TRIM21-regulated FSP1 Plasma Membrane Translocation Confers Ferroptosis Resistance In Digestive System Tumors

Objective: Ferroptosis is a form of cell death characterized by lethal iron-dependent accumulation of lipid peroxides which holds great therapeutic potential in cancers.However,cancer cells usually upregulate pivotal antiferroptotic proteins to prevent ferroptosis such as FSP1(Ferroptosis Suppressor Protein 1).At present,the upstream regulation mechanisms of FSP1,especially its post-translational modification,remain elusive.This study was to explore the role of FSP1 ubiquitination on the ferroptosis of tumors and the underlying molecular mechanisms,providing new targets for tumor treatment strategies based on ferroptosis.Methods: Immunoprecipitation and protein ubiquitination assay were used to explore the ubiquitination of FSP1 and the type of its ubiquitin chain.Cell fractionation protein extraction and confocal microscopy were used to explore the subcellular location of FSP1 after ferroptosis inducer treatment.Immunoprecipitation-mass spectrometry,Co-IP,immunofluorescence and GST-pull down assays were used to verify the interaction of FSP1 and TRIM21,together with their binding domains.Tissue microarray of pancreatic cancer and public database of digestive system tumors were used to analyze the expression of TRIM21 and its correlation with prognosis.GSEA analysis was carried out to explore the relevance of TRIM21 with ferroptosis pathways.Expression level of TRIM21 and its binding degree with FSP1 was analyzed after ferroptosis induction.Immunoprecipitation was used to explore the regulatory mechanisms of TRIM21 on ubiquitination of FSP1.Point mutation plasmids were constructed to identify the exact ubiquitination site of FSP1.Western blot was used to explore whether TRIM21 has an impact on the protein stability of FSP1.Membrane and cytosol protein extraction and confocal microscopy were used to explore the influence of TRIM21 and ubiquitination site of FSP1 on the membrane translocation of FSP1.CRISPR/Cas9 technology was used to construct TRIM21 or FSP1 knockout cell lines,respectively.In vitro and in vivo experiments were carried out to explore the influence of TRIM21 and FSP1 on ferroptosis resistance of cancer cells.Pancreatic cancer KPPC mice model was constructed and the therapeutic effect of ferroptosis inducers on KPPC mice was explored.Results: The K63 ubiquitination and plasma membrane localization of FSP1 increase significantly after ferroptosis induction.The W381/W383 sites of TRIM21 interact with the NADH oxidoreductase domain of FSP1.TRIM21 is highly expressed and correlates with poor prognosis in various digestive system tumors.GSEA analysis indicates that TRIM21 is associated with ferroptosis pathways.Expression level of TRIM21 and its binding with FSP1 increases after ferroptosis induction.TRIM21 promotes the K63 ubiquitination of FSP1 in a dose-dependent manner,which is dependent on its E3 ligase activity and binding capacity.TRIM21 doesn’t affect FSP1’s protein stability,but facilitates its translocation to the plasma membrane by K63 ubiquitination.FSP1 undergoes ubiquitination on its K322 and K366 sites,which are critical for its translocation to plasma membrane.In vitro and in vivo functional experiments confirms the antiferroptotic role of TRIM21 through FSP1 in tumors,whereas knockout of TRIM21 sensitizes tumors to ferroptosis.Pancreatic cancer KPPC mice model is successfully constructed and ascites-derived primary cell line was established.The ferroptosis inducer RSL3 has a therapeutic effect on pancreatic cancer of KPPC mice.Conclusions: The K63 ubiquitination and plasma membrane localization of FSP1 increased significantly upon ferroptosis induction.TRIM21 binds to FSP1 and catalyzes its K63 ubiquitination,which has no influence on its protein level but facilitates its translocation to plasma membrane.TRIM21 is highly expressed in various digestive system tumors and is associated with poor prognosis.TRIM21 promotes the ferroptosis resistance of digestive system tumors through FSP1.The ferroptosis inducer RSL3 has a therapeutic effect on pancreatic cancer of KPPC mice.