Skip to main content

Thank you for visiting nature.com. 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.

  • Original Article
  • Published:

In vivo and in vitro models for the therapeutic targeting of Wnt signaling using a Tet-OΔN89β-catenin system

Oncogene volume 32, pages 883–893 (2013)Cite this article

Subjects

Abstract

Although significant progress has been made in understanding the importance of Wnt signaling in the initiation of colorectal cancer, less is known about responses that accompany the reversal of oncogenic Wnt signaling. The aim of this study was to analyze in vivo and in vitro responses to an ‘ideal’ Wnt pathway inhibitor as a model for the therapeutic targeting of the pathway. A tetracycline-inducible transgenic mouse model expressing truncated β-catenin (ΔN89β-catenin) that exhibited a strong intestinal hyperplasia was analyzed during the removal of oncogenic β-catenin expression both in 3D ‘crypt culture’ and in vivo. Oncogenic Wnt signaling was rapidly and completely reversed. The strongest inhibition of Wnt target gene expression occurred within 24 h of doxycycline removal at which time the target genes Ascl2, Axin2 and C-myc were downregulated to levels below that in the control intestine. In vitro, the small molecule Wnt inhibitor CCT036477 induced a response within 4 h of treatment. By 7 days following doxycycline withdrawal, gene expression, cell proliferation and tissue morphology were undistinguishable from control animals.In conclusion, these results demonstrate that the reversal of Wnt signaling by inhibitors should ideally be studied within hours of treatment. The reversible system described, involving medium throughput in vitro approaches and rapid in vivo responses, should allow the rapid advance of early stage compounds into efficacy models that are more usually considered later in the drug discovery pipeline.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Van der Flier LG, Clevers H . Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu Rev Physiol 2009; 71: 241–260.

    Article  CAS  PubMed  Google Scholar 

  2. Amit S, Hatzubai A, Birman Y, Andersen JS, Ben-Shushan E, Mann M et al. Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev 2002; 16: 1066–1076.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Van der Flier LG, Sabates-Bellver J, Oving I, Haegebarth A, De Palo M, Anti M et al. The intestinal Wnt/TCF signature. Gastroenterology 2007; 132: 628–632.

    Article  CAS  PubMed  Google Scholar 

  4. Fevr T, Robine S, Louvard D, Huelsken J . Wnt/beta-catenin is essential for intestinal homeostasis and maintenance of intestinal stem cells. Mol Cell Biol 2007; 27: 7551–7559.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Korinek V, Barker N, Moerer P, van Donselaar E, Huls G, Peters PJ et al. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nat Genet 1998; 19: 379–383.

    Article  CAS  PubMed  Google Scholar 

  6. Kuhnert F, Davis CR, Wang HT, Chu P, Lee M, Yuan J et al. Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1. Proc Natl Acad Sci USA 2004; 101: 266–271.

    Article  CAS  PubMed  Google Scholar 

  7. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009; 459: 262–265.

    Article  CAS  PubMed  Google Scholar 

  8. Giles RH, van Es JH, Clevers H . Caught up in a Wnt storm: Wnt signaling in cancer. Biochim Biophys Acta 2003; 1653: 1–24.

    CAS  PubMed  Google Scholar 

  9. Polakis P . Wnt signaling and cancer. Genes Dev 2000; 14: 1837–1851.

    CAS  PubMed  Google Scholar 

  10. Harada N, Tamai Y, Ishikawa T, Sauer B, Takaku K, Oshima M et al. Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene. EMBO J 1999; 18: 5931–5942.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wong MH, Rubinfeld B, Gordon JI . Effects of forced expression of an NH2-terminal truncated beta-catenin on mouse intestinal epithelial homeostasis. J Cell Biol 1998; 141: 765–777.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Romagnolo B, Berrebi D, Saadi-Keddoucci S, Porteu A, Pichard AL, Peuchmaur M et al. Intestinal dysplasia and adenoma in transgenic mice after overexpression of an activated beta-catenin. Cancer Res 1999; 59: 3875–3879.

