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The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche.

Glucagon-like peptide-1-based (GLP-1-based) therapies improve glycemic control in patients with type 2 diabetes. While these agents augment insulin secretion, they do not mimic the physiological meal-related rise and fall of GLP-1 concentrations. Here, we tested the hypothesis that increasing the number of intestinal L cells, which produce GLP-1, is an alternative strategy to augment insulin responses and improve glucose tolerance. Blocking the NOTCH signaling pathway with the γ- secretase inhibitor dibenzazepine increased the number of L cells in intestinal organoid-based mouse and human culture systems and augmented glucose-stimulated GLP-1 secretion. In a high-fat diet-fed mouse model of impaired glucose tolerance and type 2 diabetes, dibenzazepine administration increased L cell numbers in the intestine, improved the early insulin response to glucose, and restored glucose tolerance. Dibenzazepine also increased K cell numbers, resulting in increased gastric inhibitory polypeptide (GIP) secretion. Using a GLP-1 receptor antagonist, we determined that the insulinotropic effect of dibenzazepine was mediated through an increase in GLP-1 signaling. Together, our data indicate that modulation of the development of incretin- producing cells in the intestine has potential as a therapeutic strategy to improve glycemic control., Introduction Glucagon-like peptide-1 (GLP-1) is a gut hormone with a powerful insulinotropic effect (1, 2). GLP-1 based therapies are widely used for the treatment of patients with type 2 diabetes [...]

The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon (1), stomach (2) and hair follicles (3). A three-dimensional culture system allows long-term clonal expansion of single [Lgr5.sup.+] stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture (2,4,5). A crucial component of the culture medium is the Wnt agonist RSPO1 (6), the recently discovered ligand of LGR5 (7,8). Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small [Lgr5-LacZ.sup.+] cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced [Lgr5.sup.+] cells generate hepatocytes and bile ducts invivo. Single [Lgr5.sup.+] cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate invitroand to generate functional hepatocytes upon transplantation into [Fah.sup.-/-] mice. These findings indicate that previous observations concerning [Lgr5.sup.+] stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation., Quiescent liver stem cells are believed to reside in biliary ducts (9). Sox9- and Fox11-based lineage tracing have proven the existence of such cells (10-13). In the adult liver, the [...]

[LGR5.sup.+] stem cells reside at crypt bottoms, intermingled with Paneth cells that provide Wnt, Notch and epidermal growth factor signals (1). Here we find that the related RNF43 and ZNRF3 transmembrane E3 ubiquitin ligases are uniquely expressed in [LGR5.sup.+] stem cells. Simultaneous deletion of the two genes encoding these proteins in the intestinal epithelium of mice induces rapidly growing adenomas containing high numbers of Paneth and [LGR5.sup.+] stem cells. In vitro, growth of organoids derived from these adenomas is arrested when Wnt secretion is inhibited, indicating a dependence of the adenoma stem cells on Wnt produced by adenoma Paneth cells. In the HEK293T human cancer cell line, expression of RNF43 blocks Wnt responses and targets surface-expressed frizzled receptors to lysosomes. In the RNF43-mutant colorectal cancer cell line HCT116, reconstitution of RNF43 expression removes its response to exogenous Wnt. We conclude that RNF43 and ZNRF3 reduce Wnt signals by selectively ubiquitinating frizzled receptors, thereby targeting these Wnt receptors for degradation., The small intestinal epithelium self renews every 4-5 days, making it the fastest proliferating mammalian tissue. Crypt base columnar (CBC) cells (2) are small cycling cells residing between the terminally [...]

Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, which are small cycling cells located at crypt bottoms (1,2). Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products such as lysozyme and cryptdins/defensins (3). Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells (4). Here we find a close physical association of Lgr5 stem cells with Paneth cells in mice, both in vivo and in vitro. [CD24.sup.+] Paneth cells express EGF, TGF-α, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells markedly improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, [CD24.sup.+] cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell., In a Matrigel-based culture system containing EGF, the Wnt agonist R-spondin 1 and the BMP inhibitor noggin (4), single Lgr5 stem cells autonomously grow into crypt-like structures with de novo [...]

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of about six cycling [Lgr5.sup.+] stem cells at the bottoms of small-intestinal crypts (1). Here we describe the establishment of long-term culture conditions under which single crypts undergo multiple crypt fission events, while simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted [Lgr5.sup.+] stem cells can also initiate these crypt-villus organoids. Tracing experiments indicate that the [Lgr5.sup.+] stem-cell hierarchy is maintained in organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche., The self-renewing epithelium of the small intestine is ordered into crypts and villi (2). Cells are newly generated in the crypts and are lost by apoptosis at the tips of [...]

Intestinal cancer is initiated by Wnt-pathway-activating mutations in genes such as adenomatous polyposis coli (APQ. As in most cancers, the cell of origin has remained elusive. In a previously established [...]

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. It is currently believed that four to six crypt stem cells reside at the +4 position immediately above [...]

Author(s): Johan H. van Es [1, 5]; Marielle E. van Gijn [1, 5]; Orbicia Riccio [2, 5]; Maaike van den Born [1]; Marc Vooijs [1]; Harry Begthel [1]; Miranda Cozijnsen [...]