Article | Published:

Cellular and Molecular Biology

Tumour cell blebbing and extracellular vesicle shedding: key role of matrikines and ribosomal protein SA

British Journal of Cancervolume 120pages453465 (2019) | Download Citation

Abstract

Background

Carcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases.

Methods

EDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA.

Results

We demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding.

Conclusions

Our data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSA binding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites.

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Acknowledgements

In memory of my father Guy Brassart (1945–2018) and my grandmother (1921–2018). The authors thank Dr Joon Kim (Korea University, South Korea) for the GFP-Hsp90 plasmid and Dr Steven Hooper (Cancer Research UK London Research Institute, United Kingdom) for the mCherry-MLC plasmid. We are grateful to Aurélie Dupont-Deshorgue, Dr Sandra Audonnet (Flow Cytometry Core URCACyt, URCA, Reims, France), Dr Frederique Nolin and Laurence Wortham (EA4682, URCA, Reims, France) for their skilful technical assistance. This work was supported by grants from the Centre National de la Recherche Scientifique (UMR 7369), the University of Reims Champagne-Ardenne, the Region Champagne-Ardenne and the Ligue Nationale contre le Cancer.

Author contributions

B.B., J.D.S., M.D., E.S. and C.T. designed, performed, and analysed all of the experiments except for the Ca2+ measurements (performed by F.H. and H.O.A.) and the transmission electron microscopy and the scanning electron microscopy (performed by F.V. and J.M). B.B. wrote the paper with S.B.P., LR., F.X.M., J.C.M. and A.H. provided essential editorial oversight and critical review.

Author information

Affiliations

  1. Université de Reims Champagne Ardenne, SFR CAP-Santé (FED 4231), Laboratoire de Biochimie Médicale et Biologie Moléculaire, Reims, France

    • Bertrand Brassart
    • , Jordan Da Silva
    • , Mélissa Donet
    • , Emeline Seurat
    • , Jean-Claude Monboisse
    • , François-Xavier Maquart
    • , Laurent Ramont
    •  & Sylvie Brassart-Pasco
  2. CNRS UMR 7369, Matrice Extracellulaire et Dynamique Cellulaire - MEDyC, Reims, France

    • Bertrand Brassart
    • , Jordan Da Silva
    • , Mélissa Donet
    • , Emeline Seurat
    • , Jean-Claude Monboisse
    • , François-Xavier Maquart
    • , Laurent Ramont
    •  & Sylvie Brassart-Pasco
  3. Université de Picardie Jules Verne, SFR CAP-Santé (FED 4231), Laboratoire de Physiologie Cellulaire et Moléculaire, EA 4667, Amiens, France

    • Frédéric Hague
    •  & Halima Ouadid-Ahidouch
  4. Plate-forme Imagerie Cellulaire et Tissulaire, Université de Reims Champagne-Ardenne, Reims, France

    • Christine Terryn
  5. Biomatériaux & inflammation en site osseux, EA 4691, SFR CAP-Santé (FED 4231), Université de Reims-Champagne-Ardenne, Reims, France

    • Fréderic Velard
  6. Laboratoire de Recherche en Nanosciences, EA 4682, SFR CAP-Santé (FED 4231), Université de Reims-Champagne-Ardenne, Reims, France

    • Jean Michel
  7. CHU Reims, Laboratoire Central de Biochimie, Reims, France

    • Jean-Claude Monboisse
    • , François-Xavier Maquart
    •  & Laurent Ramont
  8. Physiology & Experimental Medicine Program, Hospital for Sick Children, Toronto, ON, Canada

    • Aleksander Hinek
  9. Institute of Medical Science, University of Toronto, Toronto, ON, Canada

    • Aleksander Hinek
  10. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada

    • Aleksander Hinek

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The authors declare no competing interests.

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Correspondence to Bertrand Brassart.

Supplementary information

  1. Video G - Spinning disk microscopy of GFP-Hsp90 HT-1080 cell in presence of EGCG and in absence of EDPs

  2. Video H - Spinning disk microscopy of GFP-Hsp90 HT-1080 cell in presence of EGCG and EDPs

  3. S Fig 1 - EDPs stimulate cell membrane blebbing in 3D collagen matrix. GFP-Hsp90 transfected HT-1080 cells were seeded in a 3D collagen matrix in the presence of EDPs

  4. S Fig 2 - 2D Time-lapse snapshots of blebbing HT-1080 cells in presence of EDPs at t0min and at t75min

  5. S Fig 3 - Cell proliferation and cell survival tests

  6. S Fig 4 - 2D Time-lapse snapshots of cell-to-cell communication between a blebbing cell and a mesenchymal cell in presence of EDPs

  7. S Fig 5 - Extracellular vesicles were prepared from cell-conditioned medium by centrifugation and ultracentrifugation after 24h of incubation

  8. S Fig 6 - Signalling pathway immunostaining quantifications and localizations using the ImageJ software

  9. S Fig 7 - Blebbistatin and Y27632 inhibit EDP-stimulated blebbing, Hsp90 and proteinase secretions

  10. Video 1. Spinning disk microscopy of a mesenchymal GFP-Hsp90 HT-1080 cell in absence of EDP

  11. Video 2. Spinning disk microscopy of a blebbing GFP-Hsp90 HT-1080 cell in presence of EDPs

  12. Video 3. Live videomicroscopy of the reversible blebbing in presence of EDPs

  13. Video 4 - Spinning disk microscopy of cell-to-cell communication via shed extracellular vesicles in presence of EDPs

  14. Video 5 - Spinning disk microscopy of mesenchymal mCherry-MLC HT-1080 cells in absence of EDP

  15. Video 6 - Spinning disk microscopy of blebbing mCherry-MLC HT-1080 cells in presence of EDPs

  16. Video 7 - Spinning disk microscopy of blebbing GFP-Hsp90 HT-1080 cells and shed microsicles in presence of EDPs

  17. S Table 1. Immunostaining quantification and localization in HT-1080 cells using ImageJ plugin

  18. S Table 2. Blebbing quantification in HT-1080 cells in presence of different elastin receptor inhibitors

  19. S Fig 8 - Identification of the RPSA protein as the VGVAPG receptor by affinity chromatography

  20. S Fig 9 - EGCG inhibits EDP-stimulated blebbing

  21. Video A - Spinning disk microscopy of a mesenchymal GFP-Hsp90 HT-1080 cell in absence of EDP

  22. Video B - Spinning disk microscopy of blebbing GFP-Hsp90 HT-1080 cell in presence of EDPs

  23. Video C - Live videomicroscopy of blebbing HT-1080 cells in presence of EDPs

  24. Video D - Live videomicroscopy of cell-to-cell communication via shed microvesicle in presence of EDPs

  25. Video E - Spinning disk microscopy of GFP-Hsp90 HT-1080 cell in absence of EDPs

  26. Video F - Spinning disk microscopy of GFP-Hsp90 HT-1080 cell in presence of EDPs

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https://doi.org/10.1038/s41416-019-0382-0