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. 2015 Jan;21(1):62-70.
doi: 10.1038/nm.3750. Epub 2014 Dec 1.

Aberrant Epithelial GREM1 Expression Initiates Colonic Tumorigenesis From Cells Outside the Stem Cell Niche

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Free PMC article

Aberrant Epithelial GREM1 Expression Initiates Colonic Tumorigenesis From Cells Outside the Stem Cell Niche

Hayley Davis et al. Nat Med. .
Free PMC article

Abstract

Hereditary mixed polyposis syndrome (HMPS) is characterized by the development of mixed-morphology colorectal tumors and is caused by a 40-kb genetic duplication that results in aberrant epithelial expression of the gene encoding mesenchymal bone morphogenetic protein antagonist, GREM1. Here we use HMPS tissue and a mouse model of the disease to show that epithelial GREM1 disrupts homeostatic intestinal morphogen gradients, altering cell fate that is normally determined by position along the vertical epithelial axis. This promotes the persistence and/or reacquisition of stem cell properties in Lgr5-negative progenitor cells that have exited the stem cell niche. These cells form ectopic crypts, proliferate, accumulate somatic mutations and can initiate intestinal neoplasia, indicating that the crypt base stem cell is not the sole cell of origin of colorectal cancer. Furthermore, we show that epithelial expression of GREM1 also occurs in traditional serrated adenomas, sporadic premalignant lesions with a hitherto unknown pathogenesis, and these lesions can be considered the sporadic equivalents of HMPS polyps.

