DR5 Disulfide Bonding Functions as a Sensor and Effector of Protein Folding Stress

New agents are needed that selectively kill cancer cells without harming normal tissues. The TRAIL ligand and its receptors, DR5 and DR4, exhibit cancer-selective toxicity. TRAIL analogs or agonistic antibodies targeting these receptors are available but have not yet received FDA approval for cancer therapy. Small molecules for activating DR5 or DR4 independently of protein ligands may activate TRAIL receptors as a monotherapy or potentiate the efficacy of TRAIL analogs and agonistic antibodies. Previously described disulfide bond–disrupting agents activate DR5 by altering its disulfide bonding through inhibition of protein disulfide isomerases ERp44, AGR2, and PDIA1. Work presented in this article extends these findings by showing that disruption of single DR5 disulfide bonds causes high-level DR5 expression, disulfide-mediated clustering, and activation of caspase 8/caspase 3–mediated proapoptotic signaling. Recognition of the extracellular domain of DR5 by various antibodies is strongly influenced by the pattern of DR5 disulfide bonding, which has important implications for the use of agonistic DR5 antibodies for cancer therapy and as research tools. Importantly, other endoplasmic reticulum (ER) stressors, including thapsigargin and tunicamycin, also alter DR5 disulfide bonding in various cancer cell lines, and in some instances, DR5 mis-disulfide bonding is potentiated by overriding the integrated stress response (ISR) with inhibitors of the PERK kinase or the ISR inhibitor ISRIB. These observations indicate that the pattern of DR5 disulfide bonding functions as a sensor of ER stress and serves as an effector of proteotoxic stress by driving extrinsic apoptosis independently of extracellular ligands. Implications: Extreme ER stress triggers triage of transmembrane receptor production, whereby mitogenic receptors are downregulated and death receptors are simultaneously elevated.