J-Perk v5.01 serial key or number

J-Perk v5.01 serial key or number

J-Perk v5.01 serial key or number

J-Perk v5.01 serial key or number

PMC

Alvaro Avivar-Valderas

1Department of Medicine and Department of Otolaryngology, Tisch Cancer Institute, Black Family Stem Cell Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029

Eduardo Salas

2University of California San Francisco, Department of Pathology, and Helen Diller Family Comprehensive Cancer Center, San Francisco, California 94143

Ekaterina Bobrovnikova-Marjon

3Department of Cancer Biology and Abramson Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

J. Alan Diehl

3Department of Cancer Biology and Abramson Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Chandandeep Nagi

1Department of Medicine and Department of Otolaryngology, Tisch Cancer Institute, Black Family Stem Cell Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029

Jayanta Debnath

2University of California San Francisco, Department of Pathology, and Helen Diller Family Comprehensive Cancer Center, San Francisco, California 94143

Julio A. Aguirre-Ghiso

1Department of Medicine and Department of Otolaryngology, Tisch Cancer Institute, Black Family Stem Cell Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029

1Department of Medicine and Department of Otolaryngology, Tisch Cancer Institute, Black Family Stem Cell Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029
2University of California San Francisco, Department of Pathology, and Helen Diller Family Comprehensive Cancer Center, San Francisco, California 94143
3Department of Cancer Biology and Abramson Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
*Corresponding author. Mailing address: Department of Medicine and Department of Otolaryngology, Tisch Cancer Institute, Black Family Stem Cell Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Phone: (212) 241-9582. Fax: (212) 241-4096. E-mail: ude.mssm@osihg-erriuga.oiluj.
Received 2011 Feb 3; Revisions requested 2011 Mar 11; Accepted 2011 Jun 17.
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
This article has been cited by other articles in PMC.

Abstract

Mammary epithelial cells (MECs) detached from the extracellular matrix (ECM) produce deleterious reactive oxygen species (ROS) and induce autophagy to survive. The coordination of such opposing responses likely dictates whether epithelial cells survive ECM detachment or undergo anoikis. Here, we demonstrate that the endoplasmic reticulum kinase PERK facilitates survival of ECM-detached cells by concomitantly promoting autophagy, ATP production, and an antioxidant response. Loss-of-function studies show that ECM detachment activates a canonical PERK-eukaryotic translation initiation factor 2α (eIF2α)-ATF4-CHOP pathway that coordinately induces the autophagy regulators ATG6 and ATG8, sustains ATP levels, and reduces ROS levels to delay anoikis. Inducible activation of an Fv2E-ΔNPERK chimera by persistent activation of autophagy and reduction of ROS results in lumen-filled mammary epithelial acini. Finally, luminal P-PERK and LC3 levels are reduced in PERK-deficient mammary glands, whereas they are increased in human breast ductal carcinoma in situ (DCIS) versus normal breast tissues. We propose that the normal proautophagic and antioxidant PERK functions may be hijacked to promote the survival of ECM-detached tumor cells in DCIS lesions.

INTRODUCTION

The attachment of mammary epithelial cells (MECs) to the extracellular matrix (ECM) critically regulates survival, and upon ECM detachment they rapidly undergo cell death, commonly termed anoikis (11, 17). Studies of lumen formation in three-dimensional culture and of ductal elongation during mammary gland development both support a key role for anoikis in luminal clearance (10). Furthermore, anoikis resistance is proposed to promote filling of the normally hollow lumen in glandular epithelium, a hallmark of early breast cancers, such as ductal carcinomas in situ (DCIS) (11). Thus, there is significant interest in identifying the signals that induce anoikis resistance, as this may uncover new therapeutic targets that can be exploited to kill ECM-detached tumor cells.

Although the rapid activation of apoptotic pathways was originally proposed to drive anoikis, a growing body of evidence indicates that ECM-detached MECs simultaneously activate diverse cellular pathways that both positively and negatively impact anoikis (17). For example, recent work indicates that ECM-detached MECs show a rapid decrease in glucose intake, which correlates with a drop in ATP levels as well as the progressive accumulation of reactive oxygen species (ROS) (40). Thus, in order to survive the stresses of ECM detachment, epithelial cells must adapt to the concomitant reduction in energy levels and the progressive ROS-induced damage. Autophagy, a tightly regulated cellular self-digestion process, has emerged as one important adaptive mechanism that promotes cell survival during ECM detachment (16). Nonetheless, the pathways that allow detached cells to coordinate autophagy and oxidative stress relief, and thus promote the survival of ECM-detached cells, remain poorly understood.

Recently, we demonstrated that ECM-detached MECs robustly activate the endoplasmic reticulum (ER) kinase PERK (41). PERK is best studied during the accumulation of misfolded proteins in ER lumen (19), where its main function is to attenuate translation initiation by phosphorylating eIF2α at Ser51 (38). This in turn triggers a selective translation and transcription program that induces reversible growth arrest and allows cells to cope with ER stress, primarily by inducing an antioxidant response (8). Notably, PERK signaling has also been shown to induce autophagy as a survival pathway in response to several cellular insults, such as hypoxia, nutrient deprivation, or radiation (24, 33, 37, 39). Thus, a dual function is rendered by PERK, where transient growth arrest is coordinated with robust survival (2, 3, 14). This dual function is illustrated by pleiotropic roles of PERK during tumor progression. PERK can, via its growth-suppressive function, prevent or delay tumor formation (4, 41). However, when established tumors successfully bypass this growth restriction, cells take advantage of PERK activation to cope with ER stress and hypoxia present in the tumor mass (3). Accordingly, in ErbB2+ mammary epithelium, the PERK antioxidant response favors tumor development (4).

Our recent work demonstrated that the activation of the canonical PERK-eukaryotic translation initiation factor 2α (eIF2α)-ATF4-CHOP pathway contributed to cell cycle withdrawal but did not promote anoikis (41). The aforementioned studies describing dual functions for PERK in growth arrest and cell survival motivated us to more closely dissect whether PERK mediated similar functions in ECM-detached cells. Here, we demonstrate that PERK activation in ECM-detached cells is responsible for coordinating both autophagy induction and oxidative stress relief, all of which are dependent on a canonical eIF2α-ATF4-CHOP pathway. In three-dimensional (3D) culture, the enforced activation of PERK results in the aberrant accumulation of cells that resist anoikis in the luminal space, indicating that this pathway must be turned off for proper luminal clearance. The tissue-specific deletion of PERK in the mouse mammary gland tissue results in reduced autophagy and increased apoptosis, whereas both PERK phosphorylation and autophagy are concomitantly increased in human DCIS. Based on these results, we propose that the proautophagic and antioxidant functions of PERK that operate during normal mammary acinus development are subverted in breast tumor cells for them to survive oxidative stress and resist anoikis.

MATERIALS AND METHODS

Cells lines and culture conditions.

Low-passage MCF10A cells were cultured as described previously (12). For Fv2E-PERK stable cell lines, cells were treated with 100 pM AP20187 (AP) (added daily) or an equal volume of ethanol (vehicle) as a control. For anoikis assays, MCF10A cells were collected as described previously (41). After quantification, 4 × 105/ml of MCF10A cells were kept in ultra-low-attachment plates (Corning) with the appropriate growth medium at the indicated time and/or treated or transfected. Wild-type and PERK-knockout (KO) mouse embryo fibroblasts (MEFs) were kindly provided by David Ron (Cambridge University) and grown as described previously (19). For 3D cultures, cells were plated in commercially available growth factor-reduced Matrigel (Mgel) (BD Biosciences, San Diego, CA) and grown as described previously (12). For treatments and transfections during morphogenesis, 100 nM PERK or ATF4 small interfering RNA (siRNA) was supplemented every 24 h. To quantify cell viability, detached cells were washed with phosphate-buffered saline (PBS), disaggregated, and collected as a single-cell suspension using a cell-strained cap (BD Falcon) to be incubated in 1:2 trypan blue stain (BioWhittaker). Total and nonviable cells were manually determined using a counting chamber.

Reagents and plasmids.

pBABEpuro-PERKΔC (lacking the PERK kinase domain) and pBABEpuro-Fv2E-PERK (where the modified FKBP [Fv] domain is fused to the PERK cytoplasmic kinase domain) were previously described (31). The pBABEpuro-β-galactosidase and -PERK-(K618A) constructs were described previously (41). The pCAG-EGFP-LC3 plasmid was a kind gift from Zhenyu Yue (Mount Sinai School of Medicine, New York, NY). AP20187 was from ARIAD Pharmaceuticals, Cambridge, MA, and salubrinal was from Calbiochem. When indicated, 10 nM bafilomycin A (BafA) (Sigma) was added to cells 1 h prior to lysis. Chloroquine (CQ) (Sigma) was used to block autophagosome maturation. Quantification of cellular ROS was performed using 2,7-dichlorofluorescein diacetate (DCF-DA) (Invitrogen). Ethidium bromide (EtBr) (Sigma) staining of 3D cultures was performed as described previously (12).

IB.

MCF10A and MEFs were lysed, and protein was analyzed by immunoblotting (IB) as described previously (36). Membranes were blotted using antibodies against the following: P-PERK (Thr981), PERK, CHOP, and ATF4 (Santa Cruz Biotechnology); GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and BimEL (Calbiochem); LC3 (Axxora, LLC) and BiP/Grp78 (BD Biosciences); cleaved caspase-3 (Asp175), eIF2α, P-eIF2α (Ser51), p21Cip1, p27KIP, P-GCN2 (Thr898), and GCN2 (Cell Signaling Technology); laminin 5 (Chemicon International); and p62 (Progen). Bound antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and chemiluminescence assays as described previously (41).

Real-time PCR primers.

RNA was isolated from cells with TRIzol reagent following the manufacturer's instructions (Invitrogen). Reverse transcription was performed using Moloney murine leukemia virus (M-MuLV) reverse transcriptase (New England BioLabs). Real-time PCR was performed on a DNA Engine Opticon system using Power SYBR green PCR master mix. The human forward and reverse primer sequences used were as follows: ATG8, 5′-CATGAGCGAGTTGGTCAAGA-3′ and 5′-GGTTCACCAGCAGGAAGAAG-3′; ATG5, 5′-ATCCTGCAGAAGAAAATGGA-3′ and 5′-ACAGGACGAAACAGCTTCTG-3′; ATG6, 5′-GCGAGACACGTTTTTGTCTT-3′ and 5′-TGGGTTTTGATGGAATAGGA-3′; ATG7, 5′-GAACATGGTGCTGGTTTCCT-3′ and 5′-CATCCAGGGTACTGGGCTAA-3′; PERK, 5′-AGAAAAACTCCAGCCCAGTT-3′ and 5′-TCGTCCATTCATCCAGTCTT-3′; Glyt1, 5′-CAGCCCCAGCGAGGAGTACT-3′ and 5′-GAGACACCGAGGCAGCCAAG-3′; Slc3a2, 5′-ATTGGCCTGGATGCAGCTGC-3′ and 5′-ACAGCCCCTGGGATGTCAGG-3′; GAPDH, 5′-CCCCTGGCCAAGGTCATCCA-3′ and 5′-ACAGCCTTGGCAGCGCCAGT-3′; and ATF4 5′-CACTAGGTACCGCCAGAAGAAGA-3′ and 5′-AATCCGCCCTCTCTTTTAGAG-3′.

RNA interference (RNAi) and cDNA transfections.

Cells were transiently transfected using Lipofectamine RNAiMax (Invitrogen) and FuGENE HD (Roche). DNA oligonucleotides encoding short hairpin RNA (shRNA) (the target sequence against ATG7 was CCC AGC TAT TGG AAC ACT GTA) were a kind gift from Jayanta Debnath (University of California, San Francisco). shRNA transfection was performed as described previously (16). Small interfering RNAs (siRNAs) were purchased from Ambion (CHOP, PERK, and silencer negative control), Dharmacon RNA Technologies (ATG7), or Invitrogen (ATF4). Transfections of plasmids and siRNA oligonucleotides were done using 3 μg DNA mixed with 6 μl of Fugene HD or 100 nM PERK, CHOP, or ATG7 siRNA oligonucleotides with 8 μl Lipofectamine RNAiMax and incubated for 24 and 48 h.

Fluorescence-activated cell sorting (FACS) analysis.

For determination of percent apoptosis, 20,000 events were collected and the sub-G0 fraction was measured using propidium iodide staining (BD Pharmingen). The sub-G0 cell population was gated as the apoptotic fraction. Nonstained cells were used as negative control. For determination of percent ROS, cells were incubated in PBS containing 2 μM DCF-DA for 15 min, and then DCF-DA-positive cells were gated using nonstaining cells as a negative control.

Immunofluorescence and GFP-LC3 analysis and image acquisition.

