LMSU

Evaluation of EGFR Signaling Effects in Gastric Cancer Cell Lines

Targeting epidermal growth factor receptor (EGFR) with the inhibitory antibody cetuximab may affect the motile behavior of tumor cells. The basal levels of gastric cancer cell motility were determined using time-lapse microscopy and quantification of the two-dimensional (2D) migration behavior based on two parameter values for each cell. Striking differences in the percentage of motile cells were observed between the various cell lines without treatment (Fig. 1): 96.26% (AGS), 88.84% (Hs746T), 68.29% (LMSU) and 49.81% (MKN1). These differences in the proportion of motile cells were reflected by a wide range of the mean average speed of untreated cells (Fig. 2): 45.00 μm/h (AGS), 39.73 μm/h (Hs746T), 35.44 μm/h (LMSU) and 13.88 μm/h (MKN1).

Comparisons of untreated and EGF-treated cells as well as EGF-treated and EGF/cetuximab-treated cells revealed no significant changes in the percentage of motile cells in the cell lines AGS, Hs746T and LMSU (Fig. 1). In MKN1 cells, EGF treatment increased the percentage of motile cells. Two cell lines reacted to EGF treatment with increased average speed compared to untreated cells: AGS and MKN1 cells (Fig. 2). Significant reversion of the EGF-induced increase in average speed was observed in the cell line AGS at 1 μg/ml cetuximab compared to EGF-treated cells. In MKN1 cells, a concentration-dependent decrease in the average speed due to cetuximab treatment was observed but was not statistically significant. No remarkable changes in average speed were observed in Hs746T and LMSU cells upon EGF and/or cetuximab treatment.

Fig. 1 Effect of EGF and/or cetuximab treatment on the percentage of motile cells. (Keller S, et al., 2017)

Fig. 2 Effect of EGF and/or cetuximab treatment on the average speed [μm/h]. (Keller S, et al., 2017)

Enforced Expression of Mutant P53 Promotes GEA Primary Tumorigenesis

Gastric and esophageal adenocarcinoma (GEA) is one of the main causes of cancer-related mortality worldwide. Functional studies showed that mutant p53 was sufficient, but not necessary, for enhancing primary tumor growth. 3 genetically modified isogenic versions of the LMSU (endogenous R175H) cell line were generated: a double control that expressed a nontargeting sgRNA/Cas9 and GFP (sgRNA con + GFP); a mutant p53-KO cell line that expressed p53 sgRNA#1 and GFP (p53 sg#1 + GFP); and a mutant p53 cell line that expressed p53 sgRNA#1 and a cDNA construct overexpressing p53-R175H' (p53 sg#1 + R175H'). The results showed deletion of endogenous mutant p53-R175H did not significantly alter adherent proliferation of luciferase-labeled LMSU gastric cancer cells (Fig. 3A). However, enforced expression of p53-R175H' in LMSU cells that lost endogenous mutant p53 via sgRNA#1 promoted proliferation by luciferase assays (Fig. 3A). A similar pattern was observed when these genetically modified cells were cultured in ultra-low attachment and soft agar conditions (Fig. 3B and C).

Xenograft experiments in nude mice showed that although endogenous mutant p53-R175H was nonessential for the growth of LMSU gastric cancer cells in vivo, overexpression of mutant p53-R175H promoted primary tumor growth (Fig. 3D). Transcript and protein levels of mutant p53 as determined by real-time PCR (RT-PCR) and IHC were maintained throughout the experiment as assessed at the endpoint (Fig. 3E). It is noteworthy that endogenous p53-R175H was highly expressed in LMSU cells while grown in xenograft primary tumors compared with adherent culture (Fig. 3E). Given these promising findings, whether enforced expression of hotspot p53 mutants can promote primary tumor properties in HGC27 was wondered, a gastric cancer cell line with an endogenous frameshift mutation in TP53 and therefore presumably defective for WT p53 activity. Unlike in LMSU cells, overexpression of hotspot p53 mutants (Fig. 3F) did not promote the proliferation of HGC27 cells during adherent culture. By contrast, enforced expression of p53-R175H and p53-R273H promoted ultra-low attachment and soft agar colony growth (Fig. 3G and H) to a greater extent than p53-R248Q in HGC27 cells. These data indicate that although endogenous mutant p53 is not required, overexpression can enhance primary tumor properties in GEA.

Fig. 3 Enforced expression of mutant p53 in 2 gastric adenocarcinoma cell lines confers a growth advantage. (Sethi N, et al., 2019)