Article | Published:

Preventive and therapeutic effects of Smad7 on radiation-induced oral mucositis

Nature Medicine volume 19, pages 421428 (2013) | Download Citation

Abstract

We report that K5.Smad7 mice, which express a Smad7 transgene under the control of a keratin 5 promoter, were resistant to radiation-induced oral mucositis, a painful oral ulceration. In addition to nuclear factor κB (NF-κB) activation, which is known to contribute to oral mucositis, we found activated transforming growth factor β (TGF-β) signaling in cells from this condition. Smad7 dampened both pathways to attenuate inflammation, growth inhibition and apoptosis. Additionally, Smad7 promoted oral epithelial migration to close the wound. Further analyses revealed that TGF-β signaling Smads and their co-repressor C-terminal binding protein 1 (CtBP1) transcriptionally repressed Rac1, and that Smad7 abrogated this repression. Knocking down Rac1 expression in mouse keratinocytes abrogated Smad7-induced migration. Topical application of Smad7 protein conjugated with a cell-permeable Tat tag to oral mucosa showed prophylactic and therapeutic effects on radiation-induced oral mucositis in mice. Thus, we have identified new molecular mechanisms involved in oral mucositis pathogenesis, and our data suggest an alternative therapeutic strategy to block multiple pathological processes in this condition.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    Efficacy of palifermin (keratinocyte growth factor-1) in the amelioration of oral mucositis. Core Evid. 4, 199–205 (2010).

  2. 2.

    et al. Incidence and severity of oral mucositis in patients undergoing haematopoietic SCT—results of a multicentre study. Bone Marrow Transplant. 46, 727–732 (2011).

  3. 3.

    , & Oral mucositis. Oral Dis. 12, 229–241 (2006).

  4. 4.

    , & Evaluation of current and upcoming therapies in oral mucositis prevention. Future Oncol. 6, 1751–1770 (2010).

  5. 5.

    et al. Palifermin decreases severe oral mucositis of patients undergoing postoperative radiochemotherapy for head and neck cancer: a randomized, placebo-controlled trial. J. Clin. Oncol. 29, 2815–2820 (2011).

  6. 6.

    et al. Palifermin reduces severe mucositis in definitive chemoradiotherapy of locally advanced head and neck cancer: a randomized, placebo-controlled study. J. Clin. Oncol. 29, 2808–2814 (2011).

  7. 7.

    et al. R-Spondin1 protects mice from chemotherapy or radiation-induced oral mucositis through the canonical Wnt/β-catenin pathway. Proc. Natl. Acad. Sci. USA 106, 2331–2336 (2009).

  8. 8.

    et al. Therapeutic effect of recombinant human epidermal growth factor (RhEGF) on mucositis in patients undergoing radiotherapy, with or without chemotherapy, for head and neck cancer: a double-blind placebo-controlled prospective phase 2 multi-institutional clinical trial. Cancer 115, 3699–3708 (2009).

  9. 9.

    , & Pharmacokinetic evaluation of palifermin for mucosal protection from chemotherapy and radiation. Expert Opin. Drug Metab. Toxicol. 7, 505–515 (2011).

  10. 10.

    et al. Randomized phase 2 study of concomitant chemoradiotherapy using weekly carboplatin/paclitaxel with or without daily subcutaneous amifostine in patients with locally advanced head and neck cancer. Cancer 115, 4514–4523 (2009).

  11. 11.

    et al. Pharmacological protection from radiation +/− cisplatin-induced oral mucositis. Int. J. Radiat. Oncol. Biol. Phys. 83, 1284–1290 (2012).

  12. 12.

    et al. mTOR inhibition prevents epithelial stem cell senescence and protects from radiation-induced mucositis. Cell Stem Cell 11, 401–414 (2012).

  13. 13.

    & Anti-inflammatory treatment. Curr. Probl. Dermatol. 40, 58–70 (2011).

  14. 14.

    et al. Topical immunomodulators for management of oral mucosal conditions, a systematic review; part I: calcineurin inhibitors. Expert Opin. Emerg. Drugs 15, 713–726 (2010).

  15. 15.

    et al. Topical immunomodulators for management of oral mucosal conditions, a systematic review; part II: miscellaneous agents. Expert Opin. Emerg. Drugs 16, 183–202 (2011).

  16. 16.

    Off-label use of biologicals in the management of inflammatory oral mucosal disease. J. Oral Pathol. Med. 37, 575–581 (2008).

  17. 17.

    et al. Overexpression of Smad7 results in severe pathological alterations in multiple epithelial tissues. EMBO J. 21, 2580–2590 (2002).

  18. 18.

    et al. Smad7 binds to the adaptors TAB2 and TAB3 to block recruitment of the kinase TAK1 to the adaptor TRAF2. Nat. Immunol. 8, 504–513 (2007).

  19. 19.

    et al. Smad7-induced β-catenin degradation alters epidermal appendage development. Dev. Cell 11, 301–312 (2006).

