A single-cell-resolved ferroptosis gene signature with strong prognostic and therapeutic implications for ESCC is presented, showing that knockdown of CDCA3 lead to the downregulation of ferroptosis inhibitor-related genes and upregulation of ferroptosis-promoting genes, thereby enhancing the sensitivity to RSL3-induced ferroptosis.
Abstract
Background Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy with poor prognosis and limited therapeutic options. Ferroptosis, a regulated form of cell death characterized by iron-dependent lipid peroxidation, plays a crucial role in tumor progression and immune regulation. Methods We integrated single-cell RNA sequencing (scRNA-seq) and bulk transcriptomic data to identify ferroptosis-active cellular subpopulations within the ESCC tumor microenvironment. A ferroptosis-related prognostic model was constructed using LASSO-Cox regression and validated across independent cohorts from TCGA and GEO. Associations with immune infiltration, tumor mutation burden, therapeutic response, and drug sensitivity were explored. Furthermore, functional experiments were conducted in vitro using the ESCC cell lines, and the four prognostic core genes were revalidated using an independent single-cell dataset, which was also fully confirmed in clinical ESCC tissue samples. In addition, Western blot analysis was performed to examine the expression levels of ferroptosis-related proteins following CDCA3 knockdown, and to further investigate the impact of CDCA3 depletion on the cellular response to the ferroptosis inducer RSL3. Results Four ferroptosis-related genes (CBS, CDCA3, GALNT14, and IDO1) were identified used to construct a robust risk model, effectively stratifying patients into high- and low-risk groups with significant differences in survival, immune infiltration, and predicted treatment response. In vitro experiments confirmed that CDCA3 knockdown significantly inhibited the proliferation and migration of ESCC cells and induced ferroptosis. GSE188900 single-cell sequencing data further confirmed that the aforementioned genes were significantly upregulated at single-cell resolution in tumor cells, with consistent validation in clinical ESCC tissue samples, Moreover,experimental results showed that knockdown of CDCA3 lead to the downregulation of ferroptosis inhibitor-related genes and upregulation of ferroptosis-promoting genes, thereby enhancing the sensitivity to RSL3-induced ferroptosis. Conclusions Our study presents a single-cell-resolved ferroptosis gene signature with strong prognostic and therapeutic implications for ESCC. The signature was validated in clinical tissue samples, and this model lays the foundation for ferroptosis-targeted therapeutic strategies.
Diffuse large B-cell lymphoma (DLBCL) is biologically heterogeneous and is associated with variable clinical outcomes. We aimed to develop a tumor-associated macrophage (TAM)-related ferroptosis–glycolysis prognostic signature and to explore selected signature genes in preclinical models. This retrospective multi-cohort computational prognostic biomarker-development study integrated public single-cell and bulk transcriptomic datasets. GSE10846 was used for feature selection, model fitting, and parameter tuning, whereas GSE32918, GSE69051, and TCGA-DLBC were used as model-selection validation cohorts. Exploratory immune, genomic, and computational drug-sensitivity analyses were performed. Signature-gene expression was assessed in 27 archived DLBCL tissues, and GCLC and SLC1A5 were further examined in TAM-related preclinical models in vitro and in vivo. An 11-gene TAM-related ferroptosis–glycolysis signature (TAMFGS) was developed. The time-dependent AUCs (95% CIs) were 0.978 (0.966–0.990), 0.986 (0.976–0.995), and 0.982 (0.966–0.999) at 1, 3, and 5 years, respectively, in GSE10846; 0.622 (0.524–0.720), 0.608 (0.515–0.702), and 0.619 (0.524–0.715) in GSE32918; 0.516 (0.286–0.747), 0.741 (0.548–0.934), and 0.754 (0.516–0.993) at 1, 2, and 3 years, respectively, in GSE69051; and 0.929 (0.850–1.000), 0.674 (0.382–0.966), and 0.677 (0.402–0.952) at 1, 3, and 5 years, respectively, in TCGA-DLBC. In GSE10846, the continuous TAMFGS score remained associated with overall survival after adjustment for available covariates (HR, 1.094; 95% CI, 1.082–1.106; p < 0.001). Exploratory analyses identified associations between TAMFGS and immune-related transcriptomic estimates and computationally predicted drug sensitivity. GCLC or SLC1A5 knockdown was associated with ferroptosis-associated molecular changes and an M1-like inflammatory shift in the THP-1-derived TAM model and was associated with reduced DLBCL growth in preclinical models. In retrospective transcriptomic DLBCL cohorts, TAMFGS was associated with overall survival. GCLC and SLC1A5 emerged as TAM-related candidate genes that warrant further mechanistic and prospective validation.
Yingjun Wang, Lai Wei, Jie-Ting Wang et al.· European Journal of Medical...· 0 citations
This study identifies TPI1 as a key tumor-promoting factor and independent prognostic biomarker in HNSCC, providing a promising therapeutic target for overcoming ferroptosis evasion in HNSCC.
Rui Ye, Zhihua Xu, Ye-Hai Liu· Frontiers in Genetics· 0 citations
BACKGROUND
Cysteinyl-tRNA synthetase 1 (CARS1) has been included in ferroptosis-related prognostic signatures, but its clinicopathological relevance, cellular functions, and relationship with the immune microenvironment in hepatocellular carcinoma (HCC) remain incompletely characterized.
METHODS
Transcriptomic and clinical data from The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset were integrated with corresponding data from an institutional HCC tissue cohort of 60 patients. CARS1 expression was evaluated by immunohistochemistry, and immune infiltration was examined using single-sample gene-set enrichment analysis (ssGSEA) and multiplex immunofluorescence, as well as by analyzing public single-cell datasets. The effects of CARS1 depletion were evaluated in MHCC97H and Hep3B cells using Cell Counting Kit-8 (CCK-8) assays, cell-cycle profiling, wound-healing assays, Transwell migration assays, western blotting, and erlotinib-sensitivity assays.
RESULTS
CARS1 expression was elevated in HCC and was associated with adverse clinicopathological features and poor overall survival. Quantitative immunohistochemistry confirmed elevated CARS1 protein expression in tumor tissues. CARS1 depletion inhibited cell proliferation, altered cell-cycle distribution, impaired migration, and enhanced in vitro sensitivity to erlotinib. High CARS1 expression was also associated with increased infiltration of Th2-like immune cells.
CONCLUSIONS
Elevated CARS1 expression is associated with an adverse biological and immune phenotype in HCC. These clinical, histopathological, and loss-of-function findings support further investigation of CARS1 as a prognostic marker and candidate therapeutic target in HCC, although additional mechanistic and in vivo validation is required.
Ye-Zhen Tang, Ke-Jun Liu, Zhen-Ya Tan et al.· Frontiers in Bioscience· 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
This study aimed to investigate the role of ferroptosis-related long non-coding RNA CASC9 in the carcinogenic process of esophageal squamous cell carcinoma and evaluate its clinical diagnostic and prognostic value. LncRNA microarray screening was performed to identify differentially expressed genes in ESCC tissues. CASC9 expression was validated in 150 paired ESCC tissues, precancerous lesions, and cell lines by qRT-PCR. The correlation between CASC9 expression and clinicopathological characteristics as well as survival outcomes was analyzed. In vitro functional assays were conducted to examine the effects of CASC9 on ESCC cell proliferation, apoptosis, invasion, and migration. The role of CASC9 in ferroptosis was explored by detecting GSH, MDA, and ROS levels. CASC9 was significantly overexpressed in 92.7% of ESCC tissues (mean 11-fold upregulation), showing stepwise elevation during disease progression (normal mucosa→LGIN→HGIN→invasive carcinoma); High CASC9 expression correlated significantly with depth of invasion (P = 0.001) and clinical stage (P = 0.033), serving as an independent risk factor for overall survival (HR = 1.550, P = 0.026); Plasma CASC9 levels were significantly elevated in ESCC patients and markedly decreased at 14 and 30 days postoperatively. CASC9 knockdown markedly suppressed ESCC cell proliferation, invasion, and migration while promoting apoptosis. Reduced GSH and MDA/ROS accumulation indicate that CASC9 regulates ESCC progression via the ferroptosis pathway. In combination with clinicopathological features, a nomogram based on the gene signature presented strong predictive power and risk stratification for ESCC. CASC9 functions as an oncogenic lncRNA that promotes ESCC progression by modulating ferroptosis, representing a promising early diagnostic biomarker, prognostic predictor, and therapeutic target.
The findings demonstrate that MCs exhibit distinct functional states in ESCC and regulate tumor progression and support their potential relevance in immunotherapy.
Yiren Huang, Zheyi Chen, Bingqian Zhou et al.· Frontiers in Immunology· 0 citations
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