title: Putative tumour suppressor gene necdin is hypermethylated and mutated in human cancer.
abstract: BACKGROUND
  Necdin (NDN) expression is downregulated in telomerase-immortalised normal human urothelial cells. Telomerase-immortalised normal human urothelial cells have no detected genetic alterations. Accordingly, many of the genes whose expression is altered following immortalisation are those for which epigenetic silencing is reported.
  
  
  METHODS
  NDN expression was examined in normal tissues and tumour cell lines by quantitative real-time PCR and immunoblotting. Immunohistochemistry was performed on urothelial carcinoma (UC). Urothelial carcinoma and UC cell lines were subject to HumanMethylation27 BeadChip Array-based methylation analyses. Mutation screening was performed. The functional significance of NDN expression was investigated using retroviral-mediated downregulation or overexpression.
  
  
  RESULTS
  NDN protein was widely expressed in normal tissues. Loss of expression was observed in 38 out of 44 (86%) of UC cell lines and 19 out of 25 (76%) of non-UC cell lines. Loss of NDN protein was found in the majority of primary UC. Oncomine analysis demonstrated downregulation of expression in multiple tumour types. In UC, tumour-specific hypermethylation of NDN and key CpG sites where hypermethylation correlated with reduced expression were identified. Six novel mutations, including some of predicted functional significance, were identified in colorectal and ovarian cancer cell lines. Functional studies showed that NDN could suppress colony formation at low cell density and affect anchorage-independent growth and anoikis in vitro.
  
  
  CONCLUSIONS
  NDN is a novel tumour suppressor candidate that is downregulated and hypermethylated or mutated in cancer.
authors.affiliation: Section of Experimental Oncology, Cancer Research UK Centre, Leeds Institute of Molecular Medicine, St James's University Hospital, Beckett Street, Leeds LS97TF, UK.
authors.forename: L E
authors.initials: LE
authors.lastname: De Faveri
authors.forename: C D
authors.initials: CD
authors.lastname: Hurst
authors.forename: F M
authors.initials: FM
authors.lastname: Platt
authors.forename: C F
authors.initials: CF
authors.lastname: Taylor
authors.forename: J-A
authors.initials: JA
authors.lastname: Roulson
authors.forename: M
authors.initials: M
authors.lastname: Sanchez-Carbayo
authors.forename: M A
authors.initials: MA
authors.lastname: Knowles
authors.forename: E J
authors.initials: EJ
authors.lastname: Chapman
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics*; D049109:Cell Proliferation; D002478:Cells, Cultured; D019175:DNA Methylation; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D009154:Mutation / Q000235:genetics*; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D019459:Urothelium / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; C069766:necdin
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