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p53 Protein Regulates Hsp90 ATPase Activity and TherebyWnt Signaling by Modulating Aha1 Expression*

Received for publication, November 4, 2013, and in revised form, January 21, 2014 Published, JBC Papers in Press, January 22, 2014, DOI 10.1074/jbc.M113.532523

Sachiyo Okayama‡, Levy Kopelovich‡, Gabriel Balmus¶, Robert S. Weiss¶, Brittney-Shea Herbert§,Andrew J. Dannenberg‡, and Kotha Subbaramaiah‡1

From the ‡Department of Medicine, Weill Cornell Medical College, New York, New York 10065, the ¶Department of BiomedicalSciences, Cornell University, Ithaca, New York 14853, and the §Department of Medical and Molecular Genetics, Indiana UniversitySimon Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana 46202

Background: The mechanism by which p53, a tumor suppressor gene, regulates Wnt signaling is incompletely understood.Results: p53 modulates Hsp90 ATPase activity via effects on Aha1 leading to changes in the expression of Wnt target genes.Conclusion: p53 regulates Hsp90 ATPase activity and thereby Wnt signaling.Significance: We describe a new mechanism by which p53 affects Wnt signaling.

The p53 tumor suppressor gene encodes a homotetramerictranscription factor which is activated in response to a variety ofcellular stressors, including DNA damage and oncogene activa-tion. p53 mutations occur in >50% of human cancers. Althoughp53 has been shown to regulate Wnt signaling, the underlyingmechanisms are not well understood. Here we show that silenc-ing p53 in colon cancer cells led to increased expression of Aha1,a co-chaperone of Hsp90. Heat shock factor-1 was important formediating the changes in Aha1 levels. Increased Aha1 levelswere associated with enhanced interactions with Hsp90, result-ing in increased Hsp90 ATPase activity. Moreover, increasedHsp90 ATPase activity resulted in increased phosphorylation ofAkt and glycogen synthase kinase-3� (GSK3�), leading toenhanced expression of Wnt target genes. Significantly, levels ofAha1, Hsp90 ATPase activity, Akt, and GSK3� phosphorylationand expression of Wnt target genes were increased in the colonsof p53-null as compared with p53 wild type mice. Using p53heterozygous mutant epithelial cells from Li-Fraumeni syn-drome patients, we show that a monoallelic mutation of p53 wassufficient to activate the Aha1/Hsp90 ATPase axis leading tostimulation of Wnt signaling and increased expression of Wnttarget genes. Pharmacologic intervention with CP-31398, a p53rescue agent, inhibited recruitment of Aha1 to Hsp90 and sup-pressed Wnt-mediated gene expression in colon cancer cells.Taken together, this study provides new insights into the mech-anism by which p53 regulates Wnt signaling and raises theintriguing possibility that p53 status may affect the efficacy ofanticancer therapies targeting Hsp90 ATPase.

The development and progression of colon cancer are part ofa multistep process in which growth control is increasinglyimpaired. Activation of the Wnt pathway plays a major role incolon cancer initiation. In normal cells, the levels of cytoplas-

mic �-catenin are controlled by a multiprotein destructioncomplex that targets �-catenin for degradation by the protea-some (1, 2). The destruction complex consists of Axin, APC,2

and the glycogen synthase kinase-3� (GSK3�) (1, 2). Wnt sig-naling stimulates the dissociation of the �-catenin destructioncomplex leading to the accumulation and nuclear translocationof �-catenin which binds, in turn, to the T cell factor/lympho-cyte enhancer binding factor family (TCF/LEF) and regulatesWnt target genes (3). Truncating APC mutations are found in�80% of sporadic colon carcinomas and represent the mostcommon cause for activation of Wnt signaling (4). In additionto mutant APC, a variety of other mechanisms including muta-tion of the �-catenin gene, increased expression of Wnt ligands,and mutational inactivation of the p53 tumor suppressor havebeen shown to affect Wnt target gene expression (5– 8).Because p53 mutations and activation of Wnt signaling arecommon in colorectal cancer (4), a detailed understanding ofthe mechanisms by which p53 modulates Wnt signaling isimportant.

The p53 tumor suppressor gene encodes for a homotetra-meric transcription factor which is activated in response to avariety of cellular stressors, including DNA damage, hypoxia,metabolic stress, and oncogene activation (9 –12). Under theseconditions, the p53 protein is stabilized, initiating a transcrip-tional program that results in DNA repair, cell cycle arrest,senescence, or apoptosis (11). Mutations affecting p53 are pres-ent in �50% of cancers (13). Wild type p53 has been suggestedto inhibit Wnt signaling by different mechanisms including theinduction of microRNA-34 (6 – 8). Moreover, Wnt signalinghas been reported to be activated in cells derived from Li-Frau-meni syndrome (LFS) patients who carry germ line, monoallelicp53 mutations (14). In Wnt-1-overexpressing mice, p53 defi-ciency results in accelerated tumorigenesis relative to Wnt-1transgenic mice that are wild type for p53 (15). Although the

* This work was supported, in whole or in part, by National Institutes of HealthGrant R01 CA108773. This work was also supported by the New YorkCrohn’s Foundation and the Tokyo Clinical Surgical Association.

1 To whom correspondence should be addressed: Weill Cornell Medical Col-lege, 525 E. 68th St., Rm. F-203A, New York, NY 10065. Tel.: 212-746-4402;Fax: 212-746-4885; E-mail: [email protected].

2 The abbreviations used are: APC, adenomatous polyposis coli; GSK3�, gly-cogen synthase-3�;17-AAG, 17-allylamino-17-demethoxygeldanamycin;Aha1, activator of Hsp90 ATPase1; HSF-1, heat shock factor-1; Hsp, heatshock protein; LFS, Li-Fraumeni syndrome; TCF/LEF, T cell factor/lympho-cyte enhancer-binding factor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 10, pp. 6513–6525, March 7, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

MARCH 7, 2014 • VOLUME 289 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6513

This article has been withdrawn by the authors. In Fig. 1D, the first lane of the p53 immunoblot was reused as actin in the same figure panel. In Fig. 2B, lanes 1 and 2 of the actin immunoblot were reused in lanes 5 and 6. Lane 2 of the actin immunoblot in Fig. 4K was reused in lanes 3 and 4. The HOP immunoblot in Fig. 4J was reused in Fig. 4 (K and L) as actin. The actin immunoblot in Fig. 4H was reused in Fig. 4 (L and I) as actin. The actin immunoblot in Fig. 5A was reused in Fig. 5B. In Fig. 5H, the c-Myc and Naked-1 immunoblots are the same. There are undeclared gel splices in Figs. 5F, 7C, 7F, and 8I. A portion of the actin immunoblot in Fig. 10A was reused in Fig. 10B as Aha1.

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link between p53 and Wnt signaling is established, the interac-tion between these pathways is incompletely understood.

Here we have investigated the effect of p53 on Wnt signalingin human colorectal cancer cell lines and LFS-derived epithelialcells. To translate these in vitro findings, the effect of p53 onWnt-signaling including target gene expression was also com-pared in wild type versus p53-null mice. Using these comple-mentary model systems, we show that p53 modulates Wnt sig-naling via effects on Hsp90. Specifically, loss of p53 functionwas associated with increased levels of Aha1, a co-chaperone ofHsp90. The changes in Aha1 levels were mediated by HSF-1.Increased interaction of Aha1 and Hsp90 led to enhancedHsp90 ATPase activity, which stimulated the Akt/GSK3� path-way. This led, in turn, to increased nuclear translocation of�-catenin and enhanced Wnt target gene expression. Consis-tent with these findings, we also show that pharmacologic inter-vention with CP-31398, a p53 rescue compound (16), inhibited theAha1/Hsp90 axis and thereby suppressed Wnt signaling. Takentogether, this study provides new insights into the mechanism bywhich p53 regulates Wnt signaling, which may be important forunderstanding the progression of colon cancer.

EXPERIMENTAL PROCEDURES

Materials—CP-31398 was provided by the National CancerInstitute Chemopreventive Agent Repository. Zinc chloride,dimethyl sulfoxide, G418, phosphoenolpyruvate, pyruvatekinase, NADH, and antibodies to �-actin, HSF-1, p23, HOP,and XAP-2 were purchased from Sigma. 17-Allylamino-17de-methoxygeldanamycin (17-AAG) and LY294002 were fromCayman Chemicals. PU-H71 was from Tocris Bioscience. Anti-bodies to Akt, Akt1, Axin-2, c-Myc, GSK3�, phospho-GSK3�(Ser-9), Naked-1, p21, and TCF4 were from Cell SignalingTechnology. Antibodies to Hsp90, phospho-Akt (Ser-473), andp53 were from Santa Cruz Biotechnology. Antibody toAha1was obtained from Abcam. The antibody to �-catenin wasfrom BD Biosciences. Control siRNA and siRNAs to Aha1, HSF-1,Akt1, and p53 were purchased from Thermo Scientific. Chroma-tin immunoprecipitation (ChIP) assay kits were purchased fromSA Bioscience. The site-directed mutagenesis kit was purchasedfrom Stratagene. Western blotting detection reagents were pur-chased from PerkinElmer Life Sciences. M50 Super 8� TOP Flashand M51 Super 8� FOP Flash plasmids were obtained from Add-gene. p53 luciferase plasmid was from Panomics. Reagents for theluciferase assay and pSV�gal were from Promega.

Cell Culture—HCT15, DLD-1, and LoVo human colon can-cer cell lines were obtained from the American Type CultureCollection (ATCC). These cell lines were maintained accordingto ATCC instructions. The human colon carcinoma cell lineEB-1 was kindly provided by Dr. Arnold J. Levine (PrincetonUniversity) (17, 18). These cells were maintained in RPMI1640 medium with 10% FBS and supplemented with 0. 5g/li-ter geneticin (G418). The HME32, HME50, and IUSM/LFS/HME cells have been described previously and were pro-vided by Dr. Brittney-Shea Herbert (Indiana UniversitySchool of Medicine) (14, 19).

Immunoprecipitation—This assay was performed with a kitfrom Upstate Biotechnology according to the manufacturer’sinstructions. 500 –1000 �g of cell lysate or tissue lysate protein

was used for immunoprecipitation at room temperature. Theimmunoprecipitates were then analyzed by Western blotting.

Western Blotting—Cell and tissue lysates were preparedusing a lysis buffer (150 mM NaCl, 100 mM Tris, pH 8.0, 1%Tween 20, 50 mm diethyldithiocarbamate, 1 mM EDTA, 1 mM

phenylmethylsulfonyl fluoride, 10 �g/ml aprotinin, 10 �g/mltrypsin inhibitor, and 10 �g/ml leupeptin) followed by sonica-tion to remove particulate material. The protein concentrationwas determined according to Lowry et al. (20). SDS-PAGE wasperformed under reducing conditions on 10% polyacrylamidegels as described (21). Resolved proteins were transferred ontonitrocellulose sheets and incubated with the indicated antiserafollowed by a secondary antibody to horseradish peroxidase-conjugated IgG. The blots were then reacted with the ECLWestern blot detection system.

Quantitative Real-time PCR—Total RNA was isolated fromcolon tissues using the RNeasy mini kit (Qiagen). For tissueanalyses, poly(A) RNA was prepared with an Oligotex mRNAmini kit (Qiagen). Poly(A) RNA was reversed-transcribed usingmurine leukemia virus reverse transcriptase and oligo(dT)16primer. The resulting cDNA was then used for amplification.Primers for Aha1 and �-actin, an endogenous normalizationcontrol, were purchased from Qiagen. Real-time PCR was per-formed using 2� SYBR Green PCR master mix on a 7500 Real-time PCR system (Applied Biosystems). Relative -fold induc-tion was determined using the ��CT (relative quantification)analysis protocol.

Promoter Constructs—PCR was used to generate severaltruncated forms of the Aha1 promoter. By using genomic cloneDNA as a template, PCR was performed at 94 °C for 3 min ofdenaturing, annealing for 1 min at 60 °C, and extension for 10 minat 72 °C by using Taq polymerase. The primers used to clone Aha1promoter were: forward, 5�-GTGAGGGCCAGAAAAGCA-3�;reverse, 5�-CTGCAAAGCAGAAACAGAGC-3�. PCR prod-ucts of 5�-flanking fragments of Aha1 gene were inserted intothe KpnI site of the luciferase basic plasmid vector, pGL2(Promega). The subcloned PCR products were sequenced byusing T7 and SP6 promoter primers to confirm that the prod-ucts were the authentic promoter fragments. Site-directed mu-tagenesis was used to mutagenize HSF-1 binding element (HBEmut.) in Aha1 promoter.

Transient Transfections—Cells were grown to 60 –70% con-fluence in 6-well dishes and then transfected using Lipo-fectamine 2000 (Invitrogen) for 24 h. Subsequently, themedium was replaced with serum-free medium for another24 h. Luciferase and �-galactosidase were measured in cellularextracts. TOP Flash activity, a measure of TCF/�-catenin-me-diated gene transcription, was determined by the ratio ofpTOP-flash/pFOP-flash luciferase activity, each normalized to�-galactosidase enzymatic activity levels.

RNA Interference—The cells were seeded in keratinocytegrowth medium for 24 h before transfection. Two micrograms ofsiRNA oligonucleotides was transfected using DharmaFECT 4transfection reagent according to the manufacturer’s instructions.

ChIP Assay—ChIP assay was performed with a kit accordingto the manufacturer’s instructions. Briefly, 4 � 106 cells werecross-linked in a 1% formaldehyde solution at 37 °C for 10 min.Cells were then lysed and sonicated to generate 200 –1000-bp

p53 Regulates Hsp90 ATPase Activity

6514 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 10 • MARCH 7, 2014


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DNA fragments. After centrifugation, the cleared supernatantwas incubated with 4 �g of the HSF-1 antibody at 4 °C over-night. Immune complexes were precipitated, washed, andeluted as recommended. DNA-protein cross-links werereversed by heating at 65 °C for 4 h, and the DNA fragmentswere purified and used as a template for PCR amplification.

Quantitative real-time PCR was carried out. Aha1 promoteroligonucleotide sequences for PCR primers were: forward,5�-GCAGGGAGGTGCTTATTA-3� and reverse, 5�-TAGAT-GGCCACAAAAACG-3�. This primer set encompasses theAha1 promoter sequence from nucleotide �282 bp to �583 bp.PCR was performed at 94 °C for 30 s, 62 °C for 30 s, and 72 °C for

FIGURE 1. p53 regulates Wnt signaling. A, C, and E, cells were transfected with 0.45 �g each of TOP Flash and FOP Flash constructs and 0.2 �g of pSV�gal. Cellsalso received 0.9 �g of siRNA to GFP (control siRNA) or p53. 48 h after transfection, cells were harvested, and luciferase activity was measured. TOP Flash activitywas determined by the ratio of pTOP-flash to pFOP-flash luciferase activity, each normalized to �-galactosidase enzymatic activity levels. B, D, and F, cells weretransfected with 2 �g of siRNA (control) to GFP or p53 for 48 h. Following transfection, cells were harvested, and cell lysates were subjected to Western blotting.The blots were probed with antibodies to the indicated proteins. G and H, EB-1 cells were transfected with 1.8 �g of p53 luciferase construct (G) or 0.9 �g eachof TOP Flash and FOP Flash constructs and 0.2 �g of pSV�gal for 24 h. 24 h later, cells were treated with indicated concentrations of ZnCl2 for 12 h, and then cellswere harvested, and luciferase activity was measured. Luciferase activity was normalized to �-galactosidase activity. I, cells were treated with the indicatedconcentrations of ZnCl2 for 12 h. Cell lysates were subjected to Western blotting, and the blots were probed as indicated. A, C, E, G, and H, mean � S.D. (errorbars) are shown, n 6. *, p 0.01 compared with control siRNA-treated cells (A, C, and E) or vehicle-treated cells (G and H).




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45 s for 35 cycles, and real-time PCR was performed at 95 °C for15 s and 60 °C for 60 s for 40 cycles. The PCR product generatedfrom the ChIP template was sequenced, and the identity of theAha1 promoter was confirmed.

Hsp90 ATPase Activity—The ATPase assay was based on aregenerating coupled enzyme assay as described earlier (22, 23).Briefly, Hsp90 was immunoprecipitated from cell lysates, andthe pellet was resuspended and used for assay. Reaction wasconducted in a 1-ml assay containing 100 mM, Tris-HCl, pH 7.4,20 mM KCl, 6 mM MgCl2, 0.8 M ATP, 0.1 mM NADH, 2 mM

phosphoenolpyruvate, 0.2 mg of pyruvate kinase, 0.05 mg of L-lac-tate dehydrogenase, and Hsp90 immunoprecipitated from cell andtissue lysates. Equal amounts of Hsp90 protein were used in eachtreatment group. Sufficient NADH was added to give an initial

absorbance of 0.3 at 340 nm before addition of Hsp90 and activitywas detected as a decrease in absorbance. Hsp90 ATPase activity isexpressed as pmol/min per mg of protein.

Animal Model—p53 knock-out mice carrying theTrp53tm1Tyj allele were maintained on the 129S6 inbred strainbackground and genotyped by PCR analysis of DNA extractedfrom tail tip biopsies (24). Experimental p53�/� and sex-matched littermate control p53�/� mice at 6 –10 weeks of agewere euthanized by carbon dioxide asphyxiation, and isolatedcolon tissue was rinsed once with PBS and then snap frozen forsubsequent molecular analysis. All animal use was conducted inaccordance with federal and institutional guidelines, under aprotocol approved by the Cornell University Institutional Ani-mal Care and Use Committee.

FIGURE 2. CP-31398 inhibits Wnt signaling. A, C, and E, HCT-15 (A), LoVo (C), and DLD-1 (E) cells were transfected with 1.8 �g of p53 luciferase construct and0.2 �g of pSV�gal. 24 h later, the cells were treated with the indicated concentrations of CP-31398 for 24 h, and then luciferase activity was measured. Luciferaseactivity was normalized to �-galactosidase activity. B, D, and F, HCT-15 (B), LoVo (D), and DLD-1 (F) cells were treated with CP-313198 for 24 h, and cell lysateswere harvested for Western blot analysis. Immunoblots were probed with antibodies as indicated. A, C, E, mean � S.D. (error bars) are shown, n 6. *, p 0.01compared with vehicle-treated cells.




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Statistics—Comparisons between groups were made by Stu-dent’s t test. A difference between groups of p 0.05 was con-sidered significant.

RESULTS

p53 status has been identified as a determinant of Wnt sig-naling, but the mechanisms are incompletely understood(6 – 8). To further understand the interaction between p53 andWnt signaling, both in vitro and animal models were employed.

p53 Regulates Wnt Target Gene Expression—First, westudied the effects of p53 status on Wnt target gene expres-sion, namely Axin-2, c-Myc, and Naked-1. Silencing p53 inHCT-15 (p53�/�), DLD-1 (p53�/�), and LoVo (p53�/�) coloncancer cells led to decreased levels of p21, a downstream target

of p53, a marked increase in �-catenin/TCF/LEF transcrip-tional activity, and enhanced expression of Wnt target proteins(Fig. 1, A–F). The importance of p53 as a determinant of Wntsignaling was further evaluated in EB-1 cells, a p53-null cell linethat expresses Zn2�-inducible wild type p53 (17, 18). Inductionof p53 in EB-1 cells was associated with increased p53 tran-scriptional activity (Fig. 1G), decreased �-catenin/TCF/LEFtranscriptional activity (Fig. 1H), and reduced levels of Axin-2,c-Myc, and Naked-1 (Fig. 1I). In this context, levels of �-catenindecreased whereas TCF4 expression was unaltered (datanot shown). Moreover, pharmacological intervention withCP-31398, a p53 rescue/stabilizing compound, caused a dose-dependent increase in p53 levels and transcriptional activityand reduced expression of Wnt target genes (Fig. 2, A–F). These

FIGURE 3. p53 is a determinant of Hsp90 ATPase activity. A–C, cells were transfected with 2 �g of p53 siRNA or GFP (control) siRNA for 48 h. Cells were thenharvested. D, EB-1 cells were treated with the indicated concentrations of ZnCl2 for 12 h prior to being harvested. E and F, cells were treated with CP-31398 asindicated for 24 h before being harvested. A–F, cell lysates were used to measure Hsp90 ATPase activity using the method described under “ExperimentalProcedures.” Mean � S.D. (error bars) are shown, n 6. *, p 0.01 compared with control siRNA-treated cells (A–C) or vehicle-treated cells (D–F). Cell lysateswere also subjected to Western blotting, and the blots were probed as indicated in the insets.




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results clearly demonstrate that p53 status, whether by silenc-ing or pharmacological rescue, modulates Wnt signaling inhuman colorectal cancer cell lines.

p53 Status Is a Determinant of Hsp90 ATPase Activity—Hsp90, a chaperone molecule for many client proteins includ-ing those with oncogenic potential, is activated in a variety ofmalignancies (25–27). Here we determined whether p53 statusis a determinant of Hsp90 ATPase activity. Silencing p53caused a significant increase in Hsp90 ATPase activity in HCT-15, LoVo, and DLD-1 cells (Fig. 3, A–C). This increase wasobserved in the absence of increased Hsp90 protein levels. Incontrast, inducing p53 in EB-1 cells (Fig. 3D) or by treatmentwith CP-31398 (Fig. 3, E and F) led to a dose-dependent inhibitionof Hsp90 ATPase activity. This decrease in Hsp90 ATPase activityoccurred in the absence of corresponding changes in Hsp90 pro-tein levels. Taken together, these results indicate that p53 is adeterminant of Hsp90 ATPase activity in colon cancer cells.

p53 Modulates Hsp90 ATPase Activity and Wnt Target GeneExpression through Aha1—Previously, Hsp90 ATPase activitywas found to regulate Wnt signaling (28). Given this back-ground, we tested the effects of two inhibitors of Hsp90ATPase. Both 17-AAG (29) and PU-H71 (30), prototypic Hsp90ATPase inhibitors, caused dose-dependent suppression ofAxin-2, c-Myc, and Naked-1 in HCT15, DLD-1, and EB-1 cells(data not shown). Activation of Hsp90 is dependent on thepresence of its co-chaperones. Aha1, a co-chaperone of Hsp90,stimulates Hsp90 ATPase activity (31–33). Here we exploredthe possibility that Aha1 expression could be affected by p53.First we show that pharmacological rescue of p53 usingCP-31398 in HCT-15, LoVo, and DLD-1 cells caused a dose-de-pendent decrease in Aha1 levels (Fig. 4, A–C). This effect wasobserved within 4 h of treatment and was associated with acorresponding reduction in Hsp90 ATPase activity (data notshown). The relationship between p53 status and Aha1 levelswas further evaluated in EB-1 cells. Here, treatment with ZnCl2,which induced p53 (Fig. 1I), caused a decrease in Aha1 expres-sion (Fig. 4D). In contrast, silencing of p53 in HCT-15, LoVo,and DLD-1 cells caused a substantial increase in Aha1 levels(Fig. 4, E–G). Importantly, the effect of p53 on Hsp90 co-chap-erones appears to be specific to Aha1 because, in similar exper-iments, whether through silencing of p53 or its rescue by phar-macologic intervention with CP-31398, levels of otherco-chaperones of Hsp90, namely p23, XAP-2, and HOP, wereunaffected (Fig. 4, H–M). In addition to HCT-15 cells, levels ofco-chaperones other than Aha1 were unaffected in LoVo,DLD-1, and EB-1 cell lines (data not shown).

To test directly the role of Aha1 in regulating Hsp90 ATPaseactivity, Hsp90 ATPase activity was measured following silenc-ing of Aha1. Notably, silencing of Aha1 led to reduced Hsp90ATPase activity in HCT-15, EB1 (Fig. 5, A and B), and DLD-1cells (data not shown). Next, co-immunoprecipitation experi-ments were conducted to determine whether changes in p53levels modified the interaction between Hsp90 and Aha1. Asshown in Fig. 5, C and D, silencing p53 led to increased inter-action between Aha1 and Hsp90. Similar effects were observedin LoVo cells (data not shown). In contrast, treatment withCP-31398 or p53 induction in EB-1cells led to decreased inter-action between Aha1 and Hsp90 (Fig. 5, E and F). To evaluate

the functional significance of Aha1 in regulating Wnt targetgene expression, we investigated the effects of silencing Aha1.As shown in Fig. 5, G and H, silencing of Aha1 led to reducedlevels of Axin-2, c-Myc, and Naked-1. These results indicatethat p53 modulates the expression of Aha1 and its interactionwith Hsp90 which, in turn, affects Hsp90 ATPase activity andthereby the expression of genes regulated by Wnt signaling.

Next we investigated the mechanism by which p53 regulatedlevels of Aha1. To determine whether p53 regulated Aha1 tran-scription, transient transfections were carried out. Silencing ofp53 was associated with increased Aha1 promoter activity (Fig.6, A–C). Treatment with ZnCl2, which induced p53 (Fig. 1I),caused a corresponding dose- and time-dependent decrease inAha1 promoter activity (Fig. 6, D and E). Previously, HSF-1 wasfound to be a potential regulator of Aha1 expression (34).Therefore, we next evaluated whether the p53-dependenteffects on Aha1 were mediated by HSF-1. A potential HSF-1-binding element (�331 to �316) was identified in the humanAha1 promoter (Fig. 6F). Transient transfections were carriedout using a series of Aha1 promoter deletion constructs. Nota-bly, silencing p53 stimulated Aha1 promoter activity, an effectthat was lost when the HSF-1-binding element was deleted ormutated (Fig. 6, G and H). Consistent with these findings, thesuppressive effects of wild type p53 were lost when the HSF-1-binding element was deleted or mutated (Fig. 6, I and J). Havingestablished the importance of HSF-1 for regulating Aha1 pro-moter activity, ChIP assays were performed to evaluate the

FIGURE 4. p53 regulates Aha1 expression. A–C and K–M, cells were treatedwith indicated concentrations of CP-31398 for 24 h. D, EB-1 cells were treated withthe indicated concentrations of ZnCl2 for 12 h. E–J, cells were transfected with 2�g of p53 siRNA or control siRNA for 48 h. A–M, cells were harvested, and celllysates were subjected to immunoblotting and probed as indicated.




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binding of HSF-1 to the Aha1 promoter. As shown in Fig. 7Aand B, p53 regulated the binding of HSF-1 to the Aha1 pro-moter. More specifically, treatment with ZnCl2, which inducedp53, suppressed the recruitment of HSF-1 to the Aha1 pro-moter; silencing of p53 increased the recruitment of HSF-1 to theAha1 promoter. Moreover, silencing of HSF-1 led to reducedAha1 protein levels (Fig. 7C). Given the important effects of HSF-1on Aha1 levels, we next determined whether changes in HSF-1levels modulated Hsp90 ATPase activity. Silencing HSF-1 wasassociated with reduced Hsp90 ATPase activity in both EB-1 andHCT-15 cells (Fig. 7, D and E). Finally, silencing of HSF-1 led toreduced expression of Wnt target genes (Fig. 7F).

p53 Regulates Akt Phosphorylation—The PI3K/Akt pathwayis commonly deregulated in human cancers. Akt is a client pro-tein of Hsp90, which upon activation phosphorylates GSK3�,stabilizes �-catenin, and induces the expression of Wnt targetgenes (28, 35–38). Accordingly, we determined the effects ofLY294002, a PI3K inhibitor, on the expression of Axin-2,c-Myc, and Naked-1. Treatment with LY294002 caused a dose-dependent decrease in levels of Axin-2, c-Myc, and Naked-1(Fig. 8, A and B). Silencing p53 in these cells induced Akt phos-phorylation (data not shown). Induction of p53 in EB-1 cellsinhibited Akt phosphorylation (Fig. 8C). Silencing Aha1 inhib-ited Akt phosphorylation (Fig. 8D). Pharmacologic inhibition ofHsp90 ATPase was also associated with reduced Akt phosphor-

ylation (Fig. 8E). Silencing Akt1 in these cells caused a signifi-cant decrease in the levels of Wnt target proteins includingAxin-2, c-Myc, and Naked-1 (Fig. 8F). Together, these resultssupport the notion that p53 status regulated the Aha1/Hsp90axis resulting in changes in Akt activity that led, in turn, toaltered expression of Wnt target genes.

p53 Status Is a Determinant of GSK3� Phosphorylation—GSK3� serves a critical role in Wnt signaling during cancerdevelopment (1, 2). GSK3� can be phosphorylated by Akt, lead-ing to activation of TCF/LEF-dependent gene expression. Herewe investigated whether p53 status could affect GSK3� phos-phorylation via the Hsp90/Aha1/Akt axis. Silencing of p53caused a significant increase in GSK3� phosphorylation,whereas induction of wild type p53 in EB-1 cells or pharmaco-logic rescue of mutant p53 with CP-31398 caused a substantialreduction of GSK3� phosphorylation (Fig. 8, G–I). The effectsof CP-31398 on the Aha1/Akt/GSK3� pathway occurred withinfour h and persisted for up to 24 h (data not shown). Along similarlines, silencing Aha1 or inhibiting Hsp90 caused a marked reduc-tion in GSK3� phosphorylation (Fig. 8, J and K). These resultssuggest that p53 regulates the Aha1/Hsp90/Akt/GSK3� axis andthereby the expression of Wnt target genes (Fig. 8L).

Levels of Aha1, Hsp90 ATPase Activity, and Wnt TargetGenes Are Increased in p53-null Mice—To assess the impact ofp53 on the Aha1/Hsp90 ATPase axis in vivo, we utilized colon

FIGURE 5. Aha1 is a determinant of Hsp90 ATPase activity. A and B, HCT-15 (A) and EB-1 (B) cells were transfected with 2 �g of Aha1 or control siRNA for 48 h.Hsp90 ATPase activity was then measured as described under “Experimental Procedures.” Mean � S.D. (error bars) are shown, n 6. *, p 0.01 compared withcontrol siRNA-treated cells. Cell lysates were also subjected to Western blotting and the lysates probed as indicated (see insets). C and D, cells were transfectedwith 2 �g of p53 siRNA or control siRNA for 48 h. E, cells were treated with 15 �M CP-31398 for 24 h. F, EB-1 cells were treated with 75 �M ZnCl2 for 12 h. C–F, cellswere lysed, and Hsp90 was immunoprecipitated (IP). Immunoprecipitates were subjected to immunoblotting (WB), and the blots were probed as indicated. Gand H, cells were transfected with 2 �g of Aha1 siRNA or control siRNA for 48 h. Cell lysate were subjected to Western blotting and the blots probed as indicated.




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FIGURE 7. Wnt target gene expression is regulated by HSF-1. A, EB-1 cells were treated with 75 �M ZnCl2 for 4 h. B, HCT-15 cells were transfected with 2 �g of siRNAto GFP (control) or p53 for 48 h. A and B, ChIP assays were performed. Chromatin fragments were immunoprecipitated with antibodies against HSF-1, and the Aha1promoter was amplified by real-time PCR. DNA sequencing was carried out, and the PCR products were confirmed to be the Aha1 promoter. This promoter was notdetected when normal IgG was used or when antibody was omitted from the immunoprecipitation step. Columns, means (n 6); error bars, S.D. *, p 0.01. C–F, cellswere transfected with 2 �g of siRNA to GFP (control) or HSF-1 for 48 h. C, cell lysates were subjected to Western blotting, and the blots were probed as indicated. D andE, Hsp90 ATPase activity was measured. Cell lysates were also subjected to Western blotting and the blots probed as indicated (insets). Mean � S.D. (error bars) areshown, n 6. *, p 0.01 compared with control siRNA-treated cells. F, cell lysates were subjected to Western blotting and the blots probed as indicated.

FIGURE 6. HSF-1 is important for p53-mediated regulation of Aha1. A–C, the indicated cells were transfected with 0.9 �g of Aha1 promoter luciferase construct and0.2 �g of pSV�gal. Cells also received 0.9 �g of siRNA to GFP (control siRNA) or p53. 48 h after transfection, cells were harvested, and luciferase activity was measured.Luciferase activity was normalized to �-galactosidase activity. D and E, EB-1 cells were transfected with 1.8 �g of Aha1 promoter luciferase construct and 0.2 �g ofpSV�gal. Subsequently, the cells were treated with the indicated concentrations of ZnCl2 for 12 h (D) or with 100 �M ZnCl2 (E) for the indicated time period. Cells wereharvested, and luciferase activity was measured. Luciferase activity was normalized to �-galactosidase activity. F, schematic represents Aha1 promoter deletionconstructs that were used. HBE, represents the HSF-1-binding element. G and H, HCT-15 cells were transfected with 0.9 �g of Aha1 promoter deletion luciferaseconstructs as indicated and 0.2 �g of pSV�gal. Cells also received 0.9 �g of siRNA to GFP (control siRNA) or p53. 48 h after transfection, cells were harvested, andluciferase activity was measured. Luciferase activity was normalized to�-galactosidase activity. I and J, EB-1 cells were transfected with the 1.8 �g of the indicated Aha1promoter luciferase constructs and 0.2 �g of pSV�gal. Subsequently, cells were treated with 100 �M ZnCl2 for 12 h, and luciferase activity was measured. Luciferaseactivity was normalized to �-galactosidase activity. A–C, G, and H, mean � S.D. (error bars) are shown, n 6. *, p 0.01 compared with control siRNA-treated cells. D,E, I, and J, mean � S.D. (error bars) are shown, n 6. *, p 0.01 compared with vehicle (control)-treated cells.




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tissue from p53 wild type and p53-null mice. Compared withwild type mice, p53-null mice exhibited both increased Hsp90ATPase activity and elevated Aha1 levels (Fig. 9, A and B). Toevaluate the interaction between Hsp90 and Aha1, immuno-precipitation experiments were carried out. As shown in Fig.9C, the interaction between Hsp90 and Aha1 was markedlyincreased in p53-null mice. Moreover, Akt and GSK3� phospho-rylation were both increased in colon tissues from p53-null versuswild type mice (Fig. 9, D and E). Consistent with these findings, wefound increased expression of Wnt target genes including Axin-2,c-Myc, and Naked-1 in p53-null mice (Fig. 9, F–H).

Wnt Signaling Is Increased in Cells Derived from Li-FraumeniSyndrome Patients—LFS patients carry a germ line mutation inp53 and are prone to develop a variety of cancers (14, 39, 40).Accordingly, we compared the Aha1/Hsp90/Wnt signaling axisin epithelial cells from LFS p53 mutation carriers (HME50 and

IUSM/LFS/HME) versus epithelial cells that were wild type forp53 (HME32). Increased Hsp90 ATPase activity, Aha1 levels,interaction between Aha1 and Hsp90, along with elevated Aktand GSK3� phosphorylation were detected in LFS versus nor-mal epithelial cells (Fig. 10, A–E). A significant increase in�-catenin along with increased levels of Axin-2, c-Myc, andNaked-1 was found in LFS versus normal epithelial cells (Fig. 10,F and G). These changes in LFS-derived, p53 heterozygous epi-thelial cells appear to recapitulate alterations seen in colontumor cells.

DISCUSSION

In the present study, we have confirmed previous results sug-gesting that p53 affects Wnt signaling (6 – 8). Notably, our datademonstrate for the first time that Hsp90 plays a significant rolein mediating this effect. Several lines of evidence support thispoint. Levels of Hsp90 ATPase activity were increased in LFS-derived p53 heterozygous versus normal epithelial cells. Silenc-ing of p53 led to increased Hsp90 ATPase activity in multiplecolorectal cancer cell lines. Moreover, expression of wild typep53 in EB-1 cells, a p53-null cell line, led to gene dose-depen-dent suppression of Hsp90 ATPase activity. Similarly, use ofCP-31398, a p53 rescue compound, caused dose-dependentinduction of p53 and a reciprocal decrease in Hsp90 ATPaseactivity. The significance of Hsp90 ATPase activity in regulat-ing Wnt signaling was tested. We showed that 17-AAG andPU-H71, prototypic Hsp90 ATPase inhibitors, down-regulatedthe expression of Wnt target genes including Axin-2, c-Myc,and Naked-1 in several cell lines.

Because p53-mediated changes in Hsp90 ATPase activityoccurred in the absence of altered levels of Hsp90 protein,experiments were performed to elucidate the underlying mech-anism. Levels of Aha1, a known co-chaperone of Hsp90 (31–33), were affected by p53 status. By contrast, levels of otherHsp90 co-chaperones including p23, XAP-2, and HOP wereunaffected by changes in p53 status (41, 42). p53 deficiency wasassociated with elevated Aha1 levels whereas treatment withCP-31398 or expressing wild type p53 in EB-1 cells suppressedlevels of Aha1. These changes in Aha1 protein levels were asso-ciated with corresponding alterations in the interactionsbetween Aha1 and Hsp90 and Hsp90 ATPase activity. To fur-ther evaluate the functional significance of Aha1 for mediatingp53-dependent changes in Hsp90 ATPase activity, Aha1 wassilenced. Silencing of Aha1 inhibited Hsp90 ATPase activityand the expression of Wnt target genes. The mechanism bywhich p53 regulates Aha1 levels was previously unknown. Aha1can be regulated by HSF-1 (34). We identified an HSF-1-bind-ing element in the 5�-UTR of Aha1. Promoter analyses indi-cated that p53 regulated Aha1 expression via the HSF-1 ele-ment. For example, silencing p53 induced Aha1 promoteractivity; this effect was lost when cells were transfected with anAha1 promoter construct that did not contain the HSF-1 bind-ing site or when the HSF-1 binding site was mutagenized. Addi-tionally, ChIP assays showed increased recruitment of HSF-1 tothe Aha1 promoter when p53 was silenced and decreasedrecruitment when wild type p53 was overexpressed. Moreover,silencing HSF-1 led to reduced Aha1 levels, decreased Hsp90ATPase, and suppression of Wnt target gene expression. Taken

FIGURE 8. p53 regulates Akt and GSK3� phosphorylation via effects onAha1 and Hsp90. A and B, cells were treated with LY294002 as indicated for24 h. C and H, EB-1 cells were treated with indicated concentrations of ZnCl2 for6 h. D, cells were transfected with 2 �g of Aha1 siRNA or control siRNA for 48 h. E,cells were treated with the indicated concentrations of 17-AAG for 0.5 h. F, cellswere transfected with 2 �g of control siRNA or Akt1 siRNA for 48 h. G, cellswere transfected with 2 �g of control siRNA or p53 siRNA for 48 h. I, cells weretreated with indicated concentration of CP-31398 for 6 h. J, cells were transfectedwith 2 �g of control siRNA or Aha1 siRNA for 48 h. K, cells were treated as indi-cated with 1.0 �M 17-AAG for 0.5 h. A–K, cell lysates were subjected to Westernblot analysis. The blots were probed as indicated. L, p53 regulates Aha1 levelsleading to altered Hsp90 ATPase activity. This results in modulation of the Akt/GSK3� signaling axis and thereby the expression of Wnt target genes.




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together, these data suggest that HSF-1 is important for p53-mediated regulation of Wnt signaling. Of potential relevance,silencing HSF-1 has been reported to sensitize cancer cells toHsp90 inhibition (43).

A link between Hsp90 ATPase activity and Wnt signaling hasbeen reported (28). Akt is a known client protein of Hsp90 (35,36). Inhibition of the PI3K/Akt signaling pathway can blocknuclear localization of �-catenin (28, 37, 38). Here we demon-strate that Hsp90 ATPase activity regulates the phosphoryla-tion of Akt and thereby Wnt target gene expression. Inhibitionof Hsp90 ATPase led to reduced levels of phospho-Akt. Con-sistent with these findings, silencing of Aha1 also reduced Aktactivity. Moreover, treatment with LY294002, a PI3K inhibitoror silencing Akt1, inhibited the expression of Axin-2, c-Myc,and Naked-1. Because of the link between p53 and Hsp90ATPase activity, we also confirmed that Akt activity was p53-dependent. In support of this possibility, overexpressing wildtype p53 in colon cancer cells led to decreased Akt phosphory-lation. Levels of phospho-Akt were also increased in cells frompatients with LFS. Taken together, these data show thatincreased Hsp90 ATPase activity, associated with either muta-

tion or loss of p53, led to enhanced Akt activity and increasedWnt target gene expression. Akt can phosphorylate GSK3�,resulting in the accumulation of �-catenin (28, 37, 38). Severalexperiments were performed to evaluate whether this mecha-nism was operative in a p53-dependent manner. Modulatingp53 levels led to changes in GSK3� phosphorylation. Similarly,enhanced GSK3� phosphorylation and increased �-cateninlevels were observed in LFS cells. Silencing of Aha1 or inhib-iting Hsp90 ATPase activity also suppressed GSK3� phos-phorylation. Collectively, these in vitro findings suggest thatp53 regulates the Aha1/Hsp90 ATPase axis leading tochanges in Akt/GSK3� signaling thereby modulating Wnttarget gene expression. Recently, microRNA-34 was foundto be important for p53-mediated regulation of Wnt signal-ing (8). Although Aha1 is not a direct target of microRNA-34, its interacting partner GTP cyclohydrolase-1 is regulatedby it (44, 45). Possibly, microRNA-34 will modulate Hsp90ATPase activity via effects on GTP cyclohydrolase-1. Futurestudies are needed to determine whether microRNA-34 reg-ulates HSF-1 or Aha1 levels and thereby Hsp90 ATPaseactivity.

FIGURE 9. Wnt signaling is activated in p53-null mice. Colon tissue from p53 wild type and p53-null mice was used. Poly(A) RNA was isolated from total RNAextracted from colon tissues. Additionally, tissues were homogenized, and protein lysates were prepared. A, tissue lysates were used to measure Hsp90 ATPaseactivity using a protocol described under “Experimental Procedures.” Mean � S.D. (error bars) are shown, n 6. *, p 0.01 compared with p53�/� mice. Lysateswere also subjected to immunoprecipitation with Hsp90 or �-actin antibodies; Western blotting was carried out, and the blot was probed for Hsp90 and �-actinas indicated. B, relative expression of Aha1 was quantified by real-time PCR. Values were normalized to levels of �-actin. Mean � S.D. are shown, n 6. *, p 0.01 compared with p53�/� mice. Tissue lysates were also immunoprecipitated with Aha1 or �-actin antibody; Western blotting was carried out, and the blotswere probed for Aha1 and �-actin as indicated. C, tissue lysates were immunoprecipitated (IP) with Hsp90 antibody, and the immunoprecipitates weresubjected to Western blotting (WB) for Aha1 and Hsp90 as indicated. D and E, tissue lysates were immunoprecipitated with antibodies to Akt (D) or GSK3� (E),and the blots were probed for pAkt and Akt (D) or pGSK3� and GSK3� (E) as indicated. F–H, tissue lysates were immunoprecipitated with Axin-2, c-Myc,Naked-1, or �-actin antibodies, and the blots were probed with the same antibodies.




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In an attempt to translate the in vitro findings describedabove, the Aha1/Hsp90/Akt/GSK3� axis was evaluated incolon tissues from wild type versus p53-null mice. Consistentwith the in vitro results, increased Aha1 levels, Hsp90 ATPaseactivity, Akt and GSK3� phosphorylation and Wnt targetexpression was observed in colons of p53-null compared withp53 wild type mice. Remarkably, immunoprecipitation experi-ments indicated an increased interaction between Hsp90 andAha1 in the colons of p53-null mice. These results suggest thatcolons of p53-null mice have a heightened Hsp90 ATPase activ-ity and increased Wnt signaling. Previously, the p53 rescuecompound CP-31398 was found to reduce intestinal tumori-genesis in APCmin/� mice (46). Additionally, p53 deficiency hasbeen shown to accelerate tumorigenesis in Wnt-1 transgenicmice (15). Our observation that p53 activation suppressesWnt signaling may help to explain these prior observations.Whether inhibitors of Hsp90 ATPase will have a similar pro-tective effect should be tested. LFS patients are at increased riskfor a number of tumor types including colon cancer (47, 48).Our observation that cells from LFS patients had elevated Aha1levels, Hsp90 ATPase activity, Akt and GSK3� phosphorylationand increased Wnt target gene expression may further help toexplain the link between p53 and increased risk of tumor for-mation. Possibly, agents that disrupt this activated signalingaxis will possess chemopreventive properties. Another poten-tial implication of our findings relates to cancer therapy. If p53

status is found to be a determinant of Hsp90 ATPase activity intumors, this could contribute to chemoresistance or provide astrategy for identifying patients who are most likely to benefitfrom inhibitors of Aha1 or Hsp90 ATPase activity (34, 49 –51).REFERENCES

1. Schneikert, J., and Behrens, J. (2007) The canonical Wnt signalling path-way and its APC partner in colon cancer development. Gut 56, 417– 425

