LGR5+ stem cells reside at crypt bottoms, intermingled with Paneth cells that provide Wnt, Notch and epidermal growth factor signals1. Here we find that the related RNF43 and ZNRF3 transmembrane E3 ubiquitin ligases are uniquely expressed in LGR5+ 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+ 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.
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Sato, T. et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2011)
Cheng, H. & Leblond, C. P. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. I. Columnar cell. Am. J. Anat. 141, 461–479 (1974)
Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007)
Sato, T. et al. Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009)
Yui, S. et al. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell. Nature Med. 18, 618–623 (2012)
van der Flier, L. G. et al. Transcription factor achaete scute-like 2 controls intestinal stem cell fate. Cell 136, 903–912 (2009)
El Marjou, F. et al. Tissue-specific and inducible Cre-mediated recombination in the gut epithelium. Genesis 39, 186–193 (2004)
Ireland, H. et al. Inducible cre-mediated control of gene expression in the murine gastrointestinal tract: effect of loss of β-catenin. Gastroenterology 126, 1236–1246 (2004)
Sansom, O. J. et al. Myc deletion rescues Apc deficiency in the small intestine. Nature 446, 676–679 (2007)
van Es, J. H. et al. Wnt signalling induces maturation of Paneth cells in intestinal crypts. Nature Cell Biol. 7, 381–386 (2005)
Carmon, K. S., Gong, X., Lin, Q., Thomas, A. & Liu, Q. R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/β-catenin signaling. Proc. Natl Acad. Sci. USA 108, 11452–11457 (2011)
de Lau, W. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011)
Kazanskaya, O. et al. The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development. Development 135, 3655–3664 (2008)
Chen, B. et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nature Chem. Biol. 5, 100–107 (2009)
Koo, B. K. et al. Controlled gene expression in primary Lgr5 organoid cultures. Nature Methods 9, 81–83 (2012)
Liu, G., Bafico, A., Harris, V. K. & Aaronson, S. A. A novel mechanism for Wnt activation of canonical signaling through the LRP6 receptor. Mol. Cell. Biol. 23, 5825–5835 (2003)
Ivanov, I., Lo, K. C., Hawthorn, L., Cowell, J. K. & Ionov, Y. Identifying candidate colon cancer tumor suppressor genes using inhibition of nonsense-mediated mRNA decay in colon cancer cells. Oncogene 26, 2873–2884 (2007)
Li, V. S. et al. Wnt signaling through inhibition of β-catenin degradation in an intact Axin1 complex. Cell 149, 1245–1256 (2012)
Haglund, K. & Dikic, I. The role of ubiquitylation in receptor endocytosis and endosomal sorting. J. Cell Sci. 125, 265–275 (2012)
Mukai, A. et al. Balanced ubiquitylation and deubiquitylation of Frizzled regulate cellular responsiveness to Wg/Wnt. EMBO J. 29, 2114–2125 (2010)
Van der Flier, L. G. et al. The intestinal Wnt/TCF signature. Gastroenterology 132, 628–632 (2007)
Yagyu, R. et al. A novel oncoprotein RNF43 functions in an autocrine manner in colorectal cancer. Int. J. Oncol. 25, 1343–1348 (2004)
Hao, H. X. et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature 485, 195–200 (2012)
Niida, A. et al. DKK1, a negative regulator of Wnt signaling, is a target of the β-catenin/TCF pathway. Oncogene 23, 8520–8526 (2004)
Shimomura, Y. et al. APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex. Nature 464, 1043–1047 (2010)
Jho, E. H. et al. Wnt/β-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Mol. Cell. Biol. 22, 1172–1183 (2002)
Berndt, J. D. et al. Mindbomb 1, an E3 ubiquitin ligase, forms a complex with RYK to activate Wnt/β-catenin signaling. J. Cell Biol. 194, 737–750 (2011)
Wu, J. et al. Whole-exome sequencing of neoplastic cysts of the pancreas reveals recurrent mutations in components of ubiquitin-dependent pathways. Proc. Natl Acad. Sci. USA 108, 21188–21193 (2011)
Ong, C. K. e. t. a. l. Exome sequencing of liver fluke-associated cholangiocarcinoma. Nature Genet. 44, 690–693 (2012)
March, H. N. et al. Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis. Nature Genet. 43, 1202–1209 (2011)
Tauriello, D. V. et al. Loss of the tumor suppressor CYLD enhances Wnt/β-catenin signaling through K63-linked ubiquitination of Dvl. Mol. Cell 37, 607–619 (2010)
We thank A. A. Rolf, I. Kuper, D. V. F. Tauriello, M. van den Born, C. Kroon-Veenboer, H. Begthel, J. Korving and S. van den Brink for technical assistance, L. Lum for providing IWP1 and S. Bartfeld for the schematic drawing. This work was funded in part by grants from the European Research Council, EU/232814-StemCeLLMark and the National Research Foundation of Korea, NRF-2011-357-C00093 (B.-K.K.); EU/Health-F4-2007-200720 (M.v.d.W.); The Centre van Biomedical Genetics (D.E.S.); Ti Pharma/T3-106 (J.H.v.E.); the European Research Council, ERC-StG no.242958 (M.M.M.) and the KNAW/3V-fund.
H.C. is an inventor on several patents involving the culture system in this paper. The other authors declare no competing financial interests.
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