Adaptive Basal Phosphorylation of Eif2a Is Responsible for Resistance to Cellular Stress–Induced Cell Death in Pten-Null Hepatocytes

Published OnlineFirst October 18, 2011; DOI: 10.1158/1541-7786.MCR-11-0299

Molecular Cancer DNA Damage and Cellular Stress Responses Research

Adaptive Basal Phosphorylation of eIF2a Is Responsible for Resistance to Cellular Stress–Induced Cell Death in Pten-Null Hepatocytes

Ni Zeng1, Yang Li1, Lina He1, Xiaoling Xu2, Vivian Galicia1, Chuxia Deng2, and Bangyan L. Stiles1

Abstract The a-subunit of eukaryotic initiation factor 2 (eIF2a) is a key translation regulator that plays an important role in cellular stress responses. In the present study, we investigated how eIF2a phosphorylation can be regulated by a tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) and how such regulation is used by PTEN-deficient hepatocytes to adapt and cope with oxidative stress. We found that eIF2a was hyperphosphorylated when Pten was deleted, and this process was AKT dependent. Consistent with this finding, Pten we found that the -null cells developed resistance to oxidative glutamate and H2O2-induced cellular toxicity. We showed that the messenger level of CReP (constitutive repressor of eIF2a phosphorylation), a constitutive phosphatase of eIF2a, was downregulated in Pten-null hepatocytes, providing a possible mechanism through which PTEN/AKT pathway regulates eIF2a phosphorylation. Ectopic expression of CReP restored the sensitivity of the Pten mutant hepatocytes to oxidative stress, confirming the functional significance of the downregulated CReP and upregulated phospho-eIF2a in the resistance of Pten mutant hepatocytes to cellular stress. In summary, our study suggested a novel role of PTEN in regulating stress response through modulating the CReP/eIF2a pathway. Mol Cancer Res; 9(12); 1708–17. 2011 AACR.

Introduction mouse liver where PTEN is lost and PI3K signaling is activated, accumulation of lipids is accompanied by high The mitogenic signaling phosphoinositide 3-kinase levels of hydrogen peroxide, suggesting that the hepatocytes (PI3K)/AKT signaling pathway is negatively regulated by in the Pten-null liver are under conditions of chronic a lipid phosphatase PTEN (phosphatase and tensin homolog oxidative stress (7). We used the hepatocytes isolated from deleted on chromosome 10). Genetic studies have shown this model to investigate whether and how PTEN/PI3K that loss of PTEN functions results in growth and survival signal may provide the adaptation advantage for mutant cells phenotype and tumor development in multiple tissues and to survive stress-induced cell death. organ systems (1). In the liver, nearly 80% of hepatocel- Cancer cells are often "addictive" to their oncogenic events lular carcinoma (HCC) are correlated with activation of the such as loss of a tumor suppressor or induction of an PI3K signaling pathway including loss of PTEN (2). We oncogene and resistant to stress-induced cell death. We and others showed that PTEN loss in mice leads to lipid investigated the response of the Pten-null hepatocytes to accumulation in the hepatocytes early and tumor develop- stress and found that Pten-null hepatocytes are resistant to ment later in life (3–5). This 2-stage progression of tumor various forms of stress including oxidative glutamate and development is similar to that observed with human HCC H2O2 toxicity as well as endoplasmic reticulum (ER) stress. where underlying liver disease especially fatty liver disease is a Phosphorylation of eukaryotic initiation factor 2 (eIF2) common comorbid factor. In human patients, the develop- family of translation regulators is known for integrating ment of HCC is highly correlated with oxidative stress (6). In various cellular stress responses including oxidative and ER stress (8). This mechanism may underlie the conditioned protection against more severe injury in cells growing in Authors' Afﬁliations: 1Department of Pharmacology and Pharmaceutical chronic low levels of stress conditions like the stressed condi- Sciences, USC School of Pharmacy, Los Angeles, California; and 2Genet- Pten ics of Development and Disease Branch, NIDDK, NIH, Bethesda, Maryland tions that the -null cells are in (7). Under acute stress response, phosphorylation of eIF2a leads to shutdown of Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). protein synthesis and mediation of global stress. We showed that PTEN, through its regulation of PI3K/AKT signaling, Corresponding Author: Bangyan L. Stiles, Department of Pharmacology a and Pharmaceutical Sciences, USC School of Pharmacy, PSC 402, 1985 controls the basal phosphorylation of eIF2 . Chronic low Zonal Ave., Los Angeles, CA 90089. Phone: 323-442-2184; Fax: 323-224- level induction of eIF2a phosphorylation was reported to 7473; E-mail: [email protected] mediate an adaptive response of cells to chronic stress (9). doi: 10.1158/1541-7786.MCR-11-0299 Such mechanism may explain why tumor cells are more 2011 American Association for Cancer Research. resistant to stress yet cannot survive when the oncogenic

