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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Molecular Cancer 1/2017

PERK induces resistance to cell death elicited by endoplasmic reticulum stress and chemotherapy

Zeitschrift:
Molecular Cancer > Ausgabe 1/2017
Autoren:
Iris C. Salaroglio, Elisa Panada, Enrico Moiso, Ilaria Buondonno, Paolo Provero, Menachem Rubinstein, Joanna Kopecka, Chiara Riganti
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12943-017-0657-0) contains supplementary material, which is available to authorized users.
Iris C Salaroglio and Elisa Panada were co-first authors.
Joanna Kopecka and Chiara Riganti were co-last authors

Abstract

Background

Nutrient deprivation, hypoxia, radiotherapy and chemotherapy induce endoplasmic reticulum (ER) stress, which activates the so-called unfolded protein response (UPR). Extensive and acute ER stress directs the UPR towards activation of death-triggering pathways. Cancer cells are selected to resist mild and prolonged ER stress by activating pro-survival UPR. We recently found that drug-resistant tumor cells are simultaneously resistant to ER stress-triggered cell death. It is not known if cancer cells adapted to ER stressing conditions acquire a chemoresistant phenotype.

Methods

To investigate this issue, we generated human cancer cells clones with acquired resistance to ER stress from ER stress-sensitive and chemosensitive cells.

Results

ER stress-resistant cells were cross-resistant to multiple chemotherapeutic drugs: such multidrug resistance (MDR) was due to the overexpression of the plasma-membrane transporter MDR related protein 1 (MRP1). Gene profiling analysis unveiled that cells with acquired resistance to ER stress and chemotherapy share higher expression of the UPR sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK), which mediated the erythroid-derived 2-like 2 (Nrf2)-driven transcription of MRP1. Disrupting PERK/Nrf2 axis reversed at the same time resistance to ER stress and chemotherapy. The inducible silencing of PERK reduced tumor growth and restored chemosensitivity in resistant tumor xenografts.

