Cathepsin B Inhibition Limits Bone Metastasis in Breast Cancer › content › canres › ... · function of cathepsin B in breast cancer metastasis to bone. Molecular suppression

Therapeutics, Targets, and Chemical Biology

Cathepsin B Inhibition Limits Bone Metastasis in BreastCancer

Nimali P. Withana1,2, Galia Blum4,5, Mansoureh Sameni6, Clare Slaney1, Arulselvi Anbalagan6, Mary B. Olive7,Bradley N. Bidwell1,3, Laura Edgington5, Ling Wang5, Kamiar Moin7,8, Bonnie F. Sloane7,8,Robin L. Anderson1,2,4, Matthew S. Bogyo5,9, and Belinda S. Parker1,3,4

AbstractMetastasis to bone is a major cause of morbidity in breast cancer patients, emphasizing the importance of

identifying molecular drivers of bone metastasis for new therapeutic targets. The endogenous cysteine cathepsininhibitor stefin A is a suppressor of breast cancer metastasis to bone that is coexpressed with cathepsin B in bonemetastases. In this study, we used the immunocompetent 4T1.2 model of breast cancer which exhibitsspontaneous bone metastasis to evaluate the function and therapeutic targeting potential of cathepsin B inthis setting of advanced disease. Cathepsin B abundancy in the model mimicked human disease, both at the levelof primary tumors and matched spinal metastases. RNA interference–mediated knockdown of cathepsin B intumor cells reduced collagen I degradation in vitro and bone metastasis in vivo. Similarly, intraperitonealadministration of the highly selective cathepsin B inhibitor CA-074 reducedmetastasis in tumor-bearing animals,a reduction that was not reproduced by the broad spectrum cysteine cathepsin inhibitor JPM-OEt. Notably,metastasis suppression by CA-074 was maintained in a late treatment setting, pointing to a role in metastaticoutgrowth. Together, our findings established a prometastatic role for cathepsin B in distant metastasis andillustrated the therapeutic benefits of its selective inhibition in vivo. Cancer Res; 72(5); 1199–209. �2012 AACR.

IntroductionEarly detection of breast cancer has increased the 5-year

survival rate to more than 85%. However, progression tometastatic disease in tissues such as lung and bone reducesthe survival rate to 23% due to limited curative treatmentsavailable (1). Hence, it is important to determine the mechan-isms involved in primary tumor cell invasion and spread todistant sites such as bone, to allow identification of moleculartargets for new therapies.Proteases contribute to tumor cell invasion and angiogen-

esis and are commonly associated with metastasis. It is now

recognized that cysteine proteases play pivotal roles in cancerprogression. There are 11 members in the human cysteinecathepsin family (cathepsins B, C, H, F, K, L, O, S, V,W, X/Z) and19 in mouse (2). In normal cells, cysteine cathepsins areexpressed primarily in lysosomes and have roles in antigenpresentation, apoptosis, autophagy, and cellular homeostasis(3–6). Secreted cathepsin K is well documented to contributeto bone resorption and remodeling (7). In cancer, the cellularlocalization of lysosomal cysteine cathepsins is often altered.Intracellular, cell surface, and secreted cysteine cathepsins areinvolved in distinct tumorigenic processes in vivo, such asangiogenesis, invasion through extracellular matrices, andmetastasis (8–10).

Of the cysteine cathepsins, B and L have been implicatedmost in tumorigenesis (9, 11, 12). Cathepsins B and L areprognosticmarkers in several types of cancer, including breast,with increased primary tumor expression associated with pooroutcome (13–15). In the PyMT-induced transgenic mammarycarcinoma model, ablation of cathepsin B reduces and delayslungmetastasis (16), while increased expression of cathepsin Benhances metastasis in the same model (17). The prometa-static role of cysteine cathepsins has also been shown in amultistage transgenic model of pancreatic cancer. Treatmentof mice with a broad spectrum cysteine cathepsin inhibitordecreases tumor vascularization and invasion (18), an effectthat is enhanced when cysteine cathepsin inhibitors are com-bined with chemotherapy (19). Taken together, these dataindicate that cysteine cathepsins have important roles inmetastasis. Because bone is the most common site of distant

Authors' Affiliations: 1Research Division, Peter MacCallum CancerCentre, East Melbourne; 2Department of Pathology; 3Department of Bio-chemistry and Molecular Biology; 4Sir Peter MacCallum Department ofOncology, University of Melbourne, Parkville, Australia; 5Department ofPathology, Stanford University, School of Medicine, Stanford, California;6Institute of Drug Research, The School of Pharmacy, Faculty of Medicine,Campus Ein Karem, The Hebrew University, Jerusalem, Israel; 7Depart-ment of Pharmacology, Wayne State University, School of Medicine;8Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit,Michigan; and 9Department of Microbiology and Immunology, StanfordUniversity, School of Medicine, Stanford, California

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Belinda S. Parker, Research Division, Peter Mac-Callum Cancer Centre, St Andrews Place, East Melbourne, Victoria, 3002,Australia. Phone: 61-3-9656-1285; Fax: 61-3-96561411; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-11-2759

�2012 American Association for Cancer Research.

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metastasis in breast cancer patients, determining the roles ofcysteine cathepsins in bone metastasis is crucial.

Our previous studies support a role for cysteine cathepsinsin bone metastasis (20). Using our unique 4T1.2 syngeneicmodel of spontaneous bone metastasis (21), we identified theendogenous cysteine cathepsin inhibitor stefin A as a metas-tasis suppressor. Tumor cell stefin A significantly reducedpulmonary and bone metastasis in the murine model and wasan independent predictor of good prognosis in a cohort of 142breast cancer patients (20). Of the cysteine cathepsins, cathep-sin B was coexpressed with stefin A in primary tumors andmetastases, suggesting that stefin A suppressed metastasis viainhibition of cathepsin B. This was supportive of previousstudies that show that a shift in equilibrium between cathepsinB and its endogenous inhibitor stefin A predicts poor survivaloutcomes (22, 23).

In this study, we used our 4T1.2 model to show a criticalfunction of cathepsin B in breast cancer metastasis to bone.Molecular suppression and selective therapeutic inhibition ofcathepsin B significantly reduces pulmonary and bone metas-tasis. This study provides evidence that cathepsin B is apotential therapeutic target for treatment of breast cancerpatients with metastatic disease.

MethodsImmunohistochemistry

All tissues were fixed in 10% neutral-buffered formalin for 24hours, bones decalcified in 20%EDTA, pH8.0, and embedded inparaffin. Archived humanmaterial was used in which previousinformed consentwas obtained fromall patients. Citrate buffer(10 mmol/L, pH 6.0) high temperature and pressure antigenretrieval was required for human tissues, and immunohis-tochemistry (IHC) was then carried out on tissue sections asdescribed previously (20).

Cell maintenanceThe 4T1.2 lines (21) were derived in our laboratory from the

parentalmouse 4T1mammary tumor cell line. The 4T1 cell linewas derived by Fred Miller that, along with a series of otherlines, was originally derived from a spontaneous mammarytumor in Balb/cmice. Cells weremaintained in alpha-modifiedeagle medium (a-MEM) supplemented with 5% FBS (JRHBiosciences) and cultured at 37�C in an atmosphere of 5%CO2 for no more than 4 weeks.

Generation of stable cathepsin B knockdown and basevector control clones

Short hairpin RNA (shRNA) for cathepsin B (50-GGAT-GACCTGATTAACTA-30) along with control shRNA (50-AGTACTGCTTACGATACGG-30) lacking a murine gene targetwere inserted into pRetroSuper retroviral expression plasmids(Oligoengine) before transfection into PT67 retroviral packag-ing cells with Lipofectamine 2000 (Invitrogen) and infection of4T1.2 target cells. Stably transduced cells were selected withpuromycin (8 mg/mL). Single cells were sorted (FACSVantage-Diva, BD Biosciences), expanded in culture, and analyzed forgene expression by real-time reverse transcription (RT)-PCRand immunoblotting. Four clones of tumor cells containing

control base vector (4T1.2 BV) or cathepsin B–specific hairpins(4T1.2 CTSB kd) were pooled for subsequent analysis.

Immunocytochemistry4T1.2, 4T1.2 BV, and CTSB kd cells were grown on uncoated

glass coverslips for 24 hours, fixed in 3.7% cold paraformalde-hyde, and then blocked in PBS containing 2 mg/mL BSA. Cellswere then incubated overnight at 4�C with primary antibody,rabbit anti-human cathepsin B (24). Cathepsin B expressionwas detected with FITC Alexa Fluor 488 donkey anti-rabbitsecondary antibody (Invitrogen) and imaged on a Zeiss LSM510 META confocal microscope.

Live cell proteolysis assayProteolysis wasmeasured according to previously published

procedures (26, 27). Briefly, glass coverslips were coated with50 mL of 25 mg/mL quenched fluorescent substrate DQ-colla-gen IV (Invitrogen) mixed with reconstituted basement mem-brane (rBM) Cultrex (Trevigen). For collagen I, plastic cover-slips were coated with 100 mL of 25 mg/mL DQ-collagen I(Invitrogen) mixed with collagen I (Advanced BioMatrix). Theratio of quenched fluorescent components to nonfluorescentcomponentswas 1:40 for all experiments. Cellswere seeded at adensity of 5,000 cells per coverslip and cultured in serum-containing medium for 48 hours. Activity based probe (ABP)GB123 (1mmol/L)was added to themedia 16 to 18 hours beforeimaging. Fluorescence was then observed by confocal micros-copy on a Zeiss LSM 510 with a 40�water immersion lens. Celllysates labeled with GB123 were separated by SDS-PAGE andvisualized by scanning with a typhoon flatbed laser scanner(excitation/emission 633/680 nm). For inhibitor studies, thehighly selective cathepsin B inhibitor CA-074 was synthesizedand purified in the Bogyo laboratory, CA and used at 20mmol/L[in dimethyl sulfoxide (DMSO) vehicle] and replenished after24 hours. The fluorescent GB123 probe was produced asdescribed previously (28).

In vivo metastasis assayCells (1 � 105) in PBS were mixed 1:1 with Matrigel (BD

Biosciences) and injected orthotopically into the 4th mam-mary gland of 6 week-old female Balb/c mice (Walter andEliza Hall Institute), 20 mice per group. Tumor volume wascalculated as: volume¼ length (mm)� width (mm)2/2. Micewere injected (intra peritoneal, 200 mL/20 g mouse) dailywith 50 mg/kg JPM-OEt (Drug Synthesis and ChemistryBranch, Division of Cancer Treatment and Diagnosis,National Cancer Institute, MD), CA074, or vehicle (saline/5% DMSO), 3 days after tumor inoculation. After 30 days, allmice were culled upon any signs of distress due to metas-tasis by inhalation overdose and the organs resected. Tumorburden was measured by quantitative RT-PCR (qRT-PCR) aspreviously validated and published (Eckhardt and collea-gues, 2005) and 3 mice out of 20 were randomly selected forhistology. For qPCR, multiplex amplification of hygromycin/vimentin was used to measure tumor cell DNA signal (hygro-mycinR) relative to a marker present in all cells (vimentin).Reactions were conducted on an ABI Prism 7000 thermo-cycler. Relative tumor burden (RTB) in an organ was

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Cancer Res; 72(5) March 1, 2012 Cancer Research1200




calculated by: RTB ¼ 10;000

2DCt, in which DCt ¼ Ct (hygromycin)

– Ct (vimentin). Late treatment studies were conductedas above but with resection of primary tumors at day 15(�0.3 g) and treatment of mice with CA-074 or vehicle dailybeginning at day 20 and until signs of metastasis wereevident in either group, at which point all mice were culledand assessed for metastatic burden. Any mice with primarytumor regrowth were excluded from the study. Mousestudies were conducted only after approval by the PeterMacCallum Ethics Review Board.

Noninvasive imaging of 4T1.2 tumor-bearing burdenmice4T1.2 cells were injected into the 4th mammary fat pad of 6-

week old Balb/c mice followed 3 days later by daily intraper-itoneal injections of CA-074 (50 mg/kg) or vehicle (5% DMSO/saline.) Probes (25 nmol GB123 or 2 nmol osteosense750, VisEnImaging, Inc.) were administered through the tail vain in asolution of 67% DMSO, 33% PBS in 100 mL final volume, 24hours before imaging. Mice were then anesthetized with 3%isoflurane, and tomographic images taken with the fluores-cence tomography (FMT)2500 imaging system, using the 680 or

750 nm channels. All animal experiments were approved by theStanford Administrative Panel on Animal Care.

Statistical analysisStatistics were conducted with the data analysis package

within GraphPad Prism 5.0 forWindows (GraphPad Software).Unless otherwise stated, tests comparing 2 means are a Stu-dent t test, with equal variance assumed. Error bars indicateSEM unless otherwise stated.

ResultsExpression of cathepsin B in 4T1.2 spontaneous bonemetastasis mimics that of the human disease

To support a role for cathepsin B in bone metastasis, weevaluated protein levels in a cohort of human primary tumors(n ¼ 10) and bone metastases (n ¼ 5). In agreement withprevious reports (15, 29), cathepsin B was detected in primarytumor and stromal cells (Fig. 1A). Importantly, cathepsin Bwasalso present in bonemetastases. In all tumors, cathepsin B wasexpressed in tumor-associated stromal cells, including thelocal vasculature (Fig. 1A) and more than 60% of tumorsexpressed the protease in the tumor cells specifically. This

Figure 1. Cathepsin B levels inhuman breast tumors (A) and mouse4T1.2 tumors (B). Sections wereimmunostained for cathepsin B orwith rabbit immunoglobulin (Ig)isotype control antibodies andvisualized with DAB. All tissues werecounterstainedwith hematoxylin andeosin (H&E). Arrow indicatesvasculature. Scale bar represents50 mm.

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Cathepsin B Inhibition Reduces Bone Metastasis

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Figure 2. Cathepsin B knockdown in 4T1.2 tumor cells reduces the degradation of DQ-collagen. A,Western blot detection of cathepsin B protein expression in4T1.2 parental cells, pooled 4T1.2 BV, and CTSB kd clones. B, cathepsin B activity. C, cathepsin B expression (green) in 4T1.2 cells (blue). Framecaptures of 3D reconstructions of parental 4T1.2 andCTSB kd tumor cells (cyan nuclei staining) grown on rBMcontaining 25 mg/mLDQ-collagen IV (D; green)or collagen I containing 25 mg/mL DQ-collagen I (E; green), in the presence of 1 mmol/L GB123 (red), along with 4T1.2 cells treated with 20 mmol/L CA-074.F, quantification of GB123 cathepsin activity and DQ-collagen IV degradation products from panel D (G). H, quantification of GB123 cathepsin activityand DQ-collagen I degradation products from panel E (I). �, P < 0.05; ��, P < 0.005; ��, P < 0.0001.

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staining pattern was consistent in all primary breast tumorsand bone metastases examined.To ensure that the cathepsin B expression in human tumors

was similar in our 4T1.2 murine model (30), expression wasassessed in primary tumors and matched metastases in bone.Cathepsin B was most intense at the periphery of the primarytumors, suggestive of a role in tumor invasion through theextracellular space (ECM). In spine metastases, levels werehighest in tumor cells adjacent to bone and other stromalcomponents (Fig. 1B). This distribution of cathepsin B con-firmed the value of the 4T1.2 model to dissect the function ofcathepsin B in bone metastasis. Cathepsin B expression wasalso apparent in human and murine pulmonary metastases(Supplementary Fig. S1).

Reduced cathepsin B in tumor cells lowers spontaneousmetastasis to boneTodetermine the functional consequences of lowering cathe-

psin B levels specifically in tumor cells, we stably transduced4T1.2 cellswith cathepsinB–specific shRNA(CTSBkd) or controlhairpins (4T1.2 BV). Stable knockdown of cathepsin B wasverified by real-time RT-PCR (Supplementary Fig. S2) andimmunoblotting (Fig. 2A). Inhibition of cathepsin B activitywas also confirmed by a fluorometric assay that utilizes theselective cathepsin B substrate, Z-Arg-Arg-NHMec (25). Con-sistent with decreased levels, cathepsin B activity in cell lysateswas significantly lower in cathepsin B knockdown cells (Fig. 2Band Supplementary Fig. S3). In addition, intracellular andmembrane-associated cathepsin B staining was almost unde-tectable in the knockdown cell lines comparedwith the parentaland base vector lines (Fig. 2C). Importantly for proteolysis andmetastasis assays, cathepsin B knockdown did not alter cellproliferation (Supplementary Fig. S4).To assess proteolytic activity in the 4T1.2 clonal pool, we

used a recently established three-dimensional (3D) in vitroconfocal assay that mimics the architecture of the tumormicroenvironment and enables imaging and quantificationof proteolysis by living cells (26, 27). The degradation ofcollagen IV or collagen I by 4T1.2 parental cells, CTSB kdcells, and cells treated with the cathepsin B selective inhib-itor CA-074 (31) was assessed in the presence of the cathep-sin B selective ABP GB123. Cells were grown in rBM contain-ing DQ-collagen IV or in collagen I containing DQ-collagen I.Images of the cells grown on rBM revealed tight 3D spher-oids (Fig. 2D). In contrast, cells embedded in collagen I grewas monolayers (Fig. 2E). 3D images of cell induced proteo-lysis are available online (Supplementary Materials, movieM1–M6). Knockdown or inhibition of cathepsin B did notaffect cellular morphology on either matrix. Importantly,GB123 staining confirmed a significant reduction in cysteinecathepsin activity in 3D cultures by CTSB knockdown orinhibition by CA-074 compared with parental 4T1.2 cellsgrown on either rBM or collagen I (Fig. 2F and H respec-tively, P < 0.005). Degradation of DQ-collagen IV was reducedby cathepsin B knockdown (Fig. 2G) and to a greater extentby the CA-074 inhibitor. In addition, cathepsin B knockdownor inhibition in 4T1.2 cells led to a significant decreasein DQ-collagen I degradation (Fig. 2I, P < 0.05). Because

collagen I is the predominant bone matrix protein, this issuggestive of a role for cathepsin B in bone degradation.These studies also support the use of small-molecule cathep-sin B inhibitors to reduce proteolysis.

We next injected parental 4T1.2, pooled 4T1.2 BV controls orCTSB kd cells into the mammary gland of Balb/c mice to testthe impact of cathepsin B knockdown on metastasis. Therewas no difference in orthotopic tumor growth between theparental andCTSB kd groups (Fig. 3A) even though knockdownwas maintained (Fig. 3B). Base vector control tumorsexpressed cathepsin B at the tumor edge as seen previouslywith the 4T1.2 tumors, whereas expression in CTSB kd tumorcells was negligible. Although cathepsin B knockdown did notreduce orthotopic mammary tumor growth, lung metastasiswas significantly decreased in mice-bearing CTSB kd tumorscompared with 4T1.2 parental or 4T1.2 BV tumors (Fig. 3C, p <0.0001). Although control 4T1.2 BV tumors grew at a slowerrate than 4T1.2 parental tumors (Fig. 3A), the metastaticburden at endpoint was not altered, hence 4T1.2 BV cells wereappropriate controls for assessing the effect of cathepsin Bknockdown on metastatic burden. The decrease in lung met-astatic burden was confirmed by histology (Fig. 3D). Of par-ticular interest to this study, bone (spine) metastasis was alsoreduced dramatically in the CTSB kd group compared with4T1.2 (Fig. 3E, P < 0.005) and base vector control (Fig. 3E, P <0.05). Histologic analysis of spine from 4T1.2 BV and CTSB kdtumor-bearing mice revealed tumor deposits in the controltissue that were not detectable in the spines of mice-bearingCTSB kd tumors (Fig. 3F).

Selective inhibition of cathepsin B suppresses distantmetastasis

To test the therapeutic efficacy of cysteine protease inhibi-tors in vivo, we treated 4T1.2 tumor–bearing mice with JPM-OEt (a broad spectrum cysteine protease inhibitor; ref. 32) andthe selective CA-074 inhibitor (31). Treatment of 4T1.2 tumor–bearing mice with either inhibitor had no impact on primarytumor growth (Fig. 4A and D). However, analysis of metastasisrevealed a significant difference between these compounds.Treatment with the broad spectrum JPM-OEt inhibitor did notsignificantly reducemetastasis to lung or bonewhen comparedwith vehicle (Fig. 4B and C). In contrast, CA-074 treatmentsignificantly decreasedmetastasis to lung (Fig. 4E, P< 0.05) andbone (Fig. 4F, P < 0.05). The effect of the inhibitors in vivo wasfurther confirmed by histopathologic analysis. Visible lung andspine metastatic nodules were observed in vehicle and JPM-OEt treatment groups, whereas tumors were undetectable inthe CA-074 group (Fig. 4H and I). Importantly, measurement ofcysteine cathepsin B and L activity in primary tumor tissuelysates derived from mice treated with JPM-OEt and CA-074revealed that both inhibitors reduced tumor cathepsin Bactivity significantly (Fig. 4G, P < 0.005), yet CA-074 was moreeffective. In contrast, only JPM-OEt inhibited cathepsin Lactivity (Fig. 4G, P < 0.05). Hence, the reduced efficacy ofJPM-OEt could be due to the inhibition of other cysteinecathepsins that may have antitumorigenic functions (33)and/or due to a slightly reduced ability to suppress cathepsinB activity.






The CA-074 treatment studies were conducted from day 3until metastasis was evident. This did not specifically allowassessment of the role of cathepsin B in the later steps ofmetastasis, preventing metastatic outgrowth once tumorcells had already lodged in distant tissues. We thereforecarried out a repeat CA-074 treatment experiment aimed atrecapitulating a late-treatment setting. Primary tumors wereremoved and treatment began at day 20, a time whenmicrometastases can be detected in lung and bone. Analysis

of metastatic burden revealed that there was a significantdecrease in lung and overall bone metastases in this setting(Fig. 5), revealing a role for cathespin B in late stages ofmetastatic spread.

Noninvasive in vivo imaging of cathepsin B activity inbone metastases

We used the ABP GB123 to label active cysteine cathe-psins in vivo in the presence and absence of cathepsin B

Figure 3. CathepsinBknockdown reduces spontaneousmetastasis to lung andbone. A, primary tumor volumes. Error bar, SEM.B, IHCdetection of cathepsinB in primary tumors from 4T1.2 BV and CTSB kd tumor–bearing mice. C, real time qPCR detection of tumor burden in lung (with hygromycin-tagged 4T1.2cells). Relative tumor burden compares tumor cell hygromycin DNA signal to that of vimentin expression in all murine cells. D, hematoxylin and eosin (H&E)-stained lung sections of 4T1.2 BV and CTSB kd tumor bearing mice. E, real-time qPCR detection of tumor burden in spine (with hygromycin-tagged 4T1.2cells). F, H&E-stained spine sectionsof 4T1.2BVandCTSBkd tumor-bearingmice. T, tumor region. Scale bar, 50mm. �,P<0.05; ��,P <0.005; ��,P <0.0001

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Figure 4. Treatment of tumor-bearing mice with cathepsin inhibitors. Mice-bearing 4T1.2 tumors were treated with 50 mg/kg JPM-OEt, CA-074, orvehicle (5% DMSO/saline), 3 days after tumor inoculation. Primary tumor volumes more than 28 days in, JPM-OEt (A) and CA-074 (D) treatmentgroups. Error bar, SEM (n¼ 20). Real-time qRT-PCR of genomic DNA containing the tumor-specific hygromycin reporter gene was used for detection oftumor burden in lung (B and E) and spine (C and F) of JPM-OEt- and CA-074–treated mice, respectively. Relative tumor burden is a measure of thehygromycin signal relative to vimentin signal that is present in all cells. G, cathepsin B and L activity in vehicle-, JPM-OEt-, and CA-074–treatedprimary tumors. H&E–stained lung (H) and spine (I) sections of vehicle-, JPM-OEt-, and CA-074–treated 4T1.2 tumor–bearing mice. T, tumor region.Scale bar, 50 mm. �, P < 0.05; ��, P < 0.005; ��, P � 0.0005.






inhibitors. Mice-bearing 4T1.2 tumors were treated with CA-074 as described previously. To visualize cysteine cathepsinactivity, GB123 was injected before in vivo imaging with(FMT2500; VisEn Medical). FMT whole body imagingallowed visualization of cysteine cathepsin activity at thesite of orthotopic mammary tumors (Fig. 6A). Cysteinecathepsin activity was reduced in the CA-074-treated ani-mals even though the tumors were similar in size, suggestingthat the loss in GB123 fluorescence was due to decreasedcathepsin B activity (Fig. 6A). This loss was confirmed by exvivo imaging of the tumors (Fig. 6B), however was notsignificant and likely due to detection of other active cys-teine cathepsins such as cathepsin L in the primary tumor byGB123. The cellular localization of cysteine cathepsin activ-ity was additionally resolved by confocal microscopy (Fig.6C). Strongest activity was detected at the boundaries of the4T1.2 tumors and this was lost in CA-074–treated mice. Wenext completed whole body scans of mice injected with bothGB123 and Osteosense750 probes. Osteosense750 is a fluo-rescent diphosphonate imaging agent that detects activebone remodeling and hence allows bone visualization. Non-invasive whole body images of the spine region revealedGB123 signal in the spine of mice-bearing 4T1.2 tumors thataligned with Osteosense750 signal (Fig. 6D), and hencebone remodeling suggestive of an osteolytic tumor region.The GB123 signal was decreased in mice treated with CA-074 (Fig. 6D). Ex vivo images of the spines confirmed this(Fig. 6E and F). Quantitation of total GB123 fluorescence inspine, revealed a significant reduction in fluorescence in

CA-074–treated compared with control-treated mice(Fig. 6G, P < 0.05).

DiscussionBonemetastases occur in a high proportion of breast cancer

patientswithmetastatic disease and are associatedwith severemorbidity and eventualmortalitywhen visceral organs becomeinvolved. Dissecting the molecular mechanisms involved intumor cell survival and outgrowth in bone is essential for thedevelopment of targeted therapies to reduce patient mortality.Here, we report that tumor-derived cathepsin B is a keycontributor to bone metastasis. Our data show for the firsttime that selective inhibition of cathepsin B with small-mol-ecule inhibitors significantly reduces metastasis and has ther-apeutic potential.

There are a number of critical steps required for growth oftumors in bone. This includes collaboration between the tumorcells themselves and the bone stroma for stimulating angio-genesis, invasion through the bone matrix and growth beyondthe micrometastatic stage. Cathepsin B upregulation has beendocumented in several human cancers, including breast, pros-tate, andmelanoma (15, 34, 35). Our data show that cathepsin Bhas critical tumor-specific functions in breast cancer metas-tasis that are completely independent from primary tumorgrowth. We show that tumor cells themselves can directlydegrade the major bone matrix protein collagen I and thatcathepsin B is important, if not essential, in this process.Previous studies have implicated tumor cell cathepsin B in

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Figure 5. Late-stage treatment ofmice-bearing 4T1.2 tumors withCA-074. Five days after primarytumor resection, mice were treateddaily intraperitoneally with 50 mg/kg CA-074 or vehicle (5% DMSO/saline). Real-time qRT-PCR ofgenomic DNA containing thetumor-specific hygromycinreporter gene was used fordetection of tumor burden in lung(A), spine (B), femur (C). D,combined real-time qRT-PCRdetection in spine and femurs. Errorbar, SEM. �, P � 0.05.

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degradation of ECM proteins including fibronectin, laminin,and collagen IV (36, 37). In addition, and in support of ourstudies, inhibition of cathepsin B has been shown to reducecollagen I degradation by prostate carcinoma cell lines (38),again suggestive of a tumor cell–specific role in bone lysis.Consistent with a role in invasion, we showed a criticalfunction of tumor-derived cathepsin B in metastasis in the4T1.2 model. Although orthotopic tumor growth was notaltered by cathepsin B knockdown, pulmonary and bone(spine) metastases were significantly reduced. As outgrowthof disseminated tumor cells into macrometastases requiresmultiple steps including invasion through secondary tissues,preventing ECM degradation by cathepsin B inhibition is likelyto reduce metastatic burden.Cathepsin B has also been implicated in stromal cell–asso-

ciated protumorigenic functions. In breast cancer, thishas been shown with the MMTV-PyMT model in whichhost-derived cathepsin B promotes lung metastasis (39). Theexpression of cathepsin B predominantly in tumor-infiltratingmacrophages indicated that macrophage-derived cathepsin Bcontributed to metastasis. The expression of cathepsin B intumor-associated endothelial cells and macrophages has beenassociated with tumor progression via promotion of angio-genesis (17, 18). Cathepsin B has also been reported to beexpressed in stromalfibroblasts andmacrophages in colon and

prostate cancers (40, 41). Taken together, cathepsin B has keyroles in tumor progression, by invasion through the ECM andstimulation of angiogenesis. Our treatment of 4T1.2 tumor–bearing mice with the selective small-molecule cathepsin Binhibitor CA-074 enabled inhibition of both tumor and stromalcathepsin B and allowed assessment of the therapeutic benefitof inhibiting this protease in vivo. Consistent with cathepsin Btranscript knockdown, treatment with CA-074 did not alterprimary tumor growth yet suppression of metastasis wasimpressive. In fact, bone metastases could not be detectedhistologically in tumor-bearingmice treatedwith the inhibitor,an impressive result considering the aggressive nature of 4T1.2tumors. Due to the role of cathepsin B in degradation ofcollagen IV and I, we hypothesized that metastasis inhibitionwas caused by decreased invasion at both the primary andmetastatic site. Our results in a late treatment setting support arole for cathepsin B in metastasis posttumor cell arrest indistant tissues. Commencement of CA-074 treatment afterprimary tumor resection significantly decreased metastaticgrowth in lung and bone. The use of cathepsin inhibitors todecrease spontaneous breast cancer metastasis to bonefrom the mammary gland has not been reported previously.However, use of cathepsin K (another cysteine cathepsin;ref. 42) and cathepsin G (a serine protease; ref. 43) inhibitorshave been documented to suppress the formation and growth

Figure 6. In vivo imaging ofcathepsin B activity in tumor-burdened mice. A, fluorescenceimages of live mice-bearing 4T1.2tumors treated with vehicle or CA-074 followed by GB123. B, totaltumor fluorescence in tumorsexcised 24 hours after probeinjection and imaged ex vivo with theFMT2500 system. C, histology ofvehicle- and CA-074–treatedmammary tumor tissues from A.Nuclei were visualized with DAPI. D,fluorescence spinal images of livemice-bearing 4T1.2 tumors treatedwith vehicle or CA-074 followed byGB123 and osteosense 750. E, totalmetastases fluorescence in spinesexcised 24 hours after probeinjection and imaged ex vivo. F,histology of vehicle- and CA-074–treated spine tissues fromD. Tissueswere stained with DAPI, and imageswere taken with a 60� objective.Red, GB123 fluorescence; blue,DAPI. The colorimetric scale barsindicate nanometers offluorescence. G, quantitation of totalGB123 metastases fluorescence invehicle- and CA-074–treated spineswith the FMT2500 system. Meanfluorescence with SE is shown.�, P < 0.05. Scale bar, 50mm.






of breast cancer osteolytic lesions in experimental models ofmetastasis.

The therapeutic benefit of CA-074 treatment in the 4T1.2model was in contrast to the broad spectrum inhibitor JPM-OEt, which did not decrease lung or spine metastases. Inagreement with our studies, treatment of polyoma middle Toncogene-induced mammary carcinomas with JPM-OEt didnot alter tumor weights and lung metastasis (33). Together,these studies reveal the importance of using specific targetedtherapies in vivo. JPM-OEt targets several cysteine cathepsins,some of which could actually have tumor suppressive func-tions. For example, cathepsin L activity is decreased in highlymetastatic 4T1.2 tumors compared with weakly or nonmeta-static tumors (20) suggesting inhibitory effects of this proteaseon tumor progression, as has been suggested before in skintumorigenesis (44). The importance of using specific proteaseinhibitors is clear from early clinical trials using matrix metal-loproteinase (MMP) inhibitors in which inhibition of certainMMPs actually has deleterious effects in patients (45, 46).These studies highlight the significant effort needed to deter-mine which specific proteases contribute to advanced diseaseprogression before potential drugs can be tested in the clinic.

Current treatments available for patients with bone metas-tasis are aimed primarily at reducing the morbidity associatedwith bone lysis, a hallmark of breast cancer bone disease. Onetarget of such treatments is cathepsin K. Cathepsin K isexpressed predominantly in osteoclasts in which it is secretedand has a key role in bone proteolysis, including degradation ofcollagen I, osteonectin, andosteopontin (47). The potential role

of cathepsin K in osteolytic bone tumors is evident from itsexpression in tumor-associated osteoclasts (48), in breast andprostate metastases in bone, and in giant cell tumor of bone(49). Cathepsin K inhibitors block bone resorption (50), reduc-ing the pain associatedwith osteolytic disease, and in turn slowthe growth of bone tumors. However, as with the use ofbisphosphonates that also suppress osteolysis, treatment isnot curative. Therefore, an ideal treatment (or combination oftreatments) is one that targets both the tumor cell and theassociated stromal populations that promote osteolysis, angio-genesis, and tumor growth. The fact that cathepsin B hasessential roles throughout the metastatic cascade, in tumorand stromal cells, suggests that combining cathepsin B inhi-bitors with conventional therapies may be of clinical value.

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

Grant SupportThis work was supported by the U.S. Department of Defense BCRP Concept

AwardW81XWH-05-1-0444 (B.S. Parker), National Health and Medical ResearchCouncil (NHMRC) grant 509325 (B.S. Parker, R.L. Anderson, andM.S. Bogyo) andCareer Development Award 566553 (B.S. Parker), National Breast Cancer Foun-dation (Australia) fellowship (R.L. Anderson), NHMRC postgraduate scholarshipID 466645 (to N.P. Withana). This work was also supported, in part, by the NIH[R01CA131990 (B.F. Sloane)] and NIH/National Cancer Institute grant ROICA90291 (R.L. Anderson).

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

Received August 19, 2011; revised December 27, 2011; accepted January 3, 2012;published OnlineFirst January 19, 2012.

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2012;72:1199-1209. Published OnlineFirst January 19, 2012.Cancer Res Nimali P. Withana, Galia Blum, Mansoureh Sameni, et al. Cathepsin B Inhibition Limits Bone Metastasis in Breast Cancer

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