AP-1 Regulates Cyclin D1 and C-MYC Transcription in an AKT-Dependent Manner in Response to Mtor Inhibition: Role of AIP4/Itch-Mediated JUNB Degradation

Published OnlineFirst December 6, 2010; DOI: 10.1158/1541-7786.MCR-10-0105

Molecular Cancer Signaling and Regulation Research

AP-1 Regulates Cyclin D1 and c-MYC Transcription in an AKT-Dependent Manner in Response to mTOR Inhibition: Role of AIP4/Itch-Mediated JUNB Degradation

Raffi Vartanian1,2, Janine Masri1, Jheralyn Martin1, Cheri Cloninger1, Brent Holmes1, Nicholas Artinian1, Alex Funk1, Teresa Ruegg1, and Joseph Gera1–4

Abstract One mechanism by which AKT kinase-dependent hypersensitivity to mammalian target of rapamycin (mTOR) inhibitors is controlled is by the differential expression of cyclin D1 and c-MYC. Regulation of posttranscriptional processes has been demonstrated to be crucial in governing expression of these determinants in response to rapamycin. Our previous data suggested that cyclin D1 and c-MYC expression might additionally be coordinately regulated in an AKT-dependent manner at the level of transcription. Under conditions of relatively quiescent AKT activity, treatment of cells with rapamycin resulted in upregulation of cyclin D1 and c-MYC nascent transcription, whereas in cells containing active AKT, exposure repressed transcription. Promoter analysis identified AKT- dependent rapamycin responsive elements containing AP-1 transactivation sites. Phosphorylated c-JUN binding to these promoters correlated with activation of transcription whereas JUNB occupancy was associated with promoter repression. Forced overexpression of JunB or a conditionally active JunB-ER allele repressed cyclin D1 and c-MYC promoter activity in quiescent AKT-containing cells following rapamycin exposure. AIP4/Itch- dependent JUNB protein degradation was found to be markedly reduced in active AKT-containing cells compared with cells harboring quiescent AKT. Moreover, silencing AIP4/Itch expression or inhibiting JNK mediated AIP4 activity abrogated the rapamycin-induced effects on cyclin D1 and c-MYC promoter activities. Our findings support a role for the AKT-dependent regulation of AIP4/Itch activity in mediating the differential cyclin D1 and c-MYC transcriptional responses to rapamycin. Mol Cancer Res; 9(1); 115–30. 2010 AACR.

Introduction target of rapamycin complex 1 (mTORC1) is composed of Raptor, mLST8, PRAS40, Deptor and the catalytic subunit Inhibitors targeting the mTOR kinase are in clinical mTOR. MTORC2 components include Rictor, mSIN1, development as potential chemotherapeutic agents. MTOR Protor, Deptor, as well as mLST8 and mTOR. MTORC1 is a regulatory kinase that integrates signals linking the ability is sensitive to rapamycin and is known to regulate several cell of cells to undergo cell-cycle transit to the availability of functions including growth, size, cap-dependent mRNA nutrients (1, 2). Treatment with mTOR inhibitors typically translation and ribosomal component biogenesis (5). results in a starvation response and cell-cycle arrest, however MTORC2 has signaling outputs regulating cell growth and cells of hematopoeitic origins undergo apoptotic death follow- motility and is generally assumed to be insensitive to rapa- ing exposure (3). The mTOR kinase is a member of the serine/ mycin, but the macrolide does seem to inhibit mTORC2 threonine phosphatidyl inositol 3-like family of kinases and is assembly in some cell lines following prolonged exposure found in at least two separate complexes (4). The mammalian (6, 7). The signaling effectors of mTORC1 include the p70S6 kinase, the translational repressor 4EBP1 and the recently described activation of SGK (8). MTORC2 is the major 1 Authors' Affiliations: Department of Research & Development, Greater kinase responsible for full activation of AKT via phosphory- Los Angeles Veterans Affairs Healthcare System, Los Angeles, California; and 2Department of Medicine, David Geffen School of Medicine, 3Jonnson lation of serine 473 (9). Comprehensive Cancer Center, and 4Molecular Biology Institute, Univer- Several studies have described the hypersensitivity of dif- – sity of California Los Angeles, California ferent tumor types to mTOR inhibitors in cells whose AKT Note: Supplementary data for this article are available at Molecular Cancer activity is elevated due to gene amplification or loss of function Research Online (http://mcr.aacrjournals.org/). mutations in the tumor suppressor PTEN (10–12). We have Corresponding Author: Joseph Gera, Greater Los Angeles VA Healthcare System, 16111 Plummer Street (151), Building 1, Room C111A, Los demonstrated that differential sensitivity can be explained, in Angeles, CA 91343. Phone: 818-895-9416; Fax: 818-895-9554. E-mail: part, by the differential regulation of cyclin D1 and c-MYC [email protected] gene expression at the levels of mRNA translation initiation doi: 10.1158/1541-7786.MCR-10-0105 and stability (13, 14). Continued internal ribosome entry 2010 American Association for Cancer Research. site (IRES)-dependent translation initiation and enhanced