Conclusions

Our work demonstrates for the first time that the adaptation to ER stress in cancer cells produces a MDR phenotype. The PERK/Nrf2/MRP1 axis is responsible for the resistance to ER stress and chemotherapy, and may represent a good therapeutic target in aggressive and resistant tumors.
Zusatzmaterial
Additional file 1: IC50 (μM) of chemotherapeutic agents and ER stress inducers in HT29 and HT29/MDR cells. Data are mean ± SD (n = 3). *p < 0.02 for HT29/MDR vs. HT29 cells. (XLSX 12 kb)
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Additional file 2: Primers used in qRT-PCR experiments. (XLSX 12 kb)
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Additional file 3: Effect of oxaliplatin in 3D-cultures of colon cancer cells. Human chemosensitive HT29 cells, chemoresistant HT29/MDR cells and ER stress-resistant HT29/Tun cells were cultured 7 days embedded in Biomimesys™ matrix to generate 3D-systems. Medium was replaced with fresh medium (Ctrl) or with medium containing 10 μM oxaliplatin (oPt) for 48 h, then cells were analysed by contrast phase microscope. The images are representative of 3 independent experiments. Bar = 50 μM. (TIF 1680 kb)
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Additional file 4: Effects of chemotherapeutic drugs in human chemosensitive breast cancer cells with acquired resistance to ER stress. a. Release of the necrosis marker HMGB1 to culture media of human chemosensitive breast cancer MCF7 cells and the ER stress-resistant clone MCF7/Tun, grown in fresh medium (Ctrl) or in media containing: thapsigargin (Tg), tunicamycin (Tun), brefeldin A (Bfa), doxorubicin (Dox), cisplatin (Pt), as indicated in Methods. Data are mean ± SD (n = 3). *p < 0.001 vs MCF7 Ctrl cells; °p < 0.001 for MCF7/Tun treated cells vs MCF7 treated cells. b. Viability of cells measured by Neutral red staining. Data are mean ± SD (n = 3). *p < 0.05 vs MCF7 Ctrl cells; °p < 0.02 for MCF7/Tun treated cells vs MCF7 treated cells. c. Whole cell lysates were analyzed for the expression of MRP1. β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. d. MRP1 mRNA levels were measured by qRT-PCR. Data are mean ± SD (n = 3). *p < 0.01 vs MCF7 cells. e. MRP1 protein on cell surface was measured by flow cytometry. Left panel: data are presented as mean fluorescence intensity (MFI) ± SD (n = 3). *p < 0.02 vs MCF7 cells. Right panel: representative flow cytometry histograms. Grey peak: non immune isotypic antibody. f. Intracellular doxorubicin content, an index of MRP1 activity, measured by fluorimetry. Data are mean ± SD (n = 3). *p < 0.005 vs MCF7 cells. (TIF 1434 kb)
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Additional file 5: Effects of chemotherapeutic drugs in human chemosensitive osteosarcoma cells with acquired resistance to ER stress. a. Release of the necrosis marker HMGB1 to culture media of human chemosensitive osteosarcoma U-2OS cells and the ER stress-resistant clone U-2OS /Tun, grown in fresh medium (Ctrl) or in media containing: thapsigargin (Tg), tunicamycin (Tun), brefeldin A (Bfa), doxorubicin (Dox), cisplatin (Pt), as indicated in Methods. Data are mean ± SD (n = 3). *p < 0.005 vs U-2OS Ctrl cells; °p < 0.005 for U-2OS/Tun treated cells vs U-2OS treated cells. b. Viability of cells measured by Neutral red staining. Data are mean ± SD (n = 3). *p < 0.02 vs U-2OS Ctrl cells; °p < 0.05 for U-2OS/Tun treated cells vs U-2OS treated cells. c. Whole cell lysates were analyzed for the expression of MRP1. β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. d. MRP1 mRNA levels were measured by qRT-PCR. Data are mean ± SD (n = 3). *p < 0.005 vs U-2OS cells. e. MRP1 protein on cell surface was measured by flow cytometry. Left panel: data are presented as mean fluorescence intensity (MFI) ± SD (n = 3). *p < 0.02 vs U-2OS cells. Right panel: representative flow cytometry histograms. Grey peak: non immune isotypic antibody. f. Intracellular doxorubicin content, an index of MRP1 activity, measured by fluorimetry. Data are mean ± SD (n = 3). *p < 0.05 vs U-2OS cells. (TIF 1533 kb)
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Additional file 6: Expression of genes involved in the UPR in HT29, HT29/Tun and HT29/MDR cells. ERAD: ER-associated degradation; UPR: unfolded protein response; ERQC: ER-quality control. Fold-Change (2^(− Delta Delta Ct)) is the normalized gene expression (2^(− Delta Ct)) in HT29/Tun or HT29/MDR cells, divided the normalized gene expression (2^(− Delta Ct)) in HT29 cells (n = 4), where Ct is the threshold cycle in qRT-PCR. Fold-change values greater than 1 indicate up-regulation, fold-change values less than 1 indicate down-regulation. The p values are calculated based on a Student’s t-test of the replicate 2^(− Delta Ct) values for each gene. p < 0.05 was considered significant. Genes significantly up- or down-regulated more than 2-fold either in HT29/Tun or HT29/MDR cells are in bold characters. (XLSX 16 kb)
