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.

Nuclear factor-kappa B (NF-κB) transcription factors regulate cellular processes such as inflammation and cell survival. The NF-κB pathway is often activated with development and progression of head and neck squamous cell carcinoma (HNSCC). As such, NF-κB represents an attractive target for chemoprevention. HNSCC involves progression of lesions from premalignant to malignant, providing a window of opportunity for intervention with chemopreventive agents. Appropriate chemopreventive agents should be inexpensive, nontoxic, and target important pathways involved in the development of HNSCC. Several such agents that inhibit the NF-κB pathway have been investigated in HNSCC. Retinoids have been studied most extensively but have shown limited potential in human trials. Epidermal growth factor receptor inhibitors and PI3K-mTOR inhibitors may benefit a subset of patients. Other agents such as green tea extract and curcumin are appealing because they are generally regarded as safe. In contrast, there is evidence that Vitamin E supplementation may actually increase mortality of cancer patients. Repurposed drugs such as cyclooxygenase (COX) inhibitors and antidiabetic drugs are an emerging area of interest. Future research to develop agents with lower toxicity and higher specificity for the NF-κB pathway, and to target these therapies to individual patient genetic signatures should help to increase the utility of chemoprevention in HSNCC.

Objectives Head and neck squamous cell carcinoma (HNSCC) cells are resistant to cell death induced by Tumor Necrosis Factor ligands such as Tumor Necrosis Factor α (TNF) or TNF-Related Apoptosis-Inducing Ligand (TRAIL) and cytotoxic chemotherapies. Recently, genetic alterations in cell death pathways including inhibitor of apoptosis proteins have been demonstrated in HNSCC. We investigated the effects of birinapant, a novel SMAC-mimetic that targets inhibitor of apoptosis proteins, alone and in combination with TNFα, TRAIL or chemotherapy docetaxel. Study Design Experimental study using human HNSCC cell lines in vitro and xenograft mouse model in vivo. Methods A panel of HNSCC cell lines with varying genetic alterations in cell death pathway components were treated with birinapant ± TNFα, TRAIL and docetaxel and were assessed for effects on cell density, cell cycle and death. Synergism was determined at varying concentrations of treatments using the Chou-Talalay method. Combination studies using birinapant ± docetaxel were performed in a xenograft mouse model. Results Birinapant, alone or in combination with TNFα or TRAIL, decreased cell density in cell lines, with IC50s ranging from 0.5 nM to >1 μM. Birinapant alone or with TNF significantly increased subG0 cell death in different lines. Docetaxel showed synergism with birinapant ± TNFα in vitro. Birinapant monotherapy inhibited growth in a tumor xenograft model resistant to docetaxel, and combination treatment further delayed growth. Conclusions Birinapant alone or in combination with TNFα or TRAIL and docetaxel decreased cell density, increased cell death, and displayed anti-tumor activity in a preclinical HNSCC xenograft exhibiting aberrations in cell death pathway components and docetaxel resistance. Level of Evidence NA, Animal Studies and Basic Research

BackgroundTesticular germ cell tumors (TGCTs) are classified as seminonas or non-seminomas of which a major subset is embryonal carcinoma (EC) that can differentiate into diverse tissues. The pluripotent nature of human ECs resembles that of embryonic stem (ES) cells. Many Wnt signalling species are regulated during differentiation of TGCT-derived EC cells. This study comprehensively investigated expression profiles of Wnt signalling components regulated during induced differentiation of EC cells and explored the role of key components in maintaining pluripotency.MethodsHuman embryonal carcinoma cells were stably infected with a lentiviral construct carrying a canonical Wnt responsive reporter to assess Wnt signalling activity following induced differentiation. Cells were differentiated with all-trans retinoic acid (RA) or by targeted repression of pluripotency factor, POU5F1. A Wnt pathway real-time-PCR array was used to evaluate changes in gene expression as cells differentiated. Highlighted Wnt pathway genes were then specifically repressed using siRNA or stable shRNA and transfected EC cells were assessed for proliferation, differentiation status and levels of core pluripotency genes.ResultsCanonical Wnt signalling activity was low basally in undifferentiated EC cells, but substantially increased with induced differentiation. Wnt pathway gene expression levels were compared during induced differentiation and many components were altered including ligands (WNT2B), receptors (FZD5, FZD6, FZD10), secreted inhibitors (SFRP4, SFRP1), and other effectors of Wnt signalling (FRAT2, DAAM1, PITX2, Porcupine). Independent repression of FZD5, FZD7 and WNT5A using transient as well as stable methods of RNA interference (RNAi) inhibited cell growth of pluripotent NT2/D1 human EC cells, but did not appreciably induce differentiation or repress key pluripotency genes. Silencing of FZD7 gave the greatest growth suppression in all human EC cell lines tested including NT2/D1, NT2/D1-R1, Tera-1 and 833K cells.ConclusionDuring induced differentiation of human EC cells, the Wnt signalling pathway is reprogrammed and canonical Wnt signalling induced. Specific species regulating non-canonical Wnt signalling conferred growth inhibition when targeted for repression in these EC cells. Notably, FZD7 repression significantly inhibited growth of human EC cells and is a promising therapeutic target for TGCTs.