NF-κB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression
Margit A. Huber, Ninel Azoitei, Bernd Baumann, Stefan Grünert, Andreas Sommer, Hubert Pehamberger, Norbert Kraut, Hartmut Beug, Thomas Wirth
Margit A. Huber, Ninel Azoitei, Bernd Baumann, Stefan Grünert, Andreas Sommer, Hubert Pehamberger, Norbert Kraut, Hartmut Beug, Thomas Wirth
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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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Modulation of NF-κB activity in EpRas cells. (A and B) Expression levels...
Modulation of NF-κB activity in EpRas cells. (A and B) Expression levels of dominant interfering mutants in ΦNX producer cells (A) and in stably infected EpRas cells (B) were determined by Western blot analysis of whole-cell extracts, using IκBα- and IKK-specific antibodies CA–IKK-2, endogenous IKK-2 (comigrating with CA–IKK-2), TD-IκBα, and endogenous IκBα. Equal loading was assessed using a p65/RelA–specific antibody (B, bottom panel). (C) Extracts from stably infected EpRas mutants left untreated (–) or stimulated with TGF-β (5 ng/ml) for 2 hours (+) were analyzed by EMSA with an NF-κB–specific probe (upper panel) and with an octamer-specific probe (lower panel) as loading control. Quantified relative DNA-binding levels are indicated. (D) Stably infected cells were transiently transfected in triplicate with 3xκB.luc or β-globin-TATA reporter constructs. After 24 hours, cells were treated with TGF-β (5 ng/ml for 4 hours) (+) or were left untreated (–). Luciferase activity was measured and normalized based on Renilla luciferase expression (as described in Figure 2B). The ratio of 3xκB.luc and β-globin-TATA is shown. Expression levels of unstimulated empty vector–transfected EpRas cells were used as a reference and arbitrarily set to 1. Bars represent standard deviations. (E) EpRas mutants and EpRasXT cells were stimulated with TGF-β (5 ng/ml) for the times indicated, and RT-PCR analysis was carried out for transcript expression of MMP-13, MCP-1, cholecystokinin, and β-actin, as described in Methods. C, control (water); Empty, EpRas cells infected with empty vector control.