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Cell and Tumor Biology

Targeting Endogenous Transforming Growth Factor β Receptor Signaling in SMAD4-Deficient Human Pancreatic Carcinoma Cells Inhibits Their Invasive Phenotype 1

Gayathri Subramanian, Roderich E. Schwarz, Linda Higgins, Glenn McEnroe, Sarvajit Chakravarty, Sundeep Dugar and Michael Reiss
DOI: 10.1158/0008-5472.CAN-04-0018 Published August 2004

Abstract

Transforming growth factor-β (TGF-β) suppresses tumor formation by blocking cell cycle progression and maintaining tissue homeostasis. In pancreatic carcinomas, this tumor suppressive activity is often lost by inactivation of the TGF-β-signaling mediator, Smad4. We found that human pancreatic carcinoma cell lines that have undergone deletion of MADH4 constitutively expressed high endogenous levels of phosphorylated receptor-associated Smad proteins (pR-Smad2 and pR-Smad3), whereas Smad4-positive lines did not. These elevated pR-Smad levels could not be attributed to a decreased dephosphorylation rate nor to increased expression of TGF-β type I (TβR-I) or type II (TβR-II) receptors. Although minimal amounts of free bioactive TGF-β1 and TGF-β2 were detected in conditioned medium, treatment with a pan-specific (but not a TGF-β3 specific) TGF-β-neutralizing antibody and with anti-αVβ6 integrin antibody decreased steady-state pSmad2 levels and activation of a TGF-β-inducible reporter gene in neighboring cells, respectively. Thus, activation of TGF-β at the cell surface was responsible for the increased autocrine endogenous and paracrine signaling. Blocking TβR-I activity using a selective kinase inhibitor (SD-093) strongly decreased the in vitro motility and invasiveness of the pancreatic carcinoma cells without affecting their growth characteristics, morphology, or the subcellular distribution of E-cadherin and F-actin. Moreover, exogenous TGF-β strongly stimulated in vitro invasiveness of BxPC-3 cells, an effect that could also be blocked by SD-093. Thus, the motile and invasive properties of Smad4-deficient pancreatic cancer cells are at least partly driven by activation of endogenous TGF-β signaling. Therefore, targeting the TβR-I kinase represents a potentially powerful novel therapeutic approach for the treatment of this disease.

INTRODUCTION

Transforming growth factor-β (TGF-β) is a 25-kDa dimeric polypeptide that fulfills two major functions in epithelial tissues: first, endogenous TGF-β-signaling controls tissue homeostasis and suppresses tumor formation by maintaining the balance between cell renewal and cell differentiation and loss. Escape from this tumor-suppressive function of TGF-β is an almost universal characteristic of human epithelial tumors, although, in many cases, the underlying mechanism remains unknown (reviewed in Refs. 1 , 2 ). Secondly, TGF-β becomes strongly activated in response to tissue injury (3) . In this case, TGF-β induces epithelial cells to assume a dispersed, fibroblastoid phenotype (epithelial-to-mesenchymal transdifferentiation, EMT) and to produce extracellular matrix components of what later becomes the scar. This process is self-limited in space and time, allowing cells to eventually revert back to their cohesive epithelioid phenotype (4) .

Several recent studies have shown that this injury response to TGF-β is often preserved in rodent and human cancers and may, in fact, contribute to the tumor cells’ invasive and metastatic phenotype (5) . On the basis of these findings, we and others (2 , 6, 7, 8, 9) have proposed that targeting the TGF-β-signaling pathway may be a rational therapeutic approach in cases in which TGF-β signaling drives the malignant phenotype. TGF-β is secreted in a latent form in which the NH2-terminal domain called the latency-associated protein is noncovalently associated with the mature COOH-terminal domain of the protein (6 , 10) . After its extracellular activation, a single TGF-β1 dimer binds primarily to two type II receptors (TβR-II), followed by the recruitment of two type I receptors (TβR-I) into a heterotetrameric configuration. Once this ternary complex forms, the TβR-II kinase phosphorylates specific serine residues located within the juxtamembrane GS domain of TβR-I and, this in turn, activates the TβR-I serine-threonine kinase (11) . The postreceptor steps in TGF-β signaling are mediated by homologues of the Drosophila Mad protein (12) . In response to TGF-β, two of these proteins, Smad2 and Smad3, become transiently associated with and phosphorylated by the activated TβR-I receptor kinase at the last two serine residues of the COOH-terminal SSXS motif in the Mad-homology-2 domain (13 , 14) . Once phosphorylated, these receptor-associated R-Smads form heteromeric complexes with the common mediator Smad, Smad4, which then shuttle into the nucleus (15, 16, 17, 18) , where they interact with DNA and other components of the transcriptional machinery to regulate the expression of a wide range of TGF-β target genes (19 , 20) .

Pancreatic cancer (PC) is highly invasive and metastatic and one of the four leading causes of cancer deaths for both genders in the United States, with an overall cure rate of merely 4% (21) . Moreover, it is the type of human cancer in which the TGF-β/Smad signaling pathway is most frequently affected (22, 23, 24) . Specifically, the MADH4 locus on chromosome 18 (18q21.1) that encodes Smad4 undergoes loss of heterozygosity in >90% of PCs (24 , 25) . In over half of these cases, MADH4 is biallelically inactivated by homozygous deletion or by missense or nonsense mutations of the second allele (25, 26, 27) . The missense mutants of Smad4 found in PC are transcriptionally inactive (28) or target Smad4 to the ubiquitin-proteasome pathways for degradation (29) . Moreover, MADH4 inactivation occurs late in the neoplastic progression of PC (30) and is associated with a particularly poor prognosis (31) . Thus, not only is MADH4 inactivation particularly common in PC, but also associated with a highly aggressive metastatic tumor phenotype.

There are somewhat conflicting reports regarding the status of TβR receptors in PC. Several investigators have reported that the levels of TβR-I mRNA were markedly increased in PC specimens compared with normal pancreas, although there was no consistent correlation with TβR-I immunostaining, and no structural analysis of the gene was conducted (32 , 33) . Similarly, Jonson et al. (34) found the TβRII gene to be overexpressed in 12 of 14 PC cell lines. Although some PC cell lines seem to retain some degree of TGF-β-mediated growth suppression (34, 35, 36, 37) , others express markedly reduced levels of TβR-I and are refractory to or even growth stimulated by exogenous TGF-β (34 , 38, 39, 40) . Goggins et al. (22) recently described two cases of pancreatic carcinoma with large intragenic homozygous deletions of the TβRI gene. Thus, although genetic alterations of the TβRI gene do occur in PC, changes in the level of expression appear to be more common (39, 40, 41) .

Although it was initially believed that Smad4 was required for all of the actions of TGF-β (42, 43, 44, 45) , several recent studies demonstrate that some of the responses of TGF-β are Smad4 independent (46, 47, 48) . These findings, in conjunction with reports of increased expression of TβR receptors and TGF-β isoforms in pancreatic cancer (36 , 49) , suggested to us the possibility that Smad4 loss in PC might result in the uncoupling of the TGF-β-mediated growth-suppressive function from its pro-oncogenic effects and this may, in turn, result in a more aggressive tumor phenotype (31) .

We report here that a subset of Smad4-deficient PC cell lines display constitutive activation of the TβR receptor system as a result of autocrine production and activation of TGF-β. In vitro motility and invasiveness of these cells are strongly inhibited by blocking TβR receptor kinase activity. Thus, the hyperactive endogenous TGF-β signaling appears to be driving the invasive phenotype of Smad4-deficient cells. These results suggest that targeting TβR kinases in this particular setting might prove to be a useful new approach to the treatment of this particularly aggressive subgroup of PC.

MATERIALS AND METHODS

Cell Culture.

Human pancreatic adenocarcinoma cell lines (MiaPaCa, CaPan2, AsPC1, CFPAC1, Hs766T, BxPC-3, Panc1, and HTB-147) were obtained from the American Type Culture Collection and were grown in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (Sigma, St. Louis, MO; Refs. 50, 51, 52, 53, 54, 55 ). Mv1Lu mink lung epithelial cells were grown in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum. Mv1Lu cells stably transfected with a luciferase construct driven by a PAI-1 promoter (TMLC; kindly provided by Dr. Daniel B. Rifkin, New York University, New York, NY) were maintained in RPMI 1640 supplemented with 10% (v/v) FBS containing 200 μg/ml Geneticin (G418; Invitrogen Corporation, Grand Island, NY).

Reagents.

Human recombinant TGF-β1 was obtained from Austral Biologicals (San Ramon, CA) and dissolved in 4 mm HCl, with 1 mg/ml BSA. BMP2 was purchased from R&D Systems as a stock solution of 7.65 mm. SD-093 (provided by Scios, Inc., Fremont, CA) is a small molecular selective competitive inhibitor of the TβR-I kinase that acts by binding to the ATP binding site and keeping the enzyme in its inactive configuration (56 , 57) . SD-093 was dissolved in DMSO. The 10 mm stock solution was stored at −70°C and diluted to the working concentration immediately before use. NPC-37282, a selective p38 kinase inhibitor (Scios, Inc.) was dissolved in 0.05% (w/v) acetic acid (4 mm stock solution) and diluted to a 0.5 μm working concentration immediately before use. Y27632, a selective inhibitor of RhoC kinase (ROCK) (Calbiochem, San Diego, CA), was used at a working concentration of 10 μm.

Smad and TβR Protein Detection by Western Blot Analysis.

Cells were grown to 70% confluence in 100-mm dishes and treated with 100 pm TGF-β for 2 h. Cells were then lysed in situ using buffer composed of 150 mm NaCl, 20 mm Tris-HCl (pH 8.0), 2 mm EGTA, and 2% (v/v) Triton X-100 containing Complete Mini protease inhibitor mixture (Roche Diagnostics Corporation, Indianapolis, IN) for 40 min at 4°C. After clarification of the lysates by centrifugation, protein extracts were resolved by 12% (w/v) SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad Transblot; 0.45 μm) using a Panther Semidry Electroblotter (Owl Separation Systems, Portsmouth, NH). Duplicate filters were then incubated for 30 min at 20°C in blocking buffer [PBS, 5% (w/v) Carnation dry milk, 0.1% (v/v) Tween 20] followed by incubation for 12–16 h at 4°C in PBS containing 1 μg/ml anti-Smad peptide antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Total R-Smads were detected using rabbit polyclonal anti-Smad2 and anti-Smad3 antibodies (Zymed Laboratories, Inc., South San Francisco, CA). Smad4 was detected using a mouse monoclonal anti-Smad4 antibody (B-8; Santa Cruz Biotechnology). Phospho-Smad2 (pSmad2) and phospho-Smad3 (pSmad3) were detected using our own rabbit polyclonal anti-pSmad2 and anti-pSmad3 antibodies as described previously (58 , 59) . TβR-I and TβR-II were detected using rabbit anti-TβR-I and TβR-II antibodies (SC-399 and SC-229, respectively; Santa Cruz Biotechnology). Blots were developed using a 1:2000 dilution of horseradish peroxidase-tagged goat antirabbit or antimouse IgG (Calbiochem), and the bands were visualized using ECL Western blot detection reagent as recommended by the manufacturer (Amersham Biosciences, Piscataway, NJ). Integrated optical densities of individual bands on scanned images were determined using ImageJ v.1.29 software (NIH).

Cell Proliferation Assays.

A total of 0.8 × 104 cells was plated in duplicate wells of 24-well plates and allowed to adhere overnight. Cells were then treated with either human recombinant TGF-β1 at concentrations ranging from 0 to 200 pm or with TβR-I kinase inhibitor, SD-093. Cell numbers were determined using a Model 0039 Coulter Counter (Beckman Coulter, Fullerton, CA) counter at different time points or during mid-exponential growth.

E-Cadherin Immunostaining.

Cells were plated in 8-well tissue culture slides (BD Falcon, Franklin Lakes, NJ) or 35-mm tissue culture dishes (BD Falcon) and allowed to adhere overnight. After treatment for 24 h with TGF-β1 (100 pm), SD-093 (1 μm), both agents, or vehicle only, cells were washed with PBS and fixed using ice cold methanol at −20°C for 5 min. Air-dried slides were permeabilized by pre-incubation with 0.1% (v/v) Triton X-100 in PBS for 10 min at room temperature. After two 5-min washes with PBS, slides were incubated in 3% (w/v) Carnation dry milk powder in PBS for 30 min at 20°C, followed by incubation with 1:1000 anti-E-cadherin antibody (Transduction Laboratories, Mississuaga, Ontario, Canada) in 1% (w/v) Carnation dry milk powder in PBS. Cells were then washed three times with PBS for 5 min each and incubated with 1:100 Cy3-conjugated antimouse IgG (Sigma) for 1 h at room temperature. Slides were washed three times with PBS for 5 min each, mounted using Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA), and photographed using a Zeiss epifluorescence microscope (model 090477; Carl Zeiss, Jena, Germany) equipped with a MTI charge-coupled device camera (model DC 330E; DAGE-MTI, Inc., Michigan City, IN).

F-Actin Staining.

Cells were plated in 8-well chamber slides or 35-mm tissue culture dishes and allowed to adhere overnight. After treatment with TGF-β (100 pm) or SD-093 (1 μm), both agents, or vehicle only for 24 or 48 h, cells were washed with PBS twice and fixed with 10% Millonig’s modified phosphate-buffered formalin (Surgipath, Richmond, IL) for 10 min at room temperature. After two washes with PBS, cells were permeabilized with 0.1% (v/v) Triton X-100 in PBS for 5 min at room temperature. The cells were then washed twice with PBS and incubated in the dark with 1:40 Alexa Fluor 488 Phalloidin (Molecular Probes, Eugene, OR) diluted in PBS containing 1% (w/v) BSA. Stained slides were mounted with Vectashield and photographed using a Zeiss epifluorescence microscope (model 090477; Carl Zeiss), equipped with a MTI charge-coupled device camera (model DC 330E; DAGE-MTI, Inc.).

Cell Migration and Wound Closure Assays.

For motility assays, 1 × 105 cells were plated in the top chambers of uncoated polyethylene membranes (Transwell 24-well insert, pore size 8 μm; BD Falcon), triplicate filters per condition. TGF-β (100 pm), SD-093 (1 μm), or vehicle only were added to both top and bottom chambers. After 24-h incubation at 37°C, cells in suspension were removed by washing twice with PBS, and the cells that had adhered to the top of the inserts were removed by scraping the membrane with cotton-tip applicators. The cells that had migrated to the underside of the inserts were fixed and stained using Diff-Quik (Dade Behring, Inc., Newark, DE) staining kit. Cells in 10 random squares of 1 mm2 area were counted at ×200 magnification in each well and expressed as the total number of cells/mm2.

