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Cancer Therapy: Preclinical |
Authors' Affiliations: Departments of 1 Internal Medicine and 2 Surgery, The Cancer Institute of New Jersey, and 3 Department of Pathology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey; 4 Department of Genetics, Rutgers University, Piscataway, New Jersey; and 5 Scios, Inc., Fremont, California
Requests for reprints: Michael Reiss, Division of Medical Oncology, Department of Internal Medicine, The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Room 2007, 195 Little Albany Street, New Brunswick, NJ 08903. Phone: 732-235-6031; Fax: 815-333-3972; E-mail: michael.reiss{at}umdnj.edu.
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Abstract |
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Experimental Design: We examined the effects of selective TGF-ß type I receptor kinase inhibitors, SD-093 and SD-208, on two murine mammary carcinoma cell lines (R3T and 4T1) in vitro and in vivo.
Results: Both agents blocked TGF-ß-induced phosphorylation of the receptor-associated Smads, Smad2 and Smad3, in a dose-dependent manner, with IC50 between 20 and 80 nmol/L. TGF-ß failed to inhibit growth of these cell lines but stimulated epithelial-to-mesenchymal transdifferentiation, migration, and invasiveness into Matrigel in vitro. These effects were inhibited by SD-093, indicating that these processes are partly driven by TGF-ß. Treatment of syngeneic R3T or 4T1 tumor-bearing mice with orally given SD-208 inhibited primary tumor growth as well as the number and size of metastases. In contrast, SD-208 failed to inhibit R3T tumor growth or metastasis in athymic nude mice. Moreover, in vitro anti-4T1 cell cytotoxic T-cell responses of splenocytes 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-ß type I receptor kinase inhibitors hold promise as novel therapeutic agents for metastatic breast cancer.
In the normal adult mammary gland, TGF-ß controls the balance between cell renewal and cell differentiation and loss (15–19). It is likely through this homeostatic function that TGF-ß suppresses tumor development, and the loss of this function is an early event in epithelial carcinogenesis (20–23).
Besides its homeostatic function, TGF-ß plays a key role in tissue injury. Tissue injury results in a rapid locally increased activation of TGF-ß, which induces epithelial cells to assume a fibroblastoid and dispersed phenotype [epithelial-to-mesenchymal transdifferentiation (EMT)] and to produce extracellular matrix components of what later becomes a scar (24–27). This process is characterized by the subcellular redistribution of cell-cell cohesion molecules, such as E-cadherin, and of the F-actin cytoskeleton and is normally self-limited in space and time, allowing epithelial cells to revert back to their cohesive epithelioid phenotype (28).
Although escape from the growth-suppressive function of TGF-ß is an early event during mammary epithelial cell transformation, the tissue injury response to TGF-ß can be retained (7, 29–31). For example, in experimental models of mammary cancer, tumor cells escape from the growth-suppressive function of TGF-ß early on but retain the EMT response. Moreover, in late-stage tumors, TGF-ß signaling may become oncogenic by constitutively inducing EMT associated with a highly invasive and metastatic tumor phenotype. There is considerable evidence that tumor-derived TGF-ß plays an important role in stimulating and maintaining tumor growth by activating stromal cells, enhancing angiogenesis, and suppressing antitumor immunity (32–36). Thus, tumor-associated TGF-ß endows neoplastic cells with a selective advantage because of both the altered responsiveness of the tumor cells themselves (tumor cell-autonomous effects) and its actions on the supporting host cell infrastructure.
Based on these observations, the idea has been put forth that targeting TGF-ß signaling might represent a potentially powerful novel approach to the treatment of invasive breast cancers (32, 37–40). Recently, a series of pyridopyrimidine-based TßRI kinase inhibitors have been developed that are both potent and highly selective in vitro (41–43). Moreover, an orally bioavailable derivative compound, SD-208, inhibited the induction of pulmonary fibrosis in a rat model and blocked its progression when given transiently to animals with established disease (44). In addition, SD-208 inhibited the growth of rat gliomas in vivo (45). The current study represents the first report of the efficacy and safety of this compound in mouse models of metastatic mammary cancer in vivo. In addition, we provide evidence that SD-208 enhances antitumor immunity, inhibits tumor angiogenesis, and reduces metastatic clonogenicity in vivo.
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Materials and Methods |
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Cell culture. NMuMG, a spontaneously immortalized, nontumorigenic 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 µg/mL insulin (Sigma), and 10 µg/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-
(Invitrogen) supplemented with 8% (v/v) fetal bovine serum with the addition of 3 µg/mL puromycin (Sigma), 800 µg/mL geneticin (G418; Invitrogen), and 10 µg/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 maintained in RPMI 1640 (Life Technologies) supplemented with 10% (v/v) fetal bovine serum with the addition of 200 µg/mL geneticin (G418).
Cell proliferation assays. Cells were plated at 2 x 104 per well in 24-well cluster dishes (Corning, Inc., Corning, NY). Twenty-four hours later, cultures were treated with 1 µmol/L SD-093 or vehicle only followed 15 minutes later by addition of 100 pmol/L (2.5 ng/mL) TGF-ß1. Cells were counted 72 hours later using a Beckman Coulter counter (Model 0039, Beckman, Inc., Miami, FL).
Epithelial-to-mesenchymal transdifferentiation. For assessment of subcellular F-actin fiber distribution, cells were washed with PBS and fixed with buffered formalin for 10 minutes. Following washes with PBS, cells were permeabilized using 0.1% (v/v) Triton X-100 in PBS for 5 minutes and then incubated with 0.165 µmol/L Alexa Fluor 488–conjugated phalloidin with 1% (w/v) bovine serum albumin in PBS for 20 minutes at 20°C in the dark. For E-cadherin immunostaining, cells were washed with PBS and fixed for 5 minutes using methanol precooled to –20°C. Air-dried slides were then incubated with 5% (v/v) goat serum for 20 minutes at room temperature followed by incubation with 2 µg/mL mouse monoclonal anti-E-cadherin antibody in 2.5% (v/v) goat serum for 1 hour at room temperature. The cells were then washed 3 x 5 minutes with PBS followed by incubation with 2 µg/mL rhodamine-conjugated goat anti-mouse IgG for 45 minutes in the dark. In both cases, stained dishes were then washed 3 x 5 minutes with PBS, mounted using Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA), and viewed using a Zeiss epifluorescence microscope (model 090477, Carl Zeiss, Microimaging, Inc., Chester, VA) equipped with a MTI charge-coupled device camera (model DC 330E, DAGE-MTI, Inc., Michigan City, IN).
Western blot analysis. For detection of Smad proteins, semiconfluent cell cultures were lysed in situ using buffer composed of 150 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 protease inhibitors (Complete Mini Protease Inhibitor Cocktail Tablets, Roche Diagnostics Corp., Indianapolis, IN) for 30 minutes at 4°C. Cell lysates were subjected to Western blot analysis as described previously (42). Activated Smad2 (pSmad2) and activated Smad3 (pSmad3) were detected using our own rabbit anti-pSmad2 and anti-pSmad3 antibodies at a 1:1,000 dilution. Total Smad2, Smad3, and Smad4 were detected using rabbit anti-Smad2, rabbit anti-Smad3, and mouse anti-Smad4 antibodies, respectively, at a 1:500 dilution.
In vitro cell migration and invasion assays. For migration assays, uncoated polyethylene terephthalate track etched membrane (24-well insert; pore size, 8 µm; Becton Dickinson, Franklin Lakes, NJ) inserts were equilibrated by adding 0.5 mL cell culture medium to the upper and lower chambers followed by incubation at 37°C for 2 hours. For invasion assays, BD Biocoat Growth Factor Reduced Matrigel Invasion Chambers (24-well insert; pore size, 8 µm; BD Biosciences, Bedford, MA) were rehydrated by adding 0.5 mL warm (37°C) culture medium to the upper chambers followed by 2-hour incubation at 37°C. For both assays, medium used for equilibration was removed and 105 cells were plated in the upper chambers. TGF-ß1 (100 pmol/L, 2.5 ng/mL), SD-093 (1 µmol/L), both agents, or vehicle only were added to both upper and lower chambers. Following a 24-hour incubation at 37°C, cells in suspension were aspirated, the inserts were washed twice with PBS, and the cells adherent to the top of the inserts were removed by scraping the upper surface of the membrane with a cotton tip applicator. The cells that had migrated to the underside of the inserts were fixed and stained using the DiffQuick (Dade Behring, Newark, DE) staining kit. Cells in 10 random squares of 100 x 100 µm in each well were counted at x200 magnification using quadruplicate wells per assay condition and the results were expressed as number of cells/mm2.
Plasminogen activator inhibitor/luciferase assay. The plasminogen activator inhibitor (PAI)/luciferase assay was done as described by Abe et al. (51) with minor modifications. Briefly, 1.6 x 104 TMLC cells suspended in 100 µL medium were plated in 96-well tissue culture dishes and allowed to attach for 3 hours at 37°C in a 5% CO2 incubator. After 3 hours, the medium was replaced with 100 µL medium containing either TGF-ß at concentrations ranging from 0 to 100 pmol/L (0-2.5 ng/mL) or a suspension of 3.2 x 104 4T1 or R3T cells. The cells were lysed 14 hours later at room temperature and luciferase activity in cell lysates was determined using the Promega Luciferase Reporter Assay System following the protocol recommended by the manufacturer (Promega, Madison, WI) using a TD-20/20 Luminometer (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 x 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 postoperatively 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. Noncompartmental analysis was done using WinNonlin version 4.0.1 (Pharsight, Mountain View, CA). Because individual mice were not used to 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. Nominal time points were used for all calculations. AUC(0-8) is the area under the plasma concentration-time curve from time 0 to 8 hours for animals dosed 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 transferase–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 (x400) 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 (x400) 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 (x400) fields in areas of viable tumor.
CTL activity. Splenocytes (7 x 106) harvested from 4T1 tumor-bearing mice were restimulated with 1.4 x 105 irradiated 4T1 cells in a 24-well plate in a total of 2 mL tissue culture medium in the presence of 50 µmol/L ß-mercaptoethanol and 1 µmol/L SD-093. Cultures were maintained for 5 days at 37°C, 5% CO2, after which nonadherent effector cells were harvested. 4T1 cells were labeled in 100 µCi 51Cr (Perkin-Elmer Life and Analytical Sciences Inc., Wellesley, MA) for 1 hour at 37°C and then washed thrice with warm medium. 51Cr-labeled 4T1 target cells (1 x 104) and effector cells were then added at various E:T ratios in a total of 200 µL TCM to 96-well round-bottomed plates. Plates were incubated for 4 hours at 37°C, 5% CO2, 100 µL supernatant was removed, and released 51Cr was quantified using a gamma counter (Packard Bioscience, Meriden, CT). Percentage of specific lysis was calculated using the formula: (experimental release – spontaneous release) x 100 / (maximal release in 5% Triton X-100 – spontaneous release).
Real-time Taqman PCR. Transcript levels of individual genes were assayed in frozen R3T tumor tissue specimens using quantitative real-time PCR on an ABI Prism 7700 Sequence Detector (Applied Biosystems, 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 RNeasy mini columns (Qiagen, Valencia, CA) according to the manufacturer's instructions. The RNA concentration was adjusted to 1 µg/µL and its quality was assessed on a RNA chip using an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA). Isolated total RNA was processed as recommended by Affymetrix, Inc. (Santa Clara, CA). Biotin-labeled cRNA was size fractionated to 35 to 200 bases long using Affymetrix protocols and hybridized to the mouse genome 430 2.0 GeneChip set at 45°C for 16 hours in an Affymetrix GeneChip Hybridization Oven 320. This chip was designed using the most recent publicly available draft of the mouse genome and contains >45,000 probe sets covering >39,000 transcripts and variants representing >34,000 well-substantiated mouse genes. Each GeneChip was then washed and stained with streptavidin-phycoerythrin using an Affymetrix Fluidics Station 400 and scanned on a Affymetrix Gene Array scanner. Scanned image files were analyzed using the Microarray Suite 5.0 software (Affymetrix). Scaling and normalization were carried out using the 500 Normalization Control probe set included on the 430 2.0 GeneChip set.
Statistical analyses. For analyses of tumor growth rates, metastasis burden, microvessel densities, Ki-67 staining, apoptosis rates, and mRNA expression levels one-way ANOVA tests were done using InStat version 3.0 (GraphPad Software, Inc., San Diego, CA) or two-way repeated-measures ANOVA tests using JMP version 5.1 (SAS Institute, Inc., Cary, NC).
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TßRI kinase inhibitor affects cell migration and invasion. Several studies have suggested that tumor cell migration and invasion might be driven by TGF-ß (29, 54, 55). As shown in Fig. 1C, migration of NMuMG cells was strongly inhibited by TGF-ß in a TßRI kinase-dependent manner. In contrast, under the same culture conditions, TGF-ß stimulated migration of both mammary carcinoma lines, and this effect was also dependent on TßRI kinase activity. Moreover, treatment with SD-093 alone inhibited motility of R3T cells, indicating that the migratory phenotype of these cells is driven, at least in part, by endogenous TGF-ß signaling.
Besides cell migration, treatment with exogenous TGF-ß significantly stimulated the ability of R3T and 4T1 cells to invade Growth Factor Reduced Matrigel (3.3- and 2-fold, respectively). This effect was completely inhibited by pretreating the cells with SD-093 (Fig. 1D). Moreover, treatment of R3T cells with SD-093 alone inhibited invasion by 40%, indicating that their invasive phenotype is also partly dependent on endogenous TGF-ß signaling.
To investigate whether excessive production and/or extracellular activation of TGF-ß was responsible for activation of TGF-ß signaling, we conducted coculture experiments using TMLC cells that express a TGF-ß-inducible PAI-1 promoter driving a luciferase reporter gene construct (51). As shown in Fig. 1E, exogenous TGF-ß resulted in a concentration-dependent increase in luciferase activity in TMLC cells. Cocultivation of TMLC cells with mammary carcinoma cells induced a significant increase in luciferase activity (Fig. 1E). In both cases, the effect was abolished in the presence of pan-TGF-ß antibody, thereby showing that both mammary cancer cell lines produce biologically active TGF-ß capable of inducing specific gene responses in neighboring cells.
SD-208 inhibits mammary tumor growth and metastasis in vivo. Because of its superior oral bioavailability compared with SD-093, the SD-208 compound was used for all in vivo experiments. In a typical assay, R3T or 4T1 carcinoma cells were inoculated into the mammary fat pad, and mice were treated with 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 R3T tumor growth in syngeneic 129S1 mice in a dose-dependent manner. In addition, both the number and the size of lung metastases were significantly reduced by SD-208 treatment (Fig. 2B). The antitumor activity of SD-208 was not limited to the R3T mammary carcinoma: SD-208 also caused a dose-dependent statistically significant growth delay of 4T1 orthotopic tumors in syngeneic BALB/c mice (Fig. 2C) as well as a dose-dependent reduction in the number of metastases to the lungs (Fig. 2D) and other organs, including liver, adrenal glands, 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 higher than in 129S1 mice (Fig. 3A ). Consequently, the experiments had to be terminated sooner than in 129S1 mice, possibly accounting for the lower number of lung metastases found at autopsy (Figs. 2B and 3B). In contrast to the antitumor effects seen in syngeneic mice, SD-208 failed to retard the growth of R3T tumors in athymic nude mice (Fig. 3A). In addition, treatment with SD-208 had little effect on the size or numbers of lung metastases (Fig. 3B).
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80 nmol/L, indicating that it is slightly less potent than SD-093, which has an IC50 of 20 nmol/L. Figure 4B depicts the kinetics of SD-208 in plasma of each of the mouse strains following a single oral dose of either 20 or 60 mg/kg. Peak plasma levels as well as areas under the curve varied as function of the dose given in each of the three mouse strains. Although levels achieved at 1 hour following a dose of 60 mg/kg were not significantly different across the three mouse strains (ANOVA, P = 0.1893), trough levels at 24 hours were significantly lower in athymic than in 129S1 mice (ANOVA, P = 0.0291). Moreover, the area under the curve achieved in 129S1 mice was approximately twice as high as in athymic animals (Fig. 4B). These differences may account, in part, for the lower efficacy against R3T tumors we observed in athymic compared with 129S1 mice (see above). Plasma levels obtained at 2 hours following the penultimate dose of SD208 in treatment experiments were very similar to those achieved following a single dose, indicating that prolonged repeated dosing did not have adverse effects on pharmacokinetics (data not shown). SD-208 treatment was tolerated without observable toxicity for up to 56 days of continuous daily administration.
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To further substantiate inhibition of TGF-ß signaling by SD-208 treatment in vivo, we compared the levels of mRNA expression of several TGF-ß-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, 2), and matrix metalloproteinase-2 were all significantly reduced in tumors that had been exposed to SD-208.
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0.05) compared the mouse transcriptome in general (56). Genes that were down-regulated in SD-208-treated animals are predominantly involved in transcription, cell cycle control, intracellular signaling, and apoptosis. Several studies have examined the spectrum of TGF-ß-regulated genes in the mouse mammary epithelial cell line NMuMG (9, 11, 13) and in mouse embryo fibroblasts (12). Table 1
lists 31 genes that were repressed in the livers of SD-208-treated mice and have been reported previously to be induced by TGF-ß in NMuMG mouse mammary epithelial cells in vitro (13). Figure 5B illustrates the dose-dependent reduction of transcript levels of six of these genes by SD-208 treatment. In summary, SD-208 treatment clearly inhibited TßRI kinase activity and TGF-ß-regulated gene expression in vivo.
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Effects of SD-208 on tumor-specific CTL activity. The notable difference in therapeutic efficacy of SD-208 against R3T tumors growing in syngeneic compared with immunodeficient mice suggested that enhancing antitumor immunity might represent an 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 from each of the three treatment groups and restimulated these with irradiated 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 using a gamma counter (Fig. 6A ). Specific CTL activity was significantly increased by in vivo treatment with SD-208 in a dose-dependent manner. Moreover, this effect was only observed as long as SD-093 was present during restimulation, indicating that 4T1 produce sufficient amounts of bioactive TGF-ß to suppress CTL activity or expansion in vitro. Similar effects, although of lower magnitude, were observed using freshly isolated splenocytes without in vitro restimulation (Fig. 6A).
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Finally, treatment with SD-208 was associated with a striking infiltration of tumors with eosinophilic leukocytes (Fig. 6B). In addition, in contrast to the undifferentiated spindle-cell appearance of control tumors, some of the R3T carcinomas in SD-208-treated animals were much more differentiated with clearly recognizable ductal structures and areas of keratinization (Fig. 6B). This morphologic appearance was reminiscent of the metaplastic squamous carcinomas that develop in the mammary glands of Smad4 conditional knockout mice and in carcinogen-treated transgenic mice that express a dominant-negative TßRII receptor (20, 61). Thus, chemical or genetic inhibition of TGF-ß signaling seems to alter mammary carcinoma cell differentiation in similar ways.
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The phenotype of mammary carcinoma lines differed from that of nontransformed NMuMG cells in several important ways. First, in contrast to NMuMG cells, both R3T and 4T1 cells were refractory to TGF-ß-mediated growth suppression, consistent with previous reports (reviewed in refs. 22, 32, 33, 63). Both R3T and 4T1 cells seem to have retained a typical epithelioid morphology in vitro, although 4T1 cells were clearly less cohesive probably because they do not express E-cadherin (53). Exogenous TGF-ß clearly induced EMT in both tumor lines in vitro in a TßRI kinase-dependent manner, whereas treatment with SD-093 alone seemed to increase the cohesion of R3T cells. Thus, the growth-inhibitory effect of TGF-ß had become uncoupled from the EMT response. In contrast to NMuMG, TGF-ß not only failed to inhibit migration of R3T and 4T1 cells but also acted as a stimulus of cell motility, an effect that could be blocked by SD-093, indicating that it was mediated by the TßRI kinase. In addition, TGF-ß treatment stimulated the ability of the mammary carcinoma cells to invade Matrigel, an effect that was also inhibited by SD-093. Interestingly, TGF-ß inhibits in vitro invasion of normal trophoblast cells (64), whereas an extensive literature attests to the fact that TGF-ß signaling stimulates or even drives the invasive behavior of malignant cells (see refs. 32, 33 for reviews). Thus, TGF-ß seems to undergo a switch from being an antimigratory and anti-invasive to a promigratory and proinvasive factor during malignant transformation (29, 43, 55).
In contrast to their similar in vitro phenotype, R3T tumors in vivo were composed of highly spindle-shaped cells, whereas 4T1 tumor-derived carcinomas were more differentiated and epithelioid. This apparent discrepancy between in vitro and in vivo phenotypes is likely due to a combination of cell intrinsic factors (e.g., activation of the H-ras gene in R3T but not in 4T1 cells) and a permissive microenvironment (53, 65). Thus, one might speculate that differences in local TGF-ß activation in tumors in vivo might influence the degree of EMT. If this were correct, one would expect treatment with SD-208 to induce a reversal of the fibroblastoid to an epithelioid phenotype. This is, in fact, what we observed in R3T tumors in animals treated with the highest dose of SD-208. In contrast to the undifferentiated spindle-cell appearance of control tumors, these carcinomas were much more differentiated, with clearly recognizable ductal structures and areas of keratinization. This morphologic appearance was reminiscent of the metaplastic squamous carcinomas seen in the mammary glands of Smad4 conditional knockout mice and in carcinogen-treated transgenic mice that express a dominant-negative TßRII receptor (20, 61). Treatment with SD-208 was associated with a striking infiltration of tumors with eosinophilic leukocytes. The significance and the mechanism for this phenomenon are unclear. However, Takaku et al. (66) reported that gastric polyps that develop in Smad4 heterozygous mice are associated with an eosinophilic cell infiltrate. Conversely, a nasally delivered DNA encoding TGF-ß1 can suppress pulmonary eosinophilic responses to infectious agents (67). Thus, attenuation of TGF-ß signaling seems to promote eosinophilia, whereas increased expression of TGF-ß1 suppresses it.
As reported previously, orthotopic 4T1 or R3T invasive carcinomas efficiently metastasized to the lung parenchyma (48, 68). Most importantly, treatment of the mice with SD-208 significantly retarded primary tumor growth in both models in a dose-dependent manner. The antitumor effects of SD-208 were more pronounced against R3T than against 4T1 tumors possibly as a consequence of the higher growth rate of 4T1 tumors or the stronger TGF-ß activation by R3T cells (Fig. 1E). Alternatively, the activation of Ha-ras and/or expression of PyVMT in R3T may have conferred particular sensitivity to TGF-ß antagonists. Besides retarding the growth of primary tumors, treatment with SD-208 significantly reduced the number of lung metastases in both models. However, 4T1 metastases were significantly larger than those derived from R3T tumors, and the effect of SD-208 on their size was notably less. The therapeutic effect of SD-208 against 4T1 was entirely consistent with those observed in the 4T1 model by other investigators using different types of TGF-ß antagonists, including dominant-negative TßRII receptors (29), soluble TßRII exoreceptors (69), neutralizing anti-TGF-ß antibodies (70), or other chemical TßR kinase inhibitors (71). Our observations suggest that the effects of TGF-ß antagonists on tumor growth rates may be quite distinct from those on metastatic efficiency. Consistent with this idea, expression of a dominant-negative TßRII receptor in 4T1 or in human MCF10CA1a mammary carcinoma cells reduced their metastatic ability without affecting primary tumor growth (29, 72). Similarly, administration of soluble TßRII exoreceptor to syngeneic 4T1 or EMT6 mammary carcinoma-bearing animals had a modest inhibitory effect on primary tumor size, whereas lung metastases were inhibited by 60% to 70% (69). In addition, soluble TßRII exoreceptor inhibited the development of lung metastases in neu transgenic mice without affecting the incidence or growth of primary tumors (73). Overall, the antimetastatic effects of TGF-ß antagonists against syngeneic mouse tumors seem to be retained in human xenograft models, whereas tumor growth rates may or may not be affected. This suggests that effects on the host may primarily mediate the inhibition of tumor growth, whereas the reduction in metastatic clonal efficiency is more likely caused by cell-autonomous effects of the antagonists.
The mechanisms of the antitumor effects of SD-208 are probably multifactorial. First, although growth of R3T tumors was strongly inhibited in syngeneic host animals, SD-208 treatment failed to affect R3T tumor growth in athymic nude mice. Several factors may account, at least in part, for the difference in efficacy of SD-208 between the two experiments. First, R3T tumors grew significantly more rapidly in athymic animals compared with 129S1 mice. In addition, athymic mice seem to clear SD-208 significantly more rapidly than 129S1 mice. Thirdly, and perhaps most importantly, the difference may be due to induction of tumor-specific cytotoxic T-cell (CTL) activity (35, 36). Independent evidence for enhancement of CTL activity was provided by our finding that splenocytes isolated from 4T1 tumor-bearing SD-208-treated mice were significantly more cytotoxic against 4T1 cells in vitro than those from vehicle-treated control animals. Interestingly, this difference was uncovered only in the presence of a TßRI kinase inhibitor, indicating that active TGF-ß, presumably produced by 4T1 cells, was inhibiting CTL activity even in vitro. These results are consistent with a recent report by Suzuki et al. (74), showing that the therapeutic efficacy of a soluble TßRII exoreceptor against transplanted murine malignant mesotheliomas was largely dependent on CD8+ T cells.
Besides the effects on CTL activity, TGF-ß antagonists may stimulate antitumor natural killer cell activity. Arteaga et al. (75) showed that i.p. injections of a pan-TGF-ß-neutralizing mouse antibody, 2G7, suppressed in vivo growth and lung metastases of MDA-MB-231 human breast cancer cells in athymic nude mice with a concomitant increase in mouse spleen natural killer cell activity, although the effect that was not 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 restoration of lytic activity of polyclonal natural killer cells against glioma cells in vitro. Finally, Friese et al. (76) reported that silencing of TGF-ß1 and TGF-ß2 by small interfering RNAs in human LNT-229 malignant glioma cells suppressed their tumorigenicity in nude mice, and natural killer cells isolated from these mice showed an activated phenotype. A third immune mechanism that may play a role in the antitumor efficacy of TGF-ß antagonists is enhancement of dendritic cell function (77). In one study, the TGF-ß-neutralizing monoclonal antibody 2G7 enhanced the ability of dendritic cell vaccines to inhibit the growth of established 4T1 murine mammary tumors in vivo (78). However, in our case, we failed to observe any antitumor effect of SD-208 in athymic nude mice.
Besides the evidence in favor of an immune-mediated effect of SD-208 summarized above, we also noted a significant decrease in microvessel density in R3T tumors obtained from SD-208-treated animals compared with control tumors. Although Uhl et al. (45) failed to detect any change in microvessel density in SD-208-treated gliomas, this may be due to disparities between the tumor models, as several other studies of human tumor xenograft models have found TGF-ß antagonists to inhibit tumor angiogenesis (79–83). These observations are consistent with previous reports showing that tumor-associated TGF-ß contributes to angiogenesis and that hypoxia and TGF-ß synergistically up-regulated vascular endothelial growth factor mRNA expression (84).
That SD-208 treatment truly inhibited TGF-ß signaling in vivo was shown both by the reduction in phosphorylated Smad levels in SD-208-treated tumors and by the dose-dependent down-regulation of mRNA levels of TGF-ß target genes in tumor tissue. These results are consistent with the report by Bonniaud et al. (44), showing similar effects of SD-208 in lung tissue in a rat lung fibrosis model.
Moreover, SD-208 also affected transcription of TGF-ß target genes in normal tissue. Previous studies of the effects of short-term TGF-ß treatment of cultured cells on the global gene expression profile have shown that several hundred genes are up-regulated by TGF-ß. Although several of these previously described TGF-ß target genes were repressed in liver tissue by SD-208 treatment, the majority of repressed genes had not been previously identified as TGF-ß targets. This is, perhaps, not surprising as short-term effects of TGF-ß on cultured cells are likely to be quite different from long-term effects of a TGF-ß antagonist on animal tissue cells in vivo. Perhaps more illuminating is the fact that the genes affected by SD-208 treatment are predominantly involved in the regulation of the cell cycle, cell death, and apoptosis. Thus, our findings are highly consistent with the known biological effects of TGF-ß on normal epithelial cells in general and normal hepatocytes in particular (i.e., context-dependent inhibition of the cell cycle at the G1-S checkpoint or the induction of apoptosis; refs. 85, 86).
Finally, it is important to note that SD-208 was remarkably free of clinically observable toxicity even after prolonged treatment of mice in vivo. This extends previous observations in which SD-208 was given by gavage to rats for short periods of time (44). Although Uhl et al. (45) also gave SD-208 to mice for up to 40 days, the drug was dissolved in the drinking water. Thus, ours is the first preclinical study that shows the relative clinical safety of this agent when given at high doses by daily gavage. Moreover, we failed to detect any evidence of organ damage postmortem, including breakdown of mucosal antimicrobial defenses or inflammation. Although we did note a dose-dependent increase in relative liver weight in all three strains of mice, the histologic appearance of the livers was unremarkable. Given our finding that SD-208 treatment was associated with a dose-dependent increase in mRNA expression of a wide range of 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 protein accounted for the increase in liver weight. The majority of the induced genes are not normally regulated by TGF-ß signaling per se but seem to reflect a generalized activation of hepatic metabolic pathways in response to chronic exposure to SD-208. This spectrum of induced genes was strikingly similar to that found in rat or mouse liver tissue in response to exposure to a wide variety of chemicals (57–60, 87).
In summary, our studies show, for the first time, that the novel selective chemical inhibitor of the TßRI kinase, SD-208, is capable of inhibiting the metastatic efficiency as well as growth of mouse mammary carcinomas in vivo. The antitumor effects of SD-208 were observed in the absence of any obvious signs of toxicity, and pharmacodynamic studies of pSmad levels and TGF-ß target gene expression confirmed inhibition of the target enzyme in vivo. The inhibitory effect of SD-208 on tumor growth rates seemed to be mediated by enhancement of tumor-specific CTL activity and, to a lesser extent, by inhibition of angiogenesis, whereas the inhibition of metastatic efficiency was more likely due to cell-autonomous effects of the drug on the tumor cells themselves. The observed antitumor activity was modest in magnitude. As the oncogenic role of TGF-ß signaling seems to come into play at a relatively late stage of tumor progression, blocking this pathway by itself is unlikely to be sufficient to eradicate tumors and will have to be combined with strategies that block the primary drivers of tumor growth, such as cell cycle activators, inhibitors of apoptosis, and immortalization. Our results should help inform the clinical development of TGF-ß antagonists in general and chemical TßRI kinase inhibitors in particular.
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 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/).
Received 1/23/06; revised 4/ 7/06; accepted 5/ 4/06.
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, vehicle; 
, 20 mg/kg; 
, 60 mg/kg) were restimulated with irradiated 4T1 cells for 5 days in the presence of SD-208, after which nonadherent effector cells were harvested. 51Cr-labeled 4T1 target cells and effector cells were then incubated for 4 hours at 37°C, and released 51Cr was quantified using a gamma counter. At an E:T of 100:1, 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 4T1 tumor-bearing mice from each of the three treatment groups (