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signals are lost. We further established that downregulation Reagents and plasmids of CReP (constitutive repressor of eIF2a phosphorylation), a L-Glutamic acid and 3-morpholinosydnonimine (SIN-1) subunit of the protein phosphatase 1 (PP1) phosphatase were obtained from Sigma; H2O2 was provided by Fisher complex, is responsible for this basal phosphorylation of Scientiﬁcs; and LY294002 was purchased from Cell Signal- eIF2a. Overexpression of CReP restores the sensitivity of the ing Technology. Caspase-12 inhibitor Z-ATAD-FMK was Pten-null hepatocytes to oxidative stress–induced cytotoxic- from BioVision. ity. Together, our data suggest that basal phosphorylation of pIRES CA-AKT and WT-AKT were constructed by eIF2a induced by activation of PI3K may act as an adaptive inserting the coding sequence of the genes into the multiple response for the fast growing cells to cope with chronic stress. cloning site of pIRES-GFP vector. The original constructs for PTEN, csPTEN, CA-AKT, DN-AKT, and WT-AKT Materials and Methods were obtained from Liliental and colleagues (11). pIRE-GFP is used as control vector for all experiments where pIRES Animals constructs are used. pSG5-wtPTEN was from Addgene loxP/loxP þ loxP/loxP Pten ; Alb-Cre (Mut, Pten null) and Pten ; (plasmid 10750) and provided by Ramaswamy and collea- Alb-Cre (Con) mice were developed and characterized as gues (12). pFLAG-CReP (amino acid 24–698) was from previously described (4). All animals were kept in a 12-hour Jousse and colleagues (13). We subcloned the coding light/dark cycle controlled facility. All experimental proce- sequence into the p3xFLAG-myc-CMV-26 vector from dures are carried out following USC IACUC guidelines. Sigma.

Cell culture and transfection Immunoblotting analysis Immortalized hepatocyte cell lines were established from Cell lysate preparation and immunoblot analysis were livers of Con and Pten null (Mut) mice (5). Briefly, freshly conducted as described (14). Antibodies against phospho- isolated hepatocytes were immortalized spontaneously with eIF2a, eIF2a, phospho-ERK, phospho-AKT, PTEN, PKR, long-term culturing by a 3T3 protocol and maintained in caspase-8, and -9 were from Cell Signaling Technology; anti- Dulbecco's Modified Eagle's Medium (DMEM; Media- AKT, anti-KDEL, anti-CHOP, and anti-GRP78 antibodies tech) supplemented with 10% FBS (US Scientific), 5 mg/mL were from Santa Cruz Biotechnology; and anti-actin anti- insulin (Sigma), and 10 ng/mL epidermal growth factor body was from Sigma. Anti-fatty acid synthase antibody was (Invitrogen; ref. 5). Mouse embryonic fibroblasts (mEF; obtained from Millipore. Anti-phospho-PKR antibody was kindly provided by Dr. Hong Wu; University of California, from Abcam. Anti-caspase-3 antibody was from BD Los Angeles, CA; ref. 10), HepG2 (obtained from USC liver Biosciences. core facility), Huh-7 (a generous gift from Dr. James Ou; University of Southern California, Los Angeles, CA), and Cell survival assay PLC.PRF/5 (provided by Dr. Aiwu Ruth He; Georgetown Cells were seeded at a density of 3 103 cells per 96-well University, Washington, DC) were cultured in DMEM plate and then treated with H2O2, L-glutamic acid, or SIN-1 supplemented with 10% FBS. Hep3B cells were provided at 37 C, 5% CO2 with indicated doses. After 24 hours of by Dr. Shelly Lu (University of Southern California, Los treatment, MTT (50 mg/mL) was added into the culture and Angeles, CA) and cultured in DMEM with 10% FBS and mixed by tapping gently on the side of the tray. After 1 nonessential amino acids (Invitrogen). SNU398, incubating at 37C for 4 hours, the formazan crystals were SNU449, and SNU475 (from Dr. Aiwu Ruth He, George- dissolved by adding dimethyl sulfoxide and then incubating town University) were cultured in RPMI-1640 (Mediatech) at 37C for 30 minutes, and the absorbance at 570 nm was with 10% FBS. measured on a microplate reader. Each sample was assayed in Hepatocytes were transfected with the Lipofectamine pentad. 2000 system (Invitrogen) as described in the manufacturer's instructions. Cells were culture in 6-well plates (1 105 to Propidium iodide staining and flow cytometry 2 105 cells/well) overnight to allow attachment. Four Hepatocytes were plated in 12-well plates at the density of microgram DNA was delivered with 8 mg Lipofectamine 0.5 105 to 1 105 cells/well. After treating with 2000 in serum-free medium. Cells were harvested 24 hours 10 mmol/L H2O2 for 24 hours, cells were trypsinized and after transfection. then collected by centrifuge. As described previously (15), cells resuspended in PBS were stained with 1 mg/mL pro- Xenograft pidium iodide (PI) for 15 minutes at room temperature. Nude mice of 3 to 4 months of age were obtained from Samples were then analyzed immediately with the BD Jackson Laboratory. Single cell suspensions of Con and Mut LSR II flow cytometry system. hepatocytes were obtained and prepared at 4 different concentrations (5 105,1 106,5 106, and 1 RNA isolation and quantitative real-time PCR 107). Each mouse was injected subcutaneously with 0.1 mL Total RNA was isolated with TRIzol (Invitrogen) follow- cell suspension. Tumor growth was observed, and experi- ing the manufacturer's instructions. Reverse transcription ments were terminated 3 months later when the volume of and quantitative PCR were carried out with M-MLV reverse the largest tumor reached 1.5 cm3. transcriptase system (Promega) and the Maxima SYBR

