Disruption of glutamine transport uncouples the NUPR1 stress-adaptation program and induces prostate cancer radiosensitivity

Abstract

Background: Metabolic and stress response adaptations in prostate cancer (PCa) mediate tumor resistance to radiation therapy (RT). Our study investigated the roles of glutamine (Gln) transporters SLC1A5, SLC7A5, and SLC38A1 in regulating NUPR1-mediated stress response, PCa cell survival, metabolic reprogramming, and response to RT.

Methods: The radiosensitizing potential of GLS inhibition with CB-839 was analyzed in prostate cancer xenograft models. The level of gene expression was analyzed by RNA sequencing and RT-qPCR in the established cell lines or patient-derived tumor and adjacent non-cancerous tissues. Phosphoproteomic analysis was employed to identify the underlying signaling pathways. The publicly available PCa patient datasets, and a dataset for the patients treated with RT were analyzed by SUMO software. The key parameters of mitochondrial functions were measured by Seahorse analysis. Analysis of the general oxidative stress level and mitochondrial superoxide detection were conducted using flow cytometry. γH2A.X foci analysis was used to assess the DNA double strand break. Relative cell sensitivity to RT was evaluated by radiobiological clonogenic assays. Aldefluor assay and sphere-forming analysis were used to determine cancer stem cell (CSC) phenotype.

Results: A siRNA-mediated knockdown of Gln transporters SLC1A5, SLC7A5, and SLC38A1 resulted in significant radiosensitization of PCa cells. Consistently, the first-in-clinic glutaminase (GLS) inhibitor CB-839, combined with RT, demonstrated a synergistic effect with radiotherapy in vivo, significantly delaying tumor growth. Inhibition of Gln metabolism or knockdown of Gln transporters SLC1A5, SLC7A5, or SLC38A1 induces expression of NUPR1, a stress response transcriptional regulator, but simultaneously uncouples the NUPR1-driven metabolic stress-adaptation program. Similarly to the effect from NUPR1 knockdown, depletion of these Gln transporters led to reduced cell viability, accumulation of mitochondrial ROS, and increased PCa radiosensitivity. This effect is more pronounced in PCa cells with high dependency on OXPHOS for energy production.

Conclusions: Our work underscores the role of Gln transporters and the NUPR1-mediated stress response in PCa cell survival, oxidative stress, mitochondrial functions, and radioresistance. Our findings provide a potential therapeutic in vivo strategy to enhance the efficacy of RT and suggest a potential synergism between the depletion of Gln transporters or NUPR1 and OXPHOS inhibition.

Keywords: GLS; Glutamine transporters; Mitochondria; NUPR1; Oxidative stress; Prostate cancer; Radiation.

Fig. 1

Inhibition of glutaminase activity induces metabolic reprogramming, enhances radiotherapy efficacy and correlates with prostate cancer patient outcomes. A Schematic representation depicting the role of Gln transporters in the regulation of intracellular Gln homeostasis and mechanism of action of GLS inhibitor, CB-839, on PCa radiosensitization. Created in BioRender. Dubrovska, A. (2025) https://BioRender.com/ar8jisq. B Schematic representation of the in vivo experimental design. DU145 prostate cancer cells were subcutaneously implanted into the hind leg of Immunodeficient Rj:NMRI-Foxn1^nu/nu mice. Once tumors reached approximately 100 mm.³, mice were randomized into four treatment groups: vehicle control, CB-839 alone (oral administration, 180 mg/kg/day for 3 days), radiotherapy (RT) alone (2 Gy/day for 5 consecutive days), and combination therapy (CB-839 plus RT). C Tumor volume doubling time presented as a bar chart. Error bars represent standard deviation (SD). The combination therapy group showed a statistically significant increase in tumor doubling time compared to control and other treatment groups (*p < 0.05, **p < 0.01). D Tumor growth modeling based on measured tumor volumes, depicted as tumor volume fold change over time. The combination therapy significantly delayed tumor growth compared to other groups (*p < 0.05, **p < 0.01, ***p < 0.001). E Glutaminase (GLS) activity calculated as the plasma Glu/Gln ratio. CB-839-treated groups exhibited significantly reduced GLS activity compared to vehicle control group (**p < 0.01, ***p < 0.001). F Kaplan–Meier survival curves for prostate cancer patients (n = 67) with high and low GLS expression levels from the OncoRay cohort. Patients with high GLS expression had significantly higher rates of prostate-specific antigen (PSA) relapse compared to those with low GLS expression (*p < 0.05). G Violin plots showing glutaminase (GLS) activity (assessed by the Glu/Gln ratio) in DU145 cells treated with CB-839 (GLS inhibitor) or DMSO (control) (*p < 0.05). H qRT-PCR analysis of SLC1A5, SLC7A5, and SLC38A1 expression in DU145 cells treated with CB-839 or DMSO (control). Error bars represent SD (*p < 0.05, **p < 0.01). I qRT-PCR analysis of SLC1A5, SLC7A5, SLC38A1, MYC, and GLS expression in DU145 (left), LNCaP (middle), and PC3 (right) cells upon siRNA-mediated knockdown of MYC and GLS. Error bars represent SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 2

