Wnt/β-catenin signaling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt-hypersensitive tumors that are susceptible to anti-Wnt-based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that induce β-catenin-mediated transcription, despite exhibiting retained Wnt receptor downregulation. These mutations interfere with a ubiquitin-independent suppressor role of the RNF43 cytosolic tail that involves Casein kinase 1 (CK1) binding and phosphorylation. Mechanistically, truncated RNF43 variants trap CK1 at the plasma membrane, thereby preventing β-catenin turnover and propelling ligand-independent target gene transcription. Gene editing of human colon stem cells shows that RNF43 truncations cooperate with p53 loss to drive a niche-independent program for self-renewal and proliferation. Moreover, these RNF43 variants confer decreased sensitivity to anti-Wnt-based therapy. Our data demonstrate the relevance of studying patient-derived mutations for understanding disease mechanisms and improved applications of precision medicine.

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Cell Biology and Oncode Institute Center for Molecular Medicine University Medical Center Utrecht Utrecht The Netherlands

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Department of Molecular Biology and Oncode Institute Faculty of Science Radboud Institute for Molecular Life Sciences Radboud University Nijmegen Nijmegen The Netherlands

Hubrecht Organoid Technology Utrecht The Netherlands

Institute of Molecular Biotechnology of the Austrian Academy of Sciences Vienna Austria

Laboratory for Experimental Oncology and Radiobiology and Oncode Institute Center for Experimental and Molecular Medicine Amsterdam UMC Cancer Center Amsterdam University of Amsterdam Amsterdam The Netherlands

Zobrazit více v PubMed

Anvarian Z, Nojima H, van Kappel EC, Madl T, Spit M, Viertler M, Jordens I, Low TY, van Scherpenzeel RC, Kuper I PubMed

Bilic J, Huang YL, Davidson G, Zimmermann T, Cruciat CM, Bienz M, Niehrs C (2007) Wnt induces LRP6 signalosomes and promotes dishevelled‐dependent LRP6 phosphorylation. Science 316: 1619–1622 PubMed

Carmon KS, Gong X, Lin Q, Thomas A, Liu Q (2011) R‐spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta‐catenin signaling. Proc Natl Acad Sci USA 108: 11452–11457 PubMed PMC

Chen B, Dodge ME, Tang W, Lu J, Ma Z, Fan CW, Wei S, Hao W, Kilgore J, Williams NS PubMed PMC

Chen PH, Chen X, Lin Z, Fang D, He X (2013) The structural basis of R‐spondin recognition by LGR5 and RNF43. Genes Dev 27: 1345–1350 PubMed PMC

Cliffe A, Hamada F, Bienz M (2003) A role of Dishevelled in relocating Axin to the plasma membrane during wingless signaling. Curr Biol 13: 960–966 PubMed

Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA‐seq aligner. Bioinformatics 29: 15–21 PubMed PMC

Drost J, van Jaarsveld RH, Ponsioen B, Zimberlin C, van Boxtel R, Buijs A, Sachs N, Overmeer RM, Offerhaus GJ, Begthel H PubMed

Durinck S, Spellman PT, Birney E, Huber W (2009) Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nat Protoc 4: 1184–1191 PubMed PMC

Elyada E, Pribluda A, Goldstein RE, Morgenstern Y, Brachya G, Cojocaru G, Snir‐Alkalay I, Burstain I, Haffner‐Krausz R, Jung S PubMed

Eto T, Miyake K, Nosho K, Ohmuraya M, Imamura Y, Arima K, Kanno S, Fu L, Kiyozumi Y, Izumi D, PubMed

Fenderico N, van Scherpenzeel RC, Goldflam M, Proverbio D, Jordens I, Kralj T, Stryeck S, Bass TZ, Hermans G, Ullman C PubMed PMC

Forbes SA, Beare D, Gunasekaran P, Leung K, Bindal N, Boutselakis H, Ding M, Bamford S, Cole C, Ward S PubMed PMC

