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Protection against Anoikis and Down-regulation of Cadherin Expression by a Regulatable β-Catenin Protein

2002; Elsevier BV; Volume: 277; Issue: 21 Linguagem: Inglês

10.1074/jbc.m105331200

1083-351X

Zhigang Weng, Mei Xin, Lourdes Pablo, Dorre A. Grueneberg, Margit Hagel, Gerard Bain, Thomas Müller, Jackie Papkoff,

Kruppel-like factors research

β-Catenin signaling plays a key role in a variety of cellular contexts during embryonic development and tissue differentiation. Aberrant β-catenin signaling has also been implicated in promoting human colorectal carcinomas as well as a variety of other cancers. To study the molecular and cellular biological functions of β-catenin in a controlled fashion, we created a regulatable form of activated β-catenin by fusion to a modified estrogen receptor (ER) ligand binding domain (G525R). Transfection of tissue culture cells with expression vectors encoding this hybrid protein allows the signal transduction function of β-catenin to be induced by the synthetic estrogen, 4-hydroxytamoxifen, leading to regulated activation of a β-catenin-lymphocyte enhancer-binding factor-dependent reporter gene as well as induction of endogenous cyclin D1 expression. The activation of ER-β-catenin signaling rescues RK3E cells from anoikis and correlates with an increased phosphorylation of mitogen-activated protein kinase. The inhibition of anoikis by ER-β-catenin can be abolished by a mitogen-activated protein kinase pathway inhibitor, PD98059. Evidence is also provided to show that ER-β-catenin down-regulates cadherin protein levels. These findings support a key role for activated β-catenin signaling in processes that contribute to tumor formation and progression. β-Catenin signaling plays a key role in a variety of cellular contexts during embryonic development and tissue differentiation. Aberrant β-catenin signaling has also been implicated in promoting human colorectal carcinomas as well as a variety of other cancers. To study the molecular and cellular biological functions of β-catenin in a controlled fashion, we created a regulatable form of activated β-catenin by fusion to a modified estrogen receptor (ER) ligand binding domain (G525R). Transfection of tissue culture cells with expression vectors encoding this hybrid protein allows the signal transduction function of β-catenin to be induced by the synthetic estrogen, 4-hydroxytamoxifen, leading to regulated activation of a β-catenin-lymphocyte enhancer-binding factor-dependent reporter gene as well as induction of endogenous cyclin D1 expression. The activation of ER-β-catenin signaling rescues RK3E cells from anoikis and correlates with an increased phosphorylation of mitogen-activated protein kinase. The inhibition of anoikis by ER-β-catenin can be abolished by a mitogen-activated protein kinase pathway inhibitor, PD98059. Evidence is also provided to show that ER-β-catenin down-regulates cadherin protein levels. These findings support a key role for activated β-catenin signaling in processes that contribute to tumor formation and progression. Wnt-1 was first identified as a proto-oncogene in mouse mammary tumors (1Nusse R. Varmus H.E. 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Although the pivotal role of β-catenin in malignant transformation is well substantiated, the cell biological consequences of β-catenin signaling are not clearly defined. To study the functions of β-catenin in a controlled fashion, we created a regulatable form of activated β-catenin by fusion of the entire protein (β-catenin S37A/S45A) to a modified estrogen receptor (ER) ligand binding domain (G525R) (59Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Google Scholar). Expression of this protein in several cell lines by transfection allows the signal transduction function of β-catenin to be induced by 4-hydroxytamoxifen (4-HT), leading to activation of a β-catenin-LEF-dependent reporter construct. Using this inducible system we demonstrate that β-catenin signaling correlates with diminished cell-substrate adhesion and can rescue RK3E cells from anoikis by a process that appears to involve MAP kinase activation. We also provide evidence that ER-β-catenin down-regulates cadherin protein levels. These findings support a key role for enhanced β-catenin signaling in processes that contribute