The role of membrane-associated heat shock protein Hsp70 in migration of glioblastoma cell

Background. Membrane-associated heat shock protein Hsp70 (mHsp70) is selectively expressed in tu mor, but not in normal cells. The mHsp70 functions are not fully understood. Objective: to study the role of mHsp70 in glioblastoma cell migration. Design and methods. mHsp70-positive rat glioma C6 and human glioblastoma U251 and T98G cell lines were used. Each cell line was sorted into two subpopulations: with high (mHsp70+) and low (mHsp70-) protein expression. The contribution of mHsp70 to migration was as sessed using a wound-healing assay, manual single-cell tracking, and the Transwell analysis. We also exam ined the effect of Hsp70 inhibitors PES and JG-98 on cell motility. To identify potential protein partners of mHsp70 that regulate cell motility, proteomic analysis of lipid rafts of T98G cells was performed. Results. mHsp70+ subpopulations have a higher mean speed (according to manual tracking) and lead to complete wound healing in a shorter period of time compared to mHsp70-. The use of PES and JG-98 inhibitors helps to reduce the speed of movement, as well as the number of invasive cells, with the greatest effect observed for mHsp70+ subpopulations. Proteomic analysis of T98G cells lipid rafts revealed a relationship between mHsp70 and proteins involved in cytoskeleton and extracellular matrix remodeling, adhesion and migration. Conclusion. mHsp70 is involved in glioblastoma cell migration and can be used as a target for malignant neoplasm therapy.

1. Radons J. The human HSP70 family of chaperones: where do we stand? Cell Stress Chaperones. 2016;21(3):379 404. DOI:10.1007/s12192-016-0676-6.

2. Kohler V, Andréasson C. Hsp70-mediated quality control: Should I stay or should I go? Biol. Chem. 2020;401(11):1233–1248. DOI:10.1515/hsz-2020-0187.

3. Lang BJ, Prince TL, Okusha Y, et al. Heat shock proteins in cell signaling and cancer. Biochim. Biophys. Acta Mol. Cell Res. 2022;1869(3):119187. DOI:10.1016/j.bbamcr.2021.119187.

4. Liu Y, Zhou L, Xu Y, et al. Heat Shock Proteins and Ferroptosis. Front Cell Dev Biol. 2022;10:864635. DOI:10.3389/fcell.2022.864635.

5. Morán Luengo T, Mayer MP, Rüdiger SGD. The Hsp70-Hsp90 Chaperone Cascade in Protein Folding. Trends Cell Biol. 2019;29(2):164–177. DOI:10.1016/j. tcb.2018.10.004.

6. Vollmann-Zwerenz A, Leidgens V, Feliciello G, et al. Tumor Cell Invasion in Glioblastoma. Int J Mol Sci. 2020;21(6):1932. DOI:10.3390/ijms21061932.

7. Tagaeva R, Efimova S, Ischenko A, et al. A new look at Hsp70 activity in phosphatidylserine-enriched membranes: chaperone-induced quasi-interdigitated lipid phase. Sci Rep. 2023;13(1):19233. DOI:10.1038/s41598-023-46131-x.

8. Makky A, Czajor J, Konovalov O, et al. X-ray reflectivity study of the heat shock protein Hsp70 interaction with an artificial cell membrane model. Sci Rep. 2023;13(1):19157. DOI:10.1038/s41598-023-46066-3.

9. Gehrmann M, Liebisch G, Schmitz G, et al. Tumor specific Hsp70 plasma membrane localization is enabled by the glycosphingolipid Gb3. PLoS One. 2008;3(4):e1925. DOI:10.1371/journal.pone.0001925.

10. De Maio A, Hightower L. The interaction of heat shock proteins with cellular membranes: a historical perspective. Cell Stress Chaperones. 2021;26(5):769–783. DOI:10.1007/s12192-021-01228-y.

11. De Maio A. Extracellular heat shock proteins, cellular export vesicles, and the Stress Observation System: a form of communication during injury, infection, and cell damage. It is never known how far a controversial f inding will go! Dedicated to Ferruccio Ritossa. Cell Stress Chaperones. 2011;16(3):235–249. DOI:10.1007/s12192-010 0236-4.

12. Zhang Y, Tseng CC, Tsai YL, et al. Cancer cells resistant to therapy promote cell surface relocalization of GRP78 which complexes with PI3K and enhances PI(3,4,5) P3 production. PLoS One. 2013;8(11):e80071. DOI:10.1371/ journal.pone.0080071.