Alexa-Fluor- and rhodamine-conjugated secondary antibodies (Molecular Probes) were used for the 3D culture immunofluorescence assays. For detection of autophagosomes under adherent or suspension conditions, cells stably expressing green fluorescent protein (GFP)-LC3 MCF10A or transiently transfected cells were either grown on glass coverslips or collected by centrifugation for suspended cultures, allowed to attach on Poly-Prep slides (Sigma), and fixed with 3% paraformaldehyde following standard protocols (36). Images were captured using a Nikon Eclipse E600 microscope and an RT Slider SPOT digital camera (Diagnostic Instruments, Inc.). 3D-Matrigel MCF10A acinar structures were fixed at day 8, 10, 12, and 20 and processed for size measurement and immunofluorescence microscopy analysis as established previously (41). Confocal analyses were performed using a Leica SP5 multiphoton confocal microscope equipped with UV diode (405 nm), argon (458 nm, 476 nm, 488 nm, and 514 nm), HeNe (543 nm), and HeNe (633 nm) lasers. All images were taken using a magnification of ×63.

Animal tissues and immunohistochemistry.

Mammary gland epithelium tissues were obtained from control PERKloxP/loxP and mammary gland-specific PERK knockout (PERKΔ/Δ) mice generated as described previously (5). Immunohistochemistry on embedded paraffin sections was performed as described previously (41). The sections were processed using the VectaStain ABC Elite kit (Vector Laboratories, Burlingame, CA), and the signal was detected using the diaminobenzidine (DAB) substrate kit for peroxidase (Vector Laboratories, Burlingame, CA). P-PERK antibodies were from Santa Cruz, LC3 was from Axxora, and activated caspase-3 and P-GCN2 were from Cell Signaling.

Statistics.

Statistical analysis was performed using GraphPad Prism 5.0 software (San Diego, CA), and P values were calculated using one-way analysis of variance (ANOVA) followed by the Bonferroni multiple-comparison posttest or the unpaired t test, with a P value of <0.05 considered statistically significant.

RESULTS

PERK activation in ECM-detached cells is linked to autophagy induction.

We previously showed that PERK inhibits proliferation of ECM-bound MECs (41). However, we also discovered that PERK was strongly induced in suspended luminal cells (41), and this correlated with upregulation of CHOP. However, PERK inhibition via a PERKΔC mutant, while enhancing proliferation of ECM-attached MECs and inhibiting CHOP induction in luminal cells, did not affect the rate of luminal cell apoptosis (41). We reasoned that PERK and CHOP might have a different function in the luminal compartment. Because ECM detachment can induce autophagy (16) and PERK can activate autophagy (24, 27, 34), we first tested whether PERK activation in suspension was responsible for autophagy induction. The substratum detachment of MCF10A cells enhanced both PERK and eIF2α phosphorylation (Fig. 1A), which correlated with autophagosome formation, as evidenced by increased numbers of cells exhibiting punctate GFP-LC3 (Fig. 1A, right panel) and increased phosphatidylethanolamine (PE) lipidation of endogenous LC3 (LC3-II) (Fig. 1B). Restoration of cell-ECM interactions due to the addition of laminin-rich reconstituted basement membrane (5% Matrigel) resulted in PERK and eIF2α deactivation and the inhibition of detachment-induced autophagy (Fig. 1B). In parallel, we tested whether PERK activation was sufficient to induce autophagy, using MCF10A cells engineered to express a construct in which the Fv2E dimerization domain is fused to the cytoplasmic kinase domain of PERK (Fv2E-PERK); this system allows for controlled PERK activation in the absence of fully activated ER stress via dimerization with the divalent small molecule AP20187 (AP) (31). Upon AP addition, PERK was robustly activated, resulting in increased autophagosome formation (Fig. 1C); remarkably, at the concentration of AP used (100 pM), we did not observe apoptosis or G0-G1 arrest (Fig. 1D). Furthermore, the induction of eIF2α phosphorylation with salubrinal, a small-molecule inhibitor of the GADD34-PP1c complex, also resulted in increased punctate GFP-LC3 (Fig. 1C) but had no significant effect on apoptosis (data not shown).

PERK activation in ECM-detached cells is associated with autophagy induction. (A) Whole-cell lysates from MCF10A cells adhered (A) or suspended for 24 h (S) and immunoblotted (IB) with indicated antibodies (Abs). The graph shows the percentage of autophagic (autophagosome puncta staining) MCF10A GFP-LC3 adhered or 24 h suspended cells. (B) Adhered (A) or suspended (S) MCF10A cells were treated or not treated with 5% Matrigel (MGel), and LC3 processing was detected by IB. (C) Adhered Fv2E-ΔNPERK MCF10A cell lines were transfected with GFP-LC3 plasmid and treated or not treated (control) with 100 pM AP10287 (AP) (dimerizing molecule) for 24 h, or adhered GFP-CL3 MCF10A cells were treated or not treated (control) with 100 nM salubrinal; cells were fixed and the percentage of autophagic cells was scored and quantified using fluorescence microscopy. The right panels show representative images of autophagic Fv2E-ΔNPERK (+AP) and GFP-LC3 (+Sal) MCF10A cells. Blue, 4′,6′-diamidino-2-phenylindole (DAPI); green, GFP-LC3. Scale bars are 5 μm. (D) Adhered Fv2E-ΔNPERK MCF10A cell line transfected with GFP-LC3 plasmid and treated with 100 pM or 2 nM AP or with vehicle [Et(Oh)] for 24 h were stained with EtBr, and the percentage of apoptotic cells was further measured by FACS. Right graph, population doubling during exponential growth (from day 2 to 8) of Fv2E-ΔNPERK MCF10A cells treated with vehicle (ethanol) or 100 pM or 2 nM AP. Cells were collected, and viability was determined using trypan blue exclusion. n.s., not significant. (E) Whole-cell lysates from Fv2E-ΔNPERK MCF10A cells treated or not treated with 100 pM AP and in combination with 0.1 mM leupeptin and 20 mM NH4Cl as indicated were analyzed by IB for the indicated antigens. Leupeptin- and NH4Cl-mediated inhibition of lysosomal degradation resulted in LC3-II accumulation (lane 3). Densitometric analysis (bottom panel) for LC3 flux was determined using Image J software (n = 3). (F) MCF10A cells were transfected with 5 μg cDNA encoding GST-BHMT (glutathione S-transferase fused to betaine homocysteine S methyltransferase 1). After 24 h, the cells were cultured in full growth medium, serum-free medium (6 h), or medium with AP20185 (100 pM) in the presence or absence of 10 nM bafilomycin A1 (BafA). Whole-cell lysates were immunoblotted for the indicated antigens. Myc Ab was used to detect GFP-myc (expressed from an internal ribosome entry site [IRES] sequence) as a control for transfection efficiency.

We next evaluated the lysosomal turnover of PE-lipidated LC3 (LC3-II) upon PERK activation to verify autophagic flux. Upon inhibition of lysosomal function, using either leupeptin plus ammonium chloride (Leu/NH4Cl) or bafilomycin A (BafA), we observed enhanced LC3-II accumulation in AP-treated Fv2E-PERK MCF10A cells (Fig. 1D and E), thus corroborating that PERK activation promotes the lysosomal turnover of LC3-II. To further validate this result, we assessed the autophagy-dependent degradation of an exogenously provided cytoplasmic cargo protein (13). Fv2E-PERK cells were transfected with the macroautophagy reporter construct GST-BHMT (glutathione S-transferase fused to betaine homocysteine S methyltransferase 1) and treated with AP20187. Both serum withdrawal and AP-mediated activation of PERK promoted the lysosome-dependent cleavage of GST-BHMT (Fig. 1E). Chemical inhibition of autophagy with BafA, a vacuolar H+-ATPase inhibitor, reverted GST-BHMT cleavage. Overall, these results demonstrate that PERK activation is sufficient to mediate both the formation and lysosomal turnover of autophagosomes as well as to drive autophagic proteolysis. Furthermore, in ECM-detached cells, PERK phosphorylation strongly correlates with autophagy induction and with the adhesive state of MCF10A cells.

PERK and eIF2α are required for detachment-induced autophagy.

We subsequently assessed whether PERK activation is required for detachment-induced autophagy. As previously reported, the expression of a dominant-negative PERK mutant (PERKΔC, a kinase-dead C-terminal deletion mutant) (41) in MCF10A cells inhibited PERK autophosphorylation and eIF2α Ser51 phosphorylation during matrix detachment (Fig. 2A). Importantly, PERKΔC suspended cultures exhibited a 50% reduction in LC3-II compared to controls (β-galactosidase [β-Gal]). Thus, PERK kinase activity is required for LC3-I conversion to LC3-II (Fig. 2A). Accordingly, we also observed reduced LC3 lipidation in PERK-deficient mouse embryonic fibroblasts (Fig. 2B), suggesting that PERK regulation of autophagy is not limited to MCF10A cells. Moreover, siRNA-mediated depletion of PERK potently inhibited both eIF2α phosphorylation and detachment-induced autophagy in MCF10A cells; notably, the reduction in LC3-II was comparable to the inhibition of autophagy observed upon depletion of ATG7, a critical autophagy regulator (Fig. 2C). Thus, we conclude that both PERK kinase activity and the resultant eIF2α phosphorylation at Ser51 are at least partially responsible for detachment-induced autophagy.

PERK inhibition partially reverts suspension-induced autophagy. (A) Lysates from adhered (A) or 6 h suspended (S) β-Gal and PERKΔC MCF10A cells were analyzed by IB for the indicated antigens. Densitometric analysis for LC3 flux was done using Image J software (n = 3). (B) Lysates of PERK+/+ and PERK−/− mouse embryonic fibroblasts (MEFs) were collected at the indicated time points, and the indicated antigens were detected by IB. (C) MCF-10A lysates from attached (A) and suspended (S) cells that were transfected with ATG7, PERK, or control (C) siRNA were used for IB against the indicated antigens. Results for the ATG7 and PERK knockdown controls are shown in the right panel.

PERK-dependent induction of ATG genes in suspension is mediated by ATF4 and CHOP.

Phosphorylation of eIF2α, translation repression, and selective translation of the transcription factor (TF) ATF4 are central events in the unfolded-protein response (UPR) (20). ATF4 in turn induces the TF CHOP/GADD153 (1), a key mediator of the UPR. Notably, both ATF4 and CHOP were implicated as potential transcriptional mediators in autophagy (32, 37). Thus, we hypothesized that these PERK/eIF2α-dependent transcriptional pathways may contribute to autophagy. In support of this, RNAi-mediated depletion of PERK, ATF4, and CHOP prevented suspension-induced LC3-II formation (Fig. 3A). Furthermore, expression of the PERKΔC mutant strongly reduced the mRNA levels of ATG8/LC3 and ATG5 under attached conditions (Fig. 3B). In addition, upon 6 h of detachment, MCF10A cells expressing the PERKΔC mutant or a kinase-dead K618A-PERK mutant showed an impaired ability to induce ATG6/Beclin-1 mRNA compared to controls (Fig. 3C). A similar inhibition of ATG6/Beclin-1 mRNA induction was observed in MCF10A cells expressing an eIF2α S51A mutant (Fig. 3C), as well as during ATF4 and PERK knockdown (Fig. 3D). Notably, Fv2E-ΔNPERK activation with AP was also able to drive transcriptional upregulation of ATF4, CHOP, and ATG6/Beclin1 under adhered conditions (Fig. 3E). These data suggest that in addition to ATG8/LC3 processing, PERK and eIF2α phosphorylation result in upregulation of ATF4 and CHOP (41), which contributes to the transcription of ATG6 mRNA as well as LC3 processing in suspended cells, respectively.

Phosphorylation of PERK and eIF2α at Ser51 is required for efficient induction of ATG genes during suspension. (A) Lysates from MCF10A cells were suspended for 6 h (S) and transfected with PERK 1, PERK 2, ATF4, CHOP, and scRNA (C) siRNAs and immunoblotted for the indicated antigens. The graphs below the blots show densitometric analysis for LC3-II/LC3-I ratios as quantified using Image J software (n = 3). (B to E) Graphs showing quantitative PCR (qPCR) analysis of ATG8/LC3 and ATG5 mRNA transcript levels from β-Gal and PERKΔC MCF10A cells (B), Beclin1/ATG6 mRNA transcript levels from β-Gal, PERKΔC, PERK kinase-dead mutant K618A (K618A), and S51A-eIF2α mutant (S51A) suspended MCF10A cultures. (C) The inset shows a control IB of MCF10A cells stably expressing pBabepuro-HA-eIF2αS51A mutant or the empty vector (EV), which were immunoblotted with hemagglutinin (HA) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) Abs. (D and E) Beclin1/ATG6 mRNA transcript levels in adhered and suspended cultures transfected with PERK, ATF4, and control (C) siRNAs (D) and CHOP, ATF4, and Beclin1/ATG6 mRNA transcripts levels of Fv2E-PERK with (+) or without (−) 100 pM AP for 24 h (E). GAPDH mRNA was used for normalization.

Enforced activation of PERK induces luminal space filling.

