Epigenetic downregulation of human disabled homolog 2 switches TGF-β from a tumor suppressor to a tumor promoter
Adèle Hannigan, … , Tim Crook, Gareth J. Inman
Adèle Hannigan, … , Tim Crook, Gareth J. Inman
Published July 1, 2010
Citation Information: J Clin Invest. 2010;120(8):2842-2857. https://doi.org/10.1172/JCI36125.
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Research Article Oncology

Epigenetic downregulation of human disabled homolog 2 switches TGF-β from a tumor suppressor to a tumor promoter

Abstract

The cytokine TGF-β acts as a tumor suppressor in normal epithelial cells and during the early stages of tumorigenesis. During malignant progression, cancer cells can switch their response to TGF-β and use this cytokine as a potent oncogenic factor; however, the mechanistic basis for this is poorly understood. Here we demonstrate that downregulation of disabled homolog 2 (DAB2) gene expression via promoter methylation frequently occurs in human squamous cell carcinomas (SCCs) and acts as an independent predictor of metastasis and poor prognosis. Retrospective microarray analysis in an independent data set indicated that low levels of DAB2 and high levels of TGFB2 expression correlate with poor prognosis. Immunohistochemistry, reexpression, genetic knockout, and RNAi silencing studies demonstrated that downregulation of DAB2 expression modulated the TGF-β/Smad pathway. Simultaneously, DAB2 downregulation abrogated TGF-β tumor suppressor function, while enabling TGF-β tumor-promoting activities. Downregulation of DAB2 blocked TGF-β–mediated inhibition of cell proliferation and migration and enabled TGF-β to promote cell motility, anchorage-independent growth, and tumor growth in vivo. Our data indicate that DAB2 acts as a tumor suppressor by dictating tumor cell TGF-β responses, identify a biomarker for SCC progression, and suggest a means to stratify patients with advanced SCC who may benefit clinically from anti–TGF-β therapies.

Authors

Adèle Hannigan, Paul Smith, Gabriela Kalna, Cristiana Lo Nigro, Clare Orange, Darren I. O’Brien, Reshma Shah, Nelofer Syed, Lindsay C. Spender, Blanca Herrera, Johanna K. Thurlow, Laura Lattanzio, Martino Monteverde, Meghan E. Maurer, Francesca M. Buffa, Jelena Mann, David C.K. Chu, Catharine M.L. West, Max Patridge, Karin A. Oien, Jonathan A. Cooper, Margaret C. Frame, Adrian L. Harris, Louise Hiller, Linda J. Nicholson, Milena Gasco, Tim Crook, Gareth J. Inman

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Figure 7

DAB2 reexpression switches the TGF-β response in vitro and in vivo.

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DAB2 reexpression switches the TGF-β response in vitro and in vivo. (A) ...
(A) Western blotting analysis for DAB2 expression in A431 doxycycline-inducible cell lines. The asterisk indicates cross-reactive bands. A431 TetOn is a clonal derivative of the parental A431 cell line that stably expresses the Tet transactivator, from which the 2 DAB2-inducible cell lines were derived (A431 TDAB2#1, A431TDAB2#2). Dab2 expression was assessed after treatment with 1 μg/ml doxycycline (Dox) for 24 hours. (B and C) DAB2 expression switches the TGF-β response from promotion to inhibition of anchorage-independent growth. (B) Cells were seeded into soft agar, with or without TGF-β and/or doxycycline treatment. Data represent the mean ± SD (n = 4). (C) Stable cell lines, described in Figure 4 and Supplemental Figure 8, were seeded into soft agar and treated or not with 1 ng/ml TGF-β. Data represent the mean ± SD (n = 3). (D) Stable DAB2 expression switches the TGF-β response from promotion to inhibition of motility. Data represent the mean ± SD directional velocity of 8 cells tracked from each of 2 independent wounds. (E and F) DAB2 expression switches the TGF-β response in vivo. A431 vector control (E) and a DAB2 stable clone (F) were pretreated and mixed with 1 ng/ml TGF-β and injected into the flanks of CD1 nude mice. Tumor volumes were then calculated at the indicated time points for up to 15 days after injection. Data represent the mean volume ± SEM (n = 6). Statistical analyses were performed with paired 2-tailed t tests throughout. *P < 0.05, **P < 0.01, ***P < 0.001.