In vitro effect of cetuximab on EGFR and downstream mediators. With a focus on gliomas
Abstract:Primary Glioblastoma Multiforme (GBM) is frequently associated with amplification and/or mutation of the epidermal growth factor receptor (EGFR). EGFR is involved in regulation of cell proliferation, growth, survival and motility through, among others, the two downstream pathways Ras/Raf/ERK and PI3K/AKT. Moreover, inactivating mutations of the tumor suppressor gene PTEN occurs in 15–60% of all GBM.
The EGFR inhibitor cetuximab has shown to be effective in various cancers both in vitro and in vivo. However, we observed only moderate effect on glioma cell viability in vitro either with or without the presence of EGF. In comparison the HN5 and A431 cell lines, with known EGFR amplifications, are sensitive to increasing concentrations of cetuximab in the absence of EGF, whereas the presence of EGF almost eliminates the effect of cetuximab.
Western blotting experiments show that EGF induces phosphorylation of EGFR (pEGFR) in various glioma cell lines, as well as in HN5. Increasing concentrations of cetuximab could inhibit this phosphorylation. However, activation of AKT (pAKT) and ERK (pERK) was not affected by cetuximab in any of the cell lines. Without the presence of EGF, cetuximab induced inhibition of pEGFR in all cell lines tested, but only HN5 showed inhibition of pAKT and pERK. This could indicate that the spontaneous dimerization of EGFR, which seems to be inhibited by cetuximab, is only of importance for the AKT/PI3K and Ras/Raf/ERK pathways in HN5 but not the glioma cell lines. Moreover, as opposed to HN5, all the glioma cells tested have mutated PTEN contributing to an abnormally high activity of the PI3K/AKT pathway. The lack of effect in the viability assays for the glioma cell lines could either be caused by PTEN mutations or that EGFR is not the primary inducer of activity for the AKT/PI3K and Ras/Raf/ERK pathways.
Furthermore, both EGF and cetuximab induce internalization of the EGFR, but our results indicate that when comparing to the effect of EGF, the receptor is not degraded sufficiently in the presence of cetuximab, suggesting another explanation for the observed lack of response to cetuximab in the glioma cell lines.
Document Type: Abstract
Publication date: 2008-05-01