Novel inhibition of AKR1C3 and androgen receptor axis by PTUPB synergizes enzalutamide treatment in advanced prostate cancer

Abstract

Castration-resistant prostate cancer (CRPC) is the main driving force of mortality in prostate cancer patients. Among the parameters contributing to the progression of CRPC and treatment failure, elevation of the steroidogenic enzyme AKR1C3 and androgen receptor variant 7 (AR-V7) are frequently reported. The AKR1C3/AR-V7 complex has been recognized as a major driver for drug resistance in advanced prostate cancer. Herein we report that the level of AKR1C3 is reciprocally regulated by the full-length androgen receptor (AR-FL) through binding to the distal enhancer region of the AKR1C3 gene. A novel function of PTUPB in AKR1C3 inhibition was discovered and PTUPB showed more effectiveness than indomethacin and celecoxib in suppressing AKR1C3 activity and CRPC cell growth. PTUPB synergizes with enzalutamide treatment in tumor suppression and gene signature regulation. Combination treatments with PTUPB and enzalutamide provide benefits by blocking AR/AR-V7 signaling, which inhibits the growth of castration relapsed VCaP xenograft tumors and patient-derived xenograft organoids. Targeting of the ARK1C3/AR/AR-V7 axis with PTUPB and enzalutamide may overcome drug resistance to AR signaling inhibitors in advanced prostate cancer.

Fig. 1. AR negatively regulates AKR1C3 expression in CRPC.

A The gene expression data from TCGA database was extracted, and AR, KLK3, NKX3–1, TMPRSS2, and AKR1C3 correlations were determined by Spearman correlation. B C4–2B MDVR and C4–2B AbiR cells were treated at 10 and 20 μM concentrations of enzalutamide for 3 days. The levels of AKR1C3 mRNA were determined. C C4–2B MDVR and C4–2B AbiR cells were treated enzalutamide or abiraterone for 3 days. The protein levels of AKR1C3 were determined. D, E AR-FL or AR-V7 was knocked down in C4–2B MDVR cells, mRNA and protein levels of AR-FL, AR-V7, and AKR1C3 were determined. F Genome browser view showing DHT treatment increased AR binding on the AKR1C3 enhancer at different time points. However, enzalutamide treatment blocked the DHT induced AR binding on the AKR1C3 enhancer in VCaP AR ChIP-Seq data. G VCaP cells were treated with 10 nM DHT with or without 20 μM enzalutamide for 5 days, mRNA expression of AKR1C3, AR-FL and AR-V7 were examined by qRT-PCR. H VCaP cells were treated with different concentrations of DHT with or without enzalutamide for 5 days. AKR1C3, AR-FL, and AR-V7 were examined by western blot. I ChIP-qPCR of the AR occupancy at the AKR1C3 enhancer by DHT treatment (10 nM, 24 h) in VCaP cells. Enza: enzalutamide, Abi: abirateorne. *p < 0.05. Results are the mean of three independent experiments (±SD).

Fig. 2. A novel AKR1C3 inhibitor-PTUPB suppresses the AKR1C3 activity and intracrine androgen synthesis.

A The chemical structure of PTUPB. B The docking model of PTUPB and the previously published AKR1C3/NADP+ crystal structure (PDB 7C7F), the lowest binding energy was −10.2 Kcal/mol through AutoDock vina and PyMOL. PTUPB bound site of the ternary complex, showing the surrounding residues (green) and NADP+ (red). Close-up view of the H-bond network between PTUPB and F306, Y216 in the ternary complex. C The AKR1C3 activity was determined by oxidation of S-tetralol. Different concentrations of PTUPB, indomethacin, and enzalutamide were mixed with S-tetralol, NADP+, and AKR1C3 recombinant protein; then, the initial velocity and the AKR1C3 activity inhibition were determined. D C4–2B parental and C4–2B MDVR cells were cultured in phenol red free RPMI1640 charcoal-stripped serum medium for 5 days and treated with 100 nM Δ4-dione. C4–2B MDVR cells were treated with or without PTUPB for 2 days. Levels of steroids in the cell extracts were analyzed by LC-MS. Representative testosterone and DHT chromatograms were viewed by the chromatogram viewer. E–G The represented steroid metabolites (testosterone, DHT and 5α-Dione) between C4–2B parental and C4–2B MDVR cells were quantified by Quanlynx. *p < 0.05. Results are the mean of three independent experiments (±SD).

Fig. 3. PTUPB synergizes with the enzalutamide treatment and degrades AR/AR-V7 in vitro.

A C4–2B MDVR cells were treated with different concentrations of PTUPB, Celecoxib (Cele), TPPU, indomethacin (Indocin), and enzalutamide (Enza) for 3 days, total cell numbers were determined, and cell survival rate was calculated. B C4–2B MDVR cells were treated with different concentrations of PTUPB with or without different concentrations of enzalutamide. The drug synergy distribution map was generated by Combenefit. C C4–2B MDVR cells were treated with different concentrations of PTUPB or Indocin, with/without enzalutamide for 3 days. Total cell numbers were determined. D C4–2B MDVR cells were treated with DMSO, PTUPB (10 and 20 μM), or Indocin (10 and 20 μM) with/without 20 μM enzalutamide, then the colony formation ability was examined by the clonogenic assay, and the colony numbers were determined. E C4–2B MDVR cells were treated with DMSO, Indocin (20 μM), or PTUPB (20 μM) with/without 20 μM enzalutamide; the whole cell lysates were collected, and protein expression was determined by western blot. F CWR22Rv1 cells were treated with PTUPB (20 μM) for 3 days, followed with MG132 (5 μM) overnight, and the AR and AR-V7 protein levels were determined by western blot. G CWR22Rv1 cells were treated with or without PTUPB (20 μM) for 3 days in the presence of MG132 (5 μM) overnight. Total cell lysates were immunoprecipitated with anti-AR antibody and immunoblotted with anti-Ub. *p < 0.05. Results are the mean of three independent experiments (±SD).