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mRNA stability of cyclin D1 and c-MYC mRNAs is sufficient indicated, native AP-1 sites in the cyclin D1 and c-MYC to overcome rapamycin-induced G1 arrest. Our data however, promoters were replaced with (TATTGTA). All mutagen- also suggested coordinate regulation of cyclin D1 and c-MYC esis was confirmed by sequencing. The pMV7JUNB and transcription in addition to the posttranscriptional control pMV7JunB-ER constructs were obtained from Drs. Latifa exerted by AKT in the face of mTOR inhibition (12). How Bakiri and Moshe Yaniv (Insitut Pasteur, Paris, France). AKT activity may regulate the transcriptional responses of The HA-ubiquitin construct was provided by Dr. Ted cells to mTOR inhibitors is unknown. Dawson (Department of Neurology, Johns Hopkins Uni- In the current study, we have extended our previous versity School of Medicine). Antibodies against the follow- analysis of AKT-dependent cyclin D1 and c-MYC post- ing proteins were used: anti-HA and control IgG were from transcriptional regulation to try and understand the Santa Cruz Biotechnology; phospho-S6K, S6K, phospho- mechanisms controlling gene transcription of these deter- AKT, AKT, cyclin D1, c-MYC, JUNB, c-JUN, and JNK minants following rapamycin exposure. Tumor cells con- and antibodies were from Cell Signaling; RNA PolII phos- taining active AKT were found to repress transcription of pho-S2 CTD, phospho-c-JUN and AIP4/Itch antibodies cyclin D1 and c-MYC, whereas in cells with relatively were from Abcam; phospho-JUNB antibody was from quiescent AKT activity transcription was induced. Subse- Signalway and actin antibody from Sigma. Rapamycin quent deletion and mutational analysis of cyclin D1 and c- was obtained from LC Laboratories, MG132 was purchased MYC promoter constructs identified rapamycin responsive from Enzo Life Sciences, and the JNK inhibitor VIII was promoter elements containing AP-1 transcription factor from EMD Biosciences. Rapamycin was used at 10 nmol/L binding sites. JUNB binding to these promoter elements for 24 hours for all treatments unless otherwise indicated. correlated with transcriptional repression of cyclin D1 and c-MYC promoter activity, whereas phosphorylated c-JUN Luciferase reporter assays binding strongly activated these promoters in an AKT- The indicated reporter constructs (200 ng DNA) were dependent manner upon rapamycin treatment. Moreover, transiently transfected into U87 and U87PTEN cells. Sub- the AKT-dependent regulation of promoter activity corre- sequently, cells were exposed to 10 nmol/L of rapamycin for lated with alterations in E3 ubiquitin ligase AIP4/Itch- 24 hours after which extracts were prepared and luciferase mediated JUNB ubiquitination. These data support the activity determined. Promoterless pGL3 plasmid coding for involvement of differential AIP4/Itch-mediated JUNB firefly luciferase was used to determine basal activity and degradation in regulating the transcriptional responses of phRG-Basic cotransfected for normalization (Promega). cyclin D1 and c-MYC to mTOR inhibition in a manner Luminescence was measured using the Dual-Luciferase dependent on cellular AKT activity. Reporter Assay System (Promega).

Materials and Methods Chromatin immunoprecipitation, in vitro DNA-pull down, nuclear run on, and electrophoretic mobility Cell lines and transfections shift assays The isogenic cell lines pairs used in this study differ Chromatin immunoprecipitation (ChIP) assays and sub- significantly in their relative AKT activities by virtue of sequent real-time PCR analysis was performed as described either their PTEN status or forced expression of an activated (16). Briefly, cells were washed twice with PBS and cross- allele of AKT1. These lines were kindly provided by Ingo linked with 1% formaldehyde at room temperature for 10 Mellinghoff and Charles Sawyers and have been described minutes. Cells were rinsed with ice-cold PBS twice and þ/þ / previously (13). The isogenic Pten and Pten MEF resuspended into 100 mmol/L of Tris-HCl (pH 9.4), 10 cells were kindly provided by Hong Wu and have also been mmol/L of DTT and incubated for 15 minutes at 30C. described (15). Transient luciferase reporter transfections Cells were washed and resuspended in lysis buffer (1% SDS, were performed using FUGENE 6 (Roche) as recom- 10 mmol/L of EDTA, 50 mmol/L of Tris-HCl, pH 8.1, 1 mended by the manufacturer. To generate the JUNB protease inhibitor cocktail (Roche) and sonicated 3 times for and JunB-ER expressing lines cells were transfected similarly 10 seconds each pulse at the maximum setting (Fisher Sonic using FUGENE 6, and clones selected for G418 resistance. Dismembrator, Model 300), followed by centrifugation for 10 minutes. Immunoprecipitations were performed over- Constructs and reagents night at 4C with specific antibodies. Precipitates were The CCND1 and c-MYC promoter constructs were washed and heated at 65C to reverse cross-linking and provided by Drs. Anil Rusti (Department of Medicine, DNA fragments purified using QIAquick Spin Kit (Qiagen) University of Pennsylvania) and Linda Penn (Ontario for subsequent PCR. Detailed information regarding primer Cancer Institute, University of Toronto), respectively. sequences is available upon request. In vitro DNA pull- Mutagenesis was performed using the QuikChange site- down assays were performed as described (17) using a Directed Mutagenesis kit (Agilent Technologies) with the double-stranded oligonucleotide containing the AP-1 site appropriate mutagenic primers according to the manufac- (s) and immediately flanking sequences from either the Kip1 turer. The minimal IRES sequences from the p27 50 cyclin D1 or c-MYC promoters attached to streptavidin- UTR were inserted immediately upstream of the luciferase Sepharose beads via a 50 biotinylated plus strand according ORF in all luciferase reporter constructs (13) and where to the manufacture's recommendations (Invitrogen). Beads

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with adsorbed proteins were analyzed by SDS-PAGE fol- formed nuclear run-on analysis and observed differential lowed by immunoblotting. Nuclear run on assays were alterations in nascent transcription of cyclin D1 and c- performed as previously described (18). Electrophoretic MYC following rapamycin exposure, consistent with the PolII mobility shift assays (EMSA) were performed as described occupancy results (Fig. 1C). The relative steady-state abun- (19) and antibodies added to the binding reactions where dances of several proteins in these lines, both prior to and indicated to visualize supershifted species. following rapamycintreatmentareshown inFigure1D. As can be seen, rapamycin exposure led to inhibition of TORC1 Immunoprecipitations, immunoblot and in vitro kinase activity in all 3 paired lines and did not significantly affect and ubiquitination assays TORC2 activities, as determined via phospho-S473-AKT Immunoprecipitations and immunoblots were performed levels. This also demonstrated that the alterations in cyclin as described (13) and for displaying ubiquinated protein, D1 and c-MYC transcription are not due to increased AKT 0.1% SDS and 5 mmol/L of N-ethylmaleimide were added activation as a result of de-repressed AKT in response to to the lysis buffer to disrupt nonspecific protein interac- rapamycin via blockade of the TORC1/S6K/IRS1 feedback tions. JNK activity was assessed via an in vitro kinase assay loop. In addition, the rapamycin induced AKT-dependent kit as described by the manufacturer (Cell Signaling). In alterations in cyclin D1 and c-MYC promoter activities corre- vitro AIP4 ubiquitination reactions were performed using a lated with our previously observed effects on cell-cycle arrest in ubiquitin–protein conjugation kit as described by the man- these lines following drug treatment (12). Lines harboring Pten/ ufacturer (BostonBiochem) using immunoprecipitated active AKT (U87, LAPC4myrAKT, MEF) were mark- AIP4 and GST-JUNB as the substrate. edly hypersensitive to rapamycin resulting in G1 arrest (Fig. 1E) and concomitant inhibition of cyclin D1 and c- RNA interference analysis MYC promoter activity (Fig. 1A–C), as compared with their siRNA transfections targeting human AIP4 were performed isogenic quiescent AKT-containing counterpart lines Ptenþ/þ using double-stranded RNAs directed at sequences within (U87PTEN,LAPC4puro, MEF) which were relatively the coding region and 30 UTR (ON-TARGETplus SMART- resistant and displayed increased promoter activities following pool, Thermo Scientific). An siRNA with a scrambled se- exposure. These data demonstrate that the relative degree of quence was used as a negative targeting control. Statistical AKT activity differentially regulates the state of cyclin D1 and analysis was performed by using Student's t test and ANOVA c-MYC transcriptional activity in cells displaying AKT-depen- models using SigmaStat 4 (Aspire Software International). dent hypersensitivity to rapamycin.

