Subtype and Site Specific-Induced Metabolic Vulnerabilities in Prostate Cancer

Abstract

Aberrant metabolic functions play a crucial role in prostate cancer progression and lethality. Currently, limited knowledge is available on subtype-specific metabolic features and their implications for treatment. We therefore investigated the metabolic determinants of the two major subtypes of castration-resistant prostate cancer [androgen receptor-expressing prostate cancer (ARPC) and aggressive variant prostate cancer (AVPC)]. Transcriptomic analyses revealed enrichment of gene sets involved in oxidative phosphorylation (OXPHOS) in ARPC tumor samples compared with AVPC. Unbiased screening of metabolic signaling pathways in patient-derived xenograft models by proteomic analyses further supported an enrichment of OXPHOS in ARPC compared with AVPC, and a skewing toward glycolysis by AVPC. In vitro, ARPC C4-2B cells depended on aerobic respiration, while AVPC PC3 cells relied more heavily on glycolysis, as further confirmed by pharmacologic interference using IACS-10759, a clinical-grade inhibitor of OXPHOS. In vivo studies confirmed IACS-10759's inhibitory effects in subcutaneous and bone-localized C4-2B tumors, and no effect in subcutaneous PC3 tumors. Unexpectedly, IACS-10759 inhibited PC3 tumor growth in bone, indicating microenvironment-induced metabolic reprogramming. These results suggest that castration-resistant ARPC and AVPC exhibit different metabolic dependencies, which can further undergo metabolic reprogramming in bone.

Implications: These vulnerabilities may be exploited with mechanistically novel treatments, such as those targeting OXPHOS alone or possibly in combination with existing therapies. In addition, our findings underscore the impact of the tumor microenvironment in reprogramming prostate cancer metabolism.

Fig. 1.. Transcriptomic and proteomic analysis of ARPC and AVPC samples.

A) Schematic representation of the experiment: data from (16) were segregated in ARPC and AVPC based on AR and NE scores and GSEA analysis performed. B) GSEA enrichment plots; Normalized enrichment scores (NES) and false discovery rate (FDR)-q values are shown. C) Schematic representation of the RPPA study using PDX samples. D) Androgen expression levels in ARPC and AVPC. P value was estimated through Mann Whitney test, (***) P < 0.001. E) A UMAP shows clustering within ARPC and AVPC LuCaP PDX models. CR, Castration-resistant. F) Representative plots showing 8 different proteomic measurements in APRC and AVPC tumor lysate samples. P values are reported.

Fig. 2.. Metabolic characterization of PCa cell lines.

A) Mass spectrometry analysis of glucose-derived metabolites (lactate, pyruvate, malate and succinate) and B) glycolytic index “lactate/(lactate + pyruvate)” in C4–2B and PC3 24 hours-conditioned extracellular medium; n=3 wells/group, metabolite concentration values were normalized over cell-free culture medium metabolite-content and total cell number. C, D) Mitochondrial function parameters from Seahorse Mito Stress test (C) and Seahorse Glycolytic Rate assay (D) on C4–2B and PC3 cells; the experiments were performed 2 times, 1 representative experiment is shown, n=8 wells/group. Oxygen Consumption Rate (OCR) and glycolytic Proton Efflux Rate (glycoPER) values were normalized on the live cell area/well. E) Representative images of C4–2B and PC3 cells immunostained for glucose transporter 1 (GLUT-1) (scale bar: 50 μm); signal quantification is shown from 30 cells (10 cells/independent experiment); statistical significance was calculated on the average of the three experiments. F) Dose-response tumor cell growth curves in the presence of 2-DG; the experiments were performed 2 times, 1 representative experiment is shown, n=3 wells/group. G) Cartoon showing JC-1 probe potential-dependent accumulation and aggregation in functional mitochondria. H) Representative images of C2–2B and PC3 cells stained with JC-1 probe (scale bar: 20 μm); JC-1 polymer/monomer (red/green) fluorescence ratio is shown; n= 15 cells/group. All the values are presented as mean ± SD; p-values were estimated through unpaired Student’s t test (A-F) or one-way ANOVA, followed by Tukey’s HSD post-hoc test (H): (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (no *) not significant, as indicated.

Fig. 3.. C4–2B and PC3 cells treatment with IACS-10759 in vitro

A) Representative images of crystal violet-stained C4–2B and PC3 cells cultured in 2D and treated with different concentrations of IACS-10759 and dose-response curves; the experiments were performed 2 times, 1 representative experiment is shown, n=8 wells/group; the P values shown refer to analyses performed against control-treated samples. B) Representative images of Propidium Iodide (PI)- and Hoechst-stained C4–2B and PC3 cells after 1 or 6 days of treatment with IACS-10759 and graphs showing the percentage of dead (PI+) cells; the experiments were performed 2 times, 1 representative experiment is shown, n=4–5 wells/group. C) Mass spectrometry analysis of glucose-derived metabolites (lactate, pyruvate) and glycolytic index “lactate/(lactate + pyruvate)” in 24 hours-conditioned extracellular medium of C4–2B and PC3 C4–2B cells treated with 200 nM IACS-10759 and 10 μM Antimycin-A; n=3 wells/group, metabolite concentration values were normalized over cell-free culture medium metabolite-content and total cell number. D) Mitochondrial function parameters from Seahorse Mito Stress test and Seahorse Glycolytic Rate assay on C4–2B and PC3 cells cultured in 2D and treated with different IACS-10759 concentrations; the experiments were performed 2 times, 1 representative experiment is shown, n=8 wells/group. E) Representative images of C4–2B and PC3 cells treated with 30 nM or 10 μM IACS-10759 and immunostained for glucose transporter 1 (GLUT-1) (scale bar: 50 μm); signal quantification is shown from 30 cells (10 cells/independent experiment); statistical significance was calculated on the average of the three experiments. All the values are presented as mean ± SD; p-values were estimated through unpaired Student’s t test (B) or one-way ANOVA, followed by Tukey’s HSD post-hoc test (A,C-E): (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (n.s.) not significant, as indicated.

Fig. 4.. Macroscopic therapy response of PCa subcutaneous and intratibial tumor to IACS-10759 monitored by bioluminescence detection.

A) Schematic representation of the experimental schedule for subcutaneous tumors. 15 to 30 days after PC3/C4–2B tumor cells injection, mice were randomized and treated with IACS-10759 (10 mg/Kg in 0.5% methyl cellulose by oral gavage following a 5 days/week schedule). B) Representative images of subcutaneous tumor-derived bioluminescence detected by IVIS-200. C) Longitudinal monitoring of subcutaneous tumor growth; n=5 tumors per group. D) Schematic representation of the experimental schedule for intratibial tumors. 5 days after PC3/C4–2B tumor cells injection, mice were randomized and treated with IACS-10759 (10 mg/kg in 0.5% methyl cellulose by oral gavage following a 5 days/week schedule). E) Representative images of intratibial tumor-derived bioluminescence monitored by IVIS-200. F) Longitudinal monitoring of intratibial tumor growth; n=12 tibiae per group. Mean ± SD, p-values were estimated through unpaired Student’s T-test: (*) p < 0.05, (**) p < 0.01, as indicated. subQ = subcutaneous; random = randomization time; d = days. G) GSEA enrichment analysis performed on (24); Normalized enrichment scores (NES) and false discovery rate (FDR)-q values are shown. H) Tumor cell growth monitored by cell area of C4–2B and PC3 cells cultured in 2D in normoxic (18%) or hypoxic (1%) conditions in combination with different glucose concentrations (0.04–5 g/L) and treated with different IACS-10759 concentrations (0–10 μM, PC3; 0–3 nM, C4–2B); the experiments were performed 2 times, 1 representative experiment is shown, n=4 wells/group. All the values are presented as mean ± SD; p-values were estimated through unpaired Student’s T-test (C, F) or one-way ANOVA, followed by Tukey’s HSD post-hoc test (H): (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (no *) not significant, as indicated.