TGF-β plays a dual role in epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC). Attenuation of canonical TGF-β signaling enhances de novo tumor development, while TGF-β overexpression and signaling paradoxically promotes malignant progression. We recently observed that TGF-β-induced growth arrest response is attenuated, in association with aberrant activation of Nuclear Factor-κB (NF-κB), a transcription factor which promotes malignant progression in HNSCC. However, what role cross-talk between components of the TGF-β and NF-κB pathways plays in altered activation of these pathways has not been established. Here, we show TGF-β receptor II and TGF-β-activated kinase 1 (TAK1) are predominantly expressed in a subset of HNSCC tumors with nuclear activation of NF-κB family member RELA (p65). Further, TGF-β1 treatment induced sequential phosphorylation of TAK1, IKK, IκBα, and RELA in human HNSCC lines. TAK1 enhances TGF-β-induced NF-κB activation, as TAK1 siRNA knock-down decreased TGF-β1-induced phosphorylation of IKK, IκB, and RELA, degradation of IκBα, nuclear translocation, and DNA binding of RELA, and NF–κB-induced reporter and target gene transcription. Functionally, TAK1 siRNA inhibited cell proliferation, migration and invasion. Celastrol, a TAK1 inhibitor and anti-inflammatory used in traditional Chinese medicine, also decreased TGF-β1-induced phosphorylation of TAK1 and RELA, suppressed basal, TGF-β1- and TNFα-induced NF-κB reporter gene activity, and cell proliferation, while increasing sub-G0 DNA fragmentation and Annexin V markers of apoptosis. Furthermore, TGF-β and RELA activation promoted SMAD7 expression. In turn, SMAD7 preferentially suppressed TGF-β-induced SMAD and NF-κB reporters when compared with constitutive or TNF-α-induced NF-κB reporter gene activation. Thus, cross-talk by TGF-β via TAK1 and NF-κB promotes the malignant phenotype of HNSCC. Moreover, NF-κB may contribute to the downstream attenuation of canonical TGF-β signaling through increased SMAD7 expression. Celastrol highlights the therapeutic potential of agents targeting TAK1 as a key node in this pro-oncogenic TGF-β-NF-κB signal pathway.

3b. Laryngoscope, 126:2580-2586, 2016.

Heat shock protein 90 (HSP90) is a chaperone protein that stabilizes proteins involved in oncogenic and therapeutic resistance pathways of epithelial cancers, including head and neck squamous cell carcinomas (HNSCCs). Here, we characterized the molecular, cellular, and preclinical activity of HSP90 inhibitor SNX5422/2112 in HNSCC overexpressing HSP90. SNX2112 inhibited proliferation, induced G2/M block, and enhanced cytotoxicity, chemosensitivity, and radiosensitivity between 25 and 250 nM in vitro. SNX2112 showed combinatorial activity with paclitaxel in wild-type (wt) TP53-deficient and cisplatin in mutant (mt) TP53 HNSCC lines. SNX2112 decreased expression or phosphorylation of epidermal growth factor receptor (EGFR), c-MET, v-akt murine thymoma viral oncogene homolog 1 (AKT), extracellular signal-regulated kinases (ERK) 1 and 2, inhibitor κB kinase, and signal transducer and transcription factor 3 (STAT3), corresponding downstream nuclear factor κB, activator protein-1, and STAT3 reporter genes, and target oncogenes and angiogenic cytokines. Furthermore, SNX2112 enhanced re-expression of TP53 and targets p21WAF1 and PUMA, while TP53 inhibitor Pifithrin or siRNA attenuated the antiproliferative activity of SNX2112 in wtTP53 HNSCC in vitro. Prodrug SNX5422 similarly down-modulated key signal targets, enhanced TP53 expression and apoptosis, and inhibited proliferation, angiogenesis, and tumorigenesis in a wtTP53-deficient HNSCC xenograft model. Thus, HSP90 inhibitor SNX5422/2112 broadly modulates multiple key nodes within the dysregulated signaling network, with corresponding effects upon the malignant phenotype. Our data support investigation of SNX5422/2112 in combination with paclitaxel, cisplatin, and radiotherapy in HNSCC with different TP53 status.