The Mechanism Of TP53 Transcriptional Regulation Of PHKG2 Inhibiting NRF2/GPX4 Pathway And Promoting Ferroptosis In Head And Neck Squamous Cell Cancer

Objective:Head and neck squamous cell carcinoma（HNSCC）accounts for over 90%of malignant tumors in the head and neck region,making it the seventh most common malignant tumor worldwide.Due to its complex anatomical location and hidden onset,treatment is often challenging in the middle and late stages of the disease.The treatment methods for HNSCC include surgery,radiation therapy,chemotherapy,and targeted immunotherapy,but the overall five-year survival rate is still poor.Ferroptosis is a novel programmed cell death pathway（PCD）mediated by the accumulation of iron dependent peroxides and phospholipid peroxidation.Ferroptosis is different from other cell death pathways such as apoptosis.Its main characteristics are an increase in the content of intracellular ferrous ions and lipid peroxides,a decrease in reduced glutathione（GSH）,and a decrease in mitochondrial shrinkage and the number of mitochondrial cristae in organelle morphology,but no significant changes in the nucleus.Ferroptosis includes both exogenous and endogenous pathways:exogenous pathways activate ferroptosis by inhibiting the transmembrane protein cysteine/glutamate antagonist（system Xc^-）or activating transferrin（TF）,resulting in a decrease in intracellular GSH and iron overload of ferrous ions.The endogenous pathway is the key regulatory factor that blocks the endogenous antioxidant system,including glutathione peroxidase 4（GPX4）and nuclear factor E2 related factor 2（NRF2）,which inhibit the intracellular antioxidant system,cause redox imbalance,accumulate lipid peroxides,and activate ferroptosis.Abnormal proliferation and high-intensity metabolism of HNSCC tumor cells require activation of the antioxidant system in order to ensure that tumor cells are free from cell death caused by peroxide accumulation.The dependence of HNSCC tumor cells on the activation of this antioxidant system and the increased intracellular free iron and polyunsaturated fatty acids make them susceptible to ferroptosis.In recent years,many studies have confirmed that ferroptosis plays an important role in enhancing the sensitivity of HNSCC tumor cells to radiotherapy and chemotherapy.As a result,with the continuous deepening of research on ferroptosis,its role in HNSCC tumor cells has attracted more and more scholars’attention.Phosphorylase kinase gamma 2（PHKG2）,a gene associated with ferroptosis,is considered a driver of ferroptosis.It can activate ferroptosis,increase intracellular ferrous ion content,and is a protective factor for patient prognosis.At present,some studies have confirmed that the expression of PHKG2 is a protective factor for prognosis in patients with endometrial cancer,lung adenocarcinoma,gastric cancer,and renal clear cell carcinoma,but these studies only remain in bioinformatics analysis research.At present,research on the expression of PHKG2 in HNSCC tumor patients and its correlation with prognosis is still blank.A study published in PNAS suggests that during ferroptosis,lipoxygenase oxidizes polyunsaturated fatty acids（PUFA）on the cell membrane through the PHKG2dependent iron pool pathway.It is speculated that PHKG2 affects tumor cell iron metabolism through TP53^[1],but the specific mechanism of action is still unclear.The purpose of this study is to verify the differential expression of PHKG2 in cancer tissue and adjacent tissues through bioinformatics analysis and clinical HNSCC patient samples,and to analyze the correlation between PHKG2 expression in cancer tissue and patient prognosis,as well as the correlation with ferroptosis related protein expression.2.Verify the role of PHKG2 in ferroptosis in HNSCC cells and its regulatory relationship with TP53through in vivo and in vitro experiments.3.Verify the regulatory effect of PHKG2 on the NRF2/GPX4 pathway and its role in ferroptosis in HNSCC cells through in vivo and in vitro experiments.Methods:1.Screening ferroptosis related genes in head and neck squamous cell carcinoma through bioinformatics analysis and conducting prognostic prediction analysis.This study retrospectively analyzed the complete clinical information of patients diagnosed with HNSCC through pathology who visited the First Affiliated Hospital of China Medical University from January 2019 to January 2020.Immunohistochemistry（IHC）staining was used to investigate the differential expression of PHKG2 in HNSCC cancer and adjacent tissues,as well as the correlation between PHKG2 and patient prognosis.The correlation between PHKG2 and nuclear factor E2 related factor 2（NRF2）and glutathione peroxidase4（GPX4）was also analyzed.2.Selecting HNSCC cell lines 5-8F and Fadu,establishing PHKG2 interference cell lines using lentivirus interference and drug screening to establish PHKG2 overexpression stable transgenic cell lines.The Alamar Blu experiment was used to detect cell viability.Firstly,the effect of interference with PHKG2 on cell viability was verified by activating ferroptosis through Erastin.And detect changes in intracellular iron ions(Fe²⁺),reactive oxygen species（ROS）,glutathione（GSH）,and polyunsaturated fatty acid peroxide malondialdehyde（MDA）levels after interference and overexpression of PHKG2.Determine the occurrence of ferroptosis by observing changes in intracellular organelles under transmission electron microscopy.Predicting the correlation between PHKG2 and TP53 in ferroptosis through Ingeniity Pathway Analysis（IPA）.The regulatory effect of TP53 on PHKG2 was verified through cell chromatin immunoprecipitation（Ch IP）and dual luciferase gene reporting experiments.qRT PCR and Western blot experiments were used to detect changes in PHKG2,NRF2,and GPX4 mRNA and protein expression levels after interference and overexpression of TP53.The qRT PCR experiment was used to detect changes in ferroptosis related gene levels after interference and overexpression of PHKG2.The i GSP 1.0 software was used to predict that PHKG2 kinase may regulate NRF2 activity by activating protein phosphatase1（PP1）.The PP1 activity after interference and overexpression of PHKG2 was detected using a reagent kit,and the changes in NRF2 and GPX4 protein levels were detected using Western blot experiments.By adding PP1 activators and inhibitors,Western blotResult:1.PHKG2 is a protective factor for the prognosis of HNSCC tumor patients,and the expression level of PHKG2 is closely related to the expression levels of ferroptosis core regulatory factors NRF2 and GPX4.（1）Bioinformatics analysis shows that the ferroptosis related gene PHKG2 is closely related to patient prognosis and is a protective factor for the prognosis of HNSCC patients.（2）The IHC staining analysis of 57 HNSCC patients’cancer tissues and adjacent tissues showed that PHKG2 expression was low to moderate in cancer tissues,negative to low in adjacent tissues,and higher in cancer tissues than in adjacent tissues（p<0.001）.（3）The expression of PHKG2 is correlated with tumor T staging,lymph node metastasis,and clinical staging in HNSCC patients（p<0.001）.（4）Cox survival analysis showed that the expression of PHKG2 was a protective factor for the prognosis of HNSCC patients,with HR=0.259（（0.109-0.614）,p<0.05）.High expression of NRF2 and GPX4 was a risk factor for the prognosis of HNSCC patients,with HR=4.249（（1.572-11.480）,p<0.05）and HR=2.731（（1.071-6.968）,p<0.05）,respectively.（5）PHKG2 was negatively correlated with NRF2 protein expression（r=-0.579,p<0.001）,and negatively correlated with GPX4protein expression（r=-0.4726,p<0.001）,while NRF2 protein expression was positively correlated with GPX4 expression（r=0.679,p<0.001）.2.PHKG2 is regulated by TP53 transcription to activate ferroptosis in HNSCC tumor cells.（1）The Alma blue experiment was used to detect cell viability,confirming that interfering with PHKG2 can reverse ferroptosis caused by Erastin.（2）PHKG2 can increase the content of ROS and Fe²⁺in 5-8F and Fadu cells,while increasing the content of MDA and decreasing the content of GSH.Through transmission electron microscopy,it was found that the mitochondrial cristae disappeared and the mitochondrial membrane was broken,and the nucleus was intact,proving that PHKG2 can cause ferroptosis in 5-8F and Fadu cells.（3）Ch Ip and dual luciferase gene reporting experiments confirmed that TP53 is an upstream gene of PHKG2.TP53 can bind to the promoter of PTHG2 and activate PHKG2.qRT PCR and Western blot experiments were used to verify that TP53 promotes the expression of PHKG2 protein and inhibits the expression of NRF2 nuclear protein,but the total protein level of NRF2 remains unchanged.（4）Establish a sh-TP53 nude mouse transplanted tumor model to verify the regulatory effect of TP53 on PHKG2 and NRF2.Dynamically monitoring tumor volume and weight,using Ki67 to detect tumor proliferation ability,the results showed that tumor volume increased after interference with TP53.Western blot analysis showed a decrease in PHKG2 protein expression and an increase in NRF2 nuclear protein levels,but there was no change in total NRF2 protein levels.The regulation of NRF2 by TP53/PHKG2 is at the protein level,which inhibits the expression of NRF2 nuclear protein and exerts its function.3.PHKG2 promotes ferroptosis in HNSCC tumor cells by activating protein phosphatase PP1 and inhibiting NRF2 nuclear protein expression（1）The detection of ferroptosis related genes by qRT PCR confirmed that PHKG2 cannot regulate the expression of NRF2 mRNA at the gene level,and predicted that PHKG2 has a binding site with protein phosphatase PP1,which can reduce the expression of protein in the nucleus of NRF2.（2）Western blot experiments and PP1 kit detection results confirmed that PHKG2 reduces the nuclear protein of NRF2 by increasing PP1 activity（NRF2expression plays a functional role in the nucleus）,inhibits NRF2 function,and thus reduces the expression of GPX4 protein.（3）After activating ferroptosis with the ferroptosis activator Erastin and increasing the expression of NRF2 in the nucleus with the NRF2activator,overexpression of PHKG2 can reverse the inhibitory effect of NRF2 activator on ferroptosis,accompanied by an increase in intracellular MDA,ROS,and Fe²⁺content and a decrease in GSH content,proving that PHKG2 activates ferroptosis by inhibiting the NRF2/GPX4 pathway.Establishing a sh-PHKG2 nude mouse transplantation tumor model,ferroptosis was activated by intraperitoneal injection of Erastin,which interfered with the growth of tumor volume in the PHKG2 group.Western blot analysis showed an increase in NRF2 nuclear protein and GPX4 protein levels,a decrease in PP1 activity,and a decrease in ROS content in the tumor tissue.Conclusion:1.The expression of PHKG2 is a protective factor for the prognosis of HNSCC patients,and the expression level in cancer tissue is higher than that in adjacent tissues.The expression of PHKG2 is negatively correlated with the expression of NRF2and GPX4.2.PHKG2 is regulated by TP53 transcription to activate ferroptosis in HNSCC tumor cells and inhibit NRF2 nuclear protein expression at the protein level.3.PHKG2 activates PP1 activity,reduces NRF2 nuclear protein expression levels,inhibits the NRF2/GPX4 pathway,reduces reduced GSH content,increases ROS in tumor cells,and is accompanied by an increase in Fe²⁺content,promoting ferroptosis.