The regulatory protein GADD34 inhibits TRAIL-induced apoptosis via TRAF6/ERK-dependent stabilization of myeloid cell leukemia 1 in liver cancer cells

: GADD34 (growth arrest and DNA damage-inducible gene 34) plays a critical role in responses to DNA damage and endoplasmic reticulum stress. GADD34 has opposing effects on different stimuli-induced cell apoptosis events, but the reason for this is unclear. Here, using immunoblotting analyses and various molecular genetic approaches in HepG2 and SMMC-7721 cells, we demonstrate that GADD34 protects hepatocellular carcinoma (HCC) cells from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by stabilizing a BCL-2 family member, myeloid cell leukemia 1 (MCL-1). We found that GADD34 knockdown decreased MCL-1 levels and that GADD34 overexpression up-regulated MCL-1 expression in HCC cells. GADD34 did not affect MCL-1 transcription but enhanced MCL-1 protein stability. The proteasome inhibitor MG132 abrogated GADD34 depletion-induced MCL-1 down-regulation, suggesting that GADD34 inhibits the proteasomal degradation of MCL-1. Furthermore, GADD34 overexpression promoted extracellular signal-regulated kinase (ERK) phosphorylation through a signaling axis that consists of the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase 1 (MAP3K7)-binding protein 1 (TAB1), which mediated the up-regulation of MCL-1 by GADD34. Of note, TRAIL up-regulated both GADD34 and MCL-1 levels, and knockdown of GADD34 and TRAF6 suppressed the induction of MCL-1 by TRAIL. Correspondingly, GADD34 knockdown potentiated TRAIL-induced apoptosis, and MCL-1 overexpression rescued TRAIL-treated and GADD34-depleted HCC cells from cell death. Taken together, these findings suggest that GADD34 inhibits TRAIL-induced HCC cell apoptosis through TRAF6- and ERK-mediated stabilization of MCL-1.