b-AP15 enhances TRAIL-induced cell death in HNSCC via the induction of ROS/JNK/DR5 signalling

Therapeutic resistance to chemotherapy or radiotherapy is a significant issue in several cancers, including head and neck squamous cell carcinoma (HNSCC). One pathway associated with therapeutic resistance is the NFκB pathway, which promotes survival in response to the cytokine TNFα, a key mediator of chemotherapy and radiotherapy-induced cytotoxicity. However, direct targeting of the NFκB pathway is associated with significant toxicity and thus targeting the regulation of this pathway is a promising therapeutic target. We recently demonstrated that the USP14/UCHL5 inhibitor b-AP15 inhibits NFκB activity, inhibiting proliferation and inducing apoptosis in HNSCC cells. Furthermore, b-AP15 treatment sensitised HNSCC cells to the cytotoxic effects of TNFα, as well as TNF-inducing radiation treatment. Here, we investigated if b-AP15 sensitised HNSCC cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a cancer selective member of the TNF family. b-AP15 treatment sensitised HNSCC cells to TRAIL treatment. Mechanistically, we show that b-AP15 induced expression of the TRAIL receptor Death Receptor 5 (DR5)/TRAIL Receptor 2 (TRAILR2), which was required for b-AP15-mediated TRAIL sensitisation. b-AP15 induced reactive oxygen species (ROS) and activated the JNK signalling pathway and both ROS and JNK signalling were required for the induction of DR5 expression and TRAIL sensitisation. We further show that b-AP15-mediated reduction of the NFκB-dependent gene XIAP induced DR5 expression and TRAIL sensitisation and that combination between b-AP15 and IAP antagonists was synergistic in HNSCC cells in vitro. Our data further define the mechanism of b-AP15-mediated cytotoxicity and highlight potential combination treatments that warrant further exploration in pre-clinical studies in HNSCC.