HPV E6 Modulates Cervical Cancer Cell Metabolism By Targeting Glucose-6-phosphate Dehydrogenase

Background and purpose:Despite early screening and vaccination,the incidence of cervical cancer is still high with a high mortality rate.Cervical cancer ranks fourth among malignancies,and the mortality rate ranks the fourth.Moreover,the trend of becoming younger appears,which seriously endangers the health of women[1].At present,persistent infection with high risk human papillomavirus(HPV)is considered to be the main driver of cervical cancer.At present,there are more than 150 types of HPV virus found,according to its infection site is divided into skin type and mucous membrane type,and the mucous membrane type is divided into high-risk type and low risk type,high-risk type of HPV is the main cause of malignant tumor[2].HPV16 is the most common and dangerous type of cervical screening samples and cervical cancer tissues,which is closely related to the occurrence of cervical cancer[3-5].HPV E6(hereinafter referred to as E6)and E7 are the main factors leading to the transformation of host cells for immortality.Among them,E6 regulates cell,adhesion,differentiation,polarity,proliferation,apoptosis,gene transcription and chromosome stability by interacting with cell proteins,affecting the occurrence and development of tumor cells[6-8].Abnormal metabolism of tumor cells plays a particularly important role in tumor progression[9-10].The feature of tumor metabolism is so called aerobic glycolysis,which in short means that tumor cells tend to undergo intracellular glycolysis even when oxygen is sufficient,which is called the Warburg effect[11-13].Most glucose molecules enter cells through glucose transporters and are metabolized through the glycolysis pathway,but some enter the pentose phosphate pathway(PPP)during the intermediate stage of glycolysis[14-15].Pentose phosphate pathway also plays an important role in the metabolism of tumor cells and is closely related to the proliferation and migration of tumor cells.Meanwhile,the pentose phosphate pathway is also regulated by many tumor suppressors,cancer-related proteins and metabolites[16-17].Current studies have shown that E6 leads to p53 degradation by forming complexes with E6AP and p53[18].p53 has many effects including cell DNA repair,cycle arrest and apoptosis[19-20].In addition,recent studies support the regulation of p53 metabolism,including glycolysis and oxidative phosphorylation[21-22].p53 controls the pentose phosphate pathway by regulating glucose-6-phosphate dehydrogenase(G6PD)level[23].We speculated whether E6 could lead to abnormal metabolism of cervical cancer cells by regulating pentose phosphate pathway affecting G6PD.In this study,we studied the relationship between E6 and G6PD,as well as its mechanism in regulating the pentose phosphate pathway and promoting the metabolism of cervical cancer cells,providing evidence for the treatment of cervical cancer.Methods:Part i:1.Firstly,a stable e6-knockout CaSki cell line(sh-E6)was established and the differences in oxidative PPP flux,glucose consumption,NADPH level and G6PD enzyme activity were detected between the CaSki cell line and the control group(sh-C).Then,after down-regulating G6PD(transfected G6PD siRNA)and up-regulating G6PD(transfected Flag-G6PD),the above indicators were compared between the two groups.2.The overexpressed E6 HEK293T cell line(Flag-E6)was established,and the differences in G6PD enzyme activity were detected between the HEK293T cell line(Flag-E6)and the control group(Flag-C).The regulation and mechanism of E6 on pentose phosphate pathway were analyzed.Part ii:1.The intracellular binding of E6 protein and G6PD protein was detected by immunoprecipitation assay.2.In vitro binding of E6 protein and G6PD protein was detected by GST pull down experiment.3.The E6 protein and G6PD protein were stained by immunofluorescence,and the co-localization of E6 protein and G6PD protein in cells was scanned by confocal laser microscopy,and the interaction between E6 protein and G6PD protein was analyzed.Part iii:E6 was transfected into p53+/+ and p53-/-mef cells to detect the changes of G6PD enzyme activity and G6PD mRNA level in the two groups,and to analyze whether E6 was dependent on the p53 pathway and affected by G6PD.Part iv:1.After the G6PD gene was silenced,the differences in the proliferation,apoptosis and tumorigenesis of cervical cancer cells were compared between E6 positive and E6 negative cells,and the role of G6PD in promoting the proliferation of cervical cancer cells was analyzed.Results:1.After E6 gene knockout,oxidative PPP flux,glucose consumption,NADPH level and G6PD enzyme activity decreased,which was blocked by G6PD silencing and reversed by G6PD overexpression.On the contrary,the overexpression of E6 leads to the increase of oxidative PPP flux,glucose consumption,NADPH level and G6PD enzyme activity.2.The immunoprecipitation experiment showed that E6 protein interacts with G6PD protein in cells.GST pull down experiment and immunofluorescence experiment showed that E6 protein directly bound to G6PD protein,and their binding was located in cytoplasm.3.After E6 transfection into MEF cells with P53+/+ and P53-/-,the activity of G6PD enzyme increased and the level of G6PD mRNA increased,especially in the cells with P53+/+.4.After the silencing of G6PD gene,the proliferation and apoptosis of E6-positive cervical cancer cells decreased,and tumorigenesis slowed down in nude miceConclusion:E6 activates G6PD activity by p53 dependent or non-dependent pathway,and upregulates pentose phosphate pathway to promote the metabolism of cervical cancer cells.