Results AP-1 sites in the cyclin D1 and c-MYC promoters are required for AKT-dependent effects on transcription AKT-dependent differential regulation of cyclin D1 and To investigate these transcriptional responses we analyzed c-MYC promoter activity by rapamycin a series of promoter constructs shown inFigure 2. Towardsthis The lines used in this study have been described (12) and are end, we performed luciferase reporter gene assays utilizing isogenic lines which differ in their degrees of constitutive AKT constructs under the control of either the human cyclin D1 or activity.TheglioblastomaU87-MGcelllinecontainsaPTEN- c-MYC promoter sequences. To avoid confounding effects null mutation and has heightened AKT activity. This line was of inhibiting cap-dependent translation of the reporter fol- stably transfected with a wild-type PTEN construct which lowing rapamycin exposure, luciferase translation was driven Kip1 markedly downregulated AKT activity (10). Similarly, the via the p27 IRES sequences placed upstream of the luci- LAPC-4 prostate cancer cell line, which possesses relatively ferase open reading frame. This IRES promotes translation of quiescent AKT activity, was stably transfected with a myris- downstream ORFs following mTOR inhibition irrespective of AKT tylated allele (or empty vector control, LAPC-4puro). The AKT activity (12, 13). As illustrated in Figure 2, transient differential AKT/mTOR cascade activation of these paired transfectionofU87cellswitheitherthefull-lengthcyclinD1or lines has been described (10, 12). We also utilized murine c-MYC constructs (1,749wt and 2,052HBMwt, respec- embryonic fibroblasts (MEF) in which the Pten gene had been tively) resulted in marked decreases in luciferase activities disrupted (15). The Akt activity in these MEFs is markedly following rapamycin exposure in cells containing high levels Ptenþ/þ higherascomparedwith MEFs(15,20).Ourprevious of AKT activity. In U87PTEN cells containing relatively quies- studies (12)demonstrated prominent differences in thesteady- cent AKT activity, these full-length constructs showed sig- state mRNA levels of the cyclin D1 and c-MYC transcripts in nificant increases in luciferase activities following rapamycin these cell lines in response to mTOR inhibition. To study treatment. Thus, these promoter constructs recapitulated the potentialtranscriptionalcontributions,weanalyzedRNAPolII AKT-dependent responses of the native cyclin D1 and c-MYC promoter occupancy of these promoters via chromatin immu- promoters following mTOR inhibitor exposure (Fig. 1). noprecipitation assays (21).As showninFigure1Aand B, both Progressive deletion of the cyclin D1 promoter revealed promotersdemonstratedreducedPolIIassociationinlineswith that an AKT-dependent rapamycin responsive element lies Pten/ elevated AKT activity (U87, LAPC-4myrAkt, MEFs), somewhere between 1,095 and 770 bp upstream of the whereas those lines with relatively quiescent AKT activity transcription start site. Removal of this region abrogated the Ptenþ/þ (U87PTEN, LAPC-4puro, MEFs) displayed increased AKT-dependent response of cyclin D1 promoter activity to Pol II content following rapamycin exposure. We also per- rapamycin in U87 and U87PTEN cells. Similarly, deletion of

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Figure 1. AKT-dependent alterations in cyclin D1 and c-MYC promoter activities following rapamycin exposure. A, cyclin D1 and (B) c-MYC AKT-dependent RNA polymerase II (PolII) association following rapamycin treatment. The indicated cell lines were treated without or with C rapamycin and PolII occupancy determined. ChIP-quantitative PCR data are expressed as a ratio of cyclin D1 or c-MYC to tubulin. Mean SD are shown; n ¼ 3. C, nascent transcription of cyclin D1 and c-MYC mRNAs from the indicated lines treated without or with rapamycin. Actin and pBlueScript KS probes are also shown. Band intensities were quantified by densitometry and fold differences relative to nontreated control values are D shown in parentheses below. D, relative levels of the indicated proteins from the indicated cell lines treated without or with rapamycin. E, AKT-dependent effects on cell-cycle distributions of the indicated cell lines following rapamycin exposure. Mean SD are shown; n ¼ 3.

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Figure 2. Deletion analysis of the cyclin D1 and c-MYC promoters. The indicated constructs were transfected into U87 or U87PTEN cells, treated without or with rapamycin and luciferase activities determined. The indicated AP-1 sites within the constructs -1,749wt and -2,052HBMwt were mutated (shown in red) as described in Materials and Methods. The relative fold change in luciferase activity is shown as compared with untreated controls. Mean SD are shown; n ¼ 3. a region between 1,846 and 1,256 bp upstream of the that a 325-bp fragment of the cyclin D1 and a 590-bp transcription start site within the c-MYC promoter resulted fragment of the c-MYC promoters were responsible for in a complete loss of AKT-dependent promoter activity in regulating the responses of these 2 promoters to rapamycin response to rapamycin in both cell lines. This demonstrated in an AKT-dependent manner.