To gain greater insight into PERK control of autophagy, we monitored the kinetics of PERK activation during MCF10A acinus morphogenesis. Both the total protein levels and phosphorylation of PERK became detectable at day 4 of 3D culture, peaked at day 6, and then significantly diminished at day 8 and thereafter; importantly, these changes were associated with increased phosphorylation of eIF2α at Ser51 (Fig. 4A). Furthermore, at these time points, we did not detect any changes in the PERK mRNA levels (Fig. 4A) or observe any changes in other ER-resident protein such as Bip/Grp78 (data not shown). We did observe a slight decrease in GCN2 and calnexin protein levels at later time points of acinus morphogenesis (Fig. 4A, right panel). The timing of PERK activation and upregulation coincided with luminal clearance and with Beclin1/ATG6 induction, which was strongly induced at day 4 of morphogenesis (Fig. 4A, upper right panel). ECM-detached luminal cells (arrow) strongly induced autophagy as measured by LC3-GFP imaging in 3D cultures (Fig. 4A, lower right panel). We also found that ECM-bound acinar basal cells (arrowheads) also had basal autophagy (Fig. 4A, lower right panel). It is possible that this is basal autophagy or that those cells are in the process of detachment. Next, we tested the consequence of enforced PERK activation during acinus morphogenesis using the Fv2E-ΔNPERK MCF10A system to sustain PERK activation beyond day 8. Fv2E-ΔNPERK activation with 100 pM AP resulted in a significant increase in overall acinus cell numbers and filling of the luminal space (Fig. 4B). However, this did not significantly enhance acinus size (Fig. 4B, upper right panel) or profoundly disrupt the spherical architecture of individual acini (data not shown).

Unscheduled activation of PERK induces luminal space filling. (A) Whole-cell lysates from 2- to 12-day-old MCF10A acini were immunoblotted with the indicated Abs. The upper right panel shows PERK and Beclin1/ATG6 mRNA transcript levels normalized to GAPDH mRNA from 3D Matrigel acini collected at the indicated time points. Confocal equatorial images from GFP-LC3 (the distribution of LC3-positive events is quantified in Fig. 5A) MCF10A acini fixed at day 8 show punctate basal (arrowheads) and luminal (arrow) LC3 staining (green). Blue, DAPI. Scale bars are 25 μm. (B) Confocal images of day 12 MCF10A Fv2E-ΔNPERK acini treated from day 6 to day 12 (+) or not treated (−) with 100 pM AP, showing stacked sections to reveal increased cellularity and compaction (upper panels) or equatorial confocal sections of acini (lower panels). The graph (upper right panel) shows the distribution and mean size of day 12 MCF10A Fv2E-ΔNPERK acini treated from day 6 (+) or not treated (−) with 100 pM AP. Size was calculated using SPOT software following the equation [(length × width2)/2 = acinus volume (mm3)] (n = 50). The total number of cells per acinus (lower right panel) was also calculated (n = 50). (C) Fv2E-ΔNPERK acini treated from day 6 to days 9 and 10 (+) or not treated (−) with 100 pM AP were stained with 1 μg/ml EtBr (day 10) or cleaved caspase-3 (Cl-c3) at day 9. Magnifications show intraluminal Cl-c3 staining in nontreated acini versus negative Cl-c3 staining in AP-treated acini. The percentage of apoptotic cells was scored (right graphs) (n = 40). (D) Confocal equatorial images from Fv2E-ΔNPERK MCF10A acini treated from day 6 to day 12 (+) or not treated (−) with 100 pM AP, showing intraluminal BimEL staining (red). The graph shows the distribution and mean number of intraluminal BimEL-positive cells per acinus. (E) Confocal equatorial images from Fv2E-ΔNPERK acini treated from day 6 to day 12 (+) or not treated (−) with 100 pM AP and transfected with ATF4 or control (scRNA) siRNA. The right graph show the distribution and mean number of intraluminal cells for each equatorial section of a single acinus (n = 20). Scale bar = 10 μm. The right blot shows controls for ATF4 knockdown in the 3D cultures. (F) Equatorial confocal section images of day 20 MCF10A Fv2E-ΔNPERK acini treated from day 6 to day 20 (+) or not treated (−) with 100 pM AP, showing intraluminal filling (left panels). Graphs (right panels) show the mean number and distribution of cells in the outer ring per section (upper panel) and the total number of intraluminal cells per section (lower panel) (n = 50). In all of the images blue shows DAPI staining of nuclear DNA, and unless otherwise indicated, scale bars indicate 25 μm. P values were determined by the t test.

Next, we tested whether PERK-induced luminal occupancy correlated with decreased apoptosis in luminal cells. Fv2E-ΔNPERK-mediated activation of PERK correlated with a consistent reduction of ethidium bromide (EtBr)-, cleaved-caspase-3- and BimEL-positive acinar luminal cells (Fig. 4C and D). PERK-induced survival was dependent on ATF4, because RNAi-mediated reduction of this TF restored the formation of hollow acini in the setting of AP-mediated PERK activation (Fig. 4E). This suggests that PERK-ATF4 signaling transiently inhibits anoikis, allowing for transient survival of luminal cells devoid of ECM attachment.

Following lumen formation, excess cells are produced due to low-level proliferation within acini that continue to be cleared via luminal apoptosis (11). To test whether PERK activation enhanced the survival of ECM-detached cells during these late stages of morphogenesis, we treated Fv2E-ΔNPERK MCF10A acini with AP after day 12, a time point when luminal clearance is complete (21). Following 8 days of AP treatment, these structures displayed high numbers of intraluminal cells compared to nontreated controls (Fig. 4F). Remarkably, we also observed a significant increase in the number of basal-layer cells (Fig. 4F, right panels). This was not due to increased luminal cell proliferation or general proliferation, because in 2D cultures 100 pM AP had no significant effect on cell proliferation (Fig. 1D). Furthermore, at day 20, when acini normally exhibit proliferation arrest, no phospho-RB-positive cells were detected in either control or AP-treated Fv2E-ΔNPERK MCF10A acini (data not shown), thus supporting the idea that enhanced proliferation does not contribute to increased acinus size (Fig. 4F). Importantly, recent work demonstrates that MECs in the basal compartment are motile; they can detach into the luminal space and subsequently reattach to the ECM (35). Based on our results, we speculate that MECs that survive and persist in the luminal space due to PERK activation may be repositioned to the basal layer. Overall, the persistent unscheduled activation of PERK promotes lumen occupancy and increased numbers of cells in the basal ECM-attached layer.

Autophagy contributes to luminal filling mediated by PERK activation.

Autophagy is a prosurvival mechanism that can transiently protect cells from ECM detachment, provided that they reattach in a timely manner (30). Thus, we tested whether PERK-induced autophagy contributes to its ability to promote the luminal cell survival mechanism during acinus morphogenesis. Using Fv2E-ΔNPERK MCF10A cells stably expressing GFP-LC3, we found that AP-mediated activation of Fv2E-ΔNPERK significantly enhanced punctate GFP-LC3 within cells (Fig. 5A). Notably, PERK-stimulated LC3 processing was confined to cells occupying the luminal space (Fig. 5A, middle panel). In addition to LC3 processing, Fv2E-ΔNPERK activation in the acini increased the mRNA expression of several ATG genes, such as ATG7/8 and ATG6/Beclin1 (Fig. 5A, right panel). Importantly, treatment with the autophagy inhibitor chloroquine (CQ), which prevents the fusion of autophagosomes and lysosomes (25), completely reversed PERK-mediated accumulation of luminal cells and filling of this compartment in the AP-treated acini (Fig. 5B). Furthermore, shRNA-mediated downregulation of ATG7, which was previously shown to mediate luminal cell survival (16) and is induced by PERK (Fig. 5A), also resulted also in the reversion of luminal cell accumulation mediated by PERK (Fig. 5C). These results support the idea that autophagy contributes to the accumulation of luminal cells observed upon PERK activation.

PERK promotes survival in the luminal compartment via autophagy. (A) Representative confocal images of day 8 Fv2E-ΔNPERK and GFP-LC3 MCF10A acini treated from day 6 to 8 (+) or or not treated (−) with 100 pM AP, showing intraluminal LC3-puncta staining within cells (green). Middle panel, percentage of intraluminal LC3-puncta staining cells (n = 50); right panel, graph showing ATG gene mRNA transcript levels (middle panel) normalized to GAPDH. (B and C) Confocal equatorial images from Fv2E-ΔNPERK acini treated from day 6 to day 12 (+) or or not treated (−) with 100 pM AP and/or 20 μM chloroquine (CQ) (B) or shRNA ATG7 or control empty vector (EV) (C). The lower graphs show the distribution and mean number of intraluminal cells for each equatorial section of a single acinus (n = 20). Scale bar = 10 μm. Right blots show controls for CQ inhibition of autophagy (B) and ATG7 knockdown (C) in the 3D cultures. In all of the images, blue indicates DAPI staining of nuclear DNA, and unless otherwise indicated, scale bars indicate 25 μm. P values were determined by the t test.

Luminal cell autophagy and survival induced by PERK is accompanied by oxidative stress relief.

Both suspension-induced phosphorylation of PERK and autophagosome formation were abrogated by treatment with the reducing agent N-acetyl-l-cysteine (NAC) (Fig. 6A and B), suggesting that ROS production was a trigger for PERK activation as well as autophagy. Recent publications indicate that competing ROS-induced-death-promoting (40) and autophagy-dependent survival (16) signals are simultaneously activated in luminal ECM-detached cells. Thus, we hypothesized that PERK activation in response to increased ROS may simultaneously coordinate autophagy induction and ROS detoxification in ECM-detached cells. ROS production upon ECM-detachment was reversed by adding 5% Matrigel (Mgel) to suspension cultures (Fig. 6C, left panel). Thus, the disruption of adhesion signaling is responsible for both ROS production and PERK activation. Accordingly, inhibition of the canonical PERK/eIF2α signaling pathway via expression of the PERKΔC or S51D-eIF2α mutant strongly increased ROS levels as measured by DCF-DA fluorescence in suspended MCF10A cells (Fig. 6C, right panel). Because ROS accumulation in ECM-detached MECs is associated with reduced ATP levels, we assessed whether PERK activity modulated ATP levels in suspended MECs (40). Upon siRNA-mediated knockdown of PERK, we uncovered a more precipitous drop in ATP levels than in control suspension cultures (Fig. 6D, left panel). In contrast, AP-mediated activation of PERK was able to partially maintain ATP levels in ECM-detached cells (Fig. 6D, right panel). Importantly, we found that ROS were detectable in both basal and centrally localized cells of control Fv2E-ΔNPERK 3D acinar structures (Fig. 6E). However, AP-mediated activation of PERK drastically reduced ROS production in both compartments (Fig. 6E). Overall, these results indicated that PERK/eIF2α signaling is important to reduce oxidative stress due to the loss of adhesion. In further support of this, two ATF4 target genes specifically implicated in ROS detoxification, Glyt1 and Slc3a2, were induced in AP Fv2E-PERK-treated acini (Fig. 6E, lower right panel). Expression of the mRNA of Slc3a2 peaked at day 6 of morphogenesis, when PERK activity peaks (Fig. 4A and ​and6F),6F), and remained high until day 12. Overall, these data support an antioxidant role for PERK during morphogenesis and strongly suggest that the ROS-detoxifying function of PERK→ATF4 signaling is induced concomitantly with autophagy in mammary acini.

PERK-eIF2α protects luminal cells from anoikis in part by relieving oxidative stress. (A) Whole-cell lysates from adhered (A) and 24-h-suspended (S) β-Gal and PERKΔC MCF10A cells were treated (+) or not treated (−) with 5 mM NAC and immunoblotted with the indicated Abs. GAPDH was used as a loading control. (B) Cell lysates from adhered (A) and 6-h-suspended (S) MCF10A cells were treated (+) or not treated (−) with 5 mM NAC and immunoblotted with the indicated Abs. Note that LC3 processing in suspension is inhibited by NAC treatment. Densitometric analysis (right panel) for LC3 flux was determined using Image J software (n = 3). (C) ROS levels of 6-h-suspended MCF10A cells cultured with (+) or without (−) 5% Matrigel and β-Gal. Levels for PERKΔC (ΔC) or eIF2α Ser51A (S/A) MCF10A cells were measured with DCF-DA and FACS. (D) ATP levels in adhered and 6-h-suspended MCF10A cells transfected with a PERK siRNA (+) or scrambled siRNA control (−) and in Fv2E-PERK MCF10A cells treated (+) or not treated (−) with 100 pM AP. (E) Confocal images from Fv2E-ΔNPERK MCF10A acini treated from day 6 to day 12 (+) or not treated (−) with 100 pM AP. At 15 min before confocal analysis, cells were treated with 2 μM DCF-DA. The lower panels show differential interference contrast (DIC) microscopy plus DCF-DA fluorescence. Upper right graph, quantification of DCF-DA-positive cells per equatorial section (n = 7). Lower right graph, qPCR analysis of Glyt1 and Slc3a2 mRNA transcript levels from Fv2E-ΔNPERK MCF10A acini treated from day 6 to day 12 (+) or not treated (−) with 100 pM AP and normalized to GAPDH mRNA. (F) qPCR analysis of Slc3a2 mRNA transcript levels from Fv2E-ΔNPERK MCF10A acini treated from day 6 to day 12 (+) or not treated (−) with 100 pM AP at the indicated time points. (G) β-Gal- and PERKΔC-MCF10A 6-h-suspended cultures grown in full or serum (HS)-free medium were analyzed for viability using the trypan blue (TB) exclusion test. (H) β-Gal, PERKΔC (ΔC), or K618A PERK MCF10A 6-h-suspended cultures were collected and assayed for plating efficiency. PERKΔC or K618A PERK mutants inhibited plating efficiency (left panel). Fv2E-ΔNPERK MCF10A 6-h-suspended cultures (right panel) treated with 100 pM AP (+) exhibited a higher plating efficiency than nontreated cultures (−). P values were determined by the t test.