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Green qPCR Master Mix (Fermentas) following the manu- Results facturer's instructions. Gene-specific primers are: CReP: forward 50-AGTCTC- Deletion of Pten in hepatocytes results in high oxidative TGAGTTCACTGCGGC-30, reverse 50-GGCGCTGCA- stress GAGTCTAAAGC-30 and glyceraldehyde-3-phosphate de- Oxidative stress occurring with inflammation is often hydrogenase (GAPDH): forward 50-GCACAGTCAA implicated in promoting the development of tumors (18). GGCCGAGAAT-30, reverse 50-GCCTTCTCCATGGT- Free radicals produced with oxidative stress can be both GGTGAA-30. The cycling condition was 95C for 5 min- genotoxic and cytotoxic, causing some cells to acquire utes followed by amplification for 40 cycles at 95C for 30 survival advantages whereas other cells to undergo cell death. seconds, 60C for 30 seconds, and 72C for 30 seconds in We studied a gene that is commonly dysregulated in human the Bio-Rad iCycler. Relative expression of mRNA levels liver cancers, PTEN and its role in oxidative stress response in was determined (using GAPDH as a standard) with the the liver. We have shown previously that mice lacking PTEN D D C PtenloxP/loxP Alb-Creþ Pten - t method (16, 17). in the liver ( ; ; null) develops liver steatosis (Fig. 1A and Supplementary Fig. S1) as well as liver Statistical analysis cancer (4, 7). The steatosis in the liver is accompanied by The data are presented as means the SEM. Differences high H2O2 contents indicating that the hepatocytes are between individual groups were analyzed by the Student t experiencing high oxidative stress conditions (Fig. 1B, left). test, with 2-tailed P values less than 0.05 considered statis- We also investigated enzymes responsible for reducing tically significant. oxidative stress in the cells. The expression of these enzymes

Figure 1. High oxidative stress conditions in Pten-null liver. A, deletion of Pten leads to lipid accumulation in liver hepatocytes. Images are hematoxylin and eosin (H&E)- stained liver sections from Pten loxP/loxP control (Con, Pten ; Alb-Cre ) and Pten null (Pten null, loxP/loxP þ Pten ; Alb-Cre ). Pten-null liver sections show lipid vacuoles. B, high oxidative stress in Pten- null liver. Left, H2O2 levels are signiﬁcantly higher in Pten-null livers versus controls (Con). Expression of enzymes responsible for scavenging free radicals, glutathione peroxidase (GPx), and glutathione-S- transferase (GST) are both increased (right 2 panels). n ¼ 5; , P 0.05. C, immunohistostaining for lipid peroxidation indicates high oxidative stress in Pten-null liver. Staining for 4-HNE (green) indicates higher lipid peroxidation products in Pten-null liver versus controls. Blue indicates 4',6- diamidino-2-phenylindole (DAPI) staining for nuclei.