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Present within these promoter sequences were previously lowing rapamycin exposure (13), we initially investigated characterized AP-1 or AP-1–like binding sites, which we whether these site(s) within the cyclin D1 and c-MYC pro- reasonedmayplayaroleintheobserved effects on promoter moters differentially bound c-JUN in an AKT-dependent activity (22–24). Within the cyclin D1 promoter a single AP-1 manner following rapamycin exposure. As shown in Figure site was mutated and within the c-MYC promoter 3 AP-1 sites 3A, EMSAs utilizing the AP-1 sites from either the cyclin D1 were sequentially mutated (shown in red). These constructs (left) or c-MYC (right) promoters and adjacent sequences did were transiently transfected into U87 and U87PTEN cells and differentially bind c-JUN in nuclear extracts prepared from relative luciferase activity determined before and after rapamy- either U87 or U87PTEN cells following rapamycin exposure. cin treatment. As shown, mutating the cyclin D1 AP-1 binding c-JUN binding was only observed in extracts prepared from site ( 954 bp) completely abolished the rapamycin-induced quiescent AKT-containing U87PTEN cells, whereas in U87 effects on promoter activity. Similarly, mutating all 3 AP-1 sites cells containing active AKT no c-JUN binding was detectable. within the c-MYC promoter abrogated AKT-dependent pro- Similar analyses interrogating other AP-1 family members moter activity in response to rapamycin, while mutating either a (FRA-1, FRA-2, FOS A, FOS B, JUN D, JAB1) demon- single or 2 AP-1 sites resulted in partial responses. This stratedsignificant differential bindingonly forJUNB. Binding suggested that AP-1 transcription factor binding was respon- of JUNB to the cyclin D1 promoter has also been associated sible for the AKT-dependent changes in cyclin D1 and c-MYC with the repression of transcriptional activity (25) and we promoter activities following rapamycin exposure. observed significant binding of JUNB only in active AKT- containing U87 cells to the cyclin D1 and c-MYC AP-1 sites c-JUN and JUNB bind differentially to the cyclin D1 (Fig. 3B; cyclin D1, left; c-MYC, right) where promoter and c-MYC promoters in an AKT-dependent manner activity was inhibited following rapamycin treatment. We following rapamycin exposure validated this EMSA data by chromatin immunoprecipitation Within the cyclin D1 promoter, the AP-1 site has been experiments, followed by quantitative PCR analysis (16). previously demonstrated to bind protein complexes contain- Following rapamycin exposure, the AP-1 sites within the ing activated c-JUN which promoted cyclin D1 transcription cyclin D1 and c-MYC promoters bound c-JUN in cells with (25). Furthermore, activation of promoter activity via this quiescent AKT (U87PTEN) and demonstrated no appreciable AP-1 site had been shown to be driven by ERK phosphory- binding in cells with elevated AKT activity (U87; Fig. 3C, left lation of c-JUN (26). Since, we had previously demonstrated and right top). In a reciprocal fashion, JUNB was found a differential activation of ERK in our paired cells lines fol- associated with these AP-1 sites exclusively in cells containing

Figure 3. AKT-dependent c-JUN and JUNB differential binding to the cyclin D1 and c-MYC promoters following rapamycin treatment. A, EMSA analysis using 32P-labeled cyclin D1 (left) or c-MYC (right) DNA probes containing native AP-1 binding sites were carried out using the indicated nuclear extracts in the absence or presence of control (IgG) or antibody against c-JUN. B, as in (A) except antibody to JUNB was used. Arrows indicate super-shifted species. Experiments were carried out 3 times with similar results.

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relatively active AKT (U87) compared with cells containing U87 cells (Fig. 3D, left). Consistent with the chromatin quiescent AKT (U87PTEN; Fig. 3C, left and right, bottom). immunoprecipitation experiments, we observed high levels The phosphorylation states of functionally bound c-JUN and of JUNB bound to the cyclin D1 AP-1 promoter element JUNB to the cyclin D1and c-MYC AP-1 elements were also linked beads, only from nuclear extracts from active AKT- assessed in a series of in vitro DNA binding pull-down assays containing U87 cells following rapamycin exposure. No (Fig. 3D). High levels of both unbound c-JUN and JUNB are detectable amount of Ser79 phosphorylated JUNB was found present in nuclear extracts from both U87 and U87PTEN cells in U87 or U87PTEN nuclear extracts or associated with the in the absence of rapamycin, however following rapamycin cyclin D1 AP-1 element–linked beads irrespective of rapa- exposure markedly elevated levels of Ser63 phosphorylated c- mycin exposure. Similar binding data were observed in DNA JUN is associated with the cyclin D1 AP-1 promoter element pull-down experiments using the AP-1 sites and flanking linked to sepharose beads (D1-beads) in quiescent AKT- sequences from the c-MYC promoter (Fig. 3D, right; 63 containing U87PTEN cells but not in active AKT-containing MYC-beads). Again, high levels of Ser phosphorylated

rapamycin rapamycin P-S63-c-JUN P-S63-c-JUN c-JUN c-JUN P-S79-JUNB P-S79-JUNB JUNB JUNB

Figure 3. (Continued) C, extracts from U87 and U87PTEN cells untreated or treated with rapamycin were chromatin immunoprecipitated with the indicated antibodies (c-JUN, top; JUNB, bottom) and fragments were subjected to quantitative real-time PCR analysis using various primer sets spanning the promoter regions (cyclin D1, left; c-MYC, right) as indicated by the gray bars. Primer sets 1 correspond to the AP-1 binding site(s) in the cyclin D1 and c-MYC promoters. At least 2 independent experiments were performed and the mean SD are shown. D, sepharose beads were conjugated with cyclin D1 promoter element DNA (D1-beads, left) or c-MYC promoter element (MYC-beads, right) containing the native AP-1 binding site(s) or beads without linked DNA were incubated with nuclear extracts from U87 or U87PTEN cells treated without or with rapamycin as indicated. Following recovery by centrifugation and washing of the beads, bound material was analyzed by immunoblot for the indicated proteins. These experiments were performed twice with similar results.