Next, we investigated the effects of PERK on cell survival during ECM detachment. PERK inhibition using the PERKΔC mutant significantly reduced viability after 6 h in suspension (Fig. 6G), and this correlated with induction of BimEL and increased caspase-3 processing (data not shown). PERK-mediated regulation of BimEL was not associated with changes in ERK1/2 activation (data not shown). ATF4 RNAi also induced BimEL expression and caspase-3 activation in suspension, while Fv2E-PERK activation attenuated these effects (data not shown). Similarly, PERK inhibition using the ΔC and K618A mutants also resulted in reduced clonogenic plating efficiency of cells after 6 h in suspension (Fig. 6H). In contrast, Fv2E-ΔNPERK activation enhanced the plating efficiency of cells following detachment (Fig. 6H, right panel). Notably, these prosurvival functions of PERK during ECM detachment were temporally limited; beyond 8 h in suspension, both PERKΔC mutant and Fv2E-ΔNPERK activation showed no significant differences in BimEL levels and in plating efficiency versus β-Gal or inactive Fv2E-ΔNPERK controls (data not shown). We conclude that in ECM-detached MECs, the canonical PERK/eIF2α/ATF4 pathway promotes autophagy and ROS reduction, which transiently protects MECs lacking cell-ECM contact, provided that they reattach in a timely manner.

PERK promotes autophagy and survival in lactating mammary glands.

Collectively, our data suggest that PERK-induced autophagy, ROS detoxification, and survival of luminal MECs may be important during mammary morphogenesis. To test this possibility, we examined the expression levels of LC3 and BimEL using immunohistochemistry in mammary gland sections from lactating female mice (L12) that carried wild-type (wt) PERK (PERKloxP/loxP) or in which PERK was conditionally deleted (PERKΔ/Δ) in the mammary tissue. We focused our studies on this developmental stage because the expanded ducts of lactating mammary glands facilitate the detection of luminal cells undergoing anoikis, which would be difficult to detect in virgin tissues or masked by the massive wave apoptosis observed during involution. As expected, a strong P-PERK signal in wt PERK tissues was lost in PERKΔ/Δ mammary glands (Fig. 7A). Strikingly, we found that P-PERK levels were higher in both detached (Fig. 7A, middle row) and luminal (Fig. 7A, lower row) PERKloxP/loxP cells. Isotype controls demonstrated no noticeable immunohistochemical signal, supporting that the staining observed in Fig. 7 is specific (data available upon request). Phospho-GCN2 staining showed no difference between wt PERK and PERKΔ/Δ tissues, suggesting that there is no compensatory increase in GCN2 activation in response to PERK KO (data available upon request).

PERK promotes autophagy and survival in lactating mammary glands. Detection of P-PERK (A), LC3 (B), p62 (C), and BimEL and cleaved caspase-3 (D) in day 12 lactating mammary glands sections from control PERKloxP/loxP and mammary gland-specific PERK knockout (PERKΔ/Δ) mice was done by immunohistochemistry (A, B, and D) and immunofluorescence (C). (A) Images showing enhanced P-PERK staining in the control tissues along with strong staining in detaching (middle panels and insets) and luminal cells (arrow, lower panels), which is lost in PERKΔ/Δ tissues. (B) Images showing enhanced LC3 staining (upper panels and insets) and detachment-induced LC3 expression (lower panels) in PERKloxP/loxP control tissues, which is lost in PERKΔ/Δ tissues. (C) Images showing enhanced p62 punctate staining in PERKΔ/Δ versus control tissues. Insets show magnification to illustrate the accumulation of p62 in autophagosomes. Arrows show p62 punctate staining in both basal (arrowheads) and ECM-detached (arrow) cells in PERKΔ/Δ tissues. (D) Upper and middle rows, images showing enhanced BimEL staining in PERKΔ/Δ versus control tissues. Insets show magnification details. The middle panels show strong BimEL staining of PERKΔ/Δ detached cells located within the lumen (arrows). Lower panels show cleaved caspase-3 staining in PERKΔ/Δ and control tissues, which displayed more cellularity than the KO mammary epithelium. Numbers in the lower left corners are the means ± standard errors of the means (SEM) of the percentages of cleaved caspase-3-positive luminal cells per field; ∼1,000 total luminal cells were scored. Blue, hematoxylin and eosin (H&E) staining.

Importantly, the levels of LC3 were dramatically reduced in PERKΔ/Δ in comparison to PERKloxP/loxP tissues (Fig. 7B), suggesting that PERK deletion reduces the activation of autophagy in the mammary tissue. Paralleling P-PERK activation, LC3 levels were significantly enhanced in luminal PERKloxP/loxP cells (Fig. 7B, lower row); isotype-matched IgG controls showed no staining (data available upon request). In agreement with an impaired autophagic flux in PERKΔ/Δ mammary epithelium, we detected the strong punctate accumulation of p62/SQSTM1 (Fig. 7C), an established autophagy cargo receptor that accumulates as cytoplasmic bodies when autophagy is inhibited (26). In addition, we observed increased BimEL staining throughout the mammary epithelium in PERKΔ/Δ compared to PERKloxP/loxP tissues, which was most evident in luminal cells within PERKΔ/Δ ducts that were strongly stained for BimEL (Fig. 7D, upper and middle rows). This pattern suggested that PERK deletion is accompanied by increased cell death due to BimEL induction. Accordingly, PERKΔ/Δ tissues displayed significantly increased numbers of cleaved-caspase-3-positive luminal cells (Fig. 7D, lower rows). Collectively, these results support that PERK activation promotes autophagy as well as inhibits the induction of BimEL, which together promote the survival of MECs.

Enhanced PERK phosphorylation and LC3 expression in DCIS.

The PERK antioxidant response and autophagy were shown to be advantageous for tumors (4, 7). Because Fv2E-ΔNPERK activation caused luminal filling reminiscent of that observed during DCIS, we evaluated whether autophagy and PERK phosphorylation were differentially regulated in normal breast and DCIS tissues. We analyzed P-PERK and LC3 expression using immunohistochemistry in normal breast tissue and in DCIS and its benign adjacent tissue. Negative controls showed no staining (data available upon request). The P-PERK signal was low and homogeneous in normal breast epithelium or benign adjacent normal breast tissue (Fig. 8A and B), although it was upregulated in luminal cells (Fig. 8A, right panel). In contrast to the case for normal tissue, the P-PERK signal was highly increased and heterogeneous in DCIS tissue (Fig. 8C). The enhanced P-PERK signal was limited primarily to the epithelial tissue, and some regions of the DCIS lesions or individual cells showed dramatically increased P-PERK levels (Fig. 8C, right panel). The LC3 signal was also homogeneous in normal breast epithelium or benign adjacent normal breast tissue and, as predicted by our studies, it was found to be upregulated in luminal cells (Fig. 8D and E). In agreement with a previous report (15), DCIS tissue showed a clear enhancement in the overall LC3 signal, and this increased signal was more prominent in centrally located cells in the lesions (Fig. 8F). These data indicate that PERK activation and LC3 upregulation are concordant in normal, benign adjacent, and DCIS tissues; moreover, both of these signals are increased in DCIS tissues, with the highest levels of expression present in the centrally located cells of these preinvasive lesions.

PERK phosphorylation and LC3 expression in normal and DCIS tissues (A and F) and a model summarizing the findings (G). (A to C) Representative images of benign (A), benign adjacent (B), and DCIS (C) human mammary gland tissues embedded in paraffin blocks, sectioned, and stained with P-PERK Ab. The insets show detailed magnifications of lumens and detaching cells (arrow in panel C) with strong P-PERK. (D to F) Representative images of benign (D), benign adjacent (E), and DCIS (F) human mammary gland tissues embedded in paraffin blocks, sectioned, and stained with LC3 Ab. The insets show detailed magnifications of lumens and detaching cells (arrows in panel F) with enhanced LC3 staining. Blue, H&E staining. Total samples tested, n = 5; samples shown, n = 2. Patterns were similar in all 5 control or tumor tissues. (G) Model of PERK-induced survival during lumen formation. During mammary acinar development or tissue maintenance, MECs that become detached (A, green cells) activate a “survival license”-dependent PERK activation (B, pathway). Upon loss of adhesion, ROS accumulate due to low ATP production. PERK senses this signal, possibly due to misfolding on nascent proteins in the ER, and phosphorylates eIF2α. This in turn results in ATF4 and CHOP upregulation of autophagy genes such as ATG7 (B, autophagy), which promotes survival of suspended cells. Concomitantly, PERK, possibly through eIF2α→ATF4 or other transcription factors such as NRF2, induces genes that allow ROS detoxification via the upregulation of GSH (B, antioxidant response). Whether autophagy and antioxidant responses are interdependent is unclear at this time. During normal acinar development the “survival license” activated by PERK, which expires after 6 to 8 h, provides a window of opportunity for some luminal cells to resist anoikis, reattach, and contribute to the timely organization of a polarized acinus (C, hollow lumen). However, signals (e.g., oncogenes) that maintain activated PERK might hijack this “survival license” to make it permanent (D). This results in anoikis resistance and luminal occupancy, which might favor the progressive development of cancer lesions such as DCIS.

DISCUSSION

Here we demonstrate that activation of the ER kinase PERK promotes the survival of luminal MECs detached from the ECM. Two recent studies have shown that upon loss of adhesion, MECs activate survival via autophagy (16) but also accumulate lethal ROS levels in association with reduced ATP production (40). We now demonstrate that suspension-induced PERK coordinately contributes to the induction of autophagy, the maintenance of ATP production, and the stimulation of a ROS detoxification response. The last most likely allows for autophagy to proceed and protect cells until adhesion is restored. In a 3D morphogenesis model, the enforced activation of PERK results in the aberrant survival and accumulation of cells in the luminal space, indicating that this pathway must be turned off during morphogenesis for proper luminal clearance. The tissue-specific deletion of PERK in the mouse mammary gland tissue results in reduced autophagy and increased apoptosis, whereas both PERK phosphorylation and autophagy are concomitantly increased in human DCIS. Based on these results, we propose that the proautophagic and antioxidant functions of PERK that operate during normal mammary acinar development are subverted in breast tumor cells to survive oxidative stress and resist anoikis.

Autophagy was found to protect luminal MECs lacking ECM contact in an ATG5- and ATG7-dependent manner (16), but the precise signals triggering this prosurvival mechanism in ECM-detached cells remain largely unclear (9). Here we show that PERK is an upstream inducer of detachment-induced autophagy. Interestingly, our data implicate the canonical PERK-eIF2α signaling axis as an important transcriptional regulator of multiple autophagy regulators (ATGs) involved in the early steps of autophagosome formation, including ATG5, ATG6/Beclin-1, and ATG8 expression. Among these, ATG6/Beclin1 was the most responsive ATG gene induced by PERK and its downstream effectors. Because transcription of these ATG genes is likely not sufficient to drive autophagy, it is possible that these core components of the autophagy machinery are also subject to posttranscriptional control downstream of PERK activation. Nonetheless, in addition to the induction of these ATG transcripts, our results also demonstrate that PERK activation (using a control dimerization strategy to activate this ER kinase) is sufficient to promote bona fide autophagic degradation. Notably, PERK may directly or indirectly affect other pathways important in autophagy induction, such as the energy-sensing LKB1-AMPK pathway, which is activated when AMP levels increase and consequently leads to both mTOR inhibition (18) and autophagy activation (29). Further examination of this salient issue remains a important topic for future study.

In MCF10A 3D cultures, enforced PERK activation during the late stages of morphogenesis allowed viable MECs devoid of any ECM (i.e., surface bound laminin-5) to persistently occupy the luminal space. This phenotype was dependent on ATF4 and ATG7 and ultimately resulted in increased acinar basal cell numbers, suggesting that surviving MECs reattached to the ECM in the basal layer of cells. Luminal MEC survival was reversed by chloroquine, which blocks fusion of autophagosomes to lysosomes (25). Thus, drugs that interrupt PERK-induced autophagy might have antitumor effects. In agreement with this, chloroquine was shown to limit the survival of breast cancer cells (15). We conclude that PERK can protect against anoikis and that oncogenic signals that perpetuate PERK activation might promote unwanted MEC survival.

Based on these results, one can speculate that PERK ensures that a MEC inefficiently attached survives via autophagy until proper adhesion is restored. Importantly, we found that these functions of PERK appear to be operational in vivo. During lactation, mouse mammary epithelial tissue where PERK was conditionally deleted showed an absence of P-PERK signal that was associated with reduced autophagosomes (e.g., LC3 staining). In further support of this, we found that in normal reduction mammoplasty and benign adjacent human tissues, PERK activation was enhanced and LC3-associated autophagy was increased primarily in luminal cells, once again suggesting an anoikis resistance mechanism. Furthermore, in DCIS lesions, where anoikis resistance is thought to lead to luminal filling (11), strong PERK phosphorylation was associated with prominent LC3 staining. In agreement with our findings, a recent study demonstrated that autophagy is upregulated in DCIS lesions, but the link to PERK was not delineated (15).

