Smad7 induces tumorigenicity by blocking TGF-β-induced growth inhibition and apoptosis

Experimental Cell Research 307 (2005) 231 – 246 www.elsevier.com/locate/yexcr

Smad7 induces tumorigenicity by blocking TGF-h-induced growth inhibition and apoptosis Sunil K. Halder a,b, R. Daniel Beauchampa,b, Pran K. Dattaa,b,T b

a Department of Surgery, Vanderbilt University School of Medicine, 1161 21st Avenue South, D5230 MCN, Nashville, TN 37232, USA Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

Received 26 June 2004, revised version received 9 March 2005 Available online 19 April 2005

Abstract Smad proteins play a key role in the intracellular signaling of the transforming growth factor h (TGF-h) superfamily of extracellular polypeptides that initiate signaling to regulate a wide variety of biological processes. The inhibitory Smad, Smad7, has been shown to function as intracellular antagonists of TGF-h family signaling and is upregulated in several cancers. To determine the effect of Smad7mediated blockade of TGF-h signaling, we have stably expressed Smad7 in a TGF-h-sensitive, well-differentiated, and non-tumorigenic cell line, FET, that was derived from human colon adenocarcinoma. Smad7 inhibits TGF-h-induced transcriptional responses by blocking complex formation between Smad 2/3 and Smad4. While Smad7 has no effect on TGF-h-induced activation of p38 MAPK and ERK, it blocks the phosphorylation of Akt by TGF-h and enhances TGF-h-induced phosphorylation of c-Jun. FET cells expressing Smad7 show anchorage-independent growth and enhance tumorigenicity in athymic nude mice. Smad7 blocks TGF-h-induced growth inhibition by preventing TGF-h-induced G1 arrest. Smad7 inhibits TGF-h-mediated downregulation of c-Myc, CDK4, and Cyclin D1, and suppresses the expression of p21Cip1. As a result, Smad7 inhibits TGF-h-mediated downregulation of Rb phosphorylation. Furthermore, Smad7 inhibits the apoptosis of these cells. Together, Smad7 may increase the tumorigenicity of FET cells by blocking TGF-h-induced growth inhibition and by inhibiting apoptosis. Thus, this study provides a mechanism by which a portion of human colorectal tumors may become refractory to tumorsuppressive actions of TGF-h that might result in increased tumorigenicity. D 2005 Elsevier Inc. All rights reserved. Keywords: Smad7; TGF-h (transforming growth factor-h); FET; Colon cancer; Tumorigenicity; Apoptosis

Introduction The transforming growth factor-h (TGF-h) family of polypeptides regulates a wide variety of biological functions including cell proliferation, differentiation, matrix formation, and apoptosis. The multifunctional effects of TGF-h are elicited through an oligomeric complex formation between the type I and type II serine-threonine kinase receptors. TGFh initiates signals by binding to the type II receptor (ThRII) and stabilizes the heteromeric complex with the type I

* Corresponding author. Department of Surgery, Vanderbilt University School of Medicine, 1161 21st Avenue South, T2104 MCN, Nashville, TN 37232, USA. Fax: +1 615 343 1355. E-mail address: [email protected] (P.K. Datta). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.03.009

receptor (ThRI), and as a result, ThRI is transphosphorylated and activated by ThRII. The activated ThRI then propagates the signals through interaction with and phosphorylation of receptor-associated Smads [1]. Smad proteins are divided into three distinct classes based upon their structure and function in signaling by TGF-h family members. Receptor-regulated Smads (RSmads) are phosphorylated on two serine residues at the Cterminus and thus activated in a ligand-specific manner. Smad2 and Smad3 mediate signaling by TGF-h and activin, whereas Smad1, Smad5, and presumably Smad8 are known to be involved in BMP signaling. Smad4 functions as a common mediator of TGF-h, activin, and BMP signaling pathways [2]. Upon phosphorylation by type I receptors, R-Smads form heteromeric complexes with Smad4 and translocate to the nucleus where they

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modulate transcription of TGF-h target genes [3]. A distinct class of distantly related Smads, including Smad6 and Smad7, has been identified as consisting of inhibitors of these signaling pathways, and these inhibitors function by interfering with the activation of R-Smads. Smad7 forms stable association with activated type I receptors, thereby preventing R-Smads from binding to and being activated by these receptors [4]. A short C-terminal region of Smad7 is required for interaction with the receptor and for its inhibitory function. STRAP associates with Smad7, recruits it from the cytosol to the activated receptor complex, and stabilizes the heteromeric complex with the receptors to inhibit TGF-h signaling synergistically with Smad7 [5]. Smad7 expression is rapidly induced by TGF-h family members in several cell types, indicating a key role of Smad7 in feedback regulation of TGF-h signaling [6]. Dysregulation of this feedback regulation of TGF-h activity results in disease states. Aberrant expression of Smad7 has recently been shown to cause uncontrolled TGF-h activity, which is associated with pathology of inflammatory bowel disease (IBD) and sclerosis [7]. Therefore, disruption of normal TGF-h balance in cell by an overexpression of Smad7 can lead to develop TGF-h-associated human disease [8]. Smad7 is expressed in very low level in epithelial tissues, but it is upregulated in human pancreatic cancers [9]. Significant expression of Smad7 is detected in colorectal cancer and amplification of Smad7 is associated with a worse prognosis [10,11]. Smad7 expression is increased in chemically induced mouse skin cancers [12]. The expression of Smad7 is also increased by pathways that negatively regulate TGF-h signaling [13]. Perhaps one of the most important biological effects of TGF-h is its ability to inhibit proliferation of many cell types, including most epithelial cells. TGF-h inhibits progression of cells from G1 into the S phase of the cell cycle. Cell cycle progression is mostly governed by CDKs, which are activated by Cyclins binding and inhibited by the CDK inhibitors. Several lines of evidence suggest that Smad signaling is functionally connected directly or indirectly in suppressing the growth of epithelial cells. The primary event that initiates the TGFh-induced growth arrest may be associated with increased expression of p15INK4B, p21Cip1, p27Kip1 and suppression of c-Myc expression [6]. Deregulation of CDK inhibitors may contribute to TGF-h resistance in cancer. In epithelial cells from the skin, lung, and breast, TGF-h rapidly elevates expression of the CDK4/6 inhibitor p15INK4B [6]. In keratinocytes, colon, and ovarian epithelial cells, TGFh elevates the expression of p21Cip1 [14]. Smad signaling is required for TGF-h-mediated induction of p15INK4B [15] and p21Cip1 [16]. TGF-h causes downregulation of Cyclin A, Cyclin E [17], and Cyclin D1 [18], depending on the cell type. TGF-h stimulation of epithelial cells induces the formation of a Smad complex that specifically recognizes a TGF-h inhibitory element in the c-Myc promoter, and this response may be critical for TGF-h-

induced c-Myc downregulation and growth arrest [19]. Dominant-negative Smad3 or the inhibitory Smad, Smad7, blocks TGF-h-induced growth inhibition in epithelial cells [6]. Multiple lines of evidence suggest that neoplastic transformation can result in loss of growth inhibitory response to TGF-h, and human colorectal cancers are in general functionally resistant to TGF-h-induced antiproliferative response. Resistance to TGF-h in colorectal cancers may occur through a variety of mechanisms such as reduced expression of either the ThRI or ThRII or mutational inactivation of TGF-h signaling mediators such as, ThRII, Smad2, and Smad4 [6,20]. However, downregulation or functional inactivation of TGF-h signaling molecules does not explain TGF-h unresponsiveness in all colorectal cancers. Smad7 is upregulated in several cancers. However, the potential role of altered expression of Smad7 in cellular proliferation and tumorigenicity in colorectal cancer has not been determined. We now report that overexpression of Smad7 leads to loss of transcriptional responses and growth inhibition induced by TGF-h in FET cells, a well-differentiated and non-tumorigenic human colon adenocarcinoma cell line. Smad7 blocks TGF-h-mediated down-regulation of c-Myc, Cyclin D1, and CDK4 expression, and suppresses the expression of p21Cip1. In addition, we show that Smad7-overexpressing clones exhibit enhanced anchorage-independent growth and accelerated tumor growth in nude mice. Our results suggest that blockade of TGF-h anti-proliferative effects by inhibitory Smad, Smad7, enhances the tumorigenicity of colon cancer cells.

Materials and methods Reagents and antibodies TGF-h1 was purchased from R&D Systems (Minneapolis, MN). The cell death ELISA kit was purchased from Roche Diagnostic Corporation (Indianapolis, IN). The Propidium iodide (cat# P-4170) and the monoclonal antiFlag and anti-h-actin antibodies were purchased from Sigma Biochemicals (St. Louis, MO). Rabbit anti-Smad2 and anti-Smad3 were from Zymed Laboratories Inc. (San Francisco, CA), and anti-Smad4, anti-phospho-ERK, antiERK, anti-p21Cip1, anti-Rb, anti-cMyc, anti-CDK4, antiCDK2, anti-Cyclin D1, anti-Bcl-2, anti-Bcl-xl, anti-Bcl-w, anti-Bad, anti-Bax, anti-Bid, and anti-PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Smad2 and anti-Apaf-1 antibodies were from Upstate Biotechnology (Lake Placid, NY), anti-phospho-c-Jun, anti-c-Jun, anti-phospho-p38, anti-p38, anti-phospho-Akt, anti-Akt, and anti-Caspase-3 antibodies were from Cell Signaling Technology (Beverly, MA), and anti-cytochrome C was from BD Biosciences (San Diego, CA).

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Cell lines and transfection FET cells (a kind gift from Dr. Brattain) were maintained at 37-C in a humidified atmosphere of 5% CO2 in DMEM (Invitrogen Life Technologies, Carlsbad, CA) containing 10% FBS supplemented with 50 units/ml penicillin and 50 Ag/ml streptomycin solution. Phoenix packaging cells, mink lung epithelial cells (Mv1Lu), and COS7 cells were maintained in regular DMEM as mentioned above. Transient transfections were performed using either Lipofectamine 2000 (Invitrogen Life Technologies) or FuGENE 6 (Roche Diagnostic Corporation, Indianapolis, IN), according to the manufacturer’s specifications. Retroviral construct and isolation of stable Smad7 clones To generate the retroviral expression construct, the pCDNA3-Flag-Smad7 vector containing a mouse fulllength Smad7 cDNA was digested with Xho1 and the linear vector was blunt ended by Klenow polymerase. The linear plasmid was then digested with BamH1 and the Flag-Smad7 fragment was purified. In parallel, the retroviral vector pBabe-Hygro was digested with EcoR1, blunt ended, and then digested with BamH1. The digested vector was then ligated with Flag-Smad7 cDNA and the resulting construct pBabe-Hygro-Flag-Smad7 was transiently transfected into COS7 cells to test the expression by Western blot analysis using anti-FLAG antibody. Phoenix packaging cells were transfected with 10-Ag retroviral construct/10-cm plate with Lipofectamine 2000. Twenty hours after transfection, new media were added (6 ml/plate) and cells were cultured for another 24 h to produce retrovirus into the culture media. Viral suspension was filtered through a 0.45-AM filter after mixing the suspension with polybrene (8 Ag/ml). Viruses were used to infect FET cells, and 24 h after infection, the media were changed to 10% FBS containing medium and cultured for 24 h. Infected cells were then selected for 2 weeks in the presence of 250 Ag/ml Hygromycin B (Invitrogen Life Technologies). Polyclonal population of cells was used to isolate clones and the expression of Smad7 in each clone was verified by Western blot analysis using anti-Flag antibody. Clones that expressed higher level of Smad7 were selected for the experiments and maintained in DMEM containing 10% FBS in the presence of 200 Ag/ml Hygromycin B. Transcriptional response assays Cells (0.35  106) from each pool of FET, vector control, and three stable Smad7 clones (Smad7 #2, Smad7 #6, and Smad7 #15) were seeded into each well of 12-well plates the day before transfection. Cells were then transiently transfected with CMV-h-gal and the TGF-h-sensitive reporter plasmid using Lipofectamine 2000. Equal amount of total DNA was used for each transfection. Twenty hours after transfection, cells were incubated in 0.2% FBS with 5 ng/ml

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of TGF-h for an additional 20 h. Cell lysates were used to measure both luciferase and h-gal activity. Luciferase activity was normalized to h-galactosidase activity and the resulting relative luciferase activity was expressed as the mean T SD of triplicate measurements. Cell growth inhibition assays 0.5  105 cells from each pool of FET, vector control, and three stable Smad7 clones were seeded in each well of 24-well plates and cultured for 20 h. Cells were then treated with increasing doses of TGF-h for 25 h as indicated. 4 ACi of [3H]-thymidine (NEN, Boston, MA) was then added in each well and incubated at 37-C for 2 h. Cells were then fixed in 10% cold trichloroacetic acid (TCA), washed, and lysed in 300 Al of 0.2 N NaOH for 30 min. 100 Al of each lysate was mixed with 4 ml of liquid scintillation fluid (NEN), and radioactivity incorporated into TCA-insoluble [3H]-thymidine was determined by scintillation counting. Results are expressed as the mean T SD of triplicate measurements. For cell-counting assay, 0.5  105 cells from each pool were seeded in each well of 12-well plates, treated with 5 ng/ml of TGF-h1 for a total of 5 days. At the end, cells were trypsinized, counted, and normalized results are plotted as the mean T SD of triplicate measurements. Western blot and immunoprecipitation analyses Cells (2  106) from each pool of FET, vector control, and three stable Smad7 clones were seeded in 60-mm plates and allowed to attach for at least 20 h. Cells were treated with 5 ng/ml of TGF-h1 and solubilized in lysis buffer (50 mM Tris –HCl [pH 7.5], 150 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5 mM dithiothreitol, 5 mM sodium fluoride, 0.5 mM sodium orthovanadate, 1.0 mM phenylmethylsulfonyl fluoride, 3 Ag/ml each of leupeptin, pepstatin, and aprotinin). Cell lysates were briefly sonicated and centrifuged at 14,000 rpm for 20 min at 4-C. Equal amounts of clear lysates were resolved by SDSpolyacrylamide gel electrophoresis (PAGE), electro-transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA), and immunoblotted with antibodies. For immunoprecipitation (IP), 4  106 cells from the above cell lines were seeded in 10-cm plates and cultured overnight. Cells were preincubated in serum-free medium for 2 h and then treated with 12.5 ng/ml of TGFh1 as indicated. Equal amount of each clear cell lysate was incubated with both anti-Smad2 and anti-Smad3 polyclonal antibodies for 2 h at 4-C, followed by incubation with 20 Al of protein G-Sepharose (Sigma Biochemicals, St. Louis, MO) for an additional 1 h. Immunoprecipitates were washed five times (1 ml each) with lysis buffer. The immune complexes were resolved in SDS-PAGE and then analyzed by Western blotting with mouse anti-Smad4 antibodies.

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Immunoprecipitation and kinase assays Cells from vector control and three stable Smad7 clones were serum starved and treated with 5 ng/ml TGF-h for different time points. CDK4 kinase assay was performed as described previously [21]. Briefly, 4 Ag of anti-CDK4 antibody was added to 1 mg of each cell extract and incubated for 2.5 h at 4-C. 25 Al of protein GSepharose beads was then added and incubated for an additional 1 h at 4-C. The beads were washed three times with cell lysis buffer and twice with kinase assay buffer (50 mM Tris –HCL, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 0.1 mM NaF, and 0.1 mM sodium orthovanadate), and then incubated in 25 Al of kinase reaction mixture (0.5 Ag of glutathione S-transferase-Rb (Santa Cruz Biotechnology Inc.), 1 Al (10 ACi) of [g-32P] ATP, 0.5 Al of 0.1 mM ATP, and 23 Al of kinase buffer) for 1 h at room temperature. The reaction was stopped by boiling the reactions in 15 Al of 5 SDS-PAGE sample buffer for 4 min. The reaction mixture was divided into two parts and resolved by 10% SDS-PAGE. Phosphorylated Rb in the gel was detected by autoradiography and quantitation of Rb phosphorylation was performed using a PhosphorImager (Molecular Dymamics). To verify uniform CDK4 immunoprecipitation, the second gel was used for immunoblotting with anti-CDK4 antibody. The same membrane was immunoprobed with anti-Rb antibody to verify the presence of equal amount of GST-Rb protein in each kinase reaction. Soft agarose assay To test the effects of Smad7 on anchorage-independent growth, vector controls and three stable Smad7 clones were compared for clonogenic potential in semisolid medium. 103 to 105 cells from each pool were suspended in 1 ml of 0.4% in sea plaque agarose containing 10% FBS medium and then plated on the top of 1 ml of semisolidified 0.8% agarose in the same medium in 35-mm plates. Plates were incubated for 17 days at 37-C in the presence of 5% CO2 in a humidified incubator. Colonies grown on soft agarose were counted by automated colony counter and pictures of colonies were taken under the inverted microscope. Results from inoculums of 0.5  105 cells are shown in Figs. 3A and B.

curves for tumors were plotted from the mean volume T SD of tumors from six mice. Flow cytometric analysis DNA cell cycle phase was analyzed by FACScan (Becton Dickinson) flow cytometer emitting 488-nm laser light for fluorochrome excitation. Briefly, 1  106 cells from each pool of FET, vector control, and three stable Smad7 clones were seeded in 60-mm plates and allowed to attach for 24 h. Cells were then treated with 5 ng/ml of TGF-h1 in 10% FBS containing medium for 35 h. Cells were harvested, washed in cold PBS, and suspended in 200 Al of cold PBS. One-milliliter solution containing 50 Ag/ml propidium iodide (PI), 1 mg/ml sodium citrate, 5 Al/ml Triton X-100, and 5 Ag/ml RNase A in PBS was added to each cell suspension and incubated on ice for 30 min. Cells were analyzed for red (PI) fluorescence through a 620-nm LP filter. Red fluorescence was used as a marker of DNA content and cell-cycle status. Percentage of cells present in different phases of the cell cycle was measured and analyzed. Apoptosis assay Apoptosis was measured quantitatively using the cell death detection ELISAplus kit following the manufacturer’s protocol. Cells (0.2  105) from each pool of FET, vector control, and three stable Smad7 clones were seeded in each well of 12-well plates and allowed to attach for at least 20 h. Cells were preincubated for 12 h in serum-free medium and then treated with 5 ng/ml of TGF-h1 for another 48 h. Cells (adherent and floating cells) were collected, lysed in 200 Al of lysis buffer, centrifuged, and clear supernatant (10 Al) was applied into streptavidin-coated microplates. A mixture of biotinylated anti-DNA antibody and peroxidase-conjugated anti-histone antibody was added to the wells and allowed binding for 2 h at room temperature. Plates were washed thoroughly, and then followed by the addition of 2,2V-azido-di[3-sulfonate] (ABTS) substrate. Color development was monitored spectrophotometrically at 405 nm. Apoptotic cells are directly correlated with the absorbance measured at 405 nm. Each data point is a representative of the mean T SD of three individual measurements.

Growth in nude mice

Results

FET cells, vector control, and three stable Smad7 clones were assayed for tumorigenicity in 7-week-old athymic nude mice. 0.5  106 cells were injected subcutaneously behind the anterior fore limb of each mouse as indicated. The animals were monitored for tumor formation twice a week. Tumors were measured by slide calipers and tumor volume was calculated by the equation: V = L  W 2  0.5, where V = volume, L = length, and W = width. Growth

Stable expression of Smad7 in FET cells inhibits TGF-b-induced Smad2 phosphorylation and prevents complex formation between Smad2/Smad3 and Smad4 To determine the potential role of high levels of Smad7 in cancer cells in vivo, we used FET cells, a colon adenocarcinoma-derived cell line, which is non-tumorigenic. These cells are sensitive to TGF-h-induced tran-

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scriptional responses and growth arrest induced by TGF-h (data not shown and [22]). In order to express high levels of Smad7 in FET cells, we generated retroviruses containing Flag-Smad7 by transfecting the viral construct into the amphitropic Phoenix packaging cell line. These retroviruses were then used for infection of FET cells, and infected cells were selected with Hygromycin B to generate stable clonal cell lines. Exogenous Smad7 protein expression in clones was detected by Western blot analyses using anti-Flag M2 monoclonal antibody (Fig. 1A), and three clones that expressed higher level of Smad7 (Smad7 #2, Smad7 #6, and Smad7 #15) were selected for further experiments. To test whether overexpressed Smad7 is functional, we first

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analyzed the phosphorylation of endogenous Smad2. One vector control clone and three stable Smad7 clones were treated with 5 ng/ml TGF-h in the absence of serum at different time points. Protein lysates were analyzed by Western blotting with antibodies as shown in Fig. 1B. We found that TGF-h-induced Smad2 phosphorylation was significantly reduced in the Smad7 clones when compared with the vector control, whereas Smad2, Smad3, and Smad4 protein levels were similar (Fig. 1B). To test whether reduced phosphorylation of R-Smads by Smad7 can inhibit the complex formation between Smad2/Smad3 and Smad4 in vivo, we performed immunoprecipitation experiments after treating the clones with TGF-h for 30 min or 90 min. Equal amounts of cell lysates were immunoprecipitated with both anti-Smad2 and anti-Smad3 antibodies. The immune complexes were analyzed by Western blot with anti-Smad4 monoclonal antibody as shown in Fig. 1C. TGF-h-induced heteromeric complex formation between Smad2/Smad3 and Smad4 was reduced in Smad7-expressing clones. These results suggest that Smad7 blocks Smad2/Smad3 phosphorylation and thereby prevents complex formation between Smad2/Smad3 and Smad4 induced by TGF-h. In addition, we examined whether stable expression of Smad7 can block TGF-hmediated transcriptional responses. We observed that both parental cells and vector control clone demonstrate a higher activity of the reporter (CAGA)9MLP-Luc in the presence of TGF-h, and this TGF-h-induced reporter response was downregulated in all three stable Smad7 clones (Fig. 1D). Similar inhibition of PAI-1 natural promoter activity by Smad7 was observed (data not shown), confirming that Smad7 inhibits TGF-h-induced transcriptional responses by blocking the activation of Smad pathway.

Fig. 1. Overexpression of Smad7 blocks TGF-h-induced phosphorylation of Smad2, complex formation between Smad2/3 and Smad4, and transcriptional responses. (A) Cell lysates from vector control clone and stable Smad7 clones were subjected to immunoblotting with anti-Flag M2 antibodies. Expression of Smad7 in individual clones is shown. Equal amount of protein loading was verified by immunoblotting the membrane with anti-h-actin monoclonal antibody. (B) Vector control and stable Smad7 clones were preincubated in serum-free medium for 16 h and then treated with TGF-h1 (5 ng/ml) as indicated. Cell lysates were subjected to immunoblotting with anti-phospho Smad2, anti-Smad2, anti-Smad3, and anti-Smad4 antibodies. Equal amount of protein loading was tested by immunoblotting the membrane with anti-h-actin antibody. N/S indicates non-specific band. (C) Vector control and stable Smad7 clones were preincubated for 2 h in serum-free medium and then treated with TGF-h1 (12.5 ng/ml) for the indicated times. Cell lysates were subjected to immunoprecipitation with anti-Smad2 and anti-Smad3 polyclonal antibodies and the immunoprecipitates were analyzed by immunoblotting with anti-Smad4 antibodies. (D) Smad7 blocks TGF-h1-induced reporter gene response. FET cells, vector control, and stable Smad7 clones were transiently transfected with CMV-h-gal and the p3TP-Lux reporter plasmids. Transfected cells were then treated with TGF-h1 (5 ng/ml) for 20 h. Luciferase activity was normalized to h-gal activity, and the relative luciferase activity was expressed as the mean T SD of triplicate measurements. These experiments were performed four times with similar results.

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Smad7 has no effect on TGF-b-induced phosphorylation of ERK and p38 MAPK, but it inhibits TGF-b-induced phosphorylation of Akt TGF-h activates various mitogen-activated protein kinases (MAPKs), including the extracellular signal-regulated kinase (ERK), the mitogen activated protein kinase p38MAP kinase, and c-Jun N-terminal kinase (JNK) [23]. To determine whether overexpression of Smad7 in FET cells can alter the activation of these non-Smad signaling pathways, we tested the phosphorylation of ERK, p38MAPK, and JNK by Western blot analyses using phosphospecific antibodies. TGF-h induces phosphorylation of ERK within 30 min, and thereafter the level is reduced and sustained for at least 12 h of TGF-h treatment in both vector control and stable Smad7 clones (Fig. 2A). We did not see any significant change in the total ERK level. We found a lower level of c-Jun phosphorylation in vector control than in stable Smad7 clones (Fig. 2A). TGF-h treatment increases phosphorylation of c-Jun in a time-dependent manner in both control and Smad7 clones. However, phosphorylation of c-Jun is more pronounced in Smad7 clones in response to TGF-h. To test the effect of Smad7 on TGF-h-mediated activation of p38MAP kinase, we performed Western blot analyses using a phospho-p38MAPK

antibody. As expected, exogenous TGF-h stimulated the phosphorylation of p38 MAP kinase in a time-dependent manner, whereas we did not see any effect of Smad7 on TGF-h-stimulated phosphorylation of p38 MAP kinase in stable Smad7 clones when compared to vector control clone (Fig. 2B). We further tested the effect of Smad7 on TGF-hmediated activation of PI3 kinase/Akt pathway. In consistent with previous studies, TGF-h treatment induces phosphorylation of Akt in vector control, whereas Smad7 significantly blocked TGF-h-induced phosphorylation of Akt in stable Smad7 clones (Fig. 2B). Therefore, these data suggest that Smad7 has no effect on TGF-h-induced activation of ERK and p38MAPK, whereas it can modulate the activation of c-Jun and Akt by TGF-h. Stable expression of Smad7 in FET cells induces tumorigenicity The upregulation of Smad7 in human tumors raises the possibility of involvement of Smad7 in carcinogenesis. Anchorage-independent growth in semisolid medium and the formation of xenografts in immunocompromised mice are generally considered to be useful parameters in assessing the tumorigenicity of human cells. FET cells had previously been shown to be non-tumorigenic as they

Fig. 2. Effect of Smad7 on TGF-h-induced Smad-independent pathways. (A) Cells form vector control and three stable Smad7 clones were serum starved and treated with 5 ng/ml TGF-h1 for different time points as indicated. Cell lysates were subjected to Western blot analysis using antibodies specific to phosphoERK, total ERK, phospho-c-Jun, and total c-Jun. (B) Cells were also serum starved and treated with 5 ng/ml TGF-h1 for different time points as indicated. Cell lysates were probed with antibodies specific to phospho-p38, total p38, phospho-Akt, and total Akt. Equal amount of protein loading was verified by immunoblotting with anti-h-actin antibody.

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Fig. 3. Smad7 induces tumorigenicity of FET cells. (A) Smad7 induces ancorage-independent growth. Soft agar assays were performed in 35-mm dishes. 0.5  105 cells from three stable Smad7 clones and two vector control clones were plated in 0.4% agarose over an underlayer of 0.8% agarose with Hygromycin B (200 Ag/ml) and cultured for 17 days. Pictures were taken with a magnification of 200. (B) Total number of colonies grown on soft agarose was counted by automated colony counter. Each data point is a representative of an average of three values determined from three independent plates. These experiments were performed four times in triplicate with similar results. (C) Ectopic expression of Smad7 enhances tumorigenicity in nude mice. 0.5  106 cells from each clone was injected subcutaneously behind the anterior fore limb of BALB/c athymic nude mice (7-week-old females). The animals were monitored for tumor formation two times every week. Tumors were measured externally on the indicated days in two dimensions by using slide calipers and tumor volume was calculated. Growth curves for tumors were plotted from the mean volume T SD of six tumors for each group. (D) Tumor tissues were isolated from nude mice and protein lysates were prepared. For vector control mice, protein lysate was made from the small nodule at the site of inoculation. Equal amounts of lysates from each tumor and from corresponding cells that were used for injection were resolved by SDS-PAGE and immunoblotted with anti-FLAG antibodies. The expression of exogenous Smad7 in tumor is shown (top). Equal protein loading was tested by immunoblotting with anti-h-actin antibodies (bottom).

grew poorly in soft agar assays and did not readily form tumors in nude mice [24]. To determine whether blockade of TGF-h/Smad signaling by Smad7 can alter the tumorigenic properties of cells, we performed assays for anchorage-independent growth and tumorigenicity in nude mice with Smad7-expressing FET clones and vector clones. We compared growth in soft agarose for stable Smad7 clones and vector control clones from inoculums of 103 to 105 cells. Results from inoculums of 0.5  105 cells are shown in Figs. 3A and B. We demonstrated that all three stable Smad7 clones produced significantly higher number of larger colonies on soft agar when compared to vector control clones. However, we noticed that Smad7 #6 clone produced larger colonies compared with other two stable Smad7 clones (Fig. 3A). We also observed that Smad7 #15 clone produced a higher number of colonies when compared with other Smad7 clones. These data suggest that stable expression of Smad7 enhances the anchorage-independent growth of FET cells. As we demonstrated that stable Smad7 clones showed aggressive colony formation on soft agar, we further tested their ability to induce tumorigenicity in athymic nude

mice. Cells (0.5  106) from each pool of FET, vector control clone, and three stable Smad7 clones were injected subcutaneously into athymic nude mice and the results are shown in Fig. 3C. Inoculation of all stable Smad7 clones resulted in palpable tumor formation within 15 days and they grew continuously over the period of observation. In contrast, FET cells did not give rise to any tumors within 42 days, and the vector control clone formed only a small nodule at the site of inoculation after 30 days that regressed within 45 days (Fig. 3C). To verify whether exogenous Smad7 expression is maintained in the tumors of nude mice, we sacrificed the mice and isolated the tumors. Protein lysates were prepared from tumor samples and were analyzed for the expression of Smad7 by Western blot using anti-Flag M2 monoclonal antibody (Fig. 3D). Similar levels of Smad7 expression were detected in both stable Smad7 cells that were injected, and the corresponding tumors. However, as expected, we did not detect exogenous Smad7 expression in vector control clone and in the corresponding nodule. These results indicate that stable expression of Smad7 in FET cells induces tumorigenicity.

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Smad7 blocks TGF-b-induced growth inhibition by accumulating cells in S-phase of the cell cycle To understand the mechanism by which Smad7 induces tumorigenicity of FET cells, we first performed [3H]thymidine incorporation assay to examine whether Smad7

can block TGF-h-induced growth inhibition. FET cells, vector control, and three stable Smad7 clones were treated with increasing concentrations of TGF-h and the incorporation of [3H]-thymidine was measured by liquid scintillation counter (Fig. 4A). TGF-h treatment reduced the rate of [3H]-thymidine incorporation (DNA synthesis) in a

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dose-dependent manner. We found 60 – 84% inhibition of DNA synthesis in control cells (FET and vector control clone) at 2 ng/ml of TGF-h1, whereas only 27 – 39% inhibition of DNA synthesis was observed in Smad7 stable clones. Further addition of TGF-h did not inhibit DNA synthesis significantly in these cell lines. We found that stable expression of Smad7 in FET cells increases incorporation of [3H]-thymidine as compared to control cells in the absence of TGF-h (Fig. 4B). We further tested the effect of stable expression of Smad7 on TGF-hmediated inhibition of cell proliferation by a cell counting assay. FET cells and clones were treated with 5 ng/ml of TGF-h for 5 days and then counted (Fig. 4C). We found that in response to TGF-h both FET and vector control cells showed 75% growth inhibition, whereas Smad7 clones showed 25 –40% growth inhibition. Interestingly, Smad7 induces basal proliferation of FET cells as evident from Fig. 4D, which is consistent with the results of thymidine incorporation assay (Fig. 4B). These data suggest that stable expression of Smad7 induces proliferation of FET cells by blocking TGF-h-mediated inhibition of cell growth. We then analyzed the distribution of cells in each phase of cell cycle using above cell lines. Cell proliferation and programmed cell death are tightly regulated in normal cells. Disruption of this balance by loss of cell cycle control may eventually lead to develop tumor [25]. The transition of cells from G1 to S phase of the cell cycle in response to mitogens takes place with an irreversible commitment to a new cycle. TGF-h inhibits cell proliferation by arresting the cells in the G1 phase of the cell cycle. To examine whether increased expression of Smad7 promotes cells from G0-G1 to S phase by blocking TGF-h-induced G1 arrest, we performed flow cytometric analyses by propidium iodide staining of FET cells, vector control, and Smad7 clones (Fig. 4E). 94% FET cells accumulated in the G0-G1 phase, whereas only 3.5% cells accumulated in the S phase of the cell cycle in response to TGF-h. However, a significantly higher number of cells from Smad7 clones (14 to 20%) were found to be in the S phase of the cell cycle with subsequent decrease of the cells in the G0-G1 phase (69 to 79%) in the presence of TGF-h as shown in Fig. 4E. Interestingly, in the absence of TGFh, we found a similar trend in increase of cells in the S phase and decrease of cells in the G0-G1 phase of the cell cycle in case of stable Smad7 clones when compared with FET cells. However, the differences in percentage of cells

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present in S phase between parental and Smad7 clones in the absence of TGF-h are less than that in the presence of TGF-h (Fig. 4E). Together, these data suggest that Smad7 promotes cell proliferation by blocking TGF-h-induced G1 arrest. Smad7 blocks TGF-b-induced repression of c-Myc, CDK4, and Cyclin D1 expression, and decreases p21Cip1 expression Perhaps one of the most important biological effects of TGF-h is its ability to inhibit proliferation of many cell types, including most epithelial cells. TGF-h inhibits progression of cells from G1 into the S phase of the cell cycle. Proposed mechanisms have suggested that TGF-hinduced G1 arrest involves the suppression of gene expression, including c-Myc, Cyclins A and D, and the CDKs, cdc25A [6]. Other proposed mechanisms have included TGF-h induction of the CDK inhibitors, p15INK4B, p21Cip1, and p27Kip1. Although there is not much data linking the known TGF-h signaling pathways with the regulators of cell cycle progression implicated in TGF-h-induced growth arrest, several lines of evidence suggest that Smad signaling is functionally connected directly or indirectly in suppressing the growth of epithelial cells. Deregulation of the pro-oncoprotein c-Myc can overcome the cell cycle arrest to promote cellular proliferation. To understand the mechanism of Smad7mediated blockade of TGF-h-induced growth inhibition in FET cells, we examined whether stable expression of Smad7 can alter the cellular levels of c-Myc. FET cells and the clones were treated with TGF-h for several time points and the cell lysates were subjected to Western blot analyses using anti-c-Myc antibody (Fig. 5A). c-Myc expression was significantly downregulated in parental FET and vector control cells by 5 ng/ml of TGF-h, and little c-Myc protein was detected after 2 h of TGF-h treatment. However, TGFh-induced downregulation of c-Myc expression was blocked in all three stable Smad7 clones, and its expression is maintained up to 12 h after treatment. These results suggest that stable expression of Smad7 inhibits TGF-hinduced repression of c-Myc expression. To test the effect of Smad7 on the expression of cell cycle regulatory molecules induced by TGF-h, we analyzed the expression levels of p21Cip1 in vector control and three stable Smad7 clones by Western blot analyses (Fig. 5B). We found reduction in p21Cip1 protein expression in all three Smad7

Fig. 4. Smad7 blocks TGF-h-induced growth inhibition and induces cell-cycle progression. (A) FET cells, vector control, and stable Smad7 clones were cultured in DMEM containing 10% serum and treated with increasing doses of TGF-h1. Cells were then labeled with [3H]-thymidine for 2 h and the radioactivity incorporated was counted by liquid scintillation counter. Radioactivity incorporated by cells without TGF-h treatment is considered as 100%, and the results are expressed as the mean T SD for triplicate measurements. (B) [3H]-thymidine incorporation by FET cells, vector control, and stable Smad7 clones was plotted and the results are expressed as the mean T SD for triplicate measurements. (C) FET cells, vector control, and stable Smad7 clones were treated with TGF-h for 5 days and then counted. The average cell number in the absence of TGF-h1 in each set was considered as 100% and the relative cell number in the presence of TGF-h was calculated. Individual data points are the mean T SD of triplicate determinations. (D) Cell numbers from the above experiment cultured for 5 days without TGF-h treatment were plotted. Individual data points are the mean T SD of triplicate determinations. (E) FET cells, vector control, and stable Smad7 clones were treated with TGF-h1 (5 ng/ml) for 35 h. Cells were collected and evaluated for DNA content by flow cytometric analyses as described in Materials and methods. Results are expressed as percentage of cells in different phases of the cell cycle. Each experiment was repeated three times.

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Fig. 5. Effects of Smad7 on TGF-h-induced downregulation of c-Myc and CDK4 expression, and p21Cip1 expression. (A) FET cells, vector control, and stable Smad7 clones were preincubated in serum-free medium for 2 h and then treated with TGF-h1 (5 ng/ml) at different time points as indicated. Expression of c-Myc protein was tested by Western blot analysis with anti-c-Myc antibodies (top). Equal amount of lysate loading was verified by immunoblotting h-actin (bottom). (B) Cell lysates from the vector and Smad7 transfectants without TGF-h treatment were tested for p21Cip1 expression. (C) Vector control and stable Smad7 clones were transiently transfected with CMV-h-gal and p21-Luc reporter, which contains the complete p21Cip1 promoter region. Transfected cells were then treated with TGF-h1 (5 ng/ml) for 22 h in 0.2% serum containing medium. Luciferase activity was normalized to h-gal activity, and the relative luciferase activity was expressed as the mean T SD of triplicate measurements. (D) Vector control and stable Smad7 clones were preincubated in serum-free medium for 2 h and then treated with increasing doses of TGF-h1 as indicated. Cell lysates were subjected to immunoblotting with anti-CDK4, anti-Cyclin D1, and antiCDK2 antibodies. Equal amount of protein loading was verified by immunoblotting with anti-h-actin antibody (bottom). These experiments were repeated at least three times.

clones when compared with the vector control. However, p21Cip1 expression was weakly induced by TGF-h in this cell line (data not shown). We further tested whether the repression of p21Cip1 protein in stable Smad7 clones is due to the reduced activation of p21Cip1 promoter by transient transfection of a 2.3-kb p21Cip1 promoter reporter (Fig. 5C). TGF-h induced the promoter activity by only 1.7fold, which is consistent with our protein data (not shown). We found a 3- to 4-fold repression of p21Cip1 promoter

activity in all Smad7 clones when compared with the vector control clone. We also noticed that the TGF-h treatment did not induce the p21Cip1 promoter activity in the Smad7 clones. These results demonstrate that stable expression of Smad7 reduces p21Cip1 protein level. We further examined whether Smad7 can alter the expression of cyclins and cyclin-dependent kinases (CDK) by Western blot analyses. CDK4 levels were similar in vector control and Smad7 clones. We observed a significant reduction in

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the CDK4 and Cyclin D1 levels by TGF-h after 10 h in vector clone, whereas the expression of these proteins was maintained over 30 h of TGF-h treatment in stable Smad7 clones (Fig. 5D). Under similar conditions, we do observe a decrease in CDK2 protein level in vector control cells, but we do not see the maintenance of the CDK2 level in a consistent manner in Smad7 clones (Fig. 5D). The levels of Cyclin A and Cyclin E were unchanged by Smad7 (data not shown). Together, these data suggest that Smad7mediated alterations of these cell cycle regulatory proteins play an important role in blocking TGF-h-induced G1 arrest and growth inhibition. Smad7 inhibits TGF-b-mediated suppression of CDK4 kinase activity Since we have observed that TGF-h-induced downregulation of CDK4 and Cyclin D1 is inhibited by Smad7 and that the expression of p21Cip1 is decreased by Smad7, we tested the kinase activity of CDK-Cyclin complex in Smad7 clones. Cell lysates from vector and Smad7 clones were used for immunoprecipitation with anti-CDK4 anti-

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body and the immune complexes were analyzed for Rbassociated CDK4 kinase activity using GST-Rb protein as substrate. TGF-h treatment strongly reduced CDK4 kinase activity in a time-dependent manner in vector control clone, whereas overexpression of Smad7 inhibited TGF-hinduced downregulation of CDK4 kinase activity (Figs. 6A, top panel, and B). Immunoprecipitation of equal amount of CDK4 protein in each kinase reaction was verified by Western blotting with anti-CDK4 antibody (Fig. 6A, middle panel). In addition, we verified the presence of equal amount of GST-Rb protein in each kinase reaction by Western blotting with anti-Rb antibody (Fig. 6A, bottom panel). Phosphorylation of endogenous Rb by CDK-Cyclin complex in the clones was confirmed by Western blot analyses with Phospho-Rb antibody (data not shown), and the results are in consistent with the kinase assay data. To confirm whether or not the CDK4 kinase activity is altered with time without treatment, cells from vector control clone were treated in the presence or absence of TGF-h for 16 h and 30 h. We observed that the CDK4 activity remained unchanged up to 30 h without treatment, whereas TGF-h decreases the CDK4 activity as mentioned above

Fig. 6. Effect of Smad7 on TGF-h-mediated repression of CDK4 kinase activity. (A) Vector control and stable Smad7 clones were serum starved and treated with 5 ng/ml TGF-h for different time points as indicated. CDK4 kinase activity was determined by in-bead GST-Rb fusion protein kinase assay using immunoprecipitated CDK4 from total cell lysates as mentioned in Materials and methods. The labeled substrate was subjected to SDS-PAGE, and the gel was dried and exposed to X-ray film. The phosphorylated GST-Rb was shown in the top panel. A portion of the kinase reaction was subjected to immunoblot analyses using antibodies specific to CDK4 and Rb (middle and bottom panel, respectively). (B) Quantitative analysis of CDK4 kinase activity. Normalized CDK4 kinase activity was plotted and activity in each cell line without TGF-h treatment is considered as 100%. (C) CDK4 kinase activity using extract from vector control clones that are treated with or without TGF-h for 16 h and 30 h. The kinase activity was determined as above. The above experiments were performed three times with similar results. IP, immunoprecipitation; WB, Western blot.

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(Fig. 6C). We performed similar kinase assays after immunoprecipitating CDK2 and using histone H1 as substrate. TGF-h marginally decreases CDK2 kinase activity in vector control and Smad7 has no effect on TGF-h-induced repression of CDK2 activity in FET cells (data not shown). This is probably because CDK2 and CDK4 kinase activities are differentially regulated in FET cells, which is similar to previous reports with MCF-7 human breast cancer cells [26]. Together, these data suggest that overexpression of Smad7 in FET cells inhibits TGF-h-induced repression of CDK4 kinase activity and may provide a mechanism by which Smad7 enhances cell proliferation in colorectal cancer. Smad7 inhibits apoptosis in FET cells Previous studies have demonstrated that ectopic expression of Smad7 protects WEHI 231 B-lymphocytes from TGF-h-induced growth inhibition and apoptosis [27]. Smad7 has been reported to be induced in response to activin and to suppress activin-mediated apoptosis in B-cell hybridoma [28]. It is also known that Smad7 induces apoptosis in normal cells by inhibiting the activity of the cell survival factor NF-kB [29] or by activating the JNK

signaling pathway [30]. Therefore, the effect of Smad7 on apoptosis is dependent on cell type. To evaluate the role of Smad7 in apoptosis in FET cells, we performed a quantitative determination of apoptosis by cell death ELISA assay using FET cells, control, and stable Smad7 clones in the presence or absence of TGF-h. Both Smad7 #2 and Smad7 #6 clones reduced the basal levels of apoptosis by 6to 7-fold, whereas Smad7 #15 reduces the basal levels of apoptosis by around 15-fold when compared with FET cells and vector control (Fig. 7A). However, we observed a slight decrease in apoptosis by TGF-h in both control cells and Smad7 clones. We did not observe any TGF-h-induced apoptosis in FET cells, probably because the TGF-hinduced apoptosis is cell type specific. Under similar conditions, sodium butyrate induced apoptosis of FET cells in a dose-dependent manner (Fig. 7B). The Bcl-2 family of genes has long been known to play an important role in the apoptotic pathway in mammalian cells and may contribute to cancer development [31]. Since Bcl-2, Bcl-xl, and Bcl-w proteins are anti-apoptotic, these proteins are regulated by TGF-h, and Smad7 inhibits apoptosis in FET cells, we investigated whether these proteins are involved in regulating apoptosis in stable Smad7 clones. We found that the expression of Bcl-2,

Fig. 7. Overexpression of Smad7 inhibits apoptosis in FET cells. (A) FET cells, vector control, and stable Smad7 clones were pre-incubated for 12 h in serumfree medium and then treated with TGF-h1 (5 ng/ml) for 48 h in serum-free condition. Cells (both adherent and floating) were lysed, and 10 Al of each clear cell lysate was used for ELISA according to the manufacturer’s protocol. Each data point represents the mean T SD of three individual measurements. These experiments were performed four times in triplicate with similar results. (B) FET cells were treated with increasing doses of Na-Butyrate for 48 h and cell lysates were used for ELISA assay as indicated above. (C) Cell lysates from FET cells, vector control, and stable Smad7 clones were subjected to immunoblotting with antibodies against Bcl-2, Bcl-xl, Bcl-w, Bad, Bax, Bid, Apaf1, Caspase-3, cytochrome C, and PARP. Equal amount of protein loading was verified by immunoblotting with anti-h-actin antibody (bottom). These experiments were repeated at least three times.

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Bcl-xl (bcl-2 like-1), and Bcl-w proteins was induced in stable Smad7 clones when compared with vector control and FET cells that expressed very low levels of these proteins (Fig. 7C). We did not observe any change in the levels of these proteins in response to TGF-h (data not shown). As the survival or death of cells is critically dependent upon molecular balance between the antiapoptotic and pro-apoptotic protein factors, we further investigated whether stable expression of Smad7 in FET cells decreases the expression of pro-apoptotic family proteins. Interestingly, we found that pro-apoptotic Bid (BH3 interacting domain death agonist) is down-regulated in stable Smad7 clones, whereas the expression of other proapoptotic proteins including Bax (bcl-2-associated X protein) and Bad (bcl-2 antagonist of cell death) was not changed (Fig. 7C). Cellular apoptosis may involve the disruption of mitochondrial function through the release of cytochrome C from mitochondria into the cytosol. Cytosolic cytochrome C can bind to apoptotic protease activating factor-1 (Apaf-1) and activates caspase 9 in the apoptosome in response to diverse inducers of cell death, which may lead to the activation of caspase-3 and subsequently to the final apoptosis event through the degradation of nuclear poly (ADP-ribose) polymerase (PARP) [32]. To further investigate the downstream effects, we first tested the protein expression of cytochrome C by Western blotting. We did not observe any change in the level of cytochrome C in stable Smad7 clones when compared with vector control and FET cells as expected (Fig. 7C). Apaf-1 expression was also not changed in stable Smad7 clones when compared with vector control and FET cells (Fig. 7C). In order to test caspase-3 activation, we found similar levels of procaspase3 and cleaved caspase-3 in stable Smad7 clones when compared with vector control and FET cells. In addition, we did not observe any change in degradation of nuclear PARP (Fig. 7C). Therefore, these results suggest that stable expression of Smad7 protects FET cells from apoptosis through the induction of anti-apoptotic Bcl2, Bcl-xl, and Bcl-w expression and the down-regulation of pro-apoptotic Bid expression.

Discussion There is compelling evidence indicating that TGF-h has a predominant growth inhibitory effect in normal epithelial cells and serves as a tumor suppressor. Cell growth is controlled by negative and positive regulatory signals. Any disruption of these signals may cause diseases. Loss of negative growth constraints may contribute to oncogenic processes. Such perturbations may occur as a consequence of the loss of TGF-h tumor suppressor functions. It has been well documented that most human cancers, including colorectal cancer, are resistant to TGF-h-mediated growth inhibition. Resistance to TGF-h growth inhibition in colorectal cancer can occur through a variety of mechanisms.

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First, mutations or functional inactivation of ThRII may cause loss of TGF-h responsiveness in at least 28% of human colorectal cancers [33,34]. Second, decreased expression of either ThR1 or ThRII has been implicated as a mechanism for TGF-h resistance in human tumors [35]. Third, inactivating mutations in components of the TGF-h signaling pathway, such as Smad2 (7%) or Smad4 (20%), lead to a TGF-h-resistant state [2,36,37]. Therefore, inactivating mutations of the TGF-h receptor and selected TGF-h signal transducers (Smad2/4) are not enough for the high frequency of insensitivity to TGF-h-induced antitumor activity in colon cancer. We have hypothesized that abrogation of TGF-h-induced growth arrest by inhibitory signaling molecules may provide a mechanism by which human colon tumors become non-responsive to TGF-h. The inhibitory Smad, Smad7, has been shown to function as an intracellular antagonist of TGF-h signaling. Smad7 is upregulated in pancreatic cancer [9]. In colorectal tumors, strong expression of Smad7 is detected [10] and amplification of Smad7 is associated with a worse prognosis [11]. Carcinoma cells are known to increase the production of TGF-h that may increase the expression of Smad7. However, little is known about how blockade of TGF-h/Smad signals by Smad7 plays a role in the development and progression of human colon cancer. Our present study demonstrates that over-expression of Smad7 in human colon cancer-derived FET cells enhances tumorigenicity by blocking TGF-hinduced growth inhibition and apoptosis. Stable expression of Smad7 in FET cells significantly overcomes TGF-hinduced G1 arrest and subsequent growth inhibition. In an attempt to understand the mechanism of blockade of TGF-hinduced growth inhibition in FET cells, we have observed that Smad7 inhibits TGF-h-mediated downregulation of cMyc, CDK4, and Cyclin D1, and suppresses TGF-h-induced expression of p21Cip1. As a result, Smad7 inhibits TGF-hmediated downregulation of Rb phosphorylation. This study may provide a mechanism by which human colon tumors become refractory to TGF-h-induced tumor suppression function. Although FET cells are derived from well-differentiated colon adenocarcinoma, there are several advantages to study the tumorigenic potential of a protein in these cells. These cells are relatively less aggressive when compared with other colon cancer cell lines. These cells grow poorly in semisolid medium and do not readily form tumors in nude mice [22]. To determine the consequences of blocking TGF-h/Smad signaling on tumorigenicity, we stably expressed full-length Smad7 in FET cells. These cells are strongly responsive to TGF-h-induced transcriptional induction as determined by TGF-h-responsive reporters, p3TP-Lux and (CAGA)9MLPLuc (data not shown), suggesting the presence of an intact TGF-h/Smad signaling pathway [22]. Stable expression of Smad7 blocks phosphorylation and activation of R-Smads that results in reduced complex formation with Smad4 (Figs. 1B and C). Thus, Smad7-overexpressing clones show strong inhibition of transcriptional responses induced by TGF-h,

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suggesting that ectopically expressed Smad7 is functional. In vitro anchorage-independent growth showed dramatic differences between the vector control clones and the stable Smad7 clones (Figs. 3A and B). A higher cloning efficiency was shown by stable Smad7 clones, and in addition, a substantial difference in size was also observed between the vector control and the stable Smad7 clones. Therefore, blockade of Smad signaling by Smad7 leads to the generation of an aggressive phenotype of colon carcinoma cells. This result prompted us to further investigate the in vivo tumor xenograft formation in nude mice (Fig. 3C). Repression of TGF-h/Smad signaling by stable expression of Smad7 led to a higher level of tumorigenicity in athymic nude mice and the expression of Smad7 was maintained in the tumor. Taken together, these observations demonstrate that enhanced expression of Smad7 in FET cells induces tumorigenicity in nude mice. In an attempt to understand the mechanism of Smad7induced tumorigenicity in FET cells, we observed that Smad7 blocked TGF-h-mediated growth inhibition as determined by [3H]-thymidine incorporation assays and cell-counting experiments. [3H]-thymidine incorporation in FET cells is strongly inhibited by TGF-h. In contrast, Smad7 clones showed only weak inhibition in [3H]-thymidine incorporation in response to TGF-h (Fig. 4A). This result is further supported by the cell-counting experiments in which the growth of parental and vector control cells was strongly inhibited by TGF-h, whereas Smad7 clones were significantly resistant to TGF-h-induced growth suppression. From these results, it is clear that Smad7 cannot completely block TGF-h-induced growth inhibition. This is consistent with the incomplete inhibition of complex formation between Smad2/Smad3 and Smad4 (Fig. 1C). It is also possible that other non-Smad pathway might be involved in the TGF-h-induced growth inhibition of FET cells. TGF-h suppresses growth by arresting cells in the G1 phase of the cell cycle. In parental FET and vector control cells, we observed a significant increase in the number of cells in the G0-G1 phase with a corresponding decrease in the S phase cells in response to TGF-h. This G1 arrest induced by TGF-h is abrogated in Smad7 clones (Fig. 4E). Interestingly, Smad7 increased the number of cells in the S phase in the absence of TGF-h with a corresponding decrease of cells in the G0-G1 phase. This is consistent with the fact that Smad7 induces cell proliferation independent of TGF-h (Figs. 4B and D). These results suggest that Smad7 may have an additional effect on cell growth independent of TGF-h/Smad pathway or Smad7 may also block the effect of endogenous TGF-h. Although Smad proteins are identified as key regulators of TGF-h-induced growth suppression, Smad2, Smad3, and Smad4 are known to be potent inducers of apoptosis. The inhibitory Smad, Smad7, is known to block TGF-h-dependent apoptosis [38]. Smad7 protects B-lymphocytes from TGF-h-induced apoptosis [27] and has been reported to suppress activin-mediated apoptosis [28]. In contrast, Smad7

induces apoptosis by inhibiting the activity of the cell survival factor NF-kB [29] or by activating the JNK signaling pathway [30]. Therefore, Smad7 has dual effects on apoptosis depending on the cell type and on the mutation status of a particular cell line. In an attempt to determine the effect of blocking TGF-h/Smad signaling by smad7 on apoptosis in FET cells, we observed that Smad7 inhibits apoptosis significantly in the presence or absence of TGF-h (Fig. 7A). Interestingly, TGF-h also inhibits apoptosis weakly in this cell line. Although, Smad7 inhibits TGF-hmediated induction of Akt phosphorylation and enhances TGF-h-induced phosphorylation of c-Jun, it appears that these pathways do not influence the effect of Smad7 on apoptosis in this cell line. However, Smad7 enhances the expression of anti-apoptotic proteins like Bcl-2, Bcl-xl, and Bcl-w, and down-regulates the pro-apoptotic protein Bid expression (Fig. 7C) that might contribute to Smad7mediated inhibition of apoptosis. Although cytochrome C release from mitochondria to cytosol is a critical step in the activation of downstream caspase protease cascade, we did not see any change in the level of cytochrome C expression by Western blot analysis as expected (Fig. 7C). The antiapoptotic proteins Bcl-2, Bcl-xl, and Bcl-w function by blocking cytochrome C release from mitochondria to cytosol, whereas the pro-apoptotic proteins Bid, Bax, and Bad promote the release of cytochrome C from mitochondria into the cytosol [39]. It is possible that the inhibition of apoptosis by Smad7 is a resultant effect of pro-apoptotic and anti-apoptotic protein expression regulated by Smad7. The increased expression of anti-apoptotic Bcl-2, Bcl-xl, and Bcl-w proteins and reduced expression of pro-apoptotic Bid in stable Smad7 clones might block the cytochrome C release from the mitochondria into the cytosol and as a result inhibit the downstream caspase activation that leads to inhibit apoptosis. However, Na-butyrate shows apoptosis of these cells in a dose-dependent manner (Fig. 7B). Smad7 has little effect on Na-butyrate-induced apoptosis (data not shown). One could speculate that Smad7-mediated blockade of apoptosis might contribute to the increased tumorigenicity in Smad7-expressing clones. In order to understand the mechanism of Smad7-mediated blockade of TGF-h-induced growth inhibition, we have tested the regulation of several cell cycle regulatory proteins that are involved in the progression of cells from G1 into the S phase. It has been demonstrated that ectopic expression of c-Myc promotes cell cycle progression and induces shortening of the G1 phase [40]. In addition, the ability of c-Myc to repress CDK inhibitors and induce D-type Cyclins and CDKs may contribute to its ability to promote cell proliferation and oncogenesis [41]. Inhibition of the cell cycle by TGF-h is mediated at least in part by downregulation of proliferative protein, c-Myc, and upregulation of cell cycle inhibitory protein, p21Cip1. Stable expression of Smad7 blocks TGF-h-mediated downregulation of c-Myc and sustained its expression up to 12 h of TGF-h treatment (Fig. 5A). p21Cip1 level is weakly induced in FET cells in

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response to TGF-h (data not shown), which is consistent with a small increase in p21Cip1 promoter activity by TGF-h. p21Cip1 is downregulated in Smad7 clones in the absence of TGF-h (Fig. 5B). However, we did not observe any significant change of TGF-h-induced p21Cip1 level in Smad7 clones when compared with the vector control clones (data not shown). We have observed a marked attenuation of the p21Cip1 promoter activity in Smad7 clones in the presence or absence of TGF-h (Fig. 5C). p27Kip1 was not regulated either by TGF-h or by Smad7 (data not shown). Thus, our results demonstrate that Smad7 can trigger the FET cells to oncogenic cell phenotype by stimulation of oncogenic cMyc expression and downregulation of Cyclin-dependent kinase inhibitor p21Cip1. TGF-h is known to downregulate Cyclin D1 and Cyclin-dependent kinase, CDK4, and these are frequently overexpressed in colon carcinogenesis [42]. We have observed that TGF-h-induced downregulation of Cyclin D1 and CDK4 is inhibited by Smad7 (Fig. 5D), whereas Smad7 does not have a significant effect on CDK2 (Fig. 5D) and other Cyclins in FET cells. It is possible that the regulation of c-Myc level by Smad7 may contribute to the sustained levels of Cyclin D1 and CDK4. As a result, we have found that Smad7 strongly inhibited the TGF-hinduced downregulation of CDK4 kinase activity that leads to sustained Rb phosphorylation (Fig. 6). In contrast, we did not see any change in TGF-h-induced repression of kinase activity of CDK2 by Smad7 (data not shown). We have seen that the CDK2 and CDK4 kinase activity is regulated differentially in FET cells, which is supported by the previous report where the CDK2 and CDK4 kinase activity was regulated differentially in retinoic acid (RA)-treated human breast cancer cells [26]. However, recent literature demonstrates that although p21Cip1 and p27Kip1 can inhibit the activity of CDK2 and CDK4/6, p21Cip1 can also serve as an assembly factor for CyclinD-CDK4 complexes, increasing the efficiency of complex formation and CDK4 activity. At low concentrations, p21 promotes the assembly of active kinase complexes, whereas at higher concentrations, it inhibits activity [43 – 45]. Therefore, the activities of CDK4 and CDK2 are regulated by Cyclins and Cyclin-dependent kinase inhibitors at different levels and by complex mechanisms. It is possible that the CDK2 and CDK4 kinase activity is regulated differentially depending on cell types and mutation status. Taken together, these results indicate that enhanced expression of Smad7 can abrogate the tumor suppressor function of TGF-h in colon cancer cells that may be involved in inducing tumorigenic phenotype. In conclusion, our studies demonstrate that Smad7 overexpression in colorectal cancer cells in vivo may enhance tumor growth by rendering the tumor cells resistant to the antiproliferative effects of TGF-h. Thus, this study provides a mechanism by which a portion of human colorectal tumors may become unresponsive to tumorsuppressive actions of TGF-h. Several lines of evidence suggest that carcinoma cells increase the production of TGFh that may increase the expression of Smad7. Increased level

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of Smad7 may inhibit tumor suppressor functions of TGF-h by blocking Smad signaling, and may keep TGF-h-inducible pro-oncogenic pathways like ERK, p38 MAPK, PI3K/Akt, and RhoA unaffected. Thus, overexpression of Smad7 in human colorectal cancer may promote tumor growth, and Smad7 may be an important therapeutic target for the future development of anti-cancer drug.

Acknowledgments We thank Dr. Michael G. Brattain (Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 14263) for providing us FET cells for experiments. This work was supported by R01 CA95195 and The Charlotte Geyer Foundation Grant (to P.K.D.), and CA69457 and DK52334 (to R.D.B.).

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