DNA Damage–Induced Protein 14-3-3 S Inhibits Protein Kinase B/Akt

Activation and Suppresses Akt-Activated Cancer

Huiling Yang,1Yu-Ye Wen,

1,4Ruiying Zhao,

1,4Yu-Li Lin,

1Keith Fournier,

5Heng-Yin Yang,

1

Yun Qiu,6Jose Diaz,

5Christine Laronga,

5and Mong-Hong Lee

1,2,3,4

1Department of Molecular and Cellular Oncology and 2Breast Cancer Research Program, The University of Texas M.D. Anderson CancerCenter; Programs in 3Cancer Biology and 4Genes and Development, The University of Texas Graduate School of Biomedical Sciencesat Houston, Houston, Texas; 5Department of Surgery, Eastern Virginia Medical School, Norfolk, Virginia; and 6Pharmacology andExperimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland

Abstract

14-3-3 S is induced by tumor suppressor protein p53 inresponse to DNA damage. p53 can directly transactivate theexpression of 14-3-3 S to cause a G2 cell cycle arrest when cellDNA is damaged. The expression of 14-3-3 S protein is down-regulated in various tumors, but its function has not been fullyestablished. Protein kinase B/Akt, a crucial regulator ofoncogenic signal involved in cell survival and proliferation, isderegulated in many types of cancer. Akt activation canenhance p53 degradation, but its role in DNA damage responseis not clear. Here, we show that Akt activation is diminishedwhen p53 and 14-3-3 S is up-regulated in response to DNAdamage. Evidence is provided that 14-3-3 S binds and inhibitsAkt. In keeping with this concept, Akt-mediated cell survival isinhibited by 14-3-3 S. Significantly, we show that 14-3-3 S

inhibits Akt-mediated cell growth, transformation, and tumor-igenesis. Low expression of 14-3-3 S in human primary breastcancers correlates with Akt activation. These data provide aninsight into Akt regulation and rational cancer gene therapy byidentifying 14-3-3 S as a molecular regulator of Akt and as apotential anticancer agent for Akt-activated cancers. (CancerRes 2006; 66(6): 3096-105)

Introduction

Protein kinase B (also called Akt) is the cellular homologue ofthe oncogene of the AKT8 oncovirus (v-Akt). Akt is activatedwhen particular extracellular signals activate receptor tyrosinekinases to enhance phosphatidylinositide 3-OH kinase (PI3K)activity on phospholipids. The oncogene is a crucial regulator of avariety of cellular processes, including cell survival and prolifer-ation. Importantly, Akt activity is elevated in several types ofhuman malignancy, including ovarian, breast, lung, and thyroidcancers (1). The kinase activity of Akt is constitutively activatedin human cancer as a result of dysregulation of its regulators,including the tumor suppressor PTEN (2) and the amplification ofthe catalytic subunit of PI3K (3). Recently, PTEN, a negativeregulator of Akt activation, can down-regulate Mdm2 andincrease p53 stability (4). In addition, PTEN�/� cells have highAkt activity and are defective in checkpoint control in response to

DNA damage (5). In addition, Akt can mediate phosphorylation ofMdm2, promotes Mdm2 nuclear localization, and inhibitsinteraction between Mdm2 and p19ARF (6), thereby potentiatingMdm2’s activity in degrading p53. These observations suggest thatAkt is involved in DNA checkpoint control. Thus far, it is notclear how Akt is regulated in response to DNA damage. Our studyhas indicated that Akt activation is diminished by the expressionof a p53-inducible protein, 14-3-3 j.14-3-3 j is a member of 14-3-3 family proteins that have

critical roles in signal transduction pathways and cell cycleregulation (7–9). The 14-3-3 family is highly conserved over awide range of mammalian species, including seven isotypes h, q,D, g, H (also called u), ~ , and j. Among the family members, 14-3-3 j is unique. 14-3-3 j is the only 14-3-3 isoform induced bytumor suppressor protein p53 in response to g irradiation andother DNA-damaging agents (10). 14-3-3 j was characterized as ahuman mammary epithelial-specific marker (HME1; ref. 11) thatis down-regulated in mammary carcinoma cells. 14-3-3 jregulates the cell cycle by interacting with cyclin-dependentkinases (cdks; ref. 12) and serving as a target of p53 (10) andBRCA1 (13, 14). BRCA1 is a tumor suppressor for breast andovarian cancers and has important roles in DNA repair andtranscription (15, 16). Mutations identified in the COOH terminusof BRCA1 from patients with breast cancer cannot activatetranscription of 14-3-3 j (14), suggesting that the tumor-suppressive function of BRCA1 involves 14-3-3 j. 14-3-3 jsequesters cyclin B1/CDC2 complexes in the cytoplasm to causeG2 arrest in response to DNA damage (12, 17). During this DNAdamage process, 14-3-3 j also positively regulates p53 stabilityand potentiates p53 transcriptional activity (18). In addition,down-regulation of 14-3-3 j can make primary human epithelialcells grow indefinitely in a single step without the need ofexogenous oncogenes and/or oncoviruses, suggesting that thisimmortality caused by 14-3-3 j inhibition may lead to tumorformation (19). Importantly, 14-3-3 j is down-regulated in severaltypes of cancer, including breast cancer (20). Overexpression of14-3-3 j suppresses the anchorage-independent growth of severalbreast cancer cell lines (12). These observations suggest that thetumor suppressor function of 14-3-3 j is compromised duringtumorigenesis. However, the mechanisms of 14-3-3 j’s role intumorigenesis and signal transduction have not been fullyelucidated. Here, we show that 14-3-3 j up-regulation correlateswith Akt inactivation in response to DNA damage. We show that14-3-3 j negatively regulates Akt and inhibits Akt-mediated cellsurvival, cell proliferation, transformation, and tumorigenicity.Our studies in human breast cancers show that low expression of14-3-3 j is associated with Akt activation, providing a mechanis-tic role for 14-3-3 j down-regulation in breast cancer formation.

