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Molecular Cancer Therapeutics
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An Autocrine Loop between TGF-β1 and the Transcription Factor Brachyury Controls the Transition of Human Carcinoma Cells into a Mesenchymal Phenotype

Cecilia Larocca, Joseph R. Cohen, Romaine I. Fernando, Bruce Huang, Duane H. Hamilton and Claudia Palena
Cecilia Larocca
1Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, NIH; and 2HHMI-NIH Research Scholar, Bethesda, Maryland
1Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, NIH; and 2HHMI-NIH Research Scholar, Bethesda, Maryland
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Joseph R. Cohen
1Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, NIH; and 2HHMI-NIH Research Scholar, Bethesda, Maryland
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Romaine I. Fernando
1Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, NIH; and 2HHMI-NIH Research Scholar, Bethesda, Maryland
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Bruce Huang
1Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, NIH; and 2HHMI-NIH Research Scholar, Bethesda, Maryland
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Duane H. Hamilton
1Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, NIH; and 2HHMI-NIH Research Scholar, Bethesda, Maryland
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Claudia Palena
1Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, NIH; and 2HHMI-NIH Research Scholar, Bethesda, Maryland
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DOI: 10.1158/1535-7163.MCT-12-1007 Published September 2013
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    Figure 1.

    TGF-β1 induces Brachyury and EMT in human carcinoma cells. A, bright-field images of DU145 cells left untreated or treated for 72 hours with increasing concentrations of TGF-β1. Expression of E-cadherin and fibronectin in DU145 cells treated with TGF-β1 for 72 hours by (B) real-time PCR or (C) Western blot analysis. D, Brachyury expression in DU145 cells treated with TGF-β1 by real-time PCR or (E) Western blot analysis. Real-time PCR analysis of Brachyury expression in human prostate (F) and lung cancer (G) cell lines treated with indicated concentrations of TGF-β1 for 72 hours. Error bars indicate SEM for triplicate measurements; *, P < 0.05; **, P < 0.005; ***, P < 0.001.

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    Figure 2.

    TGF-β1–mediated Brachyury upregulation is relevant to tumor invasiveness. A, immunofluorescent analysis of Brachyury expression in DU145 cells treated as indicated for 72 hours. Green corresponds to Brachyury expression; blue indicates 4′, 6-diamidino-2-phenylindole (DAPI)-stained nuclei (magnification, ×40). B, DU145 cells treated as indicated for 48 hours were evaluated for cell migration and invasion. C, DU145 cells were left untransfected or transfected with control- versus Brachyury-specific siRNA for 24 hours and subsequently left untreated or treated for 48 hours with TGF-β1. At the end of the cytokine treatment, cells were harvested for Brachyury mRNA analysis or (D) invasion assays. E, real-time PCR analysis of Brachyury, Snail, and Slug in DU145 cells treated for 72 hours as indicated. Expression of Snail and Slug by real-time PCR (F) or Western blot analysis (G) in DU145 cells untransfected versus cells transfected with control- versus Brachyury-specific siRNA and subsequently incubated with TGF-β1 for 48 hours. Error bars indicate SEM for triplicate measurements. *, P < 0.05; **, P < 0.005; ***, P < 0.001, ****; P < 0.0001.

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    Figure 3.

    Brachyury induces TGF-β secretion. ELISA for TGF-β1 in culture supernatants of human cancer cell pairs obtained by (A) Brachyury overexpression in DU145 cells (pBrachyury) or (B) via stable silencing with a Brachyury-specific shRNA (Brac.shRNA) in SW620 and H460 cells. C, levels of TGF-β1 secreted by H460 cells silenced for Brachyury expression and subsequently transfected with an empty vector (pcDNA) or a pBrachyury vector. D, immunohistochemical analysis of TGF-β1 in xenografts of H460 control.shRNA versus H460 Brachyury.shRNA cells. Brown corresponds to TGF-β1 expression; slides were counterstained with hematoxylin. (magnification: left, ×10; right, ×20). E, secretion of TGF-β2 in culture supernatants of indicated tumor cells. Error bars indicate SEM for triplicate measurements. *, P < 0.05; **, P < 0.005; ***, P < 0.001.

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    Figure 4.

    Brachyury activates TGF-β1 promoter transcription. A and B, real-time PCR analysis of TGF-β1 in tumor cells with various levels of Brachyury. C, TGF-β1 promoter reporter activity normalized to GAPDH promoter in H460 control.shRNA versus Brachyury.shRNA. Error bars indicate SEM for triplicate measurements. *, P < 0.05; **, P < 0.005.

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    Figure 5.

    SD-208 reverses TGF-β1–induced EMT. A, chemical structure of SD-208. B, expression of E-cadherin, fibronectin, and Brachyury mRNA in DU145 cells untreated (−) or pretreated with SD-208 for 30 minutes followed by treatment with TGF-β1 for 72 hours. C, Western blot analysis of total Smad-2, phospho-Smad-2, and β-actin in protein lysates from DU145 cells pretreated with or without SD-208 for 30 minutes in the presence or absence of TGF-β1 for an additional 30 minutes. D, cell migration and ECM invasion assay with DU145 cells treated with TGF-β1 for 48 hours in the absence or presence of indicated doses of SD-208. Results from a representative experiment are shown. E, Western blot analysis for ZO-1, fibronectin, and β-actin in H460 cells treated for 72 hours as indicated. F, immunofluorescent analysis of Plakoglobin and fibronectin (green) in H460 cells; blue corresponds to nuclei stained with DAPI (original magnification, ×40). G, cell migration and ECM invasion assay with H460 cells treated as indicated for 48 hours. Error bars indicate SEM for triplicate measurements; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. phospho-Smad-2, P-Smad-2.

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    Figure 6.

    SD-208 decreases Brachyury expression and enhances chemotherapy sensitivity of human carcinoma cells. A, H460 cells were treated with increasing concentrations of SD-208 for 72 hours and evaluated for Brachyury and β-actin expression by Western blot analysis. Bottom graph shows Brachyury levels normalized to β-actin. B, chemical structure of chemotherapeutics used. C, H460 cells were left untreated (0) or pretreated for 48 hours with indicated concentrations of SD-208 and subsequently exposed to chemotherapeutics (165/0.12 ng/mL cisplatin/vinorelbine, 165 ng/mL cisplatin, 0.12 ng/mL vinorelbine, and 0.07 ng/mL docetaxel). Survival was evaluated after 4 days by the MTT assay. D, expression of Brachyury and GAPDH in indicated tumor cells incubated for 48 hours with increasing doses of SD-208. E, survival of H460 control.shRNA and Brachyury.shRNA cells pretreated with indicated doses of SD-208 and subsequently treated as above with cisplatin/vinorelbine. Cis, cisplatin; vin, vinorelbine (ng/mL).

Additional Files

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    • Supplementary Figure 1 - PDF file - 89K, TGF-β1 secretion in LNCap and H520 cells.
    • Supplementary Figure 2 - PDF file - 326K, Treatment with SD-208 reverts the mesenchymal phenotype of colon carcinoma cell lines.
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Molecular Cancer Therapeutics: 12 (9)
September 2013
Volume 12, Issue 9
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An Autocrine Loop between TGF-β1 and the Transcription Factor Brachyury Controls the Transition of Human Carcinoma Cells into a Mesenchymal Phenotype
Cecilia Larocca, Joseph R. Cohen, Romaine I. Fernando, Bruce Huang, Duane H. Hamilton and Claudia Palena
Mol Cancer Ther September 1 2013 (12) (9) 1805-1815; DOI: 10.1158/1535-7163.MCT-12-1007

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An Autocrine Loop between TGF-β1 and the Transcription Factor Brachyury Controls the Transition of Human Carcinoma Cells into a Mesenchymal Phenotype
Cecilia Larocca, Joseph R. Cohen, Romaine I. Fernando, Bruce Huang, Duane H. Hamilton and Claudia Palena
Mol Cancer Ther September 1 2013 (12) (9) 1805-1815; DOI: 10.1158/1535-7163.MCT-12-1007
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