The use of EGFR TKIs is a clinically validated therapeutic option in NSCLC, especially for those tumors that harbor a sensitizing EGFR kinase domain mutation. However, single agent TKI treatment does not completely abrogate the oncogenic action of the receptor on cell growth and apoptosis induction. Furthermore, initial responders with mutant EGFR invariably develop secondary resistance to first generation TKIs [4
]. Several strategies are being investigated for improving this therapeutic efficacy, by either combining EGFR TKI with other agents aimed at inhibiting other growth factor pathways that are responsible for EGFR TKI resistance, such as over-expressed c-Met. Another strategy is to target the EGFR with other agents that can suppress the oncogenic function, independent of the type of mutation. An example is cetuximab. Recently, the addition of cetuximab to afatinib has yielded impressive results in the treatment of EGFR reversible TKI resistant lung cancer due to T790M mutation [41
EGFR-specific siRNAs may be good candidates for cancer therapy because of their specificity, efficiency, and endurance in gene-specific silencing and ability to suppress EGFR expression independent of the mutation status of the gene. Currently, there are only a few reports on the biological effects of EGFR siRNAs on lung cancer cells [31
]. Sordella et al.
] used a commercial EGFR wild-type siRNA pool that effectively induced the apoptotic enzyme caspase 3 at 96 h post transfection. The siRNA treatment also suppressed viability in H1975 cells expressing a T790M mutant EGFR and H1650 cells harboring a downstream PTEN mutation, but not in H358 cells that are wild-type for EGFR. In the present study, we have shown that an EGFR specific siRNA is very effective at suppressing the expression of EGFR in all cell lines tested, independent of the EGFR mutation status. We have also shown that all cell lines were variably inhibited in their growth by the siRNA and that the siRNA induced apoptosis in a dose- and time-dependent manner, upon transfection with siRNAs targeting wild type EGFR. Our results are partly in discordance with the data of Sordella et al.
who, albeit using different siRNA sequences (commercially from Dharmacon, the concentration of siRNA was not reported) and detecting assays, found no biological effects in wild-type cells. These differences might reside in the respective concentration of the siRNAs used and the ability of the siRNAs to suppress gene expression which was high and uniform across cell lines in our experiments. Our results are in line with the report of Rothenberg et al.
], which showed that lentivirus-based shRNA constructs targeting wild-type EGFR mRNA could promote cell death. Furthermore, a reduction in cell viability was observed in EGFR wild-type cells by Yamanaka et al.
] who studied the effect of an EGFR siRNA (5'-GUCAGCCUGAACAUAACAU-3'), in different set of lung adenocarcinoma cell lines harboring a spectrum of EGFR wild-type, mutant, and KRAS mutant cell lines (PC-9 [EGFR 15-bp exon 19 deletion]; H3255 [EGFR L858R]; HT-1 [wt EGFR and wt KRAS]; 11-18 [wt EGFR and wt KRAS]; LK87 [KRAS G12D]; A549 [KRAS G12S]).
