Specificity of U2 and GOL1 aptamers to EGFR-positive human glioblastoma cells in vitro

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

Overexpression of the epidermal growth factor receptor (EGFR) or its mutations mediate signaling pathways leading to proliferation, invasion of tumor cells, as well as to an increase in their survival. Despite the success of the clinical use of antibodies against EGFR in patients with colorectal cancer and squamous cell carcinoma of the head and neck, their low effectiveness in glioblastoma has been shown. Therefore, for the treatment of gliomas, a specific EGFR drug is needed, capable of penetrating into the tumor focus in the brain, and having low immunogenicity.

In this work, aptamers – single-stranded DNA oligonucleotides specific to EGFR, U2 and Gol1 are presented as such a preparation. In this study, we obtained a cellular model of human glioma with EGFR and EGFRvIII overexpression, which showed the specificity of U2 and Gol1 aptamers to these receptors using classical methods, as well as the method of aptaimmunocytochemistry. A study of the effect of binding of the Gol1 aptamer to the EGFRvIII receptor on the next steps of the signaling pathway showed a change in the expression levels of genes associated with cell proliferation and survival (JUN, FOS, CCND1, PI3K and AKT3), while the U2 aptamer did not demonstrate a significant effect on cells in vitro.

These results showed that the Gol1 aptamer has therapeutic potential against human glioblastoma tumor cells overexpressing the EGFRvIII mutant type receptor.

Full Text

Restricted Access

About the authors

F. M. Dzarieva

Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences (IHNA)

Author for correspondence.
Email: dz.fatima@mail.ru
Russian Federation, Moscow

D. V. Shamadykova

Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences (IHNA)

Email: dz.fatima@mail.ru
Russian Federation, Moscow

O. V. Sluchanko

Pirogov Russian National Research Medical University

Email: dz.fatima@mail.ru
Russian Federation, Moscow

S. A. Pavlova

Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences (IHNA)

Email: dz.fatima@mail.ru
Russian Federation, Moscow

L. V. Fab

Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences (IHNA)

Email: dz.fatima@mail.ru
Russian Federation, Moscow

A. V. Ryabova

Prokhorov General Physics Institute

Email: dz.fatima@mail.ru
Russian Federation, Moscow

D. Yu. Panteleev

Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences (IHNA)

Email: dz.fatima@mail.ru
Russian Federation, Moscow

A. M. Kopylov

M.V. Lomonosov Moscow State University (MSU)

Email: dz.fatima@mail.ru
Russian Federation, Moscow

D. Yu. Usachev

N.N. Burdenko National Medical Research Center of Neurosurgery, Ministry of Healthcare of Russia

Email: dz.fatima@mail.ru
Russian Federation, Moscow

A. V. Golovin

M.V. Lomonosov Moscow State University (MSU); I.M. Sechenov First Moscow State Medical University (MSMU)

Email: dz.fatima@mail.ru
Russian Federation, Moscow; Moscow

G. V. Pavlova

Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences (IHNA); N.N. Burdenko National Medical Research Center of Neurosurgery, Ministry of Healthcare of Russia; I.M. Sechenov First Moscow State Medical University (MSMU)

