title: Epigenetic regulation and role of metastasis suppressor genes in pancreatic ductal adenocarcinoma.
abstract: BACKGROUND
  Pancreatic ductal adenocarcinoma (PDAC) is distinguished by rapid dissemination. Thus, genetic and/or epigenetic deregulation of metastasis suppressor genes (MSG) is a likely event during early pancreatic carcinogenesis and a potential diagnostic marker for the disease. We investigated 9 known MSGs for their role in the dissemination of PDAC and examined their promoters for methylation and its use in PDAC detection.
  
  
  METHODS
  MRNA expression of 9 MSGs was determined in 18 PDAC cell lines by quantitative RT-PCR and promoter methylation was analyzed by Methylation Specific PCR and validated by Bisulfite Sequencing PCR. These data were compared to the cell lines' in vivo metastatic and invasive potential that had been previously established. Statistical analysis was performed with SPSS 20 using 2-tailed Spearman's correlation with P < 0.05 being considered significant.
  
  
  RESULTS
  Complete downregulation of MSG-mRNA expression in PDAC cell lines vs. normal pancreatic RNA occurred in only 1 of 9 investigated genes. 3 MSGs (CDH1, TIMP3 and KiSS-1) were significantly methylated. Methylation only correlated to loss of mRNA expression in CDH1 (P < 0.05). Bisulfite Sequencing PCR showed distinct methylation patterns, termed constant and variable methylation, which could distinguish methylation-regulated from non methylation-regulated genes. Higher MSG mRNA-expression did not correlate to less aggressive PDAC-phenotypes (P > 0.14).
  
  
  CONCLUSIONS
  Genes with metastasis suppressing functions in other tumor entities did not show evidence of assuming the same role in PDAC. Inactivation of MSGs by promoter methylation was an infrequent event and unsuitable as a diagnostic marker of PDAC. A distinct methylation pattern was identified, that resulted in reduced mRNA expression in all cases. Thus, constant methylation patterns could predict regulatory significance of a promoter's methylation prior to expression analysis and hence present an additional tool during target gene selection.
authors.affiliation: Comprehensive Cancer Center Muenster, University Hospital of Muenster, Albert-Schweitzer-Campus 1, Muenster, 48149, Germany.
authors.forename: Wolf Arif
authors.initials: WA
authors.lastname: Mardin
authors.forename: Joerg
authors.initials: J
authors.lastname: Haier
authors.forename: Soeren Torge
authors.initials: ST
authors.lastname: Mees
mesh_terms: D000818:Animals; D021441:Carcinoma, Pancreatic Ductal / Q000235:genetics*; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000235:genetics*; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D006801:Humans; D008297:Male; D051379:Mice; D008819:Mice, Nude; D010190:Pancreatic Neoplasms / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D023041:Xenograft Model Antitumor Assays