Super-enhancers mediates SLC7A11 via FOXA1 to regulate disulfidptosis in prostate cancer

Abstract

Prostate cancer (PCa) remains a major therapeutic challenge due to aberrant androgen receptor signaling and a remodeled tumor microenvironment. Disulfidptosis, a recently identified form of cell death characterized by cytoskeletal collapse under conditions of glucose deprivation and elevated SLC7A11 expression, presents a potential novel avenue for intervention. In this study, we integrated TCGA and GEO data and employed machine learning techniques to identify disulfidptosis-related genes in prostate cancer. Functional analyses using SLC7A11-overexpressing and knockout cell lines demonstrated that SLC7A11 promotes cellular proliferation, migration, and invasion, while its overexpression under glucose-starved conditions triggers disulfidptosis, also inducible pharmacologically using the glucose uptake inhibitor BAY-876. Through CUT&Tag, ChIP-seq, and luciferase assays, we identified FOXA1 as a key transcriptional regulator of SLC7A11, driven by a super-enhancer located at chr14:37583488-37589585. CRISPR-Cas9 deletion of this super-enhancer reduced FOXA1 and SLC7A11 expression, thereby protecting cells from disulfidptosis. These findings highlight the critical role of the SE/FOXA1/SLC7A11 regulatory axis in driving both disulfidptosis and tumor progression, suggesting that targeting this pathway, particularly in glucose-deprived tumor environments, may offer promising therapeutic strategies for PCa.

Fig. 1. Analysis of biomarkers in prostate cancer.

A Concordance index (C-index) values of multiple machine learning models across different datasets. B Differential expression of ferroptosis-related genes between prostate cancer tissues and normal samples. C–F Kaplan–Meier curves comparing progression-free survival between high and low expression groups of RPN1, OXSM, NDUFA11, and SLC7A11. G Protein–protein interaction network of 15 key ferroptosis-related proteins. H Bubble plot of KEGG pathway enrichment for the selected gene set. I Chromosomal locations of ferroptosis-related genes in the human genome. J Correlation network of ferroptosis-related genes before and after machine learning selection; edges represent correlations between risk and protective factors, with color and thickness indicating correlation strength. K UMAP dimensionality reduction analysis of the GSE141445 dataset showing spatial clustering of different tumor cell subtypes. L–O Expression patterns of RPN1, OXSM, NDUFA11, and SLC7A11 across various tumor cell clusters. P Immunohistochemical staining of SLC7A11 in benign prostate (BP) tissues and prostate cancer tissues with different Gleason scores (<7, =7, >7). Q Quantitative analysis of SLC7A11-positive areas in immunohistochemistry; higher Gleason scores correlate with significantly increased SLC7A11 expression. R–T Expression levels of SLC7A11 in different prostate cell lines detected by qRT-PCR and Western blot assays.

Fig. 2. Effects of SLC7A11 on prostate cancer cell viability.

A Western blot showing SLC7A11 expression in different treatment groups. B–F Wound healing and Transwell assays showing enhanced migration/invasion in SLC7A11-overexpressing (OE) cells and reduced abilities in knockout cells (sgRNA#1, sgRNA#2). G Tumor morphology after subcutaneous injection of DU145 cells (OE, NC, sgRNA#1, sgRNA#2) in nude mice. H Tumor weight comparison. I Tumor growth curves. J Ki67 immunohistochemistry in DU145 tumors. K Tumor morphology after PC-3 cell injection (OE, NC, sgRNA#1, sgRNA#2). L Tumor weight comparison. M Tumor growth curves for PC-3 xenografts. N Ki67 immunohistochemistry in PC-3 tumors. O Annexin V/7-AAD flow cytometry plots of PC-3 and DU145 cells under glucose-sufficient (+Glucose) and glucose-deprived (−Glucose) conditions. P–S Apoptosis rate statistics for PC-3 and DU145 cells. T F-actin staining in DU145 and PC-3 cells under +Glucose and −Glucose conditions.

Fig. 3. Regulatory effects of iBET-151 on prostate cancer cells.

A, B IC₅₀ values of iBET-151 in PC-3 and DU145 cells. C, D IC₅₀ values of JQ-1 in PC-3 and DU145 cells. E Flow cytometry analysis of apoptosis in PC-3 and DU145 cells treated with increasing concentrations of iBET-151 and JQ-1. F, G Quantification of apoptotic cells in PC-3 and DU145 following treatment. H Effects of iBET-151 and JQ-1 on colony formation in PC-3 and DU145 cells. I, J Quantification of colony numbers in treated and control groups. K Co-expression analysis of SLC7A11 and 10 predicted transcription factors in prostate tissue, based on TCGA and GTEx transcriptomic datasets. L Spatial transcriptomic analysis showing FOXA1 and SLC7A11 expression patterns in prostate cancer tissues. M Spatial transcriptomic profiles of FOXA1 and SLC7A11 in normal prostate tissues. N–V Changes in FOXA1 protein and mRNA levels in PC-3 and DU145 cells treated with iBET-151 (300 μM and 150 μM) and JQ-1 (1 μM and 0.5 μM), as assessed by Western blot and qPCR.

Fig. 4. Identification of super-enhancer target genes.

A–E Secondary analysis of ChIP-seq data from the ENCODE public database showing super-enhancer (SE) target genes across various prostate cell lines. F–K Super-enhancer target genes identified in DU145, PC-3, and RWPE-1 cell lines based on CUT&Tag experimental data. L–O Changes in SE-associated genes in PC-3 and DU145 cells following iBET-151 treatment. P Overlap of SE target genes identified by CUT&Tag and ChIP-seq analyses; FOXA1 and IER2 were the only genes consistently identified. Q SE enrichment surrounding the FOXA1 locus across different cell lines based on ENCODE ChIP-seq data. R CUT&Tag sequencing results showing SE enrichment at the FOXA1 downstream region; genomic locations and sequences targeted by SE-sgRNA constructs. S–U In DU145 cells, transfection with SE-sgRNA-1, SE-sgRNA-2, or SE-sgRNA-3 significantly reduced FOXA1 mRNA and protein expression levels. V–X In PC-3 cells, transfection with SE-sgRNA-1, SE-sgRNA-2, or SE-sgRNA-3 also led to marked downregulation of FOXA1 transcription and protein expression.

Fig. 5. Interaction between FOXA1 and the SLC7A11 promoter and its role in gene regulation.

A Predicted binding sites of FOXA1 on the SLC7A11 promoter based on AlphaFold-3 modeling. B Molecular docking simulation illustrating the interaction between FOXA1 protein and the SLC7A11 promoter region. C Dual-luciferase reporter assays show that FOXA1 enhances SLC7A11 promoter activity, while mutation of the binding sites disrupts this regulatory effect. D CUT&Tag analysis reveals DNA amplification bands in the FOXA1 immunoprecipitated (IP) group, indicating specific enrichment. E qPCR analysis confirms significant enrichment of the SLC7A11 promoter region in the FOXA1-IP group compared to controls. F–J In PC-3 cells, sequential disruption of the super-enhancer (SE) or promoter region significantly reduces FOXA1 and SLC7A11 expression. K–O In DU145 cells, similar SE and promoter targeting also leads to a marked downregulation of FOXA1 and SLC7A11, demonstrating consistent regulatory effects.

Fig. 6. Effects of SLC7A11 on cellular metabolism.

A, B Cysteine level in PC-3 and DU145 cells with varying SLC7A11 expression levels. C, D NADP⁺/NADPH ratio in SLC7A11-overexpressing (OE) cells under glucose-deprived conditions (−Glc) at different time points. E Reducing and non-reducing Western blot analysis showing the migration patterns of cytoskeletal proteins in SLC7A11-OE cells under different culture conditions. F–H Flow cytometry analysis of apoptosis in DU145-OE and PC-3-OE cells under glucose and/or cystine deprivation. I, J BAY-876 significantly inhibits glucose uptake in DU145 and PC-3 cells. K, L BAY-876 treatment leads to increased NADPH consumption, as reflected by elevated NADP⁺/NADPH ratios in DU145 and PC-3 cells. M–O In vivo, BAY-876 treatment results in significantly reduced tumor volume, weight, and growth rate in DU145-OE xenografts compared to DU145-NC controls. P Immunohistochemistry reveals a marked reduction in Ki67-positive area in DU145-OE tumors compared to DU145-NC. Q–S BAY-876 also significantly suppresses tumor volume, weight, and growth rate in PC-3-OE xenografts relative to PC-3-NC controls. T Immunohistochemical staining indicates decreased Ki67 expression in PC-3-OE tumors compared to PC-3-NC tumors.

Fig. 7. Effects of FOXA1-associated super-enhancers on metabolic regulation in PC-3 and DU145 prostate cancer cells.

A–C Changes in FOXA1 and SLC7A11 expression following FOXA1-SEs deletion in PC-3 and DU145 cells. D, E Impact of SE-sgRNA-mediated FOXA1-SEs deletion on BAY-876 sensitivity in PC-3 and DU145 cells. F, G Effects of SE-sgRNA on cysteine level in PC-3 and DU145 cells. H, I Effects of SE-sgRNA on NADP⁺/NADPH ratios in PC-3 and DU145 cells (1 µM). J, K SE-sgRNA-mediated alterations in intracellular GSH levels in PC-3 and DU145 cells. L F-actin and DAPI staining showing FOXA1-SEs influence on cytoskeletal morphology under glucose deprivation. M, N Apoptosis rates in PC-3 and DU145 cells under different treatments, including SE-sgRNA and various regulated cell death inhibitors.

Fig. 8

The molecular regulatory mechanism of SEs-FOXA1-SLC7A11 in disulfidptosis (By Figdraw).