Overexpression of HER2 (erbB2) in Human Breast Epithelial Cells Unmasks Transforming Growth Factor β-induced Cell Motility
2004; Elsevier BV; Volume: 279; Issue: 23 Linguagem: Inglês
10.1074/jbc.m400081200
ISSN1083-351X
AutoresYukiko Ueda, Shizhen Wang, Nancy Dumont, Jae Youn Yi, Yasuhiro Koh, Carlos L. Arteaga,
Tópico(s)Cell Adhesion Molecules Research
ResumoWe have examined overexpression of the human epidermal growth factor receptor 2 (HER2) to determine if it modifies the anti-proliferative effect of transforming growth factor (TGF)-β against MCF-10A human mammary epithelial cells. Exogenous TGF-β inhibited cell proliferation and induced Smad-dependent transcriptional reporter activity in both MCF-10A/HER2 and MCF-10A/vector control cells. Ligand-induced reporter activity was 7-fold higher in HER2-overexpressing cells. In wound closure and transwell assays, TGF-β induced motility of HER2-transduced, but not control cells. The HER2-blocking antibody trastuzumab (Herceptin) prevented TGF-β-induced cell motility. Expression of a constitutively active TGF-β type I receptor (ALK5T204D) induced motility of MCF-10A/HER2 but not MCF-10A/vector cells. TGF-β-induced motility was blocked by coincubation with either the phosphatidylinositol 3-kinase inhibitor LY294002, the mitogen-activated protein kinase (MAPK) inhibitor U0126, the p38 MAPK inhibitor SB202190, and an integrin β1 blocking antibody. Rac1 activity was higher in HER2-overexpressing cells, where both Rac1 and Pak1 proteins were constitutively associated with HER2. Both exogenous TGF-β and transduction with constitutively active ALK5 enhanced this association. TGF-β induced actin stress fibers as well as lamellipodia within the leading edge of wounds. Herceptin blocked basal and TGF-β-stimulated Rac1 activity but did not repress TGF-β-stimulated transcriptional reporter activity. These data suggest that 1) overexpression of HER2 in nontumorigenic mammary epithelial is permissive for the ability of TGF-β to induce cell motility and Rac1 activity, and 2) HER2 and TGF-β signaling cooperate in the induction of cellular events associated with tumor progression. We have examined overexpression of the human epidermal growth factor receptor 2 (HER2) to determine if it modifies the anti-proliferative effect of transforming growth factor (TGF)-β against MCF-10A human mammary epithelial cells. Exogenous TGF-β inhibited cell proliferation and induced Smad-dependent transcriptional reporter activity in both MCF-10A/HER2 and MCF-10A/vector control cells. Ligand-induced reporter activity was 7-fold higher in HER2-overexpressing cells. In wound closure and transwell assays, TGF-β induced motility of HER2-transduced, but not control cells. The HER2-blocking antibody trastuzumab (Herceptin) prevented TGF-β-induced cell motility. Expression of a constitutively active TGF-β type I receptor (ALK5T204D) induced motility of MCF-10A/HER2 but not MCF-10A/vector cells. TGF-β-induced motility was blocked by coincubation with either the phosphatidylinositol 3-kinase inhibitor LY294002, the mitogen-activated protein kinase (MAPK) inhibitor U0126, the p38 MAPK inhibitor SB202190, and an integrin β1 blocking antibody. Rac1 activity was higher in HER2-overexpressing cells, where both Rac1 and Pak1 proteins were constitutively associated with HER2. Both exogenous TGF-β and transduction with constitutively active ALK5 enhanced this association. TGF-β induced actin stress fibers as well as lamellipodia within the leading edge of wounds. Herceptin blocked basal and TGF-β-stimulated Rac1 activity but did not repress TGF-β-stimulated transcriptional reporter activity. These data suggest that 1) overexpression of HER2 in nontumorigenic mammary epithelial is permissive for the ability of TGF-β to