ER stress-mediated autophagy promotes Myc-dependent transformation and tumor growth

The proto-oncogene c-Myc paradoxically activates both proliferation and apoptosis. In the pathogenic state, c-Myc-induced apoptosis is bypassed via a critical, yet poorly understood escape mechanism that promotes cellular transformation and tumorigenesis. The accumulation of unfolded proteins in the ER initiates a cellular stress program termed the unfolded protein response (UPR) to support cell survival. Analysis of spontaneous mouse and human lymphomas demonstrated significantly higher levels of UPR activation compared with normal tissues. Using multiple genetic models, we demonstrated that c-Myc and N-Myc activated the PERK/eIF2α/ATF4 arm of the UPR, leading to increased cell survival via the induction of cytoprotective autophagy. Inhibition of PERK significantly reduced Myc-induced autophagy, colony formation, and tumor formation. Moreover, pharmacologic or genetic inhibition of autophagy resulted in increased Myc-dependent apoptosis. Mechanistically, we demonstrated an important link between Myc-dependent increases in protein synthesis and UPR activation. Specifically, by employing a mouse minute (L24+/-) mutant, which resulted in wild-type levels of protein synthesis and attenuation of Myc-induced lymphomagenesis, we showed that Myc-induced UPR activation was reversed. Our findings establish a role for UPR as an enhancer of c-Myc-induced transformation and suggest that UPR inhibition may be particularly effective against malignancies characterized by c-Myc overexpression.

Activation of AMP-activated protein kinase inhibits oxidized LDL-triggered endoplasmic reticulum stress in vivo

The oxidation of LDLs is considered a key step in the development of atherosclerosis. How LDL oxidation contributes to atherosclerosis remains poorly defined. Here we report that oxidized and glycated LDL (HOG-LDL) causes aberrant endoplasmic reticulum (ER) stress and that the AMP-activated protein kinase (AMPK) suppressed HOG-LDL-triggered ER stress in vivo.

Survival or death: a dual role of autophagy in stress-induced pericyte loss in diabetic retinopathy

Aims/hypothesis
Intra-retinal extravasation and modification of LDL have been implicated in diabetic retinopathy: autophagy may mediate these effects.
Methods
Immunohistochemistry was used to detect autophagy marker LC3B in human and murine diabetic and non-diabetic retinas. Cultured human retinal capillary pericytes (HRCPs) were treated with in vitro-modified heavily-oxidised glycated LDL (HOG-LDL) vs native LDL (N-LDL) with or without autophagy modulators: green fluorescent protein–LC3 transfection; small interfering RNAs against Beclin-1, c-Jun NH(2)-terminal kinase (JNK) and C/EBP-homologous protein (CHOP); autophagy inhibitor 3-MA (5 mmol/l) and/or caspase inhibitor Z-VAD-fmk (100 μmol/l). Autophagy, cell viability, oxidative stress, endoplasmic reticulum stress, JNK activation, apoptosis and CHOP expression were assessed by western blots, CCK-8 assay and TUNEL assay. Finally, HOG-LDL vs N-LDL were injected intravitreally to STZ-induced diabetic vs control rats (yielding 50 and 200 mg protein/l intravitreal concentration) and, after 7 days, retinas were analysed for ER stress, autophagy and apoptosis.
Results
Intra-retinal autophagy (LC3B staining) was increased in diabetic vs non-diabetic humans and mice. In HRCPs, 50 mg/l HOG-LDL elicited autophagy without altering cell viability, and inhibition of autophagy decreased survival. At 100–200 mg/l, HOG-LDL caused significant cell death, and inhibition of either autophagy or apoptosis improved survival. Further, 25–200 mg/l HOG-LDL dose-dependently induced oxidative and ER stress. JNK activation was implicated in autophagy but not in apoptosis. In diabetic rat retina, 50 mg/l intravitreal HOG-LDL elicited autophagy and ER stress but not apoptosis; 200 mg/l elicited greater ER stress and apoptosis.
Conclusions
Autophagy has a dual role in diabetic retinopathy: under mild stress (50 mg/l HOG-LDL) it is protective; under more severe stress (200 mg/l HOG-LDL) it promotes cell death.

Effects of modified LDL and HDL on retinal pigment epithelial cells: a role in diabetic retinopathy?

Aims/hypothesis: Blood–retina barrier leakage in diabetes results in extravasation of plasma lipoproteins. Intra-retinal modified LDLs have been implicated in diabetic retinopathy (DR), but their effects on retinal pigment epithelial (RPE) cells and the added effects of extravasated modified HDLs are unknown.

Methods: In human retinas from individuals with and without diabetes and DR, immunohistochemistry was used to detect ApoB, ApoA1 and endoplasmic reticulum (ER) stress markers. In cell culture, human RPE cells were treated with native LDL (N-LDL) or heavily-oxidised glycated LDL (HOG-LDL) with or without pretreatment with native HDL (N-HDL) or heavilyoxidised glycated HDL (HOG-HDL). Cell viability, oxidative stress, ER stress, apoptosis and autophagy were assessed by Cell Counting Kit-8 assay, dichlorofluorescein assay, western blotting, immunofluorescence and TUNEL assay. In separate
experiments, RPE cells were treated with lipid oxidation products, 7-ketocholesterol (7-KC, 5–40 µmol/l) or 4-hydroxynonenal (4-HNE, 5–80 µmol/l), with or without pretreatment with N-HDL or HOG-HDL.

