Human Blood Serum Counteracts EGFR/HER2-Targeted Drug Lapatinib Impact on Squamous Carcinoma SK-BR-3 Cell Growth and Gene Expression.

Lapatinib is a targeted therapeutic inhibiting HER2 and EGFR proteins. It is used for the therapy of HER2-positive breast cancer, although not all the patients respond to it. Using human blood serum samples from 14 female donors (separately taken or combined), we found that human blood serum dramatically abolishes the lapatinib-mediated inhibition of growth of the human breast squamous carcinoma SK-BR-3 cell line. This antagonism between lapatinib and human serum was associated with cancelation of the drug induced G1/S cell cycle transition arrest. RNA sequencing revealed 308 differentially expressed genes in the presence of lapatinib. Remarkably, when combined with lapatinib, human blood serum showed the capacity of restoring both the rate of cell growth, and the expression of 96.1% of the genes expression of which were altered by the lapatinib treatment alone. Co-administration of EGF with lapatinib also restores the cell growth and cancels alteration of expression of 95.8% of the genes specific to lapatinib treatment of SK-BR-3 cells. Differential gene expression analysis also showed that in the presence of human serum or EGF, lapatinib was unable to inhibit the Toll-Like Receptor signaling pathway and alter expression of genes linked to the Gene Ontology term of Focal adhesion.

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

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.

Members of the EGFR family of tyrosine kinase receptors are major regulators of cellular proliferation, differentiation, and survival. In humans, abnormal activation of EGFR is associated with the development and progression of many cancer types, which makes it an attractive target for molecular-guided therapy. Two classes of EGFR-targeted cancer therapeutics include monoclonal antibodies (mAbs), which bind to the extracellular domain of EGFR, and tyrosine kinase inhibitors (TKIs), which mostly target the intracellular part of EGFR and inhibit its activity in molecular signaling. While EGFR-specific mAbs and three generations of TKIs have demonstrated clinical efficacy in various settings, molecular evolution of tumors leads to apparent and sometimes inevitable resistance to current therapeutics, which highlights the need for deeper research in this field. Here, we tried to provide a comprehensive and systematic overview of the rationale, molecular mechanisms, and clinical significance of the current EGFR-targeting drugs, highlighting potential candidate molecules in development. We summarized the underlying mechanisms of resistance and available personalized predictive approaches that may lead to improved efficacy of EGFR-targeted therapies. We also discuss recent developments and the use of specific therapeutic strategies, such as multi-targeting agents and combination therapies, for overcoming cancer resistance to EGFR-specific drugs.

Human Blood Serum Inhibits Ductal Carcinoma Cells BT474 Growth and Modulates Effect of HER2 Inhibition.

Trastuzumab, a HER2-targeted antibody, is widely used for targeted therapy of HER2-positive breast cancer (BC) patients; yet, not all of them respond to this treatment. We investigated here whether trastuzumab activity on the growth of HER2-overexpressing BT474 cells may interfere with human peripheral blood endogenous factors. Among 33 individual BC patient blood samples supplemented to the media, BT474 sensitivity to trastuzumab varied up to 14 times. In the absence of trastuzumab, human peripheral blood serum samples could inhibit growth of BT474, and this effect varied ~10 times for 50 individual samples. In turn, the epidermal growth factor (EGF) suppressed the trastuzumab effect on BT474 cell growth. Trastuzumab treatment increased the proportion of BT474 cells in the G0/G1 phases of cell cycle, while simultaneous addition of EGF decreased it, yet not to the control level. We used RNA sequencing profiling of gene expression to elucidate the molecular mechanisms involved in EGF- and human-sera-mediated attenuation of the trastuzumab effect on BT474 cell growth. Bioinformatic analysis of the molecular profiles suggested that trastuzumab acts similarly to the inhibition of PI3K/Akt/mTOR signaling axis, and the mechanism of EGF suppression of trastuzumab activity may be associated with parallel activation of PKC and transcriptional factors ETV1-ETV5.

Human blood serum can donor-specifically antagonize effects of EGFR-targeted drugs on squamous carcinoma cell growth.

Many patients fail to respond to EGFR-targeted therapeutics, and personalized diagnostics is needed to identify putative responders. We investigated 1630 colorectal and lung squamous carcinomas and 1357 normal lung and colon samples and observed huge variation in EGFR pathway activation in both cancerous and healthy tissues, irrespectively on EGFR gene mutation status. We investigated whether human blood serum can affect squamous carcinoma cell growth and EGFR drug response. We demonstrate that human serum antagonizes the effects of EGFR-targeted drugs erlotinib and cetuximab on A431 squamous carcinoma cells by increasing IC50 by about 2- and 20-fold, respectively. The effects on clonogenicity varied significantly across the individual serum samples in every experiment, with up to 100% differences. EGF concentration could explain many effects of blood serum samples, and EGFR ligands-depleted serum showed lesser effect on drug sensitivity.