GPX3-Mediated Oxidative Stress Affects Pyrimidine Metabolism Levels in Stomach Adenocarcinoma via the AMPK/mTOR Pathway

Background: Amino acid metabolism, including ATP production, nucleotide synthesis, and redox homeostatic processes, are associated with proliferation and differentiation of tumor cells. This study aimed to identify novel prognostic biomarkers and potential therapeutic targets of amino acid metabolism-related genes for stomach adenocarcinoma (STAD).

Methods: RNA sequencing transcriptome data in the TCGA-STAD (training set) and GTEx datasets (validation set) were used. The LIMMA R program enabled the differentially expressed amino acid metabolism-related genes (AAMRGs) to be found. A prognostic risk score model based on clinical phenotypic features was built using LASSO regression and step multi-Cox analyses. Gene set enrichment analysis (GSEA) was used to find potential molecular pathways associated with STAD. Hierarchical cluster analysis was used to evaluate pyrimidine metabolism. Cultured STAD cells assessed the proliferation of STAD and upregulation of GPX3 expression by CCK8 and flow cytometry. Transwell and wound healing assays assessed the impact of GPX3 on invasion and migration of STAD cells. Western blot and qRT-PCR were used to measure changes in pyrimidine metabolism-related markers and active molecules involved in the AMPK/mTOR signaling pathway.

Results: Three AAMRGs, DNMT1, F2R, and GPX3, could independently predict the course of STAD. Pyrimidine metabolism appeared to be significantly associated with these by GSEA and clustering analyses. Pyrimidine metabolism was negatively correlated with GPX3. Functional studies using an overexpressed GPX3 plasmid showed an enhanced migration and invasion of STAD cells as well as the expression of genes associated with pyrimidine metabolism and the AMPK/mTOR signaling pathway. By using a CAD siRNA, it was found that that GPX3 affected 5-fluorouracil resistance during de novo synthesis of pyrimidine through the CAD-UMPS signaling axis.

Conclusions: GPX3 which regulates the level of pyrimidine metabolism through the AMPK/mTOR pathway was found to be closely associated with STAD. Our findings demonstrate GPX3 is a reliable biomarker for the prognosis of amino acid metabolism and a probable target for STAD therapy.