J Bacteriol Virol.  2019 Dec;49(4):221-229. 10.4167/jbv.2019.49.4.221.

p90RSK Activation Promotes Epithelial-Mesenchymal Transition in Cisplatin-Treated Triple-Negative Breast Cancer Cells

Affiliations
  • 1College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon, South Korea. kheo@cnu.ac.kr

Abstract

p90 ribosomal S6 kinase (p90RSK), one of the downstream effectors in ERK1/2 pathways, shows high expression in human breast cancer tissues. However, its role in breast cancer development and drug resistance is not fully understood. Here, we demonstrate that Cis-DDP treatment failed to increase cytotoxicity in MDA-MB-231 cells compared to MCF-7 cells and p90RSK activation was involved in Cis-DDP-resistance to MDA-MB-231 cells. In the study, we found that inhibition of p90RSK expression or activation using a small interfering RNA (siRNA) or dominant-negative kinase mutant (DN-p90RSK) plasmid overexpression increased Cis-DDP-induced cytotoxicity of MDA-MB-231 cells, respectively. Mechanistically, we found that Cis-DDP resistance was associated with up-regulation of epithelial growth factor (EGF) expression and EGF treatment induced cancer survival signaling pathway including activation of ERK1/2, p90RSK, and Akt. We also examined the expression of epithelial-mesenchymal transition (EMT)-associated proteins using a reverse transition-quantitative PCR analysis. Cis-DDP treatment induced EMT by increasing the expression levels of N-cadherin, Snail, and Twist, while decreasing the expression levels of E-cadherin. Furthermore, we examined the epithelial marker, Zonula occludens-1 (ZO-1) using immunofluorescence analysis and found that Cis-DDP-inhibited ZO-1 expression was recovered by p90RSK deactivated condition. Therefore, we conclude that Cis-DDP resistance is involved in EMT via regulating the EGF-mediated p90RSK signaling pathway in MDA-MB-231 cells.

Keyword

Cisplatin; Epithelial-mesenchymal transition; p90RSK; Triple-negative breast cancer cells; Breast Cancer; Cisplatin; Epithelial-mesenchymal transition; p90RSK

MeSH Terms

Breast Neoplasms
Cadherins
Cisplatin
Drug Resistance
Epidermal Growth Factor
Epithelial-Mesenchymal Transition*
Fluorescent Antibody Technique
Humans
MCF-7 Cells
Phosphotransferases
Plasmids
Polymerase Chain Reaction
Ribosomal Protein S6 Kinases, 90-kDa
RNA, Small Interfering
Snails
Triple Negative Breast Neoplasms*
Up-Regulation
Cadherins
Cisplatin
Epidermal Growth Factor
Phosphotransferases
RNA, Small Interfering
Ribosomal Protein S6 Kinases, 90-kDa

Figure

  • Figure 1 Effect of Cis-DDP on cell viability of breast cancer cells MDA-MB-231 and MCF-7 cells were treated with indicated various concentrations of Cis-DDP for 36 h. cell viability was assessed using the Muse cell count & viability. Data are the mean ± SEM of experiments in triplicate (n=3). *P<0.05, **P<0.01 compared with 0 sample.

  • Figure 2 Involvement of p90RSK activation in cell viability of MDA-MB-231 cells (A) MDA-MB-231 cells transfected with si-Cont or si-RSK1 for 48 h followed by treatment with the indicated concentrations of Cis-DDP for 5 min. Whole cell lysates were subjected to western blot analysis against the indicated antibodies. (B) MDA-MB-231 cells were transfected with si-Cont or si-RSK1 for 18 h followed by treatment with the indicated concentrations of Cis-DDP for 24 h and cell viability was determined by an MTT assay. (C) MDA-MB-231 were transfected with WT-p90RSK or DN-p90RSK for 18 h followed by treatment with the indicated concentrations of Cis-DDP for 24 h and cell viability was determined by flow cytometry. The data are presented as means ± SEM (n = 3). ***P<0.001 compared with 0 sample; ###P<0.001 compared with each control in the siCont or WT-p90RSK, respectively.

  • Figure 3 Effect of p90RSK activation on hEGF-induced cell survival pathways (A) MDA-MB-231 cells were stimulated with Cis-DDP 6 h and mRNA expression level was determined by qRT-PCR. (B) MDA-MB-231 cells were pretreated with 10 nM of FMK for 1 h followed by treatment of 10 ng/ml hEGF for selected time intervals (0~120 min). Whole cell lysates were subjected to western blot analysis against the indicated antibodies. The data are presented as means ± SEM (n = 3). **P<0.01 compared with 0 sample.

  • Figure 4 Effect of p90RSK activation on EMT markers(A) E-cadherin; (B) N-cadherin; (C) Snail and (D) Twist mRNA level in MD A-MB-231 cells. MDA-MB-231 cells were transfected with si-Cont or si-RSK1 for 48 h followed by treatment with 0, 10, an d 20 ug/ml of Cis-DDP for 6 h. Data are the mean ± SEM of experiments in triplicate (n=3). The data are presented as mean s ± SEM (n = 3). *P<0.05, **P<0.01 compared with 0 sample; #P<0.5, ##P<0.01 compared with each control. (E) MDA-MB-2 31 cells were treated with FMK for 3 h followed by treatment with 20 ug/ml of Cis-DDP for 6 h. The ZO-1 expression in cell s was observed using a laser scanning confocal spectral microscope (K1-Fluo, Nanoscope systems). DAPI staining was used t o determine the nuclei localization. Bars indicate 30 µm.


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