Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Small molecule promotes β-catenin citrullination and inhibits Wnt signaling in cancer


Wnt (wingless)/β-catenin signaling is critical for tumor progression and is frequently activated in colorectal cancer as a result of the mutation of adenomatous polyposis coli (APC); however, therapeutic agents targeting this pathway for clinical use are lacking. Here we report that nitazoxanide (NTZ), a clinically approved antiparasitic drug, efficiently inhibits Wnt signaling independent of APC. Using chemoproteomic approaches, we have identified peptidyl arginine deiminase 2 (PAD2) as the functional target of NTZ in Wnt inhibition. By targeting PAD2, NTZ increased the deamination (citrullination) and turnover of β-catenin in colon cancer cells. Replacement of arginine residues disrupted the transcriptional activity, and NTZ induced degradation of β-catenin. In Wnt-activated colon cancer cells, knockout of either PAD2 or β-catenin substantially increased resistance to NTZ treatment. Our data highlight the potential of NTZ as a modulator of β-catenin citrullination for the treatment of cancer patients with Wnt pathway mutations.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: NTZ inhibits Wnt/β-catenin signaling.
Figure 2: NTZ promotes β-catenin degradation.
Figure 3: PAD2 is the direct target of NTZ.
Figure 4: PAD2 directly binds to and citrullinates β-catenin.
Figure 5: NTZ stabilizes PAD2 and increases protein citrullination.
Figure 6: PAD2 inhibits the growth of Wnt activated cancer cells.


  1. 1

    Li, V.S. et al. Wnt signaling through inhibition of β-catenin degradation in an intact Axin1 complex. Cell 149, 1245–1256 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature 487, 330–337 (2012).

  3. 3

    Ding, L. et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature 455, 1069–1075 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Grasso, C.S. et al. The mutational landscape of lethal castration-resistant prostate cancer. Nature 487, 239–243 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Jia, D. et al. Exome sequencing of hepatoblastoma reveals novel mutations and cancer genes in the Wnt pathway and ubiquitin ligase complex. Hepatology 60, 1686–1696 (2014).

    CAS  PubMed  Google Scholar 

  6. 6

    Jones, D.T. et al. Dissecting the genomic complexity underlying medulloblastoma. Nature 488, 100–105 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Kandoth, C. et al. Cancer Genome Atlas Research Network. Integrated genomic characterization of endometrial carcinoma. Nature 497, 67–73 (2013).

    PubMed  Google Scholar 

  8. 8

    Reya, T. & Clevers, H. Wnt signalling in stem cells and cancer. Nature 434, 843–850 (2005).

    CAS  PubMed  Google Scholar 

  9. 9

    Harada, N. et al. Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene. EMBO J. 18, 5931–5942 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Kahn, M. Can we safely target the WNT pathway? Nat. Rev. Drug Discov. 13, 513–532 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Kandoth, C. et al. Mutational landscape and significance across 12 major cancer types. Nature 502, 333–339 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Sato, N., Meijer, L., Skaltsounis, L., Greengard, P. & Brivanlou, A.H. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med. 10, 55–63 (2004).

    CAS  PubMed  Google Scholar 

  13. 13

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

    PubMed  PubMed Central  Google Scholar 

  14. 14

    Heckl, D. et al. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nat. Biotechnol. 32, 941–946 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Chan, T.A., Wang, Z., Dang, L.H., Vogelstein, B. & Kinzler, K.W. Targeted inactivation of CTNNB1 reveals unexpected effects of beta-catenin mutation. Proc. Natl. Acad. Sci. USA 99, 8265–8270 (2002).

    CAS  PubMed  Google Scholar 

  16. 16

    Hudson, C., Kawai, N., Negishi, T. & Yasuo, H. β-Catenin-driven binary fate specification segregates germ layers in ascidian embryos. Curr. Biol. 23, 491–495 (2013).

    CAS  PubMed  Google Scholar 

  17. 17

    Schneider, S.Q. & Bowerman, B. β-Catenin asymmetries after all animal/vegetal- oriented cell divisions in Platynereis dumerilii embryos mediate binary cell-fate specification. Dev. Cell 13, 73–86 (2007).

    CAS  PubMed  Google Scholar 

  18. 18

    Watanabe, K. et al. Integrative ChIP-seq/microarray analysis identifies a CTNNB1 target signature enriched in intestinal stem cells and colon cancer. PLoS One 9, e92317 (2014).

    PubMed  PubMed Central  Google Scholar 

  19. 19

    Su, L.K. et al. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science 256, 668–670 (1992).

    CAS  PubMed  Google Scholar 

  20. 20

    Theodos, C.M., Griffiths, J.K., D'Onfro, J., Fairfield, A. & Tzipori, S. Efficacy of nitazoxanide against Cryptosporidium parvum in cell culture and in animal models. Antimicrob. Agents Chemother. 42, 1959–1965 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Bajaj, J., Zimdahl, B. & Reya, T. Fearful symmetry: subversion of asymmetric division in cancer development and progression. Cancer Res. 75, 792–797 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Su, Y. et al. APC is essential for targeting phosphorylated β-catenin to the SCFbeta-TrCP ubiquitin ligase. Mol. Cell 32, 652–661 (2008).

    CAS  PubMed  Google Scholar 

  23. 23

    Lomenick, B. et al. Target identification using drug affinity responsive target stability (DARTS). Proc. Natl. Acad. Sci. USA 106, 21984–21989 (2009).

    CAS  PubMed  Google Scholar 

  24. 24

    Wienken, C.J., Baaske, P., Rothbauer, U., Braun, D. & Duhr, S. Protein-binding assays in biological liquids using microscale thermophoresis. Nat. Commun. 1, 100 (2010).

    PubMed  Google Scholar 

  25. 25

    Martinez Molina, D. et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013).

    PubMed  Google Scholar 

  26. 26

    Fuhrmann, J., Clancy, K.W. & Thompson, P.R. Chemical biology of protein arginine modifications in epigenetic regulation. Chem. Rev. 115, 5413–5461 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Lewallen, D.M. et al. Chemical proteomic platform to identify citrullinated proteins. ACS Chem. Biol. 10, 2520–2528 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Slade, D.J. et al. Protein arginine deiminase 2 binds calcium in an ordered fashion: implications for inhibitor design. ACS Chem. Biol. 10, 1043–1053 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Bang, H. et al. Mutation and citrullination modifies vimentin to a novel autoantigen for rheumatoid arthritis. Arthritis Rheum. 56, 2503–2511 (2007).

    CAS  PubMed  Google Scholar 

  30. 30

    Ashiru, O., Howe, J.D. & Butters, T.D. Nitazoxanide, an antiviral thiazolide, depletes ATP-sensitive intracellular Ca2+ stores. Virology 462–463, 135–148 (2014).

    PubMed  Google Scholar 

  31. 31

    Huels, D.J. et al. E-cadherin can limit the transforming properties of activating β-catenin mutations. EMBO J. 34, 2321–2333 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Anderson, V.R. & Curran, M.P. Nitazoxanide: a review of its use in the treatment of gastrointestinal infections. Drugs 67, 1947–1967 (2007).

    CAS  PubMed  Google Scholar 

  33. 33

    Di Santo, N. & Ehrisman, J. A functional perspective of nitazoxanide as a potential anticancer drug. Mutat. Res. 768, 16–21 (2014).

    CAS  PubMed  Google Scholar 

  34. 34

    Hsu, K.W. et al. The activated Notch1 receptor cooperates with alpha-enolase and MBP-1 in modulating c-myc activity. Mol. Cell. Biol. 28, 4829–4842 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Senkowski, W. et al. Three-dimensional cell culture-based screening identifies the anthelmintic drug nitazoxanide as a candidate for treatment of colorectal cancer. Mol. Cancer Ther. 14, 1504–1516 (2015).

    CAS  PubMed  Google Scholar 

  36. 36

    Rossignol, J.F., Elfert, A., El-Gohary, Y. & Keeffe, E.B. Improved virologic response in chronic hepatitis C genotype 4 treated with nitazoxanide, peginterferon, and ribavirin. Gastroenterology 136, 856–862 (2009).

    CAS  PubMed  Google Scholar 

  37. 37

    Yue, X. et al. Hepatitis B virus-induced calreticulin protein is involved in IFN resistance. J. Immunol. 189, 279–286 (2012).

    CAS  PubMed  Google Scholar 

  38. 38

    Korba, B.E., Elazar, M., Lui, P., Rossignol, J.F. & Glenn, J.S. Potential for hepatitis C virus resistance to nitazoxanide or tizoxanide. Antimicrob. Agents Chemother. 52, 4069–4071 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Ramírez, G., Valck, C., Ferreira, V.P., López, N. & Ferreira, A. Extracellular Trypanosoma cruzi calreticulin in the host-parasite interplay. Trends Parasitol. 27, 115–122 (2011).

    PubMed  Google Scholar 

  40. 40

    Müller, J. et al. Thiazolides inhibit growth and induce glutathione-S-transferase Pi (GSTP1)-dependent cell death in human colon cancer cells. Int. J. Cancer 123, 1797–1806 (2008).

