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.

Bone morphogenetic protein induces bone invasion of melanoma by epithelial–mesenchymal transition via the Smad1/5 signaling pathway

Abstract

Oral malignant melanoma, which frequently invades the hard palate or maxillary bone, is extremely rare and has a poor prognosis. Bone morphogenetic protein (BMP) is abundantly expressed in bone matrix and is highly expressed in malignant melanoma, inducing an aggressive phenotype. We examined the role of BMP signaling in the acquisition of an aggressive phenotype in melanoma cells in vitro and in vivo. In five cases, immunohistochemistry indicated the phosphorylation of Smad1/5 (p-Smad1/5) in the nuclei of melanoma cells. In the B16 mouse and A2058 human melanoma cell lines, BMP2, BMP4, or BMP7 induces morphological changes accompanied by the downregulation of E-cadherin, and the upregulation of N-cadherin and Snail, markers of epithelial–mesenchymal transition (EMT). BMP2 also stimulates cell invasion by increasing matrix metalloproteinase activity in B16 cells. These effects were canceled by the addition of LDN193189, a specific inhibitor of Smad1/5 signaling. In vivo, the injection of B16 cells expressing constitutively activated ALK3 enhanced zygoma destruction in comparison to empty B16 cells by increasing osteoclast numbers. These results suggest that the activation of BMP signaling induces EMT, thus driving the acquisition of an aggressive phenotype in malignant melanoma.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: The comparison of the expression pattern of phosphorylated-Smad1/5 in malignant melanoma and benign oral nevus patient samples.
Fig. 2: BMP signaling is activated in B16 melanoma cells.
Fig. 3: BMP2 induces epithelial–mesenchymal transition (EMT) in B16 melanoma cells.
Fig. 4: BMP2 induces epithelial–mesenchymal transition (EMT) in A2058 human melanoma cells.
Fig. 5: BMP2 promotes B16 melanoma cell migration and invasion.
Fig. 6: Generation of B16 cells expressing constitutively activated BMP receptor.
Fig. 7: Constitutively activated BMP signaling enhances bone invasion by B16 melanoma cells.

Data availability

Data are available on request from the corresponding author.

References

  1. 1.

    Tas, F. Metastatic behavior in melanoma: timing, pattern, survival, and influencing factors. J. Oncol. 2012, 647684 (2012).

    PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Sandru, A., Voinea, S., Panaitesu, E. & Blidaru, A. Survival rates of patients with metastatic malignant melanoma. J. Med. Life 7, 572–576 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Rapini, R. P., Golitz, L. E., Greer, R. O. Jr, Krekorian, E. A. & Poulson, T. Primary malignant melanoma of the oral cavity. A review of 177 cases. Cancer 55, 1543–1551 (1985).

    CAS  PubMed  Article  Google Scholar 

  4. 4.

    Barker, B. F. et al. Oral mucosal melanomas, the WESTOP Banff workshop proceedings. Western Society of Teachers of Oral Pathology. Oral Surg. Oral. Med. Oral Pathol. Oral Radiol. Endod. 83, 672–679 (1997).

    CAS  PubMed  Article  Google Scholar 

  5. 5.

    Broomhall, C. Malignant melanoma of the oral cavity in Ugandan Africans. Br. J. Surg. 54, 581–584 (1967).

    CAS  PubMed  Article  Google Scholar 

  6. 6.

    Takagi, M., Ishikawa, G. & Mori, W. Primary malignant melanoma of the oral cavity in Japan. With special reference to mucosal melanosis. Cancer 34, 358–370 (1974).

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Padhye, A. & D’souza, J. Oral malignant melanoma: a silent killer? J. Indian Soc. Periodontol. 15, 425–428 (2011).

    PubMed  PubMed Central  Article  Google Scholar 

  8. 8.

    Ferreira, L., Jham, B., Assi, R., Readinger, A. & Kessler, H. P. Oral melanocytic nevi: a clinicopathologic study of 100 cases. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 120, 358–367 (2015).

    PubMed  Article  Google Scholar 

  9. 9.

    Kumar, V. et al. Primary malignant melanoma of oral cavity: a tertiary care center experience. Natl J. Maxillofac Surg. 6, 167–171 (2015).

    PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Tchernev, G., Lotti, T. & Wollina, U. Palatal melanoma: “The Silent Killer”. Open Access Maced J. Med. Sci. 6, 364–366 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Hsu, M. Y., Rovinsky, S., Penmatcha, S., Herlyn, M. & Muirhead, D. Bone morphogenetic proteins in melanoma: angel or devil? Cancer Metastasis Rev. 24, 251–263 (2005).

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Jimi, E. The role of BMP signaling and NF-κB signaling on osteoblastic differentiation, cancer development, and vascular diseases-Is the activation of NF-κB a friend or foe of BMP Function? Vitam. Horm. 99, 145–170 (2015).

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    Katagiri, T. & Watabe, T. Bone morphogenetic proteins. Cold Spring Harb. Perspect. Biol. 8, pii: a021899 (2016).

    Article  CAS  Google Scholar 

  14. 14.

    Katagiri, T., Tsukamoto, S., Nakachi, Y. & Kuratani, M. Discovery of heterotopic bone-inducing activity in hard tissues and the TGF-β superfamily. Int. J. Mol. Sci. 19, 3586 (2018).

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  15. 15.

    De Bosscher, K., Hill, C. S. & Nicolás, F. J. Molecular and functional consequences of Smad4 C-terminal missense mutations in colorectal tumour cells. Biochem. J. 379, 209–216 (2004).

    PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Zhou, X. P. et al. Germline mutations in BMPR1A/ALK3 cause a subset of cases of juvenile polyposis syndrome and of Cowden and Bannayan-Riley-Ruvalcaba syndromes. Am. J. Hum. Genet. 69, 704–711 (2001).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17.

    Jin, Y. et al. Overexpression of BMP-2/4, -5 and BMPR-IA associated with malignancy of oral epithelium. Oral Oncol. 37, 225–233 (2001).

    PubMed  Article  Google Scholar 

  18. 18.

    Bhowmick, N. A. et al. Transforming growth factor-β1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism. Mol. Biol. Cell 12, 27–36 (2001).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Saika, S. et al. Epithelial–mesenchymal transition as a therapeutic target for prevention of ocular tissue fibrosis. Endocr. Metab. Immune Disord. Drug Targets 8, 69–76 (2008).

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Voon, D. C., Huang, R. Y., Jackson, R. A. & Thiery, J. P. The EMT spectrum and therapeutic opportunities. Mol. Oncol. 11, 878–891 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  21. 21.

    Katagiri, T. et al. Identification of a BMP-responsive element in Id1, the gene for inhibition of myogenesis. Genes Cells 7, 949–960 (2002).

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Akiyama, S. et al. Constitutively active BMP type I receptors transduce BMP-2 signals without the ligand in C2C12 myoblasts. Exp. Cell Res. 235, 362–369 (1997).

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Furuta, H. et al. Selective inhibition of NF-κB suppresses bone invasion by oral squamous cell carcinoma in vivo. Int. J. Cancer 131, E625–E635 (2012).

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Tada, Y. et al. The novel IκB kinase β inhibitor IMD-0560 prevents bone invasion by oral squamous cell carcinoma. Oncotarget 5, 12317–12330 (2014).

    PubMed  PubMed Central  Article  Google Scholar 

  25. 25.

    Shin, M. et al. The inhibition of RANKL/RANK signaling by osteoprotegerin suppresses bone invasion by oral squamous cell carcinoma cells. Carcinogenesis 32, 1634–1640 (2011).

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    Rothhammer, T. et al. Bone morphogenic proteins are overexpressed in malignant melanoma and promote cell invasion and migration. Cancer Res. 65, 448–456 (2005).

    CAS  PubMed  Google Scholar 

  27. 27.

    Hsu, M. Y. et al. Aggressive melanoma cells escape from BMP7-mediated autocrine growth inhibition through coordinated Noggin upregulation. Lab. Invest. 88, 842–855 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Braig, S. & Bosserhoff, A. K. Death inducer-obliterator 1 (Dido1) is a BMP target gene and promotes BMP-induced melanomaprogression. Oncogene 32, 837–848 (2013).

    CAS  PubMed  Article  Google Scholar 

  29. 29.

    Park, W. Y., Hong, B. L., Lee, J., Choi, C. & Kim, M. Y. H3K27 Demethylase JMJD3 employs the NF-κB and BMP signaling pathways to modulate the tumor microenvironment and promote melanoma progression and metastasis. Cancer Res. 76, 161–170 (2016).

    CAS  PubMed  Article  Google Scholar 

  30. 30.

    Capasso, M. et al. A predicted functional single-nucleotide polymorphism of bone morphogenetic protein-4 gene affects mRNA expression and shows a significant association with cutaneous melanoma in Southern Italian population. J. Cancer Res. Clin. Oncol. 135, 1799–1807 (2009).

    CAS  PubMed  Article  Google Scholar 

  31. 31.

    Bilodeau, M. L. et al. BMP-2 stimulates tyrosinase gene expression and melanogenesis in differentiated melanocytes. Pigment Cell Res. 14, 328–336 (2001).

    CAS  PubMed  Article  Google Scholar 

  32. 32.

    Tian, H., Zhao, J., Brochmann, E. J., Wang, J. C. & Murray, S. S. Bone morphogenetic protein-2 and tumor growth: diverse effects and possibilities for therapy. Cytokine Growth Factor Rev. 34, 73–91 (2017).

    CAS  PubMed  Article  Google Scholar 

  33. 33.

    Ghosh-Choudhury, N. et al. Bone morphogenetic protein-2 blocks MDA MB 231 human breast cancer cell proliferation by inhibiting cyclin-dependent kinase-mediated retinoblastoma protein phosphorylation. Biochem. Biophys. Res. Commun. 272, 705–711 (2000).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34.

    Ghosh-Choudhury, N. et al. Bone morphogenetic protein-2 induces cyclin kinase inhibitor p21 and hypophosphorylation of retinoblastoma protein in estradiol-treated MCF-7 human breast cancer cells. Biochim. Biophys. Acta 21, 186–196 (2000).

    Article  Google Scholar 

  35. 35.

    Dumont, N. & Arteaga, C. L. A kinase-inactive type II TGF-β receptor impairs BMP signaling in human breast cancer cells. Biochem Biophys. Res. Commun. 301, 108–112 (2003).

    CAS  PubMed  Article  Google Scholar 

  36. 36.

    Kleeff, J. et al. Bone morphogenetic protein 2 exerts diverse effects on cell growth in vitro and is expressed in human pancreatic cancer in vivo. Gastroenterology 116, 1202–1216 (1999).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  37. 37.

    Langenfeld, E. M. et al. The mature bone morphogenetic protein-2 is aberrantly expressed in non-small cell lung carcinomas and stimulates tumor growth of A549 cells. Carcinogenesis 24, 1445–1454 (2003).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  38. 38.

    Varley, J. E., McPherson, C. E., Zou, H., Niswander, L. & Maxwell, G. D. Expression of a constitutively active type I BMP receptor using a retroviral vector promotes the development of adrenergic cells in neural crest cultures. Dev. Biol. 196, 107–118 (1998).

    CAS  PubMed  Article  Google Scholar 

  39. 39.

    Wang, Y., Zheng, Y., Chen, D. & Chen, Y. Enhanced BMP signaling prevents degeneration and leads to endochondral ossification of Meckel’s cartilage in mice. Dev. Biol. 381, 301–311 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Kawamura, C., Kizaki, M. & Ikeda, Y. Bone morphogenetic protein (BMP)-2 induces apoptosis in human myeloma cells. Leuk. Lymphoma 43, 635–639 (2002).

    CAS  PubMed  Article  Google Scholar 

  41. 41.

    Liu, H., Bao, D., Xia, X., Chau, J. F. & Li, B. An unconventional role of BMP-Smad1 signaling in DNA damage response: a mechanism for tumorsuppression. J. Cell Biochem. 115, 450–456 (2014).

    CAS  PubMed  Article  Google Scholar 

  42. 42.

    Venkatesan, A. M. et al. Ligand-activated BMP signaling inhibits cell differentiation and death to promote melanoma. J. Clin. Investig. 128, 294–308 (2018).

    PubMed  Article  Google Scholar 

  43. 43.

    McCormack, N., Molloy, E. L. & O’Dea, S. Bone morphogenetic proteins enhance an epithelial-mesenchymal transition in normal airway epithelial cells during restitution of a disrupted epithelium. Respir. Res. 19, 14–36 (2013).

    Google Scholar 

  44. 44.

    Davis, H., Raja, E., Miyazono, K., Tsubakihara, Y. & Moustakas, A. Mechanisms of action of bone morphogenetic proteins in cancer. Cytokine Growth Factor Rev. 27, 81–92 (2017).

    Article  CAS  Google Scholar 

  45. 45.

    Fang, R. et al. Nodal promotes aggressive phenotype via Snail-mediated epithelial-mesenchymal transition in murine melanoma. Cancer Lett. 333, 66–75 (2013).

    CAS  PubMed  Article  Google Scholar 

  46. 46.

    Hofmann, U. B., Houben, R., Bröcker, E. B. & Becker, J. C. Role of matrix metalloproteinases in melanoma cell invasion. Biochimie 87, 307–314 (2005).

    CAS  PubMed  Article  Google Scholar 

  47. 47.

    Iida, J. & McCarthy, J. B. Expression of collagenase-1 (MMP-1) promotes melanoma growth through the generation of active transforming growth factor-β. Melanoma Res. 17, 205–213 (2007).

    CAS  PubMed  Article  Google Scholar 

  48. 48.

    Rothhammer, T., Braig, S. & Bosserhoff, A. K. Bone morphogenetic proteins induce expression of metalloproteinases in melanoma cells and fibroblasts. Eur. J Cancer 44, 2526–2534 (2008).

    CAS  PubMed  Article  Google Scholar 

  49. 49.

    Huang, Q. et al. IL-1β-induced activation of p38 promotes metastasis in gastric adenocarcinoma via upregulation of AP-1/c-fos, MMP2 and MMP9. Mol. Cancer 13, 18 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  50. 50.

    Gonzalez, D. M. & Medici, D. Signaling mechanisms of the epithelial-mesenchymal transition. Sci. Signal. 7, re8 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  51. 51.

    Davis, H., Raja, E., Miyazono, K., Tsubakihara, Y. & Moustakas, A. Mechanisms of action of bone morphogenetic proteins in cancer. Cytokine Growth Factor Rev. 27, 81–92 (2016).

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgements

We thank Dr. Takenobu Katagiri (Saitama Medical University, Saitama, Japan) for providing the d-WT4F-luciferase reporter and the V5-Tagged constitutively active form of BMPRIA (V5-CaALK3/pDEF). This work was supported by a research grant for the OBT Research Center from Kyushu University (to E.J.), and Fukuoka Public Health Promotion Organization Cancer Research Fund (to J.G.)

Author information

Affiliations

Authors

Contributions

J.G., R.M., H.F., M.S., M.I., S.C., and E.J. performed the experiments. K.A., Y.T., T.Y., and T. Kukita performed the radiological assessments. K.N., R.M., R.N., T.Y., M.M., and T. Kiyoshima prepared the histological samples. Y.T. and S.F. analyzed TCGA dataset. J.G., T. Kiyoshima, K.O., and Y.M. reviewed the intermediate draft. E.J. designed the study, performed the literature review, prepared the initial, final versions, and revised versions of the paper, and submitted the documents.

Corresponding author

Correspondence to Eijiro Jimi.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All experimental procedures conducted in this study were reviewed and approved by the Kyushu University Research Ethics Committee (approval numbers 27–362 and 30–235), and by the Council on Animal Care and Use Committee of Kyushu University (approval number A30-362).

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gao, J., Muroya, R., Huang, F. et al. Bone morphogenetic protein induces bone invasion of melanoma by epithelial–mesenchymal transition via the Smad1/5 signaling pathway. Lab Invest (2021). https://doi.org/10.1038/s41374-021-00661-y

Download citation

Search

Quick links