Transforming growth factor β (TGF-β) cytokines are critical for embryonic development, normal homeostasis and human diseases including chronic fibrosis and cancer.1 Although TGF-β1 is a potent tumor suppressor, malignant cancers frequently express high levels of TGF-β1.2, 3, 4 The experimental evidence implicate TGF-β1 signaling in promoting tumor progression and metastasis via induction of epithelial to mesenchymal transition (EMT), cell migration and matrix proteolysis.1
EMT and cell migration play critical roles during morphogenesis and organogenesis, as well as in wound healing, chronic fibrosis and cancer progression (reviewed in Ref.5). EMT is associated with disintegration of the polarized epithelial architecture leading to dissociation of cellular contacts and remodeling the cellular filamentous structures such as actin filaments. Depending on cellular context, TGF-β can induce EMT alone6, 7 or in cooperation with other factors including oncogenic Ras8 and TNF-alpha.9 The mechanism of EMT in response to TGF-β1 requires de-novo protein synthesis and transcription.10, 11 Signaling pathways mediated by Smads, PI3 kinase and mitogen-activated protein (MAP) kinases (p38 MAPK, ERK) have been implicated in EMT induced by TGF-β1 (reviewed in Ref.12). It is generally considered that EMT increases motility and invasiveness of cells. TGF-β1-mediated EMT in mammary epithelial cells expressing active Ras increases cell motility and invasion.8 However, several studies indicate that TGF-β1 induces EMT and inhibits motility of normal kidney epithelial cells13, 14 and NBT-II hepatocytes,15 as well as TGF-β1 does not increase motility of nontransformed kidney fibroblasts.16 Apparently, TGF-β1 can induce EMT in both normal and tumor cells,11, 17 whereas the effect of TGF-β1 on cell migration may depend on the cellular context. Interestingly, our recent studies indicate that the ability of TGF-β1 to stimulate cell motility inversely correlate with formation of actin stress fibers.18
The actin cytoskeleton plays key roles in a variety of cell functions including motility and invasion.19 Reorganization of actin filaments and formation of focal adhesions are hallmarks of TGF-β1-mediated EMT and tissue fibrosis.12, 20 TGF-β1-mediated actin remodeling involves RhoA-Rho kinase signaling,21 p38MAPK22, 23 and Rac1/CDC42.22, 24 TGF-β1 induces a fast cycle of activation/deactivation of the RhoA-Rho kinase pathway21 and CDC42-Lim kinase signaling.24 However, formation of stress fibers in response to TGF-β1 takes a prolong time6, 17, 22, 24, 25 and requires a de novo protein synthesis.11 Our studies indicate that Smad3 and Smad4 mediate the stress fiber response via upregulation of actin-stabilizing proteins such as α-actinin (ACTN1), calponin h2 (CNN2) and high-molecular weight (HMW) tropomyosins (HMW-tropomyosins), encoded by TPM1 and TPM2.11 Among these proteins, HMW-tropomyosins are essential for TGF-β1 induction of stress fibers. Suppression of HMW-tropomyosins by small interfering RNA (siRNA) inhibits stress fibers, whereas over-expression of HMW-tropomyosin induces stress fibers.11
Tropomyosins are actin-stabilizing proteins consisting of 2 α-helical chains arranged as a coiled-coil that bind along the actin filaments26 and protect them from binding of the actin-destablilizing proteins such as ADF/cofilins and gelsolin.27, 28, 29 Tropomyosins also regulate actin filament branching and nucleation by affecting the Arp2/3 complex.30 Tropomyosin is essential for formation of cable-like filaments and cytokinesis in yeast.31 Four genes encode multiple isoforms of tropomyosin in a tissue specific manner with at least 20 different isoforms expressed widely in vertebrates.26 Fibroblasts and epithelial cells express HMW-tropomyosin isoforms TM2, TM3 and TM6 encoded by the TPM1 gene and TM1 encoded by TPM2.32 Early studies have shown that neoplastic transformation leads to downregulation of the HMW-tropomyosin levels.33, 34, 35, 36, 37 High-grade tumors of breast, prostate, bladder and brain express significantly lower levels of tropomyosin compared to normal tissues.38, 39, 40, 41, 42 Overexpression of HMW-tropomyosin in transformed or tumor cells has been shown to suppress anchorage-independent cell growth and even lead to anoikis.41, 43, 44 Our recent studies have shown that the TPM1 gene is silenced by promoter hypermethylation in metastatic breast and colon cell lines.18 Reactivation of TPM1 by demethylating agent blocks TGF-β-induced motility of MDA-MB-231 cells.18 Given that TGF-β signaling contributes to invasion and metastasis of MDA-MB-231 cells,45, 46, 47, 48, 49 we hypothesized that HMW-tropomyosins may control the ability of TGF-β1 to regulate cell migration and invasion.11
The present study investigates the role of HMW-tropomyosin in TGF-β-mediated regulation of cell motility, invasion and metastasis. We show that TGF-β1 reduces motility of normal epithelial cells while inducing EMT and tropomyosin-mediated stress fibers and focal adhesions. Suppression of HMW-TMs by siRNA in normal cells alleviates TGF-β1 control of cell motility. Re-expression of HMW-tropomyosin in MDA-MB-231 cells using Tet-Off system suppresses the tumor cell invasiveness by increasing cell adhesion to matrix and reducing matrix proteolysis. Moreover, tumor cells expressing tropomyosin are impaired in formation of lung-surface metastases in SCID mice. These findings demonstrate that HMW-tropomyosins are important components of the TGF-β1 pathway in control of cell motility and invasion.
Material and methods
Mouse mammary epithelial NMuMG and human breast normal MCF10A and cancer cell lines MDA-MB-231, MDA-MB-435, MDA-MB-468, T-47D and A549 were purchased from American Tissure Culture Collection (ATCC, Manassas, VA). Human breast cancer MCF-7 cells were purchased from BD Biosciences (Palo Alto, CA). MDA-MB-231 was maintained in improved Minimum Essential Medium (IMEM) (Cellgro, Kansas City, MO). MCF10A cells were cultured as recommended by ATCC. All other cell lines were cultured in Dulbecco's Modified Eagles's medium (DMEM) (Invitrogen, Carlsbad, CA) at 37°C under an atmosphere of 5% carbon dioxide. The medium was supplemented with 10% fetal bovine serum (FBS) (Cellgro), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Invitrogen).
Antibodies, plasmids and other reagents
TGF-β1 was purchased from R&D Systems (Minneapolis, MN). Doxycycline (DOX) was obtained from BD Biosciences. Puromycin and hygromycin were purchased from Sigma (St. Louis, MO). The following antibodies were used: mouse monoclonal to β1 integrin from BD Transduction Laboratories (BD Biosciences); mouse monoclonals to vinculin, α-catenin and to tropomyosin (TM311) from Sigma; and rabbit polyclonal to phospho-Smad2/3 from Cell Signaling (Beverly, MA), rabbit polyclonal to GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody 4G10 to phospho-Tyr was a gift of Dr. Ray Mernaugh, Vanderbilt University. Alexa Green and Texas Red conjugated phalloidins were from Molecular Probes (Eugene, OR).
Generation of Tet-Off MDA-MB-231 cells
Human breast carcinoma MDA-MB-231 cells were co-transfected with pBabe-Puro and ptTA-IRES-Neo plasmid (tTA, tet activator, IRES, internal ribosome entry site) described in Ref.50. Puromycin-resistant clones exhibiting TGF-β responses equal to parental cells were selected in the presence of 1 μg/ml of puromycin. Individual MDA-MB-231-tTA clones with a tight regulation of the tet-responsive pTRE-lux reporter (BD Bioscience) and intact TGF-β responses were chosen to generate inducible cell lines. Rat tropomyosin 3 (TM3) cDNA43 was subcloned in pBluescript II KS(+) (Stratagene) at BamHI/XbaI sites and then shuttled into pTRE2hyg (BD Biosciences Clontech, Palo Alto, CA) at NotI/SalI sites to generate pTRE2hyg-TM3. The MDA-MB-231-tTA clone 4 was transfected with pTRE2hyg-TM3 encoding un-tagged rat TM3 and cells retaining TGF-β responses were selected in the presence of 200 μg/ml hygromycin and 2 μg/ml DOX. To induce expression of TM3, MDA-MB-231-TO-TM3 cells were washed twice with 1× PBS, detached with trypsin-EDTA and given an additional wash with 1× PBS while in suspension. Cells were plated onto fresh tissue culture dishes and media was replenished the next day. Induction of TM3 was confirmed by immunoblotting with TM311 antibody.
The transcriptional activities were measured as described in Ref.11 using the following luciferase reporters: SBE-Lux with 12 repeats of Smad binding sequence was provided by J.-M. Gauthier, Laboratoire Glaxo Wellcome, Les Ulis Cedex, France; the tet-responsive pTRE-lux reporter (BD Bioscience). MDA-MB-231 cells (3 × 104/well) were transfected with a luciferase reporter and pCMV-Rl (Promega, Madison, WI) in 24-well plates using FuGENE6 reagent (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's protocol. Cells were treated with 2 ng/ml TGF-β1 for 16 hr. Firefly luciferase (Luc) and Renilla reniformis luciferase (Rl) activities in cell lysates were determined using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol in a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Luc activity was normalized to Renilla activity and presented as Relative Luciferase Units. All assays were done in triplicate wells and each experiment was repeated at least twice.
Cells were grown in medium containing 10% FBS with or without DOX on glass coverslips (22 mm × 22 mm) for 24 hr. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X-100, and blocked with 3% milk in PBS for 30 min at room temperature (RT). The cells were incubated for 1 hr with antibody to vinculin (1/5,000 dilution) or phospho-Tyr 4G10 antibody (1/500) in 1% milk/PBS, followed by incubation for 30 min with fluorescent secondary antibody (1/500) at RT. Actin filaments were stained with phalloidin conjugated to Alexa Green or Texas Red. Fluorescent images were captured using Nikon TE2000-E inverted microscope equipped with Roper CoolSnap HQ CCD camera.
