Epigenetic downregulation of human disabled homolog 2 switches TGF-β from a tumor suppressor to a tumor promoter
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
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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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 4

DAB2 represses TGF-β–mediated Smad2 activation.

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DAB2 represses TGF-β–mediated Smad2 activation. (A and B) Low-level DAB2...
(A and B) Low-level DAB2 expression does not preclude Smad2 and Smad3 phosphorylation. VSCC cell lines (A) and HNSCC cell lines (B) were treated for 1 hour with or without 1 ng/ml TGF-β prior to Western blotting for activation of Smad2 and Smad3, using PO4-Smad2 and PO4-Smad3 antibodies. DAB2, Smad2, and Smad3 Western blotting analysis is also shown. (C) siRNA-mediated knockdown of DAB2 enhances TGF-β–mediated Smad2 activation. Western blotting analysis of TGF-β–mediated activation of Smad2 and Smad3 phosphorylation in UMSCV1B cells, following transient siRNA transfection with nonsilencing control (negative) and DAB2-specific siRNA (DAB2 s#1) and TGF-β treatment (1 ng/ml) for the indicated time points. Knockdown of DAB2 was assessed by DAB2 Western blotting, and analysis of PO4-Smad2, PO4-Smad3, total Smad2, and Smad3 levels is shown. (D) DAB2 stable reexpression inhibits TGF-β–mediated Smad2 activation. Western blotting analysis of A431 vector control (V) and Dab2 stable cell lines (A431D2#1 [D] analyzed as in C). (E and F) DAB2 protein levels inversely correlate with PO4-Smad2 levels. Serial sections of a HNSCC TMA were stained with DAB2 and PO4-Smad2 antibodies, respectively, and protein levels were determined by histoscore. DAB2 low samples (histoscore, <70) have a higher level of PO4-Smad2 (*P < 0.05) than DAB2 high samples. In box plots, the 75th and 25th percentiles are represented by the top and bottom of the box, respectively. The horizontal lines refer to the mean. (F) Regions of serial sections of 3 example tumor TMA cores stained with the indicated antibodies, revealing mutually exclusive staining. Original magnification, ×20.