Human Papillomavirus-16 E6 Activates The Pentose Phosphate Pathway To Promote Cervical Cancer Cell Proliferation By Inhibiting G6PD Lactylation

BackgroundCervical cancer is one of the most common cancers in women,with persistent highrisk HPV infection playing a key role in its development.HPV oncogenes E6 and E7 integrate into host cell chromosomes during viral infection and function as oncogenes.E6 oncoproteins disrupt cell growth arrest,inhibit apoptosis,induce genomic instability and somatic mutations,activate telomerase and telomerase reverse transcriptase,promote immortalization,disrupt cell polarity,prevent loss-of-nest apoptosis,and allow cells to grow in the absence of extracellular matrix attachment.E7 oncoproteins bind to a variety of cellular proteins and interfere with cellular physiological processes,resulting in cell cycle dysregulation,centrosome amplification,DNA damage,inhibition of apoptosis-and node-loss-induced cell death,noncontact-dependent cell growth,immune escape,and persistent infections.The E7 and E6 oncoproteins bind to multiple cellular targets,including the tumor suppressors p RB and P53,leading to the dysregulation of multiple signaling pathways and immortalization.However,the molecular events surrounding HPV-induced progression of precancerous lesions to invasive cancers remain largely unknown.Thus,identifying new cellular targets is crucial to understanding the oncogenic functions of E6 and E7.Metabolic reprogramming is a hallmark of cancer.Tumor cells prefer to generate energy via glycolysis,even in the presence of oxygen(Warburg effect).The rapid proliferation of tumor cells generates large amounts of ROS,and high ROS levels are counteracted by upregulated antioxidant defense mechanisms.One key mechanism to counter oxidative stress is the diversion of glycolysis intermediates to the oxidative pentose phosphate pathway(PPP),which is commonly overactivated in tumor cells.Glucose-6-phosphate dehydrogenase(G6PD),the rate-limiting enzyme in the PPP,mediates the oxidative PPP to produce NADPH and maintain intracellular redox homeostasis.The nonoxidative PPP provides substrates for nucleotide synthesis.PPP intermediates are converted to fructose 6-phosphate and glyceraldehyde 3-phosphate in conjunction with glycolysis.Thus,the PPP contributes to the anabolic needs and removal of ROS stress in highly glycolyzed cancer cells.However,whether HPV can activate the PPP to promote cervical cancer progression remains unclear.Lactylation is a novel posttranslational modification achieved through the addition of lactyl groups to lysine(K)residues.Histone lactylation modifications play important roles in regulating macrophage polarization and inducing the expression of pluripotency genes during the reprogramming of senescent cells into pluripotent stem cells.Non-histone lactylation modifications stimulate HMGB1 release from macrophages.Notably,global lactylation mapping analysis,conducted using mass spectrometry,revealed lactylation modifications in several enzymes involved in the PPP,glycolysis,and tricarboxylic acid cycle.However,whether G6 PD can be modified by lactylation and the specific site of G6 PD lactylation remain unknown.ObjectiveInvestigate the impact of HPV infection on cellular PPP;specify the role of PPP metabolic alterations in HPV carcinogenic activity;elucidate the mechanisms by which HPV oncogenes induce these metabolic abnormalities;evaluate the significance of metabolic abnormalities as therapeutic targets for cervical cancer treatment.Methods Part Ⅰ: HPV16 E6 reprogramming PPP promotes cervical cancer cell proliferation1.Detection of metabolic differences in PHKs transduced with HPV16 E6E7 and Vector using untargeted metabolomics.2.Detection of PPP-related indicators(NADPH,GSH,Ed U),intracellular ROS levels,and Nile Red staining in PHKs and C33 A cells expressing HPV16 E6E7 and Vector.3.Detection of PPP-related indicators(NADPH,GSH,Ed U),intracellular ROS levels,and Nile Red staining in PHKs and C33 A cells expressing HPV16 E6,HPV16 E7,or Vector.4.CCK8 cell viability assay after G6 PD knockdown or inhibition of G6 PD enzyme activity by 6-An in PHKs and C33 A cells expressing HPV16 E6 and HPV16 E7.5.Construction of a subcutaneous xenograft tumor model in nude mice using C33A-E6,C33A-E7,C33A-E6-sh G6 PD,C33A-E7-sh G6 PD,and C33A-Vector cells.The G6PD inhibitor(6-An)was injected intraperitoneally to detect the tumorigenic capacity of C33A-E6 and C33A-E7 cells.Part Ⅱ: Mechanistic of HPV16 E6 increasing G6 PDase activity to reprogram the PPP1.Detection of G6 PD enzyme activity by DSS cross-linking in PHKs and C33 A cells exposed to different treatments.2.Western blot detection of G6 PD lactylation modification levels in PHKs and C33A cells.3.Molecular docking prediction of potential G6 PD lactylation modification sites and generation of mutation plasmids(K45A,K46 A,K47A,K171 A,K205A,K238 A,K288A,K403 A,K408A,and K508A).Part Ⅲ: G6 PD K45-mediated lactylation modification inhibits HPV16E6-induced cell proliferation1.Transduction of cells with endogenous G6 PD knockdown using mutation plasmids and detection of G6 PD enzyme activity,GSH,and ROS.2.Construction of a subcutaneous xenograft tumor model using Si Ha and C33 A cells to investigate whether G6 PD K45-mediated lactylation modification inhibits tumor growth.Results Part Ⅰ: HPV16 E6 reprogramming PPP promotes cervical cancer cell proliferation1.HPV16 E6E7 reduces intracellular oxidative stress in tumor cells,increases the expression of intracellular NADPH and GSH,and promotes DNA and lipid synthesis by reprogramming the PPP.2.HPV16 E6 activates the PPP,leading to cell proliferation and tumor growth.Part Ⅱ: Mechanistic of HPV16 E6 increasing G6 PDase activity to reprogram the PPP1.HPV16 E6 promotes the formation of active G6 PD dimers.2.HPV16 E6 promotes G6 PD dimer formation by inhibiting G6 PD lactylation.Part Ⅲ: G6 PD K45-mediated lactylation modification inhibits HPV16E6-induced cell proliferation1.K45 is a key site for G6 PD lactylation regulated by the HPV16 E6 oncoprotein.2.Inhibiting G6 PD enzyme activity with 6-An or re-expressing G6 PD K45T inhibited tumor proliferation.Conclusion1.HPV16 E6 upregulates G6 PD enzymatic activity to activate the PPP,thereby promoting the proliferation of cervical cancer cells.2.HPV16 E6 upregulates G6 PD enzyme activity by inhibiting G6 PD K45 lactylation.3.Inhibiting G6 PD enzyme activity with 6-An or re-expressing K45T(mimicking sustained G6 PD lactylation modification)significantly reduced tumor growth.