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Additional file 7: MRP1 expression on cell surface of sensitive and resistant colon cancer cells upon PERK/Nrf2 inhibition. a, b. Representative flow cytometry histograms of MRP1 protein in human chemoresistant colon cancer HT29/MDR cells and ER stress-resistant clones (HT29/Tg, HT29/Tun, HT29/Bfa), stably and inducibly transduced with a silencing vector for PERK, or treated with PD98059 (10 μM, 72 h), which blocks Nrf2 nuclear translocation. HT29 were included as control of chemosensitive/ER stress-sensitive cells. Grey peaks: non immune isotypic antibody. (TIF 1205 kb)
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Additional file 8: Expression of PERK- and Nrf2-upstream and downstream genes in chemoresistant and ER stress-resistant cells. a. Schematic representation of upstream and downstream targets of PERK and Nrf2. Blue box: genes upstream PERK; red boxes: genes downstream PERK; orange boxes: genes upstream Nrf2; green boxes: genes downstream Nrf2. b. Relative expression, indicated in a colorimetric scale, of the indicated PERK- and Nrf2-upstream and downstream genes, in HT29/MDR and HT29/Tun cells, grown un fresh medium (Ctrl), transduced with a silencing vector for PERK, or treated with PD98059 (PD; 10 μM, 72 h), measured by RT-PCR. The expression of each gene in HT29 cells, used as internal control, was considered equal to 1. Data are mean ± SD (n = 4). *p < 0.05 for untreated (Ctrl) HT29/MDR or HT29/Tun cells vs. HT29 cells; °p < 0.05 for significantly reduced genes in siPERK- and PD-treated cells vs. respective untreated (Ctrl) HT29/MDR or HT29/Tun cells. GRP78: glucose-regulated protein 78; EIF2S1: eukaryotic translation initiation factor 2 subunit 1; ATF4: activating transcription factor 4; GSK3β: glycogen synthase kinase 3β; JNK1: c-Jun N-terminal kinase 1; MAPK1: mitogen activated kinase 1; PI3KCD: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit δ; PRKCA: protein kinase Cα; NFKB: nuclear factor-kB; GSR: glutathione-disulfide reductase; G6PD: glucose-6-phosphate dehydrogenase; TXNRD1: thioredoxin reductase 1; SOD1: superoxide dismutase 1; HMOX1: heme oxygenase 1; NQO1: NAD(P)H quinone dehydrogenase 1; STIP1: stress induced phosphoprotein 1. (TIF 836 kb)
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Additional file 9: Nrf2 inhibition reverses the resistance to chemotherapy and ER stress. Human chemoresistant colon cancer HT29/MDR cells and ER stress-resistant clones (HT29/Tg, HT29/Tun, HT29/Bfa) were grown in the absence or in the presence of PD98059 (10 μM, 72 h), which blocks Nrf2 nuclear translocation. a, b. Release of the necrosis marker HMGB1 to culture media of the indicated cells following incubation in fresh medium (Ctrl), or in media containing: thapsigargin (Tg), tunicamycin (Tun), brefeldin A (Bfa), oxaliplatin (oPt), 5-fluorouracil (5FU), doxorubicin (Dox), as indicated in Methods. Data are mean ± SD (n = 3). *p < 0.001 vs HT29 Ctrl cells; °p < 0.001 for HT29/MDR, HT29/Tg, HT29/Tun, HT29/Bfa vs HT29 cells; # p < 0.001 for PD98059-treated cells vs PD98059-untreated cells. c, d. Viability of cells measured by Neutral red staining. *p < 0.005 vs HT29 Ctrl cells; °p < 0.01 for HT29/MDR, HT29/Tg, HT29/Tun, HT29/Bfa vs HT29 cells; # p < 0.02 for PD98059-treated cells vs PD98059-untreated cells. (TIF 3494 kb)
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Additional file 10: Correlation between PERK or NRF2 expression and patient clinical outcome in different tumors. Patient overall survival was calculated by Cox's proportional hazard model and Kaplan-Meier method using the GSEA software. Z score: correlation score between gene expression and survival. FDR: false discovery rate. ACC: adrenocortical carcinoma; BRCA: breast invasive carcinoma; BLCA: bladder urothelial carcinoma; CESC: cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: cholangiocarcinoma; COAD: colon adenocarcinoma; COADREAD: colorectal adenocarcinoma; DLBC: diffuse large B-cell lymphoma; GBM: glioblastoma multiforme; HNSC: head and neck squamous cell carcinoma; KICH: chromophobe renal cell carcinoma; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; LGG: lower grade glioma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; MESO: mesothelioma; OV: ovarian serous cystadenocarcinoma; PAAD: pancreatic adenocarcinoma; PCPG: pheochromocytoma and paraganglioma; PRAD: prostate adenocarcinoma; READ: rectum adenocarcinoma; SARC: sarcoma; SKCM: skin cutaneous melanoma; TGCT: testicular germ cell tumor; THCA: thyroid carcinoma; THYM: thymoma; UCEC: uterine corpus endometrial carcinoma; UCS: uterine carcinosacoma; UVM: uveal melanoma. Significant p values are in bold characters. (XLSX 17 kb)
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Additional file 12: Migration of sensitive and resistant colon cancer cells. Migration ability of HT29, HT29/MDR and HT29/Tun cells was evaluated as capacity to close the wound over a period of 24 h. a. Representative images of 1 out of 3 experiments. Bar: 200 μm. b. Quantification of migration rate. Data are mean ± SD (n = 3). *p < 0.002 vs HT29 cells; °p < 0.001 vs HT29/MDR cells. (TIF 2275 kb)
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