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

Report23 April 2021Open Access Source DataTransparent process Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2 Nathalie Vaes Nathalie Vaes orcid.org/0000-0001-9571-3449 Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Simone L Schonkeren Simone L Schonkeren orcid.org/0000-0003-4715-4173 Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Glenn Rademakers Glenn Rademakers Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Amy M Holland Amy M Holland Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Alexander Koch Alexander Koch Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Marion J Gijbels Marion J Gijbels Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Department of Molecular Genetics, Cardiovascular Research Institute Maastricht (CARIM), Maastricht, The Netherlands Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands Search for more papers by this author Tom G Keulers Tom G Keulers Department of Radiotherapy, GROW-School for Oncology and Developmental Biology and Comprehensive Cancer Center Maastricht MUMC+, Maastricht University, Maastricht, The Netherlands Search for more papers by this author Meike de Wit Meike de Wit Department of Medical Oncology and Oncoproteomics Laboratory, Cancer Center Amsterdam, Vrije Universiteit Amsterdam, Amsterdam UMC, Amsterdam, The Netherlands Department of Pathology, Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Laura Moonen Laura Moonen Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Jaleesa R M Van der Meer Jaleesa R M Van der Meer Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Edith van den Boezem Edith van den Boezem Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Tim G A M Wolfs Tim G A M Wolfs Department of Pediatrics, GROW-School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands Search for more papers by this author David W Threadgill David W Threadgill Department of Molecular and Cellular Medicine, Texas A&M University Health Science Center, College Station, TX, USA Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA Search for more papers by this author Jeroen Demmers Jeroen Demmers Proteomics Center, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Remond J A Fijneman Remond J A Fijneman Department of Pathology, Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Connie R Jimenez Connie R Jimenez Department of Medical Oncology and Oncoproteomics Laboratory, Cancer Center Amsterdam, Vrije Universiteit Amsterdam, Amsterdam UMC, Amsterdam, The Netherlands Search for more papers by this author Pieter Vanden Berghe Pieter Vanden Berghe Laboratory for Enteric Neuroscience (LENS) and Translational Research Center for Gastrointestinal Disorders (TARGID), Department of Chronic Diseases, Metabolism and Ageing, KU Leuven, Leuven, Belgium Search for more papers by this author Kim M Smits Kim M Smits Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Kasper M A Rouschop Kasper M A Rouschop Department of Radiotherapy, GROW-School for Oncology and Developmental Biology and Comprehensive Cancer Center Maastricht MUMC+, Maastricht University, Maastricht, The Netherlands Search for more papers by this author Werend Boesmans Werend Boesmans orcid.org/0000-0002-2426-0451 Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Biomedical Research Institute (BIOMED), Hasselt University, Hasselt, Belgium Search for more papers by this author Robert M W Hofstra Robert M W Hofstra Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Veerle Melotte Corresponding Author Veerle Melotte [email protected] orcid.org/0000-0002-9459-123X Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Nathalie Vaes Nathalie Vaes orcid.org/0000-0001-9571-3449 Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Simone L Schonkeren Simone L Schonkeren orcid.org/0000-0003-4715-4173 Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Glenn Rademakers Glenn Rademakers Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Amy M Holland Amy M