Exploiting epigenetic targets to overcome taxane resistance in prostate cancer

Abstract

The development of taxane resistance remains a major challenge for castration resistant prostate cancer (CR-PCa), despite the effectiveness of taxanes in prolonging patient survival. To uncover novel targets, we performed an epigenetic drug screen on taxane (docetaxel and cabazitaxel) resistant CR-PCa cells. We identified BRPF reader proteins, along with several epigenetic groups (CBP/p300, Menin-MLL, PRMT5 and SIRT1) that act as targets effectively reversing the resistance mediated by ABCB1. Targeting BRPFs specifically resulted in the resensitization of resistant cells, while no such effect was observed on the sensitive compartment. These cells were successfully arrested at the G₂/M phase of cell cycle and underwent apoptosis upon BRPF inhibition, confirming the restoration of taxane susceptibility. Pharmacological inhibition of BRPFs reduced ABCB1 activity, indicating that BRPFs may be involved in an efflux-related mechanism. Indeed, ChIP-qPCR analysis confirmed binding of BRPF1 to the ABCB1 promoter suggesting direct regulation of the ABCB1 gene at the transcriptional level. RNA-seq analysis revealed that BRPF1 knockdown affects the genes enriched in mTORC1 and UPR signaling pathways, revealing potential mechanisms underlying its functional impact, which is further supported by the enhancement of taxane response through the combined inhibition of ABCB1 and mTOR pathways, providing evidence for the involvement of multiple BRPF1-regulated pathways. Beyond clinical attributes (Gleason score, tumor stage, therapy outcome, recurrence), metastatic PCa databases further supported the significance of BRPF1 in taxane resistance, as evidenced by its upregulation in taxane-exposed PCa patients.

Fig. 1. Upregulation of ABCB1 in taxane-resistant cells and its clinical relevance in advanced PCa.

A Gene set enrichment (GSEA) analysis using hallmark gene sets from the MSigDB revealed the upregulation of genes involved in MYC signaling, unfolded protein response (UPR), NF-κB and E2F signaling (FDR < 0.05, and Log₂FC ≥ 0.5 or ≤ −0.5). GSEA was performed using the h.all.v2023.1.Hs.symbols.gmt dataset in the MsigDB database. B DEGs (FDR < 0.05, and Log₂FC ≥ 1 or ≤−1) from resistant cells were further distributed based on their expression changes in the same direction (up or down). The red and purple scatters indicate up- and downregulated DEGs, respectively, in resistant Du145 cells. C Expression levels of ABCB1 mRNA were determined by qRt-PCR. Data is the mean ± SEM. D ABCB1 protein expression levels in parental and taxane resistant PCa cells were determined by western blotting. E Comparison of patient survival based on ABCB1 expression levels using a Kaplan-Meier plot (Metastatic Prostate Adenocarcinoma, MCTP). F ABCB1 mRNA levels in clinical specimens (TCGA-PRAD) were investigated by using cBioPortal and its relationship with the occurrence of local recurrence and metastasis (Pan-cancer Analysis of Advanced and Metastatic Tumors, BCGSC); (G) and gleason score (Metastatic Prostate Adenocarcinoma, SU2C/PCF Dream Team) were shown as scatter plots. Uncropped western blot images corresponding to Fig. 1D were shown in Supplemental Material.

Fig. 2. Epigenetic drug screening reveals vulnerabilities for taxane-resistant PCa cells.

A Schematic view of the epidrug library screening. The figure was generated using BioRender software. B Screening of 145 targeted drugs to identify epigenetic modulators capable of overcoming taxane resistance was performed using the SRB assay and the result obtained in Du145-DtxR cells is shown representatively. Purple dots show the cell viabilities upon the standalone use of each epidrugs, while the red dots indicate the cell-killing effect of their combination with Dtx. Re-sensitizers ranked in the top 5 groups are highlighted in circles. C Pie Chart illustrates the epigenetic proteins targeted by the small molecules (epidrugs) in the library. Epidrugs were screened for resistance reverter activity which resulted in the identification of 5 classes of epigenetic targets (PRMT5, Menin-MLL, SIRT1, BRPF, and CBP/p300) that induced the most pronounced resensitization when applied to resistant cells. D Molecular structure of BRPF inhibitors. E Cartoon representation of the BRPF family and (F) demonstration of the efficacy of BRPF inhibition on histone acetylation by western blotting. Uncropped western blot images corresponding to F were shown in Supplemental Material.

