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Epithelial calcineurin controls microbiota-dependent intestinal tumor development

Nature Medicine volume 22, pages 506–515 (2016)Cite this article

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Abstract

Inflammation-associated pathways are active in intestinal epithelial cells (IECs) and contribute to the pathogenesis of colorectal cancer (CRC). Calcineurin, a phosphatase required for the activation of the nuclear factor of activated T cells (NFAT) family of transcription factors, shows increased expression in CRC. We therefore investigated the role of calcineurin in intestinal tumor development. We demonstrate that calcineurin and NFAT factors are constitutively expressed by primary IECs and selectively activated in intestinal tumors as a result of impaired stratification of the tumor-associated microbiota and toll-like receptor signaling. Epithelial calcineurin supports the survival and proliferation of cancer stem cells in an NFAT-dependent manner and promotes the development of intestinal tumors in mice. Moreover, somatic mutations that have been identified in human CRC are associated with constitutive activation of calcineurin, whereas nuclear translocation of NFAT is associated with increased death from CRC. These findings highlight an epithelial cell–intrinsic pathway that integrates signals derived from the commensal microbiota to promote intestinal tumor development.

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Figure 1: Intestinal epithelial calcineurin promotes intestinal tumor development.
Figure 2: Calcineurin regulates tumor development through NFAT.
Figure 3: TLR stimulation promotes NFAT activation in CRC.
Figure 4: Calcineurin-mediated tumor development is dependent on the microbiota.
Figure 5: Calcineurin and NFATc3 regulate cancer stem cells.
Figure 6: Activation of NFATc3 is associated with increased death from CRC.

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Acknowledgements

The authors thank L.H. Glimcher (Weill Cornell Medical College), M.B. Greenblatt (Weill Cornell Medical College) and A.O. Aliprantis (Brigham and Women's Hospital, Harvard Medical School) for Nfatc1fl/fl mice and for helpful discussions; G.R. Crabtree (Center for Regenerative Therapies, Dresden) for Ppp3r1fl/fl and Nfatc3fl/fl mice; L. Niederreiter (Addenbrooke Hospital) for help with in situ hybridization; K. Schmidt, A. Gerneth, M. Pein (University Medical Center Schleswig-Holstein), S. Krüger (University Medical Center Schleswig-Holstein) and M. Jäger (Stanford University) for assistance with immunofluorescence, immunohistochemistry and TMA stainings; K. Cloppenborg-Schmidt (Institute for Experimental Medicine) for assistance with NGS library preparation; S. Robine (Institut Curie CNRS) for villin-CreERT2 mice; S. Akira (Research Institute for Microbial Diseases) for Myd88−/− mice; R.H. Whitehead (Vanderbilt University Medical Center) for the YAMC, IMCE and IMCE-Ras cells; C.M. Norris (Molecular and Biomedical Pharmacology) for the Ad5-CMV-VIVIT construct; R. Moon (University of Washington) for the TOPFlash plasmid; A. Rao ( La Jolla Institute for Allergy and Immunology) for the NFAT-Luc and NFATc3-eGFP plasmids; and W. Garrett (Harvard T.H. Chan School of Public Health), S. Schölch and D.E. Stange (University Medical Center Dresden) for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants ZE814/5-1 (S.Z.), BA2863/5-1 (J.F.B.) and CH279/5-1 (T.C.), the European Research Council (ERC) starting grant 336528 (S.Z.), a Postdoctoral Fellowship Award from the Crohn's and Colitis Foundation of America (S.Z.), the European Commission (Marie Curie International Reintegration grant 256363; S.Z.), the DFG Excellence Cluster 'Inflammation at Interfaces' (S.Z. and J.F.B.), the DFG Excellence Cluster 'Center for Regenerative Therapies' (S.Z.); the US National Institutes of Health grants DK044319 (R.S.B.), DK051362 (R.S.B.), DK053056 (R.S.B.) and DK088199 (R.S.B.), the Harvard Digestive Diseases Center (HDDC) grant DK0034854 (R.S.B.), and the AIRC grant IG-14233 (M.E.B.).

