Energy‑stress‑mediated activation of AMPK sensitizes MPS1 kinase inhibition in triple‑negative breast cancer

Abstract

Monopolar spindle 1 kinase (Mps1, also known as TTK protein kinase) inhibitors exert marked anticancer effects against triple‑negative breast cancer (TNBC) by causing genomic instability and cell death. As aneuploid cells are vulnerable to compounds that induce energy stress through adenosine monophosphate‑activated protein kinase (AMPK) activation, the synergistic effect of Mps1/TTK inhibition and AMPK activation was investigated in the present study. The combined effects of CFI‑402257, an Mps1/TTK inhibitor, and AICAR, an AMPK agonist, were evaluated in terms of cytotoxicity, cell‑cycle distribution, and in vivo xenograft models. Additional molecular mechanistic studies were conducted to elucidate the mechanisms underlying apoptosis and autophagic cell death. The combination of CFI‑402257 and AICAR showed selective cytotoxicity in a TNBC cell line. The formation of polyploid cells was attenuated, and apoptosis was increased by the combination treatment, which also induced autophagy through dual inhibition of the PI3K/Akt/mTOR and mitogen‑activated protein kinase (MAPK) signaling pathways. Additionally, the combination therapy showed strongly improved efficacy in comparison with CFI‑402257 and AICAR monotherapy in the MDA‑MB‑231 xenograft model. The present study suggested that the combination of CFI‑402257 and AICAR is a promising therapeutic strategy for TNBC.

Keywords: AMPK; MPS1 kinase; TNBC; TTK protein kinase; breast cancer; energy stress.

Figure 1.

Selective cytotoxicity on a combination of CFI-402257 and AICAR in MDA-MB-231 cells. (A) Cytotoxicity of CFI-402257 and AICAR against three breast cell lines. Cells were treated with various concentrations of CFI-402257 (0, 30, 100, 300 and 1,000 nM) and AICAR (150, 300, 600, 900 and 1,200 µM) for 72 h. (B) Cytotoxicity of monotherapy and combination therapy (CFI-402257: 100 nM, AICAR: 300 µM) in the three breast cell lines for 72 h. The values represent the mean ± SEM. ***P<0.001 vs. the control group. (C) Basal TTK expression levels in the three breast cell lines were determined by western blot analysis.

Figure 2.

Effects of CFI-402257 and AICAR on the main target proteins in MDA-MB-231 cells. (A) Western blot analysis of p-TTK and TTK. Cells were pretreated with 50 ng/ml nocodazole for 17 h before treatment with CFI-402257 and AICAR and their combination for 4 h. (B) Western blot analysis of p-AMPK and AMPK. Cells were treated with CFI-402257 and AICAR and their combination for 24 h. (C) Representative histogram of protein expression levels. The values represent the mean ± SEM. ^###P<0.001 vs. nocodazole group; **P<0.01 and ***P<0.001 vs. the control group. p-, phosphorylated.

Figure 3.

Cell-cycle distribution of MDA-MB-231 cells after treatment with the combination of CFI-402257 and AICAR. Cells were treated with CFI-402257 and AICAR for 72 h and stained with propidium iodide and analyzed by Muse™ Cell Analyzer. (A and B) The bar diagrams show the number of cells in the G0/G1, S, G2/M and polyploidy phases in the MDA-MB-231 cell line. Polyploidy was defined by the presence of more than 4n chromosomes. (C) The proportion of dead cells (%) in the ungated part of the samples. Data are shown as the mean ± SEM (n=3). **P<0.01 and ***P<0.001 vs. the control group; ^##P<0.01 vs. the CFI-402257 treatment group.

Figure 4.

Effects of the combination of CFI-402257 and AICAR on cell cycle related proteins in MDA-MB-231 cells. (A) Western blot analysis of cyclin protein, CDK-related protein and p27. (B) Representative histogram of protein expression level. Data are shown as the mean ± SEM (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. the control group.

Figure 5.

Effects of the combination of CFI-402257 and AICAR on apoptotic cell death in MDA-MB-231 cells. The cells were treated with CFI-402257 and AICAR for 72 h and evaluated by the annexin V-7AAD double-staining method. (A) Histogram showing the percentage of total apoptosis. (B) Table showing the percentage of live cells, early apoptosis, late apoptosis and dead cells. (C) Expression levels of apoptosis-related proteins were analyzed by western blotting. (D) Representative histogram of protein expression levels. The values represent the mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 vs. the control group; ^##P<0.01 vs. the CFI-402257 treatment group. PARP, poly (ADP-ribose) polymerase.

Figure 6.

Autophagic cell death in MDA-MB-231 cells by the combination of CFI-402257 and AICAR. (A) Cells were stained with acridine orange after treatment with CFI-402257 and AICAR for 72 h. Images observed under a confocal laser scanning microscope (K1-Fluo; magnification, ×200) (B) Expression levels of autophagy-related proteins analyzed by western blots. (C) Representative histogram of protein expression levels. The values represent the mean ± SEM. **P<0.01 and ***P<0.001 vs. the control group.

Figure 7.

Inhibition of the PI3K/Akt/mTOR signaling pathway by the combination of CFI-402257 and AICAR. (A) Western blot analysis of the expression levels of PI3K/Akt/mTOR-related proteins in MDA-MB-231 cells. (B) Representative histogram of the protein expression levels. The values represent the mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 vs. the control group. p-, phosphorylated.

Figure 8.

Inhibition of the MAPK signaling pathway by combined treatment with CFI-402257 and AICAR. (A) Western blot analysis of the expression levels of MAPK-related proteins in MDA-MB-231 cells. (B) Representative histogram of the protein expression level. The values represent the mean ± SEM. *P<0.05 and ***P<0.001 vs. the control group. p-, phosphorylated.

Figure 9.

Anticancer effect of the combination of CFI-402257 with AICAR on an in vivo tumor MDA-MB-231 ×enograft model. (A) Athymic nude mice bearing MDA-MB-231 ×enografts were administered CFI-402257 (1 mg/kg, QD, P.O.), AICAR (100 mg/kg, QD, I.P.) and their combination for 28 days. Tumor volume was measured once every 2 days. (B) Representative image of the tumor. (C) Body weight changes in nude mice. (D) Tumor weight changes in each group. The values represent the mean ± SD (n=5). **P<0.01 and ***P<0.001 vs. the control group; ^##P<0.01 vs. the CFI-402257 treatment group.