The Heat Shock Protein 90 Inhibitor, 17-Allylamino-17-

demethoxygeldanamycin, Enhances Osteoclast

Formation and Potentiates Bone Metastasis

of a Human Breast Cancer Cell Line

John T. Price,1Julian M.W. Quinn,

2Natalie A. Sims,

2,5Jessica Vieusseux,

1Kelly Waldeck,

1

Susan E. Docherty,1Damian Myers,

6Akira Nakamura,

2Mark C. Waltham,

3

Matthew T. Gillespie,2and Erik W. Thompson

4,7

1Tumour Cell Migration and Metastasis Laboratory, 2Bone Joint and Cancer Laboratory, 3Pharmacogenomics Laboratory, and 4VictorianBreast Cancer Research Consortium Invasion and Metastasis Laboratory, St. Vincent’s Institute; and Departments of 5Medicine,6Physiology, and 7Surgery, University of Melbourne, Melbourne, Australia

Abstract

Breast cancer metastasis to the bone occurs frequently,causing numerous complications including severe pain,fracture, hypercalcemia, and paralysis. Despite its preva-lence and severity, few effective therapies exist. To addressthis, we examined whether the heat shock protein 90(Hsp90) inhibitor, 17-allylamino-17-demethoxygeldanamycin(17-AAG), would be efficacious in inhibiting breast cancermetastasis to bone. Utilizing the human breast cancersubline, MDA-MB-231SA, previously in vivo selected for itsenhanced ability to generate osteolytic bone lesions, wedetermined that 17-AAG potently inhibited its in vitroproliferation and migration. Moreover, 17-AAG significantlyreduced MDA-MB-231SA tumor growth in the mammary-fatpad of nude mice. Despite these findings, 17-AAG enhancedthe incidence of bone metastasis and osteolytic lesionsfollowing intracardiac inoculation in the nude mouse.Consistent with these findings, 17-AAG enhanced osteoclastformation 2- to 4-fold in mouse bone marrow/osteoblastcocultures, receptor activator of nuclear factor KB ligand(RANKL)–stimulated bone marrow, and RAW264.7 cellmodels of in vitro osteoclastogenesis. Moreover, the drugenhanced osteoclastogenesis in human cord blood progen-itor cells, demonstrating that its effects were not limitedto mouse models. In addition to 17-AAG, other Hsp90inhibitors, such as radicicol and herbimycin A, alsoenhanced osteoclastogenesis. A pro-osteolytic action of 17-AAG independent of tumor presence was also determinedin vivo , in which 17-AAG–treated tumor-naı̈ve mice hadreduced trabecular bone volume with an associatedincrease in osteoclast number. Thus, HSP90 inhibitors canstimulate osteoclast formation, which may underlie theincreased incidence of osteolysis and skeletal tumorincidence causedby 17-AAG in vivo. These data suggest animportant contraindication to the Hsp90 targeted cancertherapy currently undergoing clinical trial. (Cancer Res 2005;65(11): 4929-38)

Introduction

The dissemination of tumor cells from their primary site ofgrowth to distant organs is the major cause of morbidity and deathamong cancer patients (1, 2). It is well recognized that ratherthan being a random process, different cancer types display apredilection for metastasis to particular organs (3). In the case ofbreast cancer, the great majority of patients with advanced diseasedevelop osteolytic metastases (4). This aspect of breast cancercauses a number of major complications for patients, includingsevere pain, pathologic fractures, hypercalcemia, and paralysis dueto nerve compression (4–6). Moreover, once tumors have spread tothe bone, they frequently do not respond to therapy, with only 20%of breast cancer patients surviving for 5 years after the discovery ofbone metastasis (7).The growth of metastases at distant sites is known to depend

upon interactions between tumor cells and the host microenvi-ronment (8). It is unknown why breast cancer cells preferentiallymetastasize to bone; however, it is known that the complexinterplay between tumor cells and the bone microenvironmentplays an important role (5, 9). Although breast cancer metastasescan grow in the marrow cavity, the tumors normally promote thedestruction and invasion of the bone tissue itself, whichcompromises the structural integrity of the bone. To achieve this,the invading tumors recruit osteoclasts, highly specialized boneresorbing cells that form rapidly from myelomonocytic progenitorspresent locally in large numbers in bone marrow and thecirculation. This bone destruction itself can release factors thatencourage tumor growth (4, 10). The role of the osteoclast in bonemetastasis has led to the use of agents that target bone resorption,such as bisphosphonates, in the treatment of patients with bonemetastasis (11). However, although the bisphosphonates haveprovided promising results (4, 12), these treatments are oftenpalliative and do not provide substantial life-prolonging benefits topatients suffering from bone metastasis (11, 12). Therefore, there isa need to identify new approaches that would provide usefuladjunct therapies for the treatment of cancers that metastasizeto bone.Heat shock protein 90 (Hsp90) is a molecular chaperone that is

ubiquitously and abundantly expressed. Numerous proteinsinvolved in the control of physiologic and pathophysiologic pro-cesses require Hsp90 for their biogenesis, regulation, and func-tionality (13, 14). In cancer, the expression of Hsp90 is increasedwhen compared with that of normal tissues. Moreover, withmany of its ‘‘client’’ proteins, such as Akt, Her2/Neu, and Raf-1,

Note: J. Price and J. Quinn contributed equally to this work.Requests for reprints: John Price, Tumour Cell Migration and Metastasis

Laboratory, St. Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy,3065 Melbourne, Victoria, Australia. Phone: 61-3-9288-2480; Fax: 61-3-9416-2676;E-mail: jprice@svi.edu.au.

I2005 American Association for Cancer Research.

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important participants in pathways driving tumor cell survival,proliferation, and progression, Hsp90 is believed to be an excellentmolecular target in cancer therapy (15–17). Moreover, a number ofHsp90 clients, such as c-Src, epidermal growth factor receptor, andmatrix metalloproteinase-2 (14, 18), have been shown experimen-tally to play a role in bone metastasis, suggesting that Hsp90inhibition may not only prove effective in primary tumor growthbut could also be an effective inhibitor of bone metastasis (19–22).In addition, we have recently identified that Hsp90 expressioncorrelates with bone metastasis in an in vivo mouse model (23).Naturally occurring compounds that inhibit Hsp90 action,

such as geldanamycin and radicicol, have provided evidence ofthe importance of Hsp90 in cancer cell growth and progression(24–26). These compounds bind to the ATP/ADP binding pocket inthe NH2-terminal domain with high affinity, inhibiting Hsp90, andthus are potent inhibitors of cancer cell growth and the malignantphenotype in a number of cancer cell types, including breast(27–29). In addition, analogue compounds, such as the geldana-mycin derivative 17 allylamino-17-demethoxygeldanamycin (17-AAG), have been shown to possess antitumor activity in severalhuman xenograft models, including colon, breast, and prostatecancer (30–32) and have been used in patient clinical trials(15, 25, 33). 17-AAG has completed multi-institution phase I clinicaltrials in which different schedules of drug administration havebeen examined and phase II trials have commenced (17, 34).The aim of this study was to investigate whether pharmacologic

inhibition of Hsp90 by the geldanamycin analogue, 17-AAG, wouldbe effective in inhibiting the incidence and growth of osteolyticbone metastasis in an animal model of experimental bonemetastasis using the human breast cancer subline, MDA-MB-231SA.

Materials and Methods

Drugs and reagents. 17-AAG was kindly provided by Dr. E. Sausville

(Drug Synthesis and Chemistry Branch, Developmental Therapeutics

Program, National Cancer Institute, Bethesda, MD); geldanamycin, radicicol,

herbimycin A, human collagen IV, and prostaglandin E2 (PGE2) wereobtained from Sigma Chemical, Co. (Castle Hill, NSW, Australia). Collagen I

(Vitrogen 100) was obtained from Cohesion (Palo Alto, CA) and the growth

factors insulin-like growth factor I (IGF-I) and epidermal growth factor(EGF) were obtained from Becton Dickinson (Bedford, MA). Antibodies

toward Hsp90a and Hsp90h were purchased from Stressgen (San Diego,

CA) and antibodies to actin were purchased from Neomarkers (Freemont,

CA). Recombinant murine GST-RANKL158-316 (receptor activator of nuclearfactor nB ligand, RANKL) was produced in Escherichia coli BL21 cells using

a protein expression construct kindly provided by Prof. F. Patrick Ross

(Washington University School of Medicine, St. Louis, MO). Human

macrophage colony stimulating factor (M-CSF) was obtained from R&DSystems (Minneapolis, MN), whereas 1,25(OH)2 vitamin D3 [1,25(OH)2D3]

was purchased from Wako Pure Chemical, Co. (Osaka, Japan). The C57BL/6

and BALB/c-nu/nu (nude) mice strains were obtained from Animal

Resource Centre (Perth, WA, Australia).Cell lines. The estrogen-independent human breast cancer cell subline,

MDA-MB-231SA, was kindly provided by T. Yoneda (University of Texas

Health Science Center at San Antonio, San Antonio, TX). These cells hadbeen previously generated from MDA-MB-231 cells by intracardiac

inoculation and in vivo selection of cells displaying the ability to spread

and grow in bone. The MDA-MB-231SA cells were isolated after being in vivo

selected by passaging and reselecting seven times through nude mice asdescribed previously (35) and displayed an enhanced onset and severity of

bone metastasis. MDA-MB-231SA cells were cultured routinely in DME

with 10% fetal bovine serum (FBS, JHR Biosciences, Lenexa, KS). MDA-

MB-231SArfp (red fluorescent protein, RFP) cells were generated through

stable transfection of the pDsRed2-N1 construct (Clontech). Briefly,pDsRed2-N1 was transfected into MDA-MB-231SA using LipofectAMINE

2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruc-

tions. Twenty-four hours after transfection, RFP-positive cells were isolated

by fluorescence-activated cell sorting (FACS, DIVA, Becton Dickinson) andgrown in culture with G-418 selection (0.75 mg/mL). After 2 weeks of

selection, RFP-positive cells were once again sorted by FACS analysis and

cells expressing the highest levels of RFP (10% of the total population) were

retained. Cells were selected for a further 2 weeks in G-418 (Invitrogen) afterwhich time a final round of FACS-based cell sorting was done in which 10%

of the highest RFP-expressing cells were selected. The resulting cells were

termed MDA-MB-231SArfp.

RAW264.7 cells and mouse primary osteoblasts were cultured in a

minimal essential medium (Life Technologies, Gaithersburg, MD) contain-

ing 10% FBS (CSL Biosciences, Parkville, Australia; MEM/FBS). All

osteoclast formation assays utilized this medium. RPMI 1640 (LifeTechnologies) containing 10% heat-inactivated FBS (RPMI/HIFBS) was

used for the preparation of bone marrow macrophages (BMM) as indicated.

Western blot analysis. Cell lysates were prepared in modified

radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 7.4), 1%

NP40, 0.25% Na deoxycholate, 150 mmol/L NaCl] with EDTA free Complete

Protease Inhibitors (Roche, Kew, Victoria, Australia). Resulting cell lysates

were sonicated and protein concentrations determined by BCA protein

assay following the manufacturer’s protocol (Pierce, Rockford, IL). Cell

lysates were electrophoresed on a 10% SDS-PAGE gel under reducing

conditions. Proteins were transferred to polyvinylidene difluoride mem-

branes and probed with appropriate antibodies and incubated with

horseradish peroxidase–conjugated secondary antibodies. Western blots

were visualized by ECL detection system according to manufacturer’s

instructions.Tumor cell proliferation analysis. Cell growth was determined by the

sulforhodamine B assay in a 96-well format as previously described by

Skehan et al. (36).

Tumor cell migration analysis. Cell migration was determined using a

48-well microchemotaxis chamber assay using collagen type I–coated 8 Ampolycarbonate membranes (Neuroprobe, Gaithersburg, MD) as previously

described (37). Briefly, IGF-I and EGF were used as chemoattractants at the

concentrations indicated and added to the lower wells. Cells (1 � 106/mL)

were resuspended in serum-free DME containing 0.1% bovine serum

albumin (DME/BSA) and added to the upper chambers (56 AL/well).Chambers were incubated at 37jC in a humidified incubator in an

atmosphere of 5% CO2/95% air for 4 hours, after which the filters were

removed, fixed, and stained with Diff-Quik (Baxter Scientific, McGaw

Park, IL) and mounted on glass slides. Nonmigrated cells were removed by

wiping with a cotton swab. At least four random fields per well (�20

objective) were counted for quantitation of cell migration. Triplicate wells

were done in each assay and the assay was repeated at least thrice.Intracardiac inoculation model. Intracardiac injection of the MDA-

MB-231, MDA-MB-231SA, and MDA-MB-231SArfp cells was done as

previously described (22, 38). Briefly, subconfluent cells were fed with freshmedia 24 hours before intracardiac injections. Cells were removed

nonenzymatically (PBS-EDTA), resuspended in DME/10% FBS medium,

and were then washed thrice with PBS and resuspended in PBS at

106 cells/mL. Mice (4-5-week-old female BALB/c-nu/nu ; Animal ResourceCentre) were anesthetized with ketamine (40 mg/kg/mouse)/xylazine

(16 mg/kg/mouse) and 105 cells were inoculated into the left heart ventricle

using a 27-gauge needle. Mice were allowed to recover on a heat pad, then

returned to their cages and maintained under pathogen-free conditions.Mouse health was monitored daily and mice were sacrificed at the first

signs of discomfort or after 4 to 6 weeks. Animals were weighed twice

weekly. All animal experiments were conducted with full approval of theSt. Vincent’s Hospital Animal Ethics Committee and in accordance with the

National Health and Medical Research Council (Australia) and NIH

(United States) Guidelines for the Care and Use of Laboratory Animals.

Treatment schedules. 17-AAG was reconstituted in 10% DMSO/0.05%Tween 80 in PBS. Mice were treated on a 4-day treatment/3-day rest

schedule with the daily dose of 70 mg/kg/mouse being given as a split dose

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of 35 mg/kg/mouse separated by a period of 8 hours. Administration of thedrug was by i.p. injection and drug treatments were commenced 1 day after

intracardiac or mammary-fat pad inoculation of tumor cells.

Analysis of bone metastases. Radiographic analysis and fluorescent

imaging were used to determine tumor incidence in the bones of mice. For

radiographic analysis, animals were anesthetized, laid in the prone position

against film (22 � 27 cm; X-OMAT AR; Eastman Kodak, Co., Rochester, NY),

and exposed to an X-ray at 35 kV for 10 seconds using a Faxitron instrument

(Model MX-20-20 Am focal source; Faxitron, Corp., Buffalo, IL). Films were

developed and inspected for visible bone lesions. For fluorescent imaging, at

the time of harvest, culled mice were skinned and soft organs removed to

enhance fluorescent sensitivity. The mouse was then imaged under

fluorescence using the LAS1000plus instrument (Fujifilm Scientific, Tokyo,

Japan) using excitation-dominant wavelength of 530 nm and green emission

filter of 585LP. Images were captured using Science Lab 99 software

(Fujifilm Scientific).

Mammary-fat pad inoculation. Groups of 8 to 10 mice (4-5-week-old

female BALB/c-nu/nu ; Animal Resource Centre) received mammary-fat padinoculation of MDA-MB-231SA cells in a mixture (1:1) of PBS and Matrigel

(1.5 � 106 cells/15 AL) as previously described (36). Tumor growth was

assessed by measuring the length and width of tumors with electronic

calipers every 3 to 4 days continuously after the tumor became palpable.Volumes were calculated using the formula (length) � (width)2 / 2, where

the length was determined as the larger measurement. Mice were sacrificed

when tumors approached 1,500 mm3.

Murine osteoclast formation assays. Bone marrow cells were flushedfrom bisected long bones of C57BL/6 mice with PBS using a 10 mL syringe;

cells were centrifuged and resuspended (106 cells/mL) in MEM/FBS. For

BMM preparation, bone marrow cells (106 cells/mL) were suspended inRPMI/HIFBS supplemented with 30% L-cell–conditioned medium (an

impure source of secreted murine M-CSF prepared as described in ref. 39)

and incubated at 37jC for 3 days after which time the nonadherent cell

fraction was removed, centrifuged, and resuspended in MEM/FBS. Primaryosteoblasts were prepared by sequential collagenase digestion of neonatal

mouse calvaria as previously described (40).

All osteoclast assays were done in 10-mm-diameter tissue culture wells.

Primary osteoblasts (4 � 104 cells/well) were cocultured with bone marrow

cells (105 cells/well) in the presence of 10 nmol/L 1,25(OH)2D3 and

100 nmol/L PGE2 in the presence or absence of 17-AAG or vehicle for

7 days. Cells were fixed and histochemically stained for tartrate-resistant

acid phosphatase (TRAP) as described previously (41). TRAP-positive cells

with three or more nuclei were counted as osteoclasts. Osteoclasts were

also generated from bone marrow cells (105 cells/well) in the absence of

osteoblasts using stimulation by 100 ng/mL RANKL and 25 ng/mL M-CSF

(with or without 17-AAG or vehicle) for 7 days. Similarly, osteoclasts were

generated from RANKL- and M-CSF–stimulated BMMs (105 cells/well).

Experiments were done four times, each with five replicate cultures per

experiment. Final representations of results were pooled data from the

four individual experiments.

Human osteoclast formation assays. Human cord blood–derived

progenitors were stimulated to form osteoclasts as previously described(42). Briefly, cord blood mononuclear cells were cultured in semisolid

medium containing interleukin (IL)-3, granulocyte M-CSF, and stem cell

factor (42) for 7 days. The resulting expanded progenitors were cultured in

6-mm-diameter wells (4 � 104 cells/well) in MEM/FBS with RANKL andM-CSF (with or without 17-AAG or vehicle) and TRAP-positive multinu-

cleated (>3 nuclei) osteoclasts were counted at day 7.

Human cord blood was obtained in accordance with full ethical approvalof the human ethics committee of the University of Melbourne. Experiments

were done thrice, each with five replicate cultures per experiment. Final

representations of results were pooled data from the three individual

experiments.Histologic analysis. Hind limbs were fixed in 10% neutral buffered

formalin for 48 hours, decalcified in EDTA (pH 7.2) for 2 weeks, and

processed for conventional paraffin-embedded H&E staining.

Histomorphometric analysis of long bones. Tibial specimens werefixed in 4% paraformaldehyde in PBS and embedded in methylmethacrylate

as described previously (43). Undecalcified 5 Am sections of the proximaltibial were stained with toluidine blue and the secondary spongiosa was

analyzed by histomorphometry according to standard procedures using the

Osteomeasure system (OsteoMetrics, Inc, Decatur, GA; ref. 43).

Statistical analyses. Results are represented as mean F SD unlessotherwise stated. m2 analysis and Fisher’s exact probability test were used

for tumor incidence, whereas the significance of MFP growth was

determined by two-way repeated measures ANOVA (GraphPad Prism, San

Diego, CA). Cell migration and osteoclast generation assays were analyzedby Student’s t test.

Results

Selection of MDA-MB-231 cells enhances incidence of bonelesions in the intracardiac inoculation nude mouse model. Tostudy bone metastasis (i.e., the seeding and growth of tumor cellsin the bone microenvironment), the intracardiac inoculation modelusing BALB/c-nu/nu (nude) mice was utilized (22). This method-ology directly seeds cells into the arterial circulation of micethrough inoculation into the left ventricle, thus allowing the stagesof metastasis postintravasation to be examined. Although thismodel results in bone lesions, seeding to visceral organs is alsoachieved (21). However, to examine bone metastasis more precisely,we obtained the human breast cancer cell subline, MDA-MB-231SA(T. Yoneda). This line, previously generated by serial in vivo passageof MDA-MB-231 cells through the bones of nude mice, displays ahigher incidence, severity, and specificity for bone metastasis. Inour hands, when MDA-MB-231SA cells were compared with astandard MDA MB 231 cell line in the intracardiac inoculationmodel, the MDA-MB-231SA displayed a bone lesion incidence ofbetween 90% and 100% as determined by hind limb X-ray analysisafter 4 weeks (Fig. 1A). In contrast, parental MDA-MB-231 cellsonly displayed a 20% to 30% incidence of bone metastasis after asimilar period of time (Fig. 1A). Moreover, bone lesions generatedby the MDA-MB-231SA cells were much larger in size whencompared with those produced by MDA-MB-231 cells (Fig. 1B).Previously, we have identified two subclones of the MDA-MB-231cell line, namely, MDA-MB-231#16 and MDA-MB-231#17, whichhave a high and low bone metastatic potential in the intracardiacinoculation model, respectively. Gene array profiling of these twosubclones and subsequent postarray validation by Western blotanalysis (Fig. 1C) showed that Hsp90h was expressed at higherlevels in the highly bone metastatic MDA-MB-231#16 subclone (23).Therefore, we examined whether Hsp90 protein levels would behigher in the highly bone metastatic MDA-MB-231SA cells whencompared with the parental cell line. Western blot analysis showedthat Hsp90a and Hsp90h isoforms were both found to be expressedat higher levels in the MDA-MB-231SA cells (Fig. 1C), recapitulatingour findings within the MDA-MB-231#16 and MDA-MB-231#17model.17-Allylamino-17-demethoxygeldanamycin treatment

inhibits MDA-MB-231SA proliferation and chemotactic mi-gration. To determine whether the MDA-MB-231SA cells weresensitive to Hsp90 inhibition by 17-AAG with respect to theirgrowth, we examined the effects of varying concentrations of thedrug in an adhesion-dependent proliferation assay (36). At lowerconcentrations of 17-AAG (0.01 and 0.1 Amol/L), there was nodetectable effect upon the proliferation of the MDA-MB-231SAcells (Fig. 2A). However, when the concentration of the drug wasincreased to 1.0 Amol/L, a significant reduction in the rate ofcellular growth was observed (Fig. 2A). In addition to 17-AAG,the Hsp90 inhibitors, radicicol and herbimycin A, were also

17-AAG Potentiates Bone Metastasis

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effective in inhibiting growth of the MDA-MB-231SA cells (datanot shown). In addition to the MDA-MB-231SA cell line, we havealso found that other human breast cancer lines, namely MDA-MB-231, MCF-7, MDA-MB-435, and BT-474 cells, are also growthinhibited by 17-AAG (data not shown). In addition to growth, wealso examined the effect of Hsp90 inhibition upon anotherimportant parameter of tumor metastasis, that of cellularmigration (44). MDA-MB-231 cells have been previously shownto migrate toward a number of growth factors and cytokines(37, 45). Using a standard microchemotaxis assay, we examinedthe effect of 17-AAG on MDA-MB-231SA cell migration towardEGF and IGF-I. IGF-I–induced chemomigration of MDA-MB-231SAwas significantly decreased at all concentrations of 17-AAG,while only the highest concentration of 17-AAG was effective atinhibiting EGF-induced or the random background migration of

the cells (Fig. 2B). MDA-MB-231 cells have been shown to besensitive to effects of Hsp90 inhibition upon the IGF-I signalingpathways with reduction of IRS-1 and IRS-2 phosphorylation andthe degradation of the IGF-I type I receptor (46). Therefore, 17-AAG is effective in inhibiting MDA-MB-231SA cell proliferation andIGF-I– and EGF-induced migration in vitro .17-Allylamino-17-demethoxygeldanamycin treatment

enhances bone metastasis to the long bones as determinedby X-ray analysis. Although it is well accepted that 17-AAG is apotent inhibitor of xenograft tumor growth in a number ofmodels, including those of breast, prostate, and colon cancers(30–32), the efficacy of the drug has yet to be assessed for itsability to reduce tumor spread to and growth in bone. Toaddress this, we used the intracardiac inoculation model,administering 17-AAG (70 mg/kg/mouse) to the mice as a splitdose on a 4-day treatment, 3-day rest schedule commencing theday after the inoculation of tumor cells. Similar dose schedulesas well as higher dose schedules of 17-AAG have been previouslyused to show 17-AAG efficacy in breast, prostate, melanona, andcolon tumor models (30–32, 47). Moreover, in our hands, thisschedule is well tolerated in nontumor and mammary-fat padtumor-bearing mice, being below the maximum tolerated dose of80 mg/kg/d (48).Contrary to our expectations, mice treated with 17-AAG

displayed a 2-fold increase in bone lesion incidence after 21 dayswhen compared with the vehicle control group (Fig. 2C). Due to thegeneral poor health and weight loss of the mice in the 17-AAGtreatment group, the experiment was terminated at this point,10 to 14 days earlier than typical. No gross histomorphologicdifferences were observed in the tumors of treated and nontreatedgroups (Fig. 2D).17-Allylamino-17-demethoxygeldanamycin enhances spread

to skeletal sites other than the long bones of nude mice. Toenhance our ability to observe tumor cell spread to other sites ofthe skeleton that are generally more difficult to detect by X-rayanalysis alone, we generated a stably transfected cell line from theMDA-MB-231SA cells that constitutively expressed RFP allowingthe in vivo fluorescent imaging of tumors. The MDA-MB-231SArfpcells were generated in a nonclonal manner and displayed similarmorphologic and growth characteristics as the parental MDA-MB-231SA cells (Fig. 3A and B). These cells were also sensitive to 17-AAG with respect to their in vitro proliferation (data not shown).Intracardiac inoculation of these cells into nude mice, which weresubsequently treated with 17-AAG as previously described, resultedin enhanced bone metastasis as observed in the MDA-MB-231SAcells (Fig. 3C). Once again, the experiment had to be terminatedafter 21 days because of the poor health of the animals.The use of full body fluorescent imaging showed the presence of

the tumor cells not only in the long bones of the mice but also inthe jaw, spine, and skull (Fig. 4). In addition, examination of tumorincidence in the skeleton by fluorescent imaging revealed asignificant increase in the incidence of metastasis in the longbones and in the jaws of the mice (Fig. 4), the latter providing apossible explanation for the weight loss in the 17-AAG–treatedmice. Although there was only a trend to an increased incidence ofbone metastases in the spine, the spinal lesions detected weremuch larger in size (Fig. 4).17-Allylamino-17-demethoxygeldanamycin reduces mam-

mary-fat pad MDA-MB-231SArfp tumor growth in nude mice.To determine if 17-AAG inhibited MDA-MB-231SA tumor cell

growth at the orthotopic site, MDA-MB-231SArfp cells were

Figure 1. In vivo selection of the MDA-MB-231 cell line enhances the bonemetastatic incidence of the cells. A, incidence of bone metastasis in theMDA-MB-231SA subline is enhanced over 28 days. Hind limb X-ray analysis wasdone at 28 days after tumor cell inoculation. B, severity of lesions generatedby the MDA-MB-231SA subline was significantly greater than that of theMDA-MB-231 cell line. Osteolytic bone lesions were observed as opaque regionson the X-ray image (arrows ). C, Western blot analysis of total cell lysates (5 Ag)of MDA-MB-231#17 (lane 1), MDA-MB-231#16 (lane 2 ), MDA-MB-231 (lane 3),and MDA-MB-231SA (lane 4 ) demonstrating protein levels of Hsp90a andHsp90h in each of the cell types. Actin (pan) was used as a loading control.Increased expression of Hsp90h correlates with enhanced bone metastaticpropensity in vivo .

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inoculated into the mammary-fat pads of nude mice and treatedwith 17-AAG. As has been previously shown in breast and prostatexenograft models (31, 32), 17-AAG significantly inhibited thegrowth of tumors at the mammary-fat pad (Fig. 4H). Therefore,depending upon the microenvironment, 17-AAG had differentialeffects.17-Allylamino-17-demethoxygeldanamycin enhances in vitro

osteoclastogenesis. To colonize bone, tumors recruit osteoclasts,which are formed from local hemopoietic progenitors. In addition,it has previously been shown that resorption of bone by osteoclastsis important in the process of bone metastasis in the intracardiacinoculation model (49). Therefore, we initially determined whether17-AAG would affect osteoclastogenesis in vitro . Treatment ofosteoblasts with osteolytic factors, such as 1,25(OH)2D3, showenhanced expression of RANKL and decreased expression ofosteoprotegerin, permitting osteoclast formation (50). We thusexamined the effect of 17-AAG upon osteoclastogenesis in acoculture assay in which a primary calvarial osteoblast feeder layerand the addition of PGE2 and 1,25(OH)2D3 provided the stimulusfor osteoclastogensis from bone marrow cells. Treatment of thesecocultures with 17-AAG significantly enhanced the number ofosteoclasts after 7 days, although at concentrations higher than100 nmol/L, 17-AAG resulted in no osteoclast formation due to itscellular toxicity (Fig. 5A). Whereas this data indicates that 17-AAGcan enhance osteoclastogenesis, it does not define whether this isvia a direct action upon the osteoclast progenitors or by an indirectmechanism through the osteoblasts. Thus, bone marrow cells werestimulated with recombinant RANKL and M-CSF to generateosteoclasts over a 7-day period. 17-AAG treatment of these culturesresulted in a significant increase in osteoclast numbers over theperiod of the assay (Fig. 5B). Once again, concentrations of 17-AAG>100 nmol/L resulted in cellular toxicity and decreased the number

of osteoclasts generated. 17-AAG also increased osteoclastformation in RANKL- and M-CSF–stimulated BMM, which are ahighly enriched population of osteoclast/macrophage progenitors(Fig. 5C). Moreover, the osteoclast formation of the RAW264.7 cellline when these were utilized as the osteoclast progenitor cells wasalso increased by 17-AAG (data not shown). We have determinedthat 17-AAG has maximal activity upon osteoclast formationwhen present over the entire culture period of 7 days. However,when cultures were treated with 17-AAG for 0 to 3 or 4 to 7 days,increases in osteoclast numbers were noted although a submax-imal effect was observed (data not shown).Therefore, although this does not rule out an indirect action of