2. Polakis, P. (2000) Wnt signaling and cancer. Genes Dev. 14, 1837–18513. Bienz, M., and Clevers, H. (2000) Linking colorectal cancer to Wnt signal-

ing. Cell 103, 311–3204. Kinzler, K. W., and Vogelstein, B. (1996) Lessons from hereditary colorec-

tal cancer. Cell 87, 159 –1705. Mikels, A. J., and Nusse, R. (2006) Wnts as ligands: processing, secretion

and reception. Oncogene 25, 7461–74686. Sadot, E., Geiger, B., Oren, M., and Ben-Ze’ev, A. (2001) Down-regulation

of �-catenin by activated p53. Mol. Cell. Biol. 21, 6768 – 67817. Levina, E., Oren, M., and Ben-Ze’ev, A. (2004) Down-regulation of

�-catenin by p53 involves changes in the rate of �-catenin phosphoryla-tion and Axin dynamics. Oncogene 23, 4444 – 4453

8. Kim, N. H., Kim, H. S., Kim, N. G., Lee, I., Choi, H. S., Li, X. Y., Kang, S. E.,Cha, S. Y., Ryu, J. K., Na, J. M., Park, C., Kim, K., Lee, S., Gumbiner, B. M.,Yook, J. I., and Weiss, S. J. (2011) p53 and microRNA-34 are suppressors ofcanonical Wnt signaling. Sci. Signal. 4, ra71

9. Muller, P. A., and Vousden, K. H. (2013) p53 mutations in cancer. Nat. CellBiol. 15, 2– 8

10. Gaglia, G., Guan, Y., Shah, J. V., and Lahav, G. (2013) Activation andcontrol of p53 tetramerization in individual living cells. Proc. Natl. Acad.Sci. U.S.A. 110, 15497–15501

11. Vousden, K. H., and Prives, C. (2009) Blinded by the light: the growing

FIGURE 10. Wnt signaling is activated in cells derived from in Li-Fraumeni syndrome patients. LFS epithelial cells (HME50, IUSM/LFS/HME) and wild typeepithelial cells (HME32) were compared. A, cell lysates were prepared, and Hsp90 ATPase activity was measured as described under “Experimental Procedures.”Mean � S.D. (error bars) are shown, n 6. *, p 0.01 compared with HME32 cells. Upper panel, cell lysates were subjected to immunoblotting, and the blot wasprobed for Hsp90 and �-actin. B, cell lysates were subjected to immunoblotting and probed for Aha1 or �-actin. C, cell lysates were immunoprecipitated withHsp90 antibody, and the blot was probed for Aha1 and Hsp90 as indicated. D and E, cell lysates were subjected to immunoblotting and the blots probed asindicated. F and G, cell lysates were subjected to Western blotting and probed as indicated.




ww

.jbc.org/D

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complexity of p53. Cell 137, 413– 43112. Vogelstein, B., Lane, D., and Levine, A. J. (2000) Surfing the p53 network.

Nature 408, 307–31013. Soussi, T., Ishioka, C., Claustres, M., and Béroud, C. (2006) Locus-specific

mutation databases: pitfalls and good practice based on the p53 experi-ence. Nat. Rev. Cancer 6, 83–90

14. Herbert, B. S., Chanoux, R. A., Liu, Y., Baenziger, P. H., Goswami, C. P., Mc-Clintick, J. N., Edenberg, H. J., Pennington, R. E., Lipkin, S. M., and Kopelov-ich, L. (2010) A molecular signature of normal breast epithelial and stromalcells from Li-Fraumeni syndrome mutation carriers. Oncotarget 1, 405–422

15. Donehower, L. A., Godley, L. A., Aldaz, C. M., Pyle, R., Shi, Y. P., Pinkel, D.,Gray, J., Bradley, A., Medina, D., and Varmus, H. E. (1995) Deficiency ofp53 accelerates mammary tumorigenesis in Wnt-1 transgenic mice andpromotes chromosomal instability. Genes Dev. 9, 882– 895

16. Athar, M., Elmets, C. A., and Kopelovich, L. (2011) Pharmacological acti-vation of p53 in cancer cells. Curr. Pharm. Des. 17, 631– 639

17. Shaw, P., Bovey, R., Tardy, S., Sahli, R., Sordat, B., and Costa, J. (1992)Induction of apoptosis by wild-type p53 in a human colon tumor-derivedcell line. Proc. Natl. Acad. Sci. U.S.A. 89, 4495– 4499

18. Zhao, R., Gish, K., Murphy, M., Yin, Y., Notterman, D., Hoffman, W. H., Tom,E., Mack, D. H., and Levine, A. J. (2000) Analysis of p53-regulated gene ex-pression patterns using oligonucleotide arrays. Genes Dev. 14, 981–993

19. Shay, J. W., Tomlinson, G., Piatyszek, M. A., and Gollahon, L. S. (1995)Spontaneous in vitro immortalization of breast epithelial cells from a pa-tient with Li-Fraumeni syndrome. Mol. Cell. Biol. 15, 425– 432

20. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265–275

21. Laemmli, U. K. (1970) Cleavage of structural proteins during the assemblyof the head of bacteriophage T4. Nature 227, 680 – 685

22. Ali, J. A., Jackson, A. P., Howells, A. J., and Maxwell, A. (1993) The 43-kilodalton N-terminal fragment of the DNA gyrase B protein hydrolyzesATP and binds coumarin drugs. Biochemistry 32, 2717–2724

23. Mohebati, A., Guttenplan, J. B., Kochhar, A., Zhao, Z. L., Kosinska, W., Sub-baramaiah, K., and Dannenberg, A. J. (2012) Carnosol, a constituent of Zyfla-mend, inhibits aryl hydrocarbon receptor-mediated activation of CYP1A1and CYP1B1 transcription and mutagenesis. Cancer Prev. Res. 5, 593–602

24. Jacks, T., Remington, L., Williams, B. O., Schmitt, E. M., Halachmi, S.,Bronson, R. T., and Weinberg, R. A. (1994) Tumor spectrum analysis inp53-mutant mice. Curr. Biol. 4, 1–7

25. Taipale, M., Jarosz, D. F., and Lindquist, S. (2010) HSP90 at the hub ofprotein homeostasis: emerging mechanistic insights. Nat. Rev. Mol. CellBiol. 11, 515–528

26. Zhang, H., and Burrows, F. (2004) Targeting multiple signal transductionpathways through inhibition of Hsp90. J. Mol. Med. 82, 488 – 499

27. Garcia-Carbonero, R., Carnero, A., and Paz-Ares, L. (2013) Inhibition ofHSP90 molecular chaperones: moving into the clinic. Lancet Oncol. 14,e358 – e369

28. Kurashina, R., Ohyashiki, J. H., Kobayashi, C., Hamamura, R., Zhang, Y.,Hirano, T., and Ohyashiki, K. (2009) Anti-proliferative activity of heatshock protein (Hsp) 90 inhibitors via �-catenin/TCF7L2 pathway in adultT cell leukemia cells. Cancer Lett. 284, 62–70

29. Maloney, A., and Workman, P. (2002) HSP90 as a new therapeutic targetfor cancer therapy: the story unfolds. Expert Opin. Biol. Ther. 2, 3–24

30. He, H., Zatorska, D., Kim, J., Aguirre, J., Llauger, L., She, Y., Wu, N.,Immormino, R. M., Gewirth, D. T., and Chiosis, G. (2006) Identification ofpotent water soluble purine-scaffold inhibitors of the heat shock protein90. J. Med. Chem. 49, 381–390

31. Panaretou, B., Siligardi, G., Meyer, P., Maloney, A., Sullivan, J. K., Singh, S.,Millson, S. H., Clarke, P. A., Naaby-Hansen, S., Stein, R., Cramer, R., Mol-lapour, M., Workman, P., Piper, P. W., Pearl, L. H., and Prodromou, C.(2002) Activation of the ATPase activity of Hsp90 by the stress-regulatedcochaperone Aha1. Mol. Cell 10, 1307–1318