Holland Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Alexander Koch Alexander Koch Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Marion J Gijbels Marion J Gijbels Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Department of Molecular Genetics, Cardiovascular Research Institute Maastricht (CARIM), Maastricht, The Netherlands Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands Search for more papers by this author Tom G Keulers Tom G Keulers Department of Radiotherapy, GROW-School for Oncology and Developmental Biology and Comprehensive Cancer Center Maastricht MUMC+, Maastricht University, Maastricht, The Netherlands Search for more papers by this author Meike de Wit Meike de Wit Department of Medical Oncology and Oncoproteomics Laboratory, Cancer Center Amsterdam, Vrije Universiteit Amsterdam, Amsterdam UMC, Amsterdam, The Netherlands Department of Pathology, Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Laura Moonen Laura Moonen Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Jaleesa R M Van der Meer Jaleesa R M Van der Meer Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Edith van den Boezem Edith van den Boezem Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Tim G A M Wolfs Tim G A M Wolfs Department of Pediatrics, GROW-School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands Search for more papers by this author David W Threadgill David W Threadgill Department of Molecular and Cellular Medicine, Texas A&M University Health Science Center, College Station, TX, USA Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA Search for more papers by this author Jeroen Demmers Jeroen Demmers Proteomics Center, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Remond J A Fijneman Remond J A Fijneman Department of Pathology, Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Connie R Jimenez Connie R Jimenez Department of Medical Oncology and Oncoproteomics Laboratory, Cancer Center Amsterdam, Vrije Universiteit Amsterdam, Amsterdam UMC, Amsterdam, The Netherlands Search for more papers by this author Pieter Vanden Berghe Pieter Vanden Berghe Laboratory for Enteric Neuroscience (LENS) and Translational Research Center for Gastrointestinal Disorders (TARGID), Department of Chronic Diseases, Metabolism and Ageing, KU Leuven, Leuven, Belgium Search for more papers by this author Kim M Smits Kim M Smits Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Search for more papers by this author Kasper M A Rouschop Kasper M A Rouschop Department of Radiotherapy, GROW-School for Oncology and Developmental Biology and Comprehensive Cancer Center Maastricht MUMC+, Maastricht University, Maastricht, The Netherlands Search for more papers by this author Werend Boesmans Werend Boesmans orcid.org/0000-0002-2426-0451 Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Biomedical Research Institute (BIOMED), Hasselt University, Hasselt, Belgium Search for more papers by this author Robert M W Hofstra Robert M W Hofstra Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Veerle Melotte Corresponding Author Veerle Melotte [email protected] orcid.org/0000-0002-9459-123X Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Author Information Nathalie Vaes1, Simone L Schonkeren1, Glenn Rademakers1, Amy M Holland1, Alexander Koch1, Marion J Gijbels1,2,3, Tom G Keulers4, Meike de Wit5,6, Laura Moonen1, Jaleesa R M Van der Meer1, Edith van den Boezem1, Tim G A M Wolfs7, David W Threadgill8,9, Jeroen Demmers10, Remond J A Fijneman6, Connie R Jimenez5, Pieter Vanden Berghe11, Kim M Smits1, Kasper M A Rouschop4, Werend Boesmans1,12, Robert M W Hofstra13 and Veerle Melotte *,1,13 1Department of Pathology, GROW–School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands 2Department of Molecular Genetics, Cardiovascular Research Institute Maastricht (CARIM), Maastricht, The Netherlands 3Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands 4Department of Radiotherapy, GROW-School for Oncology and Developmental Biology and Comprehensive Cancer Center Maastricht MUMC+, Maastricht