Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2

Nathalie Vaes, Simone L. Schonkeren, Glenn Rademakers, Amy M. Holland, Alexander Koch, Marion J. Gijbels, Tom G. Keulers, Meike de Wit, Laura Moonen, Jaleesa R.M. Van der Meer, Edith van den Boezem, Tim G.A.M. Wolfs, David W. Threadgill, Jeroen Demmers, Remond J.A. Fijneman, Connie R. Jimenez, Pieter Vanden Berghe, Kim M. Smits, Kasper M.A. Rouschop, Werend BoesmansRobert M.W. Hofstra, Veerle Melotte*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

The N-Myc Downstream-Regulated Gene 4 (NDRG4), a prominent biomarker for colorectal cancer (CRC), is specifically expressed by enteric neurons. Considering that nerves are important members of the tumor microenvironment, we here establish different Ndrg4 knockout (Ndrg4−/−) CRC models and an indirect co-culture of primary enteric nervous system (ENS) cells and intestinal organoids to identify whether the ENS, via NDRG4, affects intestinal tumorigenesis. Linking immunostainings and gastrointestinal motility (GI) assays, we show that the absence of Ndrg4 does not trigger any functional or morphological GI abnormalities. However, combining in vivo, in vitro, and quantitative proteomics data, we uncover that Ndrg4 knockdown is associated with enlarged intestinal adenoma development and that organoid growth is boosted by the Ndrg4−/− ENS cell secretome, which is enriched for Nidogen-1 (Nid1) and Fibulin-2 (Fbln2). Moreover, NID1 and FBLN2 are expressed in enteric neurons, enhance migration capacities of CRC cells, and are enriched in human CRC secretomes. Hence, we provide evidence that the ENS, via loss of Ndrg4, is involved in colorectal pathogenesis and that ENS-derived Nidogen-1 and Fibulin-2 enhance colorectal carcinogenesis.

Original languageEnglish
Article numbere51913
JournalEMBO Reports
Volume22
Issue number6
DOIs
Publication statusPublished - 23 Apr 2021

Bibliographical note

Funding Information:
We are grateful to Dr. Ir. J. Cleutjens for the custom‐made program in the Leica QWIN V3 software for measuring IEO circumference, cell confluence, and relative wound density. We thank I. de Lange, a PhD student within the group of Dr. T.G.A.M. Wolfs for her technical support to set up our IEO and HIO cultures. We are highly appreciative of Ir. K. Bezstarosti for his support with the murine proteomics analysis and Dr. S. Piersma and Dr. A. Henneman for generating the source data files containing the human NID1&FBLN2 proteomics data. We are profoundly thankful to Dr. M.H.F.M. Lentjes for the help with the characterization of the mouse and to K.L.J. Daenen and K.A.D. Wouters for their assistance with sectioning of intestinal tissues and the performance of immunostainings. The schematic figure file in our synopsis was created with BioRender.com. This work was financially supported by the KWF Kankerbestrijding grant (UM 2013‐6075) and VENI‐NWO grant (016.186.124) obtained by Dr. V. Melotte, and by the VIDI‐NWO grant (016.196.367) obtained by Dr. W. Boesmans. Further support was granted by the VUmc Cancer Center Amsterdam for the proteomics infrastructure. Ndrg4 −/−

Funding Information:
We are grateful to Dr. Ir. J. Cleutjens for the custom-made program in the Leica QWIN V3 software for measuring IEO circumference, cell confluence, and relative wound density. We thank I. de Lange, a PhD student within the group of Dr. T.G.A.M. Wolfs for her technical support to set up our IEO and HIO cultures. We are highly appreciative of Ir. K. Bezstarosti for his support with the murine proteomics analysis and Dr. S. Piersma and Dr. A. Henneman for generating the source data files containing the human NID1&FBLN2 proteomics data. We are profoundly thankful to Dr. M.H.F.M. Lentjes for the help with the characterization of the Ndrg4?/? mouse and to K.L.J. Daenen and K.A.D. Wouters for their assistance with sectioning of intestinal tissues and the performance of immunostainings. The schematic figure file in our synopsis was created with BioRender.com. This work was financially supported by the KWF Kankerbestrijding grant (UM 2013-6075) and VENI-NWO grant (016.186.124) obtained by Dr. V. Melotte, and by the VIDI-NWO grant (016.196.367) obtained by Dr. W. Boesmans. Further support was granted by the VUmc Cancer Center Amsterdam for the proteomics infrastructure.

Publisher Copyright:
© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license

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