The E3 ubiquitin ligase Itch regulates death receptor and cholesterol trafficking to affect TRAIL-mediated apoptosis

The activation of apoptosis signalling by TRAIL (TNF-related apoptosis-inducing ligand) through receptor binding is a fundamental mechanism of cell death induction and is often perturbed in cancer cells to enhance their cell survival and treatment resistance. Ubiquitination plays an important role in the regulation of TRAIL-mediated apoptosis, and here we investigate the role of the E3 ubiquitin ligase Itch in TRAIL-mediated apoptosis in oesophageal cancer cells. Knockdown of Itch expression resulted in resistance to TRAIL-induced apoptosis, caspase-8 activation, Bid cleavage and also promoted cisplatin resistance. Whilst the assembly of the death-inducing signalling complex (DISC) at the plasma membrane was not perturbed relative to the control, the TRAIL-R2 receptor was mis-localised in the Itch-knockdown cells. Further, we observed significant mitochondrial widening with an increased cholesterol content. An inhibitor of cholesterol trafficking, U18666A, was able to replicate some of the effects of Itch knockdown, including protection from TRAIL-induced apoptosis, reduced caspase-8 activation, Bid cleavage and Cisplatin resistance. This study highlights the importance of Itch in regulating the crosstalk between mitochondrial cholesterol and TRAIL-induced apoptosis.