Note: Y. Wen and R. Zhao contributed equally to this work.H. Yang is presently at the Department of Pathophysiology, Zhongshan University,

China.Requests for reprints: Mong-Hong Lee, The University of Texas M.D. Anderson

Cancer Center, Box 79, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-794-1323; Fax: 713-792-6059; E-mail: mhlee@mdanderson.org.

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-3620

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Materials and Methods

Cell lines, viruses, plasmids, kinase inhibitors, and antibodies. R1B/L17 (the mink lung epithelial cell line; ref. 21), Rat-1 cells, A549 cells(American Type Culture Collection, Rockville, MD), and Rat1-akt cells(Binhua Zhou, M.D. Anderson Cancer Center) were cultured in DMEMmedia containing 10% fetal bovine serum. Tet-o-Flag-14-3-3 j cells wereconstructed as previously described (22). Human HCT116 cell lines deficientin 14-3-3 j were kindly provided by Dr. Vogelstein (17). Ad-14-3-3 j and Ad-Ad-h-gal viruses (10) were produced as previously described (23). Plasmidsof Akt-PHD and Akt D11-60 (Philip Tsichlis, Tufts University) and Akt D4-129 have been previously described (24, 25). Adriamycin is obtained fromSigma (St. Louis, MO). For immunoprecipitation and immunoblotting, thefollowing antibodies were used: monoclonal antibodies against FLAG,hemagglutinin (HA; 12CA5, Babco), tubulin from Sigma; antibodies againstAkt, phospho-Akt (Ser473) 4E2, phospho-(serine/threonine) Akt substrateantibody from Cell Signaling (Beverly, MA); antibody against poly(ADP-ribose) polymerase (PARP) from BD Biosciences (San Jose, CA); andantibody against 14-3-3 j from RDI (Flanders, NJ).

Immunoprecipitation and in vitro binding assay. For the immuno-precipitation assay, the cells were lysed in NP40 lysis buffer (22). A549 celllysates were immunoprecipitated with anti-Akt (Cell Signaling) andimmunoblotted with anti-14-3-3 j (RDI). Tet-o-Flag-14-3-3 j cell lysateswere immunoprecipitated with anti-Flag (M2, Sigma) and immunoblottedwith anti-Akt (Cell Signaling). In addition, R1B/L17 cells cotransfectedwith expressing vectors of Flag-14-3-3 j and HA-tagged CA-Akt wereimmunoprecipitated with anti-Flag (M2, Sigma) and immunoblotted withanti-HA (12CA5), or immunoprecipitated with anti-HA (12CA5) andimmunoblotted with anti-Flag (M2, Sigma) to detect association. For thein vitro binding assay, a T7 RNA polymerase-driven pET vector containingthe coding region of the 14-3-3 j domain cDNA was transcribed in vitroand translated using a TNT kit (Promega, Madison, WI). These productswere labeled with [35S]methionine and were then incubated withimmobilized GST-Akt (Cell Signaling). The retained proteins were detectedby autoradiography.

Kinase assays. R1B/L17 or Rat1-akt cells were either left uninfected orinfected with Ad-14-3-3 j [multiplicity of infection (MOI) = 5] or Ad-h-gal(MOI = 5). Cell lysates were immunoprecipitated with anti-Akt. Immunecomplexes were incubated with 0.04 Ag GSK3h (Cell Signaling) and 10 ACi[g-32P]ATP (Amersham, Arlington Heights, IL) for a kinase assay asdescribed (22). In other in vitro kinase assays, 1 AL of baculovirus-producedactive recombinant Akt1 (Cell Signaling), which is activated (T308D andS473D) and is isolated and purified from Sf9 cells, was incubated withpurified recombinant 14-3-3 j, 14-3-3 j NH2-terminal domain, 14-3-3 jCOOH-terminal domain, 14-3-3 g, or 14-3-3 D (bacterially produced by usingexpression vector pET21a and purified) for kinase activity againstrecombinant GSK3h (Cell Signaling). Phosphorylated substrate wasvisualized or quantitated using a STORM840 PhosphorImager.

Fluorescence-activated cell sorting assay, soft agar colony forma-tion assay, and bromodeoxyuridine incorporation assay. Rat1-akt cellsor Rat1 cells were infected with Ad-HA-14-3-3 j (MOI = 5) or Ad-h-gal(MOI = 5) and compared with untreated cells (PBS control) for the assays.These assays were done as described (22).

Apoptosis assays. Rat1-akt cells or Rat1 cells were treated with Ad-HA-14-3-3 j (MOI = 5 or 10), Ad-h-gal (MOI = 10), or apoptotic stimulus (0%FCS) in DMEM and compared with untreated cells (PBS control). Afterinduction of apoptosis, cell extracts were used for the cell death ELISA,which was done as previously described (26) and according to themanufacturer’s protocol (Roche, Nutley, NJ).

Tumor growth in nude mice. Female 4- to 5-week-old nude mice(Charles River Laboratories, Wilmington, MA) were divided into threeexperimental groups, six for each. Rat1-akt cells were left uninfected(control) or infected with Ad-h-gal (MOI = 5) or Ad-14-3-3 j (MOI = 5) for48 hours. Cells (2 � 106) were harvested and injected s.c. into the right flankof mice. Tumor volumes were measured as described (22). At the end of2 weeks, the mice were sacrificed and the tumors were removed fordetection of HA-14-3-3 j, phospho-Akt (Ser473), and phospho-(serine/threonine) Akt substrate. In a dose dependence study, mice were divided

into five experimental groups, six for each. Rat1-akt cells were harvestedand injected (1.5 � 106). Animals were treated by injection with Ad-HA-14-3-3 j (MOI = 5), Ad-h-gal (MOI = 5), or PBS (0.2 mL/site). Ad-HA-14-3-3 jwas administrated every 1, 3, or 15 days. Tumor volumes were measured.Tumor volumes were measured every 2 days from day 3 of cell inoculationfor 80 days.

Immunohistochemistry. Sections from paraffin blocks of 37 invasivebreast tumors were deparaffinized in serial grades of xylene followed by

rehydration in sequential increasing dilutions of ethanol. Antigen retrieval

was facilitated by heating in 10 mol/L Na Citrate buffer (pH 6). Slides were

incubated with 14-3-3 j (C-18; Santa Cruz Biotechnology, Santa Cruz, CA;1:50, room temperature, 1 hour) or phospho-Akt (Ser473; Cell Signaling;

1:100). The general immunohistochemical staining scheme was done as

described previously (27). Antibody detection was done with the avidin-biotin complex substrate kit (Vector Laboratories, Burlingame, CA), and

slides were counterstained with hematoxylin. Statistical comparisons were

done using Fisher’s exact test.

Results

DNA damage response results in p53/14-3-3 S induction andAkt inactivation. We previously showed that 14-3-3 j has apositive feedback effect on p53 by antagonizing Mdm2-mediatedp53 ubiquitination (18). Mdm2 is a substrate of Akt, and Aktphosphorylates Mdm2 to induce localization of Mdm2 to thenucleus (6, 28), thus enhancing Mdm2 activity to degrade p53. It ispossible that Akt is regulated when 14-3-3 j mediates p53stabilization in response to DNA damage. To address thishypothesis, we first examined whether Akt activity is down-regulated in response to DNA damage. To this end, we treated A549cells, which has wild-type p53, with DNA-damaging agentAdriamycin. The cell lysates were subsequently collected atdifferent time points and immunoblotted with anti-p53, anti-14-3-3 j, and anti-Akt-p at Ser473. Akt activation (Akt-p at Ser473) wasclearly diminished when p53 is stabilized and when 14-3-3 j is up-regulated, as shown by reduced level of phospho-Akt at Ser473

(Fig. 1A). It is possible that 14-3-3 j can negatively regulate the Aktactivation. Because 14-3-3 j may change catalytic activity of thebinding partner, we hypothesize that 14-3-3 j may physically bindAkt and regulate Akt activity to stabilize p53. To test thishypothesis, we examined whether 14-3-3 j interacted with Akt incoimmunoprecipitation experiments. Importantly, 14-3-3 j wasdetected in the anti-Akt immunoprecipitation complex in A549cells (Fig. 1A), suggesting that endogenous 14-3-3 j associatedphysically with endogenous Akt. In addition, there was anincreasing binding between endogenous 14-3-3 j and Akt followingDNA damage time courses, as shown by the increasing amounts of14-3-3 j in the Akt immunoprecipitation complex (Fig. 1A). Theincreasing binding between 14-3-3 j and Akt correlates withdecreased Akt activation (level of Akt-p at Ser473) following DNAdamage time course, suggesting that the increased amounts of 14-3-3 j may reach a threshold enough to cause Akt inactivation.14-3-3 S associates with Akt. To further confirm the

association between 14-3-3 j and Akt in cells, we cotransfectedcells with the expression vectors of Flag-tagged 14-3-3 j and HA-tagged Akt. Cell lysates were subjected to reciprocal immunopre-cipitation with antibodies to Flag or HA. A Western blot using HAmonoclonal antibody confirms the presence of HA-Akt in the Flag-14-3-3 j–immunoprecipitated complex (Fig. 1B). Reciprocalimmunoprecipitation using an anti-HA antibody also precipitatesFlag-14-3-3 j (Fig. 1B). In addition, we generated 14-3-3 jtetracycline-regulated cells (22) and investigated whether 14-3-3j interacted with Akt. 14-3-3 j was expressed in the absence of

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tetracycline, and Akt was detected in the 14-3-3 j immunoprecip-itation complex (Fig. 1B). Thus, compelling evidence indicates thatthe two proteins were associated in the cells.To define Akt-binding domains in 14-3-3 j, we constructed

deletion mutants and analyzed their binding to Akt bycoimmunoprecipitation. Cells were cotransfected with the expres-

sion vectors of Flag-tagged 14-3-3 j deletion mutants and HA-tagged Akt. The NH2 terminus of 14-3-3 j (residues 1-161) did notbind to Akt (Fig. 1C). However, the COOH terminus of 14-3-3 j(residues 152-248) did interact with Akt (Fig. 1C). We confirmedthis result using a glutathione S-transferase (GST) pull-downassay in vitro (Fig. 1C). GST-tagged Akt was able to bind to

Figure 1. Akt inactivation and increased interaction of Akt with 14-3-3 j following DNA damage. A, Akt is inactivated in response to DNA damage. A549 cellswere treated with 0.2 Ag/mL of Adriamycin for the indicated time. Equal amounts of cell lysates were immunoblotted (IB ) with anti-p53 antibody, anti-14-3-3 j, oranti-phospho-Akt (p-Akt ; Ser473). Equal amounts of cell lysates were immunoprecipitated (IP ) with anti-Akt antibody and then immunoblotted with anti-14-3-3 j antibodyto observe the increased association or immunoblotted with anti-Akt antibody to observe the equal immunoprecipitation of Akt. B, 14-3-3 j interacts with Akt. R1B/L17cells were cotransfected with Flag-tagged 14-3-3 j and HA-tagged Akt. Cell lysates were subjected to immunoprecipitation with either control mouse IgG (lane 1, top )or anti-Flag (lane 2, top ). The resulting anti-Flag immunoprecipitation complex was subjected to immunoblot analysis with anti-HA to detect the association betweenHA-tagged Akt and Flag-tagged 14-3-3 j. Expression of HA-tagged Akt in the lysates (lane 3, top ). In addition, cell lysates were subjected to immunoprecipitationwith either control mouse IgG (lane 1, bottom ) or anti-HA (lane 2, bottom ). The resulting anti-HA immunoprecipitation complex was subjected to immunoblot analysiswith anti-Flag to detect the association between Flag-tagged 14-3-3 j and HA-tagged Akt. Expression of Flag-tagged 14-3-3 j in the lysates (lane 3, bottom ).Tetracycline-regulated tet-o-Flag-14-3-3 j cells were treated with (+) or without (�) tetracycline. Immunoprecipitated complex of anti-Flag antibody was immunoblottedwith anti-Akt antibody to observe the association between endogenous Akt and Flag-tagged 14-3-3 j. Expression of Flag-14-3-3 j (bottom ). C, interaction of thedomains in 14-3-3 j with Akt. Lysates of R1B/L17 cells transfected with the indicated schematic Flag-tagged domain of 14-3-3 j were immunoprecipitated with anti-Flagand analyzed by immunoblotting using anti-Akt to observe the interaction. Immunoprecipitated amounts of Flag-14-3-3 j domains (bottom ). GST-Akt immobilizedon GST beads was incubated with indicated in vitro -transcribed, translated, and 35S-labeled 14-3-3 j domains (amino acids 1-161 or 135-248), to observe theinteraction between domains of 14-3-3 j and Akt. Ten percent of the in vitro -translated 35S-labeled 14-3-3 j domain inputs (bottom ). D, interaction of the Akt domainswith 14-3-3 j. Lysates of 293T cells transfected with the indicated HA-tagged domain of Akt and Flag-14-3-3 j were immunoprecipitated with anti-Flag and analyzedby immunoblotting using anti-HA to observe the interaction between Akt domains and 14-3-3 j. The same anti-Flag immunoprecipitates were immunoblotted withanti-Flag to observe equal amounts of immunoprecipitated Flag-14-3-3 j (middle ). Expression of HA-tagged Akt domains (bottom). wt, wild type.

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in vitro translated 35S-labeled 14-3-3 j (153-248 amino acids) butnot 14-3-3 j (1-161 amino acids; Fig. 1C), suggesting that theCOOH-terminal sequence comprising residues 153-248 of 14-3-3 jmight contain the Akt-interaction domain. It is notable that the14-3-3 family usually uses the carboxyl terminal domain tointeract with targeted proteins (7), and 14-3-3 j seems to use theCOOH-terminal domain to interact and inhibit Akt. To determinethe binding site of 14-3-3 j on Akt, we did a coimmunopreci-pitation assay to observe the interaction between Akt domainsand 14-3-3 j. Akt D11-60 (deletion 11-60 amino acids) and AktD4-129 (deletion 4-129 amino acids) are two proteins containingthe kinase domain (150-408 amino acids), whereas Akt PHDcontains only pleckstrin homology domain (PH/AH domain, 1-147amino acids) but no kinase domain (24). We found that 14-3-3 jwas able to bind to Akt D11-60, Akt D4-129 but not Akt PHD,

suggesting that COOH-terminal Akt that contains kinase domainis involved in 14-3-3 j binding (Fig. 1D). Together, these resultsindicate that 14-3-3 j can specifically interact with Akt.14-3-3 S inhibits Akt activity. To address the hypothesis that

14-3-3 j binds Akt and inhibits Akt kinase activity, we firstexamined whether Akt activity is inhibited when 14-3-3 j isoverexpressed. Akt can phosphorylate GSK3h (29), so GSK3h isused as an Akt substrate in a kinase assay. R1B/L17 Cells wereinfected with Ad-HA-14-3-3 j or Ad-h-gal (control). Akt wasimmunoprecipitated and assayed for its activity to phosphorylaterecombinant GSK3h substrate. The Akt-mediated GSK3h phos-phorylation of control cells was normal, but that of Ad-HA-14-3-3j–infected cells was severely reduced (Fig. 2A). To confirm thenegative regulation of Akt kinase activity by 14-3-3 j in a definedsystem, we incubated recombinant Akt, which is activated (T308D

Figure 2. 14-3-3 j inhibits Akt kinase activity. A, kinase assay performed with immunoprecipitated (IP ) Akt from R1B/L17 cells infected with Ad-Ad-h-gal orAd-14-3-3 j. Cell lysates immunoprecipitated with control IgG were used as negative control for kinase assay. Purified recombinant GSK3h served as substrates foran Akt kinase assay. Autoradiographs show the phosphorylated GSK3h (p-GSK3b) bands. B, inhibition of Akt kinase activity in vitro . Baculovirus-produced activerecombinant Akt1 was incubated with indicated amounts of bacterially produced and purified recombinant 14-3-3 j protein for kinase activity against recombinantGSK3h or p27 substrate. Autoradiographs show the phosphorylated GSK3h and 14-3-3 j bands. Active recombinant Akt1 was incubated with 0.3 Ag of bacteriallyproduced and purified recombinant 14-3-3 j, 14-3-3 g, or 14-3-3 D for kinase activity against recombinant GSK3h. Amounts of purified recombinant 14-3-3 j, 14-3-3 g,or 14-3-3 D used in the Akt kinase reaction. C, effects of 14-3-3 j domains on Akt kinase activity. Active recombinant Akt1 was incubated with 0.3 Ag of bacteriallyproduced and purified recombinant 14-3-3 j NH2-terminal domain (N ), 14-3-3 j COOH-terminal domain (C), or without (�) for kinase activity against recombinantGSK3h. Phosphorylation of GSK3h. Amounts of purified recombinant 14-3-3 j NH2-terminal domain (N ) and 14-3-3 j COOH-terminal domain (C ) used in the Aktkinase reaction. Baculovirus-produced active recombinant Akt1 was incubated with indicated amounts of bacterially produced and purified recombinant 14-3-3 jNH2-terminal domain (N ), 14-3-3 j COOH-terminal domain (C ), or without (control ) for kinase activity against recombinant GSK3h substrate. Phosphorylation levelsof GSK3h were quantitated by imagequant program and are plotted as the relative percentage of phosphorylation observed in reaction without 14-3-3 j (control, wasset at 100%). D, immunoblot analysis of phospho-Akt (p-Akt ) in 14-3-3 j–deficient cells. Cell lysates from HCT116 cells containing wild-type 14-3-3 j (+/+) or deficientin 14-3-3 j (�/� ) were immunoblotted with anti phospho-Akt (Ser473). Amounts of 14-3-3 j are also indicated. In addition, 14-3-3 j (�/� ) cells were infected withAd-h-gal (control, MOI = 5) and Ad-14-3-3 j (MOI = 5) for 12 hours followed by immunoblotting with the above same antibodies.

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and S473D), with increasing amounts of purified 14-3-3 j for kinaseactivity against purified recombinant wild-type GSK3h substrate.14-3-3 j inhibited Akt-mediated GSK3h phosphorylation in a dose-dependent manner (Fig. 2B). In addition, 14-3-3 j was notphosphorylated during the kinase assay (Fig. 2B). These resultsindicated that 14-3-3 j directly inhibited Akt kinase activitytowards GSK3h substrate and was not a competitive substrate forAkt. To address the specificity of 14-3-3 j in inhibiting Akt kinaseactivity, we used other bacterially produced and purified 14-3-3isotypes, including 14-3-3 D and 14-3-3 g, to perform the same kindof kinase assay. 14-3-3 D and 14-3-3 g proteins did not inhibit Aktkinase activity (Fig. 2B), suggesting that 14-3-3 j is specific in termsof inhibiting the kinase activity of Akt. To determine which domainof 14-3-3 j is involved in inhibiting Akt kinase activity, we usedbacterially produced and purified recombinant 14-3-3 j NH2-terminal domain (N), 14-3-3 j COOH-terminal domain (C), orwithout for kinase activity against recombinant GSK3h substrate(Fig. 2C). 14-3-3 j COOH-terminal domain inhibited Akt kinaseefficiently, as shown by reduced GSK3h phosphorylation (Fig. 2C).In addition, 14-3-3 j COOH-terminal domain inhibited Akt-mediated GSK3h phosphorylation in a dose-dependent manner,whereas recombinant 14-3-3 j NH2-terminal domain had littleinhibitory activity (Fig. 2C). To confirm 14-3-3 j’s negative effect onAkt activity in a physiologic situation, we investigated Akt signalingin the absence of 14-3-3 j expression using 14-3-3 j null cells.These 14-3-3 j–deficient cells were created by homologousrecombination in the background of human colorectal cancer cellHCT116 (17). The disruption of 14-3-3 j had a marked effect on theactivity of Akt. As shown in Fig. 2D , Akt activation is increased in14-3-3 j–deficient HCT 116 cells (14-3-3 j�/�) when comparedwith parental 14-3-3 j wild-type HCT116 cells (14-3-3 j+/+), asshown by increased Akt phosphorylation on Ser473. As expected,reintroducing 14-3-3 j back to 14-3-3 j null cells by adenoviraldelivery (Ad-14-3-3 j) led to decreased Akt phosphorylation onSer473 when compared with cells infected with Ad-h-gal control(Fig. 2D). Thus, 14-3-3 j has negative effect on Akt activity.14-3-3 S inhibits Akt-mediated cell survival. Akt has been

implicated in the control of cell survival. For example, mice withtargeted disruption of the akt1 gene are more sensitive toapoptosis-inducing stimuli (30). Because 14-3-3 j inhibits Aktactivity, we tested whether 14-3-3 j could specifically block theAkt-mediated survival activity in Akt-activated cells. Rat1-aktcells, which have constitutive Akt, were left uninfected (control)or infected with Ad-HA-14-3-3 j or Ad-h-gal and subjected tofluorescence-activated cell sorting (FACS) analysis. Ad-14-3-3 j–infected cells had a higher sub-G1 population (26%) than controlcells (0.68%) or Ad-h-gal–infected cells (1.57%), indicating that theoverexpression of 14-3-3 j can overcome the survival signal of Aktto induce apoptosis, as evident in the increased number of sub-G1

cells (Fig. 3A). Rat1 cells were included as a control. The datashowed that Rat1 cells infected with Ad-14-3-3 j had only anincreased G2-M population but no sub-G1 population (Fig. 3A),suggesting that overexpression of 14-3-3 j did not induce apoptosisin non–Akt-activated cells.One possible explanation is that 14-3-3 j blocks Akt-mediated

survival by activating caspase (31) and causes PARP cleavage. Ad-14-3-3 j–infected Rat1-akt cells had decreased Akt-mediatedGSK3h phosphorylation and showed more PARP cleavage thanAd-h-gal–infected cells (Fig. 3B ), indicating that caspase-3activation were involved in 14-3-3 j–mediated apoptosis in Akt-activated cells.

To determine whether the14-3-3 j–induced apoptosis in Rat1-aktcells is dose dependent and/or expression dependent, we infectedthe cells with various MOIs following different time courses. Wemeasured apoptosis by quantitating DNA fragmentation. Bothincreased MOIs and longer infection time of Ad-14-3-3 j led to 3- to6-fold increases in DNA fragmentation over that seen in cells thatwere induced to apoptosis by serum deprivation (0% FCS; Fig. 3C),indicating that 14-3-3 j overexpression caused apoptosis in Akt-activated cells. In contrast, 14-3-3 j overexpression did not lead toany apoptosis in Rat1 cells (Fig. 3C). The data are consistent with theFACS analysis result that 14-3-3 j did not cause apoptosis in Rat1cells (Fig. 3A). Thus, Akt’s activity in providing protection fromapoptosis was abolished by 14-3-3 j expression. Because 14-3-3 jCOOH-terminal domain inhibited Akt kinase efficiently (Fig. 2C),we then determined whether this domain is also sufficient to inhibitcell survival in Rat1-Akt cells. Rat1-Akt cells were equally transfectedwith wild-type 14-3-3 j or 14-3-3 j COOH-terminal domain andassayed for the degree of DNA fragmentation. We found that 14-3-3 jCOOH-terminal domain caused apoptosis as efficiently as wild-type14-3-3 j (Fig. 3D). Thus, 14-3-3 j promoted apoptosis in cellscontaining activated Akt.14-3-3 S blocks Akt-mediated cell proliferation, transfor-

