Extended Data Fig. 1: Elevated MYC expression is required for Wnt-driven cancer in the liver and single mutant clones do not express senescence markers or the translational machinery required for tumorigenesis.
From: Hepatic zonation determines tumorigenic potential of mutant β-catenin
a, Mouse model recombining alleles at a low clonal density in the hepatocyte epithelium. The illustrations of the mouse and adenovirus were adapted from Medical Art Servier (https://servier.com) under a CC BY 4.0 licence. b, Representative image of RFP IHC; R26LSL-tdTomato liver 30 days post administration of AAV8.TBG.Cre (GC/ml = genome copy per ml), n = 3. c, Liver-to-body weight ratio (LW/BW). Biological replicates: Apcfl/fl (A) n = 6, Ctnnb1ex3/WT (B) n = 12, Rnf43fl/fl;Znrf3fl/fl (RZ) n = 6, R26LSL-MYC (M) n = 8, Ctnnb1ex3/WT;R26LSL-MYC (BM) n = 11. Bars are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons test. d, Tumour scoring. Biological replicates: A n = 6, B n = 12, RZ n = 6, M n = 9, BM n = 10. Bars are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons test. e, Whole-tissue normalised RNA-Seq read counts for Myc in indicated tissues from wild-type and Apcfl/fl mice. Box plots: centre line = median, upper (25th percentile) and lower quartiles (75th percentile) (box limits), and 1.5× interquartile range (whiskers) Biological replicates, moving left to right on the x-axis: n = 4,4,4,3,4,4,5,5,5,3,4,5. f, Top reactome pathways enriched in genes differentially upregulated in: AAV8.TBG.Cre-treated livers 10 days post induction (2 × 1011 GC/ml) BM versus B; and VillinCreERApcfl/fl versus VillinCreER (WT) intestines at day 4 post induction with tamoxifen. Dots are coloured according to their adjusted P-values, with the size of the dots representing the number of differentially expressed. Statistical significance was determined with the enrichPathway() function in R, using a two-sided hypergeometric model to determine the probability of geneset overlap occurring by chance. Multiple testing correction was applied using the Benjamini-Hochberg method. Biological replicates: liver, n = 3; intestinal, n = 16 WT and n = 15 Apcfl/fl. g-k, Ctnnb1ex/WT;R26LSL-MYC liver, 60 days post AAV8.TBG.Cre (6.4 × 108 GC/ml). g, Representative images of CTNNB1, BrdU, p21 and p16 IHC on serial sections, n = 3. Arrowheads represent single mutant clones and dashed black lines indicate a lesion. h, Representative images of MYC IHC, black arrowheads highlight MYCpos nuclei i, Spatial transcriptomics GSEA. Normalised Enrichment Score (NES) was calculated by normalising to the mean enrichment of random samples, and two-sided permutation testing with a Benjamini-Hochberg test was applied, p-adj = p adjusted value. j-k, In situ hybridisation for Notum and IHC for peEF2, p4E-BP1 (Thr37/46), pS6(Ser235/236), pS6(Ser240/244), and CCND1. Dashed outline highlights lesion. Representative images of n = 4 per group. All black scale bars = 100 μm. All red scale bars = 20 μm.