Depletion of amino acid transporters reduces cell viability and enhances radiosensitivity in prostate cancer cells. A Clonogenic survival curves for DU145 (left), LNCaP (middle), and PC3 (right) cells following transporter knockdown and exposure to increasing doses of radiation (0–6 Gy). Survival fractions were calculated, and curves were fitted using the linear-quadratic model S(D)/S(0) = exp(–αD – βD²). Error bars represent SD (*p < 0.05, **p < 0.01, ***p < 0.001). B Cell viability of DU145 and LNCaP cells upon siRNA-mediated knockdown of SLC1A5, SLC7A5, and SLC38A1, measured by CellTiter-Glo assay. Data are presented as a percentage normalized to control (siSCR). Error bars represent SD (*p < 0.05, **p < 0.01, ***p < 0.001). C Analysis of the expression changes of 84 apoptosis-related genes listed in Supplementary Table 10 in response to SLC1A5, SLC7A5 or SLC38A1 knockdown compared to Scr siRNA control. D Bar chart showing the number of γH2AX foci per nucleus in DU145 and LNCaP cells following transporter depletion under sham-irradiated and 4 Gy irradiated conditions. Error bars represent SD (*p < 0.05). E Representative examples of the γH2A.X foci staining in DU145 and LNCaP cells transfected with SLC1A5, SLC7A5, and SLC38A1 siRNAs. Cells transfected with Scr siRNA were used as a control. The images were taken 24 h after cell irradiation with 4 Gy of X-rays. The scale bar is 20 µm

Fig. 3

Depletion of amino acid transporters affects stem cell-like properties in PCa cells. A Schematic representation depicting the role of Gln transporters in the regulation of intracellular α-KG levels as a result of GLS-driven catabolism of Gln. Created in BioRender. Dubrovska, A. (2025) https://BioRender.com/f2qoteo. B Expression of epigenetic regulators in response to SLC1A5, SLC7A5 or SLC38A1 knockdown compared to Scr siRNA control. Asterisks represent the statistically significant changes. C Inverse cumulative distribution plots showing sphere size versus number in DU145 cells under sham, and 4 Gy irradiation following transporter knockdown. Error bars represent SEM. The bar chart represents the percentage of spherogenic cells capable of forming spheres for each cell line. Error bars represent SD (*p < 0.05, ***p < 0.001). D Inverse cumulative distribution plots showing sphere size versus number in LNCaP cells under sham, and 4 Gy irradiation following transporter knockdown. Error bars represent SEM. The bar chart represents the percentage of spherogenic cells capable of forming spheres for each cell line. Error bars represent SD. E Aldefluor assay results showing the percentage of ALDH-positive cells in DU145 cells following transporter knockdown. Error bars represent SD (*p < 0.05, **p < 0.01). F Aldefluor assay results showing the percentage of ALDH-positive cells in LNCaP cells following transporter knockdown. Error bars represent SD