In addition to the induction of autophagy, PERK senses the accumulation of ROS in suspended cells and induces an antioxidant response dependent on eIF2α phosphorylation (41). During ECM detachment, this response includes the induction of the glycine transporter Glyt1 and the x(c)-cystine/glutamate exchanger Slc3a2, which is required to transport cystine that upon reduction is converted into the glutathione (GSH) precursor cysteine (22, 23

Источник: [https://torrent-igruha.org/3551-portal.html]
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ItsPersonal 2.1h : Name: Phrozen Crew s/n: RKS-3802174
It's Personal v2.8 : Name: Versus s/n: RKS-2170130 or Name: n03l s/n: RKS-1125087
Its Personal v2.8b Win9XNT : Name: Blackstar/TRPS98 s/n: RKS-5122928
Its Personal v2.8e Win9xNT : Name: Blackstar/TRPS98 s/n: RKS-5122928
Its Personal v2.9d : Name: draXXter[Faith2000] s/n: RKS-6199757
It's Time v2.1d : Company: TheForceTeam Key: RKS-6378405
Its Time v2.5a : Name: RAGGER/CORE Key: RKS-6165365
It's Time v2.8 : Name: Versus Key: RKS-3509202 or Name: n03l Key: RKS-1819319
iTrack Enterprise v1.3d : Go to the System Settings form and enter: Code Part A: dg Code Part B: khj3h7cs
iTrack Enterprise v1.3e : Go to the System Settings form and enter: Code Part A: dg Code Part B: khj3h7cs
iTrack Enterprise v1.3f : Part A: dg Part B: khj3h7cs
iTrack Enterprise v1.3g : Part A: dg Part B: khj3h7cs
iTrack Enterprise v1.3h : Part A: dg Part B: khj3h7cs
iTrack Workgroup v1.3c : Code A: kw Code B: dsnchv47
IWare Connect : Key: GKY$YKYKYTSK s/n: 20696
IWare Internet Suite : Key: D38E92SE9R3E s/n: 25005
iWrite v1.0 : Name: Annette S. Alvis Company: (blank) s/n: grvj1980
Ixla Explorer v1.2 : RegCode: TKL and click "Buy Now"
I Do Windows v3.0 - Name - Finn Mac CooL - Serial - 1K102L101M119Q84N

i publish v2.0 - Serial - 0123-456-789-0404AL


IACS Feature Manager v6.00.02 - Serial - 12345678004220
IACS Report Manager v6.00.02 - Serial - 12345678004221
IACS Rules Manager v6.00.02 - Serial - 12345678004597
IACS Specification Manager v6.00.02 - Serial - 12345678004219
IACS Symbolization Manager v6.00.02.02 - Serial - 12345678004596
ICCD v3.x - Name - Finn Mac CooL - Serial - ICCD37679938074853
125% Service-Providers Marketing Software for Win95 : s/n: 14106-124-0145506
ImTOO MP4 Video Converter v2.1.59.0303b

Added: 2006-03-23

Rating - 86%, Yes - 13, No - 2

Name: www.serials.ws Code: 1299-3423-6429-2279-B724-408E-9005-C018


ImTOO 3GP Video Converter v2.1.54.918b-BRD
Name: www.serialnews.com
Serial: BRD-Y79YAOFQHPS6CTB-1683-3F18-A89D-22AF
J
Jetforms Central v5.1 for NT : s/n: 1100-10479-6
Jetforms Design v5.1 : s/n: 1100-10479-6
Jetforms Transformation Builder v5.1 : s/n: 1100-10479-6
JeWLinr 1.7 : name: ZiLLY s/n: Pszz7wYPO6
JewlLinr V2.0 : Name: EzD s/n: 7Ywa1wYPO6
Jewl Linr v2.3 : Name: davy - blizzard s/n: NvEJvP7Cy5
JewlLinr v2.4 : Name: Matter of Darkness s/n: 7vGG3z7NsL
jForge v2.6 : Key: steven anger Code: 6yCBtY89ox
JForge v2.61 : Key: steven anger Code: 6yCBtY89ox
JigSaw-It v1.0 for Windows : s/n: 7104401272
JigSoft v1.2g for Windows : s/n: 503-331
Job Track v5.4 : Key: house'
JobTrack v5.4e : Name: Azrael [PC] Code: house
JOC Downloader v1.2 beta : Name: MISTERE[INSIDE] s/n: 5798544489
JOC Email Checker v1.20 : Name: Dazzler s/n: 8501CR1737
JOC Web Promote v1.0 : Name: MISTERE[INSIDE] s/n: 9798544499
JOC Web Promote v1.20 : Name: Ringer s/n: 9774004827PSCO6299
JOC Web Promote v1.21 : Name: MISTERE[INSIDE] s/n: 9798544499
Joggler v1.04 : Name: Audrey Jones Address: Brooklyn, NY 11234 email: slidah@hotmail.com RegNum: C1F 5451C145C1F492477
Joggler v1.06 : Name: jog [DNG] Code: C1F 1A75145C1F378558
Joggler 1.06a : Name: Warp Code: C1F-809145C1F376050
Joker Wild Video Poker 2.6 : Name: fungus / blizzard Code: LLB6034
Joker Wild Video Poker v2.7 : Name: Warp Code: DJM0416
Joker Wild Video Poker v2.75 : Name: Warp Code: DJM0416
Jones in the fast lane : name: Gummy s/n: (leave blank)
Jot+ Notes v2.0.1 build 59 32bit : Name: Fully Licensed User s/n: 8937 Code: 19FH1GW0AQP
Jot+ Notes 2.11 Build 134 : Name: BaMa/MANiFEST s/n: 6666 Code: G621E5JM2N
Jot+ Notes v2.1.1 : Name: Norway/Revolt98 s/n: 1129 Key: 152A1BFJ14YI
Jot plus Notes v2.2.0 build 156 : Name: dustie of blizzard s/n: 1998 Code: N9Z7BN6V1
Joystick's Friend v1.0 : Name: L!M!T / The Exterminators s/n: 11114115763377
JPad v3.1.3 build 99a : User ID: 10000000#400101 KeyCode: 808565617
JPadPro v3.2 build 99a : User ID: 20000139#400101 KeyCode: 420631652
JPadPro v3.7.297 : Name: BaRT SiMPSoN s/n: 733253764
JPEG Optimizer v1.32 : Code: JO1638
Jpeg Optimizer v2.00 : Code: qo7012
JPEG Optimizer v2.02 : Code: kj7246
JPEG Optimizer v3.03 : Code: FY9281
JPEG Optimizer v3.06 : Code: RW3418
JPEG Optimizer v3.07 : Code: AY8124
JProbe Profiler v1.1 : s/n: 40011FS-M9-314086
JProbe Profiler v1.1.1 : s/n: 40011FS-M9-314086
JRun Pro v2.3 : s/n: _MuFFiN_[uCF]-99999999-99-JSM.UL-1-0-5f37-154d
JR Ware Complete v2.0 : s/n: 86911260
JSB Surf Control Scout PLus : Code: 0w1g001751lalxsaikh
JSB Surf Control Scout Proxy : Code: 0w1g001751lalxsaikh
JSB Surf Control Scout : Code: 0w1g001751lalxsaikh
JSB Surf Control Super Scout : Code: 0w1g001751lalxsaikh
JShop Professional v2.0 : s/n: 01184011631
JShop Professional v2.1a : Name: (Anything) s/n: 01184011631
JSMail v3.56.1556 : s/n: C07463U00T00F4DBB436001800009A0200000405
JS Office v1.2 Win95/NT : Name: FALLEN s/n: 666777666 Reg: 1BF5A492
JS Office v1.3 : Name: Versace Theking s/n: 11002855 Reg: 57A6B0D3
JS Office v1.4 : Name: EinZtein s/n: 9D3FE627 Reg: E0696352
JSource V1.1 Win95/NT : Code: Bark!-0001-0001-0002
jSQL v1.31 : Name: jman s/n: N1HM1S
Jumpers v1.1 : s/n: JUMP4986
Just Add OS/2 Warp : s/n: GDLIT224
just!Audio v1.0 : s/n: 01294-80802579s
Just! Audio v2.0 (build 1.92) : s/n: 01246-99014884b
Just between Friends v2.1 for Windows : s/n: epngdfho
Just Checking v1.8 : s/n: 15386217
Just The Fax v2.8 for Win9x/NT : Name: Versus Code: RKS-3388178 or Name: n03l Code: RKS-1756575
Just The Fax v2.8g : Name: REKiEM / PCY '99 s/n: RKS-6116770
Just the Fax v2.8b : Name: Warp Code: RKS-2180098
Just the Fax v2.8i : Name: AzzYRiAN Code: RKS-3858352
Juxto v1.1 : Name: DSI Code: 459$9267
J-Perk v4.2 : Hit Unlock/Register and enter: Password: 1$2876558201
J-Perk v5.0 : Password: 2$F48W6299T7
J-Perk v5.01 : Password: 2$F48W6299T7
J-Write v2.4 : Name: Ringer Code: 7-007192
J v4.0a : Key: softwar (No "e")
Jaba Com Web Browser v1.9.98 : s/n: 0k7u0z6y1a981-3t6r3a5m7s24
Jaba Com Web Browser v1.9.99 : s/n: 999-9999999999999999
JackHammer 1.5 : Order #: SAVAGE RegKey: 78224938464138085318640458370445
Jack Hammer v2.1 : Name: RyDeR_H00k! Email: ryder@ucf.com ID: 1234-1234-1234-1234 s/n: BA91-7345-0000
Jackpot Slots v1.5 : Key: 274955

