Targeting endoplasmic reticulum stress and nitroso-redox imbalance in neuroendocrine prostate cancer: the therapeutic role of nitric oxide

Abstract

Neuroendocrine prostate cancer (NEPC) is an aggressive and therapy-resistant subtype of prostate cancer characterized by high levels of endoplasmic reticulum (ER) stress and metabolic dysregulation. The subsequential metabolic adaptations in the cancer cells reinforce survival mechanisms that contribute to therapy resistance and metastasis. The oncogenic driver neuroblastoma-derived MYC (MYCN) exacerbates ER stress by increasing calcium ion efflux from the ER into mitochondria, promoting glycolytic and oxidative stress. Here, we demonstrate that nitric oxide (NO) signaling is dysregulated in NEPC, thus allowing impaired S-nitrosylation of MYCN and uncontrolled ER stress. We show that exogenous NO supplementation restores MYCN S-nitrosylation at Cys4, Cys186, and Cys464. This re-establishment significantly reduces ER stress markers, inhibits the unfolded protein response (UPR), and suppresses NEPC cell proliferation and colony formation in vitro. In an orthotopic NEPC murine model, NO treatment led to a substantial reduction in tumor burden and metastasis to the liver and brain, with corresponding decreases in chromogranin and synaptophysin expression. Additionally, NO supplementation attenuated glycolytic stress by limiting calcium-mediated mitochondrial dysfunction and modulating metabolic pathways. Our findings uncover a direct mechanistic link between MYCN-driven ER stress and NEPC progression and highlight NO supplementation as a potential therapeutic strategy to counteract lineage plasticity and metabolic adaptations in NEPC. These results provide a compelling rationale for further investigation into NO-based therapies as a novel intervention for NEPC, a cancer subtype with limited treatment options and poor prognosis.

Fig. 1. An enrichment analysis showing ER stress biomarkers correlate with prostate cancer (PCa) progression and survival outcomes.

A The expression of ER stress markers in PCa patients was obtained from the Decipher Grid. The box plots span the 25th to 75th percentiles, and the expression is represented as log10log10 (reads per kilobase million, RPKM). The median (midline of box plots) and maximum and minimum (upper and lower bounds of whiskers) values are depicted. Significance was determined by a non-parametric Wilcoxon signed-rank test (two-sided), and P values correspond to the comparison between Gleason score groups with other subgroups of PCa patients. B The plots show a significant positive correlation between ER stress markers (IRE1, BiP, PDI, PERK, CHOP, and XBP1) with NOS3 in the Decipher Grid data. C Kaplan–Meier survival curves segregated by BiP, CHOP, CANX, ERO1A, MYCN, NOS3, and PDI expression. Overall survival is significantly diminished for individuals with high expression. P values were calculated using a log-rank (Mantel–Cox) test.

Fig. 2. MYC enhances ER stress responses and nitroso-redox imbalance in PCa Cells.

A Western blot analysis showing androgen receptor (AR) levels in MyCaP, MyCaPshAR, and MyCaPAPIPC cell lines. GAPDH serves as a loading control. B Bar graph depicting percent Mitomass in various PCa cell lines (LNCaP, 22Rv1, H660, MyCaP, MyCaPshAR, and MyCaPAPIPC). C Western blot comparing ER stress markers between LNCaP and H660 cell lines. GAPDH serves as a loading control. D Western blot showing ER stress marker expression in 22Rv1 cells with and without stable MYCC or MYCN overexpression. GAPDH serves as a loading control. E Bar graphs comparing GSNOR activity between LNCaP and H660 cells (left panel) and between MyCaP and MyCaPAPIPC cells (right panel). Data represent mean ± standard deviation from three independent biological replicates (p < 0.001, two-way ANOVA).

Fig. 3. Nitric oxide supplementation inhibits MYC and inhibits ER stress responses in prostate cancer cells.

