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Small-molecule inhibition of Wnt signaling through activation of casein kinase 1α

Nature Chemical Biology volume 6, pages 829–836 (2010)Cite this article

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Abstract

Wnt/β-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote β-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC50 of ∼10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and β-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or β-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.

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Figure 1: Xenopus egg extract screen identifies pyrvinium as an inhibitor of Wnt signaling.
Figure 2: Pyrvinium inhibits Wnt signaling in vivo.
Figure 3: CK1α is the critical target of pyrvinium.
Figure 4: Pyrvinium promotes Pygopus degradation.
Figure 5: Pyrvinium selectively decreases cell viability of colon cancer cells with activating mutations in the Wnt pathway.

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References

  1. Yamamoto, H. et al. Phosphorylation of axin, a Wnt signal negative regulator, by glycogen synthase kinase-3beta regulates its stability. J. Biol. Chem. 274, 10681–10684 (1999).

    Article  CAS  Google Scholar 

  2. Tolwinski, N.S. et al. Wg/Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity. Dev. Cell 4, 407–418 (2003).

    Article  CAS  Google Scholar 

  3. Kofron, M. et al. Wnt11/beta-catenin signaling in both oocytes and early embryos acts through LRP6-mediated regulation of axin. Development 134, 503–513 (2007).

    Article  CAS  Google Scholar 

  4. Cselenyi, C.S. et al. LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3's phosphorylation of beta-catenin. Proc. Natl. Acad. Sci. USA 105, 8032–8037 (2008).

    Article  CAS  Google Scholar 

  5. Barker, N. & Clevers, H. Mining the Wnt pathway for cancer therapeutics. Nat. Rev. Drug Discov. 5, 997–1014 (2006).

    Article  CAS  Google Scholar 

  6. Kinzler, K.W. et al. Identification of FAP locus genes from chromosome 5q21. Science 253, 661–665 (1991).

    Article  CAS  Google Scholar 

  7. Klaus, A. & Birchmeier, W. Wnt signalling and its impact on development and cancer. Nat. Rev. Cancer 8, 387–398 (2008).

    Article  CAS  Google Scholar 

  8. Salic, A., Lee, E., Mayer, L. & Kirschner, M.W. Control of beta-catenin stability: reconstitution of the cytoplasmic steps of the wnt pathway in Xenopus egg extracts. Mol. Cell 5, 523–532 (2000).

    Article  CAS  Google Scholar 

  9. Hempelmann, E. Hemozoin biocrystallization in Plasmodium falciparum and the antimalarial activity of crystallization inhibitors. Parasitol. Res. 100, 671–676 (2007).

    Article  Google Scholar 

  10. Downey, A.S., Chong, C.R., Graczyk, T.K. & Sullivan, D.J. Efficacy of pyrvinium pamoate against Cryptosporidium parvum infection in vitro and in a neonatal mouse model. Antimicrob. Agents Chemother. 52, 3106–3112 (2008).

    Article  CAS  Google Scholar 

  11. Clevers, H. Wnt/beta-catenin signaling in development and disease. Cell 127, 469–480 (2006).

    Article  CAS  Google Scholar 

  12. Xu, Q. et al. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair. Cell 116, 883–895 (2004).

    Article  CAS  Google Scholar 

  13. Lustig, B. et al. Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors. Mol. Cell. Biol. 22, 1184–1193 (2002).

    Article  CAS  Google Scholar 

  14. Jho, E.H. et al. Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Mol. Cell. Biol. 22, 1172–1183 (2002).

    Article  CAS  Google Scholar 

  15. de la Roche, M., Worm, J. & Bienz, M. The function of BCL9 in Wnt/beta-catenin signaling and colorectal cancer cells. BMC Cancer 8, 199 (2008).