Results: ApoB, ApoA1 staining and RPE ER stress were increased in the presence of DR. HOG-LDL but not N-LDL significantly decreased RPE cell viability and increased reactive oxygen species generation, ER stress, apoptosis and autophagy. Similarly, 4-HNE and 7-KC decreased viability and induced ER stress. Pretreatment with N-HDL mitigated these effects, whereas HOG-HDL was less effective by most, but not all, measures.

Conclusions/interpretation: In DR, extravascular modified LDL may promote RPE injury through oxidative stress, ER stress, autophagy and apoptosis. N-HDL has protective effects, but HOG-HDL is less effective. Extravasation and modification of HDL may modulate the injurious effects of extravasated modified LDL on the retinal pigment epithelium.

Mechanisms of modified LDL-induced pericyte loss and retinal injury in diabetic retinopathy

Aims/hypothesis: In previous studies we have shown that extravasated, modified LDL is associated with pericyte loss, an early feature of diabetic retinopathy (DR). Here we sought to determine detailed mechanisms of this LDLinduced pericyte loss.

Methods: Human retinal capillary pericytes (HRCP) were exposed to ‘highly-oxidised glycated’ LDL (HOG-LDL) (a model of extravasated and modified LDL) and to 4-hydroxynonenal or 7-ketocholesterol (components of oxidised LDL), or to native LDL for 1 to 24 h with or without 1 h of pretreatment with inhibitors of the following: (1) the scavenger receptor (polyinosinic acid); (2) oxidative stress (N-acetyl cysteine); (3) endoplasmic reticulum (ER) stress (4-phenyl butyric acid); and (4) mitochondrial dysfunction (cyclosporin A). Oxidative stress, ER stress, mitochondrial dysfunction, apoptosis and autophagy were assessed using techniques including western blotting, immunofluorescence, RT-PCR, flow cytometry and TUNEL assay. To assess the relevance of the results in vivo, immunohistochemistry was used to detect the ER stress chaperon, 78 kDa glucose-regulated protein, and the ER sensor, activating transcription factor 6, in retinas from a mouse model of DR that mimics exposure of the retina to elevated glucose and elevated LDL levels, and in retinas from human participants with and without diabetes and DR.

Results: Compared with native LDL, HOG-LDL activated oxidative and ER stress in HRCP, resulting in mitochondrial dysfunction, apoptosis and autophagy. In a mouse model of diabetes and hyperlipidaemia (vs mouse models of either condition alone), retinal ER stress was enhanced. ER stress was also enhanced in diabetic human retina and correlated with the severity of DR.

Conclusions/interpretation: Cell culture, animal, and human data suggest that oxidative stress and ER stress are induced by modified LDL, and are implicated in pericyte loss in DR.

Antitumor alkyl-lysophospholipid analog edelfosine induces apoptosis in pancreatic cancer by targeting endoplasmic reticulum

Pancreatic cancer remains as one of the most deadly cancers, and responds poorly to current therapies. The prognosis is extremely poor, with a 5-year survival of less than 5%. Therefore, search for new effective therapeutic drugs is of pivotal need and urgency to improve treatment of this incurable malignancy. Synthetic alkyl-lysophospholipid analogs (ALPs) constitute a heterogeneous group of unnatural lipids that promote apoptosis in a wide variety of tumor cells. In this study, we found that the anticancer drug edelfosine was the most potent ALP in killing human pancreatic cancer cells, targeting endoplasmic reticulum (ER). Edelfosine was taken up in significant amounts by pancreatic cancer cells and induced caspase-and mitochondrial-mediated apoptosis. Pancreatic cancer cells show a prominent ER and edelfosine accumulated in this subcellular structure, inducing a potent ER stress response, with caspase-4, BAP31 and c-Jun NH 2-terminal kinase (JNK) activation, CHOP/GADD153 upregulation and phosphorylation of eukaryotic translation initiation factor 2 a-subunit that eventually led to cell death. Oral administration of edelfosine in xenograft mouse models of pancreatic cancer induced a significant regression in tumor growth and an increase in apoptotic index, as assessed by TUNEL assay and caspase-3 activation in the tumor sections. The ER stress-associated marker CHOP/GADD153 was visualized in the pancreatic tumor isolated from edelfosine-treated mice, indicating a strong in vivo ER stress response. These results suggest that edelfosine exerts its pro-apoptotic action in pancreatic cancer cells, both in vitro and in vivo, through its accumulation in the ER, which leads to ER stress and apoptosis. Thus, we propose that the ER could be a key target in pancreatic cancer, and edelfosine may constitute a prototype for the development of a new class of antitumor drugs targeting the ER. © 2012 Macmillan Publishers Limited All rights reserved.

