Integrated stress response is critical for gemcitabine resistance in pancreatic ductal adenocarcinoma.

L R Palam,J L Wilson,K E Craven,J Gore,J Gore,M Korc,M Korc

doi:10.1038/cddis.2015.264

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with marked chemoresistance and a 5-year survival rate of 7%. The integrated stress response (ISR) is a cytoprotective pathway initiated in response to exposure to various environmental stimuli. We used pancreatic cancer cells (PCCs) that are highly resistant to gemcitabine (Gem) and an orthotopic mouse model to investigate the role of the ISR in Gem chemoresistance. Gem induced eIF2 phosphorylation and downstream transcription factors ATF4 and CHOP in PCCs, and these effects occurred in an eIF2α-S51 phosphorylation-dependent manner as determined using PANC-1 cells, and wild type and S51 mutant mouse embryo fibroblasts. Blocking the ISR pathway in PCCs with the ISR inhibitor ISRIB or siRNA-mediated depletion of ATF4 resulted in enhanced Gem-mediated apoptosis. Polyribosomal profiling revealed that Gem caused repression of global translation and this effect was reversed by ISRIB or by expressing GADD34 to facilitate eIF2 dephosphorylation. Moreover, Gem promoted preferential mRNA translation as determined in a TK-ATF4 5′UTR-Luciferase reporter assay, and this effect was also reversed by ISRIB. RNA-seq analysis revealed that Gem upregulated eIF2 and Nrf2 pathways, and that ISRIB significantly inhibited these pathways. Gem also induced the expression of the antiapoptotic factors Nupr1, BEX2, and Bcl2a1, whereas ISRIB reduced their expression. In an orthotopic tumor model using PANC-1 cells, ISRIB facilitated Gem-mediated increases in PARP cleavage, which occurred in conjunction with decreased tumor size. These findings indicate that Gem chemoresistance is enhanced by activating multiple ISR-dependent pathways, including eIF2, Nrf2, Nupr1, BEX2, and Bcl2A1. It is suggested that targeting the ISR pathway may be an efficient mechanism for enhancing therapeutic responsiveness to Gem in PDAC.

Highlights

The integrated stress response (ISR) is a signaling pathway initiated upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 at serine 51 in response to diverse stress conditions
There are four mammalian eIF2 kinases that phosphorylate eIF2α: general control nonderepressible 2 (GCN2), which is upregulated by amino acid starvation; protein kinase R (PKR), which is activated by viral infections; PKR-like endoplasmic reticulum (ER) kinase (PERK), which is upregulated by ER stress; and hemeregulated eIF2α kinase (HRI), which is induced upon oxidative stress or heme deprivation.[3,4,5,6]
When PANC-1 cells were transfected with a GADD34 cDNA, there was a marked reduction in eIF2 phosphorylation which was associated with marked decreases in ATF4 protein levels (Figure 1b)

Summary

Introduction

The integrated stress response (ISR) is a signaling pathway initiated upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) at serine 51 in response to diverse stress conditions. There are four mammalian eIF2 kinases that phosphorylate eIF2α: general control nonderepressible 2 (GCN2), which is upregulated by amino acid starvation; protein kinase R (PKR), which is activated by viral infections; PKR-like endoplasmic reticulum (ER) kinase (PERK), which is upregulated by ER stress; and hemeregulated eIF2α kinase (HRI), which is induced upon oxidative stress or heme deprivation.[3,4,5,6] Phosphorylated eIF2α (p-eIF2α) markedly attenuates translation initiation and overall protein synthesis, allowing for conservation of cellular resources. P-eIF2α facilitates the preferential translation of certain mRNAs, most notably ATF4, whose upregulation promotes the expression of genes involved in oxidative stress (OS), metabolism, and nutrient uptake.[7,8,9] gene reprograming by p-eIF2α allows cells to recover from stress-induced damage facilitating survival in response to mild stress and promoting apoptosis in response to chronic stress.[9,10]. We report that ISRIB enhances Gem chemosensitivity in PCCs by suppressing ISR and its downstream antiapoptotic pathways

Methods

Results

Conclusion