title: NT5E (CD73) is epigenetically regulated in malignant melanoma and associated with metastatic site specificity.
abstract: BACKGROUND
  Novel prognostic biomarkers and therapeutic strategies are urgently required for malignant melanoma. Ecto-5-prime-nucleotidase (NT5E; CD73) overexpression has been reported in several human cancers. The mechanism(s) underlying deregulated expression and the clinical consequences of changes in expression are not known.
  
  
  METHODS
  We used RT-PCR, qPCR, methylation-specific PCR and pyrosequencing to analyse expression and regulation of NT5E in malignant melanoma cell lines and primary and metastatic melanomas.
  
  
  RESULTS
  NT5E is subject to epigenetic regulation in melanoma. NT5E mRNA is downregulated by methylation-dependent transcriptional silencing in the melanoma cell lines SKMel2, SKMel23, WM35, Mel501, Mel505 and C81-61 and expression is reactivated by azacytidine. In contrast, the CpG island is unmethylated and the gene expressed in cultured normal melanocytes. In clinical cases of melanoma, methylation in the NT5E CpG island occurs in both primary and metastatic melanomas and correlates with transcriptional downregulation of NT5E mRNA. Relapse with metastatic disease, particularly to the visceral sites and brain, is more common in primary melanomas lacking NT5E methylation. Primary melanomas with methylation in NT5E show limited metastatic potential or more commonly metastasise predominantly to nodal sites rather than viscera and brain (P=0.01).
  
  
  CONCLUSIONS
  Deregulation of NT5E expression in melanoma occurs via epigenetic changes in the NT5E CpG island. Confirmation of our results in larger clinical series would support the candidacy of NT5E as a clinical biomarker in melanoma, which could be applied in both primary and relapsed disease. Inhibition of NT5E may have therapeutic potential in melanoma, particularly in patients with more aggressive disease metastatic to viscera or the brain.
authors.affiliation: Wellcome Trust Centre for Molecular Medicine, Division of Medical Sciences, Clinical Research Centre, Dundee, UK.
authors.forename: H
authors.initials: H
authors.lastname: Wang
authors.forename: S
authors.initials: S
authors.lastname: Lee
authors.forename: C Lo
authors.initials: CL
authors.lastname: Nigro
authors.forename: L
authors.initials: L
authors.lastname: Lattanzio
authors.forename: M
authors.initials: M
authors.lastname: Merlano
authors.forename: M
authors.initials: M
authors.lastname: Monteverde
authors.forename: R
authors.initials: R
authors.lastname: Matin
authors.forename: K
authors.initials: K
authors.lastname: Purdie
authors.forename: N
authors.initials: N
authors.lastname: Mladkova
authors.forename: D
authors.initials: D
authors.lastname: Bergamaschi
authors.forename: C
authors.initials: C
authors.lastname: Harwood
authors.forename: N
authors.initials: N
authors.lastname: Syed
authors.forename: P
authors.initials: P
authors.lastname: Szlosarek
authors.forename: E
authors.initials: E
authors.lastname: Briasoulis
authors.forename: A
authors.initials: A
authors.lastname: McHugh
authors.forename: A
authors.initials: A
authors.lastname: Thompson
authors.forename: A
authors.initials: A
authors.lastname: Evans
authors.forename: I
authors.initials: I
authors.lastname: Leigh
authors.forename: C
authors.initials: C
authors.lastname: Fleming
authors.forename: G J
authors.initials: GJ
authors.lastname: Inman
authors.forename: E
authors.initials: E
authors.lastname: Hatzimichael
authors.forename: C
authors.initials: C
authors.lastname: Proby
authors.forename: T
authors.initials: T
authors.lastname: Crook
mesh_terms: D015720:5'-Nucleotidase / Q000235:genetics* / Q000378:metabolism; D001932:Brain Neoplasms / Q000235:genetics* / Q000556:secondary*; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics; D044127:Epigenesis, Genetic / Q000235:genetics*; D058851:GPI-Linked Proteins / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000473:pathology*; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D058851:GPI-Linked Proteins; D012333:RNA, Messenger; D015720:5'-Nucleotidase; C548241:NT5E protein, human