Hypoxia and aspirin additively increase intracellular glutamine accumulation in PIK3CA-mutated colorectal cancer cells

Aspirin exhibits potential as a repurposed agent for preventing and treating cancer, specifically PIK3CA-mutated (MT) colorectal cancer. However, the fundamental biological reasons for the particular antitumor activity of aspirin against PIK3CA-MT colorectal cancer are not yet fully elucidated. We have previously established that aspirin relates to glutamine metabolism in PIK3CA-MT colorectal cancer. In evaluating the antitumor effects of aspirin, it is essential to consider the tumor growth environment, such as hypoxia. The impact of the tumor microenvironment on aspirin efficacy remains unknown. Therefore, we aimed to investigate the mechanisms through which hypoxia influences aspirin- associated intracellular glutamine accumulation in PIK3CA-MT colorectal cancer cell lines. Connectivity Map analysis was used to explore the associations of aspirin with biological pathways in HT-29 (PIK3CA-MT) cells. RNA sequencing was performed on PIK3CA-wild type (WT)/mutant isogenic SW48 cell lines to assess aspirin’s effects under hypoxic conditions. Targeted metabolomics was employed to measure intracellular glutamine levels in PIK3CA-MT HCT116 cells following aspirin treatment and hypoxic stimulation. Bioinformatic analysis revealed associations of aspirin with amino acid metabolism and hypoxic pathways. Under hypoxic conditions, aspirin enhanced amino acid uptake in PIK3CA-MT cells. Targeted metabolomics showed that aspirin treatment and hypoxic stimulation additively upregulated intracellular glutamine levels in PIK3CA-MT cells. This increase was reversed by V-9302, an inhibitor of the glutamine transporter ASCT2, which demonstrated antitumor potential in combination with aspirin. Our findings suggest that the combination of aspirin and hypoxia significantly enhances intracellular glutamine accumulation in PIK3CA-MT colorectal cancer under hypoxic conditions, emphasizing glutamine metabolism as a potential therapeutic target to enhance aspirin’s efficacy.