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, 52 (12), 706-711

Inhibition of p90RSK Activation Sensitizes Triple-Negative Breast Cancer Cells to Cisplatin by Inhibiting Proliferation, Migration and EMT

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Inhibition of p90RSK Activation Sensitizes Triple-Negative Breast Cancer Cells to Cisplatin by Inhibiting Proliferation, Migration and EMT

Yujin Jin et al. BMB Rep.

Abstract

Cisplatin (Cis-DDP) is one of the most widely used anti-cancer drugs. It is applicable to many types of cancer, including lung, bladder, and breast cancer. However, its use is now limited because of drug resistance. p90 ribosomal S6 kinase (p90RSK) is one of the downstream effectors in the extracellular signalregulated protein kinases 1 and 2 (ERK1/2) pathway and high expression of p90RSK is observed in human breast cancer tissues. Therefore, we investigated the role of p90RSK in the Cis-DDP resistance-related signaling pathway and epithelialmesenchymal transition (EMT) in breast cancer cells. First, we discovered that MDA-MB-231 cells exhibited more Cis-DDP resistance than other breast cancer cells, including MCF-7 and BT549 cells. Cis-DDP increased p90RSK activation, whereas the inactivation of p90RSK using a small interfering RNA (siRNA) or dominant-negative kinase mutant plasmid overexpression significantly reduced Cis-DDP-induced cell proliferation and migration via the inhibition of matrix metallopeptidase (MMP)2 and MMP9 in MDA-MB-231 cells. In addition, p90RSK activation was involved in EMT via the upregulation of mRNA expression, including that of Snail, Twist, ZEB1, N-cadherin, and vimentin. We also investigated NF-κB, the upstream regulator of EMT markers, and discovered that Cis-DDP treatment led to NF-κB translocation in the nucleus as well as its promoter activity. Our results suggest that targeting p90RSK would be a good strategy to increase Cis-DDP sensitivity in triple-negative breast cancers. [BMB Reports 2019; 52(12): 706-711].

Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no conflicting interests.

Figures

Fig. 1
Fig. 1
Inhibition of p90RSK decreases cell proliferation in MDAMB-231 cells. (A) MDA-MB-231, BT549, and MCF-7 breast cancer cells were stimulated with Cis-DDP for 24 h and cell viability was determined by an MTT assay. (B) Cells were treated with the Cis-DDP for 36 h and cell proliferation was determined by FACS using a Ki-67 proliferation kit. (C) MDA-MB-231 cells were stimulated with 20 μg/ml of Cis-DDP or 5 μg/ml of Dox for 24 h and the cell cycle was investigated using FACS. (D) MDA-MB-231 cells were stimulated with Cis-DDP for 24 h and the protein and mRNA expression of p90RSK were determined by western blotting (upper panel) and qPCR (lower bar graph). (E) MDA-MB-231 cells were stimulated with Cis-DDP for 5 min and whole-cell lysates were subjected to western blot analysis against the indicated antibodies. (F, G) MDA-MB-231 cells were treated with 10 μM of FMK for 3 h (F) or transfected with pcDNA or DN-RSK1 for 18 h (G) followed by treatment with the indicated dose of Cis-DDP for 24 h and cell viability was determined by an MTT assay. The data are presented as means ± SEM (n = 3). *P < 0.05, **P < 0.01 or ***P < 0.001 compared with 0 sample; #P < 0.05, ##P < 0.01 or ###P < 0.001 compared with each control.
Fig. 2
Fig. 2
Effect of p90RSK activation on cell migration in Cis-DDP-induced MDA-MB-231 cells. (A) Cells were pretreated with 10 μM of FMK for 3 h or transfected with DN-RSK for 18 h followed by treatment with 20 μg/ml of Cis-DDP. After 36 h, cells were fixed, stained with crystal violet, and photographed using an Olympus microscope. The bar indicates 200 μm. (B) The migration area was measured using Image J software and indicated as fold change compared to the control (0) sample. (C–E) MDA-MB-231 cells were treated with the same conditions as above (Figure 2A) and whole protein lysates and RNA samples were subjected to western blotting (C) or qPCR (D, E) against MMP2 and MMP9. (F–I) MDA-MB-231 cells were transfected with siRNA from the control (si-Cont) or RSK1 (si-RSK1) for 48 h followed by treatment with 10 or 20 μg/ml of Cis-DDP for 24 h. (F) p90RSK protein expression was determined by western blotting. (G–I) Total RNA samples were subjected to qPCR against RSK1, MMP2, and MMP9 primers. The data are presented as means ± SEM (n = 3). *P < 0.05, **P < 0.01 or ***P < 0.001 compared with 0 sample; #P < 0.05, ##P < 0.01 or ###P < 0.001 compared with each control.
Fig. 3
Fig. 3
Involvement of p90RSK activation in Cis-DDP-induced EMT. (A–F) MDA-MB-231 cells were pretreated with 10 μM of FMK for 1 h followed by treatment with 20 μg/ml of Cis-DDP for 6 h. (G–L) Cells were transfected with WT-RSK1 or DN-RSK1 plasmids for 18 h followed by treatment with 20 μg/ml of Cis-DDP for 6 h. Total RNA samples were subjected to qPCR against E-cadherin (A, G), N-cadherin (B, H), vimentin (C, I), Snail (D, J), Twist (E, K), and ZEB1 (F, L) primers. The data are presented as means ± SEM (n = 3). *P < 0.05, **P < 0.01 or ***P < 0.001 compared with 0 sample; #P < 0.05, ##P < 0.01 or ###P < 0.001 compared with each control.
Fig. 4
Fig. 4
Effect of p90RSK activation on NF-κB transcriptional activity. (A–C) MDA-MB-231 cells were pretreated with 10 μM of FMK for 1 h followed by treatment with 20 μg/ml of Cis-DDP for 1 h. (A) The p65 expression in cells was observed using a laser scanning confocal spectral microscope (Nanoscope systems). Bars indicate 30 μm. (B) Many nuclear p65 NF-κBs were indicated as fold change compared to the control (0) sample. (C) Total protein lysates were subjected to western blotting using anti-phospho or -total p65 antibodies. (D) MDA-MB-231 cells were co-transfected with the pNF-κB-luc and pRL-Renilla reporter construct. At 18 h after transfection, cells were treated with 10 μM of FMK for 1 h followed by treatment with Cis-DDP or TNF-α (positive control) for 12 h. The luciferase activities of the extracts were determined and normalized based on Renilla luciferase expression. The data are presented as means ± SEM (n = 3). ***P < 0.001 compared with 0 sample; ##P < 0.01 compared with each control.

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