Abstract 655: Genome-wide methylation profiling and the PI3K-AKT pathway analysis associated with smoking in urothelial cell carcinoma .

Abstract

Abstract Urothelial Cell Carcinoma (UCC) is the second most common genitourinary malignant disease in the USA, and tobacco smoking is the major known risk factor for UCC development. Exposure to carcinogens, such as those contained in tobacco smoke, is known to directly or indirectly damage DNA, causing mutations, chromosomal deletion events and epigenetic alterations in UCC. Molecular studies have shown that chromosome 9 alterations and P53, RAS, RB and PTEN mutations are among the most frequent events in UCC. Recent studies suggested that continuous tobacco carcinogen exposure drives and enhances the selection of epigenetically altered cells in UCC, predominantly in the invasive form of the disease. However, the sequence of molecular events that lead to UCC after exposure to tobacco smoke is not well understood. To elucidate molecular events that lead to UCC oncogenesis and progression after tobacco exposure, we developed an in vitro cellular model for smoking induced UCC. SV-40 immortalized normal HUC1 human bladder epithelial cells were continuously exposed to 0.1% cigarette smoke extract (CSE) until transformation occurred. Morphological alterations and increased cell proliferation of non-malignant urothelial cells were observed after 4 months of treatment with CSE. Anchorage-independent growth assessed by soft agar assay and increase in the migratory and invasive potential was observed in urothelial cells after 6 months of CSE treatment. By performing a PCR mRNA expression array specific to the PI3K-AKT pathway, we found 26 genes were up-regulated and 22 genes were down-regulated after 6 months of CSE exposure of HUC cells. Among the altered genes, PTEN, FOXO1, MAPK1 and PDK1 were down-regulated in the transformed cells while AKT1, AKT2, HRAS, RAC1 were up-regulated. Validation by RT-PCR and western blot analysis was then performed. Furthermore, genome-wide methylation analysis revealed MCAM, DCC and HIC1 are hypermethylated in CSE treated urothelial cells when compared to non-CSE exposed cells. The methylation status of these genes was validated using Quantitative Methylation Specific PCR (QMSP), confirming an increase in methylation of CSE treated urothelial cells compared untreated controls. Therefore, our findings suggest that a tobacco signature could emerge from distinctive patterns of genetic and epigenetic alterations and can be identified using an in vitro cellular model for the development of smoking induced cancer. Citation Format: Enrico Munari, Mariana Brait, Cynthia LeBron, Maartje G. Noordhuis, Shahnaz Begum, Christina Michailidi, Nilda Gonzalez-Roibon, Leonel Maldonado, Tanusree Sen, Rafael Guerrero-Preston, Leslie Cope, Paola Parrella, Vito Michele Fazio, Patrick K. Ha, George Netto, David Sidransky, Mohammad O. Hoque. Genome-wide methylation profiling and the PI3K-AKT pathway analysis associated with smoking in urothelial cell carcinoma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 655. doi:10.1158/1538-7445.AM2013-655