ABSTRACT: Transforming growth factor-beta (TGF-?) has a dual role in epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC). Attenuation of canonical TGF-? signaling enhances de novo tumor development, whereas TGF-? overexpression and signaling paradoxically promotes malignant progression. We recently observed that TGF-?-induced growth arrest response is attenuated, in association with aberrant activation of nuclear factor-?B (NF-?B), a transcription factor, which promotes malignant progression in HNSCC. However, what role cross-talk between components of the TGF-? and NF-?B pathways plays in altered activation of these pathways has not been established. Here, we show TGF-? receptor II and TGF-?-activated kinase 1 (TAK1) are predominantly expressed in a subset of HNSCC tumors with nuclear activation of NF-?B family member RELA (p65). Further, TGF-?1 treatment induced sequential phosphorylation of TAK1, IKK, I?B? and RELA in human HNSCC lines. TAK1 enhances TGF-?-induced NF-?B activation, as TAK1 siRNA knockdown decreased TGF-?1-induced phosphorylation of IKK, I?B and RELA, degradation of I?B?, RELA nuclear translocation and DNA binding, and NF-?B-induced reporter and target gene transcription. Functionally, TAK1 siRNA inhibited cell proliferation, migration and invasion. Celastrol, a TAK1 inhibitor and anti-inflammatory compound used in traditional Chinese medicine, also decreased TGF-?1-induced phosphorylation of TAK1 and RELA, and suppressed basal, TGF-?1- and tumor necrosis factor-alpha (TNF-?)-induced NF-?B reporter gene activity. Celastrol also inhibited cell proliferation, while increasing sub-G0 DNA fragmentation and Annexin V markers of apoptosis. Furthermore, TGF-? and RELA activation promoted SMAD7 expression. In turn, SMAD7 preferentially suppressed TGF-?-induced SMAD and NF-?B reporters when compared with constitutive or TNF-?-induced NF-?B reporter gene activation. Thus, cross-talk by TGF-? via TAK1 and NF-?B promotes the malignant phenotype of HNSCC. Moreover, NF-?B may contribute to the downstream attenuation of canonical TGF-? signaling through increased SMAD7 expression. Celastrol highlights the therapeutic potential of agents targeting TAK1 as a key node in this pro-oncogenic TGF-?-NF-?B signal pathway.