17-AAG acting through the osteoblast, it does confirm that 17-AAGcan act directly upon osteoclast progenitors to enhance osteoclas-togenesis. To rule out a nonspecific mechanism by whichosteoclastogenesis is enhanced before cell death, cultures weretreated with a number of metabolic inhibitors, proteosomalinhibitors, and rapamycin, each of which resulted in inhibition ofosteoclastogenesis and, at higher concentrations, cell death. Noneof these agents were observed to increase osteoclastogenesis beforecellular toxicity (data not shown). Moreover, use of other Hsp90inhibitors, herbimycin A (Fig. 5D), or radicicol (data not shown),also resulted in enhanced osteoclastogenesis in RANKL/M-CSFBMM cultures, demonstrating that this effect was not specific to17-AAG but a more general phenomenon of Hsp90 inhibitors. Itwas also observed that these inhibitors not only enhancedosteoclast numbers but also enhanced osteoclast fusion, increasingthe nuclear number and size of the osteoclasts (Fig. 5E). Thus, theeffect of the drugs on osteoclast numbers may be greater thansuggested by the osteoclast numbers alone.We next evaluated whether the actions of 17-AAG were limited

to mouse models of osteoclastogenesis or whether the drug had the

Figure 2. MDA-MB-231SA subline proliferation andchemotactic migration are inhibited by 17-AAG. Effects of17-AAG (0.01-1.0 Amol/L) upon cell growth andchemotactic migration were analyzed byadhesion-dependent growth assay and microchemotaxisassay, respectively. A, significant inhibition of cell growthwas observed at 1.0 Amol/L 17-AAG over a 6-day period;cellular growth was unaffected at other concentrations of17-AAG. B, significant inhibition of chemotactic cellmigration toward IGF-I (10 ng/mL) was observed at allconcentrations of 17-AAG tested. However, thebackground migration (0.1% BSA) and migration towardEGF (10 ng/mL) were only significantly affected at1.0 Amol/L 17-AAG. Cells were preincubated for 20 to 24hours with 17-AAG before cell migration analysis; viablecell counts were done before seeding into the 4-hourmicrochemotaxis assay, ensuring cell death did not have amajor influence upon the assay. Columns, mean; bars,SD. Statistical significance was determined using theStudent’s t test; ***P < 0.001, **P < 0.01; *P < 0.05.17-AAG treatment of mice intracardially inoculated withMDA-MB-231SA cells. X-ray analysis of the hind limbs ofmice was done at 21 days. C, a significant increase in theincidence of bone metastasis was observed in the 17-AAGtreatment group as determined by m2 analysis; *P < 0.05.D, H&E staining of hind limbs showed no gross changes intumor morphology. B, bone, TC, tumor cells.

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potential to enhance the generation of osteoclasts in a humanmodel. Human cord blood–derived osteoclast progenitors werecultured in the presence of recombinant RANKL and M-CSF, withand without 17-AAG. As observed in the mouse models ofosteoclastogenesis, 17-AAG significantly enhanced the number ofhuman osteoclasts generated over the 7-day period (Fig. 5F).Hence, the effects of 17-AAG upon osteoclastogenesis were notlimited to mouse models but also translated to human models ofosteoclastogenesis. Although the stimulation of osteoclast forma-tion was not as striking as that observed in the mouse cells, thismay be due to the greater toxicity of 17-AAG in these cells as thedrug was observed to be toxic at concentrations above 30 nmol/Lwith extensive cell death being observed at 100 nmol/L 17-AAG.17-Allylamino-17-demethoxygeldanamycin treatment

enhances osteoclast numbers and decreases bone massin vivo . To identify the effects of 17-AAG on the bonemicroenvironment in the absence of tumor cells, nude mice were

treated with 17-AAG with no prior inoculation of tumor cells,using the same treatment regime of 17-AAG as in tumor-challenged mice. Despite the already low bone mass of thesemice (Fig. 6A), histomorphometric analysis of the long bones after21 days of treatment showed a significant reduction in thetrabecular bone volume (Fig 6A). However, the very low bone massin these mice meant that osteoclast and osteoblast numbers couldnot be properly analyzed. For this reason, and because of theimmunocompromised nature of the nude mice, we examined theeffects of 17-AAG upon the C57BL/6 mouse strain. As observed inthe nude mice, the trabecular bone volume was significantlydecreased in the C57BL/6 mice (Fig. 6A). Moreover, due to thehigher bone mass in the untreated C57BL/6 mice relative to thenude mice, more meaningful data could be obtained with respectto osteoblast and osteoclast numbers. No difference was observedin osteoblast numbers between the 17-AAG– and vehicle-treatedgroups (Fig. 6C ); however, as observed in vitro , 17-AAGsignificantly enhanced the number of osteoclasts (Fig. 6B).Therefore, in the absence of tumor cells, 17-AAG treatmentresulted in loss of bone volume with a concomitant increase inosteoclast numbers.

Discussion

The identification of several naturally occurring anticancerantibiotics, including geldanamycin, radicicol, and herbimycin A,which selectively inhibit the function of the molecular chaperoneHsp90, has led to the candidature of this molecule as a noveltarget for cancer drug therapy. These agents and derivatives haveproven highly effective at inhibiting the growth of xenograftmodels of colon, breast, and prostate cancer (30–32), as well asenhancing conventional therapies, such as Taxol, when used incombination (33).However, it is the ability of a tumor cell to metastasize, rather

than the growth of the primary tumor, that is the major cause oftreatment failure, morbidity, and death in breast cancer patients.Despite this, the majority of preclinical investigations haveexamined the effects of 17-AAG upon tumor growth in subcuta-neous or orthotopic models, with little being done to investigate itsefficacy in the inhibition of tumor cell metastasis. To address this,we utilized an intracardiac inoculation model of tumor spread,focussing on metastasis and invasion of the bone, the mostcommon and often the earliest site of breast cancer spread.Moreover, the bone is a tissue that represents a very differentmicroenvironment to that of the primary tumor site, often allowingtumors to be refractory to conventional therapies (7, 51).Our initial in vitro investigations of the effects of 17-AAG upon

the proliferation and chemotactic migration of MDA-MB-231SAcells are entirely consistent with previous findings in which 17-AAG inhibited proliferation and reduced IGF-I–mediated signalingin MDA-MB-231 cells (46, 53). Additionally, our observation that17-AAG inhibits the growth of MDA-MB-231SArfp tumors at theorthotopic site are in accordance with findings in other breastcancer models of tumor growth (27, 28). However, in contrast tothese very positive indications, we have made the surprisingobservation that 17-AAG increases the incidence of osteolytic bonemetastases and, consistent with this, had the ability to enhance theformation of osteoclasts, the specialized bone-resorbing cellsessential for the destruction of bone necessary for tumorcolonization. Furthermore, and consistent with this pattern of17-AAG osteolytic action we observed, 17-AAG caused an increase

