P4HA3 promotes gastric cancer cell proliferation and invasion via the PI3K/AKT/mTOR signaling pathway and regulates glycolytic metabolic reprogramming

Abstract: Background: Dysregulated expression of prolyl 4-hydroxylase subunit alpha 3 (P4HA3) has been identified in Colon cancer, breast cancer, head and neck cancers, etc. and is closely associated with tumor progression. However, its specific roles in gastric cancer (GC), particularly regarding alterations in metabolic pathways, are remain unknown. Objectives: This study investigated how P4HA3 influences glycolytic metabolic reprogramming and the proliferation and invasion of gastric cancer cells via the PI3K/AKT/mTOR signaling pathway. Methods: To determine P4HA3 levels in GC tissues and corresponding cell lines, bioinformatics methods, immunohistochemical (IHC) staining and Western blotting (WB) were conducted. Furthermore, gain- and loss-of-function experiments, including cell viability assays (CCK-8), colony-forming tests, scratch wound assays and Transwell invasion experiments, were utilized to explore the influence of P4HA3 on GC cell growth, motility and invasive capacity. Changes in cellular metabolism were assessed using the Seahorse system (under normoxic conditions), including measurements of basal metabolism, glycolytic capacity and mitochondrial respiration (oxidative phosphorylation), as well as Western blot analysis of key glycolytic proteins. Additionally, the involvement of the PI3K/AKT/mTOR signaling axis was examined by gene set enrichment analysis (GSEA), WB analysis and pharmacological inhibition studies. Results: GC tissues and cell lines exhibited markedly increased levels of P4HA3, correlating significantly with decreased overall survival (OS). Experimental data indicated that P4HA3 facilitated GC cell proliferation, migration and invasion. In addition, P4HA3 enhanced glycolytic activity and increased the expression of key glycolysis-related proteins. Unexpectedly, P4HA3 also increased mitochondrial respiration under the assay conditions, suggesting that P4HA3 may induce a broader metabolic activation rather than an exclusively glycolytic shift. Mechanistically, the glycolysis-enhancing effects of P4HA3 were found to require PI3K/AKT/mTOR signaling (as shown by inhibitor studies). Conclusion: P4HA3 promotes glycolysis via the PI3K/AKT/mTOR signaling pathway, thereby promoting the progression of gastric cancer.