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Cancer Therapy: Preclinical

Brachyury, a Driver of the Epithelial–Mesenchymal Transition, Is Overexpressed in Human Lung Tumors: An Opportunity for Novel Interventions against Lung Cancer

Mario Roselli, Romaine I. Fernando, Fiorella Guadagni, Antonella Spila, Jhessica Alessandroni, Raffaele Palmirotta, Leopoldo Costarelli, Mary Litzinger, Duane Hamilton, Bruce Huang, Joanne Tucker, Kwong-Yok Tsang, Jeffrey Schlom and Claudia Palena
Mario Roselli
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Romaine I. Fernando
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Fiorella Guadagni
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Antonella Spila
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Jhessica Alessandroni
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Raffaele Palmirotta
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Leopoldo Costarelli
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Mary Litzinger
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Duane Hamilton
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Bruce Huang
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Joanne Tucker
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Kwong-Yok Tsang
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Jeffrey Schlom
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Claudia Palena
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DOI: 10.1158/1078-0432.CCR-11-3211 Published July 2012
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  • Figure 1.
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    Figure 1.

    Detection of Brachyury protein in lung tumor cells and tissues. Western blot analysis using anti-Brachyury mAb for detection of (A) recombinant His6-Brachyury fusion protein derived from insect cells versus purified human serum albumin (HSA; Sigma-Aldrich) or (B) Brachyury protein in H460 lung carcinoma cells stably transfected with a control- or a Brachyury-specific shRNA-encoding vector. β-Actin was used as protein loading control; shown in the graph is the ratio Brachyury/β-actin. C, representative tissue sections stained with anti-Brachyury mAb corresponding to a grade 3 adenocarcinoma, patient 2 (i); a grade 1 squamous carcinoma, patient 12 (ii); and a grade 1 bronchioloalveolar carcinoma, mucinous type, patient 16 (iii). Tumor cells positive for Brachyury expression are indicated with (t); positive cells in the stroma adjacent to the tumor are indicated with (s). iv, staining of a grade 2 lung adenocarcinoma (patient 10) showing Brachyury-positive tumor cells invading a blood vessel. Arrows indicate endothelial cells. v, staining of a grade 2 adenocarcinoma (patient 9) showing Brachyury positivity in a fraction of tumor cells (t), and cells in the stroma directly adjacent to the tumor (s). Indicated with (n) are tumor cells negative for Brachyury expression (magnification 20×: panels i, ii; 40×: panels, iii–v). D, Western blot analysis of Brachyury expression in nuclear protein lysates prepared from primary lung tumor tissue (tumor) and corresponding normal lung adjacent to the tumor from patients 7 and 17.

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    Figure 2.

    Expression of EMT markers and mechanism of Brachyury overexpression. A, representative tissue sections stained for Brachyury, E-cadherin, and Vimentin. In one example (adenocarcinoma patient 2, top), the epithelial tumor compartment (indicated with white arrowheads) showed intense expression of Brachyury and simultaneous expression of epithelial E-cadherin and mesenchymal Vimentin. Another example of an adenocarcinoma (patient 6, bottom) showed expression of Brachyury in the epithelial tumor compartment (white arrowheads), with weak expression of E-cadherin and no expression of Vimentin in the tumor cells. In both cases, single disseminated cells within the adjacent stroma (indicated with black arrowheads) stained positive for Brachyury but negative for E-cadherin (magnification 20×). B, results of qPCR analysis for Brachyury copy number in indicated tumor specimens. Pos = positive control reaction with genomic DNA; neg = negative control reaction without DNA. SM = size marker.

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    Figure 3.

    Expression of Brachyury in human normal tissues. A, representative pictures of normal (i) positive testis, (ii) positive thyroid, (iii) negative lung alveoli, (iv) negative lung bronchioles, (v) negative spleen, and (vi) negative skeletal muscle (magnification 40×). B, real-time PCR analysis of Brachyury, Snail, and Twist mRNA expression in commercial panels of cDNA (*, normal tissue adjacent to tumor). C, Western blot analysis of Brachyury, Snail, and Twist protein expression in commercially available whole-tissue protein lysates obtained from normal testis and normal lung. The following antibodies were used: Snail clone H-130, Twist clone H-81, GAPDH clone 0411 (Santa Cruz Biotechnology). D, real-time PCR analysis of Brachyury, PAP, and CEA mRNA expression in cDNA from normal testis and thyroid tissues.

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    Figure 4.

    Brachyury and susceptibility to EGFR inhibition. A, real-time PCR analysis of Brachyury mRNA in human primary lung tumors and lung carcinoma lines. B, A549 and H460 lung carcinoma cell pairs were treated with various doses (μmol/L) of the EGFR kinase inhibitor AG1478 for 2 days and assayed for survival by the MTT assay. C, real-time PCR analysis of Brachyury expression in A549 cells grown for 2 weeks in the presence of control DMSO- or 1 μmol/L AG1478-containing medium. D, extracellular matrix invasion assay of A549 cells untreated (parental) or treated as indicated for 2 weeks in culture. *, P < 0.05; ***, P < 0.005.

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    Figure 5.

    Brachyury as a target for antitumor interventions. A, CTL-mediated lysis of H226 and H441 lung carcinoma cells with a normal donor–derived Brachyury-specific T-cell line I (25:1 effector-to-target ratio). B, lysis of H441 tumor cells with a Brachyury-specific T-cell line II derived from a prostate cancer patient in the presence of cold, competitor K562 A2.1 cells unpulsed or pulsed with the specific Brachyury peptide (Bra pep). C, lysis of H441 and H1703 lung carcinoma and control ASPC1 cells by tetramer-isolated, CD8+ Brachyury-specific T-cell line III derived from a different prostate cancer patient.

Tables

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  • Table 1.

    Brachyury protein expression analyzed by IHC in human primary lung carcinoma tissues

    Lung tumor tissuesBrachyury positive (%)
    Adenocarcinoma10/21 (48)
    Squamous carcinoma3/12 (25)
    Undifferentiated carcinoma2/4 (50)
    Bronchioloalveolar carcinoma1/1 (100)
    Small cell lung carcinoma0/1 (0)
    Total16/39 (41)
  • Table 2.

    Lung tumor tissues positive for Brachyury protein expression analyzed by IHC

    Tumor cells
    NuclearCytoplasmicAdjacent tissue
    #HistologyGradeStage% positiveintensity% positiveintensity% positiveintensity
    1AdenocarcinomaG2pT2N085+++85+negneg
    2AdenocarcinomaG3pT2N280+++80+30+++
    3AdenocarcinomaG2pT1aN080++80+10+
    4AdenocarcinomaG2pT1aN060++60+negneg
    5AdenocarcinomaG3pT2N160+60+50+
    6AdenocarcinomaG2pT2N040++20+negneg
    7AdenocarcinomaG3pT2N030+30+negneg
    8AdenocarcinomaG2pT1bN030+negnegnegneg
    9AdenocarcinomaG2pT2N010++10++30+++
    10AdenocarcinomaG2pT2N110+10+negneg
    11Squamous cell carcinomaG2pT3N080++negnegnegneg
    12Squamous cell carcinomaG1pT3N050++negneg40+
    13Squamous cell carcinoma—rec10+10+negneg
    14Undifferentiated carcinomaG3pT2N180++60+10+
    15Undifferentiated carcinomaG3pT2N125+15+50+
    16Bronchioloalveolar carcinomaG1pT1bN090++90+negneg

    NOTE: rec = recurrence; neg = negative.

    Additional Files

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    • Supplementary Data

      Files in this Data Supplement:

      • Supplementary Table 1 - PDF file, 62KB, Analysis of Brachyury gene copy number and promoter methylation status.
      • Supplementary Table 2 - PDF file, 55KB, Number and percentage of tissues from adult non-cancer subjects that were positive for Brachyury expression by immunohistochemistry.
      • Supplementary Table 3 - PDF file, 113KB, Patient characteristics for samples in human lung cancer cDNA arrays.
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    Clinical Cancer Research: 18 (14)
    July 2012
    Volume 18, Issue 14
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    Brachyury, a Driver of the Epithelial–Mesenchymal Transition, Is Overexpressed in Human Lung Tumors: An Opportunity for Novel Interventions against Lung Cancer
    Mario Roselli, Romaine I. Fernando, Fiorella Guadagni, Antonella Spila, Jhessica Alessandroni, Raffaele Palmirotta, Leopoldo Costarelli, Mary Litzinger, Duane Hamilton, Bruce Huang, Joanne Tucker, Kwong-Yok Tsang, Jeffrey Schlom and Claudia Palena
    Clin Cancer Res July 15 2012 (18) (14) 3868-3879; DOI: 10.1158/1078-0432.CCR-11-3211

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    Brachyury, a Driver of the Epithelial–Mesenchymal Transition, Is Overexpressed in Human Lung Tumors: An Opportunity for Novel Interventions against Lung Cancer
    Mario Roselli, Romaine I. Fernando, Fiorella Guadagni, Antonella Spila, Jhessica Alessandroni, Raffaele Palmirotta, Leopoldo Costarelli, Mary Litzinger, Duane Hamilton, Bruce Huang, Joanne Tucker, Kwong-Yok Tsang, Jeffrey Schlom and Claudia Palena
    Clin Cancer Res July 15 2012 (18) (14) 3868-3879; DOI: 10.1158/1078-0432.CCR-11-3211
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