to tumor formation and progression. Human β-catenin was first amplified from a cDNA library by PCR, and site-directed mutagenesis was then used to generate the activated mutant form of β-catenin (S37A/A45A). Restriction sites were engineered into PCR primers which allowed fusion of the modified hormone binding domain of the murine estrogen receptor (ER-HBD) in-frame with β-catenin S37A/A45A. To generate ER-β-catenin, ER-HBD G525R was amplified by PCR and cloned into an in-house mammalian expression vector under the control of the human cytomegalovirus enhancer and promoter at the XbaI-BamHI sites. The human β-catenin S37A/A45A was also amplified by PCR and fused in-frame at the C terminus of ER-HBD using the restriction sites ClaI and BamHI. A similar strategy was used to generate β-catenin-ER. Both constructs were sequenced to ensure that no mutations had been introduced into the constructs during PCR amplification. Both chimeras were expressed with an N-terminal epitope tag encoding a 16-amino acid portion of the Haemophilus influenzae hemagglutinin (HA) gene. A β-catenin-LEF-responsive reporter gene was constructed by linking the coding sequences for a firefly luciferase gene to the interleukin-2 minimal promoter following five copies of LEF binding sites. For the control vector used to normalize the transfections and reporter assays the Renilla luciferase gene is under the control of a constitutive thymidine kinase promoter (Promega, Madison, WI). 293T cells were obtained from GenHunter Corporation (Nashville, TN), and RK3E cells were purchased from American Type Culture Collection. Cells were cultured in Dulbecco's modified Eagle's medium (with 4.5 g/liter glucose) supplemented with 10% fetal bovine serum. 105 cells were seeded into each well of a 12-well plate and transfected ∼18 h later. RK3E cells were transfected with 1 μg of DNA using FuGENE 6 (Roche Molecular Biochemicals); 293T cells were transfected with 2 μg of DNA using Effectene (Qiagen) according to the manufacturer's recommendation. For generation of stable transfected cell lines, RK3E cells were transfected with the expression plasmid encoding ER-β-catenin along with a selection plasmid pcDNA3.1 (−) (Invitrogen) encoding a neomycin resistance gene at a ratio of either 10:1 or 50:1 (ER-β-catenin:pcDNA3.1). Two days later, the cells were split at a 1:10 dilution, and the following day, the standard culture medium was replaced with standard medium containing either 300 or 800 μg/ml Geneticin (Invitrogen). The selective medium was changed every 3–4 days, and 2 weeks later 60 clones were picked and expanded. To select for clones expressing ER-β-catenin, the clonal lines were transfected individually with the β-catenin-LEF reporter plasmid, cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum in the presence or absence of 1 μm 4-HT (Sigma) for 2 days and then analyzed using a dual luciferase assay to identify those with optimal basal and induced β-catenin activity. A dual luciferase assay was carried out according to the manufacturer's suggestions (Promega). RK3E and 293T cells were transfected with test plasmids of interest along with the β-catenin-LEF firefly luciferase reporter plasmid and the thymidine kinase-Rennila luciferase control plasmid. Two days post-transfection, the cells were harvested and assayed for firefly and Renilla luciferase activities using the Stop and Glow assay (Promega) according to the manufacturer's suggestions. Briefly, the cells were lysed with 1× passive lysis buffer, and 10 μl of each cell lysate sample was transferred to a 96-well plate. 100 μl of luciferase assay reagent II was first injected to each well to measure the firefly luciferase activity followed by injection of 100 μl of Stop and Glow reagent for the measurement of the Renilla luciferase activities using a MicroLumat LB96P (Wallac). RK3E cells expressing ER-β-catenin (clones 27 and 96) and a control clone with no expression of ER-β-catenin were grown with or without 1 μm 4-HT for 2 days. Cell extracts were prepared using RIPA lysis buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 50 mm Tris-HCl, pH 8.0) supplemented with appropriate amounts of complete protease inhibitor mixture tablets (Roche). Approximately 10 μg of each sample was resolved in a Tris-glycine polyacrylamide gel (Novex), transferred to nitrocellulose followed by Western blot analysis using antisera directed against phospho-specific MAP kinase (New England Biolab), MAP kinase (New England Biolab), E-cadherin (Transduction Laboratories), β-catenin (Transduction Laboratories), HA tag (Covance), vimentin (Lab Vision Corporation), and keratin (Lab Vision Corporation) as described previously (60Weng Z. Thomas S.M. Rickles R.J. Taylor J.A. Brauer A.W. Seidel-Dugan C. Michael W.M. Dreyfuss G. Brugge J.S. Mol. Cell. Biol. 1994; 14: 4509-4521Crossref PubMed Scopus (205) Google Scholar). For immunoprecipitation, RK3E cells expressing ER-β-catenin (clones 27 and 96) and a control clone with no expression of ER-β-catenin were grown with or without 1 μm 4-HT for 2 days. Cell extracts were prepared using Nonidet P-40 lysis buffer (137 mm NaCl, 1% Nonidet P-40, 10% glycerol, and 20 mm Tris-HCl, pH 7.4). After centrifugation at 15,000 × g for 15 min, the supernatant was collected as the Nonidet P-40-soluble fraction. The pellet of each sample was then washed three times with Nonidet P-40 lysis buffer and solubilized in RIPA buffer and analyzed as the Nonidet P-40-insoluble fraction. Approximately 250 μg of the Nonidet P-40-soluble fraction was precipitated with 5 μg of anti-E-cadherin (Transduction Laboratories) or anti-α-catenin (Santa Cruz Biotechnology) antibodies in a total volume of 1.2 ml. After incubation at 4 °C for 2 h, 50 μl of γ-bind beads (AmershamBiosciences) was added to each sample. After a 1-h incubation at 4 °C, the beads were washed five times with Nonidet P-40 lysis buffer plus 1% Triton X-100 (Sigma). Western blot analysis was performed as described above. An RK3E cell line expressing ER-β-catenin (clone 27) and a control RK3E cell line (clone 152) with no expression of ER-β-catenin were first grown in eight-well Lab-Tek II chamberslides (Nalgen Nunc International) on coverslips to allow cell attachment overnight and then cultured in standard growth medium with or without 1 μm 4-HT for either 1 or 2 days. The cells were then fixed with ice-cold methanol:acetone (1:1) at –20 °C for 10 min. After incubation in blocking buffer (1% bovine serum albumin in phosphate-buffered saline) for 10 min, the cells were incubated with antiserum against β-catenin (1:500, Transduction Laboratories), E-cadherin (1:50, Transduction Laboratories) and HA (1:50, Covance) diluted in blocking buffer for 45 min at room temperature. After washing, the coverslips were then incubated with fluorescein isothiocyanate-conjugated donkey anti-mouse antibodies (Jackson Immune Research) at a dilution of 1:200 for 45 min at room temperature. After brief washing, the localization of β-catenin was visualized by fluorescence microscopy. Control RK3E cells (clone 4) or ER-β-catenin-expressing RK3E cells (clone 27) were grown with or without 4-HT treatment for 1 or 2 days. RNA was prepared (RNeasy mini kit from Qiagen), and contaminating genomic DNA was removed by DNase I treatment. The expression of cyclin D1, a gene known to be transcriptionally activated by β-catenin signaling (53Tetsu O. McCormick F. Nature. 1999; 398: 422-426Crossref PubMed Scopus (3129) Google Scholar, 54Shtutman M. Zhurinsky J. Simcha I. Albanese C. D'Amico M. Pestell R. Ben-Ze'ev A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5522-5527Crossref PubMed Scopus (1812) Google Scholar), was measured in the various RNA samples by TaqMan quantitative PCR. Specific oligonucleotide primers and a fluorogenic probe were designed using Primer Express (Applied Biosystems). Relative expression levels, normalized to the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene, were calculated using the standard curves method as described by the manufacturer of the TaqMan instrument (Applied Biosystems). 12-well tissue culture plates were coated with 200 μl (6 mg/ml in 95% ethanol) of poly-(2-hydroxyethyl methacrylate) (poly-HEME; Sigma) by incubation overnight at 37 °C. To perform the anoikis assay, control or ER-β-catenin-expressing RK3E cells were trypsinized into a single cell suspension, and 2.5 ml was cultured in poly-HEME-coated plates at a density of ∼105 cells/ml (total of 2.5 × 105 cells) in the absence or presence of 1 μm4-HT in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. As a positive control, parental RK3E cells were infected with a retroviral construct coexpressing β-catenin S37A/A45A and a marker green fluorescence protein. Five days after infection the cells were harvested and plated as described above in poly-HEME-coated plates. Suspended cells were incubated at 37 °C for ∼18 h. Cells were then harvested, washed, and stained with annexin V-phycoerythrin antibodies (PharMingen) and analyzed by flow cytometry using FACScalibur (Becton Dickinson). Because the pool of positive control cells infected with the β-catenin S37A/A45A-expressing retrovirus contained both expressing and nonexpressing cells, the percentage of viable RK3E cells that expressed β-catenin S37A/A45A (green fluorescence protein-positive population) was compared with the percentage of viable RK3E cells that did not express β-catenin S37A/A45A (green fluorescence protein-negative population). To study the role of activated β-catenin in signal transduction and cellular transformation we developed a system in which the activity of β-catenin could be rapidly and conditionally regulated. For this purpose an ER fusion strategy was utilized where the HBD of ER is fused to a protein of interest, thereby creating an estrogen-regulated protein activity (61Mattioni T. Louvion J.F. Picard D. Methods Cell Biol. 1994; 43: 335-352Crossref PubMed Google Scholar). These ER fusion proteins are generally inactive and can be induced by estrogen or synthetic steroids such as 4-HT. Although not applicable to all proteins, such a strategy has been successful in generating a variety of functionally hormone-dependent proteins including cytoplasmic enzymes (Src, Raf, and MAP kinase kinase) (62Aziz N. Cherwinski H. McMahon M. Mol. Cell. Biol. 1999; 19: 1101-1115Crossref PubMed Scopus (51) Google Scholar, 63Samuels M.L. Weber M.J. Bishop J.M. McMahon M. Mol. Cell. Biol. 1993; 13: 6241-6252Crossref PubMed Google Scholar) and transcription factors (Myc and LEF) (64Eilers M. Picard D. Yamamoto K.R. Bishop J.M. Nature. 1989; 340: 66-68Crossref PubMed Google Scholar, 65Aoki M. Hecht A. Kruse U. Kemler R. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 139-144Crossref PubMed Scopus (145) Google Scholar, 66Hermeking H. Eick D. Science. 1994; 265: 2091-2093Crossref PubMed Google Scholar). ER-β-catenin fusion constructs were made by designing expression vectors in which sequences encoding an activated form of β-catenin, with a serine to alanine substitution at positions 37 and 45 (β-catenin S37A/A45A), were linked in-frame to sequences encoding a modified ER HBD (ER-G525R), which is unresponsive to estrogen yet can still be specifically activated by a synthetic estrogen, 4-HT (59Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Google Scholar). Previous studies have shown that the altered specificity of ER-G525R can prevent constitutive activation of ER fusion proteins by estrogen and/or estrogen agonists present in culture media (59Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Google Scholar). Because the functional effects of creating a hybrid protein between ER and β-catenin were unknown, two different chimeras were generated to place the ER sequences at either the N terminus (ER-β-catenin) or at the C terminus (β-catenin-ER) of β-catenin. Both versions were engineered to contain an N-terminal influenza HA epitope tag and were subcloned into a mammalian expression vector with a cytomegalovirus promoter-enhancer (Fig. 1A). To verify protein expression from these constructs, 293T cells were transfected using standard methods, and 2 days later cell extracts were examined by Western blot analysis using anti-HA antibody. Both constructs expressed β-catenin fusion proteins of expected size (Fig. 1B). To determine whether the ER-β-catenin and β-catenin-ER fusion proteins still retained transcriptional activity that could now be regulated by 4-HT, these proteins were analyzed using a dual luciferase reporter assay. A β-catenin-LEF-responsive reporter gene, similar to those described by others (67Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2814) Google Scholar), was constructed which consists of a firefly luciferase gene under the control of an interleukin-2 minimal promoter following five copies of a consensus LEF binding site. A control reporter plasmid was employed which expresses a Renilla luciferase gene whose expression is driven by a constitutive thymidine kinase promoter for transfection normalization (Fig. 2A). RK3E cells, which contain components require