The PC3, LNCaP, and DU145 cell lines were sourced from the Department of Laboratory Medicine, Shanghai East Hospital. DU145 and PC3 cells were cultured in DMEM (BasaIMedia) and RPMI 1640 (BasaIMedia), respectively, each supplemented with 10% fetal bovine serum (FBS). The LNCaP cells were cultured in RPMI 1640 medium supplemented with 15% fetal bovine serum (FBS). All cells were grown in normal culture medium for at least 1 day prior to each experiment.

To prepare the lentivirus, plasmids containing p53 (MIAOLING BIOLOGY) and packaging (psPAX2) and envelope (pMD2.G) vectors were transfected into 293T cells using Hieff TransTM Liposomal Transfection Reagent (Yeasen) according to the manufacturer’s instructions. Approximately 16 h later, the transfection medium was replaced by the fresh complete medium. After 48 h of transfection, the supernatant was collected, filtered through a 0.45-μm filter, and ultracentrifuged at 28,000 rpm for 2 h at 4°C. The recovered viral particles were used to infect cells in the presence of 8 μg/mL polybrene.

A total of 1 × 10⁷ cells were harvested and washed with PBS and lysed with lysis buffer (50 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 10% glycerol). The cell lysates were centrifuged at 16,000 g for 10 min, and protein concentration was determined. Equal amounts of protein were loaded onto 8%–15% SDS-PAGE gels, followed by electrophoresis and transfer to a nitrocellulose membrane. After blocking the membrane with 5% non-fat milk in PBS for 1 h, the membrane was incubated with primary antibodies against PLK1 (Millipore, 05-844), PARP (Cell Signaling Technology, 9532S), Caspase-3 (Cell Signaling Technology, 9661S), p53 (Beyotime Biotechnology, AF0255 and AF7671), ubiquitin (Proteintech, 10201-2-AP), Topors (Santa Cruze,sc-101182),α-tubulin (Sigma-Aldrich, ABT171), or β-actin (Santa Cruze,sc-47778). Signals were detected using a chemiluminescence HRP detection kit (Zetalife, 310208) according to the manufacturer’s instructions.

Seed 2,000 cells/well into a 6-well plate and incubated for 14 days. Fix in 4% paraformaldehyde for 30 min, stain with crystal violet for 30 min, wash, dry, and capture images.

Following the manufacturer’s instructions, 20 µL of CCK-8 solution (Beyotime Biotechnology) was added to each well of a 96-well plate containing 2 × 10³ cells. After incubation for 2 h at 37°C, the absorbance was measured at 450 nm.

p53 overexpression and knockdown were achieved by transient transfection of cells with pcDNA-P53 (MIAOLING BIOLOGY) and P53 siRNA (RIBOBIO), respectively. Cells were transfected with negative control or pcDNA-P53 or siP53 using Hieff TransTM Liposomal Transfection Reagent (Yeasen) according to the manufacturer’s protocol. siNC refers to a negative control siRNA with no homologous sequence in the human or mouse transcriptome. The target sequences for the three P53 siRNAs used in this study are as follows:

siP53-1: 5′-GTACCACCATCCACTACAA -3’.

siP53-2: 5′-AGAGAATCTCCGCAAGAAA -3’.

siP53-3: 5′-GGAGTATTTGGATGACAGA -3’.

Data on STAR counts, mutation MAF, and corresponding clinical information for prostate cancer were obtained from the TCGA database (https://portal.gdc.cancer.gov). Subsequently, data were extracted from the STAR counts in TPM format, followed by log2 (TPM+1) normalization. After filtering for samples that contained RNA sequencing data, MAF mutation data, and clinical information, a total of 494 samples were included for further analysis. Somatic mutation data from prostate cancer patients were obtained and visualized utilizing the maftools package within the R software environment. Statistical analyses were conducted using R software version 4.0.3. Results were considered statistically significant at a P-value threshold of less than 0.05.

The Gene_Mutation module, utilizing Timer2.0 (http://timer.comp-genomics.org/) (Li T. et al., 2020), was used to compare the expression levels of the P53 gene in both wild-type and mutant states of the PLK1 gene.

Statistical analysis was performed using Excel or GraphPad Prism 9.5 software. Quantitative data are presented as mean ± SEM, and p < 0.05 was considered statistically significant.

Cells were harvested and washed with pre-chilled PBS, then lysed in NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris, 0.5% Nonidet P-40) supplemented with protease inhibitors (Beyotime Biotechnology). The lysates were sonicated and clarified by centrifugation at 12,000 × g for 5 min at 4°C, repeated three times.After centrifugation, the supernatant was incubated with the appropriate antibody at 4°C for 16 h, followed by incubation with Protein A/G PLUS-Agarose beads (Santa Cruze,sc-2003) for 2 h at 4°C. The beads were then collected by centrifugation at 2,000 × g for 5 min, and the supernatant was discarded. Beads were washed at least five times with NETN lysis buffer and then lysed with lysis buffer.

Although many studies have investigated the effect of p53 on the efficacy of PLK1 inhibitors, the results have been inconsistent. Previous studies mainly used tumor cells with wild-type p53 and did not investigate the effect of p53 on BI6727-induced apoptosis in tumor cells with long-term p53 loss. PC3 cells are prostate cancer cells with long-term p53 loss due to P53 gene mutation (Isaacs et al., 1991) (Figure 1A). BI6727 has been shown to exert cytotoxic effects on various tumor cells (Gjertsen and Schöffski, 2015). The analysis of CCK-8 assay showed that BI6727 could inhibit the proliferation of PC3 cells. The IC50 of BI6727 in PC3 cells was 25.21 nM (Figure 1B). Additionally, the colony formation assay showed decreased cell proliferation in PC3 cells treated with BI6727, especially in PC3 cells treated with 50 nM BI6727 (Figure 1C). In order to explore if p53 affects the sensitivity of PC3 to BI6727, p53 was successfully transiently expressed in PC3 cells (Figure 1D). After BI6727 treatment, the activation form of caspase-3 was higher in PC3-p53 cells than in PC3-Vector cells (Figure 1E). Then, the PC3 cell lines stably expressing Vector or wild-type p53 were established (Figure 1F). The same results were obtained. After BI6727 treatment, the activation form of caspase-3 was elevated and the cleavage of its substrate PARP increased (Figure 1G). These data indicate that, in PC3 cells with long-term p53 loss, the presence of functional p53 increased the sensitivity of them to the PLK1 inhibitors BI6727 (Zhu et al., 2002; Kruse and Gu, 2009; Ando et al., 2004).

LNCaP cells are androgen-dependent prostate cancer cells with functional p53 (Isaacs et al., 1991; Hierowski et al., 1987) (Figure 1A). The IC50 of BI6727 in LNCaP cells is 13.61 nM (Figure 2A). As the concentration increased, BI6727 became more effective in inhibiting LNCaP cell proliferation, and at 50 nM, no colonies were formed (Figure 2B), consistent with PC3 cells. BI6727 exerts different levels of cytotoxicity in PC3 and LNCaP cells, with the IC50 in LNCaP cells being significantly lower than in PC3 cells. Since LNCaP cells express wild-type p53, we hypothesized that the presence of p53 may influence the cytotoxic effect of BI6727. Therefore, we knocked down endogenous p53 in LNCaP cells to investigate its role in BI6727-induced apoptosis. We used three different siP53s to knock down p53, with siP53-1 showing a much lower knockdown efficiency than siP53-2 and siP53-3 (Figure 2C). Therefore, in subsequent experiments, we used a 1:1 mixture of siP53-2 and siP53-3 (siP53-2,3) to knock down p53. The results showed that p53 expression was significantly reduced in LNCaP-siP53-2,3 cells compared to LNCaP-siNC (Figure 2D). Cells were treated with siP53-2,3 for 16 h and then cultured in normal medium for 40 h. The PLK1 inhibitor BI6727 or DMSO was then added, and cells were incubated for 48 h before harvesting and protein extraction for Western blot. After BI6727 treatment, p53 knockdown in LNCaP-siP53-2,3 cells significantly decreased the activation form of caspase-3 and downstream PARP cleavage (Figure 2E). Compared to the control, BI6727 treatment increased p53 expression in the cells (Figure 2E), which is consistent with reports that PLK1 inhibition induces apoptosis by increasing p53 expression and activating p53-dependent transcriptional activity (Wang et al., 2024). Therefore, BI6727 induces apoptosis through a p53-dependent pathway in wild-type p53 cells. Overall, in LNCaP cells, p53 promotes apoptosis induced by BI6727.

As we know, we can detect p53 in DU145 cells (Figure 1A), but it is a mutant. We next investigated the effect of BI6727 on p53-mutant DU145 prostate cancer cells. In the colony formation assay, with the concentration of BI6727 at 20 nM, only a few small clones were observed; and at 50 nM, there was nearly no colony formation (Figure 3A). To evaluate apoptosis effect of BI6727 on DU145 cells, cells were treated with 10 nM and 50 nM BI6727 for 48 h, PARP degradation was observed (Figure 3B). It has been suggested that mutant p53 is associated with the dominant negative effect of in DU145 cells (Zhu et al., 2002). DU145 cells were treated with siP53-2,3, the effect of siRNA was detected by Western blot (Figure 3C). The results showed that mutant p53 can be knocked down effectively in DU145 cells. When cells were treated with BI6727, the activation form of caspase-3 was increased and PARP cleavage was enhanced in DU145-siP53-2,3 cells compared to DU145-siNC (Figure 3D). In conclusion, in p53-mutant DU145 cells, the expression of mutant p53 reduces PLK1 inhibitor-induced apoptosis.

Considering that p53 mutation affects the effect of the PLK1 inhibitor BI6727 in inducing apoptosis in prostate cancer cells, we next investigated the mutation status of p53 in prostate cancer specimens. In this study, we performed a comprehensive gene mutation analysis on 494 prostate cancer samples and found that there is a 12% mutation frequency of the P53 gene in prostate cancer (Figure 4A). Among the P53 gene mutations, we observed various types, including missense mutations, splice site mutations, nonsense mutations, frameshift insertions, and frameshift deletions. The distribution of these mutations highlights the diversity and complexity of p53 in prostate cancer samples (Figure 4B). In addition, missense mutations were the most common type of p53 mutation. Further analysis of the cohort revealed that patients with high PLK1 expression also had a significantly increased mutation rate of p53 (Figure 4C). More intriguingly, in the prostate cancer specimens with p53 mutation, PLK1 expression was significantly increased (Figure 4D). Overall, the informatics assay showed that 12% of the prostate cancer specimens had a p53 mutation.

Topors has been reported to ubiquitinate p53 and significantly reduce its protein levels (Rajendra et al., 2004). Phosphorylation of Topors by PLK1 enhances its activity, promoting p53 ubiquitination and subsequent degradation (Yang et al., 2009). Meanwhile, PLK1 phosphorylates Topors to promote its degradation (Yang et al., 2010).To determine whether the ubiquitin–proteasome system is involved in BI6727-induced cell death, we assessed both p53 ubiquitination and global protein ubiquitination. In 293T cells overexpressing exogenous p53 and ubiquitin, immunoblotting showed that BI6727 reduced p53 ubiquitination, with minimal effect on overall protein ubiquitination (Figure 5A).We also examined endogenous p53 ubiquitination in 293T cells and obtained similar results (Figure 5B). Furthermore, in PC3 prostate cancer cells stably expressing p53, BI6727 likewise reduced p53 ubiquitination with little effect on overall ubiquitination levels (Figure 5C).Since BI6727 reduced p53 ubiquitination, we next investigated whether this effect was mediated by Topors. We found that BI6727 treatment markedly attenuated Topors degradation, leading to increased Topors protein levels (Figure 5D). These findings suggest that the PLK1 inhibitor BI6727 enhances p53 stability by inhibiting Topors degradation and thereby reducing p53 ubiquitination.