title: Epigenetic inactivation of ST6GAL1 in human bladder cancer.
abstract: BACKGROUND
  Posttranslational protein modifications are known to modulate key biological processes like proliferation and apoptosis. Accumulating evidence shows that ST6GAL1, an enzyme that catalyzes the transfer of sialic acid onto galactose-containing substrates, is aberrantly expressed in various cancers and may affect cell motility and invasion. This is the first study to describe ST6GAL1 expression and regulation in human bladder cancer.
  
  
  METHODS
  ST6GAL1 mRNA expression levels in human cell lines (UROtsa, RT4, RT112 and J82) and tissue samples (n = 15 normal urothelium (NU), n = 13 papillary non-invasive tumors (pTa), n = 12 carcinoma in situ (CIS), n = 26 muscle invasive tumors (pT2-4)) were assessed using real-time PCR. In addition, ST6GAL1 protein expression was evaluated using immunohistochemistry. Promoter methylation analysis was performed using methylation-specific PCR (MSP) in cell lines (n = 4) and patient samples (n = 23 NU, n = 12 CIS, n = 29 pTa, n = 41 pT2-4). Epigenetic ST6GAL1 gene silencing was confirmed by in vitro demethylation of bladder cell lines. Data were validated by analysis of an independent bladder tumor data set (n = 184) based on The Cancer Genome Atlas (TCGA) portal.
  
  
  RESULTS
  Semi-quantitative ST6GAL1 real-time PCR expression analysis showed two distinct trends: In muscle-invasive tumors ST6GAL1 expression was downregulation by 2.7-fold, while papillary non-invasive tumors showed an increased ST6GAL1 mRNA expression compared to normal urothelium. ST6GAL1 loss in muscle-invasive tumors was associated with increasing invasiveness. On the protein level, 69.2% (n = 45/65) of all tumors showed a weak ST6GAL1 protein staining (IRS ≤ 4) while 25.6% (16/65) exhibited a complete loss (IRS = 0) of ST6GAL1 protein. Tumor-specific DNA methylation of the ST6GAL1 promoter region was frequently found in pT2-4 tumors (53.6% (22/41)), whereas only 13.8% (4/29) of pTa tumors showed ST6GAL1 promoter methylation. Normal urothelium remained unmethylated. Importantly, we significantly revealed an inverse correlation between ST6GAL1 mRNA expression and ST6GAL1 promoter merthylation in primary bladder cancer. These findings were clearly verified by the TCGA public data set and in vitro demethylation assays functionally confirmed ST6GAL1 promoter methylation as a potential regulatory factor for ST6GAL1 gene silencing.
  
  
  CONCLUSIONS
  Our study characterizes for the first time ST6GAL1 expression loss caused by aberrant ST6GAL1 promoter methylation potentially indicating a tumor suppressive role in bladder carcinogenesis.
authors.forename: Pia
authors.initials: P
authors.lastname: Antony
authors.forename: Michael
authors.initials: M
authors.lastname: Rose
authors.forename: Axel
authors.initials: A
authors.lastname: Heidenreich
authors.forename: Ruth
authors.initials: R
authors.lastname: Knüchel
authors.forename: Nadine T
authors.initials: NT
authors.lastname: Gaisa
authors.affiliation: Molecular Oncology Group, Institute of Pathology, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany. edahl@ukaachen.de.
authors.forename: Edgar
authors.initials: E
authors.lastname: Dahl
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D002278:Carcinoma in Situ / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002291:Carcinoma, Papillary / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D012799:Sialyltransferases / Q000235:genetics* / Q000378:metabolism; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D015703:Antigens, CD; D012799:Sialyltransferases; C093493:ST6GAL1 protein, human; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase