pten regulates aurora-a and cooperates with fbxw7 in

Signaling and Regulation

Pten Regulates Aurora-A and Cooperates with Fbxw7 inModulating Radiation-Induced Tumor Development

Yong-Won Kwon1, Il-Jin Kim2,3, Di Wu3, Jing Lu5, William A. Stock Jr1,6, Yueyong Liu1, Yurong Huang1,Hio Chung Kang3, Reyno DelRosario3, Kuang-Yu Jen3,4, Jesus Perez-Losada7, Guangwei Wei1,5,Allan Balmain3, and Jian-Hua Mao1

AbstractThe Aurora-A kinase gene is frequently amplified and/or overexpressed in a variety of human cancers, leading to

major efforts to develop therapeutic agents targeting this pathway. Here, we show that Aurora-A is targeted forubiquitination and subsequent degradation by the F-box protein FBXW7 in a process that is regulated by GSK3b.Using a series of truncated Aurora-A proteins and site-directed mutagenesis, we identified distinct FBXW7 andGSK3b-binding sites in Aurora-A. Mutation of critical residues in either site substantially disrupts degradation ofAurora-A. Furthermore, we show that loss of Pten results in the stabilization of Aurora-A by attenuating FBXW7-dependent degradation of Aurora-A through the AKT/GSK3b pathway. Moreover, radiation-induced tumorlatency is significantly shortened in Fbxw7þ/�Ptenþ/� mice as compared with either Fbxw7þ/� or Ptenþ/� mice,indicating that Fbxw7 and Pten appear to cooperate in suppressing tumorigenesis. Our results establish a novelposttranslational regulatory network inwhich the Pten and Fbxw7pathways appear to converge on the regulation ofAurora-A level. Mol Cancer Res; 10(6); 834–44. �2012 AACR.

IntroductionAurora-A kinase, a key regulator of chromosome segre-

gation and cytokinesis (1), is frequently amplified in avariety of human carcinomas, including breast, colorectal,pancreatic, and bladder cancers (2–5). Overexpression ofAurora-A in tumors is correlated with clinically aggressivedisease (6). A wealth of functional data exists showing thatoverexpression of Aurora-A leads to centrosome amplifica-tion, chromosomal instability, and oncogenic transforma-tion (5, 7). Furthermore, overexpression of Aurora-A intransgenic mouse models result in the development ofmammary gland tumors (8). Taken together, these dataindicate that Aurora-A possesses oncogenic activity and maybe a valuable therapeutic target in cancer therapy (6, 9).

Consequently, several small-molecule inhibitors of Aurora-A kinase have been developed and are currently undergoingclinical trials.FBXW7 is a p53-dependent tumor suppressor that under-

goes deletion and/or mutation in a number of humanneoplasms, including those from the breast, colon, endo-metrium, stomach, lung, ovary, pancreas, and prostate (10).The FBXW7 gene encodes an F-box protein essential forubiquitination of several well-defined oncoproteins includ-ing c-Myc (11), cyclin E (12), Notch (13), c-Jun (14), andmTOR (15). Previous studies have shown that FBXW7binds Aurora-A in cell lines (16) and that depletion of Fbxw7results in increased Aurora-A expression (17); however, themechanism by which Fbxw7 controls Aurora-A levelremains unknown. In the present study, we show that Fbxw7physically binds Aurora-A and facilitates the ubiquitinationand degradation of Aurora-A through the proteasome path-way. Furthermore, we show that this process occurs in aGsk3b-dependent manner, similar to what has beenobserved for several oncoproteins that undergo FBXW7-mediated ubiquitination.Prior studies indicate that GSK3b is inactivated through

phosphorylation at the Ser-9 position (18), which occursthrough PI3K/AKT signaling. Because the PI3K/AKT path-way is crucial in tumorigenesis and is regulated by the tumorsuppressor PTEN, we further investigated whether PTEN isable to regulate Aurora-A via the PI3K/AKT/GSK3b path-way. Our results reveal that similar to Gsk3b inhibition,downregulation of Pten by short hairpin RNA (shRNA)increased the level and half-life of Aurora-A by blocking itsubiqutination. Finally, we show that the deletion of one copyof either Fbxw7 or Pten leads to the increase in protein levels

Authors' Affiliations: 1Life Sciences Division, Lawrence BerkeleyNational Laboratory, University of California, Berkeley; 2Thoracic Oncol-ogy Laboratory, Department of Surgery, 3Helen Diller Family CancerCenter, 4Department of Pathology, University of California at San Fran-cisco, San Francisco, California; 5Department of Anatomy, ShandongUniversity School of Medicine, Jinan, Shandong, PR China; 6Under-graduate Program, Department of Biomedical Engineering, WashingtonUniversity in St. Louis, St. Louis, Missouri; and 7Instituto de BiologíaMolecular y Celular del C�ancer (IBMCC), Instituto Mixto Universidad deSalamanca/CSIC, Campus Miguel de Unamuno s/n, Salamanca, Spain

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Y.W. Kwon and I.J. Kim contributed equally to this work.

Corresponding Author: Jian-Hua Mao, Life Sciences Division, LawrenceBerkeley National Laboratory, University of California, Berkeley, CA 94720.Phone: 1-510-4866204; Fax: 1-510-4952535; E-mail: [email protected].

doi: 10.1158/1541-7786.MCR-12-0025

�2012 American Association for Cancer Research.

MolecularCancer

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of Aurora-A in vivo, and mice heterozygous for both Fbxw7and Pten (Ptenþ/�Fbxw7þ/�) display significantly shortenedlatency for tumor development when compared with eitherPtenþ/� mice or Fbxw7þ/� mice, indicating that simulta-neous loss of Pten and Fbxw7 has a synergic effect intumorigenesis. In summary, our data establish a novelregulatory pathway in which interactions between Fbxw7,Pten, and Gsk3b ultimately regulate Aurora-A levels in aposttranslational manner. We also show that disruption ofmultiple components of this regulatory pathway can accel-erate tumor development.

Materials and MethodsCells and cell cultureNIH3T3 (mouse embryonic fibroblast), C5N (mouse

keratinocyte cell line), and HEK293T were maintained inDulbecco's Modified Eagle's Medium (Invitrogen) supple-mented with 10% fetal calf serum (Invitrogen). HCT116andHCT116 FBXW7�/� cell lines were a gift fromDr. BertVogelstein.

Antibodies and reagentsThe antibodies and reagents used were from the following

sources: anti-PTEN rabbit polyclonal (1:1,000), anti-Aktrabbit polyclonal (1:1,000), anti-phospho-Akt (Ser473)rabbit polyclonal (1:1,000), anti-Gsk3b rabbit polyclonal(1:1,000), and anti-phospho-Gsk3b (Ser9) rabbit polyclonal(1:1,000) antibody were from Cell Signaling; anti-Aurora-A(IAK1)mousemonoclonal antibody (1:2,000) was fromBDSciences; anti-Flag-M2 Affinity gel, anti-HA-Affinity gel,and anti-b-actin mouse monoclonal antibody (1:5,000)were from Sigma; cycloheximide (C4859) was from Sigma;MG132 and Gsk3b inhibitor VIII were from Calbiochem.

Plasmids construction and site-directed mutagenesispCG-N-HA-FBXW7 was a gift from Prof. K.I.

Nakayama, and pCG-N-HA-Ubiquitin was a gift from Dr.O. Tetsu. Full-length and truncated Aurora-A were ampli-fied by PCR and subcloned into BglII/XhaI sites of p3xFlag-CMV-10 (Sigma). The Aurora-A mutants (T217A/E221A,S226A/E230A, S245A, S387A, and S245A/S387A) weregenerated by site-directed mutagenesis (Stratagen). Theprimers for constructing and mutagenesis were listed inSupplementary Table S1. All the constructs were validatedby DNA sequencing.

shRNA constructs and retrovirus productionAn shRNA with high Pten knockdown efficiency was

cloned into pSuper.Retro-puro (Oligoengine). The shRNAsequence for Pten is 50-GACCATAACCCACCACAGC-30.NIH3T3 and C5N cell lines were infected with high-titerretroviral stocks produced by the transient transfection of293T ecotropic Phoenix cells. After infection with thepSuper.Retro.puro retrovirus, which allowed the expressionof the shRNA, cells were selected with 1 to 2 mg/mL ofpuromycin in the culture medium. pSuper.Retro.puro vec-tor without shRNA was used as control.

Immunoblotting and immunoprecipitationCells were collected by washing with cold PBS at 4�C.

Total protein extracts were prepared from cells and normaland tumor tissues with RIPA lysis buffer (1% Triton X-100,0.1% SDS, 50 mmol/L Tris pH 7.5, 150 mmol/L NaCl,0.5% sodium deoxycholate, 10 mmol/L NaF) supplemen-ted with 2 mmol/L phenylmethylsulfonylfluoride (PMSF),2 mmol/L Na3VO4, and complete protease inhibitors(Roche). The lysates were fractionated by SDS-PAGE andelectoblotted on to polyvinylidene fluoride (PDVF) mem-brane (Millipore). The membranes were then incubated for1 hour in TBS/Tween-20 (0.1%) containing 5% nonfatskim milk, then with primary antibodies for 1 hour at roomtemperature or overnight at 4�C, and the target antigenswere identified with the appropriate horseradish peroxidase–labeled secondary antibody (Amersham) in the presence ofSuperSignal West Pico Chemoluminiscent substrate(Pierce).For immunoprecipitation, 293T cells were transiently

transfected using Lipofectamine 2000 with p3xFlag-Auro-ra-A and pCGN-HA-Fbxw7. Forty-eight hours after trans-fection, MG132 (10 mmol/L) and Gsk3b inhibitor (25mmol/L) were added to the media. Six hours later, cells werelysed on ice inNG lysis buffer (50mmol/LTris, pH7.5, 150mmol/L NaCl, 10% glycerol, 1% NP-40, 10 mmol/L NaF,2 mmol/L PMSF, 2 mmol/L Na3VO4, plus "completeprotease inhibitors"; Roche). The lysates were incubatedwith anti-Flag-M2 affinity gel (Sigma) or anti-HA (3F10,Roche) with protein-G-sepharose (GE Healthcare). Immu-nocomplexes were isolated, fractionated by SDS-PAGE, andidentified by immunoblot using anti-Flag, anti-HA, or anti-Gsk3b.

Ubiquitination assayCells were transfected with a plasmid encoding a HA-

ubiquitin. Twenty-four hours after transfection, the protea-some inhibitor MG132 (10 mmol/L) was added. Six hourslater, anti-HA immunoprecipitates were recovered andimmunoblotted with anti-Aurora-A antibody.

Analysis of mRNATotal RNA was purified with TRIzol (Invitrogen) accord-

ing to the manufacturer's instructions and then 5 mg of eachsample was reverse transcribed using the SuperScript IIRNaseH first-strand synthesis system (Invitrogen). PCRwascarried out with 2 mL of cDNA from each sample usingExtaqDNApolymerase (TAKARA). Primers for Flag-taggedAurora-A were as follows: sense 50-GACTACAAAGAC-CATGACGGT-30 (for Flag) and antisense 50-TGGTGCA-TATTCCAGAATTAGG-30 (Aurora-A 621–642).

Mice and tumor inductionFbxw7 and Pten single or double heterozygous knockout

mice were generated by crossing Fbxw7þ/� heterozygousknockout mice (C57BL/6J) with Ptenþ/� heterozygousknockout mice (C57BL/6J) purchased from JAX Labora-tories. Five-week-old mice of both sexes were exposed to asingle dose of 4 Gy whole body g-irradiation and monitored

Cooperative Role of Pten and Fbxw7 in Tumorigenesis

www.aacrjournals.org Mol Cancer Res; 10(6) June 2012 835




daily until moribund, then killed and autopsied. Mice werebred and treated under UCSF LARC regulations.

Statistical analysisThe Kaplan–Meier method was used to compare the

survival time postradiation between different genotypicgroups of mice using the SPSS statistical package.

ResultsFbxw7 binds to and targets Aurora-A for ubiquitin-mediated degradationWe previously showed that depletion of Fbxw7 resulted

in increased Aurora-A expression (17); however, the mech-

anism by which this regulation occurs is unknown. Previ-ous study suggests that FBXW7 binds Aurora-A in immu-noprecipitation assays (16). We hypothesize that FBXW7may physically interact with Aurora-A to regulate itsubiquitination and turnover by proteasomal degradation.To address this, 293T cells were cotransfected with Flag-tagged Aurora-A and hemagglutinin (HA)-tagged FBXW7.Next, immunoprecipitation and subsequent immunoblotanalyses using antibodies directed against the Flag or HAtag were conducted and revealed that HA-tagged FBXW7coimmunoprecipitated with Flag-tagged Aurora-A, indicat-ing a physical interaction between the 2 proteins (Fig. 1Aand B). Moreover, we found that HA-tagged FBXW7

Figure 1. Aurora-A interacts with FBXW7 and GSK3b. A and B, co-immunoprecipitation of Flag-tagged Aurora-A and HA-tagged FBXW7 in transientlytransfected 293T cells. Immunoprecipitations were carried out with either (A) anti-Flag or (B) anti-HA antibodies. Immunoprecipitates were fractionatedby SDS-PAGE and subsequently immunoblotted with (A) anti-HA or (B) anti-Flag antibodies. Co-immunoprecipitation of endogenous GSK3b was alsodetected using anti-GSK3b antibody. Bottom shows the input levels of FBXW7,GSK3b, andAurora-A in the lysates. C, co-immunoprecipitation ofHA-taggedFBXW7 with endogenous Aurora-A. 293T cells were transiently transfected with HA-tagged FBXW7. Immunoprecipitation was carried out with anti-HAantibody and subsequently immunoblotted with anti-Aurora-A antibody. Bottom shows the input levels of FBXW7 and Aurora-A in the lysates. All resultsare representative of 3 independent experiments. WB, Western blotting.

Figure 2. Aurora-A ubiquitinationis Fbxw7 dependent. A, NIH3T3and C5N cells show increasedendogenous Aurora-A levels upontreatment with proteasomeinhibitor MG132. B, HCT116FBXW7WT and HCT116 FBXW7�/

� cells were transfected with HA-ubiquitin. Immuno-precipitation ofHA followedby Western blot (WB) analysisof Aurora-A showed thatubiquitination of Aurora-Awas onlyseen in the HCT116 FBXW7 WTcells (column 2) and not in HCT116FBXW7�/� cells (column 4). Allresults are representative of 3independent experiments.

Kwon et al.

Mol Cancer Res; 10(6) June 2012 Molecular Cancer Research836




also coimmunoprecipitated with endogenous Aurora-A(Fig. 1C).Next, we determined whether the regulation of Aurora-A

by Fbxw7 is through the proteasome-dependent degradationpathway. NIH3T3 and C5N cells were treated with theproteasome inhibitor MG132. Proteasome inhibitioncaused a significant increase in Aurora-A levels in both celllines (Fig. 2A), suggesting that Aurora-A is regulated in aposttranslational manner. To examine whether the ubiqui-tination status of Aurora-A is Fbxw7 dependent, HCT116FBXW7 wild-type (WT) and FBXW7�/� cells were trans-fected with a vector containing cytomegalovirus (CMV)promoter-driven HA-ubiquitin. Immunoprecipitation of

HA followed by immunoblot analysis of Aurora-A showedthat Aurora-A ubiquitination was only found in HCT116FBXW7 WT cells, but not in HCT116 FBXW7�/� cells(Fig. 2B), consistent with previous findings (19). In sum-mary, these results suggest that FBXW7 binds to Aurora-Aand targets Aurora-A for ubiquitin-mediated degradationthrough the proteasome.

Fbxw7 mediates ubiquitination of Aurora-A in aGsk3b-dependent mannerBecause Fbxw7-mediated ubiquitination of most sub-

strates (such as cyclin E, c-Jun, and Myc) occurs in aGsk3b-dependent manner, we examined whether Fbxw7

Figure 3. GSK3b regulates the ubiquitination and degradation of Aurora-A by Fbxw7. A, NIH3T3 andC5N cells show increasedAurora-A levels upon treatmentwith Gsk3b inhibitor VIII. B, NIH3T3 cells transfected with Flag-tagged Aurora-A show increased endogenous and exogenous Aurora-A levels upontreatment with MG132. Further increase in Aurora-A levels is observed when cells were treated with both MG132 and Gsk3b inhibitor VIII. C, Gsk3b inhibitorVIII treatment stabilizes Aurora-A protein. CHX indicates hours after cycloheximide treatment. D, quantification of Aurora-A protein levels shown in (C).E, Gsk3b inhibitor VIII treatment dramatically reduces ubiquitination of Aurora-A. All results are representative of 3 independent experiments.






A

3xFlag

3xFlag

1 403383131Aurora-A

WT (1-403)

Catalytic domain

ΔN2 (90-403)

3xFlag

3xFlag

3xFlag

3xFlag

3xFlag

3xFlag

3xFlag

3xFlag

ΔN3 (101-403)

ΔN4 (134-403)

ΔN5 (173-403)

ΔN6 (202-403)

ΔN7 (229-403)

ΔC3 (1-230)

ΔC2 (1-244)

ΔC1 (1-386)

Interact ion withFBXW7 GSK3ββ

+

+

+

+

+

+

+

–

3xFlagΔN1 (71-403)

+

+

+

+

–

+

++

+

+

+

+

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–

B

Fbxw7 degron consensus: I/L-I/L/P-S/T-P-XXXX

202 DATRVYLILEYAPLGTVYRELQKLSKFDE 230Aurora-A

T217A/E221A

S226A/E230A

202 DATRVYLILEYAPLGAVYRALQKLSKFDE 230

202 DATRVYLILEYAPLGTVYRELQKLAKFDA 230

C D

Figure 4. Identification of the FBXW7-binding site within Aurora-A. A, schematic representation of full-length Aurora-A and its truncated forms thatwere tested for their ability to bind Fbxw7. Numbers in brackets indicate the amino acids that comprise the various truncated Aurora-A proteins."þ" indicates that the truncated Aurora-A is able to bind to FBXW7 or GSK3b whereas "�" indicates that the truncated Aurora-A is unable tobind to FBXW7 or GSK3b. B, co-immunoprecipitation of the truncated forms of Flag-tagged Aurora-A with either HA-tagged Fbxw7 or GSK3b.HA-tagged FBXW7 and truncated forms of Flag-tagged Aurora-A were expressed in 293T cells, followed by immunoprecipitation (IP) with anti-Flag

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ubiquitination of Aurora-A is also dependent on Gsk3bactivity. First, Gsk3b coimmunoprecipitated with bothFlag-tagged Aurora-A and HA-tagged Fbxw7 (Fig. 1A andB), suggesting that Gsk3b may exist in a complex withAurora-A and Fbxw7. In addition, in the presence of Gsk3binhibitor, the binding between Fbxw7 and Aurora-A wasdramatically reduced (Fig. 1A and B, lane 3), indicating thatphysical interaction between Fbxw7 and Aurora-A is depen-dent on Gsk3b activity. Moreover, Gsk3b inhibitor treat-ment of both NIH-3T3 and C5N cell lines resulted in asignificant increase in Aurora-A levels (Fig. 3A). The proteinlevels of both endogenous and exogenous Aurora-Aincreased even further in cells treated with both the protea-some and Gsk3b inhibitors (Fig. 3B). Gsk3b inhibitortreatment also dramatically increased the stability andhalf-life of Aurora-A (Fig. 3C and D). Finally, we investi-gated whether Gsk3b inhibitor treatment blocked Aurora-Aubiquitination by Fbxw7. 293T cells were transfected with avector containing CMV promoter-drivenHA-ubiquitin andwere then treated with Gsk3b inhibitor. Immunoprecipita-tion of HA followed by immunoblot analysis of Aurora-Ashowed that Aurora-A ubiquitination was dramaticallyreduced in the cells following Gsk3b inhibitor treatment(Fig. 3E). Taken together, these results indicate that Gsk3bis able to physically interact with both Fbxw7 and Aurora-Aand that Gsk3b is required for Fbxw7-mediated ubiquitina-tion of Aurora-A.

Mapping the FBXW7-binding site within Aurora-AThe consensus CDC4 phosphodegron (CPD) sequence I/

L-I/L/P-S/T-P-XXXX (where lysine and arginine are unfa-vorable in the X locations) of FBXW7 was not found inAurora-A using bioinformatics analysis. To identify theregion within Aurora-A that is responsible for interactionwith FBXW7, we made serial truncations of Aurora-A (Fig.4A), coexpressed them with HA-tagged Fbxw7 inHEK293T cells, and tested the ability of the truncatedproteins to bind to FBXW7 by immunoprecipitation.FBXW7 bound to DN1-DN6 and DC1-DC3, but not toDN7 (Fig. 4A and B, Supplementary Fig. S1), implicatingresidues 202 to 230 of Aurora-A as the critical regionnecessary for interaction with FBXW7. Closer inspectionof this region showed that 2 potential sites are similar to theFBXW7 CPD (Fig. 4C). To investigate the significance ofeach individual site for binding FBXW7, we generated 2mutants: T217A/E221A and S226A/E230A. S226A/E230A retained the ability to bind FBXW7 although thisinteraction was significantly reduced as compared with wild-type Aurora-A (Fig. 4D). On the other hand, FBXW7binding capability was completely abolished in T217A/E221A, indicating that the T217E221 site is largely respon-

sible for the physical interaction between Aurora-A andFBXW7.

Mapping the GSK3b-binding site within Aurora-ATo map the GSK3b-binding region in Aurora-A, we used

the same serial truncated proteins of Aurora-A describedearlier to test their ability to bind to GSK3b. Only DC2 andDC3 were not able to bind GSK3b, indicating that the 173C-terminal residues of Aurora-A are required for interactionwith GSK3b (Fig. 4A and B, Supplementary Fig. S1). Usingthe PhosphoMotif Finder software, we found that this C-terminal region of Aurora-A (amino acids 230–403) harbors2 potential GSK3b phosphorylation sites (Fig. 5A). Toinvestigate the significance of each individual phospho-rylation site, we systematically disrupted each potentialsites with alanine substitutions (Fig. 5A). Although bothsingle site Aurora-A mutants S245A and S387A interactedwith GSK3b, the S245A/S387A double mutant complete-ly lost the capacity to bind to GSK3b (Fig. 5B). Inter-estingly, the S245A/S387A double mutant also displayedsignificantly reduced binding capability to FBXW7(Fig. 4D).

