Cell surface heparan sulfate proteoglycans are involved in the extracellular Hsp90-stimulated migration and invasion of cancer cells

  • Anastasiya V. Snigireva
  • Veronika V. Vrublevskaya
  • Yuri Y. Skarga
  • Oleg S. MorenkovEmail author
Original Paper


The extracellular heat shock protein 90 (Hsp90) is known to participate in cell migration and invasion. Recently, we have shown that cell surface heparan sulfate proteoglycans (HSPGs) are involved in the binding and anchoring of extracellular Hsp90 to the plasma membrane, but the biological relevance of this finding was unclear. Here, we demonstrated that the digestion of heparan sulfate (HS) moieties of HSPGs with a heparinase I/III blend and the metabolic inhibition of the sulfation of HS chains by sodium chlorate considerably impair the migration and invasion of human glioblastoma A-172 and fibrosarcoma HT1080 cells stimulated by extracellular native Hsp90. Heparin, a polysaccharide closely related to HS, also reduced the Hsp90-stimulated migration and invasion of cells. Phorbol 12-myristate 13-acetate, an intracellular inducer of cell motility bypassing the ligand activation of receptors, restored the basal migration of heparinase- and chlorate-treated cells almost to the control level, suggesting that the cell motility machinery was insignificantly affected in cells with degraded and undersulfated HS chains. On the other hand, the downstream phosphorylation of AKT in response to extracellular Hsp90 was substantially impaired in heparinase- and chlorate-treated cells as compared to untreated cells. Taken together, our results demonstrated for the first time that cell surface HSPGs play an important role in the migration and invasion of cancer cells stimulated by extracellular Hsp90 and that plasma membrane-associated HSPGs are required for the efficient transmission of signal from extracellular Hsp90 into the cell.


Cell surface HSPGs Extracellular Hsp90 Hsp90-stimulated cell migration Hsp90-stimulated cell invasion 



We thank A.O. Shepelyakovskaya for the help with flow cytometry.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interest.

Supplementary material

12192_2018_955_MOESM1_ESM.pdf (95 kb)
Fig. S1 Purified native mouse Hsp90 was subjected to SDS-PAGE (a) and Western blot analysis with Hsp90α- and Hsp90β-specific antibodies (b). (PDF 95 kb)
12192_2018_955_MOESM2_ESM.pdf (44 kb)
Fig. S2 The stimulation of migration and invasion of cells by native Hsp90s from different animal species (a) and the analysis of cytotoxicity and antiproliferative activity of native mouse Hsp90 (b). (a) The migration and invasion of cells were evaluated in the transwell migration/invasion assays in the absence and presence of mouse, swine, and bovine Hsp90 (50 μg/ml). The migration and invasion of cells were expressed in percent relative to that of control cells without Hsp90. Each bar represents the mean ± SD (n = 5). *p < 0.05 and **p < 0.01 indicate statistically significant differences. The representative results are shown. (b) For the determination of cytotoxicity, A-172 and HT1080 cells were grown in wells of 96-well plates till confluence, then mouse Hsp90 diluted at different concentrations in DMEM-FBS was added to cells, and the plates were incubated for 48 h at 37 °C in an atmosphere of 5% CO2. After incubation, 50 μl of an MTT solution (5 mg/ml) was added to each well, and the plates were incubated at 37 °C for 2 h. The medium was removed from the wells, precipitated crystals were dissolved with DMSO, and the optical density was measured at 495 nm (OD495). To determine the antiproliferative activity, the cells were placed in wells of 96-well plates (5.0–7.0 × 103 cells per well) in DMEM-FBS containing native mouse Hsp90 at different concentrations and incubated for 48 h. Staining with MTT and measurements of OD495 values were performed as described above. The absorbance of wells expressed in percent is presented, and OD495 values of control wells without Hsp90 is taken as 100%. Each figure represents the mean ± SD (n = 5). (PDF 43 kb)