Fig. 4. Synergism of the PTUPB and enzalutamide combination treatment reprograms gene signature regulation and suppresses AR/AR-V7 signaling pathways in resistant prostate cancer.

A Venn diagram of up-regulated and down-regulated gene numbers analyzed from RNA sequence data by three comparisons: PTUPB (10 μM or 20 μM) vs. DMSO, enzalutamide vs. DMSO, and PTUPB + enzalutamide vs. DMSO in C4–2B MDVR cells (FPKM > 1 and fold change>1.2). B GSEA of top enriched gene sets in C4–2B MDVR cells treated by PTUPB. The upregulated and down-regulated gene sets from the Hallmark and KEGG platforms were output by GSEA. C Heatmap and hierarchical clustering of the differentially expressed genes (DEGs) between treatments (PTUPB, enzalutamide, and combination) in C4–2B MDVR cells with FPKM > 1 and fold change >1.2, as compared to vehicle (DMSO). The genes were displayed in rows and the normalized counts per sample were displayed in columns. Red indicates up-regulated, and blue designates down-regulated expression levels. Right: AR-FL/AR-V7 targeted genes and cell growth/survival genes that were altered in expression are displayed. D GSEA of the AR-V7 associated gene signature in C4–2B MDVR cells treated with PTUPB, as compared to DMSO (left). GSEA of the AR-V7 up-regulated gene signatures in C4–2B MDVR cells treated with PTUPB (middle). The signature was defined by genes that are preferentially upregulated by AR-V7 [44]. GSEA of the cancer cell growth/survival gene signatures in C4–2B MDVR cells treated with PTUPB (right). E C4–2B MDVR cells were transiently transfected with PSA E/P-luciferase plasmid, treated with different concentrations of PTUPB overnight, and the PSA luciferase activity was examined. F qRT-PCR analysis of the indicated genes in C4–2B MDVR cells treated with DMSO or PTUPB (10 μM or 20 μM) alone or with 20 μM enzalutamide for 48 h. Enza enzalutamide, PB PTUPB. *p < 0.05. Results are the mean of three independent experiments (±SD).

Fig. 5. PTUPB improves enzalutamide treatment in LuCaP35CR organoid and relapsed VCaP xenograft tumor models.

A, B LuCaP35CR organoids were seeded in a 96 well plate in a format of the 3D Matrigel and then treated with PTUPB, enzalutamide, or combination and cultured for 14 days. The viability of the organoids was visualized by LIVE/DEAD^™ Cell Imaging Kit staining and analyzed by CellTiter Glo. Green (live cells), Red (dead cells). C Mice bearing VCaP xenografts were castrated, and relapsed tumors were treated with vehicle control, enzalutamide (25 mg/Kg, p.o.), PTUPB (30 mg/Kg, p.o.), or the combination for 21 days (n = 6). Tumor volumes were measured twice weekly. Tumor pictures were taken. D Tumor weight was calculated. E Tumors were processed, and the testosterone level was determined by LC/MS. F IHC staining and quantification of Ki67 and AR-V7 in representative tumors. *p < 0.05.

Fig. 6. PTUPB suppresses the growth of patient-derived xenograft tumor cells and organoids.

A Scheme of the CRCs/PDX/Organoid model establishment. B Tumor tissues from UCD1172 patient were processed and CRCs were established. AKR1C3, AR, human mitochondria (Mito) and DAPI were stained. C The UCD1172 PDX castration-resistant (CR) model establishment and the expression of AR variants were determined by qRT-PCR. D UCD1172 and UCD1172CR cells were treated with enzalutamide for 3 days, AKR1C3 mRNA expression was determined (left). The AR, AR-V7, and AKR1C3 protein expression in UCD1172 and UCD1172CR cells were determined by western blot (right). E UCD1172CR cells were infected with lenti-shControl or lenti-shAKR1C3 for 5 days. The cell growth and AR/AR-V7 expression were determined. F UCD1172CR cells were treated with different concentrations of PTUPB or indomethacin with/without enzalutamide for 3 days. Total cell numbers were determined. G UCD1172CR cells were treated with DMSO, PTUPB (5 μM, 10 μM and 20 μM), indomethacin (10 and 20 μM) with/without 20 μM enzalutamide, and the colony formation ability was examined by the clonogenic assay. H UCD1172CR cells were treated with DMSO, indomethacin (20 μM), PTUPB (20 μM) with/without 20 μM enzalutamide, then the whole cell lysates were collected, and protein expression was determined by western blot. I UCD1173 and UCD1178 organoids generated from PDX tumors were seeded in a 96 well plate in a format of the 3D Matrigel, and then treated with different concentrations of PTUPB and cultured for 14 days. The viability of the organoids was visualized by LIVE/DEAD^™ Cell Imaging Kit staining. Green (live cells), Red (dead cells). *p < 0.05. Results are the mean of three independent experiments (±SD).