activated tyrosine kinase ack1 promotes prostate ... · (cancer res 2005; 65(22): 10514-23)...

Activated Tyrosine Kinase Ack1 Promotes Prostate Tumorigenesis:

Role of Ack1 in Polyubiquitination of Tumor Suppressor Wwox

Nupam P. Mahajan,1Young E. Whang,

1James L. Mohler,

2,3and H. Shelton Earp

1

1Lineberger Comprehensive Cancer Center, Department of Medicine and Pharmacology, and 2Departments of Surgery and Pathologyand Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina and 3Department ofUrologic Oncology, Roswell Park Cancer Institute, New York State University at Buffalo, Buffalo, New York

Abstract

Aberrant activation of tyrosine kinases is linked causally tohuman cancers. Activated Cdc42-associated kinase (Ack1), anintracellular tyrosine kinase, has primarily been studied for itssignaling properties but has not been linked to specificpathologic conditions. Herein, we report that expression ofactivated Ack1 in LNCaP cells, while minimally increasinggrowth in culture, enhanced anchorage-independent growthin vitro and dramatically accelerated tumorigenesis in nudemice. Molecular chaperone heat shock protein 90B (Hsp90B)–bound Ack1 and treatment of cells with geldanamycin, aHsp90 inhibitor, inhibited Ack1 kinase activity and suppressedtumorigenesis. Further, we identify the tumor suppressor WWdomain containing oxidoreductase (Wwox) as an Ack1-interacting protein. Activated Ack1 tyrosine phosphorylatedWwox, leading to rapid dissociation of the Ack1-Wwoxcomplex and concomitant Wwox polyubiquitination followedby degradation. Tyrosine phosphorylation of Wwox wascritical for its degradation, as splice variant Wwox#5-8 thatwas not phosphorylated by Ack1 failed to undergo polyubi-quitination and degradation. It has been reported thatphosphorylation of Wwox at Tyr33 stimulated its proapoptoticactivity. We observed that Y33F Wwox mutant was stilltyrosine phosphorylated and polyubiquitinated by Ack1action. Site-directed mutagenesis revealed that activatedAck1 primarily phosphorylated Wwox at Tyr287, suggestingthat phosphorylation of distinct tyrosine residues activate ordegrade Wwox. Primary androgen-independent prostatetumors but not benign prostate showed increased tyrosine-phosphorylated Ack1 and decreased Wwox. Taken together,these data indicate that Ack1 stimulated prostate tumorigen-esis in part by negatively regulating the proapoptotic tumorsuppressor, Wwox. Further, these findings suggest that Ack1could be a novel therapeutic target for prostate cancer.(Cancer Res 2005; 65(22): 10514-23)

Introduction

Tyrosine kinases are critical regulators of intracellular signaltransduction pathways and their activity is tightly controlled.Dysregulation of tyrosine kinase signaling by chromosomaltranslocation, overexpression, or activating mutation may resultin perturbation of the kinase activity and malignant transformation

(1–3). Constitutive tyrosine kinase activation can lead to growthfactor–independent cell growth, suppression of apoptosis, angio-genesis, invasion, and metastasis. Thus, tyrosine kinases haveproven to be specific molecular targets for the cancer therapeuticsdevelopment, and inhibitors of the tumor-relevant tyrosine kinasesthat drive oncogenic behavior have emerged as a novel paradigm forcancer therapy (4, 5).Prostate cancer is the second leading cause of cancer deaths in

American men (6). Androgen deprivation through surgical ormedical castration is the current treatment for advanced metastaticdisease (7). However, the disease invariably relapses with amedian of24 months, after which it is considered androgen independent(AICaP) or hormone refractory, with no curative treatment. Cancerprogression through the various stages presumably requires alteredexpression of oncogenes and tumor suppressor genes in the initiatedcell (8). Growing evidence suggests that tyrosine kinases play asignificant role in prostate cancer progression (9–12), and thusidentification of the disease-associated tyrosine kinases, and themechanism by which they enhance prostate tumorigenesis shouldproduce additional therapeutic targets.Our previous work identified a transmembrane receptor tyrosine

kinase Mer (also known as NYK; ref. 13), belonging to the Mer/Axl/Tyro3 receptor family (14). We observed expression of Mer inmacrophages, epithelial cells, and cells of reproductive origin (e.g.,testis, ovary, and prostate; refs. 14, 15). Further, we showed thatMersignaling was involved in monocytic ingestion of apoptotic cells (16)in part by regulating Vav1-Rac/Cdc42–mediated cytoskeletalremodeling (15). Gas6 was identified as a ligand for Mer/Axl/Tyro3family (17, 18) and we have shown that Gas6 binding activates Merleading to autophosphorylation, tyrosine phosphorylation of gua-nine nucleotide exchange factors, and accumulation of GTP-boundRac and Cdc42 (15). Three human prostatic adenocarcinoma celllines, LNCaP, CWR-R1, and DU-145, express the cell surface 175-kDaMer protein (13).4 To identify Mer-activated tyrosine phosphopro-tein, we used proteomics approach and identified activated Cdc42-associated kinase (Ack1) as a Mer-responsive intracellular tyrosinekinase. To determine what role Ack1 might play in prostatemalignancy, we generated constitutively active Ack1 (caAck) andkinase-inactive Ack1 constructs and expressed them in LNCaP cells.Although caAck only modestly increased LNCaP proliferation inculture, we observed dramatic acceleration of xenograft growth ofactivated Ack1-expressing LNCaP cells. This was suppressed by aheat shock protein 90 (Hsp90) inhibitor that abolished Ack1 kinaseactivity. Further, we identified one novel mechanism of Ack1-mediated tumorigenesis, tyrosine phosphorylation of the tumorsuppressor WW domain containing oxidoreductase (Wwox) leadingNote: Supplementary data for this article are available at Cancer Research Online

(http://cancerres.aacrjournals.org/).Requests for reprints: H. Shelton Earp, Lineberger Comprehensive Cancer Center,

University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295. Phone: 919-966-2335; Fax: 919-966-3015; E-mail: [email protected].

I2005 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-1127 4 N.P. Mahajan and H.S. Earp, unpublished data.

Cancer Res 2005; 65: (22). November 15, 2005 10514 www.aacrjournals.org

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to its polyubiquitination and degradation. Although others haveidentified stimulation of Wwox proapoptotic activity by Tyr33

phosphorylation, the primary Ack1 site of Wwox phosphorylationwas at Tyr287. Our analysis of human androgen-independentprostate tumors corroborated this inverse relationship, showingincreased tyrosine-phosphorylated Ack1 and decreased Wwoxprotein. This and the accelerated experimental tumorigenesis defineAck1 as a novel therapeutic target in prostate cancer.

Materials and Methods

Plasmids and site-directed mutagenesis. The full-length Mer cDNA

was subcloned into pcDNA3.1 vector (Invitrogen, Carlsbad, CA). The myc-

tagged constructs caAck, kinase-dead Ack1 (kdAck), and wAck and Flag-tagged Wwox construct were generated using PCR amplification and

subcloned into pcDNA4(A)Myc-His vector (Invitrogen). Mutagenesis was

done using GeneEditor system (Promega, Madison, WI). All plasmidconstructs were sequenced.

Antibodies, cell lines, virus production, and infection. A synthetic

peptide containing the 20 amino acids of Ack1 was used to generate rabbit

polyclonal antibody. Anti-phosphotyrosine (RC20; Transduction Laboratories,San Jose, CA), anti-Hsp90 (Stressgen, San Diego, CA), anti-Wwox polyclonal

(Santa Cruz Biotechnology, Santa Cruz, CA), and anti-myc (Invitrogen)

antibodies were purchased. Anti-Wwox monoclonal was kindly provided

by Dr. C. Croce (Cancer Center, Ohio State University, Columbus, OH). Togenerate LNCaP stable lines, respective retrovirus constructs, subcloned

into pMSCV (Invitrogen) vector, were transfected in A293T cells with a

packaging plasmid pCMV-VSV-G and pUMVC3-gagpol using Fugene (Roche,Indianapolis, IN). LNCaP cells were infected for 5 to 6 hours with viral

supernatants containing 8 Ag/mL polybrene. The transfected cells were

selected in the presence of puromycin (2 Ag/mL).

Mass spectrometry. Coomassie, silver, or Pro-Q diamond stained gelbands were excised and digested overnight with trypsin using a ProGest

digestor (Genomic Solutions, Ann Arbor, MI). The peptide extract was

lyophilized, resuspended, and spotted on the matrix-assisted laser

desorption/ionization target. The spectra were database searched usingABI’s Data Explorer software package (Foster City, CA). The peptide mass

fingerprinting and sequence tag data from the time of flight (TOF)/TOF

were ranked according to ABI’s GPS Explorer scores, and confident hits

were reported.Immunoprecipitation and immunoblot analysis. HEK293T cells were

transfected using Fugene as per manufacturer’s protocol. Thirty-two hours

after transfection, cells were lysed in receptor lysis buffer containing25 mmol/L Tris (pH 7.5), 225 mmol/L NaCl, 1% Triton X-100, 1 mmol/L DTT,

10% glycerol, phosphatase inhibitors (10 mmol/L NaF, 1 mmol/L Na2VO4),

and protease inhibitor mix (Roche). Equivalent amounts of protein were

incubated with respective primary antibodies and protein A/G-Sepharose(Santa Cruz Biotechnology) for 2 hours or overnight at 4jC. Immunopre-

cipitates were washed and prepared for immunoblot analysis. The blots

were stripped and reprobed with a second set of antibodies to confirm the

presence of respective proteins.Purification of glutathione S -transferase fusion proteins and

immunoaffinity assay. Glutathione S-transferase (GST)-caAck and GST-

kdAck constructs were transformed into BL21(DE3) cells and plated ontoampicillin plates. Colonies were picked and grown overnight in 10 mL LB

containing ampicillin. Culture grown overnight was added to 300 mL fresh

LB containing ampicillin and grown for 2 hours, which was followed by

isopropyl-L-thio-h-D-galactopyranoside addition (0.1 mmol/L final concen-tration). Culture was grown for 4 more hours, and cells were harvested and

lysed in lysis buffer containing 25 mmol/L Tris (pH 7.5), 150 mmol/L NaCl,

0.4% Triton X-100, 1 mmol/L DTT, 15% glycerol, phosphatase inhibitors

(10 mmol/L NaF, 1 mmol/L Na2VO4), and protease inhibitor mix. Lysateswere incubated with glutathione beads for 2 hours followed by washing the

beads with lysis buffer. Affinity purification method was employed to

identify Ack1-interacting proteins. LNCaP cell lysate (made using receptor

lysis buffer) was incubated with glutathione beads for 2 hours. Glutathione

beads were washed and bound proteins were eluted in 10 mmol/Lglutathione, subjected to SDS-PAGE, and stained with Coomassie brilliant

blue. Ack1-interacting protein was identified using mass spectrometry.

Anchorage-independent and cell growth assay. LNCaP cells (1 � 104)

that stably expressed caAck, kdAck, wAck, and vector were seeded in12-well plates in quadruplets in 1 mL top agar. Colonies were stained with

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma,

St. Louis, MO) after 3 to 4 weeks. For cell growth assay, LNCaP cells

(1 � 103) stably expressing Ack1 variants were seeded in 96-well plates.Solution of MTT was added to cells and incubated for 2 hours, and cell

growth as a function of mitochondrial activity in living cells was measured

spectrophotometrically at a wavelength of 570 nm.

Kinase assay. Ack1 kinase activity was determined using phospho-cellulose filter binding assay. Reaction mixtures contained 20 mmol/L

Tris-HCl (pH 7.4), 10 mmol/L MgCl2, 0.1 mmol/L Na2VO4, 0.5 mmol/L DTT,

0.25 mmol/L ATP, varying concentrations of peptide substrate (EAIYAAP-FAKKKG), and [g-32P]ATP (200-400 counts/min/pmol). Reactions were

terminated and the samples were spotted on p81 phosphocellulose filter.

Incorporation of 32P into peptide was determined using liquid scintillation

counting.Tumorigenicity assay. LNCaP cells (2 � 106) that expressed Ack1

variants were suspended in 100 AL PBS and 100 AL Matrigel (Discovery

Labware, Bedford, MA) and injected s.c. into flanks of male nude mice.

Tumor volume was measured twice weekly using calipers.In vivo ubiquitination assays. HEK293T cells were transfected and

pretreated with 25 Amol/L MG-132 for 3 to 4 hours. Cells were lysed in RLB

buffer containing 1% SDS. Protein samples were diluted 1:5 andimmunoprecipitated with anti-Flag beads. Immunoprecipitates were

washed and prepared for immunoblot analysis.

Patient tissue samples. Androgen-independent prostate cancer samples

were obtained from transurethral resection specimens from men whodeveloped urinary retention from local recurrence during androgen

deprivation therapy. Androgen-stimulated benign prostate samples were

obtained from radical prostatectomy specimens from men with clinically

localized prostate cancer. Histologic diagnoses were confirmed byexamination of frozen and corresponding formalin-fixed, paraffin-embed-

ded tissue specimens. Tissue procurement and subsequent studies were

done with institutional review board and HIPPA approval.

Results

Ack1 is a major prostate cell phosphoprotein induced by theMer receptor tyrosine kinase. To identify tyrosine-phosphorylat-ed proteins responding to Mer activation, we treated large-scalecultures of the prostate cancer cell line LNCaP with Gas6 ligand.Protein lysates were affinity purified using phosphotyrosineantibody covalently immobilized on beads. After elution, sampleswere subjected to SDS-PAGE. Western analysis using anti-phospho-tyrosine antibody revealed increased tyrosine phosphorylation off120-kDa protein on Gas6 treatment (Fig. 1A). The f120-kDaband was excised and subjected to in-gel trypsin digestion andliquid chromatography/tandem mass spectrometry sequencing.Database search for the peptide sequences revealed that the120-kDa protein was Ack1 (Fig. 1B , sequenced peptides shown inbold). Ack1 is a 120-kDa soluble protein (Fig. 1C) with a NH2-terminal kinase domain, a Src homology 3 (SH3) domain, a Cdc42/Rac interactive binding domain (CRIB) domain, and a proline-richdomain at COOH terminus (19). Ack1 specifically interacts withGTP-bound Cdc42 (activated Cdc42) and inhibits both intrinsic andGTPase-activating protein–stimulated GTPase activities of Cdc42(19). Ack1 activity has been implicated in cell spreading (20),vesicle trafficking (21), axonal guidance (22), and dorsal closure inDrosophila melanogaster (23). However, the physiologic role of Ack1and its molecular mechanism of action are not fully understood, norhas Ack1 been linked to any specific pathologic condition (24).

Ack1 Promotes Prostate Tumorigenesis

www.aacrjournals.org 10515 Cancer Res 2005; 65: (22). November 15, 2005



To ascertain whether endogenous Mer and Ack1 interact inprostate cells, coimmunoprecipitation was done on LNCaP lysatesusing Mer antibodies followed by an immunoblotting with Ack1antibodies. Gas6 treatment resulted in tyrosine phosphorylation ofendogenous Mer (Fig. 1D , 4) and Ack1 (Fig. 1D , 2). EndogenousMer and Ack1 specifically associated in phosphotyrosine-dependentmanner following Gas6 treatment (Fig. 1D , 1).Ack1 autophosphorylates by an intramolecular mechanism.

To elucidate the molecular mechanism by whichMer regulates Ack1

tyrosine phosphorylation, three myc-tagged Ack1 constructs, wAck,kdAck, and caAck, were generated, each consisted of the kinasedomain, SH3 domain, CRIB domain, and proline-rich domain. Eachlacked the COOH-terminal region (788-1,036 amino acids; Fig. 2A),as we and others have observed that the full-length Ack1 construct isexpressed poorly (25); therefore, we used the truncated constructs.The kdAck construct possesses a point mutation, K158R, in the ATPacceptor site and does not autophosphorylate (Fig. 2A). Nuclearmagnetic resonance spectroscopy has shown details of molecularinteraction between Ack1 and GTP-bound Cdc42, wherein Leu487 ofAck1 interacts with Leu67 of Cdc42 (26, 27). Interestingly, Leu487

also interacts with residues in its own kinase domain, maintainingan inactive Ack1 (26, 27). Based on this intramolecular interaction

Figure 2. Mer facilitates Ack1 autotyrosine phosphorylation and activation.A, domain structure of Ack1 and myc-tagged Ack1variants. Location of pointmutations are indicated. B, lysates of HEK293T cells transfected withmyc-tagged Ack1 variants and Mer were subjected to immunoprecipitationfollowed by immunoblotting as indicated. Mer activation results in wAck tyrosinephosphorylation but does not directly phosphorylate kdAck (lane 4), indicatingthat Mer facilitates Ack1 autophosphorylation. C, lysates of LNCaP cells stablyexpressing myc-tagged Ack1 variants were subjected to immunoprecipitationfollowed by immunoblotting as indicated. D, lysates of LNCaP cells stablyexpressing myc-tagged Ack1 variants were subjected to immunoprecipitationfollowed by kinase assay. caAck-expressing cells exhibit significantly higherkinase activity compared with kdAck.

Ack1kinase SH PRC

Figure 1. Mer interacts with Ack1 in vivo. A, LNCaP cells were treated withGas6 (150 nmol/L) ligand and lysates were immunoprecipitated usinganti-phosphotyrosine (pTyr ) resin followed by electrophoresis. Westernanalysis revealed a band of 120 kDa to be a major phosphorylated protein.B, 120-kDa band was excised, proteolyzed, and subjected to mass spectrometrysequencing leading to identification of tyrosine kinase Ack1. The sequencedpeptides are shown in bold. C, domain structure of Ack1. SH, SH3 domain;C, CRIB domain; PR, proline-rich domain. D, lysates of LNCaP cells weresubjected to immunoprecipitation (IP ) followed by immunoblotting (IB ) asindicated. Endogenous Ack1 interacts with endogenous Mer inphosphotyrosine-dependent manner.

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(27, 28), we made the L487F mutation, which essentially disruptsautoinhibition and results in a caAck (Fig. 2A).To determine if tyrosine phosphorylation of Ack1 is mediated by

an intermolecular or intramolecular mechanism, HEK293T cellswere transfected with myc-tagged Ack1 variants with either thefull-length Mer or and empty vector. If tyrosine phosphorylationoccurs in trans (i.e., intermolecular), then kdAck would bephosphorylated when coexpressed with Mer . In contrast, ifphosphorylation occurs via an intramolecular (or cis) mechanism,then K158R mutation in the ATP acceptor site would disrupttyrosine phosphorylation of Ack1. Earlier we have noticed (15) thatfull-length Mer overexpression resulted in Gas6-independent Meractivation (Fig. 2B , 3). caAck was tyrosine phosphorylated in theabsence or presence of active Mer (Fig. 2B , 1, lanes 5 and 6). ActiveMer induced tyrosine phosphorylation of wAck but not kdAck(Fig. 2B , 1 , lanes 2 and 4), suggesting that activated Mer does notdirectly phosphorylate Ack1 but rather facilitates Ack1 autophos-phorylation. These results suggest that Ack1 phosphorylationoccurs by an intramolecular mechanism.To assess the potential relevance of Ack1 activation in prostate

cancer, LNCaP cells were infected with myc-tagged Ack1-expressing retroviral constructs and stable lines were generatedthat showed tyrosine-phosphorylated Ack1 in caAck-expressingcells but not kdAck- or vector-expressing cells (Fig. 2C). Longerexposure shows some autophosphorylation of wAck. To determinewhether Ack1 autophosphorylation correlated with kinase activa-tion, kinase assays were done. It has been shown that purifiedAck1 kinase prefers a peptide substrate also preferred by the Ablkinase (25). This 13–amino acid peptide (EAIYAAPFAKKKG) wassynthesized and used for kinase assays (Fig. 2D). Ack1 immuno-precipitates from LNCaP cells expressing wAck, kdAck, and vectorexhibited low kinase activity. In contrast, caAck immunoprecipi-tates had 7-fold higher kinase activity than kdAck (Fig. 2D),indicating that Ack1 autophosphorylation correlated with kinaseactivation. The disparity between minimal wAck autophosphor-ylation (Fig. 2C) and more readily detectable kinase activity seenin vitro after immunoprecipitation (Fig. 2D) is probably the resultof activation of wAck as it is concentrated in the immunoprecip-itate by the divalent Ack1 antibodies.Activated Ack1 promotes anchorage-independent growth

and tumor growth in vivo . To determine biological effects of Ack1constructs, growth characteristics of the stably transfected LNCaPcells were monitored. A modest increase in growth rate of thecaAck- and wAck-expressing cells was observed compared with thekdAck- and vector-expressing cells (Fig. 3A). We next assessedability of LNCaP cells expressing Ack1 constructs to form coloniesin soft agar. LNCaP cells expressing caAck exhibited a substantialincrease in anchorage-independent growth in soft agar. Fewercolonies were observed in kdAck-, wAck-, and vector-expressingcells (Fig. 3B). To further examine the effect of Ack1 activation,we injected wAck-, kdAck-, and vector-expressing LNCaP cells s.c.into flank of nude mice and monitored the increase in tumorvolume over a 3-month period. LNCaP cells are poorly tumorigenicwhen injected in nude mice. Strikingly, caAck-expressing LNCaPcells formed large tumors in all the mice within 23 to 24 days(Fig. 3C). Tumors were undetectable at this stage in wAck-, kdAck-,and vector-expressing LNCaP cells (compare tumor size of kdAckand caAck; Fig. 3D), although small tumors were observed in somecases when the animals were followed for 90 days. These dataindicate that kinase activation of Ack1 results in enhancementof prostate cancer cell tumorigenic properties.

Figure 3. Activated Ack1 enhances anchorage-independent growth andaccelerates tumor formation in vivo. A, cell growth assay of the LNCaP cells(1 � 103 per well) stably expressing myc-tagged Ack1 variants was done asdescribed in Materials and Methods. B, soft agar colony formation of LNCaPcells (1 � 104 per well) expressing Ack1 variants. Cells were plated inquadruplicate as described in Materials and Methods. Three weeks later,colonies were stained with the colorimetric dye MTT. C, LNCaP cells(2 � 106 per injection) stably expressing Ack1 variants were injected s.c. intomale nude mice (n = 10). Data represent growth of tumors. Points, mean tumorvolume; bars, SE. caAck-expressing cells exhibit substantial xenograft growth.Similar results were obtained in three independent experiments. D, LNCaP cellsstably expressing Ack1 variants were injected s.c. into male nude mice.caAck-expressing LNCaP cells exhibit robust tumor formation in 23 days; incontrast, kdAck-expressing cells exhibit no tumor growth.





Heat shock protein 90B maintains Ack1 activity. We nextattempted to identify Ack1-interacting proteins by biochemicalaffinity purification. GST-tagged caAck, kdAck, and GST (Fig. 4A)expressed in Escherichia coli were purified using glutathione beads.These were then incubated with LNCaP cell lysates. After extensivewashing, bound proteins were eluted using glutathione andseparated using SDS-PAGE. A band of f90 kDa isolated fromGST-caAck1 complex, not seen in GST alone, was microsequenced(Supplementary Fig. S1) and identified as Hsp90h (Fig. 4B). Hsp90 isa molecular chaperone that plays a critical role in conformationalmaturation of protein kinases, steroid hormone receptors, growthfactor receptors, and transcription factors involved in signaltransduction (29). Ack1-Hsp90h association was further confirmedby coimmunoprecipitation experiment. Hsp90h was coprecipitatedby myc affinity beads from LNCaP cells that stably express all thethree myc-tagged Ack1 constructs but not in vector-expressing cells(Fig. 4C). Immunoprecipitation of untransfected LNCaP cell lysateswith Ack1 antibodies also revealed the presence of Hsp90h,indicating endogenous Ack1-Hsp90h interaction (data not shown).Thus, in vivo interaction between Hsp90h and Ack1 seems to beconstitutive in LNCaP cells, but Hsp90h may prefer tyrosine-phosphorylated Ack1 (Fig. 4C , lane 3).We investigated whether Hsp90-specific inhibitors could sup-

press caAck kinase activity and caAck accelerated tumorigenesis.Geldanamycin binds to a conserved binding pocket inhibiting

ATP-dependent chaperone activity of Hsp90; this destabilizesmany Hsp90 client proteins, including Raf-1 and HER2 (30, 31).LNCaP cells expressing Ack1 variants were treated with geldana-mycin for 8 hours and kinase activity was monitored (Fig. 4D). ThecaAck kinase activity was reduced to basal level on geldanamycintreatment (Fig. 4D), with no significant decrease in total Ack1protein, in 8 hours (Fig. 4E , 2), indicating that Hsp90h wasinvolved in maintaining the active Ack1 kinase conformation. Thismight differ from the proposed mechanism of Hsp90 action forHER2, whereby geldanamycin treatment lead to significant HER2degradation at 8 hours (Fig. 4E , 3 ; ref. 32). The apparent increase inHER2 levels between vector and all three Ack1-transfected celllines was not reproducible (i.e., Ack1 expression does not changeHER2 expression levels).We further tested the role of Ack1 kinase activity in promoting

tumorigenesis in vivo by treating stably transfected LNCaP celllines with geldanamycin. caAck1 LNCaP cells were treated withgeldanamycin or DMSO for 8 hours and injected into nude mice.DMSO-treated caAck cells formed large tumors in all mice within24 days, but the preimplantation geldanamycin treatment substan-tially delayed caAck LNCaP cell tumorigenesis (Fig. 4F). Theseresults suggest that the mechanism underlying Ack1-mediatedtumorigenesis in vivo includes Hsp90h-stabilized Ack1 kinaseactivity, although we cannot rule out the involvement of additionalHsp90 clients.

Figure 4. Inhibition of Ack1 kinase activity by Hsp90 inhibitor suppress tumorigenesis. A, schematic of GST-Ack1 fusion proteins expressing constructs. B, lysatesof LNCaP cells were incubated with glutathione beads immobilized with GST-caAck, GST-kdAck, and GST alone. Following electrophoresis, tryptic digestion of 90-kDaexcised band, and mass spectrometry, Hsp90h was identified (see Supplementary Fig. S1). The sequenced peptides are shown in bold. C, lysates of LNCaP cellsstably expressing myc-tagged Ack1 variants were subjected to immunoprecipitation followed by immunoblotting as indicated. Ack1 is constitutively associated withHsp90h. D, LNCaP cells stably expressing myc-tagged Ack1 variants were treated with 5 Amol/L geldanamycin (GA ) for 8 hours and immunoprecipitation/kinaseassays were done. Geldanamycin treatment resulted in abrogation of Ack1 kinase activity. E, LNCaP cells were treated with geldanamycin (5 Amol/L) for 8 hoursand subjected to immunoprecipitation followed by immunoblotting as indicated. F, Hsp90 inhibition results in loss of tumorigenicity. LNCaP cells stably expressing caAckwere treated with geldanamycin (5 Amol/L) for 8 hours and injected s.c. into male nude mice (n = 10). Data represent growth of tumors. Points, mean tumor volume;bars, SE. Geldanamycin treatment delayed caAck-mediated xenograft growth of LNCaP cells. Representative of two independent experiments.

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Ack1 associates with the tumor suppressor Wwox. Toelucidate mechanisms underlying activated Ack1-mediated tumor-igenesis, mass spectrometric sequencing was used again to identifyadditional Ack1-interacting proteins in prostate cancer cells.LNCaP cell stably expressing myc-tagged caAck and vector werelysed and immunoaffinity purified using myc antibody resin. Afterextensive washing, bound proteins were eluted with the mycpeptide followed by separation by SDS-PAGE. A f36-kDa proteinwas detected in cells expressing myc-tagged caAck but not invector (Supplementary Fig. S2). Microsequencing revealed it to beWwoxD5-8 (Fig. 5A , peptides are shown in bold). This alternatelyspliced Wwox lacks exons 5 to 8 and has a COOH terminus whosesequence diverges from full-length Wwox due to translation of adifferent reading frame (Fig. 5B ; refs. 33–35).High frequency of loss of heterozygosity (LOH) of chromo-

some 16q23 is observed in prostate, breast, ovarian, and othercancers (36–38). Wwox gene, composed of nine exons, has beenmapped to this region (38). Wwox, a 46-kDa protein, possessingtwo NH2-terminal WW domains and a COOH-terminal short-chain alcohol dehydrogenase (ADH) domain (Fig. 5B), is found inadult hormonally regulated tissues, such as testis, ovary, andprostate (38). Wwox was characterized as a putative tumorsuppressor based on studies in which ectopic Wwox expressioninhibited anchorage-independent growth in soft agar andtumorigenicity of breast cancer cells in vivo (33). Wwox interactswith p53 and its homologue p73 enhancing Wwox proapoptoticactivity (35, 39).WwoxD5-8 possessing both WW domains but lacking a

functional ADH domain (Fig. 5B) is not proapoptotic and isprimarily expressed in ovarian and breast cancer cell lines (33, 34).It was intriguing that WwoxD5-8, not Wwox, was isolated as caAck-interacting protein, as reverse transcription-PCR (data not shown)

and immunoblot analysis revealed the presence of both Wwox andWwoxD5-8 in untransfected LNCaP cells (Fig. 5C).To examine caAck1-Wwox interaction, Flag-tagged Wwox or

WwoxD5-8 and myc-tagged Ack1 variants were cotransfected intoHEK293T cells. Wwox was detected in complexes with both wAckand kdAck but not with caAck (Fig. 5D , 1). Kinase-competentwAck1 minimally increased Wwox tyrosine phosphorylation, butcaAck1 was far more effective (Fig. 5D , 2, lane 3). Wwox tyrosinephosphorylation correlated with abrogation of binding andcoimmunoprecipitation with caAck1 (Fig. 5D , 1, lane 3). Incontrast, WwoxD5-8 associated with caAck1 and was not tyrosinephosphorylated to any great extent in caAck-expressing cells(Fig. 5D , 1 and 2 , compare lanes 3 and 7). The release of tyrosine-phosphorylated Wwox from caAck1 explains the absence of Wwoxin the myc affinity-purified complex from caAck1-expressingLNCaP cells.Activated Ack1 promotes Wwox polyubiquitination and

degradation. Recently, two groups have reported that Wwoxprotein expression was reduced or lost in 63% and 56% of invasivebreast carcinoma, respectively, suggesting that Wwox loss maycause aggressive tumors (40, 41). Similarly, loss of Wwox proteinwas observed in 65% of gastric adenocarcinoma and 33% of gastriccancer cell lines, indicating that high-grade tumors were likely toexhibit lower levels of Wwox protein or lack the expression entirely(42). We observed that caAck promotes both Wwox tyrosinephosphorylation and tumorigenesis in vivo and speculated thatactivated Ack1 might negatively regulate Wwox protein levels,promoting in vivo tumor growth. To test this supposition, weexamined Wwox ubiquitination and protein levels in HEK293T cellstransfected with myc-tagged Ack1 variants and Flag-tagged Wwoxor WwoxD5-8. Substantial Wwox polyubiquitination was observedin cells cotransfected with caAck, suggesting that Ack1-dependent

- + - + CHX Wwox 5-8

Figure 5. Activated Ack1 phosphorylates andinduces polyubiquitination of Wwox. A, lysatesof LNCaP cells stably expressing caAck variantor empty vector were incubated with anti-mycresin and eluted using myc peptide. Followingelectrophoresis, tryptic digestion of excised bandof 36 kDa, and mass spectrometry, WwoxD5-8was identified (see Supplementary Fig. S2). Thesequenced peptides of WwoxD5-8 are shown inbold. B, domain structure of Wwox andWwoxD5-8 variant. WW, WW domains; ADH,a COOH-terminal short-chain ADH domain.C, immunoblot analysis of untransfected LNCaPcells. Both Wwox and WwoxD5-8 are expressedin LNCaP cells. D, following cotransfection ofFlag-tagged-Wwox with myc-tagged-Ack1variants, HEK293T lysates were subjected toimmunoprecipitation followed by immunoblottingas indicated. HC, immunoglobulin heavy chain.E, following cotransfection of Flag-tagged-Wwoxor WwoxD5-8 with myc-tagged Ack1 variants,HEK293T cells were treated with cyclohexamide(CHX ) for 3 hours or untreated and cell lysateswere subjected to immunoprecipitation followedby immunoblotting as indicated. Wwox but notWwoxD5-8 is degraded when coexpressed withcaAck. F, polyubiquitination of endogenousWwox is mediated by activated Ack1. Followingtransfection of myc-tagged caAck1, HEK293Tand LNCaP cells were treated with MG-132 for3 hours or untreated and cell lysates weresubjected to immunoprecipitation followed byimmunoblotting as indicated.





tyrosine phosphorylation promotes Wwox polyubiquitination(Fig. 5D , 6 ). In contrast, WwoxD5-8 was neither tyrosinephosphorylated nor polyubiquitinated when coexpressed withcaAck (Fig. 5D , 6, lane 7), suggesting that tyrosine phosphorylationis a prerequisite for Wwox polyubiquitination.To investigate the potential of activated Ack1 to modulate

Wwox steady-state levels, HEK293T cells were cotransfected withmyc-tagged caAck1 and Flag-tagged Wwox or WwoxD5-8. After36 hours, cells were treated with cyclohexamide for 3 hours andlevels of Wwox were determined by immunoprecipitation followedby Western analysis (Fig. 5E). Coexpression of caAck1 resulted inreduction in Wwox protein (Fig. 5E , 1, lane 2). In contrast,WwoxD5-8 was not degraded (Fig. 5E , 1, lane 4), indicating thatactivated Ack1 mediated Wwox phosphorylation and polyubiqui-tination resulting in degradation.To ascertain whether endogenous Wwox is targeted for poly-

ubiquitination, HEK293T and LNCaP cells were transfected withcaAck and treated with proteasome inhibitor MG-132 for 3 hours.The cells were harvested and lysates were subjected to immunopre-cipitation using Wwox antibodies followed by an immunoblottingwith ubiquitin antibodies. MG-132 treatment resulted in detection ofpolyubiquitinated endogenous Wwox (Fig. 5F, lanes 2 and 4). Theseresults show that Ack1 activation results in polyubiquitination ofendogenous Wwox protein in prostate-derived cells.Ack1 tyrosine phosphorylates Wwox predominantly at

Tyr287 and not Tyr33. It has been reported that, during ‘‘cell stress,’’Wwox undergoes tyrosine phosphorylation at Tyr33, located in firstWW domain, stimulating its proapoptotic activity (39, 43). Toinvestigate whether the Ack1-dependent Wwox tyrosine phosphor-ylation occurs at Tyr33, we created a Y33F Wwox mutant (Fig. 6A)and assessed caAck-dependent tyrosine phosphorylation oncotransfection. The Y33F mutant was tyrosine phosphorylated andpolyubiquitinated by caAck1 (Fig. 6B , 1), indicating that the‘‘ubiquitination targeting’’ tyrosine phosphorylation occurs at adifferent site from the ‘‘apoptosis activating’’ Y33F tyrosinephosphorylation.

The fact that WwoxD5-8 is neither phosphorylated norpolyubiquitinated by caAck1 and has a different COOH-terminalsequence suggests that the ‘‘ubiquitination targeting’’ tyrosinephosphorylation site resides in the COOH terminus of Wwox. Sitedirected mutagenesis was done to identify tyrosine residue inWwox that is phosphorylated by activated Ack1. The COOH-terminal region of Wwox possess seven tyrosine residues; thus,seven distinct point mutants were generated, each having singleTyr-to-Phe substitution (Fig. 7A). One Wwox mutant, Y287F,exhibited the most significant loss of tyrosine phosphorylationwhen coexpressed with activated Ack1 (Fig. 7B), although tyrosinephosphorylation of Y287F mutant was not completely lost (Fig. 7B ,lane 2). This indicates that Tyr287 is the predominant but perhapsnot the only phosphorylation site.Androgen-independent prostate tumor specimens exhibit

increased tyrosine-phosphorylated Ack1 and decreased Wwox.To investigate the correlation between activated, tyrosine-phosphorylated Ack1 and Wwox levels in human prostate cancer,immunoprecipitation and immunoblotting were used to examineand compare 16 samples of primary androgen-independent prostatecancers (AICaP) and 16 samples of benign prostate. Whereas somebenign prostate specimens exhibited tyrosine-phosphorylated Ack1(Fig. 8A), AICaP had significantly higher tyrosine-phosphorylatedAck1 content per milligram of tissue lysate (Fig. 8A , 1). Densito-metric quantitation from all samples showed that AICaP and benignprostate samples had similar total Ack1, but AICaP exhibited 4- to5-fold higher levels of tyrosine-phosphorylated Ack1 (Fig. 8B). Incontrast, compared with benign prostate samples, Wwox levels werereduced significantly in AICaP (Fig. 8A , 3), suggesting thatautophosphorylated (i.e., activated) Ack1 regulates Wwox bytyrosine phosphorylation, polyubiquitination, and degradation.

Figure 6. Activated Ack1-mediated phosphorylation and degradation of Y33Fmutant of Wwox. A, domain structure of Wwox and its Y33F mutant. Positionof point mutation is indicated. B, following cotransfection of Flag-tagged-Wwoxor Y33F mutant with myc-tagged-Ack1 variants, HEK293T lysates weresubjected to immunoprecipitation followed by immunoblotting as indicated.Y33F mutant of Wwox was also phosphorylated and polyubiquitinated by caAck.

Figure 7. Activated Ack1 phosphorylates Wwox at Tyr287. A, domainstructure of Wwox and Tyr-to-Phe point mutants. Position of point mutationsis indicated. B, following cotransfection of Flag-tagged-Wwox or pointmutants with myc-tagged caAck, HEK293T lysates were subjected toimmunoprecipitation followed by immunoblotting as indicated.

Cancer Research




Discussion

Activated Ack1 accelerated prostate cancer cell tumorigenesisin vivo in part through a novel mechanism, stimulation ofpolyubiquitination and degradation of a tumor suppressor Wwox.Moreover, primary tumor specimen provided correlative evidencethat these two processes occur clinically. Questions remain, forexample, how is Ack1 stimulated in AICaP? One possibility is thatAck1 could acquire somatic mutations within its kinase domain orautoinhibitory activation loop leading to autoactivation as hasbeen recently detected for a portion of lung cancers (epidermalgrowth factor receptor and HER4) and hematopoietic tumor types(Janus-activated kinase 2). Tumor-specific point mutations in Ack1kinase domain or the autoinhibitory activation loop could play arole in AICaP progression, and we are in the process of examiningAICaP samples for Ack1 autoactivating mutations. However, evenwhen mutations are found, these may not account for the majorityof enhanced kinase activity. Therefore, other mechanisms, such asbone stromal cell Gas6-activating Mer, explain the high level ofAck1 activity in AICaP.We have observed Mer-dependent Vav2 and Vav3 tyrosine

phosphorylation.5 Because Mer does not directly phosphorylateAck1 (Fig. 2B), it may regulate autophosphorylation and activationindirectly by activating Cdc42. Additionally Ack1 could be activatedby other receptor tyrosine kinases (e.g., platelet-derived growthfactor receptor). We have shown that Mer interacts with andphosphorylates a guanine nucleotide exchange factor, Vav1,resulting in activation of Cdc42 (15). Thus, other signals, including

those from G-protein-coupled or cytokine receptors that activateCdc42 GDP-to-GTP exchange, could also regulate Ack1 activity.kdAck does not accelerate xenograft growth, and caAck1-

induced tumorigenesis is reversed with geldanamycin-mediatedkinase inhibition; both indicate that Ack1 tyrosine kinase activity isnecessary. The involvement of Hsp90h chaperone in activity oflarge number of potentially oncogenic protein kinases hasengendered substantial interest in Hsp90 as a cancer chemotherapytarget (44). Geldanamycin, a naturally occurring ansamycinantibiotic, and its clinical analogue 17-allylamino-17-demethox-ygeldanamycin (17-AAG) have significant anticancer activities.Recently, it has been shown that Hsp90h derived from tumor cellsbinds to 17-AAG up to 100 times more tightly that those isolatedfrom normal cells (45). In examining Ack1-Hsp90h complexes, wenoted increased amount of Hsp90h in caAck immunoprecipitatescompared with wAck or kdAck. This may suggests that AICaPtumors with their elevated levels of activated Ack1 might be moresensitive to 17-AAG to the extent that Ack1 is a major target of17-AAG. Hsp90h chaperone activity is required for maturation andstability of number of proteins involved in mediating prostatecancer progression (e.g., androgen receptor, HER2, Raf-1, and AKT;ref. 46). In murine models of prostate cancer, 17-AAG causesinhibition of hormone-naive and castration-resistant tumors (47).In our experiments, geldanamycin/17-AAG–mediated suppressionof tumor progression may be primarily due to kinase inactivationof Ack1 but may also involve a combinatorial effect, includingdegradation of androgen receptor, HER2, Raf-1, and AKT.Ack1 binds to a tumor suppressor Wwox and to at least one of its

