Modulation of NFκB Activity and E-cadherin by the Type III Transforming Growth Factor β Receptor Regulates Cell Growth and Motility
2007; Elsevier BV; Volume: 282; Issue: 44 Linguagem: Inglês
10.1074/jbc.m704434200
ISSN1083-351X
AutoresTracy Criswell, Carlos L. Arteaga,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoTransforming growth factor β is growth-inhibitory in non-transformed epithelial cells but becomes growth-promoting during tumorigenesis. The role of the type I and II receptors in tumorigenesis has been extensively studied, but the role of the ubiquitously expressed type III receptor (TβRIII) remains elusive. We developed short hairpin RNAs directed against TβRIII to investigate the role of this receptor in breast cancer tumorigenesis. Nontumorigenic NMuMG mouse cells stably expressing short hairpin RNA specific to mouse TβRIII (NM-kd) demonstrated increased cell growth, motility, and invasion as compared with control cells expressing shRNA to human TβRIII (NM-con). Reconstitution of TβRIII expression with rat TβRIII abrogated the increased growth and motility seen in the NM-kd cells. In addition, the NM-kd cells exhibited marked reduction in the expression of the adherens junction protein, E-cadherin. This loss of E-cadherin was due to increased NFκB activity that, in turn, resulted in increased expression of the transcriptional repressors of E-cadherin such as Snail, Slug, Twist, and Sip1. Finally, NMuMG cells in which TβRIII had been knocked down formed invasive tumors in athymic nude mice, whereas the control cells did not. These data indicate that TβRIII acts as a tumor suppressor in nontumorigenic mammary epithelial cells at least in part by inhibiting NFκB-mediated repression of E-cadherin. Transforming growth factor β is growth-inhibitory in non-transformed epithelial cells but becomes growth-promoting during tumorigenesis. The role of the type I and II receptors in tumorigenesis has been extensively studied, but the role of the ubiquitously expressed type III receptor (TβRIII) remains elusive. We developed short hairpin RNAs directed against TβRIII to investigate the role of this receptor in breast cancer tumorigenesis. Nontumorigenic NMuMG mouse cells stably expressing short hairpin RNA specific to mouse TβRIII (NM-kd) demonstrated increased cell growth, motility, and invasion as compared with control cells expressing shRNA to human TβRIII (NM-con). Reconstitution of TβRIII expression with rat TβRIII abrogated the increased growth and motility seen in the NM-kd cells. In addition, the NM-kd cells exhibited marked reduction in the expression of the adherens junction protein, E-cadherin. This loss of E-cadherin was due to increased NFκB activity that, in turn, resulted in increased expression of the transcriptional repressors of E-cadherin such as Snail, Slug, Twist, and Sip1. Finally, NMuMG cells in which TβRIII had been knocked down formed invasive tumors in athymic nude mice, whereas the control cells did not. These data indicate that TβRIII acts as a tumor suppressor in nontumorigenic mammary epithelial cells at least in part by inhibiting NFκB-mediated repression of E-cadherin. The transforming growth factor (TGF) 3The abbreviations used are: TGF, transforming growth factor; TβRIII, TGFβ type III receptor; shRNA, small hairpin RNA; EMT, epithelial to mesenchymal transition; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CA, constitutively active; GFP, green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; dn, dominant negative; RLU, relative luciferase unit(s).3The abbreviations used are: TGF, transforming growth factor; TβRIII, TGFβ type III receptor; shRNA, small hairpin RNA; EMT, epithelial to mesenchymal transition; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CA, constitutively active; GFP, green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; dn, dominant negative; RLU, relative luciferase unit(s). βs belong to a family of pleitropic cytokines that are involved in cell growth and proliferation, differentiation, deposition of extracellular matrix, and motility (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3946) Google Scholar). The three TGFβ isoforms (β1, β2, and β3) bind to transmembrane receptor serine/threonine kinases. TGFβ1 and TGFβ3 can bind with high affinity to the TGFβ type II receptor, resulting in activation of Smad 2/3 and downstream target genes. In contrast, TGFβ2 binds with low affinity to the type II receptor. The type III receptor (TβRIII), or betaglycan, binds with high affinity to all three TGFβ isoforms and is required for presenting TGFβ2 to the type II receptor (2Lopez-Casillas F. Kim J.L. Massague J. Cell. 1993; 73: 1435-1444Abstract Full Text PDF PubMed Scopus (764) Google Scholar). TβRIII has been shown to play an essential role in the formation of the atrioventricular cushion in the development of the heart (3Brown C.B. Kim A.S. Runyan R.B. Barnett J.V. Science. 1999; 283: 2080-2082Crossref PubMed Scopus (325) Google Scholar). Consistent with this observation, the TβRIII null mouse is embryonically lethal because of heart and liver defects (4Stenvers K.L. Kim M.L. Harder K.W. Kountouri N. Amatayakul-Chantler S. Grail D. Small C. Weinberg R.A. Sizeland A.M. Zhu H.J. Mol. Cell. Biol. 2003; 23: 4371-4385Crossref PubMed Scopus (198) Google Scholar). The role for TβRIII in cancer is less clear. Increased expression of TGFβ1 and all three TGFβ receptors was found in higher grade lymphomas (5Woszczyk D. Kim J. Jurzak M. Mazurek U. Mykala-Ciesla J. Wilczok T. Med. Sci. Monit. 2004; 10: CR33-CR37PubMed Google Scholar). Conversely, reduced expression of TβRIII was found associated with advanced stage neuroblastomas and ovarian carcinomas (6Bristow R.E. Kim R.L. Yamada S.D. Korc M. Karlan B.Y. Cancer. 1999; 85: 658-668Crossref PubMed Scopus (60) Google Scholar, 7Iolascon A. Kim L. Borriello A. Carbone R. Izzo A. Tonini G.P. Gambini C. Della Ragione F. Br. J. Cancer. 2000; 82: 1171-1176Crossref PubMed Scopus (26) Google Scholar). Similarly, a recent report using cDNA microarrays demonstrated that low TβRIII expression correlates with higher grade among a cohort of breast cancers (8Dong M. Kim T. Kirkbride K.C. Gordon K.J. Lee J.D. Hempel N. Kelly P. Moeller B.J. Marks J.R. Blobe G.C. J. Clin. Investig. 2007; 117: 206-217Crossref PubMed Scopus (191) Google Scholar). Additionally, overexpression of TβRIII in MDA-231 human breast cancer cells and DU145 prostate cancer cells resulted in decreased tumor invasion in vitro and in vivo (9Turley R.S. Kim E.C. Hempel N. How T. Fields T.A. Blobe G.C. Cancer Res. 2007; 67: 1090-1098Crossref PubMed Scopus (143) Google Scholar, 10Sun L. Kim C. J. Biol. Chem. 1997; 272: 25367-25372Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The reasons for this apparent contradictory role for TβRIII in these different tumor types have not been elucidated. Epithelial to mesenchymal transition (EMT) is a process by which TGFβ can promote tumorigenesis. EMT is characterized by a decrease in epithelial cell markers, such as E-cadherin and ZO-1, and an increase in mesenchymal markers including N-cadherin, vimentin, and fibronectin. This is associated with a decrease in cell-cell adhesion and changes in the actin cytoskeleton. Loss of E-cadherin expression, either through genetic or epigenetic alterations, is the hallmark of EMT in epithelial cells. Several proteins (i.e. Snail, Slug, Twist, and Sip1) have been identified as transcriptional repressors of E-cadherin (11Vandewalle C. Kim J. De Craene B. Vermassen P. Bruyneel E. Andersen H. Tulchinsky E. Van Roy F. Berx G. Nucleic Acids Res. 2005; 33: 6566-6578Crossref PubMed Scopus (419) Google Scholar, 12De Craene B. Kim F. Berx G. Cell. Signal. 2005; 17: 535-547Crossref PubMed Scopus (179) Google Scholar, 13Yang J. Kim S.A. Donaher J.L. Ramaswamy S. Itzykson R.A. Come C. Savagner P. Gitelman I. Richardson A. Weinberg R.A. Cell. 2004; 117: 927-939Abstract Full Text Full Text PDF PubMed Scopus (3009) Google Scholar). NFκB is a family of hetero- or homodimeric transcription factors involved in cell survival and regulation of the immune response (14Wu J.T. Kim J.G. J. Surg. Res. 2005; 123: 158-169Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). The NFκB signaling pathway appears to be a critical mediator of EMT (15Bachelder R.E. Kim S.O. Franci C. de Herreros A.G. Mercurio A.M. J. Cell Biol. 2005; 168: 29-33Crossref PubMed Scopus (334) Google Scholar, 16Vasko V. Kim A.V. Scouten W. He H. Auer H. Liyanarachchi S. Larin A. Savchenko V. Francis G.L. de la Chapelle A. Saji M. Ringel M.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 2803-2808Crossref PubMed Scopus (255) Google Scholar). Additionally, it has been reported that NFκB is required for EMT during breast cancer progression (17Huber M.A. Kim N. Baumann B. Grunert S. Sommer A. Pehamberger H. Kraut N. Beug H. Wirth T. J. Clin. Investig. 2004; 114: 569-581Crossref PubMed Scopus (806) Google Scholar). NFκB also appears to be a mediator of Snail expression (15Bachelder R.E. Kim S.O. Franci C. de Herreros A.G. Mercurio A.M. J. Cell Biol. 2005; 168: 29-33Crossref PubMed Scopus (334) Google Scholar, 18Kim H.J. Kim B.C. Cui X. Delgado D.A. Grabiner B.C. Lin X. Lewis M.T. Gottardis M.M. Wong T.W. Attar R.M. Carboni J.M. Lee A.V. Mol. Cell. Biol. 2007; 27: 3165-3175Crossref PubMed Scopus (190) Google Scholar). A recent report demonstrated that expression of E-cadherin can down-regulate NFκB activity in melanoma cells, suggesting a direct link between these two pathways (19Kuphal S. Kim A.K. Oncogene. 2006; 25: 248-259Crossref PubMed Scopus (145) Google Scholar). The reported conflicting roles of TβRIII in tumorigenesis lead us to investigate its role in mammary cell transformation. For this purpose, we used a loss of function approach with short hairpin RNAs (shRNAs) specific to TβRIII in nontumorigenic NMuMG mammary epithelial cells. In this study we show that knock-down of TβRIII expression in NMuMG cells results in increased growth, motility, invasion, and tumor formation in vivo using a xenograft mouse model. In addition, we demonstrate that these changes are due to an increase in NFκB signaling that, in turn, results in transcriptional repression of E-cadherin. These results were not limited to NMuMG cells because we also observed a similar phenotype in EMT6 mouse mammary tumor cells in which TβRIII was knocked down by stable RNA interference. Cell Culture, Viral Infections, and shRNA—All of the cells were purchased from American Type Culture Collection (Manassas, VA). NMuMG cells were grown in Dulbecco's modified Eagle's medium (DMEM; Cambrex) supplemented with 10% fetal bovine serum (FBS) and 10 μg/ml insulin. EMT-6, Phoenix-Ampho, and 293A cells were grown in DMEM containing 10% FBS in a humidified 5% CO2 incubator at 37 °C. To generate retroviruses expressing short hairpin RNAs specific to mouse TβRIII, the complimentary oligonucleotides 5′-GAAAUGACAUCCCUUCCAC and 5′-GUGGAAGGGAUGUCAUUUG were annealed and ligated into the BglII/HindIII site of the pSuper vector. Retrovirus expressing shRNA specific to human TβRIII (5′-GAGAUGACAUUCCUUCAAC and 5′-GUUGAAGGAAUGUCAUCUC) was used as a control. The resulting pSuper plasmids were transfected into Phoenix-Ampho cells using Superfect transfection reagent (Qiagen) per the manufacturer's directions. Supernatant containing viral particles was collected 72 h after transfection and filtered through a 20-μm syringe filter. Retrovirus containing mouse or human short hairpin RNA specific to the type III receptor were used to infect NMuMG cells in the presence of 4 μg/ml polybrene (Sigma). Stably expressing cells were selected with 1 μg/ml puromycin. The pGEM-4Z rat TβRIII plasmid was a gift from Dr. Joan Massague (Memorial Sloan Kettering Cancer Center, New York, NY). Rat TβRIII was digested out of pGEM-4Z using EcoRI and cloned into the EcoRI site of the LZRS-MS-neo retroviral plasmid. NM-kd cells were infected with retrovirus containing rat TβRIII and selected using 600 μg/ml G418 (Invitrogen). Adenovirus containing constitutively active IKK2 (CA-IKK2) or dominant negative IκBα (dn IκBα) were provided by Dr. Timothy Blackwell (Vanderbilt University, Nashville, TN). Adenovirus containing GFP was used as a vector control. 293A cells were infected with adenovirus to produce a concentrated stock of virions. For adenoviral infection, NMuMG cells were plated to ∼70% confluency in 100-mm dishes. Adenovirus was added to the cells in 3 ml of serum-free medium for 3 h, at which point 7 ml of medium containing 10% FBS was added. The cells were allowed to grow for 48 h before being subjected to further treatment. Antibodies and Reagents—TGFβ1 and TGFβ2 were purchased from R & D Systems (Minneapolis, MN). Growth factor-reduced Matrigel was purchased from Clontech (Mountainview, CA). Antibodies to E-cadherin and Smad 2/3 were from Transduction Laboratories (Lexington, KY); p-Smad 2 was from Cell Signaling (Danvers, MA); actin was from Sigma. Phalloidin-fluorescein isothiocyanate (actin) and goat anti mouse Oregon Green 488 antibodies were purchased from Molecular Probes (Eugene, OR). Horseradish peroxidase-conjugated secondary antibodies used for immunoblots were from Promega (Madison, WI). 125I-TGFβ1 Affinity Cross-linking Assays—125I-TGFβ1 affinity cross-linking assays were performed as described (20Dumont N. Kim A.V. Arteaga C.L. J. Biol. Chem. 2003; 278: 3275-3285Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Cross-linked cell lysates were separated on a 3-12% gradient SDS-PAGE and visualized by autoradiography. RNA Isolation and Quantitative PCR—Total RNA was extracted using the RNeasy Mini-kit (Qiagen) per the manufacturer's directions. RNA (5 μg) was reverse transcribed in a 100-μl reaction. Quantitative PCR was carried out on 500 ng of cDNA using the iQ SYBR Green Supermix from Bio-Rad in a Bio-Rad iCycler iQ multicolor real time PCR detection system. Primers were designed using the Universal Probe Library from Roche Applied Science. Primer sequences are listed in Table 1. A standard curve was generated by amplifying known concentration of cDNA using actin primers. All of the reactions were performed in triplicate.TABLE 1Quantitative PCR primer sequencesNameSequenceMouse E-cadherin5′-AAT GGC GGC AAT GCA ATC CCA AGA5′-TGC CAC AGA CCG ATT GTG GAG ATAMouse Snail5′-TCC AAA CCC ACT CGG ATG TGA AGA5′-TTG GTG CTT GTG GAG CAA GGA CATMouse Slug5′-CAC ATT CGA ACC CAC ACA TTG CCT5′-TGT GCC CTC AGG TTT GAT CTG TCTMouse Sip15′-ATG GCA ACA CAT GGG TTT AGT GGC5′-ATT GGA CTC TGA GCA GAT GGG TGTMouse Twist5′-AAT TCA CAA GAA TCA GGG CGT GGG5′-TCT ATC AGA ATG CAG AGG TGT GGGActin5′-GGG GTG TTG AAG GTC TCA AA5′-AGA AAA TCT GGC ACC CC Open table in a new tab Cell Growth and Motility Assays—The cells (1 × 104 cells/well) were seeded in 12-well plates in medium containing 10% FBS. The cells were harvested every other day for 7 days, and the cell numbers in each well were measured using a Coulter Counter. Three-dimensional growth assays were carried out in growth factor reduced Matrigel (BD Biosciences) as described (21Xiang B. Kim S.K. Methods Enzymol. 2006; 406: 692-701Crossref PubMed Scopus (40) Google Scholar). The cells were dissolved from the Matrigel using Cell Recovery Solution (BD Biosciences), and their numbers were measured in a Coulter Counter. For motility assays, confluent sheets of cells were "wounded" by scraping with a pipette tip at time of treatment. Wound closure in the presence of added ligand was assessed over time as described previously (22Wang S.E. Kim F.Y. Shin I. Qu S. Arteaga C.L. Mol. Cell. Biol. 2005; 25: 4703-4715Crossref PubMed Scopus (72) Google Scholar). Transwell assays were performed as previously described (23Bakin A.V. Kim A.K. Bhowmick N.A. Moses H.L. Arteaga C.L. J. Biol. Chem. 2000; 275: 36803-36810Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). In brief, the cells (1.5 × 105/well) were plated in serum-free medium in the upper chamber of 8-μm transwells (Costar) and incubated with or without 2 ng/ml TGFβ. After 24 h, cells that had migrated to the underside of the transwell filters were fixed and stained utilizing Diff-Quick Stain Set from Dade Behring AG (Dudingen, Switzerland). The cells in five random fields at 200× magnification were counted. Immunoblot Analysis—The cells were plated in 60-mm plates and allowed to grow overnight. The cells were then placed in low serum medium (1% FBS) for 16 h, after which the medium was replaced with low serum medium containing either 2 ng/ml TGFβ1 or TGFβ2 for 6 h. The cells were lysed in Nonidet P-40 lysis buffer (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 20 mm NaF) as previously described (22Wang S.E. Kim F.Y. Shin I. Qu S. Arteaga C.L. Mol. Cell. Biol. 2005; 25: 4703-4715Crossref PubMed Scopus (72) Google Scholar). Protein concentrations were determined using BCA protein assay reagent (Pierce); 50 μg of protein was separated by 9% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in TBST containing 5% bovine serum albumin for 1 h and then incubated with primary antibody overnight at 4 °C. This was followed by incubation with secondary antibody for 1 h at room temperature. The membranes were washed three times in TBST, and the bands were visualized using ECL (Amersham Biosciences). Transcriptional Reporter Assays—The cells were seeded in 60-mm plates and transfected with 5 μg of either 3TP-Lux (provided by Dr. Joan Massague, Memorial Sloan Kettering Cancer Center), NFκB-Luc (provided by Dr. Timothy Blackwell, Vanderbilt University), E-cad-Luc (provided by Dr. Amparo Cano, Universidad Autonoma de Madrid, Madrid, Spain), or 0.5 μg pCMV-Renilla (Promega) using Superfect transfection reagent (Qiagen) according to the manufacturer's protocol. The next day, the cells were split equally into 48-well plates and incubated overnight in DMEM, 1% FBS, after which the medium was replaced with fresh DMEM, 1% FBS containing either 2 ng/ml TGFβ1 or TGF β2 for 24 h. Firefly luciferase and Renilla reniformis luciferase activity was measured using a dual luciferase reporter system (Promega) according to the manufacturer's published protocol in a Monolight 3010 luminometer (Analytical Luminescence Laboratory). ELISAs—Serum-free conditioned medium was removed from growing cells after 72 h and tested using the TGFβ1 or the TGFβ2 Quantikine ELISA kit (R & D Systems) following acid activation as indicated in the manufacturer's protocol. A standard curve using 31.5-2,000 pg/ml human recombinant TGFβ was generated using the kit reagents and used to calculate the TGFβ equivalents in the conditioned medium. Each sample was examined in triplicate for a total of three times as described (24Biswas S. Kim M. Rinehart C. Dugger T.C. Chytil A. Moses H.L. Freeman M.L. Arteaga C.L. J. Clin. Investig. 2007; 117: 1305-1313Crossref PubMed Scopus (278) Google Scholar). All of the ELISA data are corrected for cell number (pg/ml/cell number). Immunofluorescent Microscopy—Immunofluorescent microscopy was performed as previously described (23Bakin A.V. Kim A.K. Bhowmick N.A. Moses H.L. Arteaga C.L. J. Biol. Chem. 2000; 275: 36803-36810Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). Briefly, the cells were grown in 8-well chamber slides for 48 h, fixed in 3% paraformaldehyde, incubated with either phalloidin (1:40 dilution) or E-cadherin (1:500 dilution) primary antibodies for 1 h, and then incubated with fluorescent secondary antibodies for 30 min. Fluorescent images were captured using a Princeton Instruments cooled CCD digital camera from a Zeiss Axio-phot upright microscope. Electrophoretic Mobility Shift Assays—Electrophoretic mobility shift assays were performed using the Promega Gel Shift Assay Kit (Promega) according to the manufacturer's protocol. NM-con and NM-kd cells were grown in DMEM, 1% FBS in the presence or absence of 2 ng/ml TGFβ2 for 1 h. Nuclear extracts were harvested as described previously (25Schreiber E. Kim P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3903) Google Scholar). Nuclear extracts (10 μg) were incubated with 32P-labeled NFκB oligonucleotides, separated by 6% SDS-PAGE, and visualized by autoradiography. Unlabeled NFκB oligonucleotides (cold NFκB) were used as a competitive inhibitor, and unlabeled Oct-1 oligonucleotides were used as a negative control. Xenograft Assays—The cells (1 × 106 cells) were resuspended in 200 μl of phosphate-buffered saline and injected with a 22-gauge needle into the right inguinal mammary gland (number 4) of anesthetized 6-week old athymic nude mice (Harlan Sprague-Dawley, Indianapolis, IN) and allowed to grow for 10 weeks. Fat pads, tumors, and lungs were collected, fixed in 10% formalin, and embedded in paraffin. Knock-down of TβRIII in NMuMG Cells Impairs Response to TGFβ—We developed shRNAs specific to TβRIII to determine the role of this receptor in mammary epithelial cells. Nontumorigenic NMuMG mouse epithelial cells were infected with a virus containing the shRNA specific to human (NM-con) or mouse (NM-kd) TβRIII, and individual clones were isolated through serial dilution. The clones were initially screened using semi-quantitative reverse transcription PCR with primers designed to amplify TβRIII and actin (data not shown). Positive clones were confirmed by receptor cross-linking with 125I-TGFβ1. As a control, mouse NMuMG cells were also infected with shRNA to human TβRIII. Affinity cross-linking with 125I-TGFβ1 showed loss of the type III receptor protein only in NM-kd cells but not in cells transduced with the control shRNA specific to human TβRIII (Fig. 1A). To determine the effect of TβRIII knock-down on TGFβ signaling, we examined p-Smad2 by Western blot after TGFβ1 and TGFβ2 treatment. NM-kd cells had significantly reduced p-Smad2 levels after treatment with 2 ng/ml TGFβ2 compared with control cells (Fig. 1B). Additionally, the ability of the NM-kd cells to activate the 3TP-Lux TGFβ-responsive promoter after TGFβ treatment was markedly inhibited, whereas reconstitution of the receptor with a retrovirus encoding rat TβRIII (NM-kd&RIII) restored ligand-induced 3TP-Lux reporter activity (Fig. 1C). The transduced rat TβRIII was also detectable by affinity cross-linking with 125I-TGFβ1 (Fig. 1A). Knock-down of TβRIII in NMuMG Cells Results in Increased Growth, Motility, and Invasiveness—TGFβ is an inhibitor of epithelial cell growth (26Siegel P.M. Kim J. Nat. Rev. Cancer. 2003; 3: 807-821Crossref PubMed Scopus (1320) Google Scholar). Thus, we examined proliferation and migration of cells in which TβRIII has been reduced by RNA interference. NM-kd cells grew significantly faster than NM-con cells, and re-expression of rat TβRIII in the NM-kd cells reduced their growth rate (Fig. 2A). Additionally, the NM-kd cells were able to migrate and close a wound in a wound closure assay under reduced serum conditions, whereas control cells and NM-kd cells reconstituted with TβRIII did not close the wound (Fig. 2B). The NM-kd cells were also more invasive than the NM-con cells as determined by their ability to migrate through transwell filters in the presence of low serum. TGFβ1 and TGFβ2 inhibited transwell migration of control cells but not of the NM-kd cells (Fig. 2C). This result is consistent with the dampened transcriptional response to added ligands observed in NM-kd cells (Fig. 1C). NMuMG cells are nontumorigenic and form small rounded acini when grown in Matrigel. In contrast, the NM-kd cells form invasive branching structures in three-dimensional Matrigel (Fig. 2D). Reconstitution of TβRIII abrogated the ability of the NM-kd cells to form these structures in Matrigel. To determine whether the changes in observed cell behavior could involve the production of autocrine ligands, we investigated the secretion of TGFβ ligands by NMuMG cells. ELISAs were used to determine the amount of total TGFβ1 and TGFβ2 secreted from these cells. The NM-kd cells secret significantly less TGFβ1 and TGFβ2 compared with control cells (Fig. 2E). Loss of TβRIII Results in Down-regulation of E-cadherin—The increased motility and invasiveness of the NM-kd cells was suggestive of EMT. Changes in the content and localization of E-cadherin at adherent cell junctions is a hallmark of EMT (27Thiery J.P. Curr. Opin. Cell Biol. 2003; 15: 740-746Crossref PubMed Scopus (1417) Google Scholar). Western blot analyses, real time quantitative PCR, and immunocytochemistry demonstrated that the NM-kd cells express greatly reduced levels of E-cadherin (Fig. 3, A-C). The ability of the NM-kd cells to transcriptionally activate an E-cadherin promoter luciferase reporter was also diminished as compared with NM-con cells (Fig. 3D). E-cadherin promoter activity is restored in the NM-kd&RIII cells, where the type III receptor had been re-expressed. The E-cadherin gene can be regulated by a variety of mechanisms including epigenetic changes, such as hypermethylation, as well as transcriptional repression. Therefore, we examined the mRNA levels of the E-cadherin transcriptional repressors Snail, Slug, Sip1, and Twist by quantitative PCR. mRNA levels of Snail, Slug, Sip1, and Twist were significantly increased in NM-kd cells compared with NM-con cells (Fig. 4). This increase correlated with the reduced expression of E-cadherin (Fig. 3). Further, E-cadherin expression was restored with a corresponding decrease in Snail, Slug, Twist, and Sip1 in NM-kd&RIII cells. These data suggest a role for TβRIII in maintaining the epithelial phenotype through control of the transcriptional repressors of E-cadherin. EMT-6 Cells, with Reduced Expression of TβRIII, Demonstrated an Increased Growth Rate and Decreased E-cadherin Expression—To confirm that these results were specific to loss of TβRIII expression and not specific to NMuMG cells or caused by clonal variation, we knocked down TβRIII expression in EMT-6 mouse mammary cancer cells. EMT6-con and EMT6-kd cell lines were generated as described for the NMuMG cells (Fig. 1). The clones were screened by semi-quantitative PCR (Fig. 5A), and EMT6-kd clone C5 was used in the remaining experiments. The EMT6-kd cells demonstrated a muted response to TGFβ2, but not TGFβ1, as determined by 3TP-Lux reporter assays (Fig. 5B). Similar to NMuMG cells, EMT6-kd cells grew better in Matrigel compared with EMT6-con cells (Fig. 5C). In addition, these cells showed decreased E-cadherin protein expression and E-cadherin luciferase activity (Fig. 5, D and E) with a corresponding increase in Snail mRNA (Fig. 5E). These data demonstrate that the effect of TβRIII loss on growth and E-cadherin expression is not limited to NNuMG cells. NFκB Activity Is Higher in TβRIII Knock-down Cells—NFκB is a known regulator of Snail (28Barbera M.J. Kim I. Dominguez D. Julien-Grille S. Guaita-Esteruelas S. Peiro S. Baulida J. Franci C. Dedhar S. Larue L. Garcia de Herreros A. Oncogene. 2004; 23: 7345-7354Crossref PubMed Scopus (294) Google Scholar), and NFκB activity has been shown to be regulated (positively and negatively) by TGFβ2 (29Lu T. Kim L.G. Swiatkowski S.M. Boiko A.D. Howe P.H. Stark G.R. Gudkov A.V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7112-7117Crossref PubMed Scopus (89) Google Scholar, 30Kaltschmidt B. Kim C. Mech. Dev. 2001; 101: 11-19Crossref PubMed Scopus (21) Google Scholar). Therefore, we examined NFκB activity using a NFκB-responsive luciferase reporter in transiently transfected NMuMG cells. The NM-kd cells showed high basal levels of NFκB reporter activity as compared with NM-con cells. Re-expression of the type III receptor in NM-kd cells reduced NFκB transcription to the same levels as those seen in NM-con cells (Fig. 6A). The ability of NFκB to bind to DNA was confirmed by electrophoretic mobility shift assays using 32P-radiolabeled oligonucleotides containing the putative NFκB-binding site (Fig. 6B). Interestingly, TGFβ2 blocked DNA binding in both cell lines. To examine the role of NFκB in the invasive phenotype seen in the NM-kd cells, we used an adenovirus containing either dominant negative IκBα (dn IκBα S32A/S36A) to abrogate NFκB function or a constitutively active IKK2 (CA-IKK2 D177E/D181E) to induce NFκB signaling. NM-kd and NM-con cells were transduced with adenoviruses, plated in growth factor reduced Matrigel 24 h later, and allowed to grow for 8 days. NM-kd cells infected with dn IκBα containing adenovirus showed a significant reduction of growth in Matrigel (Fig. 6, C and D). Transduction of adenoviruses encoding dn IκBα into NM-kd cells significantly diminished NFκB promoter activity. On the other hand, NM-kd cells infected with CA-IKK2 showed a significant increase in NFκB reporter activity that was not seen in NM-control cells (Fig. 6, E and F). This result implies that the endogenous type III TGFβ receptor may counteract the NFκB-inducing effect of CA-IKK2. It is significant to note that the reduction in NFκB activity did not reduce the ability of the NM-kd cells to grow in Matrigel down to that of the NM-con cells and that these cells still retain their invasive phenotype, suggesting the involvement of additional signaling pathways on the cells invasive behavior. The decreased ability to grow in Matrigel corresponded to a significant increase in base-line E-cadherin promoter activity in NM-con versus NM-kd cells. Infection with an adenovirus containing dn IκBα up-regulated the low basal E-cadherin promoter activity in NM-kd but not in NM-con cells (Fig. 7, A and C). Additionally, NM-con cells infected with adenovirus containing constitutively active IKK2 showed a decrease in E-cadherin promoter reporter activity (Fig. 7, A and B), consistent with the role of NFκB signaling on EMT. In accordance with these results, E-cadherin mRNA, as measured by quantitative PCR, is significantly increased in NM-kd cells transduced with dn IκBα (Fig. 7C) with a corresponding decrease in Snail (Fig. 7D). These changes were not observed in the control cells (Fig. 7, C and D). NM-kd Cells Form Invasive Tumors in Vivo—NMuMG cells are not able to grow
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