Hypoxia drives aspirin-induced glutaminolysis in PIK3CA-mutated colorectal cancer cells

Aspirin exhibits potential as a repurposed agent for the prevention and treatment of cancer, particularly in PIK3CA-mutated colorectal cancer. However, when evaluating the anti-tumor ef-ficacy of aspirin, it is crucial to consider the tumor microenvironment, including hypoxia, which is common in colorectal cancer. To investigate the impact of hypoxia on aspirin's effectiveness against PIK3CA-mutated colorectal cancer, we conducted bioinformatics approach utilizing the public database Connectivity Map and transcriptome analysis of PIK3CA isogenic cell lines. Ad-ditionally, we employed targeted metabolomics focusing on the tricarboxylic acid (TCA) cycle and metabolic inhibitors. Connectivity Map analysis revealed that aspirin exhibited associations with amino acid metabolism and hypoxic pathways in HT-29, PIK3CA-mutated colorectal cancer cells. Furthermore, RNA-seq analysis of SW48 PIK3CA isogenic cell lines indicated that under hypoxic conditions, aspirin enhances amino acid uptake in PIK3CA-mutated cells. Targeted metabolomics demonstrated that aspirin treatment and hypoxic stimulation synergistically upregulated intra-cellular glutamine levels in PIK3CA-mutated HCT116 cells. This increase was abolished with V-9302, an inhibitor of the glutamine transporter ASCT2, thus revealing the anti-tumor potential of combining V-9302 with aspirin. Our results suggest that aspirin drives glutamine uptake in PIK3CA-mutated colorectal cancer under hypoxia, thereby highlighting its therapeutic signifi-cance as a potential target.