Inhibition of the PI3K/AKT pathway potentiates cytotoxicity of EGFR kinase inhibitors in triple-negative breast cancer cells

Abstract

Triple-negative breast cancers (TNBCs) are known to be intrinsically resistant to inhibitors for epidermal growth factor receptor (EGFR). Until now, clinical trials for TNBCs using EGFR inhibitors (EGFRis) as single agents have yielded disappointing results. Here, we report that combinatorial treatment using EGFRis, such as gefitinib or erlotinib, with PI3K/AKT pathway inhibitors (PI3K/AKTis) demonstrated a synergistic, anti-proliferative effect in cell lines of the basal-like (BL) subtype, a subtype of TNBC. Western blot analysis revealed that the gefitinib/PI-103 combination significantly reduced the level of both phospho-AKT and phospho-ERK in two susceptible BL subtype cell lines, SUM149PT and MDA-MB-468, whereas it had little or no effect on the level of phospho-ERK in two non-susceptible cell lines (HS578T and MDA-MB-231) of mesenchymal stem-like (MSL) TNBC subtype. The gefitinib/PI-103 combination also significantly induced caspase-3/7-mediated PARP cleavage and reduced two anti-apoptotic proteins, XIAP and Bcl-2 in the susceptible cell lines. In addition, the level of myeloid cell leukemia 1 (Mcl-1) protein was markedly decreased by gefitinib/PI-103 combination in the BL TNBC cells, but showed no significant change by this combination in MSL subtype cells. These results suggest that pharmacological inhibition of EGFR used in combination of PI3K/AKTis is a potential therapeutic approach to treat a subtype of TNBCs.

Fig. 1

EGFR is overexpressed in TNBC cell lines. Cells were harvested the day after subculture and cell lysates were analysed by western blot with the indicated antibodies. α-tubulin was used as a loading control. MSL: mesenchymal stem-like; BL: basal-like.

Fig. 2

Co-treatments of PI-103 and EGFR inhibitors enhance cytotoxicity in SUM149PT (A) and MDA-MB-468 (B) but not MDA-MB-231 (C) cells. Cells were treated with 0.3 μM of PI-103 in combination with different concentrations (0.1 and 1 μM) of EGFR inhibitors for ∼72 hrs. Cell viability was measured by MTT assay as described in Materials and methods. Data from two independent experiments performed in triplicate are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3

Combinations of PI3K/AKT inhibitors and EGFR inhibitors exert synergistic cytotoxicity in the susceptible cells. (A) SUM149PT cells were treated with various combinations of PI3K/AKT inhibitors and EGFR inhibitors for ∼72 hrs. (B) MDA-MB-468 cells were treated with gefitinib in combination with either PI-103 or MK-2206 for ∼72 hrs. (C) The non-susceptible cells (HS578T, MDA-MB-231, MDA-MB-436) were treated with gefitinib in combination with either PI-103 or MK-2206 for ∼72 hrs. (A–C) Viable cells were measured by MTT assay as described in Materials and methods. Data from two independent experiments performed in triplicate are shown as mean ± SEM. Abbreviations: Erlo, erlotinib; Gef, gefitinib; PI, PI-103; MK, MK-2206. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4

Combination of PI-103 with gefitinib abolishes AKT and ERK pathways in the susceptible cells. (A) Cells were treated with either gefitinib (3 μM), PI-103 (3 μM) alone or a combination of both drugs for 2 or 24 hrs. α-tubulin (for 2-hr treatment) or β-actin (for 24-hr treatment) was used as a loading control. (B) Two susceptible cell lines (SUM149PT and MDA-MB-468) were treated with increasing amounts of gefitinib, PI-103 or in combination of both drugs for 24 hrs. β-actin was used as a loading control. (A–B) Western blot analysis was performed with the indicated antibodies. Representative data from two independent experiments are shown.

Fig. 5

Combination of PI-103 with gefitinib synergistically induces apoptotic cell death in the susceptible cells. (A) Cells were treated with the indicated amounts of compounds for 30 hrs and caspase-3/7 activities were measured as described in Materials and methods. Data from three independent experiments are shown as mean ± SEM. *P < 0.05; ***P < 0.001. (B) MDA-MB-468 cells were treated as indicated and both attached and floating cells were harvested and stained with annexin V and PI. (Left) Representative flow cytograms are shown. (Right) Data are shown as mean ± SD performed in triplicate. ***P < 0.001. (C) Cells were treated with the indicated amounts of compounds for 30 hrs and cell lysates were analysed by western blot with the indicated antibodies. Representative data from three independent experiments are shown. HSP90 was used as a loading control.