TC-N19, a novel dual inhibitor of EGFR and cMET, efficiently overcomes EGFR-TKI resistance in non-small-cell lung cancer cells

Abstract

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) show a clinical benefit when used to treat patients with EGFR-mutated non-small-cell lung cancer (NSCLC), but this treatment unfortunately fails in patients with TKI-resistant tumors. We here provide evidence that TC-N19 (N19), a novel dual inhibitor of EGFR and cMET, efficiently overcomes the EGFR-TKI resistance in EGFR-mutated NSCLC cells via simultaneous degradation of both proteins by ubiquitin proteasomes. Comparison with HSP90 inhibitor treatment and knockdown of EGFR and cMET by small hairpin RNAs reveal that the reduction of EGFR and cMET expression by N19 is responsible for overcoming the intrinsic TKI resistance mediated by paxillin (PXN) in high PXN-expressing cells, PXN-overexpressing PC9 cells (PC9-PXN), the EGFR-T790M-mediated TKI resistance in H1975 and CL97 cells, and the acquired resistance to gefitinib in gefitinib-resistant PC9 cells (PC9GR). Annexin V-PI staining assay showed that the induction of apoptosis in NSCLC cells by N19 depended on the reduction in levels of both proteins. Xenograft tumor formation in nude mice induced by a PC9-PXN-stable clone and by PC9GR cells was nearly completely suppressed by N19 treatment, with no changes in animal body weight. MTT assays of normal lung cells and reticulocytes showed no cytotoxicity responses to N19. In summary, N19 may act as a novel dual inhibitor of EGFR and cMET that induces apoptosis in TKI-resistant EGFR-mutated NSCLC cells and suppresses xenograft tumor formation. We suggest that N19 may be a potential new-generation TKI or HSP90 inhibitor used for treatment of NSCLC patients who show resistance to current TKI-targeting therapies.

Figure 1

N19 treatment inhibits cell growth and induces apoptosis on EGFR-mutated NSCLC cells. (a) EGFR-mutated lung cancer cells (H1975, H1650, CL97, PC9-PXN, PC9GR, and PC9) were treated with gefitinib at the indicated concentrations for 48 h and cell viability and IC₅₀ (half-maximal inhibitory concentration) was analyzed by the MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Error bars are shown as mean±S.D.; n=3. (b) Normal lung cells (WI38 and Beas-2B) and EGFR-mutated lung cancer cells (H1975, H1650, CL97, PC9-PXN, PC9GR, and PC9) were treated with N19 at the indicated concentrations for 48 h and cell viability and IC₅₀ was analyzed by the MTT assay. (c and d) Colony formation (c) and annexinV-PI staining assay (d) were performed in six EGFR-mutated lung cancer cell types subjected to gefitinib or N19 treatment. (e) The relative colony formation ability and percentage of apoptotic cells modulated by gefitinib or N19 were summarized in these six cell types. All data were collected from three independent experiments. The mean value and S.D. were indicated as the column with error bars

Figure 2

N19 promotes ubiquitination to suppress the expression of EGFR and cMET proteins and their downstream signaling. (a) The effect of N19 on EGFR and cMET signaling. At 48 h after gefitinib (10 μM) or N19 (10 μM) treatment, the cell lysates were harvested and analyzed for the signaling alteration by western blot with indicated antibodies. (b) Inhibition of the proteasome pathway stabilized EGFR and cMET after N19 treatment. Cells were treated with or without MG132, or N19, or both, and analyzed by western blot. (c) N19 treatment decreased the half-life of endogenous EGFR and cMET proteins. PC9-PXN, H1650, and PC9GR cells treated with 100 μg/ml of cycloheximide for the indicated hours with or without 10 μM N19. The protein levels of EGFR and cMET were assessed by western blot. (d) The effect of N19 treatment on the polyubiquitination of EGFR and cMET. Cells were treated with N19 at 10 μM in PC9-PXN, H1650, and PC9GR cells, respectively, for 5 h. Polyubiquitination of EGFR and cMET were assessed by western blot in the IP. (e) The effect of EGFR or/and cMET knockdown on the apoptosis induced by N19 treatment. PC9-PXN, H1650, and PC9GR cells transfected with EGFR or/and cMET shRNAs were treated with 10 μM N19 for 24 h. The cell apoptosis was measured by annexinV-PI staining assay using a flow cytometry. All data were collected from three independent experiments. The mean value and S.D. were indicated as the column with error bars

Figure 3

N19 promotes the EGFR and cMET protein degradation via likely acting as HSP90 inhibition. (a) The effect of N19 on EGFR and cMET degradation via HSP90 inhibition. At 48 h after 17-AAG (10 μM) or N19 (10 μM) treatment, the cell lysates were harvested and analyzed for the signaling alteration by western blot with indicated antibodies. (b) PC9-PXN and H1650 cells were treated with gefitinib, SU11274, gefitinib+SU11274, 17-AAG or N19 for 48 h. The cell apoptosis was measured by annexinV-PI staining assay using a flow cytometry. The cell lysates were harvested and analyzed for the signaling alteration by western blot with indicated antibodies. (c) The effect of N19 on tumor growth. The tumor volume was measured as described in Materials and Methods (n=5, each group). Photographs show the representative tumor burdens in mice after drug administration for 27 days via intraperitoneal injection. Representative samples of tumor excised on day 27. The effect of N19 on the body weights of mice. Mean±S.D. values were calculated from the tumor volume and body weights of five nude mice in each group