NDRG4, an early detection marker for colorectal cancer, is specifically expressed in enteric neurons

• Published in: Neurogastroenterology and motility Vol. 29; no. 9
• Main Authors: Vaes, N., Lentjes, M. H. F. M., Gijbels, M. J., Rademakers, G., Daenen, K. L., Boesmans, W., Wouters, K. A. D., Geuzens, A., Qu, X., Steinbusch, H. P. J., Rutten, B. P. F., Baldwin, S. H., Sharkey, K. A., Hofstra, R. M. W., Engeland, M., Vanden Berghe, P., Melotte, V.
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01-09-2017
• Subjects: Animals; biomarker; Biomarkers, Tumor - analysis; Colorectal cancer; Colorectal carcinoma; Colorectal Neoplasms - metabolism; Confocal microscopy; DNA methylation; Enteric nervous system; Enteric Nervous System - metabolism; Glial cells; Glial fibrillary acidic protein; Humans; Hybridization; Immunofluorescence; Immunohistochemistry; Intestine; Mice; Mice, Inbred C57BL; Mice, Knockout; mRNA; Muscle Proteins - analysis; Muscle Proteins - biosynthesis; Myc protein; myenteric plexus; NDRG4; Nerve Tissue Proteins - analysis; Nerve Tissue Proteins - biosynthesis; Nervous system; Neuronal-glial interactions; Neurons; Neurons - metabolism; Neuropeptide Y; Nitric oxide; Nitric-oxide synthase; Rodents; Western blotting; NDRG4; colorectal cancer; enteric nervous system; biomarker; myenteric plexus
• ISSN: 1350-1925; 1365-2982
• DOI: 10.1111/nmo.13095

Background
Promoter methylation of N‐myc Downstream‐Regulated Gene 4 (NDRG4) in fecal DNA is an established early detection marker for colorectal cancer (CRC). Despite its connection to CRC, NDRG4 is predominantly studied in brain and heart, with little to no knowledge about its expression or role in other organs. In this study, we aimed to determine the whole‐body expression of NDRG4, with a focus on the intestinal tract.
Methods
We investigated NDRG4 expression throughout the body by immunohistochemistry, Western Blotting and in situ mRNA hybridization using tissues from NDRG4 wild‐type, heterozygous and knockout mice and humans. In addition, we explored cell‐specific expression of NDRG4 in murine whole‐mount gut preparations using immunofluorescence and confocal microscopy.
Key Results
NDRG4 is specifically expressed within nervous system structures throughout the body. In the intestinal tract of both mouse and man, NDRG4 immunoreactivity was restricted to the enteric nervous system (ENS), where it labeled cell bodies of the myenteric and submucosal plexuses and interconnecting nerve fibers. More precisely, NDRG4 expression was limited to neurons, as NDRG4 always co‐localized with HuC/D (pan‐neuronal marker) but never with GFAP (an enteric glial cell marker). Furthermore, NDRG4 was expressed in various neuropeptide Y positive neurons, but was only found in a minority (~10%) of neurons expressing neuronal nitric oxide synthase.
Conclusions and Inferences
NDRG4 is exclusively expressed by central, peripheral and enteric neurons/nerves, suggesting a neuronal‐specific role of this protein. Our findings raise the question whether NDRG4, via the ENS, an understudied component of the tumor microenvironment, supports CRC development and/or progression.
Despite the well‐described biomarker performance of NDRG4, little is known about the expression and role of NDRG4 in the gut. Studying the whole‐body expression of NDRG4, we revealed that NDRG4 is specifically expressed in nervous system structures throughout the body and that the expression in the gut is limited to enteric neurons.