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often increases as a result of increased oxidative stress. The in control (Con) hepatocytes in a dose-dependent manner. mRNA expressions of 2 of such enzymes, hydrogen peroxide The cell survival rate decreased from 88% to 12% as the scavenger glutathione peroxidase and glutathione-S-trans- doses increased from 1.25 to 10 mmol/L (Fig. 2A). The Pten- ferase are also significantly higher in Pten-null mice than in null hepatocytes (Pten null), however, survived 1.25 mmol/L controls (Fig. 1B, right 2 panels). Together, these data H2O2 for 24 hours without detectable cell death. At high suggest that the hepatocytes in the Pten-null mice are under dose (10 mmol/L), more than twice as much Pten-null cells high oxidative stress conditions. In addition, we analyzed survived (29%) than the Con cells (12%). The improved liver tissues for trans-4-hydroxy-2-nonenal (4-HNE), a lipid survival of Pten-null cells was also noted when cells were peroxidation product (Fig. 1C). Immunostaining with treated with SIN-1 (Fig. 2B), a peroxinitrite donor used to 4-HNE antibody identified dramatically more 4-HNE aggre- generate nitric oxide and superoxide radical (19), as well as gates in the Pten-null livers versus the controls (Fig. 1C), L-glutamic acid (Fig. 2C), which depletes intracellular cys- further supporting the presence of oxidative stress conditions teine and glutathione, leading to oxidative stress (20, 21). in the Pten-null livers. Both treatments resulted in a dose-dependent cytotoxicity in the Con cell line but a significantly diminished effect in the Resistance of Pten-null hepatocytes to cellular stress Pten-null cell line. Pten To investigate how -null cells cope with the high We further evaluated H2O2-induced cell death by staining oxidative stress conditions that they are exposed to in vitro, the unpermeablized cells with PI. Dead cells with compro- we established isogenic cell lines from the livers of control mised membranes incorporate PI and thus can be detected loxP/loxP loxP/loxP (Pten ; Alb-Cre ) and Pten-null (Pten ; with flow cytometer. Treatment of Con hepatocytes with 10 Alb-Creþ ) mice with a standard 3T3 protocol (Supplemen- mmol/L H2O2 for 24 hours resulted in the appearance of a tary Fig. S2). As expected, the Pten-null (Mut) hepatocytes distinct cell population that are high for PI staining (Fig. 2D). are transformed and capable of forming colonies on soft agar This cell population was not present in the untreated Con and in nude mice xenograft assays (Supplementary Fig. S3). cells, suggesting that this cell population is the dead cells The control cell lines (Con) are also capable of forming induced by H2O2. Under the indicated treatment conditions, colonies and graft tumors (Supplementary Fig. S3A). The approximately 30% of Con hepatocytes became positive for tumor graft derived from the control cell lines resembles the PI labeling, indicating that they are dead cells with compro- normal liver structure, suggesting that the control cell lines at mised membranes. Under the same conditions, only 1% of least partially retain the properties of the wild-type hepato- the Pten-null hepatocytes incorporated PI. This represents a cytes in vivo. The grafted tumors formed from the Pten-null 30-fold decrease in cell death when PTEN is lost. Together cell lines morphologically resemble the tumors observed with the cytotoxic analysis, these data suggest that PTEN loss loxP/loxP þ in vivo in the Pten ; Alb-Cre mice. Significantly more protects hepatocytes from oxidative stress–induced cell death. colonies were formed in the Pten-null cell cultures as compared with the controls when plated on soft agar (Supple- Enhanced basal eIF2a phosphorylation in Pten-deficient mentary Fig. S3B). These data suggest that the Pten-null cells cells may have better survival potentials under the stressful The integrated stress response (ISR) mechanism may condition of soft agar culturing than the control cells. underlie the conditioned protection against more severe On the molecular level, the isogenic hepatic cell lines injury in cells growing in chronic low levels of stress condi- recapitulated the molecular signaling profiles observed in tions like the stressed conditions the Pten-null cells are freshly isolated livers (Supplementary Fig. S3C). Both cell described in the work of Galicia and colleagues (7). We lines express albumin indicating that the origin of the cells is analyzed phosphorylation of eIF2a, the central regulator of liver hepatocytes. The molecular signaling pathways such as the ISR response mechanism and found that p-eIF2a is mTOR were activated in the Pten-null cell line as predicted. induced in unstressed Pten-null hepatocytes comparing with Phospho-AKT is robustly induced when Pten is deleted. We the control cells with intact PTEN (Fig. 3A, left). Similarly, also observed enhanced expression of fatty acid synthase in this basal level hyperphosphorylation of eIF2a is also the Pten-null cell line versus the controls, similar to what we observed in vivo in Pten-null liver lysates versus control lysate have seen previously in mouse liver lysates (Supplementary (Fig. 3A, right). We observed a consistent 2-fold increase of Fig. S3C; ref. 4). eIF2a phosphorylation in Pten-null livers and hepatocytes The preliminary observation from the colony-forming versus that of controls. This difference in eIF2a phosphor- assay suggests that the Pten-null hepatocytes are more ylation is correlated with an increase of p-AKT but not resistance to the stressful culture conditions as more colonies extracellular signal–regulated kinase (p-ERK; Fig. 3A, left). formed with the Pten-null versus Con cell lines. To deter- To test whether the change in eIF2a phosphorylation mine whether loss of PTEN protects the hepatocytes from also alters the cellular response to other stress, we tested cell death induced by high oxidative stress condition, we theresponseofthePten-null hepatocytes to induced stress treated the control and Pten-null cells with 3 oxidative of the ER and compared this response to the control cells. stressors: hydrogen peroxide (H2O2), SIN-1, and L-glutamic Thapsigargin is a chemical used to induce stress of the acid. Hydrogen peroxide is widely regarded as a cytotoxic ERandaccumulationofGRP78(22).InbothConand agent that induces cell death through oxidative stress. Pten-null cell lines, thapsigargin treatment induced the Twenty-four hours exposure to H2O2 induced cell death expected increase of GRP78. No difference in GRP78

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Figure 2. Improved survival and decreased cell death in Pten-null hepatocytes when exposed to oxidative stress. Control (Con) and Pten-null hepatocytes are treated with H2O2 (A), SIN-1 (B), and L-glutamic acid (C) at the indicated concentrations. Hundred percent of survival rate is deﬁned as the values in cells that were treated with vehicles only. n ¼ 3; , P < 0.05. D, cell death analysis via PI staining. Control (Con) and Pten-null hepatocytes

are treated with 10 mmol/L H2O2 or vehicle for 24 hours, harvested, stained, and analyzed on ﬂow cytometry. The y-axis is PI area, the x-axis is PI width. Insets, PI-positive dead cells.

