early lesion in lung tumor pathogenesis. Toxicol. Appl. Pharmacol. 156: 70-77, 1999.

Miller, M.S., Gressani, K.M, Leone-Kabler, S., Townsend, A.J., Malkinson, A.M., and O’Sullivan, M.G.: Differential sensitivity to lung tumorigenesis following transplacental exposure of mice to polycyclic hydrocarbons, heterocyclic amines, and lung tumor promoters. Exp. Lung Research 26: 709-730, 2000.

Witschi, H., Espiritu, I., Dance, S.T., and Miller, M.S.: A mouse lung tumor model of tobacco smoke carcinogenesis. Toxicol. Sci. 68: 322-330, 2002.

Xu, M., Floyd, H.S., Greth, S.M., Chang, W.-C.L., Lohman, K., Stoyanova, R., Kucera, G.L., Kute, T.E., Willingham, M.C., and Miller, M.S.: Perillyl alcohol-mediated inhibition of lung cancer cell line proliferation: potential mechanisms for its chemotherapeutic effects. Toxicol. Appl. Pharmacol. 195: 232-246, 2004.

Miller, M.S.: Transplacental lung carcinogenesis: molecular mechanisms and pathogenesis. Toxicol. Appl. Pharmacol. 198: 95-110, 2004.

Xu, M. and Miller, M.S.: Determination of murine fetal Cyp1a1and 1b1 expression by real-time fluorescence reverse transcription-polymerase chain reaction. Toxicol. Appl. Pharmacol. 201: 295-302, 2004.

Xu, M., Nelson, G.B., Moore, J.E., McCoy, T.P., Dai, J., Manderville, R.A., Ross, J.A., and Miller, M.S.: Induction of Cyp1a1 and Cyp1b1 and formation of DNA adducts in C57BL/6, Balb/c, and F1 mice following in utero exposure to 3-methylcholanthrene. Toxicol. Appl. Pharmacol. 209: 28-38, 2005.

Floyd, H.S., Farnsworth, C.L., Kock, N.D., Mizesko, M.C., Little, J.L., Dance, S.T., Everitt, J., Tichelaar, J., Whitsett, J.A., and Miller, M.S.: Conditional expression of the mutant Ki-ras^G12C allele results in formation of benign lung adenomas: development of a novel mouse lung tumor model. Carcinogenesis 26: 2196-2206, 2005.

Floyd, H.S., Jennings-Gee, J., Kock, N.D., and Miller, M.S.: Genetic and epigenetic alterations in lung tumors from bitransgenic Ki-ras^G12C expressing mice.  Molec. Carcinogenesis 45: 506-517, 2006.

Xu, M., Moore, J.E., Leone-Kabler, S., McCoy, T.P., Swank, A., Ross, J.A., Townsend, A.J., and Miller, M.S.: Expression of glutathione S-transferases in fetal lung and liver tissue from C57BL/6, Balb/c, and F1 mice following in utero exposure to 3-methylcholanthrene. Biochem. Pharmacol. 72: 115-123, 2006.

Jennings-Gee, J.E., Moore, J.E., Xu, M., Dance, S.T., Kock, N.D., McCoy, T.P., Carr, J.J., and Miller, M.S.: Strain specific induction of murine lung tumors following in utero exposure to 3-methylcholan­threne. Molec. Carcinogenesis 9: 676-684, 2006.

Petty, W.J., Knight, S.N., Mosley, L., Lovato, J., Capellari, J., Tucker, R., Blackstock, W., Miller, M.S., and Miller, A.A.: A pharmacogeno­mic study of docetaxel and gemcitabine for the initial treatment of advanced non-small cell lung cancer. J. Thoracic Oncol. 2: 197-202, 2007.

Van Emburgh, B.O., Hu, J.J., Levine, E.A., Mosley, L.J., Case, D.L., Lin, H.-Y., Knight, S.N., Perrier, N.D., Rubin, P., Sherrill, G.B., Shaw, C.S., Carey, L.A., Sawyer, L.R., Allen, G.O., Willingham, M.C., and Miller, M.S.: Polymorphisms in drug metabolism genes, smoking, and p53 mutations in breast cancer. Molec. Carcinogenesis 47: 89-99, 2008.

Van Emburgh, B.O., Allen, G.O., Hu, J.J., Levine, E.A., Mosley, L.J., Perrier, N.D., Freimanis, R.I., Lin, H.-Y., Rubin, P., Sherrill, G.B., Shaw, C.S., Carey, L.A., Sawyer, L.R., and Miller, M.S.: Polymor­phisms in CYP1B1, GSTM1, GSTT1, and GSTP1 and susceptibility to breast cancer.  Oncology Reports 19: 1311-1321, 2008.

Contact Information

Phone: 336-716-0795 E-mail: msmiller@wfubmc.edu

Updated 6/10/2008