Although all cell lines tested in the present study were sensitive to our EGFR siRNA, some differences were noted. First of all, the differential sensitivity towards inhibition of cell growth versus apoptosis induction was not the same. The effect of an siRNA upon important aspects of the malignant phenotype, cell growth, and survival is a measure of the specific amplitude of the oncogenic potency and quality of the different mutations. The H1650 and HCC827 cell lines with an exon 19 deletion were the most sensitive, both for growth inhibition and apoptosis induction, confirming that the exon 19 mutation is the most oncogenic and addictive. H1650 cells have been described as resistant to TKIs due the loss of a functional PTEN suppressor [28
]. Our results indicate that the EGFR mutation in H1650 cells at least partially bypasses the PTEN deficiency in driving cell growth and survival and that such a downstream mutation does not confer an absolute resistance to EGFR inhibition. To the contrary, upon siRNA treatment, this cell line was the second most sensitive to both growth and apoptosis induction. The lesser sensitivity of H1975 cells to EGFR siRNA treatment despite an equally high inhibition of EGFR protein expression indicates that the EGFR carrying a T790M mutation in combination with an exon 21 mutation is a less potent driver of cell growth and survival, which could also help to explain the clinical resistance to TKI inhibition of that receptor. Our siRNA results also confirm that in EGFR wild-type cells the receptor contributes the least to the malignant phenotype if at all, especially for cell survival. While there were anti-proliferative effects in the H292 cell line with a wild-type status, this cell line was relatively resistant to apoptosis induction. This is in concordance with the clinical experience that such cancers do not really benefit from TKI treatment. The most puzzling of our results are in the H358 cell line that has a wild-type EGFR receptor and carries a homozygous KRAS mutation that normally subverts the signaling emanating from the EGFR and creates resistance to inhibition of the receptor with TKIs or monoclonal antibodies [46
]. In our experiments this cell line was the most sensitive to apoptosis induction and growth inhibition by siRNA EGFR inhibition. This result could not be explained by a higher EGFR mRNA knockdown in this cell line. H358 cells were found to be "KRAS-addicted" cells in which ablation of KRAS expression by shRNA interference results in apoptosis induction [48
]. Inhibition of growth by EGFR siRNA has also been observed in KRAS mutant cell lines A549 and LK87 [45
]. Our hypothesis is that the strong reduction of EGFR induced by EGFR-specific RNA interference, also induces a large depletion of GRB2-SOS complexes necessary to load GTP into normal or mutant KRAS and hence interferes with KRAS signaling. However, there are other, non-mutually exclusive possibilities. H358 cells were found to secrete increased levels of the EGFR ligand amphiregulin [49
]. Knocking down EGFR expression would interrupt the amphiregulin/EGFR positive feedback loop and this could induce apoptosis. Thirdly, H358 cells were found to have a high ErbB3 expression [50
], and since EGFR links to PI3K signaling via ErbB3, the PI3/AKT pathway may also be a major source of malignant growth in these cells (in addition to KRAS). The removal of PI3K/AKT signals by EGFR RNAi might then also lead to apoptosis. Moreover, others have reported observations that might point in the same direction as the present study: Sunaga et al.
] found that cell survival is not much affected by KRAS knockdown in KRAS mutant NSCLC cell lines and hypothesized that a feedback signal to EGFR and Akt leads to increased stimulation. An additional mechanism for the observed effect might be an off-target effect of erlotinib on the Janus kinase 2 (JAK2). Erlotinib was shown to decrease phosphorylation of JAK2 and STAT-5 in EGFR-negative myelodysplastic syndrome (MDS) cell lines KG-1 [50
] and erlotinib can disrupt signaling of the JAK2/STAT-5 pathway. JAK2 is activated by mutant p53 (or p53 deficiency) [51
]. Thus, some of the survival pathways emanating from EGFR bypass KRAS in the cell line H358, and the KRAS mutation is more important for resistance to proliferation and less for apoptosis induction. Our and others' results suggest that the presence of a KRAS mutation (or other unknown alterations in these cells) could render H358 cells dependent on EGFR signaling and that EGFR would be a candidate therapeutic target in such cancers. In the current work we have explored the effects of a near maximal elimination of EGFR using siRNA. Although our experiments do provide an estimate of the relative oncogenic potency of the various EGFR mutations and downstream mutations, currently we do not know whether it will be possible to attain similar concentrations of a therapeutic equivalent of our siRNA in vivo
and in patients and thus obtain similar efficacy.