were observed between Con and Pten-null cell lines at any level of phospho-eIF2a remained for 8 hours before return- time points after thapsigargin treatment (Fig. 3B). Sim- ing to baseline levels. By 24 hours, the phospho-eIF2a levels ilarly, no changes were observed with other ER proteins in Con cells are reduced back to control levels whereas such as GRP94 and PDI either, suggesting that the phosphorylation of eIF2a is again higher in the Pten-null response of ER to unfolded protein (UPR) accumulation cells versus control ones. Thus, phosphorylation of eIF2a is not altered by PTEN loss. Similar to the response to seems to respond to PTEN signal and ER stress indepen- Pten a H2O2-induced stress, the -null hepatocytes survived dently. Basal phosphorylation of eIF2 is altered with ER stress–induced cell death much better as indicated by PTEN status where phosphorylation of eIF2a is also the attenuated cyclophosphamide-Adriamycin-vincris- robustly increased transiently (lasting 8 hours) when UPR tine-prednisone (Oncovin; CHOP) induction in response is induced. The former event, basal phosphorylation of to thapsigargin treatment. These data suggest that PTEN- eIF2a changes with PTEN status, but is independent of regulated signals may confer resistance to stress-induced ER stress signals. Consistent with this UPR independent role cell death regardless of the type of stress. of PTEN regulated eIF2a phosphorylation, eIF2a phos- During thapsigargin-induced ER stress response, the phorylation correlates with induction of AKT but not differences of p-eIF2a observed between control and GRP78 expression in vivo when PTEN is lost (Fig. 3C). Pten-null cells disappeared, as acute stress response in control These data are consistent with the notion that PTEN and cells led to enhanced phosphorylation of eIF2a (Fig. 3B). PI3K signal regulates all integrated stress response at the site Immediately following treatment with thapsigargin at of eIF2a regardless of the source of the stress. 1 hour, we observe that the phosphorylation of eIF2a is The caspases are known targets of PTEN and PI3K induced to a similar level in Con and Pten-null cell lines. This signaling pathway. We evaluated the potential involvement

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Figure 3. Elevated phosphorylation of eIF2a in PTEN-deficient hepatocytes and livers. A, immunoblotting analysis of p-eIF2a, eIF2a, p-ERK, and p-AKT in hepatocytes (left) and mouse liver (right). Actin is detected as loading control. Bottom, quantification of Western blots. , P < 0.05. B, UPR stress is induced by treatment with 20 nmol/L thapsigargin in control (C) and Pten-null (M) hepatocytes to assess the response of the cells to ER stress. In response to thapsigargin treatment, ER chaperone protein expressions (GRP78, 94 and PDI) increase. No consistent differences are detected between control (C) and Pten-null (M) hepatocytes. Apoptotic factor CHOP is induced as a result of thapsigargin treatment. Loss of Pten significantly attenuates the induction of CHOP. At basal level, phospho-eIF2a is higher in Pten- null (M) hepatocytes versus controls (C). This difference diminishes in thapsigargin-treated cells (at 1, 2, and 8 hours) and returns 16 hours after thapsigargin treatment. C, p-eIF2a correlated with p-AKT but not GRP78 in mouse liver. No UPR stress is detected in Pten-null livers. GRP78 levels are the same in liver lysates from Pten control (Con) and null mice. D, treatment of control and Pten-null hepatocytes with caspase-12 inhibitor (C12) does not result in alteration in p-eIF2a. Veh, vehicle.

of the caspase cascades in PTEN and PI3K signal–regulated Pten-null cells to evaluate whether this introduction can stress response. We are unable to observe any cleaved diminish phosphorylation of eIF2a observed with PTEN caspase-3, the ﬁnal step of the caspase cascade, with loss. Compared with the GFP-transfected cells, introduc- a H2O2 treatment (Supplementary Fig. S4). Similarly, we tion of wtPTEN led to reduction in phospho-eIF2 also did not observe changes of caspase-8, which is involved (Fig. 4A and Supplementary Fig. S5). The phosphatase in the extrinsic apoptotic pathway, or caspase-9, involved in dead mutant of Pten (csPTEN) did not induce phosphor- a a the intrinsic apoptotic pathway with our H2O2 treatment ylationofeIF2 , suggesting that this basal eIF2 phos- conditions (data not shown). Thus, cell death induced by phorylation depends on the phosphatase activity of a H2O2 in the hepatocytes is likely mediated by caspases- PTEN. The enhanced phosphorylation of eIF2 is also independent pathways such as the lysosomal protease path- observed in the isogenic mEFs where PTEN is lost as well ways (23). Caspase-12 is reported to mediate ER stress– as HepG2 cells where PTEN expression is relatively lower induced cell death. We treated the control and Pten-null cells than the Huh-7 cells (Fig. 4B). with an inhibitor for caspase-12 (24). Inhibition of caspase- To further interrogate the downstream signaling of 12 had no effect on the phosphorylation of eIF2a (Fig. 3D), PTEN, we manipulate the PI3K signaling pathway with further conﬁrming that ER-mediated events are unlikely to chemical inhibitors. LY294002 is an inhibitor for PI3K and be involved in ISR response regulation by PTEN loss. blocks the pathways downstream of PI3K signaling. We treated the Pten-null cells with an activated PI3K signaling PI3K/AKT signaling regulates the phosphorylation of pathway with LY294002 (20 mmol/L). This treatment led eIF2a to a time-dependent reduction in the phosphorylation of To explore the signaling events leading to the accumu- eIF2a that follows the inhibition of p-AKT (Fig. 4C). The lation of phospho-eIF2a, we introduced PTEN into the phosphorylated AKT is dramatically decreased 30 minutes

