BACKGROUND: The epidermal growth factor receptor (EGFR) is a validated 
therapeutic target in non-small cell lung cancer (NSCLC). However, current 
single agent receptor targeting does not achieve a maximal therapeutic effect, 
and some mutations confer resistance to current available agents. In the current 
study we have examined, in different NSCLC cell lines, the combined effect of 
RNA interference targeting the EGFR mRNA, and inactivation of EGFR signaling 
using different receptor tyrosine kinase inhibitors (TKIs) or a monoclonal 
antibody cetuximab.
METHODS: NSCLC cells (cell lines HCC827, H292, H358, H1650, and H1975) were 
transfected with EGFR siRNA and/or treated with the TKIs gefitinib, erlotinib, 
and afatinib, and/or with the monoclonal antibody cetuximab. The reduction of 
EGFR mRNA expression was measured by real-time quantitative RT-PCR. The 
down-regulation of EGFR protein expression was measured by western blot, and the 
proliferation, viability, caspase3/7 activity, and apoptotic morphology were 
monitored by spectrophotometry, fluorimetry, and fluorescence microscopy. The 
combined effect of EGFR siRNA and different drugs was evaluated using a 
combination index.
RESULTS: EGFR-specific siRNA strongly inhibited EGFR protein expression almost 
equally in all cell lines and inhibited cell growth and induced cell apoptosis 
in all NSCLC cell lines studied, albeit with a different magnitude. The effects 
on growth obtained with siRNA was strikingly different from the effects obtained 
with TKIs. The effects of siRNA probably correlate with the overall oncogenic 
significance of the receptor, which is only partly inhibited by the TKIs. The 
cells which showed weak response to TKIs, such as the H1975 cell line containing 
the T790M resistance mutation, were found to be responsive to siRNA knockdown of 
EGFR, as were cell lines with downstream TKI resistance mutations. The cell line 
HCC827, harboring an exon 19 deletion mutation, was more than 10-fold more 
sensitive to TKI proliferation inhibition and apoptosis induction than any of 
the other cell lines. Cetuximab alone had no relevant in vitro activity at 
concentrations obtainable in the clinic. The addition of EGFR siRNA to either 
TKIs or cetuximab additively enhanced growth inhibition and induction of 
apoptosis in all five cell lines, independent of the EGFR mutation status 
(wild-type or sensitizing mutation or resistant mutation). The strongest 
biological effect was observed when afatinib was combined with an EGFR-specific 
siRNA.
CONCLUSIONS: EGFR knockdown by siRNA further decreases the cell growth of lung 
cancer cells that are treated with TKIs or cetuximab alone, confirming that 
single agent drug targeting does not achieve a maximal biological effect. The 
siRNA inhibits EGFR oncogenic activity that bypasses downstream "resistance" 
mutations such as KRAS and PTEN. The combined treatment of siRNA and EGFR 
inhibitory agents is additive. The combination of a potent, irreversible kinase 
inhibitor such as afatinib, with EGFR-specific siRNAs should be further 
investigated as a new strategy in the treatment of lung cancer and other EGFR 
dependent cancers, including those with downstream resistance mutations.