Skip to main content

Advertisement

SpringerLink
Log in

Survivin is released from cancer cells via exosomes

  • Original Paper
  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Inhibitor of apoptosis (IAP) and Heat shock proteins (HSPs) provide assistance in protecting cells from stresses of hypoxia, imbalanced pH, and altered metabolic and redox states commonly found in the microenvironmental mixture of tumor and nontumor cells. HSPs are upregulated, cell-surface displayed and released extracellularly in some types of tumors, a finding that until now was not shared by members of the IAP family. The IAP Survivin has been implicated in apoptosis inhibition and the regulation of mitosis in cancer cells. Survivin exists in a number of subcellular locations such as the mitochondria, cytoplasm, nucleus, and most recently, the extracellular space. Our previous work showing that extracellular survivin was able to enhance cellular proliferation, survival and tumor cell invasion provides evidence that Survivin might be secreted via an unidentified exocytotic pathway. In the present study, we describe for the first time the exosome-release of Survivin to the extracellular space both basally and after proton irradiation-induced stress. To examine whether exosomes contributed to Survivin release from cancer cells, exosomes were purified from HeLa cervical carcinoma cells and exosome quantity and Survivin content were determined. We demonstrate that although proton irradiation does not influence the exosomal secretory rate, the Survivin content of exosomes isolated from HeLa cells treated with a sublethal dose of proton irradiation (3 Gy) is significantly higher than control. These data identify a novel secretory pathway by which Survivin can be actively released from cells in both the basal and stress-induced state.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

IAP:

Inhibitor of apoptosis

HSPs:

Heat shock proteins

AChE:

Acetylcholinesterase

LAMP1:

Lysosomal-associated membrane protein 1

cytoD:

Cytochalasin D

XIAP:

X-linked inhibitor of apoptosis

ATCC:

American Type Culture Collection

IL2R:

Interleukin-2 receptor

DTT:

Dithiothreitol

CM:

Conditioned medium

References

  1. Aznavoorian S, Stracke ML, Krutzsch H, Schiffmann E, Liotta LA (1990) Signal transduction for chemotaxis and haptotaxis by matrix molecules in tumor cells. J Cell Biol 110:1427–1438

    Article  CAS  PubMed  Google Scholar 

  2. Ohtani H (1998) Stromal reaction in cancer tissue: pathophysiologic significance of the expression of matrix-degrading enzymes in relation to matrix turnover and immune/inflammatory reactions. Pathol Int 48:1–9

    Article  CAS  PubMed  Google Scholar 

  3. Graner MW, Cumming RI, Bigner DD (2007) The heat shock response and chaperones/heat shock proteins in brain tumors: surface expression, release, and possible immune consequences. J Neurosci 27:11214–11227

    Article  CAS  PubMed  Google Scholar 

  4. Eustace BK, Jay DG (2004) Extracellular roles for the molecular chaperone, hsp90. Cell Cycle 3:1098–1100

    Article  CAS  PubMed  Google Scholar 

  5. Radons J, Multhoff G (2005) Immunostimulatory functions of membrane-bound and exported heat shock protein 70. Exerc Immunol Rev 11:17–33

    PubMed  Google Scholar 

  6. Altieri DC (2003) Survivin, versatile modulation of cell division and apoptosis in cancer. Oncogene 22:8581–8589

    Article  CAS  PubMed  Google Scholar 

  7. Li F, Ambrosini G, Chu EY et al (1998) Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396:580–584

    Article  CAS  PubMed  Google Scholar 

  8. Altieri DC (2006) The case for survivin as a regulator of microtubule dynamics and cell-death decisions. Curr Opin Cell Biol 18:609–615

    Article  CAS  PubMed  Google Scholar 

  9. Fortugno P, Wall NR, Giodini A et al (2002) Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function. J Cell Sci 115:575–585

    CAS  PubMed  Google Scholar 

  10. Khan S, Aspe JR, Asumen MG et al (2009) Extracellular, cell-permeable survivin inhibits apoptosis while promoting proliferative and metastatic potential. Br J Cancer 100:1073–1086

    Article  CAS  PubMed  Google Scholar 

  11. Dohi T, Beltrami E, Wall NR, Plescia J, Altieri DC (2004) Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis. J Clin Invest 114:1117–1127

    CAS  PubMed  Google Scholar 

  12. Ghosh JC, Dohi T, Kang BH, Altieri DC (2008) Hsp60 regulation of tumor cell apoptosis. J Biol Chem 283:5188–5194

    Article  CAS  PubMed  Google Scholar 

  13. Fortugno P, Beltrami E, Plescia J et al (2003) Regulation of survivin function by Hsp90. Proc Natl Acad Sci USA 100:13791–13796

