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

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.

Figure 1

Gemcitabine induces the integrated stress pathway in pancreatic cancer cells. (a) AsPC-1 and PANC-1 cells were incubated with 0.5 μM ISRIB or 10 μM Gem for indicated times and protein lysates were analyzed by immunoblotting for phospho-eIF2, total eIF2, and ATF4 using specific antibodies. ERK2 served as loading control. (b) Empty vector or GADD34 expression plasmid DNA was transfected into PANC-1 cells, which were incubated 24 h later with 10 μM Gem. Protein lysates were analyzed by immunoblotting. (c) PANC-1 cells were incubated for 36 h with 0.5 μM ISRIB or 10 μM Gem and protein lysates were analyzed by immunoblotting for ATF4, ATF3, GADD34, and CHOP using specific antibodies. ERK2 served as loading control. (d) PANC-1 cells were incubated with 0.5 μM ISRIB or 10 μM Gem for 24 h. ATF4, CHOP, ATF3, and GADD34 mRNA levels were measured using qRT-PCR. Relative actin mRNA levels were used for normalization. Data are the means±S.D. from three experiments. *P<0.05, **P<0.001 compared with control, ^##P<0.01 compared with ISRIB, and ^$P<0.05 compared with Gem+ISRIB. (e) MEFs were incubated for 6 h with 10 μM Gem in the absence and presence of ISRIB. As a positive control, MEFs were also incubated for 6 h with thapsigargin (0.1 μM). (f) Wild-type MEFs or MEFs mutated for eIF2α at serine 51 (to alanine) were incubated with 10 μM Gem for indicated time point and analyzed by immnuoblotting. Panels (a, b, d and e) show representative data from three independent experiments

Figure 2

Suppression of integrated stress pathway enhances gemcitabine chemosensitivity. (a) AsPC-1 cells were incubated with ISRIB alone or (b) Gem alone, or ISRIB and Gem for 72 h as indicated, and MTT assays were performed. Data are the means±S.D. from three experiments. *P<0.001 compared with control, **P<0.005 compared with Gem alone, ^#P<0.005 compared with Gem (1 μM)+ISRIB (250 nM). (c) PANC-1 cells were incubated with ISRIB alone or (d) with Gem alone or with Gem+ISRIB for 72 h as indicated, and MTT assays were performed. Data are the means±S.D. from three experiments. *P<0.001 compared with control, **P<0.005 compared with Gem alone, ^$P<0.005 compared with 0.1 μM Gem+250 nM ISRIB. (e and g) AsPC-1 cells were incubated with 0.5 μM ISRIB alone or in combination with 10 μM Gem. (f and h) PANC-1 cells were incubated with 0.5 μM ISRIB alone or in combination with 1 μM Gem. Cell lysates were immunoblotted for cleaved caspase-3 protein in (e and f) and caspase-3/7 activity was measured in (g and h). Data shown are from three independent experiments in (e and f). Data are the means±S.D. from three experiments in ( g and h). ^##P<0.01 compared with control and ISRIB, ***P<0.01 compared with control, ISRIB, and Gem

Figure 3

Gemcitabine causes translation repression in pancreatic cancer cells. (a) PANC-1 cells were incubated for 12 h in the absence or presence of 10 μM Gem or 0.5 μM ISRIB. Cell lysates were subjected to sucrose gradient centrifugation, and gradients were fractionated in-line with 254 nm UV absorbance measurement. Top fractions containing free ribosomal subunits are labeled as 40/43S, 60S. Mono ribosomes are labeled as 80S. Ribosomes bound to RNA fractions were labeled as polysomes. (b) PANC-1 cells were transfected with an empty vector or the GADD34 plasmid DNA, and incubated 24 h later with 10 μM Gem for 12 h. Cell lysates were analyzed for polysome profiles as in (a). (c) PANC-1 cells were incubated in the absence or presence of Gem or ISRIB+Gem and subjected to sucrose gradient centrifugation as in (a). Fractions were collected and luciferase (10 ng/ml) spiked into each fraction. RNA Isolated and ATF4 transcript levels were quantitated using qRT-PCR, and normalized to spike-in luciferase control. The percent total ATF4 transcript for each fraction is represented. Fractions 5, 6, and 7 corresponds to fractions with polysomes rich in translation. (d) PANC-1 cells were transfected with the TK-ATF4-Luc plasmid construct that with illustrated features: ATF4 5′UTR harboring uORF1 and uORF2, TK promoter and Luciferase coding region. At 24 h post transfection cells were incubated for 12 h in the absence or presence of 10 μM Gem and 0.5 μM ISRIB. Firefly luciferase units were measured and normalized to internal control Renilla luciferase activity. Data are the means ±S.D. of three experiments. *P<0.05, **P<0.005 compared with control, ^##P<0.001 compared with Gem+ISRIB. Panels a and b show representative data from three independent experiments

