Development of a novel treatment based on PKMYT1 inhibition for cisplatin-resistant bladder cancer with miR-424-5p-dependent cyclin E1 amplification

Abstract

Background: Chemotherapy including cisplatin is recommended for the treatment of advanced bladder cancer, but its effectiveness is limited due to the acquisition of drug resistance. Although several mechanisms of cisplatin resistance have been reported, there are still many unknowns, and treatment of cisplatin-resistant bladder cancer remains difficult. Accordingly, in this study, we aimed to identify and characterize microRNAs involved in cisplatin resistance.

Methods: Small RNA sequencing analysis was performed to search for microRNAs related to cisplatin resistance. The identified microRNAs were then characterized using gain-of-function studies, sensitivity analysis, target gene analysis, and cellular assays.

Results: We identified miR-424-5p as a candidate microRNA that was downregulated in cisplatin-resistant strains compared with parental strains. Notably, in gain-of-function studies, miR-424-5p suppressed the proliferative ability of cisplatin-resistant bladder cancer (CDDP-R BC). Furthermore, miR-424-5p restored sensitivity to cisplatin. RNA sequence analysis revealed seven candidate genes targeted by this microRNA. Among them, cyclin E1 (CCNE1) was chosen for subsequent analyses because its expression was upregulated in cisplatin-resistant cells compared with parental cells and because recent studies have shown that CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition. Therefore, we performed functional analysis using the PKMYT1 inhibitor RP-6306 and demonstrated that RP-6306 inhibited cell growth through suppression of mitotic entry and restored cisplatin sensitivity in CDDP-R BC.

Conclusions: Overall, our findings provided insights into the development of novel therapeutic strategies for CDDP-R BC.

Keywords: miR-424-5p; Cisplatin-resistant bladder cancer; Cyclin E1; PKMYT1; Synthetic lethal.

Fig. 1

Targeted genes were extracted and analyzed using TCGA database. A RNA-seq analysis of previously established CDDP-R BC was performed to compare parental and CDDP-R BC. Five microRNAs were extracted that were commonly downregulated in two cell lines, BOY and T24 (fold change CDDP-R-BOY /BOY: 0.25, fold change CDDP-R-T24 /T24: 0.74). B Comparison of miR-424-5p expression in parental and CDDP-R BC cell lines by RT-qPCR (fold change CDDP-R-BOY /BOY: 0.36, fold change CDDP-R-T24 /T24: 0.35). * P < 0.05. C Candidate microRNAs were transfected into CDDP-R BC cells, respectively, and their proliferative ability was assessed using the XTT assay. P = 0.5

Fig. 2

Effects of transfection with miR-424-5p. A, B, BOY, CDDP-R BOY, T24, and CR-T24 cells were transfected with miR424-5p, and proliferative ability was measured using XTT assays. * P < 0.001. C, Colony formation was performed by transfection of CDDP-R BOY and CDDP-R T24 cells with miR-424-5p, and colony-forming ability was measured

Fig. 3

Improvement of CDDP resistance by miR-424-5p and additive effects. A, CDDP-R BOY and CDDP-R T24 cells were treated with 4 µM CDDP and transfected with miR424-5p, and proliferative ability was measured using XTT assays. * P < 0.0001. B, CDDP-R BOY and CDDP-R T24 cells were treated with 10 µM CDDP and transfected with miR-424-5p, and proliferative ability was measured using XTT assays. * P < 0.0001

Fig. 4

RNA-seq analysis was performed using CDDP-R BOY and CDDP-R T24 cells transfected with miR-424-5p. A, Seven genes were commonly downregulated in the two cell lines. B, Expression of each gene was measured by RT-qPCR in parental and CDDP-R BC cells. * P < 0.05. C, CCNE1 expression was compared in normal tissues and BC cells using data from TCGA database (normal: n = 19, tumor: n = 404). * P < 0.0001. D, PKMYT1 expression was compared in normal tissues and BC cells using data from TCGA database (normal: n = 19, tumor: n = 404). * P < 0.0001. E, Expression of CCNE1 and PKMYT1 in CDDP-R BC cells transfected with miR-424-5p was examined by RT-qPCR. * P < 0.05. F, Analysis of CCNE1 and PKMYT1 correlation using the TCGA database. P < 0.001, r = 0.34

Fig. 5

Effect of RP6306 on CDDP-R BOY and CDDPR-T24 cells. A, CDDP-R BOY and CDDP-R T24 cells were treated with RP-6306, and proliferative ability was measured using XTT assays. CDDP (10 µM) was also used in combination. * P < 0.0001. B, CDDP-R BOY and CDDP-R T24 cells were treated with miR-424-5p, RP-6306 and CDDP in cell cycle assay. * P < 0.05