Fig. 3. Restoration of taxane susceptibility by BRPF Inhibition.

A Validation dose-response curves of BRPF inhibitors (PFI4, GSK5959 and OF1) on Dtx-resistant cells. Cells were co-treated with indicated drugs (Dtx; 1–250 nM and BRPF inhibitors; 1.25–5 µM) and the results were obtained by SRB viability assay (72 h). The data is expressed as mean ± SEM. B Heat map representation of the Combination Index (CI) values, with red color indicating a synergistic effect. CI was calculated using the CalcuSyn software. C Clonogenic images were obtained by treating cells with indicated drugs for 72 h and the colony formation ability was analyzed 10–15 days after drug exposure. D Flow cytometry analysis of cell death (48 h) and (E) cell cycle distribution (24 h) in resensitized Du145-DtxR cells. F The expression of BRPF1 showed a positive correlation with MKi67 expression in metastatic PCa. G Calcein retention assay was performed in the absence or presence of BRPF inhibitors (5 µM, 24 h). H CETSA for in-cell ABCB1 engagement. Western blots showing thermostable ABCB1 following indicated heat shocks (44 °C, 46 °C, 48 °C, 50 °C and 52 °C) in the presence of Zosuquidar (5 µM) and GSK5959 (5 µM) in Du145-DtxR cells. I The expression of ABCB1 was evaluated by qRt-PCR at the indicated time points following treatment with GSK5959 (5 µM). P parental, R resistant. Uncropped western blot images corresponding to H were shown in Supplemental Material.

Fig. 4. BRPF1 regulates ABCB1 expression and interferes with multiple signalings in resistant cells.

A Taxane response of siBRPF1 treated resistant cells evaluated by colony formation assay. Representative images and (B) quantifications are shown. C The expression of ABCB1 was evaluated by qRt-PCR at the indicated time points following treatment with siBRPF1 in Du145-DtxR cells. D ChIP-qPCR showing BRPF1 enrichment at the ABCB1 promoter in Du145-DtxR cells expressing endogenous BRPF1 (left panel) and exogenous HA-tagged BRPF1 (right panel). BRPF1 enrichment at a control region (Chr12 gene desert) is also shown. Data are shown as percentage of ChIP input; dots represent individual biological replicates; bars represent mean replicates. E BRPF1 mRNA levels of RNA-seq samples from siControl and siBRPF1 in Du145-DtxR cells. F Venn diagram showing the number of genes (intersection, 461) whose expression decreased after silencing of BRPF1 among genes with increased expression in Du145-DtxR cells (vs Du145-P). G Computed overlaps of the 461 genes in the Hallmark Collection of GSEA (MSigDB) database. H The efficacy of Torin1 (mTORC1/2 inhibitor, 8–500 nM) on Du145-DtxR cells was determined by CTG assay and represented as a heat map. I The efficacy of Ceapin-A7 (ATF6α inhibitor, 0.6–10 µM) on Du145-DtxR cells was determined by SRB assay and represented as a heat map. The Combination Index (CI) was calculated using the CalcuSyn program. Statistical significance denoted as *p < 0.05, **p < 0.01, and ***p < 0.0001.

Fig. 5. Clinical significance of BRPF1 expression.

A BRPF1 mRNA levels were analyzed in the Pan-Cancer Analysis dataset (ICGC/TCGA, Nature 2020) from cBioPortal. Kaplan-Meier plot showing the comparison of patient survival based on BRPF1 expression levels. The patients were classified into Low BRPF1 and High BRPF1 groups based on the expression median. The distribution of BRPF1 expression (B) across different cancers and (C) sample types. The numbers within the column bars correspond to the sample size. D BRPF1 mRNA levels in clinical PCa specimens were examined for their correlation with neoplasm status (Prostate Adenocarcinoma, TCGA, PanCancer Atlas), recurrence (Prostate Adenocarcinoma, TCGA, Firehose Legacy), sample type (Prostate Adenocarcinoma, Fred Hutchinson CRC, Nat Med 2016) (E) Gleason score (Prostate Adenocarcinoma, TCGA, Firehose Legacy), tumor stage (Prostate Adenocarcinoma, TCGA, Firehose Legacy), (F) therapy outcome (Prostate Adenocarcinoma, TCGA, Firehose Legacy), and taxane exposure status (Metastatic Prostate Adenocarcinoma, SU2C/PCF Dream Team, PNAS 2019). The results were presented as scatter plots.

Fig. 6. Graphical abstract.

The figure illustrates the major findings of the study.