mation, and tumorigenicity. Akt is known to promote cellproliferation, transformation, and tumor formation. To assess thebiological consequences of the impairment of Akt activity by 14-3-3j, we investigated the effect of 14-3-3j on Akt-mediated cellproliferation, transformation, and tumorigenesis. We determinedthe S-phase progression using a bromodeoxyuridine (BrdUrd)incorporation in Rat1-akt cells (Fig. 4A). The BrdUrd incorporationof Rat1-akt cells infected with Ad-14-3-3 j decreased drastically afterthe infection. Ad-14-3-3 j –infected cells had fewer BrdUrd-positivecells (about 45%) than control (which was set at 100%). However, Ad-h-gal–infected cells had a high percentage of BrdUrd-positive cells(98%). Thus, these data suggest that the overexpression of 14-3-3 jcan inhibit growth in Akt-activated cells. We next investigatedwhether 14-3-3 j overexpression affected Akt-mediated transfor-mation. The Akt proto-oncogene can induce transformation byenabling cells to grow in soft agar (anchorage-independent growth).Rat1-Akt cells were left uninfected (control) or infected with Ad-HA-14-3-3 j or Ad-h-gal and subjected to a soft agar colony formationassay. Adenoviral delivery of 14-3-3 j into Rat1-akt cells resulted infewer colonies than control and Ad-hgal infection (Fig. 4B), showingthat the overexpression of 14-3-3 j can suppress the in vitrotransformation phenotype of Akt-activated cells.Next, we used Rat1-Akt cells as a model system to explore the

tumor suppressive activity of 14-3-3 j. Rat1-Akt cells were leftuninfected (control) or infected with Ad-HA-14-3-3 j or Ad-h-gal.The cells were then implanted into nude mice. Tumor growth wasobserved in control mice and Ad-h-gal–treated mice; however,tumor volume was dramatically decreased in Ad-HA-14-3-3 j–treated mice (Fig. 4C), suggesting that 14-3-3 j inhibits Akt-mediated tumorigenicity. In addition, we found an efficientinhibition of tumor growth in mice treated with frequent Ad-HA-14-3-3 j administration (Fig. 4C). Tumor growth in mice treatedwith Ad-14-3-3 j every day was inhibited and remained at <50mm3 of tumor volume after 15 days, whereas the tumor growth inmice treated with Ad-HA-14-3-3 j every 3 or 15 days was notefficiently suppressed (120 mm3 tumor volume in every 3 days oftreatment and 180 mm3 tumor volume in every 15 days oftreatment compared with 250 mm3 tumor volume in controlmice), suggesting that a minimal level of 14-3-3 j expression is

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required for suppressing Akt-activated tumors efficiently (Fig. 4C).Levels of activated Akt (P-S473Akt) and phospho-Akt substrates intumors obtained from Ad-HA-14-3-3 j–treated mice weremarkedly less than those in control mice and Ad-h-gal–treatedmice (Fig. 4D). As expected, transduced HA-14-3-3 j proteinswere present in small tumors of Ad-HA-14-3-3 j–treated mice butwere absent in larger tumors of control mice and Ad-h-gal–treated mice (Fig. 4D), suggesting that the expression of 14-3-3 jis directly involved in inhibiting tumor growth. Together, theseresults indicate that 14-3-3 j–mediated Akt inhibition leads toreduced cell proliferation, transformation, and tumorigenicity incells containing activated Akt.

Down-regulation of 14-3-3 S correlates with Akt activationin breast cancer. 14-3-3 j is down-regulated in many types ofcancer, including breast cancer (20). To further investigate thecross-regulation between 14-3-3 j and Akt activation in vivo , weinvestigated the expression of 14-3-3 j and activated Akt (P-S473Akt) in 37 primary breast cancer tissue using immunohisto-chemistry. We found a strong inverse relationship between 14-3-3 jexpression and the activation of Akt (Fig. 5; Table 1). In tumors, inwhich 14-3-3 j expression is low, P-S473Akt is high (representativecase I in Fig. 5), whereas in tumors in which 14-3-3 j expression ishigh, signals of P-S473Akt is low (representative case II in Fig. 5).These results indicate that down-regulation of 14-3-3 j correlates

Figure 3. 14-3-3 j inhibits Akt-mediated cell survival. A, flow cytometric analysis of Rat1 and Rat1-Akt cells infected with no virus (C ), Ad-h-gal, or Ad-14-3-3 j.The percent distribution in different cell cycle compartment is shown. B, immunoblot analysis of PARP cleavage in Rat1-Akt cells infected with Ad-h-gal (C) or Ad-14-3-3 j.Akt was immunoprecipitated (IP ) with anti-Akt from cell lysates to assay for GSK3h kinase activity. Autoradiograph shows the phosphorylated GSK3h (p-GSK3b ).C, Akt-activated cells were sensitized to apoptosis by adenoviral transduction of 14-3-3 j. Rat1-akt cells or Rat1 cells were infected with Ad-HA-14-3-3 j at variousMOIs for the indicated hours or infected with Ad-h-gal at the indicated MOIs. Uninfected cells were used as controls (C ). Rat1-Akt cells were also cultured in theabsence of FCS (0% FCS) for 48 hours. Induced apoptosis was determined by measuring DNA fragmentation using the cell death ELISA kit (Roche). The resultswere expressed as the value of A405 reading. The absorbance (OD ) is directly proportional to the apoptosis. Bars, SD. Columns, average of three independentexperiments; bars, SE. ELISA agents only were used as negative controls (NC ). Rat1 cells were infected with Ad-HA-14-3-3 j or Ad-h-gal at various MOIs for theindicated hours. Uninfected cells were used as controls (C). Rat1 cells cultured in the absence of FCS (0% FCS) for 48 hours were used as positive control (PC )for apoptosis. Induced apoptosis was determined as above using cell death ELISA kit. D, COOH-terminal domain of 14-3-3 j–induced apoptosis in Rat1-Akt cells.Rat1-Akt cells were equally transfected with wild-type 14-3-3 j or 14-3-3 j COOH-terminal domain. Cells transfected with empty vectors were used as controls (C ).Induced apoptosis was determined as above using cell death ELISA kit.