Email: lkorochkin@mail.ru
Russian Federation, Moscow; Moscow; Moscow

References

  1. Каприн А. Д. Злокачественные новообразования в России в 2021 г. МНИОИ им. П. А. Герцена – филиал ФГБУ «НМИЦ радиологии» Минздрава России. 2022.
  2. Babic I., Anderson E. S., Tanaka K., Guo D., Masui K., Li B., Zhu S., Gu Y., Villa G. R., Akhavan D., Nathanson D., Gini B., Mareninov S., Li R., Camacho C. E., Kurdistani S. K., Eskin A., Nelson S. F., Yong W. H., Cavenee W. K.,
  3. Cloughesy T. F., Christofk H. R., Black D. L., Mischel P. S. EGFR mutation-induced alternative splicing of max contributes to growth of glycolytic tumors in brain cancer. Cell Metabolism. 2013. 17 (6): 1000–1008. https://doi.org/10.1016/j.cmet.2013.04.013
  4. Brand T. M., Iida M., Luthar N., Starr M. M., Huppert E. J., Wheeler D. L. Nuclear EGFR as a molecular target in cancer. Radiother. Oncol. 2013. 108 (3): 370–377. https://doi.org/10.1016/j.radonc.2013.06.010
  5. Ciznadija D., Liu Y., Pyonteck S. M., Holland E. C., Koff A. Cyclin D1 and Cdk4 mediate development of neurologically destructive oligodendroglioma. Cancer Res. 2011. 71 (19): 6174–6183. https://doi.org/10.1158/0008– 5472.CAN-11–1031.
  6. Cooper A. J., Sequist L. V., Lin J. J. Third-generation EGFR and ALK inhibitors: mechanisms of resistance and management. Nat. Rev. Clin. Oncol. 2022. 19 (8): 499– 514. https://doi.org/10.1038/s41571–022–00639–9
  7. Cvrljevic A. N., Akhavan D., Wu M., Martinello P., Furnari F. B., Johnston A. J., Guo D., Pike L., Cavenee W. K., Scott A. M., Mischel P. S., Hoogenraad N. J., Johns T. G. Activation of Src induces mitochondrial localisation of de 2–7EGFR (EGFRvIII) in glioma cells: implications for glucose metabolism. Journal of Cell Science. 2011. 124 (17): 2938–2950. https://doi.org/10.1242/jcs.083295
  8. David J.-P., Mehic D., Bakiri L., Schilling A. F., Mandic V., Priemel M., Idarraga M. H., Reschke M. O., Hoffmann O., Amling M., Wagner E. F. Essential role of RSK2 in c-Fos–dependent osteosarcoma development. J Clin. Invest. 2005. 115 (3): 664–672. https://doi.org/10.1172/ JCI22877
  9. Feng H., Hu B., Jarzynka M. J., Li Y., Keezer S., Johns T. G., Tang, C. K., Hamilton, R. L., Vuori, K., Nishikawa, R., Sarkaria, J. N., Fenton, T., Cheng, T., Furnari, F. B., Cavenee, W. K., Cheng, S.-Y. Phosphorylation of dedicator of cytokinesis 1 (Dock180) at tyrosine residue Y722 by Src family kinases mediates EGFRvIII-driven glioblastoma tumorigenesis. Proc. Nat. Acad. Sci. 2012. 109 (8): 3018–3023. https://doi.org/10.1073/pnas.1121457109
  10. Fiano V., Ghimenti C., Schiffer D. Expression of cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors in oligodendrogliomas in humans. Neurosci. Letters. 2003. 347 (2): 111–115. https://doi.org/10.1016/ S0304–3940(03)00615–3
  11. Goto N., Suzuki, H., Tanaka T., Ishikawa K., Ouchida T., Kaneko M. K., Kato Y. EMab-300 detects mouse epidermal growth factor receptor-expressing cancer cell lines in flow cytometry. Antibodies (Basel, Switzerland). 2023. 12 (3): 42. https://doi.org/10.3390/antib12030042
  12. Hammer S., Tschiatschek B., Flamm C., Hofacker I. L., Findeiß S. RNAblueprint: flexible multiple target nucleic acid sequence design. Bioinformatics. 2017. 33 (18): 2850–2858. https://doi.org/10.1093/bioinformatics/ btx263
  13. Hammer S., Wang W., Will S., Ponty Y. Fixed-parameter tractable sampling for RNA design with multiple target structures. BMC Bioinformatics. 2009. 20 (1): 209. https://doi.org/10.1186/s12859–019–2784–7
  14. Hanahan D. Hallmarks of cancer: new dimensions. Cancer Discovery. 2022. 12 (1): 31–46. https://doi.org/ 10.1158/2159–8290.CD-21–1059
  15. Hanahan D., Weinberg R. A. Hallmarks of cancer: the next generation. Cell. 2011. 144 (5): 646–674. https://doi. org/10.1016/j.cell.2011.02.013
  16. Horvath S., Zhang B., Carlson M., Lu K. V., Zhu S., Felciano R. M., Laurance M. F., Zhao W., Qi S., Chen Z., Lee Y., Scheck A. C., Liau L. M., Wu H., Geschwind D. H., Febbo P. G., Kornblum H. I., Cloughesy T. F., Nelson S. F., Mischel P. S. Analysis of oncogenic signaling networks in glioblastoma identifies ASPM as a molecular target. Proceedings of the National Academy of Sciences. 2006. 103(46: 17402–17407. https://doi.org/10.1073/ pnas.0608396103
  17. Kappelmann M., Bosserhoff A., Kuphal S. AP-1/c-Jun transcription factors: Regulation and function in malignant melanoma. Eur. J. Cell Biol. 2014. 93 (1–2): 76–81. https://doi.org/10.1016/j.ejcb.2013.10.003
  18. Kopylov A. M., Fab L. V., Antipova O., Savchenko E. A., Revishchin A. V., Parshina V. V., Pavlova S. V., Kireev I. I., Golovin A. V., Usachev D. Y., Pavlova G. V. RNA aptamers for theranostics of glioblastoma of human brain. Biochemistry (Moscow). 2021. 86(8): 1012–1024. https://doi.org/10.1134/S0006297921080113
  19. Lao Y.-H., Phua K. K.L., Leong K. W. Aptamer nanomedicine for cancer therapeutics: barriers and potential for translation. ACS Nano. 2015. 9 (3), 2235–2254. https://doi. org/10.1021/nn507494p