Jackpot Slots v2.0 : Key: 274955


Jackpot Super Slots v1.55 : Name: Warezpup [KAC 99] Password: 182E43481723 Code: 5605F-05FB67910-169B3
Jackpot Super Slots v1.6.3 : Name: Harlem Password: Niggah Code: 2182E-82E434B34-3F265
Jacobs Image Browser v2.0.0 : Name: accz of blizzard Code: 4D2
James Gleick's Chaos v1.01 : s/n: 900000-01101-05-0107
Jammer SongMaker : s/n: JWS11413
Janus GridEX v1.6a : s/n: CSGDX-31255-YUKKM-12103-56422
Janus GridEX v1.6b : s/n: CSGDX-31255-YUKKM-12103-56422
Java Embedded Server v1.0 : s/n: JS0876939287244113
JarHelper v3.5.1 : s/n: 60035GE-AJ-314314
Java Browser v2.1 : Name: forcekill Company: infra Code: 454353453
Java Draw : name: Steve Hsu s/n: Rw|Äe_M}q
JavaPC v1.1 : s/n: JS8266699581686405
Java Safe v1.0 : s/n: JS 0243 8494 8708 4565
Java Script It! v1.3 : First Name: LOMAX Last Name: DSI s/n: 16@vh7ujs
JavaScript Scrambler v1.1 : name: LOMAX s/n: 4438-716-8
JavaScript Scrambler v1.11 : name: LOMAX s/n: 4438-716-8
Java Script It v1.4 : Name: LOMAX Company: DSI Code: 16@vh7ujs
Java Script it v1.5 : First Name: LOMAX Last Name: DSI s/n: 16@vh7ujs
Java Web Server v1.1.3 : s/n: JS2671414474923375
Java Workshop : s/n: JWS100-DAAESU-906052896
Java WorkShop v1.0 Win : s/n: JWS100-AT4072-888641479 or s/n: JWS100-CO6222-048623471 or s/n: JWS100-DA2082-879883481 or s/n: JWS100-DAAES4-906052896 or s/n: JWS100-DAAESU-906052896 or s/n: JWS100-IF2902-901345480 or s/n: JWS100-JS1212-119673481 or s/n: JWS100-KN4162-088731469 or s/n: JWS100-PC9797-666666676 or s/n: JWS100-VY0272-953819465
Java WorkShop v2.0 Win95/NT : s/n: JSW100-RV9797-929569987 or s/n: JWS100-DAAESU-906052896
Java Workshop v2.1 : s/n: JWS100-RV9797-929569987
JayTrax v1.0 : s/n: BOB-28701-FCJ
JayTrax v1.1b : s/n: BOB-28701-FCJ
JayTrax v2.0 : Name: Azrael [PC] s/n: 13956064
Jbuilder Pro v3.0 : s/n: 300-000-0071 Key: 9bx6-x0x0
JClass BWT v3.0 : s/n: 75010FS-GP-314394
JClass BWT v3.5 : s/n: 70035FS-QF-315151
JClass Chart v3.0 : s/n: 79010FS-WB-314356
JClass Chart v3.5 : s/n: 79035FS-JL-314748
JClass Chart Bytecode v3.6j : s/n: 79036FS-KG-315563
JClass DataSource v3.5 : s/n: 87035FS-LJ-314038
JClass DataSource v3.5.1 : s/n: 87035FS-VY-314208
JClass Enterprise Suite v3.6.1 : s/n: 62036GE-8V-314602
JClass Field v3.0 : s/n: 83010FS-EY-314381
JClass Field v3.5 : s/n: 83035FS-S7-314715
JClass Field Bytecode v3.6j : s/n: 83036FS-NS-315241
JClass HiGrid v3.5 : s/n: 85035FS-LS-314038
JClass HiGrid v3.5.1 : s/n: 85035FS-XV-314189
JClass JarHelper v3.5 : s/n: 79035FS-JL-314748
JClass LiveTable v3.0 : s/n: 70030FS-BY-314675
JClass LiveTable v3.5 : s/n: 70035FS-QF-315151
JClass LiveTable Bytecode v3.6j : s/n: 70036FS-VF-315802
JClass PageLayout v1.1.0 : s/n: 91011FS-PF-314084
JClass Standard Suite v3.5.1 : s/n: 60035GE-AJ-314314
JClass Standard Suite v3.5.1 Upgrade : s/n: 60035GE-AJ-314314
JClass Swing Suite v1.1 : ByteCode: 89011FS-S9-314018 SourceCode: 90011FS-JD-314016
Jeek v1.1 : Name: tYruS@c4n.edu s/n: O4ICWO3G0XX07XOR
JellyFish32 v3 : name: Plushmm [PC97] s/n: 101313506404208
Jennifer vs Sarah v1.0 : s/n: 8JG-7NT-000521
Jeta Reyes For MAX 2.x : Name: Kashmir s/n: 41025
K
Kohesion v1.2 : s/n: 1865271321
Kokarev The MusicArt v1.00 : Name: dustie of blizzard Code: 2821641
Kommunicate RightFAX NT Enterprise Server 5.20 : s/n: SNR056549 BumpCode: 3G877++0+++++++8C++I++++ (the 2 characters in between the +'s are a zero and the letter "eye")
Komorebi v1.5 : Name: Concept s/n: L92hD9Gfy!!7NFWtD9KqCpynBFmkAVahA8?ezoObyEC8xUq5LH SYK
Komorebi v1.7 : Name: forcekill s/n: Cp2dD9GkBFej!VWtD9KqCpynBFmkAVahA8?ezoObyEC8xUq5LH SYK
Komorebi v1.8 : Name: SiraX/CORE s/n: H9qdDGvgLb2JKL?tD9KqCpynBFmkAVahA8?ezoObyEC8xUq5LH SYK
Komorebi v1.9 : Name: SiraX/CORE s/n: H9qdDGvgLb2JKL?tD9KqCpynBFmkAVahA8?ezoObyEC8xUq5LH SYK
Komorebi v1.91 : Name: Versus s/n: GpGdz8Gc!Yz7NFWtD9KqCpynBFmkAVahA8?ezoObyEC8xUq5LH SYK
Komorebi v2.05 Win9xNT : Name: DSi s/n: KWOm!YLaOsz7NFWtD9KqCpynBFmkAVahA8?ezoObyEC8xUq5LH SYK
Komorebi v2.20 : Name: CokeBottle98 s/n: L92QCHSgGUKPCIr7!VKqCpynBFmkAVahA8?ezoObyEC8xUq5LH SYK
KonMei 32 v2.07 : ID: KXN@986
KostenTeller v6.0 for Dos : id-Code: 173596222933 s/n: 173596222933
Kremlin Domestic v2.21 : Code: 9999632666
Kremlin Encryption and Security Suite v2.11 : s/n: 5173111659
Kremlin Encryption and Security Suite v2.21 : s/n: 9797708151
Kremlin International v2.21 : Code: 9999632666
KS Calculator v1.02 : Name: dLLord/DNG Code: 353851C4A5CC
KS-Soft Calculator v1.02 : Name: tYruS@c4n.edu s/n: DBC0277CE134
KSP Software Solitaire II v2.1a : Name: (Anything) Code: 2960K1S8
KSP Solitare For Windows v2.1a : Name: (Anything) Code: 2Q6K2135
Kwik Grader v3.3 : Name: IBH-RiP [Blizzard] Printout Name: IBH-RiP [Blizzard] s/n: KG-495840
Kwik Grader v4.4 : Name: (Anything) s/n: KG-1377036
Kwik Grader v4.8 : UserName: Spider] Name as you want it to appear: Spider's Professional Code: KG-1681776
KWQ MailReader +v1.2i for OS/2 : Name: Buxton Bailey s/n: 8163-05F3-313B or Name: Cheng Li s/n: 1504-034C-408C
Kyodai Mahjongg v4.28 : Name: knoweffex Code: 771003431
Kyodai Mahjongg v5.0 Win95NT : Name: 10851-Brooks Code: 7ab55
Kyodai Mahjongg v5.28 : Name: zaarnik-BLiZZARD Code: 0941452212150570
Kyodai v5.36 : Name: Naglfar [DDT98] Code: 150109163962139
Kyodai Mahjongg v6.0 : Name: zaarnik-BLiZZARD Code: 0941452212150570
Kyodai Mahjongg v6.21 : Name: AlpHaz - Fluke Code: 63049947728548
Kyodai Mahjongg v6.42 : Name: AlpHaz - Fluke Code: 63049947728548
Kyodai Mahjongg v7.0 : Name: Blackstar TRPS Code: 94566213166623
Kyodai Mahjongg v7.21 : Name: TCPGC1999 Code: 117186644
Kyodai Mahjongg v7.42 : Name: Blackstar TRPS Code: 94566213166623
Kyodai Mahjongg v8.42 : Name: CoKeBoTtLe99 Code: 13812800
Kyodai Mahjongg v8.52 : Name: Mr_GReeN [WkT!] Code: 019633290384176
Kyodai Mahjongg v9.0 Beta 3 : Press F9 and enter: Name: _ERaD_ [Laxity] s/n: 841953737375174
Kyodai Mahjongg v80.75 : Name: Jimmy Trockly s/n: 5948036850659
K-Chess Elite v2.5 : Name: (Anything) s/n: KE700000 Unlock code: 7293
K-Chess Elite v2.5d : Name: (Anything) s/n: KE700000
Kabcam v1.0 : Code: KC-CORE6J7
KABcam v1.0A : Code: KC-@J@JAJA
KABcam v1.1 : Code: KC-@J@JAJA
KABcam v2.0 Beta 6 : Code: KC-@Y@d@!B@
Kahn 97 1.1 : Code: 0000000000000000 Name: Riz la+ RegNo: E8264ABF7BB0FD40
KaleidaGraph v3.09 : Name: PREMiERE Org: (Anything) s/n: 8000234
Kaleidoscope '95 : Name: tHATDUDE! Reg: HYSAK
Kaleidoscope 95 : Name: Dan Tauber s/n: TUTAJ
Kaleidoscope95(Click on "Syntrillumn") : Name: Byte Ripper s/n: NEVAD
Kalkulator v2.22 : Name: CoKeBoTtLe99 License: 6669 s/n: 807461 693494
Kamorebie v2.25 : Name: aerosmith s/n: DFGdA8OiBEKn!VWtD9KqCpynBFmkAVahA8?ezoObyEC8xUq5LH SYK
KanjiWeb v1.0 : s/n: 5162345-34
Kansmen Little Brother v3.1 : s/n: 978334-5J2I6A
Katiesoft Scroll V3.0.300 : Code: 5783-125-1931-9481
KatieSoft Scroll v3.11 : Code: 5783-125-1931-9481
Kawa 2.0 : Name: BLiTZ / Phrozen Crew s/n: AADHA
KAWA v2.5 : Name: CORE/JES Key: AGGB
Kawa 2.52.01 : Key: Phrozen Crew Code: IGFF or Key: _RudeBoy_ Code: EAHC
Kawa v3.1 : Key: Drone [F4CG] Code: heif
Kawa v3.11 : Code: _RudeBoy_ s/n: EAHC
Kawa v3.13 : Name: LOMAX s/n: @@AA
Kawa v3.20 : Name: darkstar s/n: FF@C
KazStamp v7.5c : Name: EinZtein Company: USC s/n: 123456789012345 Code: 19191
KDriVe v2.05 for Windows : (Type at command prompt) Brand Kdrivew.exe 4711 0815
KDrive v2.51 for Dos : (Type at command prompt) Brand Kdrive.exe 4711 0815
KDriVe v2.51 for OS/2 : (Type at command prompt) Brand Kdriveos2.exe 4711
Kea XServer 3.05 For Windows95 : Password: access
Keep It Compact v1.10 : Name: Registered s/n: 1842152
Keep It Compact v1.10 (2) : Name: tHATDUDE s/n: 1381784
Keep It Compact v2.1 : Name: zaarnik Code: 751686471111036

Keep It Compact v2.2.01 : Name: LOMAX [DSi] s/n: 034014B4


Keep it Compact v2.2.02 : Name: LOMAX [JiOO] s/n: 2709184D
Keep it Compact v2.2.03 : Name: LOMAX [DSi] s/n: 034014B4
Keep it Compact v2.2.04 : Name: LOMAX [DSi] s/n: 034014B4
Keep it Compact v2.26 : Name: aip35[Tbc] s/n: 4FE5CEF6
Keep It Safe 2.1 : s/n: 45198933
Keep Track v2.0.1 : Name: madcease Code: 682004
Kerropi Bigtop Organizer for Windows 95/NT: s/n: TSO6-7775-P9DT
KeyBoard Calculator v1.0 : Name: (Anything) Code: SUMMA Number: (Anything that starts with a #)
Keyboard Layout Manager Pro Upgrade : Name: Lisa Prince Company: TRPS s/n: KLM32-48EB-3434
Keyboard Navigator v2.0 : Name/Company: 1 s/n: 1
KeyChain v1.0 : Name: Sune Code: 017-739
Key Click v1.1 : s/n: gmbrdybv
Key Information DBMS v2.01.01 : s/n: 11023021543210
Key It v1.0 : Email: ehaden@netcom.ca Code: CC6CC3EO
Key It v1.011 : Name: GZI Code: QI7EA0E
KeyKey v1.0 : Name: MisterE Company: [iNSiDE] s/n: K100-67-901-0-00000AC0003046-51
KeyKey v1.00b : Name: SiraX Company: CORE s/n: K100-43-100-0-0567882234D141-56
Key Key 1.00c : Subject: KeyKey Code: K100-00-001-1-00000AA080004D-22
KeyKey v1.12 : Name: SiraX Company: CORE Code: K100-43-100-0-0567882234D141-56
KeyKey v1.15 : s/n: K100-67-901-0-00000AC0003046-51
KeyKey v1.12 : Name: SiraX Company: CORE s/n: K100-43-100-0-0567882234D141-56
Keylogic Team Leader v6.0a : s/n: 010199
KeyMouse v5 : s/n: GorgAMAIGHND
KeyNote Music Drills : s/n: 867578755
Key-Note v1.0 : Name: FALLEN Code: ID-115115961-ZKRY
KeyText v1.02 : Name: Vizion/CORE s/n: 60630565I
KeyText v1.12 : Name: SiraX/CORE s/n: 5053946J
Key Text v1.17 : Name: MANiFEST DESTiNY s/n: 4245-38YC-8EMU
KeyText v1.2 : Name: MANiFEST DESTiNY s/n: 4245-38YC-8EMU
KeyText v1.21 : Name: DEGEN[FCRP] s/n: 7496-79YM-8EMW
KeyText v1.22 : Name: MANiFEST DESTiNY s/n: 4245-38YC-8EMU
Key unlock for Webedit 1.4 : 1FDBBL3P6NBP
Key unlock for Webedit 2.9 pro : 846633B89C1E
KeyView for Win95 : s/n: TrcfLc03929
KeyView v4.2 for Windows 95 : s/n: 4000-0144-5297
KeyView v6.5 : s/n: 4000-0144-5297
KGL Unlocker 1.11 : Name: William Anderson Password: 391516876297
KGL UNLOCKER : Name: William Anderson Password: 391516876297
Kid Cad v1.1c for Windows : s/n: 51581-26830
Kimiko HTML Editor v3.2 : Name: Pamela J. Annonson s/n: KIMLJ18-76ENOXM-PRO45B
Kimiko HTML Editor v3.4 : Name: Shawn Robinson s/n: KIMLJ18-OF12FR2-PRO45B
Kinemorphics v1.0.165 : Code: 64222
Kinemorphic 3D ScreenSaver v3.0 : Code: 13181
KingCom ComPort Manager v2.0c for Windows: s/n: 654-16203
Kings Corners v3.0c : s/n: 9072163093
Kiplinger's Taming the Paper Tiger v1.0 : s/n: PTS-1023-1065-1141
Kismet v1.00 : Name: Delphic Code: 705-412-304
Kitchen Assistant/2 v1.2 for OS/2 : Name: EVIL UNCLE s/n: 0130200
KiWi Enveloppes v3.0 : s/n: KE301120RA or s/n: KE304244RC
Klik Play v1.0 for Windows : s/n: KPE10001143 or s/n: KPG10002097 or s/n: KPU10006512 or s/n: KPE10048950
KMMC : 79965827338
Kniffel'98 v6.0 : Code: 30051978
Know-It Xpert_Trak_net v2.5 : Code: 2670289503
Know-it Xpertrak Net v2.6 : Name: MAGNUM98 Code: 2200958503
Know Your Bible Deluxe Edition v2.1 : Name: Gorgeous Ladies Of Warez Code: 6F6E694D-3C74614D-5C6E764D
KoalaTerm International Edition V2.7f : s/n: 98765432 Licenses: 98765432101234567890 Code: B05F2001326A5F01


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Tumor cells rely on the thiol oxidoreductase PDI for PERK signaling in order to survive ER stress

Abstract

Upon ER stress cells activate the unfolded protein response through PERK, IRE1 and ATF6. Remarkable effort has been made to delineate the downstream signaling of these three ER stress sensors after activation, but upstream regulation at the ER luminal site still remains mostly undefined. Here we report that the thiol oxidoreductase PDI is mandatory for activation of the PERK pathway in HEK293T as well as in human pancreatic, lung and colon cancer cells. Under ER stress, depletion of PDI selectively abrogated eIF2α phosphorylation, induction of ATF4, CHOP and even BiP. Furthermore, we could demonstrate that PDI prevented degradation of activated PERK by the 26S proteasome and therefore contributes to maintained PERK signaling. As a result of decreased PERK activity, PDI depleted cells showed an increased vulnerability to ER stress induced by chemicals or ionizing radiation in 2D as well as in 3D culture models. We conclude that PDI is an obligatory regulator of the PERK pathway with future therapy implications.

