EGFRvIII does not affect radiosensitivity with or without gefitinib treatment in glioblastoma cells

Abstract

BackgroundGlioblastomas (GBM) are often characterized by an elevated expression of the epidermal growth factor receptor variant III (EGFRvIII). We used GBM cell lines with native EGFRvIII expression to determine whether this EGFR variant affects radiosensitivity with or without EGFR targeting.MethodsExperiments were performed with GBM cell lines lacking (LN229, U87MG, U251, CAS-1) or endogenously expressing EGFRvIII (BS153, DKMG). The two latter cell lines were also used to establish sublines with a low (−) or a high proportion (+) of cells expressing EGFRvIII. EGFR signaling and the cell cycle were analyzed using Western blot and flow cytometry; cell survival was assessed by colony forming assay and double-strand break repair capacity by immunofluorescence.ResultsDKMG and BS153 parental cells with heterogeneous EGFRvIII expression were clearly more radiosensitive compared to other GBM cell lines without EGFRvIII expression. However, no significant difference was observed in cell proliferation, clonogenicity or radiosensitivity between the EGFRvIII− and + sublines derived from DKMG and BS153 parental cells. Expression of EGFRvIII was associated with decreased DSB repair capacity for BS153 but not for DKMG cells. The effects of EGFR targeting by gefitinib alone or in combination with irradiation were also found not to depend on EGFRvIII expression. Gefitinib was only observed to influence the proliferation of EGFRvIII− BS153 cells.ConclusionThe data indicate that EGFRvIII does not alter radiosensitivity with or without anti-EGFR treatment.

Cell line with endogenous EGFRvIII expression is a suitable model for research and drug development purposes

“…Interestingly, proliferation rate of the DK-MG high line in serum free media is trending to be higher than that of DK-MG low line, although statistical significance has not been reached. Those results find confirmation in the molecular studies, which show similar levels of PI3K/Akt signaling pathway activation in both DK-MG cell lines (Figure 4A-4D), pathway that is associated with the proliferation and survival processes, a result identical to the one observed by Struve and colleagues [55, 57, 58]. It should be noted that PI3K/Akt signaling pathway is often referred to as the main signal transducer of the EGFR vIII signaling, which is contradictory to our data [6, 22, 41, 59].…”

“…Of particular importance is the fact that EGFR vIII mRNA levels as well as amplicon number did not change over prolonged culturing periods (Sup.Figure 6), even though loss of EGFR vIII amplicons during in vitro adaptation process of primary tumor cells has been observed by us (Figure 1C) and consistently reported in the literature [26, 27]. Stability of the DK-MG line, also confirmed by Struve and colleagues [55], is of particular importance in scientific research and drug development, where model's stability is a basic requirement.…”

“…Two potential causes underlie such discrepancy - either the methods employed are not sufficiently sensitive to detect changes on the protein level resulting from the small number of amplicons (below 10), or expression of EGFR vIII is somehow regulated post-transcriptionally or epigenetically (e.g. miRNA, protein folding or methylation), which has been reported previously [36, 50, 54, 55]. With regards to the amplicon number and protein levels per cell, the number of EGFR vIII -positive cells in the DK-MG high line was much higher compared to the DK-MG low line, however no distinctive populations could be observed in FISH and immunofluorescence studies, indicative of high heterogeneity (Figure 2B and 2F, respectively).…”

“…Controversial is the role of EGFR vIII in the processes such as populational growth, cell proliferation and viability, with contradicting results reported [25, 40, 44, 55, 56]. However, only a small subset of publications recognizes that populational growth depends on the proliferation rate as well as cell viability, which have opposite effects.…”

F25P preproinsulin abrogates the secretion of pro-growth factors from EGFRvIII cells and suppresses tumor growth in an EGFRvIII/wt heterogenic model

“…Camorani and colleagues [31] reported that aptamers could target EGFRvIII and affect the migration, invasion, and proliferation of GBM cells by hampering the constitutive autophosphorylation of EGFRvIII. Struve and colleagues [32] performed experiments to confirm that EGFRvIII did not alter radiosensitivity with or without gefitinib during anti-EGFR treatment in glioblastoma cells. However, these two studies both focused on uniform populations of EGFRvIII-expressing cancer cells, and surrounding cells were not taken into account.…”