The HDAC inhibitor, MPT0E028, enhances erlotinib-induced cell death in EGFR-TKI-resistant NSCLC cells

Abstract

Epidermal growth factor receptor (EGFR), which promotes cell survival and division, is found at abnormally high levels on the surface of many cancer cell types, including many cases of non-small cell lung cancer. Erlotinib (Tarceva), an oral small-molecule tyrosine kinase inhibitor, is a so-called targeted drug that inhibits the tyrosine kinase domain of EGFR, and thus targets cancer cells with some specificity while doing less damage to normal cells. However, erlotinib resistance can occur, reducing the efficacy of this treatment. To develop more effective therapeutic interventions by overcoming this resistance problem, we combined the histone deacetylase inhibitor, MPT0E028, with erlotinib in an effort to increase their antitumor effects in erlotinib-resistant lung adenocarcinoma cells. This combined treatment yielded significant growth inhibition, induced the expression of apoptotic proteins (PARP, γH2AX, and caspase-3), increased the levels of acetylated histone H3, and showed synergistic effects in vitro and in vivo. These effects were independent of the mutation status of the genes encoding EGFR or K-Ras. MPT0E028 synergistically blocked key regulators of the EGFR/HER2 signaling pathways, attenuating multiple compensatory pathways (e.g., AKT, extracellular signal-regulated kinase, and c-MET). Our results indicate that this combination therapy might be a promising strategy for facilitating the effects of erlotinib monotherapy by activating various networks. Taken together, our data provide compelling evidence that MPT0E028 has the potential to improve the treatment of heterogeneous and drug-resistant tumors that cannot be controlled with single-target agents.

Figure 1

Chemical structures and cytotoxicity of MPT0E028. Chemical structures of erlotinib (a), vorinostat/SAHA (b), and MPT0E028 (c). (d) Effects of MPT0E028 on the viability of A549, H1299, H1975, CL97, and PC9/IR cells. Cells were treated with the indicated drugs for 72 h and cell viability was determined by the MTT assay, as described in the Materials and Methods section. Results are representative of at least three independent experiments

Figure 2

MPT0E028 enhances EGFR inhibitor-induced cytotoxicity in erlotinib-resistant NSCLC cells. Erlotinib-resistant A549 (a), H1299 (b), H1975 (c), PC9/IR (d), and CL97 (e) cells were incubated with increasing concentrations of erlotinib (E) and MPT0E028 (M) alone or concurrently for 72 h. (f) MPT0E028 and erlotinib together synergistically suppress colony formation. Clonogenic survival was assessed as described in the Materials and Methods section, and cell viability was determined by MTT assay. The results are expressed as the percentage surviving cells in drug-treated cultures relative to DMSO-treated control cells. Error bars represent S.D. CI values for the combination of erlotinib and MPT0E028 were calculated using the Calcusyn software (Cambridge, UK), as described in the Materials and Methods section

Figure 3

Assessment of apoptosis by propidium iodide in A549 cells. (a) Induction of apoptosis (subG1 phase) in A549 cells treated with MPT0E028 (M) in combination with erlotinib (E). Cells were treated with the indicated concentrations (μM) for 72 h, stained with propidium iodide, and assessed by flow cytometry. (b and c) The percentage of cells in sub-G1 at 72 h after treatment with erlotinib/MPT0E028 (b) or erlotinib/SAHA (c). Data are representative of the results from at least three independent experiments. Error bars represent S.D.

Figure 4

MPT0E028/erlotinib co-treatment induces acetylated histone and non-histone proteins and upregulates apoptotic proteins in lung adenocarcinoma cells. (a) Effect of erlotinib/MPT0E028 co-treatment on drug-induced DNA fragmentation in A549 cells. Cells were treated with the indicated concentrations of MPT0E028 and/or erlotinib for 72 h, and DNA fragmentation was quantitatively measured using a cell death detection ELISA kit. Columns, mean (n=4); bars, S.D. Symbols: *P<0.05; **P<0.01; and ***P<0.001 compared with the control group. (b and c) Western blot analysis of γH2AX, caspase 3, and PARP in CL97 (b) and PC9/IR (c) cells. Cells were treated with MPT0E028 and/or erlotinib for 72 h and cell lysates were subjected to immunoblotting using the indicated antibodies. (d and e) Comparison of co-treatment with erlotinib plus MPT0E028 (d) or SAHA (e) in A549 cells. Cells were treated with the indicated drugs for 72 h, and cell lysates were subjected to immunoblotting using the indicated antibodies

Figure 5

Significant suppression of EGFR and downstream signaling molecules is seen following co-treatment with MPT0E028 and erlotinib. (a) Effect of MPT0E028 and erlotinib on the activity of the EGFR/PI3K/AKT pathway in A549 cells. (b) Co-treatment with erlotinib and MPT0E028 downregulates phospho-EGFR and EGFR protein levels in CL97 and PC9/IR cells. (c) Western blot analysis of the effect of erlotinib/MPT0E028 on IGF-1R, c-met, and HER2 protein expression in A549 cells. Cells were treated with MPT0E028 and/or erlotinib for 72 h, and cell lysates were subjected to immunoblotting using the indicated antibodies

Figure 6

Evidence that EGFR involved in erlotinib/MPT0E028-mediated cell death in erlotinib-resistant cells. (a) mRNA expression level of EGFR in A549 cells was determined by qRT-PCR. Cells were treated with MPT0E028 (M) and/or erlotinib (E) for 72 h, and mRNA expression was analyzed by qRT-PCR as described in the Materials and Methods section. Ectopic EGFR expression protects cells against erlotinib/MPT0E028-induced cytotoxicity and apoptosis. A549 cells (b) and PC9/IR cells (c) were transfected with control vector or Flag-EGFR plasmids and treated with MPT0E028 (M) and/or erlotinib (E) as indicated for 72 h. Cell lysates were subjected to immunoblotting using the indicated antibodies (left panel) and cell viability was measured by the MTT assay (right panel). Error bars represent S.D. Symbols: *P<0.05; **P<0.01; and ***P<0.001 compared with the control group

Figure 7

MPT0E028 in combination with erlotinib suppresses the growth of EGFR inhibitor-resistant tumor xenografts in vivo. (a) Kaplan–Meier survival curves are shown for each treatment group. Survival in the co-treated group was significantly extended (P<0.05) compared with the control groups. The log-rank test was used to calculate P-values. (b) Mice bearing established A549 tumors (∼100 mm³) were dosed by gavage with vehicle (Veh), MPT0E028 (M) 50 or 100 mg/kg QD, erlotinib (E) at 50 mg/kg QD, or MPT0E028 plus erlotinib at 50+50 mg/kg QD or 50+100 mg/kg QD (combination). Eight mice per group were used in the xenograft experiment. The tumor volumes of mice were measured. Symbols: *P<0.05, **P<0.01, and ***P<0.001 for comparisons with MPT0E028 alone. (c) The drug treatments did not cause significant body weight loss in the tested animals. (d) Effects of treatments on intratumoral biomarkers of drug activity in A549 xenograft tumors. Athymic nude mice bearing established subcutaneous (s.c.) A549 xenograft tumors were randomized to six groups (n=8 per group) that received the indicated treatments by gavage. The experimental details are described in the Materials and Methods section