Endoplasmic reticulum targeting in Ewing’s sarcoma by the alkylphospholipid analog edelfosine

Ewing's sarcoma (ES) is the second most common bone cancer in children and young people. Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) is the prototype of a family of synthetic antitumor compounds, collectively known as alkylphospholipid analogs (APLs). We have found that APLs ranked edelfosine>perifosine>erucylphosphocholine>miltefosine for their capacity to promote apoptosis in ES cells. Edelfosine accumulated in the endoplasmic reticulum (ER) and triggered an ER stress response that eventually led to caspase-dependent apoptosis in ES cells. This apoptotic response involved mitochondrial-mediated processes, with cytochrome c release, caspase-9 activation and generation of reactive oxygen species. Edelfosine-induced apoptosis was also dependent on sustained c-Jun NH2-terminal kinase activation. Oral administration of edelfosine showed a potent in vivo antitumor activity in an ES xenograft animal model. Histochemical staining gave evidence for ER stress response and apoptosis in the ES tumors isolated from edelfosine-treated mice. Edelfosine showed a preferential action on ES tumor cells as compared to non-transformed osteoblasts, and appeared to be well suited for combination therapy regimens. These results demonstrate in vitro and in vivo antitumor activity of edelfosine against ES cells that is mediated by caspase activation and ER stress, and provide the proof of concept for a putative edelfosine-and ER stress-mediated approach for ES treatment.

Down-regulation of PERK enhances resistance to ionizing radiation

Although, ionizing radiation (IR) has been implicated to cause stress in endoplasmic reticulum (ER), how ER stress signaling and major ER stress sensors modulate cellular response to IR is unclear. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is an ER transmembrane protein which initiates unfolded protein response (UPR) or ER stress signaling when ER homeostasis is disturbed. Here, we report that down-regulation of PERK resulted in increased clonogenic survival, enhanced DNA repair and reduced apoptosis in irradiated cancer cells. Our study demonstrated that PERK has a role in sensitizing cancer cells to IR. 

UAP1 is overexpressed in prostate cancer and is protective against inhibitors of N-linked glycosylation

Prostate cancer is the second most common cause of cancer-associated deaths in men, and signaling via a transcription factor called androgen receptor (AR) is an important driver of the disease. Consequently, AR target genes are prominent candidates to be specific for prostate cancer and also important for the survival of the cancer cells. Here we assess the levels of all hexosamine biosynthetic pathway (HBP) enzymes in 15 separate clinical gene expression data sets and identify the last enzyme in the pathway, UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1), to be highly overexpressed in prostate cancer. We analyzed 3261 prostate cancers on a tissue microarray and found that UAP1 staining correlates negatively with Gleason score (P=0.0039) and positively with high AR expression (P<0.0001). Cells with high UAP1 expression have 10-fold increased levels of the HBP end-product, UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is essential for N-linked glycosylation occurring in the endoplasmic reticulum (ER) and high UAP1 expression associates with resistance against inhibitors of N-linked glycosylation (tunicamycin and 2-deoxyglucose) but not with a general ER stress-inducing agent, the calcium ionophore A23187. Knockdown of UAP1 expression re-sensitized cells towards inhibitors of N-linked glycosylation, as measured by proliferation and activation of ER stress markers. Taken together, we have identified an enzyme, UAP1, which is highly overexpressed in prostate cancer and protects cancer cells from ER stress conferring a growth advantage.

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress-the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor thapsigargin (TG)-potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.

Inhibition of Secretion of Interleukin (IL)-12/IL-23 Family Cytokines by 4-Trifluoromethyl-celecoxib Is Coupled to Degradation via the Endoplasmic Reticulum Stress Protein HERP

Interleukin-12 (IL-12), p80, and IL-23 are structurally related cytokines sharing a p40 subunit. We have recently demonstrated that celecoxib and its COX-2-independent analogue 4-trifluoromethyl-celecoxib (TFM-C) inhibit secretion but not transcription of IL-12 (p35/p40) and p80 (p40/p40). This is associated with a mechanism involving altered cytokine-chaperone interaction in the endoplasmic reticulum (ER). In the present study, we found that celecoxib and TFM-C also block secretion of IL-23 (p40/p19 heterodimers). Given the putative ER-centric mode of these compounds, we performed a comprehensive RTPCR analysis of 23 ER-resident chaperones/foldases and associated co-factors. This revealed that TFM-C induced 1.5-3-fold transcriptional up-regulation of calreticulin, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29. However, more significantly, a 7-fold up-regulation of homocysteine-inducible ER protein (HERP) was observed. HERP is part of a high molecular mass protein complex involved in ER-associated protein degradation (ERAD). Using co-immunoprecipitation assays, we show that TFM-C induces protein interaction of p80 and IL-23 with HERP. Both HERP siRNA knockdown and HERP overexpression coupled to cycloheximide chase assays revealed that HERP is necessary for degradation of intracellularly retained p80 by TFM-C. Thus, our data suggest that targeting cytokine folding in the ER by small molecule drugs could be therapeutically exploited to alleviate in appropriate inflammation in autoimmune conditions.