Cigarette smoke enhances oncogene addiction to c-MET and desensitizes EGFR-expressing non-small cell lung cancer to EGFR TKIs

Abstract

Cigarette smoking is one of the leading risks for lung cancer and is associated with the insensitivity of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, it remains undetermined whether and how cigarette smoke affects the therapeutic efficacy of EGFR TKIs. In this study, our data showed that chronic exposure to cigarette smoke extract (CSE) or tobacco smoke-derived carcinogen benzo[α]pyrene, B[α]P, but not nicotine-derived nitrosamine ketone (NNK), reduced the sensitivity of wild-type EGFR-expressing NSCLC cells to EGFR TKIs. Treatment with TKIs almost abolished EGFR tyrosine kinase activity but did not show an inhibitory effect on downstream Akt and ERK pathways in B[α]P-treated NSCLC cells. CSE and B[α]P transcriptionally upregulate c-MET and activate its downstream Akt pathway, which is not inhibited by EGFR TKIs. Silencing of c-MET reduces B[α]P-induced Akt activation. The CSE-treated NSCLC cells are sensitive to the c-MET inhibitor crizotinib. These findings suggest that cigarette smoke augments oncogene addiction to c-MET in NSCLC cells and that MET inhibitors may show clinical benefits for lung cancer patients with a smoking history.

Keywords: EGFR-TKI; benzo[α]pyrene; c-MET; cigarette smoke; lung cancer.

Figure 1

Cigarette smoke extract treatment rendered wtEGFR‐ but not EGFR mutant‐expressing NSCLC more resistant to EGFR tyrosine kinase inhibitors. Whole‐cell extracts prepared from H292/CSE, HCC827/CSE and PC9/CSE. Stable cells were subjected to western blot analysis to determine p65 phosphorylation at ser536 (A). CSE‐treated H292 (B), HCC827 (C) and PC9 (D) were treated with gefitinib (left) or erlotinib (right) for 3 days and their viabilities were determined in MTT assays.

Figure 2

Treatment with B[α]P but not NNK reduces the sensitivity of H292 cells to EGFR tyrosine kinase inhibitors. H292 (A, B), HCC827 (C, D), and PC9 (E) selected with 1 μm B[α]P (A, C, E) or 10 μm NNK (B, D) were treated with gefitinib (left) or erlotinib (right) for 3 days, and their viabilities were determined in MTT assays.

Figure 3

Activities of Akt and Erk were higher in CSE‐ and B[α]P‐selected H292 cells. The activities and protein levels of EGFR and its downstream signaling Akt and ERK were detected in H292 (A) and HCC827 (B) by western blot analysis with indicated antibodies.

Figure 4

EGFR TKI failed to inhibit activation of Akt and Erk in H292/B[α]P cells but not in HCC827/ B[α]P cells. H292 (A, B) and HCC827 (C) cells selected with B[α]P or NNK were pretreated with 1 μm EGFR TKIs for 2 h followed by 50 ng·mL ⁻¹ EGF stimulation as indicated. Whole‐cell extracts were prepared and subjected to western blot analysis to determine the expression of EGFR signaling pathway with indicated antibodies.

Figure 5

Proto‐oncogene c‐MET expression and activity were higher in H292/B[α]P cells than in control cells. (A) Whole‐cell extract from H292/B[α]P cells was subjected to western blot analysis with anti‐phosphotyrosine antibody. (B) Total lysates prepared from the H292/DMSO and H292/B[α]P were incubated with RTK antibody arrays, and phosphotyrosine was detected by anti‐phospho‐tyrosine‐HRP. The changes in tyrosine phosphorylation of RTK were labeled as indicated. The upregulation (C) and downregulation (D) of RTK tyrosine phosphorylations in H292/B[α]P cells shown in (B) were quantitated using imagej software. (E) The RTK activities and protein levels in H292/B[α]P cells determined by western blot analysis with indicated antibodies. (F) The mRNA level of c‐MET in indicated cells was detected by q‐RT‐PCR. (G) The c‐MET mRNA levels in non‐smoker and smoker NSCLC patients were analyzed from the GSE31210 GEO dataset.

Figure 6

Cigarette smoke extract and B[α]P induced c‐MET expression through promoter de‐methylation. Parental H292 cells were treated with conditioned medium of (A) H292/CSE or (B) H292/B[α]P cells for 6 h. The whole‐cell lysates were then prepared and subjected to western blot analysis with indicated antibodies. (C) The CpG island on c‐MET promoter and primer sets used in ChIP analysis were illustrated. Parental H292 cells and their CSE and B[α]P clones were subjected to ChIP analysis with 5‐methylcytosine (5‐mC) (D) or anti‐MeCP2 (E) antibodies followed by quantitative PCR with primers targeting c‐MET promoter. ‘Fold of Input’ is expressed as the ratio between the amount of total immunoprecipitated DNA (bound) and the amount of input DNA. Data are expressed as means ± SD. **P < 0.005; ***P < 0.001.

Figure 7

The activity of Akt was reduced by inhibition of c‐MET in H292/CSE and H292/B[α]P cells. c‐MET expression was knocked down by shRNA in H292/CSE (A) and H292/B[α]P (B) cells for 3 days. (C,D) These stable clones were treated with 1 μm crizotinib for 3 days. Total lysates were collected and subjected to western blots with indicated antibodies (C) and the cell viability was analyzed in MTT assay (D).

Figure 8

Current hypothetic model of this study. Cigarette smoke de‐represses c‐MET expression through reduction of promoter methylation. The induced c‐MET induced drug resistance to EGFR TKIs by maintaining Akt activity in wtEGFR‐expressing lung cancer cells.