  20. Linder M., Glitzner E., Srivatsa S., Bakiri L., Matsuoka K., Shahrouzi P., Dumanic M., Novoszel P., Mohr T., Langer O., Wanek T., Mitterhauser M., Wagner E. F., Sibilia M. EGFR is required for FOS‐dependent bone tumor development via RSK2/CREB signaling. EMBO Mol. Med. 2018. 10: e9408. (11). https://doi.org/10.15252/ emmm.201809408
  21. Mayer I. A., Arteaga C. L. The PI3K/AKT pathway as a target for cancer treatment. Ann. Rev. Med. 2016. 67 (1): 11–28. https://doi.org/10.1146/annurev-med-062913–051343
  22. Odajima T., Sasaki Y., Tanaka N., Kato-Mori Y., Asanuma H., Ikeda T., Satoh M., Hiratsuka H., Tokino T., Sawada N. Abnormal β-catenin expression in oral cancer with no gene mutation: correlation with expression of cyclin D1 and epidermal growth factor receptor, Ki-67 labeling index, and clinicopathological features. Hum. Pathol. 2005. 36 (3): 234–241. https://doi.org/10.1016/j. humpath.2004.12.009
  23. Oprita A., Baloi S.-C., Staicu G.-A., Alexandru O., Tache D. E., Danoiu S., Micu E. S., Sevastre A.-S. Updated insights on EGFR signaling pathways in glioma. Int. J. Mol. Sci. 2021. 22 (2): 587. https://doi.org/10.3390/ijms22020587
  24. Pavlova G., Kolesnikova V., Samoylenkova N., Drozd S., Revishchin A., Usachev D., Kopylov A. Novel weapon to conquer human glioblastoma: G-quadruplexes and neuro-inducers. 2021.
  25. Pawson T. Specificity in signal transduction. Cell. 2004. 116 (2): 191–203. https://doi.org/10.1016/S0092–8674(03) 01077–8
  26. Peng L., Liang Y., Zhong X., Liang Z., Tian Y., Li S., Liang J., Wang R., Zhong Y., Shi Y., Zhang X. Aptamer-conjugated gold nanoparticles targeting epidermal growth factor receptor variant III for the treatment of glioblastoma. Int. J. Nanomed. 2020. 15: 1363– 1372. https://doi.org/10.2147/IJN.S238206
  27. Peurala E., Koivunen P., Haapasaari K.-M., Bloigu R., Jukkola-Vuorinen A. The prognostic significance and value of cyclin D1, CDK4 and p16 in human breast cancer. Breast Cancer Res.. 2013. 15 (1): R5. https://doi. org/10.1186/bcr3376
  28. Ratushny, V., Astsaturov, I., Burtness, B.A., Golemis, E.A., & Silverman, J. S. Targeting EGFR resistance networks in head and neck cancer. Cell. Signal. 2009. 21 (8): 1255–1268. https://doi.org/10.1016/j.cellsig.2009.02.021
  29. Reuter J. S., Mathews D. H. RNA structure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics. 2010. 11 (1): 129. https://doi.org/ 10.1186/1471–2105–11–129
  30. Shen Q., Uray I. P., Li Y., Krisko T. I., Strecker T. E., Kim H.-T., Brown P. H. The AP-1 transcription factor regulates breast cancer cell growth via cyclins and E2F factors. Oncogene. 2008. 27 (3): 366–377. https://doi.org/ 10.1038/sj.onc.1210643
  31. Sugawa N., Ekstrand A. J., James C. D., Collins V. P. Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas. Proc. Nat. Acad. Sci. 1990. 87 (21): 8602–8606. https://doi.org/10.1073/pnas.87.21.8602
  32. Wan Y., Kim Y., Li N., Cho S. K., Bachoo R., Ellington A. D., Iqbal S. M. Surface-immobilized aptamers for cancer cell isolation and microscopic cytology. Cancer Res. 2010. 70 (22): 9371–9380. https://doi.org/10.1158/ 0008–5472.CAN-10–0568
  33. Ward A. F., Braun B. S., Shannon K. M. Targeting oncogenic Ras signaling in hematologic malignancies. Blood. 2012. 120 (17): 3397–3406. https://doi.org/10.1182/ blood-2012–05–378596
  34. Wilson T., Karajannis M., Harter D. Glioblastoma multiforme: State of the art and future therapeutics. Surg. Neurol. Int. 2014. 5 (1): 64. https://doi.org/10.4103/ 2152–7806.132138
  35. Wu X., Liang H., Tan Y., Yuan C., Li S., Li X., Li G., Shi Y., Zhang X. Cell-SELEX aptamer for highly specific radionuclide molecular imaging of glioblastoma in vivo. PLoS ONE. 2014. 9 (3): e90752. https://doi.org/10.1371/ journal.pone.0090752
  36. Xia W., Wei Y., Du Y., Liu J., Chang B., Yu Y.-L., Huo L.-F., Miller S., Hung M.-C. Nuclear expression of epidermal growth factor receptor is a novel prognostic value in patients with ovarian cancer. Mol. Carcinogen. 2009. 48 (7): 610–617. https://doi.org/10.1002/mc.20504
  37. Yu J. S. L., Cui W. Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination. Development. 2016. 143 (17): 3050–3060. https://doi.org/10.1242/dev.137075
  38. Zhang X., Zhao M., Huang A., Fei Z., Zhang W., Wang X. The effect of cyclin D expression on cell proliferation in human gliomas. J. Clin. Neurosci. 2005. 12 (2), 166–168. https://doi.org/10.1016/j.jocn.2004.03.036
  39. Zhou J., Wu Z., Wong G., Pectasides E., Nagaraja A., Stachler M., Zhang H., Chen T., Zhang H., Liu J. Bin, Xu X., Sicinska E., Sanchez-Vega F., Rustgi A. K., Diehl J. A., Wong K.-K., Bass A. J. CDK4/6 or MAPK blockade enhances efficacy of EGFR inhibition in oesophageal squamous cell carcinoma. Nat. Commun. 2017. 8 (1): 13897. https://doi.org/10.1038/ncomms13897
  40. Zhu G., Chen X. Aptamer-based targeted therapy. Adv. Drug Del. Rev. 2018. 134: 65–78. https://doi.org/ 10.1016/j.addr.2018.08.005
  41. Zok T., Antczak M., Zurkowski M., Popenda M., Blazewicz J., Adamiak R. W., Szachniuk M. RNApdbee 2.0: multifunctional tool for RNA structure annotation. Nucl. Acids Res. 2018. 46 (W1): W30–W35. https://doi.org/ 10.1093/nar/gky314

