Affiliations of authors: Department of Urology (HA, TS, SK, Y. Kiyama, TU, T. Inamoto, Y. Katsuoka) and Department of Anatomy and Biology (YI, YO), Osaka Medical College, Takatsuki, Osaka, Japan; Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan (SE, HM, TW, KM); Department of Urology, Juntendo University School of Medicine, Tokyo, Japan (SE); Department of Urology, Teikyo University School of Medicine, Tokyo, Japan (OM, SH); Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo Japan (SE, T. Imamura, KM); Department of Urology, Osaka University, School of Medicine, Osaka, Japan (ST)
Correspondence to: Kohei Miyazono, MD, Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (e-mail: miyazono-ind{at}umin.ac.jp).
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ABSTRACT |
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INTRODUCTION |
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Members of the TGF- superfamily, which include TGF-
, activin, and bone morphogenetic proteins (BMPs), transmit their signals through Smad proteins (4,10). Signals from TGF-
and activin are transduced through two receptor-regulated Smad proteins (R-Smads), Smad2 and Smad3, whereas signals from BMPs are transduced through other R-Smads (i.e., Smad1, Smad5, and Smad8). Smad4 is a common-partner Smad (Co-Smad) that forms complexes with activated R-Smads. The R-Smad and Co-Smad complexes translocate into the nucleus, where they regulate transcription of target genes (e.g., plasminogen activator inhibitor 1 [PAI-1]). By contrast, Smad6 and Smad7 inhibit intracellular signaling by the TGF-
superfamily proteins, mainly by interacting with activated type I receptors for the TGF-
superfamily proteins. Smad6 predominantly inhibits BMP signaling, whereas Smad7 inhibits both TGF-
and BMP signaling.
Positive and negative regulation of target gene transcription by TGF- is mediated by the binding of Smads to transcriptional coactivators and corepressors, respectively (11). The protooncoprotein c-Ski is a transcriptional corepressor that interacts strongly with Smad2, Smad3, and Smad4 but only weakly with Smad1 and Smad5 (1214). c-Ski suppresses signaling of both TGF-
and BMPs through its binding to Smad proteins and recruitment of histone deacetylases by means of N-CoR and mSin3A (1517). A mutant form of c-Ski, c-Ski (ARPG), which has an insertion of four amino acids (AlaArgProGly), inhibits signaling by TGF-
but not by BMPs because of its lack of binding to Smad4 (17,18).
In this study, we used adenovirus-mediated gene transfer to modulate TGF- signaling in an established mouse model of breast cancer to elucidate the roles of TGF-
superfamily signals in cancer growth and metastasis (19).
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MATERIALS AND METHODS |
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JygMC(A) cells, which spontaneously metastasize to lung and liver when inoculated subcutaneously into nude mice, were originally isolated from a mammary carcinoma that arose in a Chinese wild mouse (Mus musculus Sub-Jyg) (19). The cells were cultured in Dulbecco's modified Eagle's minimal essential medium (Sigma, St. Louis, MO) containing 10% fetal calf serum (FCS) and 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO, Grand Island, NY). We established JygMC(A) cells that stably overexpressed mouse Smad6, Smad7, or neither by transfecting the cells with expression plasmids pCAG-IP-Smad6, pCAG-IP-Smad7, or pCAG-IP-empty (20), respectively, using FuGENE6 reagent according to the manufacturer's instructions (Roche, Indianapolis, IL). Transfected cells were cultured for 3 weeks in medium containing 5 µg/mL puromycin (Sigma), and the puromycin-resistant JygMC(A) cells that remained in culture were then subcloned and maintained in medium containing 5 µg/mL puromycin. Recombinant human TGF-3 and BMP-4 were obtained from R&D Systems (Minneapolis, MN).
Adenoviruses
Recombinant E1-deleted adenoviral vectors carrying mouse cDNAs encoding Smad6 (AdCMVSmad6), Smad7 (AdCMVSmad7), c-Ski (AdCMVcSki), c-Ski (ARPG) (AdCMVc-Ski[ARPG]), or the -galactosidase (LacZ) reporter gene (AdCMVLacZ) under control of cytomegalovirus (CMV) promoters were generated and purified as previously described (17,21,22). We used AdCMVLacZ as control (LacZ), and we also used virus buffer only as another control group (Control).
Adenovirus-Mediated Gene Transfer Into Cells In Vitro and Into Tissues In Vivo
For in vitro experiments, JygMC(A) cells (106) were plated and incubated with each adenovirus at various concentrations (1, 10, or 100 multiplicity of infection) for 2 hours with gentle agitation. The cells were incubated for 12 hours after the addition of fresh medium containing 1% FCS. Cells incubated with virus buffer served as controls. For in vivo gene transfer, intravenous administration of each virus was performed through the penile vein. We detected expression of -galactosidase by incubating adenovirus-infected tissues or cells in 0.2% glutaraldehyde for 10 minutes followed by incubation at 37 °C for at least 4 hours in 1 mg/mL 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal; Sigma) and observation with a phase-contrast microscope.
In Vivo Experiment With Adenovirus-Mediated Smad6 or Smad7 Expression
JygMC(A) cells (107 cells, unless otherwise specified) were inoculated subcutaneously into the flanks of 5-week-old male BALB/c nu/nu mice (Nihon Slc, Shizuoka, Japan). Ten days after the inoculation of the cells, when the primary tumors were approximately 10 mm x 10 mm, mice were randomly assigned to six groups; mice in each group received an intravenous administration of a virus or virus buffer once weekly (109 plaque-forming units/week) until the animals died (30 mice per group for the Smad6, Smad7, LacZ, and Control groups, and 20 mice per group for the c-Ski and c-Ski[ARPG] groups). On days 7, 21, 28, 36, and 42 after adenovirus administration was initiated, we randomly selected mice from each group (total n > 9 per group), killed them by administering xylazine (20 mg/kg of body weight) and ketamine (120 mg/kg) in one intraperitoneal injection, and harvested their lungs, livers, and primary tumors for histologic and molecular analyses. The remaining mice from each group (at least 10 mice per group) were used to evaluate survival, which was defined as the time from inoculation of JygMC(A) cells to the time of death. None of the mice had to be killed because of criteria specified in the experimental protocols. All experimental protocols were conducted in accordance with the policies of the Animal Ethics Committee at the Osaka Medical College.
In Vivo Experiment Using JygMC(A) Cells that Stably Express Smad6 or Smad7
To investigate tumor growth and metastasis in mice bearing tumors derived from JygMC(A) cells that stably express Smad6 or Smad7 (Jyg-Smad6 cells and Jyg-Smad7 cells, respectively), we injected 107 JygSmad6 cells (JygSmad6 group), JygSmad7 cells (JygSmad7 group), JygEmpty cells (JygEmpty group), or parental JygMC(A) cells into the flanks of male 5-week-old BALB/c nu/nu mice (n > 40 mice/group). In vivo tumor growth, metastasis, and survival times of animals were examined in a same way as they were in the adenoviral experiment.
Analyses of Tumor Growth and Metastasis
Tumors were measured every 3 days in two dimensions: the longest axis (a) and the shortest perpendicular axis (b). We calculated tumor volume (TV) using the formula 0.4 ab2 and transformed the resulting absolute values for tumor volume into relative values (v) using the formula Vx/V0, where V0 was the tumor volume on the day adenovirus administration was initiated and Vx was the tumor volume on day x thereafter (23). We evaluated tumor metastasis by counting the number of metastatic colonies in one histologic section of the midportion of each sample of liver and lung from each mouse, by measuring lung and liver weights, and by determining the ratio of the metastatic area to the total area in histologic sections from the midportion of each organ harvested on day 36 after the initiation of the virus administration (n > 9/group). The ratio of metastatic area to total area in the histologic section was calculated by using a public-domain image analysis program (NIH Image; written by Wayne Rasband at National Institutes of Health and available at http://rsb.info.nih.gov/nih-image/download.html). The result was expressed as a percentage.
Reverse TranscriptionPolymerase Chain Reaction Analysis
Total RNAs isolated from JygMC(A) cells and from mouse tissues by using an RNeasy Mini kit (QIAGEN, Hilden, Germany) were used for first-strand synthesis of cDNAs by using an Omniscript RT kit (QIAGEN). We used a Lightcycler system (Roche Diagnostics), as previously described (24), to perform quantitative real-time reverse transcriptionpolymerase chain reaction (RTPCR) analyses of RNA levels for the mouse genes encoding Smad6 and Smad7, and of target genes of TGF- superfamily signals, including PAI-1 and inhibitor of differentiation/inhibitor of DNA binding 1 (Id-1), and various cell adhesion molecules, including E-cadherin (Cdh-1), N-cadherin (Cdh-2),
-catenin (Catn-
),
-catenin (Catn-
), nexillin (Nexn), profilin 1 (Pfn-1), gelsolin (Gsn), Occludin (Ocln), and zonula occludens 2 (ZO-2). The primer sequences and PCR conditions are described in Supplemental Table 1 (available at: http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol97/issue23).
Western Blot Analysis
JygMC(A) cell pellets or mouse tissues were homogenized and sonicated three times for 3 seconds each on ice in radioimmunoprecipitation assay (RIPA) buffer. The lysates were centrifuged (at 12 000g for 15 minutes), and their protein concentrations were determined using a bicinchoninic acid (BCA) assay kit (Pierce Biotechnology, Rockford, IL). Proteins (equal amounts of lysate loaded per lane) were resolved by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) and transferred onto the polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA). All gels were stained with Coomassie blue to confirm equal protein loads and homogenous protein transfer. Membranes were incubated with blocking solution (20 mM TrisHCl [pH 7.5], 100 mM NaCl, 0.1% Triton-X-100, and 3% bovine serum albumin) overnight at 4 °C. The filters were then incubated overnight with the following primary antibodies: rabbit antimouse phospho-Smad1/5 antibody, which recognizes the phosphorylated forms of Smad1 and Smad5, (1 : 200 dilution; Cell Signaling Technology, Beverly, MA); rabbit antimouse Smad1/5 antibody, which recognizes the phosphorylated and unphosphorylated forms of Smad1 and Smad5, (1 : 200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit antimouse phospho-Smad2 antibody, which recognizes the phosphorylated form of Smad2, (1 : 200 dilution; United Biomedical, Hauppauge, MA), and rabbit antimouse Smad2/3 antibody, which recognizes the phosphorylated and unphosphorylated forms of Smad2 and Smad3 (1 : 200 dilution; Transduction Laboratories, Lexington, KY). The filters were washed extensively with Tris-buffered saline0.1% Triton-X (TBST), and immunoreactive bands were visualized by using an enhanced chemiluminescence (ECL) blotting system (Amersham Pharmacia Biotech, Uppsala, Sweden).
PowerBlot Western Array Analysis
Total cell extracts from JygMC(A) cells and from the primary tumors from mice in each group harvested on the day 36 after initiation of the virus injection were processed by the PowerBlot facility (Becton Dickinson Biosciences, Bedford, MA), which determined the expression levels of 47 different signal transduction proteins using a combination of SDSPAGE (5%15% gradient acrylamide gels); immunoblotting with specific monoclonal antibodies as revealed by a horseradish peroxidaseconjugated goat antimouse secondary antibody; capture of chemiluminescence data by a charge-coupled device camera; and computerized processing of densitometric data (for details about the methodology, see http://www.clontech.co.jp/custom/powerblot). Data were normalized by dividing the signal obtained for each protein by the sum of signals obtained for all 47 proteins for one given sample. Proteins whose expression levels in two initial PowerBlot analyses varied by at least 20% were subjected to a third independent analysis to verify their expression levels.
Cell Growth, Migration, and Invasion Assays
For cell growth assays, we plated JygMC(A) cells, JygSmad6 cells, or JygSmad7 cells in six-well plates in fresh medium (104 cells/well), incubated the cells at 37 °C for 72 hours, and then determined the number of cells per well. For migration assays, we prepared 80% confluent monolayers of each cell line and infected cells with each adenovirus. A wound was incised in cultured cells of each dish for 24 hours after infection with each virus, and the edge of the wound was marked on the bottom of each well, as previously described (25). After another 24 hours, photographs were taken while viewing the cells by phase-contrast microscopy. We counted only the cells that had migrated into the midportion of each trisected wound area to exclude the influence of cell growth at the edge of wounded area. In vitro invasion assays were performed using BioCoat Matrigel Invasion Chambers (Becton-Dickinson Bioscience) according to the manufacturer's instructions. In brief, JygMC(A) cells were cultured for 24 hours in medium containing 1% FCS and then seeded into the transwell insert chamber with a filter coated with Matrigel, and the inserts were placed in the lower chambers, which were filled with medium containing 5% FCS. The cells were incubated for 12 hours, after which we counted the cells that had invaded the lower side of the filter in at least 10 fields of view of a phase-contrast microscope. All assays (i.e., the cell growth, migration, and invasion assays) were done in triplicate, and two independent experiments were performed.
Statistical Analysis
We used the KruskalWallis H statistic to compare tumor metastasis data from multiple groups of mice, including organ weight, number of metastatic colonies, and the ratio of metastatic area to total area, as well as in vitro data from JygMC(A) cells infected with each adenovirus tested in the cell growth, migration, and invasion assays. When the P values for the overall comparisons were less than .05, we performed posthoc pairwise comparisons by using Wilcoxon's rank sum test. Unpaired Student's t tests were performed for parametric comparisons between two groups (assumptions of the test were verified). Parametric comparisons among more than two groups were subjected to one-way analysis of variance (ANOVA) without replication. When the ANOVA result was statistically significant, posthoc pairwise comparisons were performed by using the Scheffe test (26). Mouse survival was evaluated by KaplanMeier analysis and the log rank test. P values less than .05 were considered statistically significant. All statistical tests were two-sided.
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RESULTS |
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Mice bearing tumors derived from injection of JygMC(A) cells were treated with intravenous administration of AdCMVSmad7 (Smad7 group, n = 30), AdCMVSmad6 (Smad6 group, n = 30), AdCMVc-Ski (c-Ski group, n = 20), AdCMVc-Ski (ARPG) (c-Ski [ARPG] group, n = 20), AdCMVLacZ (LacZ group, n = 30), or virus buffer (Control group, n = 30). More than half of the mice in the Control and LacZ groups developed metastatic lesions in lung and liver by 3 weeks after virus or virus buffer administration. In these two groups of control mice, metastatic lesions progressed aggressively and diffusely thereafter, and almost all mice developed metastases in these organs by 5 weeks (Fig. 1, A and B).
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Representative macroscopic and microscopic images of hematoxylineosin-stained sections of lung and liver from mice that were killed after 5 weeks of treatment with AdCMVSmad7 or AdCMVSmad6 and from Control (virus buffer) mice are shown in Fig. 1, E. In control mice, we observed more than 100 metastatic lesions (i.e., hematoxylineosin-stained areas) in a lung section, and more than 20 metastatic lesions in a liver section from each mouse (n = 10 or more sections/group). Microscopic observation revealed aggressive cancer growth, with structural deformity in each organ (Fig. 1, E, left panels). By contrast, in the mice treated with AdCMVSmad7, only minute metastatic lesions were observed macroscopically, and only a few cancer cells were observed at higher resolution, both in lung and liver (Fig. 1, E, middle panels). However, mice treated with AdCMVSmad6 showed many metastases (Fig. 1, E, right panels), similar to what we observed in the control mice. There were no statistically significant differences in primary tumor growth among all groups of mice examined, and in all mice, subcutaneously inoculated tumor cells became bulky masses by 35 days after inoculation (Fig. 1, F).
Effect of Systemic Gene Transfer of Smad7 or c-Ski on Mouse Survival
We evaluated the survival of mice that received the various systemic treatments from the time of JygMC(A) cell inoculation to the time of death due to the lung and liver metastasis (n > 10 mice per group). Among the control mice (LacZ and Control), more than 50% of the mice in each group (LacZ: 13 of 15 mice; Control: 10 of 15 mice) died within 43 days of JygMC(A) cell inoculation with marked body weight loss and cachexia; no mice survived beyond 50 days (Fig. 1, G). There was no difference in median survival between mice treated with AdCMVLacZ and Control mice (i.e., mice treated with virus buffer; P = .37). By contrast, mice systemically treated with AdCMV-Smad7 or AdCMVc-Ski lived statistically significantly longer than mice in each of the control groups (Smad7 versus LacZ: median survival = 55 days versus 41 days, difference = 14 days [95% CI = 6 days to 22 days], P<.001; Smad7 versus Control: median survival = 55 days versus 43 days, difference = 12 days [95% CI = 6 days to 18 days] P<.001; c-Ski versus LacZ: median survival = 51 days versus 41 days, difference = 10 days [95% CI = 5 days to 15 days], P<.001; c-Ski versus Control: median survival = 51 days versus 43 days, difference = 8 days [95% CI = 3 days to 13 days], P<.001); at least 50% of the mice in the Smad7 (7 of 12 mice) and c-Ski (5 of 10 mice) groups survived for 50 days with healthy appearance, and two (17%) of the 12 mice treated with AdCMVSmad7 ultimately survived for more than 70 days. By contrast, there was no statistically significant difference in survival time between AdCMV-Smad6treated or AdCMV-c-Ski (ARPG)treated mice and control mice (Smad6 versus LacZ: median survival = 43 days versus 41 days, difference = 2 days [95% CI = 1 day to 5 days], P = .11; Smad6 versus Control: median survival = 43 days versus 43 days, difference = 0 days [95% CI = 3 day to 3 days], P = .30; c-Ski(ARPG) versus LacZ: median survival = 44 days versus 41 days, difference = 3 days [95% CI = 1 day to 5 days], P<.18; c-Ski(ARPG) versus Control; median survival = 44 days versus 43 days, difference = 1 day [95% CI = 3 day to 5 days], P = .70). These results suggest that adenovirus-mediated gene transfer of Smad7 or c-Ski, but not of Smad6 or c-Ski (ARPG), prolonged mouse survival through the inhibition tumor metastasis.
Effect of Delayed Gene Transfer of Smad7 on Metastasis of JygMC(A) Cells
We also examined the effect of Smad7 expression on the growth of tumor cells after the initial development of metastases at the target organs by delaying the initiation of Smad7 adenovirus administration until 3 weeks after inoculation of the cancer cells (Fig. 1). Compared with mice treated with virus buffer only (Control), mice bearing tumors with delayed Smad7 treatment (Smad73wks) developed many but statistically significantly fewer metastases in the lung and liver (Smad73wks versus Control: median number of lung colonies = 49 versus 128, difference = 79 [95% CI = 57 to 99], P = .0022; median lung weight = 0.623 g versus 1.026 g, difference = 0.403 g [95% CI = 0.118 g to 0.635 g], P = .001; median ratio of metastatic area in lung = 31.7% versus 48.3%, difference = 16.6% [95% CI = 2.12% to 26.6%], P = .0357; Smad73wks versus Control: median number of liver colonies = 9.5 versus 13, difference = 3.5 [95% CI = 1.0 to 14.0], P = .0171; median liver weight = 1.786 g versus 2.297 g, difference = 0.511 g [95% CI = 0.314 g to 0.744 g], P = .0021; ratio of metastatic area in liver = 12.7% versus 15.0%, difference = 2.3% [95% CI = 0.26% to 11.0%], P = .0498) (Fig. 1, AD) and statistically significantly longer survival (Smad73wks versus Control: median survival = 43 days versus 49 days, difference = 6 days [95% CI = 3.4 days to 8.6 days], P = .014) (Fig. 1, G). These results suggest that Smad7 expression inhibits the development of new metastatic colonies but does not influence the growth or progression of existing metastatic colonies.
Efficacy of Systemic Adenovirus Administration in Tumors and Host Tissues
To determine the efficiency of adenovirus-mediated gene transfer into mice bearing tumors, we evaluated the LacZ expression in lung, liver, and primary tumor tissue after systemic administration of AdCMVLacZ using X-Gal staining methods. We observed a strong LacZ expression in the liver and weaker expression in the lung and primary tumors of mice on day 4 after adenovirus administration (Fig. 2, A).
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Response of JygMC(A) Cells to TGF- and BMP
We next examined whether TGF- and BMP signals are transduced in JygMC(A) cells by treating cells in culture with human recombinant TGF-
3 (3 ng/mL) or BMP-4 (50 ng/mL) for 1 hour and examining their effects on phosphorylation of Smad proteins using antiphospho Smad antibodies. TGF-
3, but not BMP-4, induced phosphorylation of Smad2 (Fig. 3, A), and BMP-4, but not TGF-
3, induced phosphorylation of Smad1 and Smad5 (Fig. 3, B) in JygMC(A) cells. Moreover, TGF-
3, but not BMP-4, increased PAI-1 mRNA levels (Fig. 3, C, upper panel), and BMP-4, but not TGF-
3, increased Id-1 mRNA levels compared with the untreated controls (Fig. 3, C, lower panel), in JygMC(A) cells. These findings suggest that JygMC(A) cells can respond to both TGF-
and BMP.
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We next examined whether Smad7 inhibits metastasis through a direct effect on the JygMC(A) cells from which the primary tumors were derived or through an indirect effect on the microenvironment for metastasis in the target organs. We first generated JygMC(A) cells that stably express Smad6 or Smad7 (JygSmad6 cells and JygSmad7 cells, respectively) or that contained the empty expression vector (JygEmpty) as a control. Western blot analyses confirmed that equivalent levels of Smad6 and Smad7 genes were expressed by the respective stable cell lines (data not shown). We injected the flanks of nude mice with each newly constructed stable cell line, JygSmad6, JygSmad7 or JygEmpty (n = 40 mice/group) or with parental JygMC(A) cells (n = 30 mice). Mice bearing tumors derived from cells stably transfected with JygEmpty and from parental cells (Control) served as the control groups. Similar to the results in the adenovirus-mediated gene transfer experiment (Fig. 1), there were no statistically significant differences in tumor growth among mice injected with any of the three stable cell lines or with parental cell line (Supplemental Fig. 1; available at: http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol97/issue23). However, mice bearing tumors derived from JygSmad7 cells displayed statistically significantly less metastasis than control mice, as determined by a decrease in the number of metastatic colonies (Fig. 4, B), in organ weight (Fig. 4, C), and in the ratio of metastatic area to total area (Fig. 4, D) in both lung and liver. Also, the JygSmad7 mice survived statistically significantly longer than mice from any of the other groups (JygSmad7 versus JygEmpty: median survival = 95 days versus 45 days, difference = 50 days [95% CI = 39 days to 61 days], P<.001; JygSmad7 versus Control: median survival = 95 days versus 42 days, difference = 53 days [95% CI = 42 days to 64 days], P<.001; JygSmad7 versus JygSmad6: median survival = 95 days versus 43 days, difference = 52 days [95% CI = 41 days to 63 days], P<.001); no mice in the JygSmad7 group died before 40 days after tumor cell injection, and 12 (57%) of the 21 mice in this group survived for more than 90 days with a healthy appearance. Two mice ultimately survived for more than 130 days (Fig. 4, E). All of the mice injected with JygSmad7 cells died because of enlarged primary tumors but had little evidence of metastasis. By contrast, mice bearing tumors derived from JygSmad6 cells showed neither a reduction in cancer metastasis (Fig. 4, AD) nor a statistically significant difference in survival compared with the control mice (Fig. 4, E; P = .34 for JygSmad6 versus JygEmpty; P = .16 for JygSmad6 versus Control).
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To examine whether JygMC(A) cells that stably express Smad7 influence the microenvironment of surrounding tissues (possibly through the production of soluble factors), we evaluated tumor growth, metastasis, and survival of 5-week-old male nude mice that were subcutaneously injected with a mixture of 5 x 106 untransfected parental JygMC(A) cells and 5 x 106 JygSmad7 cells (Cont-Smad7-combined group, n = 30). Mice injected with 5 x 106 parental JygMC(A) cells (Cont-1/2 group, n = 30) or with 5 x 106 Jyg-Smad7 cells (JygSmad71/2 group, n = 30) were used as controls. We observed no statistically significant differences between mice bearing tumors derived from the combined cell lines (the Cont-Smad7-combined group) and mice bearing tumors derived from JygMC(A) cells alone (the Cont-1/2 group) with respect to the growth of the primary tumors (Supplemental Fig. 1; available at: http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol97/issue23), metastasis to lung and liver (Fig. 4, AD), or animal survival (Fig. 4, E). These findings strongly suggest that the Smad7 protein directly modulates the cancer cells in which it is expressed to reduce their metastatic potential rather than acting through intercellular mediators.
Effect of Smad7 Expression on the Molecular Signature of JygMC(A) Cells
We next attempted to identify proteins whose expression is differentially modulated by Smad7 expression. For this purpose, we used a high-throughput western blotting method that uses mixtures of monoclonal antibodies to evaluate differences in levels of cellular signaling proteins in total cell extracts among different cells or tissues. We were particularly interested in changes in the expression of proteins that are associated with cell adhesion because in the first steps of metastasis, tumor cells lose cellcell adhesiveness and gain motility (27).
We prepared extracts of JygMC(A) cells, JygMC(A) cells that had been infected with AdCMVLacZ or AdCMVSmad7, JygEmpty cells, and JygSmad7 cells, and subjected the extracts to a PowerBlot analysis to examine the relative expression levels of 47 signal transducing molecules involved in cellcell adhesion. Analyses of the resulting antibody array data indicated that expression of nine proteins was increased whereas expression of one protein was decreased in JygMC(A) cells treated with AdCMVSmad7 relative to parental JygMC(A) as well as in JygSmad7 cells relative to JygEmpty cells. Proteins whose expression increased included components of adherens junctions (i.e., E-cadherin, -catenin,
-catenin, nexillin, profilin, gelsolin, and phosphorylated FAK) and tight junctions (i.e., occludin and ZO-2). However, expression of the mesenchymal marker N-cadherin was statistically significantly lower in cells that expressed exogenous Smad7 than in cells that did not (Smad7 versus Control: mean relative RNA level = 1.15 versus 16.2, difference = 15.05 [95% CI = 12.4 to 17.9], P = .003; JygSmad7 versus JygEmpty: mean relative RNA level = 1.23 versus 7.54, difference = 6.31 [95% CI = 4.54 to 8.94], P<.001). The PowerBlot results were confirmed by western blot analyses of cell extracts (data not shown) as well as by quantitative RTPCR of RNA isolated from JygMC(A) cells infected with AdCMVLacZ or AdCMVSmad7, JygEmpty cells, and JygSmad7 cells (Fig. 5). These findings indicate that Smad7 may increase the expression of proteins associated with cell adhesion properties and that Smad7 increases E-cadherin expression but inhibits N-cadherin expression.
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Molecular alterations of JygMC(A) cells by Smad7 prompted us to examine the effects of Smad7 expression on cell morphology. JygMC(A) cells exhibited spindlelike, fibroblastic morphology with stress fibers oriented longitudinally (Supplemental Fig. 2; available at: http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol97/issue23). However, expression of Smad7, but not Smad6, changed the morphology of JygMC(A) cells to a flat, epithelial shape, and markedly increased the number of tight junctions compared with parental cells (Supplemental Fig. 2; available at: http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol97/issue23).
Effect of Smad7 Expression on JygMC(A) Cell Motility and Invasion In Vitro
We examined the effects of Smad6 or Smad7 expression on the behavior of JygMC(A) cells in vitro. We used JygMC(A) cells, JygMC(A) cells that had been infected with AdCMVSmad6 (Smad6), AdCMVSmad7 (Smad7), or AdCMVLacZ (LacZ), and JygEmpty, JygSmad6, and JygSmad7 cells in these assays. Cell growth assays demonstrated that Smad6 or Smad7 expression by both adenovirus-mediated and stable methods did not affect cancer cell proliferation. There was no statistically significant difference in cancer cell growth ratios among any of the cancer cell lines we used [JygMC(A) cells, JygMC(A) cells that had been infected with AdCMVSmad6, AdCMVSmad7, or AdCMVLacZ, and JygEmpty, JygSmad6, and JygSmad7 cells]. (Fig. 6, A). These results are consistent with our in vivo data showing that Smad7 gene transfer did not influence primary tumor growth.
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DISCUSSION |
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The spread of tumor cells from a primary site to distant organs is a critical stage in cancer metastasis that involves both a loss of cellcell adhesion and a gain of invasive properties (28). Many studies in experimental systems as well as in human patients have revealed that alterations in the adhesive properties of tumor cells are associated with tumor progression and metastasis (2933). Decreased expression or mutation of major components of adherens junctions is associated with the initiation of cancer metastasis (29,31,32,3443). Moreover, increases in or reconstitution of such adhesive properties has been shown to suppress metastasis in many different neoplastic tissues (4447). Here we have shown that Smad7 expression substantially increased the expression of major components of adherens junctions, such as E-cadherin, and tight junctions, such as occludin. Thus, Smad7 expression may enhance the adhesive properties of cancer cells through the formation of adherens junctions and tight junctions in cancer cells, and thereby contribute to the inhibition of metastasis.
We also found that expression of N-cadherin was statistically significantly lower in JygMC(A) cells that expressed Smad7 than in JygMC(A) cells that did not. N-cadherin enhances the motility and invasive ability of various types of tumor cells (4852). It has been repeatedly reported that de novo expression of N-cadherin in tumor cells is associated with the loss of functional E-cadherin (5355). Also, TGF- has been implicated in the switching of cadherin properties from E-cadherin to N-cadherin. This cadherin switch has been found to occur during the epithelial-to-mesenchymal transition (EMT) and may promote cancer metastasis (5658). Our finding that cancer cells with Smad7 expression have increased expression of N-cadherin and decreased expression of E-cadherin (Fig. 5) raises the intriguing possibility that a Smad7-mediated "cadherin switch" from expression of mesenchymal, promigratory cadherins (e.g., N-cadherin) to expression of epithelial, proadhesive cadherins (e.g., E-cadherin) inhibits tumor invasion and metastasis, and that this cadherin switch may be associated with Smad7-mediated inhibition of TGF-
signaling.
In agreement with the above findings, morphologic analysis of cancer cells with Smad7 expression revealed that Smad7 expression changed the morphology of JygMC(A) cells from a spindlelike, fibroblastic shape to a flat, epithelial shape, and markedly strengthened the cellcell adhesion with increased numbers of tight junctions. In vitro migration and invasion assays revealed that JygMC(A) cell motility was statistically significantly lower in cells that expressed exogenous Smad7 than in cells that did not. These results, taken together with our data on the expression of adhesion properties (Fig. 5) and delayed gene transfer of Smad7, suggest that Smad7 gene expression inhibits the development of new metastases but does not influence the growth or progression of already-existing metastatic colonies by increasing the cellcell interactions within primary tumors.
Several studies (2,6,59) have shown that the extracellular domain of TR-II, a soluble TGF-
antagonist, suppresses metastasis in mouse breast cancer models, suggesting that inhibition of TGF-
signaling is essential for the prevention of metastasis. In agreement with these findings, we found that only Smad7 and c-Ski, which effectively inhibit TGF-
regulated signals (6061), but not Smad6, which does not, prevented metastasis in our mouse model. Although Smad7, c-Ski, and the extracellular domain of T
R-II all block TGF-
signaling, their modes of action differ. The extracellular domain of T
R-II blocks binding of TGF-
to its specific receptors. Because TGF-
acts in an autocrine fashion in some cancer cells (8) and might bind to its receptors on the surface of the cells, TGF-
that is secreted in autocrine fashion may escape from the inhibitory effect of the extracellular domain of T
R-II. By contrast, Smad7 and c-Ski may efficiently block TGF-
superfamily signaling inside the cells. Therefore, Smad7 and c-Ski may exhibit their effects more efficiently than the extracellular domain of T
R-II. However, Smad7 and c-Ski primarily inhibit Smad-mediated signaling, but not other types of signaling (62). Furthermore, Smad7 and c-Ski have been reported to elicit biologic activities that are independent of TGF-
signals, such as the induction of apoptosis in prostate carcinoma cells (63). However, we detected no differences in apoptosis between the control JygMC(A) cells and Smad7-expressing JygMC(A) cells (data not shown). It will be interesting to compare the effects on the inhibition of metastasis of Smad7 and c-Ski with those of the extracellular domain of T
R-II in a future study.
We found that the c-Ski (ARPG) mutant, which was previously shown to inhibit TGF- signaling (17), did not inhibit cancer metastasis. Although it is not known whether BMP signaling can facilitate the progression of certain tumors in a similar fashion to TGF-
signaling, BMPs regulate the growth of some cancers (64,65). Our results show that Smad7 and c-Ski, which inhibit both TGF-
and BMP signaling, efficiently inhibited the development of metastasis, suggesting that the blocking of both TGF-
and BMP signals is required for the inhibition of cancer metastasis. However, our study is limited because we have not shown directly that inhibition of TGF-
signaling is sufficient to inhibit metastasis of JygMC(A) cells.
In conclusion, our results indicate that TGF- superfamily signals are important for regulating cellcell interactions and cancer metastasis, and that blocking these signals by systemic expression of Smad7 may be a novel strategy for the prevention of cancer metastasis, especially among patients with advanced-stage disease.
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Supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Science, Sports and Technology of Japan. The study sponsor had no role in the design of the study or in the collection, analysis, or interpretation of the data.
Funding to pay the Open Access publication charges for this article was provided by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Science, Sports, and Technology of Japan.
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Manuscript received February 24, 2005; revised September 22, 2005; accepted October 7, 2005.
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