FBXW7- and GSK3b-binding sites within Aurora-A arecrucial for proteasome-dependent degradation ofAurora-ABecause FBXW7 seems to mediate ubiquitination of

Aurora-A in a GSK3b-dependent manner, we investigatedwhether disruption of the interactions between FBXW7/Aurora-A and GSK3b/Aurora-A would block proteasome-dependent degradation of Aurora-A. First, 293T cells weretransfected with either flag-tagged wild-type Aurora-A,T217A/E221A, S226A/E230A, or S245A/S387A mutants.Subsequent immunoblot analysis with anti-flag antibodyrevealed that while wild-type and the S226A/E230Amutantexhibited low levels of expression, the T217A/E221A andS245A/S387A mutants displayed significantly greater pro-tein levels (Fig. 5C, left), suggesting that disruption of eitherFBXW7- or GSK3b-binding site within Aurora-A substan-tially stabilizes (or decreases turnover of) Aurora-A. In con-trast, when these cells were treated with the proteasomeinhibitor MG132, all groups displayed comparably highlevels of Aurora-A protein (Fig. 5C, right). When comparingM132 treated and untreated cells, a significant increase inlevels of wild-type Aurora-A and the S226A/E230A mutantoccurred following M132 treatment, whereas levels of theT217A/E221A and S245A/S387A mutants did not change.To verify that the observed differences in Aurora-A levelsresulted from posttranslational regulation, transcript levelsof the wild-type and mutant forms of Aurora-A were mea-sured by semiquantitative reverse transcription-PCR, which

antibodies and immunoblotting with antibodies against HA or GSK3b. Bottom shows the input levels of GSK3b, FBXW7, and Aurora-A proteins in thelysates. C, potential FBXW7-binding sites within Aurora-A. Numbers indicate the amino acid residues that were substituted with alanines to generatethe corresponding mutant Aurora-A. D, co-immunoprecipitation of HA-tagged Fbxw7 with wild-type or mutant forms of Flag-tagged Aurora-A intransiently transfected 293T cells. Immunoprecipitations were carried out with anti-Flag antibody and subsequently immunoblotted with anti-HAantibodies. Bottom shows the input levels of FBXW7 and Aurora-A proteins in the lysates.






showed no differences between any of the Aurora-A variants(Supplementary Fig. S2). As a whole, these data suggest thatthe interactions of Aurora-A with FBXW7 and GSK3b arecrucial for proteasome-dependent turnover of Aurora-A.

Loss of Pten stabilizes Aurora-A via the Akt/Gsk3bpathwayPten is a negative regulator of the Pi3k/Akt pathway (20).

Activation of Pi3k/Akt signaling leads to the phosphoryla-tion (Ser-9) and inactivation of Gsk3b (18). Thus, weinvestigated whether Pten could regulate Aurora-A via theAkt/Gsk3b pathway. To examine the effects of Pten down-regulation on Aurora-A level and stability, we designed ashRNA against Pten (shPten) and generated stable transfec-tants in NIH3T3 and C5N cells. The shRNA effectivelyreduced Pten protein expression in both cell lines (Fig. 6A).Downregulation of Pten led to the increase in Aurora-Alevels as compared with controls (Fig. 6A). We also analyzedlevels of other Fbxw7 substrates that were shown to beubiquitinated in a Gsk3b-dependent manner. Levels of c-Jun were unaffected in both cell lines (Fig. 6A). c-Myc wasundetectable in NIH3T3 cells even after downregulation ofPten, whereas levels of c-Myc increased in C5N cells withshPten (Fig. 6A). The expression levels of p-Akt (serine-473)were also significantly increased in shPten-transfected cellsand presumably were responsible for the inactivation ofGsk3b. Indeed, the level of p-Gsk3b (Ser-9) increaseddramatically with downregulation of Pten. In contrast, thetotal level of Gsk3b did not change. As expected, the level ofp-mTOR (Ser2448) increased whereas the level of totalmTOR did not change (Fig. 6A). Furthermore, in NIH3T3cells with shPten, Gsk3b inhibitor treatment did not causean increase in Aurora-A levels, indicating that Pten regulatesAurora-A via the Akt/Gsk3b pathway (Fig. 6B).The stability of Aurora-A was also examined in shPten

cells after treatment with cycloheximide. Aurora-A was

much more stably maintained in shPten cells than in thecontrol cells (Fig. 6C and D). Furthermore, downregulationof Pten blocked Aurora-A ubiquitination (Fig. 6E). Insummary, these findings are similar to those that wereobserved withGsk3b inhibitor treatment, further suggestingthat loss of Pten attenuates the degradation of Aurora-A byFbxw7 through the Akt/Gsk3b pathway.

Pten synergizes with Fbxw7 in radiation-induced tumordevelopmentWe have previously used a mouse model of radiation-

induced lymphoma to show that haploinsufficiency for P53,Pten, or Fbxw7 confers susceptibility to lymphoma devel-opment (21–23). Because we have established that Pten andFbxw7 pathways both impact Aurora-A levels and mTORsignaling; therefore, we speculate that simultaneous loss ofPten and Fbxw7 may result in a synergistic effect on tumordevelopment in vivo. To investigate this possibility, wecrossed Fbxw7þ/� mice with Ptenþ/� mice to generatePtenþ/�Fbxw7þ/� mice. Twenty-four Ptenþ/�Fbxw7þ/�,26 Fbxw7þ/�, 31 Ptenþ/�, and 30 wild-type mice wereexposed to a single dose of 4 Gy whole body g-irradiationat 5 weeks of age. Within 65 weeks, none of wild-typemice developed any tumors (Fig. 7A). In contrast, a signi-ficant portion of Fbxw7þ/� (40%) and Ptenþ/� (95%)mice developed tumors by 65 weeks postirradiation (P <0.0001; Fig. 7). Interestingly, the double heterozygousFbxw7þ/�Ptenþ/� mice developed tumors much fasterthan either Fbxw7þ/� or Ptenþ/�mice (P < 0.0001; Fig. 7).These data indicate that Pten synergizes with Fbxw7 insuppressing radiation-induced tumorigenesis.Next, we investigated the effects of loss of either Fbxw7 or

Pten on Aurora-A protein levels in the normal and tumortissues byWestern blotting. One consistent observation wasthat normal tissues from Fbxw7þ/�mice had higher proteinlevels of Aurora-A than the equivalent tissue from wild-type

GSK3 phosphorylation motif: SXXXS

238 TELANALSYCHSKRVIHRD 256

381 PWITANSSKPSNCQNKE 397

GSK3 phosphorylation site Priming phosphorylation site

Aurora-A

Aurora-A

S245A

S387A

238 TELANALAYCHSKRVIHRD 256

381 PWITANASKPSNCQNKE 397

A

B C

Figure 5. Identification of GSK3b-binding sites within Aurora-A. A,potential GSK3b-binding siteswithin Aurora-A. Numbers indicatethe amino acid residues that weresubstituted with alanines togenerate the correspondingmutant Aurora-A. B, co-immunoprecipitation ofendogenousGSK3bwith wild-typeor mutant forms of Flag-taggedAurora-A in transiently transfected293T cells. Immunoprecipitations(IP) were carried out with anti-Flagantibody and subsequentlyimmunoblotted with anti-GSK3bantibodies. Bottom shows theinput levels ofGSK3bandAurora-Aproteins in the lysates. C,mutations at the Fbxw7- andGSKb-binding sites within Aurora-A result in increased levels ofAurora-A. This effect is abolished inthe presence of proteasomeinhibitor MG132.

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mice (Fig. 7B). Similar observations were made with thetissues from Ptenþ/� mice (Fig. 7B). It is also found thattumors retained the higher starting levels of endogenousAurora-A protein, but few tumors from Ptenþ/� miceshowed the lower Aurora-A level (Fig. 7C). These datatogether with our previous findings (15) suggests a novelinteraction between the Fbxw7 and Pten tumor suppressorpathways that controls the levels of the oncoprotein Aurora-A kinase and mTOR, but we could not fully exclude thepossibility of other oncogenes, such as Myc. Additionalstudies will be granted.

DiscussionAurora-A controls chromosome segregation during mito-

sis and is amplified and/or overexpressed in a large propor-tion of human tumors. However, the frequency of Aurora-Aoverexpression in human cancers is much higher than that of

its amplification. For example, in breast cancers, Aurora-A isamplified in 12% of cases whereas it is overexpressed in 98%of cases. The discrepancy between overexpression and ampli-fication suggests that elevated Aurora-A levels are likelymediated through other mechanisms such as posttransla-tional modifications. In this study, we show that 2 tumorsuppressor genes, Fbxw7 and Pten, regulate Aurora-A levelsin a posttranslational manner.Our results reveal that Aurora-A is targeted for ubiquitin-

dependent degradation by FBXW7 in a process that isregulated by GSK3b. A recent study showed that over-expression of Aurora-A in AGS and SNU1 gastric cancercells led to increased phosphorylation and inactivation ofGsk3b (24), suggesting that a positive feedback loop existsbetween Gsk3b and Aurora-A. This process may be medi-ated through Akt as it has been shown that Aurora-A canactivate Akt through phosphorylation (24, 25). As a result, it

Figure 6. Pten regulates Aurora-A by attenuating Aurora-A degradation by Fbxw7. All results are representative of 3 independent experiments. A, shRNAknockdown of PTEN results in higher levels of Aurora-A in both NIH3T3 and C5N cells. B, upon treatment with Gsk3b inhibitor VIII, Aurora-A levels remainthe same following shRNA knockdown of PTEN in NIH3T3 cells. C, shRNA knockdown of PTEN stabilizes Aurora-A. CHX indicates hours postcycloheximidetreatment. D, quantification of Aurora-A levels shown in (C). E, shRNA knockdown of PTEN abolishes ubiqutination of Aurora-A.






is possible that Aurora-A activates Akt followed by Aktinactivation of Gsk3b resulting in the increase of Aurora-A levels. Furthermore, on the basis of our results that Ptennegatively regulates Aurora-A through AKT/GSK3b,together with the recent report by Taga and colleagues(26) that long-term overexpression of Aurora-A in cells leadsto degradation of Pten, it appears that yet another layer ofpositive feedback may be operating within this complexsignaling network (Supplementary Fig. S3).Unlike other FBXW7 targets, the consensus CDC4

phosphodegron motif was not found in Aurora-A. Usinga series of Aurora-A truncated proteins, we have nowidentified residues 202 to 230 of the Aurora-A as theminimal region required for binding to FBXW7, which islocated within Aurora-A catalytic domain. Moreover, theresidues T217/E221 have been defined critical for binding

to FBXW7 as our results showed that mutation of these 2residues totally abolished the interaction between Aurora-A and FBXW7. Interestingly, recent studies showed thatT217 is an important residue for developing small inhi-bitors that are highly selective for targeting Aurora-A(27, 28).In this study, we also found 2 GSK3b-binding sites in

Aurora-A. Our results indicate that both sites are equiva-lently important for the interaction between Aurora-A andGSK3b. Mutation of either site alone is insufficient to blocktheir interaction. Surprisingly, we found that the GSK3b-binding sites in Aurora-A are distinct from the FBXW7binding site, which differs from what is found in otherFBXW7 targets, such as c-Myc and CCNE1. Mutation ofthe GSK3b-binding sites within Aurora-A or pharmacologicinhibition of GSK3b reduces the binding affinity between

A

n+/–

+/– +

/–

+/–

+/–

+/–

+/–

+/–

+/–

+/–

+/–

etP+/–7wxbF

1 2 3 4 5 6

Aurora-A

ββ-Actin

Thymus

Fbxw7+/–Pten+/–

1 2 3 4 5 6 1 2 3 4 5 6

Thymic Lymphoma

B

C

Aurora-A

β-Actin

Thymus Spleen Lung Liver

Aurora-A

β-Actin

Mouse

#1

#2

Figure 7. Pten interacts with Fbxw7in suppressing radiation-inducedtumorigenesis. A, radiation-induced tumor latency issignificantly shortened in Fbxw7þ/