    Article  Google Scholar 

  16. He, T.C. et al. Identification of c-MYC as a target of the APC pathway. Science 281, 1509–1512 (1998).

    Article  CAS  Google Scholar 

  17. Miyamoto, D.T., Perlman, Z.E., Burbank, K.S., Groen, A.C. & Mitchison, T.J. The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles. J. Cell Biol. 167, 813–818 (2004).

    Article  CAS  Google Scholar 

  18. Larabell, C.A. et al. Establishment of the dorso-ventral axis in Xenopus embryos is presaged by early asymmetries in beta-catenin that are modulated by the Wnt signaling pathway. J. Cell Biol. 136, 1123–1136 (1997).

    Article  CAS  Google Scholar 

  19. De Robertis, E.M. & Kuroda, H. Dorsal-ventral patterning and neural induction in Xenopus embryos. Annu. Rev. Cell Dev. Biol. 20, 285–308 (2004).

    Article  CAS  Google Scholar 

  20. Bhanot, P. et al. A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 382, 225–230 (1996).

    Article  CAS  Google Scholar 

  21. Whangbo, J. & Kenyon, C. A Wnt signaling system that specifies two patterns of cell migration in C. elegans. Mol. Cell 4, 851–858 (1999).

    Article  CAS  Google Scholar 

  22. Gleason, J.E., Korswagen, H.C. & Eisenmann, D.M. Activation of Wnt signaling bypasses the requirement for RTK/Ras signaling during C. elegans vulval induction. Genes Dev. 16, 1281–1290 (2002).

    Article  CAS  Google Scholar 

  23. Korswagen, H.C. et al. The Axin-like protein PRY-1 is a negative regulator of a canonical Wnt pathway in C. elegans. Genes Dev. 16, 1291–1302 (2002).

    Article  CAS  Google Scholar 

  24. Lewis, J.A., Wu, C.H., Berg, H. & Levine, J.H. The genetics of levamisole resistance in the nematode Caenorhabditis elegans. Genetics 95, 905–928 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Liu, C. et al. Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108, 837–847 (2002).

    Article  CAS  Google Scholar 

  26. Gao, Z.H., Seeling, J.M., Hill, V., Yochum, A. & Virshup, D.M. Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex. Proc. Natl. Acad. Sci. USA 99, 1182–1187 (2002).

    Article  CAS  Google Scholar 

  27. Pierre, M. & Nunez, J. Multisite phosphorylation of tau proteins from rat brain. Biochem. Biophys. Res. Commun. 115, 212–219 (1983).

    Article  CAS  Google Scholar 

  28. Knight, Z.A. & Shokat, K.M. Features of selective kinase inhibitors. Chem. Biol. 12, 621–637 (2005).

    Article  CAS  Google Scholar 

  29. Price, M.A. CKI, there's more than one: casein kinase I family members in Wnt and Hedgehog signaling. Genes Dev. 20, 399–410 (2006).

    Article  CAS  Google Scholar 

  30. Bidère, N. et al. Casein kinase 1alpha governs antigen-receptor-induced NF-kappaB activation and human lymphoma cell survival. Nature 458, 92–96 (2009).

    Article  Google Scholar 

  31. Huang, S.M. et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461, 614–620 (2009).

    Article  CAS  Google Scholar 

  32. Chen, B. et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nat. Chem. Biol. 5, 100–107 (2009).

    Article  CAS  Google Scholar 

  33. Sparks, A.B., Morin, P.J., Vogelstein, B. & Kinzler, K.W. Mutational analysis of the APC/beta-catenin/Tcf pathway in colorectal cancer. Cancer Res. 58, 1130–1134 (1998).

    CAS  PubMed  Google Scholar 

  34. Polakis, P. Wnt signaling and cancer. Genes Dev. 14, 1837–1851 (2000).

    CAS  PubMed  Google Scholar 

  35. Aoki, M., Hecht, A., Kruse, U., Kemler, R. & Vogt, P.K. Nuclear endpoint of Wnt signaling: neoplastic transformation induced by transactivating lymphoid-enhancing factor 1. Proc. Natl. Acad. Sci. USA 96, 139–144 (1999).

