Inhibition of non-small cell lung cancer (NSCLC) growth by a novel small molecular inhibitor of EGFR

Abstract

The epidermal growth factor receptor (EGFR) is a therapeutic target (oncotarget) in NSCLC. Using in vitro EGFR kinase activity system, we identified a novel small molecule, WB-308, as an inhibitor of EGFR. WB-308 decreased NSCLC cell proliferation and colony formation, by causing G2/M arrest and apoptosis. Furthermore, WB-308 inhibited the engraft tumor growths in two animal models in vivo (lung orthotopic transplantation model and patient-derived engraft mouse model). WB-308 impaired the phosphorylation of EGFR, AKT, and ERK1/2 protein. WB-308 was less cytotoxic than Gefitinib. Our study suggests that WB-308 is a novel EGFR-TKI and may be considered to substitute for Gefitinib in clinical therapy for NSCLC.

Keywords: EGFR; NSCLC; TKIs; tumor growth.

Figure 1. WB-308 suppresses the EGFR signaling pathway in NSCLC cells

(A) The chemical structure of WB-308 (left) and Gefitinib (right). (B) The binding mode of WB-308 and Gefitinib bound with the pocket of the EGFR kinase domain. WB-308 inserts into the binding site of EGFR (right). The binding cavity is shown as achromatic surface. (C) In vitro EGFR kinase inhibition. Half maximal inhibitory concentration (IC₅₀) values of WB-308 and positive control Gefitinib. (D) Suppression by WB-308 of EGF–induced phosphorylation of AKT and ERK1/2 in NSCLC cell lines A549, PC-9 and NCI-H1975. Cancer cells were pretreated with indicated concentrations of WB-308 for 4 hours, and then incubated with 20 ng/ml of EGF for another hour. Cell lysates were subjected to Western blotting analysis with indicated antibodies. (E) WB-308′s inhibition to the phosphorylation of AKT and ERK1/2 was directly through EGFR but not Insulin receptor. PC-9 cells were pretreated with indicated concentrations of WB-308 for 4 hours, and then incubated with 20 ng/ml of EGF and 100 nM Insulin, respectively, for another hour. Cell lysates were subjected to Western blotting analysis with antibodies as indicated.

Figure 2. WB-308 exerts the same molecular mechanism with Gefitinib in EGFR mutated NSCLC cells

(A) Establishment of the EGFR-WT, EGFR-L858R and the vehicle over-expressing stable lines. The 3 stable cell lysates were subjected to Western blotting analysis with antibodies against p-EGFR (Tyr1068) and EGFR. Anti-β-actin antibody (1:10 000 dilution) was used as a loading control. (B) WB-308 increases NSCLC cell's sensitivity EGFR mutation. H1299 (EGFR-WT) and H1299 (EGFR-L858R) stable cell lines were exposed to indicated concentrations of WB-308 for 4 hours and cell lysates were subjected to Western blotting analysis with antibodies against p-EGFR (Tyr1068) and EGFR. (C) WB-308 suppresses NSCLC cell's proliferation in EGFR mutation. The cell viability assay stained by SRB was performed as described in Materials and Methods. Columns, mean; bars, SE (n = 3; t-test, P < 0.05).

Figure 3. WB-308 exhibits evidences to be a better EGFR inhibitor than Gefitinib

11 NSCLC cell lines (HCC-827, PC-9, NCI-H1650, SPC-A1, A549, NCI-H1975, HCC-827GR, MRC-5, HFL1, IMR-90 and BEAS-2B) were subjected to the cell viability assay stained by SRB as described in Materials and Methods. Columns, mean; bars, SE (n = 3; t-test, P < 0.05).

Figure 4. WB-308 inhibits colony formation of NSCLC cells

(A) NSCLC cell line SPC-A1 was seeded in 6-well plates for 7 days after the treatment of WB-308 in according concentrations and fixed with 4% paraformaldehyde, and stained with 0.2% crystal violet. (B) Statistic results of 2-D colony formation. Columns, mean; bars, SE (n = 3; t-test, P < 0.05). (C) NSCLC cell line SPC-A1 was subjected to the 3-D colony formation assay described in Materials and Methods and the result were photographed. (D, E) Statistic results of 3-D colony formation. Columns, mean; bars, SE (n = 3; t test, P < 0.05).

Figure 5. WB-308 induces NSCLC cell apoptosis and arrests NSCLC cell at G2/M phase

(A) Five representative genes with expression levels after the treatments of indicated concentrations of WB-308. Effects of WB-308 on transcription of five cell apoptosis and cell cycle checkpoint-associated genes were analyzed through the real-time PCR. (B) MRC-5 and PC-9 cell lines were exposed to indicat concentrations of WB-308 for 4 hours and cell lysates were subjected to Western blotting analysis with indicated antibodies. Anti-β-actin antibody was used as a loading control. (C) Cell cycle analysis of PC-9 cells. PC-9 cells were treated with Nocodazole (100 ng/ml) for 16 hrs aiming to synchronize cell cycle at the same phase and then release, then cells were exposed to different concentration of WB-308 for according time. After PI staining, cells were analyzed by flow cytometry. (D) Statistic results of cell cycle arrested in G2/M phase. Columns, mean; bars, SE (n = 3; t-test, P < 0.05).

Figure 6. WB-308 inhibits PC-9 orthotopic tumor growth in vivo

(A) Tumor growth in the orthotopic lungs over a 28-day period was detected by bioluminescence analysis every 7 days. (B) Quantitative analysis of growing cells in lung bioluminescence analysis every 5 days. The means and 95% confidence intervals (error bars) are presented; ***P < 0.001, **P = 0.004. P values were calculated using a two sided Student's t-test. p/sec/cm²/sr = photons/second/cm²/steradian. (C) To detect the inhibitory effect of WB-308 on tumor cell proliferation and EGFR signaling pathway in PC-9-luc orthotopic tumor growth model, primary tumors and lungs were sectioned and probed with anti-pEGFR (1:100 dilution), anti-pERK1/2 (1:200 dilution), anti-Ki-67 (1:200 dilution) and anti-Bcl-xL (1:100 dilution) antibodies. Representative images are shown. Brown color indicates positive cells. Scale bar = 30 μm.

Figure 7. WB-308 inhibits the growth of patient-derived NSCLC tumor xenografts

(A) The patient-derived spinal metastasized tumor cells were injected subcutaneously into the nude mice. The tumors model was established according to the steps described in Materials and Methods. After mice sacrificed, tumors were removed and images taken with a Nikon camera. (B) Statistic results of quantitative analysis of growing tumor volume in mice back subcutaneous every 3 days. The means and 95% confidence intervals (error bars) were presented (***P < 0.001, **P = 0.004). (C) Effect of WB-308 on mouse body weight. WB-308 did not affect the body weight of mice when recorded every 3 days. Columns, mean (n = 10); bars, SE. **P < 0.01 versus control.