NF-κB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression
Margit A. Huber, … , Hartmut Beug, Thomas Wirth
Margit A. Huber, … , Hartmut Beug, Thomas Wirth
Published August 16, 2004
Citation Information: J Clin Invest. 2004;114(4):569-581. https://doi.org/10.1172/JCI21358.
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Article Oncology

NF-κB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression

Abstract

The transcription factor NF-κB is activated in a range of human cancers and is thought to promote tumorigenesis, mainly due to its ability to protect transformed cells from apoptosis. To investigate the role of NF-κB in epithelial plasticity and metastasis, we utilized a well-characterized in vitro/in vivo model of mammary carcinogenesis that depends on the collaboration of the Ha-Ras oncoprotein and TGF-β. We show here that the IKK-2/IκBα/NF-κB pathway is required for the induction and maintenance of epithelial-mesenchymal transition (EMT). Inhibition of NF-κB signaling prevented EMT in Ras-transformed epithelial cells, while activation of this pathway promoted the transition to a mesenchymal phenotype even in the absence of TGF-β. Furthermore, inhibition of NF-κB activity in mesenchymal cells caused a reversal of EMT, suggesting that NF-κB is essential for both the induction and maintenance of EMT. In line with the importance of EMT for invasion, blocking of NF-κB activity abrogated the metastatic potential of mammary epithelial cells in a mouse model system. Collectively, these data provide evidence of an essential role for NF-κB during distinct steps of breast cancer progression and suggest that the cooperation of Ras- and TGF-β–dependent signaling pathways in late-stage tumorigenesis depends critically on NF-κB activity.

Authors

Margit A. Huber, Ninel Azoitei, Bernd Baumann, Stefan Grünert, Andreas Sommer, Hubert Pehamberger, Norbert Kraut, Hartmut Beug, Thomas Wirth

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Suppression of NF-κB activity in EpRas cells leads to apoptosis and prev...
Suppression of NF-κB activity in EpRas cells leads to apoptosis and prevents EMT. (A) EpRas cells expressing the empty vector control, TD-IκBα, or CA–IKK-2 were seeded into collagen gels, were allowed to form structures for 3 days, and were treated with TGF-β for 6 days or were left untreated. Collagen cultures were subjected to in situ TUNEL staining (red) and DAPI staining (blue, indicating living cells). Light microscopy images of collagen gels from the same experiment photographed prior to TUNEL staining are also shown. Arrows indicate TUNEL-positive nuclei. Inset, right middle panel: another structure with TUNEL-positive nuclei. Original magnification, ×200. (B) Quantification of TUNEL-positive cells from collagen gel structures shown in A. TUNEL staining of nuclei was assessed in at least 300 cells from three to six randomly chosen fields. The average from two to three collagen gels was used to calculate the apoptotic index and standard deviation. Average percentage of apoptotic cells, assessed in 2–3 collagen gels (at least 300 cells per gel) for each cell type, are indicated above bars. (C) EpRas cells expressing the empty vector control or TD-IκBα were cultivated for 5 days on porous support in the presence or absence of 25 μM Z-VAD-FMK (Z-VAD; days 0–5) and/or TGF-β (5 ng/ml; days 1–5). The quantification of the area on porous support covered by mesenchymal strands is shown as the percentage relative to the total area covered by adherent cells (at day 4, after 3 days with or without TGF-β treatment). (D) Cells as indicated (with or without 25 μM Z-VAD-FMK; days 0–5) were cultivated on porous support for 5 days in the presence or absence of TGF-β (5 ng/ml; days 1–5). Cells were immunostained for E-cadherin or vimentin (red) plus DAPI counterstaining for DNA (blue) after 5 days of culture on porous support. Original magnification, ×400.