Blocking Ubiquitin-Specific Protease 7 Induces Ferroptosis in Gastric Cancer via Targeting Stearoyl-CoA Desaturase

Abstract

Gastric cancer (GC) presents a formidable global health challenge, and conventional therapies face efficacy limitations. Ubiquitin-specific protease 7 (USP7) plays pivotal roles in GC development, immune response, and chemo-resistance, making it a promising target. Various USP7 inhibitors have shown selectivity and efficacy in preclinical studies. However, the mechanistic role of USP7 has not been fully elucidated, and currently, no USP7 inhibitors have been approved for clinical use. In this study, DHPO is identified as a potent USP7 inhibitor for GC treatment through in silico screening. DHPO demonstrates significant anti-tumor activity in vitro, inhibiting cell viability and clonogenic ability, and preventing tumor migration and invasion. In vivo studies using orthotopic gastric tumor mouse models validate DHPO's efficacy in suppressing tumor growth and metastasis without significant toxicity. Mechanistically, DHPO inhibition triggers ferroptosis, evidenced by mitochondrial alterations, lipid Reactive Oxygen Species (ROS), Malondialdehyde (MDA) accumulation, and iron overload. Further investigations unveil USP7's regulation of Stearoyl-CoA Desaturase (SCD) through deubiquitination, linking USP7 inhibition to SCD degradation and ferroptosis induction. Overall, this study identifies USP7 as a key player in ferroptosis of GC, elucidates DHPO's inhibitory mechanisms, and highlights its potential for GC treatment by inducing ferroptosis through SCD regulation.

Keywords: SCD; USP7; USP7 inhibitor; ferroptosis; gastric cancer; ubiquitination.

Figure 1

USP7 is an oncogenic driver in gastric cancer (GC). A,B) Barplots showing the USP7 essentiality in GC cell lines from publicly available RNAi screening (A) and CRISPR‐Cas9 screening (B). C) mRNA expression of USP7 in human GC tissues and normal tissues from the TCGA and GTEx cohort. D) Correlation analysis of mRNA expression of USP7 and Ki‐67 in human GC tissues in the TCGA, ACRG, and Zhejiang cohorts. E) The representative IHC images and statistical results of USP7 expression in para‐cancerous and tumor tissues of GC patients. F) Survival analysis of 128 GC patients with high USP7 expression compared to low expression.

Figure 2

Identification of a novel USP7 inhibitor DHPO. A) Overview of in silico screening of small‐molecule inhibitors of USP7. B) Structure of DHPO. C) Molecular docking of DHPO in the allosteric site of human USP7. D) MicroScale Thermophoresis (MST) assay showing binding affinity of DHPO and purified USP7 protein. E) The anti‐proliferative effects of DHPO on MGC803 and MKN1 were assessed using CCK‐8 assay after 72 h of treatment. F) Linear MS spectra of USP7 without or with DHPO incubation show a mass shift of USP7 with the addition of DHPO. G) MS/MS analysis of DHPO‐bound peptide 294–301.

Figure 3

The USP7 inhibitor DHPO exerts anti‐gastric cancer (GC) activity in vitro. A) RNA‐seq and pathway enrichment analysis in GC cells treated with DHPO or DMSO. B) Colony formation experiments were performed to quantify and analyze cell colonies in MGC803 and MKN1 cell lines treated with DHPO at concentrations of 0, 1, and 2 µm. C) Wound healing assays were performed to evaluate cell migration after 12 h and 36 h of DHPO treatment (0, 1, and 2 µM) in MGC803 and MKN1 cell lines, with wound closure percentage calculated. D) Transwell assays were performed to quantify the effects of DHPO (0, 1, and 2 µm) on the invasive ability of MGC803 and MKN1 cell lines.

Figure 4

The USP7 inhibitor DHPO demonstrates anti‐cancer efficacy in vivo without causing significant host toxicity. A) A schema depicting the assessment of in vivo efficacy and toxicity of DHPO using orthotopic tumor mouse models derived from MGC803‐Luc and MKN1‐Luc cell lines. B) Bioluminescence imaging demonstrated the efficacy of vehicle and DHPO (5 or 10 mg kg⁻¹) in mice bearing MGC803‐Luc orthotopic tumors. C) Quantification of fluorescence intensity was conducted for in vivo imaging of mice treated with vehicle or DHPO (5 and 10 mg kg⁻¹). D) Body weight changes monitored in SCID mice receiving vehicle or DHPO treatment. E) Bioluminescence imaging illustrated the differences between mice bearing MKN1‐Luc orthotopic tumors in the vehicle group and those treated with DHPO (10 mg kg⁻¹). F) Quantification of fluorescence intensity performed for in vivo imaging of mice in the vehicle and DHPO (10 mg kg⁻¹) treatment groups. G) Body weight changes were recorded over time in tumor‐bearing mice treated with vehicle and DHPO (10 mg kg⁻¹). H) Statistical analysis presented the numbers of mice having metastasis to liver (LM), spleen (SM), and peritoneum (PM) or no significant metastasis (NM). I) Histological examination of major organs (heart, liver, spleen, lungs, kidneys, and brain) in the vehicle‐ and DHPO (5 or 10 mg kg⁻¹)‐treated mice bearing orthotopic tumors.

Figure 5

USP7 inhibitor DHPO promotes ferroptosis in gastric cancer. A) TMT proteomic analysis revealed significant dysregulation of ferroptosis in MGC803 cells treated with DHPO compared to DMSO after 24 h. B) Transmission electron microscopy (TEM) images showed alterations in mitochondria morphology in NUGC4 cells upon DHPO treatment. C) Lipid ROS level in MGC803 cells was measured with the treatment of DHPO at different concentrations. D) Lipid ROS level in AZ521 cells was measured with the treatment of DMSO, DHPO, FT671, and P5091. E) Immunoblots for USP7 in MGC803 cells transfected with scrambled or USP7 siRNAs. (F) The representative images (left) and statistical results (right) of lipid ROS after USP7 knockdown in MGC803 cells. G) Immunoblots for USP7 in AZ521 cells transfected with control or USP7 shRNA. H) Lipid ROS measurement after USP7 knockdown with or without treatment of Fer‐1 in AZ521 cells. I) MDA content was determined in MGC803 and AZ521 cells treated with DMSO, DHPO, FT671, and P5091. J) Total iron content was determined in the AZ521 cells treated with DMSO, DHPO, FT671, and P5091. K) Total iron content was determined after USP7 knockdown with or without treatment of Fer‐1 in the MGC803 cells.

Figure 6

USP7 inhibition induces ferroptosis by targeting the putative substrate SCD. A) Heatmap analysis depicted the differential expression of proteins between DHPO‐ and DMSO‐treated MGC803 cells, with marked downregulation of SCD in the DHPO group. B) Western blot analysis showed the reduced SCD protein levels in MGC803, HGC27, and AGS cell lines treated with escalating concentrations of DHPO for 24 h. C) qRT‐PCR results exhibited a concentration‐dependent increase in SCD mRNA expression in MGC803 and AZ521 cell lines treated with increasing concentrations of DHPO. D) DHPO treatment resulted in an accelerated degradation rate of SCD protein compared to the non‐pretreated group, as assessed by Western blot analysis. E) Treatment of MGC803 cells with MG132 attenuated the degradation of SCD protein by DHPO, as evidenced by Western blot analysis. F) The schematic diagram illustrates that USP7 inhibitor DHPO induces ferroptosis by degrading SCD. G) MGC803 and AZ521 cell lines were harvested for co‐immunoprecipitation assay by using control IgG or USP7 antibody; the immunoprecipitates were then blotted to detect the protein levels of USP7 and SCD. H) The protein level of SCD was detected using Western blot analysis in HGC27 and AGS cell lines transfected with control siRNA or USP7 siRNA. I) The protein level of SCD was detected using Western blot analysis in MGC803 and AZ521 cell lines infected with USP7 shRNA and control shRNA. J,K) USP7 knockdown and control MGC803 cells were treated with 40 µm cycloheximide (CHX) at the indicated time points. The protein degradation rate of SCD was detected using Western blotting and quantified using Image J. L) Knockdown of USP7 using shRNA in AZ521 cells promoted the downregulation of SCD protein expression upon DHPO treatment, as detected by Western blot analysis. M) Control or USP7 knockdown MGC803 cells transfected with HA‐Ub were subjected to co‐immunoprecipitation experiments with HA antibodies, and then Western blotting was used to detect the ubiquitination level of SCD.

Figure 7

USP7‐SCD axis is negatively correlated with overall survival of PDX‐bearing mice. (A‐C) Tumor volume A), survival time B), and body weight C) were evaluated in mice bearing patient‐derived xenografts (PDX) treated with vehicle, DHPO (5 and 10 mg kg⁻¹), or cisplatin (5 mg kg⁻¹). D) SCD protein expression was assessed by Western blot analysis in PDXs from control and DHPO‐treated groups. E) Immunohistochemistry (IHC) images demonstrated USP7, 4‐HNE, and SCD expression, and Hematoxylin and Eosin staining (HE) in PDX from the control and DHPO‐treated groups. F) scRNA‐seq analyses showed that USP7 and SCD were highly expressed in gastric cancer cells.