Combined Epidermal Growth Factor Receptor Targeting With The Tyrosine Kinase Inhibitor And The Monoclonal Antibody In No-Small Cell Lung Cancer

ObjectiveAlthough non-small cell lung cancer (NSCLC) cells with somatic mutations in their epidermal growth factor receptors (EGFR) initially show a dramatic response to tyrosine kinase inhibitor (TKI), these cells eventually develop resistance to TKI. This resistance may be caused by a secondary T790M mutation in the EGFR tyrosine kinase, which leads to the substitution of methionine for threonine in 790.and non-small cell lung cancer (NSCLC) cells without somatic mutations in their epidermal growth factor receptors (EGFR) do not show a dramatic response to tyrosine kinase inhibitor (TKI), In this study, we show whether a combination of erlotinib and cetuximab overcomes the drug resistance in NSCLC with the T790M mutation,and enhance the effects in NSCLC without somatic mutations in their epidermal growth factor receptors (EGFR).MethodsWe studied the effects of the combination of erlotinib and cetuximab in T790M and L858R mutation lung cancer cells lines (H1975), and not EGFR mutation lung cancer cell lines(A-549,LETP). in the primary NSCLC cells with the T790M and L858R mutation and without somatic mutations in their epidermal growth factor receptors (EGFR). in an TKI-resistant EGFR mutations(H1975) and not EGFR mutation lung cancer cell lines(A-549,LETP)human tumor xenograft model, The effects of these two agents on EGFR signaling, proliferation and apoptosis, were evaluated.Result1. We examined the antiproliferative effects of cetuximab in combination with erlotinib using the MTT assay in EGFR TKI-resistant NSCLC cell line(H1975)with T790M and L858R mutation and lung cancer cell lines(A-549,LETP) without EGFR mutation. we observed that the growth inhibition effects on H1975, A-549,LETP cells was further inhibited in a dose-dependent manner. Combining cetuximab with erlotinib resulted in more pronounced growth inhibition than single-agent treatment, in the H1975 cells (P< 0.05),not in the A-549,LETP (P>0.05). 2. We evaluated whether dual anti-EGFR inhibitors can augment cellular apoptotic response in EGFR TKI-resistant NSCLC cell line(H1975)with T790M and L858R mutation and lung cancer cell lines(A-549, LETP) without EGFR mutation. treatment with a combination of erlotinib and cetuximab resulted in a greater induction of apoptosis in EGFR TKI-resistant NSCLC cell line(H 1975)with T790M and L858R mutation(P<0.05) but not in lung cancer cell lines(A-549,LETP) without EGFR mutation.3. We examined the effect of combining cetuximab with erlotinib on the growth of human tumor xenografts. H1975, A-549, LETP cells were inoculated s.c. into athymic mice and allowed to grow until they had achieved a mean volume of 100 mm3 before treatments. Cetuximab was administered via i.p. injection at a dose of 0.2 mg twice per week for 4 consecutive weeks, and erlotinib was given by oral gavage at a dose of lmg,5 days per week for 4 consecutive weeks. Using the same control and erlotinib-treated mice as a reference, the combination of cetuximab and erlotinib resulted in significant inhibition of tumor growth and growth delay in the H1975 tumor xenografts (P<0.05),but not in A-549,LETP tumor xenografts.4. We tested the effect of drug treatment on EGFR downstream molecules. P-STAT3/STAT3,, p-AKT/AKT, p-MAPK/MAPK were more reduced as a result of combination treatment in H1975-bearing xenografts but not in A-549, LETP tumor xenografts, suggesting that the antitumor activity of the combination approach is mediated by inhibition of EGFR signaling. Taken together, these data show that treatment with a combination of erlotinib and cetuximab significantly inhibited the growth of T790M H1975 cells.5. The expression of molecular markers of tumor proliferation was further examined in histopathologic sections taken from H1975 tumor xenografts. Immunohistochemical staining with PCNA, Ki-67 demonstrated the number of proliferating cells to be greatest in the control and erlotinib group; intermediate in the groups receiving single-modality treatment with cetuximab; and least in the group receiving dual EGFR inhibitor treatment. These results complement previous data and suggest that combining distinct classes of EGFR inhibitors may augment one another via inhibition of cellular proliferation.6. We examined the Effect of Cetuximab in Combination with Erlotinib on the Growth of EGFR TKI-resistant primary NSCLC Cells with T790M mutation and without EGFR mutation suspension in vitro. Combining cetuximab with erlotinib resulted in more pronounced growth inhibition than single-agent treatment, in the primary NSCLC Cells with T790M mutation(P<0.05), but not in the primary NSCLC cell without EGFR mutation.ConclusionsThese data suggest that treatment with a combination of erlotinib and cetuximab, which induces dimeric dissociation and EGFR downregulation, appears to be an effective strategy for treatment of patients with EGFR TKI-resistant NSCLC and support combined EGFR targeting as a clinically exploitable strategy.