  1. Baeuerle PA, Huttner WB (1986) Chlorate—a potent inhibitor of protein sulfation in intact cells. Biochem Biophys Res Commun 141:870–877CrossRefGoogle Scholar
  2. Basu S, Binder RJ, Ramalingam T, Srivastava PK (2001) CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity 14:303–313CrossRefGoogle Scholar
  3. Becker B, Multhoff G, Farkas B, Wild PJ, Landthaler M, Stolz W, Vogt T (2004) Induction of Hsp90 protein expression in malignant melanomas and melanoma metastases. Exp Dermatol 13:27–32CrossRefGoogle Scholar
  4. Chen JS, Hsu YM, Chen CC, Chen LL, Lee CC, Huang TS (2010) Secreted heat shock protein 90α induces colorectal cancer cell invasion through CD91/LRP-1 and NF-kB-mediated integrin αV expression. J Biol Chem 285:25458–25466CrossRefGoogle Scholar
  5. Chen WS, Chen CC, Chen LL, Lee CC, Huang TS (2013) Secreted heat shock protein 90α (HSP90α) induces nuclear factor-κB-mediated TCF12 protein expression to down-regulate E-cadherin and to enhance colorectal cancer cell migration and invasion. J Biol Chem 288:9001–9010CrossRefGoogle Scholar
  6. Cheng CF, Fan J, Fedesco M, Guan S, Li Y, Bandyopadhyay B, Bright AM, Yerushalmi D, Liang M, Chen M, Han YP, Woodley DT, Li W (2008) Transforming growth factor alpha (TGFalpha)-stimulated secretion of HSP90alpha: using the receptor LRP-1/CD91 to promote human skin cell migration against a TGFbeta-rich environment during wound healing. Mol Cell Biol 28:3344–3358CrossRefGoogle Scholar
  7. Cheng CF, Sahu D, Tsen F, Zhao Z, Fan J, Kim R, Wang X, O'Brien K, Li Y, Kuang Y, Chen M, Woodley DT, Li W (2011) A fragment of secreted Hsp90α carries properties that enable it to accelerate effectively both acute and diabetic wound healing in mice. J Clin Invest 121:4348–4361CrossRefGoogle Scholar
  8. Chiodelli P, Mitola S, Ravelli C, Oreste P, Rusnati M, Presta M (2011) Heparan sulfate proteoglycans mediate the angiogenic activity of the vascular endothelial growth factor receptor-2 agonist gremlin. Arterioscler Thromb Vasc Biol 31:e116–e127CrossRefGoogle Scholar
  9. Clayton A, Turkes A, Navabi H, Mason MD, Tabi Z (2005) Induction of heat shock proteins in B-cell exosomes. J Cell Sci 118:3631–3638CrossRefGoogle Scholar
  10. Correia AL, Mori H, Chen EI, Schmitt FC, Bissell MJ (2013) The hemopexin domain of MMP3 is responsible for mammary epithelial invasion and morphogenesis through extracellular interaction with HSP90β. Genes Dev 27:805–817CrossRefGoogle Scholar
  11. Crowe LB, Hughes PF, Alcorta DA, Osada T, Smith AP, Totzke J, Loiselle DR, Lutz ID, Gargesha M, Roy D, Roques J, Darr D, Lyerly HK, Spector NL, Haystead TAJ (2017) A fluorescent Hsp90 probe demonstrates the unique association between extracellular Hsp90 and malignancy in vivo. ACS Chem Biol 12:1047–1055CrossRefGoogle Scholar
  12. Deakin JA, Lyon M (1999) Differential regulation of hepatocyte growth factor/scatter factor by cell surface proteoglycans and free glycosaminoglycan chains. J Cell Sci 112:1999–2009PubMedGoogle Scholar
  13. Dreyfuss JL, Regatieri CV, Jarrouge TR, Cavalheiro RP, Sampaio LO, Nader HB (2009) Heparan sulfate proteoglycans: structure, protein interactions and cell signaling. An Acad Bras Cienc 81:409–429CrossRefGoogle Scholar
  14. El Hamidieh A, Grammatikakis N, Patsavoudi E (2012) Cell surface Cdc37 participates in extracellular HSP90 mediated cancer cell invasion. PLoS One 7:e42722CrossRefGoogle Scholar
  15. Ernst S, Langer R, Cooney CL, Sasisekharan R (1995) Enzymatic degradation of glycosaminoglycans. Crit Rev Biochem Mol Biol 30:387–444CrossRefGoogle Scholar
  16. Gallagher J (2015) Fell-Muir lecture: Heparan sulphate and the art of cell regulation: a polymer chain conducts the protein orchestra. Int J Exp Pathol 96:203–231CrossRefGoogle Scholar