32. Retzlaff, M., Hagn, F., Mitschke, L., Hessling, M., Gugel, F., Kessler, H.,Richter, K., and Buchner, J. (2010) Asymmetric activation of the Hsp90dimer by its cochaperone Aha1. Mol. Cell 37, 344 –354

33. Lotz, G. P., Lin, H., Harst, A., and Obermann, W. M. (2003) Aha1 binds to

the middle domain of Hsp90, contributes to client protein activation, andstimulates the ATPase activity of the molecular chaperone. J. Biol. Chem.278, 17228 –17235

34. Holmes, J. L., Sharp, S. Y., Hobbs, S., and Workman, P. (2008) Silencing ofHSP90 cochaperone AHA1 expression decreases client protein activationand increases cellular sensitivity to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin. Cancer Res. 68, 1188 –1197

35. Sato, S., Fujita, N., and Tsuruo, T. (2000) Modulation of Akt kinase activityby binding to Hsp90. Proc. Natl. Acad. Sci. U.S.A. 97, 10832–10837

36. Basso, A. D., Solit, D. B., Chiosis, G., Giri, B., Tsichlis, P., and Rosen, N.(2002) Akt forms an intracellular complex with heat shock protein 90(Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function.J. Biol. Chem. 277, 39858 –39866

37. Anderson, E. C., and Wong, M. H. (2010) Caught in the Akt: regulation ofWnt signaling in the intestine. Gastroenterology 139, 718 –722

38. Nakayama, M., Hisatsune, J., Yamasaki, E., Isomoto, H., Kurazono, H.,Hatakeyama, M., Azuma, T., Yamaoka, Y., Yahiro, K., Moss, J., and Hi-rayama, T. (2009) Helicobacter pylori VacA-induced inhibition of GSK3through the PI3K/Akt signaling pathway. J. Biol. Chem. 284, 1612–1619

39. Srivastava, S., Zou, Z. Q., Pirollo, K., Blattner, W., and Chang, E. H. (1990)Germ line transmission of a mutated p53 gene in a cancer-prone familywith Li-Fraumeni syndrome. Nature 348, 747–749

40. Evans, D. G., Birch, J. M., Thorneycroft, M., McGown, G., Lalloo, F., andVarley, J. M. (2002) Low rate of TP53 germ line mutations in breast can-cer/sarcoma families not fulfilling classical criteria for Li-Fraumeni syn-drome. J. Med. Genet. 39, 941–944

41. Li, J., Soroka, J., and Buchner, J. (2012) The Hsp90 chaperone machinery:conformational dynamics and regulation by co-chaperones. Biochim. Bio-phys. Acta 1823, 624 – 635

42. Cox, M. B., and Johnson, J. L. (2011) The role of p23, Hop, immunophilins,and other co-chaperones in regulating Hsp90 function. Methods Mol. Biol.787, 45– 66

43. Chen, Y., Chen, J., Loo, A., Jaeger, S., Bagdasarian, L., Yu, J., Chung, F.,Korn, J., Ruddy, D., Guo, R., McLaughlin, M. E., Feng, F., Zhu, P., Steg-meier, F., Pagliarini, R., Porter, D., and Zhou, W. (2013) Targeting HSF1sensitizes cancer cells to HSP90 inhibition. Oncotarget 4, 816 – 829

44. Swick, L., and Kapatos, G. (2006) A yeast 2-hybrid analysis of human GTPcyclohydrolase I protein interactions. J. Neurochem. 97, 1447–1455

45. Chen, D. D., Zhao, T., Li, J., and Chen, A. F. (2011) GTP cyclohydrolase Irescues microRNA-34a impairment of endothelial progenitor cell angio-genesis in aging. Circulation 124, A17288

46. Rao, C. V., Swamy, M. V., Patlolla, J. M., and Kopelovich, L. (2008) Sup-pression of familial adenomatous polyposis by CP-31398, a TP53 modu-lator, in APCmin/� mice. Cancer Res. 68, 7670 –7675

47. Wong, P., Verselis, S. J., Garber, J. E., Schneider, K., DiGianni, L., Stock-well, D. H., Li, F. P., and Syngal, S. (2006) Prevalence of early onset colo-rectal cancer in 397 patients with classic Li-Fraumeni syndrome. Gastro-enterology 130, 73–79

48. Ruijs, M. W., Verhoef, S., Rookus, M. A., Pruntel, R., van der Hout, A. H.,Hogervorst, F. B., Kluijt, I., Sijmons, R. H., Aalfs, C. M., Wagner, A., Aus-ems, M. G., Hoogerbrugge, N., van Asperen, C. J., Gomez Garcia, E. B.,Meijers-Heijboer, H., Ten Kate, L. P., Menko, F. H., and van ’t Veer, L. J.(2010) TP53 germ line mutation testing in 180 families suspected of Li-Fraumeni syndrome: mutation detection rate and relative frequency ofcancers in different familial phenotypes. J. Med. Genet. 47, 421– 428

49. Maloney, A., Clarke, P. A., and Workman, P. (2003) Genes and proteinsgoverning the cellular sensitivity to HSP90 inhibitors: a mechanistic per-spective. Curr. Cancer Drug Targets 3, 331–341

50. Armstrong, H., Wolmarans, A., Mercier, R., Mai, B., and LaPointe, P.(2012) The co-chaperone Hch1 regulates Hsp90 function differently thanits homologue Aha1 and confers sensitivity to yeast to the Hsp90 inhibitorNVP-AUY922. PLoS One 7, e49322

51. Zurawska, A., Urbanski, J., Matuliene, J., Baraniak, J., Klejman, M. P., Fili-pek, S., Matulis, D., and Bieganowski, P. (2010) Mutations that increaseboth Hsp90 ATPase activity in vitro and Hsp90 drug resistance in vivo.Biochim. Biophys. Acta 1803, 575–583




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Herbert, Andrew J. Dannenberg and Kotha SubbaramaiahSachiyo Okayama, Levy Kopelovich, Gabriel Balmus, Robert S. Weiss, Brittney-Shea

Modulating Aha1 Expressionp53 Protein Regulates Hsp90 ATPase Activity and Thereby Wnt Signaling by

doi: 10.1074/jbc.M113.532523 originally published online January 22, 20142014, 289:6513-6525.J. Biol. Chem.

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VOLUME 289 (2014) PAGES 6513– 6525DOI 10.1074/jbc.W119.012134

Withdrawal: p53 protein regulates Hsp90 ATPaseactivity and thereby Wnt signaling by modulating Aha1expression.Sachiyo Okayama, Levy Kopelovich, Gabriel Balmus, Robert S. Weiss,Brittney-Shea Herbert, Andrew J. Dannenberg, and Kotha Subbaramaiah

This article has been withdrawn by the authors. In Fig. 1D, the firstlane of the p53 immunoblot was reused as actin in the same figurepanel. In Fig. 2B, lanes 1 and 2 of the actin immunoblot were reusedin lanes 5 and 6. Lane 2 of the actin immunoblot in Fig. 4K was reusedin lanes 3 and 4. The HOP immunoblot in Fig. 4J was reused in Fig. 4(K and L) as actin. The actin immunoblot in Fig. 4H was reused in Fig.4 (L and I) as actin. The actin immunoblot in Fig. 5A was reused inFig. 5B. In Fig. 5H, the c-Myc and Naked-1 immunoblots are thesame. There are undeclared gel splices in Figs. 5F, 7C, 7F, and 8I. Aportion of the actin immunoblot in Fig. 10A was reused in Fig. 10B asAha1.

WITHDRAWALS/RETRACTIONS

J. Biol. Chem. (2020) 295(1) 289 –289 289© 2020 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

p53proteinregulateshsp90atpaseactivityandthereby ...were purified and used as a template for pcr...

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