Figure 3. MDA-MB-231SA cells were engineered to constitutively expressRFP (rfp ). A, phase contrast microscopy showed no morphologic changes inthe MDA-MB-231SArfp when compared with parental MDA-MB-231SA cells.B, in addition, no significant alteration in the in vitro proliferation of theMDA-MB-231SArfp cells was identified as determined by an adhesion-dependent cell growth assay. C, X-ray analysis of the hind limbs of mice wasdone at 21 days postinoculation of MDA-MB-231SArfp cells. 17-AAG treatmentof mice inoculated with the MDA-MB-231SArfp cells showed a significantincrease in hind limb metastasis as determined by X-ray analysis. Significancewas determined by m2 analysis.

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in osteoclast numbers in both nude and C57BL/6 mice in theabsence of any tumor challenge. As a result, 17-AAG reduced thebone mass in these mice, an osteopenic effect that in itself couldpose a significant problem for patients undergoing therapy with17-AAG, regardless of the presence or progression of their cancer.This risk may not be limited to 17-AAG, as enhanced osteoclastformation, albeit in vitro , was observed with herbimycin A and thestructurally unrelated Hsp90 inhibitor, radicicol, suggesting thatthe osteopenic effect may be a more general phenomenon ofHsp90 inhibitors.In the osteolytic metastasis that are typically associated with

breast cancer, it is believed that complex cellular interactions occurwithin the bone. Tumor cells release factors, such as parathyroidhormone–related protein, that stimulate the local mesenchymalcells of bone (osteoblasts) to produce RANKL, a tumor necrosisfactor-related molecule required for osteoclast formation andactivation (4). The resulting bone resorption by the osteoclastrelease factors, such as transforming growth factor h (TGFh) andIGF-I, which can further stimulate tumor growth, forming apositive feedback loop or ‘‘vicious cycle’’ of local pro-osteolytic andtumor proliferation stimuli (4). It is not clear whether thismechanism is important in the establishment of tumor metastasesin all models or that it is critical in the human disease, and indeedthere is emerging evidence of the importance of other tumor-derived factors such as IL-8, IL-11, and connective tissue growthfactor (52, 53). However, this and other evidence suggest that notonly are the bone and bone marrow a conducive microenviron-ment or ‘‘soil’’ for metastatic breast cancer growth, but that boneundergoing high turnover or breakdown may be particularlypermissive to tumor establishment and growth (48). Therefore,

our finding that 17-AAG reduces bone mass in mice in the absenceof tumor invasion may indicate that, in addition to any effects onthe behavior of the tumor cells themselves, 17-AAG can act as apro-osteolytic factor, enhancing the capability of tumor cells toestablish and grow in the bone.Although the precise mechanism of the reduction in bone mass

after 17-AAG administration (even in the absence of tumorchallenge) is difficult to determine, the in vitro data suggests thatit may be the result of enhanced osteoclast differentiation and/orsurvival. The increased numbers of osteoclasts in the absence of anaccompanying increase in osteoblast numbers in vivo indicate thatthis is indeed likely to be the major cause of the lower bone mass inthese mice. Moreover, because this was evident in nude mice,which already have low bone mass, it suggests that 17-AAG haspotent effects upon the osteoclast lineage and that this is notdependent on the presence or absence of lymphocytes.To date, the in vivo effects of 17-AAG in the tumor models and

the tumor naı̈ve mice have only been examined at a single doseusing a single treatment schedule. Designed to observe maximalbenefit of the drug, it was, therefore, surprising to see a strongenhancement of bone metastasis at a drug concentration close tothe maximal tolerated dose. Further dosing and treatmentschedules will be required to further define the potential severityof the effects of 17-AAG upon the bone microenvironment and itsimpact upon tumor growth.Our studies in vitro are in strong accord with our in vivo findings.

Bone marrow cells (containing the hematopoietic progenitors fromwhich osteoclasts derive) were cocultured with osteoblasts in thepresence of a hormonal osteolytic stimulus provided by highconcentrations of 1,25(OH)2D3 and PGE2, a type of in vitro model

Figure 4. The presence of tumors at sites of the skeleton otherthan that of the hind limbs was determined by fluorescentimaging. A, X-ray analysis of the hind limb showed thepresence of osteolytic lesions (arrows ) after 21 days in a17-AAG–treated mouse. B, corresponding fluorescent image ofthe hind limb osteolytic tumor shown by fluorescent imaging.Fluorescent imaging also revealed metastases to the jaws (C ),skull (D ), and spine (E) of 17-AGG–treated nude mice.F, spine metastases were also identified in the vehicle controlgroup as previously reported for this model; however, thesewere less numerous and smaller in size. G, graphicalrepresentation of the incidence of bone metastasis to varioussites as determined by fluorescent imaging. A significantincrease in metastasis to the hind limbs and the jaws wasobserved as determined by Fischer exact probability test.*P < 0.05. H, 17-AAG treatment of mice that had beenorthotopically inoculated with MDA-MB-231SArfp cells showeda significant decrease in tumor growth when compared with thevehicle control mice (8 mice/gp). Columns, mean; bars, SD.

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thought to recapitulate the cell-to-cell interactions at the bonesurface and used to elucidate the mechanisms controllingosteoclast formation. 17-AAG greatly increased the osteoclastformation in this model; however, this action of 17-AAG did notrequire the presence of osteoblasts: RANKL-stimulated bonemarrow cells also formed more osteoclasts in the presence of 17-AAG. Furthermore, BMM, a highly enriched source of osteoclast/macrophage progenitors, and the RAW 264.7 pre-osteoclast cell line(data not shown) also formed more osteoclasts with 17-AAGtreatment. This suggests that 17-AAG pro-osteolytic effects requireonly the presence of hematopoietic-derived cells and may indeedact directly on osteoclast progenitors to increase their response toRANKL. The only other exogenous factor known to do this isTGFh (and, less potently, the related bone morphogenic protein-2),and whereas we cannot exclude an induction of TGFh secretionby 17-AAG, TGFh strongly inhibits osteoclast formation in the

coculture model as it suppresses RANKL stimulation by theosteoblasts (55); this is inconsistent with our results with 17-AAG.A number of endogenous factors are known to increase osteoclastformation, e.g., thioredoxin expression (56), and it is possible that17-AAG might induce such factors in osteoclasts. This is supportedby previous findings that heat shock and oxidative stress in thepremonocytic line U937 increases thioredoxin expression viaactivation of HSF (57). It is also known that 17-AAG and otherHsp90 inhibitors, such as herbimycin A and radicicol, induce apotent heat shock response in cells through the release andsubsequent activation of HSF-1 from the Hsp90 molecularchaperone complex (58). Therefore, Hsp90 inhibitors may havean indirect effect on osteoclast formation via HSF-1–mediated up-regulation of gene transcription. It is also conceivable that 17-AAGmay have a direct effect upon osteoclast formation in thatendogenous repressors of the process may exist and that these

Figure 5. The effects of Hsp90 inhibitors upon in vitroosteoclast formation was determined using a number ofmodel systems. A, 17-AAG significantly enhancedosteoclast formation at 30 and 100 nmol/L in mouse bonemarrow/osteoblast cocultures over a 7-day period. Cellulartoxicity was observed at higher concentrations of 17-AAG.B, 17-AAG (30 nmol/L, 100 nmol/L) significantly enhancedosteoclastogenesis in mouse bone marrow cultures in thepresence of soluble RANKL and M-CSF before cellulartoxicity. C, use of the highly purified osteoclast progenitorcells, BMMs, in the presence of soluble RANKL andM-CSF also showed a significant increase in osteoclastformation when stimulated with 17-AAG. D, in addition to17-AAG, the Hsp90 inhibitor, herbimycin A significantlyenhanced osteoclast formation in the BMM cultures at 30and 100 nmol/L concentrations before cellular toxicity.E, 17-AAG enhanced the size of the osteoclasts in vitrowhen compared with the positive control and the vehiclecontrol. Arrows, examples of osteoclasts in vitro illustratingthe size differences. F, a model of human osteoclastformation was done using human cord blood–derivedprogenitors cultured with soluble RANKL and M-CSF.A significant increase in human osteoclast formation wasobserved with 17-AAG treatment at 3, 10, and 30 nmol/Lconcentrations before cellular toxicity. Columns, mean;bars, SD. Significance was determined by the Student’st test. *P < 0.05, **P < 0.01. #, cellular toxicity.

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molecules are Hsp90 ‘‘client’’ proteins. Upon Hsp90 inhibition by17-AAG or other Hsp90 inhibitors, these proteins may losefunctionality and/or are degraded resulting in the release of therepression leading to enhanced osteoclast formation.

Although our results clearly define an action of 17-AAG uponthe host cells of the bone, it is not known at this time whether17-AAG can act directly upon the tumor cells, modifying theirphenotype and enabling them to survive and grow moreefficiently in bone. 17-AAG is known to have profound effectsupon cancer cell gene expression (59), increasing the expressionof genes that may aid in tumor cell survival and/or growth inbone. Thus, in different microenvironments with differing growthpressures, tumor cells may escape 17-AAG–mediated toxicity,allowing the drug to modify the gene expression profile of thetumor cell and ultimately aid in the generation of bonemetastases.These findings come at a critical time in the clinical

development of 17-AAG as well as the preclinical development ofother Hsp90 inhibitors. Although phase I clinical trials have servedas a proof of principle that Hsp90 pharmacologic inhibition canbe achieved with tolerable toxicity in humans, its longer-termapplication will determine whether our experimental findings willbe translated to the clinical situation. If so, the identification of apro-osteolytic action of 17-AAG in the bone microenvironmentneeds to be addressed, especially in patients with a high likelihoodof bone metastasis, such as in breast and prostate cancer andmultiple myeloma. However, the potential deleterious effects of17-AAG upon the bone may be overcome by the administration ofexisting effective anti-osteolytic therapies, such as the bisphosph-onates, which are currently clinically approved and being usedroutinely (2). The use of such approaches deserves carefulconsideration for those investigating HSP90 inhibitors, andsuggests that its evaluation using in vivo murine models isdesirable.

Acknowledgments

Received 12/13/2004; revised 2/21/2005; accepted 3/4/2005.Grant support: Association for International Cancer Research grant 99-081 (J.T.

Price); Susan G. Komen Foundation Basic, Clinical, and Translational Research grant0100915 (J.T. Price and E.W. Thompson); Victorian Breast Cancer ResearchConsortium (E.W. Thompson); and Thomaiy Breast Cancer Research Fund andNational Health and Medical Research Council Program grant 003211 (M.T. Gillespie).

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

We thank T. Yoneda for the provision of the MDA-MB-231SA cells; E. Sausville forthe 17-AAG; N. Rosen for helpful technical discussions; Nicolle Bruengger and MarijaMikasinovic for valuable animal technical support; Bernard Jeffreys (Berthold Imaging,Melbourne Victoria, Australia) and Sarah Fraser (Victoria University, Melbourne,Victoria, Australia) for the in vivo fluorescent imaging support; and Prof. T.J. Martinfor the critical reading of the manuscript.

Figure 6. Tumor-naı̈ve nude and C57BL/6 mice were treated for 3 weeks with17-AAG (70 mg/kg/mouse) and bones were processed for histomorphometricanalysis. A, a significant decrease in percentage of trabecular bone volume wasidentified in both the nude and C57BL/6 mice when the 17-AAG–treated groupwere compared with the vehicle control group. B, no significant alteration inosteoclast surface as a percentage of trabecular bone surface was observedin nude mice. However, a significant increase in osteoclast surface was observedin the C57BL/6 mice. C, no difference in osteoblast number was observed ineither the nude or the C57BL/6 mice with 17-AAG treatment. Columns, mean;bars, SD (n = 10). Significance was determined by ANOVA, *P < 0.05.

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