| Background:Gastric cancer is one of the most common cancers worldwide,most patients are already in the progressive stage when diagnosed in China.Chemotherapy improves survival and quality of life for patients with locally advanced unresectable or metastatic gastric cancer.Among commonly used chemotherapy drugs in patients with gastric cancer,cisplatin is one of the earliest discovered and most effective platinum-based chemotherapeutic agents.The clinical application of cisplatin is largely limited by primary resistance or acquired resistance developed during treatment.In our previous study on PFKFB3 regulation of trastuzumab resistance in gastric cancer,we unexpectedly found that PFKFB3 was able to mediate the resistance to cisplatin toxicity in gastric cancer cells in vitro.This study attempted to elucidate the mechanism of PFKFB3-mediated cisplatin resistance in gastric cancer,with the aim of enhancing chemotherapy efficacy,overcoming chemoresistance and improving the prognosis for gastric cancer patients treated with cisplatin.Methods:1.Differential analyses were done with the help of TNMplot online tool and TCGA-STAD RNA-seq data,and surgical specimens from gastric cancer patients in our center were collected for immunohistochemical staining to verify the expression of PFKFB3 in gastric cancer tissues;3.GEPIA and K-M plotter online tools were utilized to verify the relationship between PFKFB3 and the prognosis of gastric cancer patients;4.GSEA enrichment analyses were performed to explore the relationship between PFKFB3 and cisplatin resistance and the potential downstream mechanisms;5.Exogenous PFKFB3,xCT,PFKFB3 segments,xCT mutations overexpression and PFKFB3 siRNA sequences were designed to construct a series of overexpression and knockdown gastric cancer cell lines by plasmid transfection,siRNA transfection and lentiviral transfection;6.Wesrn blot and qPCR were used to detect the protein level and mRNA level of gene expression;7.CCK-8,PI staining and subcutaneous tumor formation model in nude mice were operated to verify the growth and survival status of gastric cancer cells;8.Cellular immunofluorescence and Co-IP were performed to validate the interaction between PFKFB3 and xCT;9.Phosphorylated proteomics were applied to search for PFKFB3 downstream targets;10.Cys,GSH,ROS and MDA were detected to characterize the ferroptosis levels in gastric cancer cells.Results:1.PFKFB3 mediated cisplatin resistance in gastric cancer in vitro and in vivo.2.PFKFB3 was upregulated in gastric cancer tissues,which indicated poor prognosis.3.PFKFB3 was significantly associated with ferroptosis pathway4.Cisplatin induced ferroptosis in gastric cancer cells.5.PFKFB3 mediated cisplatin resistance in gastric cancer via ferroptosis inhibition.6.PFKFB3 interacted with xCT through its phosphatase structural domain.7.PFKFB3 regulated the level of xCT phosphorylation.8.PFKFB3 and xCT did not affect each other in terms of gene expression.9.S26 phosphorylation of xCT was closely associated with ferroptosis.10.PFKFB3 mediated cisplatin resistance in gastric cancer through regulation of S26 phosphorylation of xCT.11.Erastin reversed PFKFB3-mediated cisplatin resistance in gastric cancer in vitro and in vivo.Conclusions:1.PFKFB3 is upregulated in gastric cancer tissues and is associated with poor prognosis of gastric cancer patients.2.PFKFB3 interacts with xCT through its phosphatase structural domain to induce the S26 dephosphorylation of xCT,thereby enhancing xCT transport function,upregulating intracellular Cys and GSH levels and downregulating intracellular ROS and MDA levels,finally inhibiting cisplatin-induced ferroptosis in gastric cancer cells.3.The combination of cisplatin and xCT inhibitor Erastin could reverse PFKFB3mediated cisplatin resistance in gastric cancer,which is expected to provide a potential chemoresistant treatment strategy for clinical practice. |
PDF Full Text Request