title: Epigenetic regulation of L1CAM in endometrial carcinoma: comparison to cancer-testis (CT-X) antigens.
abstract: BACKGROUND
  L1CAM was originally identified as an adhesion molecule involved in neural development. In many human carcinomas L1CAM is over-expressed and is associated with a bad prognosis. We previously reported that L1CAM was absent in the vast majority of endometrioid endometrial carcinomas (ECs) (type 1) but was strongly expressed in the more aggressive serous and clear-cell ECs (termed type 2). The differential regulation of L1CAM in ECs is not well understood. Recent evidence suggests that it can be regulated by epigenetic mechanisms. Here we investigated the role of DNA-methylation of the L1CAM promoter for expression. We also studied the relationship to cancer testis (CT-X) antigens that co-localize with L1CAM on chromosome Xq28, a region that is often activated in human tumors.
  
  
  METHODS
  We used EC cell lines and primary tumor tissues for our analysis. For expression analysis we employed RT-PCR and Western blotting. DNA-Methylation of the L1CAM promoter was determined after bisulfite conversation and DNA sequencing. Tumor tissues were examined by immunohistochemical (IHC) staining.
  
  
  RESULTS
  We demonstrate that the treatment of L1CAM low/negative expressing EC cell lines with 5'-Azacytidine (5-AzaC) or knock-down of DNMT1 (DNA methyltransferase 1) as well as the HDAC (histone deacetylase) inhibitor Trichostatin A (TSA) up-regulated L1CAM at the mRNA and protein level. The L1CAM gene has two promoter regions with two distinct CpG islands. We observed that the expression of L1CAM correlated with hypermethylation in promoter 1 and 5-AzaC treatment affected the DNA-methylation pattern in this region. The CT-X antigens NY-ESO-1, MAGE-A3 and MAGE-A4 were also strongly up-regulated by 5-AzaC or knock-down of DNMT1 but did not respond to treatment with TSA. Primary EC tumor tissues showed a variable methylation pattern of the L1CAM promoter. No striking differences in promoter methylation were observed between tumor areas with L1CAM expression and those without expression.
  
  
  CONCLUSIONS
  L1CAM expression correlated with methylation of the L1CAM promoter in EC cell lines. In negative cell lines L1CAM expression is up-regulated by epigenetic mechanism. Although genes localized on Xq28 are often re-expressed by human tumors, L1CAM and CT-X antigens show distinct regulation in response to HADC inhibitors and 5-AzaC.
authors.affiliation: Department of Translational Immunology, German Cancer Research Center, D015, Heidelberg, D 69120, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Schirmer
authors.forename: Heidi
authors.initials: H
authors.lastname: Fiegl
authors.forename: Marco
authors.initials: M
authors.lastname: Pfeifer
authors.forename: Alain G
authors.initials: AG
authors.lastname: Zeimet
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Müller-Holzner
authors.forename: Peter K
authors.initials: PK
authors.lastname: Bode
authors.forename: Verena
authors.initials: V
authors.lastname: Tischler
authors.forename: Peter
authors.initials: P
authors.lastname: Altevogt
mesh_terms: D000328:Adult; D000368:Aged; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D018899:CpG Islands; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D016889:Endometrial Neoplasms / Q000235:genetics* / Q000378:metabolism; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D039842:Neural Cell Adhesion Molecule L1 / Q000235:genetics*; D011401:Promoter Regions, Genetic; D013737:Testis / Q000378:metabolism; D055815:Young Adult
chemical_list: D000951:Antigens, Neoplasm; C104948:CTAG1B protein, human; C072013:MAGEA3 protein, human; C095204:MAGEA4 protein, human; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D039842:Neural Cell Adhesion Molecule L1; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human