Restriction of intestinal stem cell expansion and the regenerative response by YAP

Abstract

A remarkable feature of regenerative processes is their ability to halt proliferation once an organ's structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)--a protein known for its powerful growth-inducing and oncogenic properties--has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN-APC-GSK-3β complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.

Figure 1. YAP overabundance inhibits Wnt-mediated intestinal regeneration

a, H&E staining of doxycycline induced YAP-S127A small intestine at 2, 4 and 7 days. Inset of Ki67 stain representative of crypt proliferation. b, c, Wnt pathway activity and ISC presence at 2, 4 and 7 days post dox induction represented by CD44 (b) and Olfm4 (in-situ) (c). d, Heatmap of crypts isolated from control (n=3) and day 2 dox (n=3) treated mice displaying 540 downregulated genes in rank order. Labeled genes are examples known to be involved in Wnt signaling and the ISC niche. Original magnifications in each panel are 20x (a, b), 10x (c).

Figure 2. Loss of YAP leads to hyperactive Wnt signaling and expansion of the stem cell niche after injury or stimulation with RSpo1

a, H&E, CD44, SOX9 and Lysozyme staining of small intestinal crypts in control and cKO mice 1 week after irradiation. b, H&E of inert virus treated control and cKO small intestine. c, d, H&E staining (c) and CD44 IHC (d) in control and cKO mice 1 week after administration of adenovirus expressing RSpo1. e, GSEA of the ISC gene signature and intestine-specific β-catenin target gene sets. Black bars represent individual genes in rank order. f, Q-PCR validating several upregulated Wnt/ISC markers. g, h, IHC on small intestine of control and cKO small intestine for EGFP (expressed from the Lgr5 locus, inset is an untreated mouse) (g) and Lysozyme marking Paneth cells (h). i, Ectopic crypt formation in cKO mice treated with RSpo1 stained for H&E or the Wnt target CD44. Original magnifications in each panel are 20x (a), 10x (b-d), 20x (g) and 10x (h, i). Graphed data represent the mean and S.E.M. of 3 individual mice per genotype.

Figure 3. YAP restricts Wnt signaling by blocking DVL nuclear translocation

a, b, DVL2 IHC in control and cKO small intestine treated with Ad-Fc (a) and Ad-RSpo1-Fc (b). Insets are higher magnifications showing subcellular localization of DVL2. c, Immunofluorescent staining for DVL2 in confluent DLD1 cells after transfection with indicated siRNA. d, Expression analysis of Wnt target genes in DLD1 cells after transfection with siRNAs against YAP, DVL2+DVL3 (D2/3) or YAP + D2/3. e, Expression analysis of organoids infected with lentivirus encoding Dox-inducible DVL-NLS alone, or in a Villin-rtTA TetO-YAP-S127A background. Dox was given in culture medium for 4 days. All graphed data represent the mean and standard deviation of triplicate cultures.

Figure 4. YAP function in human colorectal cancer

a, Xenograft tumor formation of DLD1 cells infected with lentivirus encoding Dox-inducible YAP-WT or YAP-S127D mutant. Tumor photos are representative images. b, Heatmap representing the top 328 genes downregulated by YAP-S127D induction in xenograft assays (n=3 tumors per treatment group). Labeled genes are known β-catenin targets in human CRC. c, GSEA of TCF4-dependent CRC target genes. d, Overall survival analysis of patients with specific YAP staining patterns. YAP IHC was placed into 4 groups of staining patterns correlating with subcellular localization from 672 colorectal cancer patients: Cytoplasmic and nuclear (C (+) N (+)), Cytoplasmic (C (+) N (−)), Nuclear positive (C (−) N (+)) and Complete loss of staining (C (−) N (−)). e, YAP staining in low and high grade tumors. f, YAP loss is significantly associated with high grade tumors and stage IV disease (CI, confidence interval).