    CAS  PubMed  Google Scholar 

  13. Beard C, Hochedlinger K, Plath K, Wutz A, Jaenisch R . Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis 2006; 44: 23–28.

    Article  CAS  PubMed  Google Scholar 

  14. Bocker R, Estler CJ, Maywald M, Weber D . Comparison of distribution of doxycycline in mice after oral and intravenous application measured by a high-performance liquid chromatographic method. Arzneimittelforschung 1981; 31: 2116–2117.

    CAS  PubMed  Google Scholar 

  15. Rubinfeld B, Robbins P, El-Gamil M, Albert I, Porfiri E, Polakis P . Stabilization of beta-catenin by genetic defects in melanoma cell lines. Science 1997; 275: 1790–1792.

    Article  CAS  PubMed  Google Scholar 

  16. Ewan K, Pajak B, Stubbs M, Todd H, Barbeau O, Quevedo C et al. A useful approach to identify novel small-molecule inhibitors of Wnt-dependent transcription. Cancer Res 2010; 70: 5963–5973.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kinzler KW, Nilbert MC, Su LK, Vogelstein B, Bryan TM, Levy DB et al. Identification of FAP locus genes from chromosome 5q21. Science 1991; 253: 661–665.

    Article  CAS  PubMed  Google Scholar 

  18. Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B et al. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 1997; 275: 1787–1790.

    Article  CAS  PubMed  Google Scholar 

  19. Andreu P, Colnot S, Godard C, Gad S, Chafey P, Niwa-Kawakita M et al. Crypt-restricted proliferation and commitment to the paneth cell lineage following Apc loss in the mouse intestine. Development 2005; 132: 1443–1451.

    Article  CAS  PubMed  Google Scholar 

  20. Sansom OJ, Reed KR, Hayes AJ, Ireland H, Brinkmann H, Newton IP et al. Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration. Genes Dev 2004; 18: 1385–1390.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Logan CY, Nusse R . The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 2004; 20: 781–810.

    Article  CAS  PubMed  Google Scholar 

  22. Niida A, Hiroko T, Kasai M, Furukawa Y, Nakamura Y, Suzuki Y et al. DKK1, a negative regulator of Wnt signaling, is a target of the beta-catenin/TCF pathway. Oncogene 2004; 23: 8520–8526.

    Article  CAS  PubMed  Google Scholar 

  23. Minke KS, Staib P, Puetter A, Gehrke I, Gandhirajan RK, Schlosser A et al. Small molecule inhibitors of WNT signaling effectively induce apoptosis in acute myeloid leukemia cells. Eur J Haematol 2009; 82: 165–175.

    Article  CAS  PubMed  Google Scholar 

  24. He B, You L, Uematsu K, Xu Z, Lee AY, Matsangou M et al. A monoclonal antibody against Wnt-1 induces apoptosis in human cancer cells. Neoplasia 2004; 6: 7–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Scholer-Dahirel A, Schlabach MR, Loo A, Bagdasarian L, Meyer R, Guo R et al. Maintenance of adenomatous polyposis coli (APC)-mutant colorectal cancer is dependent on Wnt/beta-catenin signaling. Proc Natl Acad Sci USA 2011; 108: 17135–17140.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Gunther EJ, Moody SE, Belka GK, Hahn KT, Innocent N, Dugan KD et al. Impact of p53 loss on reversal and recurrence of conditional Wnt-induced tumorigenesis. Genes Dev 2003; 17: 488–501.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Coste I, Freund JN, Spaderna S, Brabletz T, Renno T . Precancerous lesions upon sporadic activation of beta-catenin in mice. Gastroenterology 2007; 132: 1299–1308.

