title: N-Myc downstream-regulated gene 4 (NDRG4): a candidate tumor suppressor gene and potential biomarker for colorectal cancer.
abstract: BACKGROUND
  Identification of hypermethylated tumor suppressor genes in body fluids is an appealing strategy for the noninvasive detection of colorectal cancer. Here we examined the role of N-Myc downstream-regulated gene 4 (NDRG4) as a novel tumor suppressor and biomarker in colorectal cancer.
  
  
  METHODS
  NDRG4 promoter methylation was analyzed in human colorectal cancer cell lines, colorectal tissue, and noncancerous colon mucosa by using methylation-specific polymerase chain reaction (PCR) and bisulfite sequencing. NDRG4 mRNA and protein expression were studied using real-time-PCR and immunohistochemistry, respectively. Tumor suppressor functions of NDRG4 were examined by colony formation, cell proliferation, and migration and invasion assays in colorectal cancer cell lines that were stably transfected with an NDRG4 expression construct. Quantitative methylation-specific PCR was used to examine the utility of NDRG4 promoter methylation as a biomarker in fecal DNA from 75 colorectal cancer patients and 75 control subjects. All P values are two-sided.
  
  
  RESULTS
  The prevalence of NDRG4 promoter methylation in two independent series of colorectal cancers was 86% (71/83) and 70% (128/184) compared with 4% (2/48) in noncancerous colon mucosa (P < .001). NDRG4 mRNA and protein expression were decreased in colorectal cancer tissue compared with noncancerous colon mucosa. NDRG4 overexpression in colorectal cancer cell lines suppressed colony formation (P = .014), cell proliferation (P < .001), and invasion (P < .001). NDRG4 promoter methylation analysis in fecal DNA from a training set of colorectal cancer patients and control subjects yielded a sensitivity of 61% (95% confidence interval [CI] = 43% to 79%) and a specificity of 93% (95% CI = 90% to 97%). An independent test set of colorectal cancer patients and control subjects yielded a sensitivity of 53% (95% CI = 39% to 67%) and a specificity of 100% (95% CI = 86% to 100%).
  
  
  CONCLUSIONS
  NDRG4 is a candidate tumor suppressor gene in colorectal cancer whose expression is frequently inactivated by promoter methylation. NDRG4 promoter methylation is a potential biomarker for the noninvasive detection of colorectal cancer in stool samples.
authors.affiliation: Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center, The Netherlands.
authors.forename: Veerle
authors.initials: V
authors.lastname: Melotte
authors.forename: Marjolein H F M
authors.initials: MH
authors.lastname: Lentjes
authors.forename: Sandra M
authors.initials: SM
authors.lastname: van den Bosch
authors.forename: Debby M E I
authors.initials: DM
authors.lastname: Hellebrekers
authors.forename: Joep P J
authors.initials: JP
authors.lastname: de Hoon
authors.forename: Kim A D
authors.initials: KA
authors.lastname: Wouters
authors.forename: Kathleen L J
authors.initials: KL
authors.lastname: Daenen
authors.forename: Iris E J M
authors.initials: IE
authors.lastname: Partouns-Hendriks
authors.forename: Filip
authors.initials: F
authors.lastname: Stessels
authors.forename: Joost
authors.initials: J
authors.lastname: Louwagie
authors.forename: Kim M
authors.initials: KM
authors.lastname: Smits
authors.forename: Matty P
authors.initials: MP
authors.lastname: Weijenberg
authors.forename: Silvia
authors.initials: S
authors.lastname: Sanduleanu
authors.forename: Carolina A J
authors.initials: CA
authors.lastname: Khalid-de Bakker
authors.forename: Frank A
authors.initials: FA
authors.lastname: Oort
authors.forename: Gerrit A
authors.initials: GA
authors.lastname: Meijer
authors.forename: Daisy M A E
authors.initials: DM
authors.lastname: Jonkers
authors.forename: James G
authors.initials: JG
authors.lastname: Herman
authors.forename: Adriaan P
authors.initials: AP
authors.lastname: de Bruïne
authors.forename: Manon
authors.initials: M
authors.lastname: van Engeland
mesh_terms: D000236:Adenoma / Q000737:chemistry* / Q000175:diagnosis / Q000235:genetics / Q000517:prevention & control; D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics; D002277:Carcinoma / Q000737:chemistry* / Q000175:diagnosis / Q000235:genetics / Q000517:prevention & control; D016022:Case-Control Studies; D002465:Cell Movement; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000737:chemistry* / Q000175:diagnosis / Q000235:genetics / Q000517:prevention & control; D019175:DNA Methylation; D055088:Early Detection of Cancer; D005243:Feces / Q000737:chemistry*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D006801:Humans; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000737:chemistry*; D008297:Male; D008875:Middle Aged; D009124:Muscle Proteins / Q000032:analysis* / Q000235:genetics*; D009361:Neoplasm Invasiveness; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015995:Prevalence; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D014410:Tumor Stem Cell Assay; D015854:Up-Regulation
chemical_list: D014408:Biomarkers, Tumor; D009124:Muscle Proteins; C431705:NDRG4 protein, human; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger