RRM2 protects against ferroptosis and is a tumor biomarker for liver cancer

Abstract

Background Ferroptosis is the process of cell death triggered by lipid peroxides, and inhibition of glutathione (GSH) synthesis leads to ferroptosis. Liver cancer progression is closely linked to ferroptosis suppression. However, the mechanism by which inhibition of GSH synthesis suppresses potential ferroptosis of liver cancer cells and whether ferroptosis-related liver cancer biomarkers have a promising diagnostic value remain unknown. Methods Ribonucleotide reductase regulatory subunit M2 (RRM2) levels were measured using an enzyme linked immunosorbent assay (ELISA), quantitative RT-PCR (qPCR), immunoblotting (IB) and immunochemistry (IHC). Cell viability and cell death were measured by a CellTiter-Glo luminescent cell viability assay and staining with SYTOX Green followed by flow cytometry, respectively. Metabolites were measured using the indicated kits. The Interaction between glutathione synthetase (GSS) and RRM2 was measured using immunofluorescence (IF), co-immunoprecipitation (co-IP) and the proximal ligation assay (PLA). The diagnostic value was analyzed using the area under the receiver operating characteristic curve (AUC-ROC). Bioinformatics analysis was performed using the indicated database. Results RRM2 showed specifically elevated levels in liver cancer and inhibited ferroptosis by stimulating GSH synthesis via GSS. Mechanistically, phosphorylation of RRM2 at the Threonine 33 residue (T33) was maintained at normal levels to block the RRM2–GSS interaction and therefore protected RRM2 and GSS from further proteasome degradation. However, under ferroptotic stress, RRM2 was dephosphorylated at T33, thus the RRM2–GSS interaction was promoted. This resulted in the translocation of RRM2 and GSS to the proteasome for simultaneous degradation. Clinically, serum RRM2 was significantly associated with serum alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT), albumin (ALB) and total bilirubin. The AUC-ROC for the combination of RRM2 with AFP was 0.947, with a sensitivity of 88.7% and a specificity of 97.0%, which indicates better diagnostic performance compared to either RRM2 or AFP alone. Conclusion RRM2 exerts an anti-ferroptotic role in liver cancer cells by sustaining GSH synthesis. Serum RRM2 will be useful as a biomarker to evaluate the degree to which ferroptosis is suppressed and improve diagnostic efficiency for liver cancer.

The Activation of Endothelial Cells Relies on a Ferroptosis-Like Mechanism: Novel Perspectives in Management of Angiogenesis and Cancer Therapy

“…So far, there are no specific markers of ferroptosis as a cell death mechanism and because we were exploring the non-lethal effect of ferroptosis, in this work we evaluated the levels of ROS-induced lipids peroxides underlying ferroptosis and GSH dynamics (67). The expression of genes pointed as being decreased upon ferroptosis activation, PTGS2, GPX4 and GSS was also assessed (10,(68)(69)(70). In our experimental conditions, we observed that Erastin significantly decreases cyst(e)ine uptake (Figure 1E) and promotes the increase of intracellular ROS (Figure 1A) and lipid peroxide levels (Figure 1C), without affecting the mitochondrial ROS content (Figure 1B).…”

Analysis of Ferroptosis-Mediated Modification Patterns and Tumor Immune Microenvironment Characterization in Uveal Melanoma

“…Glutathione synthetase (GSS), which plays a key role in metabolism, catalyzes the last step of glutathione (GSH) synthesis, which is an important antioxidant that protects cancer cells against potential ferroptosis ( Proneth and Conrad, 2019 ; Chen et al, 2020 ). In liver cancer, researchers found that RRM2 was capable of sustaining intracellular GSH by protecting GSS from degradation ( Yang et al, 2020 ). To the best of our knowledge, this is the first study to explore the correlation between the expression of GSS and the carcinogenic environment of UVM.…”

Prognostic Role and Potential Mechanisms of the Ferroptosis-Related Metabolic Gene Signature in Hepatocellular Carcinoma

“…As for the top ten hub Fer-MRGs, few studies have investigated their role in ferroptosis, while RRM2 has been identified as an anti-ferroptotic regulator in HCC by promoting the GSH synthesis in a recent study. 23 Hence, these findings need further investigation.…”

“… 24 , 25 RRM2 has been found a role in GSH synthesis and ferroptosis inhibition in HCC. 23 Besides, RRM2 was also found as a core gene in the p53 regulation pathway in hepatitis B virus-related HCC. 26 TXNRD1 was identified as a key metabolic reprogramming-associated gene, and could participate in the regulation of oxidative stress and lipid peroxidation in HCC.…”