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very well with the activation of Akt in tumor tissues, whichsuggests that down-regulation of 14-3-3 j plays important role inAkt-mediated tumorigenesis of breast cancer.

Discussion

The high frequency of down-regulation of 14-3-3 j in severaltypes of cancer shows that the dysregulation of 14-3-3 j plays animportant role in human tumorigenesis. Akt also plays a pivotalrole in cancer formation, and its oncogenic activity arises fromthe activation of both proliferative and antiapoptotic signaling.

Obviously, Akt is an important molecular target for rationalcancer therapy. This study shows that 14-3-3 j binds andnegatively regulates Akt. This important biological functionantagonizes Akt-mediated cell survival to promote apoptosis.Moreover, primary breast cancer studies showed that down-regulation of 14-3-3 j results in Akt activation. Importantly,overexpression of 14-3-3 j inhibits tumorigenicity mediated byAkt activity in cancer models. As a cellular inhibitor of Akt, 14-3-3j should prove useful in the treatment of tumors with elevatedAkt activity.

Figure 4. 14-3-3 j inhibits the cell growth, transformation, and tumoigenicity of Akt-activated cells. A, 14-3-3 j expression blocked cell cycle entry into theS phase in Rat1-Akt cells. Rat1-Akt cells were left uninfected (control) or infected with Ad-h-gal or Ad-14-3-3 j. Incorporation of BrdUrd was examined under afluorescence microscope using FITC-conjugated anti-BrdUrd. BrdUrd-positive cells were counted in a pool of cells. Three hundred cells were counted for BrdUrdstaining in each group of cells. The number of BrdUrd-positive cells from the control group was set as 100%. The relative % BrdUrd-positive cells in cells infectedwith Ad-h-gal or Ad-14-3-3 j shown. From a typical experiment conducted in triplicate. B, soft agar colony formation assay. Rat1-Akt cells were left uninfected (control )or infected with Ad-h-gal or Ad-14-3-3 j. The Rat1-Akt cells were then measured for anchorage-independent growth in soft agar. The relative % colony formationfrom cells infected with Ad-HA-14-3-3 j or Ad-h-gal. The number of colonies from uninfected cells was set as 100%. Bars, SD. Soft-agar colony formation assay.C, 14-3-3 j inhibited Akt-mediated tumorigenicity. Rat1-Akt cells were infected with Ad-HA-14-3-3 j, or Ad-h-gal, or left uninfected (control). Cells (1 � 106) wereharvested and s.c. injected into the flank region of female nude mice. Tumor volumes were monitored for 13 days. Change in tumor volume over a 13-day period. Bars,SD. Rat1-Akt cells (1 � 106) were harvested and s.c. injected into the flank region of female nude mice. Ad-HA-14-3-3 j or Ad-h-gal was injected at the sites ofimplantation for treatment process. Mice were injected with Ad-h-gal every day. Other mice received no injections and served as controls. Three groups of micewere injected with Ad-HA-14-3-3 j every 1, 3, or 15 days. Tumor volumes were monitored for 15 days. Change in tumor volume over the 15-day period. Points, meanvalue of six treated mice; bars, SD. D, protein expression in tumor tissues. Tumor tissues from the sites of implantation from experiment in (A) were assessedfor expressed proteins by immunoblotting with anti-phospho-Akt (p-Akt ; Ser473), anti-Akt phosphorylated substrate, anti-HA (for delivered 14-3-3 j expression),and anti-tubulin. Representative tumors.

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Recent studies show that Akt have negative effects on DNAdamage response. It was shown that activated Akt can phosphor-ylate Chk1 and reduce the nuclear localization of Chk1, therebyinterfering with Chk1-mediated p53 phosphorylation and subse-quent p53 stabilization (5). In addition, activated Akt can enhanceMdm2-mediated p53 degradation (6, 28, 32). In addition, activatedAkt can phosphorylate Miz1, thus blocking Miz1-activatedtranscription of p21, a cdk inhibitor, in response to DNA damage(33). Miz1 is a zinc-finger protein that can form a complex withc-Myc and is implicated in p21 transcriptional control (34). Giventhat activated Akt can have these mentioned effects on DNAdamage response, Akt activity must be properly restrained inresponse to DNA damage. Our results show that increasingassociation between 14-3-3 j and Akt correlates with inactivationof Akt in response to DNA damage, suggesting that 14-3-3 j mayserve as a regulator to inhibit Akt to maintain intact DNA damageresponse. Indeed, we have shown that 14-3-3 j is an importantregulator of Akt. Thus, it is clear that Akt must be controlled whencells have DNA damages, and our study provide important insightregarding how Akt is restrained in response to DNA damage.14-3-3 protein family interact with proteins involved in a wide

variety of signaling pathways of cell cycle and apoptosis, includingraf-1, Bad, forkhead transcription factors, and Cdc25 (3). However,the interacting proteins of 14-3-3 j are not well characterized,although 14-3-3 j is implicated in cell cycle, apoptosis, andtumorigenesis. The known proteins interacting with 14-3-3 j,including Cdks (12, 17), p53 (18), and Efp (35), all play importantroles in tumorigenesis. We found that Akt is a new member of 14-3-3 j-associated protein. It was shown that other 14-3-3 isoforms,including 14-3-3 u, 14-3-3 h, and 14-3-3 ~ , do not interact with Akt(36). However, another study shows that 14-3-3 ~ is able to bind Aktbut has no effect on Akt activity (37). Instead, it is phosphorylatedby Akt at Ser58 (37). This phosphorylation leads to formation of