  20. 20.

    , , & Acute radiation reactions in oral and pharyngeal mucosa: tolerable levels in altered fractionation schedules. Radiother. Oncol. 69, 161–168 (2003).

  21. 21.

    et al. Targeted disruption of the mouse transforming growth factor-β 1 gene results in multifocal inflammatory disease. Nature 359, 693–699 (1992).

  22. 22.

    et al. Transforming growth factor β 1 null mutation in mice causes excessive inflammatory response and early death. Proc. Natl. Acad. Sci. USA 90, 770–774 (1993).

  23. 23.

    et al. Overexpression of transforming growth factor β1 in head and neck epithelia results in inflammation, angiogenesis, and epithelial hyperproliferation. Cancer Res. 64, 4405–4410 (2004).

  24. 24.

    & Smad7: not only a regulator, but also a cross-talk mediator of TGF-β signalling. Biochem. J. 434, 1–10 (2011).

  25. 25.

    et al. Rac1 is required for epithelial stem cell function during dermal and oral mucosal wound healing but not for tissue homeostasis in mice. PLoS ONE 5, e10503 (2010).

  26. 26.

    et al. Incisional wound healing in transforming growth factor-β1 null mice. Wound Repair Regen. 8, 179–191 (2000).

  27. 27.

    & Loss of Smad3 modulates wound healing. Cytokine Growth Factor Rev. 11, 125–131 (2000).

  28. 28.

    Non-Smad pathways in TGF-β signaling. Cell Res. 19, 128–139 (2009).

  29. 29.

    & The logic of TGFβ signaling. FEBS Lett. 580, 2811–2820 (2006).

  30. 30.

    , & Smad transcription factors. Genes Dev. 19, 2783–2810 (2005).

  31. 31.

    et al. HGF upregulation contributes to angiogenesis in mice with keratinocyte-specific Smad2 deletion. J. Clin. Invest. 120, 3606–3616 (2010).

  32. 32.

    , & Tat peptide-mediated cellular delivery: back to basics. Adv. Drug Deliv. Rev. 57, 559–577 (2005).

  33. 33.

    , , & Tuning the transport properties of HIV-1 Tat arginine-rich motif in living cells. Traffic 9, 528–539 (2008).

  34. 34.

    et al. Enhancement of gene targeting in human cells by intranuclear permeation of the Saccharomyces cerevisiae Rad52 protein. Nucleic Acids Res. 38, e149 (2010).

  35. 35.

    , & Effects of keratinocyte growth factor (palifermin) administration protocols on oral mucositis (mouse) induced by fractionated irradiation. Radiother. Oncol. 75, 99–105 (2005).

  36. 36.

    , & Reduction of oral mucositis by palifermin (rHuKGF): dose-effect of rHuKGF. Int. J. Radiat. Biol. 81, 557–565 (2005).

  37. 37.

    et al. Molecular cytogenetic characterization of head and neck squamous cell carcinoma and refinement of 3q amplification. Cancer Res. 61, 4506–4513 (2001).

  38. 38.

    , , & Disruption of transforming growth factor β–Smad signaling pathway in head and neck squamous cell carcinoma as evidenced by mutations of SMAD2 and SMAD4. Cancer Lett. 245, 163–170 (2007).

  39. 39.

    TGFβ in Cancer. Cell 134, 215–230 (2008).

  40. 40.

    , , & Regulation of cell proliferation by Smad proteins. J. Cell Physiol. 191, 1–16 (2002).

  41. 41.

    & Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature 425, 577–584 (2003).

  42. 42.

    et al. TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2. Nature 417, 861–866 (2002).

  43. 43.

    , & Radioprotective gene therapy. Expert Opin. Biol. Ther. 11, 1135–1151 (2011).

  44. 44.

    et al. Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation. J. Clin. Invest. 119, 3408–3419 (2009).

  45. 45.

    et al. Loss of transforming growth factor-β type II receptor promotes metastatic head-and-neck squamous cell carcinoma. Genes Dev. 20, 1331–1342 (2006).

  46. 46.

    & Targeting the TGFβ signalling pathway in disease. Nat. Rev. Drug Discov. 11, 790–811 (2012).

  47. 47.

    , & Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics. Br. J. Pharmacol. 157, 195–206 (2009).

  48. 48.

    et al. Prevention of radiation-induced oral mucositis after adenoviral vector–mediated transfer of the keratinocyte growth factor cDNA to mouse submandibular glands. Clin. Cancer Res. 15, 4641–4648 (2009).

  49. 49.

    , , & Temporal smad7 transgene induction in mouse epidermis accelerates skin wound healing. Am. J. Pathol. 179, 1768–1779 (2011).

  50. 50.

    , , , & Homeodomain interacting protein kinase 2 promotes apoptosis by downregulating the transcriptional corepressor CtBP. Cell 115, 177–186 (2003).

  51. 51.