A Bar graph showing overall calcium levels in LNCaP, 22Rv1, and H660 cells with or without GSNO treatment (50 μM) within 2 h of treatment. B Bar graph depicting the number of mitochondria in LNCaP, 22Rv1, and H660 cells with or without GSNO treatment (50 μM), assessed using mitochondrial dye. C Results of Seahorse XF Mito Stress Tests on LNCaP and H660 cells with or without GSNO treatment. The graph shows a double plot of Extracellular Acidification Rate (ECAR) from three independent biological replicates. D Western blot analysis showing the inhibitory effects of GSNO treatment on ER stress markers in H660 cells. GAPDH serves as a loading control. E Quantitative real-time PCR results demonstrating the inhibitory effects of GSNO treatment on neuroendocrine prostate cancer (NEPC) markers in H660 cells. Data represent mean ± standard deviation from three independent biological replicates (p < 0.001). F Western blot analysis showing the inhibitory effects of GSNO treatment on ER stress markers in MYCN overexpressing 22Rv1 cells. GAPDH serves as a loading control.

Fig. 4. RNA sequencing reveals GSNO-induced transcriptional changes in H660 cells.

A Schematic illustrating the RNA sequencing workflow for H660 cells treated with 50 μM GSNO for 48 h. B Heatmap depicting significantly differentially expressed genes (fold change > 2, q-value < 0.05) following 48-h GSNO treatment. C Visualization of differentially expressed genes (DEGs) with fold change > 1.5 and adjusted P < 0.05. Upregulated genes are shown in red, down-regulated genes in blue. D Bar graph highlighting key downregulated pathways, including ER stress signaling (featuring genes EXT1, TRPS2, CHDS, CDC42, and RAC), oxidative stress, cell cycle regulation, DNA damage response, L1CAM interactions, and TGF-beta signaling. E Bubble plot illustrating upregulated molecular processes. F Bubble plot showing enriched cellular processes. G Bubble plot depicting upregulated biological processes, including protein synthesis and metabolic pathways such as eukaryotic translation elongation, ribosomal protein synthesis, protein processing in the ER, fatty acid oxidation, and amino acid metabolism.

Fig. 5. Exogenous induction of NO treatment demonstrates an inhibitory role.

A bar graph showing the inhibitory effects of GSNO treatment on the colony-forming capabilities of DU145, PC3, and H660 cells. B Line graph illustrating the inhibitory effects of GSNO on cell proliferation (MTT assay) in DU145, PC3, and H660 cells over time. C effects of GSNO (50 μM) treatment on H660, DU145, and PC3 3D tumoroids upon 9 days of treatment. D Western blot results show neuroendocrine markers such as chromogranin, synaptophysin, and MYCN levels in PC3, DU145, and H660 protein lysates before and after GSNO treatment. E Bar graph demonstrating the pro-apoptotic effects of GSNO treatment on H660 cells and 22Rv1 cells overexpressing MYC. Data represent mean ± standard deviation from three independent biological replicates (p < 0.001, two-way ANOVA).

Fig. 6. Exogenous Nitric Oxide Inhibits NEPC Tumor Growth and Metastasis.

A Schematic illustrating the protocol used to evaluate the effects of NO stimulation on NEPC tumor burden. B Left: Representative bioluminescence imaging (BLI) of NOD-SCID mice (n = 8) orthotopically injected with H660-Luciferase tagged cells at week 0 and week 4. Right: Line graph showing mean tumor volumes over time for GSNO-treated and control groups. C Bar graphs depicting: Tumor volumes, Animal weights, Tumor weights in mice treated with or without GSNO (10 mg/kg/day, intraperitoneal). D Ex-vivo Imaging of Metastases: Left: Representative ex-vivo bioluminescence images of liver, brain, bone, and lung tissues highlighting metastatic areas. Right: Bar graph showing mean normalized photon flux/second (±SEM) from metastases in each tissue type. Student’s t-test, **p < 0.001. E Hematoxylin and eosin (H&E)-stained sections of liver and brain tissues. Arrows indicate metastatic lesions. F Images showing immunohistochemical staining for chromogranin (CHG) neuroendocrine markers in liver and brain tissue sections. G Images showing immunohistochemical staining for pIRE1-α, synaptophysin (SYP), and chromogranin A (CHG) in tumor sections from each group. Scale bar: 20 μm. H Western blot results showing NEPC and ER stress markers levels in tumor lysates from GSNO-treated and control mice. I Western blot results showing levels of EMT markers in tumor lysates from GSNO-treated and control mice.