It is within that window of a maximal effect of EGFR inhibition that we have to analyze the results with TKI or cetuximab inhibition, which are strikingly different. The effect of TKI inhibition on the malignant phenotype is indeed the integration of several variables: the oncogenic potency of the targeted receptor, the significance of the kinase activity to this oncogenic potency, the variable sensitivity of the receptor to kinase inhibitors and the relative potency of kinase inhibitors to shut down this enzymatic activity. The action of monoclonal antibodies is even more complex and more difficult to relate to the mutational status of the receptor. By analogy to what is observed in the clinical studies, the exon 19 deletion HCC827 cell line conferred by far the highest sensitivity to TKI which is consistent with earlier reports [4
]. This is also consistent with the high dependency of this cell line on this mutant receptor for cell growth and survival in our siRNA experiments. Comparatively, all other cell lines are to be considered to be relatively resistant to TKI inhibition. The striking difference with the siRNA results for the two cell lines with downstream TKI resistance mutations (PTEN and KRAS) indicates that the kinase activity of the receptor is not the sole mediator of the oncogenic activity of EGFR, although we observed some reflection of the siRNA results in the KRAS mutant H358 cells, especially with higher concentrations of erlotinib with regard to apoptosis induction. None of the cell lines had a relevant sensitivity to cetuximab alone under 10% FBS culture condition, and even the TKI sensitive cell line HCC827 cells showed limited response. This might be explained by the absence of an oncogenic significance of the wild-type receptor and insensitivity of mutant receptors to inhibition by monoclonal antibodies. Activating mutations indeed confer hypersensitivity to TKIs, but not necessarily to inhibition by monoclonal antibodies [56
]. The failure to detect a significant activity for cetuximab agrees with the absence of a significant activity as single agent or very modest added benefit in clinical lung cancer in association with chemotherapy [21
Although EGFR is clearly a valid target in NSCLC treatment, the efficacy demonstrated by EGFR-targeted agents is not maximal as shown in preclinical models and more recently in clinical trials [40
]. One approach to improve responsiveness to EGFR inhibitors may be to simultaneously target multiple HER family members. Afatinib is currently the most advanced compound in this class. Afatinib is an irreversible EGFR/HER2 inhibitor, with activity against wild-type and mutant forms of EGFR [57
]. Afatinib was more potent than gefitinib, erlotinib, and lapatinib in inducing the cell death of NSCLC cell lines, including those harboring wild-type EGFR, and the erlotinib-resistant T790M mutation [57
]. It was also found in the present study (Figure ) that the molar potency of afatinib against these cells was significantly higher than either gefitinib or erlotinib. HCC827 cells harboring the activating E746_A750 deletion were highly sensitive to afatinib, whereas other NSCLC cell lines were moderately sensitive, which is in agreement with other reports [57
]. The activity against the resistance mutation T790M and cell lines with downstream resistance mechanisms was, however, only slightly better than the reversible TKIs.
The several EGFR-targeting approaches differ in action mechanisms. TKIs compete with ATP to bind to the EGFR kinase, hence inhibiting EGFR autophosphorylation and activation of downstream signaling. Anti-EGFR antibodies prevent receptor dimerization and hence activation [59
]. However, none of these agents alone does maximally suppress EGFR signaling or the effect of mutant EGFR in the malignant phenotype, as also shown in our experiments. The combination of cetuximab with the different TKI has already been tested [60
]. The in vitro
and in vivo
results showed that the combined treatment can augment the potency of EGFR signaling inhibition. Ramalingam et al.
] used a combination of cetuximab and gefitinib for patients with advanced/metastatic lung cancer who were previously treated with platinum-based chemotherapy. It was concluded that dual inhibition is feasible and safe, and may have modest activity in advanced/metastatic NSCLC. The combination of afatinib and cetuximab can even overcome resistance due to the T790M mutation both preclinically [40
] as well as clinically [41
]. In the present study, the combined treatment of EGFR siRNA and TKIs or antibody achieved increased tumor cell growth suppression (about 30% more at the most) (Figures , , , , and ) in all the five NSCLC cell lines and increased apoptosis as high as by 100% (Figures and ). The effect with the different agents in the different cell lines was additive, not synergistic, as calculated by a combination index (CalcuSyn software). Again, the differential sensitivity of the cell lines to the combination mimicked their sensitivity to TKI alone: the cell lines that demonstrated the most sensitivity to siRNA had the largest effect from the combination, including the cell lines with downstream TKI-resistance mutations (which also in this context displayed the highest sensitivity) or the T790M mutation. The least added effect was seen with afatinib associated to EGFR siRNA in the cell line with the TKI sensitive exon 19 deletion mutation, in which afatinib alone is already highly active at very low molar concentrations.