Multi-omics screening and functional validation identify SLC5A6 as a candidate disulfidptosis-related gene and prognostic biomarker in hepatocellular carcinoma
Aug 2026· Frontiers in Oncology· Vol 16· 0 citations· 29 references
Medicine
TL;DR
The findings suggest that SLC5A6 may participate in glucose-deprivation-induced disulfide stress, while its direct role in regulating disulfidptotic cell death remains to be established.
Abstract
Objective Hepatocellular carcinoma (HCC) is characterized by frequent recurrence, therapeutic resistance, and marked metabolic adaptability. Disulfidptosis is a recently described form of regulated cell death associated with glucose deprivation and disulfide stress. This study aimed to identify disulfidptosis-related genes associated with HCC progression and to investigate the potential biological role of SLC5A6. Methods Single-cell RNA sequencing and TCGA-LIHC transcriptomic data were integrated. A literature-derived, non-directional disulfidptosis-related gene-set enrichment score was calculated using ssGSEA, and copy-number alterations were inferred using inferCNV. WGCNA, differential expression analysis, and the SLC-family gene list were integrated to identify candidate genes. Bayesian deconvolution, ESTIMATE, TIDE, and GSVA were used to evaluate tumor-microenvironment-related features and pathway signatures. The biological effects of SLC5A6 silencing were assessed using proliferation, migration, invasion, apoptosis, and xenograft assays. Glucose-deprivation-induced disulfide stress was further evaluated by measuring protein disulfide content, the NADP+/NADPH ratio, and FLNA and FLNB band patterns under non-reducing conditions. Results Single-cell analysis showed that malignant hepatocytes with higher inferCNV-derived CNV scores exhibited greater enrichment of the disulfidptosis-related gene signature. Integration of glucose-deprivation-associated DEGs, WGCNA modules, and SLC-family genes identified SLC5A6 as a candidate disulfidptosis-related gene that was upregulated in HCC and associated with poor prognosis. Bayesian deconvolution, ESTIMATE, TIDE, and GSVA analyses linked elevated SLC5A6 expression to advanced disease, stromal and immunosuppressive cell enrichment, higher T-cell exclusion scores, and activation of Wnt/mTOR-related signaling signatures. In SLC7A11-high HCC cells, glucose deprivation increased protein disulfide content and the NADP+/NADPH ratio and altered non-reducing FLNA and FLNB band patterns, whereas these changes were partially attenuated by SLC5A6 silencing. Under conventional culture conditions, SLC5A6 silencing inhibited proliferation, migration, invasion, and xenograft growth and increased apoptosis. Conclusion SLC5A6 is a candidate disulfidptosis-related gene and prognostic biomarker associated with malignant progression in HCC. The findings suggest that SLC5A6 may participate in glucose-deprivation-induced disulfide stress, while its direct role in regulating disulfidptotic cell death remains to be established. Its associations with immune-exclusion-related features also require further functional validation.
INTRODUCTION
Sorafenib is a first-line therapy for hepatocellular carcinoma (HCC), but its clinical benefit is limited by significant interpatient variability in response. Identifying reliable candidate biomarkers associated with sorafenib treatment is critical for optimizing treatment strategies and improving patient outcomes.
METHODS
Samples in the GSE109211 dataset were divided into sorafenib-treated and control groups. Differentially expressed genes (DEGs) were screened using the limma package and filtered via LASSO and SVM-RFE algorithms to identify potential HCC biomarkers. CIBERSORT and ESTIMATE algorithms were used to evaluate immune infiltration and its correlation with candidate genes, followed by GSEA to explore enriched functional pathways. Regulatory networks were constructed using hTFtarget and ENCORI, and potential drugs were predicted by molecular docking. The expression and knockdown effects of RPS21 were validated in THLE-2 and HuH7 cells using qRTPCR, wound-healing, and Transwell assays.
RESULTS
Five sorafenib-treatment-associated candidate biomarkers were identified, including glutamic-oxaloacetic transaminase 1 (GOT1), out at first homolog (OAF), Xprolyl aminopeptidase 2 (XPNPEP2), cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2), and ribosomal protein S21 (RPS21). These genes were related to immune cell infiltration. In particular, the expression of OAF and CRISPLD2 was associated with immune scores. GSEA showed enrichment in pathways such as OXIDATIVE_ PHOSPHORYLATION and MYC_TARGETS. GOT1 stably bound to sorafenib, carbamazepine, and diclofenac. RPS21 was upregulated in HuH7 cells, and its knockdown impaired migration and invasion, validating its oncogenic role.
DISCUSSION
This study identified candidate biomarkers related to sorafenib treatment for HCC through computational analyses, experimentally validated the oncogenic function of RPS21, and predicted carbamazepine and diclofenac as potential combination therapies for precision treatment of HCC.
CONCLUSION
We identified HCC biomarkers associated with sorafenib treatment and the tumor immune microenvironment, predicted targeted drugs, and expanded the paradigm for precision therapy in HCC.
Mengru Zhan, Peng-Hui Li, Wan-Yi Wang et al.· Current Medicinal Chemistry· 0 citations
Background Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with poor prognosis and limited treatment options. Disulfidptosis, a novel cell death pathway driven by disulfide bond accumulation, has emerged as a potential mechanism in cancer biology; however, its role in ICC remains unclear. Methods We integrated single−cell RNA sequencing (GSE138709) with bulk transcriptomic datasets (TCGA−CHOL, GSE107943, GSE32225) to systematically characterize the ICC cellular landscape. Analyses included CNV inference, stemness scoring, disulfidptosis activity assessment, and cell−cell communication profiling. A diagnostic model was constructed using LASSO−logistic regression with 10−fold cross−validation and validated in independent cohorts. TME characterization, survival analysis, and drug−target screening were also performed. Experimental validation included HPA immunohistochemistry, qRT−PCR, and functional assays following TMED3 knockdown. Results Seven major cell types were identified, with malignant cholangiocytes exhibiting high aneuploidy (74%), elevated stemness, upregulated disulfidptosis activity, and extensive communication via SPP1−CD44 and IGFBP3−TMEM219 networks. A five−gene signature (TMED3, TMEM184B, MAPK13, MFSD10, GRB7) demonstrated robust diagnostic performance. Survival analysis showed borderline prognostic value for TMED3 (adjusted HR = 2.37, P = 0.073), while TMEM184B emerged as an independent prognostic factor (adjusted HR = 4.79, P = 0.028). PPI and co−expression analyses established links between signature genes and disulfidptosis regulators. Functional experiments confirmed that TMED3 knockdown suppressed ICC cell proliferation, migration, and enhanced sensitivity to glucose deprivation−induced disulfidptosis. Network−based drug screening identified eight high−priority candidates for therapeutic repurposing. Conclusion This study provides a comprehensive single−cell atlas of ICC, identifies TMED3 as a key regulator of a disulfidptosis−related diagnostic signature, and demonstrates its functional role in promoting ICC malignancy. The five−gene signature shows diagnostic and prognostic promise, and the drug screening offers preliminary leads for therapeutic repurposing, providing a foundation for precision diagnosis and targeted therapy in ICC.
Wan-Jia Qiao, Yixiang He, Jing Li et al.· Frontiers in Immunology· 0 citations
Atherosclerosis (AS) is a chronic inflammatory vascular disease characterized by immune dysregulation, metabolic disturbance, and progressive vascular remodeling. Although anoikis resistance has been extensively investigated in cancer biology, its involvement in immune-cell persistence and plaque progression in AS remains poorly understood.
We integrated two publicly available bulk transcriptomic datasets (GSE100927 and GSE28829) to identify differentially expressed genes (DEGs) and performed weighted gene co-expression network analysis (WGCNA) to determine disease-associated gene modules. Anoikis-related candidate genes were obtained by intersecting DEGs, WGCNA-derived module genes, and a curated anoikis-related gene set, and were further prioritized using least absolute shrinkage and selection operator (LASSO) regression and random forest algorithms. Immune infiltration and cell type–specific expression patterns were evaluated using CIBERSORT and single-cell RNA sequencing (scRNA-seq) analyses. The expression of the leading candidate was validated using both
in vitro
and
in vivo
experiments.
We identified eight core anoikis-related hub genes—HAVCR2, CD36, PLAU, TNF, PYCARD, SERPINA1, BLNK, and CHI3L1—that were closely associated with AS. These genes were enriched in inflammatory and metabolic pathways and showed strong diagnostic performance across validation analyses, with AUC values greater than 0.83. Immune deconvolution and single-cell transcriptomic analyses revealed that the anoikis-related signature was predominantly localized to macrophage and T-cell compartments within atherosclerotic lesions. Among the identified candidates, HAVCR2 showed the most consistent disease-associated pattern. HAVCR2 expression was markedly increased in oxLDL-stimulated THP-1-derived macrophages and was enriched in macrophage-rich atherosclerotic lesions in high-fat diet–fed ApoE
−/−
mice, supporting its association with lipid-induced macrophage activation and vascular inflammation.
This study defines a macrophage-centered anoikis-related immune signature in atherosclerosis and identifies HAVCR2, together with CD36, PLAU, TNF, PYCARD, SERPINA1, BLNK, and CHI3L1, as potential biomarkers and candidate targets for future diagnostic and therapeutic investigation.
Qiang Shen, Zheng-Feng Fan, Jin-Cheng Hou et al.· Frontiers in Immunology· 0 citations
The six-gene ubiquitination-related signature may provide a valuable reference for potential therapeutic targets and prognosis of BC, however, prospective and experimental validation is still required.
Zhi-Biao Li, Ting Yan, Chuxiang Hu et al.· Current Medicinal Chemistry· 0 citations
Background and Aims Dysregulated lipid metabolism contributes to hepatocellular carcinoma (HCC) progression, but the prognostic value and mechanistic roles of lipid metabolism-related long noncoding RNAs (LRLs) remain insufficiently characterized. This study aimed to construct and validate an LRL-based prognostic model and to investigate the biological function and metabolic mechanism of AC026412.3 in HCC. Methods Transcriptomic and clinical data from the The Cancer Genome Atlas Liver Hepatocellular Carcinoma cohort were analyzed to identify LRLs based on their correlation with curated lipid metabolism genes. Differential expression, univariate Cox, least absolute shrinkage and selection operator (LASSO), and multivariate Cox analyses were performed to construct a prognostic signature, which was evaluated using Kaplan–Meier survival and time-dependent receiver operating characteristic (ROC) analyses. Functional enrichment analyses Gene Ontology [GO], Kyoto Encyclopedia of Genes and Genomes [KEGG] and gene set enrichment analysis [GSEA], mutation profiling, tumor mutational burden, immune infiltration estimation, and consensus clustering were applied to characterize associated features. A key LRL was identified through integrated bioinformatic screening and prioritization. Its biological role was assessed by quantitative reverse transcription polymerase chain reactionq (RT-PCR), western blotting, BODIPY staining, colony formation, Transwell assays, and xenograft models. RNA sequencing followed by pathway enrichment analysis was conducted to explore underlying mechanisms. Results A three-LRL signature (AL031985.3, NRAV, and AC026412.3) stratified HCC patients into distinct risk groups with significantly different survival outcomes and demonstrated independent prognostic value. AC026412.3 was markedly upregulated in HCC and associated with poor prognosis. Functional assays demonstrated that AC026412.3 promoted proliferation, invasion, and tumor growth while reducing lipid accumulation. Mechanistically, AC026412.3 upregulated solute carrier family 22 member 5 (SLC22A5), enhanced fatty acid β-oxidation, and increased adenosine triphosphate (ATP) production, thereby driving metabolic reprogramming. Conclusions This study establishes a robust LRL-based prognostic model and identifies AC026412.3 as a key regulator of lipid metabolic reprogramming via the SLC22A5–fatty acid β-oxidation axis, highlighting its potential as a biomarker and therapeutic target in HCC.
Li-Xin Liu, Yu-Hao Fan, Hao Zou et al.· Journal of Clinical and Tran...· 0 citations
Background Clear cell renal cell carcinoma (ccRCC) is metabolically primed for ferroptosis, yet the prognostic relevance and mechanistic contribution of ferroptosis-related genes remain incompletely defined. This study aimed to identify ferroptosis-associated biomarkers with prognostic value and to clarify their functional relevance in ccRCC progression. Methods We integrated single-cell RNA sequencing, bulk RNA-seq, spatial transcriptomics, and machine-learning-based feature selection to identify ferroptosis-related prognostic genes in ccRCC. A four-gene risk model and an integrated nomogram were constructed and evaluated in independent cohorts. PANX2 was prioritized for experimental validation using stable knockdown models, RNA sequencing, lipid peroxidation and iron probes, redox assays, Western blot, luciferase reporter assays, xenografts, and an immunocompetent murine renal carcinoma model. Results A four-gene prognostic signature (CA9, PVT1, RRM2, and PANX2) was identified and used to construct a risk model with consistent predictive performance in the training and external validation cohorts. Among these genes, PANX2 was predominantly enriched in epithelial tumor compartments and had not been functionally characterized in ccRCC. PANX2 knockdown inhibited ccRCC cell proliferation, reduced antioxidant capacity, increased intracellular Fe2+ accumulation and lipid peroxidation, and sensitized cells to erastin-induced ferroptotic death. Mechanistically, PANX2 loss was associated with reduced Akt/mTOR pathway activity and diminished SLC7A11 expression; rescue with an Akt activator or SLC7A11 overexpression attenuated ferroptosis-associated phenotypes. In an immunocompetent murine renal carcinoma model, PANX2 knockdown was accompanied by increased infiltration of CD45+ leukocytes, CD3+ T cells, and CD8+ T cells, supporting a potential link between PANX2-dependent ferroptosis resistance and the tumor immune contexture. Conclusions This study identifies PANX2 as a ccRCC-relevant suppressor of ferroptosis and supports the involvement of a PANX2-Akt/mTOR-SLC7A11-associated signaling axis in redox homeostasis and tumor progression. The ferroptosis-related prognostic model and nomogram may support risk stratification, while PANX2 represents a candidate therapeutic vulnerability that warrants further mechanistic and translational validation.
Xing-Lin Li, Yiqi Xiong, Ji-Yin Wang et al.· Frontiers in Immunology· 0 citations
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