Fig. 7. NO supplementation mitigates ER stress and inhibits tumor growth in AR-knockout prostate cancer models.

A Comparison of ER stress marker expression (PERK, IRE1α, XBP1, and HSPA5) between LNCaP and LNCaPAPIPC cells, demonstrating significantly elevated expression in the AR-knockout (LNCaPAPIPC) model. B Gene expression analysis of ER stress markers following GSNO treatment (0, 50, and 100 µM) in LNCaPAPIPC cells, showing a dose-dependent reduction in expression. C, D Tumor burden analysis in NSG mice subcutaneously grafted with LNCaP (non-castrated) and LNCaPAPIPC (castrated) cells. Mice were treated with GSNO (10 mg/kg/day, IP) for four weeks, resulting in a significant reduction in tumor size in both groups. E, F Western blot and immunohistochemistry (IHC) analysis of tumor tissues from LNCaPAPIPC-grafted mice, revealing increased expression of ER stress markers (BiP, ERO1α, and PDI) in untreated tumors, which was significantly inhibited following GSNO treatment. Together, these findings reinforce the role of NO supplementation in modulating ER stress and reducing tumor burden in AR-deficient prostate cancer models, complementing observations from the NEPC model.

Fig. 8. S-nitrosylation of MYCN by GSNO and its effects on protein binding and ER stress in NEPC cells.

A GPS-SNO 1.0 software predicted 16 cysteine residues on the MYCN protein susceptible to S-nitrosylation, with Cys4, Cys186, and Cys464 exhibiting the highest thresholds for nitrosylation (cutoff 2.443). B Biotin-switch assay confirmed S-nitrosylation of MYCN upon GSNO treatment, specifically affecting the heavy chain of MYCN. C Western blot showing the protein expression of ER stress markers (PDI, BIP, and CHOP) in 22Rv1, DU145, and H660 cells, post-transfection with MYCN plasmid-containing mutations at Cys4, Cys186, and Cys464 sites, followed by GSNO treatment (50 μg) for 48 h.

Fig. 9. Proteomic analysis of GSNO treatment effects on protein interactions in NEPC tumors.

A Schematic illustration of the immunoprecipitation followed by mass spectrometry (IP-MS) protocol used to analyze protein interactions in control and GSNO-treated mice. B Comparison of protein interactions between control and GSNO-treated mice. NO supplementation via GSNO inhibited the binding of proteins involved in key cellular processes such as RNA/DNA metabolism, ER protein import, Protein folding, and Chromatin remodeling. C–F Gene Ontology and pathway analyses were performed on down-regulated proteins in nuclear lysates from tumor and GSNO-treated tumor samples. The results are presented in bubble plots, where bubble size represents the number of genes and color intensity indicates statistical significance. C Human Cyt 2016: Bubble plot illustrating enriched cytological terms associated with down-regulated proteins. This analysis provides insights into the cellular compartments and structures affected by GSNO treatment. D GO Biological Process: Bubble plot showing enriched cellular processes impacted by GSNO treatment. This analysis highlights the biological functions most significantly altered in response to NO supplementation. E KEGG Pathway Analysis: Bubble plot depicting enriched cellular pathways affected by GSNO treatment. This analysis reveals the broader cellular systems and networks influenced by NO-mediated protein interaction changes. F MSigDB Hallmark: Bubble plot showing enrichment of hallmark gene sets, providing a high-level overview of the cellular states and processes altered by GSNO treatment.