Human Blood Serum Can Diminish EGFR-Targeted Inhibition of Squamous Carcinoma Cell Growth through Reactivation of MAPK and EGFR Pathways

Abstract

Regardless of the presence or absence of specific diagnostic mutations, many cancer patients fail to respond to EGFR-targeted therapeutics, and a personalized approach is needed to identify putative (non)responders. We found previously that human peripheral blood and EGF can modulate the activities of EGFR-specific drugs on inhibiting clonogenity in model EGFR-positive A431 squamous carcinoma cells. Here, we report that human serum can dramatically abolish the cell growth rate inhibition by EGFR-specific drugs cetuximab and erlotinib. We show that this phenomenon is linked with derepression of drug-induced G1S cell cycle transition arrest. Furthermore, A431 cell growth inhibition by cetuximab, erlotinib, and EGF correlates with a decreased activity of ERK1/2 proteins. In turn, the EGF- and human serum-mediated rescue of drug-treated A431 cells restores ERK1/2 activity in functional tests. RNA sequencing revealed 1271 and 1566 differentially expressed genes (DEGs) in the presence of cetuximab and erlotinib, respectively. Erlotinib- and cetuximab-specific DEGs significantly overlapped. Interestingly, the expression of 100% and 75% of these DEGs restores to the no-drug level when EGF or a mixed human serum sample, respectively, is added along with cetuximab. In the case of erlotinib, EGF and human serum restore the expression of 39% and 83% of DEGs, respectively. We further assessed differential molecular pathway activation levels and propose that EGF/human serum-mediated A431 resistance to EGFR drugs can be largely explained by reactivation of the MAPK signaling cascade.

Targeted Inhibitors of EGFR: Structure, Biology, Biomarkers, and Clinical Applications

“…The addition of 5% human blood serum to cells contributed to a decrease in the antiproliferative activity of cetuximab in the EGFR-overexpressing A431 cell line [222], and this effect correlated with a decreased activity of ERK1/2 proteins and repression of cetuximab-induced G1S cell cycle transition The expression of 75% differently expressed genes, obtained by RNA sequencing, restores to the no-drug level when human serum is added along with cetuximab. The analysis of molecular pathways revealed that the addition of human serum reactivated MAPK signaling pathways inhibited by cetuximab alone [90]. Interestingly, human serum also modulated the effect of the HER2 monoclonal antibody trastuzumab on the HER2overexpressing cell line BT-474 [223].…”

“…Despite the proven efficacy of erlotinib in vitro, it has been shown that human blood serum of healthy donors can donor-specifically dramatically abolish the cell growth rate inhibition by erlotinib, and this effect correlates with a decreased activity of ERK1/2 proteins and abolishment of drug-induced G1S cell cycle transition arrest. Bioinformatic analysis revealed that EGF/human serum-mediated A431 resistance to EGFR drugs can be largely explained by the reactivation of the MAPK signaling pathway [ 90 ].…”

“…The expression of 75% differently expressed genes, obtained by RNA sequencing, restores to the no-drug level when human serum is added along with cetuximab. The analysis of molecular pathways revealed that the addition of human serum reactivated MAPK signaling pathways inhibited by cetuximab alone [ 90 ]. Interestingly, human serum also modulated the effect of the HER2 monoclonal antibody trastuzumab on the HER2-overexpressing cell line BT-474 [ 223 ].…”