monomer instead of dimmer (38). Conversely, 14-3-3 j does nothave Ser58 and is not a substrate of Akt (Fig. 2B), thereby preservingdimeric formation. Previous studies indicate that monomer of 14-3-3 is unable to regulate functions of target proteins, althoughmonomeric forms of 14-3-3 are capable of binding to targetproteins (39, 38). Furthermore, we have found that 14-3-3 j, but notother isoforms of 14-3-3 (g, H , and ~), inhibits Akt-mediated kinaseactivity (Fig. 2B ; data not shown). These observations indicate that14-3-3 j and other 14-3-3 members are not equivalent functionally.Structure studies have supported such a concept: 14-3-3 j hasunique amino acids (Met202, Asp204, and His206) that may beresponsible for binding particular ligands that are not recognizedby other 14-3-3 members (40, 41).The observation that 14-3-3 j reduces the activity of Akt in kinase

assays is very intriguing. It is possible that several aspects could beinvolved in 14-3-3 j–mediated Akt inhibition: (a) phosphorylationchange in the activation loop of the catalytic domain, (b) controllingsubcellular localization, (c) protein-protein interactions, such asCOOH-terminal modulator protein (CTMP), a plasma membraneprotein involved in negative regulation of Akt (42). Obviously, 14-3-3j inhibits Akt activity, but we have not found that 14-3-3 j caninteract with 3-phosphoinositide-dependent protein kinase-1(PDK1; data not shown), a kinase involved in phosphorylation of

Table 1. Summary of breast cancer tissue samples(P = 0.0024)

14-3-3 j (low) 14-3-3 j (high) Total

Akt-p (high) 11 (33.3%) 7 (21%) 18 (54.5%)Akt-p (low) 3 (9.1%) 12 (36.4%) 15 (45.5%)

Total 14 (42.4%) 19 (57.6%) 33 (100%)

Figure 5. Down-regulation of 14-3-3 jexpression correlates with activated Aktin primary breast carcinomas.Immunohistochemical studies of primarybreast adenocarcinomas. Human primarybreast cancer tissues were immunostainedwith anti-14-3-3 j and anti-P-S473 Akt(p-Akt) antibodies. Representative tissuesections from tumor I, a low 14-3-3j–expressing ductal carcinoma containinga high level of P-S473 Akt. Representativesections from tumor II, a 14-3-3j–abundant cancer with a low level ofP-S473 Akt signal.

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Akt at the activation loop (Thr308) to affect the activation of Akt andsubsequent phosphorylation at Ser473, although some of the 14-3-3members (u and D) are known to bind and affect PDK1 activity (36).Because 14-3-3 j blocks Cdc2 activity by sequestering Cdc2from the nucleus, 14-3-3 j may regulate Akt kinase activity throughcontrolling subcellular localization. However, we found that 14-3-3j did not dramatically affect the distribution of Akt (43).The detailed mechanism behind CTMP-mediated Akt inhibitionhas not been determined yet (42). Given that CTMP can also bindand reduce the phosphorylation of Akt on Ser473 (42), 14-3-3 j mayregulate CTMP to inhibit Akt kinase activity.The ability of Akt to promote survival was dependent on its

kinase activity (30, 31). The fact that 14-3-3 j inhibits Akt andpromotes apoptosis in Akt-activated cells (Fig. 3A) is reminiscentof the increased sensitivity to apoptotic stimuli in Akt knockoutmice (30). Akt1�/� mouse embryo fibroblasts (MEF) are moresusceptible to apoptosis stimuli than wild-type Akt MEF cells (30).Akt regulates Bad (44, 45), forkhead transcription factor (46, 47),and inhibition of Ced3/interleukin 1h converting enzyme–likeproteases (caspases; ref. 31) to inhibit apoptosis. Given that 14-3-3j inhibits Akt kinase activity, 14-3-3 j may be involved inregulating some of these proapoptotic signals. Indeed, we havefound that 14-3-3 j caused the PARP cleavage in Akt-activated cells(Fig. 3B), suggesting that 14-3-3 j antagonizes the inhibitoryactivity of Akt toward caspase activation to promote apoptosis. Incontrast, several isotypes of 14-3-3 are involved in suppressingapoptosis by binding to the phosphorylated death agonist BAD(48), inhibiting apoptosis signal-regulating kinase 1 (49), or bindingto the forkhead transcription factor to block gene expression

involved in apoptosis (46), suggesting that some 14-3-3 familymembers are indeed mediators of antiapoptotic signals.Based on negative regulatory activity of 14-3-3 j toward Akt, we

propose that 14-3-3 j has tumor-suppressive activity in Akt-activated cancer cells. Our studies indicated that 14-3-3 j inhibitedthe tumorigenicity of Akt-transformed cells, highlighting thetumor-suppressive role of 14-3-3 j. Importantly, the significanceof our studies is that Akt activities are suppressed by 14-3-3 jexpression. Given that Akt promotes cell growth and survival and isactivated in several types of cancers as a result of dysregulation inthe PI3K/Akt pathway, our findings in defining the negative role of14-3-3 j in Akt signaling and in exploring 14-3-3 j as an anticanceragent have important clinical relevance. Recently, we showed that14-3-3 j is efficient in inhibiting the tumorigenicity of nasopha-ryngeal carcinoma cells (50). Thus, targeting Akt by theadministration of 14-3-3 j could be an excellent therapeuticregime for the treatment of cancers in which the PI3K/Akt pathwayis constitutively activated, including cancer cells with themutations of the PTEN tumor suppressor.

Acknowledgments

Received 10/6/2005; revised 12/9/2005; accepted 1/10/2006.Grant support: NIH grant RO1CA 089266, Cancer Center Core grant CA16672,

Flemin and Davenport (M-H. Lee), and the Susan G. Koman Breast Cancer Foundation(M-H. Lee).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Vogelstein (The Johns Hopkins Oncology Center, Baltimore, MD)and Hung (UT MD Anderson Cancer Center, Houston, TX) for valuable reagents andDrs. Lozano, Legerski, and Behringer for critical reading and comments.

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2006;66:3096-3105. Cancer Res   Huiling Yang, Yu-Ye Wen, Ruiying Zhao, et al.   CancerKinase B/Akt Activation and Suppresses Akt-Activated

Inhibits ProteinσInduced Protein 14-3-3 −DNA Damage

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