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level of phospho-eIF2a recovered 6 hours after treatment following the complete recovery of phospho-AKT. These data suggest that phosphorylation of eIF2a may be regulated by AKT signaling. To substantiate the potential regulation of eIF2a by PI3K/AKT and determine whether AKT is indeed involved in this regulation, we introduced constitutively active myristylated AKT (CA), dominant negative AKT (DN), wild-type (WT) AKT, and vector to the Con and Pten-null hepatocytes (Fig. 4D). In the control cells, introduction of caAKT moderately induced phospho- eIF2a. Inhibition of AKT activity with dominant negative AKT signiﬁcantly reduced phospho-eIF2a, suggesting that this basal level of phospho-eIF2a is at least partially dependent on AKT activity. In Pten-null cells, a similar trend is observed though more moderate, likely due to the already induced hyperphosphorylation of AKT and the inability of DN-AKT to signiﬁcantly reduce the activity of this hyperactive AKT.

CReP downregulation mediates the hyperphosphorylation of eIF2a in Pten-null cells The delayed response of phospho-eIF2a to LY294002 treatment comparing with phospho-AKT suggests that the regulation of eIF2a phosphorylation by PI3K/AKT signaling is not direct. We found that the stress-related kinases such as the ER stress–induced PERK (8) are unlikely the mediators for the observed basal level increase of eIF2a phosphorylation as ER stress–induced response on phospho-eIF2a dose not differ between the 2 cell lines (data not shown). RNA-dependent protein kinase (PKR) Figure 4. PI3K/AKT signal regulates eIF2a phosphorylation. A, expression was previously reported to regulate PTEN-mediated of wtPTEN in Pten-null hepatocytes results in downregulation of eIF2a eIF2a independent of PI3K (25). We determined whether phosphorylation. GFP, wild-type (wt)PTEN, and csPTEN containing PKR may regulate the basal eIF2a phosphorylation we plasmids are transfected into Pten-null hepatocytes to evaluate their effects on p-eIF2a. wtPTEN induces downregulation of p-eIF2a as observed with the unstressed hepatocytes. We found that compared with GFP controls but not csPTEN. Actin and eIF2a were PKR is not detected in the untransfected and unstressed detected as loading controls. B, hyperphosphorylation of eIF-2a is hepatocytes cell lines (data not shown), suggesting that observed in several cell lines lacking PTEN: hepatocyte cell lines isolated this kinase is unlikely to be responsible for the enhanced from control (C) and Pten-null (M) mice, mEFs established from the Pten basal phosphorylation of eIF2a observed with PTEN loss. null (M) and control (C) mice and human hepatocytes cell lines with differential levels of PTEN expression (Huh-7 and HepG2). C, treatment of We also were unable to observe an appreciated change in Pten-null hepatocytes with 20 mmol/L PI3K inhibitor LY294002 leads to the phosphatase GADD34 between Con and Pten-null downregulation of p-eIF2a. Phospho-AKT is blotted to confirm the cells in the unstressed conditions (data not shown). inhibition. Phospho-eIF2a and eIF2a are also analyzed on the same GADD34 is induced under ER stress whereas not detect- membrane. D, AKT constructs (CA, constitutive active; WT, wild-type; and DN, dominant negative) are transfected into Control (Con) and Pten- able in unstressed cells (13, 26, 27). The lack of response null hepatocytes to assess the role of AKT in the regulation of p-eIF2a. in GADD34 is consistent with the observation that UPR The transfection efficiency is tested by examining p-AKT level. In both response did not differ between control and Pten-null cells control (Con) and Pten-null cell lines, transfection of CA-AKT (CA) results (Fig.3B).AhomologueofGADD34,CRePisthoughtto in induction of p-eIF2a. DN-AKT (DN) significantly downregulates p-AKT regulate the phosphorylation of eIF2a at basal level (13) and leads to almost 2-fold decrease in p-eIF2a in control cells compared with vector (Vec)-transfected cells. In Pten-null cells, AKT activity is whereas GADD34 responds to stress-induced recovery of moderately inhibited, leading to moderate attenuation of eIF2a phospho-eIF2a.CRePisalsofoundtohaveagrowth phosphorylation. regulatory role (28), thus a more likely candidate to integratethechronicstressresponsewithadaptivegrowth response. In the Pten-null cells, we observed a significant after LY294002 treatment and started to recover at 1 hour decrease of CReP transcript level (Fig. 5A). The mRNA posttreatment. Phospho-eIF2a started to reduce at 30 levels of CReP were 40% less in the Pten-null cells versus minutes but reached the lowest levels 2 hours after treatment the controls. Treatment of Pten control hepatocytes cell with LY294002. This long delay suggests that the effect of line with insulin-like growth factor (IGF-1) to induce PI3K/AKT on eIF2a phosphorylation is likely indirect. The PI3K activity led to a significant downregulation of

