NDUFS6 promotes neuroblastoma progression and represents a potential therapeutic target

Introduction

Neuroblastoma (NB) is the most common malignant extracranial solid tumor in children. Primary tumors typically arise along the sympathetic nervous system, particularly in the adrenal medulla, and are believed to originate from sympathetic neuron precursors and chromaffin cell precursors (1). Approximately half of NB cases are classified as high-risk NB (HR-NB) (2). Despite advances in intensive multimodal therapy, including multi-agent chemotherapy, surgical resection, high-dose chemotherapy with autologous stem cell transplantation, and GD2-targeted immunotherapy, the prognosis for HR-NB remains poor, with a 5-year event-free survival rate of only 51% (2). Outcomes are particularly dismal for patients who relapse or exhibit refractory disease, with a median progression-free survival of 6.4 months (3,4). HR-NB thus continues to represent a major clinical challenge and a significant burden in pediatric oncology (5-7), underscoring the urgent need to identify novel therapeutic targets to improve patient outcomes.

Recent studies have revealed that HR-NB undergoes metabolic reprogramming characterized by elevated oxidative phosphorylation (OXPHOS), enabling tumor cells to meet increased bioenergetic and biosynthetic demands (8-10). This metabolic adaptation may represent a therapeutic vulnerability. Inhibiting key components of the OXPHOS pathway has been shown to suppress tumor growth (8). NADH:ubiquinone oxidoreductase subunit S6 (NDUFS6), a structural subunit of mitochondrial respiratory chain complex I (CI), is essential for maintaining CI integrity and function (11). NDUFS6 has been implicated in the development and progression of multiple malignancies, with several oncogenic pathways acting through its regulatory network (12-16). In multiple myeloma, protein arginine methyltransferase 1 (PRMT1) enhanced oxidative phosphorylation by upregulating NDUFS6, thereby promoting cellular proliferation and tumorigenesis (12). Moreover, in breast cancer, retinoid orphan nuclear receptor alpha interacts with NDUFS6 and suppresses its expression, leading to reduced mitochondrial reactive oxygen species (ROS) production and attenuation of ROS-driven mammary tumor progression (14). Nevertheless, the specific role of NDUFS6 and its mechanistic contributions to NB biology remain largely unexplored.

In this study, elevated NDUFS6 expression was observed in HR-NB tumor cells in the single-cell RNA sequencing (scRNA-seq) dataset. To further investigate its clinical relevance, NDUFS6 expression was analyzed using public transcriptomic datasets and assessed in NB tumor tissues via immunohistochemistry. Functional assays were conducted in NB cell lines with stable NDUFS6 overexpression or knockdown to investigate its effects on tumor cell proliferation, migration, and invasion. Furthermore, transcriptomic profiling was performed to elucidate downstream signaling pathways regulated by NDUFS6. Finally, structure-based virtual screening and preliminary drug sensitivity testing were undertaken to identify candidate small-molecule inhibitors targeting NDUFS6 protein, thereby assessing its potential as a therapeutic target in NB. We present this article in accordance with the MDAR reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2026-1-0095/rc).

Methods

Specimen collection

Formalin-fixed, paraffin-embedded (FFPE) tumor specimens from 34 patients with NB were obtained from the Department of Oncology at the Children’s Hospital of Fudan University. 13 patients were classified as HR-NB, while 21 were categorized as intermediate- or low-risk NB. The inclusion and exclusion criteria are as follows; inclusion criteria: (I) pathologically confirmed diagnosis of NB; (II) availability of FFPE tumor tissue specimens suitable for analysis; exclusion criteria: (I) inadequate specimen quality: FFPE specimens with insufficient tissue quantity, low tumor cellularity precluding meaningful analysis, or severe degradation due to improper pre-analytical handling; (II) missing key clinical data: absence of essential clinicopathological information required for analysis, including but not limited to age, disease stage, risk group, MYCN status, treatment details, and follow-up data; (III) prior neoadjuvant therapy: administration of chemotherapy or radiotherapy before the acquisition of the tumor specimen used in this study. Informed consent was obtained from the legal guardians of all participants prior to enrollment. The detailed information is presented in Table S1. The study protocol was approved by the Institutional Review Board of Children’s Hospital of Fudan University [No. 2020(169)]. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

NB cell lines

NB cell lines SK-N-BE(2) (RRID: CVCL_0528) and SH-SY5Y (RRID: CVCL_0019) were purchased from the Cell Resource Center, Chinese Academy of Sciences. The cells were cultured in Minimum Essential Medium (Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (Gibco), supplemented with sodium glutamate (Gibco), pyruvate (Gibco), and a mixture of non-essential amino acids (Gibco), and maintained in a humidified incubator at 37 °C with 5% CO₂.