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c-JUN bound AP-1 promoter linked beads in quiescent lated c-JUN to the AP-1 sites could be correlated with U87PTEN extracts following rapamycin exposure, whereas differential activation of JNK. Shown in Supplementary JUNB specifically associatedwith AP-1promoterlinked beads Figures 1B and 1C, JNK activity was determined by in vitro in active AKT-containing U87 extracts under similar condi- kinase analysis and was induced similarly following exposure tions. We further confirmed that JUNB was not significantly to rapamycin in U87, U87PTEN,LAPC-4myrAKT,and phosphorylated at other residues via phosphatase treatment of LAPC-4puro cells. This was consistent with the findings of nuclear extracts from U87 and U87PTEN cells before and after others (27) and suggested that additional mechanisms rapamycin exposure and found no significant electrophoretic mediated the differential transcriptional responses to rapamy- mobility changes following in vitro phosphatase treatment cin. These results taken together demonstrate that c-JUN and (not shown). In addition, we examined whether heterodimers JUNB differentially bind to AP-1 sites present in the cyclin containing c-JUN and JUNB were present following rapa- D1 and c-MYC promoters in an AKT-dependent manner mycin exposure. Nuclear extracts from both cell line pairs, following rapamycin treatment. prior to and following rapamycin treatment, were coimmuno- precipitated using either c-JUN or JUNB antibodies followed JUNB governs cyclin D1 and c-MYC promoter by immunoblot analysis. As shown in supplementary Fig- activities in an AKT-dependent manner in response to ure 1A, we were able to detect modest amounts of heterodimer rapamycin formation prior to treatment in all lines, which increased in In our in vitro DNA pull-down experiments we noted a lines containing active AKT (U87, LAPC-4myrAKT)following relatively large change in JUNB abundance following rapa- rapamycinexposure. However, wewereunable todetectstable mycin exposure as compared with c-JUN levels (see Fig. 3D) co-complexes in lines with quiescent AKT activity (U87PTEN, suggestingthattheabsolutelevelsoractivityofJUNBmayplay LAPC-4puro) following treatment consistent with the absence a role in regulating AKT-dependent cyclin D1 and c-MYC of JUNB under these conditions (see Discussion). We also promoter activity. To test this hypothesis, we initially deter- determined whether the differential binding of phosphory- mined whether forced expression of JUNB or activation of a

Figure 4. Modulation of JUNB regulates AKT-dependent cyclin D1 and c-MYC promoter activity and rapamycin sensitivity. A, control or JUNB-overexpressing

cells (U87 and U87PTEN, shaded bars; LAPC-4myrAKT and LAPC-4puro, open bars) were treated without or with rapamycin and PolII promoter occupancy determined for cyclin D1 (left) and c-MYC (right) as in Figure 1. Mean SD are shown; n ¼ 3. B, effects of rapamycin on PolII promoter occupancy of

cyclin D1 (left) or c-MYC (right) in the indicated lines (U87 and U87PTEN, shaded bars; LAPC-4myrAKT and LAPC-4puro, open bars) expressing the JunB-ER fusion in the absence or presence of 4OHT (1 mmol/L). Mean SD are shown; n ¼ 3.

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JunB-ER conditional allele would alter the AKT-dependent D1 and c-MYC promoter activity following rapamycin expo- cyclinD1andc-MYCpromoteractivitiespreviouslyobserved. sure in lines containing elevated levels of active AKT. We U87 and LAPC-4 cell line pairs, which stably overexpressed employedan siRNA strategyutilizing JUNB targeting siRNAs JUNB (Supplementary Fig. 2A, left) were treated with rapa- to specifically inhibit expression (Supplementary Fig. 2B). As mycin and promoter activities determined via ChIP assays as shown in Figure 4C, JUNB siRNA-treated cells displayed a before. As shown in Figure 4A, quiescent AKT-containing significant derepression of both cyclin D1 and c-MYC pro- U87PTEN and LAPC-4puro cellsoverexpressingJUNBdemon- moter activity in active AKT-containing cell lines. We also strated significantly lower levels of cyclin D1 (left) and c-MYC examined the degree to which modulation of JUNB expres- (right) promoter activities as compared with controls. Similar sion affected rapamycin-induced G1 arrest in these cell lines. effects were observed in U87 and LAPC-4 paired lines which As shown in Figure 4D, both the U87 and LAPC-4 cell line expressed comparable levels of a conditionally active JunB-ER pairs that stably overexpressed JUNB demonstrated protein fusion (Supplementary Fig. 2A, right; ref. 25). As increased G1 arrest of quiescent AKT-containing cells shown in Figure 4B, both in control and nonactivated JunB- (U87PTEN, LAPC-4puro) following rapamycin exposure as ER expressing cells, rapamycin exposure led to an increase in compared with controls. In a reciprocal fashion, JUNB cyclin D1 (left) and c-MYC (right) promoter activity only in siRNA-treated U87 and LAPC-4 lines which contained high quiescent AKT-containing U87PTEN and LAPC-4puro cells. levels of active AKT (U87, LAPC-4myrAKT) displayed However, in U87PTEN and LAPC-4puro cells expressing the reduced G1 arrest following rapamycin treatment JunB-ER fusion protein activated by the ligand hydroxyta- (Fig. 4E). These data taken together, demonstrate that moxifen, rapamycin-induced cyclin D1and c-MYC promoter modulation of JUNB profoundly effects rapamycin-induced activity was markedly reduced. We also attempted to deter- alterations in cyclin D1 and c-MYC promoter activities in an mine whether JUNB was required for the inhibition of cyclin AKT-dependent manner.

Figure 4. (Continued) C, effects of JUNB knockdown on rapamycin-induced alterations in cyclin D1 (left) and c-MYC (right) PolII promoter activity in the indicated cell lines. Mean SD are shown; n ¼ 3. D, S-phase cell-cycle analysis of JUNB overexpressing U87 and LAPC-4 cell line pairs following rapamycin exposure. Mean SD; n ¼ 3. E, cell-cycle analysis of JUNB siRNA-treated U87 and LAPC-4 cell line pairs following rapamycin exposure. Mean SD; n ¼ 3.