Fig. 4

Depletion of amino acid transporters induces metabolic reprogramming. A Analysis of the expression changes of 79 genes related to the mitochondrial energy metabolism listed in Supplementary Table 10 in response to SLC1A5, SLC7A5 or SLC38A1 knockdown compared to Scr siRNA control. B Gene Set Enrichment Analysis (GSEA) for genes significantly up- or down-regulated upon SLC1A5, SLC7A5, SLC38A1 knockdown revealed that deregulated genes are associated with an activation of oxidative phosphorylation. C The oxygen consumption rate (OCR) was measured using a Seahorse XF Analyzer in DU145 and LNCaP cells. Error bars represent SEM. D The ratio of oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) in DU145 and LNCaP cells. The OCR/ECAR ratio is a metabolic indicator of the balance between mitochondrial respiration and glycolysis. Error bars represent SD (*p < 0.05, **p < 0.01). E The ratio of oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) in DU145 and LNCaP cells in response to SLC1A5, SLC7A5 or SLC38A1 knockdown compared to Scr siRNA control. Error bars represent SD (*p < 0.05, **p < 0.01, ***p < 0.001). F Mitochondrial superoxide levels in DU145 and LNCaP cells measured by MitoSOX Red staining following transporter knockdown under sham and 6 Gy-irradiated conditions. Error bars represent SD (*p < 0.05). G The ratios between mitochondrial ROS measured by MitoSOX and cytoplasmic ROS measured by CM-H2DCFDA in LNCaP and DU145 cells in response to SLC1A5, SLC7A5 or SLC38A1 knockdown compared to Scr siRNA control. Cells were either sham irradiated (0 Gy) or irradiated with 6 Gy of X-rays. Error bars represent SD (*p < 0.05). H Differential expression of genes involved in cellular antioxidant defense and redox homeostasis in LNCaP vs. DU145 cells. Asterisks represent the statistically significant changes. I Effect of the SLC1A5, SLC7A5, SLC38A1 knockdown in the expression of genes involved in cellular antioxidant defense and redox homeostasis in LNCaP and DU145 cells. Asterisks represent the statistically significant changes. J Mitochondrial respiration parameters in DU145 and LNCaP cells following transporter knockdown, measured using the SeaHorse XF MitoStress test kit. Parameters include non-mitochondrial oxygen consumption, ATP production, basal respiration, spare respiratory capacity, maximal respiration, and proton leak. Error bars represent SD (*p < 0.05, **p < 0.01, ***p < 0.001). K Scheme demonstrating the effects of AATs on the mitochondrial adaptability of PCa cells against ROS. Created in BioRender. Dubrovska, A. (2025) https://BioRender.com/v3uublo