Introduction

The endoplasmic reticulum (ER) acts as the major calcium storage site in eukaryotic cells and is indispensable for steroid hormone and lipid synthesis1. Furthermore, protein synthesis and protein folding of secretory and membrane proteins take place at the rough ER. Depending on the origin of the cell and its specific function, 30 up to 50% of all cellular proteins pass this organelle to achieve disulfide bonding and proper folding2,3. This is executed through the highly oxidative environment in the ER and supported by classes of specialized enzymes like chaperones and ER oxidoreductases4. The capacities of this organelle are easily exceeded by intrinsic (genetic aberrations, ROS, high demand of secretory proteins, cell division etc.) and extrinsic factors (hypoxia, chemo- and radiotherapy, lack of nutrients etc.) which then result in unfolded protein burden and ER stress5. In these situations, cells are able to activate an evolutionary conserved program named the unfolded protein response (UPR). The UPR is carried out through the three sensor proteins the inositol-requiring enzyme 1 (IRE1), the activating transcription factor 6 (ATF6) and the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), all of them trying to decrease protein burden at the ER and increasing the folding capacity by upregulating specific UPR target genes6. Downstream signaling of IRE1, ATF6 and PERK has been studied extensively throughout the past decades, but how these three sensors are activated from the luminal site of the ER is still under investigation7. So far, some members of the protein disulfide isomerase family (PDIs) have been identified as regulators for ATF6, IRE1 and in parts even for PERK8,9,10,11,12.

In the competition model, it is thought that upon ER stress, PERK is released from binding immunoglobulin protein (BiP), forms dimers and oligomers which are then able to autophosphorylate themselves7. PERK then transmits a signal to the cytoplasm by phosphorylation of translation initiation factor-2α (eIF2α), which leads to an immediate translational repression to prevent further unfolded protein burden in the ER. Cap independent translation is increased in return and results in elevated translation of activating transcription factor 4 (ATF4). ATF4 upregulates a set of UPR targets genes like GRP94, BiP and PDI to increase the folding capacity and cope with the stress. After longer exposure to ER stress, ATF4 is responsible for an increase in proapoptotic C/EBP-homologous protein (CHOP) expression and therefore contributes to cell death observed under chronic ER stress6.

PERK has been shown to support tumor growth, metastasis, autophagy and radiation resistance and was therefore proposed as a future therapy target to overcome therapy failure13,14,15,16,17,18,19,20. Small molecule inhibitors were designed to inhibit PERK phosphorylation and its downstream signaling. In preclinical studies they were successfully tested in antitumor treatment but showed severe side effects21,22,23. Therefore, identifying new necessary upstream regulators of PERK at the ER luminal side could be an opportunity to limit PERK activity in tumors while sparing adverse effects. PDIA1 (afterwards referred to as PDI) is one of the most abundant proteins in the ER and participates in disulfide bond reduction, oxidation and isomerization. Although the disulfide bonding function of PDI has been shown in numerous studies24,25, client proteins which rely on PDI’s function are rare and loss of PDI can be compensated by other PDI family members (e.g. ERp57 or Erp46)26,27,28. These observations presume further functions in regulating ER homeostasis or other undiscovered cellular roles for PDI. Previously, we could show in a colorectal cancer cell model, that upon depletion of the thiol oxidoreductase ERp57, PERK gets activated in a PDI dependent manner and reduces proliferation, induces cell death and sensitizes cancer cells to ionizing radiation29,30. Thus, PDI represents a promising target to overcome tumor cell resistance. Here we demonstrate the general validity of a PDI dependency for PERK signaling during acute and prolonged ER stress in a set of various human cancer cell lines. We also expand PDI’s role as a PERK activator to that of a maintainer of PERK signaling and thus offer a new therapeutic strategy to inhibit PERK signaling in tumor cells.

Results

PDI is mandatory for activation of the PERK pathway during acute and chronic ER stress

To assess the importance of PDI for PERK activation under ER stress, HCT116, HEK293T, A549 and BxPC3 cells harboring a stable inducible shRNA to knockdown (KD) PDI were generated by lentiviral transduction. Since scrambled shRNA showed major impairment in cell proliferation in various cell lines (data not shown), cells without doxycycline treatment were used as controls. Cells were exposed to thapsigargin up to 3 h and ER stress response was monitored by phosphorylation of eIF2α and PERK by western blotting. PDI KD almost completely abrogated eIF2α phosphorylation in HCT116 and HEK293T cells, indicating impaired PERK activity, although phosphorylation of PERK itself was still present (Fig. 1A, S1C). To ensure this phenotype was not only related to a disruption in calcium homeostasis, a second ER stressor tunicamycin, an inhibitor of N-linked glycosylation, was used in A549 and HEK293T PDI KD cells up to 5 h. In line with thapsigargin treatment, depletion of PDI resulted in impaired PERK signaling as shown by decreased eIF2α phosphorylation and BiP induction (Fig. 1B). The amount of total PERK protein was reduced during longer exposure times to tunicamycin, suggesting impaired stability or decreased expression (Fig. 1B). Treatment with the reducing agent DTT showed similar results (Fig. 1C).

ER stress is known to induce a pro-apoptotic signaling pathway through CHOP after long time exposure to ER stress inducing agents. To check if PERK activity was also decreased after PDI depletion in situations of long term exposure to ER stress, BxPC3, HCT116 and HEK293T cells were cultured in the presence of thapsigargin for 24 h. Depletion of PDI led to decreased eIF2α phosphorylation in all three cell lines, lower BiP induction in BxPC3 and declined levels of total PERK in HCT116 and HEK293T cells (Fig. 2A, S1C). Furthermore, CHOP and ATF4 were less induced without PDI in HEK293T and A549 cells after 6 h of tunicamycin treatment, indicating the necessity of PDI to induce full PERK downstream signaling (Fig. 2B). Luciferase assays were performed to validate transcriptional activity of ATF4 after PDI KD and 20 h of thapsigargin treatment in HEK293T cells. PDI KD significantly decreased ATF4 transcriptional activity already under basal conditions. Thapsigargin treatment did not lead to an adequate increase of ATF4 activity when PDI was depleted compared to the control cells, confirming the western blots results (Fig. 2C, S1A).

PERK activation has been shown to increase the survival of cells during ER stress by limiting protein translation and upregulation of important UPR target genes. Depletion of PDI combined with tunicamycin or thapsigargin treatment resulted in massive decrease of cell survival in A549, HEK293T and HCT116 cells as shown by bright-field microscopy, indicating higher vulnerability to ER stress which is in line with abrogation of the PERK pathway (Fig. 2D). KD of PERK in A549 cells showed comparable results (Fig. 2D).

During the UPR, PDI specifically stabilizes PERK and does not promote IRE1 or ATF6 signaling

Luciferase assays were performed to assess if KD of PDI also affects IRE1 and ATF6 signaling (Fig. 3A). Under non-stressed conditions, we observed a minor increase in ATF6 activity and a slight decrease of XBP1 activity after KD of PDI. It became evident that KD of PDI alone does not induce full UPR signaling. However, under ER stress triggered by thapsigargin, ATF6 and IRE1 activity remained mainly unchanged through KD of PDI, indicating an exceptional role for PDI in activating the PERK pathway (compare Figs. 3A and 2C).

PDI is one of the most abundant proteins in the ER and is thought to contribute to disulfide bond formation of client proteins. To exclude that an overload of these client proteins without properly formed disulfide bonds is the cause for the increased vulnerability to ER stress, the activity of secreted gauss luciferase was monitored in the supernatant of PDI KD cells. Gauss luciferase contains five mandatory disulfide bonds to maintain luciferase function and therefore represents a good model for measuring protein folding and secretion ability of the ER. Notably, depletion of PDI did not significantly alter the activity of Gauss luciferase indicating that neither disulfide bond formation nor the secretory capacity was compromised (Fig. 3B).

PERK protein levels were decreased in PDI KD cells after short as well as long time exposure to ER stress inducing agents, indicating decreased PERK expression or increased PERK degradation when PDI is depleted (Figs. 1A-C and 2A,B). RT-PCR was performed to investigate differences in expression levels of PERK mRNA with and without tunicamycin treatment. However, no significant differences in transcriptional levels of PERK could be observed, strongly indicating a translational or posttranslational event (Fig. 3C). Since ER membrane proteins are usually degraded by the 26S proteasome in a p97 dependent manner 31, proteasomal degradation was blocked by MG132 prior to tunicamycin treatment and PERK protein levels were determined by western blotting. Indeed, MG132 treatment partly restored PERK protein levels after PDI KD in A549 cells, indicating a stabilizing function of PDI for PERK after its activation (Fig. 3D). Stabilization of PERK presumes a direct interaction between PDI and PERK which can occur by PDIs active cysteines at the a and a’ domain or its hydrophobic binding motif in the b’ domain. Co-immunoprecipitation experiments were performed in HEK293T cells expressing either the luminal domain of WT-PERK or Cys-null-PERK (4 luminal cysteines of PERK mutated to serine) together with WT-PDI or the trap mutant form C400S-PDI. These experiments showed no strong interaction between PERK and PDI, irrespective of the cysteines of PERK or expression of the PDI trap mutant which has been shown to enhance co-immunoprecipitation of PDI substrates (Fig. 3E). Anyhow, these results don’t exclude a possible transient interaction between PDI and PERK through the b’ motif during ER stress or an unknown necessary protein important for PERK activation which relies on functional PDI.

Role of PDI in radiation sensitivity

Radiotherapy has been shown to induce ER stress and activation of the PERK pathway. To elucidate the role of PDI and its activating function for PERK during radiotherapy, colony formation assays were performed. KD of PDI decreased colony formation ability of A549 cells already under control conditions and was further reduced after different doses of radiation (Fig. 4A, S1D). The same tendencies were observed using an shRNA against PERK, although the effect was even more pronounced (Fig. 4B). To simulate in vivo tumor conditions, spheroid growth experiments were carried out. KD of PDI combined with radiation decreased the spheroid volume during the first days after radiation and resulted in later regrowth compared to the control spheroids (Fig. 4C). These results highlight the mandatory protective function of PDI and PERK under ER stress conditions and strongly suggest PDI as a target to reduce adaptation to ER stress through the PERK pathway.

Discussion

The most common theory how ATF6, IRE1 and PERK are activated in response to ER stress to date is the dissociation of BiP, which then allows dimerization and autophosphorylation for IRE1 and PERK or cleavage of ATF6 respectively. Nevertheless, there is still uncertainty about the binding of BiP, its dissociation, ATP dependence, mandatory cofactors and redox regulation of luminal cysteines regarding to the activation and fine tuning of these sensors7,32.

In this study we were able to almost completely abrogate PERK signaling by depletion of PDI in HEK293T, BxPC3, A549 and HCT116 cells. This phenotype was explained by decreased stability of activated PERK and increased degradation of the UPR sensor by the 26S proteasome after loss of PDI. Expression level of PERK mRNA remained unchanged and the induction during ER stress appeared to be similar after depletion of PDI. It is possible that due to missing eIF2α phosphorylation the cap independent translation of PERK mRNA is not as effective after PDI KD as compared to control cells. This could also contribute to the decreased PERK protein level we observed after long time exposure to chemical ER stress inducers.

PERK possesses 4 luminal cysteines, but their roles in activation, inactivation or stability of PERK remain elusive. Although we could not show a direct interaction between PERK and PDI, it is not unlikely that PDI contributes directly or indirectly to proper disulfide arrangement in the luminal domain of PERK which in turn prevents degradation. The significance of luminal cysteines has been already shown for IRE1 and ATF6. Eletto et al. could prove that PDIA6 limits the duration of IRE1 signaling by direct binding to cysteine 148 to adjust IRE1 activity and thereby preventing apoptosis8. Furthermore, they showed a direct binding between PERK and PDIA6 regulating the strength and duration of PERK activity, which could be the missing link between PDI and PERK in our observed phenotype8. ATF6 exists in monomers as well as disulfide bonded dimers and oligomers in unstressed cells11. The level of intramolecular disulfide reduction correlates with ATF6 transcriptional activity and is therefore a mandatory event to activate this sensor11. Additionally, PDIA5 has been identified to be mandatory for this disulfide bond rearrangement during ER stress, leading to ATF6 export from the ER and enhanced chemoresistance10. A recent study identified CNPY2 as a key factor for activating and maintaining PERK function. CNPY2 is released from BiP during ER stress and engages PERK signaling by direct binding in mice and mice cells12. CNPY2 exists in disulfide bonded dimers and therefore could be a PDI client protein, although the binding to PERK seemed to be independent of CNPY2 cysteines12. However, we did not see a decrease in CNPY2 expression or dimer formation after depletion of PDI (data not shown). In our hands, BiP levels were decreased in PDI KD cells during ER stress and therefore CNPY2 should be able to bind PERK to a higher extend and enhance PERK activity even more, which was clearly not the case. Furthermore, we were unable to detect CNPY2 expression in A549 cells and therefore excluded it as the key factor for our observed results.

