Halofuginone inhibits tumor growth in the polyoma middle T antigen ...

[Cancer Biology & Therapy 5:2, 218-224, February 2006]; ©2006 Landes Bioscience

Halofuginone Inhibits Tumor Growth in the Polyoma Middle T Antigen Mouse via a Thrombospondin-1 Independent Mechanism Research Paper

ABSTRACT

Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/abstract.php?id=2419

KEY WORDS breast cancer, angiogenesis, TGF-β, collagen, thrombospondin-1

ABBREVIATIONS

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Halofuginone is a plant-derived alkaloid that inhibits fibrosis by decreasing type I collagen synthesis.1-3 In culture, halofuginone attenuated collagen synthesis by skin fibroblasts from scleroderma and chronic graft-versus-host disease (cGvHD) patients.4 In animal models of fibrosis in which excess collagen is the hallmark of the disease, administration of halofuginone prevented the increase in collagen α1(I) gene expression and collagen synthesis. These models included mice afflicted with cGvHD and tight skin (Tsk+) mice,5,6 rats with pulmonary fibrosis,7 rats that had developed adhesions at various locations,8-10 rats with liver fibrosis,11-13 and mice with radiation-induced fibrosis.14 Clinical efficacy was demonstrated by topical dermal halofuginone administration in a cGvHD patient15 and in a scleroderma phase II clinical study.16 In light of the involvement of the extracellular matrix (ECM) in general and of collagen type I in particular in angiogenesis,17,18 the efficacy of halofuginone in inhibiting angiogenesis and tumor growth was evaluated. Halofuginone was found to inhibit microvessel formation in vitro and in vivo resulting in decreased tumor growth.19-21 This includes transplantable and chemically induced bladder carcinoma,20 glioma,22 Hippel-Lindau associated pheochromocytoma,23 prostate cancer,24 hepatocellular carcinoma25 and Wilms tumor.26 Several groups have shown that halofuginone blocks TGF-β mediated collagen synthesis by decreasing activation of Smad 3 through c-Jun27,28 and increasing expression of Smad 7, an inhibitor of Smad 2/3 activation.14 One of the matrix proteins involved in fibrosis, angiogenesis and tumorigenesis is thrombospondin-1 (TSP-1).29,30 TSP-1 is a 450kDa homotrimeric extracellular matrix protein expressed by both normal and tumor cells and has been shown to inhibit tumorigenesis, angiogenesis and experimental metastasis.30-36 TSP-1 expression is suppressed by some oncogenes and induced by some tumor suppressor genes.35,37-39 Overexpression of TSP-1 suppresses the growth of tumor cells in vivo.33,40 Recently, low dose chemotherapy has been shown to inhibit tumor growth by increasing systemic levels of TSP-1.41,42 TSP-1

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ACKNOWLEDGEMENTS

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Thanks to Carole Perruzzi for isolating the human microvascular endothelial cells for us and to Mark Duquette and Eric Galardi for technical assistance. This work was supported by National Institute of Health grants HL68003 and CA92644. Karen O. Yee was supported by a post-doctoral fellowship from the Aid for Cancer Research (Boston, MA).

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INTRODUCTION

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Thrombospondin-1 Polyoma middle T antigen Transforming growth factor β Type I Repeat of thrombospondin-1 Extracellular signal-regulated kinase Jun N-Terminal Kinase mitogen-activated protein kinase kinase 1

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Received 05/19/05; Accepted 12/19/05

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*Correspondence to: Jack Lawler; Beth Israel Deaconess Medical Center; Department of Pathology; 99 Brookline Avenue; Research North Room 270C; Boston, Massachusetts 02215 USA; Tel.: 617.667.1694; Fax: 617.667.3591; Email: [email protected]

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of Animal Science; Agricultural Research Organization; The Volcani Center; Bet Dagan, Israel

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1Department of Pathology; Beth Israel Deaconess Medical Center; Boston, Massuchusetts USA

TSP-1 PyT TGF-β TSR ERK JNK MEKK

Halofuginone inhibits fibrosis by decreasing type I collagen synthesis and tumor growth through an anti-angiogenic mechanism. In vitro data suggested that halofuginone inhibits angiogenesis through upregulating thrombospondin-1 (TSP-1) expression and by inhibiting cell proliferation. To determine whether thrombospondin-1 (TSP-1) is necessary for inhibition of tumor growth and angiogenesis by halofuginone, we tested the effect of halofuginone on mammary tumor growth in polyoma middle T antigen, TSP-1 null (TSP-1-/-PyT) transgenic mice. After 30 days of treatment, we found a significant decrease in tumor weight in these mice and the extent of tumor growth inhibition was comparable to that found in TSP-1 expressing PyT mice (TSP-1+/+PyT). However, no significant difference in tumor weight was observed after 60 days of halofuginone treatment between control and treated mice in both genotypes. Interestingly, type I collagen level was lower in the halofuginone treated TSP-1+/+PyT tumors at 30 days, but this was not observed in the TSP-1-/-PyT mice. Levels of type I collagen did not correlate with blood vessel number as a decrease in the number of vessels was observed in the halofuginone treated tumors from both the TSP-1+/+PyT and TSP-1-/-PyT mice as compared to control tumors. Because halofuginone has been shown to inhibit type I collagen synthesis by inhibiting the TGF-β signaling pathway, we measured Smad 2/3 phosphorylation levels and found that halofuginone inhibited Smad 2/3 phosphorylation in cells derived from TSP-1+/+PyT tumors. We also found that it inhibited Smad 2/3 phosphorylation in cells treated with the TGF-β activating sequence of TSP-1, TSR2+RFK. Our data demonstrate that halofuginone inhibits mammary tumor growth in a transgenic mouse model via a TSP-1 independent pathway, by decreasing tumor angiogenesis and by inhibiting TGF-β signaling.

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Karen O Yee1 Caitlin M. Connolly1 Mark Pines2 Jack Lawler1,*

Cancer Biology & Therapy

2006; Vol. 5 Issue 2

Halofuginone Inhibits Early Breast Cancer Progression

is also significantly upregulated in herceptin-treated experimental human breast tumors as compared to untreated tumors.43 The three type-1 repeats (TSRs) of TSP-1 have been shown to confer the majority of the anti-tumorigenic and anti-angiogenic properties.31,44-47 The TSRs inhibit tumor growth by inhibiting matrix metalloproteinase-dependent mobilization of vascular endothelial growth factor (VEGF), inhibiting endothelial cell migration and inducing endothelial cell apoptosis.45,48-51 The TSRs of TSP-1 have a sequence (RFK) that activates TGF-β by a yet-to-be-determined mechanism.52 The TGF-β that is activated by the TSRs can inhibit tumor growth if the tumor cells are responsive to this cytokine.45,53 We previously observed a five-fold increase in TSP-1 mRNA expression upon treating human prostrate cancer cells in vitro with halofuginone for 24 hours (M. Pines, unpublished data). To both evaluate the efficacy of halofuginone in a genetic model of breast cancer and to ask whether TSP-1 is necessary for inhibition of tumor growth by halofuginone, we treated mice that express the polyoma middle T antigen (PyT) gene in the mammary epithelium but are null for TSP-1. We found that halofuginone was an effective inhibitor of early tumor growth in this transgenic breast cancer model and that TSP-1 was not required for its action.

MATERIALS AND METHODS

Materials. Halofuginone bromhydrate was obtained from Collgard Biopharmaceuticals Ltd (Tel Aviv, Israel). Anti-type-1 collagen-biotin antibody was purchased from Southern Biotech (Birminghan, AL). Anti-CD31 antibody was purchased from BD Biosciences Pharmingen (San Jose, CA). Polyoma Middle T antigen transgenic mice were purchased from Jackson Laboratories (Bar Harbor, ME). Animal model and treatment. Generation of the TSP-1-/- mice on the 129Sv background has already been described.54 These mice were backcrossed to wild-type FVB mice (Taconic Farms) eight times and then intercrossed to produce TSP-1-/- FVB mice. Female TSP-1-/- mice were crossed with male, PyT mice to generate the F1 generation. The F2 generation was produced by crossing male TSP-1 +/- PyT mice with female TSP-1-/- mice (unpublished data). Test animals were injected interperitoneal with 5µg of halofuginone every other day starting at 30 days of age while control mice received 100µl of water. The treated mice were euthanized after 30 days or 60 days of treatment. Tumors were pooled and weighed. All animal experiments were approved by the Beth Israel Deaconess Medical Center Research Animal Care Committee. Immunohistochemistry. Tumors were embedded in OCT compound, frozen in liquid nitrogen and 7 µm sections were cut using a cyrostat. Slides were fixed in precooled acetone for 2 minutes at -20˚C, fixed in 80% methanol for an additional 5 minutes at 4˚C and washed twice in PBS for 5 minutes each. Slides were then placed in 0.6% hydrogen peroxide/PBS for 20 minutes and then washed three times in PBS for 5 minutes each. Tumor sections were blocked with 5% rabbit serum (CD 31Ab) or BSA (collagen Ab) in PBS for 30 minutes. Either anti-CD31 antibody at 1:200 dilution in 5% rabbits serum/PBS or anti-type-1 collagen-biotin antibody at 1:50 dilution in 5% BSA/PBS was placed on the tumor sections overnight at 4˚C. For the collagen I staining, slides were washed with PBS three times for 5 minutes each, incubated with the ABC kit solution and stained with DAB. Two investigators graded the slides from 1–5 with five having the most staining. For the CD31 staining, biotinylated rabbit anti-rat, mouse absorbed, at 1:200 dilution in 5% rabbit serum/PBS was placed on the slides for 1 hour at room temperature. Slides were washed three times in PBS before adding Vectastain ABC solution (Vector Laboratories, Burlingame, CA) for 45 minutes at room temperature. Slides were washed twice in PBS before a DAB solution was applied (Vector Laboratories, Burlingame, CA) and then washed in water before dehydration through graded ethanol and xylene.

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Computer-assisted morphometric analysis of tumor vessels. Cryostat sections of the tumors were stained with the anti-mouse CD31 monoclonal antibody and representative sections obtained from at least four tumors for each group were analyzed using a Nikon E-600 microscope (Nikon, Melville, NY). Images were captured using a spot digital camera (Diagnostic Instruments, Sterling Heights, IL). Morphometric analyses were carried out as previously described using the IP LAB software (Scanalytics Inc., Fairfax, VA).46 Isolation of tumor cells and microvascular endothelial cells. Mammary tumors were excised from three month old TSP-1+/+PyT mice, minced, incubated in 0.2% collagenase for 1 hr with rocking at 37˚C spun down at 2000RPM and supernatant was placed in 100 mm tissue culture treated plates containing DMEM media, anti-biotics and anti-mycotics. The media was changed the next day and the cells were allowed to propagate until 80% confluence at which time they were either frozen down for storage or used for experiments. The plate contained a mixed population of cells that include tumor cells and fibroblasts. Human dermal microvascular endothelial cells were isolated as previously described.55 Proliferation assay. Cells (1.6 x 105) were seeded into six-well tissue culture treated plates and allowed to adhere to the plate overnight. Eighteen hours later, the media was replaced with either full growth media alone or full growth media containing halofuginone (10 nM) or TGF-β1 (10 ng/ml). At 24, 48 and 72 hrs, cells in two wells were trypsinized and counted using a hemacytometer. Two measurements per well were collected for a total of four measurements. TGF-β1 was added to the media again at 48 and 72 hrs post plating. Halofuginone, TGF-β, TSR2+RFK and TSR2 treatment of tumor cells. Halofuginone (10 nM), human recombinant TGF-β1 (10 ng/ml; R&D Systems) or peptides to the second type-1 repeat of TSP-1 without (TSR2) and with the RFK sequence (TSR2 + RFK) (10ng/ml)45 in 1% FCS/ DMEM media were added to a mixed population of cells derived from TSP-1+/+PyT tumors. Sixteen hours later, either RNA or protein lysates was isolated from the cells. RNA was isolated using the Qiagen RNAeasy Mini columns following manufacturer’s directions. Protein was isolated by adding PBS containing 1% SDS, 1% Triton-X-100 and protease inhibitors to the plates. Cells were scraped off, cellular debris was removed by centrifugation and total protein concentration was measured using DC Protein Assay (BioRad, Hercules, CA). Real Time PCR reactions. Using 0.5 µg of RNA, cDNAs were created through a reverse transcription reaction using random hexamers, the Taqman Reverse Transcription Kit from Applied Biosystems and following manufacturer’s directions. TSP-1 oligos and a FAM-labeled probe were purchased from Applied Biosystems (Mm00449022_A1 Thbs1) as well as the Rodent GAPDH Control reagents. They were used with the Taqman Fast Universal PCR master mix (2X) from Applied Biosystems. All reactions were carried out in duplicates and repeated once to confirm accuracy. To calculate the fold difference between the groups, first the CT values from each group were averaged, and the GADPH average CT value was subtracted from the TSP-1 average CT value to derive the ∆CT value. To determine the ∆∆CT value, the control ∆Ct value was subtracted from the halofuginone treated ∆CT value. To calculate fold difference between the groups, the equation, 2^ (-∆∆CT), was used. Western blot analysis. Protein was loaded onto a 12% SDS-PAGE-gel (Smad 2/3) and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA) overnight. The membrane was blocked with 5% milk in Tris Buffered Saline with 0.5% Tween (TBST) for one hour and Smad 2/3 antibody (BD Transduction Laboratories), Phospho Smad 2/3 antibody (Cell Signaling Technology) or actin antibody (Sigma) in 5% milk/TBST were added to the blots for two hours (actin antibody) or overnight. Secondary antibodies (anti-mouse horseradish peroxidase or anti-rabbit horseradish peroxidase at 1:5000; (Amersham) in 5% milk-TBST were added to the blots for two hours and proteins were visualized using SuperSignal West Pico Chemilluminescent kit (Pierce, Rockford, IL). Statistical analysis. The InStat for Macintosh program was used to calculate the P values for the data. If the data set had standard deviations that


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Figure 1. Effect of Halofuginone treatment on polyoma middle T tumor cells in vitro. Cells were counted after 24, 48 and 72 hours of exposure to either halofuginone (10nM) or TGF-β1 (10 ng/ml). Experiment was repeated twice with one representative data set shown. Values are mean ± SD n = 4.

were similar, the Analysis of Variance, unpaired test was carried out. A data set that had standard deviations that were significantly different, the Kruskal-Wallis Nonparametric ANOVA Test and Dunn’s Multiple Comparisons Test were carried out.

RESULTS Halofuginone inhibits polyoma middle T tumor cells proliferation and ppregulates TSP-1 expression in vitro. TSP-1+/+PyT cells were isolated from the tumors and grown in culture in 10% FCS/DMEM media. Cells (1.6 x 105) were plated and after 18 hours, were exposed to either 10 nM halofuginone or 10 ng/ml TGF-β1. Twenty-four hours after the initial exposure to the reagents, there was significantly fewer cells in the halofuginone treated dishes than either the control (p-value < 0.01) or the TGF-β1 treated dishes (p-value < 0.01) (Fig. 1). This difference became much larger after 48 hours and 72 hours of exposure to halofuginone. While the control cells had a linear growth curve, the halofuginone growth curve was relatively flat. The TGF-β1-treated cells grew equally as well as the control cells for the first 24 hours, but after that, the growth curve also flattened out. By 72 hours, the total number of control cells, the TGFβ1-treated cells and the halofuginone-treated cells were significantly different from one another (p-value < 0.05). Because we previously noted that TSP-1 expression was upregulated upon treatment of human prostrate cancer cells with halofuginone, we also isolated RNA from halofuginone treated and control mouse breast cancer cells and carried out reverse transcription/real-time PCR reactions using oligos and probes specific for mouse TSP-1 and GAPDH. We found an average 1.69-fold increase in TSP-1 message levels after two days of treatment with 1 nM of halofuginone (n = 4) and an average 0.89-fold change with 10 nM of halofuginone treatment (n = 4). The difference between the number of cells in the control-treated wells and the 1 nM halofuginone-treated wells was statistically significant (data not shown; p-value < 0.01). We next decided to test whether halofuginone would effect tumor growth in vivo. Halofuginone inhibit early tumor growth in polyoma middle T antigen transgenic mice. PyT mice exhibit onset of mammary epithelial hyperplasia 21 days after birth and develop palpable tumors 35 days after birth.56,57 By 60 days of age, we observed nests of tumor cells throughout the fat pad,56 and at 90 days of age, the tumor burden is obviously much greater and all 10 mammary glands have developed large tumors that overlap one another. The overall pattern of tumor progression is comparable in the TSP-1+/+PyT and TSP-1-/-PyT strains. Starting at 30 days of age, mice were injected with 5µg of halofuginone every other day for 30 or 60 days. Control mice received vehicle solution. In this model, tumors that form in the six mammary glands that reside in the thoracic cavity grow more rapidly than those

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that develop in the lower four mammary glands of the body (inguinal mammary glands). We therefore pooled these two populations and quantitated tumor growth through weight. After 30 days of halofuginone treatment, tumors from TSP-1+/+PyT mice were 30% lighter than tumors from the control mice (Fig. 2A; p-value = 0.09). Halofuginone-treated tumors from TSP-1-/-PyT mice were 44% lighter than the control (Fig. 2A; p-value < 0.05). Interestingly, untreated tumors from TSP-1 -/- PyT control mice were heavier than those from the untreated TSP-1+/+ PyT mice indicating that TSP-1 inhibits tumor growth in this mouse model of breast cancer (p-value = 0.056). After 60 days of halofuginone treatment, tumor weight did not significantly differ between treated and control mice (Fig. 2B). This will be discussed in more details below. The reduction in tumor volume was accompanied by reduction in vessel formation. Staining the cells for CD31 and using IP lab to count the vessels, we found a decrease in the number of blood vessels in the halofuginone treated tumors at 30 days in both the TSP-1+/+ PyT and TSP-1-/- PyT mice (p < 0.001; Fig. 3A). TSP-1+/+ PyT control tumors contained an average of 57.9 blood vessels per field, which differed significantly from the halofuginone treated TSP-1+/+PyT tumors that contained 30.4 vessels per field. The average number of vessels per field in the TSP-1-/- PyT tumors untreated and treated with halofuginone was 57.7 and 28.7, respectively. Due to the involvement of the extracellular matrix, in general, and of collagen type I, in particular, in angiogenesis, and because reduction of collagen synthesis is reportedly part of halofuginone’s mode of action, we assessed the level of type I collagen in mammary tumors using immunohistochemistry. We stained and graded the presence of type I collagen in the tissues with 1 being the least collagen and 5 being the most collagen. We averaged the grade of each group and found that less type I collagen was present in the halofuginone treated TSP-1+/+PyT tumors than in the control tumors after 30 days of treatment (Fig. 3B). TSP-1+/+PyT tumors treated with halofuginone had an average grade of 2.9 while the control tumors had an average grade of 4.4 (p-value < 0.05). A halofuginone-dependent reduction in collagen synthesis was observed in the TSP-1-/-PyT tumors as well (average grade for control was 3.2 and for halofuginone-treated, it was 2.5) but this reduction did not achieve statistical significant because of the lower level of collagen synthesis in the untreated TSP-1-/-PyT mice. Decreased collagen is also observed within wounds in TSP-1-null mice as compared to wild-type mice.58 The decrease in collagen synthesis in TSP-1-null mice may be due to decreased TGF-β activation in the absence of TSP-1. After 60 days of halofuginone treatment, there was not a significant difference between the tumor weight of the treated and the control groups both in the TSP-1+/+ and TSP-1-/- mice (Fig. 2B; TSP-1+/+: 3.02 g vs. 2.64 g; TSP-1-/-: 3.31 g vs. 2.62 g). As seen in the control tumors harvested after 30 days of treatment, the T+/+PyT tumors were lighter than the TSP-1-/tumors suggesting that TSP-1 by itself is inhibiting tumor growth. However, those tumors that were treated for 60 days with halofuginone did not differ either in the blood vessel numbers or in the collagen content as compared to vehicle-treated mice in either genotype except for one case (Fig. 4A and B). The number of blood vessels was significantly different between the PyT TSP-1-/- halofuginone treated mice as compared with the control PyT T-/mice (p-value < 0.001). At this timepoint, the blood vessel density is higher in the untreated PyT TSP-1-/- mice as compared to the untreated PyT TSP-1+/+ mice presumably due to the anti-angiogenic activity of TSP-1. Halofuginone affects TGFβ pathway via inhibition of Smad 2/3 activation. To determine if halofuginone is inhibiting mammary tumor growth by inhibiting TGF-β signaling, we measured total Smad 2/3 and phosphorylated Smad 2/3 by Western Blot analysis. In the control untreated cells isolated from TSP-1+/+ PyT tumors, Smad 2/3 phosphorylation levels were low and a slight increase was observed after halofuginone treatment (Fig. 5). Treating the cells either with TGF-β1 or TSR2+RFK increased Smad 2/3 phosphorylation level, which was partly prevented by halofuginone. The changes in phosphorylation was not due to changes in Smad 2/3 synthesis since its level was not significantly changed in all treatments tested (see densitometry readings below figure). The inhibition of TGFβ-dependent Smad 2/3 phosphorylation by halofuginone was not specific to the tumor




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Figure 2. Effect of Halofuginone treatment on tumor weight after (A) 30 days of treatment and (B) 60 days of treatment. Values are mean ± SE. A single star (*) indicates p < 0.05. T+/+ represents TSP-1+/+PyT mice and T-/represents TSP-1-/-PyT mice.

Figure 3. Effect of 30 days of Halofuginone treatment on (A) Blood vessel number per field and (B) Collagen type 1 level. Values are mean ± SD. A single star (*) indicates p < 0.001 and two stars (**) indicates that p < 0.05. T+/+ represents TSP-1+/+PyT mice and T-/- represents TSP-1-/-PyT mice.

cells. Human dermal endothelial cells were also able to respond to TGF-β by increasing Smad 2/3 phosphorylation level and this was also partly prevented by halofuginone (Fig. 6).

TGFβ, the major promoter of collagen type I gene expression.62 Transcriptional activity of the α1(I)-collagen promoter has been correlated with the formation of capillary-like structures by endothelial cells,63 and with the stimulation of microvascular endothelial cells to form solid cords that resemble the precapillary structures found during angiogenesis.64 We previously demonstrated that halofuginone, which inhibits collagen type I synthesis on the transcriptional level,2,3,15 was a potent inhibitor of angiogenesis21 and led to inhibition of tumor growth in bladder carcinoma, C6 glioma and prostrate.20,22,24 In the data presented here, halofuginone inhibited collagen synthesis in mammary tumors from PyT mice after 30 days of treatment. Furthermore, halofuginone had no effect on Smad 2/3 phosphorylation in the untreated tumor cells while it inhibited the TGFβ or TSR2+RFK-dependent Smad 2/3 phosporylation in the tumor cells (Fig. 5), endothelial cells (Fig. 6) and fibroblasts.27 Thus, halofuginone mainly affects stimulated collagen synthesis, such as by TGF-β, and when administered systemically, it will be effective in only fibrotic location without affecting collagen synthesis in other locations. In most animal models of fibrosis, regardless of the tissue, halofuginone had a minimal effect on collagen content in the control, nonfibrotic animals, whereas it exhibited a profound inhibitory effect in the fibrotic organs.2,6,12,14,16 These results suggest separate mechanisms for the maintenance of the usual low level of expression of collagen type I genes and the overexpression induced by fibrogenic stimuli such as TGFβ, an aggressive and a rapid process. Our in vitro data demonstrates that halofuginone not only inhibits tumor growth by decreasing collagen type 1 synthesis, but may also do so in a more direct manner, by inhibiting cell proliferation. TSP-1+/+PyT cells do not proliferate in the presence of halofuginone when followed for three days (Fig. 1). Unlike the control cells, the

DISCUSSION Halofuginone has been used as an effective anti-tumorigenic reagent in several xenografts and chemically-induced models of cancer.20,21,24,25 This study is the first to evaluate the efficacy of halofuginone in a transgenic, spontaneous mouse model of breast cancer. The PyT mouse closely recapitulates the human, breast cancer disease process in that it progresses from hyperplasia to malignancy and eventually to metastatic disease.57,59 During the earlier stages of tumor development, halofuginone treatment inhibited tumor growth in all mammary glands both in the TSP-1+/+PyT and in the TSP-1-/-PyT mice. The reduction in tumor weight was accompanied by a reduction in collagen synthesis and blood vessel number in agreement with other studies describing the effect of halofuginone in various malignancies.19-26 Although TSP-1 is known to be involved in inhibiting angiogenesis, it is probably not required for halofuginone’s action as halofuginone was able to reduce tumor volume in the TSP-1 null mice. It is well established that collagen type I, the major component of the ECM, not only provides a scaffold for the tissue, but also regulates many fundamental cellular processes that are essential for angiogenesis such as migration and proliferation.18 Drugs that promote early and marked angiogenesis enhanced collagen type I deposition60 whereas suppression of angiogenesis by anti-tumor drugs was associated with decreased collagen synthesis.61 Delayed neovascularization was also associated with a decrease in collagen type I and www.landesbioscience.com


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B Figure 5. Western Blot demonstrating expression levels of phospho Smad 2/3, total Smad 2/3 and actin. The lysates (100 µg) of cells extracted from a tumor that formed in a TSP-1+/+PyT mouse (100 µg of protein) were electrophoresed through a 10% SDS-polyacrylamide gel, transferred onto nitrocellulose paper and probed for protein expression using antibodies against phospho Smad 2/3, Smad 2/3 and actin.

Figure 4. Effect of 60 days of Halofuginone treatment on (A) Blood vessel number per field and (B) Collagen type 1 level. Values are mean ± SD. A single star(*) indicates p < 0.001. T+/+ represents TSP-1+/+PyT mice and T-/- represents TSP-1-/- PyT mice.

Figure 6. Western Blot demonstrating expression levels of phospho Smad 2/3, total Smad 2/3 and actin. Lysates derived from cultured human dermal endothelial cells were electrophoresed through a 10% SDS-polyacrylamide gel, transferred onto nitrocellulose paper and probed for protein expression using antibodies against phospho Smad 2/3, Smad 2/3 and actin.

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halofuginone-treated cells exhibited a relatively flat growth curve with the same number of cells remaining in the dish. A similar profile was seen using human MDA-MB-435 breast cancer cells (Pines M, unpublished data) and Wilm’s tumor cell lines.26 Interestingly, we saw that TGF-β1 also inhibited cell proliferation, but unlike halofuginone whose effect was seen after 24 hours, it took 72 hours for TGF-β to significantly decrease cell proliferation. On the surface, it may seem like halofuginone is promoting cell proliferation by inhibiting TGF-β signaling, but the time course of it’s action suggests that its anti-proliferative effect would dominate over TGF-β’s anti-proliferative effect. Furthermore, halofuginone would have a greater effect on angiogenesis than cell proliferation by inhibiting TGF-β mediated collagen synthesis. Although TSP-1 has an important role in the activation of TGFβ, potent stimuli for TSP-1 synthesis are TGFβ165-67 and TGFβ2 which in turn activate Smad 2 and 3.68,69 Indeed, we saw a 3-fold increase in TSP-1 RNA expression upon 48 hour exposure to TGF-β1 in the TSP-1+/+PyT cells (Yee K, unpublished data). Thus, in some cells, a positive feedback loop exists with activation of TGFβ stimulating the synthesis of one of its activators. It was shown that induction of TSP-1 is mediated by Smad 2 but not Smad 3, while the TGFβ-dependent synthesis of VEGF by endothelial cells was dependent on Smad 3 activation and not by a Smad 2-dependent mechanism.66 Although the antibodies used in our study cannot distinguish between phosphorylated Smad 2 and 3, a previous study demonstrated that halofuginone specifically inhibited Smad 3 but not Smad 2 phosphorylation.14,70 The data suggest that halofuginone inhibits TGF-β dependent synthesis of VEGF and collagen and this can explain the halofuginone-dependent reduction in vessel number in the TSP-1 null mice. Thus, in our model, the inhibition of angiogenesis by halofuginone probably depends more upon the downregulation of the pro-angiogenic side of the angiogenic balance, rather than the upregulation of anti-angiogenic activity. It is interesting to note that halofuginone treatment in the absence of TGF-β inhibits phosphorylation of Smad2/3 in endothelial cells (Fig. 5), but had little effect in the tumor cells. Only upon pretreatment with TGF-β was halofuginone able to downregulate phosphorylation in the tumor cells. Moreover, halofuginone decreased phosphorylated Smad 2/3 levels to a greater extent in the tumor cells




than in the endothelial cells. This suggest that there may be two pathways involved in the phosphorylation of Smad 2/3 by TGF-β which are cell-specific. In both cases, the overall result would be decreased collagen synthesis and decreased deposition of collagen in the tumor matrix. This could lead to a decrease in tumor angiogenesis. Also noteworthy is the observation that TSR2 also caused an increase in the level of phosphorylation of Smad 2/3 despite the absence of the sequence, RFK. In our previous work, we saw that TSR2 alone can cause an increase in active TGF-β levels.46 Although it did not reach statistical significance, the mean weight of the halofuginone-treated, mammary tumors in the TSP-1+/+PyT or in the TSP-1-/-PyT mice was still smaller than the control tumors after 60 days of treatment. Sixty days of halofuginone treatment had no effect on collagen content and only a minor reduction in vessel number was observed in the TSP-1-/-PyT mice. These results suggest that once the tumors are robustly growing, halofuginone loses its effectiveness in inhibiting tumor growth. Moreover, during this window of time, the blood vessel bed could be already established and halofuginone would be less effective in inhibiting tumor growth during this later stage. Indeed, it was observed in this mouse model that after this time point, blood vessels were not observed to increase in number.57 In conclusion, our study has demonstrated that halofuginone inhibits early tumor progression in a transgenic mouse model of breast cancer through a TSP-1-independent mechanism, but once the tumors are rapidly growing, it is no longer effective in halting tumor growth. Furthermore, halofuginone inhibits mammary tumor growth by decreasing TGFβ signaling in tumor cells, fibroblasts and endothelial cells, by decreasing expression of type I collagen and decreasing cell proliferation. References 1. Granot I, Halevy O, Hurwitz S, Pines M. Halofuginone: An inhibitor of collagen type 1 synthesis. Biochem Biophys Acta 1993; 1156:107-12. 2. Pines M, Vlodavsky I, Nagler A. Halofuginone-a novel anti-fibrotic therapy. Gen Pharmacol 1997; 30:445-50. 3. Pines M, Vlodavsky I, Nagler A. Halofuginone: From veterinary use to human therapy. Drug Develop Res 2000; 50:371-8. 4. Halevy O, Nagler A, Levi-Schaffer F, Genina O, Pines M. Inhibition of collagen type 1 synthesis by skin fibroblasts of graft versus host disease and scleroderma patients: Effect of halofuginone. Biochem Pharmocol 1996; 52:1057-63. 5. Levi-Schaffer F, Nagler A, Slavin S, Knopov V, Pines M. Inhibition of collagen synthesis and changes in skin morphology in murine graft-versus-host disease and tight skin mice: Effect of halofuginone. J Invest Dermatol 1996; 106:84-8. 6. Pines M, Domb A, Ohana M, Inbar J, Genina O, Alexiev R, Nagler A. Reduction in dermal fibrosis in the tight-skin (Tsk) mouse after local application of halofuginone. Biochem Pharmocol 2001; 62:1221-7. 7. Nagler A, Firman N, Feferman R, Cotev S, Pines M, Shoshan S. Reduction in pulmonary fibrosis in vivo by halofuginone. Am J Resp Critical Care Med 1996; 154:1082-6. 8. Nagler A, Rivkind AI, Raphael J, Levi-Schaffer F, Genina O, Lavelin I, Pines M. Halofuginone–an inhibitor of collagen type I synthesis–prevents postoperative formation of abdominal adhesions. Ann Surg 1998; 227:575-82. 9. Nagler A, Genina O, Lavelin I, Ohana M, Pines M. Halofuginone-an inhibitor of collagen type I synthesis-prevents formation of post-operative adhesions formation in the rat uterine horn model-prevents formation of post-operative adhesions formation in the rat uterine horn model. Am J Obstet Gyn 1999; 180. 10. Nagler A, Gofrit O, Ohana M, Pode D, Genina O, Pines M. The effect of halofuginone,an inhibitor of collagen type I synthesis, on urethral stricture formation in vivo and in vitro study in a rat model. J Urol 2000; 164:1776-80. 11. Pines M, Knopov V, Genina O, Lavelin I, Nagler A. Halofuginone, a specific inhibitor of collagen type I synthesis, prevents dimethylnitrosamine-induced liver cirrhosis. J Hepatol 1997; 27:391-8. 12. Bruck R, Genina O, Aeed H, Alexiev R, Nagler A, Pines M. Halofuginone to prevent and treat thioacetamide-induced liver fibrosis in rats. Hepatology 2001; 33:379-86. 13. Gnainsky Y, Spira G, Paizi M, Bruck R, Nagler A, Abu-Amara SN, Geiger B, Genina O, Monsonego-Ornan E, Pines M. Halofuginone, an inhibitor of collagen synthesis by rat stellate cells, stimulates insulin-like growth factor binding protein-1 synthesis by hepatocytes. J Hepatol 2004; 40:269-77.

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