�Ptenþ/� mice as compared witheither Fbxw7þ/� or Ptenþ/� mice.Wild-type (open squares; n ¼ 30),Fbxw7þ/�Ptenþ/� (open circles; n¼ 24), Fbxw7þ/�Ptenþ/þ (opentriangles; n ¼ 26), and Fbxw7þ/

þPtenþ/� (open diamonds; n ¼ 31)micewere exposed to asingle doseof 4Gywhole body g irradiation at 5weeks of age and followed up for65 weeks postradiation. B,Western blot analysis showingincreased Aurora-A level whendeletion of one copy of eitherFbxw7 or Pten in normal thymus,spleen, lung, and liver tissue. C,detection of Aurora-A protein levelin radiation-induced tumors fromFbxw7þ/�, Ptenþ/� and Fbxw7þ/

�Ptenþ/�mice byWestern blotting.

Kwon et al.





Aurora-A and FBXW7, suggesting that GSK3b binding toAurora-A promotes the interaction between Aurora-A andFBXW7. Nevertheless, both GSK3b- and FBXW7-bindingsites play an equivalent role in degradation of Aurora-A.Mutation of critical residues in either sites substantiallystabilizes Aurora-A.In conclusion, we have identified a novel interaction

between the Fbxw7 and Pten tumor suppressor pathwaysthat controls the levels of the oncoprotein Aurora-Akinase. We show that Fbxw7 degrades Aurora-A throughphysical interaction. Like most of the other known Fbxw7substrates, ubiquitination of Aurora-A by Fbxw7 is aGSK3b-dependent event. Furthermore, we show that lossof Pten also increases the stability of Aurora-A through theAkt/GSK3b pathway. Using a genetic mouse model, wereport that mice heterozygous for both Fbxw7 and Ptenhave significantly reduced tumor latency compared withmice heterozygous for either Fbxw7 or Pten, and normaltissues from Fbxw7 þ/� or Ptenþ/� mice have higherprotein levels of Aurora-A than the equivalent tissue fromwild-type mice. Moreover such higher starting levels ofendogenous Aurora-A protein sustained in the tumorsgenerated from Fbxw7 þ/� and Fbxw7 þ/�Ptenþ/� miceand in most of tumors from Ptenþ/� mice. In view of thetoxicity of presently available inhibitors of Aurora-Akinase in clinical trials, these findings may lead to thedesign of alternative approaches to manipulate the levels ofAurora-A kinase protein for cancer therapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y.W. Kwon, I.J. Kim, D. Wu, Y. Liu, J. Perez-Losada, G.Wei, J.-H. MaoDevelopment of methodology: Y.W. Kwon, I.J. Kim, D. Wu, J. Lu, H.C. Kang,J.-H. MaoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y.W. Kwon, D. Wu, J. Lu, Y. Huang, R. DelRosario,J.-H. MaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): Y.W. Kwon, D. Wu, J. Lu, W.A. Stock Jr, Y. Liu, K.-Y. Jen,J. Perez-Losada, G. Wei, J.-H. MaoWriting, review, and/or revision of the manuscript: Y.W. Kwon, I.J. Kim,D. Wu, W.A. Stock Jr, H.C. Kang, K.-Y. Jen, J. Perez-Losada, G. Wei,A. Balmain, J.-H. MaoAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): Y.W. Kwon, D. Wu, W.A. Stock Jr, G. Wei, A. Balmain,J.-H. MaoStudy supervision: Y.W. Kwon, D. Wu, G. Wei, A. Balmain, J.-H. Mao

AcknowledgmentsThe authors thank B. Vogelstein for providing us with the HCT116 WT and

HCT116 FBXW7�/� cell lines; K.I. Nakayama for providing Fbxw7 knockout miceand vectors (HA-FBXW7 and HA-FBXW7DF); and O. Tetsu for vector encodingHA-ubiquitin.

Grant SupportJ.-H. Mao was supported by the NIH, National Cancer Institute grant R01

CA116481, the Low Dose Scientific Focus Area, Office of Biological & Envi-ronmental Research, of the U.S. Department of Energy under contract no. DE-AC02-05CH11231, and Laboratory Directed Research & Development Program(LDRD). A. Balmain was supported by the Department of Energy Low DoseRadiation Research Program DESC0003679 and the National Cancer Institutegrant U01 CA141455. J. Losada-Losada was partially supported by FEDER,MICINN (PLE2009-119), FIS (PI10/00328), and "Fundaci�on Eugenio Rodrí-guez-Pascual."

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received January 18, 2012; revised April 6, 2012; accepted April 9, 2012;published OnlineFirst April 18, 2012.

References1. Fu J, BianM, Jiang Q, Zhang C. Roles of Aurora kinases in mitosis and

tumorigenesis. Mol Cancer Res 2007;5:1–10.2. Sen S, Zhou H, Zhang RD, Yoon DS, Vakar-Lopez F, Ito S, et al.

Amplification/overexpression of a mitotic kinase gene in human blad-der cancer. J Natl Cancer Inst 2002;94:1320–9.

3. Li D, Zhu J, Firozi PF, Abbruzzese JL, Evans DB, Cleary K, et al.Overexpression of oncogenic STK15/BTAK/Aurora A kinase in humanpancreatic cancer. Clin Cancer Res 2003;9:991–7.

4. Sakakura C, Hagiwara A, Yasuoka R, Fujita Y, Nakanishi M,Masuda K,et al. Tumour-amplified kinaseBTAK is amplified and overexpressed ingastric cancers with possible involvement in aneuploid formation. Br JCancer 2001;84:824–31.

