title: Genome-wide methylation analysis and epigenetic unmasking identify tumor suppressor genes in hepatocellular carcinoma.
abstract: OBJECTIVE
  Epigenetic silencing of tumor suppressor genes contributes to the pathogenesis of hepatocellular carcinoma (HCC). To identify clinically relevant tumor suppressor genes silenced by DNA methylation in HCC, we integrated DNA methylation data from human primary HCC samples with data on up-regulation of gene expression after epigenetic unmasking.
  
  
  METHODS
  We performed genome-wide methylation analysis of 71 human HCC samples using the Illumina HumanBeadchip27K array; data were combined with those from microarray analysis of gene re-expression in 4 liver cancer cell lines after their exposure to reagents that reverse DNA methylation (epigenetic unmasking).
  
  
  RESULTS
  Based on DNA methylation in primary HCC and gene re-expression in cell lines after epigenetic unmasking, we identified 13 candidate tumor suppressor genes. Subsequent validation led us to focus on functionally characterizing 2 candidates, sphingomyelin phosphodiesterase 3 (SMPD3) and neurofilament, heavy polypeptide (NEFH), which we found to behave as tumor suppressor genes in HCC. Overexpression of SMPD3 and NEFH by stable transfection of inducible constructs into an HCC cell line reduced cell proliferation by 50% and 20%, respectively (SMPD3, P = .003 and NEFH, P = .003). Conversely, knocking down expression of these genes with small hairpin RNA promoted cell invasion and migration in vitro (SMPD3, P = .0001 and NEFH, P = .022), and increased their ability to form tumors after subcutaneous injection or orthotopic transplantation into mice, confirming their role as tumor suppressor genes in HCC. Low levels of SMPD3 were associated with early recurrence of HCC after curative surgery in an independent patient cohort (P = .001; hazard ratio = 3.22; 95% confidence interval: 1.6-6.5 in multivariate analysis).
  
  
  CONCLUSIONS
  Integrative genomic analysis identified SMPD3 and NEFH as tumor suppressor genes in HCC. We provide evidence that SMPD3 is a potent tumor suppressor gene that could affect tumor aggressiveness; a reduced level of SMPD3 is an independent prognostic factor for early recurrence of HCC.
authors.affiliation: Cancer Genome Center, Cold Spring Harbor Laboratory, Woodbury, New York; Mount Sinai Liver Cancer Program, Division of Liver Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York. Electronic address: kate.revill@mssm.edu.
authors.forename: Kate
authors.initials: K
authors.lastname: Revill
authors.forename: Tim
authors.initials: T
authors.lastname: Wang
authors.forename: Anja
authors.initials: A
authors.lastname: Lachenmayer
authors.forename: Kensuke
authors.initials: K
authors.lastname: Kojima
authors.forename: Andrew
authors.initials: A
authors.lastname: Harrington
authors.forename: Jinyu
authors.initials: J
authors.lastname: Li
authors.forename: Yujin
authors.initials: Y
authors.lastname: Hoshida
authors.forename: Josep M
authors.initials: JM
authors.lastname: Llovet
authors.forename: Scott
authors.initials: S
authors.lastname: Powers
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D006528:Carcinoma, Hepatocellular / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics*; D057890:Epigenomics / Q000379:methods*; D005260:Female; D016147:Genes, Tumor Suppressor; D055106:Genome-Wide Association Study / Q000379:methods*; D006801:Humans; D008113:Liver Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D016900:Neurofilament Proteins / Q000235:genetics; D011379:Prognosis; D012008:Recurrence; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics
chemical_list: D004273:DNA, Neoplasm; D016900:Neurofilament Proteins; C051774:neurofilament protein H; C491484:SMPD3 protein, human; D013108:Sphingomyelin Phosphodiesterase
keywords: 5-aza-2-deoxycitidine; 5-aza-2′deoxycitidine; ACTL6B; CHTN; CpG; DAC; DGKI; ELOVL fatty acid elongase 4; ELOVL4; GSTP1; HCC; LDHB; LRAT; ND; NEFH; PRPH; SMPD3; Sphingomyelin Phosphodiesterase; TSA; actin-like 6B; cooperative tissue network; cytosine preceding guanine; diacylglycerol kinase, iota; glutathione S-transferase 1; hepatocellular carcinoma; lactate dehydrogenase B; lecithin retinol acyltransferase (phosphatidylcholine−retinol O-acyltransferase); nSMase2; neurofilament, heavy polypeptide; nondiseased; peripherin; shRNA; short hairpin RNA; sphingomyelin phosphodiesterase 3; trichostatin A