  17. Gopal U, Bohonowych JE, Lema-Tome C, Liu A, Garrett-Mayer E, Wang B, Isaacs JS (2011) A novel extracellular Hsp90 mediated co-receptor function for LRP1 regulates EphA2 dependent glioblastoma cell invasion. PLoS One 6:e17649CrossRefGoogle Scholar
  18. Hance MW, Dole K, Gopal U, Bohonowych JE, Jezierska-Drutel A, Neumann CA, Liu H, Garraway IP, Isaacs JS (2012) Secreted Hsp90 is a novel regulator of the epithelial to mesenchymal transition (EMT) in prostate cancer. J Biol Chem 287:37732–37744CrossRefGoogle Scholar
  19. Higashiyama S, Abraham JA, Klgsbrun M (1993) Heparin-binding EGF-like growth factor stimulation of smooth muscle cell migration: dependence on interactions with cell surface heparan sulfate. J Cell Biol 122:933–940CrossRefGoogle Scholar
  20. Hunter MC, O'Hagan KL, Kenyon A, Dhanani KC, Prinsloo E, Edkins AL (2014) Hsp90 binds directly to fibronectin (FN) and inhibition reduces the extracellular fibronectin matrix in breast cancer cells. PLoS One 9:e86842CrossRefGoogle Scholar
  21. Jockheck-Clark AR, Bowers EV, Totonchy MB, Neubauer J, Pizzo SV, Nicchitta CV (2010) Re-examination of CD91 function in GRP94 (glycoprotein 96) surface binding, uptake, and peptide cross-presentation. J Immunol 185:6819–6830CrossRefGoogle Scholar
  22. Kanekiyo T, Zhang J, Liu Q, Liu CC, Zhang L, Bu G (2011) Heparansulphate proteoglycan and the low-density lipoprotein receptor-related protein 1 constitute major pathways for neuronal amyloid-beta uptake. J Neurosci 31:1644–1651CrossRefGoogle Scholar
  23. Lagarrigue F, Dupuis-Coronas S, Ramel D, Delsol G, Tronchère H, Payrastre B, Gaits-Iacovoni F (2010) Matrix metalloproteinase-9 is upregulated in nucleophosmin-anaplastic lymphoma kinase-positive anaplastic lymphomas and activated at the cell surface by the chaperone heat shock protein 90 to promote cell invasion. Cancer Res 70:6978–6987CrossRefGoogle Scholar
  24. Lambaerts K, Wilcox-Adelman SA, Zimmermann P (2009) The signalling mechanisms of syndecan heparan sulphate proteoglycans. Curr Opin Cell Biol 21:662–669CrossRefGoogle Scholar
  25. Lei H, Romeo G, Kazlauskas A (2004) Heat shock protein 90alpha-dependent translocation of annexin II to the surface of endothelial cells modulates plasmin activity in the diabetic rat aorta. Circ Res 94:902–909CrossRefGoogle Scholar
  26. Li J, Buchner J (2013) Structure, function and regulation of the hsp90 machinery. Biom J 36:106–117Google Scholar
  27. Li W, Li Y, Guan S, Fan J, Cheng CF, Bright AM, Chinn C, Chen M, Woodley DT (2007) Extracellular heat shock protein-90alpha: linking hypoxia to skin cell motility and wound healing. EMBO J 26:1221–1233CrossRefGoogle Scholar
  28. Lindahl U, Li JP (2009) Interactions between heparin sulphate and proteins—design and functional implications. Int Rev Cell Mol Biol 276:105–159CrossRefGoogle Scholar
  29. Lisov A, Vrublevskaya V, Lisova Z, Leontievsky A, Morenkov O (2015) A 2,5-Dihydroxybenzoic acid–gelatin conjugate: the synthesis, antiviral activity and mechanism of antiviral action against two alphaherpesviruses. Viruses 7:5343–5360CrossRefGoogle Scholar
  30. Mahley RW, Ji ZS (1999) Remnant lipoprotein metabolism: key pathways involving cell-surface heparan sulfate proteoglycans and apolipoprotein E. J Lipid Res 40:1–16PubMedGoogle Scholar
  31. McCready J, Sims JD, Chan D, Jay DG (2010) Secretion of extracellular hsp90α via exosomes increases cancer cell motility: a role for plasminogen activation. BMC Cancer 10:294CrossRefGoogle Scholar
  32. Mitsou I, Multhaupt HAB, Couchman JR (2017) Proteoglycans, ion channels and cell-matrix adhesion. Biochem J 474:1965–1979CrossRefGoogle Scholar
  33. Nomura N, Nomura M, Takahira M, Sugiyama K (2007) Phorbol 12-myristate 13-acetate-activated protein kinase C increased migratory activity of subconjunctival fibroblasts via stress-activated protein kinase pathways. Mol Vis 13:2320–2327PubMedGoogle Scholar
