Autocrine EGF and TGF-α promote primary and acquired resistance to ALK/c-Met kinase inhibitors in non-small-cell lung cancer

Abstract

Drug resistance severely limits the clinical therapeutic value of molecularly targeted drugs. Growth factors gain a tremendous amount of focus due to the ability to promote drug resistance in non-small-cell lung cancer (NSCLC). However, whether tumor cells themselves can mediate drug resistance by secreting growth factors needs further clarification. Here, we first screened growth factors to identify autocrine epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α) that caused primary resistance to the ALK inhibitor TAE684 in H3122 cells and the c-MET-specific inhibitor SGX-523 in EBC-1 cells. Next, we discovered increased autocrine production of EGF and TGF-α in established acquired resistant H3122/TR and EBC-1/SR cells. Importantly, overexpression of EGF and TGF-α in two NSCLC cell lines produced resistance to TAE684 and SGX-523. Clinically, NSCLC patients with high expression of EGF and TGF-α developed primary resistance to crizotinib. Mechanistically, autocrine EGF and TGF-α activated EGFR signaling pathways to survive targeted c-Met and ALK inhibition. Furthermore, combined treatment with gefitinib circumvented EGF- and TGF-α-mediated primary and acquired resistance to TAE684/SGX-523. Taken together, these results suggested increased autocrine EGF and TGF-α conferred primary and acquired resistance to ALK/c-Met kinase inhibitors in NSCLC.

Keywords: acquired resistance; autocrine growth factor; non-small-cell lung cancer; primary resistance.

FIGURE 1

Transient exposure to EGF and TGF‐α promoted primary resistance to ALK/c‐Met TKI in H3122 and EBC‐1 cells. (A) Summary of results from H3122 and EBC‐1 cells treated with eight widespread growth factors in the presence of TAE684 (1 μM) or SGX‐523 (1 μM). CR, complete resistance; PR, partial resistance; NR, no rescue. (B, C) H3122 cells (B) or EBC‐1 cells (C) were treated with growth factors in the presence of TAE684 (1 μM) or SGX‐523 (1 μM) for 72 h and cell viability was assessed by MTT assay. Data are shown as mean ± SD from three independent experiments. **p < .01, ***p < .001. (D, E) H3122 cells (D) or EBC‐1 cells (E) were treated with TAE684 (1 μM) or SGX‐523 (1 μM) for 12 h and incubated with growth factors for additional 30 min followed by western blotting analysis with indicated antibodies.

FIGURE 2

Increased autocrine EGF and TGF‐α activated EGFR pathway in acquired resistant H3122/TR and EBC‐1/SR cells. (A, B) Sensitive (H3122, EBC‐1) and resistant (H3122/TR, EBC‐1/SR) cells were treated with different concentrations of TAE684 (A) or SGX‐523 (B) for 72 h and cell viability was assessed by MTT assay. Data are shown as mean ± SD from three independent experiments. (C, D) Sensitive and resistant cells were treated with TAE684 (1 μM) (C) or SGX‐523 (1 μM) (D) for 3 h and the whole cell lysates was subjected to western blotting with indicated antibodies. (E, F) ELISA of HGF, EGF and TGF‐α secretion by sensitive or resistant cells. Data are shown as mean ± SD from three independent experiments. *p < .05, **p < .01, ***p < .001. (G, H) Western blotting analysis of protein phosphorylation in sensitive and resistant cells with indicated antibodies. Results of three independent experiments are shown.

FIGURE 3

High expression of EGF and TGF‐α promoted ALK/c‐Met TKI resistance in vitro and in clinical samples. (A) ELISA of EGF and TGF‐α secretion by NSCLC cells transfected with EGF (H3122/EGF, EBC‐1/EGF), TGF‐α (H3122/TGF‐α, EBC‐1/TGF‐α) or an empty vector (H3122/Vec, EBC‐1/Vec). Data are means ± SD from three independent experiments. *p < .05, **p < .01. (B, C) H3122 cells (B) or EBC‐1 cells (C) transfected with EGF or TGF‐α were treated with different concentrations of TAE684 or SGX‐523 for 72 h and cell viability was assessed by MTT assay. Data are means ± SD from three independent experiments. (D) Characteristics of NSCLC patients and their response to crizotinib treatment. (E) Immunohistochemistry analysis of EGF and TGF‐α expression in specimens taken from NSCLC patients before crizotinib treatment. Images taken at 20 × magnification (scale bar −50 μm).

FIGURE 4

Combination of ALK/c‐Met inhibitor with EGFR inhibitor circumvented EGF‐ and TGF‐α‐driven primary resistance. (A, B) H3122 cells treated with EGF (100 ng/ml) (A) or TGF‐α (100 ng/ml) (B) were incubated with different concentrations of TAE684 alone or in combination with gefitinib (1 μM) for 72 h and cell proliferation was detected by MTT assay. Data are shown as mean ± SD from three independent experiments. (C, D) EBC‐1 cells treated with EGF (100 ng/ml) (C) or TGF‐α (100 ng/ml) (D) were incubated with different concentrations of SGX‐523 alone or in combination with gefitinib (1 μM) for 72 h and cell proliferation was detected by MTT assay. Data are shown as mean ± SD from three independent experiments. (E, F) H3122 cells (E) or EBC‐1 cells (F) were treated with TKIs (1 μM) alone or in combination for 12 h and incubated with EGF (100 ng/ml) or TGF‐α (100 ng/ml) for additional 30 min followed by western blotting analysis with indicated antibodies. Results of three independent experiments are shown.

FIGURE 5

Combination treatment of c‐Met/ALK inhibitor and EGFR inhibitor circumvented EGF‐ and TGF‐α‐mediated acquired resistance. (A, B) H3122/TR cells (A) or EBC‐1/SR cells (B) were treated with TKIs (1 μM) alone or in combination for 72 h and cell proliferation was detected by MTT assay. Data are shown as mean ± SD from three independent experiments. **p < .01. (C, D) H3122/TR cells (C) or EBC‐1/SR cells (D) were treated with TKIs (1 μM) alone or in combination for 3 h and the whole cell lysates was detected by western blotting with indicated antibodies. Results of three independent experiments are shown. (E) Graphical summary of autocrine EGF and TGF‐α promoting primary and acquired resistance to ALK−/c‐Met‐targeted TKI by activation of EGFR signal pathway in non‐small cell lung cancer.