TRIM21‐Promoted FSP1 Plasma Membrane Translocation Confers Ferroptosis Resistance in Human Cancers

Abstract Ferroptosis, an iron‐dependent form of regulated cell death driven by excessive accumulation of lipid peroxides, has become a promising strategy in cancer treatment. Cancer cells exploit antioxidant proteins, including Ferroptosis Suppressor Protein 1 (FSP1), to prevent ferroptosis. In this study, it is found that the E3 ubiquitin ligase TRIM21 bound to FSP1 and mediated its ubiquitination on K322 and K366 residues via K63 linkage, which is essential for its membrane translocation and ferroptosis suppression ability. It is further verified the protective role of the TRIM21‐FSP1 axis in RSL3‐induced ferroptosis in cancer cells and a subcutaneous tumor model. Moreover, TRIM21 is highly expressed in multiple gastrointestinal (GI) tumors, and its expression is further stimulated upon ferroptosis induction in cancer cells and the KPC mouse model. In summary, This study identifies TRIM21 as a negative regulator of ferroptosis through K63 ubiquitination of FSP1, which can serve as a therapeutic target to enhance the chemosensitivity of tumors based on ferroptosis induction.


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Figure S3 .
Figure S3.TRIM21 doesn't affect the protein level of FSP1.A-B) Expression level of FSP1 was determined by immunoblotting in PANC-1 (A) or A549 (B) cells transfected with increasing amounts of Flag-TRIM21.C-D) Expression level of FSP1 was determined by immunoblotting in MHCC-97H (C) or Hela (D) cells transfected with HA-FSP1 and increasing amount of Flag-TRIM21.E) MHCC-97H cells were transfected with HA-FSP1 with or without co-transfection of Flag-TRIM21 and treated with the proteasome inhibitor MG132 (10 μM, 6 h) or lysosome inhibitor CQ (20 μM, 6 h) or 3-MA (4 mM, 6 h).Expression level of FSP1 was determined by immunoblotting.F) MHCC-97H cells were transfected with HA-FSP1 and Flag-TRIM21 and treated with CHX (50 μM) for 0-12 h.Expression level of FSP1 was determined by immunoblotting.Representative images were shown in the left panel and quantitative analysis results were shown in the right panel (n = 3).Data were presented as means ± SD.P values were calculated by two-way ANOVA.ns, not significant.

Figure S4 .
Figure S4.Construction of knockout cell lines using CRISPR/Cas9 technology.A) Schematic description of sgRNA target in the genome of human TRIM21 (upper panel) and amino acid sequence of isolated cell subclones verified by sanger sequencing (left panel).Immunoblotting was used to confirm the completely knockout of TRIM21 in the right panel.B) Schematic description of sgRNA target in the genome of human FSP1 (upper panel) and amino acid sequence of isolated cell subclones verified by sanger sequencing (left panel).Immunoblotting was used to confirm the completely knockout of FSP1 in the right panel.

Figure S5 .
Figure S5.The protective role of TRIM21 against ferroptosis in GI tumor cells.A-C) KPC-1A cells transfected with indicated plasmids were treated with DMSO or 0.1 μM RSL3 for 1 h (A) or 1 μM RSL3 for 4 h (B and C) prior to C11-BODIPY staining.Flowcytometry were used to analyze the level of lipid peroxides as indicated by FITC fluorescence.Representative images of flowcytometry were shown in the upper panel.C11-BODIPY positive rate were counted in the lower panel (n = 3).Data were presented as means ± SD.D) MHCC-97H cells transfected with indicated plasmids were treated with 1 μM RSL3 for 6 h, and then were subjected to TEM analysis.Representative images were shown in the left panel.Mitochondrial shrinkage and mitochondrial ridge disappearance could be seen in cells with higher level of ferroptosis.Scale bars, 1 μm.Mitochondrial lengths along the long axis in each group were measured and summarized in the right panel.E-G) Cells were transfected with vector or sgTRIM21 plasmid and treated with DMSO or 1

Figure S6 .
Figure S6.TRIM21 is associated with ferroptosis in multiple GI tumors.A-E) GSEA was conducted to explore the correlation between TRIM21 and ferroptosis-related genes based on LIHC (A), COAD (B), STAD (C), CHOL (D) and ESCA (E) dataset in TCGA database.F) Friends analysis was conducted to explore whether TRIM21 functions as an important hub gene in ferroptosis-related genes.Bar plot in the left panel shows the average score of each gene.Higher score indicates a closer relationship of the gene with other genes in the group based on GO annotation.Heatmap in the right panel shows the exact score of each gene with other genes in the group.