Fig. 5

Disrupting Gln metabolism by chemical inhibition of GLS, by Gln starvation or by knockdown of AATs induces NUPR1-mediated stress response, alters the NUPR1-target gene response. A qRT-PCR analysis of NUPR1 expression in DU145 cells treated with CB-839 or DMSO (control). Error bars represent SD (*p < 0.05). B CB-839 treatment of patient-derived tumor (T) tissue (n = 1) and adjacent patient-derived benign prostatic hyperplasia (BPH) (n = 1) increased expression of SLC1A5, SLC7A5, SLC38A1, and NUPR1. Error bars represent the SD of technical replicates. The upper scheme is created in BioRender. Dubrovska, A. (2025) https://BioRender.com/82t9ncl. C Effect of the SLC1A5, SLC7A5, SLC38A1 knockdown on the NUPR1 expression in DU145 and LNCaP cells. Asterisks represent the statistically significant changes. D Pearson correlation of mRNA expression levels of SLC1A5, SLC7A5, SLC38A1 and NUPR1 genes in the TCGA PRAD patient cohort (n = 498), MSKCC PRAD patient cohort (n = 150), and OncoRay patient cohort (n = 68), *p < 0.05. E Gene Set Enrichment Analysis (GSEA) revealed negative enrichment of the NUPR1 target genes (n = 261) in response to SLC1A5, SLC7A5, and SLC38A1 knockdown. F Gene Set Enrichment Analysis (GSEA) revealed negative enrichment of the NUPR1 target genes (n = 261) in response to Gln starvation. G A g:Profiler (g:GOSt) pathway enrichment analysis identified enrichment for REAC Cell Cycle for the NUPR1-responsive genes that are upregulated in response to the SLC1A5, SLC7A5, SLC38A1 knockdown and enrichment for KEGG HIF1 signaling pathway for the NUPR1-responsive genes that are downregulated in response to the SLC1A5, SLC7A5, SLC38A1 knockdown. The heatmaps for these genesets are provided in Supplementary Fig. 9D. H Gln starvation or knockdown of Gln transporters SLC1A5, SLC7A5, or SLC38A1 uncouples the NUPR1 stress-adaptation program and induces prostate cancer radiosensitivity. The knockdown of SLC1A5, SLC7A5, and SLC38A1 upregulates some NUPR1-mediated defensive mechanisms, including genes involved in cell cycle progression and DNA repair. At the same time, the knockdown of these AATs downregulates a set of NUPR1-regulated genes, which are key regulators of glycolysis. These metabolic changes upregulate the expression of the OXPHOS-driven genes, cell dependence on OXPHOS, and the accumulation of mitochondrial ROS. Thus, AAT knockdown or NUPR1 inhibition results in tumor radiosensitization

Fig. 6

Targeting NUPR1 reduces cell viability, modulates mitochondrial superoxide levels, and enhances radiosensitivity in prostate cancer cells. A Schematic representation depicting the role of Gln transporters in the regulation of intracellular Gln homeostasis and mechanism of action of GLS inhibitor, CB-839, and NUPR1 inhibitor, ZZW-115, on PCa radiosensitization. Created in BioRender. Dubrovska, A. (2025) https://BioRender.com/qxq81ll. B A top 20 enriched Gene Ontology (GO) terms in the set of the NUPR1 target genes (n = 261). The gene list is provided in Supplementary Table 7. The statistical data for GO analysis is provided in Supplementary Table 8. C Cell viability of DU145 and LNCaP cells upon NUPR1 knockdown, measured by CellTiter-Glo assay. Error bars represent SD (*p < 0.05, **p < 0.01). D Mitochondrial superoxide levels in DU145 and LNCaP cells measured by MitoSOX Red staining following NUPR1 knockdown under sham and irradiated conditions. Error bars represent SD (*p < 0.05). E Bar chart showing the number of γH2AX foci per nucleus in DU145 and LNCaP cells following NUPR1 depletion under sham-irradiated and 4 Gy irradiated conditions. Error bars represent SD (**p < 0.01). F Representative examples of the γH2A.X foci staining in DU145 and LNCaP cells transfected with NUPR1 siRNAs. Cells transfected with Scr siRNA were used as a control. The images were taken 30 min after cell irradiation with 4 Gy of X-rays. The scale bar is 20 µm. G Clonogenic survival curves for DU145 and LNCaP cells following NUPR1 knockdown and radiation exposure. Survival fractions were calculated, and curves were fitted using the linear-quadratic model. Error bars represent SD (*p < 0.05, ***p < 0.001). H Clonogenic survival curves for DU145 and LNCaP cells following ZZW-115 inhibitor treatment and radiation exposure. Survival fractions were calculated, and curves were fitted using the linear-quadratic model. Error bars represent SD (**p < 0.01). I qRT-PCR analysis of the indicated gene expression in DU145 and LNCaP cells upon siRNA-mediated knockdown of NUPR1 ± 6 Gy irradiation. Error bars represent SD (*p < 0.05, **p < 0.01, ***p < 0.001). J Cell-line specific consequences of NUPR1 depletion on the radiation response. Simplified cartoon integrating our qPCR, ROS, and clonogenic data for LNCaP and DU145 cell lines following NUPR1 knockdown combined with irradiation. Created in BioRender. Dubrovska, A. (2025) https://BioRender.com/sog8o4l