5. Nishida N, Nagasaka T, Kashiwagi K, Boland CR, Goel A. High copyamplification of the Aurora-A gene is associated with chromosomalinstability phenotype in human colorectal cancers. Cancer Biol Ther2007;6:525–33.

6. Lassmann S, Shen Y, Jutting U, Wiehle P, Walch A, Gitsch G, et al.Predictive value of Aurora-A/STK15 expression for late stage epithelialovarian cancer patients treated by adjuvant chemotherapy. Clin Can-cer Res 2007;13:4083–91.

7. Zhou H, Kuang J, Zhong L, Kuo WL, Gray JW, Sahin A, et al. Tumouramplified kinase STK15/BTAK induces centrosome amplification,aneuploidy and transformation. Nat Genet 1998;20:189–93.

8. Zhang D, Hirota T, Marumoto T, Shimizu M, Kunitoku N, Sasayama T,et al. Cre-loxP-controlled periodic Aurora-A overexpression inducesmitotic abnormalities and hyperplasia in mammary glands of mousemodels. Oncogene 2004;23:8720–30.

9. Harrington EA, Bebbington D, Moore J, Rasmussen RK, Ajose-Adeo-gun AO, Nakayama T, et al. VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth invivo. Nat Med 2004;10:262–7.

10. Tan Y, Sangfelt O, Spruck C. The Fbxw7/hCdc4 tumor suppressor inhuman cancer. Cancer Lett 2008;271:1–12.

11. YadaM,HatakeyamaS,Kamura T,NishiyamaM, TsunematsuR, ImakiH, et al. Phosphorylation-dependent degradation of c-Myc ismediatedby the F-box protein Fbw7. EMBO J 2004;23:2116–25.

12. Strohmaier H, Spruck CH, Kaiser P, Won KA, Sangfelt O, Reed SI.Human F-box protein hCdc4 targets cyclin E for proteolysis andis mutated in a breast cancer cell line. Nature 2001;413:316–22.

13. Tetzlaff MT, YuW, LiM, ZhangP, FinegoldM,MahonK, et al. Defectivecardiovascular development and elevated cyclin E and Notch proteinsin mice lacking the Fbw7 F-box protein. Proc Natl Acad Sci U S A2004;101:3338–45.

14. Wei W, Jin J, Schlisio S, Harper JW, Kaelin WG Jr. The v-Jun pointmutation allows c-Jun to escape GSK3-dependent recognition anddestruction by the Fbw7 ubiquitin ligase. Cancer Cell 2005;8:25–33.

15. Mao JH, Kim IJ,WuD, Climent J, KangHC, DelRosario R, et al. FBXW7targets mTOR for degradation and cooperates with PTEN in tumorsuppression. Science 2008;321:1499–502.

16. Otto T, Horn S, BrockmannM, Eilers U, Schuttrumpf L, Popov N, et al.Stabilization of N-Myc is a critical function of Aurora A in humanneuroblastoma. Cancer Cell 2009;15:67–78.






17. Mao JH, Perez-Losada J, Wu D, Delrosario R, Tsunematsu R,NakayamaKI, et al. Fbxw7/Cdc4 is a p53-dependent, haploinsufficienttumour suppressor gene. Nature 2004;432:775–9.

18. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA.Inhibitionof glycogen synthase kinase-3by insulinmediatedbyproteinkinase B. Nature 1995;378:785–9.

19. Fujii Y, Yada M, NishiyamaM, Kamura T, Takahashi H, Tsunematsu R,et al. Fbxw7 contributes to tumor suppression by targeting multipleproteins for ubiquitin-dependent degradation. Cancer Sci 2006;97:729–36.

20. Salmena L, Carracedo A, Pandolfi PP. Tenets of PTEN tumor sup-pression. Cell 2008;133:403–14.

21. Kemp CJ, Wheldon T, Balmain A. p53-deficient mice are extremelysusceptible to radiation-induced tumorigenesis. Nat Genet 1994;8:66–9.

22. Mao JH, Wu D, Perez-Losada J, Nagase H, DelRosario R,Balmain A. Genetic interactions between Pten and p53 in radi-ation-induced lymphoma development. Oncogene 2003;22:8379–85.

23. Mao JH, To MD, Perez-Losada J, Wu D, Del Rosario R, Balmain A.Mutually exclusive mutations of the Pten and ras pathways in skintumor progression. Genes Dev 2004;18:1800–5.

24. DarAA,Belkhiri A, El-RifaiW. Theaurora kinaseA regulatesGSK-3betain gastric cancer cells. Oncogene 2009;28:866–75.

25. Yang H, He L, Kruk P, Nicosia SV, Cheng JQ. Aurora-A induces cellsurvival and chemoresistance by activation of Akt through a p53-dependent manner in ovarian cancer cells. Int J Cancer 2006;119:2304–12.

26. Taga M, Hirooka E, Ouchi T. Essential roles of mTOR/Akt pathway inAurora-A cell transformation. Int J Biol Sci 2009;5:444–50.

27. Sloane DA, Trikic MZ, ChuML, Lamers MB, Mason CS, Mueller I, et al.Drug-resistant aurora A mutants for cellular target validation of thesmall molecule kinase inhibitors MLN8054 and MLN8237. ACS ChemBiol 2010;5:563–76.

28. Dodson CA, Kosmopoulou M, Richards MW, Atrash B, Bavetsias V,Blagg J, et al. Crystal structure of an Aurora-A mutant that mimicsAurora-B bound toMLN8054: insights into selectivity and drug design.Biochem J 2010;427:19–28.


Kwon et al.




2012;10:834-844. Published OnlineFirst April 18, 2012.Mol Cancer Res Yong-Won Kwon, Il-Jin Kim, Di Wu, et al. Radiation-Induced Tumor DevelopmentPten Regulates Aurora-A and Cooperates with Fbxw7 in Modulating

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