University, Maastricht, The Netherlands 5Department of Medical Oncology and Oncoproteomics Laboratory, Cancer Center Amsterdam, Vrije Universiteit Amsterdam, Amsterdam UMC, Amsterdam, The Netherlands 6Department of Pathology, Netherlands Cancer Institute, Amsterdam, The Netherlands 7Department of Pediatrics, GROW-School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands 8Department of Molecular and Cellular Medicine, Texas A&M University Health Science Center, College Station, TX, USA 9Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA 10Proteomics Center, Erasmus University Medical Center, Rotterdam, The Netherlands 11Laboratory for Enteric Neuroscience (LENS) and Translational Research Center for Gastrointestinal Disorders (TARGID), Department of Chronic Diseases, Metabolism and Ageing, KU Leuven, Leuven, Belgium 12Biomedical Research Institute (BIOMED), Hasselt University, Hasselt, Belgium 13Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands *Corresponding author. Tel: +31 43 3872210; Fax: +31 43 3876613; E-mail: [email protected] EMBO Reports (2021)22:e51913https://doi.org/10.15252/embr.202051913 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. SYNOPSIS Loss of enteric neuronal N-Myc Downstream-Regulated Gene 4 (Ndrg4) increases the release of the extracellular matrix proteins, Nidogen-1 and Fibulin-2, which accelerates intestinal growth in vitro and promotes colorectal cancer progression in vivo. Loss of Ndrg4 is associated with a more aggressive tumor behavior in murine models of colorectal cancer. Medium derived from primary Ndrg4-/- enteric nervous system cells accelerates the growth of murine intestinal organoids. Ndrg4-/- medium is enriched for two extracellular matrix components: Nidogen-1 and Fibulin-2. Soluble Nidogen-1 and Fibulin-2 stimulate human intestinal organoid proliferation and CRC cell migration in vitro. Introduction Colorectal cancer (CRC) is one of the most common lethal malignancies in the world even with major advances in screening and therapeutic strategies. This is partly attributed to the incomplete knowledge regarding the pathogenesis of CRC. Notably, it is well established that CRC not only arises from (epi-)genetic events in epithelial cells, since also cells and signals from the tumor micro-environment (TME) have been highlighted to be important for the initiation and progression of CRC (Colangelo et al, 2017). Several cell types within the gastrointestinal (GI) tract, e.g., fibroblast, immune, nerve, and endothelial cells, constitute the TME. Even though the significant impact of immune cells on (colorectal) carcinogenesis has been recognized for several years (Gutting et al, 2018), research into other TME components is still in its infancy. Interest in the role of the nervous system in tumorigenesis recently took the foreground (Venkatesh, 2019; Zahalka & Frenette, 2020) with the publication of several landmark papers describing the functional importance of nerves in skin (Peterson et al, 2015), prostate (Ayala et al, 2008; Magnon et al, 2013), breast (Kamiya et al, 2019), pancreatic (Bapat et al, 2011; Stopczynski et al, 2014), and gastric (Zhao et al, 2014; Hayakawa et al, 2017) cancer. These studies showed that surgical or pharmacological denervation of autonomic or (para-) sympathetic nerves suppresses cancer development. Remarkably, even with perineural invasion and neoneurogenesis being recognized as unfavorable prognostic factors in CRC (Albo et al, 2011; Knijn et al, 2016), knowledge regarding the contribution of the intrinsic nervous system of the gut: the enteric nervous system (ENS (Furness, 2012)) to CRC is sparse (Rademakers et al, 2017; Duchalais et al, 2018). The ENS is a complex neuroglial network embedded in the gut wall along the entire digestive tract. It communicates with other intestinal cells by transmitting signals like neurotransmitters, (neuro-) peptides, and hormones, in order to orchestrate GI functions (e.g., intestinal motility, blood flow and intestinal epithelial barrier integrity) and to