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Preclinical Development

Breast Cancer–Derived Bone Metastasis Can Be EffectivelyReduced through Specific c-MET Inhibitor Tivantinib(ARQ 197) and shRNA c-MET Knockdown

Sara Previdi1, Giovanni Abbadessa2, Francesca Dal�o1, Dennis S. France2, and Massimo Broggini1

AbstractBreast cancer exhibits a propensity to metastasize to bone, resulting in debilitating skeletal complications

associated with significant morbidity and poor prognosis. The cross-talk between metastatic cancer cells and

bone is critical to the development and progression of bonemetastases.We have shown the involvement of the

HGF/c-MET system in tumor–bone interaction contributing to human breast cancer metastasis. Therefore,

disruption ofHGF/c-MET signaling is a potential targeted approach to treatingmetastatic bone disease. In this

study, we evaluated the effects of c-MET inhibition by both an oral, selective, small-molecule c-MET inhibitor,

tivantinib, and a specific short hairpin RNA (shRNA) against c-MET in amousemodel of human breast cancer.

Tivantinib exhibited dose-dependent antimetastatic activity in vivo, and the 120 mg/kg dose, proven to be

suboptimal in reducing subcutaneous tumor growth, induced significant inhibition of metastatic growth of

breast cancer cells in bone and a noteworthy reduction of tumor-induced osteolysis. shRNA-mediated c-MET

silencing did not affect in vitro proliferation of bone metastatic cells, but significantly reduced their migration,

and this effect was further enhanced by tivantinib. Both observations were confirmed in vivo. Indeed, more

pronounced tumor growth suppression with concomitant marked decreases of lytic lesions and prolongation

of survival were achieved by dual c-MET inhibition using both tivantinib and RNA interference strategies.

Overall, our findings highlighted the effectiveness of c-MET inhibition in delaying the onset andprogression of

bone metastases and strongly suggest that targeting c-MET may have promising therapeutic value in the

treatment of bone metastases from breast cancer. Mol Cancer Ther; 11(1); 214–23. �2011 AACR.

Introduction

Breast cancer, along with prostate, thyroid, and kidneycancer, displays a remarkable predilection to metastasizeto bone. At least 80% of patients with metastatic breastcancer will develop bone metastases during the course oftheir disease (1, 2). The pathologic manifestations ofosteolytic lesions can have devastating effects, includingpain, pathologic fractures, spinal compression, andhypercalcemia. An increased understanding of the cellu-lar and molecular mechanisms that contribute to bonemetastasis is necessary for improving clinical manage-ment. Breast cancer cells secrete various factors that stim-ulate osteoblasts, osteoclasts, and other cells of the bone

microenvironment favoring bone resorption; bone cells,in turn, secrete factors that stimulate tumor cell growth.These interactions between tumor cells and the bonemicroenvironment result in a vicious cycle that increasesboth bone destruction and tumor burden, nurturing thedevelopment and propagation of bone metastasis (3–8).Therapeutic targeting of tumor–bone interaction is underintensive investigation (9). A potential target candidate isc-MET, the tyrosine kinase receptor for the hepatocytegrowth factor (HGF). Primarily expressed on epithelialcells, c-MET drives different intracellular signaling path-ways that are essential for the development and progres-sion of many human cancers (refs. 10–14; additionalinformation available at: http://www.vai.org/met). TheHGF/c-MET pathway regulates diverse biological activ-ities, ranging from proliferation, motility, and invasion tosurvival and angiogenesis, many of which are hallmarksof cancer (15). Aberrant signaling of the c-MET pathway,identified in a wide variety of human malignancies, hasbeen associated with a poor prognosis, aggressive phe-notype, increased metastasis, and shortened patient sur-vival (10).

However, the role of c-MET signaling in human breastcancer bone metastasis has scarcely been investigated.Recently, we have reported that the c-MET receptor actsas an important mediator of the cross-talk between

Authors' Affiliations: 1Laboratory of Molecular Pharmacology, Istituto diRicerche Farmacologiche "Mario Negri," Milan, Italy; and 2ArQule, Inc.,Woburn, Massachusetts

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

Corresponding Author:Massimo Broggini, Laboratory of Molecular Phar-macology, Istituto di Ricerche Farmacologiche "Mario Negri", via G. LaMasa 19, 20156 Milan, Italy. Phone: 39-02-39014585; Fax: 39-02-39014734; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0277

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 11(1) January 2012214

on June 14, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst October 25, 2011; DOI: 10.1158/1535-7163.MCT-11-0277

http://mct.aacrjournals.org/

epithelial breast cancer cells andmesenchymal cells of thebone microenvironment, contributing to progression ofosteolytic bone metastases in vivo (16). Given the impor-tance and the potential therapeutic role of the c-METreceptor in bone metastasis progression, we examinedthe effects of inhibiting c-METusing both a specific c-METinhibitor (tivantinib) and RNA interference technology inan in vivo murine model of breast cancer bone metastasis(17). Tivantinib is a novel, orally available, small-mole-cule, non–ATP-competitive c-MET inhibitor that is highlyspecific for the c-MET receptor (18–21).Herewe show thattreatment with different concentrations of tivantinibaffected bonemetastasis progression in a dose-dependentmanner. Moreover, treatment with tivantinib in combi-nation with the silencing of c-MET protein expression byspecific short hairpinRNA (shRNA) led to an even greaterreduction in bone metastasis progression and cancer-induced bone destruction as well as an increase in overallsurvival.

Materials and Methods

CompoundTivantinib [(-)-trans-3-(5,6-dihydro-4H-pyrrolo [3,-

2,1-ij] quinolin-1-yl)-4(1H-indol-3-yl) pyrrolidine-2, 5-dione; ref. 22] was synthesized and separated by chiralpurification at ArQule, Inc. Tivantinib, also known asARQ 197, has been shown to selectively inhibit c-MET(Ki ¼ 355 nmol/L) via a non–ATP-competitive mecha-nism and to have minimal inhibitory activity against themajority of a panel of 229 human kinases (20, 21).Tivantinib also showed antiproliferative activity in sev-eral human cancer cell lines expressing c-MET (GI50values ranging from 0.30 to 0.66 mmol/L) and showedantitumor activity in murine xenograft models of colon,gastric, and breast cancer (21).

Cell lines and cultureThe human MDA-MB-231 breast cancer cell line was

obtained from theAmerican TypeCulture Collection. Thebone-seeking clone wild-type (1833) and retrovirallytransfected with the triple reporter construct (1833/TGL;ref. 23), and the parental MDA-MB-231 genetically mod-ified to express TGL reporter protein (MDA-MB-231/TGL; ref. 17) were obtained from Professor J. Massagu�e(Memorial Sloan-Kettering Cancer Center, New York,NY). Cell line authentication was not carried out by theauthors within the last 6 months. Cells were cultured inDulbecco’s Modified Eagle’s Medium supplementedwith 10% FBS (Sigma-Aldrich) and 1% L-glutamine.

In vitro cytotoxic assayTumor cells were seeded in 130 mL of medium at 3,500

cells per well in 96-well microplates. After 24 hours,different concentrations of tivantinib were added for 72hours. At the end of the treatment period, MTS reagent(Promega) was added to each well and the plates wereincubated at 37�C for 4 hours in 5%CO2. Cell proliferation

was evaluated by measuring the absorbance at 490 nmusing an infinite M200 Microplate Reader (Tecan). Anonlinear regression method was used to calculate GI50values (compound concentration required to produce50% growth inhibition).

Inducible MET RNA interference constructs andtransfection

An inducible downregulation of c-MET was devel-oped by using pSuperior retrovirus-based vector system(OligoEngine) expressing inducible shRNA. Detailsand sequences used are available in SupplementaryMethods.

Western blotThirty micrograms of total cellular proteins were sep-

arated on a 6% to 8% SDS-PAGE gel and then transferredto a nitrocellulose membrane (Whatman). The mem-braneswere incubatedwith the different antibodies usingthe dilutions and conditions as specified in Supplemen-tary Methods.

In vitro wound healing assayThe shMET D and MutB 1833 cells (2 � 105 cells per

well) were plated on a 12-well culture plate (BectonDickinson) in the presence or absence of both doxycycline(2 mg/mL) and tivantinib (0.2 mmol/L). Upon reachingconfluence, a single wound was created in the center ofthe cell monolayers by gentle removal of the attached cellswith a sterile plastic pipette tip. The platewas placed underan IX81 motorized inverted microscope (Olympus) fittedwith an incubator to maintain 37�C, 5% CO2, and 60%humidity (Okolab). Cells were followed for 24 hours, andevery 30 minutes an image was captured using an ORCA-ER CCD camera (Hamamatsu). Phase-contrast pictureswere analyzed by ImageJ software (Wayne Rasband). Theextent of wound closure was determined by calculatingthe ratio between the surface area of the wound for eachtime point and the surface of the initial wound. These datawere then expressed as the percentage of wound closureand plotted against the hours after wounding. The exper-iment was conducted 3 times independently.

In vivo studiesFemale 4-week-old athymic nude (nu/nu) mice were

used for all experiments. Mice were obtained from Har-lan-Italy and maintained under specific pathogen-freeconditions with food and water provided ad libitum.Procedures involving animals and their care were con-ducted in conformitywith institutional guidelines that arein compliance with national (Legislative Decree 116 ofJanuary 27, 1992, Authorization n.169/94-A issuedDecember 19, 1994, by Ministry of Health) and interna-tional laws andpolicies (EECCouncilDirective 86/609,OJL 358. 1, December 12, 1987; Standards for the Care andUse of Laboratory Animals, United States NationalResearch Council, Statement of Compliance A5023-01,November 6, 1998).

c-MET Inhibition Reduces Breast Cancer Bone Metastasis

www.aacrjournals.org Mol Cancer Ther; 11(1) January 2012 215




Experimental subcutaneous xenograft modelA total of 7.5 � 106 tumor cells in 200 mL 1:1 medium/

Matrigel (BD Biosciences) were injected subcutaneouslyinto the left flank of mice; when tumors reached approx-imately 100mm3,micewere treatedwith tivantinib (orallyat the dose of 120mg/kg in a volume of 10mL/kg of bodyweight daily until the end of the experiment) or withvehicle [PEG 400:20% vitamin E TPGS solution (60:40)].Tumor diameters were measured with a caliper twiceweekly until the animals were sacrificed. Tumor weightwas derived from tumor volume (assuming a density of 1)calculated by the formula: (length � width2)/2. Bodyweights were measured weekly during the treatmentperiod.

Experimental bone metastasis modelA total of 5 � 105 tumor cells per 100 mL of PBS were

injected into the left cardiac ventricle of 3% isoflurane-anesthetized mice. Animals were randomized intogroups of 10 mice each. Tivantinib was administeredorally at the doses of 30, 60, or 120 mg/kg. Controlanimals were treated with vehicle. In the preventive andtherapeutic protocols, treatments started 2 or 12 daysafter implant, respectively, and, in both cases, continueddaily until the end of the experiment. Tetracycline(2 mg/mL; Sigma) was provided in the drinking waterand replaced every 2 days, starting from the day of cellimplant. Body weights were measured weekly. Cancercell dissemination to bone and extent of bone lesionswere monitored by weekly analysis of bioluminescenceimaging (BLI) technology and micro-computed tomog-raphy (micro-CT), respectively, as described in Supple-mentary Methods. Animals were euthanized when con-trols started to show signs of suffering. After sacrifice,hindlimb specimens (tibia and femora) were removedduring autopsy and collected for histologic and immu-nohistologic analysis.

Bone histology and immunohistochemistryBones were fixed for 90 minutes at room temperature

and decalcified inMielodec (Bio-Optica) for 4 days, dehy-drated, embedded in paraffin, and cut into 5 mm sections.Bone slides were stained with Mayer’s hematoxylin andeosin (H&E) by standard protocols. Bone sections weredeparaffinized, rehydrated, and subjected to antigenretrieval by water-bath heating (95�C, 20 minutes) inantigen unmasking solution (Vector Laboratories) atpH ¼ 6. After blocking endogenous peroxidase activitywith 1% H2O2, slides were probed with different anti-bodies as specified in Supplementary Methods.

Statistical analysisDifferences in bioluminescence intensity between the

control and treatment groups were evaluated by 1-wayANOVA followed by Bonferroni’s multiple comparisontest using the GraphPad Prism software package (version5.03). All data were expressed as the mean � SE anddifferences were considered statistically significant at alevel of P < 0.05.

Results

Cell viabilityThe effect of tivantinib (Fig. 1A) on the viability of

the parental MDA-MB-231/TGL and the derived bone-seeking clone 1833/TGL human breast cancer cell linesis shown in Fig. 1B. Treatment of cells with tivantinibresulted in a comparable concentration-dependent de-crease in cell viability with GI50 values of 1.2 and3.7 mmol/L obtained in MDA-MB-231/TGL and 1833/TGL cells, respectively.

Bone metastasis progressionThe efficacy of tivantinib to inhibit bone metastasis

progression is shown in Fig. 2A. The appearance of cancer

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Previdi et al.

Mol Cancer Ther; 11(1) January 2012 Molecular Cancer Therapeutics216




cells in the leg bones varied from 11 to 14 days after cellimplant and increased over time, both in control andtivantinib-treated animals. In particular, the signal fromthe hindlimbs of tivantinib-treated (30 mg/kg) mice wasvery similar to that of control mice, although treatmentwith tivantinib at thedoses of 60 and 120mg/kg induced adose-dependent delay and a reduction of bone metastaticgrowth. In fact, tivantinib at a dose of 60 mg/kg caused atemporary reduction of the tumor-related luciferase sig-nal starting from17 to 21 days after cell implant. Fromday21 to the end of the experiment, the mean value of the BLIsignal intensity from this groupwas comparablewith thatof controls. On the contrary, the dose of 120 mg/kg oftivantinib significantly inhibited BLI signal and, conse-quently, tumor burden in the bone of treated animalscompared with the controls, starting from 14 to 21 daysafter cell injection. This inhibition was maintained untilthe end of the experiment. These BLI results were furtherconfirmed by micro-CT data, underlining that increasingdoses of tivantinibwerewell correlatedwith a decrease inthe number and the extent of osteolytic lesions. Repre-sentative images of ventral views (Fig. 2B) from opticalimaging and micro-CT showed that treatment of 1833/TGL-injected mice with increasing doses of tivantinibresulted in adose-dependent decrease inbioluminescence(Fig. 2B, top panels) and in tumor-induced osteolysis

(Fig. 2B, bottom panels), representing a reduction in bonemetastasis progression compared with control mice.

On the basis of these results, further studies wereconducted with the 120 mg/kg dose of tivantinib. Resultsfrom 2 separate experiments showed an exponentialincrease of photon emission associated with an increasein tumor burden, clearly evident from day 14 onward, inthe hindlimbs of control mice (Fig. 3A). Treatment withtivantinib (120 mg/kg) resulted in a significant reductionof bioluminescence signal starting from 14 days afterimplant (Fig. 3A). As evident in Fig. 3B, animals treatedwith tivantinib exhibited a significant inhibition of photoncounts and, therefore, of tumor growth in the leg bonescomparedwith vehicle-treated group. The bodyweight ofanimals also showed the effects of tivantinib in injectedmice. The relative bodyweight curve of controls started todecrease at day 17 (Fig. 3C), coincident with the rapidprogression of metastatic bone disease. In animals receiv-ing tivantinib (120 mg/kg), weight loss started from day21andwas less than that observed in controls. The effect oftivantinib on 1833/TGL-induced osteolysis was assessedby micro-CT analysis (Fig. 3D). Three-dimensional (3D)reconstructed micro-CT images of the hindlimbs of rep-resentative control animals revealed severe bone destruc-tion on day 21 following cancer cell transplantation. Incontrast, bones from tivantinib-treated (120 mg/kg) mice

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Figure 2. Noninvasive BLI and micro-CT to monitor the effects of tivantinib at different concentrations on bone metastases development and progression.1833/TGL cells were injected into the left cardiac ventricle of immunodeficient mice. A, at the indicated times after xenografting, the bioluminescencesignalwas captured and the growth kinetics of right and left hindlimbmetastasis for eachgroupwere expressed in the histogramasmean value of total photoncounts from the hindlimbs of animals. Columns, mean value � SE; �, P < 0.05; ��, P < 0.01. B, representative optical and micro-CT scanning in thesupine position for each group 21 days after tumor implant are shown. The intensity of the BLI signal is shown as a pseudocolor scale bar (top panels). For thesame animals, representative volume-rendered micro-CT images of osteolytic bone metastases obtained 21 days after xenografting are shown (bottompanels; R, right hindlimb, L, left hindlimb).






were apparently intact or showed only limited signs ofosteolysis at day 21 (arrows).At the endof the experiment,histologic examination of representative tibial and femo-ral sections from control and treated mice confirmed thepresence of metastatic 1833/TGL breast cancer cells with-in the bone marrow cavity (Supplementary Fig. S1).

The same bone metastatic clone 1833/TGL wasinjected subcutaneously into the left flanks of athymicnude mice. Chronic treatment with vehicle or tivantinib(120 mg/kg) was initiated when tumors were estab-lished and reached a defined size of 100 mm3. Dailydosing for 25 days of tivantinib did not affect the growthof subcutaneously growing tumors compared with con-trols (Fig. 4A). Daily drug administration was welltolerated by the animals, as there was no significantdifference between the body weight of the tivantinib-treated and control mice (Fig. 4B). Similar results wereobtained with the bone metastatic model when tivanti-nib 120 mg/kg administration started 12 days aftertumor cell implant, when metastasis were already wellestablished (Supplementary Fig. S2).

Silencing c-MET gene expressionTo further investigate the role of the c-MET receptor in

bonemetastatic breast cancer cells, c-MET protein expres-sion was silenced (in a doxycycline-inducible manner) bytransfection of cells with shRNA. The results showed thatthe presence of doxycycline induced a decrease in theexpression of c-MET receptor in shMET D 1833 cells (Fig.5A, top left panels). In these cells, reduction in c-METprotein was found to be time dependent, as indicated bydensitometric analysis (Fig. 5A, bottom left panels). By 96hours after doxycycline administration, the c-MET pro-tein levelswere reducedby 90% in shMETD1833 cells.Onthe contrary, in control Mut B 1833 cells carrying mutatedshRNA, c-MET protein expression was not affected bydoxycycline addition (Fig. 5A top right panels). shRNA-mediated downregulation of c-MET protein expressionhad no effect on either cell growth of shMET D 1833 (Fig.5B) or Mut B 1833 cells (Supplementary Fig. S3).

To study the effects of c-MET silencing on HGF-medi-ated signaling events downstream of c-MET activation,specific phosphorylation of c-MET, AKT, and ERK1

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Figure 3. In vivo effects of tivantinib at 120mg/kg dose on bonemetastasis. The effects of tivantinib treatment on bonemetastasis progressionweremonitoredover time by in vivo BLI and micro-CT. A and B, in vivo BLI. A, average growth of bone metastasis in the hindlimbs of control and tivantinib-treatedmice—meanphotoncounts from the hindlimb (right and left) regions ofmicewerequantifiedanddisplayedover time (�,P<0.05; ��,P<0.01). B, ventral imagesfrom 3 representative mice 21 days after implant are shown. Numbers at the bottom of the images represent number of mice. C, relative body weight ofcontrol and tivantinib-treated mice. The relative body weight was calculated as Bt/B0, where Bt is body weight at the day of measurement and B0 isbodyweight at the day of tumor cell injection.D, in vivomicro-CT analysis; representativemicro-CTof the hindlimbsof both control and tivantinib-treatedmiceat day 21 after xenografting. Osteolytic lesions are indicated by arrows; arrow size is proportional to the extent of bone lesion. Numbers at the bottomof the images represent number of mice.

Previdi et al.





proteins was analyzed (Fig. 5C). In the absence of HGF,no phosphorylation of c-MET was observed in bothshMET D and Mut B 1833 cells (SupplementaryFig. S4). HGF-mediated induction of tyrosine phosphor-ylation (pY1234/1235) of c-MET receptor was evident inMut B 1833 cells both in presence and absence of doxy-cycline and in shMET D 1833 cells without doxycycline.Upon HGF stimulation, the doxycycline-induced c-METdownregulation correlated with a reduction of c-METtyrosine phosphorylation status in shMET D 1833 cells.Moreover, in Mut B 1833 cells, phosphorylation of ERK(Tyr204) and AKT (Thr308) was markedly induced afterHGF stimulation, independent of the presence of doxy-cycline. Phosphorylation of Tyr204 of ERK1 and Thr308 ofAKT was evident in response to HGF in doxycycline-untreated shMET D 1833 cells; although in the same cells,doxycycline induced moderate decreases in the phos-phorylation and, presumably, the activation state of thesedownstream c-MET–related effectors. Neither HGF stim-ulationnordoxycycline treatment affected the total steadystate of ERK1 and AKT levels.An in vitrowound healing assay was done to assess the

effects of c-MET silencing alone or in combination withtivantinib on the migration of both shMET D 1833 andMut B 1833 cells. Figure 5D (top panel) shows represen-tativephotographs of cellsmigrating into scratchwounds.Quantitative analysis revealed that the combination ofdoxycycline-induced c-MET downregulation and tivanti-nib treatment significantlydecreased themigrationpoten-tial of shMETD1833 (Fig. 5D, bottompanel). As expected,the presence or absence of doxycycline did not affect themigration of Mut B 1833 cells (data not shown).The effects of c-MET silencing, alone or in combination

with tivantinib treatment, was also investigated in the invivo breast cancer–derived bone metastasis model. Ani-mals transplanted with shMET D 1833 cells were treatedwith either vehicle alone (shMET D 1833 TET� group),

vehicle plus administration of tetracycline (shMETD1833TETþ group), tivantinib (120 mg/kg) alone (shMET D1833 tivantinibþ group), or tivantinib (120 mg/kg) plustetracycline (shMET D 1833 TETþ/tivantinibþ group).

Bone metastases in the hindlimbs became detectable atday 27 after tumor cell injection (Fig. 6A). c-MET silencinginduced a reduction in bonemetastasis growth comparedwith controls. The antimetastatic efficacy of tivantinib(120 mg/kg) was also confirmed in this bone metastaticmodel. Interestingly, the combination of tivantinib andshRNA-mediated c-MET silencing induced a significantdecrease in tumor burden in the femoral/tibial bonesof treated mice compared with control mice starting from35 days after tumor implant. Representative biolumines-cence ventral images taken 38 days after cell implantindicated that c-MET silencing in combination with inhi-bition of c-MET activity by tivantinibmay strongly reduceskeletal tumor burden (Fig. 6B, top panels). It is notewor-thy that BLI photon counts fromhindlimbs ofmice treatedwith tetracycline plus tivantinib remained constant orincreased slowly over time (Fig. 6A). Control animals hadto be sacrificed at day 38 because of considerable re-ductions in body weight and generalized deteriorationof their health status. The group of animals treated withthe tivantinib/shMET combination survived until day 58after implantation. As in the 1833/TGL-derived model,bone destruction assessed by micro-CT was again mainlyevident at the ends of distal femora and proximal tibiae,and the analysis of skeletal changes 38 days after tumorcell implant closely correlated with the bioluminescenceresults (Fig. 6B; bottom panels). In vehicle-treated mice atday 38 after implantation, bone destruction was massive,with sizable holes in the tumor-bearing bones. In thetetracycline-treated group, the extent of osteolysis wascomparable with controls, evenwhen the number of bonelesions was reduced. In contrast, osteolytic lesions intivantinib-treated animals were significantly reduced as

Figure 4. Activity of tivantinibagainst subcutaneous breastcancer xenografts. A, tumor growthinhibition. Results are expressed asmean�SE. B,mean bodyweight ofmice bearing subcutaneous tumorxenografts and treated with vehicleor tivantinib. Body weights weremeasured twice weekly.

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Previdi et al.





compared to both control and c-MET–silenced animals.Finally, bones from shMET/tivantinib-treated mice wereintact, with only early signs of lysis. BLI and micro-CTdata were confirmed by histologic and immunohisto-chemical examinations of tumors in the bone sections atday 38 after cancer cell inoculation (Fig. 6C). As revealedbyH&E and pan-cytokeratin immunostaining, bone mar-rowof distal femora from control and tetracycline-admin-istered groups was completely invaded by metastatictumor cells. Otherwise, only small colonies of cytokera-tin-positive 1833 cells were detectable in the bones oftivantinib-treated and shMET/tivantinib-treated mice.In vivo downregulation of c-MET activation was eval-

uated through immunohistochemical staining. The pres-ence of phospho-c-MET positive cells was clear in thebone metastatic sections from vehicle-treated animals(Fig. 6D). Phospho-c-MET positive cells were also

detected in shMET D 1833 TETþ-derived sections,although the number of positive cells was greatly reducedas comparedwith the vehicle-treated group, in agreementwith a reduction of shRNA-induced total c-MET proteinexpression. Otherwise, no phospho-c-MET–positive cellswere seen in bone sections from tivantinib-treated mice.These findingshighlighted the in vivo efficacyof tivantinibtreatment in inhibiting c-MET phosphorylation, and thusits functional activation.

Discussion

Management of bone metastasis is an important issuefor the improvement of quality of life and survival inbreast cancer patients. Therapeutic strategies aimed atinterrupting the vicious cycle, impeding the developmentand progression of bone metastases, and improving bone

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Figure 6. In vivo BLI to monitor the effects of c-MET silencing in shMET D 1833–derived bone metastasis. A, metastatic growth curve in untreatedmice (shMETD1833TET�), mice expressing shRNAagainst c-MET (shMETD1833TETþ), mice treatedwith tivantinib 120mg/kg (shMETD1833þ tivantinib),and mice treated with tivantinib plus c-MET shRNA (shMET D 1833 TETþ tivantinibþ). Data are expressed as mean value� SE (�, P < 0.05). B, representativewhole-body BLI images of mice injected with shMET D 1833 (ventral views). Signals are displayed as pseudocolor image. Images were recorded atday 38 after cell implant (top panels). Numbers at the bottom of the images represent the number of mice. Representative 3D reconstruction of micro-CTimages of hindlimbs of shMET D 1833–injected mice from different treatment groups at day 38 from intracardiac injection (bottom panels). Arrows indicatetumor-induced osteolytic lesions; arrow size is proportional to the extent of bone lesion. C, H&E staining of sections from leg bones at day 38 followingintracardiac injection: 1, �4 magnification; 2, �100 magnification; 3, immunohistochemistry for human cytokeratin (magnification, �100). Cellsimmunoreactive with the antihuman pan-cytokeratin antibody are stained in brown. Representative images of 5 analyzed leg bone sections from 3 differentmice are shown. D, immunohistochemistry for phospho-cMET on bone sections 38 days after shMET D 1833 cells implant. Cells immunoreactive withthe antihuman phospho-c-MET antibody exhibit brown staining. BM, bone marrow; Tu, tumor cell population.






integrity are under intensive investigation (24). A vastamount of compelling in vitro and in vivo evidence sup-porting the role of the HGF/c-MET pathway in tumorprogression and metastasis suggests that this pathwaycould represent an attractive therapeutic target for met-astatic breast cancer (25–29).

On the basis of these considerations, our bone meta-static model established with 1833 cells represents anoptimal tool to assess the antimetastatic activity of tivan-tinib. Our results show that the c-MET inhibitor tivantinibexhibits dose-dependent activity and, at a daily dose of120 mg/kg, significantly delays the formation and pro-gression of bone metastases and cancer-induced osteoly-sis. Interestingly, this dose was ineffective in inhibitinggrowth of the same cells implanted subcutaneously, sug-gesting that the effects of tivantinib against bone metas-tasis in this model were not due to a direct cytotoxic effectof the compound. It could be hypothesized that theobserved antimetastatic effects by tivantinib occurred inthe bone microenvironment. The results obtained withcells, in which c-MET has been downregulated usingshRNA, further corroborate these data and strongly sup-port the evidence that tivantinib exerts its antimetastaticaction through c-MET inhibition. c-MET silencing in 1833cells didnot affect cell growth in vitrobut strongly reducedmigration potential. The pharmacodynamic effects ofc-MET silencing in 1833 cells clearly showed that c-METphosphorylation is reduced and that the functionof downstream pathways involved in c-MET–inducedcell proliferation (i.e., ERK, MAPK, JNKs, NF-kB, andPI3K/AKT; ref. 30) were only marginally affected, inagreement with the lack of in vitro cell growth inhibitoryactivity. Overall, these in vitro results provide evidence fora potential role of c-MET in breast cancer metastasis tobone, assuming a role in cellularmotilitywithout affectingcell proliferation, and could explain the data obtained invivo. In our bone metastasis model, c-MET silencingshowed high antimetastatic activity. It is difficult to deter-mine a direct comparison of activity of c-MET silencingcompared with that induced by tivantinib. Notably, bothstrategies proved to be effective, and in our experiments,we used lower doses of tivantinib compared with thoseused in other studies (21), in an effort to show activitypossibly not linked to a direct cytotoxic effect. Further-more, the combinationofpharmacologic c-MET inhibition

(through tivantinib) and genetic downregulation of c-MET protein expression (through shRNA) seemed toproduce an enhanced inhibition of bone metastasis pro-gression. These data are in strong agreement with theobserved inhibition of in vitro migration of cells simulta-neously treatedwith tivantinib and c-MET shRNA. Takentogether, our results revealed that c-MET inhibition iseffective in delaying the onset and progression of tumorgrowth in bone and in greatly diminishing osteolyticlesions. Thus, inhibition of c-MET may offer significantbenefits for the prevention of bone metastasis develop-ment in patients with breast malignancies. Finally, ourfinding that tivantinib is active as an antimetastatic agentat well-tolerated noncytotoxic doses suggests that tivan-tinib may be a good candidate for combination studieswith cytotoxic agents.

In summary, this study showed that treatment withtivantinib inhibited bone metastasis progression and can-cer-induced bone destruction, with an increase in thesurvival of treated mice. These findings suggest thattivantinib has significant therapeutic potential for themanagement of metastasis and provide further evidencethat c-MET represents a promising therapeutic target forprevention of bone metastases from breast cancer.

Disclosure of Potential Conflicts of Interest

G. Abbadessa and D.S. France are employed by ArQule, Inc. that isdeveloping tivantinib.

Acknowledgments

The authors thank Jeffrey Riegel of Accuverus, Beachwood, OH, formedical editorial support and Drs. Enrico Radaelli and Marco Losa fromthe Fondazione Filarete (Milan, Italy) for their expert advice withimmunostaining.

Grant Support

Grant support was provided to M. Broggini by the Italian AssociationforCancer Research (AIRC), and S. Previdi is recipient of a fellowship fromMonzino Foundation, Milan, Italy.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 13, 2011; revised September 9, 2011; accepted October 8,2011; published OnlineFirst October 25, 2011.

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2012;11:214-223. Published OnlineFirst October 25, 2011.Mol Cancer Ther Sara Previdi, Giovanni Abbadessa, Francesca Dalò, et al. shRNA c-MET Knockdown

andReduced through Specific c-MET Inhibitor Tivantinib (ARQ 197) Derived Bone Metastasis Can Be Effectively−Breast Cancer

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