USF2 Negatively Regulates Ferroptosis In Pancreatic Cancer Cells Through Transcriptional Regulation Of PKM2 And Preliminary Research On Molecular Imaging

Objective:Pancreatic cancer（PC）is a highly malignant tumor that is the fourth leading cause of cancer-related death.Due to the lack of early symptoms,80%of patients are already at a late stage when being diagnosed.Besides,PC is poorly in response to chemotherapy and radiotherapy,resulting in a low five-year survival rate,which was only 9%.Therefore,searching for the potential therapeutic targets for the treatment of PC is of great significance.Ferroptosis is a kind of programmed cell death that is different from necrosis and apoptosis,and it is mainly triggered by the accumulation of iron-dependent lipid peroxides.Emerging evidence has revealed the anticancer effects of ferroptosis inducers.Upstream stimulatory factor 2（USF2）is a transcription factor implicated in different cancers acting as a tumor promoter or suppressor.Pancreatic iron overload was observed in Usf2 knockout mice and USF2 played an essential role in maintaining the level of intracellular ROS and mitochondrial homeostasis.Given that iron accumulation plays an important role in the induction of ferroptosis,we speculated that there was a potential relationship between USF2 and ferroptosis.The enhanced aerobic glycolysis plays a pivotal role in the survival and growth of cancer cells.As one of the key enzymes in glycolysis,pyruvate kinase M2（PKM2）is involved in regulating biosynthesis and energy production.It promotes the accumulation of intermediate products of glycolysis,and reduces the oxidative stress produced by excessive cell proliferation.Also,PKM2 inhibits autophagy in tumor cells,which induces ferritin degradation and ferroptosis.In addition,we found that there were several potential binding sites between USF2 and the region of PKM2 promotor by using PROMO and JASPAR transcription databases,indicating the potential transcriptional regulation of PKM2 expression by USF2.In the present study,we mainly focused on the functions of USF2 and PKM2 in ferroptosis in PC cells and USF2-mediated transcriptional regulation of PKM2 expression.In addition,by adopting molecular imaging technology ¹⁸F-FDG PET/CT,we attempted to predict their expression levels in tumor tissues noninvasively.This work will help investigate the regulatory mechanism of ferroptosis on PC and provide new insight into the management of PC.Methods:1.GEPIA database was used to analyze the transcriptional levels of USF2and PKM2 in PC patients.Bx PC-3 cells were transfected with si RNA against USF2（si-USF2）or negative control（si-NC）.As PC-1 cells were transfected with USF2 overexpressing plasmid（USF2 OE）or empty vector（vector）.The efficiencies of the si RNA and gene overexpression were verified by Western Blot 48 hours after transfection.After incubation with erastin for 24 hours,the cell viability was evaluated using the CCK-8 according to the manufacturer’s instructions.Lipid reactive oxygen species（ROS）were measured using the C11-BODIPY-based Image-i T^TMLipidPeroxidation Kit.The concentrations of glutathione（GSH）,malondialdehyde（MDA）,and iron were measured using commercial kits according to the manufacturers’ instructions.Western Blot was used to measure the expression of SLC7A11,GPX4,and FTH.2.Si-PKM2 was transfected into As PC-1 cells,and CCK-8 was used to measure the cell viability after incubation with erastin.The concentrations of lipid ROS and iron were measured using commercial kits.Western Blot was used to measure the expression of GPX4 and FTH.The USF2 OE plasmid and si-PKM2 were co-transfected into As PC-1cells.After incubation with erastin,the cell viability was measured by CCK-8,and the concentrations of lipid ROS,GSH,and iron were measured by commercial kits.Western Blot was used to measure the expression of GPX4.PCR was adopted to measure the transcriptional levels of PKM2 after being transfected with USF2 OE.Luciferase reporter,Ch IP,and DNA pull down assays were used to prove the binding of USF2 and the PKM2 promoter.3.The selection of the subjects was divided into two parts.27 cases of patients with pancreatic cancer who underwent ¹⁸F-FDG PET/CT imaging before surgery;30 cases of patients who underwent pancreatic cancer surgery.Clinical information of the total 57cases was collected and radiomics indexes of PET/CT images were extracted.57 cases of pancreatic carcinoma tissue and 30 cases of adjacent pancreatic tissues were obtained.The expression and correlation of USF2 and PKM2 in cancer and adjacent tissues were analyzed by immunohistochemical analysis and their corresponding clinical features as well as PET/CT radiomics indexes were statistically analyzed.Results:Bioinformatic analysis showed that USF2 and PKM2 were highly expressed in PC tissues and the level of USF2 was positively correlated with that of PKM2.Erastin-induced cell death was promoted by USF2 knockdown and inhibited by USF2 overexpression.USF2 knockdown increased lipid reactive oxygen species and malonaldehyde generation and decreased glutathione concentration,indicating the enhanced lipid peroxidation.USF2 knockdown also increased iron levels and ferritin heavy chain expressions and reduced glutathione peroxidase 4 and solute carrier family 7member 11 expressions.However,USF2 overexpression reversed these changes.Interestingly,PKM2 also negatively regulated ferroptosis,and PKM2 knockdown markedly impaired the effects of USF2 on lipid peroxidation and erastin-induced ferroptosis.Furthermore,dual-luciferase reporter assay,chromatin immunoprecipitation assay,and DNA pull down assay validated that USF2 transcriptionally regulated PKM2expression through binding to its promoter.Immunohistochemical analysis showed that USF2 and PKM2 were highly expressed in PC tissues and the level of USF2 was positively correlated with PKM2.There were no statistical differences between the expression levels and clinical features.¹⁸F-FDG PET/CT imaging parameters have predictive value for their differential expression and statistical differences in Dependence Variance and Busyness were found between different expression level groups of USF2.Conclusion:This study demonstrated that USF2 negatively regulated erastin-induced ferroptosis in PC cells through transcriptional regulation of PKM2,providing new evidence for uncovering the regulatory mechanism of ferroptosis on PC.Besides,USF2and PKM2 were highly correlated in human pancreatic cancer tissues.¹⁸F-FDG PET/CT imaging parameters have predictive value for their differential expression.