maintain GI homeostasis (Furness, 2012). The importance of the ENS is highlighted by the wide range of enteric neuropathies (e.g., Hirschsprung disease) and inflammatory conditions that may arise from defective ENS development and/or functioning (Obermayr et al, 2013; Margolis & Gershon, 2016) and depicts some potential importance in colorectal carcinogenesis (Rademakers et al, 2017). Previously, we revealed that the N-Myc Downstream-Regulated Gene 4 (NDRG4), one of the most accurate DNA methylation-based biomarkers for the early detection of CRC (Melotte et al, 2009; Imperiale et al, 2014), is specifically expressed in the ENS (Vaes et al, 2017; Vaes et al, 2018). Even though NDRG4 is an established DNA methylation-based biomarker (Melotte et al, 2009; Imperiale et al, 2014), knowledge regarding its functional importance is sparse, yet seems to affect key developmental and carcinogenic processes, including cellular proliferation and differentiation (Melotte et al, 2010; Vaes et al, 2018; Schonkeren et al, 2019). Moreover, Ndrg4 has been shown to be involved in vesicle trafficking and thus possibly secretory actions (Benesh et al, 2013; Fontenas et al, 2016). Given the extensive crosstalk between the ENS and the intestinal epithelium, we here investigate whether enteric neuronal NDRG4 influences the intestinal (tumor) epithelium. Results and Discussion Loss of Ndrg4 has no major influence on intestinal morphology and physiology Prior to addressing the role of Ndrg4 in CRC, we sought to explore its overall impact in the intestinal tract. Therefore, we first performed in-depth histological analyses of intestinal segments of Ndrg4−/− and Ndrg4+/+ mice. Focusing on the epithelial cell layer, these analyses revealed a normal intestinal epithelial architecture: i.e., uniform presence of alkaline phosphatase in the enterocyte brush border and a similar number and distribution of Paneth cells (Lysozyme; P = 0.537) in the small intestine, and of neuroendocrine (chromogranin A; P = 0.304), goblet (PAS; P = 0.958), and proliferating cells (Ki67; P = 0.543) in the colon (Fig 1A). Within the small intestine, we observed comparable numbers and distributions of neuroendocrine, goblet, and proliferating cells (Fig EV1, EV2). The number of Lgr5-positive cells detected within small and large intestinal segments (Fig EV1D and E) were similar as previously described (Dehmer et al, 2011; Fernandez Vallone et al, 2020). However, no differences in the number of Lgr5-positive cells were observed between Ndrg4+/+ and Ndrg4−/− intestines. Figure 1. Loss of Ndrg4 does not alter intestinal morphology or physiology A. Representative microscopic views of Ndrg4+/+ and Ndrg4−/− murine intestinal sections (n = 4, 12 months of age) reveal an even distribution of alkaline phosphatase along the enterocyte brush border, and a similar number and distribution of Paneth cells (Lysozyme) in the small intestine; and neuroendocrine (chromogranin A), goblet (PAS+), and proliferating (Ki67) cells in the colon. Scale bars, 50 µm. B, C. Representative microscopic views of the myenteric plexus of Ndrg4+/+ and Ndrg4−/− murine colonic sections labeled with either TuJ1 and S100 (B, n = 3) or HuC/D (C, n = 6) did not reveal structural or organizational differences in the ganglionic network of Ndrg4+/+ and Ndrg4−/− mice. Scale bars, 100 µm. D. Quantification of the enteric neuronal cell number (HuC/D) shows a similar number of enteric neurons in the proximal and distal colon of Ndrg4+/+ and Ndrg4−/− mice (n = 6). E–I. Gastrointestinal motility assays reveal a similar whole-gut transit time (E; n = 12 versus 12), with a similar weight per stool and stool water content of the fecal pellets (F, G; n = 11 versus 10), a comparable small intestinal transit (H; n = 10 versus 12) and colonic propulsion time (I; n = 7 versus 6) in Ndrg4−/− compared to Ndrg4+/+ mice. Data information: All data are presented as mean ± SEM, with P-values determined using a two-tailed, unpaired t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Loss of Ndrg4 