Slit-2 Induces a Tumor-suppressive Effect by Regulating β-Catenin in Breast Cancer Cells
2008; Elsevier BV; Volume: 283; Issue: 39 Linguagem: Inglês
10.1074/jbc.m800679200
ISSN1083-351X
AutoresAnil Prasad, Vikram Paruchuri, Anju Preet, Farida Latif, Ramesh K. Ganju,
Tópico(s)Hedgehog Signaling Pathway Studies
ResumoSLIT-2 is considered as a candidate tumor suppressor gene, because it is frequently inactivated in various cancers due to hypermethylation of its promoter region and allelic loss. However, the exact mechanism of its tumor-suppressive effect has not been elucidated. Here, we observed that Slit-2-overexpressing breast cancer cells exhibited decreased proliferation and migration capabilities compared with control cells under in vitro conditions. These results were confirmed in vivo in mouse model systems. Mice injected with MCF-7/Slit-2 cells showed a 60–70% reduction in tumor size compared with mice injected with MCF-7/VC cells both in the absence and presence of estrogen. Upon further elucidation, we observed that Slit-2 mediates the tumor-suppressive effect via a coordinated regulation of the β-catenin and PI3K signaling pathways and by enhancing β-catenin/E-cadherin-mediated cell-cell adhesion. Our study for the first time reveals that Slit-2-overexpressing breast cancer cells exhibit tumor suppressor capabilities through the novel mechanism of β-catenin modulation. SLIT-2 is considered as a candidate tumor suppressor gene, because it is frequently inactivated in various cancers due to hypermethylation of its promoter region and allelic loss. However, the exact mechanism of its tumor-suppressive effect has not been elucidated. Here, we observed that Slit-2-overexpressing breast cancer cells exhibited decreased proliferation and migration capabilities compared with control cells under in vitro conditions. These results were confirmed in vivo in mouse model systems. Mice injected with MCF-7/Slit-2 cells showed a 60–70% reduction in tumor size compared with mice injected with MCF-7/VC cells both in the absence and presence of estrogen. Upon further elucidation, we observed that Slit-2 mediates the tumor-suppressive effect via a coordinated regulation of the β-catenin and PI3K signaling pathways and by enhancing β-catenin/E-cadherin-mediated cell-cell adhesion. Our study for the first time reveals that Slit-2-overexpressing breast cancer cells exhibit tumor suppressor capabilities through the novel mechanism of β-catenin modulation. Slit-2, a protein that belongs to the Slit family of large extracellular matrix-secreted glycoproteins, has been shown to exhibit tumor-suppressive effects in various human cancers (1Brose K. Bland K.S. Wang K.H. Arnott D. Henzel W. Goodman C.S. Tessier-Lavigne M. Kidd T. Cell. 1999; 96: 795-806Abstract Full Text Full Text PDF PubMed Scopus (962) Google Scholar, 2Sundaresan V. Chung G. Heppell-Parton A. Xiong J. Grundy C. Roberts I. James L. Cahn A. Bench A. Douglas J. Minna J. Sekido Y. Lerman M. Latif F. Bergh J. Li H. Lowe N. Ogilvie D. Rabbitts P. 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Smith K. Holmes G.P. Chenevix-Trench G. Ioannou P.A. Little M.H. Cytogenet. Cell Genet. 1999; 86: 246-247Crossref PubMed Google Scholar). Slit consists of a family of 3 genes (SLIT-1, SLIT-2, and SLIT-3), and these genes are candidate ligands for the repulsive guidance receptors, members of the ROBO gene family (1Brose K. Bland K.S. Wang K.H. Arnott D. Henzel W. Goodman C.S. Tessier-Lavigne M. Kidd T. Cell. 1999; 96: 795-806Abstract Full Text Full Text PDF PubMed Scopus (962) Google Scholar, 9Kidd T. Bland K.S. Goodman C.S. Cell. 1999; 96: 785-794Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar, 10Yuan W. Zhou L. Chen J.H. Wu J.Y. Rao Y. Ornitz D.M. Dev. Biol. 1999; 212: 290-306Crossref PubMed Scopus (253) Google Scholar, 11Li H.S. Chen J.H. Wu W. Fagaly T. Zhou L. Yuan W. Dupuis S. Jiang Z.H. Nash W. Gick C. Ornitz D.M. Wu J.Y. Rao Y. Cell. 1999; 96: 807-818Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 12Piper M. Georgas K. Yamada T. 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These studies suggest that Slit/Robo may be involved in the regulation of organogenesis. epidermal growth factor Roundabout-1 phosphatidylinositol 3-kinase Dulbecco's modified Eagle' medium small interference RNA phosphate-buffered saline T-cell factor lymphoid enhancer factor vector control glycogen synthase kinase-3β severe combined immunodeficiency disease 4′,6-diamidino-2-phenylindole. epidermal growth factor Roundabout-1 phosphatidylinositol 3-kinase Dulbecco's modified Eagle' medium small interference RNA phosphate-buffered saline T-cell factor lymphoid enhancer factor vector control glycogen synthase kinase-3β severe combined immunodeficiency disease 4′,6-diamidino-2-phenylindole. SLIT-2 promoter region hypermethylation has been detected in various cancers such as breast cancer, non-small cell lung cancer, small cell lung cancer, colorectal carcinoma, and gliomas (2Sundaresan V. Chung G. Heppell-Parton A. Xiong J. Grundy C. Roberts I. James L. Cahn A. Bench A. Douglas J. Minna J. Sekido Y. Lerman M. Latif F. Bergh J. Li H. Lowe N. Ogilvie D. Rabbitts P. Oncogene. 1998; 17: 1723-1729Crossref PubMed Scopus (92) Google Scholar, 3Dallol A. Da Silva N.F. Viacava P. Minna J.D. Bieche I. Maher E.R. Latif F. Cancer Res. 2002; 62: 5874-5880PubMed Google Scholar, 4Dallol A. Krex D. Hesson L. Eng C. Maher E.R. Latif F. Oncogene. 2003; 22: 4611-4616Crossref PubMed Scopus (115) Google Scholar, 5Dallol A. Morton D. Maher E.R. Latif F. Cancer Res. 2003; 63: 1054-1058PubMed Google Scholar, 6Dickinson R.E. Dallol A. Bieche I. Krex D. Morton D. Maher E.R. Latif F. Br. J. Cancer. 2004; 91: 2071-2078Crossref PubMed Scopus (115) Google Scholar, 7Astuti D. Da Silva N.F. Dallol A. Gentle D. Martinsson T. Kogner P. Grundy R. Kishida T. Yao M. Latif F. Maher E.R. Br. J. Cancer. 2004; 90: 515-521Crossref PubMed Scopus (51) Google Scholar, 28Werbowetski-Ogilvie T.E. Seyed Sadr M. Jabado N. Angers-Loustau A. Agar N.Y. Wu J. Bjerkvig R. Antel J.P. Faury D. Rao Y. Del Maestro R.F. Oncogene. 2006; 25: 5103-5112Crossref PubMed Scopus (59) Google Scholar). It has been also shown that, in various cancer types, Robo expression is also altered (19Xian J. Clark K.J. Fordham R. Pannell R. Rabbitts T.H. Rabbitts P.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15062-15066Crossref PubMed Scopus (134) Google Scholar, 29Grone J. Doebler O. Loddenkemper C. Hotz B. Buhr H.J. Bhargava S. Oncol. Rep. 2006; 15: 1437-1443PubMed Google Scholar). This indicates that Slit/Robo signaling may play important roles in cancer development. Mutation studies and expression analysis of the SLIT-2 gene have revealed loss of heterozygosity, a couple of missense substitutions in one of the EGF-like domains, and rearrangement at its genomic locus (4p15.2) in a significant proportion of human cancers (3Dallol A. Da Silva N.F. Viacava P. Minna J.D. Bieche I. Maher E.R. Latif F. Cancer Res. 2002; 62: 5874-5880PubMed Google Scholar, 4Dallol A. Krex D. Hesson L. Eng C. Maher E.R. Latif F. Oncogene. 2003; 22: 4611-4616Crossref PubMed Scopus (115) Google Scholar, 5Dallol A. Morton D. Maher E.R. Latif F. Cancer Res. 2003; 63: 1054-1058PubMed Google Scholar, 6Dickinson R.E. Dallol A. Bieche I. Krex D. Morton D. Maher E.R. Latif F. Br. J. Cancer. 2004; 91: 2071-2078Crossref PubMed Scopus (115) Google Scholar). Moreover, primary breast tumors and a majority of breast tumor cell lines have been reported to exhibit reduced or absent SLIT-2 expression (3Dallol A. Da Silva N.F. Viacava P. Minna J.D. Bieche I. Maher E.R. Latif F. Cancer Res. 2002; 62: 5874-5880PubMed Google Scholar). Furthermore, analysis of the Slit-2 promoter region in these tumor cells showed the presence of extensive hypermethylation of the SLIT-2 5′-cytosine-guanine (CpG) island (3Dallol A. Da Silva N.F. Viacava P. Minna J.D. Bieche I. Maher E.R. Latif F. Cancer Res. 2002; 62: 5874-5880PubMed Google Scholar). Lack or reduced expression of SLIT-2 was correlated with CpG hypermethylation, and further treatment with the demethylating agent 5′-azacytidine restored SLIT-2 expression (3Dallol A. Da Silva N.F. Viacava P. Minna J.D. Bieche I. Maher E.R. Latif F. Cancer Res. 2002; 62: 5874-5880PubMed Google Scholar). In addition, Slit-2-overexpressing breast cancer cells or Slit-2-treated breast cancer cells showed decreased colony formation (3Dallol A. Da Silva N.F. Viacava P. Minna J.D. Bieche I. Maher E.R. Latif F. Cancer Res. 2002; 62: 5874-5880PubMed Google Scholar). Although these studies indicate that SLIT-2 can act as a tumor suppressor gene, its tumor-suppressive effect under in vivo conditions and the exact mechanisms of its anti-tumor property are not yet known. 