13. Jagadish N, Agarwal S, Gupta N, et al. Heat shock protein 70-2 (HSP70-2) overexpression in breast cancer. J Exp Clin Cancer Res. 2016;35(1):150. DOI:10.1186/s13046 016-0425-9.

14. Jagadish N, Parashar D, Gupta N, et al. Heat shock protein 70-2 (HSP70-2) is a novel therapeutic target for colorectal cancer and is associated with tumor growth. BMC Cancer. 2016;16:561. DOI:10.1186/s12885-016-2592-7.

15. Kobayashi K, Matsushima-Nishiwaki R, Yamada N, et al. Heat shock protein 70 positively regulates trans forming growth factor-α-induced hepatocellular carcinoma cell migration via the AKT signaling pathway. Heliyon. 2020;6(9):e05002. DOI:10.1016/j.heliyon.2020.e05002.

16. Liu CC, Jan YJ, Ko BS, et al. 14-3-3σ induces heat shock protein 70 expression in hepatocellular carcinoma. BMC Cancer. 2014;14:425. DOI:10.1186/1471-2407-14-425.

17. Bobkov D, Yudintceva N, Lomert E, et al. Lipid raft integrity is required for human leukemia Jurkat T-cell migratory activity. Biochim Biophys Acta Mol Cell Biol Lipids. 2021;1866(6):158917. DOI:10.1016/j.bbalip.2021.158917.

18. Yudintceva N, Bobkov D, Sulatsky M, et al. Mesenchymal stem cells-derived extracellular vesicles for therapeutics of renal tuberculosis. Sci Rep. 2024;14(1):4495. DOI:10.1038/s41598-024-54992-z.

19. Ma B, Zhang K, Hendrie C, et al. PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Commun Mass Spectrom. 2003;17(20):2337–2342. DOI:10.1002/rcm.1196.

20. Vostakolaei MA, Hatami-Baroogh L, Babaei G, et al. Hsp70 in cancer: A double agent in the battle between survival and death. J Cell Physiol. 2021;236(5):3420–3444. DOI:10.1002/jcp.30132.

21. Alhasan B, Gladova YA, Sverchinsky DV, et al. Hsp70 Negatively Regulates Autophagy via Governing AMPK Activation, and Dual Hsp70-Autophagy Inhibition Induces Synergetic Cell Death in NSCLC Cells. Int J Mol Sci. 2024;25(16):9090. DOI:10.3390/ijms25169090.

22. Tagaeva RB, Bobkov DE, Nechaeva AS, et al. Membrane-bound heat shock protein mHsp70 as a marker for malignant brain tumors. Russian neurosurgical journal named after professor A. L. Polenov. 2023;15(2):98–101. In Russian.

23. De P, Aske JC, Dey N. RAC1 Takes the Lead in Solid Tumors. Cells. 2019;8(5):382. DOI:10.3390/cells8050382.

24. Seclì L, Fusella F, Avalle L, et al. The dark side of the outside: how extracellular heat shock proteins promote cancer. Cell Mol Life Sci. 2021;78(9):4069–4083. DOI:10.1007/s00018-021-03764-3.

25. Sims JD, McCready J, Jay DG. Extracellular heat shock protein (Hsp)70 and Hsp90α assist in matrix metalloproteinase-2 activation and breast cancer cell migration and invasion. PLoS One. 2011;6(4):e18848. DOI:10.1371/journal.pone.0018848.

26. Hunter MC, O’Hagan KL, Kenyon A, et al. Hsp90 binds directly to fibronectin (FN) and inhibition reduces the extracellular fibronectin matrix in breast cancer cells. PLoS One. 2014;9(1):e86842. DOI:10.1371/journal.pone.0086842.

27. McCready J, Sims JD, Chan D, et al. Secretion of extracellular hsp90alpha via exosomes increases cancer cell motility: a role for plasminogen activation. BMC Cancer. 2010;10:294. DOI:10.1186/1471-2407-10-294.

28. McCready J, Wong DS, Burlison JA, et al. An Impermeant Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell Migration that Involves Lysyl Oxidase 2-like Protein. Cancers (Basel). 2014;6(2):1031 1046. DOI:10.3390/cancers6021031.

29. Amissah HA, Combs SE, Shevtsov M. Tumor Dormancy and Reactivation: The Role of Heat Shock Proteins. Cells. 2024;13(13):1087. DOI:10.3390/cells13131087.

30. Walsh N, Larkin A, Swan N, et al. RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation. Cancer Lett. 2011;306(2):180 189. DOI:10.1016/j.canlet.2011.03.004.