Dual Inhibition of AKT and MEK Pathways Potentiates the Anti-Cancer Effect of Gefitinib in Triple-Negative Breast Cancer Cells

Abstract

There is an unmet medical need for the development of new targeted therapeutic strategies for triple-negative breast cancer (TNBC). With drug combination screenings, we found that the triple combination of the protein kinase inhibitors (PKIs) of the epidermal growth factor receptor (EGFR), v-akt murine thymoma viral oncogene homolog (AKT), and MAPK/ERK kinase (MEK) is effective in inducing apoptosis in TNBC cells. A set of PKIs were first screened in combination with gefitinib in the TNBC cell line, MDA-MB-231. The AKT inhibitor, AT7867, was identified and further analyzed in two mesenchymal stem-like (MSL) subtype TNBC cells, MDA-MB-231 and HS578T. A combination of gefitinib and AT7867 reduced the proliferation and long-term survival of MSL TNBC cells. However, gefitinib and AT7867 induced the activation of the rat sarcoma (RAS)/ v-raf-1 murine leukemia viral oncogene homolog (RAF)/MEK/ extracellular signal-regulated kinase (ERK) pathway. To inhibit this pathway, MEK/ERK inhibitors were further screened in MDA-MB-231 cells in the presence of gefitinib and AT7867. As a result, we identified that the MEK inhibitor, PD-0325901, further enhanced the anti-proliferative and anti-clonogenic effects of gefitinib and AT7867 by inducing apoptosis. Our results suggest that the dual inhibition of the AKT and MEK pathways is a novel potential therapeutic strategy for targeting EGFR in TNBC cells.

Keywords: AKT; EGFR resistance; MEK; anti-cancer; combination; protein kinase inhibitor (PKI); synergism; triple-negative breast cancer (TNBC).

Figure 1

Treatment with AT7867 induced triple-negative breast cancer (TNBC) cells’ sensitivity to gefitinib. (A) PKI + gefitinib screening results in MDA-MB-231 cells. The numbers of combination points with CI > 1.3 was indexed (see the Materials and Methods). The numbers on the right of each bar are the mean CI values of indicated drugs. (B) MTT screening results for AT7867 with gefitinib. MDA-MB-231 cells were treated with an increasing concentrations of AT7867 and gefitinib for 72 hr. The viable cells was measured by MTT assay. (C) The combination effect of gefitinib with AT7867 in two MSL TNBC cells. Data represent mean ± SD from at least three independent experiments performed in triplicate. * p < 0.05 and ** p < 0.01.

Figure 2

A combination of AT7867 and gefitinib-induced tetraploid gap 1 (G1) arrest in MDA-MB-231 cells. (A) Representative histograms of cell cycle analysis in MDA-MB-231 cells in the presence of each drug or gefitinib and AT7867 (Gefi+AT7867). (B) Relative distribution of cell cycle phases.

Figure 3

Gefitinib and AT7867 reduced the survival of MSL subtype TNBC cells. (A) MDA-MB-231 and HS578T cells were treated with 10 μM Gefitinib, 5 μM AT7867, or 10 μM Gefitinib + 5 μM AT7867 (Gefi+AT) for 24 h in normal growth media. Then, cells were washed and cultivated for additional 10–14 days in normal growth media. The colonies were stained as described in the Materials and Methods. Representative images are shown from three independent experiments performed in triplicate (B) The relative colony number was determined and presented as mean ± SEM from three independent experiments performed in triplicate. * p < 0.05 and ** p < 0.01.

Figure 4

Cell signaling in Gefi+AT7867-treated TNBC cells. Cells were treated with different drug combinations for indicated times. Cell lysates were subjected to Western blot (Figure S1) analysis with antibodies for indicated proteins.

Figure 5

Antibody array analysis. (A) Fluorescence images of the antibody arrays. (B) Scatter plots of fluorescence signals; intensities of the control sample were plotted on the x-axis, and intensities of the test sample were plotted on the y-axis. Red and green lines indicate a 1.5-fold increase and decrease in fluorescence intensities, respectively, compared with the control sample. (C) The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway map for MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway identified by antibody array analysis. Blue color indicates upregulated molecules. The KEGG pathway map was kindly provided by Kanehisa Laboratories (Kyoto University, Kyoto, Japan).

Figure 5

Antibody array analysis. (A) Fluorescence images of the antibody arrays. (B) Scatter plots of fluorescence signals; intensities of the control sample were plotted on the x-axis, and intensities of the test sample were plotted on the y-axis. Red and green lines indicate a 1.5-fold increase and decrease in fluorescence intensities, respectively, compared with the control sample. (C) The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway map for MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway identified by antibody array analysis. Blue color indicates upregulated molecules. The KEGG pathway map was kindly provided by Kanehisa Laboratories (Kyoto University, Kyoto, Japan).

Figure 6

Synergistic anti-proliferative effect in TNBC cells via additional inhibition of the MEK/ERK pathway in the presence of Gefi+A7867. (A) Results of the MEK/ERK inhibitor screening with Gefi+AT7867 in MDA-MB-231 cells. The numbers of combination points with a CI > 1.3 are depicted, and the numbers on the top of each bar are the mean CI values of the indicated drugs. (B) The combination effects of PD-0325901 in the presence of Gefi+AT7867 in two TNBC cells. Cells were treated with serially diluted concentrations of PD-0325901 in the presence of Gefi+AT7867 for 72 h. Data represent mean ± SD from at least three independent experiments performed in triplicate. ** p < 0.01.

Figure 7

Cell cycle analysis of MDA-MB-231 cells treated with a triple combination of gefitinib and AT7867 and PD-0326901(A–H). Representative histograms of cell cycle analysis in MDA-MB-231 cells in the presence of each drug or combinations of drugs as indicated. Abbreviations: AT, AT7867; Gefi, gefitinib; and PD-0325901.

Figure 8

Reduced colony formation of MSL TNBC cells treated with gefitinib andAT7867 and PD-0326901. (A) Representative images of the colonies; cells were treated with 10 μM of gefitinib, 2.5 μM of AT7867, and 10 μM of PD-0325901, or other drug combinations (as indicated) for 24 h and further cultivated for 10–14 days to form colonies in normal growth media. The surviving colonies were stained and scanned using an image scanner. Three independent experiments were performed in triplicate and representative images are shown. (B) Relative colony numbers were determined and presented as mean ± SEM. ** p < 0.01.

Figure 9

Western blot analysis of TNBC cells treated with different drug combinations (Figure S2). The cells were treated with drug combinations for 24 h, and the cell lysates were resolved on SDS-PAGE and proved with antibodies for indicated proteins.

Figure 10

Putative Pathways affected by small-molecule inhibitors in this study. The PI3K/AKT/mTORC1 and RAS/RAF/MEK/ERK pathways cross-talk each other. Small-molecule PKIs used in this study would inhibit their target molecules, which results in synergistic anti-cancer effects. Abbreviations: 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; AKT, v-akt murine thymoma viral oncogene homolog; c-Myc, cellular myelocytomatosis; CREB, cAMP responsive element-binding protein; EGFR, epidermal growth factor receptor; eIF4E, eukaryotic translation initiation factor 4E; ELK-1, E twenty-six (ETS) like-1; ERK, extracellular signal-regulated kinase; GF, growth factor; MEK, MAPK/ERK kinase; mTORC1/2, mammalian target of rapamycin complex 1/2; PDK1, 3-phosphoinositide-dependent protein kinase-1; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PKC, protein kinase C; RAF, v-raf-1 murine leukemia viral oncogene homolog; RAS, rat sarcoma; RPS6, ribosomal protein S6; RSK, ribosomal S6 kinase; RTK, receptor tyrosine kinase; S6K, S6 kinase; TSC1/2, Tuberous sclerosis 1/2.