Yang Yueyue, Lin Jiafei, Guo Susu, Xue Xiangfei, Wang Yikun, Qiu Shiyu, Cui Jiangtao, Ma Lifang, Zhang Xiao, Wang Jiayi
Department of Clinical Laboratory, Shanghai Tenth People's Hospital of Tongji University, Shanghai, 200072, China.
Department of Clinical Laboratory, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
Cancer Cell Int. 2020 Dec 7;20(1):587. doi: 10.1186/s12935-020-01689-8.
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.
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.
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.
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.
铁死亡是由脂质过氧化物引发的细胞死亡过程，抑制谷胱甘肽（GSH）合成会导致铁死亡。肝癌进展与铁死亡抑制密切相关。然而，抑制GSH合成抑制肝癌细胞潜在铁死亡的机制以及铁死亡相关的肝癌生物标志物是否具有良好的诊断价值仍不清楚。
使用酶联免疫吸附测定（ELISA）、定量逆转录聚合酶链反应（qPCR）、免疫印迹（IB）和免疫组化（IHC）检测核糖核苷酸还原酶调节亚基M2（RRM2）水平。分别通过CellTiter-Glo发光细胞活力测定和SYTOX Green染色后流式细胞术检测细胞活力和细胞死亡情况。使用指定试剂盒测量代谢物。使用免疫荧光（IF）、免疫共沉淀（co-IP）和邻近连接测定（PLA）检测谷胱甘肽合成酶（GSS）与RRM2之间的相互作用。使用受试者工作特征曲线下面积（AUC-ROC）分析诊断价值。使用指定数据库进行生物信息学分析。
RRM2在肝癌中特异性升高，并通过GSS刺激GSH合成来抑制铁死亡。机制上，RRM2苏氨酸33残基（T33）处的磷酸化维持在正常水平，以阻断RRM2-GSS相互作用，从而保护RRM2和GSS免于进一步的蛋白酶体降解。然而，在铁死亡应激下，RRM2在T33处去磷酸化，从而促进RRM2-GSS相互作用。这导致RRM2和GSS易位至蛋白酶体进行同时降解。临床上，血清RRM2与血清甲胎蛋白（AFP）、癌胚抗原（CEA）、丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、碱性磷酸酶（ALP）、γ-谷氨酰转肽酶（γ-GT）、白蛋白（ALB）和总胆红素显著相关。RRM2与AFP联合检测的AUC-ROC为0.947，灵敏度为88.7%，特异性为97.0%，这表明与单独的RRM2或AFP相比，具有更好的诊断性能。
RRM2通过维持GSH合成在肝癌细胞中发挥抗铁死亡作用。血清RRM2将作为一种生物标志物，用于评估铁死亡被抑制的程度并提高肝癌的诊断效率。
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