CRB2 Activates an Epigenetic Axis to Promote Ferroptosis in Head and Neck Squamous Cell Carcinoma

Abstract

Therapeutic activation of ferroptosis is a potential strategy to induce cell death in cancer. Deciphering the epigenetic regulation of ferroptosis could provide insights into effective approaches for enhancing ferroptosis in cancer cells. In this study, we identified CRB2 as an epigenetic modulator of ferroptosis in head and neck squamous cell carcinoma (HNSCC). CRB2 expression correlated with the expression of ferroptosis-related genes and improved prognosis in patients with HNSCC. Ferroptosis inducers erastin and RSL3 significantly increased CRB2 expression, and overexpression of CRB2 sensitized HNSCC to ferroptosis, suppressing growth in vitro and in vivo. CRB2 upregulation led to an increase in the dimethylation of histone H4 lysine 20 in the promoter region of the ferroptosis inhibitor SLC7A11, which epigenetically inhibited its transcription and induced ferroptosis in HNSCC. Mechanistically, CRB2 hindered the interaction between the E8A isoform of the histone lysine demethylase LSD1 and the deubiquitinase USP7, thereby facilitating the degradation of LSD1(E8A) and subsequently increasing histone H4 lysine 20 dimethylation levels. Taken together, these results indicate that CRB2 stimulates ferroptosis by activating an epigenetic axis, suggesting that the upregulation of CRB2 is a potential therapeutic strategy for HNSCC.

Significance: CRB2 epigenetically inhibits SCL7A11-GPX4 signaling to promote ferroptosis and suppress tumor growth in head and neck squamous cell carcinoma, providing insights that could guide ferroptosis-activating treatment approaches.

Figure 1.

CRB2 as a potential epigenetic modifier of ferroptosis. A, Flowchart of the study strategy. ERG, epigenetic relatation gene; GSVA, gene set variation analysis. B, Heatmap of 19 differentially expressed epigenetic regulators (red genes are epi-regulatory factors with high expression in paracancerous tissues and good prognostic value in the TCGA-HNSCC tissues). C, Ten epigenetic genes exhibited significant differential expression between tumors and adjacent epithelial tissues in ULACAN. D, Kaplan–Meier analyses of ZBTB7C, SATB1, SCML4, and CRB2 in patients with HNSCC. E, mRNA expression of CRB2, ZBTB7C, SATB1, and SCML4 was determined by qRT-PCR in HN8 and Fadu cells treated with RSL3 (4 µmol/L) and erastin (20 µmol/L) for 24 hours (n = 3). F, CRB2 protein was determined by Western blotting in HN8 and Fadu cells treated with RSL3 and erastin. P values were determined by unpaired Student t tests.

Figure 2.

CRB2 promotes ferroptosis and inhibits growth in HNSCC in vitro and in vivo. A, CRB2 protein levels in HNSCC cell lines, including Tu686, HN8, JHU011, SCC17B, SCC4, Fadu, and the noncancerous oral mucosa cell line DOK were quantified by Western blotting assays. B and C, The forced expression efficiency of CRB2 was verified by qRT-PCR (B) and Western blotting assays (C). D, CCK8 assays showed the cell survival ratios of the vector control group and the CRB2 overexpression (OE) group in the presence of different concentrations of erastin for 72 hours [IC₅₀: negative control (NC; vector) 8.614 ± 1.018 µmol/L vs. overexpression (CRB2) 4.822 ± 0.312 µmol/L]. E–H, CRB2-overexpressed Fadu cells were treated with erastin, with or without Fer-1, Z-VAD, and 3-methyladenine (3-MA). E, Viability of Fadu cells. F, Representative phase contrast images. Scale bar, 100 μm. G, Flow cytometry detected the lipid peroxidation levels. H, GSH/oxidized glutathione (GSSG) levels. I, CRB2-overexpressed Fadu cells were treated with erastin and analyzed by TEM. Scale bars, 2 μm (columns 1 and 3) and 500 nm (columns 2 and 4). J, CRB2-overexpressed Fadu cells were treated with or without erastin and stained with rhodamine 123, a known mitochondria-tracking probe, and analyzed by structured illumination microscopy. Scale bars, 5 μm (columns 1 and 3) and 1 μm (columns 2 and 4). K, Workflow of constructing the subcutaneous xenograft model of nude mice with Fadu cells (n = 5). L, The tumor size is shown in the subcutaneous xenograft mouse. M, The dry weight of the final dissected tumor is shown. P values were determined by unpaired Student t tests (B, E, G, H, and M) or two-way ANOVA (D and L). .

Figure 3.

CRB2 induces ferroptosis through the inhibition of the SLC7A11-GPX4 pathway. A, Gene Ontology (GO) enrichment analysis of DEGs in transcriptome sequencing between CRB2-overexpressing and vector control Fadu cells (n = 3). B, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs in transcriptome sequencing. C, Venn diagram analyzes the DEGs (n = 623) between CRB2-overexpressing and vector control Fadu cells and the intersection of ferroptosis database genes (http://www.datjar.com:40013/bt2104/; n = 259). D, The mRNA expression levels of DRD5, SESN2, CHAC1, TSC22D3, and SLC7A11 in Fadu and SCC4 cells with vector and overexpression (OE) of CRB2. NC, negative control. E, Protein expression levels of SLC7A11, GPX4, FSP1, and DHODH in Fadu and SCC4 cells with vector and overexpression of CRB2. F, IHC staining for CRB2, SLC7A11, GPX4, FSP1, and DHODH in subcutaneous xenografts of mice with CRB2-overexpressing Fadu cells. Magnification, ×400. Scale bar, 50 μm. P values were determined by unpaired t tests.

Figure 4.

CRB2 enriches H4K20me2 at the promoter region of SLC7A11. A, The protein levels of H4K20me1, H4K20me2, and H4K20me3 in Fadu and SCC4 cells with vector and overexpression (OE) of CRB2. NC, negative control. B, IHC staining for H4K20me1, H4K20me2, and H4K20me3 in subcutaneous xenografts of mice with CRB2-overexpressed Fadu cells. C, The protein levels of ferroptosis regulatory factors and H4K20 methylation in Tu686 and HN8 cells with negative control and knockdown of CRB2. D and F, ChIP-level antibodies of H4K20me2 were used to pull down the bound DNA in CRB2-overexpressed Fadu cells, and further qPCR experiments confirmed H4K20me2-bound SLC7A11 (Promotor-463∼-338, Promotor-808∼-688, Promotor-1929∼-1730) and GPX4 (Promotor-127∼-13, Promotor-947∼-752, Promotor-1916∼-1813) promoter fragments (<200 bp). E and G, Agarose gel electrophoresis of ChIP-qPCR products (qPCR using enzyme-free water as the blank group, input group, negative control IgG group, positive control RNA pol II group, and IP experimental group). H, Dual luciferase reporter gene assay detected SLC7A11 promoter transcriptional activity in CRB2-overexpressed and vector control Fadu and SCC4 cells, and SLC7A11 transcriptional activity (RLU) was evaluated in the ratio of Gaussia luciferase/secreted alkaline phosphatase. The difference between groups was analyzed by unpaired t tests.

Figure 5.

CRB2 promotes ferroptosis and elevates the level of H4K20me2 by suppressing LSD1. A, The mRNA levels of genes related to H4K20me2 methylation were detected by quantitative real-time PCRB2. B, Potential CRB2-interacting H4K20me2 methyltransferases were identified by mass spectrometry analysis. C, Immunoblot analysis of the indicated antibodies in Fadu and SCC4 cells overexpressing CRB2. D, Analysis of LSD1(E8A) mRNA expression in the indicated cell lines through PCR amplification and DNA gel electrophoresis. E, Immunoblot analysis of whole cell lysates derived from Fadu and SCC4 cells subjected to the indicated treatments. F–I, Cell death analysis of Fadu and SCC4 cells overexpressing CRB2 and LSD1(E8A) after 24 hours of treatment with 20 µmol/L erastin. F, Cell viability analysis. G and H, lipid peroxidation levels. I, GSH levels. P values were determined by unpaired t tests. NC, negative control; OE, overexpression.

Figure 6.

CRB2 suppresses the deubiquitination of LSD1 by inhibiting the interaction between USP7 and LSD1. A, Western blot analysis of whole cell lysates derived from Fadu and SCC4 cells treated with 50 µg/mL cycloheximide (CHX) at the indicated time points after being transfected with LSD1(E8A)-His and either negative control (NC; vector) or overexpression (OE; CRB2). B, Cells were treated with 10 µmol/L MG132 for 10 hours, and LSD1(E8A) protein levels were detected by Western blotting. C, Fadu cells were transfected with Ub-HA and LSD1(E8A)-His, with or without CRB2-Flag. After treatment with 10 µmol/L MG132 for 10 hours, LSD1(E8A) ubiquitination was measured. D, Tu686 cells were transfected with Ub-HA and LSD1(E8A)-His, with or without shCRB2. Following treatment with 10 µmol/L MG132 for 10 hours, LSD1(E8A) ubiquitination was assessed. E, The indicated plasmids were transfected into Fadu and SCC4 cells for 48 hours. The cells were then harvested and subjected to coimmunoprecipitation using Flag magnetic beads. F, The subcellular localization of CRB2 and LSD1(E8A) was determined by immunofluorescence in Fadu cells. G, Coimmunoprecipitation analysis of the interaction between CRB2 and USP7. H, Fadu cells were transfected with USP7-HA, with or without LSD1(E8A)-His and CRB2-Flag. Western blotting was performed using the indicated antibodies. I, Fadu cells were transfected with or without USP7-HA and CRB2-Flag. Western blotting was conducted using the indicated antibodies. J–N, Fadu cells overexpressing CRB2 and USP7 were treated with the indicated treatments. J, Cell viability analysis. K and L, lipid peroxidation levels. M and N, GSH and oxidized glutathione (GSSG) levels. P values were determined by two-way ANOVA (A) or unpaired t tests (J–N).

Figure 7.

CRB2 overexpression is associated with a favorable prognosis in HNSCC. A, qRT-PCR analysis of 20 matched fresh-frozen HNSCC tissues for CRB2 mRNA expression (P = 0.0021). B, Representative IHC staining for CRB2 protein in HNSCC tissues (n = 114). Scale bars, 50 μm. C, Kaplan–Meier analysis of overall survival in all patients according to CRB2 protein level.