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JUNB is posttranslationally regulated via AIP4/Itch LAPC-4puro cells (see Supplementary Fig. 3). JUNB ubiqui- ubiquitin ligase activity in an AKT-dependent manner tin (Ub)-mediated turnover has been demonstrated to be following rapamycin exposure induced via the JNK-dependent E3 ligase AIP4/Itch (29). To gain further insight as to how AKT-dependent JUNB This prompted us to examine whether AIP4 activity was expression might be regulated following rapamycin exposure involved in this regulation. We initially examined the effects we examined whether JUNB may be subject to differential of rapamycin exposure on AIP4 expression in the paired cell ubiquitin mediated proteosomal degradation. JUNB expres- lines. As shown in Figure 5B, a time-dependent increase in sion is known to be regulated posttranslationally via this AIP4 protein levels was observed following rapamycin treat- mechanism (28). The U87 and LAPC-4 cell line pairs were ment irrespective of AKT activity. JUNB levels increased in transfected with a HA-tagged ubiquitin construct and treated active AKT-containing U87 and LAPC-4myrAKT cells, with rapamycin as indicated in Figure 5A. Extracts were pre- whereas displayed reductions in protein amounts in quies- pared, immunoprecipitated with JUNB antibodies and sub- cent AKT-containing U87PTEN and LAPC-4puro lines, jected to immunoblot analysis using anti-HA sera. As can be consistent with the large alterations in JUNB levels pre- seen, increased endogenous JUNB ubiquitination was ob- viously observed in the in vitro DNA-pull down assays served in quiescent AKT-containing U87PTEN and LAPC- following rapamycin exposure (see Fig. 3D). Increases in 4puro cells following rapamycin exposure in these ubiquination phosphorylated c-JUN were observed in all lines correlating assays relative to their active AKT-containing isogenic coun- with increased JNK activity following rapamycin treatment terpart lines. We also assessed the affects of the proteasome (see Supplementary Fig. 1B) whereas total c-JUN levels did inhibitor MG132 in both paired lines following rapamycin not appreciably change. We then examined whether rapa- exposure. MG132 blocked therapamycin-induced decreasein mycin exposure resulted in alterations of AIP4 activity in JUNB levels in quiescent AKT-containing U87PTEN and extracts prepared from the cell line pairs. AIP4 immune

Figure 5 AKT-dependent variations in JUNB protein ubiquitination, AIP4 protein levels and activity following rapamycin exposure. A, JUNB ubiquitination

in U87, U87PTEN, LAPC-4myrAKT, and LAPC-4puro cells following rapamycin exposure as indicated. Cells were transfected with an HA- ubiquitin construct, treated without or with rapamycin and extracts immunoprecipitated B using JUNB antibodies and immunoprecipitates subsequently immunoblotted for the extent of HA-ubiquitination using a-HA antibody and total JUNB. Relative densitometric values are shown in parentheses below. B, relative AIP4, JUN and actin protein levels following rapamycin exposure (10 nmol/L) for various time points from the indicated cell lines. C,

AIP4 activity in U87, U87PTEN, LAPC-4myrAKT, and LAPC-4puro C cells following rapamycin exposure. Cells were treated with rapamycin as indicated and AIP4 immunoprecipitates utilized in in vitro ubiquitination reactions using GST-JUNB as a substrate. Poly- ubiquinated JUNB was visualized by immunoblotting using a-JUNB antibodies.

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complexes were used in in vitro ubiquitination assays from AIP4/Itch knockdown abrogates rapamycin-induced the indicated cells lines and rapamycin treatments using AKT-dependent cyclin D1 and c-MYC promoter GST-JUNB as a substrate. As shown in Figure 5C, AIP4 regulation activity was significantly increased in both quiescent AKT- Our results demonstrating AKT-dependent differential containing lines, U87PTEN and LAPC-4puro relative to AIP4 activity and JUNB ubiquitination motivated us to deter- their active AKT-containing counterpart lines following mine whether knockdown of AIP4 would have effects on rapamycin treatment. These results demonstrate a differ- cyclin D1 and c-MYC promoter activity in the paired cell lines ential AKT-dependent increase in ubiquitin-conjugation of upon mTOR inhibition. As shown in Figure 6C, treatment of JUNB and AIP4 activity in response to rapamycin. lines with siRNAs targeting AIP4 resulted in inhibition of

Figure 6. Knockdown of AIP4 abrogates AKT-dependent cyclin D1 and c-MYC promoter activity following mTOR inhibition. A, U87, U87PTEN, LAPC-4myrAKT, or LAPC-4puro cells were treated with siRNA targeting AIP4 or a scrambled (scr) nontargeting control siRNA in the presence of rapamycin and extracts immunoblotted for the indicated proteins. B, cyclin D1 (left) and c-MYC (right) promoter PolII occupancy in the paired cell lines (U87 and U87PTEN, shaded bars; LAPC-4myrAKT and LAPC-4puro, open bars) following treatment with the indicated siRNAs and rapamycin. Mean SD are shown; n ¼ 3. C, S-phase cell-cycle analysis of AIP4 siRNA-treated treated cell lines in response to rapamycin exposure. Mean SD; n ¼ 3.

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AIP4 expression to below detectable levels whereas actin levels displayed the AKT-dependent alterations in cyclin D1 and c- remainedunchanged. Control treatments ortreating cellswith MYC promoter activity previously observed however, in cells a nontargeting scrambled siRNA did not effect AIP4 or actin in which AIP4 expression was silenced, rapamycin induced expression. We then treated the lines with the indicated cyclin D1 and c-MYC PolII association was markedly reduced siRNAs, as shown in Figure 6B, and determined the effects in quiescent AKT-containing cells (U87PTEN,LAPC-4puro). of AIP4 knockdown on cyclin D1 (left) and c-MYC (right) In addition, knockdown of AIP4 also resulted in a marked Pol II association following rapamycin exposure as before (see increase in G1 arrest of quiescent AKT-containing lines – Figs. 1 and 4). In control and nontargeting scrambled siRNA (U87PTEN and LAPC4puro) as compared with control or treated cultures, both the U87 and LAPC-4 cell line pairs nontargeting siRNA–treated cells in response to rapamycin

Figure 7. JNK activity is required for differential AKT-dependent AIP4 activity, cyclin D1 and c-MYC transcriptional responses, and G1 arrest in response to rapamycin. A, effects of JNK inhibitor (JNKi VIII) on in vitro AIP4 activity. The paired cell lines were treated with rapamycin and JNKi VIII (4 mmol/L) as indicated and AIP4 immune complexes used in in vitro ubiquitination reactions as in Figure 5 (C). B, cyclin D1 (left) and c-MYC (right) PolII association in

the cell lines (U87 and U87PTEN, shaded bars; LAPC-4myrAKT and LAPC-4puro, open bars) following treatments with JNKi VIII (4 mmol/L) and rapamycin as indicated. Mean SD are shown; n ¼ 3. C, cyclin D1 and c-MYC PolII occupancy in U87, U87PTEN, LAPC-4myrAKT, and LAPC-4puro cells treated with nontargeting scrambled (scr) or JNK siRNA as indicated in the presence of rapamycin (U87 and U87PTEN, shaded bars; LAPC-4myrAKT and LAPC-4puro, open bars). Mean SD are shown; n ¼ 3.

