Targeting multiple oncogenes simultaneously improves response to therapy and circumvents acquired resistance to single target tyrosine kinase inhibitors

Introduction
Epidermal growth factor receptors 1 and 2 (EGFR, ErbB2), and cytosolic Src (c Src) are tyrosine kinases that stimulate cell survival, proliferation and migration and if dysregulated can act as oncogenes. Several tyrosine kinase inhibitors (TKIs) have recently gained approval, but long term treatment with TKIs against a single target often lead to acquired resistance. The aim of this study was to investigate the effect of targeting multiple signalling pathways simultaneously in naïve and TKI-resistant cell lines.
Materials and Methods
The ErbB2 and c-Src genes were sequenced and small interfering RNAs (siRNAs) targeting them were developed. Effective siRNAs as well as ErbB2, Egfr and c-Src TKIs (AG828, gefitinib, GW583340 and saracatinib) were evaluated in canine and feline carcinoma cell lines. The effects of combined RNAi targeting of Egfr, ErbB2 and c-Src were assessed.
Results
Inhibition of c-Src and Egfr inhibited cell proliferation, migration and colony formation in feline and canine carcinoma cell lines while ErbB2 targeting exhibited no effect. Chronic drug exposure with gefitinib and saracatinib lead to resistance which was not associated with ATP binding site mutations. RNA interference (RNAi) strategies targeting Egfr plus ErbB2 and c Src plus either Egfr or ErbB2 showed a synergistic inhibition of cell proliferation. When targeting multiple areas of an oncogene or multiple oncogenes simultaneously significantly lower concentrations of siRNAs were required. Cells rendered resistant by chronic drug exposure were still sensitive to RNAi.
Conclusions
Targeting multiple pathways simultaneously improves growth inhibition, reduces the siRNA concentrations required and are still effective in cells rendered resistant to TKIs.