    Article  CAS  PubMed  Google Scholar 

  14. Plescia J, Salz W, Xia F et al (2005) Rational design of shepherdin, a novel anticancer agent. Cancer Cell 7:457–468

    Article  CAS  PubMed  Google Scholar 

  15. Farsad K (2002) Exosomes: novel organelles implicated in immunomodulation and apoptosis. Yale J Biol Med 75:95–101

    CAS  PubMed  Google Scholar 

  16. Yu X, Harris SL, Levine AJ (2006) The regulation of exosome secretion: a novel function of the p53 protein. Cancer Res 66:4795–4801

    Article  CAS  PubMed  Google Scholar 

  17. Hao QL, Shah AJ, Thiemann FT, Smogorzewska EM, Crooks GM (1995) A functional comparison of CD34 + CD38- cells in cord blood and bone marrow. Blood 86:3745–3753

    CAS  PubMed  Google Scholar 

  18. Wang CY, Azzo W, Al-Katib A, Chiorazzi N, Knowles DM 2nd (1984) Preparation and characterization of monoclonal antibodies recognizing three distinct differentiation antigens (BL1, BL2, BL3) on human B lymphocytes. J Immunol 133:684–691

    CAS  PubMed  Google Scholar 

  19. Ogawa H, Ishiguro K, Gaubatz S, Livingston DM, Nakatani Y (2002) A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells. Science 296:1132–1136

    Article  CAS  PubMed  Google Scholar 

  20. Shi Y, Sawada J, Sui G et al (2003) Coordinated histone modifications mediated by a CtBP co-repressor complex. Nature 422:735–738

    Article  CAS  PubMed  Google Scholar 

  21. Eng JK, McCormack AL, Yates JR III (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5:976–989

    Article  CAS  Google Scholar 

  22. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567

    Article  CAS  PubMed  Google Scholar 

  23. Craig R, Beavis RC (2004) TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20:1466–1467

    Article  CAS  PubMed  Google Scholar 

  24. Keller A, Nesvizhskii AI, Kolker E, Aebersold R (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74:5383–5392

    Article  CAS  PubMed  Google Scholar 

  25. Nesvizhskii AI, Keller A, Kolker E, Aebersold R (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75:4646–4658

    Article  CAS  PubMed  Google Scholar 

  26. Savina A, Furlan M, Vidal M, Colombo MI (2003) Exosome release is regulated by a calcium-dependent mechanism in K562 cells. J Biol Chem 278:20083–20090

    Article  CAS  PubMed  Google Scholar 

  27. Thery C, Clayton A, Amigorena S, Raposo G (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol (Suppl 30): 3.22.21–23.22.29

  28. Savina A, Vidal M, Colombo MI (2002) The exosome pathway in K562 cells is regulated by Rab11. J Cell Sci 115:2505–2515

    CAS  PubMed  Google Scholar 

  29. Clayton A, Court J, Navabi H et al (2001) Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry. J Immunol Methods 247:163–174

    Article  CAS  PubMed  Google Scholar 

  30. Liao DF, Jin ZG, Baas AS et al (2000) Purification and identification of secreted oxidative stress-induced factors from vascular smooth muscle cells. J Biol Chem 275:189–196

    Article  CAS  PubMed  Google Scholar 

  31. Caroni P, Rothenfluh A, McGlynn E, Schneider C (1991) S-cyclophilin. New member of the cyclophilin family associated with the secretory pathway. J Biol Chem 266:10739–10742

    CAS  PubMed  Google Scholar 

  32. Sherry B, Yarlett N, Strupp A, Cerami A (1992) Identification of cyclophilin as a proinflammatory secretory product of lipopolysaccharide-activated macrophages. Proc Natl Acad Sci USA 89:3511–3515

    Article  CAS  PubMed  Google Scholar 

  33. Iero M, Valenti R, Huber V et al (2008) Tumour-released exosomes and their implications in cancer immunity. Cell Death Differ 15:80–88

    Article  CAS  PubMed  Google Scholar 

  34. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795

    Article  PubMed  Google Scholar 

  35. Lancaster GI, Febbraio MA (2005) Exosome-dependent trafficking of HSP70: a novel secretory pathway for cellular stress proteins. J Biol Chem 280:23349–23355

    Article  CAS  PubMed  Google Scholar 

  36. Johnstone RM (2006) Exosomes biological significance: a concise review. Blood Cells Mol Dis 36:315–321

    Article  CAS  PubMed  Google Scholar 

  37. Clayton A, Turkes A, Navabi H, Mason MD, Tabi Z (2005) Induction of heat shock proteins in B-cell exosomes. J Cell Sci 118:3631–3638

