Antitumor activity of selective MEK1/2 inhibitor AZD6244 in combination with PI3K/mTOR inhibitor BEZ235 in gefitinib-resistant NSCLC xenograft models

Abstract

Purpose: Although the EGF receptor tyrosine kinase inhibitors (EGFR-TKI) gefitinib have shown dramatic effects against EGFR mutant lung cancer, patients become resistant by various mechanisms, including gatekeeper EGFR-T790M mutation, MET amplification, and KRAS mutation, thereafter relapsing. AZD6244 is a potent, selective, and orally available MEK1/2 inhibitor. In this study, we evaluated the therapeutic efficacy of AZD6244 alone or with BEZ235, an orally available potent inhibitor of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR), in gefitinib-resistant non-small cell lung carcinoma (NSCLC) models.

Experimental design: NCI-H1975 with EGFR-T790M mutation, NCI-H1993 with MET amplification and NCI-H460 with KRAS/PIK3CA mutation human NSCLC cells were subcutaneous injected into the athymic nude mice respectively. Mice were randomly assigned to treatment with AZD6244, BEZ235, AZD6244 plus BEZ235, or control for 3 weeks, then all mice were sacrificed and tumor tissues were subjected to western blot analyses and immunohistochemical staining.

Results: AZD6244 could inhibit the tumor growth of NCI-H1993, but slightly inhibit the tumor growth of NCI-1975 and NCI-H460. Combining AZD6244 with BEZ235 markedly enhanced their antitumor effects and without any marked adverse events. Western blot analysis and immunohistochemical staining revealed that AZD6244 alone reduced ERK1/2 phosphorylation, angiogenesis, and tumor cell proliferation. Moreover, MEK1/2 inhibition resulted in decreased AKT phosphorylation in NCI-H1993 tumor model. BEZ235 also inhibited AKT phosphorylation as well as their downstream molecules in all three tumor models. The antiangiogenic effects were substantially enhanced when the agents were combined, which may due to the reduced expression of matrix metallopeptidase-9 in tumor tissues (MMP-9).

Conclusions: In this study, we evaluated therapy directed against MEK and PI3K/mTOR in distinct gefitinib-resistant NSCLC xenograft models. Combining AZD6244 with BEZ235 enhanced their antitumor and antiangiogenic effects. We concluded that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor was effective in suppressing the growth of gefitinib-resistant tumors caused by EGFR T790M mutation, MET amplification, and KRAS/PIK3CA mutation. This new therapeutic strategy may be a practical approach in the treatment of these patients.

Figure 1

Anti-proliferative effects of AZD6244 and BEZ235 in NCI-H1993, NCI-H1975 and NCI-H460 gefitinib-resistant cell lines. Cells were treated with varying concentrations of AZD6244 (A) or BEZ235 (B) alone for 72 h. Doses ranged from 0.01 μM to 100 μM. Mean ± SD, n = 5.

Figure 2

Synergistic effects of AZD6244-BEZ235 combination therapy on cell viability. NCI-H1993 (A), NCI-H1975 (B) and NCI-H460 (C) cells were treated with AZD6244 alone, BEZ235 alone or AZD6244-BEZ235 in combination for 72 h. Results were analyzed according to the Chou-Talalay method [19]. The combination index (CI) values were calculated by using CalcuSyn software. Mean ± SD, n = 5.

Figure 3

Antitumor activity of AZD6244 and/or BEZ235 in mouse xenograft models of human tumors. Nude mice-bearing NCI-H1993 (A), NCI-H1975 (B), and NCI-H460 (C) tumors were administered 25 mg/kg AZD6244 twice daily and/or 20 mg/kg BEZ235 once daily up to 21 days. Tumors were resected from nude mice on day 21 (D). Tumor volume was measured using calipers on the indicated days. Mean ± SD, n = 10. *, P < 0.05 vs control group. **, P < 0.01 vs control group.

Figure 4

Changes in body weight of mice treated with control group or AZD6244 and/or BEZ235. Nude mice-bearing NCI-H1993 (A), NCI-H1975 (B), and NCI-H460 (C) tumors were administered 25 mg/kg AZD6244 twice daily and/or 20 mg/kg BEZ235 once daily up to 21 days. Body weight was measured on the indicated days. Mean ± SD, n = 10.

Figure 5

Effects of AZD6244-BEZ235 combination therapy on PI3K/AKT and MEK/ERK pathways. All three gefitinib-resistant tumor xenograft models were treated with the AZD6244 and/or BEZ235 for 2 h on Day 21 of the efficacy study, tumor tissues were then harvested to detect p-AKT (S473)/AKT, p-ERK (T202/Y204)/ERK, p-S6 (S240/244)/S6 and p-4E-BP1 (S65)/4E-BP1.

Figure 6

Effects of AZD6244 and/or BEZ235 on the expressions of Ki-67 (A) and CD31 (B) in NCI-1993, NCI-1975 and NCI-H460 xenograft models. All three gefitinib-resistant tumor xenograft models were treated with the AZD6244 and/or BEZ235 for 2 h on Day 21 of the efficacy study, tumor tissues in each group were resected and immunostained with anti-Ki67 and anti-CD31 antibodies. N = 10, *, P < 0.05 vs vehicle group. **, P < 0.01 vs vehicle group.

Figure 7

Effects of AZD6244 and/or BEZ235 on the activities of caspase-3, 8 and 9 in NCI-1993, NCI-1975 and NCI-H460 xenograft models. All three gefitinib-resistant tumor xenograft models were treated with the AZD6244 and/or BEZ235 for 2 h on Day 21 of the efficacy study, tumor tissues in each group were resected and measured by caspase colorimetric protease kits. N = 10.