N-Myc downstream-regulated gene 4 (NDRG4): a candidate tumor suppressor gene and potential biomarker for colorectal cancer.
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.
Melotte
Veerle
V
Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center, The Netherlands.
Lentjes
Marjolein H F M
MH
van den Bosch
Sandra M
SM
Hellebrekers
Debby M E I
DM
de Hoon
Joep P J
JP
Wouters
Kim A D
KA
Daenen
Kathleen L J
KL
Partouns-Hendriks
Iris E J M
IE
Stessels
Filip
F
Louwagie
Joost
J
Smits
Kim M
KM
Weijenberg
Matty P
MP
Sanduleanu
Silvia
S
Khalid-de Bakker
Carolina A J
CA
Oort
Frank A
FA
Meijer
Gerrit A
GA
Jonkers
Daisy M A E
DM
Herman
James G
JG
de Bruïne
Adriaan P
AP
van Engeland
Manon
M
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
D014408:Biomarkers, Tumor; D009124:Muscle Proteins; C431705:NDRG4 protein, human; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger