InhibitionofGrowthandMetastasisofMouseMammaryCarcinoma ...PBS for 20 minutes at 20jC in the dark.For E-cadherin immunostain-ing, cells were washed with PBS and fixed for 5 minutes

Inhibitionof GrowthandMetastasis ofMouseMammary Carcinomaby Selective Inhibitor ofTransforming Growth Factor-B Type IReceptor Kinase In vivoRongrong Ge,1Vaishali Rajeev,1Partha Ray,2 Edmund Lattime,2 Susan Rittling,4 SatyaMedicherla,5

Andy Protter,5 AlisonMurphy,5 Jit Chakravarty,5 Sundeep Dugar,5 George Schreiner,5

Nicola Barnard,3 andMichael Reiss1

Abstract Purpose: Transforming growth factor-h (TGF-h) suppresses tumor development by inhibitingcellular proliferation, inducing differentiation and apoptosis, and maintaining genomic integrity.However, once tumor cells escape from the tumor-suppressive effects ofTGF-h, they often con-stitutively overexpress and activateTGF-h, which may promote tumor progression by enhancinginvasion, metastasis, and angiogenesis and by suppressing antitumor immunity. The purpose ofthis study was to test this hypothesis usingTGF-h pathway antagonists.Experimental Design:We examined the effects of selectiveTGF-h type I receptor kinase inhib-itors, SD-093 and SD-208, on two murine mammary carcinoma cell lines (R3Tand 4T1) in vitroand in vivo.Results: Both agents blocked TGF-h-induced phosphorylation of the receptor-associatedSmads, Smad2 and Smad3, in a dose-dependent manner, with IC50 between 20 and 80 nmol/L.TGF-h failed to inhibit growth of these cell lines but stimulated epithelial-to-mesenchymal trans-differentiation, migration, and invasiveness into Matrigel in vitro. These effects were inhibited bySD-093, indicating that these processes are partly driven byTGF-h. Treatment of syngeneic R3Tor 4T1tumor-bearingmicewith orally given SD-208 inhibitedprimary tumor growth aswell as thenumber and size of metastases. In contrast, SD-208 failed to inhibit R3T tumor growth ormetas-tasis in athymic nude mice. Moreover, in vitro anti-4T1cell cytotoxicT-cell responses of spleno-cytes from drug-treated animals were enhanced compared with cells from control animals.In addition, SD-208 treatment resulted in a decrease in tumor angiogenesis.Conclusion:TGF-h type I receptor kinase inhibitors hold promise as novel therapeutic agents formetastatic breast cancer.

Transforming growth factor-h (TGF-h) is a multifunctionalcytokine that plays a key role in embryonic development,wound healing, hematopoiesis, and immunity as well as diseasestates, such as cancer and chronic inflammatory conditions(1, 2). The TGF-h signal is transduced by a pair of transmem-brane serine/threonine kinase receptors (3). Binding of biolog-ically active TGF-h to type II receptor (ThRII) homodimersresults in recruitment of two type I receptor (ThRI) moleculesinto heterotetrameric complexes, which in turn results inactivation of the ThRI kinase by the ThRII kinase. In responseto receptor activation, two cytosolic proteins, Smad2 andSmad3, become transiently associated with and phosphorylatedby the ThRI kinase, allowing them to form heteromericcomplexes with a third homologue, Smad4. These complexesare translocated to the nucleus and bind to DNA, which resultsin the transcriptional regulation of wide range target genesinvolved in cell differentiation, proliferation, apoptosis, migra-tion, and extracellular matrix production (4–14).

In the normal adult mammary gland, TGF-h controls thebalance between cell renewal and cell differentiation and loss(15 – 19). It is likely through this homeostatic function

Cancer Therapy: Preclinical

Authors’Affiliations:Departments of1InternalMedicine and 2Surgery,The CancerInstitute of NewJersey, and 3Department of Pathology, University of Medicine andDentistry of NewJersey-RobertWood Johnson Medical School, New Brunswick,New Jersey; 4Department of Genetics, Rutgers University, Piscataway, NewJersey; and 5Scios, Inc., Fremont, CaliforniaReceived1/23/06; revised 4/7/06; accepted 5/4/06.Grant support: National Cancer Institute, USPHS awards CA-41556 andCA-94431and Department of Defense Breast Cancer Research Program IDEAaward DAMD17-00-1-0510 (M. Reiss), National Institutes of Diabetes, Digestiveand Kidney Diseases grant DK-67685 (S. Rittling), National Cancer Institute grantCA-42908 (E. Lattime), and National Cancer Institute Cancer Center supportgrant CA-72720.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 accordancewith18 U.S.C. Section1734 solely to indicate this fact.Note: Current address for V. Rajeev: University of Maryland Medical Center,Baltimore, MD. Current address for S. Rittling:The Forsyth Institute, Boston, MA.Supplementary data for this article are available at Clinical Cancer Research Online(http://clincancerres.aacrjournals.org/).Requests for reprints:Michael Reiss, Division of Medical Oncology, Departmentof Internal Medicine,The Cancer Institute of NewJersey, University ofMedicine andDentistry of NewJersey-Robert Wood Johnson Medical School, Room 2007, 195Little Albany Street, New Brunswick, NJ 08903. Phone: 732-235-6031; Fax:815-333-3972; E-mail: [email protected].

F2006 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-06-0162

www.aacrjournals.org Clin Cancer Res 2006;12(14) July15, 20064315

that TGF-h suppresses tumor development, and the loss ofthis function is an early event in epithelial carcinogenesis(20–23).

Besides its homeostatic function, TGF-h plays a key role intissue injury. Tissue injury results in a rapid locally increasedactivation of TGF-h, which induces epithelial cells to assumea fibroblastoid and dispersed phenotype [epithelial-to-mesenchymal transdifferentiation (EMT)] and to produceextracellular matrix components of what later becomes a scar(24–27). This process is characterized by the subcellular redis-tribution of cell-cell cohesion molecules, such as E-cadherin,and of the F-actin cytoskeleton and is normally self-limited inspace and time, allowing epithelial cells to revert back to theircohesive epithelioid phenotype (28).

Although escape from the growth-suppressive function ofTGF-h is an early event during mammary epithelial celltransformation, the tissue injury response to TGF-h can beretained (7, 29–31). For example, in experimental modelsof mammary cancer, tumor cells escape from the growth-suppressive function of TGF-h early on but retain the EMTresponse. Moreover, in late-stage tumors, TGF-h signaling maybecome oncogenic by constitutively inducing EMT associatedwith a highly invasive and metastatic tumor phenotype. Thereis considerable evidence that tumor-derived TGF-h plays animportant role in stimulating and maintaining tumor growthby activating stromal cells, enhancing angiogenesis, andsuppressing antitumor immunity (32–36). Thus, tumor-asso-ciated TGF-h endows neoplastic cells with a selective advantagebecause of both the altered responsiveness of the tumor cellsthemselves (tumor cell-autonomous effects) and its actions onthe supporting host cell infrastructure.

Based on these observations, the idea has been put forth thattargeting TGF-h signaling might represent a potentiallypowerful novel approach to the treatment of invasive breastcancers (32, 37–40). Recently, a series of pyridopyrimidine-based ThRI kinase inhibitors have been developed that areboth potent and highly selective in vitro (41–43). Moreover, anorally bioavailable derivative compound, SD-208, inhibited theinduction of pulmonary fibrosis in a rat model and blocked itsprogression when given transiently to animals with establisheddisease (44). In addition, SD-208 inhibited the growth of ratgliomas in vivo (45). The current study represents the firstreport of the efficacy and safety of this compound in mousemodels of metastatic mammary cancer in vivo. In addition, weprovide evidence that SD-208 enhances antitumor immunity,inhibits tumor angiogenesis, and reduces metastatic clonoge-nicity in vivo .

Materials andMethods

Reagents and antibodies. Human recombinant TGF-h1 (1 Ag/mL;Austral Biologicals, San Ramon, CA) was dissolved in 4 mmol/L HCland 1 mg/mL bovine serum albumin (Sigma, St. Louis, MO). SD-093and SD-208 are selective chemical inhibitors of the ThRI receptor kinasethat inhibit cellular responses to TGF-h with an IC50 of 20 and80 nmol/L, respectively (42, 44, 45). For in vitro studies, SD-093 (Scios,Inc., Sunnyvale, CA) was dissolved in DMSO (Sigma) and stored at�70jC. For in vivo studies, SD-208 was suspended in 1% (w/v)methylcellulose in water. Rabbit polyclonal antibodies directed againstSmad2 and Smad3 were obtained from Zymed Laboratories (South SanFrancisco, CA), and a mouse monoclonal antibody directed against

Smad4 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).Rabbit polyclonal antibodies directed against phosphorylated Smad2(pSmad2) and phosphorylated Smad3 (pSmad3) were produced in ourlaboratory (46, 47). A monoclonal antimouse E-cadherin antibody wasobtained from BD Transduction Laboratories (Bedford, MA). AlexaFluor 488–conjugated phalloidin was obtained from Molecular Probes(Eugene, OR).

Cell culture. NMuMG, a spontaneously immortalized, nontumori-

genic cell line derived from a normal murine mammary gland, was

obtained from Dr. Daniel DiMaio (Yale University, New Haven, CT).

NMuMG cells were maintained in high-glucose (4.5 g/L) DMEM (Life

Technologies, Grand Island, NY) supplemented with 10% (v/v) fetal

bovine serum (Sigma), 10 Ag/mL insulin (Sigma), and 10 Ag/mL

gentamicin (Life Technologies). MDA-MB-435 human breast carcinoma

cells (derived from a malignant pleural effusion) were maintained in

DMEM/F-12 (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v)

fetal bovine serum, pyruvate (10 mmol/L; Life Technologies), and

nonessential amino acids (1 mmol/L; Life Technologies). R3T

mammary carcinoma cells (48) were maintained in MEM-a (Invitro-

gen) supplemented with 8% (v/v) fetal bovine serum with the addition

of 3 Ag/mL puromycin (Sigma), 800 Ag/mL geneticin (G418;

Invitrogen), and 10 Ag/mL gentamicin (Invitrogen). 4T1 cells (refs.

49, 50; kindly provided by Dr. Fred Miller, Michigan Cancer

Foundation, Detroit, MI) were maintained in DMEM supplemented

with 10% (v/v) fetal bovine serum. TMLC cells (kindly provided by

Dr. Daniel Rifkin, New York University, New York, NY) were main-

tained in RPMI 1640 (Life Technologies) supplemented with 10% (v/v)

fetal bovine serum with the addition of 200 Ag/mL geneticin (G418).Cell proliferation assays. Cells were plated at 2 � 104 per well in 24-

well cluster dishes (Corning, Inc., Corning, NY). Twenty-four hourslater, cultures were treated with 1 Amol/L SD-093 or vehicle onlyfollowed 15 minutes later by addition of 100 pmol/L (2.5 ng/mL) TGF-h1. Cells were counted 72 hours later using a Beckman Coulter counter(Model 0039, Beckman, Inc., Miami, FL).

Epithelial-to-mesenchymal transdifferentiation. For assessment ofsubcellular F-actin fiber distribution, cells were washed with PBSand fixed with buffered formalin for 10 minutes. Following washeswith PBS, cells were permeabilized using 0.1% (v/v) Triton X-100 inPBS for 5 minutes and then incubated with 0.165 Amol/L Alexa Fluor488–conjugated phalloidin with 1% (w/v) bovine serum albumin inPBS for 20 minutes at 20jC in the dark. For E-cadherin immunostain-ing, cells were washed with PBS and fixed for 5 minutes usingmethanol precooled to �20jC. Air-dried slides were then incubatedwith 5% (v/v) goat serum for 20 minutes at room temperaturefollowed by incubation with 2 Ag/mL mouse monoclonal anti-E-cadherin antibody in 2.5% (v/v) goat serum for 1 hour at roomtemperature. The cells were then washed 3 � 5 minutes with PBSfollowed by incubation with 2 Ag/mL rhodamine-conjugated goatanti-mouse IgG for 45 minutes in the dark. In both cases, staineddishes were then washed 3 � 5 minutes with PBS, mounted usingVectashield mounting medium (Vector Laboratories, Inc., Burlingame,CA), and viewed using a Zeiss epifluorescence microscope (model090477, Carl Zeiss, Microimaging, Inc., Chester, VA) equipped with aMTI charge-coupled device camera (model DC 330E, DAGE-MTI, Inc.,Michigan City, IN).

Western blot analysis. For detection of Smad proteins, semicon-fluent cell cultures were lysed in situ using buffer composed of150 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA,1 mmol/L EGTA, and 1% (v/v) Triton X-100 in the presence of proteaseinhibitors (Complete Mini Protease Inhibitor Cocktail Tablets, RocheDiagnostics Corp., Indianapolis, IN) for 30 minutes at 4jC. Cell lysateswere subjected to Western blot analysis as described previously (42).Activated Smad2 (pSmad2) and activated Smad3 (pSmad3) weredetected using our own rabbit anti-pSmad2 and anti-pSmad3 anti-bodies at a 1:1,000 dilution. Total Smad2, Smad3, and Smad4 weredetected using rabbit anti-Smad2, rabbit anti-Smad3, and mouseanti-Smad4 antibodies, respectively, at a 1:500 dilution.


www.aacrjournals.orgClin Cancer Res 2006;12(14) July15, 2006 4316

In vitro cell migration and invasion assays. For migration assays,uncoated polyethylene terephthalate track etched membrane (24-wellinsert; pore size, 8 Am; Becton Dickinson, Franklin Lakes, NJ) insertswere equilibrated by adding 0.5 mL cell culture medium to the upperand lower chambers followed by incubation at 37jC for 2 hours. Forinvasion assays, BD Biocoat Growth Factor Reduced Matrigel InvasionChambers (24-well insert; pore size, 8 Am; BD Biosciences, Bedford,MA) were rehydrated by adding 0.5 mL warm (37jC) culture mediumto the upper chambers followed by 2-hour incubation at 37jC. Forboth assays, medium used for equilibration was removed and 105 cellswere plated in the upper chambers. TGF-h1 (100 pmol/L, 2.5 ng/mL),SD-093 (1 Amol/L), both agents, or vehicle only were added to bothupper and lower chambers. Following a 24-hour incubation at 37jC,cells in suspension were aspirated, the inserts were washed twice withPBS, and the cells adherent to the top of the inserts were removed byscraping the upper surface of the membrane with a cotton tipapplicator. The cells that had migrated to the underside of the insertswere fixed and stained using the DiffQuick (Dade Behring, Newark,DE) staining kit. Cells in 10 random squares of 100 � 100 Am ineach well were counted at �200 magnification using quadruplicatewells per assay condition and the results were expressed as number ofcells/mm2.

Plasminogen activator inhibitor/luciferase assay. The plasminogenactivator inhibitor (PAI)/luciferase assay was done as described by Abeet al. (51) with minor modifications. Briefly, 1.6 � 104 TMLC cellssuspended in 100 AL medium were plated in 96-well tissue culturedishes and allowed to attach for 3 hours at 37jC in a 5% CO2

incubator. After 3 hours, the medium was replaced with 100 ALmedium containing either TGF-h at concentrations ranging from 0 to100 pmol/L (0-2.5 ng/mL) or a suspension of 3.2 � 104 4T1 or R3Tcells. The cells were lysed 14 hours later at room temperature andluciferase activity in cell lysates was determined using the PromegaLuciferase Reporter Assay System following the protocol recommendedby the manufacturer (Promega, Madison, WI) using a TD-20/20Luminometer (Turner Designs, Sunnyvale, CA).

Animal experiments. Viral antibody-free 8- to 9-week-old female

athymic (Harlan Laboratory, Indianapolis, IN) 129S1 and BALB/cJ (The

Jackson Laboratory, Bar Harbor, ME) were weighed and randomly

assigned to different treatment groups. Tumor cells (1 � 106) were

injected orthotopically into the right second or third fat pads (52). Mice

were then treated with single daily 0.2 mL doses of vehicle [1% (w/v)

methylcellulose, SD-208 (20 mg/kg), or SD-208 (60 mg/kg)] by gavage

beginning 1 day following tumor cell inoculation. Animal body weight

and tumor sizes were measured thrice weekly. R3T cells were inoculated

into 129S1 or athymic nude mice, whereas 4T1 cells were injected into

syngeneic BALB/cJ mice. Primary tumors were resected from athymic

nude mice 18 days after R3T cell injection or from BALB/cJ mice 18 days

after 4T1 cell injection, and animals continued to be treated postoper-

atively for the indicated periods. Blood was collected 2 hours following

the penultimate dose to determine plasma levels of SD-208. At sacrifice,

liver and body weight were measured. Lungs, liver, kidneys, adrenal

glands, and major lymph node groups were visually inspected for the

presence of tumor metastases and then processed for routine histology.

The burden of lung metastases was estimated by digitally imaging H&E-

stained cross-sections of fixed and paraffin-embedded lung preparations

and measuring the areas of individual metastases using IP Lab (version

3.6.5, Scanalytics, Inc., Billerica, MA) image analysis software.Pharmacokinetic analysis. Descriptive pharmacokinetic variables

were determined by standard model-independent methods. Non-compartmental analysis was done using WinNonlin version 4.0.1(Pharsight, Mountain View, CA). Because individual mice were not usedto describe a full profile, variables were calculated using mean data.Samples with SD-208 concentration below quantifiable limits(10 ng/mL) were assigned the value of 0 for the analysis. Nominaltime points were used for all calculations. AUC(0-8) is the area under theplasma concentration-time curve from time 0 to 8 hours for animalsdosed with SD-208.

Cell proliferation, apoptosis, and angiogenesis. Tissue sections were

deparaffinized, rehydrated, and stained with H&E, rat antimouse

monoclonal CD34 IgG2a (1:100; CL8927AP; Cedarlane, Hornby,

British Columbia, Canada), or rabbit polyclonal anti-Ki-67 (1:100;

ab833-500; Novus Biologicals, Littleton, CO). Biotinylated secondary

antibodies (1:150; Zymed Laboratories) were used for detection.

Apoptotic cells were identified by terminal deoxynucleotidyl transfer-

ase–mediated dUTP nick end labeling (TUNEL) assay using the In situ

Cell Death Detection kit (Roche Molecular Biochemicals, Palo Alto,

CA). Streptavidin-alkaline phosphatase (1:100) was added, and the

staining was developed with naphtol as substrate and levamisole as

inhibitor of endogenous alkaline phosphatase (Fast Red Tablets; Roche

Molecular Biochemicals). The negative control for CD34 was normal rat

IgG2a (CBL605; Chemicon International, Temecula, CA). The negative

control for Ki-67 staining was normal rabbit IgG (SC-2027; Santa Cruz

Biotechnology). The total number of CD34+ microvessels were counted

in five randomly selected high-power (�400) fields in areas of viable

tumor. To assess the percentage of proliferating cells, the proportion of

Ki-67-positive nuclei was determined. At least 600 nuclei were counted

in five randomly selected high-power (�400) fields in areas of viable

tumor. To assess the degree of apoptosis, TUNEL-positive cells were

counted in the tumor in five randomly selected high-power (�400)

fields in areas of viable tumor.CTL activity. Splenocytes (7 � 106) harvested from 4T1 tumor-

bearing mice were restimulated with 1.4 � 105 irradiated 4T1 cells in a24-well plate in a total of 2 mL tissue culture medium in the presence of50 Amol/L h-mercaptoethanol and 1 Amol/L SD-093. Cultures weremaintained for 5 days at 37jC, 5% CO2, after which nonadherent ef-fector cells were harvested. 4T1 cells were labeled in 100 ACi 51Cr (Perkin-Elmer Life and Analytical Sciences Inc., Wellesley, MA) for 1 hour at 37jCand then washed thrice with warm medium. 51Cr-labeled 4T1 target cells(1� 104) and effector cells were then added at various E:T ratios in a totalof 200 AL TCM to 96-well round-bottomed plates. Plates were incubatedfor 4 hours at 37jC, 5% CO2, 100 AL supernatant was removed, andreleased 51Cr was quantified using a gamma counter (PackardBioscience, Meriden, CT). Percentage of specific lysis was calculatedusing the formula: (experimental release � spontaneous release) � 100 /(maximal release in 5% Triton X-100 � spontaneous release).

Real-time Taqman PCR. Transcript levels of individual genes wereassayed in frozen R3T tumor tissue specimens using quantitative real-time PCR on an ABI Prism 7700 Sequence Detector (AppliedBiosystems, Foster City, CA) as described previously (44).

Microarray gene expression profiling. RNA was extracted from snap-frozen livers using Trizol reagent (Invitrogen) and purified using RNeasymini columns (Qiagen, Valencia, CA) according to the manufacturer’sinstructions. The RNA concentration was adjusted to 1 Ag/AL and itsquality was assessed on a RNA chip using an Agilent Bioanalyzer (AgilentTechnologies, Palo Alto, CA). Isolated total RNA was processed asrecommended by Affymetrix, Inc. (Santa Clara, CA). Biotin-labeledcRNA was size fractionated to 35 to 200 bases long using Affymetrixprotocols and hybridized to the mouse genome 430 2.0 GeneChip set at45jC for 16 hours in an Affymetrix GeneChip Hybridization Oven 320.This chip was designed using the most recent publicly available draft ofthe mouse genome and contains >45,000 probe sets covering >39,000transcripts and variants representing >34,000 well-substantiated mousegenes. Each GeneChip was then washed and stained with streptavidin-phycoerythrin using an Affymetrix Fluidics Station 400 and scanned on aAffymetrix Gene Array scanner. Scanned image files were analyzed usingthe Microarray Suite 5.0 software (Affymetrix). Scaling and normaliza-tion were carried out using the 500 Normalization Control probe setincluded on the 430 2.0 GeneChip set.

Statistical analyses. For analyses of tumor growth rates, metastasisburden, microvessel densities, Ki-67 staining, apoptosis rates, andmRNA expression levels one-way ANOVA tests were done using InStatversion 3.0 (GraphPad Software, Inc., San Diego, CA) or two-wayrepeated-measures ANOVA tests using JMP version 5.1 (SAS Institute,Inc., Cary, NC).

TargetingTGF-b Signaling inMammary Cancer


Results

Effects of TbRI kinase inhibitor on mammary carcinoma cellgrowth. TGF-h is a known inhibitor of cell cycle progression ofepithelial cells. As shown in Fig. 1A, TGF-h strongly inhibitedgrowth of NMuMG cells in a ThRI kinase-dependent manner.In contrast, growth of R3T and 4T1 mammary carcinoma cellswas not significantly inhibited by exogenous TGF-h. Moreover,treatment with the ThRI kinase inhibitor SD-093 by itself didnot affect growth of NMuMG or R3T cells and caused only asmall but statistically significant inhibition of 4T1 cell growth(unpaired t test with Welch correction).

TbRI kinase inhibitor blocks TGF-b-induced EMT. As shownin Fig. 1B, TGF-h induced EMT in NMuMG cells as manifested

by spindle-cell morphology, reduced cell-cell cohesion, andcellular redistribution of F-actin and E-cadherin. These effectswere blocked by pretreatment with the ThRI kinase inhibitorSD-093. Both mammary cancer cell lines displayed some degreeof EMT even in the absence of exogenous TGF-h. 4T1 cells wereless cohesive than R3T cells presumably because they expresslittle or no E-cadherin at their cell surface (Fig. 1B; ref. 53). Astreatment with SD-093 by itself caused cells to assume a moreepithelioid phenotype than untreated cells, the basal level ofEMT seems to be dependent on endogenous TGF-h signaling.Conversely, EMT became more pronounced with the additionof exogenous TGF-h, an effect that was blocked by treatmentwith SD-093. EMT was most clearly detectable at 24 to 36 hoursfollowing the addition of TGF-h. Thus, in these two mammary



carcinoma cell lines, the ability of TGF-h to induce EMThas been retained, whereas its ability to suppress growth hasbeen lost.

TbRI kinase inhibitor affects cell migration and invasion. Sev-eral studies have suggested that tumor cell migration andinvasion might be driven by TGF-h (29, 54, 55). As shown inFig. 1C, migration of NMuMG cells was strongly inhibited byTGF-h in a ThRI kinase-dependent manner. In contrast, underthe same culture conditions, TGF-h stimulated migration of bothmammary carcinoma lines, and this effect was also dependenton ThRI kinase activity. Moreover, treatment with SD-093 aloneinhibited motility of R3T cells, indicating that the migratoryphenotype of these cells is driven, at least in part, by endogenousTGF-h signaling.

Besides cell migration, treatment with exogenous TGF-hsignificantly stimulated the ability of R3T and 4T1 cells toinvade Growth Factor Reduced Matrigel (3.3- and 2-fold,respectively). This effect was completely inhibited by pretreat-ing the cells with SD-093 (Fig. 1D). Moreover, treatment of R3Tcells with SD-093 alone inhibited invasion by 40%, indicatingthat their invasive phenotype is also partly dependent onendogenous TGF-h signaling.

To investigate whether excessive production and/or extracel-lular activation of TGF-h was responsible for activation of TGF-h signaling, we conducted coculture experiments using TMLCcells that express a TGF-h-inducible PAI-1 promoter driving aluciferase reporter gene construct (51). As shown in Fig. 1E,exogenous TGF-h resulted in a concentration-dependentincrease in luciferase activity in TMLC cells. Cocultivation ofTMLC cells with mammary carcinoma cells induced asignificant increase in luciferase activity (Fig. 1E). In bothcases, the effect was abolished in the presence of pan-TGF-hantibody, thereby showing that both mammary cancer cell linesproduce biologically active TGF-h capable of inducing specificgene responses in neighboring cells.

SD-208 inhibits mammary tumor growth and metastasis invivo. Because of its superior oral bioavailability comparedwith SD-093, the SD-208 compound was used for all in vivoexperiments. In a typical assay, R3T or 4T1 carcinoma cells wereinoculated into the mammary fat pad, and mice were treatedwith single daily doses of vehicle, SD-208 (20 mg/kg), or SD-

208 (60 mg/kg) beginning 1 day after tumor cell inoculation.As shown in Fig. 2A, SD-208 treatment inhibited primary R3Ttumor growth in syngeneic 129S1 mice in a dose-dependentmanner. In addition, both the number and the size of lungmetastases were significantly reduced by SD-208 treatment (Fig.2B). The antitumor activity of SD-208 was not limited to theR3T mammary carcinoma: SD-208 also caused a dose-dependent statistically significant growth delay of 4T1 ortho-topic tumors in syngeneic BALB/c mice (Fig. 2C) as well as adose-dependent reduction in the number of metastases to thelungs (Fig. 2D) and other organs, including liver, adrenalglands, stomach, and retroperitoneal lymph nodes (Fig. 2D,inset). When R3T cells were inoculated into athymic nude mice,the rate of growth of primary tumors was substantially higherthan in 129S1 mice (Fig. 3A). Consequently, the experimentshad to be terminated sooner than in 129S1 mice, possiblyaccounting for the lower number of lung metastases found atautopsy (Figs. 2B and 3B). In contrast to the antitumor effectsseen in syngeneic mice, SD-208 failed to retard the growth ofR3T tumors in athymic nude mice (Fig. 3A). In addition,treatment with SD-208 had little effect on the size or numbersof lung metastases (Fig. 3B).

Pharmacokinetic and pharmacodynamic properties of SD-208. Asshown in Fig. 4A, SD-208 inhibited TGF-h-induced Smad2phosphorylation in cultured cells in a dose-dependent manner,with an IC50 of f80 nmol/L, indicating that it is slightlyless potent than SD-093, which has an IC50 of 20 nmol/L.Figure 4B depicts the kinetics of SD-208 in plasma of each ofthe mouse strains following a single oral dose of either 20 or60 mg/kg. Peak plasma levels as well as areas under the curvevaried as function of the dose given in each of the three mousestrains. Although levels achieved at 1 hour following a dose of60 mg/kg were not significantly different across the threemouse strains (ANOVA, P = 0.1893), trough levels at 24 hourswere significantly lower in athymic than in 129S1 mice(ANOVA, P = 0.0291). Moreover, the area under the curveachieved in 129S1 mice was approximately twice as high asin athymic animals (Fig. 4B). These differences may account, inpart, for the lower efficacy against R3T tumors we observed inathymic compared with 129S1 mice (see above). Plasma levelsobtained at 2 hours following the penultimate dose of SD208

Fig. 1. A, effects ofThRI kinase inhibitor on anchorage-dependent growth. R3Tand 4T1cells were treated with vehicle control, 100 pmol/L (2.5 ng/mL) TGF-h, 1 Amol/LSD-093, or100 pmol/LTGF-h + 1 Amol/LSD-093 for 72 hours after attachment.TGF-h strongly inhibited the growth of NMuMG cells, and this effect was effectively blockedby pretreatment with SD-093. In contrast,TGF-h had no significant effect on the growth of R3Tand 4T1compared with vehicle control. However, treatment with SD-093alone caused a small but statistically significant inhibition of 4T1cell growth. Columns, mean of four separate experiments, triplicate wells per condition; bars, SE. Resultsfor each cell line were analyzed by one-wayANOVA (NMuMG: P < 0.0001, R3T: P = 0.2469, and 4T1: P = 0.0074).B, effects ofThRI kinase inhibitor on EMT. Phase-contrastpictures (magnification, �100) show that the R3Tand 4T1become spindeloid followingTGF-h (100 pmol/L, 2.5 ng/mL) treatment, which can be completely blocked bypretreatment with SD-093 (1 Amol/L).Treatment withTGF-h resulted in acquisition of actin stress fibers (magnification,�400) and loss of E-cadherin (magnification,�200)from cell margins. Pictures were taken using a Zeiss epifluorescence microscope attached to a MTI charge-coupled device camera. C, effects ofThRI kinase inhibitoron cell migration in vitro. Cells were plated onto polyethylene terephthalate membranes as described in Materials and Methods and incubated for 24 hours withTGF-h(100 pmol/L, 2.5 ng/mL), SD-093 (1 Amol/L), or both.TGF-h strongly inhibited motility of NMuMG cells, an effect that was completely blocked by pretreatment of cells withSD-093. In contrast,TGF-h significantly stimulated motility of both R3Tand 4T1cells, and pretreatment with SD-093 effectively blocked this effect. In fact, in R3Tcells,SD-093 treatment strongly inhibited cell motility compared with vehicle treatment, independently of the presence of exogenousTGF-h (P < 0.001). Columns, mean numberof cells/mm2 of four separate experiments, triplicate wells per condition; bars, SE. Results for each cell line were analyzed by one-wayANOVA (NMuMG: P < 0.0001,R3T: P < 0.0001, and 4T1: P < 0.0001). D, effects ofThRI kinase inhibitor on invasion in vitro. Cells were plated onto (Growth Factor Reduced) Matrigel Invasion cellculture inserts as described in Materials and Methods and incubated for 24 hours withTGF-h (100 pmol/L, 2.5 ng/mL), SD-093 (1 Amol/L), or both.TGF-h significantlystimulated the invasive capacity of both R3Tand 4T1cells, an effect that was blocked by preincubation of the cells with SD-093. Columns, mean number of cells/mm2 ofthree separate experiments, triplicate wells per condition; bars, SE. Results for each cell line were analyzed by one-wayANOVA (R3T: P = 0.0034 and 4T1: P = 0.0001).E, mammary carcinoma cells activate PAI-1promoter activity in neighboringTMLC cells.To generate a standard curve, 1.6� 104 TMLC cells were plated in 96-well tissueculture dishes and allowed to attach for 3 hours at 37jC in a 5% CO2 incubator. After 3 hours, the mediumwas replaced with100 AL medium containingTGF-h at theindicated concentrations. Luciferase activity was determined in cell lysates 14 hours later. Left,TGF-h induced activation of the PAI-1promoter and luciferase expression in adose-dependent manner.To determine whether mammary carcinoma cells produce and/or activateTGF-h in their microenvironment,TMLC cells were coincubated with 4T1or R3Tcells, or vehicle only, in the presence or absence of1D11murine pan-TGF-h-neutralizing antibody. Luciferase activity was determined in cell lysates 14 hours later.Points, mean of two independent experiments, triplicate wells per condition; bars, SD. Conditions were compared pairwise by Student’s t test. Both cell lines inducedactivation of the PAI-1promoter and luciferase expression in neighboringTMLC cells. In both cases, this effect was blocked in the presence ofTGF-h-neutralizing antibody.



in treatment experiments were very similar to those achievedfollowing a single dose, indicating that prolonged repeateddosing did not have adverse effects on pharmacokinetics (datanot shown). SD-208 treatment was tolerated without observabletoxicity for up to 56 days of continuous daily administration.

To determine whether treatment with SD-208 resulted indetectable inhibition of ThRI kinase activity in vivo , wemeasured pSmad2 and pSmad-3 levels in protein extracts fromindividual mammary fat pad tumors harvested and flash frozenwithin 2 hours of the penultimate SD-208 dose (Fig. 4C). 4T1

Fig. 2. Effects of SD-208 on R3Tand 4T1mammary carcinomas in vivo. R3T (A and B) or 4T1 (C andD) cells (1�106) were injected orthotopically into the right secondor third mammary fat pads of 8- to 9-week-old129S1or BALB/c mice, respectively (8-10 mice per group). Mice were treated with single 0.2 mL daily doses of vehicle[1% (w/v) methylcellulose], SD-208 (20 mg/kg), or SD-208 (60 mg/kg) by gavage starting1day after tumor cell inoculation. Fat pad tumor sizes were measured thriceweekly. Primary tumors were resected when they reached a size off1,000 mg. Median tumor weights for each treatment group. Bars, SE. Animals continued to be treatedpostoperatively and were autopsied at the end of the experiment. At that time, lungs, liver, kidneys, adrenal glands, and major lymph node groups were visually inspectedfor the presence of tumormetastases and thenprocessed for routine histology. Lungmetastases were quantified by digitally imaging H&E-stained cross-sections of fixed andparaffin-embedded lung preparations and measuring the areas of individual metastases using IP Lab version 3.6.5 image analysis software. SD-208 treatment inhibitedboth primary andmetastatic tumor growth in129S1mice.A, R3T primary tumor growth.Treatment with SD-208 caused significant primary tumor growth delay (P = 0.0026,univariate test of drug effect over time, repeated-measures multivariateANOVA). B, R3T lung metastases.The number of metastases per group of surviving mice (left) andthe sizes (right, box plot ; boxes, median values withupper and lower quartiles; filled square, mean;whiskers, 90th and10thpercentiles) of lungmetastases in SD-208-treatedmice were significantly smaller than in vehicle-treated control animals (P < 0.001for either 20 or 60 mg/kg/d SD-208 compared with vehicle, Kruskal-Wallis nonparametricANOVA). C, 4T1primary tumor growth.Treatment with SD-208 caused significant primary tumor growth delay (P = 0.017, univariate test of drug effect over time,repeated-measures multivariateANOVA). D, 4T1metastases.The total number of metastases per group of surviving mice (top left) as well as the sizes of lung metastases(top right) in high-dose SD-208-treated mice were significantly smaller than in low-dose or vehicle-treated control animals (P = 0.0016 and P < 0.0001, respectively,for 60 mg/kg/d SD-208 compared with either vehicle or 20 mg/kg/d SD-208, Kruskal-Wallis nonparametric ANOVA). In addition, the total number of macroscopically visiblemetastases to other sites also varied per treatment group (bottom).



tumors seemed to express slightly higher steady-state levels ofpSmad2 and pSmad3 than R3T tumors. However, in bothcases, pSmad levels were significantly reduced in tumorsobtained from SD-208-treated animals compared with thosefrom control animals.

To further substantiate inhibition of TGF-h signaling bySD-208 treatment in vivo , we compared the levels of mRNAexpression of several TGF-h-regulated target genes in snap-frozen R3T tumors by quantitative reverse transcription-PCR.As shown in Fig. 5A, the levels of mRNA of Serpine1 (PAI-1),connective tissue growth factor, Col1a2 (procollagen I, a2),and matrix metalloproteinase-2 were all significantly reduced intumors that had been exposed to SD-208.

In addition to measuring the expression of TGF-h targetgenes in tumor tissue, we also examined the effects of SD-208treatment on the gene expression profile of normal mousetissue. Because liver tissue is easily accessible and relativelyhomogeneous in terms of its cellular composition, wecompared the gene expression profiles in livers obtained within2 hours of the last dose of SD-208 from 129S1 mice in each ofthe three treatment groups. RNA extracted from four individuallivers was pooled, and two such pools from different groups ofmice were subjected to Affymetrix GeneChip analysis. Theexperiment was repeated using pooled RNAs obtained from thesecond 129S1 in vivo treatment study. Genes (n = 12,612) werescored as ‘‘present’’ on all GeneChips, indicating that mRNAwas expressed under all 12 conditions (3 dose levels, 2 pooledRNA samples per experiment, and 2 separate experiments). Aswe ended up with four replicates for each condition, expressionlevels for each feature could be compared across the threetreatment groups by ANOVA. A total of 1,693 Affymetrix IDsrepresenting 1,539 unique genes (79% with annotation) were

significantly differentially expressed between the three treat-ment groups (ANOVA, P < 0.05). These included 729 genesthat were down-regulated and 810 genes that were up-regulatedin livers of SD-208-treated animals compared with vehicle-treated control animals. We used the DAVID 2.0 softwaretool (56) to assign the genes to biological processes and mole-cular functions (Supplementary Fig. 1A and B). A modificationof the Fisher’s exact test (EASE score) was used to determinewhether biological categories to which genes from our listwere assigned were overrepresented (P V 0.05) compared themouse transcriptome in general (56). Genes that weredown-regulated in SD-208-treated animals are predominantlyinvolved in transcription, cell cycle control, intracellularsignaling, and apoptosis. Several studies have examined thespectrum of TGF-h-regulated genes in the mouse mammaryepithelial cell line NMuMG (9, 11, 13) and in mouse embryofibroblasts (12). Table 1 lists 31 genes that were repressed in thelivers of SD-208-treated mice and have been reported previ-ously to be induced by TGF-h in NMuMG mouse mammaryepithelial cells in vitro (13). Figure 5B illustrates the dose-dependent reduction of transcript levels of six of these genes bySD-208 treatment. In summary, SD-208 treatment clearlyinhibited ThRI kinase activity and TGF-h-regulated geneexpression in vivo.

In contrast to the repressed genes, the vast majority of genesthat were up-regulated in SD-208-treated animals encodedenzymes and cofactors involved in the intermediary metabo-lism of carbohydrates, proteins, and lipids, oxidative phos-phorylation, or detoxification reactions (Supplementary Fig.1C and D). Rather, the spectrum of induced genes wasstrikingly similar to that found in rat or mouse liver tissue inresponse to a variety of toxins (57–60). Thus, the profile of

Fig. 3. Effects of SD-208 on R3T mammary carcinoma in athymic nude mice in vivo. R3Tcells (1�106) were injected orthotopically into the right second or third mammaryfat pads of 8- to 9-week-old athymic nude mice, 8 to10 mice per group.Treatment and evaluation as described in Fig. 2. A, R3T primary tumor growth.Treatment withSD-208 failed to affect primary tumor growth (P = 0.9766, univariate test of drug effect over time, repeated-measures multivariateANOVA). B, lung metastases.The size(right) and number (left) of lung metastases were similar across treatment groups (P = 0.5946 and 0.4602, respectively, Kruskal-Wallis nonparametric ANOVA).



SD-208-induced genes likely reflects a generalized activationof hepatic metabolic pathways in response to chronic expo-sure to SD-208.

Effects of SD-208 on tumor-specific CTL activity. The notabledifference in therapeutic efficacy of SD-208 against R3T tumorsgrowing in syngeneic compared with immunodeficient micesuggested that enhancing antitumor immunity might representan important mechanism of action of this agent in vivo (45).To test this hypothesis in a second independent model, we

harvested splenocytes from 4T1 tumor-bearing mice fromeach of the three treatment groups and restimulated these withirradiated 4T1 cells for 5 days in the presence of SD-093.Nonadherent effector cells were then incubated with 51Cr-labeled 4T1 target cells, and 51Cr release was quantified usinga gamma counter (Fig. 6A). Specific CTL activity wassignificantly increased by in vivo treatment with SD-208 in adose-dependent manner. Moreover, this effect was onlyobserved as long as SD-093 was present during restimulation,

Fig. 4. Properties of SD-208.A, pretreatment of MDA-MB-435 breastcancer cells withThRI kinase inhibitorsinhibitedTGF-h-induced Smad2phosphorylation in a dose-dependentmanner, with an IC50 off20 and 80 nmol/Lfor SD-093 and SD-208, respectively.B, levels of SD-208 in plasma collectedfollowing a single oral dose of either20 or 60 mg/kg were determined byhigh-performance liquid chromatography.Points, average of three mice per group;bars, SD. Area under the curve wasestimated as described in Materials andMethods. C, determination of pSmad2 andpSmad3 levels in protein extracts fromindividual flash frozen carcinomas growingin mammary fat pads byWestern blotanalysis. A, control tumors; B, tumors fromanimals treated with 20 mg/kg/d SD-208;C, tumors from animals treated with60 mg/kg/d SD-208. Endogenous pSmad2and pSmad3 levels in R3Tand 4T1tumorswere clearly decreased by SD-208treatment.



indicating that 4T1 produce sufficient amounts of bioactiveTGF-h to suppress CTL activity or expansion in vitro. Similareffects, although of lower magnitude, were observed usingfreshly isolated splenocytes without in vitro restimulation(Fig. 6A).

To further elucidate the mechanisms of the antitumor effectsof SD-208 in vivo , we examined the rates of cell proliferation

and apoptosis of R3T tumors as well as their neovascularization(Fig. 6B and C). We failed to detect any significant differencesin the rate of tumor cell proliferation (Ki-67 staining) orapoptosis (TUNEL) between R3T tumors of untreated and SD-208-treated animals (Fig. 6C). However, tumor angiogenesis, asreflected by CD34+ microvessel density, was significantlyreduced in the tumors from animals treated with the highest

Fig. 5. Effects of SD-208 onTGF-h-regulated genes in vivo.A, real-time PCR: mRNA levels for the Serpine1 (PAI-1),connective tissue growth factor, Col1a1, and matrixmetalloproteinase-2 genes were decreased in R3T tumorsgrowing in129S1mice treated with 20 mg/kg SD-208compared with control animals (Student’s t test). B, effectsof SD-208 onmRNA transcript levels of individual geneswere compared byANOVA. Six genes that were repressedby SD-208 treatment in a dose-dependent manner areshown as example. Boxes, median values with upper andlower quartiles; filled square, mean;whiskers, 90th and10thpercentiles.



dose of SD-208 (Fig. 6C). This finding suggests that, besidesenhancing antitumor immunity, SD-208 also has modestantiangiogenic properties in vivo .

Finally, treatment with SD-208 was associated with a strikinginfiltration of tumors with eosinophilic leukocytes (Fig. 6B).In addition, in contrast to the undifferentiated spindle-cellappearance of control tumors, some of the R3T carcinomas inSD-208-treated animals were much more differentiated with

clearly recognizable ductal structures and areas of keratiniza-tion (Fig. 6B). This morphologic appearance was reminiscentof the metaplastic squamous carcinomas that develop in themammary glands of Smad4 conditional knockout mice and incarcinogen-treated transgenic mice that express a dominant-negative ThRII receptor (20, 61). Thus, chemical or geneticinhibition of TGF-h signaling seems to alter mammarycarcinoma cell differentiation in similar ways.

Table1. Effects of SD-208 treatment on gene expression in normal liver

Gene name Genbank Unigene ID Gene Ontologybiological process

Gene Ontologymolecular function

Actin-related protein 2/3complex, subunit1B

BC010275.1 Q91Z25 Regulation of actincytoskeleton

WD repeat

BC003441.1AF162768.1NM___023142.1

Actinin, a1 BE853286 Q99LJ3 Biological process unknown Actin bindingAnaphase-promotingcomplex subunit 5

AI842295 ANC5____MOUSE Ubiquitin cycle Molecular function unknown

BCL2-associatedtranscription factor1

BB833538 BCF1____MOUSE Negative regulation oftranscription

DNA binding

Caldesmon1 BC019435.1 Q8VCQ8 Regulation of smoothmuscle contraction

Actin binding

Calmodulin 3 NM____007589.1 O93622 G-protein-coupled receptorprotein signaling pathway

Calcium ion binding

AW543361AI256814

Core promoterelement-binding protein

BG800611 KLF6____MOUSE Regulation of transcription,DNA dependent

Zinc ion binding

BC020042.1NM____011803.1

Damage-specificDNA-binding protein1

NM____015735.1 Q99LV3 DNA repair Damaged DNA binding

AB026432.1AF159853.1

E26 avian leukemiaoncogene1, 5¶ domain

BB151715 Q8BVW8 Cellular process Binding

Erythrocyte proteinband 4.1-like 2

BG075070 Q80UE5 Cortical actin cytoskeletonorganization and biogenesis

Spectrin binding

Forkhead box P1 BM220880 Q6P221 Regulation of transcription,DNA dependent

Zinc ion binding

BB479687Glucosaminyl (N-acetyl)transferase 2, I-branchingenzyme

BM236768 BGIB____MOUSE Null N-acetyllactosaminide b-1,6-N-acetylglucosaminyltransferase activity

AB037596.1Glutathione synthetase NM____008180.1 GSHB____MOUSE Glutathione biosynthesis Glutathione synthase activity

BB125219BC003784.1U35456.1

Highmobility group box1 BF166000 HMG1____RAT Cell organization andbiogenesis

Heparin binding

NM____010439.1BC008565.1

(Continued on the following page)



Discussion

The discovery that many cancers produce or induce bioactiveTGF-h, which, in turn, acts as a tumor-promoting oncogene,has generated a great deal of enthusiasm for targeting tumor-associated bioactive TGF-h as cancer therapy (see refs. 37–40for recent reviews). Our study describes, for the first time, theinhibition of tumor growth and metastatic efficiency in vivo of

mouse mammary carcinomas by a novel selective chemicalinhibitor of the ThIR kinase, SD-208. This agent belongs to aclass of highly selective and potent pyridopyrimidine typeThRI kinase inhibitors that block TGF-h-induced Smadphosphorylation, reporter gene activation, and cellularresponses at submicromolar concentrations (42). These chem-icals bind to the ATP-binding site of the ThRI kinase andmaintain the enzyme in its inactive configuration (62).

Table1. Effects of SD-208 treatment on gene expression in normal liver (Cont’d)

Gene name Genbank Unigene ID Gene Ontologybiological process

Gene Ontologymolecular function

Hydroxyacyl-CoAdehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoAhydratase, a subunit

AW107842 Q5U5Y5 Fatty acid h-oxidation Enoyl-CoA hydratase activity

Integrin b1 (fibronectin receptor b) U37029.1 Q61126 G1-S transition ofmitotic cell cycle

Receptor activity

Jumonji domain containing1C AV293164 Q8C4I5 Null NullLIM and SH3 protein1 BB105164 LAS1____MOUSE Null Zinc ion binding

AV027151MARCKS-like protein NM____010807.1 Q5U2A3 Null Calmodulin binding

BC006757.1AV215438

Myeloid cell leukemia sequence1 NM____008562.1 P97287 Apoptosis Protein bindingBC003839.1U35623.1BC021638.1BC005427.1BB374534AV274748

Neuropilin1 NM____008737.1 NRP1____MOUSE Angiogenesis Receptor activityD50086.1AK002673.1AK011144.1

Peroxisome proliferative-activatedreceptor, c, coactivator1a

BB752393 Q8CCT1 Cellular lipidmetabolism Transcription coactivator activity

BB745167Rap guanine nucleotide exchange factor 2 AK018008.1 Q6NXI4 Protein kinase cascade Metal ion bindingRing finger protein (C3H2C3 type) 6 BI738010 Q8K565 Positive regulation of transcription,

DNA dependentDNA binding

RNA-bindingmotif, single-strandedinteracting protein1

AW541585 RBS1____MOUSE Regulation of translation ssDNA binding, dsDNA binding

SEC63-like (Saccharomyces cerevisiae) BC019366.1 Q8K2U5 Protein folding Unfolded protein bindingSyndecan1 NM____011519.1 SDC1____MOUSE Null Obsolete molecular function

BB533095Thyroid hormone receptor-associatedprotein 3

BC012655.1 Q8R353 Positive regulation of transcriptionfrom Pol II promoter

Transcription coactivator activity

BB531820Tight junction protein 2 BB758095 Q95M46 Null Protein bindingTGF-b1-induced transcript 4 AU016382 Q5U2A0 Regulation of transcription,

DNA dependentTranscription factor activity

BB357514AF201285.1

V-ral simian leukemia viral oncogenehomologue B (ras related)

BB465250 RALB____MOUSE Small GTPase-mediatedsignal transduction

GTPase activity

NOTE: Genes that were found to be significantly repressed in mouse livers by SD-208 treatment were compared with the profile of genes reported previously to beinduced byTGF-b in NMuMG mouse mammary epithelial cells in vitro (13). The table lists the 31genes that were represented in both sets along with their assignedmolecular function and biological process (Gene Ontology database).



Fig. 6. Mechanisms of action of SD-208 in vivo. A, effects of treatment with SD-208 on tumor-specific CTL activity. Day 5: Splenocytes harvested from 4T1tumor-bearingmice from each of the three treatment groups (n, vehicle; ., 20 mg/kg; y, 60 mg/kg) were restimulated with irradiated 4T1cells for 5 days in the presence of SD-208, afterwhich nonadherent effector cells were harvested. 51Cr-labeled 4T1target cells and effector cells were then incubated for 4 hours at 37jC, and released 51Cr was quantifiedusing a gamma counter. At an E:Tof100:1, 4T1-specific CTL activity was significantly increased in mice treated with 20 and 60 mg/kg/d SD-208 (P = 0.04 and 0.02,respectively, Student’s t test). Points, mean from two independent experiments, triplicate wells per condition; bars, SD. Day 0: Splenocytes harvested from 4T1tumor-bearingmice from each of the three treatment groups (n, vehicle; ., 20 mg/kg; y, 60 mg/kg) were immediately incubated with 51Cr-labeled 4T1target cells for 4 hours at 37jC,and released 51Cr quantified using a gamma counter. 4T1-specific CTL activity was increased as a function of the SD-208 dose the animals had received. B, sections of R3Ttumors from SD-208-treated129S1mice were stained by H&E as well as for markers of cell proliferation (Ki-67), apoptosis (TUNEL), and capillaries (CD34). Magnification,�200. R3T tumors are undifferentiated spindle-cell carcinomas. Moreover, R3T tumors have a high proliferative cell fraction and a low rate of apoptosis. However, in someR3T tumors, SD-208 induced adenosquamous differentiation. Moreover, SD-208 treatment was associated with a striking eosinophil infiltrate. C, treatment with SD-208did not affect tumor cell proliferation (Ki-67; P = 0.3066, one-wayANOVA) or apoptosis (TUNEL assay; P = 0.5474, one-wayANOVA). However, microvessel density,as determined by anti-CD34 immunostaining, was significantly reduced in tumors of animals treated with 60 mg/kg/d SD-208 compared with either vehicle-treated controlanimals or animals treated at the 20 mg/kg/d dose level (P = 0.0143, one-wayANOVA). For each of these analyses, a total of 20 randomly selected high-power (�400)fields in areas of viable tumor were examined in tumor sections from each treatment group.



The phenotype of mammary carcinoma lines differed fromthat of nontransformed NMuMG cells in several importantways. First, in contrast to NMuMG cells, both R3T and 4T1cells were refractory to TGF-h-mediated growth suppression,consistent with previous reports (reviewed in refs. 22, 32, 33,63). Both R3T and 4T1 cells seem to have retained a typicalepithelioid morphology in vitro, although 4T1 cells wereclearly less cohesive probably because they do not expressE-cadherin (53). Exogenous TGF-h clearly induced EMT inboth tumor lines in vitro in a ThRI kinase-dependent manner,whereas treatment with SD-093 alone seemed to increase thecohesion of R3T cells. Thus, the growth-inhibitory effect ofTGF-h had become uncoupled from the EMT response. Incontrast to NMuMG, TGF-h not only failed to inhibitmigration of R3T and 4T1 cells but also acted as a stimulusof cell motility, an effect that could be blocked by SD-093,indicating that it was mediated by the ThRI kinase. Inaddition, TGF-h treatment stimulated the ability of themammary carcinoma cells to invade Matrigel, an effect thatwas also inhibited by SD-093. Interestingly, TGF-h inhibitsin vitro invasion of normal trophoblast cells (64), whereas anextensive literature attests to the fact that TGF-h signalingstimulates or even drives the invasive behavior of malignantcells (see refs. 32, 33 for reviews). Thus, TGF-h seems toundergo a switch from being an antimigratory and anti-invasive to a promigratory and proinvasive factor duringmalignant transformation (29, 43, 55).

In contrast to their similar in vitro phenotype, R3T tumorsin vivo were composed of highly spindle-shaped cells, whereas4T1 tumor-derived carcinomas were more differentiated andepithelioid. This apparent discrepancy between in vitro andin vivo phenotypes is likely due to a combination of cellintrinsic factors (e.g., activation of the H-ras gene in R3T butnot in 4T1 cells) and a permissive microenvironment (53, 65).Thus, one might speculate that differences in local TGF-hactivation in tumors in vivo might influence the degree of EMT.If this were correct, one would expect treatment with SD-208 toinduce a reversal of the fibroblastoid to an epithelioidphenotype. This is, in fact, what we observed in R3T tumorsin animals treated with the highest dose of SD-208. In contrastto the undifferentiated spindle-cell appearance of controltumors, these carcinomas were much more differentiated, withclearly recognizable ductal structures and areas of keratiniza-tion. This morphologic appearance was reminiscent of themetaplastic squamous carcinomas seen in the mammary glandsof Smad4 conditional knockout mice and in carcinogen-treatedtransgenic mice that express a dominant-negative ThRIIreceptor (20, 61). Treatment with SD-208 was associated witha striking infiltration of tumors with eosinophilic leukocytes.The significance and the mechanism for this phenomenon areunclear. However, Takaku et al. (66) reported that gastricpolyps that develop in Smad4 heterozygous mice are associatedwith an eosinophilic cell infiltrate. Conversely, a nasallydelivered DNA encoding TGF-h1 can suppress pulmonaryeosinophilic responses to infectious agents (67). Thus, atten-uation of TGF-h signaling seems to promote eosinophilia,whereas increased expression of TGF-h1 suppresses it.

As reported previously, orthotopic 4T1 or R3T invasivecarcinomas efficiently metastasized to the lung parenchyma(48, 68). Most importantly, treatment of the mice with SD-208significantly retarded primary tumor growth in both models in

a dose-dependent manner. The antitumor effects of SD-208were more pronounced against R3T than against 4T1 tumorspossibly as a consequence of the higher growth rate of 4T1tumors or the stronger TGF-h activation by R3T cells (Fig. 1E).Alternatively, the activation of Ha-ras and/or expression ofPyVMT in R3T may have conferred particular sensitivity toTGF-h antagonists. Besides retarding the growth of primarytumors, treatment with SD-208 significantly reduced thenumber of lung metastases in both models. However, 4T1metastases were significantly larger than those derived fromR3T tumors, and the effect of SD-208 on their size was notablyless. The therapeutic effect of SD-208 against 4T1 was entirelyconsistent with those observed in the 4T1 model by otherinvestigators using different types of TGF-h antagonists,including dominant-negative ThRII receptors (29), solubleThRII exoreceptors (69), neutralizing anti-TGF-h antibodies(70), or other chemical ThR kinase inhibitors (71). Ourobservations suggest that the effects of TGF-h antagonists ontumor growth rates may be quite distinct from those onmetastatic efficiency. Consistent with this idea, expression of adominant-negative ThRII receptor in 4T1 or in humanMCF10CA1a mammary carcinoma cells reduced their meta-static ability without affecting primary tumor growth (29, 72).Similarly, administration of soluble ThRII exoreceptor tosyngeneic 4T1 or EMT6 mammary carcinoma-bearing animalshad a modest inhibitory effect on primary tumor size, whereaslung metastases were inhibited by 60% to 70% (69). Inaddition, soluble ThRII exoreceptor inhibited the developmentof lung metastases in neu transgenic mice without affecting theincidence or growth of primary tumors (73). Overall, theantimetastatic effects of TGF-h antagonists against syngeneicmouse tumors seem to be retained in human xenograftmodels, whereas tumor growth rates may or may not beaffected. This suggests that effects on the host may primarilymediate the inhibition of tumor growth, whereas the reductionin metastatic clonal efficiency is more likely caused by cell-autonomous effects of the antagonists.

The mechanisms of the antitumor effects of SD-208 areprobably multifactorial. First, although growth of R3T tumorswas strongly inhibited in syngeneic host animals, SD-208treatment failed to affect R3T tumor growth in athymic nudemice. Several factors may account, at least in part, for thedifference in efficacy of SD-208 between the two experiments.First, R3T tumors grew significantly more rapidly in athymicanimals compared with 129S1 mice. In addition, athymicmice seem to clear SD-208 significantly more rapidly than129S1 mice. Thirdly, and perhaps most importantly, thedifference may be due to induction of tumor-specific cytotoxicT-cell (CTL) activity (35, 36). Independent evidence forenhancement of CTL activity was provided by our findingthat splenocytes isolated from 4T1 tumor-bearing SD-208-treated mice were significantly more cytotoxic against 4T1 cellsin vitro than those from vehicle-treated control animals.Interestingly, this difference was uncovered only in thepresence of a ThRI kinase inhibitor, indicating that activeTGF-h, presumably produced by 4T1 cells, was inhibiting CTLactivity even in vitro. These results are consistent with a recentreport by Suzuki et al. (74), showing that the therapeuticefficacy of a soluble ThRII exoreceptor against transplantedmurine malignant mesotheliomas was largely dependent onCD8+ T cells.



Besides the effects on CTL activity, TGF-h antagonists maystimulate antitumor natural killer cell activity. Arteaga et al.(75) showed that i.p. injections of a pan-TGF-h-neutralizingmouse antibody, 2G7, suppressed in vivo growth and lungmetastases of MDA-MB-231 human breast cancer cells inathymic nude mice with a concomitant increase in mousespleen natural killer cell activity, although the effect that wasnot seen in natural killer–deficient mice. Similarly, Uhl et al.(45) recently reported that the in vivo antitumor effects of SD-208 against murine SMA-560 gliomas correlated with restora-tion of lytic activity of polyclonal natural killer cells againstglioma cells in vitro. Finally, Friese et al. (76) reported thatsilencing of TGF-h1 and TGF-h2 by small interfering RNAs inhuman LNT-229 malignant glioma cells suppressed theirtumorigenicity in nude mice, and natural killer cells isolatedfrom these mice showed an activated phenotype. A thirdimmune mechanism that may play a role in the antitumorefficacy of TGF-h antagonists is enhancement of dendritic cellfunction (77). In one study, the TGF-h-neutralizing monoclo-nal antibody 2G7 enhanced the ability of dendritic cell vaccinesto inhibit the growth of established 4T1 murine mammarytumors in vivo (78). However, in our case, we failed to observeany antitumor effect of SD-208 in athymic nude mice.

Besides the evidence in favor of an immune-mediated effectof SD-208 summarized above, we also noted a significantdecrease in microvessel density in R3T tumors obtained fromSD-208-treated animals compared with control tumors. Al-though Uhl et al. (45) failed to detect any change inmicrovessel density in SD-208-treated gliomas, this may bedue to disparities between the tumor models, as several otherstudies of human tumor xenograft models have found TGF-hantagonists to inhibit tumor angiogenesis (79–83). Theseobservations are consistent with previous reports showing thattumor-associated TGF-h contributes to angiogenesis and thathypoxia and TGF-h synergistically up-regulated vascular endo-thelial growth factor mRNA expression (84).

That SD-208 treatment truly inhibited TGF-h signaling in vivowas shown both by the reduction in phosphorylated Smadlevels in SD-208-treated tumors and by the dose-dependentdown-regulation of mRNA levels of TGF-h target genes intumor tissue. These results are consistent with the report byBonniaud et al. (44), showing similar effects of SD-208 in lungtissue in a rat lung fibrosis model.

Moreover, SD-208 also affected transcription of TGF-h targetgenes in normal tissue. Previous studies of the effects of short-term TGF-h treatment of cultured cells on the global geneexpression profile have shown that several hundred genes areup-regulated by TGF-h. Although several of these previouslydescribed TGF-h target genes were repressed in liver tissue bySD-208 treatment, the majority of repressed genes had not beenpreviously identified as TGF-h targets. This is, perhaps, notsurprising as short-term effects of TGF-h on cultured cells arelikely to be quite different from long-term effects of a TGF-hantagonist on animal tissue cells in vivo . Perhaps more

illuminating is the fact that the genes affected by SD-208treatment are predominantly involved in the regulation of thecell cycle, cell death, and apoptosis. Thus, our findings arehighly consistent with the known biological effects of TGF-h onnormal epithelial cells in general and normal hepatocytes inparticular (i.e., context-dependent inhibition of the cell cycle atthe G1-S checkpoint or the induction of apoptosis; refs. 85, 86).

Finally, it is important to note that SD-208 was remarkablyfree of clinically observable toxicity even after prolongedtreatment of mice in vivo. This extends previous observationsin which SD-208 was given by gavage to rats for short periodsof time (44). Although Uhl et al. (45) also gave SD-208 to micefor up to 40 days, the drug was dissolved in the drinking water.Thus, ours is the first preclinical study that shows the relativeclinical safety of this agent when given at high doses by dailygavage. Moreover, we failed to detect any evidence of organdamage postmortem, including breakdown of mucosal antimi-crobial defenses or inflammation. Although we did note a dose-dependent increase in relative liver weight in all three strains ofmice, the histologic appearance of the livers was unremarkable.Given our finding that SD-208 treatment was associated with adose-dependent increase in mRNA expression of a wide rangeof genes that encode enzymes involved in carbohydrate,protein, and fat metabolism as well as detoxification reactions,it is likely that the resulting global increases in proteinaccounted for the increase in liver weight. The majority of theinduced genes are not normally regulated by TGF-h signalingper se but seem to reflect a generalized activation of hepaticmetabolic pathways in response to chronic exposure to SD-208.This spectrum of induced genes was strikingly similar to thatfound in rat or mouse liver tissue in response to exposure to awide variety of chemicals (57–60, 87).

In summary, our studies show, for the first time, that thenovel selective chemical inhibitor of the ThRI kinase, SD-208, iscapable of inhibiting the metastatic efficiency as well as growthof mouse mammary carcinomas in vivo . The antitumor effectsof SD-208 were observed in the absence of any obvious signs oftoxicity, and pharmacodynamic studies of pSmad levels andTGF-h target gene expression confirmed inhibition of the targetenzyme in vivo . The inhibitory effect of SD-208 on tumorgrowth rates seemed to be mediated by enhancement of tumor-specific CTL activity and, to a lesser extent, by inhibition ofangiogenesis, whereas the inhibition of metastatic efficiencywas more likely due to cell-autonomous effects of the drug onthe tumor cells themselves. The observed antitumor activity wasmodest in magnitude. As the oncogenic role of TGF-h signalingseems to come into play at a relatively late stage of tumorprogression, blocking this pathway by itself is unlikely to besufficient to eradicate tumors and will have to be combinedwith strategies that block the primary drivers of tumor growth,such as cell cycle activators, inhibitors of apoptosis, andimmortalization. Our results should help inform the clinicaldevelopment of TGF-h antagonists in general and chemicalThRI kinase inhibitors in particular.

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InhibitionofGrowthandMetastasisofMouseMammaryCarcinoma ...PBS for 20 minutes at 20jC in the dark.For E-cadherin immunostain-ing, cells were washed with PBS and fixed for 5 minutes