Preparation of whole-cell extracts and immunoblotting were performed as described in Ref.22. Cells were lysed in buffer containing 20 mM Tris, pH 7.4, 137 mM NaCl, 1% NP-40, 10% glycerol, 20 mM NaF, 1 mM Na orthovanadate, 1 mM PMSF, 2 μg/ml aprotinin and 2 μg/ml leupeptin. Protein concentrations were measured using Bio-Rad DC protein assay (BioRad, Hercules, CA). Membranes were incubated overnight at 4°C with primary antibodies. Anti-rabbit or anti-mouse IgG antibodies conjugated to Horseradish Peroxidase (HRP) (Amersham, Piscataway, NJ) were used as secondary antibodies. Immune complexes were visualized using the west pico chemiluminescent substrate from Pierce (Rockford, IL).
Total RNA was extracted as described previously.51 Amplification of transcripts was performed using the one-step RT-PCR system (Invitrogen) with 50 ng of total RNA as described in Refs.11 and52. The optimal number of the PCR cycles was determined for each primer set to ensure a linear range of amplification, typically 21–26 cycles. Primer sequences: human TPM1, GenBankAcc# NM_000366, forward: 5′-GCTGGTGTCACTGCAA AAGA-3′, reverse: 5′-CTGCAGCCATTAATGCTTTC-3′; human TPM2, GenBankAcc# NM_003289, forward: 50-AAGGAGGCCCAGGAGAAACT-3′, reverse: 5′ CTTCCTTCAGCTGCATC TCC-3′; human ACTB (β-actin), GenBankAcc# NM_001101, forward: 5′-GCTCGTCGTCGACAACGGCTC-3′, reverse: 5′-CAAA CATGATCTGGGTCATCTTCTC-3′; human MMP-9, Acc#NM_ 004994, forward: 5′-TTCATCTTCCAAGGCCAATC-3′, reverse 5′-CAGAAGCCCCACTTCTTGTC-3′; human MMP14, Acc# NM_004995, forward: 5′-CATTGGAGG AGACACCCACT-3′; reverse: 5′-TGGGGTTTTTGGGTTTATCA-3′. Human ITGB1, Acc#NM_002211, forward: 5′-TAAGATCAGGGGAGCCACAG-3′; reverse: 5′-TCACTTGTGCAAGGGTTCCT-3′. The product sizes for human TPM1, TPM2 and β-actin were 506, 258 and 353 bp, respectively.
MTS cell growth assay
Cell proliferation was evaluated using colorimetric MTS assay (Promega) that measures restoration of 3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) to formazan by metabolically active cells. The absorbance of the formazan at 490 nm was determined in tissue culture medium following 48 hr incubation with or without DOX according to manufacturer instructions using a microplate reader (BioRad). The results are expressed mean values ± standard deviation for 2 independent experiments.
Migration and matrigel invasion
Migration and invasion assays were performed using polycarbonate 8 μm-pore membranes with and without Matrigel coating (Costar, High Wycombe Bucks, UK) as described in Ref.22. For Tet-Off regulation of TM3 expression, 50,000 cells in 100 μl of serum-free DMEM were seeded in the upper chamber and incubated alone or with 2 μg/ml DOX. The bottom wells received 600 μl of DMEM supplemented with 1% FBS as a chemoattractant. After 3, 6 hr incubation at 37°C in 5% CO2, the non-migrated cells on the top surface of membrane were wiped off with a cotton swab. Cells that migrated onto the bottom surface of membrane were fixed and stained with Diff-quick stain (VWR Scientific). Membranes were mounted on microslides and migrating cells were counted from at least 5 fields at 200× magnification. Experiments were performed in duplicates and repeated at least 3 times.
Short interference RNA
RNA duplexes against tropomyosin (target sequence: AAGCAGCTGGAAGATGAGC) were designed using the siDESIGN program at the Dharmacon siDESIGN center. A second set of siRNA to tropomyosin (SMARTpool, mouse Tpm1, Accession #NM_024427) was obtained from Dharmacon (Lafayette, CO). A scramble control RNA duplex labeled with rhodamine was obtained from Qiagen (Chatsworth, CA). Cells were transfected with RNA duplexes using Oligofectamine reagents (Invitrogen) following the manufacturers protocol. The media was replenished after 6 hr and cells were grown for 1 day before proceeding with further experiments. For the cell migration assay, 5 × 104 cells were seeded in the upper chamber. The bottom wells received 600 μl of DMEM supplemented with TGF-β1 as a chemoattractant. After 16 hr incubation at 37°C in 5% CO2, the cells were process as described in the migration assay.
Wound closure assay
The assay was performed as described in Ref.22. Cells (1–2 × 105/well) were seeded in 12-well plates and preincubated for 24 hr in serum-free IMEM (Invitrogen) prior to wounding with plastic tip across the cell monolayer. The cells were left untreated or treated with 2 ng/ml TGF-β1 for 16 hr. The wound closure was estimated as the ratio of the remaining open area relative to the initial area. Experiments were repeated at least 2 times.
Cell culture supernatants were collected and mixed with 2× sample buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 0.005% Bromophenol Blue). Zymography was performed using 10% polyacrylamide gels with 0.2% gelatin, renaturing buffer (2.5% Triton X-100) and developing buffer (50 mM Tris-HCl, pH 7.4, 5 mM CaCl2, 1% Triton X-100, 0.2 M NaCl, 0.02% Brij 35). Gelatinase activities were visualized by staining zymograms with Coomassie Brilliant Blue G250 (0.25% Coomassie Brilliant Blue G250, acetic acid 30%, methanol 10%) and destained in acetic acid–methanol–H2O (1:3:6).
Extracellular matrix degradation
Oregon green® 488-labeled gelatin was purchased from Molecular Probes. Gelatin-coated coverslips were prepared essentially as described in Ref.53. Briefly, 0.2 mg/ml Oregon green® 488-labeled gelatin was coated onto glass coverslips and immediately crosslinked with ice-cold 0.5% glutaraldehyde (Sigma) in PBS for 15 min at 4°C. Coverslips were gently washed 3 times with PBS and incubated with 5 mg/ml sodium borohydride (Sigma) in PBS for 3 min. After washing in PBS, the coverslips were sterilized in 70% ethanol for 15 min, washed in PBS and then incubated in serum free medium for 1 hr at 37°C. Cells were grown on the coverslips for various time periods and processed to Fluorescence Microscopy procedures.
Fibronectin and type I collagen were purchased from Calbiochem (La Jolla, CA). The 96-well plates were coated by incubating for 1 hr at RT with 100 μl fibronectin or collagen (10 μg/ml in PBS). Wells were blocked with 10 mg/ml BSA and washed with PBS. Cells were seeded to the wells at 2 × 104/well in warm serum-free IMEM in triplicates. After 1 hr, nonadherent and loosely attached cells were removed from wells by gently washing with DPBS (Invitrogen), attached cells were fixed by adding 100 μl of 5% gluaraldehyde, and then washed with DPBS. Cells were counted from at least 3 fields at 200× magnification. The results were expressed mean value ± standard deviation from 2 independent experiments.
Lung metastasis after tail-vein injection
The experiments were done as described in Ref.54. Female SCID/CB17 mice, 8-weeks of age, were obtained from a colony of SCID/CB17 mice that were bred and maintained at the Department of Laboratory Animal Resources (DLAR) facility at the Roswell Park Cancer Institute (RPCI). All animals were kept 3 to 5 mice per cage in microisolator units and provided with water and food adlibitum according to a protocol approved by the Institute Animal Care and Use Committee at RPCI. The facility has been certified by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulation and standards of the U.S. Department of Agriculture and the U.S. Department of Health and Human Services. Exponentially growing breast cancer cells (2.5 × 106) in 0.2 ml of sterile Hank's solution were injected using 28-G needle into a tail vein of 8-weeks old female SCID/CB17 mice. Mice were sacrificed 4 weeks thereafter. Lungs were stained with 15% black Indian ink and fixed for 24 hr in Fekete's solution (20:2:1 of 70% ethanol, 35% formaldehyde and acetic acid). Tumor colonies on lung surfaces were counted and statistical analysis was performed using Student's t-test.
TGF-β1 induces EMT in normal epithelial cells but reduces cell motility
TGF-β1 can induce EMT in normal and tumor epithelial cells6, 17, 25, 55 and stimulate migration of human and mouse carcinoma cells.55, 56, 57, 58, 59 Here, we examined EMT and regulation of cell motility by TGF-β1 in nontumorigenic mouse mammary NMuMG cell line. Treatment of NMuMG cells with TGF-β1 for 24 hr resulted in loss of epithelial cell polarity and reorganization of the actin cytoskeleton (Fig. 1a). Immuno-fluorescence microscopy showed loss of ZO-1 staining in TGF-β-treated cells, indicating disruption of tight junctions (Fig. 1a). Staining of actin (phalloidin) and focal adhesions (vinculin) showed the epithelial organization of actin filaments at the adhesion belts in untreated cells and formation of stress fibers attached to vinculin-containing focal contacts upon TGF-β1 treatment (Fig. 1a). Tropomyosin is essential for TGF-β-mediated induction of stress fibers in epithelial cells.11 Immunoblot analysis showed upregulation of HMW-tropomyosins in TGF-β-treated cells, whereas vinculin was not affected (Fig. 1b). Together these results showed induction of EMT by TGF-β1 in NMuMG cells. We next examined the effect of TGF-β1 on cell motility using a transwell-migration assay. Migration of cells to the bottom chamber containing 5% FBS was reduced when TGF-β1 was added to a lower chamber (Fig. 1c) or to both chambers (data not shown). The effect of TGF-β1 on cell migration was also examined in human lung adenocarcinoma A549 cells, that respond to TGF-β1 with EMT-like transition25, 60 and formation of extensive stress fibers.18 TGF-β1 induced expression of tropomyosin (Fig. 1d) and reduced by nearly 50% transwell migration of A549 cells (Fig. 1e). To determine whether pretreatment with TGF-β1 affects cell motility, NMuMG cells were treated with TGF-β1 for 24 hr and then reseeded onto fresh plastic dishes or used in transwell assays. The levels of tropomyosin were increased by pretreatment with TGF-β1 and reduced upon withdrawal of TGF-β1 for 24 hr (Fig. 2a). Addition of TGF-β1 to lower chamber reduced transwell motility of cells that were untreated or pretreated with TGF-β1 (Fig. 2b). Cell migration towards 5% FBS was also reduced in the presence of TGF-β1 (data not shown). To determine whether downregulation of HMW-tropomyosins modulate cell migration, NMuMG cells were transfected with control-scrambled siRNA or siRNA against tropomyosin isoforms TM1, TM2/3 and TM6 (si-TPM). Immunoblotting showed a nearly 80% reduction of HMW-tropomyosins by si-TPM (Fig. 2c). Transwell migration assays showed an alleviation of the TGF-β1 inhibitory effect and a 3-fold increase in motility of cells transfected with si-TPM (Fig. 2d). Thus, these results indicate that TGF-β1 can inhibit cell motility by inducing actin stress fibers mediated by HMW-tropomyosins and anchored to focal adhesions.
TGF-β1 stimulates motility of metastatic breast cancer cells that express low levels of HMW-tropomyosins and stress fibers
To investigate the link between HMW-tropomyosins and the metastatic potential of cancer cells, protein levels of HMW-tropomyosins were analyzed in human breast cancer cell lines using the TM311 monoclonal antibody that recognizes the N-terminal epitope in tropomyosin isoforms 1 (TM1) encoded by TPM2, and TM2, 3 and 6 encoded by TPM1.32 The tropomyosin levels were normalized to GAPDH. Immunoblot analysis showed that protein levels of HMW-tropomyosins encoded by TPM1 and TPM2 genes were significantly lower in cancer cell lines compared to normal MCF10A (Figs. 3a, and 3b). Metastatic cell lines MDA-MB-231 and MDA-MB-435 expressed extremely low tropomyosin levels, in agreement with our previous findings that in these cells the TPM1 gene is silenced by promoter hypermethylation.18 Microscopic analysis of metastatic cell line MDA-MB-231 showed fibroblast-like morphology with disorganized actin filaments (Fig. 3c) compared to normal epithelial cells (Fig. 1). Treatment with TGF-β1 did not induce significant morphological changes and did not increase stress fibers (Fig. 3c). Similar results were found for MDA-MB-435 cells (data not shown). The effect of TGF-β1 on cell motility was examined in MDA-MB-231 cells, which are competent in the TGF-β signal transduction47 and TGF-β1 contributes to the metastatic potential of these cells.61 In contrast to NMuMG, migration of MDA-MB-231 cells was enhanced by TGF-β1 (Fig. 3d). Together, these results demonstrate that the metastatic potential of cells and the ability of TGF-β1 to stimulate cell migration inversely correlate with the level of HMW-tropomyosins.
Effects of TM3 on actin cytoskeleton and cell migration
To investigate the role of HMW-tropomyosins in regulation of the metastatic properties of tumor cells, we generated MDA-MB-231 cells expressing TM3 encoded by the TPM1 gene under control of the Tet-Off system. Immunoblot analysis showed that removal of DOX upregulates TM3 to the levels that are roughly 2-fold lower than in MCF7 (Fig. 4a). The MTS assay showed no significant effect of TM3 on cell growth (data not shown). TGF-β1-mediated phosphorylation of Smad2/3 and the luciferase- reporter activity in the Tet-Off-TM3 cells were similar to parental cells (data not shown). Phase-contrast microscopy showed that in the absence of DOX, when TM3 is expressed, MDA-MB-231 cells exhibit less refractive, larger and flatter cell morphology compared to cells incubated in the presence of DOX (Fig. 4b). Phalloidin staining of actin filaments revealed the presence of actin stress fibers in cells expressing TM3 (no DOX) and actin ruffles in cells incubated with DOX, when TM3 is not expressed (Fig. 4b). Thus, a moderate expression of TM3 in MDA-MB-231 cells is sufficient to increase actin stress fibers and change cell morphology without inhibition of cell growth. The motility of MDA-MB-231-Tet-Off-TM3 cells was investigated in transwell assays. Expression of TM3 reduced chemotactic migration of cells towards FBS after 3 and 6 hr of incubation (Fig. 4c). The effect of TM3 on TGF-β1-mediated regulation of cell migration was assessed in wound closure assay. In the absence of DOX, when TM3 is expressed, the closure of wounds was delayed independent of TGF-β1 (Fig. 4d). Reduction of TM3 expression by DOX enhanced wound closure in the absence and presence of TGF-β1 (Fig. 4d). Thus, expression of TM3 upregulates stress fibers and reduces motility of MDA-MB-231 cells.
TM3 reduces cell invasion but does not affect secretion of MMP-9
The effect of TM3 on tumor cell invasion was measured using matrigel-coated transwells. The Tet-Off-TM3 cells were incubated without or in the presence of 0.03 and 2 μg/ml DOX. Expression of TM3 (no DOX) decreased tumor cell invasion of Matrigel by nearly 5-fold (Fig. 5a). The reduction of invasion (80%) by TM3 was more pronounced than inhibition of cell motility (35%). This effect of TM3 was similar to a 4-fold reduction of Matrigel invasion in the presence of GM6001, a general inhibitor of matrix metalloproteinases (MMPs) (Fig. 5b). To determine whether TM3 affects expression of MMPs, we performed RT-PCR analysis of MMP-9/gelatinase-B and membrane-associated MMP-14 (MT1-MMP), major MMPs expressed by MDA-MB-231 cells.48 The analysis showed no effect of TM3 on these MMPs (Fig. 5c). Furthermore, gelatin zymography revealed no significant effect of TM3 on secreted proMMP-9 (Fig. 5d). Collectively, these findings indicate that TM3 reduces invasiveness of tumor cells without suppression of MMP expression.
Tropomyosin-mediated actin fibers reduce matrix proteolysis at the cell-matrix contacts
To further investigate the mechanisms by which TM3 inhibits tumor cell invasiveness, we assessed the effect of TM3 on matrix proteolysis at cell-matrix contacts that is critical for cell invasion.62 The ability of live cells to degrade extracellular matrix (ECM) at the focal contacts was evaluated using an assay that visualizes activity of invadopodia and podosomes, the cellular structures that promote cell invasion.63, 64 Cells were cultured on glass cover-slips coated with gelatin conjugated to fluorescent dye Oregon Green® 488. Matrix proteolysis was detected under a fluorescence microscope as black nonfluorescent spots. Cell nuclei and actin filaments were visualized by staining with DAPI and phalloidin Texas Red, respectively. Parental MDA-MB-231 cells showed black spots or “footprints” at the focal contacts (Fig. 6a), which is consistent with previous studies.54, 64 Incubation of cells with MMP inhibitor GM6001 significantly reduced a number of “footprints” (Fig. 6a), confirming importance of MMPs for focal matrix proteolysis. In the presence of DOX, MDA-MB-231-TetOff-TM3 cells exhibited “footprints” comparable to parental cells (Fig. 6b, plus DOX), whereas cells expressing TM3 (no DOX) showed a significant reduction in “footprints” (Fig. 6b). To determine whether stress fibers are involved in control of ECM degradation, the TM3-expressing cells were treated with cytochalasin D that disrupts actin filaments. Phalloidin staining showed that treatment with 250 nM cytochalasin D reduced actin stress fibers and increased actin at the cell periphery (Fig. 6c). Similar changes in the actin cytoskeleton were observed when TM3 was modulated by DOX (Fig. 4b). Importantly, cytochalasin D increased focal matrix proteolysis (Fig. 6c). Gelatin zymography studies showed no effect of 250–500 nM cytochalasin D on secretion of MMP-9 and MMP2 (data not shown). Our recent studies have shown that autocrine TGF-β1 signaling enhances focal matrix proteolysis inMDA-MB-231 cells.54 Here, we compared regulation of ECM-degrading activity by TGF-β1 in MDA-MB-231 parental cells, tet-activator-expressing cells (tTA) and Tet-Off-cells expressing TM3 (no DOX, TM3). TGF-β1 stimulated matrix degradation in parental and tTA cells, but not in TM3-expressing cells (Fig. 6d). Together these findings indicate that HMW-tropomyosin reduces matrix proteolysis at focal contacts and the ability of TGF-β1 to stimulate this matrix-degrading activity via a mechanism involving actin stress fibers.
TM3 increases cell adhesion to ECM
The inhibitory effect of TM3 on focal matrix proteolysis raised a question whether TM3 regulates focal adhesions. Immunofluorescence microscopy was used to analyze localization of vinculin, a marker of focal adhesions.65 In the absence of DOX, when TM3 is expressed, vinculin was localized along actin fibers and at the tip of actin filaments, indicating formation of focal adhesions (Fig. 7a). In cells treated with DOX, vinculin staining was more diffused (Fig. 7a). Immunoblot analysis showed no regulation of vinculin expression (Fig. 7b). These results suggested that TM3 promotes localization of vinculin to actin fibers and focal adhesions. The formation of focal adhesions is associated with clustering of integrins and activation of integrin signaling that increases phosphorylation of tyrosine residues of the components of the focal complexes.65 Immunofluorescence microscopy with the 4G10 antibody recognizing phospho-Tyr showed significant accumulation of phospho-Tyr staining at the focal sites in TM3-expressing cells (Figs. 7c and 7d). These findings suggested that TM3 may increase cell adhesion to matrix by enhancing focal contacts and integrin signaling.
To determine whether TM3 affects cell-matrix adhesion, we measured adhesiveness of MDA-MB-231-Tet-Off-TM3 cells onto plates coated with fibronectin or type I collagen. In the absence of DOX, when TM3 is expressed, cells adhered to type I collagen roughly 2-fold more effectively than the cells in which TM3 was suppressed by DOX (Fig. 8a). Similar results were observed for fibronectin. To address the mechanism by which TM3 enhances cell adhesion, we determined expression of β1 integrin, a receptor for fibronectin and collagen. Immunoblot analysis showed higher levels of the matured form of β1 integrin in the TM3-expressing cells compared to cells incubated with DOX (Fig. 8b). RT-PCR analysis did not reveal regulation of β1 integrin at the level of mRNA (Fig. 8c), suggesting a posttranslational regulation of β1 integrin. We then asked whether TGF-β1 affects cell-matrix adhesion and β1 integrin levels in NMuMG cells, where it also upregulates HMW-tropomyosins (Fig. 1). Pretreatment with TGF-β1 for 24 hr increased by nearly 3-fold adhesiveness of cells to fibronectin (Fig. 8d). Immunoblot analysis showed that the levels of both matured and precursor forms of β1 integrin were increased by TGF-β1 in NMuMG cells (Fig. 8e). We then examined whether downregulation of tropomyosin affects TGF-β1-mediated regulation of β1 integrin. Silencing of HMW-tropomyosins by siRNA reduced levels of the matured form of β1 integrin in untreated cells but did not affect induction of β1 integrin by TGF-β1 (Fig. 8f). These findings were confirmed with a second set of siRNA to HMW-tropomyosins (Fig. 8f). Together our studies indicate that both TGF-β1 and HMW-tropomyosin enhance cell adhesion to matrix via a mechanism involving β1 integrin and TGF-β1 upregulates β1 integrin levels independently of tropomyosin.
TM3 suppresses formation of lung metastasis
The effect of TM3 on the metastatic potential of MDA-MB-231 cells was investigated using the experimental metastasis model, when tumor cells are placed directly into the blood stream of the animal. This model overcomes the requirement for basement membrane invasion, an obvious defect in the TM3-expressing cells given the results of in vitro studies. Control MDA-MB-231-tTA-4 and MDA-MB-231-Tet-Off-TM3 cells expressing TM3 were injected into the tail vein of female SCID mice. After 4 weeks, animals were sacrificed and the lungs were stained with Indian ink to visualize the tumor colonies. The analysis showed that TM3 reduced by nearly 70% the number of tumor colonies on lung surfaces (p < 0.05, Student's t-test) (Fig. 9a). This result indicates that HMW-tropomyosins are potent suppressors of metastasis.
The present study shows that TGF-β1 can negatively regulate cell motility by upregulating HMW-tropomyosins, which enhance actin stress fibers and cell-matrix adhesion but reduce matrix proteolysis. The following findings support this conclusion. First, in normal epithelial cells, TGF-β1 reduced cell motility while promoting EMT and formation of extensive actin stress fibers linked to focal adhesions. Second, TGF-β1 upregulated HMW-tropomyosins that are required for TGF-β-mediated induction of actin stress fibers.11 Suppression of HMW-tropomyosins by siRNA enhanced cell motility. TGF-β1 stimulated migration of breast cancer MDA-MB-231 cells that express low levels of HMW-TMs and actin stress fibers. Third, expression of HMW-tropomyosin in MDA-MB-231 cells under control of the Tet-Off system reduced invasiveness of tumor cells. Tropomyosin increased stress fibers and cell adhesion to matrix but reduced matrix proteolysis at cell-matrix contacts. Conversely, disruption of actin fibers by cytochalasin D enhanced matrix proteolysis in cells expressing tropomyosin. Finally, HMW-tropomyosin impaired the ability of tumor cells to form lung metastases.
Our studies indicate that TGF-β1 can reduce migration of normal epithelial cells that also undergo mesenchymal transition (EMT). Similar findings have been recently reported for normal kidney epithelial cells and NBT-II hepatocytes in which TGF-β1 induces EMT but inhibits motility.13, 14, 15 We found that the reduction of cell motility is associated with upregulation of HMW-tropomyosins, tropomyosin-mediated actin stress fibers and cell-matrix adhesions. Suppression of HMW-tropomyosins by siRNA increased cell motility (Fig. 2). In agreement with this observation, TGF-β1 stimulated migration of breast cancer cells expressing low levels of HMW-tropomyosins and do not form stress fibers in response to TGF-β1 (Fig. 3). Conversely, Tet-Off-regulated expression of HMW-tropomyosin in these cells enhanced stress fibers and cell-matrix adhesion (Figs. 7 and 8) but reduced motility and significantly inhibited invasion (Figs. 4 and 5). Further, we made important new observations that HMW-tropomyosin upregulates β1 integrin and integrin signaling (Figs. 7 and 8). Consistent with these results, a forced expression of tropomyosin enhances focal contacts in Ras-transformed fibroblasts.43 The regulation of focal adhesions by HMW-tropomyosins is likely mediated via stabilization of stress fibers as HMW-tropomyosins are not found at the focal contacts in epithelial and mesenchymal cells.32, 66, 67 Tropomyosin may also stabilize focal adhesions by regulating actomyosin contractility,26 which contributes to the formation of focal adhesions.68
Our study indicates an important new role of HMW-tropomyosin in regulation of TGF-β1-mediated matrix proteolysis at focal cell-matrix contacts (Fig. 6d). We have recently reported that TGF-β1 increases focal matrix proteolysis and invasiveness in MDA-MB-231 cells via a mechanism involving MMP-9.54 Here, we found that HMW-tropomyosin inhibits matrix proteolysis without reducing expression of MMPs (Fig. 6). The mechanism of this effect involves actin filaments as disruption of actin fibers by cytochalasin D increased matrix proteolysis. The matrix proteolytic activity at focal contacts is required for cell migration in 3D matrices (invasion).62, 69 This activity is localized at the dynamic cell-matrix structures termed podosomes and invadopodia.70 Recent study showed that in MDA-MB-231 cells the knockdown of Tks5/Fish protein, which is required for the formation of podosomes and invadopodia, results in inhibition of focal proteolysis and invasion but not secretion of MMP-9.64 This result is strikingly similar to our findings as tropomyosin did not affect expression of MMP-9 and MMP-14, major MMPs in MDA-MB-231 cells,48 suggesting that HMW-tropomyosins may affect the assembly or function of podosomes and invadopodia.
The factors that regulate actin-cytoskeleton assembly, stability and turnover such as Src, Arp2/3 and Dia1 also regulate podosomes and invadopodia.67 For example, tropomyosin may interfere with WASP-mediated nucleation activity of Arp2/3,71 which is required for podosome assembly.70 Another possibility is that HMW-tropomyosins, by stabilizing actin fibers and focal adhesion complexes, promote formation of focal adhesions, more stable cell-matrix contacts with reduced matrix-degrading activity compared to podosomes and invadopodia.72, 73 Interestingly, TGF-β1 and tropomyosin increased cell adhesion to matrix as well as upregulated β1 integrin and focal adhesions in both tumor and normal cells (Figs. 1 and 9). Integrins and vinculin are implicated in the assembly of focal contacts65 and in the location of MMPs to the sites of matrix degradation.62 Focal adhesions are complex structures composed of more than 50 proteins.72, 74 Currently, little is known regarding the regulation of these structures by TGF-β and tropomyosins. Our studies indicate that more rigorous research is needed to delineate regulation of matrix contacts by TGF-β and tropomyosins in normal and tumor cells.
The studies with the tail-vein injection model showed that overexpression of HMW-tropomyosin in MDA-MB-231 cells impaired formation of lung colonies in SCID mice (Fig. 9a). The decrease of metastases is likely associated with tropomyosin-mediated inhibition of cell motility and invasion as HMW-tropomyosin did not affect cell growth. In addition, HMW-tropomyosin may also affect cell survival in the absence of cell-matrix contacts (anoikis).75 Consistent with our findings, loss of HMW-tropomyosins has been noted in high-grade tumors of breast, prostate, bladder and brain.38, 39, 40, 41, 42 Together these results indicate that HMW-tropomyosins play an important role in suppression of metastasis and cancer progression.
In conclusion, our findings indicate that TGF-β1 can reduce migration of normal epithelial cells that undergo EMT, suggesting that TGF-β-mediated EMT is not sufficient for acquisition of the metastatic phenotype. Consistent with this finding, a recent survey of epithelial cell lines indicates that only nontumorigenic cells undergo EMT in response to TGF-β1.25 Our study shows that TGF-β1-mediated EMT and inhibition of cell motility are associated with upregulation of HMW-tropomyosins that enhance actin stress fibers and cell-matrix adhesion but strongly reduce cell motility and invasion. Tumor suppressor Smad4 is required for induction of tropomyosins and formation of stress fibers11 as well as for TGF-β-mediated EMT.11, 17 Conversely, transformation of cells by oncogenic Ras or Src leads to downregulation of HMW-tropomyosins and disruption of actin filaments.76, 77 In addition, epigenetic mechanisms may also suppress expression of HMW-tropomyosins in metastatic cells.18 In consolidation of these findings we suggest the following 2-step model (Fig. 9b). In cancer progression, high levels of TGF-β1 may induce EMT with tropomyosin-stabilized stress fibers and low cell motility. Subsequent epigenetic mechanisms or oncogenic pathways downregulate tropomyosins leading to the acquisition of the metastastic phenotype with high cell motility and invasion.
According to this model, TGF-β1 may induce EMT in normal and tumor cells and this response is not restricted to any particular time of tumor development. The acquisition of the metastatic phenotype occurs later in cancer progression and requires additional step(s) such as downregulation of HMW-tropomyosins. The model suggests that TGF-β1 induces a reversible EMT in tumor cells at early stages of tumor development or in normal cells during, for example, wound healing or embryonic development. Withdrawal of TGF-β1 restores epithelial phenotype and reduces expression of HMW-tropomyosins (Fig. 2a). In epithelial cells HMW-tropomyosins contribute to normal morphology and function of epithelium.26 In cancer progression, however, loss of HMW-tropomyosins may result in irreversible EMT with disrupted epithelial cell architecture and highly dynamic actin filaments. This model helps to explain why carcinoma cells of primary tumors and lymph node metastasis frequently exhibit epithelial morphology whereas carcinoma cells from distal metastasis exhibit mesenchymal morphology with reduced stress fibers and adhesion.
We thank Dr. Sei-Ichi Matsui for the SKY analysis of the Tet-Off cells; Dr. David Helfman for providing reagents; Dr. Heinz Baumann for helpful discussion of the manuscript; Mrs. Erika VanDette, Dr. Dimiter Kunnev and Dr. Andrea Varga for technical assistance.