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. 2018;17(16):2069-2079.
doi: 10.1080/15384101.2018.1515550. Epub 2018 Sep 19.

AKT inhibitor MK-2206 sensitizes breast cancer cells to MLN4924, a first-in-class NEDD8-activating enzyme (NAE) inhibitor

Xiaoyu Chen  1   2 , Danrui Cui  1   2 , Yanli Bi  1   2 , Jianfeng Shu  1   2 , Xiufang Xiong  1   2 , Yongchao Zhao  1   2
Affiliations

AKT inhibitor MK-2206 sensitizes breast cancer cells to MLN4924, a first-in-class NEDD8-activating enzyme (NAE) inhibitor

Xiaoyu Chen et al. Cell Cycle. 2018.

Abstract

Breast cancer is a common type of cancer among female cancer patients and the main cause of cancer-related deaths. During the last decades, targeted therapies for breast cancer have been rapidly developing. Among them, MLN4924, a first-in-class NEDD8-activating enzyme (NAE) inhibitor, has performed antitumor activity by inactivating the cullin-RING ligases and causing the accumulation of their substrates to induce apoptosis in a number of studies. In this study, we found that MLN4924 activates the AKT pathway in both HER2-positive and triple-negative breast cancer (TNBC) cell lines. Given that AKT signaling is responsible for tumor progression and drug resistance in some types of cancers, we hypothesized that the AKT inhibitor may synergistically enhance the tumor suppression capability in breast cancer by MLN4924. To demonstrate the sensitizing effect, MK-2206 was chosen as the adjuvant treatment, and cell growth, migration and apoptosis were detected. The results showed that MLN4924 treatment inhibited cell growth and migration and induced apoptosis in both SK-BR3 and MDA-MB231 breast cancer cell lines. More importantly, the combined treatment of MLN4924 and MK-2206 indeed caused stronger cytotoxicity and inhibition of migration and a much higher induction of apoptosis compared with MLN4924 treatment alone. Our study provides the proof-of-concept evidence for strategic drug combination of MLN4924 with an AKT inhibitor for maximal killing of breast cancer cells via the enhancement of apoptosis.

Keywords: AKT inhibitor; Breast cancer; MK-2206; MLN4924; neddylation; targeted therapy.

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Figures

Figure 1.
Figure 1.
AKT signaling pathway is activated by MLN4924 treatment.(a) SK-BR3 and MDA-MB231 cells were incubated with various concentrations of MLN4924 (0, 10, 25 and 50 nM) for 24 h, followed by western blotting using antibodies against p-AKT (S473), t-AKT and NEDD8. ACTIN was used as a loading control. The levels of p-AKT (S473) were quantified and expressed as fold change, compared with the control. (b) SK-BR3 and MDA-MB231 cells were treated with MLN4924 (1 μM) for different time periods (0, 12, 24, 36, and 48 h).
Figure 2.
Figure 2.
AKT inhibitor MK-2206 enhances the suppression of growth in breast cancer cells by MLN4924.(a) SK-BR3 and MDA-MB231 were treated with indicated concentrations of MK-2206. After 24 h, cells were obtained for western blotting using the indicated antibodies. (b) Cells were treated with the indicated concentrations of MK-2206 for 24 h, 48 h, and 72 h, followed by the ATP-lite assay. (c) Cells were treated with the indicated concentrations of MLN4924 for 72 h and in combination with MK-2206 (100 nM) for 48 h.
Figure 3.
Figure 3.
AKT inhibitor MK-2206 enhances the suppression of survival in breast cancer cells by MLN4924.A total of 300 cells were seeded in triplicate in 60-mm dishes and treated with different concentrations of MLN4924 (0, 100, 200 nM) alone or in combination with MK-2206 (100 nM). After 7 days, colonies were stained (a) and counted (> 50 cells in a colony) (b). Rate of colony formation (%) = colony number/300*100% (mean± SEM, n = 3). **p < 0.01.
Figure 4.
Figure 4.
AKT inhibitor MK-2206 enhances the suppression of migration in breast cancer cells by MLN4924.(a) Cells were seeded in 6-well plates and treated with MLN4924 (1 μM) for 24 h, followed by MK-2206 treatment (1 μM) for 24 h. After serum starvation for 12–18 h, the cells were photographed at 0 h, 24 h and 48 h after a pipette tip was used to scratch across the well. Data were shown as the relative wound area normalized to the control (mean± SEM, n = 3). (b) Cells (1x105) were treated with the indicated agents and then seeded into the upper chamber containing serum-free medium. After 24 h, cells were fixed and stained, followed by photography and counting (mean± SEM). (c) Cells were treated with the drugs for 48 h, followed by western blotting using the indicated antibodies. *** p < 0.001.
Figure 5.
Figure 5.
AKT inhibitor MK-2206 enhances the induction of apoptosis induced by MLN4924.(a, b) Cells were treated with MLN4924 (0, 0.3, and 1 μM) alone or in combination with MK-2206 (1 μM). After 48 h, the cells were harvested and stained using the Annexin V-FITC apoptosis detection kit. Cells with Annexin V+ staining located in the right upper and lower quadrants were considered as apoptotic cells (mean± SEM, n = 3). (c) Cells were treated with the drugs for 48 h, followed by DNA extraction. Then, DNA samples were analyzed by agarose gel electrophoresis and were photographed. (d) Cells were incubated with various concentrations of MLN4924 (0, 0.1, 0.3, and 1 μM) for 24 h, followed by MK-2206 (0.5 or 1 μM) or DMSO for 24 h. Cells were harvested for western blotting using indicated antibodies.
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This work was supported by the National Key R&D Program of China [2016YFA0501800 to YZ and XX], the Natural Science Foundation of Zhejiang Province [Grant No. LR16C050001 to YZ], and the National Natural Science Foundation of China [Grant Nos. 31470753 and 81672728 to YZ and 81572708 and 31501129 to XX].