  34. Ornitz DM, Yayon A, Flanagan JG, Svahn CM, Levi E, Leder P (1992) Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells. Mol Cell Biol 12:240–247CrossRefGoogle Scholar
  35. Safaiyan F, Kolset SO, Prydz K, Gottfridsson E, Lindahl U, Salmivirta M (1999) Selective effects of sodium chlorate treatment on the sulfation of heparan sulfate. J Biol Chem 274:36267–33673CrossRefGoogle Scholar
  36. Sarrazin S, Lamanna WC, Esko JD (2011) Heparan sulfate proteoglycans. Cold Spring Harb Perspect Biol 3:a004952CrossRefGoogle Scholar
  37. Sidera K, Samiotaki M, Yfanti E, Panayotou G, Patsavoudi E (2004) Involvement of cell surface HSP90 in cell migration reveals a novel role in the developing nervous system. J Biol Chem 279:45379–45388CrossRefGoogle Scholar
  38. Sidera K, Gaitanou M, Stellas D, Matsas R, Patsavoudi E (2008) A critical role for HSP90 in cancer cell invasion involves interaction with the extracellular domain of HER-2. J Biol Chem 283:2031–2041CrossRefGoogle Scholar
  39. Skarga Y, Vrublevskaya V, Evdokimovskaya Y, Morenkov O (2009) Purification of the 90 kDa heat shock protein (hsp90) and simultaneous purification of hsp70/hsc70, hsp90 and hsp96 from mammalian tissues and cells using thiophilic interaction chromatography. Biomed Chromatogr 23:1208–1216CrossRefGoogle Scholar
  40. Snigireva AV, Vrublevskaya V, Afanasyev N, Morenkov O (2015) Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane. Cell Adhes Migr 9:460–468CrossRefGoogle Scholar
  41. Song X, Wang X, Zhuo W, Shi H, Feng D, Sun Y, Liang Y, Fu Y, Zhou D, Luo Y (2010) The regulatory mechanism of extracellular Hsp90 on matrix metalloproteinase-2 processing and tumor angiogenesis. J Biol Chem 285:40039–40049CrossRefGoogle Scholar
  42. Spijkers PP, Denis CV, Blom AM, Lenting PJ (2008) Cellular uptake of C4b-binding protein is mediated by heparan sulfate proteoglycans and CD91/LDL receptor-related protein. Eur J Immunol 38:809–817CrossRefGoogle Scholar
  43. Sreedhar AS, Kalma’r E, Csermely P, Shen YF (2004) Hsp90 isoforms: functions, expression and clinical importance. FEBS Lett 562:11–15CrossRefGoogle Scholar
  44. Suzuki S, Kulkarni AB (2010) Extracellular heat shock protein HSP90beta secreted by MG63 osteosarcoma cells inhibits activation of latent TGF-beta 1. Biochem Biophys Res Commun 398:525–531CrossRefGoogle Scholar
  45. Thuringer D, Hammann A, Benikhlef N, Fourmaux E, Bouchot A, Wettstein G, Solary E, Garrido CJ (2011) Transactivation of the epidermal growth factor receptor by heat shock protein 90 via toll-like receptor 4 contributes to the migration of glioblastoma cells. Biol Chem 286:3418–3428CrossRefGoogle Scholar
  46. Tsutsumi S, Scroggins B, Koga F, Lee MJ, Trepel J, Felts S, Carreras C, Neckers L (2008) A small molecule cell-impermeant Hsp90 antagonist inhibits tumor cell motility and invasion. Oncogene 27:2478–2487CrossRefGoogle Scholar
  47. Wang X, Song X, Zhuo W, Fu Y, Shi H, Liang Y, Tong M, Chang G, Luo Y (2009) The regulatory mechanism of HSP90α secretion and its function in tumor malignancy. Proc Natl Acad Sci U S A 106:21288–21293CrossRefGoogle Scholar
  48. Wilsie LC, Orlando RA (2003) The low density lipoprotein receptor-related protein complexes with cell surface heparan sulfate proteoglycans to regulate proteoglycan-mediated lipoprotein catabolism. J Biol Chem 278:15758–15764CrossRefGoogle Scholar

Copyright information

© Cell Stress Society International 2019

Authors and Affiliations

  • Anastasiya V. Snigireva
    • 1
  • Veronika V. Vrublevskaya
    • 1
  • Yuri Y. Skarga
    • 1
  • Oleg S. Morenkov
    • 1
    Email author
  1. 1.Laboratory of Cell Culture and Cell Engineering, Institute of Cell BiophysicsRussian Academy of SciencesPushchinoRussia

Personalised recommendations