Regulation of Endoplasmic Reticulum Stress-induced Cell Death by ATF4 in Neuroectodermal Tumor Cells

Abstract

The neuroectodermal tumors neuroblastoma and melanoma represent biologically aggressive and chemoresistant cancers. The chemotherapeutic agents fenretinide and bortezomib induce apoptosis through endoplasmic reticulum (ER) stress in these tumor types. The aim of this study was to test the hypothesis that the early events of ER stress signaling and response pathways induced by fenretinide and bortezomib are mediated by the eukaryotic initiation factor 2␣ (eIF2␣)-ATF4 signaling pathway. Treatment of neuroblastoma and melanoma cell lines with fenretinide, bortezomib, or thapsigargin resulted in induction of eIF2␣ signaling, characterized by increased expression of phosphorylated eIF2␣, ATF4, ATF3, and GADD34. These events correlated with induction of the pro-apoptotic protein Noxa. The cytotoxic response, characterized by up-regulation of Noxa and cell death, was dependent on ATF4, but not the ERrelated pro-death signaling pathways involving GADD153 or IRE1. Although PERK-dependent phosphorylation of eIF2␣ enhanced ATF4 protein levels during ER stress, cell death in response to fenretinide, bortezomib, or thapsigargin was not abrogated by inhibition of eIF2␣ phosphorylation through PERK knockdown or overexpression of wild-type eIF2␣. Furthermore, ATF4 induction in response to ER stress was dependent primarily on transcriptional activation, which occurred in a PERK-and phosphorylated eIF2␣-independent manner. These results demonstrate that ATF4 mediates ER stress-induced cell death of neuroectodermal tumor cells in response to fenretinide or bortezomib. Understanding the complex regulation of cell death pathways in response to ER stress-inducing drugs has the potential to reveal novel therapeutic targets, thus allowing the development of improved treatment strategies to overcome chemoresistance.The development of novel strategies to overcome chemoresistance in cancers, particularly neuroectodermal tumors, which are frequently associated with poor survival despite intensive chemotherapy, would be aided by understanding the complex regulation of cell death programs. The chemotherapeutic agents fenretinide, a synthetic derivative of retinoic acid, and bortezomib, a 26 S proteasome inhibitor, induce endoplasmic reticulum (ER) 2 stress, culminating in apoptosis of neuroblastoma and melanoma cells both in vitro (1, 2) and in vivo (3-6). These studies highlight ER stress as an intracellular stress response that can be exploited to promote cancer cell death, providing an opportunity for rational drug design programs and the development of more effective therapeutic strategies.Normal ER function is required for the regulation of intracellular calcium and correct folding of secretory or cell-surface proteins; ER stress occurs when the protein-folding capacity of the ER is exceeded. Stresses that perturb redox state, energy levels, or calcium homeostasis trigger the accumulation of unfolded proteins within the ER, eliciting a stress response termed the unfolded protein response (UPR). The principles of the UPR are now relative...

Aberrant, differential and bidirectional regulation of the unfolded protein response towards cell survival by 3′-deoxyadenosine

“…The induction of ATF4 may be regulated at both transcriptional and translational level. 24,25 Indeed, treatment with salubrinal resulted in expression of ATF4 and CHOP in NRK-52E cells. However, in contrast to salubrinal, 3 0 -deoxyadenosine did not induce and rather attenuated expression of ATF4 and CHOP.…”

“…23 The induction of ATF4 may be regulated at both transcriptional and translational levels. 24,25 However, treatment with 3 0 -deoxyadenosine did not trigger substantial expression of CHOP and ATF4, and rather suppressed induction of these genes by ER stress (Figures 6f and g). The suppressive effect of 3 0 -deoxyadenosine on ATF4 was also confirmed by western blot analysis of nuclear ATF4 (Figure 6h).…”

Transcriptional Repression of ATF4 Gene by CCAAT/Enhancer-binding Protein β (C/EBPβ) Differentially Regulates Integrated Stress Response

“…This discordant induction of the ISR was also reported in brain ischemia and non-alcoholic steatohepatitis (26,27). Along with preferential translation of ATF4, transcriptional induction of ATF4 is a contributor to its increased expression in the ISR (25,28,29). Increased ATF4 mRNA helps to replenish the labile transcript for translational expression during eIF2␣ϳP.…”

“…Regarding transcriptional activation of ATF4, stresses that can increase ATF4 mRNA levels include ER stress (6,29,62), such as that induced by thapsigargin, starvation for amino acids (28), oxidative stress (63,64), and certain anticancer agents (65,66) (Fig. 10B).…”

Neddylation Inhibition Activates the Extrinsic Apoptosis Pathway through ATF4–CHOP–DR5 Axis in Human Esophageal Cancer Cells

“…Based on the previous findings that Noxa could be transactivated by ATF4 (39,40) which was stabilized by MLN4924 (Fig. 4A and B), we next determined whether MLN4924-induced Noxa expression was mediated by ATF4 in ESCC cells.…”