Based on our results we suggest a dual role of PDI for the PERK pathway which is depicted in Fig. 5. Herein, PDI is not only indispensable for PERK activation but also prevents its degradation. Although the precise mechanism how PDI is involved in PERK signaling remains uncertain, this observation shows potential for clinical applications. Activation of the UPR and especially the PERK pathway are present in almost all tumors and support tumor growth and adaptation to nutrient deprivation and hypoxia13,15,17,20,33. It is noteworthy that loss of PERK can be compensated by GCN2 as well as GCN2 loss can be compensated by PERK in some tumor models, which in part negotiates the antitumor effects34,35. We could not observe any compensation after loss of PDI regarding to ATF4 induction and further PERK downstream signaling. This might be explained through a lesser extend of PERK blockade which not induces compensation by GCN2, maybe due to incomplete PDI KD or by also affecting GCN2 kinase activity. Furthermore, the defect in PERK activity was not accompanied by increased activation of ATF6 or IRE1.

Anyhow, depletion of PDI increased the sensitivity of all tested cells to ER stress inducing agents and ionizing radiation with comparable effects like the direct KD of PERK. This was further validated using a tumor spheroid model, which simulates the tumor growth and radiation response in vivo more precise36. While PERK inhibition or depletion in tumor cells remarkably reduced tumor growth and metastasis in mouse models21, clinical applications of PERK inhibitors are limited by pancreatic adverse effects23,37. Different PDI inhibitors in comparison were well tolerated in preclinical tumor models and showed strong antitumor activity38,39,40,41,42. The mechanisms of action of PDI inhibitors vary massively regarding to the effect on UPR pathways. While in some studies UPR activation is not addressed42, others show massive induction of all UPR pathways in multiple myeloma cells41 or no effect on the UPR at all38. It is worth noting, that although most inhibitors targeting the enzymatic activity of PDI at the a domain (PACMA31, KSC-34, CCF642), differences in chemical structure seem to alter UPR response after inhibition and therefore off target effects cannot be excluded. To our knowledge, studies comparing genetic depletion of PDI with these inhibitors have not been executed yet. It is therefore difficult to distinguish which roles the a’ and the b domains play after inhibition of the a domain and if their functions are also affected. Another PDI modulator, LOC14, which forces PDI to adapt in an oxidized conformation and thereby blocks enzymatic activity, has been reported to suppress UPR response in a mutant huntington model and supports our findings that PDI is important for activating the PERK pathway43,44. To address these indistinct results, further research using genetic knockout models and re-expressing mutant forms of PDI with dysfunctional enzymatic activity are necessary to delineate the functions of the different PDI domains regarding their involvement in sensing ER stress.

In summary, PDI presents a novel target to selectively overcome tumor cell resistance to ER stress and radiotherapy acquired by activation of the PERK pathway in solid as well as leukemic malignancies while sparing the severe side effects received by direct inhibition of PERK.

Material and methods

Cell culture, transfection and lentiviral transduction

HEK293T (ATCC, CRL-3216, human embryonic kidney) and A549 (ATCC, CCL-185 human lung adenocarcinoma) cells were cultured in DMEM high glucose (Invitrogen, Darmstadt, Germany), BxPC3 (DSMZ, ACC-760, human pancreatic adenocarcinoma) cells in RPMI 1640 (Invitrogen) and HCT116 (ATCC, CCL-247, colorectal carcinoma) cells in McCoy’s 5A (Pan-Biotech, Aidenbach, Germany) supplemented each with 10% tetracycline-free FBS and antibiotics. Transient transfections were performed using Viafect (Promega, Mannheim, Germany). Production of lentiviral particles were done in HEK293T cells as described previously (Kranz et al., 2017). For transduction, 2 × 105 cells were incubated with 2 × 106 lentiviral particles in the presence of 8 µg/ml polybrene for 24 h followed by selection with 2 µg/ml puromycin for 5 days. All experiments were performed with mycoplasma-free cells.

Plasmids, shRNA sequences and knockdown induction

Plasmids used for luciferase assays: pFLAG-XBP1u-FLuc (Addgene #31239), p5xATF6-GL3 (#11976, Addgene), pNL NlucP/ATF4-RE/Hygro (Promega), pTK-GLuc- (#N8084S, NEB, Frankfurt/Main, Germany), pGL4.74 (#E6921, Promega). pCMMP-dnPERK-IRES-eGFP (Addgene #36954) was used to create V5-tagged and Cys-null mutant (C187S, C192S, C307S, C423S) of PERK luminal domain by site directed mutagenesis45. The following shRNA sequences were cloned into Tet-pLKO vector: 5′-GTGTGGTCACTGCAAACAGTT-3′ (corresponding to 1,385–1,405 bp of human PDI mRNA, GenBank acc. no. NM_000918), #2 5′-TGCTGTTCTTGCCCAAGAGTG-3′ (corresponding to 837–857 bp of human PDI mRNA, GenBank acc. no. NM_000918), 5′-GGAACGACCTGAAGCTATAAA-3′ (corresponding to 3,383–3,403 bp of human PERK mRNA, GenBank acc. no. NM_001313915.1). Knockdown of PDI and PERK was induced by addition of 250 ng/ml doxycycline to the cell culture media.

RNA extraction, RT-primer and PCR

RNA extraction was performed with the NEB monarch RNA extraction kit (NEB) following the manufacturer’s instructions. cDNA was generated using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). For RT-PCR SampleIN Direct PCR Kit (highQu, Kraichtal, Germany) was used according to the manufacturer’s protocol. Expression of PERK was tested by using PERK primers 5′-ATCCCCCATGGAACGACCTG-3′ (forward), 5′-ACCCGCCAGGGACAAAAATG-3′ (reverse). β-Actin expression was assessed using Actin primers 5′-GCCGCCAGCTCACCAT-3′ (forward) and 5′-TCGATGGGGTACTTCAGGGT-3′ (reverse). Conditions for amplifications: 95 °C for 1 min followed by 30 cycles of 95 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s. Samples were loaded on a 2% agarose gel containing SYBR Safe DNA Gel stain (Thermo fisher scientific, Schwerte, Germany). Bands were visualized using the FX7 chemoluminescence documentation system (Peqlab, Erlangen, Germany) and quantified using ImageJ software.

Antibodies and reagents

Tunicamycin, Thapsigargin and MG132 were obtained in p.a. quality from Sigma-Aldrich (Schnelldorf, Germany) and Santa Cruz biotechnology (Santa Cruz, California, US). Antibodies against GAPDH and Actin were from Sigma-Aldrich, against PDI from RnD-Systems (Minneapolis, MN, USA), against BiP, eIF2α, P-eIF2α, PERK, PDI, CHOP, V5, Flag and ATF4 from Cell-Signaling technologies (Frankfurt/Main, Germany). Secondary HRP coupled antibodies against rabbit and mouse were from DAKO (Hamburg, Germany).

Clonogenic survival assay

400 A549 cells were plated in six-well plates and KD was induced immediately afterwards. Cells were irradiated with different doses after 40 h using an X-ray machine (X-rad 320, PXI) operated at 320 kV, 12.5 mA with a 1.65 mm aluminum filter (dose rate: 3.6 Gy/min). After 11 to 13 days of incubation, colonies were fixed and stained with 0.25% PFA, 70% EtOH and Coomassie brilliant blue (0.1 Coomassie blue, 5% acetic acid, 45% methanol). Colonies were counted automatically by using the colony area plugin for ImageJ as described previously46. Plating efficiency (PE) and survival fraction (SF) were calculated with the formulas “PE = colonies formed/number of cells seeded” and “SF = colonies formed/number of cells seeded × PE”.

Tumor spheroid growth experiments

Tumor spheroid growth was performed in 96-well round bottom plates coated with 1.5% agarose. 5 × 103 A549 shPDI cells were seeded, KD was induced immediately and plates were centrifuged with 800 rpm for 30 min. After 2 days, spheroids were measured and irradiated with 5 Gy. Spheroid area was measured using ImageJ plugin as described previously 47 and volume was calculated using the sphere formula (4/3 * π * r3). Controls had to be terminated before treated samples due to exponential growth and rapidly exceeding the size of the well.

Cell lysis, SDS-PAGE and western blotting

Cells were seeded in 6-Well or P60-dishes at densities between 7 × 104 and 2 × 105 cells per well and KD was induced directly afterwards. After 72 h cells were treated with ER-stress inducers for the indicated time and lysed in RIPA buffer (50 mM Tris pH 7.5, 2 mM EDTA, 150 mM NaCl, 1% Nonidet P40, 0.1% SDS, 0.5% sodium deoxycholate and protease/phosphatase inhibitor cocktail (Cell Signaling)). Protein concentration was measured with a BCA assay kit (Thermo fisher scientific) and 10–30 µg protein were dissolved in SDS sample buffer (62.5 mM Tris (pH 7.4)) 2% SDS, 3% β-mercaptoethanol, 10% glycerol, 0.25 mg/ml bromophenol blue, 25 mM DTT). Samples were loaded on 7.5–10% polyacrylamide gels and afterwards transferred to PVDF membranes using the Trans-Blot Turbo system (Bio Rad, Munich, Germany). Membranes were blocked in 5% skim milk in TBS-T (50 mM Tris/HCL, 150 mM, NaCl, 0.1% Tween-20, pH 7.5). Primary antibodies were diluted as recommended by the manufacturer and incubated with the membrane overnight. Secondary antibodies were diluted 1:2000 in TBS-T and incubated for 1 h. ECL kit (Thermo Fisher Scientific, Oberhausen, Germany) and FX7 chemiluminescence documentation system (Peqlab) were used for protein detection. To show results of the western blots thoroughly through the whole manuscript, we avoided presenting multiple gels in one subfigure. Therefore, it was not possible to present all targets of interest in every subfigure.

Co-immunoprecipitation

8 × 104 HEK293T cells were plated in four wells of a 24-well plate and transfected with 500 ng V5-PERK and 500 ng flag-PDI per well. After 72 h of incubation four wells were pooled together to achieve adequate protein concentration for immunoprecipitation. Cells were lysed in caspase lysis buffer (Tris (pH 7.3) 50 mM, NaCl 150 mM, NP-40 1% (v/v)) and incubated for 4 h at 4 °C with V5 antibody. Afterwards the lysate was incubated for 1 h with protein S and G magnetic beads (Cell-Signaling) for pull down. Lysates were transferred to SDS-sample buffer containing DTT and heated at 95 °C for 10 min before loaded on a 7.5% polyacrylamide gel.

Luciferase assays

7 × 104 HEK293T cells were seeded in 24-well plates, KD was induced immediately and cells were transfected with 500 ng of firefly luciferase reporter gene plasmid and 100 ng of renilla luciferase plasmid. After 72 h cells were harvested and prepared for measurement using the Dual Glo luciferase assay kit (Promega) as recommended by the manufacturer. Firefly and renilla luciferase were measured using the GloMax detection system (Promega) and normalized to renilla values to exclude variations in transfection efficiency. For testing the secretion efficiency, cells were transfected with 500 ng gaussia luciferase and 100 ng of renilla luciferase plasmid. Luciferase activity was measured in cell culture media and normalized to intracellular renilla luciferase activity.

Microscopy

For bright field microscopy images a Zeiss Axiovert 200M (Carl Zeiss, Oberkochen, Germany) was used with a 4 × magnification objective and 2.5 NA. Quantification of cell confluence was performed using ImageJ.

Statistical analysis

All results were obtained in at least two independent experiments. In bar graphs mean plus standard deviation is shown for one representative experiment run in triplicates. For comparison between groups, two-way ANOVA with Bonferroni or Sidak post-hoc test was used. Significance is presented as *P < 0.05.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

Activating transcription factor 4

Activating transcription factor 6

Binding immunoglobulin protein

C/EBP-homologous protein

Protein canopy homolog 2

Dimethyl sulfoxide

Doxycycline

Dithiothreitol

Eukaryotic initiation factor 2

Endoplasmic reticulum

ER protein 46

ER protein 57

Glyceraldehyde 3-phosphate dehydrogenase

General control nonderepressible 2

Inositol-requiring enzyme 1

Knockdown

Protein disulfide isomerase

Protein kinase R (PKR)-like endoplasmic reticulum kinase

Reactive oxygen species

Reverse transcription polymerase chain reaction

Thapsigargin

Tunicamycin

Unfolded protein response

Wildtype

X-box binding protein 1

References

  1. 1.

    Han, J. & Kaufman, R. J. The role of ER stress in lipid metabolism and lipotoxicity. J. Lipid Res.57, 1329–1338 (2016).

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