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PTEN/PI3K and eIF2a

Figure 5. CReP is downregulated in Pten-null hepatocytes. A, total RNA from Con and Pten-null hepatocytes is extracted and then reverse transcribed into cDNA. Quantitative PCR is carried out to compare the mRNA levels of CReP in Con and Pten-null cells. Each sample is assayed in triplicate and repeated 3 times. , P 0.05. B, control and Pten-null (Mut) hepatocytes are treated with IGF-I and LY294002, respectively, to induce or inhibit PI3K/AKT signaling. Induction of PI3K/AKT with IGF-I in control cells leads to attenuation of CReP mRNA expression. Inhibition of PI3K/AKT with LY294002 resulted in induction of CReP mRNA expression. C, transfection of CReP to Pten-null (Mut) hepatocytes leads to downregulation of p-eIF2. D, hepatocytes are transfected with CReP or vector as control and then treated with 5 mmol/L H2O2 for 72 hours. Cell death is evaluated by using PI staining followed by ﬂuorescence-activated cell-sorting (FACS) analysis. CReP transfection restores the sensitivity of Pten-null hepatocytes

H2O2 treatment–induced cell death. mRNA expression of CReP (Fig. 5B). Conversely, block- sible for the stress resistance phenotype observed with the ing PI3K activity in Pten-nullcelllinesresultedina Pten-null cells. signiﬁcant induction of CReP expression (Fig. 5B). Together, these data suggestthatPI3Ksignalsmayreg- Discussion ulate basal phosphorylation of eIF2a by downregulating the phosphatase CReP. The ISR is a defensive mechanism that cells developed to To evaluate the relationship between the upregulation of cope with the environmental insults they encounter (8). eIF2a and downregulation of CReP in Pten-null cells and Phosphorylation/dephosphorylation of a translation initia- their response to cellular stresses, we sought to reduce tion factor eIF2a lies at the centre of this integrated response. p-eIF2a by overexpressing CReP in the Pten-null cells. Phosphorylation of eIF2a induced by stress kinases coordi- We found that the introduction of CReP is able to reduce nates a network of translation and transcription response, the the phosphorylation of eIF2a (Fig. 5C). Consistently, we ISR mechanism to lower cellular stress. When such stress is observed that 7.7% of the Pten-null cells are stained for PI prolonged, the response switches from survival to apoptosis. in vector-transfected hepatocytes without H2O2 treatment, In this article, we found that activation of the mitogenic comparing with the 0.3% in the untransfected cells seen signaling pathway PI3K/AKT through deletion of Pten in Fig. 3. When treated with H2O2, approximately 19% of resulted in the upregulation of basal phosphorylation of the vector-transfected cells underwent cell death. Reintro- eIF2a in unstressed hepatocytes. Our results indicate that duction of CReP into Pten-null hepatocytes partially chronic induction of low level eIF2a phosphorylation restored their sensitivity to H2O2 (Fig. 5D). When CReP mediated by downregulation of the PP1 partner CReP was overexpressed, the amount of dead cells almost doubled protects the Pten-null hepatocytes against stress-induced cell (37%). Transfection alone also induced acute phosphory- death. lation of eIF2a and hepatocytes death when cells were not Oxidative stress is one of the most common forms of stress treated with H2O2, likely due to activation of stress kinases. that the cells need to cope with. Oxygen radicals accumulate These data indicate that CReP downregulation and chronic as a by-product of normal cellular processes such as metab- basal phosphorylation of eIF2a is at least partially respon- olism. A sophisticated antioxidant and oxygen radical

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Zeng et al.