For wound closure assays, cells were plated in wells of 6-well plates. Confluent cell monolayers were wounded by manually drawing a furrow across the monolayer with a 1–200-μl pipette tip (Fisherbrand). The cell culture medium was then replaced with fresh medium, and 100 pm TGF-β and/or 1 μm SD-093 or vehicle were added as required, and wound closure was monitored by phase contrast microscopy at various times. Using a grid to demarcate the wells into segments, representative photographs were taken from four different fields spanning the entire wound at various time intervals, at ×40 and ×100 magnifications. The wound area at each time point after wounding was quantified using Adobe Photoshop version 7.0 (Adobe Systems Inc.) and ImageJ version 1.29 (NIH) software. The experiments were performed in duplicates.

Invasion Assay.

BD Biocoat growth factor-reduced Matrigel Invasion Chambers (24-well insert, pore size 8 μm; BD Biosciences, Bedford, MA) were rehydrated by adding 0.5 ml of warm (37°C) culture medium to the upper chambers followed by 2 h incubation at 37°C. After rehydration, the medium was carefully removed, and 2.0 × 106 cells in 350 μl of medium were added to the upper chambers in triplicate. TGF-β (100 pm), SD-093 (1 μm), both, or vehicle alone were added to both upper and lower chambers. After a 24-h incubation at 37°C, cells in suspension were removed by washing twice with PBS, and the cells adherent to the top of the inserts were removed by scraping the upper surface of the membrane with cotton tip applicators. Cells that had invaded through the growth factor-reduced Matrigel matrix and adhered to the underside of the membrane were washed with PBS and fixed and stained using the Diff-Quick staining kit (Dade Behring, Inc.). Cells in five randomly selected squares of 1 mm2 were counted at ×200 magnification in each well and expressed as total cells/mm2.

TGF-β Neutralization Assays.

Cells were plated in 6-well plates and allowed to adhere for 8 h. They were then treated for 12 h with a neutralizing monoclonal antibody directed against TGF-β1, TGF-β2, and TGF-β3 (MAB1835) or TGF-β3 (MAB643) (R&D Systems, Minneapolis, MN) at concentrations ranging from 0 to 10 μg/ml. Cells were then lysed and subjected to Western blotting for pSmad2 and Smad2 as described above.

Detection of TGF-β1 and TGF-β2 by ELISA.

Cells were plated in duplicate wells of 6-well plates and allowed to adhere overnight. Cells were incubated in fresh medium in which FBS had been replaced with 200 μg/ml BSA. Conditioned medium was collected at 0, 12, 24, and 36 h. Half of each sample was subjected to TGF-β activation in vitro using 1 n HCl for 10 min followed by neutralization with 1.2 n NaOH/0.5 m HEPES, and these samples were used to measure the total concentration of TGF-β1 or TGF-β2, whereas the remainder was used to measure biologically active TGF-βs (60) . Concentrations of TGF-β1 and TGF-β2 were determined using Quantikine ELISA kits (R&D Systems) following directions provided by the manufacturer.

RNA Extraction and Reverse Transcription-PCR.

RNA was extracted from the pancreatic cell lines using the Qiagen RNEasy RNA Extraction kit (Qiagen, Inc., Valencia, CA). Reverse transcription-PCR was performed for TβR-I, TβR-II, Smad7, and glyceraldehyde-3-phosphate dehydrogenase using the Qiagen OneStep RT-PCR kit. The following primers were used: TβRI, forward primer, 5′-ATCCTTCAAACGTGCTGACATCTATGC-3′, and reverse primer, 5′-AATCCGCAATGCTGTAAGCCTAGCTGC-3′ (expected product size, 280 bp); Smad7, forward primer, 5′-TACCGTGCAGATCAGCTTTG-3′, and reverse primer, 5′-TTTGCATGAAAAGCAAGCAC-3′ (expected product size, 200 bp); and glyceraldehyde-3-phosphate dehydrogenase, forward primer, 5′-GAGTCAACGGATTTGGTCGT-3′, and reverse primer, 5′-TTGATTTTGGAGGGATCTCG-3′ (expected product size, 238 bp).

PAI/Luciferase Assay.

The PAI/Luciferase assay was performed as described by Abe et al. (61) with minor modifications. Briefly, 1.6 × 104 TMLC cells suspended in 100 μl of medium were plated in 96-well tissue culture dishes and allowed to attach for 3 h at 37°C in a 5% CO2 incubator. After 3 h, the medium was replaced with 100 μl of medium containing either TGF-β at concentrations ranging from 0 to 100 pm or a suspension of 1.6 or 3.2 × 104 BxPC-3 cells. The cells were lysed 14 h later at room temperature, and luciferase activity in cell lysates was determined using the Promega Luciferase Reporter Assay System following the protocol recommended by the manufacturer (Promega, Madison, WI) using a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA). For the αVβ6 integrin neutralization experiment, TMLC cells were plated in the presence or absence of 10 μg/ml anti-αVβ6 integrin antibody 10D5 (Chemicon International, Temecula, CA). After 3 h of incubation at 37°C, the medium was replaced with either fresh medium with or without the anti-integrin antibody or a suspension of BxPC-3 cells in the presence or absence of 10 μg/ml anti-αVβ6 integrin antibody. After 14 h of overnight incubation, the cells were lysed as above and assayed for luciferase activity.

RESULTS

To gain insight into the status of Smad signaling in PC, we analyzed the expression and activation of R-Smads and expression of the common mediator Smad, Smad4, in a panel of eight human PC cell lines (Fig. 1) ⇓ . Five of the PC lines (BxPC-3, Hs766T, CFPAC-1, CaPan2, and AsPC1) failed to express any detectable Smad4 protein. This is consistent with previous reports of homozygous deletion of the MADH4 gene in BxPC-3, Hs766T, and CFPAC-1 (25) , whereas AsPC-1 cells carry a missense mutation in Smad4 that targets the protein for rapid ubiquitination and degradation (29) . As CaPan2 cells express a wild-type MADH4 gene, the reason for loss of Smad4 protein in this case is unclear (26) . Each of the eight cell lines expressed comparable steady-state levels of Smad2 and Smad 3 (Fig. 1) ⇓ , which were not significantly affected by TGF-β treatment. As expected, treatment with exogenous TGF-β rapidly induced pSmad2 in four of the cell lines (CaPan-2, Panc-1, AsPC-1, and HTB-147), whereas pSmad2 was not induced in TβR-II-negative MiaPaCa cells (62) . To our surprise, the remaining three cell lines (BxPC-3, Hs766T, and CFPAC-1) constitutively expressed high steady-state levels of pSmad2, even in the absence of exogenous TGF-β (Fig. 1) ⇓ . In BxPC-3 cells, treatment with TGF-β induced a 2-fold additional increase in pSmad2 levels, whereas levels were minimally increased in Hs766T and CFPAC-1. In general, the pattern of inducibility of pSmad3 paralleled that of pSmad2. However, pSmad3 was proportionally less strongly induced than pSmad2 by TGF-β in CaPan2, AsPC1, and HTB-147 cells. In addition, the anti-pSmad3 antibody recognizes a second band at ∼58 kDa, which likely represents the phosphorylated form(s) of Smad1 and/or Smad5, because its phosphorylation was strongly induced by BMP2, particularly in Panc-1 and Mv1Lu mink lung epithelial cells (Fig. 1C) ⇓ . In Mv1Lu cells, Smad1/5 phosphorylation can also be induced by exogenous TGF-β, and this effect can be blocked by the selective TβR-I (Alk5) kinase inhibitor, SD-093. In contrast, in the PC cells lines, TGF-β fails to induce phosphorylation of Smad1/5, nor is Smad1/5 phosphorylation affected by SD-093. Thus, the phenotypic effects of TGF-β on PC cells (see below) cannot be attributed to activation of Smad1/5, either by TGF-β or via BMP receptor activation.

Fig. 1.
Fig. 1.

Smad signaling in human pancreatic cancer cell lines. A, lysates were prepared from preconfluent cultures and subjected to Western blot analyses for R-Smads Smad2 and Smad3 and pSmad2, pSmad3, and Smad4 as described in “Materials and Methods.” AsPC1, Hs766T, CFPAC1, and BxPC-3 failed to express Smad4, either because of gene deletion or inactivating mutation (25 , 26) . Steady-state levels of R-Smads were similar across the panel of cell lines and were not affected by treatment with transforming growth factor β (TGF-β). B, pSmad2 levels were expressed as the ratios of integrated absorbance of the pSmad2 and Smad2 signals obtained by Western blot analyses. Treatment with TGF-β induced pSmad2 in CaPan2, AspC1, Panc1, and HTB147 but not in MiaPaCa cells, which lack TβR-II receptors (62). Hs766T, CFPAC1, and BxPC-3 expressed increased basal levels of pR-Smads even in the absence of exogenous TGF-β. C, cells were treated with TGF-β (100 pm), BMP2 (4 nm), or vehicle only in the presence or absence of SD-093 (1 μm) and subjected to Western blot analysis using our pSmad3-directed antibody as described above. TGF-β treatment induced phosphorylation of a band of molecular weight of Mr 52,000, representing pSmad3. In addition, the anti-pSmad3 antibody recognizes a second band at Mr ∼58,000, which likely represents the phosphorylated form(s) of Smad1 and/or Smad5 because its phosphorylation was strongly induced by BMP2, particularly in Mv1Lu and Panc-1 cells. In addition, Smad1/5 phosphorylation is also induced by exogenous TGF-β in Mv1Lu cells, and this effect can be blocked by the selective TβR-I (Alk5) kinase inhibitor, SD-093. In contrast, in the pancreatic cancer cells lines, TGF-β fails to induce phosphorylation of Smad1/5, nor is Smad1/5 phosphorylation affected by SD-093.

A number of recent studies have suggested that tumor-associated TGF-β might promote the invasive and metastatic properties of tumor cells in a cell-autonomous manner (2 , 8 , 63 , 64) . To determine whether the apparent constitutive activation of the TGF-β-signaling pathway in Smad4-deficient PC lines such as BxPC-3 provided these tumor cells with a selective advantage, we examined the biological consequences of a constitutively active TGF-β-signaling pathway in these cells. Jonson et al. (34 , 48) recently reported that some PC cell lines were growth stimulated by TGF-β. As shown in Fig. 2A ⇓ , treatment with exogenous TGF-β failed to inhibit in vitro proliferation of PC cells, independently of whether they expressed Smad4, nor did it stimulate the growth of any of these cells. Moreover, treatment of PC cells with the selective TβR-I kinase inhibitor, SD-093, had no impact on cell growth, independently of whether they expressed high levels of pSmad2 or Smad4 (Fig. 2B) ⇓ . Thus, growth of these cell lines is not dependent on or stimulated by the constitutive endogenous activation of TβR receptor signaling.

Fig. 2.
Fig. 2.

Transforming growth factor β (TGF-β) signaling and anchorage-dependent growth of PC cells. A, Mv1Lu (□), Panc1 (•]), BxPC-3 (▪), Hs766T (▴), and CFPAC1 (♦) cells were plated at 8000 cells/well in 24-well tissue culture plates, treated with TGF-β at the indicated concentrations or vehicle only, and counted 72 h later. TGF-β treatment did not significantly affect pancreatic cancer cell growth while potently inhibiting the growth of Mv1Lu cells. Means and SDs of four independent experiments. B, Panc1, CFPAC1, BxPC-3, and Hs766T cells were plated at 8000 cells/well and treated with SD-093 (1 μm; •) or vehicle only (▪). Cell counts were obtained daily over the next 6 days. Growth rates of high pSmad2 expressing, Smad4-deficient cells (CFPAC1, BxPC-3, Hs766T) and of the low pSmad2 expressing, Smad4-positive Panc1 cells were unaffected by treatment with SD-093.

Besides causing cell cycle arrest, one of the physiological functions of TGF-β is to induce epithelial cells to assume a fibroblastoid and dispersed phenotype, a process referred to as EMT (4) . EMT is characterized by redistribution of the tight junction protein, Ecadherin, from the cell membranes to the cytoplasm and rearrangement of F-actin from predominantly subcortical bundles to stress fibers across the cytoplasm, thus allowing cells to dissociate from their neighbors and to migrate in response to external stimuli. As shown in Fig. 3 ⇓ , BxPC-3 cells grew as tight colonies, and E-cadherin was distributed along the cell membrane. Some of the E-cadherin was also localized to the cytoplasm in a speckled perinuclear pattern. Neither treatment with exogenous TGF-β nor blocking TβR-I kinase activity with SD-093 had any effect on E-cadherin expression or its subcellular distribution. Similarly, F-actin was arranged as subcortical bundles and as pronounced stress fibers. This distribution of F-actin also remained unaltered by treatment with either exogenous TGF-β or SD-093 (Fig. 3) ⇓ . In fact, we failed to observe any evidence of EMT up to 5 days of exposure of cells to TGF-β (data not shown). Thus, neither the subcellular distribution of E-cadherin or F-actin, nor cell-cell cohesion appear to be regulated by or dependent on TGF-β receptor signaling.

Fig. 3.
Fig. 3.

Epithelial-to-mesenchymal transdifferentiation in BxPC-3 is independent of transforming growth factor β (TGF-β) signaling. BxPC-3 cells were plated in 35-mm tissue culture dishes and treated with TGF-β (100 pm), SD-093 (1 μm), both, or vehicle only for 24 h. After fixation, E-cadherin (A) and F-actin (B) were detected by immunostaining and phalloidin staining, respectively, as described in “Materials and Methods.” E-Cadherin was expressed along cell membranes and also in a speckled perinuclear pattern and F-actin was present subcortically and as stress fibers. Neither treatment with exogenous TGF-β or with the TβR-I kinase inhibitor, SD-093, had any effect on the subcellular localization of E-cadherin and F-actin, nor on cell-cell cohesion. Thus, the epithelial-to-mesenchymal transdifferentiation phenotype was not regulated by or dependent on TGF-β receptor signaling.

Several recent studies have suggested that TGF-β might enhance cell motility, invasiveness, and metastasis of human carcinoma cells in a cell-autonomous manner (reviewed in Ref. 6 ). To determine whether the activation of endogenous TβR receptor signaling might affect these properties of PC cells, we compared the effects of exogenous TGF-β and of SD-093 treatment on motility of Panc1 cells, which do not display constitutive pSmad2, with BxPC-3 and Hs766T cells, which express high pSmad2 levels (Fig. 4A) ⇓ . Treatment with exogenous TGF-β had no effect on motility of Panc-1, BxPC-3, or Hs766T cells (data not shown; Fig. 4A ⇓ ). However, blocking endogenous TβRI-kinase activity with SD-093 inhibited BxPC-3 cell motility by 50%, whereas motility of Panc1 cells was unaffected (Fig. 4A) ⇓ . Thus, motility of BxPC-3 cells appears to be driven, at least in part, by the endogenous activation of TGF-β receptor system, while this is not the case for Panc1 cells. Because adherent BxPC-3 cells were not dispersed in response to exogenous TGF-β treatment (Fig. 3) ⇓ , we had to exclude the possibility that the observed effect of SD-093 was the result of the mechanical dispersion of cells that is required for the Transwell migration assay. Therefore, we also tested the effect of SD-093 on motility using a wound closure assay. In this assay, TGF-β-treated cells displayed the fastest rate of wound closure, whereas treatment with SD-093 slowed cell movement for at least 24 h (Fig. 4B) ⇓ . Thus, although exogenous TGF-β did not affect motility of cells that displayed constitutive activation of the signaling pathway, inhibiting endogenous TGF-β signaling with the inhibitor SD-093 clearly was able to slow migration irrespective of whether the cells were dissociated or not.

Fig. 4.
Fig. 4.

In vitro migration of BxPC-3 cells is inhibited by blocking TβR-I activity with SD-093. A, 105 cells were plated onto polyethylene terephthalate filters Transwell cell culture inserts in the presence of transforming growth factor β (TGF-β; 100 pm), SD-093 (1 μm), both, or vehicle only. After 24 h, the numbers of cells that had migrated through the filters were counted as described in “Materials and Methods.” Migration of BxPC-3 cells ( Embedded Image) was not affected by treatment with exogenous TGF-β but was strongly inhibited by the TβR-I kinase inhibitor, SD-093 (Student’s t test: TGF-β versus SD-093, P = 0.003; TGF-β versus TGF-β+SD-093, P = 0.005). In contrast, neither TGF-β treatment nor SD-093 affected motility of Panc-1 cells ( Embedded Image). Thus, in vitro migration of Smad4-deficient BxPC-3 cells is driven, at least in part, by the autocrine activation of TGF-β receptor signaling. Means and SD, triplicate wells/condition. B, confluent BxPC-3 monolayers in 6-well plates were wounded by manually drawing a furrow across the monolayer with a micropipette tip as described in “Materials and Methods.” A total of 100 pm TGF-β (•), 1 μm SD-093 (♦), TGF-β plus SD-093 (▴), or vehicle only (▪) was added. Wound closure at various time intervals following wounding was quantitated using Adobe Photoshop 6.0 and ImageJ 1.29 software and expressed graphically. TGF-β-treated cells migrated fastest while SD-093-treated cells migrated slowest during the entire course of the experiment. Vehicle, ▪; TGF-β, •; TGF-β + SD-093, ▴; SD-093, ♦. Means and SDs of two independent experiments.

To determine whether TGF-β signaling played a role in the invasive phenotype of Smad4-null PC cells, we examined their ability to invade. Exogenous TGF-β stimulated the ability of BxPC-3 cells to invade into Matrigel 2-fold (Fig. 5) ⇓ . This effect was completely negated by pretreatment with SD-093 (Fig. 5) ⇓ . Moreover, treatment with SD-093 alone was sufficient to inhibit basal invasiveness by ∼50%, indicating that invasion by these cells is indeed driven by a constitutively active endogenous TGF-β signaling.

Fig. 5.
Fig. 5.

In vitro invasiveness of BxPC-3 cells is dependent on active transforming growth factor β (TGF-β) receptor signaling. Cells (105/well) were plated onto Matrigel-precoated cell culture inserts in the presence of TGF-β (100 pm), SD-093 (1 μm), both, or vehicle only. After 48 h, the numbers of cells that had migrated through the filters were counted as described in “Materials and Methods.” BxPC-3 cells were able to invade into Matrigel. Invasiveness was additionally enhanced by TGF-β treatment and almost completely inhibited by SD-093 (Student’s t test, TGF-β versus TGF-β+SD-093, P = 0.019; TGF-β versus SD-093, P = 0.018). Thus, in vitro invasiveness of these pancreatic cancer cells is highly dependent on constitutive activation of their TGF-β receptors. Means and SDs of three independent experiments, triplicate wells/condition.

Thus, our results indicated that the constitutive activation of TβR receptor signaling in BxPC-3 cells increased their motile and invasive behavior. Moreover, this effect of TGF-β was clearly Smad4 independent because BxPC-3 cells are Smad4 deficient. Several recent studies have suggested that the effects of TGF-β on cell motility may be mediated by the p38/MAPK pathway in a Smad-independent manner (65 , 66) . However, treatment of BxPC-3 cells with the selective p38/MAPK inhibitor, NPC-37282, did not affect cell motility (data not shown). Because recent studies report that the ROCK may play a key role in regulation of cell motility in pancreatic cancer (67) , as well as mediating the effects of TGF-β on cytoskeletal organization (68 , 69) , we examined the possibility that ROCK might mediate the effects of TGF-β in pancreatic cancer cells. However, pretreatment of BxPC-3 cells with up to 10 μm of the ROCK inhibitor, Y27632, failed affect cell motility or invasion (data not shown). Hence, neither activation of the p38/MAPK pathway nor of the RhoA/C pathway appears to mediate the TβR-I-dependent motility or invasion we observed in these PC cells.

In aggregate, these findings raised the question how endogenous TGF-β signaling becomes activated in Smad4-deficient PC cell lines. First, we assessed relative levels of expression of TβR-I in the various cell lines (Fig. 6) ⇓ . Immunoblotting showed that CaPan2, AsPC1, Hs766T, and BxPC-3 cells expressed very similar steady-state levels of TβR-I protein, which were not affected by TGF-β treatment (Fig. 6A) ⇓ . MiaPaCa cells expressed much lower levels of TβR-I, whereas Panc1 and HTB-147 expressed 2–3-fold higher levels. As TβR-I mRNA levels were very similar across the panel of cell lines (Fig. 6B) ⇓ , differences in TβR-I protein levels appear to be due to posttranscriptional mechanisms. Moreover, steady-state levels of Smad7 mRNA were very similar across cell lines, except for MiaPaCa cells, in which Smad7 mRNA levels were negligible when compared with the other cell lines. This finding is consistent with reports that TβR receptor activity is required for Smad7 expression (70 , 71) . Conversely, constitutive TβR receptor activation did not seem to be associated with increased steady-state mRNA levels of Smad7, and Smad7 mRNA levels did not seem to affect TβR-I receptor expression levels. As the TβR-II kinase is responsible for activation of the TβR-I kinase, we also compared the levels of TβR-II expression across the panel of PC lines. As can be seen in Fig. 6C ⇓ , BxPC3, Panc-1, and Hs766T cells expressed comparable levels of TβR-II protein, whereas HTB-147 cells expressed significantly lower levels. Moreover, in the remaining lines, TβR-II protein levels were either extremely low or undetectable. In any event, differences in TβR-II protein levels cannot account for the differences in the endogenous activation state of the TβR system between Smad4-deficient (BxPC3 and Hs766T) and Smad4-positive Panc-1 cells.

Fig. 6.
Fig. 6.

Expression of TβR-I and Smad7 in pancreatic cancer cell lines. A, lysates prepared from preconfluent cultures incubated for 2 h in the presence and absence of 100 pm transforming growth factor β (TGF-β) were immunoblotted for TβR-1 protein and γ-actin as described in “Materials and Methods.” CaPan2, AsPC1, Hs766T, and BxPC-3 cells expressed very similar steady-state levels of TβR-I protein, which were not affected by TGF-β treatment. MiaPaCa cells expressed much lower levels of TβR-I, whereas Panc1 and HTB-147 expressed 2–3-fold higher levels. Thus, there appeared to be a positive correlation between the levels of Smad4 and TβR-I expression. B, total cellular RNA extracted from untreated pancreatic cancer cells was subjected to reverse transcription-PCR with primers for TβR1 and MADH7 cDNA sequences. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control. TβR-I mRNA levels were very similar across the panel of cell lines. Moreover, steady-state levels of Smad7 mRNA were very similar across cell lines, except for MiaPaCa cells, in which Smad7 mRNA levels were negligible when compared with the other cell lines. C. Western blot analysis of TβR-II protein expression in pancreatic cancer cells. BxPC3, Panc-1, and Hs766T cells expressed comparable levels of TβR-II protein, whereas HTB-147 cells expressed significantly lower levels. Moreover, in the remaining lines, TβR-II protein levels were either extremely low or undetectable.

Inman et al. (17) reported recently that Smad2 undergoes continuous nucleocytoplasmic shuttling, being phosphorylated at the level of TβR receptors and dephosphorylated in the nucleus. Thus, the constitutively elevated level of activated pR-Smads we observed in PC cells could, in principle, be caused by an increased rate of phosphorylation or a decrease in the rate of dephosphorylation (17) . We examined the rate of pR-Smad dephosphorylation by blocking TβRI kinase using SD-093 and following pR-Smad levels over time. As shown in Fig. 7A ⇓ , pSmad2 and pSmad3 were dephosphorylated with a t1/2 of ∼30 min in BxPC-3 cells. Moreover, pSmad2 was dephosphorylated at similar rates in cell lines with high (BxPC-3, CFPAC1) versus low (CaPan2, HTB-147) steady-state pSmad2 levels. Thus, elevated levels of pR-Smads did not appear to be caused by decreased nuclear phosphatase activity. Furthermore, these results demonstrated that R-Smad phosphorylation was indeed dependent on the activity of the TβR-I receptor kinase and are consistent with the report by Inman et al. (17) that nucleocytoplasmic shuttling of pSmad2 to and dephosphorylation in the nucleus does not require Smad4.

Fig. 7.
Fig. 7.

Dephosphorylation of activated R-Smads. A. BxPC-3 cells were treated with TGF-β (100 pm) or vehicle only for 1 h followed by addition of TβR-1kinase inhibitor, SD-093. Cells were lysed at 0, 2, 4, and 6 h and pSmad levels determined by Western blotting as described in “Materials and Methods.” In the presence of SD-093, pSmad2 (▪) and pSmad3 (•) in BxPC-3 cells were dephosphorylated with a similar t1/2 of ∼30 min. B, the rates of pSmad2 dephosphorylation were similar in cells with high [BxPC-3 (▪), CFPAC1 (•)] versus low [HTB147 (▴), CaPan2 (♦)] steady-state pSmad2 levels. Thus, the constitutively high levels of pSmad2 found in BxPC-3 and CFPAC1 cells could not be attributed to a decreased dephosphorylation rate.

We then went on to investigate the possibility that excessive production and/or extracellular activation of TGF-β was responsible for activation of the TβR receptors in an autocrine manner resulting in the increased steady-state levels of pR-Smads in BxPC-3 cells. As shown in Fig. 8 ⇓ , overnight incubation of cells with a pan-specific anti-TGF-β-neutralizing antibody resulted in a dose-dependent reduction in specific pSmad2 levels up to a maximum of 80%. Thus, extracellular biologically active TGF-β appeared to be in large part responsible for activating the TβR system in BxPC-3 cells.

Fig. 8.
Fig. 8.

Active transforming growth factor β (TGF-β) is responsible for the constitutive presence of pSmad2 in BxPC-3. A, confluent BxPC-3 cells were incubated overnight with a pan-specific anti-TGF-β-neutralizing antibody at the indicated concentrations, and pSmad2 and Smad2 levels were assayed by Western blot analysis as described in “Materials and Methods.” B, pSmad2/Smad2 ratios were determined from integrated optical densities of bands on Western blot analyses. Treatment of cultures with pan-specific TGF-β-neutralizing antibody resulted in a dose-dependent reduction in specific pSmad2 levels. In contrast, treatment with neutralizing antibody directed against TGF-β3 had no effect (data not shown).

To determine the magnitude of TGF-β production and activation by BxPC-3 and to identify the TGF-β isoforms involved, we assayed conditioned medium from cells grown in serum-free medium for the presence of TGF-β1 and TGF-β2 using ELISA kits (Table 1) ⇓ . Total versus bioactive levels of TGF-β were determined by comparing in vitro acid-activated and -nonactivated medium samples (60) . We were able to detect a steady increase in the concentration of both TGF-β1 and TGF-β2 isoforms over time, indicating that the cells actively secrete TGF-β1 and TGF-β2 (Table 1) ⇓ . However, <10% of TGF-βs detected was biologically active. To rule out that the cells might be producing bioactive TGF-β3, we repeated the neutralization experiments using a specific anti-TGF-β3 antibody. Overnight incubation of BxPC-3 cells with the anti-TGF-β3 antibody did not appreciably alter the intracellular levels of pSmad2, indicating that active secreted TGF-β3 did not play a significant role in generating pSmad2 (data not shown).

View this table:
Table 1

TGF-β1 and TGF-β2 in conditioned medium of BxPC-3 PC cellsa

To test the hypothesis that TGF-β was being activated and retained at or close to the cell surface, we conducted coculture experiments using TMLC cells that express a TGF-β-inducible PAI-1 promoter driving a luciferase reporter gene construct (61) . As shown in Fig. 9A ⇓ , exogenous TGF-β resulted in a dose-dependent increase in luciferase activity in TMLC cells, whereas treatment with SD-093 strongly inhibited basal luciferase activity. Cocultivation of TMLC cells with BxPC-3 cells induced an increase in luciferase activity, which was proportional to the number of BxPC-3 cells added (Fig. 9B) ⇓ . Moreover, Panc-1 cells induced significantly less luciferase activity than BxPC-3 cells (Fig. 9B) ⇓ . Thus, these experiments clearly demonstrate that BxPC-3 cells produce biologically active TGF-β that is capable of inducing specific gene responses in neighboring cells in a paracrine fashion.

Fig. 9.
Fig. 9.

BxPC-3 cells activate plasminogen activator inhibitor type 1 (PAI-1) promoter activity in neighboring TMLC cells. A, a total of 1.6 × 104 TMLC cells was plated in 96-well tissue culture dishes and allowed to attach for 3 h at 37°C in a 5% CO2 incubator. After 3 h, the medium was replaced with 100 μl of medium containing transforming growth factor β (TGF-β) at the indicated concentrations. Luciferase activity was determined in cell lysates 14 h later. Means and SDs of three independent experiments, triplicate wells/condition. TGF-β induced activation of the PAI-1 promoter and luciferase expression in a dose-dependent manner. B, a total of 1.6 × 104 TMLC cells was plated in 96-well tissue culture dishes and allowed to attach for 3 h at 37°C in a 5% CO2 incubator. After 3 h, the medium was replaced with 100 μl of medium containing 1.6 × 104 BxPC-3 or Panc-1 cells, SD-093 (1 μm), or vehicle only. Luciferase activity was determined in cell lysates 14 h later. Means and SDs of three independent experiments, triplicate wells/condition. BxPC-3 cells induced activation of the PAI-1 promoter and luciferase expression in neighboring TMLC cells to a significantly greater extent than Panc-1 cells (P = 0.05; paired t test). Treatment of TMLC cells with SD-093 repressed luciferase activity, indicating that basal PAI-1 promoter activity is dependent on endogenous TGF-β signaling. C, a total of 1.6 × 104 TMLC cells was plated in 96-well tissue culture dishes in the presence or absence of 10 mg/ml anti-αVβ6 integrin antibody 10D5 and allowed to attach for 3 h at 37°C in a 5% CO2 incubator. After 3 h, the medium was replaced with 100 μl of fresh medium with the anti-integrin antibody or vehicle only or with a suspension of 3.2 × 104 BxPC-3 cells in the presence of 10 mg/ml anti-αVβ6 integrin antibody or vehicle only. Luciferase activity was determined in cell lysates 14 h later. Means and SDs of two independent experiments, triplicate wells/condition. BxPC-3 cell-induced activation of the PAI-1 promoter and luciferase expression in neighboring TMLC cells was almost completely prevented in the presence of anti-αVβ6 integrin antibody.

Although the low level of TGF-β activation that mediates tissue homeostasis is mediated by an αVβ8 integrin-dependent mechanism (72) , the high level of TGF-β activation associated with tissue injury appears to be mediated by a αVβ6 integrin-dependent mechanism (73 , 74) . As shown in Fig. 9C ⇓ , treatment of TMLC/BxPC-3 cocultures with a neutralizing antibody directed against the αVβ6 integrin almost completely blocked the luciferase activation in TMLC cells. Thus, the constitutive activation of TGF-β by BxPC-3 cells appears to be primarily dependent on αVβ6 integrin (74) .

DISCUSSION

Pancreatic cancer is unique among human adenocarcinomas in that it frequently undergoes genetic inactivation of the tumor suppressor gene, MADH4, resulting in loss of expression of the TGF-β signaling intermediate, Smad4. Growth of Smad4-deficient PC cells is no longer inhibited by TGF-β, a response that can be restored by transfection of Smad4 (62) . Our study demonstrates that, in PC cell lines that have escaped from TGFβ’s tumor suppressive function because of Smad4 deletion, the steady state levels of phosphorylated R-Smads, pSmad2 and −3, is constitutively elevated. Steady state pR-Smad levels are dependent on the rate of RSmad phosphorylation by the TβR-I kinase on the one hand, and on their dephosphorylation rate on the other (17 , 38) . As the rates of pR-Smad dephosphorylation were very similar in Smad4-deficient and Smad4-positive PC lines and similar to rates previously reported for other cell types (17) , we conclude that the elevated pR-Smad levels are the result of increased TβR-I receptor kinase activity. This is legitimate for two reasons: First, in cell-free systems, the amount of pSmad2 detected by Western blot is proportional to the amount of active TβR-I kinase (57) . Secondly, in nonneoplastic epithelial cells, Smad2 phosphorylation is rapidly induced by treatment with exogenous TGF-β in a dose-dependent manner (58 , 75) , and this response can be blocked by pre-treating the cells with the selective TβR-I kinase inhibitor, SD-093 (A. Kareddula and M. Reiss, unpublished observations). The increased TβR-I kinase activity in Smad4-deficient PC cells was not due to higher levels of TβR-I protein expression. In fact, steady state TβR-I levels were generally lower in Smad4-deficient than in Smad4-expressing PC lines. These findings might help explain the seemingly contradictory reports that TβR receptor levels are either elevated or diminished in PC (33 , 34 , 39) . Moreover, the fact that TβR-I kinase activity could be easily blocked by the selective inhibitor, SD-093, and that activating mutations of the TβRI gene have not been found in these PC lines (22 , 34) essentially excludes the possibility of intrinsic activation of the enzyme. This conclusion was further substantiated by our demonstration that BxPC-3 cells produced detectable amounts of bioactive TGF-β2 and were able to induce TGFβ-dependent transcriptional responses in neighboring TMLC cells, whereas pSmad2 levels could be reduced by treating cell cultures with neutralizing anti-TGFβ antibody. These experiments demonstrate conclusively that activation of TGF-β at the cell surface results in activation of TβR receptors in an autocrine manner, thereby resulting in elevated intracellular pR-Smad levels. Moreover, the presence of TGFβ1 and -2 isoforms in conditioned medium samples is consistent with previous reports of detection of both isoforms in PC cell lines (76) and immunohistochemical studies of PC tissue specimens (49) .

Latent TGFβ1 can be activated at the cell surface by one of two integrin-mediated mechanisms. Mu et al. (72) recently described a mechanism whereby cell-surface activation of TGFβ might mediate cellular homeostasis: These investigators showed that latent TGF-β is sequestered to the cell surface by binding to αVβ8, an integrin expressed by normal epithelial and neuronal cells in vivo. This binding results in the membrane type 1 (MT1)metalloproteinase-dependent release of active TGFβ, which, in turn, leads to autocrine and paracrine effects on cell growth and matrix production to maintain cellular homeostasis. Alternatively, integrin αVβ6 binds latent TGF-β and activates it without cleaving the latency-associated peptide (73) , and is predominantly involved in the intensive TGF-β activation associated with tissue injury and inflammation (74) . As we were able to neutralize the BxPC-3-dependent activation of TMLC cells using anti-αVβ6 integrin antibody, this appears to be the predominant mechanism responsible for the constitutive activation of TGF-β by BxPC-3 cells. Because TGF-β remains associated with the latency-associated protein, αVβ6-dependent activation does not result in the release of active TGF-β into the surrounding medium and this also explains why we were unable to detect active TGF-β in our conditioned medium samples. This result is consistent with the observation that TGF-β treatment results in the up-regulation of αV integrin in pancreatic cancer cells (48) . Thus, it is possible that a feed forward loop further amplifies the autocrine production and activation of TGF-β within the cellular microenvironment.

Perhaps our most important observation is that the constitutive activation of endogenous TGFβ receptor signaling appears to drive cell migration and invasion in vitro in a cell-autonomous manner. Support for this conclusion comes primarily from the experiments demonstrating that treatment of BxPC-3 cells with the TβR-I kinase inhibitor, SD-093, significantly inhibited cellular migration and invasiveness in vitro, whereas treatment of these cells with exogenous TGFβ further stimulated their invasive behavior. The fact that exogenous TGFβ enhanced invasiveness of BxPC-3 cells without stimulating cell migration, may be due to activation of TGFβ-regulated genes specifically involved in invasion. For example, Ellenrieder et al. (77) reported that the stimulation of in vitro invasiveness of PC cell lines by exogenous TGFβ was associated with activation of matrix metalloproteinase-2 (MMP-2) and the urokinase plasminogen activator system. Moreover, induction of PAI-1 by TGFβ has been observed even in cells that lack Smad4 (46, 47, 48) . Our results are also consistent with recent reports showing that blocking TGFβ signaling using a soluble type II exoreceptor inhibits pancreatic tumor formation in a nude mouse model and that treatment of PC cell lines with antisense oligonucleotides specific to TGFβ2 inhibits cell proliferation and migration in vitro (7 , 78 , 79) . In addition to these experimental studies, several clinical observations support the idea that the metastatic phenotype of PC is driven by activation of TGFβ signaling, particularly in the context of Smad4 loss. For example, Teraoka et al. reported an association between TGFβ1 over-expression and the presence of liver metastases (80) . Moreover, loss of Smad4 is associated with a higher likelihood of metastasis and poor outcome following surgical resection of PC (31) .

As is the case for most cancer cell lines, exogenous TGFβ failed to suppress the growth of the PC cells in our study (1) . Moreover, blocking the activated endogenous TGFβ signaling in Smad4-deficient cells with the TβR-I inhibitor, SD-093, failed to stimulate their growth. This finding contrasts with a previous report that BxPC-3 and Panc-1 cells had retained partial responsiveness to TGFβ-mediated growth arrest (46 , 78 , 81) . This discrepancy may be due to clonal variation or differences in culture conditions. Furthermore, the phenotype of the PC cell lines used in our study seems to be distinct from lines described by Jonson et al. (34) , in which exogenous TGFβ paradoxically stimulated growth.

In mouse models of cutaneous and mammary carcinogenesis, transformed cells escape from TGFβ’s tumor suppressive function early on, whereas in late stage tumors, TGFβ signaling can acquire oncogenic properties by constitutively inducing EMT, resulting in a highly invasive and metastatic tumor phenotype. Moreover, Oft et al. showed that, in the mouse skin carcinogenesis model, high levels of nuclear pSmad2 cooperate with elevated H-ras levels to induce EMT of spindle cell carcinomas in vitro and a highly metastatic phenotype in vivo (82) . These observations demonstrate that TGFβ’s homeostatic (tumor suppressive) and EMT responses can become uncoupled during malignant progression (reviewed in (2 , 6) ). However, in our case, PC cells failed to display any evidence of EMT in response to exogenous TGFβ, nor was a more epithelioid phenotype induced by SD-093 treatment. Thus, even though constitutive activation of TGFβ signaling clearly drove cell motility and invasiveness of BxPC-3 cells, these responses occurred independently of EMT. In aggregate, our studies demonstrate that, at least in Smad4-deficient PC cells, the effects of TGFβ on cell migration and invasion have become uncoupled from the induction of growth arrest and EMT.

Several studies have shown that Smad4-deficient PC cell lines can retain partial transcriptional responsiveness to TGF-β (46 , 48) . Whether the induction of motility and invasiveness in BxPC-3 cells is independent of Smad signaling altogether or is still mediated by Smad2 and/or Smad3 remains to be determined. However, it is unlikely that the p38/MAPK pathway plays a significant role as treatment of the cells with a selective p38/MAPK inhibitor failed to affect their phenotype. In this respect, these PC cells appear to differ from nonneoplastic mammary epithelial cells in which the p38/MAPK pathway mediates TGF-β-induced fibronectin synthesis, EMT, and apoptosis (65 , 66) , as well as mammary cancer cells, in which p38/MAPK appears to mediate TGF-β-induced motility (5) . Similarly, the TGF-β-driven phenotype of BxPC-3 cells did not appear to be mediated by ROCK-1 activity because pretreatment with a ROCK-1 inhibitor had no effect. A number of studies have suggested that RhoC and ROCK-1 are frequently overexpressed in PC (67 , 83) and that down-regulation of ROCK-1 expression using antisense oligonucleotides can inhibit invasiveness of PC cells in vitro (67) . However, our results suggest that the effects of TGF-β on PC cell invasion are independent of RhoA/C/ROCK signaling. Although it is possible that other Smad-independent signaling pathways are involved in mediating the effects of TGF-β on the PC phenotype (84) , these were not investigated in the current study.

The factors that determine cellular response specificity to TGF-β (cell cycle arrest, apoptosis, differentiation, EMT, motility, or invasiveness) remain largely unclear. The level of receptor activation may provide one important level of control (85) . As the TβR-II kinase is constitutionally active, the level of TβR-II expression and the degree of receptor heterodimerization determine TβR-I activity, which is the final output variable of the receptor system that controls all of the downstream signaling events (65 , 86) . Given the nonenzymatic nature of Smad signaling, the concentration and subcellular localization of activated R-Smads may provide important additional controls. Recent studies have convincingly demonstrated that shuttling of Smads between cytoplasm and nucleus is a tightly regulated process that directly links receptor activity with cellular responses to TGF-β. Although Smad4 is rapidly and continuously shuttling between the nucleus and the cytoplasm (15) , TGF-β-induced phosphorylation of Smad2 causes it to get translocated to the nucleus by complexing with Smad4 and interacting with nucleoporins. In the nucleus, Smad2 is dephosphorylated, leading to export back to the cytoplasm. This continuous nucleocytoplasmic shuttling of Smad2 during active TGF-β receptor signaling seems to provide an important mechanism, whereby the intracellular transducers of the signal continuously monitor receptor activity (15, 16, 17) . Thus, the total amount of pR-Smad present over time appears to be a critical determinant of cellular response. Nicolas et al. (38) recently reported that the resistance of Smad4-positive Panc-1 PC cells to TGF-β-mediated growth suppression is the result of an attenuated pR-Smad response to TGF-β. Our current results indicate that a different mechanism applies in Smad4-deficient BxPC-3 cells. In this case, the steady-state level of pR-Smad is elevated, thus presumably providing a continuous high level signal to the nucleus. The fact that re-expressing Smad4 in BxPC-3 cells restores their sensitivity to the antiproliferative effects of TGF-β indicates that Smad4 is critical for proper cell cycle control (62) . Our results confirm previous studies that nuclear transport of pSmad2 does not require Smad4 as carrier protein because the rate of dephosphorylation was similar in Smad4-deficient and Smad4-expressing cell lines (17 , 87) . Thus, one might speculate that Smad4 nucleocytoplasmic shuttling somehow ensures the correct pR-Smad-dependent signal flow into the nucleus. If this idea is correct, the absence of Smad4 might allow excessive pR-Smad-dependent signals to reach the nucleus and hyperactivate the transcriptional machinery, thereby contributing to the motile and invasive phenotype of Smad4-negative PC cells.

In summary, our results not only show that TGF-β is incapable of suppressing the growth of Smad4-deficient PC cell lines but that it has acquired oncogenic properties that promote cell motility and invasiveness. Thus, loss of Smad4 appears to tip the balance toward the oncogenic effects of TGF-β, thereby providing the cells with a selective advantage. This role reversal of TGF-β during tumor progression supports the idea of targeting the TβR kinases as a novel approach to PC treatment (2 , 6, 7, 8, 9) .

Footnotes

  • Grant support: Public Health Service Awards CA-41556 and CA-94431 from the National Cancer Institute (M. Reiss).

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • Requests for reprints: Michael Reiss, The Cancer Institute of New Jersey, Room 2007, 195 Little Albany Street, New Brunswick, NJ 08903. E-mail: michael.reiss{at}umdnj.edu

  • Received January 7, 2004.
  • Revision received April 28, 2004.
  • Accepted June 4, 2004.

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Cancer Research: 64 (15)
August 2004
Volume 64, Issue 15

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Targeting Endogenous Transforming Growth Factor β Receptor Signaling in SMAD4-Deficient Human Pancreatic Carcinoma Cells Inhibits Their Invasive Phenotype 1
Gayathri Subramanian, Roderich E. Schwarz, Linda Higgins, Glenn McEnroe, Sarvajit Chakravarty, Sundeep Dugar and Michael Reiss
Cancer Res August 1 2004 (64) (15) 5200-5211; DOI: 10.1158/0008-5472.CAN-04-0018
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