Inactivation of tumor suppressor genes is an important step in the development of all forms of human cancer. The elucidation of altered genes and microRNAs (miRs) and the pathways they regulate that interact in early premalignancy of adenocarcinoma are largely unknown. Malignant tumors and cell lines are characterized by increased expression of cytosine DNA methyltransferase (DNMT) 1, DNMT3a and DNMT3b. While it is known that DNMTs cooperate in de novo methylation and gene silencing, very little is known about targets for DNMT3a and DNMT3b or their impact on the development of adenocarcinoma. Our group has developed an in vitro transformation model using immortalized bronchial epithelial cell lines (HBECs) to study pre-malignancy of lung cancer. Exposure to tobacco carcinogens induces morphological transformation involving induction of epithelial to mesenchymal transition in HBECs that manifests through down regulation of the microRNA (miR)- 200 family and miR-205, changes in expression of cytosine DNMTs and methylation of tumor suppressor genes. This Postdoctoral fellowship addresses the hypothesis that DNMT3a and DNMT3b (de novo DNMTs) target specific genes and miRs for methylation and chromatin silencing during transformation of HBECs, and that overexpression of DNMT3a and DNMT3b will accelerate transformation of carcinogen-treated HBECs to full malignancy. HBECs overexpressing DNMT3a or DNMT3b will be exposed to tobacco carcinogens, and the effect on time to transformation, transformation efficiency, EMT and growth in nude mice will be determined. [Since our preliminary data indicates that overexpression of DNMT3b in HBEC2 cells increased transformation efficiency by 2-fold, we will now focus on DNMT3b regulated genes]. The Illumina Infinium Human Methylation27 BeadChip array will be used to interrogate transformed DNMT3b overexpressing HBECs vs. parental HBECs or transformed HBECs with normal DNMT3b background to identify methylated genes regulated by DNMT3b. Next generation sequencing using the Ilumina platform and a custom tiling miR promoter array for ChIP-chip will be used to evaluate the distribution of chromatin marks in miRs present in transformed HBECs overexpressing DNMT3b compared to transformed HBECs with normal DNMT3b expression and parental HBECs. Promoter methylation status of 8-10 DNMT3b - regulated genes and 2-3 miRs will be determined in 100 primary adenocarcinoma samples and 17 lung tumor-derived cell lines. Two genes and two miRs that are most commonly silenced in primary tumors will be overexpressed in lung tumor-derived cell lines and the effect on phenotype determined. During the three years of this fellowship, the applicant will use whole genome approaches to interrogate the epigenome through promoter methylation arrays, the microRNAome through Next Generation sequencing, and chromatin remodeling through ChIP assays. To foster his education in bioinformatics and statistics, he will take a short course in bioinformatics and a one-semester course in Biostatistics. These activities along with the training received through the studies outlined in this application will provide Ivo with unique strengths, and the ability to integrate molecular biology, bioinformatics, and statistics.
Lung cancer is an enormous public health problem in the USA causing at least 30% of cancer deaths in the US, and over 1.5 million deaths globally. Genes involved in development of this disease are still being identified and characterized. The studies described in this application will identify new genes and microRNAs that participate in lung cancer development and will provide fundamental knowledge on the pathways affected by these genes.