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.

The Inhibitor-κB Kinase-Nuclear Factor-κB (IKK-NF-κB) and Epidermal Growth Factor Receptor-Activator Protein-1 (EGFR-AP-1) pathways are often co-activated and promote malignant behavior, but the underlying basis for this relationship is unclear. Resistance to inhibitors of IKKβ or EGFR is observed in head and neck squamous cell carcinomas (HNSCC). Here, we reveal that both IKKα and β contribute to nuclear activation of canonical and alternate NF-κB/REL family transcription factors, and overexpression of signal components enhancing co-activation of the EGFR-AP1 pathway. We observed that IKKα and IKKβ exhibit increased protein expression, nuclear localization and phosphorylation in HNSCC tissues and cell lines. Individually, IKK activity varied amongst different cell lines, but overexpression of both IKKs induced the strongest NF-κB activation. Conversely, siRNA knockdown of both IKKs significantly decreased nuclear localization and phosphorylation of canonical RELA and IκBα, and alternative p52 and RELB subunits. Knockdown of both IKKs more effectively inhibited NF-κB activation, broadly modulated gene expression, and suppressed cell proliferation and migration. Global expression profiling revealed that NF-κB, cytokine, inflammatory response, and growth factor signaling are among the top pathways and networks regulated by IKKs. Importantly, IKKα and IKKβ together promoted the expression and activity of TGFα, EGFR, and AP1 transcription factors cJun, JunB, and Fra1. Knockdown of AP1 subunits individually decreased 8/15 (53%) of IKK-targeted genes sampled, and similarly inhibited cell proliferation and migration. Mutations of NF-κB and AP1 binding sites abolished or decreased IKK-induced IL-8 promoter activity. Compounds such as wedelactone with dual IKK inhibitory activity, and geldanomycins that block IKKα/β and EGFR pathways were more active than IKKβ-specific inhibitors in suppressing NF-κB activation and proliferation, and inducing cell death. We conclude that IKKα and IKKβ cooperatively activate NF-κB and EGFR/AP1 networks of signaling pathways, and contribute to the malignant phenotype and the intrinsic or acquired therapeutic resistance of HNSCC.

Comparison of tumors from the Cancer Genome Atlas (TCGA) reveals that head and neck squamous cell carcinomas (HNSCC) harbor the most frequent genomic amplifications of Fas-associated death domain (FADD), with or without Baculovirus Inhibitor of Apoptosis repeat containing BIRC2 (cIAP1), affecting ~30% of patients in association with worse prognosis. Here, we identified HNSCC cell lines harboring FADD/BIRC2 amplifications and overexpression by exome sequencing, RT-PCR and Western blot. In vitro, FADD or BIRC2 siRNA knockdown inhibited HNSCC displaying amplification and increased expression of these genes, supporting their functional importance in promoting proliferation. Birinapant, a novel SMAC mimetic, sensitized multiple HNSCC lines to cell death by agonists TNFα or TRAIL, and inhibited cIAP1>XIAP>IAP2. Combination of birinapant and TNFα induced sub-G0 DNA fragmentation in sensitive lines, and birinapant alone also induced significant G2/M cell cycle arrest and cell death in UM-SCC-46 cells. Gene transfer and expression of FADD sensitized resistant UM-SCC-38 cells lacking FADD amplification to birinapant and TNFα, supporting a role for FADD in sensitization to IAP inhibitor and death ligands. HNSCC varied in mechanisms of cell death, as indicated by reversal by inhibitors or protein markers of caspase-dependent apoptosis and/or RIPK1/MLKL-mediated necroptosis. In vivo, birinapant inhibited tumor growth and enhanced radiation induced TNFα, tumor responses, and host survival in UM-SCC-46 and -11B xenograft models displaying amplification and overexpression of FADD+/−BIRC2. These findings suggest that combination of SMAC mimetics such as birinapant plus radiation may be particularly active in HNSCC, which harbor frequent FADD/BIRC2 genomic alterations.

SUMMARY Cell lines are important tools for biological and preclinical investigation, and establishing their relationship to genomic alterations in tumors could accelerate functional and therapeutic discoveries. We conducted integrated analyses of genomic and transcriptomic profiles of 15 human papillomavirus (HPV)-negative and 11 HPV-positive head and neck squamous cell carcinoma (HNSCC) lines to compare with 279 tumors from The Cancer Genome Atlas (TCGA). We identified recurrent amplifications on chromosomes 3q22–29, 5p15, 11q13/22, and 8p11 that drive increased expression of more than 100 genes in cell lines and tumors. These alterations, together with loss or mutations of tumor suppressor genes, converge on important signaling pathways, recapitulating the genomic landscape of aggressive HNSCCs. Among these, concurrent 3q26.3 amplification and TP53 mutation in most HPV(–) cell lines reflect tumors with worse survival. Our findings elucidate and validate genomic alterations underpinning numerous discoveries made with HNSCC lines and provide valuable models for future studies.

Purpose PI3K-mTOR pathway activation is often associated with altered expression or mutations of PIK3CA, TP53/p73, PTEN and TGFβR in head and neck squamous cell carcinomas (HNSCC). However, little is known about how these alterations affect response to PI3K-mTOR targeted agents. Experimental Design In this preclinical study, PI3K-Akt-mTOR signaling was characterized in 9 HNSCC (UM-SCC) cell lines and Human Oral Keratinocytes (HOK). We investigated the molecular and anti-cancer effects of dual PI3K/mTOR inhibitor PF-04691502(PF-502) in UM-SCC expressing PIK3CA with decreased wtTP53, mtTP53-/+mtTGFβR2, and in HNSCC of a conditional Pten/Tgfbr1 double knockout (2cKO) mouse model, displaying PI3K-Akt-mTOR activation. Results UM-SCC showed increased PIK3CA expression and Akt/mTOR activation, and PF-502 inhibited PI3K/mTORC1/2 targets. In human HNSCC expressing PIK3CA and decreased wtTP53 and p73, PF-502 reciprocally enhanced TP53/p73 expression and growth inhibition, which was partially reversible by p53 inhibitor pifithrin-α. Most UM-SCC with wtTP53 exhibited a lower IC50 than those with mtTP53 status. PF-502 blocked growth in G0/G1 and increased apoptotic subG0 DNA. PF502 suppressed tumorigenesis and showed combinatorial activity with radiation in a wtTP53 UMSCC xenograft model. PF-502 also significantly delayed HNSCC tumorigenesis and prolonged survival of Pten/Tgfbr1 deficient mice. Significant inhibition of p-Akt, p-4EBP1, p-S6, Ki67, as well as increased p53 and TUNEL were observed in tumor specimens. Conclusions PI3K-mTOR inhibition can enhance TP53/p73 expression and significantly inhibit tumor growth alone or when combined with radiation in HNSCC with wtTP53. PIK3CA, TP53/p73, PTEN and TGFβ alterations are potential modifiers of response and merit investigation in future clinical trials with PI3K-mTOR inhibitors.