    PubMed  Google Scholar 

  41. 41

    Fan-Minogue, H. et al. A c-Myc activation sensor-based high-throughput drug screening identifies an antineoplastic effect of nitazoxanide. Mol. Cancer Ther. 12, 1896–1905 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Brockmann, A. et al. Structure-function relationship of thiazolide-induced apoptosis in colorectal tumor cells. ACS Chem. Biol. 9, 1520–1527 (2014).

    CAS  PubMed  Google Scholar 

  43. 43

    Stadler, S.C. et al. Dysregulation of PAD4-mediated citrullination of nuclear GSK3β activates TGF-β signaling and induces epithelial-to-mesenchymal transition in breast cancer cells. Proc. Natl. Acad. Sci. USA 110, 11851–11856 (2013).

    CAS  PubMed  Google Scholar 

  44. 44

    Deplus, R. et al. Citrullination of DNMT3A by PADI4 regulates its stability and controls DNA methylation. Nucleic Acids Res. 42, 8285–8296 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Vossenaar, E.R., Zendman, A.J., van Venrooij, W.J. & Pruijn, G.J. PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. BioEssays 25, 1106–1118 (2003).

    CAS  Google Scholar 

  46. 46

    Wu, H.Y. et al. Structural basis of antizyme-mediated regulation of polyamine homeostasis. Proc. Natl. Acad. Sci. USA 112, 11229–11234 (2015).

    CAS  PubMed  Google Scholar 

  47. 47

    Van Lidth de Jeude, J.F., Vermeulen, J.L., Montenegro-Miranda, P.S., Van den Brink, G.R. & Heijmans, J. A protocol for lentiviral transduction and downstream analysis of intestinal organoids. J. Vis. Exp. 2015, e52531 (2015).

    Google Scholar 

  48. 48

    Qu, Y. et al. Generation of prostate tumor-initiating cells is associated with elevation of reactive oxygen species and IL-6/STAT3 signaling. Cancer Res. 73, 7090–7100 (2013).

    CAS  PubMed  Google Scholar 

Download references


We thank H.M. Hoang for DNA microarray profiling, M. Eidsheim for immunohistochemistry and S.M. Leh for clinical materials. We thank C. Lv for the calcium measurement, and X. Zu and X. Liu for the LC–Q-TOF–MS analysis. We thank the Flow Cytometry Core Facility, Department of Clinical Science, University of Bergen. We thank R. Nusse (Stanford University, Stanford, CA) for the Wnt reporter 7TGC, O.J. Sansom (Cancer Research UK Beatson Institute, Glasgow, UK) for the mutant Apc and Ctnnb1 organoids, and S. Coonrod for the PAD2 expression vector and valuable discussion. We acknowledge funding from Einar Galtung Døsvig, Espen Galtung Døsvig, Jan Einar Greve, Bjarne Rieber, Herman Friele, Trond Mohn, Thorstein Selvik, Kåre Rommetveit, Tordis and Fritz C. Rieber's legacy to K.H.K., Bergen Research Foundation to X.K., Helse Vest grants 911778 to Y.Q., 911626 and 912062 to K.H.K., 911747 to X.K., Professor of Chang Jiang Scholars Program and NSFC (81230090, 81520108030) to W.Z., the Society for Skin Cancer Research to M.P.L. and the EU Horizon 2020 Collaborative Research Project SOUND (633974) to P.F.C.

Author information




Y.Q. and X.K. designed the project, performed experiments, analyzed data, interpreted results and wrote the manuscript; J.R.O. performed experiments; X.Y. and W.Z. performed mice experiments, LC–Q-TOF–MS analysis and calcium measurement; P. F. C. and M.P.L. performed data analysis; P.S.H. provided materials; A.M.O. performed DNA microarray profiling; K.A.B. performed immunohistochemistry staining; K.-H.K. designed the project, interpreted results and wrote the manuscript.

Corresponding authors

Correspondence to Weidong Zhang or Karl-Henning Kalland or Xisong Ke.

Ethics declarations

Competing interests

X.S., Y.Q., K.H.K. & A.M.O. are listed as inventors of the patent-pending (PCT/EP2016/076171) filed by Bergen Teknologioverføring AS.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–23 (PDF 4628 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Qu, Y., Olsen, J., Yuan, X. et al. Small molecule promotes β-catenin citrullination and inhibits Wnt signaling in cancer. Nat Chem Biol 14, 94–101 (2018).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing