Resistance to TRAIL is associated with defects in ceramide signaling that can be overcome by exogenous C6-ceramide without requiring down-regulation of cellular FLICE inhibitory protein

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Abstract
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily that selectively induces apoptosis in malignant cells. However, not all cancer cells are susceptible to TRAIL and mechanisms of resistance and new strategies to enhance sensitivity are an area of intense investigation. Glucose withdrawal or paclitaxel increase intracellular ceramide, down-regulate cellular FLICE inhibitory protein (cFLIP), and sensitize cells to TRAIL. Therefore, we investigated whether TRAIL resistance is due to ceramide levels and/or defects in ceramide generation following ligand binding. Colon cancer cells isolated from the primary tumor (SW480) and a subsequent metastasis (SW620) of the same patient have different sensitivities to TRAIL. Mass spectrometry was used to compare ceramide content in untreated and TRAIL-treated cells. Overall levels of ceramide were comparable in the cell lines but TRAIL-sensitive SW480 cells contained a higher percentage of C16-, and C18-ceramide and lower C24-ceramides than TRAIL-resistant SW620 cells. Upon TRAIL treatment, ceramide (primarily C16-ceramide) increased in SW480 but not SW620 cells. The increase in ceramide occurred with slow kinetics, paralleling caspase-3/7 activation. Combination of C6-ceramide with TRAIL resulted in apoptosis of SW620 cells. However, exogenous C6-ceramide did not affect levels of cFLIP nor did pretreatment sensitize cells to TRAIL. Exposure to TRAIL prior to ceramide was required to induce apoptosis, suggesting that ceramide plays a role in enhancing or amplifying TRAIL-mediated signaling. Our results suggest that ceramide plays a role in promoting TRAIL-mediated apoptosis and that TRAIL-resistant cancers may benefit from combination therapy with ceramide or agents that enhance ceramide accumulation.