Evaluation of epidermal growth factor receptor signaling effects in gastric cancer cell lines by detailed motility-focused phenotypic characterization linked with molecular analysis

Abstract

BackgroundGastric cancers frequently overexpress the epidermal growth factor receptor (EGFR), which has been implicated in pathological processes including tumor cell motility, invasion and metastasis. Targeting EGFR with the inhibitory antibody cetuximab may affect the motile and invasive behavior of tumor cells. Here, we evaluated the effects of EGFR signaling in gastric cancer cell lines to link the phenotypic behavior of the cells with their molecular characteristics.MethodsPhenotypic effects were analyzed in four gastric cancer cell lines (AGS, Hs746T, LMSU and MKN1) by time-lapse microscopy and transwell invasion assay. Effects on EGFR signaling were detected using Western blot and proteome profiler analyses. A network was constructed linking EGFR signaling to the regulation of cellular motility.ResultsThe analysis of the effects of treatment with epidermal growth factor (EGF) and cetuximab revealed that only one cell line (MKN1) was sensitive to cetuximab treatment in all phenotypic assays, whereas the other cell lines were either not responsive (Hs746T, LMSU) or sensitive only in certain tests (AGS). Cetuximab inhibited EGFR, MAPK and AKT activity and associated components of the EGFR signaling pathway to different degrees in cetuximab-sensitive MKN1 cells. In contrast, no such changes were observed in Hs746T cells. Thus, the different phenotypic behaviors of the cells were linked to their molecular response to treatment. Genetic alterations had different associations with response to treatment: while PIK3CA mutations and KRAS mutation or amplification were not obstructive, the MET mutation was associated with non-response.ConclusionThese results identify components of the EGFR signaling network as important regulators of the phenotypic and molecular response to cetuximab treatment.Electronic supplementary materialThe online version of this article (10.1186/s12885-017-3822-3) contains supplementary material, which is available to authorized users.

Model-based analysis of response and resistance factors of cetuximab treatment in gastric cancer cell lines

“…We utilized the gastric cancer cell lines MKN1 and Hs746T as a model system to study response and resistance factors of cetuximab treatment. Based on previous results obtained by proliferation and motility analysis, the MKN1 cell line is characterized as a cetuximab responder, while Hs746T is characterized as a non-responder cell line Keller et al, 2017).…”

“…Cetuximab targets the EGFR signaling pathway which regulates growth, survival, proliferation, di↵erentiation (Oda et al, 2005) and motility (Wells, 1999;Keller et al, 2017 Cetuximab binds EGFR and blocks the binding of EGF or other EGFR ligands, such as amphiregulin (AREG) and epiregulin (EREG) (Li et al, 2005). This reduces -in the absence of resistance factors -the activity of EGFR and its downstream targets.…”

“…The dataset contains time and dose responses for EGFR, pEGFR, pERK and pAKT. In an iterative process, published data (Keller et al, 2017; were complemented by newly collected data to improve reliability of the parameter estimates and model predictions. In total, we considered 303 data points for the MKN1 cell line ( Figure 1B) and 312 data points for the Hs746T cell line ( Figure 1C).…”

“…For the establishment of the model, the human gastric cancer cell lines Hs746T and MKN1 were used and cultured as reported by Keller et al (2017).…”

“…However, a suitable biomarker for patient stratification is currently not available. Here, we aim to understand response and resistance mechanisms for cetuximab treatment in gastric cancer, to unravel causal di↵erences between cetuximab responders and non-responders (Keller et al, 2017), and to identify biomarkers for guiding targeted therapy by using a cell culture model.…”