Amplification of the EGFR gene can be maintained and modulated by variation of EGF concentrations in in vitro models of glioblastoma multiforme

Abstract

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in adults. It is known that amplification of the epidermal growth factor receptor gene (EGFR) occurs in approximately 40% of GBM, leading to enhanced activation of the EGFR signaling pathway and promoting tumor growth. Although GBM mutations are stably maintained in GBM in vitro models, rapid loss of EGFR gene amplification is a common observation during cell culture. To maintain EGFR amplification in vitro, heterotopic GBM xenografts with elevated EGFR copy number were cultured under varying serum conditions and EGF concentrations. EGFR copy numbers were assessed over several passages by quantitative PCR and chromogenic in situ hybridization. As expected, in control assays with 10% FCS, cells lost EGFR amplification with increasing passage numbers. However, cells cultured under serum free conditions stably maintained elevated copy numbers. Furthermore, EGFR protein expression positively correlated with genomic amplification levels. Although elevated EGFR copy numbers could be maintained over several passages in vitro, levels of EGFR amplification were variable and dependent on the EGF concentration in the medium. In vitro cultures of GBM cells with elevated EGFR copy number and corresponding EGFR protein expression should prove valuable preclinical tools to gain a better understanding of EGFR driven glioblastoma and assist in the development of new improved therapies.

Histone Lysine Methylation Dynamics ControlEGFRDNA Copy-Number Amplification

“…While investigating hypoxia modulation of EGFR amplification, we also tested whether EGF supplementation would affect EGFR copy gains. A recent report demonstrated that cancer cells with EGFR amplification required EGF supplementation in the media to propagate the copy gains, which raised the possibility that EGF could directly promote EGFR copy gains (54). Consistent with this possibility, we demonstrated that treating cells for 24 hours with 50 ng/mL EGF, the preferred ligand of EGFR, results in significant copynumber gains of the EGFR locus ( Fig.…”

“…This plasticity has also been illustrated in cell culture as EGFR copy gains disappear in some cell lines (e.g., glioblastoma cells). A recent study demonstrated, however, that supplementing EGF enables propagation of the EGFR amplifications (54). Therefore, there is a critical need to understand the mechanisms that promote or suppress this amplification event.…”

Double Recombinant Vaccinia Virus: A Candidate Drug against Human Glioblastoma

“…Amplification of the epidermal growth factor receptor (EGFR) gene is known to be observed in more than 50% of glioblastoma cases [22]. The addition of EGF in the medium is able to stimulate the EGFR-dependent signaling pathways, such as phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) [23,24]. Subsequently, phosphorylated p21-activated kinase 1 (PAK1) participates in the cytoskeleton transformation and microtubule dynamics process, and this mediates the blabbing of the cell membrane, the severity of which directly affects the number of viral particles entering the host cell [25,26].…”

The Genetic Landscape of Human Glioblastoma and Matched Primary Cancer Stem Cells Reveals Intratumour Similarity and Intertumour Heterogeneity

“…Regarding EGFR amplification, 5 out of 10 analysed cases exhibit this alteration in GBM, but only 2 of them retained it in GSC population. This could be due to the negative selective pressure induced by EGF addition to the medium, as described by [ 41 , 42 ]. However, this phenomenon is still controversial; indeed, not all GSCs derived from EGFR -amplified GBM lack the amplification of this marker, as reported in several studies [ 43 – 45 ].…”