Figures

Fig. 1
Fig. 1. Human HMPS polyps
(a) H&E of HMPS polyp showing mixed adenomatous, serrated and dilated cyst morphology and close up of ectopic crypts growing orthogonally to crypt axis. (b) Dysplastic cells (black arrowhead) emerging from an ectopic crypt rather than from the crypt base. (c) Immunostaining of HMPS polyps showing patchy loss of p-SMAD1,5,8 stain, Ki67 stain in proliferating ectopic crypt foci cells and ectopic lysozyme stain in dysplastic crypts (upper panels). Sox9 and EPHB2 immunostaining is increased whereas staining for the differentiation marker CK20, is lost in the ectopic crypt foci of HMPS polyps (lower panels) (n > 10 polyps for all stains). (d) Candidate gene (epi)genetic mutation spectra in HMPS polyps. (e) Laser-capture isolation of individual crypts across HMPS lesions. Spatial distinction of mutant clones allowed inference of mutation timing (see also Supplementary Fig. 2). Scale bars are 100 μm unless stated.
Fig. 2
Fig. 2. Vill-Grem1 mouse phenotype
(a) Macroscopic and microscopic phenotyping of Vill-Grem1 versus wild-type mouse intestine. Significant differences were noted in intestinal length and diameter (n = 10, P < 0.001, t-test), villus (n = 50) and colonic crypt (n = 100) cell count (P = 0.001, t-test), villus proliferating cell proportion (n = 50, P = 0.03, t-test), small bowel (n = 50, P < 0.002, t-test) and colonic goblet cells count (n = 50, P = 0.01, t-test) and proportion of lysozyme positive Paneth cells on the villus (n = 50, P = 0.038, t-test). In all cases: student t-test using two-tailed, unpaired and unequal variance was employed. The data from each group did not significantly deviate from a normal distribution (Shapiro-Wilk test). (b) Top left: Wildtype mouse small bowel 1 (SB1). Top middle: SB1 of a three-month old Vill-Grem1 mouse, with widened villi containing intravillus ectopic crypts (black arrowheads). Top right: dysplastic polyp formation in a seven-month old Vill-Grem1 animal exhibiting mixed morphology with serrated (inset), adenomatous and dilated cyst phenotypic regions. Lower left: wildtype mouse colon. Lower middle: early colonic lesion with luminal surface dysplasia distant from the crypt basal stem-cell niche. Lower right: colonic polyps in a seven-month old mice with mixed crypt morphology (serrated crypts, inset). (c) In situ hybridization (ISH) for mouse Grem1 with normal intestinal expression of Grem1 exclusively from the sub-crypt myofibroblasts (black arrowheads). Aberrant epithelial expression is seen in early small intestinal and colonic lesions from the Vill-Grem1 mouse. (d) Immunohistochemical analysis Vill-Grem1 versus wildtype small intestine shows loss of p-Smad1,5,8 throughout the crypt-villus axis, with Ki67, Sox9 and EphB2 staining all present in the villus ectopic crypts. Ck20 differentiation marker staining was lost in villus ECFs (n > 20 polyps for all stains). (e) Dysplasia arising in intravillus ectopic crypts (black arrowhead) with active cell proliferation correlating with p16Ink4a stain loss. (f) Grem1 ISH in Vill-Grem1 mouse tissue shows loss of Grem1 expression in a large polyp, which correlates with nuclear β–catenin staining, resulting from a Ctnnb1 p.T41I mutation. Scale bars are 100 μm.
Fig. 3
Fig. 3. Gene expression analysis of separated crypt/villus compartments in Vill-Grem1 mice
(a) Vill-Grem1 mice were crossed with Lgr5-EGFP-IRES-CreERT2 mice and tissues stained with anti-GFP antibody. No Lgr5/EGFP positive cells were seen in the ectopic crypts on the villi (white arrowheads) but were found as expected in the underlying crypt bases (black arrowheads and inset). (b) qRT-PCR analysis of villus stem-cell marker expression in individual Vill-Grem1 villi versus wild-type littermate villi. There was a significant increase in expression of Sox9 (n = 10, P = 0.01, t-test) in Vill-Grem1 villi. (c) Villi from Vill-Grem1 and wild-type littermates underwent gene expression microarray and GSEA analysis using established gene program sets. GSEA plots shown are for Vill-Grem1 (n = 5) versus wildtype villi (n = 6). Enrichment score is calculated using Kolomogrov-Smirnov test (Supplementary reference). P-value is calculated using a permutation test. Scale bars are 100 μm.
Fig. 4
Fig. 4. In vitro villus cell clonogenicity
