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Journal of the National Cancer Institute Advance Access published online on June 17, 2009
JNCI Journal of the National Cancer Institute, doi:10.1093/jnci/djp131
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© The Author 2009. Published by Oxford University Press.

ARTICLES

N-Myc Downstream-Regulated Gene 4 (NDRG4): A Candidate Tumor Suppressor Gene and Potential Biomarker for Colorectal Cancer

Veerle Melotte, Marjolein H. F. M. Lentjes, Sandra M. van den Bosch, Debby M. E. I. Hellebrekers, Joep P. J. de Hoon, Kim A. D. Wouters, Kathleen L. J. Daenen, Iris E. J. M. Partouns-Hendriks, Filip Stessels, Joost Louwagie, Kim M. Smits, Matty P. Weijenberg, Silvia Sanduleanu, Carolina A. J. Khalid-de Bakker, Frank A. Oort, Gerrit A. Meijer, Daisy M. A. E. Jonkers, James G. Herman, Adriaan P. de Bruïne, Manon van Engeland

Affiliations of authors: Department of Pathology (VM, MHFML, SMvdB, DMEIH, JPJdH, KADW, KLJD, IEJMP-H, FS, APdB, MvE) and Department of Epidemiology (KMS, MPW), GROW–School for Oncology and Developmental Biology, and Division of Gastroenterology and Hepatology, Department of Internal Medicine (SS, CAJK-dB, DMAEJ), Maastricht University Medical Center, the Netherlands; OncoMethylome Sciences, SA, Liège, Belgium (JL); Department of Gastroenterology (FAO) and Department of Pathology (GAM), VU University Medical Center, Amsterdam, the Netherlands; Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD (JGH)

Correspondence to: Manon van Engeland, PhD, Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, PO Box 616, 6200 MD Maastricht, the Netherlands (e-mail: manon.van.engeland{at}mumc.nl).

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.


CONTEXT AND CAVEATS

Prior knowledge

Identification of tumor suppressor gene promoter hypermethylation in fecal DNA is a promising strategy for noninvasive detection of colorectal cancer. N-Myc downstream-regulated gene 4 (NDRG4) is a potential tumor suppressor in colorectal cancer.

Study design

NDRG4 promoter methylation and expression were analyzed in human colorectal cancer cell lines, noncancerous colon mucosa, and colorectal cancer tissue. NDRG4 tumor suppressor functions were examined in colorectal cancer cells. NDRG4 promoter methylation was examined as a potential biomarker in stool from colorectal cancer patients and subjects without colorectal cancer.

Contribution

NDRG4 promoter methylation was prevalent in colorectal cancers compared with noncancerous colon mucosa. NDRG4 mRNA and protein expression were decreased in colorectal cancer tissue compared with noncancerous colon mucosa. NDRG4 overexpression in human colorectal cancer cells inhibited colony formation and cell proliferation and invasion in vitro. A methylation-specific polymerase chain reaction assay for NDRG4 promoter methylation identified colorectal cancer when it was present (sensitivity) in 53% of colorectal cancer cases and correctly categorized a subject as cancer free (specificity) 100% of the time.

Implications

NDRG4 is a candidate tumor suppressor gene in colorectal cancer. NDRG4 promoter methylation is a potential biomarker for the noninvasive detection of colorectal cancer in stool samples.

Limitations

Not all stool samples from colorectal cancer patients were collected before colonoscopy as was done for the control subjects. The colorectal cancer patients were older than the subjects without colorectal cancer.

From the Editors

 
Manuscript received September 24, 2008; revised April 7, 2009; accepted April 17, 2009.


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