The Deubiquitinase Inhibitor PR-619 Sensitizes Normal Human Fibroblasts to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-mediated Cell Death

Abstract

TNF-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapy that selectively targets cancer cell death while non-malignant cells remain viable. Using a panel of normal human fibroblasts, we characterized molecular differences in human foreskin fibroblasts and WI-38 TRAIL-resistant cells and marginally sensitive MRC-5 cells compared with TRAILsensitive human lung and colon cancer cells. We identified decreased caspase-8 protein expression and protein stability in normal fibroblasts compared with cancer cells. Additionally, normal fibroblasts had incomplete TRAIL-induced caspase-8 activation compared with cancer cells. We found that normal fibroblasts lack the ubiquitin modification of caspase-8 required for complete caspase-8 activation. Treatment with the deubiquitinase inhibitor PR-619 increased caspase-8 ubiquitination and caspase-8 enzymatic activity and sensitized normal fibroblasts to TRAIL-mediated apoptosis. Therefore, posttranslational regulation of caspase-8 confers resistance to TRAIL-induced cell death in normal cells through blockade of initiation of the extrinsic cell death pathway. TNF-related apoptosis inducing ligand (TRAIL)3 is a member of the TNF family and has cytotoxic effects against various cancer cells but causes little or no toxicity to non-malignant normal cells (1, 2). The selective killing of cancer cells has made TRAIL an exciting cancer target, and the clinical use of TRAIL is currently being explored (3, 4). TRAIL can bind to five receptors. Death receptor 4 (DR4) (5) and death receptor 5 (DR5) (6) are death domain-containing, proapoptotic TRAIL receptors. Decoy receptor 1 (DcR1) and decoy receptor 2 (DcR2) are decoy receptors that are incapable of TRAIL-induced cell death signaling because of truncated or nonfunctional death domains (7-10). Osteoprotegerin is a soluble decoy TRAIL receptor (11). TRAIL/TRAIL receptor ligation causes receptor trimerization and recruitment of the intracellular adaptor protein Fas-associated death domain. Caspase-8 is also recruited and interacts with Fas-associated death domain in a complex referred to as the death-inducing signaling complex (12). Death-inducing signaling complex activation of caspase-8 leads to activation of effector caspases, including caspase-3, resulting in cell death (12). TRAIL can also activate the intrinsic death pathway and lead to mitochondrial permeabilization and release of proapoptotic factors, including cytochrome c, and activation of initiator caspase-9. Activated caspase-9 can then activate caspase-3, causing cell death (12).Not all cancer cells are susceptible to TRAIL-mediated cell death. TRAIL sensitivity in cancer cells has been studied extensively, and several resistance mechanisms have been defined. Altered DR4 and DR5 posttranslational modifications, including glycosylation (13, 14), abnormal TRAIL receptor transport to the cell surface (15), TRAIL receptor endocytosis (16), recruitment of the enzymatically inactive caspase-8 homologue FLICE-inhibitory protein (FLIP) to the death-inducing signali...

TRAIL signals, extracellular matrix and vessel remodelling

“…They found that treatment of fibroblasts with a neutralising antibody to TGFβ1 inhibited TRAIL-mediated collagenα2a gene expression and total collagen secretion, implying a TRAIL-TGFβ1-collagen axis (42). While fibroblasts express all TRAIL receptors (43,44), it is unclear whether the TRAIL-TGFβ1-collagen axis involves a specific receptor. These suggest that TRAIL may contribute to the regulation of tensile strength in the blood vessel wall, in part, by regulating synthesis of collagen.…”

Dynamic ubiquitylation of Sox2 regulates proteostasis and governs neural progenitor cell differentiation

“…Subsequently, we tested the effect of modifications of Sox2 level on NPCs differentiation by chemical perturbations of the UB/DUB system. Given that the specific inhibitor targeting CUL4A DET1-COP1 or OTUD7B is unavailable, we used unspecific DUB inhibitors PR-619 37 , b-AP-15 38 , and the inhibitor of NEDD8-activating enzyme (NAE) MLN4924 which had been demonstrated to suppress neddylation of Cullins, and inactivate Cullin-RING ligases 39 . The results showed that compared with control, the treatment of MLN4924 promoted NPC maintenance marked by increased Sox2 level and decreased neuN level, while PR-619 and b-AP-15 enhanced cell differentiation marked by decreased Sox2 level and increased neuN level (Supplementary Fig.…”

Andrographolide sensitizes prostate cancer cells to TRAIL-induced apoptosis

“…Here, certain agents were used to improve the sensitivity of the cells to TRAIL-induced apoptosis, whereas TRAIL was responsible for the promotion of cancer cell apoptosis. 15 Studies have consistently found that several compounds and agents can improve the sensitivity of cells to TRAIL-induced apoptosis; these are defined as anticancer agents. Among them, Andro, a diterpenoid lactone compound from Andrographis paniculata, is a type of anticancer agent which is currently being widely researched.…”