The T-box transcription factor Brachyury promotes epithelial-mesenchymal transition in human tumor cells

Abstract

Metastatic disease is responsible for the majority of human cancer deaths. Understanding the molecular mechanisms of metastasis is a major step in designing effective cancer therapeutics. Here we show that the T-box transcription factor Brachyury induces in tumor cells epithelial-mesenchymal transition (EMT), an important step in the progression of primary tumors toward metastasis. Overexpression of Brachyury in human carcinoma cells induced changes characteristic of EMT, including upregulation of mesenchymal markers, downregulation of epithelial markers, and an increase in cell migration and invasion. Brachyury overexpression also repressed E-cadherin transcription, an effect partially mediated by Slug. Conversely, inhibition of Brachyury resulted in downregulation of mesenchymal markers and loss of cell migration and invasion and diminished the ability of human tumor cells to form lung metastases in a xenograft model. Furthermore, we found Brachyury to be overexpressed in various human tumor tissues and tumor cell lines compared with normal tissues. We also determined that the percentage of human lung tumor tissues positive for Brachyury expression increased with the stage of the tumor, indicating a potential association between Brachyury and tumor progression. The selective expression of Brachyury in tumor cells and its role in EMT and cancer progression suggest that Brachyury may be an attractive target for antitumor therapies.

Figure 1. Brachyury alters EMT marker expression and induces a highly migratory and invasive phenotype in human epithelial tumor cells.

(A) Bright-field images of PANC-1-pcDNA and PANC-1–pBrachyury cells grown on a plastic surface (top 2 panels: original magnification, ×10) and immunofluorescence analysis of EMT markers in cells grown on cover glasses (bottom panels: original magnification, ×40) are shown. The green signal represents the staining of the corresponding protein, and the blue signal represents the DAPI-stained nuclei. (B) Western blot analysis of Brachyury and EMT markers. β-Actin is shown as the protein loading control. (C) Real-time PCR analysis of cDNA from the above cells for Brachyury and EMT markers. (D) In vitro cell migration and ECM invasion assays. Results of 1 of 3 experiments are shown. Error bars indicate SEM of triplicate measurements. *P < 0.05, **P < 0.001, ***P < 0.0001 for pcDNA versus pBrachyury.

Figure 2. Control of E-cadherin expression in PANC-1–pBrachyury cells.

Expression of Brachyury, Snail, Slug, and E-cadherin was analyzed by real-time PCR in PANC-1-pcDNA versus PANC-1–pBrachyury cells that were untransfected (A) or transiently transfected with various single (B) or combined (C) siRNAs, as indicated. The experiment was repeated twice, with comparable results; data from 1 experiment are shown. Error bars indicate SEM of triplicate measurements.

Figure 3. Brachyury suppresses E-cadherin promoter activity.

(A) Relative E-cadherin promoter activity compared with the control for each cell line. Results from 1 of 3 experiments are shown; **P < 0.05, ***P < 0.001. Shown is a schematic representation of the reporter construct. (B) EMSA assay with recombinant His-Brachyury protein and a labeled fragment from the E-cadherin promoter. Supershift assay was performed with anti-Brachyury antibody versus control IgG. (C) Proposed model for E-cadherin control by Brachyury (Brachy).

Figure 4. Brachyury inhibition reduces expression of mesenchymal markers, migration, and invasion of human lung carcinoma cells.

(A) Bright-field images of H460-con.shRNA and H460-Br.shRNA cells grown on a plastic surface (top 2 panels: original magnification, ×10) and immunofluorescence analysis of EMT markers (bottom panels: original magnification, ×20) are shown. The green signal represents the staining of the corresponding protein, and the blue signal represents the DAPI-stained nuclei. (B) Western blot analysis of Brachyury and EMT markers. β-Actin is shown as a protein loading control. (C) Real-time PCR analysis of cDNA for Brachyury and EMT markers. (D) In vitro cell migration and ECM invasion assays. Results from 1 of 3 experiments are shown. Error bars indicate SEM of triplicate measurements. *P < 0.05, **P < 0.001, ***P < 0.0001 for con.shRNA versus Br.shRNA.

Figure 5. H460-Br.shRNA cells rescued by Brachyury or Slug overexpression.

(A) Bright field images of cells grown on plastic surface (top: original magnification, ×10) and immunofluorescence analysis of Plakoglobin (bottom: original magnification, ×20) are shown. The green signal represents the staining of the corresponding protein, and the blue signal represents the DAPI-stained nuclei. (B) Real-time PCR analysis for Brachyury and EMT markers and (C) cell migration and ECM invasion assay with H460-Br.shRNA coexpressing empty vector (pcDNA) or a human Brachyury-encoding vector (pBrachyury). (D) Expression of Brachyury and EMT markers and (E) cell migration and invasion assays with H460-Br.shRNA cells transiently transfected with control (pcDNA) or a human Slug-expressing vector (pSLUG). Error bars indicate SEM of triplicate measurements. *P < 0.05, **P < 0.01, ***P < 0.0001 for pcDNA versus pBrachyury or pSLUG.

Figure 6. Brachyury inhibition reduces tumor metastasis.

(A) H460 cells stably transfected with con.shRNA (circles) or Br.shRNA vectors (triangles) were assessed for cell proliferation and survival by MTT assay. Three independent experiments showed comparable results; results from 1 experiment are shown. ***P = 0.0003 for con.shRNA versus Br.shRNA. Western blot shows cyclin D1 and β-actin expression. (B) Subcutaneous tumors were induced by injection of 4 × 10⁶ of H460 cells in HBSS admixed with 50% (v/v) Matrigel. Graph shows volumes from 8 tumors at day 15 after tumor implantation. (C) cDNA from tumors in B were analyzed by real-time PCR for indicated markers. (D) Lungs from animals bearing subcutaneous tumors were collected at day 15 after tumor implantation, homogenized, and cultured in puromycin-containing medium. Graph shows visible colony counts. (E) Mice were inoculated with 7.5 × 10⁵ H460 cells transfected as indicated via tail vein. Forty-five days after tumor implantation, animals were euthanized, and lungs were evaluated for tumor nodules. Graph shows results from 3 independent experiments. Experiments 1–3 are denoted by black, gray, and white circles (con.shRNA) and triangles (Br.shRNA), respectively. Statistical difference between treatment groups was analyzed by pooled results of the above experiments. Two representative lungs from each group are shown for comparison. White outlines and black arrowheads indicate tumor masses.

Figure 7. Brachyury expression in human lung tumor tissues.

Real-time PCR was performed for Brachyury on lung tumor tissue cDNA from 80 lung cancer patients of the indicated stages of disease. The stage II, III, and IV cDNA samples are further represented by squares, triangles, and ×’s, respectively. As controls, 16 samples of normal lung cDNA were also analyzed, each obtained from a pathologically normal section of lung from a cancer patient. All values and the medians for each group are expressed as a ratio to the endogenous control GAPDH.