splice isoforms. On Ack1 activation, Wwox is tyrosine phosphory-lated and targeted for ubiquitination-mediated degradation.This observation provides a novel mechanism by which Ack1 couldaccelerate tumorigenesis. Wwox has been found to be ‘‘underex-pressed’’ in human cancers, which could result from distinct events(e.g., allelic loss, point mutation, or promoter hypermethylation orcombination of two or more events resulting in a loss of tumorsuppressor activity), as proposed by Knudson (48). Point mutationsare rare inWwox gene (49), but LOH ofWwox allele was observed invariety of cancers, including prostate (49, 50). Our data lead us tospeculate that another ‘‘second hit’’ diminishing functional Wwoxprotein is COOH-terminal Wwox tyrosine phosphorylation at Tyr287,targeting Wwox for polyubiquitination and degradation. It isinteresting that Wwox tyrosine phosphorylation at two differentsites (Tyr33 and Tyr287) seems to have radically different outcomes,activation of apoptosis or targeting for destruction.WW domains are protein-protein interaction modules that

recognize short proline-rich motifs and have been identified invarious signaling proteins (51). Src tyrosine kinase was initiallypredicted to be involved in tyrosine phosphorylation of WWdomain containing proteins (52). Later, it was shown that Srckinase activation and stress stimuli, including UV irradiation, couldlead to Wwox phosphorylation at Tyr33, promoting cell death(39, 43). We observed ubiquitination of Wwox but not WwoxD5-8 when coexpressed with caAck. This suggests that activated Ack1-mediated COOH-terminal tyrosine phosphorylation at Tyr287 wouldoverride Wwox proapoptotic action due to degradation, thuspromoting tumorigenesis. The inverse correlation between tyro-sine-phosphorylated Ack1 and Wwox levels in AICaP reinforces theimportance of this experimental system’s observations.Watanabe et al. (53) showed accumulation of a truncated Wwox

protein of approximate molecular weight of 26 kDa by inhibitingproteasomal degradation with MG-132 treatment. The expression

Figure 8. Androgen-independent prostate cancer specimen (AICaP) exhibitelevated levels of activated Ack1 and lower levels of Wwox. A, lysates of AICaPand benign prostate (BP) were subjected to immunoprecipitation followed byimmunoblotting as indicated. AICaP exhibit increased tyrosine-phosphorylatedAck1 and decreased Wwox. B, tyrosine-phosphorylated and total Ack1 andtotal Wwox expression in AICaP and benign prostate were quantitated usingSCION Image software (NIH). Ps (Student’s t test) for differences betweenAICaP and benign prostate samples are shown.

5 N.P. Mahajan, unpublished data.





level of full-length Wwox protein did not change, suggesting thattruncated Wwox proteins may be subjected to rapid degradationthrough proteasomal machinery. Based on sizes of truncatedproteins (WwoxD6-8 is a 26-kDa protein, whereas WwoxD5-8 is36-kDa protein), it is apparent that Watanabe et al. had noticeddegradation of WwoxD6-8 protein. Absence of higher molecularweight species on inhibition of proteasomalmachinery suggests thatWwoxD6-8 may not be polyubiquitinated. In contrast, we observedthat the full-length Wwox but not WwoxD5-8 is polyubiquitinatedand degraded. This open up the possibility that whereas Wwox isregulated by polyubiquitination the truncated protein WwoxD6-8 could be degraded by an ubiquitin-independent process.Apart from targeting tumor suppressor Wwox for polyubiquiti-

nation, Ack1 may have additional mechanisms by which itaccelerates prostate tumor growth. The fact that caAck1 modestlystimulates LNCaP growth in cell culture while dramaticallyenhancing in vivo tumorigenesis indicates that the caAck1mechanism of action is not simply pro-proliferative. For example,Ack1’s role as a potential downstream effector of GTP-boundCdc42 on cytoskeletal or integrin dynamics is a possibility, as arealterations in gene expression. Recently, it has been shown that Ras

transformants, but not normal cells, are highly reliant on the Ack1kinase for both survival and growth, suggesting that Ack1 plays arole in transducing Ras signals for transformation of mammaliancells (54). The fact that Ack1 can significantly enhance prostatetumorigenesis in vivo indicates that both upstream and down-stream Ack1 signaling pathways may provide potential targets fordrug discovery in prostate cancer and paves the way for newtherapeutic strategies in advanced prostate cancer.

Acknowledgments

Received 4/4/2005; revised 7/27/2005; accepted 9/2/2005.Grant support: National Cancer Institute grants CA-77739 (J.L. Mohler), CA-85772

and CA-100700 (Y.E. Whang), and CA-81503 (H.S. Earp).The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Carol Parker and Maria Esteban-Warren (University of North CarolinaMichael Hooker Proteomics Core Facility) for help in microsequencing, NatalieEdmund and Cathy Aiken (University of North Carolina Lineberger Animal StudiesCore Facility), Debra Hunter for technical support, Ed Manser and T. Satoh for Ack1cDNAs, John Ludes-Meyers and C. Marcelo Aldaz for DWwox cDNA, Dr. C. Croce forWwox monoclonal antibodies, N.S. Chang for anti-GST-Wwox polyclonal antibodies,Lee Graves for kinase assay, Brian Varnum (Amgen Corp., Thousand Oaks, CA) forGas6, and Kiran Mahajan for helpful discussions.

Cancer Research


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2005;65:10514-10523. Cancer Res Nupam P. Mahajan, Young E. Whang, James L. Mohler, et al. Suppressor WwoxTumorigenesis: Role of Ack1 in Polyubiquitination of Tumor Activated Tyrosine Kinase Ack1 Promotes Prostate

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