Direct Effect of Bevacizumab on Glioblastoma Cell Lines In Vitro

Abstract

Bevacizumab is a humanized monoclonal antibody directed against the pro-angiogenic factor vascular and endothelial growth factor-A (VEGF-A) used in the treatment of glioblastomas. Although most patients respond initially to this treatment, studies have shown that glioblastomas eventually recur. Several non-mutually exclusive theories based on the anti-angiogenic effect of bevacizumab have been proposed to explain these mechanisms of resistance. In this report, we studied whether bevacizumab can act directly on malignant glioblastoma cells. We observe changes in the expression profiles of components of the VEGF/VEGF-R pathway and in the response to a VEGF-A stimulus following bevacizumab treatment. In addition, we show that bevacizumab itself acts on glioblastoma cells by activating the Akt and Erks survival signaling pathways. Bevacizumab also enhances proliferation and invasiveness of glioblastoma cells in hyaluronic acid hydrogel. We propose that the paradoxical effect of bevacizumab on glioblastoma cells could be due to changes in the VEGF-A-dependent autocrine loop as well as in the intracellular survival pathways, leading to the enhancement of tumor aggressiveness. Investigation of how bevacizumab interacts with glioblastoma cells and the resulting downstream signaling pathways will help targeting populations of resistant glioblastoma cells.

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Abbreviations

CNS:

Central nervous system

ECM:

Extracellular matrix

HA:

Hyaluronic acid

IgG1:

Immunoglobulin G1

PlGF:

Placental growth factor

VEGF:

Vascular and endothelial growth factor

References

  1. Cao, Y., Zhong, W., & Sun, Y. (2009). Improvement of antiangiogenic cancer therapy by understanding the mechanisms of angiogenic factor interplay and drug resistance. Seminars in Cancer Biology, 19(5), 338–343. doi:10.1016/j.semcancer.2009.05.001.

    CAS  PubMed  Article  Google Scholar 

  2. Charles, N. A., Holland, E. C., Gilbertson, R., Glass, R., & Kettenmann, H. (2012). The brain tumor microenvironment. Glia, 60(3), 502–514.

    PubMed  Article  Google Scholar 

  3. Chauzy, C., Delpech, B., Olivier, A., Bastard, C., Girard, N., Courel, M. N., Creissard, P. (1992). Establishment and characterisation of a human glioma cell line. European journal of cancer (Oxford, England: 1990), 28A(6–7), 1129–1134.

  4. Coquerel, B., Poyer, F., Torossian, F., Dulong, V., Bellon, G., Dubus, I., et al. (2009). Elastin-derived peptides: Matrikines critical for glioblastoma cell aggressiveness in a 3-D system. Glia, 57(16), 1716–1726. doi:10.1002/glia.20884.

    PubMed  Article  Google Scholar 

  5. David, L., Dulong, V., Le Cerf, D., Cazin, L., Lamacz, M., & Vannier, J.-P. (2008). Hyaluronan hydrogel: An appropriate three-dimensional model for evaluation of anticancer drug sensitivity. Acta Biomaterialia, 4(2), 256–263. doi:10.1016/j.actbio.2007.08.012.

    CAS  PubMed  Article  Google Scholar 

  6. David, L., Dulong, V., Le Cerf, D., Chauzy, C., Norris, V., Delpech, B., et al. (2004). Reticulated hyaluronan hydrogels: A model for examining cancer cell invasion in 3D. Matrix Biology: Journal of the International Society for Matrix Biology, 23(3), 183–193. doi:10.1016/j.matbio.2004.05.005.

    CAS  Article  Google Scholar 

  7. De Groot, J. F., Fuller, G., Kumar, A. J., Piao, Y., Eterovic, K., Ji, Y., et al. (2010). Tumor invasion after treatment of glioblastoma with bevacizumab: Radiographic and pathologic correlation in humans and mice. Neuro-oncology, 12(3), 233–242. doi:10.1093/neuonc/nop027.

    PubMed Central  PubMed  Article  Google Scholar 

  8. DeAngelis, L. M. (2001). Brain tumors. The New England journal of medicine, 344(2), 114–123. doi:10.1056/NEJM200101113440207.

    CAS  PubMed  Article  Google Scholar 

  9. Ellis, L. M., & Hicklin, D. J. (2008). Pathways mediating resistance to vascular endothelial growth factor-targeted therapy. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 14(20), 6371–6375. doi:10.1158/1078-0432.CCR-07-5287.

    CAS  Article  Google Scholar 

  10. Fan, F., Samuel, S., Gaur, P., Lu, J., Dallas, N. A., Xia, L., et al. (2011). Chronic exposure of colorectal cancer cells to bevacizumab promotes compensatory pathways that mediate tumour cell migration. British Journal of Cancer, 104(8), 1270–1277. doi:10.1038/bjc.2011.81.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  11. Ferrara, N. (2009). VEGF-A: A critical regulator of blood vessel growth. European Cytokine Network, 20(4), 158–163. doi:10.1684/ecn.2009.0170.

    CAS  PubMed  Google Scholar 

  12. Ferrara, N. (2010). Binding to the extracellular matrix and proteolytic processing: Two key mechanisms regulating vascular endothelial growth factor action. Molecular Biology of the Cell, 21(5), 687–690. doi:10.1091/mbc.E09-07-0590.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  13. Ferrara, N., Hillan, K. J., & Novotny, W. (2005). Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy. Biochemical and Biophysical Research Communications, 333(2), 328–335. doi:10.1016/j.bbrc.2005.05.132.

    CAS  PubMed  Article  Google Scholar 

  14. Fischer, C., Jonckx, B., Mazzone, M., Zacchigna, S., Loges, S., Pattarini, L., et al. (2007). Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels. Cell, 131(3), 463–475. doi:10.1016/j.cell.2007.08.038.

    CAS  PubMed  Article  Google Scholar 

  15. Folkman, J. (1971). Tumor angiogenesis: Therapeutic implications. The New England Journal of Medicine, 285(21), 1182–1186. doi:10.1056/NEJM197111182852108.

    CAS  PubMed  Article  Google Scholar 

  16. Friedman, H. S., Prados, M. D., Wen, P. Y., Mikkelsen, T., Schiff, D., Abrey, L. E., et al. (2009). Bevacizumab alone and in combination with irinotecan in recurrent glioblastoma. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology, 27(28), 4733–4740. doi:10.1200/JCO.2008.19.8721.

    CAS  Article  Google Scholar 

  17. Galas, L., Garnier, M., & Lamacz, M. (2000). Calcium waves in frog melanotrophs are generated by intracellular inactivation of TTX-sensitive membrane Na+ channel. Molecular and Cellular Endocrinology, 170(1–2), 197–209.

    CAS  PubMed  Article  Google Scholar 

  18. Grau, S., Thorsteinsdottir, J., von Baumgarten, L., Winkler, F., Tonn, J.-C., & Schichor, C. (2011). Bevacizumab can induce reactivity to VEGF-C and -D in human brain and tumour derived endothelial cells. Journal of Neuro-oncology, 104(1), 103–112. doi:10.1007/s11060-010-0480-6.

    CAS  PubMed  Article  Google Scholar 

  19. Hoelzinger, D. B., Demuth, T., & Berens, M. E. (2007). Autocrine factors that sustain glioma invasion and paracrine biology in the brain microenvironment. Journal of the National Cancer Institute, 99(21), 1583–1593. doi:10.1093/jnci/djm187.

    CAS  PubMed  Article  Google Scholar 

  20. Hong, X., Jiang, F., Kalkanis, S. N., Zhang, Z. G., Zhang, X.-P., DeCarvalho, A. C., et al. (2006). SDF-1 and CXCR4 are up-regulated by VEGF and contribute to glioma cell invasion. Cancer Letters, 236(1), 39–45. doi:10.1016/j.canlet.2005.05.011.

