UNDERSTANDING THE ROLE OF CERAMIDE IN p53-MEDIATED AND p53-INDEPENDENT RESPONSES TO HYPOXIA IN HCT-116 COLON CANCER CELLS

Abstract

Hypoxia is a common feature of solid tumors resulting from the disordered vasculature developed to supply oxygen to the rapidly growing tumor. As a result, gradients of oxygen concentration emerge; regions of severe hypoxia undergo cell death through the stabilization and activation of p53. Cancer cells can evade hypoxia-induced cell death by inducing genetic alterations, such as mutations in the TP53 tumor-suppressor gene. Hypoxic stress then acts as a potent selector for aggressive mutant clones allowing their expansion at the expense of their wild-type hypoxia-sensitive counterparts. Restoration of hypoxia-induced apoptosis in tumors harboring p53 mutations has been proposed as a potential therapeutic strategy. Although several p53-targeted therapies have entered clinical trials, several limitations arose and hindered the efficacy of these approaches. Ceramide, a signaling sphingolipid, was previously reported as a potential collaborator with p53 in the stress-induced cellular response. Ceramide metabolism is quite complex and comprises a set of reactions that produce different metabolites involved in a variety of cellular responses. While ceramide (Cer) and sphingosine (Sph) act as pro-apoptotic lipids, ceramide 1-phosphate (C1P) and sphingosine 1-phosphate (S1P) are considered as oncometabolites. Slight changes in the balance between pro and anti-apoptotic sphingolipids, commanded by the expression and activity of appropriate enzymes, could dictate cell fate in response to hypoxia in both p53-proficient and p53-deficient cells. In order to define the role of ceramide in the p53-dependent and independent responses to hypoxia, HCT-116 colon carcinoma cells differentially expressing p53 were incubated under 1% O2 and the changes in their ceramide metabolism were measured at several time points. Unlike p53-proficient colonic carcinoma HCT-116 cells, p53-deficient cells resisted the apoptotic response induced by hypoxia. This resistance was accompanied by ceramide accumulation and a drop in sphingosine levels, which may have been promoted by sphingosine phosphorylation into the anti-apoptotic sphingolipid S1P. Indeed, we showed, through protein and mRNA expression studies as well as through enzymatic pharmacological modulation, that ceramide synthases and dihydroceramide desaturase 1, key players in the de novo synthesis pathway of ceramide, were orchestrating the response of HCT-116 cells to hypoxia in the absence of p53. We also demonstrated that the drop in sphingosine level upon hypoxia was accompanied by an increase in the mRNA expression of sphingosine kinase 1 in p53-deficient cells, which may have contributed to their resistance to hypoxia-induced apoptosis. Treating HCT-116 cells with exogenous C6 ceramide specifically targeted p53-deficient cells towards apoptosis, possibly by shifting the ceramide/S1P balance towards ceramide accumulation. This data supports the hypothesis that bypassing p53 loss of function through altering downstream ceramide metabolism is a potentially promising therapeutic strategy for the treatment of p53-mutated chemo-resistant solid tumors.