(a) Non-dysplastic small intestinal crypts from wild-type and Vill-Grem1 mice readily formed branching enteroids with similar efficiency in standard conditions (ENS media). Vill-Grem1 crypts also formed lasting enteroids in the absence of Noggin in the medium (ES media), although the effect of endogenous Grem1 expression could be overcome by addition of competing recombinant BMP ligands 2, 4, and 7 (ES+rBMP2,4,7 media). Culture of ApcMin polyp tissue resulted in non-branching spheroid generation in all media conditions, indicating dispensability of BMP antagonist in somatically mutant cells. Villi dissected from wild-type and ApcMin/+ mice dissociated and died regardless of media supplementation. Villi dissected from Vill-Grem1 mice (in Wnt3A supplemented (ENSW) media) and Vill-Grem1/ApcMin mice (in all media conditions) could form proliferative spheroid lesions that could be repeatedly passaged and propagated long-term. Vill-Grem1/ApcMin mice generated villus spheroids efficiently in the absence of Noggin as a media supplement and spheroid generation could not be abrogated by addition of competing BMP ligands indicating BMP antagonist independent growth. Abbreviations of media supplements E: Epidermal growth factor; N: Noggin; S: R-Spondin; W: Wnt3A. (b) qRT-PCR analysis of effect of mouse genotype on villus Wnt target gene expression (versus wild-type) showed that a single Apc hit was sufficient to increase endogenous individual villus Wnt target gene expression including the stem-cell markers Lgr5 and Ascl2. Expression of the progenitor cell markers Sox9 and EphB2 was significantly increased in Vill-Grem1 animals (n = 10 villi for each strain, P < 0.01, t-test) and emergent spheroids had a further increase in both Wnt target and progenitor marker genes. (c) Villus spheroid immunostain showing nuclear β–catenin staining, membranous EphB2 and nuclear Sox9 stain. (d) Selection of somatically mutant cells on the villus of Vill-Grem1/ApcMin/+ mice. Extracted non-dysplastic villi entering into culture retained a residual wild-type Apc allele, whilst emergent spheroids had lost this allele. (e) There was an increase in the percentage of villi transforming into clonogenic spheroids with increasing age of Vill-Grem1/ApcMin/+ mice. Histology review showed that this increase correlated with the emergence of villus ectopic crypts (black arrowheads). Scale bars are 100 μm.
Fig. 5
Fig. 5. Effect of Grem1 on conventional Wnt driven tumorigenesis and pathogenic role in human sporadic traditional serrated adenomas
(a) Double-mutant Vill-Grem1/ApcMin/+ mice had a rapidly progressive intestinal phenotype, with a greater polyp burden than the parental strains at mean 57 days (Vill-Grem1 n = 5; ApcMin/+ n = 7; Vill-Grem1/ApcMin/+ n = 8, Pinteraction<0.002 for all regions of the bowel, generalized linear regression incorporating a multiplicative interaction term between Apc mutation and Grem1 status). Polyp size was also significantly greater in Vill-Grem1/ApcMin/+ animals (Pinteraction < 0.001, linear regression, data not shown). The data from each group did not significantly deviate from a normal distribution (Shapiro-Wilk test) (b) Vill-Grem1/ApcMin/+ mouse polyps had central dysplastic areas with a sharp cut off between enclosing serrated epithelium. Laser dissection of the different morphological types revealed Apc loss of heterozygosity in the dysplastic tissue. (c) Conditional inactivation of physiological Grem1 significantly reduced conventional, Wnt-initiated tumourigenesis in CAGG-CreERT2/Grem1fl/fl/ApcMin/+ mice at mean 248 days (n = 4 mice for test and non-injected control, P = 0.027, t-test unpaired with unequal variances). The data from each group did not significantly deviate from a normal distribution (Shapiro-Wilk test). (d) Top panel: above-median expression of GREM1 in the AMC-AJCCII-90 human CRC set was associated with a significant reduction in disease-free survival (P = 0.0162, log rank test). Bottom panel: the Cancer Genome Atlas (TCGA) RNAseq data was used to classify tumours into the three colon cancer subtypes (CCS) described by De Sousa et al . A highly significant correlation was seen between CCS3 subtype cancers and high whole tumour GREM1 expression (P < 0.0001, ANOVA). (e) qRT-PCR measurement of known BMP antagonists from individual fresh TSAs (22 crypts from four different lesions) compared with surrounding normal crypts. (f) In situ hybridsation for GREM1 in archival human TSA samples showed aberrant epithelial GREM1 mRNA expression in TSA epithelium (brown dots). Scale bars are 100 μm.
Fig. 6
Fig. 6. Model summarising the proposed mechanistic consequences of disrupted GREM1 morphogen gradients
Aberrant ectopic epithelial expression of GREM1 disrupts the coupling of cell-fate determination to position along the crypt-villus axis and allows persistance and expansion of an Lgr5 negative progenitor cell pool (characterized by aberrant SOX9 and EPHB2 expression) which form orthogonal ectopic crypt foci. Aberrant cell-proliferation in this progenitor cell population within these ECFs predisposes towards somatic (epi)mutation events and gives rise to neoplastic transformation (inset boxes). In vitro, the persistence of somatically mutated progenitor cells in dissected villi gives rise to clonogenic tumour spheroid growth from cells that have exited the crypt basal stem-cell niche. Coloured bars represent morphogen and gene expression gradients in the normal and pathological states. Blue dots represent physiological Grem1 expression from peri-cryptal myofibroblasts. CBC stem cells are coloured red.

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