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 2alpha (eIF2alpha)-ATF4 signaling pathway. Treatment of neuroblastoma and melanoma cell lines with fenretinide, bortezomib, or thapsigargin resulted in induction of eIF2alpha signaling, characterized by increased expression of phosphorylated eIF2alpha, 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 ER-related pro-death signaling pathways involving GADD153 or IRE1. Although PERK-dependent phosphorylation of eIF2alpha enhanced ATF4 protein levels during ER stress, cell death in response to fenretinide, bortezomib, or thapsigargin was not abrogated by inhibition of eIF2alpha phosphorylation through PERK knockdown or overexpression of wild-type eIF2alpha. Furthermore, ATF4 induction in response to ER stress was dependent primarily on transcriptional activation, which occurred in a PERK- and phosphorylated eIF2alpha-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.
Highlights
Charity, and the British Skin Foundation. □S The on-line version of this article contains supplemental Figs. 1–5. 1 To whom correspondence should be addressed: Dermatological Sciences, 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 [3,4,5,6]
Understanding the molecular mechanisms mediating ER stress-induced apoptosis in the context of cancer therapy is essential to promote the pharmacological exploitation of ER stress responses to combat cancer. Both fenretinide and bortezomib induce hallmarks of ER stress [2, 13], and because there is evidence for a central role for eIF2␣-ATF4 signaling in ER stress-induced cell death [14, 21, 22], we hypothesize that this pathway is fundamental to the cytotoxicity induced by these agents in neuroectodermal tumor cells
PERK or ATF4 mRNA was measured by real-time PCR relative to -actin as an internal control (A). eIF2␣, phospho-eIF2␣, ATF4, ATF3, and -actin expression was determined by Western blotting (B)
Summary
Western blot analysis revealed that phosphorylation of eIF2␣ was transient, and the kinetics varied between drug treatment and cell line (Fig. 1). In SH-SY5Y cells, apoptosis induced by fenretinide, bortezomib, or thapsigargin ATF4 expression was up-regulated to the greatest extent in were tested by two-way analysis of variance (ANOVA; GLM) response to thapsigargin and to a lesser extent in response to with siRNA type (control or gene-specific) and treatment The specific weakly up-regulated in response to fenretinide in A375 and effects of siRNA type (when more than one gene-specific siRNA SK-MEL-28 cells despite strong phosphorylation of eIF2␣; this was used) or treatments were tested, when appropriate, using is consistent with weak ATF3 induction under these conditions simple contrasts
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