induce cell motility and Rac1 activity, and 2) HER2 and TGF-β signaling cooperate in the induction of cellular events associated with tumor progression. IntroductionThe transforming growth factors (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor; TβRII, TGF-β type II receptor; MMTV, murine mammary tumor virus; EGF, epidermal growth factor; MAPK, mitogen-activated protein kinase; GFP, green fluorescent protein; EGFP, enhanced GFP; HA, hemagglutinin; BrdUrd, bromodeoxyuridine; GST-PBD, glutathione S-transferase fused with the Pak family binding domain for Cdc42/Rac1; EST, Expressed Sequence Tag; IL, interleukin. are polypeptides that are part of the superfamily of structurally related ligands that include the TGF-βs, activins, and bone morphogenetic proteins. TGF-β ligands can modulate cell proliferation, lineage determination, functional differentiation, extracellular matrix production, cell motility, and apoptosis (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Google Scholar). TGF-β was originally reported to induce anchorage-independent growth of mouse fibroblasts (2Moses H.L. Branum E.L. Proper J.A. Robinson R.A. Cancer Res. 1981; 41: 2842-2848Google Scholar). Later studies indicated that TGF-β is a potent inhibitor of epithelial cell proliferation (3Roberts A.B. Anzano M.A. Wakefield L.M. Roche N.S. Stern D.F. Sporn M.B. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 119-123Google Scholar, 4Tucker R.F. Shipley G.D. Moses H.L. Holley R.W. Science. 1984; 226: 705-707Google Scholar), suggesting a role in tumor suppression. Transgenic mice expressing active TGF-β1S223/225 in the mammary gland exhibit delayed ductal morphogenesis, renewal capacity, and functional differentiation (5Pierce Jr., D.F. Johnson M.D. Matsui Y. Robinson S.D. Gold L.I. Purchio A.F. Daniel C.W. Hogan B.L. Moses H.L. Genes Dev. 1993; 7: 2308-2317Google Scholar, 6Kordon E.C. McKnight R.A. Jhappan C. Hennighausen L. Merlino G. Smith G.H. Dev. Biol. 1995; 168: 47-61Google Scholar, 7Jhappan C. Geiser A.G. Kordon E.C. Bagheri D. Hennighausen L. Roberts A.B. Smith G.H. Merlino G. EMBO J. 1993; 12: 1835-1845Google Scholar). Inhibition of autocrine TGF-β signaling in the mouse mammary gland by tissue-specific expression of a truncated dominant-negative TGF-β type II receptor (TβRII) results in accelerated lobulo-alveolar development (8Gorska A.E. Joseph H. Derynck R. Moses H.L. Serra R. Cell Growth Differ. 1998; 9: 229-238Google Scholar) and enhanced propensity for the development of carcinogen-induced lung, mammary, and skin tumors (9Bottinger E.P. Jakubczak J.L. Haines D.C. Bagnall K. Wakefield L.M. Cancer Res. 1997; 57: 5564-5570Google Scholar, 10Go C. Li P. Wang X.J. Cancer Res. 1999; 59: 2861-2868Google Scholar) as well as spontaneous invasive mammary carcinomas (11Gorska A.E. Jensen R.A. Shyr Y. Aakre M.E. Bhowmick N.A. Moses H.L. Am. J. Pathol. 2003; 163: 1539-1549Google Scholar). Mice with targeted disruption of either the Tgfb1 or the Smad3 genes develop colon adenomas and carcinomas (12Engle S.J. Hoying J.B. Boivin G.P. Ormsby I. Gartside P.S. Doetschman T. Cancer Res. 1999; 59: 3379-3386Google Scholar, 13Zhu Y. Richardson J.A. Parada L.F. Graff J.M. Cell. 1998; 94: 703-714Google Scholar). Mice heterozygous for deletion of the Tgfb1 gene express 10-30% of the TGF-β1 protein, and this partial loss is sufficient to attenuate epithelial growth restraint and inhibit tumor suppression function, resulting in enhanced carcinogen-induced liver and lung tumors (14Tang B. Bottinger E.P. Jakowlew S.B. Bagnall K.M. Mariano J. Anver M.R. Letterio J.J. Wakefield L.M. Nat. Med. 1998; 4: 802-807Google Scholar). MMTV/TGF-β1 mice are resistant to (7,12-dimethylbenz[a]-anthracene)-induced mammary tumors, and when cross-bred with mice expressing the EGF receptor ligand TGF-α in the mammary gland (MMTV/TGF-α), the bigenic (TGF-β/TGF-α) progeny fails to develop TGF-α-induced mammary hyperplasias and carcinomas while still exhibiting a hypoplastic phenotype (15Pierce Jr., D.F. Gorska A.E. Chytil A. Meise K.S. Page D.L. Coffey Jr., R.J. Moses H.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4254-4258Google Scholar). Finally, mice overexpressing active TGF-β1 under the control of the K14 keratinocyte-specific promoter are relatively protected from skin tumors induced by chemical carcinogens (16Cui W. Fowlis D.J. Bryson S. Duffie E. Ireland H. Balmain A. Akhurst R.J. Cell. 1996; 86: 531-542Google Scholar), further supporting the tumor suppressive role of TGF-β signaling.Most cancer cells lose or attenuate TGF-β-mediated antiproliferative effects either by mutational inactivation of TGF-β receptors or their signal transducers or by less known mechanisms (for review, see Refs. 17Derynck R. Akhurst R.J. Balmain A. Nat. Genet. 2001; 29: 117-129Google Scholar, 18Wakefield L.M. Roberts A.B. Curr. Opin. Genet. Dev. 2002; 12: 22-29Google Scholar, 19Siegel P.M. Massague J. Nat. Rev. Cancer. 2003; 3: 807-820Google Scholar). There is also abundant experimental evidence to support that excess production and/or activation of TGF-β in tumors, including breast cancers, can foster cancer progression by autocrine and paracrine mechanisms (for review, see Ref. 20Dumont N. Arteaga C.L. Cancer Cell. 2003; 3: 531-536Google Scholar). These include enhancement of tumor cell motility and survival, increase in tumor neo-angiogenesis and extracellular matrix production, up-regulation of peritumor metalloproteases, and inhibition of host immune surveillance mechanisms (17Derynck R. Akhurst R.J. Balmain A. Nat. Genet. 2001; 29: 117-129Google Scholar, 21Dumont N. Arteaga C.L. Breast Cancer Res. 2000; 2: 125-132Google Scholar). For example, overexpression of active TGF-β1 or an activated type I TGF-β receptor (TβRI) in the mammary gland of transgenic mice results in acceleration of metastases derived from neu-induced primary mammary tumors (22Siegel P.M. Shu W. Cardiff R.D. Muller W.J. Massague J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8430-8435Google Scholar, 23Muraoka R.S. Koh Y. Roebuck L.R. Sanders M.E. Brantley-Sieders D. Gorska A.E. Moses H.L. Arteaga C.L. Mol. Cell. Biol. 2003; 23: 8691-8703Google Scholar). Furthermore, in transgenic mice expressing the Polyomavirus middle T antigen (PyVmT) in mammary epithelium, blockade of TGF-β with a soluble fusion protein consisting of the extracellular domain of TβRII and the Fc domain of human IgG1 (sTβRII:Fc) results in increased apoptosis in primary mammary tumors and a reduction in both circulating tumor cells and lung metastases (24Muraoka R.S. Dumont N. Ritter C.A. Dugger T.C. Brantley D.M. Chen J. Easterly E. Roebuck L.R. Ryan S. Gotwals P.J. Koteliansky V. Arteaga C.L. J. Clin. Invest. 2002; 109: 1551-1559Google Scholar). Taken together, these data suggest that 1) TGF-β can suppress some but not all oncogenic signals in epithelial cells, and 2) some oncogenes can engage TGF-β signaling and utilize it for tumor maintenance and progression.We have studied the HER2 proto-oncogene product, the human homolog of erbB2 and neu, to determine if it modulates cellular responses to TGF-β in MCF-10A nontumorigenic human mammary epithelial cells. The HER2 protein is a member of the erbB family of transmembrane receptor tyrosine kinases, which also includes the EGF receptor (HER1, erbB1), HER3 (erbB3), and HER4 (erbB4). Binding of ligands to EGFR, HER3, and HER4 induces the formation of homodimeric and heterodimeric, kinase-active complexes to which HER2 is recruited as a preferred partner (25Olayioye M.A. Neve R.M. Lane H.A. Hynes N.E. EMBO J. 2000; 19: 3159-3167Google Scholar). Even though HER2 is unable to interact directly with receptor ligands, it can amplify signaling pathways activated by erbB co-receptors, which include phospholipase Cγ-1, Ras-Raf-MEK-ERK (extracellular signal-regulated kinase), phosphatidylinositol 3-kinase-Akt-p70S6 kinase, Pak-JNKK-JNK (c-Jun NH2-terminal kinase), stress-activated protein kinases, and signal transducers and activators of transcription (26Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Cell. Biol. 2001; 2: 127-137Google Scholar). Some of these pathways such as ERK, p38 MAPK, phosphatidylinositol 3-kinase, and c-Jun NH2-terminal kinase are also stimulated by TGF-β in some cells (18Wakefield L.M. Roberts A.B. Curr. Opin. Genet. Dev. 2002; 12: 22-29Google Scholar, 27Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Google Scholar), thus providing a biochemical basis for synergy between TGF-β and erbB receptor signaling.As indicated above, TGF-β can suppress TGF-α-induced transformation in the mammary gland (15Pierce Jr., D.F. Gorska A.E. Chytil A. Meise K.S. Page D.L. Coffey Jr., R.J. Moses H.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4254-4258Google Scholar). Furthermore, TGF-β can also suppress EGF-induced mitogenesis in epithelial cells (28Coffey Jr., R.J. Sipes N.J. Bascom C.C. Graves-Deal R. Pennington C.Y. Weissman B.E. Moses H.L. Cancer Res. 1988; 48: 1596-1602Google Scholar). These results imply that TGF-β is dominant over the EGF receptor. Because of the ability of HER2 to signal with increased potency than the EGF receptor (26Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Cell. Biol. 2001; 2: 127-137Google Scholar), we speculated that overexpression of HER2 may prevent TGF-β-mediated growth inhibition and/or modulate other cellular responses of non-transformed mammary epithelial cells to this ligand. In this report, the proliferation of MCF-10A cells overexpressing stably transduced HER2 was still delayed by exogenous TGF-β. However, MCF-10A/HER2 cells exhibited increased motility and Rac1 activity in response to TGF-β, suggesting that HER2 and TGF-β signals can cooperate in inducing cellular events required for tumor progression.EXPERIMENTAL PROCEDURESCell Culture and Reagents—MCF-10A cells were purchased from the American Type Culture Collection (Manassas, VA) and maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with horse serum, l-glutamine, human recombinant EGF (Calbiochem), cholera toxin (Biomol, Plymouth Meeting, PA), insulin (Calbiochem), and hydrocortisone (Sigma). Experiments were performed in serum-free Dulbecco's modified Eagle's medium /F-12 medium supplemented with 0.2% bovine serum albumin (fraction V). 293T cells were maintained in Dulbecco's modified Eagle's medium, 10% heat-inactivated fetal calf serum. All tissue culture reagents were from Invitrogen unless otherwise specified. Human recombinant TGF-β1 was from R&D Systems (Minneapolis, MN) and the humanized IgG1 HER2 antibody trastuzumab (Herceptin) was purchased from the Vanderbilt University Medical Center Pharmacy. ZD1839 (Iressa) was kindly provided by Alan Wakeling (AstraZeneca Pharmaceuticals, Macclesfield, UK). LY294002 and SB202190 were from Calbiochem, U-0126 was from Promega (Madison, WI), and the integrin β1 blocking antibody was from Chemicon (Temecula, CA).Retroviral and Adenoviral Vectors—c-Myc-tagged HER2 cDNA (provided by Dr. James Staros, Vanderbilt University) was excised using XbaI/HindIII enzymes, purified by agarose-gel electrophoresis, and converted to blunt ends with Klenow DNA polymerase. The retroviral expression vector pBMN-IRES-EGFP (29Grignani F. Kinsella T. Mencarelli A. Valtieri M. Riganelli D. Lanfrancone L. Peschle C. Nolan G.P. Pelicci P.G. Cancer Res. 1998; 58: 14-19Google Scholar) (provided by Dr. Gary Nolan, Stanford University) was digested with SnaBI and dephosphorylated at the 5′-ends with alkaline phosphatase (Promega). The blunt-ended vector and the HER2 cDNA were ligated and then replicated in DH5α (Invitrogen). To confirm that the HER2 insert had no truncations, the vector was digested with StuI and also sequenced using the primer 5′-GACCTTACACAGTCCTGC-3′. Restriction enzymes, T4-DNA ligase, and DNA polymerases were all from New England BioLabs (Beverly, MA). 293T retrovirus-packaging cells (2.5 × 106/60-mm dish; ATCC) were transfected with 5 μg of pBMN-HER2-IRES-EGFP or 5 μg of pBMN-IRES-EGFP (control) for 24 h using FuGENE 6 (Roche Applied Science). For each case, cells were cotransfected with 3 μg of pHCMV-G (30Yee J.K. Miyanohara A. LaPorte P. Bouic K. Burns J.C. Friedmann T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9564-9568Google Scholar) and 3 μg of pSV-Ψ-env-MLV (pSV-pol/gag) (31Landau N.R. Littman D.R. J. Virol. 1992; 66: 5110-5113Google Scholar) (provided by Dr. Jane Burns, University of California, San Diego, CA). Virus-containing medium was collected 48-72 h later and passed through a 45-μm filter. MCF-10A cells (105/60-mm dish) were transduced with control or HER2-encoding retroviral vectors for 2 h. After 5 passages, cells stably expressing EGFP were sorted by flow cytometry and expanded. EGFP expression was monitored with an inverted fluorescent microscope and maintained at 100% throughout all experiments.The hemagglutinin (HA)-tagged ALK5T204D adenoviral construct was provided by Dr. Kohei Miyazono (Japanese Foundation for Cancer Research, Tokyo, Japan). Stock of recombinant viruses was replicated in 293 cells and titered using the adenovirus expression vector kit (Takara Biomedicals, Tokyo). MCF-10A cells were infected with ALK5T204D or a control β-galactosidase adenovirus at a multiplicity of infection of 10 plaque-forming units per cell for 1 h, unless otherwise specified. After 48 h, β-galactosidase expression was assessed using the β-galactosidase enzyme system (Promega). Expression of HA-tagged mutant ALK5 was confirmed by HA and TβRI immunoblot analyses.Immunoblot Analysis and Immunoprecipitation—Cells were washed twice with ice-cold phosphate-buffered saline and lysed with a buffer containing 50 mm Tris (pH 7.4), 150 NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mm EDTA, 20 mm NaF, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, and 2 μg/ml each aprotinin, pepstatin, and leupeptin. Protein content was quantitated using the BCA protein assay reagent (Pierce). Protein extracts were separated by SDS-PAGE, transferred to nitrocellulose membrane, and subjected to immunoblot analysis as described previously (32Dumont N. Bakin A.V. Arteaga C.L. J. Biol. Chem. 2003; 278: 3275-3285Google Scholar). Immunoreactive bands were visualized by chemiluminescence (Roche Applied Science). Immunoprecipitations were performed as described previously (32Dumont N. Bakin A.V. Arteaga C.L. J. Biol. Chem. 2003; 278: 3275-3285Google Scholar) after preclearing cell lysates with protein A-Sepharose (Sigma) for 2 h at 4 °C. The antibodies utilized were HER2 (erbB2 Ab1) and HER3 (erbB3 C-17; NeoMarkers, Union City, CA), c-Myc (9E10; Roche Applied Science), HA, TβRI (V22), and Pak1 (Santa Cruz Biotechnology, Santa Cruz, CA), Rac1 (Transduction Laboratories, Lexington, KY), and Tyr(P) monoclonal (Upstate Biotechnology, Lake Placid, NY).Cell Cycle Analysis—Cells were harvested by trypsinization and labeled with 50 μg/ml propidium iodide (Sigma) containing 125 units/ml protease-free RNase (Calbiochem). A total of 10,000 stained nuclei were analyzed in a FACSCalibur flow cytometer (BD Biosciences) as described previously (33Shin I. Bakin A.V. Rodeck U. Brunet A. Arteaga C.L. Mol. Biol. Cell. 2001; 12: 3328-3339Google Scholar).Cell Motility Assays and BrdUrd Incorporation—Cell motility was assessed in wound closure assays as described previously (32Dumont N. Bakin A.V. Arteaga C.L. J. Biol. Chem. 2003; 278: 3275-3285Google Scholar). For wound closure experiments, cells were allowed to reach confluence in serum-containing complete growth medium and then incubated for 16 h in serum-free medium before the addition of TGF-β or other inhibitors. Wound closure was monitored by microscopy at various times. In some cases wounded monolayers were incubated with BrdUrd (Zymed Laboratories Inc., San Francisco, CA) for 3 h and then stained with a biotinylated BrdUrd antibody according to the BrdUrd staining kit protocol (Zymed Laboratories Inc.). Transwell migration was performed utilizing 5-μm pore, polycarbonate filters (Corning Costar Corp., Cambridge, MA) as described (32Dumont N. Bakin A.V. Arteaga C.L. J. Biol. Chem. 2003; 278: 3275-3285Google Scholar). Cells migrated to the underside of transwell filters were fixed, stained with Diff-Quick (Düdingen, Switzerland), and counted by bright field microscopy at 200× in five random fields.Transcription Reporter Assays—Cells were seeded in 24-well plates and transfected with 3 μg/35-mm dish of the Smad-dependent reporter construct p(CAGA)12-luciferase (provided by J.-M. Gauthier, Laboratoire GlaxoSmithKline, Les Ulis Cedex, France) along with 0.02 μg/well of pCMV-Renilla using FuGENE 6 according to the manufacturer's protocol. Firefly and Renilla reniformis luciferase activities were measured using the dual luciferase assay system (Promega), and the data were normalized utilizing the ratio of firefly to R. reniformis luciferase as described previously (32Dumont N. Bakin A.V. Arteaga C.L. J. Biol. Chem. 2003; 278: 3275-3285Google Scholar).Immunofluorescent Microscopy—Cells were treated with TGF-β1 on glass coverslips and then fixed and permeabilized as described previously (34Bakin A.V. Rinehart C. Tomlinson A.K. Arteaga C.L. J. Cell Sci. 2002; 115: 3193-3206Google Scholar) followed by incubation with phalloidin-Texas Red (2 units/ml in phosphate-buffered saline for 30 min; Molecular Probes). Fluorescent images of coverslips mounted in AquaPolyMount (Polysciences) were captured using a Princeton Instruments cooled CCD digital camera from a Zeiss Axiophot upright microscope.Rac1 Activity Assay—The pGEX plasmid containing GST-PBD (glutathione S-transferase fused with the Pak family binding domain for Cdc42/Rac1) (35Bagrodia S. Taylor S.J. Creasy C.L. Chernoff J. Cerione R.A. J. Biol. Chem. 1995; 270: 22731-22737Google Scholar) was a gift from R. Cerione (Cornell University, New York, NY). GST-PBD was expressed in Escherichia coli and immobilized by glutathione-Sepharose 4B beads after the GST gene fusion system protocol (Amersham Biosciences). Fifty μg of GST-PBD were incubated with 800 μg of protein from precleared cell lysates for 3 h at 4 °C. GST-PBD was affinity-precipitated using glutathione-Sepharose 4B beads (Amersham Biosciences), and the adsorbed proteins were eluted from the beads by suspending them in Laemmli sample buffer followed by SDS-PAGE and Rac1 immunoblot analysis as described previously (34Bakin A.V. Rinehart C. Tomlinson A.K. Arteaga C.L. J. Cell Sci. 2002; 115: 3193-3206Google Scholar).cDNA Array and Northern Analysis—Cells were incubated for 16 h in serum-free medium and then treated with TGF-β1 for 24 h. Total RNAs were isolated with Trizol reagent (Invitrogen), digested with RNase-free DNase (0.1 unit/1 μg RNA; Promega), and purified using a silica gel column (RNeasy Kit, Qiagen). Purified RNAs (50 μg) were reverse-transcribed to cDNAs, and these were labeled with Cy3-conjugated dCTP (MCF-10A/HER2) or Cy5-conjugated dCTP (MCF-10A/vector). The procedures for reverse transcription, RNA template digestion, and cDNA purification and hybridization to a human 5000 cDNA array were performed following the protocols listed in www.array.vanderbilt.edu. Cy3- and Cy5-labeled cDNAs were scanned at 532 and 635 nm, respectively, in GenePix Pro (Axon Instruments, Inc., Foster City, CA). DNA polymerase, primers, and fluorescent dye-conjugated nucleotides were from Invitrogen unless otherwise specified. For Northern analysis, RNAs were resolved by agarose gel electrophoresis, transferred onto nylon membranes (Schleicher & Schüll), and hybridized with cDNA probes using Express hybridization solution (Clontech, Palo Alto, CA) following the manufacturer's protocol. The cDNA probes were made from 3′-ESTs excised from cloning vectors, 1) TβRII 3′-EST (accession number AA487034, restriction enzymes EcoR-I/Xho-1) and 2) metallothionein I-H (H77597, EcoR-I/Pac-1). The excised 3′-ESTs were isolated by agarose gel electrophoresis, labeled with [α-32P]dCTP (PerkinElmer Life Sciences) using the Random Primer labeling system (Invitrogen), and purified with a silica gel column (Qiagen).RESULTSTGF-β Induces Motility of MCF-10A/HER2 Cells—We have examined the effects of HER2 overexpression on the response of human mammary epithelial cells to TGF-β. MCF-10A nontumorigenic mammary epithelial cells were stably transduced with a retroviral vector encoding Myc-tagged HER2 (Fig. 1A). By Western analysis with HER2 and c-Myc antibodies, protooncogene expression was confirmed in the transduced cells. Both HER3 and a 185-kDa phosphotyrosine band were identified in HER2 immunoprecipitates from the HER2-transfected cells, indicating that HER2 was constitutively phosphorylated and associated with HER3 (erbB3) in the absence of exogenous ligands (Fig. 1B). Treatment with recombinant TGF-β (for 24 h) increased the fraction of cells in G1 phase of the cell cycle in MCF-10A/HER2 (34 → 47%) and control cells (32 → 49%) with a simultaneous reduction in the S-phase fraction and a 50% reduction in cell number after 6 days (Figs. 1, C and D).We next examined the effect of TGF-β on the motility of HER2-overexpressing cells in wound closure and transwell motility assays. After 18 h, MCF-10A/HER2 cells migrated into the wounded area but did not close the wound, whereas in MCF-10A/vector cells the wound remained completely open. Similar results were observed in cells plated in transwells (Fig. 2, A and B, first column). The addition of TGF-β resulted in complete wound closure by MCF-10A/HER2 cells but had no effect on motility of control cells (Fig. 2, A and B, second column). TGF-β-induced motility was blocked by the addition of a saturating concentration of the HER2 blocking antibody Herceptin (Fig. 2, A and B), thus implying that the effect of TGF-β was HER2-specific. The EGF receptor tyrosine kinase inhibitor ZD1839 (Iressa), which has been shown to block phosphorylation of HER2 (36Moulder S.L. Yakes F.M. Muthuswamy S.K. Bianco R. Simpson J.F. Arteaga C.L. Cancer Res. 2001; 61: 8887-8895Google Scholar), also inhibited TGF-β-induced wound closure completely (data not shown).Fig. 2TGF-β induces motility of MCF-10A/HER2 cells. A, confluent cell monolayers in serum-free medium were wounded with a pipette tip. After wounding, TGF-β1 (1 ng/ml) and/or Herceptin (10 μg/ml) were added as indicated, and wound closure was monitored by microscopy at 18 h. Scale bar, 100 μm. B, a single cell suspension of 2 × 105 cells in serum-free medium was seeded onto transwell filters (6-mm diameter, 5-μm pores) in 12-well plates and allowed to migrate. After 18 h, cells on the underside of the filters were fixed, stained, and counted. Scale bar, 100 μm. The results are represented quantitatively in the bar graph below. Each data point represents the mean ± S.D. of three wells.View Large Image Figure ViewerDownload (PPT)Expression of ALK5T204D Enhances Motility in MCF-10A/HER2 Cells—To determine whether the motility effect was specific to activated TGF-β receptors, we induced TGF-β signaling in both cell types by expressing an HA-tagged adenovirus encoding a constitutively active mutant of TβRI (ALK5T204D). Mutation of threonine 204 in ALK5 to aspartic acid leads to constitutive activation of the type I receptor serine/threonine kinase, allowing it to signal in the absence of added ligand or TβRII (37Wieser R. Wrana J.L. Massague J. EMBO J. 1995; 14: 2199-2208Google Scholar). Cells infected with a β-galactosidase adenovirus at the same multiplicity of infection were used as controls. The efficiency of transduction was >90% as evaluated by in situ staining for β-galactosidase activity 48 h after infection (data not shown). Expression of ALK5T204D was confirmed by HA immunoblot analysis (Fig. 3A). The β-galactosidase-transduced MCF-10A/HER2 cells exhibited slightly higher basal motility than the non-motile MCF-10A/vector cells. However, ALK5T204D induced wound closure and transwell migration in HER2-overexpressing but not in control cells (Fig. 3, B and C).Fig. 3Expression of ALK5T204D enhances motility in MCF-10A/HER2 cells. A, MCF-10A/vector and MCF-10A/HER2 cells were infected with adenoviruses encoding β-galactosidase or an HA-tagged constitutively active mutant of TβRI (ALK5T204D) at a multiplicity of infection of 10 plaque-forming units/cell for 1 h. Forty-eight h after infection, HER2 and ALK5 expression was verified by immunoblot (IB) utilizing HER2, HA, TβRI, and actin antibodies. B, confluent monolayers of adenovirus-infected cells were wounded with a pipette tip as indicated in Fig. 2A, and wound closure was monitored 18 h later. Scale bar, 100 μm. C, adenovirus-transduced cells were plated onto transwell filters as indicated in Fig. 2B. Cell migration to the underside of the transwell filters was assessed 18 h after plating.View Large Image Figure ViewerDownload (PPT)TGF-β-induced Motility of HER2-overexpressing MCF-10A Cells Requires Multiple Signaling Pathways—TGF-β-induced cell motility in HER2 cells was blocked by the addition of mitomycin C, a compound that inhibits DNA synthesis (data not shown). Therefore, we examined if TGF-β stimulated DNA synthesis in the wounded monolayers as measured by BrdUrd incorporation. Interestingly, the addition of TGF-β clearly stimulated BrdUrd incorporation in MCF-10A/HER2 but not MCF-10A/vector cells, but this was limited to cells in the edge of the wound (Fig. 4A). Because TGF-β can activate non-Smad signaling pathways or induce molecules associated with cell motility (32Dumont N. Bakin A.V. Arteaga C.L. J. Biol. Chem. 2003; 278: 3275-3285Google Scholar, 34Bakin A.V. Rinehart C. Tomlinson A.K. Arteaga C.L. J. Cell Sci. 2002; 115: 3193-3206Google Scholar), we examined for evidence of activated (phosphorylated) Akt, ERK and p38 MAPK as well as increased integrin β1 by immunocytochemistry focusing on cells in the leading edge of the wound. Using commercially available (phospho-specific) antibodies, we were unable to detect the above-mentioned molecules. However, TGF-β-induced wound closure was completely blocked in the presence of the phosphatidylinositol 3-kinase inhibitor LY294002, the MEK1/2 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) inhibitor U0126, the p38 MAPK inhibitor SB202190, and the CD29 integrin β1 blocking antibody (Fig. 4B).Fig. 4TGF-β-induced motility of HER2-overexpressing MCF-10A cells requires multiple signaling pathways. A, wounded monolayer
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