USP8-governed GPX4 homeostasis orchestrates ferroptosis and cancer immunotherapy

Abstract

Ferroptosis is an iron-dependent type of regulated cell death resulting from extensive lipid peroxidation and plays a critical role in various physiological and pathological processes. However, the regulatory mechanisms for ferroptosis sensitivity remain incompletely understood. Here, we report that homozygous deletion of Usp8 (ubiquitin-specific protease 8) in intestinal epithelial cells (IECs) leads to architectural changes in the colonic epithelium and shortens mouse lifespan accompanied by increased IEC death and signs of lipid peroxidation. However, mice with heterozygous deletion of Usp8 in IECs display normal phenotype and become resistant to azoxymethane/dextran sodium sulfate-induced colorectal tumorigenesis. Mechanistically, USP8 interacts with and deubiquitinates glutathione peroxidase 4 (GPX4), leading to GPX4 stabilization. Thus, USP8 inhibition destabilizes GPX4 and sensitizes cancer cells to ferroptosis in vitro. Notably, USP8 inhibition in combination with ferroptosis inducers retards tumor growth and enhances CD8⁺ T cell infiltration, which potentiates tumor response to anti-PD-1 immunotherapy in vivo. These findings uncover that USP8 counteracts ferroptosis by stabilizing GPX4 and highlight targeting USP8 as a potential therapeutic strategy to boost ferroptosis for enhancing cancer immunotherapy.

Keywords: GPX4; USP8; ferroptosis; immunotherapy; ubiquitination.

Fig. 1.

Homozygous deletion of Usp8 in IECs shortens mouse lifespan accompanied by impaired colon homeostasis and increased IEC death. (A–D) Representative general appearance (A), body weight (B), representative colon and rectum appearance (C), and survival rate (D) of Usp8^fl/fl, Usp8^fl/+;Vil-Cre, and Usp8^fl/fl;Vil-Cre pups. N = 16, 16, and 7 mice per group (B). N = 20 mice per group (D). (E–G) Representative H&E (E) and TUNEL (F) staining images of distal and proximal colon from Usp8^fl/fl, Usp8^fl/+;Vil-Cre, and Usp8^fl/fl;Vil-Cre mice at P10. The blue color indicated DAPI staining of nuclei, and the green color in nuclei indicated TUNEL-positive signals. The TUNEL-positive cells were counted in three randomly selected fields per biological replicate (G). N = 5 mice (E) or 3 mice (F and G) per group. (Scale bar, 50 μm.) (H–K) Representative immunohistochemical (IHC) staining images of 4-HNE, cleaved-caspase 3, and N-terminal domain of GSDMD (GSDMD-N) in colons from Usp8^fl/fl, Usp8^fl/+;Vil-Cre, and Usp8^fl/fl;Vil-Cre mice at P10. [Scale bar, 50 μm (H).] The 4-HNE staining (I) and GSDMD-N (J) were quantified by randomly selecting three fields per biological replicate on the basis of average optical density (AOD). The cleaved-caspase-3-positive cells (K) were counted in randomly selecting three fields per independent sample. N = 3 mice per group. In (B, G, and I–K), data were presented as mean ± SD; one-way ANOVA test with Tukey’s test. In (D), log-rank (Mantel-Cox) test was employed. ns, not significant, ***P < 0.001.

Fig. 2.

Heterozygous deletion of Usp8 in IECs suppresses colorectal tumorigenesis in mice. (A) A scheme of the AOM/DSS-induced colorectal cancer murine model. i.p., intraperitoneal. (B–D) Representative images of the tumor-burned colon and rectum (B), representative H&E staining (C), and tumor number per mouse (D) of colonic tissues from female Usp8^fl/fl and Usp8^fl/+;Vil-Cre mice after AOM/DSS treatment. The tumor number per mouse was measured at the end of the experiment. [Scale bar, 1 mm (C).] N = 7 mice per group (D). (E–H) Immunoblot (IB) analysis of Usp8 expression (E) and representative IHC staining of 4-HNE (G) in colon tumors from Usp8^fl/fl and Usp8^fl/+;Vil-Cre mice after AOM/DSS treatment. Relative Usp8 protein levels were quantified using ImageJ and normalized to Gapdh (F). [Scale bar, 50 μm (G).] The relative 4-HNE staining was quantified by randomly selecting three fields per biological replicate on the basis of AOD (H). N = 4 mice per group. (I) A schematic strategy for AOM/DSS-induced colorectal tumorigenesis in Usp8^fl/fl and Usp8^fl/+;Vil-Cre mice and subsequent ferrostatin-1 (Fer-1) treatment. The mice were intraperitoneally injected with vehicle or Fer-1 (2 mg/kg) daily with a break every 7 d. i.p., intraperitoneal. (J–O) Representative images of the tumor-burned colon and rectum (J), H&E staining of colonic tissues (K), and IHC staining of 4-HNE in tumor tissues (N) from Usp8^fl/fl and Usp8^fl/+;Vil-Cre mice after AOM/DSS treatment. [Scale bar, 1 mm (K).] The tumor number per mouse was measured at the end of the experiment. N = 6 mice per group (L and M). [Scale bar, 50 μm (N).] The relative 4-HNE staining was quantified by randomly selecting three fields per biological replicate on the basis of AOD. N = 3 mice per group (O). In (D, F, and H), data were presented as mean ± SD; two-tailed unpaired t test. In (L, M, and O), data were presented as mean ± SD; one-way ANOVA test with Dunnett’s test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

USP8 inhibition sensitizes cancer cells to ferroptosis in vitro. (A) Workflow for identifying key DUBs in ferroptosis regulation. HCT116 cells were transfected with an arrayed human ON-TARGETplus siRNA library against 96 DUBs. Cell viability was measured by the CCK8 assay after 36 h treatment with DMSO or erastin (15 μM). (B) A heatmap showing the results of RNAi-based deubiquitinating enzyme screening, pinpointing knockdown of USP8 and USP50 as the top-two candidates sensitize HCT116 cells to erastin treatment. Data were presented as a mean value, N = 3 independent biological replicates. (C) The correlation between USP8 expression in colorectal cancer cell lines and drug sensitivities to individual compounds. Plotted data were mined from the CTRP database that contains correlation coefficients between gene expression and drug sensitivity for colorectal cancer cell lines treated with 545 compounds. Plotted values are z-scored Pearson’s correlation coefficients. The drug sensitivities were determined using the AUC of the dose–response curves of the indicated compounds. (D and E) Cell viability (D) and cell death (E) were measured in HCT116 cells that were treated with erastin (10 μM) or DMSO for 16 h, then added indicated inhibitors (2.5 μM) or DMSO for another 9 h. (F–I) IB analysis of WCL from shGFP- or shUSP8-HCT116 cells (F). Cell viability was measured in shGFP- or shUSP8-HCT116 cells after treatment with different concentrations of erastin for 24 h (G). Cell death was assessed using PI staining in shGFP- or shUSP8-HCT116 cells exposed to erastin (20 μM) in combination with the indicated inhibitors for 36 h (H). Lipid peroxidation was measured by BODIPY™ 581/591 C11 staining in shGFP- or shUSP8-treated HCT116 cells subjected to erastin (10 μM) in combination with the indicated inhibitors for 24 h (I). Fer-1, 10 μM; Necrostatin-1 (Nec), 2 μM; Z-VAD-FMK (Z-V), 10 μM. LE, long exposure; SE, short exposure. (J–M) IB analysis of WCL from shGFP- or shUSP8-SW620 cells (J). Cell viability was measured in shGFP- or shUSP8-SW620 cells after treatment with different concentrations of erastin for 24 h (K). Cell death was assessed in shGFP- or shUSP8-SW620 cells exposed to erastin (15 μM) in combination with the indicated inhibitors for 36 h (L). Lipid peroxidation was measured by BODIPY™ 581/591 C11 staining in shGFP- or shUSP8-treated SW620 cells subjected to erastin (10 μM) in combination with the indicated inhibitors for 24 h (M). Fer-1, 10 μM; Nec, 2 μM; Z-V, 10 μM. In (D and E), data were presented as mean ± SD; N = 3 independent biological replicates; two-tailed unpaired t test. In (G and K), data were presented as mean ± SD; N = 3 independent biological replicates; two-way ANOVA test with Dunnett’s test. In (H, I, L, and M), data were presented as mean ± SD; N = 3 independent biological replicates; one-way ANOVA test with Dunnett’ test. ***P < 0.001.

Fig. 4.

USP8 stabilizes GPX4 through removing K48-linked ubiquitination on GPX4. (A and B) IB analysis of WCL from shGFP- or shUSP8-HCT116 (A) or SW620 (B) cells. (C and D) IB analysis of WCL derived from HCT116 (C) or SW620 (D) cells treated with DUB-IN-2 (1 μM or 2 μM) for 9 h. (E and F) IB analysis of WCL from shGFP- or shUSP8-HCT116 cells with treatment at indicated time points followed by 200 μg/mL CHX treatment (E). GPX4 band intensity was normalized to vinculin and then compared to the t = 0 timepoint (F). (G) IB analysis of WCL derived from shGFP- or shUSP8-HCT116 cells treated with MG132 (10 μM), bafilomycin A1 (BafA1, 100 nM), or chloroquine (CQ, 5 μM) for 12 h. (H) IB analysis of WCL and anti-USP8 immunoprecipitates (IPs) derived from SW620 cells. (I) IB analysis of WCL and GST pull-down precipitates from recombinant His-GPX4 incubated with GST or GST-USP8 protein. (J and K) IB analysis of WCL and Ni-NTA pull-down products derived from lysates of HEK293T cells transfected with the indicated constructs. Cells were treated with MG132 (10 µM) for 12 h before harvesting. Ub, ubiquitin. In (F), data were presented as mean ± SD.; N = 3 independent biological replicates; two-tailed unpaired t test. *P < 0.05, ***P < 0.001.

Fig. 5.

USP8 inhibition in combination with ferroptosis inducer retards tumor growth and promotes tumor-infiltrating CD8⁺ T cells. (A) A schematic illustration of DUB-IN-2 and SAS treatment strategy for BALB/c mice bearing subcutaneous CT26 tumors. i.p., intraperitoneal. (B–D) The tumor growth curves (B), images of endpoint tumors (C), and endpoint tumor weight (D). N = 6 mice per group. (E and F) Representative images of 4-HNE IHC staining in CT26 tumor tissues from BALB/c mice with indicated treatment. [Scale bar, 100 μm (E).] The relative optical density of 4-HNE staining was quantified by randomly selecting three fields per independent sample on the basis of AOD. N = 4 mice per group (F). (G and H) Flow cytometry analysis of the percentage of tumor-infiltrating CD8⁺ T cells (G) or CD4⁺ T cells (H) from CT26 tumors with indicated treatments. N = 6 mice per group. (I) A schematic strategy for AOM/DSS-induced colorectal tumorigenesis in Usp8^fl/fl and Usp8^fl/+;Vil-Cre mice and subsequent SAS treatment. The Usp8^fl/fl and Usp8^fl/+;Vil-Cre mice were intraperitoneally injected with SAS (100 mg/kg) daily with a break every 7 d. (J–N) Representative images of colon and rectum (J) and 4-HNE IHC staining in tumors (M) from Usp8^fl/fl and Usp8^fl/+;Vil-Cre mice at the end of the experiment. The tumor number per mouse was measured at the end of the experiment. N = 7 mice per group (K and L). [Scale bar, 100 μm (M).] The relative 4-HNE staining was quantified by randomly selecting three fields per independent sample on the basis of AOD. N = 3 mice per group (N). (O and P) Flow cytometry analysis of the percentage of tumor-infiltrating CD8⁺ T cells (O) or CD4⁺ T cells (P) derived from colorectal cancer tissues of the AOM/DSS-induced mice with indicated treatment. N = 5 mice per group. In (B), data were presented as mean ± SD; two-way ANOVA test with Dunnett’s test. In (D, F–H, K, L, and N–P), data were presented as mean ± SD; one-way ANOVA test with Dunnett’s test. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

USP8 inhibition in combination with ferroptosis inducer sensitizes tumors to anti-PD-1 immunotherapy in vivo. (A) A schematic illustration of the treatment strategy for BALB/c mice bearing subcutaneous CT26 tumors. Mice bearing CT26 tumors were treated with control vehicle, DUB-IN-2 (1 mg/kg) plus SAS (100 mg/kg), PD-1 mAb (100 μg per mouse), or triple combined treatment (DUB-IN-2 plus SAS plus PD-1 mAb), respectively. i.p., intraperitoneal. mAb, monoclonal antibody. (B–D) The tumor growth curves (B), images of endpoint tumors (C), and endpoint tumor weight (D). N = 6 mice per group. (E–H) Flow cytometry analysis of the percentage of tumor-infiltrating CD8⁺ T cells (E), CD4⁺ T cells (F), IFNγ⁺CD8⁺ T cells (G), or TNF⁺CD8⁺ T cells (H) from CT26 tumors with indicated treatments. N = 6 mice per group. (I) A schematic strategy for AOM/DSS-induced colorectal tumorigenesis in Usp8^fl/fl and Usp8^fl/+;Vil-Cre mice and treatment with SAS and PD-1 mAb. The mice were intraperitoneally injected with PD-1 mAb (100 μg per mouse) every 3 d and SAS (100 mg/kg) daily with a break every 7 d. (J–M) Representative images of gross appearance of colon and rectum (J) and H&E staining of colorectal tumors (K) from Usp8^fl/fl and Usp8^fl/+;Vil-Cre mice at the end of the experiment. [Scale bar, 2 mm (K).] The tumor number per mouse was measured at the end of the experiment (L and M). N = 5 mice per group. In (B), data were presented as mean ± SD; two-way ANOVA test with Dunnett’s test. In (D, E–H, L, and M), data were presented as mean ± SD; one-way ANOVA test with Dunnett’s test. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.