Plk3 Enhances Cisplatin Sensitivity of Nonsmall-Cell Lung Cancer Cells through Inhibition of the PI3K/AKT Pathway via Stabilizing PTEN

Abstract

Polo-like kinase 3 (Plk3) is involved in tumor development with a tumor suppressive function. However, the effect of Plk3 on the chemoresistance remains unclear. It has been documented that activation of the PI3K/AKT signaling pathway by PTEN loss significantly enhances chemoresistance in nonsmall-cell lung cancer (NSCLC). This study aims to evaluate the PTEN regulation by Plk3 and identify targets and underlying mechanisms that could be used to relieve chemoresistance. Our results showed that silencing Plk3 reduced PTEN expression and activated PI3K/AKT signaling by dephosphorylating and destabilizing PTEN in NSCLC cells. Reducing Plk3 expression promoted drug resistance to cisplatin (DDP), while overexpressing Plk3 promoted DDP sensitivity. However, these effects were attenuated when MK2206, a PI3K/AKT inhibitor, was applied. In conclusion, upregulation of Plk3 sensitized NSCLC cells toward DDP, which provides a potential target to restore DDP chemoresponse. We provided novel evidence that the PTEN/PI3K/AKT signaling pathway could be regulated by Plk3 through phosphorylation of PTEN and highlighted the critical role of Plk3 in the DDP resistance of NSCLC.

Figure 1

Plk3 expression is downregulated in NSCLC clinical samples and cell lines. (A–C) Differential gene expression levels of Plk3, GSK3B, and CSNK2A2 between normal, LUAD, and LUSC subtypes were analyzed using GEPIA2 based on the TCGA and GTEx databases. (D) RT-qPCR assay was performed to measure the Plk3 mRNA levels in different cell lines. (E,F) Western blot analysis was conducted for the detection of Plk3 protein levels in different cell lines. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Overexpression of Plk3 enhances the DDP sensitivity of DDP-resistant NSCLC cells. (A–C) Plk3-overexpressing A549/DDP cells and H1299/DDP cells were, respectively, constructed. Results of RT-qPCR and Western blot showed that Plk3 expression was dramatically upregulated in A549/DDP cells and H1299/DDP cells after transfection with the Plk3-OE plasmid. (D,E) Plk3 suppressed the DDP resistance of A549/DDP cells and H1299/DDP cells, as shown by reduced IC₅₀ values of DDP. (F,G) Flow cytometry demonstrated that Plk3 overexpression promoted A549/DDP and H1299/DDP cell apoptosis. (H,I) The mRNA levels of ABCB1 and the protein levels of p-gp (encoded by the ABCB1 gene) were downregulated by Plk3, as shown by RT-qPCR and Western blot. **p < 0.01, ***p < 0.001.

Figure 3

Knockdown of Plk3 decreases the DDP sensitivity of NSCLC cells via regulating PI3K/AKT signaling. (A,B) Plk3-silencing A549/DDP and H1299/DDP cell lines were respectively constructed through transfection with si-Plk3. RT-qPCR and Western blot showed that Plk3 expression was dramatically downregulated after transfection with si-Plk3. (C,D) A549 and H1299 cells transfected with si-NC or si-Plk3 were incubated with MK-2206 (10 μM) for 24 h in the presence of 2 μg/mL DDP. Plk3-silencing A549 and H1299 cells showed higher levels of the p-Akt/Akt ratio, which could be partially reversed by MK2206. (E) Flow cytometry showed that the si-Plk3-caused decrease in cell apoptosis was mitigated by MK2206. (F,G) Knockdown of Plk3 caused an obvious increase in the IC₅₀ values of DDP in A549 and H1299 cells. While interfering with MK2206 blocked the effects of si-Plk3 on the IC₅₀ values of DDP. (H–J) A549 and H1299 cells transfected with si-Plk3 showed increased mRNA levels of ABCB1 and protein levels of p-gp, which could be attenuated by MK2206. **p < 0.01, ***p < 0.001.

Figure 4

Plk3 phosphorylates and stabilizes PTEN in both DDP-sensitive NSCLC cells and DDP-resistant NSCLC cells. (A,B) For CHX treatment, the cells were respectively incubated with CHX (0.1 mg/mL) for 0, 1, 4, or 8 h. A western blot was then performed to measure PTEN protein degradation. (C–E) Western blot also showed that Plk3 overexpression induced expression levels of PTEN and p-PTEN in A549/DDP cells, and Plk3 knockdown suppressed PTEN and p-PTEN expression in H1299 cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Plk3 inhibits PI3K/AKT signaling by regulating PTEN. (A,B) Decreased p-Akt/Akt ratio caused by Plk3 overexpression was elevated after transfection with si-PTEN. (C,D) Knockdown of Plk3 significantly increased the p-Akt/Akt ratio, which could be reversed by PTEN overexpression. **p < 0.01, ***p < 0.001.