    Article  CAS  PubMed  Google Scholar 

  38. Lamparski HG, Metha-Damani A, Yao JY et al (2002) Production and characterization of clinical grade exosomes derived from dendritic cells. J Immunol Methods 270:211–226

    CAS  PubMed  Google Scholar 

  39. Gastpar R, Gehrmann M, Bausero MA et al (2005) Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells. Cancer Res 65:5238–5247

    Article  CAS  PubMed  Google Scholar 

  40. Mathew A, Bell A, Johnstone RM (1995) Hsp-70 is closely associated with the transferrin receptor in exosomes from maturing reticulocytes. Biochem J 308(Pt 3):823–830

    CAS  PubMed  Google Scholar 

  41. Tajika Y, Matsuzaki T, Suzuki T et al (2005) Differential regulation of AQP2 trafficking in endosomes by microtubules and actin filaments. Histochem Cell Biol 124:1–12

    Article  CAS  PubMed  Google Scholar 

  42. Takata K (2006) Aquaporin-2 (AQP2): its intracellular compartment and trafficking. Cell Mol Biol (Noisy-le-grand) 52:34–39

    CAS  Google Scholar 

  43. Mignot G, Roux S, Thery C, Segura E, Zitvogel L (2006) Prospects for exosomes in immunotherapy of cancer. J Cell Mol Med 10:376–388

    Article  CAS  PubMed  Google Scholar 

  44. Escola JM, Kleijmeer MJ, Stoorvogel W, Griffith JM, Yoshie O, Geuze HJ (1998) Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J Biol Chem 273:20121–20127

    Article  CAS  PubMed  Google Scholar 

  45. Valentijn KM, Gumkowski FD, Jamieson JD (1999) The subapical actin cytoskeleton regulates secretion and membrane retrieval in pancreatic acinar cells. J Cell Sci 112(Pt 1):81–96

    CAS  PubMed  Google Scholar 

  46. Matter K, Dreyer F, Aktories K (1989) Actin involvement in exocytosis from PC12 cells: studies on the influence of botulinum C2 toxin on stimulated noradrenaline release. J Neurochem 52:370–376

    Article  CAS  PubMed  Google Scholar 

  47. Sandvig K, van Deurs B (1990) Selective modulation of the endocytic uptake of ricin and fluid phase markers without alteration in transferrin endocytosis. J Biol Chem 265:6382–6388

    CAS  PubMed  Google Scholar 

  48. Mita AC, Mita MM, Nawrocki ST, Giles FJ (2008) Survivin: key regulator of mitosis and apoptosis and novel target for cancer therapeutics. Clin Cancer Res 14:5000–5005

    Article  CAS  PubMed  Google Scholar 

  49. Galloway NR, Aspe JR, Sellers C, Wall NR (2009) Enhanced antitumor effect of combined gemcitabine and proton radiation in the treatment of pancreatic cancer. Pancreas 38:782–790

    Article  CAS  PubMed  Google Scholar 

  50. Mera S, Magnusson M, Tarkowski A, Bokarewa M (2008) Extracellular survivin up-regulates adhesion molecules on the surface of leukocytes changing their reactivity pattern. J Leukoc Biol 83:149–155

    Article  CAS  PubMed  Google Scholar 

  51. Bokarewa M, Lindblad S, Bokarew D, Tarkowski A (2005) Balance between survivin, a key member of the apoptosis inhibitor family, and its specific antibodies determines erosivity in rheumatoid arthritis. Arthritis Res Ther 7:R349–R358

    Article  CAS  PubMed  Google Scholar 

  52. Kapp EA, Schutz F, Connolly LM et al (2005) An evaluation, comparison, and accurate benchmarking of several publicly available MS/MS search algorithms: sensitivity and specificity analysis. Proteomics 5:3475–3490

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Grant Support: NCMHD Project EXPORT Program 5P20MD001632/Project 3 (N.R. Wall). Funding was also obtained as part of a start-up package from Loma Linda University’s Center for Molecular Biology and Gene Therapy, now the Center for Health Disparities Research and Molecular Medicine (NRW) and a National Merit Test Bed (NMTB) award sponsored by the Department of the Army under Cooperative Agreement Number DAMD17-97-2-7016 (NRW). Proton irradiation was accomplished at the Loma Linda University Radiobiology Proton Treatment Facility, now the James M. Slater, MD, Proton Treatment and Research Center. The authors would like to personally thank Dr. James Slater, Dr. Daila Gridley, Steven Rightnar and Celso Perez for all their help and we dedicate this work to our dear friend and colleague Dr. Lora Green who passed away unexpectedly.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Center for Health Disparities Research and Molecular Medicine, Loma Linda University, 11085 Campus Street, Mortensen Hall Room 162, Loma Linda, CA, 92350, USA