Figure 4

Genome-wide analysis for response to gemcitabine and ISRIB. PNAC-1 cells were incubated for 36 h in the absence or presence of 10 μM Gem 0.5 μM ISRIB, and collected RNA was used for RNA transcriptome sequencing. (a) Total number of genes with significant change in gene expression (>1.5-folds increase or <−1.5-fold decrease, P<0.001) following indicated incubations compared with control are represented in a Venn diagram. (b) Heat map for fold changes in gene expression (P<0.001) over control following indicated incubations is shown for genes involved in the ISR pathway. ISR genes and gene expression data from RNA-seq analysis are shown. (c) ISR genes with more than 2-fold change in gene expression (P<0.001) in response to Gem compared with control are shown. (d) IPA was performed on obtained RNA-seq data and major canonical signaling pathways that were modulated in response to drugs are represented. (e) Heat map for fold changes in gene expression (P<0.001) over control condition following indicated incubations is shown for genes involved in eIF2 signaling. Network of genes involved in eIF2 signaling was collected from IPA as described in Materials and Methods. (f) Top 20 genes that are differentially regulated in response to Gem alone versus Gem+ISIRB are shown (P<0.001)

Figure 5

Inhibition of ISR pathway completely reduces gemcitabine-induced Nupr1 increase in PANC-1 cells. (a) Nupr1 expression data from RNA-seq analysis and qRT-PCR is represented. **P<0.001 compared with control, ^##P<0.001 compared with ISRIB, ^$P<0.001 compared with Gem+ISRIB. PANC-1 cells were incubated with 0.5 μM ISRIB, 10 μM Gem, or ISRIB+Gem for 36 h, and RNA was analyzed for Nupr1 mRNA levels using qRT-PCR. Data are the means ±S.D. of three experiments. **P<0.001 compared with control, ^##P<0.001 compared with ISRIB, ^$P<0.001 compared with Gem+ISRIB. (b) Heat map for fold changes in gene expression in response to indicated drugs over control for genes involved in Nupr1 network, based on Nupr1 network genes collected from Ingenuity Pathway Analysis as described in Materials and Methods. (c) PANC-1 cells stably expressing either control-shRNA or PERK-shRNA and PANC-1 cells with ATF4 siRNA were incubated with 10 μM Gem for 24 h, and RNA was analyzed for Nupr1 mRNA expression levels. Data are the means ±S.D. of three experiments. **P<0.001 compared with control-shRNA and PERK-shRNA, ^##P<0.001 compared with PERK-shRNA, *P<0.001 compared with control-shRNA, ^$P<0.001 compared with PERK-shRNA in the presence of Gem. (d) PANC-1 cells were transfected with indicated siRNA, and incubated 48 h later in the absence or presence of 10 μM Gem for 24 h. Caspase-3 activity was measured using Casp3/7 glow assay. Data are the means ±S.D. from three independent experiments. **P<0.001 compared with control, ^##P<0.001 compared with sham-siRNA control and sham-siRNA with Gem, ***P<0.001 compared with sham-siRNA control, ATF4 siRNA control, and sham-siRNA with Gem, ^$P<0.001 compared with sham-siRNA control, Nupr1-siRNA control, and sham-siRNA with Gem. (e) Antiapoptotic factor BEX2 and (g) BCL2A1 expression data from RNA-seq analysis and qRT-PCR analysis are shown; **P<0.001 compared with control and ISRIB, ^##P<0.001 compared with Gem, ^$P<0.001 compared with Gem+ISRIB. (f) PANC-1 cells were incubated with 0.5 μM ISRIB or 10 μM Gem for 36 h and protein lysates were analyzed by immunoblotting for Nupr1 and BEX2 using specific antibody. ERK2 served as loading control. Nupr1 protein levels were quantified from the blots and fold changes in expression are shown. Data are the means±S.D. of three experiments. *P<0.01 compared with control, ISRIB, and Gem+ISRIB. (h and i) PANC-1 cells expressing ATF4 siRNA were incubated with 10 μM Gem for 24 h, and RNA was analyzed for (h) BEX2 mRNA and (i) BCL2A1 expression levels. Data are the means ±S.D. of three experiments. **P<0.001 compared with control siRNA and ATF4 siRNA, ^##P<0.001 compared with ATF4 siRNA, *P<0.001 compared with control siRNA, ^$P<0.001 compared with ATF4 in the presence of Gem

Figure 6

Inhibition of gemcitabine induced ISR pathway decreases PANC-1 orthotopic tumor growth. (a) PCCs were injected into the pancreas of athymic mice as described in Materials and Methods. High-resolution ultrasound images were obtained at 2 weeks post-PCC injection, and at indicated treatment times. Tumors are outlined and are representative images from six mice per group. (b) Tumor volumes were calculated using 3-D abdominal imaging. There was a step-wise decrease in tumor volumes from Vehicle-treated, to ISRIB-treated, to Gem-treated, and to Gem+ISRIB-treated mice. Data are the means±S.D.; ***P<0.05 compared with control, ^##P<0.05 compared with ISRIB. (c) H&E and PARP staining was performed on paraffin-embedded tissue sections (0.5 μm thick), bars=50 μm. (d) PARP-positive cells were counted and tabulated after normalization to percent of control. Data are the means±S.D. for four experiments. ***P<0.001, ^##P<0.001, ^$P<0.001 compared with control, ISRIB, and Gem+ISRIB treatments, respectively