    CAS  PubMed  Article  Google Scholar 

  21. Jackson, A. P., Timmerman, M. P., Bagshaw, C. R., & Ashley, C. C. (1987). The kinetics of calcium binding to fura-2 and indo-1. FEBS Letters, 216(1), 35–39.

    CAS  PubMed  Article  Google Scholar 

  22. Keunen, O., Johansson, M., Oudin, A., Sanzey, M., Rahim, S. A. A., Fack, F., et al. (2011). Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma. Proceedings of the National Academy of Sciences of the United States of America, 108(9), 3749–3754. doi:10.1073/pnas.1014480108.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  23. Knizetova, P., Darling, J. L., & Bartek, J. (2008a). Vascular endothelial growth factor in astroglioma stem cell biology and response to therapy. Journal of Cellular and Molecular Medicine, 12(1), 111–125. doi:10.1111/j.1582-4934.2007.00153.x.

    CAS  PubMed  Article  Google Scholar 

  24. Knizetova, P., Ehrmann, J., Hlobilkova, A., Vancova, I., Kalita, O., Kolar, Z., & Bartek, J. (2008b). Autocrine regulation of glioblastoma cell cycle progression, viability and radioresistance through the VEGF-VEGFR2 (KDR) interplay. Cell cycle (Georgetown, Tex.), 7(16), 2553–2561.

  25. Kwiatkowska, A., & Symons, M. (2013). Signaling determinants of glioma cell invasion. Advances in Experimental Medicine and Biology, 986, 121–141. doi:10.1007/978-94-007-4719-7_7.

    CAS  PubMed  Article  Google Scholar 

  26. Lee, S., Jilani, S. M., Nikolova, G. V., Carpizo, D., & Iruela-Arispe, M. L. (2005). Processing of VEGF-A by matrix metalloproteinases regulates bioavailability and vascular patterning in tumors. The Journal of Cell Biology, 169(4), 681–691. doi:10.1083/jcb.200409115.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  27. Lee, J., Lee, J., Yu, H., Choi, K., & Choi, C. (2011). Differential dependency of human cancer cells on vascular endothelial growth factor-mediated autocrine growth and survival. Cancer Letters, 309(2), 145–150. doi:10.1016/j.canlet.2011.05.026.

    CAS  PubMed  Article  Google Scholar 

  28. Lucio-Eterovic, A. K., Piao, Y., & de Groot, J. F. (2009). Mediators of glioblastoma resistance and invasion during antivascular endothelial growth factor therapy. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 15(14), 4589–4599. doi:10.1158/1078-0432.CCR-09-0575.

    CAS  Article  Google Scholar 

  29. Mahesparan, R., Read, T.-A., Lund-Johansen, M., Skaftnesmo, K. O., Bjerkvig, R., & Engebraaten, O. (2003). Expression of extracellular matrix components in a highly infiltrative in vivo glioma model. Acta Neuropathologica, 105(1), 49–57. doi:10.1007/s00401-002-0610-0.

    CAS  PubMed  Google Scholar 

  30. Masood, R., Cai, J., Zheng, T., Smith, D. L., Hinton, D. R., & Gill, P. S. (2001). Vascular endothelial growth factor (VEGF) is an autocrine growth factor for VEGF receptor-positive human tumors. Blood, 98(6), 1904–1913.

    CAS  PubMed  Article  Google Scholar 

  31. Mellinghoff, I. K., Lassman, A. B., & Wen, P. Y. (2011). Signal transduction inhibitors and antiangiogenic therapies for malignant glioma. Glia, 59(8), 1205–1212. doi:10.1002/glia.21137.

    PubMed  Article  Google Scholar 

  32. Miletic, H., Niclou, S. P., Johansson, M., & Bjerkvig, R. (2009). Anti-VEGF therapies for malignant glioma: Treatment effects and escape mechanisms. Expert Opinion on Therapeutic Targets, 13(4), 455–468. doi:10.1517/14728220902806444.