Construction of lentiviral vectors and establishment of stable cell lines

Lentiviral vectors containing the full-length human NDUFS6 coding sequence and short hairpin RNAs (shRNAs) targeting NDUFS6 (target sequence: 5’-CGAGGTGGAGACTCGGGTGAT-3’) were constructed to generate stable overexpression (OE) and knockdown (KD) cell lines, respectively. Corresponding empty vectors were used as negative controls. Cells were incubated with lentiviral particles in culture medium supplemented with 5 µg/mL polybrene (Sigma TR-1003) for 16 hours. Selection with 2 µg/mL puromycin (A1113803) began 48 hours post-infection and continued for approximately one week.

Quantitative real-time polymerase chain reaction (RT-qPCR)

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and RNA concentration was measured with NanoDrop 2000 spectrophotometer (Thermo Scientific). Human NDUFS6 primers were retrieved from the PrimerBank database (17). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. RT-qPCR reactions were prepared using the Universal Blue qPCR SYBR Green Master Mix Kit (Yesenbio 11184ES08) and conducted on the QuantStudio 3 Real-Time PCR System (Applied Biosystems). Melting curve analysis was performed to confirm specificity, and relative expression levels were calculated using the 2^−ΔΔCT method. Primer sequences were as follows: NDUFS6: F: 5'-TTCGCTTTGTAGGTCGTCAGA-3', R: 5'-CCATCGCACGCTATCACCC-3'; GAPDH: F: 5'-CTCATAGTCGTATCAGGGGTCG-3', R: 5'-ACACAGTCGTTTTCTGTCCAC-3'.

Immunohistochemistry

For immunohistochemistry, paraffin-embedded NB tissue samples were sectioned at 5 µm thickness. Sections were deparaffinized in xylene, rehydrated through graded ethanol, and subjected to antigen retrieval by microwave heating in Tris/EDTA buffer (pH 9.0) for 15 minutes. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 20 minutes, followed by blocking with 5% BSA for 1 hour at room temperature. Sections were incubated overnight at 4 ℃ with a recombinant anti-NDUFS6 rabbit monoclonal antibody (clone EPR15957-37, Abcam, cat# ab195808) at a dilution of 1:250. According to the manufacturer’s validation data, this antibody has been validated for IHC-P using human tissue samples and is a recombinant RabMAb^® with high batch-to-batch consistency. After washing with PBS, sections were incubated with HRP-conjugated secondary antibody for 30 minutes at room temperature. Signal was visualized using DAB substrate, and sections were counterstained with hematoxylin. Average optical density (AOD) was quantified using ImageJ software (NIH, Bethesda, MD, USA) to evaluate NDUFS6 expression levels. IHC staining was evaluated independently by two pathologists blinded to clinical outcome data.

Cell counting Kit-8 assay

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay (Yeasen, cat# 40203ES60) according to the manufacturer’s instructions. SK-N-BE(2) and SH-SY5Y cells were seeded into 96-well plates at densities of 1,700 and 2,200 cells per well, respectively. At 0, 24, 48, 72, and 96 hours, 10 µL of CCK-8 reagent was added to each well, followed by a 2-hour incubation at 37 ℃. Absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

To evaluate the response to guanosine-5'-triphosphate, NDUFS6-OE and NC cells were seeded in 96-well plates at 5,000 cells per well in 100 µL of culture medium. Each concentration condition was set up in triplicate technical wells per plate. After overnight incubation to allow cell attachment, cells were treated with increasing concentrations of guanosine-5'-triphosphate (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 10 µM) for 72 hours. Subsequently, 10 µL of CCK-8 solution was added to each well and incubated for 2 hours at 37 ℃, and absorbance was measured at 450 nm. The entire experiment was performed three independent times (three biological replicates). Cell viability was calculated as a percentage relative to untreated control cells (set as 100%). Dose-response curves and half-maximal inhibitory concentration (IC₅₀) values were generated using GraphPad Prism 11 software (GraphPad Software, San Diego, CA, USA) by nonlinear regression (log[inhibitor] vs. response, variable slope). Data are presented as mean ± SD from three independent experiments.

Colony formation assay

For colony formation assays, SK-N-BE(2) and SH-SY5Y cells were seeded in 6-well plates at 1,200 and 1,700 cells per well, respectively. Culture medium was refreshed every three days. After approximately two weeks, colonies were fixed with 4% paraformaldehyde and stained with 1% crystal violet. Colony numbers were quantified using ImageJ software.

In vitro migration and invasion assays

Transwell migration and invasion assays were performed using 24-well plates with 8 µm pore-size inserts (Corning, Corning, NY, USA). SK-N-BE(2) and SH-SY5Y cells were serum-starved for 24 hours in medium containing 0% and 3% FBS, respectively. For invasion assays, Transwell inserts were pre-coated with Matrigel diluted in serum-free medium. A total of 1×10⁵ SK-N-BE(2) cells and 1.5×10⁵ SH-SY5Y cells were suspended in serum-free medium and seeded into the upper chambers, while medium containing 10% FBS was added to the lower chambers as a chemoattractant. After 24 or 48 hours of incubation, cells that had migrated to the lower membrane surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Cells were counted in five randomly selected fields under a 200× microscope.

Bioinformatic analysis of public datasets

The single-cell RNA sequencing dataset GSE137804 (18) was obtained and analyzed using the Seurat package (v5.0.2) in R (v4.2.3). Tumor cells were isolated and reanalyzed. The expression of NDUFS6 across clinical subgroups was visualized using the DimPlot and VlnPlot functions. Statistical comparisons between groups were performed using unpaired t-tests.

Additionally, the bulk transcriptomic dataset “Tumor Neuroblastoma-SEQC-498-RPM seqcnb1 (GSE49710) (19)” from the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl) was analyzed to investigate associations between NDUFS6 expression and clinical parameters, including INSS stage, MYCN amplification status, disease progression, and overall prognosis.

Library preparation for bulk RNA-seq

RNA extraction was performed as previously described. RNA concentration was quantified using the Qubit fluorometer (Thermo Fisher Scientific), and integrity was assessed by gel electrophoresis. Strand-specific libraries were prepared from three biological replicates using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA), and sequencing was carried out on the Illumina NovaSeq 6000 platform. The raw sequencing data have been deposited in the GEO database under accession number GSE308228.

Data analysis for RNA-seq

Raw sequencing reads were trimmed for adapters using Skewer, and quality control was performed with FastQC (v0.11.2). Clean reads were aligned to the human reference genome (hg38) using STAR. Transcript assembly was conducted with StringTie (v1.3.1c), and expression levels were quantified as fragments per kilobase of transcript per million mapped reads (FPKM) using Perl.

Differentially expressed genes (DEGs) between the groups were identified using DESeq2 following the standard workflow of the DESeq2 package, with thresholds set at |log₂FC| ≥1 and FDR ≤0.05. The Benjamini-Hochberg method was applied to adjust P values for multiple testing, and we consistently reported FDR-adjusted P values (q-values) for all differential expression analyses. A threshold of FDR <0.05 was applied to define statistically significant DEGs. DEGs were subjected to Gene Ontology (GO) functional enrichment analysis using TopGO. Fisher’s exact test was employed to calculate P values, and the Benjamini-Hochberg method was used to correct for multiple testing. Pathway enrichment analyses were also conducted using Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and DisGeNET databases. Significantly enriched pathways were identified based on a threshold of P<0.05 and the inclusion of at least two associated genes. Gene Set Enrichment Analysis (GSEA) was performed using the GSEA package.

ATP production assays

Intracellular ATP levels were measured using the ATP Microplate Assay Kit (Absin, cat# abs580117) according to the manufacturer’s instructions. Briefly, NDUFS6-overexpressing and control cells were lysed in PBS containing 0.1% Triton X-100 on ice for 30 minutes. The lysates were centrifuged at 10,000 ×g for 10 minutes at 4 ℃, and the supernatants were collected. ATP standard solutions were prepared by serial dilution ranging from 0.0039 to 1 μM using the provided ATP Assay Buffer. Fifty microliters of each sample or standard were added to a white 96-well plate, followed by 50 μL of ATP detection working solution (containing ATP Assay Buffer, D-luciferin, Cofactor, and Luciferase). After a 5-minute incubation at room temperature, the relative luminescence units (RLU) were measured using a chemiluminescence microplate reader. ATP concentrations were calculated based on the standard curve generated from the RLU values of the ATP standards. All assays were performed in triplicate.

Virtual screening

Protein preparation was the initial step. The three-dimensional structure of human NDUFS6 (PDB ID: 5XTB) was obtained from the RCSB Protein Data Bank. Preprocessing was performed using the Protein Preparation Wizard and Receptor Grid Generation modules. Compounds downloaded from the MedChemExpress (MCE) Bioactive Compound Library database were prepared using the LigPrep module of the Schrödinger software suite. Virtual screening was conducted using the Virtual Screening Workflow module, and molecular docking was performed with Glide. High-throughput virtual screening, standard precision screening, and extra precision screening were sequentially applied to identify the final set of small-molecule candidates. Following manual review, the top 200 compounds based on docking scores were selected as the final candidates.

Drug sensitivity assay

The 30 selected compounds were initially diluted in DMSO at a 250× concentration and subsequently diluted in culture medium to reach a final working concentration of 10×. After enzymatic digestion and centrifugation, the cells were resuspended in culture medium at the appropriate concentration. Cells were cultured at 37 ℃ with 5% CO₂ for 48 or 72 hours. Cell viability was assessed using CellCounting-Lite 2.0^® reagent. Plates were equilibrated to room temperature, shaken at 110 rpm for 2 minutes, incubated for 10 minutes to stabilize the signal, and fluorescence intensity was measured.

Statistical analysis

Statistical analyses were conducted for RT-qPCR, Western blot densitometry, colony formation and Transwell assays across NDUFS6 OE and KD groups. The Shapiro-Wilk test was used to assess the normality of the data distribution, and the F-test was applied to evaluate the homogeneity of variance. Depending on the results, either an unpaired Student’s t-test or Welch’s t-test was applied. CCK-8 assay results were analyzed using two-way ANOVA. A P value <0.05 was considered statistically significant. GraphPad Prism (v10.1.2) was used for data visualization.

Results

NDUFS6 is upregulated in HR-NB and correlates with poor prognosis

To evaluate the expression level of NDUFS6 in NB tumor cells, we analyzed the publicly available scRNA-seq dataset GSE137804 (18). Among 160,910 cells profiled, 126,683 were identified as tumor cells and included for subsequent analysis (Figure 1A, Figure S1A). In line with the elevated energy demands of tumor cells, NB tumor cells displayed significantly higher expression of mitochondrial respiratory chain complex I (CI) genes compared to non-tumor cell types (Figure S1B). Notably, both the CI and OXPHOS pathway were significantly upregulated in tumor cells from HR-NB (Figure 1B). Among these genes, NDUFS6 emerged as a particularly tumor-enriched component (Figure S1B), with its median expression level significantly higher in HR-NB compared to non-HR-NB samples (P<0.0001). Moreover, NDUFS6 expression was significantly elevated in patients with MYCN amplification or diagnosed at an age over 18 months (P<0.0001; Figure 1C), highlighting its potential association with aggressive tumor phenotypes.

Figure 1 Expression and clinical relevance of NDUFS6 in NB tissues. (A) tSNE embedding plot of 160,910 cells from scRNA dataset GSE137804, with color coded by cell type. (B) GSEA plot showing the enrichment of WP_OXIDATIVE_PHOSPHORYLATION and WP_MITOCHONDRIAL_COMPLEX_I_ASSEMBLY_MODEL_OXPHOS_SYSTEM in HR-NB tumor cells. (C) Violin plots depicting NDUFS6 expression in tumor cells across different groups from GSE137804. (D) Box plots illustrating NDUFS6 expression across subgroups in the SEQC cohort. (E) Kaplan-Meier overall survival and event-free survival curve stratified by NDUFS6 expression in patients with NB from the SEQC cohort. (F) Forest plot showing the result of multivariate Cox regression analysis in the SEQC cohort. For the analyses shown in (D) and (F), patients with MYCN status recorded as “NA (not available)” were excluded, resulting in a total of 493 patients (from the original 498). (G) Representative IHC staining of NDUFS6 in high-risk and low-risk NB tissues at ×40 and ×200 magnification. (H) Bar plot summarizing the semi-quantitative scoring of NDUFS6 IHC staining in NB tissues across different groups. ns, no significance; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. HR, high-risk; IHC, immunohistochemistry; IR, intermediate-risk; LR, low-risk; NB, neuroblastoma; NDUFS6, NADH:ubiquinone oxidoreductase subunit S6.

Analysis of the SEQC cohort (n=498) from the R2 Genomics Platform further confirmed these findings (19). NDUFS6 expression increased significantly with advancing INSS stage (P<0.0001) and was higher in tumors with MYCN amplification (P<0.0001), HR-NB risk classification (P<0.0001), disease progression (P<0.0001), and patient mortality (P<0.001). In contrast, no significant difference was observed between male and female patients (P=0.714; Figure 1D). Kaplan-Meier survival analysis revealed that patients with high NDUFS6 expression had significantly lower overall survival (OS) (P<0.0001) and event-free survival (EFS) (P<0.0001) (Figure 1E). Importantly, multivariate Cox regression analysis adjusting for MYCN amplification status, age at diagnosis, INSS stage, and gender revealed that NDUFS6 expression remained associated with poorer OS and EFS (hazard ratio >1), indicating that it serves as an independent prognostic factor for adverse outcomes in NB (Figure 1F).

Immunohistochemical staining was subsequently performed on paraffin-embedded NB tumor specimens to evaluate NDUFS6 expression. The results also showed that NDUFS6 expression was significantly higher in the HR-NB, INSS stage 4 and metastatic tumors (P<0.05; Figure 1G,1H, Figures S2,S3, Table S1).

Collectively, these findings demonstrate that NDUFS6 upregulation is closely linked to aggressive clinicopathological features and may contribute to the malignant progression of NB.

NDUFS6 promotes the proliferation, invasion and migration of NB cells

To determine whether NDUFS6 exerts oncogenic effects in NB, we constructed NDUFS6 stable overexpression and knockdown SK-N-BE(2) and SH-SY5Y cell lines. NDUFS6 expression was confirmed by RT-qPCR (Figure 2A). The CCK-8 assay and colony formation assay were employed to evaluate cell proliferation and clonal formation capabilities. In both cell lines, NDUFS6 knockdown significantly suppressed proliferation and markedly reduced colony numbers compared with the control group (Figure 2B,2C). Conversely, NDUFS6 overexpression significantly enhanced proliferation and colony formation relative to controls (Figure 2D,2E).

Figure 2 Functional role of NDUFS6 in NB cell proliferation, migration and invasion in vitro. (A) Relative mRNA expression of NDUFS6 after OE or KD quantified by RT-qPCR. (B) CCK-8 assay showing differences in cell proliferation between NDUFS6 KD and control groups in SK-N-BE(2) and SH-SY5Y cell lines. (C) Colony formation assay stained with crystal violet comparing NDUFS6 KD and control groups in SK-N-BE(2) and SH-SY5Y cells. Macroscopic view of culture dishes. (D) CCK-8 assay demonstrating differences in cell proliferation between NDUFS6 OE and control groups in SK-N-BE(2)and SH-SY5Y cell lines. (E) Colony formation assay stained with crystal violet comparing NDUFS6 OE and control groups in SK-N-BE(2)) and SH-SY5Y cells. Macroscopic view of culture dishes. (F) Transwell assay showing differences in cell migration and invasion between NDUFS6 KD and control groups in SK-N-BE(2) cell line. (G) Trasnwell assay showing differences in cell migration and invasion between NDUFS6 KD and control groups in SH-SY5Y cell line. (H) Transwell assay showing differences in cell migration and invasion between NDUFS6 OE and control groups in SK-N-BE(2) cell line. (I) Transwell assay showing differences in cell migration and invasion between NDUFS6 OE and control groups in SH-SY5Y cell line. ns, no significance; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; KD, knockdown; NDUFS6, NADH:ubiquinone oxidoreductase subunit S6; OE, overexpression; RT-qPCR, quantitative real-time polymerase chain reaction.

In addition, Transwell assays were performed to evaluate the effect of NDUFS6 on cell motility. NDUFS6 knockdown significantly reduced the invasive and migratory capacity of both SK-N-BE(2) and SH-SY5Y cells (Figure 2F,2G). Conversely, NDUFS6 overexpression markedly enhanced invasion and migration in SK-N-BE(2) and SH-SY5Y cells (Figure 2H,2I).

Taken together, these findings indicate that NDUFS6 facilitates not only cell proliferation and clonal expansion but also invasive and migratory capacities, further supporting its function as a driver of NB progression.

Potential downstream signaling pathways regulated by NDUFS6 in NB

To further elucidate the downstream signaling pathways and targets regulated by NDUFS6, we performed RNA sequencing on SK-N-BE(2) and SH-SY5Y cells following NDUFS6 OE or KD. Principal component analysis of bulk RNA-seq data revealed distinct transcriptional profiles across the experimental groups (Figure S4A). Differential expression analysis identified 775 upregulated and 1,315 downregulated genes in SK-N-BE(2) cells with NDUFS6 OE and KD respectively, whereas SH-SY5Y cells exhibited 416 upregulated and 2,454 downregulated genes (Figure S4B, available online: https://cdn.amegroups.cn/static/public/tp-2026-1-0095-1.xlsx). Consistent with the established role of NDUFS6 in mitochondrial function, GSEA analysis demonstrated that the oxidative phosphorylation pathway was significantly activated in OE groups and repressed in KD groups (Figure 3A).

Figure 3 Transcriptional alterations following NDUFS6 overexpression or knockdown. (A) GSEA plots showing the enrichment of WP_OXIDATIVE PHOSPHORYLATION after NDUFS6 OE or KD in SK-N-BE(2) and SH-SY5Y cell lines. (B) Venn diagram showing the intersection of genes upregulated in OE and downregulated in KD, and vice versa, in SK-N-BE(2) cells. Dot plots display the GO and KEGG enrichment results of the intersecting genes. (C) Heatmap depicting the intersection genes identified in (B). (D) Venn diagram showing the intersection of genes upregulated in OE and downregulated in KD, and vice versa, in SH-SY5Y cells. Dot plots display the GO and KEGG enrichment results of the intersecting genes. (E) Heatmap depicting the intersection genes identified in (D). (F) GSEA plots showing enrichment of selected pathways in tumor cells from the GSE137804 dataset. (G) Bar plot showing the relative proportions of cell types in NDUFS6-high versus NDUFS6-low expression groups in the GSE137804 dataset. (H) Box plots displaying cell type scores based on ssGSEA analysis in the bulk RNA-seq dataset from the SEQC cohort. ****, P<0.0001. GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis; KD, knockdown; KEGG, Kyoto Encyclopedia of Genes and Genomes; NDUFS6, NADH:ubiquinone oxidoreductase subunit S6; OE, overexpression.

Putative downstream targets were identified by intersecting genes upregulated in OE and downregulated in KD, and vice versa. Although no overlapping DEGs were shared between the MYCN-amplified SK-N-BE(2) and non-MYCN-amplified SH-SY5Y cell lines, both exhibited enrichment of similar downstream signaling pathways following NDUFS6 perturbation (Figure 3B-3E). In particular, genes positively regulated by NDUFS6 OE were associated with G protein-coupled receptor signaling, amine and amphetamine response pathways, and energy metabolism (Figure 3B-3E). In SK-N-BE(2) cells, NDUFS6 OE led to enrichment of pathways involved in acetyl-CoA biosynthesis (e.g., ACSS1) and mitochondrial respiratory function (e.g., MARC2, SLC25A43) (Figure 3C), whereas in SH-SY5Y cells, enhanced expression of genes related to ATP transport (ABCC9) and linoleic acid metabolism (PLA2G4B) was observed (Figure 3E). Conversely, NDUFS6 OE was associated with repression of neuronal differentiation genes [e.g., SK-N-BE(2): ISL2, DLX1, SOX8, DLX2; SH-SY5Y: BRINP1, NLGN1] and immune-related pathways, including Toll-like receptor signaling (e.g., SK-N-BE(2): C3, CSMD3, IRF7, TLR4; SH-SY5Y: SEMA6A, CMKLR1, TLR1, NOD2) (Figure 3B-3E).

GSEA of the scRNA-seq dataset further corroborated these results, showing that tumor cells with high NDUFS6 expression exhibited robust activation of energy metabolism and ATP synthesis pathways, alongside reduced expression of neuronal differentiation markers, major histocompatibility complex (MHC) genes, and chemotaxis-associated genes (Figure 3F, Figure S4C). To assess the functional impact of NDUFS6 on mitochondrial bioenergetics, we measured intracellular ATP levels in NDUFS6-overexpressing cells versus controls. Notably, NDUFS6 overexpression led to a significant increase in ATP production (P<0.01), indicating enhanced mitochondrial metabolic activity (Figure S4D). Consistently, NB samples with high expression of NDUFS6 exhibited reduced infiltration of T cells and fibroblasts (Figure 3G). This pattern was further validated by ssGSEA analysis of the SEQC cohort, which revealed a marked decrease in both immune and stromal components in NDUFS6-high samples, indicating the presence of a “cold” tumor microenvironment associated with NDUFS6 upregulation (Figure 3H). Moreover, we examined the expression of MHC molecules following NDUFS6 KD and OE which revealed key MHC genes, including HLA-A, HLA-B, HLA-C, TAP2, TAP1, HLA-E and B2M were markedly upregulated in NDUFS6-KD cells compared to controls (Figure S4E,S4F). These findings suggest that NDUFS6 may suppress antigen presentation machinery, potentially contributing to an immune-cold phenotype.

Collectively, these results suggest that NDUFS6 overexpression drives a pro-metabolic transcriptional program in NB cells, enhancing bioenergetic capacity while suppressing neuron differentiation and immune activation pathways, thereby facilitating tumor progression.

NDUFS6-targeted drugs inhibit NB cell proliferation

Building on the transcriptomic evidence that NDUFS6 promotes a pro-metabolic, tumor-supportive program in NB, we next explored whether pharmacological targeting of NDUFS6 could suppress NB cell growth. Cryo-electron microscopy structural analysis of the human respiratory chain megacomplex-I2III2IV2 revealed that NDUFS6 interacts tightly with NDUFS12, a binding that is critical for maintaining the structural stability of CI and ensuring its proper electron transfer function (20). Therefore, we focused on the key residues of NDUFS6 (GLN44, TYR46, and GLU62) located at its binding interface with NDUFA12, and conducted computer-based virtual screening using the MCE Bioactive Compound Library Plus to identify small-molecule compounds with strong binding affinity to the target residues, thereby providing potential candidates for therapeutic strategies targeting NDUFS6 (Figure 4A,4B).

Figure 4 Evaluation of new therapeutic strategies targeting NDUFS6 in NB. (A) The protein structure of NDUFS6. (B) Workflow diagram of the virtual drug screening process. (C) Representative docking diagrams illustrating the binding of selected small-molecule compounds with the NDUFS6 protein. (D) Dose-response inhibition curves of candidate compounds targeting NDUFS6 in SK-N-BE(2). (E) Dose-response inhibition curves of candidate compounds targeting NDUFS6 in SH-SY5Y. GI₅₀, 50% growth inhibition concentration; NB, neuroblastoma; NDUFS6, NADH:ubiquinone oxidoreductase subunit S6.

A total of 200 bioactive compounds exhibited docking scores below −5 against the NDUFS6 protein (available online: https://cdn.amegroups.cn/static/public/tp-2026-1-0095-2.xlsx), indicative of favorable intermolecular binding affinity, as lower scores correspond to stronger predicted binding energies. From these, the top 30 compounds, with docking scores ranging from −9.266 to −6.709, were selected for preliminary drug sensitivity assays in SK-N-BE(2) and SH-SY5Y cells (available online: https://cdn.amegroups.cn/static/public/tp-2026-1-0095-2.xlsx, Figure 4C, Figure S5A).

In SK-N-BE(2) cells, six compounds including guanosine-5’-triphosphate (disodium salt), 1,4-β-D-xylopentaose, forsythoside I, nicotiflorin, xylotetraose, and isomaltotetraose exhibited dose-dependent cytotoxicity, with 48-hour relative inhibition rates exceeding 50% (Figure 4D, Figure S5B). Among them, guanosine-5'-triphosphate (disodium salt) and 1,4-β-D-xylopentaose achieved inhibition rates approaching 70%, with a steep concentration-dependent increase, whereas forsythoside I, xylotetraose, nicotiflorin, and isomaltotetraose maintained moderate inhibition levels (50–60%) with minimal dose responsiveness (Figure S5B).

In SH-SY5Y cells, eight compounds including guanosine-5'-triphosphate (disodium salt), deferoxamine (mesylate), Fmoc-Ala-Glu-Asn-Lys-NH₂, solasonine, fodipir, tubuloside A, GDP-α-D-mannose (disodium) and NADPH (tetrasodium salt) demonstrated 72-hour relative inhibition rates exceeding 50% in a concentration-dependent manner (Figure 4E, Figure S5C). Notably, Fmoc-Ala-Glu-Asn-Lys-NH₂, deferoxamine (mesylate), and solasonine approached complete inhibition, while fodipir, guanosine-5’-triphosphate (disodium salt), and tubuloside A exhibited pronounced, sustained increases in inhibition with rising concentrations (Figure 4E, Figure S5C).

Moreover, we performed CCK-8 assays to assess the drug response in NDUFS6-OE cells versus NC cells across different drug concentrations. As shown in the dose-response curves, NDUFS6-OE cells exhibited significantly higher sensitivity to guanosine-5'-triphosphate treatment compared to NC cells at equivalent drug concentrations, suggesting that NDUFS6 overexpression enhanced cellular responsiveness to this compound (Figure S5D).

Taken together, these results suggest that NDUFS6-targeted compounds exhibit robust anti-proliferative effects in NB cells. In particular, guanosine-5'-triphosphate (disodium salt) demonstrated potent cytotoxicity in both MYCN-amplified and non-amplified cell lines, with NDUFS6-OE cell lines showing greater sensitivity, highlighting its potential as a broadly applicable therapeutic agent for HR-NB.

Discussion

In recent years, alterations in mitochondrial activity have attracted considerable attention in the study of tumorigenesis. Mitochondria play a crucial role in tumor development and chemoresistance (21-23). NDUFS6 encodes a core subunit of CI and plays a crucial role in oxidative phosphorylation and cellular energy metabolism (20,24). Given its pivotal function, further investigation into the role of NDUFS6 in NB is warranted, particularly to uncover potential metabolic vulnerabilities that may be exploited therapeutically.

In our study, we observed that increased NDUFS6 expression was associated with more aggressive tumor phenotypes. Both single-cell and bulk RNA sequencing data demonstrated that NDUFS6 expression was significantly higher in tumor samples characterized by malignant features, including HR-NB classification, disease progression, and poor prognosis. Functional experiments further demonstrated that NDUFS6 promotes proliferation, invasion, and migration of SK-N-BE(2) and SH-SY5Y cells. Overall, these findings underscore the critical role of NDUFS6 in promoting progression of NB.

Subsequent functional enrichment analysis of RNA-seq data from the stable NDUFS6 OE and KD cell lines revealed enrichment of genes involved in energy metabolism, ATP synthesis, neuronal development, and immune-related pathways. Specifically, NDUFS6 regulated gene programs related to energy metabolism, ATP synthesis, neuronal development, and immune response. Consistent with its established function in complex I, NDUFS6 upregulation likely supports the heightened bioenergetic demands of rapidly proliferating NB cells (12-16). Beyond its metabolic function, emerging evidence suggests mitochondrial respiratory complex I subunits also contribute to the crosstalk between tumors and the immune cells (16,25). Notably, NDUFS6 inhibition has been shown to enhance MHC class I antigen presentation and to promote cytotoxic CD8⁺ T cell and NK cell responses (16). Recent studies comprehensively discussed how mitochondrial metabolism and dysfunction in the tumor microenvironment drive immune suppression, affecting both tumor cells and infiltrating immune cells (26). In line with this, our study demonstrated that NDUFS6 overexpression was associated with downregulation of MHC genes and suppression of immune activation and Toll-like receptor signaling pathways. Consistent with our findings, Estephan et al. recently demonstrated that inhibition of mitochondrial complex I improves tumor oxygenation and restores MHC-I expression, thereby enhancing CD8+ T cell recognition of hypoxic tumor cells (27). These data suggest that NDUFS6 may not only drives metabolic reprogramming but also contributes to immune evasion, fostering a “cold” tumor immune microenvironment characteristic of HR-NB.

To explore potential therapeutic strategies targeting NDUFS6, we performed a preliminary compound screening and identified guanosine-5'-triphosphate (disodium salt) as a candidate compound with marked efficacy against both SK-N-BE(2) and SH-SY5Y cell lines. Notably, NDUFS6-OE cells exhibited significantly enhanced sensitivity to this compound compared to control cells, suggesting a NDUFS6-dependent effect. Exogenous guanosine triphosphate has been reported to induce S-phase cell cycle arrest and promote differentiation marker expression in NB cells (28), while similar effects on differentiation have been observed in acute myeloid leukemia cells (29). These results suggest that guanosine-5'-triphosphate (disodium salt) may exert preferential cytotoxicity in NDUFS6-high NB cells. However, we acknowledge that direct binding between this compound and the NDUFS6 protein has not been established in this study. Future investigations employing surface plasmon resonance, cellular thermal shift assay, or drug affinity responsive target stability are warranted to confirm target engagement and specificity. Nonetheless, the current findings provide a rationale for exploring NDUFS6-associated metabolic pathways as a therapeutic vulnerability in HR-NB, and guanosine-5'-triphosphate or its derivatives could potentially be leveraged in combination with existing chemotherapeutic regimens.

Several limitations of this study should be acknowledged. First, the IHC validation cohort included only 34 HR-NBL patients, a relatively small sample size; although our IHC results were consistent with larger public transcriptomic datasets, multi-center validation in larger cohorts is needed. Second, the immune-related findings are primarily based on transcriptomic inference, and experimental validation (e.g., flow cytometry for surface MHC expression, T-cell co-culture assays) is required to confirm the immune-cold phenotype. Third, the mechanistic link between NDUFS6 and mitochondrial function is supported by ATP measurements, but additional parameters such as oxygen consumption rate and reactive oxygen species levels would provide a more comprehensive understanding. Despite these limitations, our study provides a strong rationale for targeting NDUFS6 as a potential therapeutic strategy in HR-NB. Nevertheless, further investigation is warranted, especially as pediatric oncology shifts from traditional multidisciplinary team-based approaches toward molecular tumor boards emphasizing precision medicine (30). To enhance translational relevance, future studies should incorporate additional NB cell lines, patient-derived xenograft models, and clinical biospecimens, with in vivo validation of NDUFS6 function and the therapeutic efficacy of guanosine-5’-triphosphate (disodium salt) being critical. Moreover, the potential clinical benefit of combining this compound with standard chemotherapy in HR-NB should be rigorously assessed in preclinical models.

Conclusions

Our study demonstrates that NDUFS6 is markedly upregulated in HR-NB and is strongly associated with aggressive tumor phenotypes. Functionally, NDUFS6 promotes NB cell proliferation, migration, and invasion, underscoring its potential role as a tumor driver. Transcriptomic analyses suggest that NDUFS6 not only regulates oxidative phosphorylation and ATP synthesis but also modulates immune phenotypes by downregulating MHC expression and suppressing immune activation. Moreover, we identified and preliminarily validated several small-molecule compounds targeting NDUFS6, including guanosine 5’-triphosphate (disodium salt), which exerted inhibitory effects in both MYCN-amplified and non-MYCN-amplified cell lines. Collectively, these findings highlight NDUFS6 as a promising therapeutic target and provide novel insights into pharmacological strategies for HR-NB.

Acknowledgments

The computational analysis in this work was supported by the Medical Science Data Center of Fudan University.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-1-0095/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-1-0095/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-1-0095/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2026-1-0095/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of Children’s Hospital of Fudan University [No. 2020(169)]. Informed consent was obtained from the legal guardians of all participants prior to enrollment.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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