Targeting ceramide metabolism to restore hypoxia-induced apoptosis in p53-deficient colon cancer cells.

Hypoxic stress in solid tumors triggers growth arrest and apoptosis through p53 activation and stabilization. This environment inactivates p53 and drives the expansion of p53-mutant clones, which accentuate tumor aggressiveness. Ceramide, a signaling sphingolipid, was previously identified as a downstream collaborator with p53 in the stress-induced apoptosis and cell cycle arrest. Among sphingolipids, the balance between pro- and anti-apoptotic products, dictated by the expression and activity of appropriate enzymes, helps determine cell fate in response to hypoxia. The current study aimed to understand the role of ceramide in HCT116 human colon cancer cells response to hypoxia in the presence or absence of p53, and to determine whether the modulation of ceramide metabolism could sensitize the resistant p53-deficient cells to hypoxia-induced cell death. We observed that HCT116 p53-deficient cells were resistant to hypoxic cell death. We explored the role of ceramide in this response by screening for different sphingolipid metabolites through liquid-chromatography-mass spectrometry, and by measuring the expression of key enzymes involved in ceramide biosynthesis and breakdown. We also evaluated the changes in the cellular response to hypoxia associated with introduction of sphingolipid metabolites or with modulating the activity of related sphingolipid-metabolizing enzymes. In hypoxic p53-deficient cells, ceramide was synthesized via the de novo pathway through the action of ceramide synthases and dihydroceramide desaturase (DEGS1) driving the evasion of hypoxia-induced apoptosis. Among the accumulating ceramide species in p53 deficient cells, C24-ceramide was the most abundant and possibly contributing to their resistance. Tipping the sphingolipid balance in favor of pro-apoptotic sphingolipids, through the addition of C6 ceramide or sphingosine, or through the combined pharmacologic inhibition of DEGS1 and sphingosine kinase 1, helped circumvent the cellular resistance to hypoxia-induced apoptosis in cells lacking p53. Therefore, modulating sphingolipid metabolism may be a viable approach in the treatment of solid tumors with hypoxic regions.