PGK1 Mediates Cisplatin Sensitivity In Nasopharyngeal Carcinoma Via Regulating The Mitophagy

Background and objective:Nasopharyngeal carcinoma（NPC）is a specific type of the head and neck carcinoma that occurs on the nasopharynx epithelium with highly invasion and metastasis.It is reported that approximately 75%of patients are newly diagnosed in the advanced stage.With the ongoing development of radiotherapy technology,chemotherapy,immunology and accurate cancer staging systems,the survival benefit of NPC has pronounced.Cisplatin is the fundamental treatment for advanced NPC,and the cisplatin-based chemoradiotherapy has improved the overall survival at 3years to 94.6%.However,15.3%of these patients developed recurrence or metastasis after the resistance of chemoradiotherapy.Therefore,it is urgent to improve the sensitivity of cisplatin.Mitophagy is a special type of selective autophagy.It is activated by hypoxia and DNA strand breaks to stabilize the normal function of mitochondrion through the PINK1/Parkin pathway.In recent years,multiple studies have observed that mitophagy-related molecules can be upregulated in lung cancer,sarcoma,and ovarian cancer cisplatin-resistant cells.Moreover,dysfunction and abnormal structure of mitochondrion,including imbalance in mitochondrial pressure and signal crosstalk,have verified to involved in the cisplatin resistance.However,there is no relevant study in the field of NPC.Previously,we found that glycolysis is an important pathway to regulate the microenvironment of NPC,and PGK1（Phosphoglycerate kinase 1）as the important glycolysis biomarker for explaining the heterogeneity of prognosis in NPC.Moreover,we found this gene involved in multiple mitochondrial pathways,like mitophagy.Therefore,in this study,we explored the mechanism of PGK1 regulating the cisplatin sensitivity through mitophagy pathway.Materials and methods:（1）Based on the TCGA-HNSCC database,we used the ssGSEA algorithm to calculate the scores of immune microenvironment mechanisms,and the patients were classified into diverse clusters by unsupervised clustering analysis.Then,we evaluated the differences of clinical outcomes,core pathways and biomarkers among those clusters and identified the hub gene PGK1.Finally,we validated the prognostic value of PGK1 in the GEO NPC databases.（2）We performed qRT-PCR and WB to detect the expression of PGK1 in NPC tissues and normal tissues.Then,we also detected the PGK1 in various NPC cell lines and determined the PGK1high-expression cell lines HONE-1 and C666-1 and low-expression cell line HK-1 as the subsequent model.After infecting the HONE-1 and C666-1 with sh-NC or sh-PGK1,and the HK-1 with vector or overexpressing PGK1 plasmids,we applied the CCK-8,clone formation,transwell,and wound healing assays to evaluate the effect of PGK1 on the proliferation,invasion,migration of NPC.（3）According to the transcriptome sequencing of PGK1,we clarified the relationship between PGK1 and mitochondrial function.Next,the mitochondrial membrane potential detection（MMP）was used to assess the PGK1 effect of onΔψm,and we observed the alteration on the co-staining of mitophagy detection kit Mtphagy Dye and Lyso Dye,and co-localization of autophagy marker LC3B and mitochondrial outer membrane protein TOMM20 by immunofluorescence（IF）.Finally,we verified the expression of autophagy markers P62,LC3-Ⅰ,LC3-Ⅱ,and mitophagy markers VDAC1,MFN1 by WB detection after PGK1knockdown or overexpression.（4）Firstly,HK-1 was treated with Vector,PGK1,and Vector+mitochondrial membrane uncoupler（CCCP）.Then,we examined the alteration of MMP and evaluated the co-localization of TOMM20 and PINK1,co-staining of Mtphagy Dye and Lyso Dye by IF.The expression of PINK1,Parkin,and other proteins were detected by WB.Subsequently,HK-1 was treated with Vector,PGK1,and PGK1+sh-PINK1,and the assays of MMP,IF and WB were used to verify the mechanism of PGK1inducing mitophagy through the PINK1-Parkin pathway.（5）In Co-IP and mass spectrometry,we selected the TOMM40 as the TOM complex proteins which interacted with PGK1,and we applied the Co-IP to examine the relationship.Through the IF,qRT-PCR,and WB,we assessed the effect of PGK1 on the expression,localization,and protein stability of TOMM40.After infected cell lines with Vector,PGK1,PGK1+TOMM40,we estimated the effect of TOMM40 on PGK1-mediated mitophagy by MMP,IF,and WB.Next,we set four groups treated with sh-NC,sh-PGK1,sh-PGK1+sh-TOMM40,and CCCP,and applied the IF,Co-IP to depict the co-localization of PINK1 with mitochondrial inner membrane protein Tim23 or outer membrane protein TOMM20 for exploring the mechanism of PGK1-TOMM40 axis promoting the localization change in mitochondrial membrane of PINK1.（6）Silver staining were used to explore the relationship PGK1 and E3 ubiquitin ligase,and qRT-PCR and Co-IP were used to select and identify the RNF123 which regulated PGK1 ubiquitination.We also performed qRT-PCR and WB to detect RNF123 expression in NPC and normal tissues.Next,we utilized the HDOCK SERVER and previous literatures to predict the binding sites of RNF123 and PGK1,and constructed all mutations to introduce transfected plasmids to identify the key site modifying the PGK1 ubiquitination.Finally,CCK-8,clone formation,transwell,and wound healing assays were used to evaluate the effects of RNF123 on the proliferation,invasion,migration of NPC.Furthermore,MMP,IF,WB were used to assess the effect of RNF123 on PGK1-mediated mitophagy.（7）Silver staining and Co-IP were used to select and identify the PGK1-interactive phosphorylase SRPK1.Next,we utilized the HDOCK SERVER to predict the binding sites of SRPK1 and PGK1,and constructed all mutations plasmids to identify the site of PGK1phosphorylation.Finally,CCK-8,clone formation,transwell,and wound healing assays were utilized to examine the influence of SRPK1 on the proliferation,invasion,migration of NPC.MMP,IF,WB were used to evaluate the effect on PGK1-mediated mitophagy.Moreover,through Co-IP and WB detection,we explored the relationship between phosphorylation and ubiquitination.（8）Setting a concentration gradient of cisplatin to determine the IC₂₀and IC₅₀.Through the MMP and IF,we detected the alteration of mitophagy in 48 hours after cisplatin treatment.We set up four groups as follows:DMSO,DDP,DDP+SRPIN340,and DDP+RNF123.In vitro experiments,by CCK-8,clone formation,wound healing assays,DNA damage repair proteinγH2AX detection,and WB analysis,we evaluated the effect of PGK1 phosphorylation or ubiquitination on regulating the cisplatin sensitivity.（9）Four groups were set up:DMSO,DDP,DDP+SRPIN340,and DDP+RNF123.Subcutaneously DMSO+Vector,DMSO+Vector,SRPIN340+Vector,and DMSO+RNF123 were transfected into HONE-1 cells and injected to nude mice.Next,treating the mice with DMSO or cisplatin regularly in tail vein.Finally,measuring the tumor volume,applying IHC and Tunel assay to confirm the effect of PGK1phosphorylation and ubiquitination on the cisplatin sensitivity.Results:（1）In TCGA,we used the ssGSEA to calculate 29 types of microenvironment mechanisms,and classified patients into three clusters.There was an essential difference of clinical outcomes and microenvironment scores between cluster A and C.Then,we constructed a prognostic model with high accuracy based on the different expressed genes from two clusters by edgeR and Lasso packages.Finally,hub gene PGK1 was selected by Cytoscape.Furthermore,the expression of PGK1was up-regulated in the tumor tissue and was related to poor survival in the GEO NPC databases.（2）The mRNA and protein expression of PGK1 was up-regulated in NPC tissues.Then,we chose the HONE-1,C666-1,HK-1 as optimal cell lines.The CCK-8,clone formation,transwell,and wound healing assays displayed that knockdown of PGK1 inhibited the proliferation,invasion,and migration of NPC.However,overexpression of PGK1 promoted cell proliferation,invasion,and migration.（3）Transcriptome sequencing suggested that PGK1 was involved in multiple mitochondrial-related pathways.PGK1 affected the change of MMP and promoted the co-localization of Mtphagy Dye and Lyso Dye,autophagy marker LC3B and mitochondrial outer membrane protein TOMM20.WB revealed that overexpressing PGK1 up-regulated the expression of LC3-Ⅱ/Ⅰ ratio,and reduced the p62,VDAC1,and MFN1expression.Autophagy inhibitors 3-MA or bafilomycin-A1 can inhibit this process.（4）Overexpression of PGK1 or treating with CCCP can induce MMP alteration and promoted the increasing co-localization of Mtphagy Dye and Lyso Dye,PINK1 and TOMM20.qRT-PCR and WB showed that overexpression of PGK1 or CCCP failed to affect mRNA levels of PINK1 and Parkin,but up-regulated the total and mitochondrial protein levels of PINK1 and p-Parkin（Ser65）.After knockdown of PINK1,MMP was recovered,co-localization of Mtphagy Dye and Lyso Dye was blocked,and the total and mitochondrial protein of Parkin,p-Parkin（Ser65）were all decreased.（5）Silver staining and Co-IP displayed that PGK1 interacted with TOM complex protein TOMM40.After overexpression of PGK1,Co-IP and IF revealed that the protein interaction and co-localization of PGK1and TOMM40 were enhanced,but PGK1 did not affect the intracellular distribution,mRNA level,protein expression and stability of TOMM40.Moreover,overexpression of TOMM40 can recover the change of MMP induced by PGK1,inhibit the co-localization of Mtphagy Dye and Lyso Dye,and down-regulate the mitochondrial protein level of PINK1 and p-Parkin（Ser65）.Subsequently,when PGK1 was silenced,PINK1 was transported to the inner membrane to co-localize and interact with mitochondrial inner membrane protein Tim23.After knockdown the TOMM40,the effect,which same as the CCCP treating was produced,may promote PINK1 to dissociate from Tim23 and aggregate to the mitochondrial outer membrane to interact with TOMM20.（6）Silver staining suggested that PGK1 interacted with 14ubiquitination ligases,and Co-IP and WB showed that E3 ligase RNF123can induce the ubiquitination and degradation of PGK1.Using HDOCK SERVER,five binding sites,K141,K199,K272,K279,and K406 were confirmed.Next,after infecting mutation sites,we verified the K199 as interactive site of RNF123 and PGK1.Finally,we observed the RNF123had low mRNA and protein expression in NPC tissues.Furthermore,RNF123 can inhibit the proliferation,invasion,and migration of NPC cells.（7）Based on silver staining and Co-IP,it was confirmed that PGK1interacted with phosphorylated kinase SRPK1.Using HDOCK SERVER,three binding sites,Ser203,Ser364,and Ser390 were confirmed and mutated to transfect.Ser203 was selected and validated as interactive phosphorylation site of SRPK1 and PGK1.Through MMP,IF,and WB,it revealed that SRPK1 promoted mitophagy activity via PGK1-TOMM40axis,while SRPIN340（SRPK1 inhibitor）can interrupt the process.Simultaneously,SRPK1 was up-regulated in NPC tissues and accelerated the proliferation,invasion,and migration of NPC.In addition,through Co-IP and WB,we found the ubiquitination of PGK1 was enhanced when treating with sh-SRPK1 or SRPIN340.After transfecting all phosphorylation mutation sites,the results suggested that modification of SRPK1 on PGK1 Ser203 down-regulated the ubiquitination level of PGK1.Conversely,after transfecting all ubiquitination mutation sites,the results revealed that modification of RNF123 on PGK1 K199down-regulated the phosphorylation level of PGK1.（8）In this study we determined IC₂₀as the cisplatin dose for vitro experiments.After cisplatin treatment,we observed the expression of LC3-Ⅱ/Ⅰ ratio,P62,PINK1,Parkin,and p-Parkin（Ser65）increased,MMP changed,and the co-localization of Mtphagy Dye and Lyso Dye enhanced.Moreover,inhibiting phosphorylation with SRPIN340 and promoting ubiquitination with overexpressing RNF123 can not only strengthen the inhibitory effect of cisplatin on the proliferation,invasion,and migration,but also reduce the DNA repair and mitophagy activity.（9）Subcutaneous transplantation tumor model,IHC and Tunel assay confirmed that PGK1 promoted the tumorigenesis of NPC,up-regulated Ki-67 expression,and reduced Caspase-3 expression and cell apoptosis.In addition,inhibiting phosphorylation with SRPIN340 or promoting ubiquitination with overexpressing RNF123 can further strengthen the shrinkage effect of cisplatin,causing a decrease in Ki-67 expression,an increase in Caspase-3 and cell apoptosis.Conclusion:（1）PGK1 is a potential oncogene of NPC,and PGK1 interacts with TOMM40 to affect PINK1 mitochondrial membrane ectopia and promote mitophagy.（2）RNF123 is a tumor suppressor gene and ubiquitinates the PGK1K199 to inhibit mitophagy.SRPK1 is an oncogene and phosphorylates the PGK1 S203 to promote mitophagy.（3）Mutual exclusion of PGK1 phosphorylation by SRPK1 and ubiquitination by RNF123 mediated mitophagy and the cisplatin sensitivity in NPC.