Gankyrin inhibits ferroptosis through the p53/SLC7A11/GPX4 axis in triple-negative breast cancer cells.
Gankyrin inhibits ferroptosis through the p53/SLC7A11/GPX4 axis in triple-negative breast cancer cells.
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DOI：
10.1038/s41598-023-49136-8
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发表时间：
2023-12-08
影响因子：
4.6
通讯作者：
Zheng, Wu-Ping
中科院分区：
文献类型：
--
作者：
Lei, Ming;Zhang, Yun-Long;Huang, Feng-Ying;Chen, Heng-Yu;Chen, Ming-Hui;Wu, Ri-Hong;Dai, Shu-Zhen;He, Gui-Sheng;Tan, Guang-Hong;Zheng, Wu-Ping
Gankyrin在三阴性乳腺癌（TNBC）中以高水平被发现，并且已经被确定与E3泛素连接酶MDM 2和p53形成复合物，导致肝癌细胞中p53的降解。因此，本研究试图确定Gankyrin是否可以通过TNBC细胞中的这种机制抑制铁凋亡。使用生物信息学研究Gankyrin的表达与TNBC预后的关系。然后进行免疫共沉淀和GST下拉测定以确定gankyrin和MDM 2复合物的存在。使用RT-qPCR和免疫印迹来检查与铁凋亡相关的分子，例如gankyrin、p53、MDM 2、SLC 7A 11和GPX 4。此外，使用流式细胞术检测7-AAD和乳酸脱氢酶释放试验以及脂质过氧化物C11-BODIPY评价细胞死亡。结果显示，Gankyrin在TNBC组织和细胞系中的表达显著较高，并且与患者的不良预后相关。随后的研究表明，抑制gankyrin活性触发TNBC细胞中的铁凋亡。此外，沉默gankyrin引起p53蛋白表达的增加，而不改变其mRNA表达。免疫共沉淀和GST下拉实验表明，Gankyrin和MDM 2形成复合物。在缺乏MDM 2和p53的小鼠胚胎成纤维细胞中，观察到这种gankyrin/MDM 2复合物泛素化p53，从而提高了被铁凋亡抑制的分子的表达，如SLC 7A 11和GPX 4。此外，沉默TNBC细胞中的gankyrin破坏了gankyrin/MDM 2复合物的形成，阻碍了p53的降解，增加了SLC 7A 11的表达，阻碍了半胱氨酸的摄取，并减少了GPX 4的产生。我们的研究结果表明，TNBC细胞能够通过gankyrin/p53/SLC 7A 11/GPX 4信号通路防止细胞铁凋亡，表明gankyrin可能是预测TNBC预后的有用生物标志物或潜在的治疗靶点。
Gankyrin is found in high levels in triple-negative breast cancer (TNBC) and has been established to form a complex with the E3 ubiquitin ligase MDM2 and p53, resulting in the degradation of p53 in hepatocarcinoma cells. Therefore, this study sought to determine whether gankyrin could inhibit ferroptosis through this mechanism in TNBC cells. The expression of gankyrin was investigated in relation to the prognosis of TNBC using bioinformatics. Co-immunoprecipitation and GST pull-down assays were then conducted to determine the presence of a gankyrin and MDM2 complex. RT-qPCR and immunoblotting were used to examine molecules related to ferroptosis, such as gankyrin, p53, MDM2, SLC7A11, and GPX4. Additionally, cell death was evaluated using flow cytometry detection of 7-AAD and a lactate dehydrogenase release assay, as well as lipid peroxide C11-BODIPY. Results showed that the expression of gankyrin is significantly higher in TNBC tissues and cell lines, and is associated with a poor prognosis for patients. Subsequent studies revealed that inhibiting gankyrin activity triggered ferroptosis in TNBC cells. Additionally, silencing gankyrin caused an increase in the expression of the p53 protein, without altering its mRNA expression. Co-immunoprecipitation and GST pull-down experiments indicated that gankyrin and MDM2 form a complex. In mouse embryonic fibroblasts lacking both MDM2 and p53, this gankyrin/MDM2 complex was observed to ubiquitinate p53, thus raising the expression of molecules inhibited by ferroptosis, such as SLC7A11 and GPX4. Furthermore, silencing gankyrin in TNBC cells disrupted the formation of the gankyrin/MDM2 complex, hindered the degradation of p53, increased SLC7A11 expression, impeded cysteine uptake, and decreased GPX4 production. Our findings suggest that TNBC cells are able to prevent cell ferroptosis through the gankyrin/p53/SLC7A11/GPX4 signaling pathway, indicating that gankyrin may be a useful biomarker for predicting TNBC prognosis or a potential therapeutic target.
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