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Cell Lines—The Slit-2-overexpressing MCF-7 (MCF-7/Slit-2) stable cell line and vector control cell line (MCF-7/VC) were provided by Dr. Farida Latif (University of Birmingham, Birmingham, UK). These cells were maintained in DMEM containing 10% fetal bovine serum, 50 μg/ml Zeocin, and 1% penicillin-streptomycin at 37 °C in 5% CO2 (3Dallol A. Da Silva N.F. Viacava P. Minna J.D. Bieche I. Maher E.R. Latif F. Cancer Res. 2002; 62: 5874-5880PubMed Google Scholar). MDA-MB-231 cells were generously provided by Hava Avraham (Beth Israel Deaconess Medical Center, Boston) and maintained in DMEM with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in 5% CO2. Microarray Analysis—Total RNA was collected from both MCF-7/Slit-2 and MCF-7/VC cell lines by using TRIzol reagent (Invitrogen), per the manufacturer's protocol. Microarray analysis was performed at the Beth Israel Deaconess Medical Center genomics core facility by using an Affymetrix Microarray gene chip containing 40,000 human genes. Proliferation Assay—Cells (5 × 103), seeded on 96-well plates, were cultured in DMEM supplemented with 0.2% fetal bovine serum and 50 ng/ml of EGF. The number of viable cells was quantified at various time points by using the CellTiter 96®Aqueous kit (Promega, Madison, WI), as per the manufacturer's instructions. Chemotaxis Assay—A chemotaxis assay was performed in 24-well cell culture chambers with 8-μm pore inserts, as described previously (45Prasad A. Fernandis A.Z. Rao Y. Ganju R.K. J. Biol. Chem. 2004; 279: 9115-9124Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Membranes were pre-coated with fibronectin (2.5 μg/ml). Breast cancer cells were suspended in chemotaxis buffer (DMEM/0.1% bovine serum albumin/12 mm HEPES) at 2.5 × 106 cells/ml. 150 μl of cell suspension was loaded onto the upper chamber. 0.6 ml of medium containing CXCL12 (5 nm) was then loaded onto the lower chamber. The plates were incubated for 6 h at 37 °C in 5% CO2. After incubation, the inserts were removed carefully and cells were fixed, stained, and counted using standard procedures. Soft-agar Colony Formation Assay—Cells (2 × 104) suspended in medium containing 0.4% Ultra pure agarose (Invitrogen) were layered on top of 1 ml of medium containing 0.8% agarose in 6-well culture plates. Cells were incubated with 1 ml of DMEM supplemented with 10% fetal bovine serum. After 2 weeks of incubation, the colonies were stained with 0.005% Crystal Violet for 1 h, and colonies were counted by using a Nikon Diaphot 300 inverted microscope. Immunoprecipitation and Western Blot Analysis—Immunoprecipitation and Western blot analysis were done as described elsewhere (45Prasad A. Fernandis A.Z. Rao Y. Ganju R.K. J. Biol. Chem. 2004; 279: 9115-9124Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). According to the particular experiment, equal amounts of protein (1 mg) were bound with specific antibodies. Then, the immune complexes were precipitated with Protein A-Sepharose beads (Amersham Biosciences) and were Western blotted with the relevant antibodies. Extractions of Nuclear and Cytoplasmic Fractions—Nuclear and cytoplasmic fractions were collected by using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Pierce), as per the manufacturer's protocol. The purity of fractionation was determined with Oct-1 antibody. Small Interfering RNA (siRNA)-mediated Knockdown—siRNA-mediated knockdown of β-catenin and Robo-1 was performed using β-catenin SMART pool siRNA reagents and ON-TARGETplus SMARTpool Robo-1 siRNA reagents (Dharmacon, Inc., Boulder, CO) respectively, according to the manufacturer's protocol. Briefly, MCF-7 cells were electroporated with 100 nm siRNA using the Amaxa Nucleofector™ device (Amaxa Biosystems, Cologne, Germany), as per the manufacturer's protocol. The respective non-targeted siRNA SMARTpool was used as a control. β-Catenin siRNA-mediated knockdown was estimated by detecting β-catenin expression 48 h after the initial transfection by using Western blot analysis. Robo-1 siRNA-mediated knockdown was estimated by detecting Robo-1 receptor expression 48 h after the initial transfection by using flow cytometry. Constructs and Transfections—ORF Clone of Homo sapiens slit homolog 2 (Drosophila) (SLIT2) was purchased from OriGene Technologies Inc. (Rockville, MD) and subcloned into pcDNA 3.1/V5-His Vector (Invitrogen). 2 μg of pcDNA 3.1/V5-His-SLIT-2 plasmid was mixed with the cellular suspensions, transferred to a 2.0-mm electroporation cuvette, and nucleofected using the Amaxa Nucleofector™ device (Amaxa Biosystems) as per manufacturer's protocol. Transfection efficiency was monitored by Western blotting procedures by using anti-V5 antibody (Invitrogen). Confocal Microscopy—MCF-7/VC and MCF-7/Slit-2 cells were cultured in chamber slides. Then slides were fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were washed thrice with PBS, blocked with 5% normal goat serum in PBS/Triton for 60 min, and treated with rabbit anti-β-catenin antibody (Cell Signaling Technology, Beverly, MA), and monoclonal anti-E-cadherin antibody (Chemicon, Temecula, CA) overnight at 4 °C. The slides were washed thrice with PBS and stained with Alexa Fluor 568-labeled anti-rabbit IgG antibodies, Alexa Fluor 568-labeled anti-mouse IgG antibody (Molecular Probes, Eugene, OR), or fluorescein isothiocyanate-labeled anti-rabbit IgG antibody. The cells were washed thrice in PBS, and slides were mounted by using Prolong Gold Antifade with DAPI (Invitrogen). The cells were then examined under a Zeiss confocal microscope, and the images were acquired by using LSM510 software. Nuclear staining was quantitated by using Volocity Software at the Beth Israel Deaconess Medical Center Microscopy Core Facility. Luciferase Reporter Assay—To determine luciferase reporter activity, TCF luciferase constructs (0.5 μg), containing the wild-type (pTOPFLASH) or mutant (pFOPFLASH; Upstate, Charlottesville, VA) TCF binding sites, were transfected into MCF-7/VC and MCF-7/Slit-2 cells (5 × 105 per well). Transfection experiments were carried out in triplicate using Lipofectamine 2000 (Invitrogen) following the instructions of the manufacturer. In addition, the cells were cotransfected with an internal control (0.1 μg of pRL-TK Renilla luciferase vector, Promega, Madison, WI). The cells were incubated for 48 h after the transfection and then treated with EGF (100 ng/ml) for 24 h. The cells were washed with PBS and lysed in 1× passive lysis buffer (Dual Luciferase kit, Promega). To measure the activities of firefly and Renilla luciferase, 20-μl aliquots of the supernatant were transferred into a 96-well plate and assayed in a Wallac Victor2 counter luminometer (PerkinElmer Life Sciences) using reagents from the Dual Luciferase kit (Promega). The firefly (TOPFLASH or FOPFLASH) luciferase activity was corrected for Renilla luciferase activity (pRL-TK) to control for transfection efficiency. TOPFLASH activity was also normalized to the FOPFLASH activity. Data are expressed as the mean of triplicate values of the normalized TOPFLASH activity. In Vivo Tumor Models—Female CB17/SCID and nude (nu/nu) mice, obtained from Charles River Laboratories (Wilmington, MA), were housed under specific pathogenfree conditions. The in vivo experiments were performed in accordance with the guidelines of our Institutional Animal Care and Use Committee. Cells from each stable cell line were concentrated to 3 × 106 per 200 μl and injected subcutaneously into the flank of each mouse. One group of mice was injected subcutaneously with 2.5 μg of β-estradiol 17-valerate in 50 μl of Sesame oil twice a week. Tumor size was assessed once a week, and tumor volume was calculated. At the end of the 4th week for the SCID mice and 6th week for nude mice, the animals were scanned under a Micro CT scanner (GE eXplore Locus Micro CT), and the final tumor volume was assessed. Tumors were excised, minced homogenized in modified RIPA buffer (150 mm NaCl, 50 mm Tris-HCl (pH 7.4), 1 mm EDTA, 1% Triton X-100, and a mixture protease inhibitors, Roche Applied Science), and clarified by centrifugation at 13,000 × g for 15 min at 4 °C. Supernatants were subjected to Western blot analysis. In this study, to analyze the tumor suppressor property of Slit-2 gene, we have used myc-tagged human SLIT-2 plasmid stably expressing MCF-7 breast cancer cells. Initially, we confirmed Slit-2 overexpression in two clones of Slit-2-overexpressing MCF-7 cells. Both cell lysates (Fig. 1A, upper panel) showed the overexpression of Slit-2-myc. Clone 2 exhibited more Slit-2-myc expression than clone 1. Because Slit-2 mediates its function by binding to specific receptor roundabout-1 (Robo-1), we also analyzed the expression of this receptor in control and Slit-2-overexpressing clones of MCF 7 and observed that MCF-7 cells and Slit-2-overexpressing MCF-7 cells express moderate level of Robo-1 receptor (Fig. 1A, middle panels). Because Slit-2 is a secretory protein we also confirmed its expression in cell supernatant concentrates of both clones (Fig. 1A, lower panel). Slit-2-overexpressing Cells Exhibit Inhibited Proliferation and Migration—Increased proliferation, migration, and adhesion are important properties of tumorigenesis and the metastasis of cancer cells (46Zlotnik A. Int. J. Cancer. 2006; 119: 2026-2029Crossref PubMed Scopus (338) Google Scholar). Dallol et al. (3Dallol A. Da Silva N.F. Viacava P. Minna J.D. Bieche I. Maher E.R. Latif F. Cancer Res. 2002; 62: 5874-5880PubMed Google Scholar) have shown that Slit-2-overexpressing breast cancer cells and breast cancer cells treated with Slit-2-conditioned medium have decreased colony-forming activity. When we studied the proliferation rate of Slit-2-overexpressing MCF-7 (MCF-7/Slit-2) breast cancer clones in the presence of EGF, we found that the MCF-7/Slit-2 cells showed significantly decreased proliferation as compared with the MCF-7 vector control (MCF-7/VC) cells (Fig. 1B). These clones also exhibited decreased chemotaxis toward CXCL12 (Fig. 1C). CXCL12 has been shown to play an important role in cancer metastasis. We also observed that the number and size of the colonies formed by the Slit-2-overexpressing cells were drastically reduced compared with the vector control-expressing cells (Fig. 1, D and E). These data support the notion that Slit-2-overexpression in MCF-7 cells significantly inhibits the proliferation of these cells. Consistent with Slit-2 expression, MCF-7/Slit-2 clone 2 exhibited more decreased proliferation and migration properties than clone 1, we have used clone 2 for our further experiments. Further, to analyze the role of Robo-1 in Slit-2-overexpressing MCF-7 cells, we knocked down Robo-1 by using an siRNA technique and studied the Slit-2-induced effects in MCF-7/Slit-2 cells. As shown in Fig. 2A, ∼55–60% knockdown of Robo-1 was observed in the MCF-7/Slit-2 (c2) cells transfected with the Robo-1 siRNA, as compared with cells transfected with the control (non-targeted) siRNA. We found significant increase in proliferation (Fig. 2B) of Robo-1siRNA-transfected MCF-7/Slit-2 cells compared with control-transfected cells. These results indicate that Slit-2 may function in an autocrine manner. Slit-2 Overexpression Inhibits Tumor Growth in a Xenograft Model—To further evaluate the tumor-suppressive effects of Slit-2, we used a xenograft model for tumor growth. MCF-7/Slit-2 (Clone 2) and MCF-7/VC cells (3 × 106) were injected subcutaneously into the flanks of female CB17/SCID mice. After the second week of injection, tumor volume was measured every week for up to 5 weeks. A dramatic decrease in tumor size was observed in mice, which were injected with Slit-2-overexpressing cells compared with mice injected with vector control cells (Fig. 3A). At the end of the fifth week, animals were scanned under a Micro CT scanner to calculate the exact tumor volume. Tumor volume in mice injected with the MCF-7/Slit-2 cells was ∼175–220 mm3 compared with mice injected with the MCF-7/VC cells, which had a tumor volume of ∼650–800 mm3. Because MCF-7 cells are estrogen receptor-positive, we further examined whether Slit-2 can overcome the tumor-sustaining effects of estrogen. For this study, we injected mice with 2.5 μg of β-estradiol 17-valerate twice weekly starting from 1 week prior to cell injection. Although tumor volume in mice
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