    , , , & CtBPs promote cell survival through the maintenance of mitotic fidelity. Mol. Cell Biol. 29, 4539–4551 (2009).

  52. 52.

    , & Smad7 sensitizes tumor necrosis factor induced apoptosis through the inhibition of antiapoptotic gene expression by suppressing activation of the nuclear factor-κB pathway. Cancer Res. 67, 9577–9583 (2007).

  53. 53.

    , & Clonogenic cell survival assay. Methods Mol. Med. 110, 21–28 (2005).

  54. 54.

    , , , & Smad3 knockout mice exhibit a resistance to skin chemical carcinogenesis. Cancer Res. 64, 7836–7845 (2004).

  55. 55.

    et al. Overexpression of Smad7 results in severe pathological alterations in multiple epithelial tissues. EMBO J. 21, 2580–2590 (2002).

  56. 56.

    et al. Keratinocyte-specific Smad2 ablation results in increased epithelial-mesenchymal transition during skin cancer formation and progression. J. Clin. Invest. 118, 2722–2732 (2008).

  57. 57.

    et al. Smad4-dependent desmoglein-4 expression contributes to hair follicle integrity. Dev. Biol. 322, 155–166 (2008).

Download references

Acknowledgements

This work was supported by NIH grants AR061792 and DE015953 to X.-J.W., R01CA115468 to Q.Z. and R03DA033982 to R.Z. and Q.Z. and the Colorado State/University of Colorado Bioscience Discovery Evaluation grant to X.-J.W., Q.Z. and Y.R. and was partially supported by NIH grant P30CA046934 to the University of Colorado Cancer Center. L.B. was supported by a grant (81060189) from The National Natural Science Foundation of China (NSFC). F.L. was supported by grants from the NSFC (81102596), The Shanghai Rising-Star Program (12QA1403300) and The Innovation Program of Shanghai Municipal Education Commission (12YZ067). The authors thank S. Said, C. Marshall and C. Liu for searching human oral mucositis clinical samples and C.Y. Li and G. Gang for their input on radiotherapy in patients with oral cancer. The authors also thank P. Garl for proofreading the manuscript.

Author information

Author notes

    • Gangwen Han
    • , Li Bian
    •  & Fulun Li

    These authors contributed equally to this work.

Affiliations

  1. Department of Pathology, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado, USA.

    • Gangwen Han
    • , Li Bian
    • , Fulun Li
    • , Donna Wang
    •  & Xiao-Jing Wang
  2. Department of Pathology, The First Affiliated Hospital of Kunming Medical University, Kunming, China.

    • Li Bian
    •  & Jianbo Lu
  3. Department of Dermatology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

    • Fulun Li
  4. Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health (NIH), Bethesda, Maryland, USA.

    • Ana Cotrim
    •  & J Silvio Gutkind
  5. Department of Dermatology, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado, USA.

    • Yu Deng
    • , Gregory Bird
    • , Yosef Refaeli
    •  & Qinghong Zhang
  6. Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.

    • Anastasia Sowers
    •  & James B Mitchell
  7. Department of Molecular Genetics and Biochemistry, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.

    • Rui Zhao
  8. Department of Radiation Oncology, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado, USA.

    • David Raben
  9. Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.

    • Peter ten Dijke

Authors

  1. Search for Gangwen Han in:

  2. Search for Li Bian in:

  3. Search for Fulun Li in:

  4. Search for Ana Cotrim in:

  5. Search for Donna Wang in:

  6. Search for Jianbo Lu in:

  7. Search for Yu Deng in:

  8. Search for Gregory Bird in:

  9. Search for Anastasia Sowers in:

  10. Search for James B Mitchell in:

  11. Search for J Silvio Gutkind in:

  12. Search for Rui Zhao in:

  13. Search for David Raben in:

  14. Search for Peter ten Dijke in:

  15. Search for Yosef Refaeli in:

  16. Search for Qinghong Zhang in:

  17. Search for Xiao-Jing Wang in:

Contributions

G.H., Q.Z. and X.-J.W. designed experiments and wrote the manuscript. D.R. provided input in radiotherapy dosing and regimens in oral cancer treatment and clinical care of radiation-induced oral mucositis and the MSK921 cell line. G.H., L.B., F.L. and D.W. performed the radiation-induced oral mucositis animal experiments and other histopathological and molecular analyses at the University of Colorado Denver, Anschutz Medical Campus. A.C., A.S. and J.B.M. performed radiation-induced oral mucositis animal experiments at the NIH. Y.D., G.B. and Q.Z. performed protein purification. R.Z., Y.R. and Q.Z. contributed to optimization of Tat-Smad7 protein production. J.S.G. provided NOK-SI cells. P.t.D. provided Smad7-specific antibody. L.B. and J.L. provided human samples and performed immunostaining on those samples. D.R., J.B.M. and J.S.G. provided suggestions for manuscript revisions.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Qinghong Zhang or Xiao-Jing Wang.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nm.3118

Further reading

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