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Mitogenic signalling in the absence of epidermal growth factor receptor activation in a human glioblastoma cell line

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Abstract

Epidermal growth factor receptor (EGFR) gene amplification and overexpression are commonly present in glioblastoma, and confer advantages of growth, invasiveness and radio/chemotherapy-resistance for tumour cells. Here, we assessed the role of EGFR activation for downstream mitogenic signalling in the commonly used glioblastoma cell line U251. Despite the high expression level, activation of EGFR under standard culture conditions was low. Intact EGFR function was verified by the rapid phosphorylation of EGFR and downstream mitogen-activated protein (MAP) kinase ERK1/2 upon addition of exogenous EGF to serum-starved cells. By contrast, addition of fetal bovine serum (FBS) activated downstream ERK1/2 via the MAP kinase kinase without phosphorylating EGFR. A phospho-receptor tyrosine kinase array showed FBS-induced activation of insulin-like growth factor-1 receptor (IGF-1R), and the IGF-1R inhibitor AG1024 inhibited FBS-induced phosphorylation of ERK1/2, implying IGF-1R as the major driver of FBS-associated mitogenic signalling in the absence of exogenous EGF. These findings have important implications for in vitro drug testing in glioblastoma. Moreover, activation of ERK1/2 was also strongly influenced by growth state and cell density of U251 cultures. Re-seeding exponentially growing cultures at high cell density induced p27/CDKN1B expression and suppressed P-ERK1/2 indicating a certain regulation of proliferation by contact inhibition. Strikingly, highly activated ERK1/2 signalling and cell-cycle progression occurred when cells were released from plateau phase regardless of high seeding density. This phenomenon might implicate a proliferation response in the early recurrence observed after clinical therapy in glioblastoma patients. However, whether it will recapitulate in vivo remains to be demonstrated.

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Acknowledgments

We thank Anne-Kathrin Kirchner and Nicole Guerth for excellent technical assistance. Roche Diagnostics GmbH, Penzberg, Germany is kindly acknowledged for providing erlotinib. P.M. was supported by a grant from the Deutsche Krebshilfe/Dr. Mildred Scheel Stiftung (10-2089-FI 1).

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Author notes

  1. Meng Wang

    Present address: Laboratory of Cellular & Molecular Radiation Oncology, Department of Radiation Oncology, Massachusetts General Hospital Cancer Center, Charlestown, MA, 02129, USA

Authors and Affiliations

  1. Department of Radiation Oncology, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer Ufer 1-3, 68167, Mannheim, Germany

    Meng Wang, Patrick Maier, Frederik Wenz, Frank Anton Giordano & Carsten Herskind

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  1. Meng Wang
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  2. Patrick Maier
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Correspondence to Carsten Herskind.

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Frank Anton Giordano and Carsten Herskind are the joint senior authors.

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Wang, M., Maier, P., Wenz, F. et al. Mitogenic signalling in the absence of epidermal growth factor receptor activation in a human glioblastoma cell line. J Neurooncol 115, 323–331 (2013). https://doi.org/10.1007/s11060-013-1232-1

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