    Article  CAS  PubMed  Google Scholar 

  28. Lucero OM, Dawson DW, Moon RT, Chien AJ . A re-evaluation of the ″oncogenic″ nature of Wnt/beta-catenin signaling in melanoma and other cancers. Curr Oncol Rep 2010; 12: 314–318.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 2011; 469: 415–418.

    Article  CAS  PubMed  Google Scholar 

  30. Binnerts ME, Kim KA, Bright JM, Patel SM, Tran K, Zhou M et al. R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6. Proc Natl Acad Sci USA 2007; 104: 14700–14705.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Buchert M, Athineos D, Abud HE, Burke ZD, Faux MC, Samuel MS et al. Genetic dissection of differential signaling threshold requirements for the Wnt/beta-catenin pathway in vivo. PLoS Genet 2010; 6: e1000816.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Wang Y . Wnt/Planar cell polarity signaling: a new paradigm for cancer therapy. Mol Cancer Ther 2009; 8: 2103–2109.

    Article  CAS  PubMed  Google Scholar 

  33. van Amerongen R, Mikels A, Nusse R . Alternative wnt signaling is initiated by distinct receptors. Sci Signal 2008; 1: re9.

    Article  PubMed  Google Scholar 

  34. Soriano P . Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 1999; 21: 70–71.

    Article  CAS  PubMed  Google Scholar 

  35. Wutz A, Jaenisch R . A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation. Mol Cell 2000; 5: 695–705.

    Article  CAS  PubMed  Google Scholar 

  36. Shorning BY, Jarde T, McCarthy A, Ashworth A, de Leng WW, Offerhaus GJ et al. Intestinal renin-angiotensin system is stimulated after deletion of Lkb1. Gut 2012; 61: 202–213.

    Article  CAS  PubMed  Google Scholar 

  37. Lustig B, Jerchow B, Sachs M, Weiler S, Pietsch T, Karsten U et al. Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors. Mol Cell Biol 2002; 22: 1184–1193.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Merritt AJ, Allen TD, Potten CS, Hickman JA . Apoptosis in small intestinal epithelial from p53-null mice: evidence for a delayed, p53-independent G2/M-associated cell death after gamma-irradiation. Oncogene 1997; 14: 2759–2766.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by Cancer Research UK and the Breast Cancer Campaign. We thank C Beard and R Jaenisch (The Whitehead Institute for Biomedical Research, Massachusetts) for the embryonic stem cell lines and for technical advice; K Ewan, A Offergeld and S Braun (Cardiff School of Biosciences) for their assistance in tissue staining and cell counting; P Watson (Cardiff School of Biosciences) for his assistance in fluorescence microscopy; O Sansom and K Myant for their assistance in organoid culture; B Allen and O Asby for technical assistance in blastocyst injections and chimera crosses; and D Scarborough for help with histology.

Author contributions: TCD, ARC and TJ were involved in the study concept and design; TCD and TJ were involved in funding and study supervision; TJ, RJE, KLM, LP, GJF and BA were involved in acquisition and analysis of data; TJ and TCD were involved in drafting the manuscript; ARC, RJE, LP and GJF were involved in critical revision.

Author information

Author notes

  1. T Jardé and R J Evans: These authors contributed equally to this work

Authors and Affiliations

  1. Cardiff School of Biosciences, Cardiff University, Cardiff, UK

    T Jardé, R J Evans, K L McQuillan, L Parry, G J Feng, B Alvares, A R Clarke & T C Dale

Authors
  1. T Jardé
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R J Evans
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K L McQuillan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. L Parry
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. G J Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. B Alvares
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. A R Clarke
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. T C Dale
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to T C Dale.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jardé, T., Evans, R., McQuillan, K. et al. In vivo and in vitro models for the therapeutic targeting of Wnt signaling using a Tet-OΔN89β-catenin system. Oncogene 32, 883–893 (2013). https://doi.org/10.1038/onc.2012.103

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2012.103

Keywords

This article is cited by

Search

Advanced search

Quick links