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Author notes

  1. Kenneth Peuker and Stefanie Muff: These authors contributed equally to this work.

  2. Richard S Blumberg and Sebastian Zeissig: These authors jointly directed this work.

Authors and Affiliations

  1. Department of Internal Medicine I, University Medical Center Schleswig-Holstein, Kiel, Germany

    Kenneth Peuker, Stefanie Muff, Esther Bosse, Giuseppina Luzzi, Andrea Ulbricht, Alexander Arlt, Jochen Hampe, Stefan Schreiber & Sebastian Zeissig

  2. Center for Regenerative Therapies, Technical University Dresden, Dresden, Germany

    Kenneth Peuker, Giuseppina Luzzi, Anne Strigli & Sebastian Zeissig

  3. Max Planck Institute for Evolutionary Biology, Plön, Germany

    Jun Wang, Sven Künzel & John F Baines

  4. Institute for Experimental Medicine, Christian-Albrechts University of Kiel, Kiel, Germany

    Jun Wang, Sven Künzel & John F Baines

  5. Department of General Pediatrics, University Medical Center Schleswig-Holstein, Kiel, Germany

    Yvonne Zeissig

  6. Department of Pediatrics, University Medical Center Dresden, Technical University Dresden, Dresden, Germany

    Yvonne Zeissig

  7. Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, Hannover, Germany

    Marijana Basic & André Bleich

  8. Division of Gastroenterology, Addenbrooke Hospital, University of Cambridge, Cambridge, UK

    Arthur Kaser

  9. Department of Clinical Pathobiochemistry, Faculty of Medicine, Technical University Dresden, Dresden, Germany

    Triantafyllos Chavakis

  10. Institute of Clinical Chemistry and Laboratory Medicine, Faculty of Medicine, Technical University Dresden, Dresden, Germany

    Triantafyllos Chavakis

  11. Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, the Netherlands

    Gijs R van den Brink

  12. Department of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam, the Netherlands

    Gijs R van den Brink

  13. Department of General Surgery and Thoracic Surgery, University Hospital Schleswig-Holstein, Kiel, Germany

    Clemens Schafmayer, Jan-Hendrik Egberts & Thomas Becker

  14. San Raffaele University and Scientific Institute, Milan, Italy

    Marco E Bianchi

  15. Institute of Pathology, University Medical Center Schleswig-Holstein, Kiel, Germany

    Christoph Röcken

  16. Department of Medicine I, University Medical Center Dresden, Technical University Dresden, Dresden, Germany

    Jochen Hampe & Sebastian Zeissig

  17. Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Richard S Blumberg & Sebastian Zeissig

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Contributions

K.P., S.M., E.B., G.L. and A.S. developed and analyzed the described mouse models and performed in vivo and in vitro studies. J.W., S.K. and J.F.B. performed microbiota analyses and provided GF mice. M.B. and A.B. provided GF mice. A.U. contributed to EGF and VEGF signaling studies and to the generation of adenovirus. Y.Z. and C.R. performed histopathological analyses. A.K. contributed to stem cell analyses. G.R.v.d.B. provided RAGE-KO tissues. T.C. and M.E.B. contributed to HMGB1 studies. J.H., C.S., J.-H.E. and T.B. collected and provided CRC tissues. A.A. and S.S. contributed to the coordination of experimental studies. R.S.B. and S.Z. designed the study, coordinated the experimental work and wrote the manuscript with input from the coauthors. All authors discussed the results and commented on the manuscript. S.Z. and R.S.B. contributed equally to this work and are co-corresponding authors.

Corresponding authors

Correspondence to Richard S Blumberg or Sebastian Zeissig.

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The authors declare no competing financial interests.

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Peuker, K., Muff, S., Wang, J. et al. Epithelial calcineurin controls microbiota-dependent intestinal tumor development. Nat Med 22, 506–515 (2016). https://doi.org/10.1038/nm.4072

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