    Article  CAS  Google Scholar 

  36. Faux, M.C. et al. Restoration of full-length adenomatous polyposis coli (APC) protein in a colon cancer cell line enhances cell adhesion. J. Cell Sci. 117, 427–439 (2004).

    Article  CAS  Google Scholar 

  37. Hämmerlein, A., Weiske, J. & Huber, O. A second protein kinase CK1-mediated step negatively regulates Wnt signalling by disrupting the lymphocyte enhancer factor-1/beta-catenin complex. Cell. Mol. Life Sci. 62, 606–618 (2005).

    Article  Google Scholar 

  38. Kramps, T. et al. Wnt/wingless signaling requires BCL9/legless-mediated recruitment of pygopus to the nuclear beta-catenin-TCF complex. Cell 109, 47–60 (2002).

    Article  CAS  Google Scholar 

  39. Thompson, B., Townsley, F., Rosin-Arbesfeld, R., Musisi, H. & Bienz, M. A new nuclear component of the Wnt signalling pathway. Nat. Cell Biol. 4, 367–373 (2002).

    Article  CAS  Google Scholar 

  40. Parker, D.S., Jemison, J. & Cadigan, K.M. Pygopus, a nuclear PHD-finger protein required for Wingless signaling in Drosophila. Development 129, 2565–2576 (2002).

    CAS  PubMed  Google Scholar 

  41. Knippschild, U. et al. The casein kinase 1 family: participation in multiple cellular processes in eukaryotes. Cell. Signal. 17, 675–689 (2005).

    Article  CAS  Google Scholar 

  42. Esumi, H., Lu, J., Kurashima, Y. & Hanaoka, T. Antitumor activity of pyrvinium pamoate, 6-(dimethylamino)-2-[2-(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)ethenyl]-1-me thyl-quinolinium pamoate salt, showing preferential cytotoxicity during glucose starvation. Cancer Sci. 95, 685–690 (2004).

    Article  CAS  Google Scholar 

  43. Rosenbluth, J.M., Mays, D.J., Pino, M.F., Tang, L.J. & Pietenpol, J.A. A gene signature-based approach identifies mTOR as a regulator of p73. Mol. Cell. Biol. 28, 5951–5964 (2008).

    Article  CAS  Google Scholar 

  44. Inoki, K. et al. TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell 126, 955–968 (2006).

    Article  CAS  Google Scholar 

  45. Yu, D.H. et al. Pyrvinium targets the unfolded protein response to hypoglycemia and its anti-tumor activity is enhanced by combination therapy. PLoS ONE 3, e3951 (2008).

    Article  Google Scholar 

  46. Jones, J.O. et al. Non-competitive androgen receptor inhibition in vitro and in vivo. Proc. Natl. Acad. Sci. USA 106, 7233–7238 (2009).

    Article  CAS  Google Scholar 

  47. Matschinsky, F.M. Assessing the potential of glucokinase activators in diabetes therapy. Nat. Rev. Drug Discov. 8, 399–416 (2009).

    Article  CAS  Google Scholar 

  48. Mithani, S.K. et al. Smad3 has a critical role in TGF-beta-mediated growth inhibition and apoptosis in colonic epithelial cells. J. Surg. Res. 117, 296–305 (2004).

    Article  CAS  Google Scholar 

  49. Gille, H. et al. ERK phosphorylation potentiates Elk-1-mediated ternary complex formation and transactivation. EMBO J. 14, 951–962 (1995).