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Fig. 1. (а) – Plasmid map of the vector EGFRwt/CMV3; (б) – Plasmid map of the vector EGFRvIII/CMV3; Evaluation of the efficiency of transfection of G01(в) cell culture and U87 (г) cell line by human glioblastoma, GFP – green. The cell nuclei were stained with bisbenzimide (Hoechst 33342, Sigma) – blue. (д) – Analysis of EGFR expression in control and transgenic cells of the human glioblastoma cell line U87 and in patient-derived human glioblastoma G01 cultured cells. Normalised for beta-tubulin.

Download (329KB)
Indexing metadata
3. Fig. 2. Analysis of binding of human glioblastoma cells in vitro with labeled aptamers U2-FAM and Gol1-FAM, specific for EGFR, by flow cytometry (a) and aptaimmunocytochemistry (b). Intact cells were taken as a control. Antibodies against EGFR (red), aptamers U2 and Gol1 (green), blue – staining of nuclei with bisbenzimide.

Download (347KB)
Indexing metadata
4. Fig. 3. Analysis of changes in gene expression levels of EGFR cascade molecules by qPCR in human glioblastoma cells before and after transfection (а), before and after the addition of U2 and Gol1 aptamers in U87 line cells (б) and G01 culture cells (в). Gene expression was normalized relative to the reference genes GAPDH, RPL13A and TBP. The data are presented as a logarithmic scale of the mean value ± standard error of the mean, the p-criterion was considered significant at *= p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 (ANOVA test). All experiments were carried out in three replicates.

Download (383KB)
Indexing metadata
5. Fig. 4. (a) Hierarchical model of EGFR and EGFRvIII signal transmission. Cells in human glioblastoma tumors expressing EGFRvIII can transmit signals to cells expressing EGFR, and vice versa, in a paracrine manner (Cvrljevic et al., 2011); (б) EGFR signaling pathways in glioma cells, taken and adapted from KEGG PATHWAY.

Download (229KB)
Indexing metadata

Copyright (c) 2024 Russian Academy of Sciences

This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies