Combined Erlotinib and Cetuximab overcome the acquired resistance to epidermal growth factor receptors tyrosine kinase inhibitor in non-small-cell lung cancer

Abstract

Purpose: Non-small-cell lung cancer (NSCLC) cells with somatic mutations in epidermal growth factor receptors (EGFR) are initially susceptible to tyrosine kinase inhibitor (TKI); however, eventually resistance to TKI is developed in these cells, which leads to the failure of treatment. The most common mechanism of this acquired drug resistance is development of a secondary T790M mutation in EGFR. In this study, we investigated the effects of the combination of Erlotinib and Cetuximab on T790M and L858R mutation lung cancer cells lines (H1975), in the primary NSCLC cells with the T790M mutation and TKI-resistant EGFR mutations human tumor xenograft model (H1975).

Methods: The effects of these two agents on cell proliferation, apoptosis, and EGFR-dependent signaling were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V staining, and Western blotting. Sensitivity of EGFR inhibitors was detected in the primary tumor cell suspension and human tumor xenograft model (H1975).

Results: Compared with single-agent treatment, the combination of Cetuximab and Erlotinib increased apoptosis of EGFR TKI-resistant NSCLC cells (H1975), resulting in more pronounced growth inhibition on cell proliferation and significant inhibition of EGFR-dependent signaling.

Conclusions: These data suggest that treatment with a combination of Erlotinib and Cetuximab overcomes T790M-mediated drug resistance.

Fig. 1

H1975 cells were treated with Erlotinib (0.1–10 μM), Cetuximab (1, 10, and 100 μg/ml), or a combination of both for 72 h. Cell viabilities were determined by MTT assays (group 1: erl 0.1 Μm; cet 1 μg/ml; erl + cet 0.1 μM + 1 μg/ml. group 2: erl 1 Μm; cet 10 μg/ml; erl + cet 1 μM + 10 μg/ml. group 3: erl 10 Μm; cet 100 μg/ml; erl + cet 10 μM + 100 μg/ml. erl + cet and erl, cet, P < 0.05, combination versus singles)

Fig. 2

H1975 cells were treated with Erlotinib, Cetuximab, or a combination of both at the indicated concentrations. The cells were collected, stained with Annexin V-phycoerythrin (AnnV PE), and analyzed via flow cytometry. The results were showed as X ± SD, n = 8, 6 compared to 2, 3; 7 compared to 4, 5, P < 0.05, combination versus singles

Fig. 3

H1975 cells were injected s.c. into nude mice with randomization (n = 8). Once the tumors reached >50 mm³, Erlotinib (50–150 mg/kg p.o.) was administered daily for 4 weeks. Cetuximab (10 mg/kg) was given i.p. twice a week for 4 weeks. The results were showed as X ± SE, *P < 0.05, combination versus singles

Fig. 4

Immunohistochemical staining for PCNA, Ki-67 was performed using human H1975 tumor tissue sections taken from mice treated with vehicle control or single or dual anti-EGFR agents at day 21 following tumor inoculation. Positive (brown) staining indicates expression of PCNA, Ki-67 [PCNA: control group (+++); erl group (+++); cet group (++); erl + cet group (∓)] (a). [Ki-67: control group (+++); erl group (+++); cet group (++); erl + cet group (+)] (b)

Fig. 5

Tumor samples treated with Erlotinib, Cetuximab, or a combination thereof were collected at the end point and homogenized. Western blots are shown for phosphorylated and total Stat3, Akt, and MAPK