    CAS  PubMed  Article  Google Scholar 

  33. Moreno Garcia, V., Basu, B., Molife, L. R., & Kaye, S. B. (2012). Combining antiangiogenics to overcome resistance: Rationale and clinical experience. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 18(14), 3750–3761. doi:10.1158/1078-0432.CCR-11-1275.

    CAS  Article  Google Scholar 

  34. Nakada, M., Nakada, S., Demuth, T., Tran, N. L., Hoelzinger, D. B., & Berens, M. E. (2007). Molecular targets of glioma invasion. Cellular and Molecular Life Sciences: CMLS, 64(4), 458–478. doi:10.1007/s00018-007-6342-5.

    CAS  PubMed  Article  Google Scholar 

  35. Olsson, A.-K., Dimberg, A., Kreuger, J., & Claesson-Welsh, L. (2006). VEGF receptor signalling: In control of vascular function. Nature Reviews Molecular Cell Biology, 7(5), 359–371. doi:10.1038/nrm1911.

    CAS  PubMed  Article  Google Scholar 

  36. Plate, K. H., Scholz, A., & Dumont, D. J. (2012). Tumor angiogenesis and anti-angiogenic therapy in malignant gliomas revisited. Acta Neuropathologica, 124(6), 763–775. doi:10.1007/s00401-012-1066-5.

    PubMed Central  PubMed  Article  Google Scholar 

  37. Pollo, B. (2012). Pathological classification of brain tumors. The Quarterly Journal of Nuclear Medicine and Molecular Imaging: Official Publication of the Italian Association of Nuclear Medicine (AIMN) [and] the International Association of Radiopharmacology (IAR), [and] Section of the Society of Radiopharmaceutical Chemistry and Biology, 56(2), 103–111.

    CAS  Google Scholar 

  38. Rahman, R., Smith, S., Rahman, C., & Grundy, R. (2010). Antiangiogenic therapy and mechanisms of tumor resistance in malignant glioma. Journal of Oncology, 2010, 251231. doi:10.1155/2010/251231.

    PubMed Central  PubMed  Article  Google Scholar 

  39. Red-Horse, K., Crawford, Y., Shojaei, F., & Ferrara, N. (2007). Endothelium-microenvironment interactions in the developing embryo and in the adult. Developmental Cell, 12(2), 181–194. doi:10.1016/j.devcel.2007.01.013.

    CAS  PubMed  Article  Google Scholar 

  40. Stupp, R., Hegi, M. E., Mason, W. P., van den Bent, M. J., Taphoorn, M. J. B., Janzer, R. C., et al. (2009). Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. The Lancet Oncology, 10(5), 459–466. doi:10.1016/S1470-2045(09)70025-7.

    CAS  PubMed  Article  Google Scholar 

  41. Tabatabai, G., Weller, M., Nabors, B., Picard, M., Reardon, D., Mikkelsen, T., et al. (2010). Targeting integrins in malignant glioma. Targeted Oncology, 5(3), 175–181. doi:10.1007/s11523-010-0156-3.

    PubMed  Article  Google Scholar 

  42. Takano, S., Mashiko, R., Osuka, S., Ishikawa, E., Ohneda, O., & Matsumura, A. (2010). Detection of failure of bevacizumab treatment for malignant glioma based on urinary matrix metalloproteinase activity. Brain Tumor Pathology, 27(2), 89–94. doi:10.1007/s10014-010-0271-y.

    CAS  PubMed  Article  Google Scholar 

  43. Tate, M. C., & Aghi, M. K. (2009). Biology of angiogenesis and invasion in glioma. Neurotherapeutics: The Journal of the American Society for Experimental NeuroTherapeutics, 6(3), 447–457. doi:10.1016/j.nurt.2009.04.001.

    CAS  Article  Google Scholar 

  44. Thompson, E. M., Frenkel, E. P., & Neuwelt, E. A. (2011). The paradoxical effect of bevacizumab in the therapy of malignant gliomas. Neurology, 76(1), 87–93. doi:10.1212/WNL.0b013e318204a3af.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  45. Tuettenberg, J., Friedel, C., & Vajkoczy, P. (2006). Angiogenesis in malignant glioma: A target for antitumor therapy? Critical Reviews in Oncology/Hematology, 59(3), 181–193. doi:10.1016/j.critrevonc.2006.01.004.

