The status of p53 affects the efficacy of PLK1 inhibitor BI6727 in prostate cancer cells

Abstract

As there are no effective treatments for advanced prostate cancer, exploring new therapies is crucial. BI6727(Volasertib), a PLK1 inhibitor, shows great promise as an anti-cancer drug. However, despite advancing to phase II and III trials in other cancers, BI6727 has shown limited anti-tumor activity in prostate cancer, making it crucial to investigate the underlying reasons for this discrepancy. In this study, we found that the status of p53 affects the sensitivity of prostate cancer cells to BI6727. Prostate cancer cells PC3 (long-term loss of p53 expression), DU145 (expressing mutant-type p53) and LNCaP (expressing wild-type p53) were treated with BI6727, respectively. It was found that PC3 cells were more sensitive to BI6727 when wild-type p53 was introduced into these cancer cells; while apoptosis induced by BI6727 was reduced after knockdown of p53 in LNCaP cells. In additional, in DU145 cells, the presence of points mutation in p53 exerted a dominant negative effect, attenuating BI6727-induced apoptosis. Further analysis revealed that missense mutations in the P53 gene are widespread in prostate cancer patients. Mechanistically, BI6727 reduces the degradation of Topors, thereby increasing the stability of p53 by reducing its ubiquitination. This ultimately influences the sensitivity of prostate cancer cells with different p53 statuses to BI6727.In summary, this study identifies p53 as a key factor limiting the clinical efficacy of BI6727 in prostate cancer cells.

Keywords: BI6727; PLK1; PLK1 inhibitor; p53; prostate cancer.

FIGURE 1

Wild-type p53 promotes BI6727-induced apoptosis in PC3 cells. (A) Cell lysates from PC3, DU145, and LNCaP cells were collected and analyzed by Western blot to detect endogenous p53 protein levels. (B) CCK-8 assay to evaluate the effect of BI6727 on PC3 cell growth. (C) PC3 cells were treated with DMSO, 20 nM, or 50 nM BI6727, and colony formation was assessed. (D) p53 expression was detected in PC3 cells by Western blot after transient transfection. (E) After treating PC3-Vector and PC3-p53 cells with DMSO and 50 nM BI6727 for 48 h, transient transfection cells were collected to detect activation form of caspase-3 by Western blot. (F) A stable p53-expressing PC3 cell line was constructed, and p53 protein expression was detected by Western blot. (G) After treating the stable PC3-Vector and PC3-p53 cells with DMSO or 50 nM BI6727 for 48 h, proteins were collected and detected by Western blot using specific antibodies.

FIGURE 2

Knockdown of p53 weakens BI6727-induced apoptosis in LNCaP cells. (A) CCK-8 assay to assess the effect of BI6727 on LNCaP prostate cancer cell growth; (B) LNCaP cells were treated with DMSO, 20 nM, and 50 nM BI6727 to evaluate colony formation; (C) Cells were treated with 50 nM siNC, siP53-1,siP53-2 or siP53-3, and protein lysates were prepared to assess p53 protein levels; (D) LNCaP cells were treated with 50 nM control si-NC or siP53-2and siP53-3 (siP53-2,3) for 56 h (E) LNCaP cells were treated with siRNA siP53-2,3 for 16 h, then cultured in normal medium for 40 h. Afterward, PLK1 inhibitor BI6727, or DMSO, and cells were incubated for 48 h before collection and protein extraction for Western blot.

FIGURE 3

The expression of mutant p53 inhibits BI6727-induced apoptosis in DU145 cells. (A) DU145 cells were treated with DMSO, 20 nM, and 50 nM BI6727, and colony formation was assessed. (B) DU145 cells were treated with 10 nM and 50 nM BI6727 for 48 h, then collected and analyzed by Western blot using antiPARP antibody. (C,D) DU145 cells were treated with siP53-2,3 for 16 h, then cultured in normal medium for 40 h. Afterward, PLK1 inhibitor BI6727, or DMSO, and cells were incubated for 48 h before collection and protein extraction for Western blot using the indicated antibodies.

FIGURE 4

P53 Mutation in Prostate Cancer. (A) Somatic mutation landscape of 494 prostate cancer samples. The side bar chart shows the types and frequencies of gene mutations. The waterfall plot illustrates the mutation details of each gene across samples, with different colors at the bottom indicating specific mutation types. A small section above the legend shows the mutation burden count. (B) A lollipop plot showing the distribution of P53 gene mutations, with the somatic mutation rates and names indicated in the figure’s title and subtitle. (C) P53 mRNA mutation distribution in 494 prostate cancer samples. Based on the median expression of PLK1, samples were grouped into high PLK1 expression and low PLK1 expression groups. (D) Timer2.0 analysis of PLK1 gene expression differences under different p53 mutation statuses.

FIGURE 5

BI6727 reduces p53 ubiquitination. (A) 293T cells were co-transfected with p53 and HA-ubiquitin, treated with DMSO or 50 nM BI6727 for 48 h, followed by 10 μM MG132 for 4 h. Total ubiquitinated proteins were detected in lysates, and p53 was immunoprecipitated for detection of ubiquitinated p53 by Western blot. (B,C) 293T cells (B) and PC3 cells stably expressing p53 (C) were treated with DMSO or 50 nM BI6727 for 48 h, followed by 10 μM MG132 for 4 h. Total and p53-specific ubiquitination levels were examined as described in (A). (D) 293T cells were treated with DMSO or 50 nM BI6727 for 48 h. Topors protein levels were analyzed by Western blot.* indicates degradation products of Topors.