Fuerer C, Nusse R (2010) Lentiviral vectors to probe and manipulate the Wnt signaling pathway. PLoS One 5: e9370 PubMed PMC

Fujii M, Matano M, Nanki K, Sato T (2015) Efficient genetic engineering of human intestinal organoids using electroporation. Nat Protoc 10: 1474–1485 PubMed

Fujii M, Shimokawa M, Date S, Takano A, Matano M, Nanki K, Ohta Y, Toshimitsu K, Nakazato Y, Kawasaki K PubMed

Furukawa T, Kuboki Y, Tanji E, Yoshida S, Hatori T, Yamamoto M, Shibata N, Shimizu K, Kamatani N, Shiratori K (2011) Whole‐exome sequencing uncovers frequent GNAS mutations in intraductal papillary mucinous neoplasms of the pancreas. Sci Rep 1: 161 PubMed PMC

Giannakis M, Hodis E, Jasmine Mu X, Yamauchi M, Rosenbluh J, Cibulskis K, Saksena G, Lawrence MS, Qian ZR, Nishihara R PubMed PMC

Gu Z, Eils R, Schlesner M (2016) Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32: 2847–2849 PubMed

Haber AL, Biton M, Rogel N, Herbst RH, Shekhar K, Smillie C, Burgin G, Delorey TM, Howitt MR, Katz Y PubMed PMC

Hao HX, Xie Y, Zhang Y, Charlat O, Oster E, Avello M, Lei H, Mickanin C, Liu D, Ruffner H PubMed

Janku F, Connolly R, LoRusso P, de Jonge M, Vaishampayan U, Rodon J, Argilés G, Myers A, Hsu Schmitz S‐F, Ji Y

Jiang X, Hao HX, Growney JD, Woolfenden S, Bottiglio C, Ng N, Lu B, Hsieh MH, Bagdasarian L, Meyer R PubMed PMC

Jiang X, Charlat O, Zamponi R, Yang Y, Cong F (2015) Dishevelled promotes Wnt receptor degradation through recruitment of ZNRF3/RNF43 E3 ubiquitin ligases. Mol Cell 58: 522–533 PubMed

Jiang X, Cong F (2016) Novel Regulation of Wnt Signaling at the Proximal Membrane Level. Trends Biochem Sci 41: 773–783 PubMed

Jung P, Sato T, Merlos‐Suarez A, Barriga FM, Iglesias M, Rossell D, Auer H, Gallardo M, Blasco MA, Sancho E PubMed

Kabiri Z, Greicius G, Madan B, Biechele S, Zhong Z, Zaribafzadeh H, Edison, Aliyev J, Wu Y, Bunte R PubMed

Koo BK, Stange DE, Sato T, Karthaus W, Farin HF, Huch M, van Es JH, Clevers H (2011) Controlled gene expression in primary Lgr5 organoid cultures. Nat Methods 9: 81–83 PubMed

Koo BK, Spit M, Jordens I, Low TY, Stange DE, van de Wetering M, van Es JH, Mohammed S, Heck AJ, Maurice MM PubMed

Koo BK, van Es JH, van den Born M, Clevers H (2015) Porcupine inhibitor suppresses paracrine Wnt‐driven growth of Rnf43;Znrf3‐mutant neoplasia. Proc Natl Acad Sci USA 112: 7548–7550 PubMed PMC

Lannagan TRM, Lee YK, Wang T, Roper J, Bettington ML, Fennell L, Vrbanac L, Jonavicius L, Somashekar R, Gieniec K PubMed PMC

de Lau W, Barker N, Low TY, Koo BK, Li VS, Teunissen H, Kujala P, Haegebarth A, Peters PJ, van de Wetering M PubMed

Lex A, Gehlenborg N, Strobelt H, Vuillemot R, Pfister H (2014) UpSet: visualization of Intersecting Sets. IEEE Trans Vis Comput Graph 20: 1983–1992 PubMed PMC

Li VS, Ng SS, Boersema PJ, Low TY, Karthaus WR, Gerlach JP, Mohammed S, Heck AJ, Maurice MM, Mahmoudi T PubMed

Liberzon A, Birger C, Thorvaldsdottir H, Ghandi M, Mesirov JP, Tamayo P (2015) The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst 1: 417–425 PubMed PMC

Lindeboom RG, Supek F, Lehner B (2016) The rules and impact of nonsense‐mediated mRNA decay in human cancers. Nat Genet 48: 1112–1118 PubMed PMC

Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA‐seq data with DESeq2. Genome Biol 15: 550 PubMed PMC

Lykke‐Andersen S, Jensen TH (2015) Nonsense‐mediated mRNA decay: an intricate machinery that shapes transcriptomes. Nat Rev Mol Cell Biol 16: 665–677 PubMed

Lyubimova A, Itzkovitz S, Junker JP, Fan ZP, Wu X, van Oudenaarden A (2013) Single‐molecule mRNA detection and counting in mammalian tissue. Nat Protoc 8: 1743–1758 PubMed

Mao J, Wang J, Liu B, Pan W, Farr GH 3rd, Flynn C, Yuan H, Takada S, Kimelman D, Li L PubMed

Marin O, Bustos VH, Cesaro L, Meggio F, Pagano MA, Antonelli M, Allende CC, Pinna LA, Allende JE (2003) A noncanonical sequence phosphorylated by casein kinase 1 in beta‐catenin may play a role in casein kinase 1 targeting of important signaling proteins. Proc Natl Acad Sci USA 100: 10193–10200 PubMed PMC

van Noort M, Clevers H (2002) TCF transcription factors, mediators of Wnt‐signaling in development and cancer. Dev Biol 244: 1–8 PubMed

Nusse R, Clevers H (2017) Wnt/beta‐catenin signaling, disease, and emerging therapeutic modalities. Cell 169: 985–999 PubMed

Ocadiz‐Ruiz R, Photenhauer AL, Hayes MM, Ding L, Fearon ER, Merchant JL (2017) ZBP‐89 function in colonic stem cells and during butyrate‐induced senescence. Oncotarget 8: 94330–94344 PubMed PMC

Ong CK, Subimerb C, Pairojkul C, Wongkham S, Cutcutache I, Yu W, McPherson JR, Allen GE, Ng CC, Wong BH PubMed

Peng WC, de Lau W, Forneris F, Granneman JC, Huch M, Clevers H, Gros P (2013a) Structure of stem cell growth factor R‐spondin 1 in complex with the ectodomain of its receptor LGR5. Cell Rep 3: 1885–1892 PubMed

Peng WC, de Lau W, Madoori PK, Forneris F, Granneman JC, Clevers H, Gros P (2013b) Structures of Wnt‐antagonist ZNRF3 and its complex with R‐spondin 1 and implications for signaling. PLoS One 8: e83110 PubMed PMC

Perez‐Riverol Y, Csordas A, Bai J, Bernal‐Llinares M, Hewapathirana S, Kundu DJ, Inuganti A, Griss J, Mayer G, Eisenacher M, PubMed PMC

Polakis P (2012) Wnt signaling in cancer. Cold Spring Harb Perspect Biol 4: a008052 PubMed PMC

Ramesh P, Kirov AB, Huels DJ, Medema JP (2018) Isolation, propagation, and clonogenicity of intestinal stem cells. Methods Mol Biol 2002: 61–73 PubMed

Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR‐Cas9 system. Nat Protoc 8: 2281 PubMed PMC

Rodon J, Argilés G, Connolly RM, Vaishampayan U, Jonge MD, Garralda E, Giannakis M, Smith DC, Dobson JR, McLaughlin M PubMed PMC

Roux KJ, Kim DI, Raida M, Burke B (2012) A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol 196: 801–810 PubMed PMC

Ryland GL, Hunter SM, Doyle MA, Rowley SM, Christie M, Allan PE, Bowtell DD, Australian Ovarian Cancer Study G , Gorringe KL, Campbell IG (2013) RNF43 is a tumour suppressor gene mutated in mucinous tumours of the ovary. J Pathol 229: 469–476 PubMed

