PHGDH-Mediated Glycolysis Is Required For Cd-Induced Cell Proliferation

Objective:According to the 2023 US cancer statistics,609,820 cancer deaths are expected,with lung cancer accounting for 21%of these deaths,which equates to approximately 350 deaths per day and is the leading cause of cancer deaths.While smoking is the main cause of lung cancer,heavy metal exposure is also considered an important causative factor.Among these,cadmium（Cd）exposure is also strongly associated with lung cancer,and Cd exposure leads to a weakening of antioxidant defense mechanisms and thus causes cancer.PHGDH,a key enzyme in the serine synthesis pathway,is highly expressed in many cancers.In our previous study,we found that Cd was able to induce a rise in the expression of PHGDH,and that glycolysis was involved in Cd-induced cell proliferation.Therefore,the aim of this study was to investigate the role of PHGDH-mediated glycolysis in Cd-induced cell proliferation.Methods:In this study,human lung adenocarcinoma A549,human embryonic lung fibroblasts HELF,human normal hepatocytes L-02,human lung cancer H1299,human ovarian cancer A2780 and OVCAR3,human liver cancer Hep G2 and human breast cancer MCF-7 cells were used as in vitro experimental models.BALB/c mice were used as an in vivo animal model and the expression of PHGDH was observed by Western blot when BALB/c mice were treated with CdCl₂ and transfected with HMGA2 plasmid.Wound healing assay were used to observe changes in cell proliferation and migration capacity induced by CdCl₂ treatment after transfection with small interfering RNA（si PHGDH）.Glucose and lactate content assay kits were used to determine the glucose and lactate content in CdCl₂-treated media after transfection with si PHGDH.Hoechst assay was used to observe the morphological changes in apoptosis induced by CdCl₂treatment after transfection with si PHGDH.Western blot was used to observe the effects of aerobic glycolysis and OXPHOS-related proteins induced by CdCl₂ treatment after transfection with si PHGDH.Western blot was used to observe the expression of aerobic glycolysis and OXPHOS-related proteins after transfection with si PHGDH and HMGA2plasmids.Chromatin immunoprecipitation（Ch IP）assays were used to observe the effect of HMGA2 on PHGDH gene expression.The expression of HMGA2 and PHGDH was observed by Western blot after treatment with CdCl₂ and HIF-1αinhibitor YC-1,and treatment with HIF-1αactivator CoCl₂ and transfection of si PHGDH.A xenograft tumor model was established in BALB/c mice to observe the effect of PHGDH and CdCl₂treatment on xenograft tumor formation.Results:The results of PHGDH expression in different cell lines showed that PHGDH expression was higher in A549 and HELF cells.PHGDH was elevated in CdCl₂-treated A549 cells,HELF cells and lung tissues of BALB/c mice.The results of Wound healing assay and Hoechst assay showed that transfection of si PHGDH inhibited CdCl₂-induced cell proliferation and migration and promoted apoptosis,indicating that PHGDH plays an important role in CdCl₂-induced cell proliferation,migration and apoptosis.The results of glucose and lactate content assays showed that si PHGDH did not reduce CdCl₂-induced glucose uptake,but significantly reduced CdCl₂-induced lactate production.Western blot assays showed that transfection with si PHGDH decreased CdCl₂-induced expression of PKM2 and LDHA and increased expression of repressed COX IV and ND1,but did not decrease CdCl₂-induced GLUT1,suggesting that PHGDH is involved in CdCl₂-induced glycolysis and repressed OXPHOS.Western blot assays showed that si PHGDH inhibited HMGA2 plasmid-induced reduction in the expression of aerobic glycolysis-related proteins and increased expression of OXPHOS-related proteins.Ch IP experiments showed that HMGA2 protein could bind to the PHGDH gene promoter,suggesting that HMGA2 regulates glycolysis and OXPHOS through transcriptional regulation of PHGDH.Western blot assays showed that the expression of HMGA2 and PHGDH induced by CdCl₂ was significantly reduced using YC-1.In contrast,si PHGDH did not reduce CoCl₂-induced expression of HIF-1αand HMGA2,indicating that HIF-1αis involved in CdCl₂-induced expression of HMGA2 and PHGDH.The xenograft tumor assay showed that si PHGDH inhibited the weight and volume of transplanted tumors in CdCl₂-treated BALB/c mice.Conclusion:In summary,HMGA2/PHGDH-mediated glycolysis played an important role in CdCl₂-induced cell proliferation in the presence of CdCl₂-induced HIF-1α elevation.