Fig. 5. Effect of Grem1 on conventional Wnt driven tumorigenesis and pathogenic role in human sporadic traditional serrated adenomas.

(a) Double-mutant Vill-Grem1/Apc^Min/+ mice had a rapidly progressive intestinal phenotype, with a greater polyp burden than the parental strains at mean 57 days (Vill-Grem1 n = 5; Apc^Min/+ n = 7; Vill-Grem1/Apc^Min/+ n = 8, P_interaction<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/Apc^Min/+ animals (P_interaction < 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/Apc^Min/+ 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-CreER^T2/Grem1^fl/fl/Apc^Min/+ 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 ²⁸. 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.