Sato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, Van Houdt WJ, Pronk A, Van Gorp J, Siersema PD PubMed

Tamai K, Zeng X, Liu C, Zhang X, Harada Y, Chang Z, He X (2004) A mechanism for Wnt coreceptor activation. Mol Cell 13: 149–156 PubMed

Tauriello DV, Haegebarth A, Kuper I, Edelmann MJ, Henraat M, Canninga‐van Dijk MR, Kessler BM, Clevers H, Maurice MM (2010) Loss of the tumor suppressor CYLD enhances Wnt/beta‐catenin signaling through K63‐linked ubiquitination of Dvl. Mol Cell 37: 607–619 PubMed

Tauriello DV, Jordens I, Kirchner K, Slootstra JW, Kruitwagen T, Bouwman BA, Noutsou M, Rudiger SG, Schwamborn K, Schambony A PubMed PMC

Tsukiyama T, Fukui A, Terai S, Fujioka Y, Shinada K, Takahashi H, Yamaguchi TP, Ohba Y, Hatakeyama S (2015) Molecular role of RNF43 in canonical and noncanonical Wnt signaling. Mol Cell Biol 35: 2007–2023 PubMed PMC

Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, Mann M, Cox J (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13: 731–740 PubMed

Wu J, Jiao Y, Dal Molin M, Maitra A, de Wilde RF, Wood LD, Eshleman JR, Goggins MG, Wolfgang CL, Canto MI PubMed PMC

Yan HHN, Lai JCW, Ho SL, Leung WK, Law WL, Lee JFY, Chan AKW, Tsui WY, Chan ASY, Lee BCH PubMed

Zebisch M, Xu Y, Krastev C, MacDonald BT, Chen M, Gilbert RJ, He X, Jones EY (2013) Structural and molecular basis of ZNRF3/RNF43 transmembrane ubiquitin ligase inhibition by the Wnt agonist R‐spondin. Nat Commun 4: 2787 PubMed PMC

Zebisch M, Jones EY (2015) Crystal structure of R‐spondin 2 in complex with the ectodomains of its receptors LGR5 and ZNRF3. J Struct Biol 191: 149–155 PubMed PMC

Zeng X, Tamai K, Doble B, Li S, Huang H, Habas R, Okamura H, Woodgett J, He X (2005) A dual‐kinase mechanism for Wnt co‐receptor phosphorylation and activation. Nature 438: 873–877 PubMed PMC

Zhan T, Rindtorff N, Boutros M (2017) Wnt signaling in cancer. Oncogene 36: 1461–1473 PubMed PMC

Zhang L, Shay JW (2017) Multiple roles of APC and its therapeutic implications in colorectal cancer. J Natl Cancer Inst 109: djw332 PubMed PMC

Zhang LS, Lum L (2018) Chemical modulation of WNT signaling in cancer. Prog Mol Biol Transl Sci 153: 245–269 PubMed

Zhou Y, Lan J, Wang W, Shi Q, Lan Y, Cheng Z, Guan H (2013) ZNRF3 acts as a tumour suppressor by the Wnt signalling pathway in human gastric adenocarcinoma. J Mol Histol 44: 555–563 PubMed

Zou Y, Wang F, Liu FY, Huang MZ, Li W, Yuan XQ, Huang OP, He M (2013) RNF43 mutations are recurrent in Chinese patients with mucinous ovarian carcinoma but absent in other subtypes of ovarian cancer. Gene 531: 112–116 PubMed

PRICKLE3 protects VANGL proteins from CK1-mediated phosphorylation and RNF43-mediated degradation

Targeting Oncogenic Pathways in the Era of Personalized Oncology: A Systemic Analysis Reveals Highly Mutated Signaling Pathways in Cancer Patients and Potential Therapeutic Targets

RNF43 inhibits WNT5A-driven signaling and suppresses melanoma invasion and resistance to the targeted therapy

Zobrazit více v PubMed

GEO
GSE129288