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(Fig. 6C).These datademonstrate that AIP4 isrequiredfor the AKT-dependent cyclin D1 and c-MYC transcriptional AKT-dependent effects on cyclin D1 and c-MYC promoter responses following mTOR inhibition. To inhibit JNK activity and G1 arrest following rapamycin exposure. activity we utilized the selective pyridinylamide inhibitor (referred to as JNKi VIII; ref. 30), which effectively inhib- JNK activity is required for AIP4-mediated JUNB ited activity in both cell line pairs (see Supplementary ubiquination and stimulation of cyclin D1 and c-MYC Fig. 1B). Moreover, exposure to JNKi VIII did not affect promoter activity in cells with quiescent AKT activity AKT activity in either cell line pair as determined by following rapamycin exposure phospho-serine473 levels (see Supplementary Fig. 4A). We As AIP4/JUNB signaling is known to be regulated by subsequently assessed in vitro AIP4 activity in extracts of JNK (29), we were curious as to whether pharmacological cells treated without or with JNKi VIII following exposure inhibition of JNK activity would lead to dramatic effects on to rapamycin as before (see Fig. 5C). As shown in Figure 7A,

Figure 7. (Continued) D, S-phase cell-cycle analysis of paired cell lines treated with nontargeting (scr) or JNK siRNAs in the presence of rapamycin. Mean SD; n ¼ 3. E, regulation of cyclin D1 and c-MYC transcription by AP-1 in response to rapamycin. In this model, rapamycin-FKBP12 inactivation of TORC1 results in sustained activation of the JNK cascade via increased ASK1 E activity (27, 52). This leads to AIP4-mediated JUNB ubiquitination and degradation in quiescent AKT-containing cells and is inhibited in tumor cells whose AKT activity is elevated. As a result, accumulated JUNB and c-JUN-JUNB heterodimers bind to AP-1 elements within the cyclin D1 and c-MYC promoters and inhibits transcription. In contrast, in cells with relatively quiescent AKT, rapamycin exposure results in activation of JNK/AIP4 signaling, degradation of JUNB and stimulation of cyclin D1 and c-MYC promoters via c-JUN.

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JNK inhibition resulted in no significant increase in the PI3K/AKT signaling is known to regulate MYC-mediated levels of detectable ubiquinated JUNB in both the quiescent transcription via posttranslational effects on the negative AKT-containing lines U87PTEN and LAPC-4puro relative to regulator MAD1 by promoting its degradation (33). AKT their active AKT-containing matched lines. We then pre- activity also regulates basic-loop-helix transcription factor– treated both cell line pairs with JNKi VIII and the deter- cofactor complex formation to promote transcriptional mined the effects on cyclin D1 and c-MYC promoter PolII activity (34). In addition, AKT activity stimulates CREB association following rapamycin exposure as before (see Figs. activity (35) and contributes to IkB degradation resulting in 1 and 6). As shown in Figure 7B, exposure of both paired NFkB induction (36). However, there are also examples lines to rapamycin resulted in reduced PolII occupancy of wherein AKT signaling has been reported to negatively cyclin D1 (left) and c-MYC (right) promoters in U87 and regulate transcription. For example, Forkhead transcription Pol LAPC-4myrAKT cells while increasing II occupancy of factors are negatively regulated by phosphorylation via AKT these promoters in U87PTEN and LAPC-4puro cells as pre- (37) and Mdm2 phosphorylation by AKT promotes viously observed. However, when the cell line pairs were increases ubiquitination of p53 leading to reductions in treated in combination with both JNKi VIII and rapamycin, p53-mediated transcriptional responses (38). In addition, both the cyclin D1 and c-MYC promoters displayed following myostatin treatment of skeletal muscle cells, reduced PolII association irrespective of AKT activity. We cyclin D1 expression is silenced via effects on p300 through also examined the effects of JNK knockdown on the cell- AKT signaling (39). Our data demonstrates the ability of cycle distributions of the paired cells following rapamycin AKT to regulate transcription of the cyclin D1 and c-MYC exposure. As shown in Figure 7D, siRNAs targeting JNK promoters following rapamycin exposure and illustrates effectively inhibited its expression (see Supplementary how the same, or similar stimuli, can lead to differential Fig. 4B) and resulted in significant increases in G1 arrest transcriptional responses. To our knowledge, our work is following rapamycin treatment in quiescent AKT-contain- also the first to demonstrate that JUNB binds to the c-MYC ing cell lines (U87PTEN and LAPC-4myrAKT) as compared promoter and results in inhibition of its promoter activity with controls. These data suggest that JNK activity is and collectively provides another example of a physiologi- required for the differential AKT-dependent effects on cally relevant antagonism between c-JUN and JUNB (40). cyclin D1 and c-MYC promoter activity and relative cell The observation that AKT regulates E3 ligase activity is resistance following rapamycin exposure. supported by several studies (41–43), yet how it may control AIP4-mediated JUNB degradation is unclear. It Discussion is possible that AKT indirectly controls an effector of AIP4 to govern overall ligase function. Alternatively, AKT may We previously demonstrated that the AKT-dependent phosphorylate AIP4 to influence activity or regulate ubi- control of cyclin D1 and c-MYC mRNA translational quitin-dependent AIP4 stability (44). It is interesting to efficiency and stability regulate the responses of these 2 note that an AKT consensus phosphorylation site is pre- critical cell-cycle determinants to rapamycin (13, 14, 31). dicted in AIP4 under conditions of limited stringency using Here, we have expanded on these studies and demonstrated Scansite (45), and experiments to test whether a physiolo- that AKT-dependent transcriptional regulation contributes gically relevant phosphorylation event in this regard may to the coordinate responses of these genes to rapamycin. The regulate AIP4 activity are underway. It is of interest that differential promoter activities appear to be regulated by Panner et al. (42), reported a PTEN/AKT/AIP4 signaling distinct AP-1 family member binding within the specific cascade which regulates FLIPs protein stability in glioblas- contexts of the cyclin D1 and c-MYC promoters. Moreover, tomas. Under conditions of quiescent AKT activity, AIP4, our data demonstrate that posttranslational regulation of although found in a highly ubiquinated form, correlated JUNB plays a major role in this response and suggests that with high levels of ubiquinated FLIPs and decreased overall AKT activity may directly or indirectly regulate AIP4- protein stability. However, in cells with elevated levels of mediated JUNB degradation. Our data are consistent with active AKT, FLIPs ubiquination was reduced and increased a model in which rapamycin-induced stress leads to activa- steady-state expression was observed. Our data support tion of JNK-mediated phosphorylation of c-JUN (27), while these findings in that increased AKT activity was found concomitantly JNK phosphorylation of AIP4 and subse- to correlate with decreased JUNB ubiquitination (see quent ubiquination of JUNB is inhibited via AKT function Fig. 5A) and suggests that AKT negatively regulates in cells containing elevated levels of active AKT. However, in AIP4. AKT may also regulate a deubiquitination process cells with relatively quiescent AKT activity, following rapa- so as to exert its effects on JUNB stability. More recently, mycin-induced activation of JNK, AIP4 activity leads to Panner et al. (46) have reported that the deubiquinase USP8 rapid degradation of JUNB. The relative ratio of phosphory- may link AKT activity to downstream AIP4 function in the lated c-JUN to JUNB bound to either the cyclin D1 or c- regulation of FLIPs protein stability. MYC promoters functions as a rheostat leading to either We observed that AIP4 expression was induced following PTEN transcriptional repression or activation resulting in G1 arrest rapamycin exposure irrespective of or AKT status, or progression, respectively (see Fig. 7E). yet AIP4 activity was blunted in cells containing active AKT Many studies have reported on the ability of AKT (see Fig. 5B and C). These data suggest AIP4 activity is signaling to positively influence promoter activities (32). regulated posttranslationally. MTOR is known to have