    Salma Khan, Jessica M. S. Jutzy, Jonathan R. Aspe, Dalmor W. McGregor & Nathan R. Wall

  2. Department of Basic Sciences, Division of Biochemistry and Microbiology, Loma Linda University, Loma Linda, CA, 92350, USA

    Salma Khan, Jessica M. S. Jutzy, Jonathan R. Aspe, Dalmor W. McGregor, Jonathan W. Neidigh & Nathan R. Wall

Authors
  1. Salma Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jessica M. S. Jutzy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jonathan R. Aspe
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dalmor W. McGregor
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jonathan W. Neidigh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nathan R. Wall
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nathan R. Wall.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Table S1.

The Scaffold score for peptides and proteins represents the probability of a correct assignment as calculated by PeptideProphet (24) and ProteinProphet (25), respectively. The PeptideProphet probability represents a consensus of SEQUEST, Mascot, and X!Tandem scores when available for a given peptide. The Sequest Xcorr values of greater than 1.73 and 1.97 for single and double charged peptides, respectively, have been empirically shown to represent a 95% probability of a correct assignment (52). The expected number of hits for each peptide that will occur by random chance during a Mascot search of the database is shown in the last column (PDF 125 kb)

Figure S1.

HeLaS/POZnSurvivin cells secrete Survivin and Hsp70-containing exosomes. (a) Isotype controls were used to show the specificity of the immunogold-conjugated antibodies. Exosomes were characterized from HeLaS/POZnSurvivin conditioned medium using electron microscopy. Immunoelectron microscopy on (b) anti-Hsp70- and (c) anti-Survivin- stained cryosections of HeLaS/POZnSurvivin cells shows that Survivin and Hsp70 localization in the exosome. Immunogold nanoparticles of 10 nm were conjugated to anti-Survivin pAb while 5 nm nanoparticles were conjugated to anti-Hsp70 mAb. Exosomes are cup-shaped and 50-150 nm in size. Bar, 100 nm (PDF 3,023 kb)

Figure S2.

Cancer cell lines disproportionately release Survivin in their exosomes compared with non cancer cells. (a) Western blotting shows that the cancer cell lines PozN-WT, Panc1 and PC3 have Survivin in their exosomes compared with cells and cell lines representing non-cancer cells: Peripheral Blood Mononuclear Cells (PBMC), Human Bone Marrow Stroma (HBMS), Prostate Stromal Cells (PrSC) and human embryonic kidney epithelial (A293) cells. Protein concentrations for Western blot loading were determined using the AChE activity. Exosome loading is controlled for by the Lysosome Associated Membrane Protein (LAMP1). Molecular-weight markers in kilodaltons (KDa) are shown on the left. Exosomes were collected after 24h of serum depletion. Protein quantitation and loading was accomplished by AChE for exosomes and BCA for whole cell lysates (WCL). (b) Exosomes were purified from control media after which AChE activity was determined as described under Methods and Materials. Values represent the means of three samples with measurements at 30 min recorded (TIFF 95 kb)

Figure S3.

Cytochalasin D disrupts actin microfilaments in HeLaS/POZnSurvivin cells. (a) HeLaS/POZnSurvivin cells and those treated with cytochalasin D were fixed and stained using alpha actin antibodies as described in the methods and materials and were visualized using fluorescence or phase-contrast light microscopy. Magnification under fluorescence and phase-contrast was x1000. (b) Propidium iodide (PI) staining was accomplished to define and quantitate the cytoD-induction of apoptosis in these cells. (c) Exosome presence was measured as AChE activity. Measured at 30 min, the addition of cytochalasin D (cytoD) or monensin (Mon.) to 3 Gy proton irradiation resulted in a marked reduction in exosome presence. (d) Western blots of Survivin and Hsp70 in the conditioned medium of exosome isolations (top) and whole cell lysates (bottom). Protein concentrations were determined using the BCA assay. Molecular-weight markers in kilodaltons (kDa) are shown on the left. AChE activity values represent the means of three samples. Exosomes were collected after 24 h of treatment. Abbreviations: CytoD = cytochalasin D, Mon. = Monensin (TIFF 260 kb)

(TIFF 119 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Khan, S., Jutzy, J.M.S., Aspe, J.R. et al. Survivin is released from cancer cells via exosomes. Apoptosis 16, 1–12 (2011). https://doi.org/10.1007/s10495-010-0534-4

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-010-0534-4

Keywords

Search

Navigation