Letter | Published:

Tumour-cell-induced endothelial cell necroptosis via death receptor 6 promotes metastasis

Nature volume 536, pages 215218 (11 August 2016) | Download Citation

Abstract

Metastasis is the leading cause of cancer-related death in humans. It is a complex multistep process during which individual tumour cells spread primarily through the circulatory system to colonize distant organs1,2,3. Once in the circulation, tumour cells remain vulnerable, and their metastatic potential largely depends on a rapid and efficient way to escape from the blood stream by passing the endothelial barrier4,5,6,7,8,9. Evidence has been provided that tumour cell extravasation resembles leukocyte transendothelial migration7,8,9. However, it remains unclear how tumour cells interact with endothelial cells during extravasation and how these processes are regulated on a molecular level. Here we show that human and murine tumour cells induce programmed necrosis (necroptosis) of endothelial cells, which promotes tumour cell extravasation and metastasis. Treatment of mice with the receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-inhibitor necrostatin-1 or endothelial-cell-specific deletion of RIPK3 reduced tumour-cell-induced endothelial necroptosis, tumour cell extravasation and metastasis. In contrast, pharmacological caspase inhibition or endothelial-cell-specific loss of caspase-8 promoted these processes. We furthermore show in vitro and in vivo that tumour-cell-induced endothelial necroptosis leading to extravasation and metastasis requires amyloid precursor protein expressed by tumour cells and its receptor, death receptor 6 (DR6), on endothelial cells as the primary mediators of these effects. Our data identify a new mechanism underlying tumour cell extravasation and metastasis, and suggest endothelial DR6-mediated necroptotic signalling pathways as targets for anti-metastatic therapies.

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.

    & Tumor metastasis: molecular insights and evolving paradigms. Cell 147, 275–292 (2011)

  2. 2.

    , & Tumor metastasis: moving new biological insights into the clinic. Nature Med. 19, 1450–1464 (2013)

  3. 3.

    & Origins of metastatic traits. Cancer Cell 24, 410–421 (2013)

  4. 4.

    et al. Genes that mediate breast cancer metastasis to the brain. Nature 459, 1005–1009 (2009)

  5. 5.

    et al. TGFβ primes breast tumors for lung metastasis seeding through angiopoietin-like 4. Cell 133, 66–77 (2008)

  6. 6.

    et al. Mediators of vascular remodelling co-opted for sequential steps in lung metastasis. Nature 446, 765–770 (2007)

  7. 7.

    , & Crossing the endothelial barrier during metastasis. Nature Rev. Cancer 13, 858–870 (2013)

  8. 8.

    & The initial hours of metastasis: the importance of cooperative host-tumor cell interactions during hematogenous dissemination. Cancer Discov . 2, 1091–1099 (2012)

  9. 9.

    & Microenvironmental regulation of metastasis. Nature Rev. Cancer 9, 239–252 (2009)

  10. 10.

    et al. Essential versus accessory aspects of cell death: recommendations of the NCCD 2015. Cell Death Differ. 22, 58–73 (2015)

  11. 11.

    & Necroptosis and its role in inflammation. Nature 517, 311–320 (2015)

  12. 12.

    & Necroptosis in health and diseases. Semin. Cell Dev. Biol. 35, 14–23 (2014)

  13. 13.

    , & The diverse role of RIP kinases in necroptosis and inflammation. Nature Immunol. 16, 689–697 (2015)

  14. 14.

    et al. Catalytic activity of the caspase-8–FLIPL complex inhibits RIPK3-dependent necrosis. Nature 471, 363–367 (2011)

  15. 15.

    et al. RIP3 mediates the embryonic lethality of caspase-8-deficient mice. Nature 471, 368–372 (2011)

  16. 16.

    , , & Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods 44, 205–221 (2008)

  17. 17.

    et al. Identification and functional characterization of DR6, a novel death domain-containing TNF receptor. FEBS Lett. 431, 351–356 (1998)

  18. 18.

    , & Death receptor signaling. J. Cell Sci. 118, 265–267 (2005)

  19. 19.

    et al. Enhanced CD4+ T cell proliferation and Th2 cytokine production in DR6-deficient mice. Immunity 15, 23–34 (2001)

  20. 20.

    et al. Death receptor 6 (DR6) antagonist antibody is neuroprotective in the mouse SOD1G93A model of amyotrophic lateral sclerosis. Cell Death Disease 4, e841 (2013)

  21. 21.

    , , & APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature 457, 981–989 (2009)

  22. 22.

    , , , & The crystal structure of DR6 in complex with the amyloid precursor protein provides insight into death receptor activation. Genes Dev. 29, 785–790 (2015)

  23. 23.

    et al. Amyloid precursor protein in human breast cancer: an androgen-induced gene associated with cell proliferation. Cancer Sci. 104, 1532–1538 (2013)

  24. 24.

    et al. Amyloid precursor protein is a primary androgen target gene that promotes prostate cancer growth. Cancer Res. 69, 137–142 (2009)

  25. 25.

    , , , & Amyloid precursor protein as a potential marker of malignancy and prognosis in papillary thyroid carcinoma. Oncol. Lett. 3, 1227–1230 (2012)

  26. 26.

    et al. Acute function of secreted amyloid precursor protein fragment APPsα in synaptic plasticity. Acta Neuropathol. 129, 21–37 (2015)

  27. 27.

    et al. Membrane-bound Fas ligand only is essential for Fas-induced apoptosis. Nature 461, 659–663 (2009)

  28. 28.

    , & Necroptosis: the release of damage-associated molecular patterns and its physiological relevance. Immunity 38, 209–223 (2013)

  29. 29.

    et al. Autocrine regulation of TGF-β1-induced cell migration by exocytosis of ATP and activation of P2 receptors in human lung cancer cells. J. Cell Sci. 125, 5051–5060 (2012)

  30. 30.

    , , , & Platelet-derived nucleotides promote tumor-cell transendothelial migration and metastasis via P2Y2 receptor. Cancer Cell 24, 130–137 (2013)

  31. 31.

    , & In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nature Protocols 2, 329–333 (2007)

  32. 32.

    et al. Anaphylactic shock depends on endothelial Gq/G11. J. Exp. Med. 206, 411–420 (2009)

  33. 33.

    et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011)

  34. 34.

    et al. Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc. Natl Acad. Sci. USA 101, 18206–18211 (2004)

  35. 35.

    et al. A critical function for β-amyloid precursor protein in neuronal migration revealed by in utero RNA interference. J. Neurosci. 27, 14459–14469 (2007)

  36. 36.

    et al. G13 controls angiogenesis through regulation of VEGFR-2 expression. Dev. Cell 25, 427–434 (2013)

Download references

Acknowledgements

We thank S. Fulda and our friends and colleagues for comments on the manuscript. We also thank S. Hümmer for secretarial help and C. Ringel, J. Hoffmann, I.-M. Gross, D. Magalei and M. Winkels for technical help. This work was supported by the German Cancer Aid and the Max Planck Society. K.H. was supported by the China Scholarship Council. U.C.M. was supported by a grant from the Deutsche Forschungsgemeinschaft (MU 1457/9-2). M.P. received funding from the European Research Council (grant agreement 323040), the Deutsche Forschungsgemeinschaft (SFB670, SFB829), Worldwide Cancer Research (grant 15-0228) and the Helmholtz Alliance Preclinical Comprehensive Cancer Center.

Author information

Affiliations

  1. Max Planck Institute for Heart and Lung Research, Department of Pharmacology, Ludwigstrasse 43, 61231 Bad Nauheim, Germany

    • Boris Strilic
    • , Lida Yang
    • , Julián Albarrán-Juárez
    •  & Stefan Offermanns
  2. University of Cologne, Institute for Genetics, Center for Molecular Medicine (CMMC), and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Joseph-Stelzmann-Strasse 26, 50931 Cologne, Germany

    • Laurens Wachsmuth
    •  & Manolis Pasparakis
  3. University of Heidelberg, Department of Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany

    • Kang Han
    •  & Ulrike C. Müller
  4. J. W. Goethe University Frankfurt, Medical Faculty, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany

    • Stefan Offermanns

Authors

  1. Search for Boris Strilic in:

  2. Search for Lida Yang in:

  3. Search for Julián Albarrán-Juárez in:

  4. Search for Laurens Wachsmuth in:

  5. Search for Kang Han in:

  6. Search for Ulrike C. Müller in:

  7. Search for Manolis Pasparakis in:

  8. Search for Stefan Offermanns in:

Contributions

B.S. performed most of the experiments and analysed the data. L.Y. generated mice with a conditional Ripk3 allele and contributed to in vitro and in vivo studies. J.A.J. contributed to in vitro experiments. L.W. and M.P. generated MLKL-deficient animals. K.H. and U.C.M. purified APPsα and performed APP-related experiments. B.S. and S.O. designed the study, discussed data and wrote the manuscript. All authors commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Boris Strilic or Stefan Offermanns.

Reviewer Information Nature thanks C. Betsholtz, S. Tavazoie and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figures

    This file contains Supplementary Figures 1-2, original source images for data obtained by electrophoretic separation.

Excel files

  1. 1.

    Supplementary Table 1

    This table contains information regarding the identification of endothelial receptors involved in the regulation of tumour cell-induced endothelial cell death (expression data, siRNA target sequences).

Videos

  1. 1.

    Examples of endothelial cells undergoing necroptosis after contact with a tumour cell

    Time lapse imaging of HUVEC upon contact with MDA-MB-231 tumour cells (GFP, green). Hoechst33342 labels all cell nuclei (blue) and EthD-III (red) labels necroptotic cells. Imaging shows endothelial cells that undergo necroptotic cell death (arrow head) several hours after contact with a tumour cell.

  2. 2.

    Examples of endothelial cells undergoing necroptosis after contact with a tumour cell

    Time lapse imaging of HUVEC upon contact with MDA-MB-231 tumour cells (GFP, green). Hoechst33342 labels all cell nuclei (blue) and EthD-III (red) labels necroptotic cells. Imaging shows endothelial cells that undergo necroptotic cell death (arrow head) several hours after contact with a tumour cell.

  3. 3.

    Examples of endothelial cells undergoing necroptosis after contact with a tumour cell

    Time lapse imaging of HUVEC upon contact with MDA-MB-231 tumour cells (GFP, green). Hoechst33342 labels all cell nuclei (blue) and EthD-III (red) labels necroptotic cells. Imaging shows endothelial cells that undergo necroptotic cell death (arrow head) several hours after contact with a tumour cell.

  4. 4.

    Morphological criteria for the distinction between living, apoptotic and necrotic endothelial cells

    Time lapse imaging of HUVEC cultured in the presence of PBS (control) or in the presence of H2O2 (1 mM) to induce necrosis or TNF (100 ng/ml) to induce apoptosis. Cell nuclei were stained with Hoechst33342 (blue). Nuclei of necrotic cells stained positive for the membrane-impermeant nuclear dye EthD-III (red). Living cells appear with normal round to kidney-shaped nuclei and are negative for EthD-III. Necrotic cells appear with normal round to kidney-shaped nuclei or with minor degrees of nuclear shrinkage (no condensation and no fragmentation) and are positive for EthD-III. Apoptotic cells appear with strong condensed and frequently fragmented nuclei and are negative for EthD-III. No late apoptotic cells are shown in the videos.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature19076

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.