Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors

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Abstract

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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Figure 1: LGR5+ stem cell genes: Rnf43 and Znrf3.
Figure 2: Strong proliferation of the Rnf43Znrf3 compound mutant intestine is accompanied by Wnt/β-catenin activation as well as stem cell and Paneth cell metaplasia.
Figure 3: RNF43 suppresses the Wnt/β-catenin pathway by reducing surface levels of frizzled receptors.
Figure 4: RNF43 promotes ubiquitin-mediated endocytosis of frizzled receptors.

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Primary accessions

Gene Expression Omnibus

Data deposits

The data for the microarray analysis have been deposited to the Gene Expression Omnibus under accession number GSE36497.

Change history

  • 29 August 2012

    An addition was made to the Acknowledgements.

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Acknowledgements

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.

Author information

B.-K.K., M.M.M. and H.C. conceived and designed the experiments. B.-K.K., M.S., I.J., D.E.S., M.v.d.W. and J.H.v.E. performed the experiments. T.Y.L., S.M. and A.J.R.H. performed the mass spectrometry analysis. B.-K.K., M.S., I.J., M.M.M. and H.C. analysed the data. B.-K.K., M.M.M. and H.C. wrote the manuscript.

Correspondence to Madelon M. Maurice or Hans Clevers.

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Competing interests

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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