    Article  CAS  Google Scholar 

  50. Goenka, S. & Boothby, M. Selective potentiation of Stat-dependent gene expression by collaborator of Stat6 (CoaSt6), a transcriptional cofactor. Proc. Natl. Acad. Sci. USA 103, 4210–4215 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank members of the Lee laboratory, K. Gould, V. Siegel and R. Roberts-Galbraith for critical reading of the manuscript; J. Merkle for help with Drosophila S2 cell culture; members of the Institute of Chemistry and Cell Biology–Longwood for assistance with screening; M. Beinz, K. Basler, J. Nathans, R. Nusse, S. Hiebert, R. Coffey, B. Gumbiner and M. Lenardo for reagents. Nematode strains were obtained from the C. elegans Genetics Center, which is supported by the US National Institutes of Health National Center for Research Resources. This work was supported by American Cancer Society Research Scholar Grant RSG-05-126-01, American Cancer Society Institutional Research Grant IRG-58-009-46, National Cancer Institute Grant GI SPORE P50 CA95103, Mouse Models of Human Cancers Consortium (US National Institutes of Health–National Cancer Institute) 5U01 CA084239, US National Institutes of Health Grant 1 R01 GM081635-01 (E.L.); US National Institutes of Health Grant 1 R01 NS26115 (D.M.M.); American Heart Association Predoctoral Fellowship 0615279B, Molecular Endocrinology Training Grant 5 T 32 DK007563, Training Program in Developmental Biology 5 T32 HD007502 (National Institute of Child Health and Human Development) (C.A.T.); US National Institute of General Medical Studies Medical-Scientist Training Grant 5 T32 GM007347 (C.S.C. and A.J.H.); American Heart Association Predoctoral Fellowships 0615162B, US National Institutes of Health Cancer Biology Training Grant T32 CA09592 (K.K.J.). E.L. is a recipient of a Pew Scholarship in the Biomedical Sciences.

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  1. Victor P Ghidu & Bonnie LaFleur

    Present address: Present addresses: Division of Epidemiology and Biostatistics, Mel and Enid Zuckerman College of Public Health, Tucson, Arizona (B.L.); Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, Philadelphia (V.P.G.).,

Authors and Affiliations

  1. Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee, USA

    Curtis A Thorne, Alison J Hanson, Judsen Schneider, Emilios Tahinci, Christopher S Cselenyi, Kristin K Jernigan, Kelly C Meyers, Brian I Hang, Laura A Lee, David M Miller III & Ethan Lee

  2. Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee, USA

    Darren Orton, Alex G Waterson, Kwangho Kim, Bruce Melancon, Victor P Ghidu, Gary A Sulikowski & Ethan Lee

  3. StemSynergy Therapeutics Inc., Lauderdale by the Sea, Florida, USA

    Darren Orton

  4. Department of Biostatistics, Vanderbilt University, Nashville, Tennessee, USA

    Bonnie LaFleur

  5. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA

    Adrian Salic

  6. Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Laura A Lee & Ethan Lee

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Contributions

C.A.T. designed, performed and analyzed biochemical, extract and cell culture experiments. C.A.T. and E.T. performed screen under E.L. and A.S.'s guidance. J.S. designed and performed C. elegans experiments under D.M.M.'s guidance. E.T. and A.J.H. performed Xenopus embryo experiments. B.L. provided bioinformatics support. D.O., A.G.W., K.K., B.M., V.P.G. and G.A.S. designed and performed chemical synthesis. C.S.C., K.K.J., A.J.H., B.I.H. and L.A.L. provided essential reagents and discussions. K.C.M. provided technical assistance. C.A.T., E.L. and L.A.L. wrote the manuscript with advice from all authors. E.L. guided all aspects of study.

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Correspondence to Ethan Lee.

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Competing interests

Ethan Lee is cofounder of StemSynergy Therapeutics Inc., a company that aims to develop inhibitors of major signaling pathways (including the Wnt pathway) as potential chemotherapeutic agents. Darren Orton is an employee of StemSynergy Therapeutics Inc.

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Thorne, C., Hanson, A., Schneider, J. et al. Small-molecule inhibition of Wnt signaling through activation of casein kinase 1α. Nat Chem Biol 6, 829–836 (2010). https://doi.org/10.1038/nchembio.453

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