scavenger systems are integrated with the ISR to reduce of eIF2a in all cases. Thus, eIF2a phosphorylation may be oxidative stress and allow cells to survive (29). Cancer cells differentially regulated at stressed and unstressed conditions. with high growth and metabolic rates are often under higher Phosphorylation/dephosphorylation of eIF2a can be con- oxidative stress than normal cells. Yet, cancer cells are able to trolled by several enzymes including kinases and phospha- survive the highly stressful conditions and develop into tases (8). The 4 kinases responding to different stresses act as tumors. It is well established that cancer cells evoke survival sensors to integrate stresses with cellular response by phos- mechanisms to evade growth/survival regulation (30). How- phorylating eIF2a. The major phosphatase involved in ever, whether and how these mechanisms may interact with extinguishing this signal is GADD34 (27), the phosphatase stress response to adapt to the stressful environment is not that is induced by stress through selective translation by known. Our study here shows that the cell growth/survival eIF2a. These kinases and GADD34 respond transiently to signaling, PI3K/AKT pathway activation promotes the acute stress conditions but cannot explain the adaptation adaptive survival of hepatocytes to oxidative stress. In liver response under low chronic stress. CReP controls the basal cancer model where PI3K signaling is upregulated because of level of eIF2a phosphorylation in unstressed cells and may loss of its negative regulator PTEN, the development of be significant for the adaptive response of cells under chronic tumors requires the underlying fatty liver disease in the Pten- low levels of stress (28). Our study indicates that over- null mice (7, 31). High levels of reactive oxygen species expression of CReP can reduce the basal phosphorylation accompany this fatty liver condition. Our data here clearly of eIF2a and restore the sensitivity of PTEN-deficient cells showed that the Pten-null hepatocytes are more advantage at to hydrogen peroxide–induced cell death. Thus, CReP- being able to grow and survive in this high reactive oxygen mediated phosphorylation of eIF2a likely acts as an adaptive species (as well as other stress) environment. response for the Pten-null hepatocytes to cope with high Our data also indicated that the interaction of PI3K levels of cellular stress. Genetic studies targeting GADD34 signaling with the ISR defense mechanism is at the level of and CReP, respectively, also support a role CReP and not eIF2a, the translation regulator that controls all stress- GADD34 in integrating growth signal with stress response related ISR. The unphosphorylated form of eIF2a is avail- (28). Loss of CReP led to growth retardation (28) whereas able to form the eIF2a-GTP-tRNAmet complex and initiates mutants lacking GADD34 is phenotypically indistinguish- translation (8). In response to cellular stress, eIF2a can be able from the wild-type controls (32, 33). We found a transiently phosphorylated by various stress-induced kinases positive correlation between the expression of CReP and (PERK, HRI, PKR, and GCN) responding to different PTEN expression in several human HCC cell lines (Sup- stimuli. The phosphorylated eIF2a cannot participate in plementary Fig. S6). Together, these data suggest that translation initiation, resulting in repression of global trans- CReP-mediated basal phosphorylation of eIF2a may under- lation and activation of selective gene expression to alleviate lie the adaptive stress response observed in cancer cells. stress conditions. The activation of PI3K signaling by Cellular adaptation to environmental stress is a major chronic loss of PTEN resulted in hyperphosphorylation of mechanism for tumor cells to respond to its stressful envi- eIF2a at basal conditions when cells are not under stress. ronment. The molecular mechanism for such response is not This hyperphosphorylation is similar to conditions where understood. Our study provided a novel molecular mech- phosphorylation of eIF2a is uncoupled from upstream stress anism on how activation of PI3K/AKT signaling may allow kinases (9). When uncoupled with the stress kinases, the cells to deal with the stress conditions and ultimately adapt to hyperphosphorylation of eIF2a at basal unstressed condi- and survive the new environment. This adaptive response of tions enhanced the cytoprotection response to lethal stress. the Pten-null hepatocytes to oxidative (and other) stress may Consistent with this observation, eIF2a phosphorylation allow them to survive the stressful environment of fatty liver in vivo was found previously to protect cells against oxidative H2O2 and play a role in the development of tumors. Our and glutamate toxicity (3). Thus, chronic phosphorylation study uncovered a novel role of PTEN in regulating the of eIF2a resulting from activation of PI3K signaling may adaptive response of cancer cells to chronic stress through represent an adaptive response of fast growing cells to stress. modulating the CReP/eIF2a pathway. The major contradictory to this observation is the report that PTEN, independently of PI3K upregulated eIF2a and Disclosure of Potential Conflicts of Interest control translation (25). Under doxycycline induction (likely high stress) conditions, PTEN was found to enhance No potential conflicts of interest were disclosed. phosphorylation of eIF2a by inducing the stress kinase PKR through its C2 domain. Through this phosphorylation, Grant Support PTEN is thought to control translation independent of PI3K. This action, involves acute stress response at the stress This work is supported from USC liver cancer seed fund (to B.L. Stiles). N. Zeng is supported by the NIH-sponsored Predoctoral Research Training Program in Cellular, sensing and not ISR response as PKR is induced in cells Biochemical, and Molecular Sciences in USC. where PTEN levels are manipulated through transfection The costs of publication of this article were defrayed in part by the payment of page and doxycycline induction. Our observation with vector, charges. This article must therefore be hereby marked advertisement in accordance with GFP, PTEN, and AKT construct transfected experiments 18 U.S.C. Section 1734 solely to indicate this fact. supported this possibility. Regardless of the gene introduced, Received June 23, 2011; revised September 29, 2011; accepted October 6, 2011; introduction of plasmids resulted in hyperphosphorylation published OnlineFirst October 18, 2011.

1716 Mol Cancer Res; 9(12) December 2011 Molecular Cancer Research

PTEN/PI3K and eIF2a

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www.aacrjournals.org Mol Cancer Res; 9(12) December 2011 1717

Adaptive Basal Phosphorylation of eIF2α Is Responsible for Resistance to Cellular Stress−Induced Cell Death in Pten-Null Hepatocytes

Ni Zeng, Yang Li, Lina He, et al.

Mol Cancer Res 2011;9:1708-1717. Published OnlineFirst October 18, 2011.

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