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effects on the control of autophagy and rapamycin exposure Our data support the notion that tumors cells coordi- induces this process in yeast, as well as in a variety of nately regulate cyclin D1 and c-MYC expression in an mammalian cell types (47). Recent data also provides AKT-dependent manner, at both the transcriptional and evidence for a role for ubiquitin in the selective degradation posttranscriptional levels, as an intrinsic mechanism of of proteins in response to starvation (48). The increase in resistance to mTOR inhibitors. Several genes are known AIP4 expression would be consistent with the global upre- to exhibit similar coordinately regulated gene expression gulation of E3 ligase activity following rapamycin exposure programs in response to various stimuli with alterations and the induction of selective autophagy. We speculate that occurring in transcription, mRNA stability and translation the induction of selective autophagy, as a consequence of (51). To our knowledge, this is the first report showing that mTOR inhibition, may be a result of rapamycin mimicking mTOR inhibitors have marked effects on AIP4. a starvation-like response in cells. It is possible that enzymes An increased understanding of the mechanistic details from both major classes of E3 ligases may play a role in such regulating the AKT-dependent hypersensitivity of tumor cells a response including AIP4. to mTOR inhibitors may lead to the identification of addi- Because c-JUN-JUNB heterodimer formation also con- tional therapeutically amenable targets whose modulation tributes to AP-1–mediated transcriptional regulation (49), may have synergistic antitumor effects with these inhibitors. we examined whether heterodimers were present following rapamycin exposure. The observation that rapamycin sig- Disclosure of Potential Conflicts of Interest nificantly increased heterodimer formation in active AKT- No potential conflicts of interest were disclosed. containing cells, where cyclin D1 and c-MYC promoter activity is inhibited, is consistent with previous data demon- Acknowledgments strating that JUNB can attenuate trans-activation by c-JUN. Thus, it is likely that heterodimer formation of these We thank Drs. Anil Rustgi, Linda Penn, Latifa Bakiri, Moshe Yaniv, Ted transcriptional regulators contributes to the differential Dawson, and Edward Yeh for the reagents. We also thank Drs. Robert Nishimura and Alan Lichtenstein for comments on the manuscript and Johnathan Lee for regulation of cyclin D1 and c-MYC transcription in an technical assistance. We are grateful to Ardella Sherwood for excellent adminis- AKT-dependent manner in response to rapamcyin. We also trative assistance. observed that rapamycin-induced significant accumulation of phosphorylated c-JUN independent of AKT activity, Grant Support which via a positive autoregulatory mechanism acting on its This work was supported, in whole or in part, by VA MERIT and NIH own promoter (50), would be predicted to result in elevated R01CA109312 grants. levels total c-JUN. However, c-JUN levels remained rela- The costs of publication of this article were defrayed in part by the payment of page tively constant following rapamycin exposure (Fig. 5B). charges. This article must therefore be hereby marked advertisement in accordance This suggests that additional regulatory mechanisms inde- with 18 U.S.C. Section 1734 solely to indicate this fact. pendent of the autoregulatory circuit result in the overall Received March 12, 2010; revised September 10, 2010; accepted December 01, unchanged levels of total c-JUN. 2010; published OnlineFirst December 6, 2010.

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130 Mol Cancer Res; 9(1) January 2011 Molecular Cancer Research

AP-1 Regulates Cyclin D1 and c-MYC Transcription in an AKT-Dependent Manner in Response to mTOR Inhibition: Role of AIP4/Itch-Mediated JUNB Degradation

Raffi Vartanian, Janine Masri, Jheralyn Martin, et al.

Mol Cancer Res 2011;9:115-130. Published OnlineFirst December 6, 2010.

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