title: Genome-wide methylation profiling and the PI3K-AKT pathway analysis associated with smoking in urothelial cell carcinoma.
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 leads 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 (mo) 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 mo of CSE treatment. By performing a PCR mRNA expression array specific to the PI3K-AKT pathway, we found that 26 genes were upregulated and 22 genes were downregulated after 6 mo of CSE exposure of HUC1 cells. Among the altered genes, PTEN, FOXO1, MAPK1 and PDK1 were downregulated in the transformed cells, while AKT1, AKT2, HRAS, RAC1 were upregulated. 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 with 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 to 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.
authors: Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors: Mariana
authors: M
authors: Brait
authors: Enrico
authors: E
authors: Munari
authors: Cynthia
authors: C
authors: LeBron
authors: Maartje G
authors: MG
authors: Noordhuis
authors: Shahnaz
authors: S
authors: Begum
authors: Christina
authors: C
authors: Michailidi
authors: Nilda
authors: N
authors: Gonzalez-Roibon
authors: Leonel
authors: L
authors: Maldonado
authors: Tanusree
authors: T
authors: Sen
authors: Rafael
authors: R
authors: Guerrero-Preston
authors: Leslie
authors: L
authors: Cope
authors: Paola
authors: P
authors: Parrella
authors: Vito Michele
authors: VM
authors: Fazio
authors: Patrick K
authors: PK
authors: Ha
authors: George J
authors: GJ
authors: Netto
authors: David
authors: D
authors: Sidransky
authors: Mohammad O
authors: MO
authors: Hoque
mesh_terms: D002295:Carcinoma, Transitional Cell / Q000378:metabolism / Q000473:pathology; D018797:Cell Cycle Proteins; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000187:drug effects; D000075389:DCC Receptor; D019175:DNA Methylation; D015536:Down-Regulation / Q000187:drug effects; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000378:metabolism; D015894:Genome, Human; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010936:Plant Extracts / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D012907:Smoking; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D001749:Urinary Bladder Neoplasms / Q000378:metabolism / Q000473:pathology; D014026:Nicotiana; D000092468:Bromodomain Containing Proteins
chemical_list: D018797:Cell Cycle Proteins; D000075389:DCC Receptor; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases; D010936:Plant Extracts; D051057:Proto-Oncogene Proteins c-akt; D011956:Receptors, Cell Surface; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C501909:DCC protein, human; C085126:FOXO1 protein, human; C499808:HIC1 protein, human
keywords: Urothelial cell carcinoma; bladder cancer; cigarette smoke extract; epigenetics; in vitro transformation; smoking