    CAS  PubMed  Article  Google Scholar 

  46. Videira, P. A., Piteira, A. R., Cabral, M. G., Martins, C., Correia, M., Severino, P., et al. (2011). Effects of bevacizumab on autocrine VEGF stimulation in bladder cancer cell lines. Urologia Internationalis, 86(1), 95–101. doi:10.1159/000321905.

    CAS  PubMed  Article  Google Scholar 

  47. Vredenburgh, J. J., Desjardins, A., Herndon, J. E, 2nd, Dowell, J. M., Reardon, D. A., Quinn, J. A., et al. (2007). Phase II trial of bevacizumab and irinotecan in recurrent malignant glioma. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 13(4), 1253–1259. doi:10.1158/1078-0432.CCR-06-2309.

    CAS  Article  Google Scholar 

  48. Wade, A., Robinson, A. E., Engler, J. R., Petritsch, C., James, C. D., & Phillips, J. J. (2013). Proteoglycans and their roles in brain cancer. The FEBS Journal, 280(10), 2399–2417. doi:10.1111/febs.12109.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  49. Watkins, S., & Sontheimer, H. (2012). Unique biology of gliomas: Challenges and opportunities. Trends in Neurosciences, 35(9), 546–556. doi:10.1016/j.tins.2012.05.001.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  50. Wen, P. Y., & Kesari, S. (2008). Malignant gliomas in adults. The New England Journal of Medicine, 359(5), 492–507. doi:10.1056/NEJMra0708126.

    CAS  PubMed  Article  Google Scholar 

  51. Xu, T., Chen, J., Lu, Y., & Wolff, J. E. (2010). Effects of bevacizumab plus irinotecan on response and survival in patients with recurrent malignant glioma: A systematic review and survival-gain analysis. BMC Cancer, 10, 252. doi:10.1186/1471-2407-10-252.

    PubMed Central  PubMed  Article  Google Scholar 

  52. Xu, L., Duda, D. G., di Tomaso, E., Ancukiewicz, M., Chung, D. C., Lauwers, G. Y., et al. (2009). Direct evidence that bevacizumab, an anti-VEGF antibody, up-regulates SDF1alpha, CXCR4, CXCL6, and neuropilin 1 in tumors from patients with rectal cancer. Cancer Research, 69(20), 7905–7910. doi:10.1158/0008-5472.CAN-09-2099.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  53. Yamagishi, N., Teshima-Kondo, S., Masuda, K., Nishida, K., Kuwano, Y., Dang, D. T., et al. (2013). Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells. BMC Cancer, 13(1), 229. doi:10.1186/1471-2407-13-229.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  54. Zimmermann, D. R., & Dours-Zimmermann, M. T. (2008). Extracellular matrix of the central nervous system: From neglect to challenge. Histochemistry and Cell Biology, 130(4), 635–653. doi:10.1007/s00418-008-0485-9.

    CAS  PubMed  Article  Google Scholar 

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Acknowledgments

The authors would like to thank Catherine Buquet, Wiem Khelil, Laure Klosek, and Elisabeth Legrand for their technical help. The authors are very grateful to Dr. Flore Gouel and Pr. Isabelle Dubus for fruitful discussions. T. Simon is recipient of a fellowship from the “Conseil Régional de Haute-Normandie.” A. Petit is recipient of a fellowship from “Ministère de l’Enseignement supérieur et de la Recherche”.

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The authors declare that they have no conflict of interest.

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Simon, T., Coquerel, B., Petit, A. et al. Direct Effect of Bevacizumab on Glioblastoma Cell Lines In Vitro. Neuromol Med 16, 752–771 (2014). https://doi.org/10.1007/s12017-014-8324-8

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Keywords

  • Anti-angiogenic therapies
  • Autocrine loop
  • Brain extracellular matrix
  • Glioblastoma
  • VEGF-A