does not affect intestinal cell populations A–C. Quantification of intestinal cells within the ileum reveals a similar distribution and number of neuroendocrine (Chrom A, A), goblet (PAS+, B), and proliferating (Ki67, C) cells in Ndrg4+/+ (n = 3) and Ndrg4−/− mice (n = 3/4). D, E. Quantification of the Lgr5-positive cell population within the intervillus (IV) region in the small intestine (D) and per crypt in the colon (E) shows a similar number of intestinal stem cells in Ndrg4+/+ (n = 3) and Ndrg4−/− mice (n = 3/4). Data information: Data are represented as mean ± SEM and P-values calculated using the Mann–Whitney U-test (IBM SPSS statistics 25). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Deletion of Ndrg4 has no influence on mucosal innervation A. Representative images of intestinal Swiss roll sections of Ndrg4+/+ (n = 3) and Ndrg4−/− (n = 3/4) mice stained with anti-PGP9.5. Scale bars, 100 µm. B, C. Quantification of the percentage of villi with anti-PGP9.5 reactivity (B) and of the number of PGP9.5-positive cells/fibers per villus or crypt (C) shows a similar mucosal innervation in Ndrg4+/+ (n = 3) and Ndrg4−/− (n = 3/4) mice. Data information: Data are represented as mean ± SEM and P-values determined with the Mann–Whitney U-test (IBM SPSS statistics 25). Download figure Download PowerPoint The organization of the ENS was evaluated by antibody labeling for the neuron-specific class III beta-tubulin (Tuj1) and the S100 calcium-binding protein (S100). As shown in Fig 1B, this labeling did not reveal structural or organizational differences of the colonic myenteric plexus between Ndrg4+/+ and Ndrg4−/− mice (n = 3). Labeling with the pan-neuronal marker HuC/D showed that the density of enteric neurons in the myenteric plexus of the proximal and distal colon was similar between Ndrg4−/− and Ndrg4+/+ mice (Fig 1C and D, P = 0.906 and P = 0.811, respectively). Finally, also anti-PGP9.5 immunohistochemistry did not reveal any differences in the mucosal innervation of the small and large intestine of Ndrg4−/− and Ndrg4+/+ mice (Fig EV2). Even though loss of Ndrg4 did not impact intestinal morphology, we further assessed whether Ndrg4 knockdown affects intestinal physiology. Gastrointestinal motor activity was analyzed by determining the whole-gut and small intestinal transit using non-absorbable carmine red solution and colonic motility through assessment of the colonic propulsion of a glass bead. Consistent with their similar whole-gut transit time (Fig 1E; 194.3 min versus 149.5 min, P = 0.128), Ndrg4+/+ and Ndrg4−/− mice excreted a similar number of fecal pellets within the first hour of the assay (Mean of 7.60 versus 6.00 pellets, P = 0.276), with a similar weight and stool water content (Fig 1F; mean of 19.24 mg versus 18.14 mg, P = 0.665; Fig 1G; mean of 60.48% versus 64.34%, P = 0.328). Similarly, we did not observe statistically significant differences in the small intestinal transit (Fig 1H; 52.4% versus 71.7%, P = 0.094) or colonic propulsion (Fig 1I; 299.0 s versus 246.9 s, P = 0.449) between Ndrg4+/+ and Ndrg4−/− mice. Together these data indicate that loss of Ndrg4 has no major effects on intestinal morphology and physiology in healthy conditions. Deletion of Ndrg4 enhances intestinal adenoma growth Consequently, the influence of Ndrg4 on CRC was studied during colorectal carcinogenesis using a genetic (APCMin/+) (Heyer et al, 1999) and azoxymethane (AOM)-induced (Neufert et al, 2007) model. In both models, Ndrg4−/− mice are similar to Ndrg4+/+ mice with respect to their physical appearance and body weight (Fig 2A and B, PAPCMin/+=0.802, PAOM = 0.352). The homozygous deletion of Ndrg4 did not change the tumor incidence in the small intestine of APCMin/+ mice (Fig 2C, P = 0.764) nor in the colon of AOM-treated mice (Fig 2D, P = 0.903). However, the adenomas in the small intestine of Ndrg4−/−-APCMin/+ mice and in the colon of AOM-treated Ndrg4−/− mice were significantly enlarged compared to those of Ndrg4+/+-APCMin/+ mice (Fig 2E–G, P = 0.008) and AOM-treated