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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 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)
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)
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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