Ndrg4+/+ mice (Fig 2F–H, P = 0.043). Moreover, the small intestinal adenomas of Ndrg4−/−-APCMin/+ mice and colonic adenomas of AOM-treated Ndrg4−/− mice were characterized by higher levels of an aggressiveness marker: i.e., a non-statistically significantly higher mean nuclear β-catenin immunoreactivity (APCmin/+=15.28 ± 2.75; AOM = 44.84 ± 13.11), than adenomas of Ndrg4+/+-APCMin/+ (8.90 ± 1.64) and AOM-treated Ndrg4+/+ (16.34 ± 4.74) mice, respectively (pAPCMin/+=0.060; pAOM = 0.085 (Wong et al, 2004)). The more aggressive phenotype of intestinal adenomas in Ndrg4−/− mice reflects the human situation, where de-differentiated tumor cells with increased nuclear β-catenin levels are known to have aggressive morphological features, which is associated with a poor prognosis (Wong et al, 2004). Interestingly, while AOM treatment normally mainly induces colonic adenoma formation (Neufert et al, 2007), 83.3% (10/12) of the AOM-treated Ndrg4−/− mice also developed small intestinal adenomas compared to just 31.1% (5/16) of Ndrg4+/+ mice (P = 0.019). Figure 2. Enhanced intestinal adenoma progression in Ndrg4−/− mice crossed with APCMin/+ mice (A, C, E, G) or treated with azoxymethane (AOM; B, D, F, H), but not after epithelial loss of Ndrg4 (I–L) A, B. Ndrg4+/+ and Ndrg4−/− mice have a similar bodyweight (n = 17 versus 14 in (A) and n = 25 versus 19 in (B). C, D. The total number of small intestinal (SI) and colonic adenomas that develops remains the same after deletion of Ndrg4 (n = 18 versus 15 in (C) and n = 25 versus 19 in (D)). E–H. Ndrg4−/− mice develop significantly enlarged small intestinal (SI) polyps (E, G) and colon adenomas (F, H). N = 10 versus 13 in (E) and n = 20 versus 17 in (F). I–L. In contrast, Ndrg4fl/fl and Ndrg4fl/fl-VillinCre mice have a similar bodyweight (I) and develop an equal number of adenomas (J) with an equivalent diameter (K, L). N = 13 versus 12 in (I), n = 13 versus 11 in (J) and n = 10 versus 8 in (K). Data information: All data are presented as mean ± SEM with P-values determined using a two-tailed, unpaired t-test. Scale bars in (G, H, L), 50 µm. Intestinal adenomas are delineated with a blue dotted line in (G, H, L). Download figure Download PowerPoint Even though intestinal expression of Ndrg4 is limited to the ENS (Vaes et al, 2017), we have to keep in mind that in this constitutive Ndrg4 knockout model, not only enteric neurons, but also peripheral nerves are deficient for Ndrg4. Given that extrinsic denervation can reduce carcinogenesis, as seen in other cancer types (Magnon et al, 2013; Stopczynski et al, 2014; Zhao et al, 2014), it may be speculated that Ndrg4 deficiency in peripheral nerves could contribute to the observed phenotype. Moreover, we aimed to ensure that the above-described effects were not mediated by low or undetectable levels of Ndrg4 in the intestinal epithelium using a conditional epithelial-specific Ndrg4 knockdown model: i.e., Ndrg4fl/fl-VillinCre mice crossed with APCMin/+ mice. We observed that Ndrg4fl/fl-VillinCre-APCMin/+ and Ndrg4fl/fl-APCMin/+ mice have a comparable body weight (Fig 2I, P = 0.717) and developed an equal number of adenomas in their small intestine (Fig 2J, P = 0.730). In contrast to the observations in the constitutive knockout model, the epithelial-specific knockdown of Ndrg4 did not alter the size (Fig 2K and L, P = 0.554) or aggressiveness (Ndrg4fl/fl-VillinCre-APCMin/+=18.16 ± 1.45 versus Ndrg4fl/fl-APCMin/+=18.59 ± 2.34; P = 0.879) of the small intestinal adenomas, confirming that the observed effects in the constitutive Ndrg4−/− models are caused by loss of non-epithelial Ndrg4. Medium from Ndrg4−/− ENS cultures enhances IEO growth and is enriched for Nidogen-1 and Fibulin-2 In view of the link between Ndrg4 and the vesicle-associated proteins Vamp3 (Benesh et al, 2013) and Rabac1 (Kim et al, 2012), and the role of Ndrg4 in vesicle trafficking in peripheral nerve cells affecting, e.g., Snap25 and Vamp2 (Fontenas et al, 2016), we aimed to identify whether Ndrg4 also affects vesicle transpo