Like many solid cancers, in head and neck cancers (HNC) the presence of metastases is a well-known prognostic factor associated with worst survival. Studies to date indicate that chronic stimulation with tumor necrosis factor alpha (TNF-α) has the potential to enhance several tumor cell characteristics, such as cell survival, proliferation, and migration, promoting metastasis. It is believed that TNF-α stimulation leads to the activation of different inflammatory pathways, such as the nuclear factor-kappa B (NF-kB) and AKT pathway, inhibiting GSK-3β-mediated phosphorylation and degradation of Snail and β-catenin, stabilizing them and contributing to the process of epithelial-mesenchymal transition (EMT), through which metastasis occurs. By targeting distinct components mediating these pathways, microRNAs can post-transcriptionally regulate these biologic processes, playing an important role in cancer progression. By using quantitative fluorescence microscopy (High-Content Analysis--HCA), we sought to investigate the roles of the aforementioned signaling pathways and of selected microRNAs, in EMT of HNC. To do so, FADU cells derived from an oropharynx HNC were transfected with microRNAs or siRNAs against different components of inflammatory pathways (P65/RelA, AKT1, GSK3β, and beta-catenin), followed by stimulation with TNF-α (20 ng/mL) for a period of 72 h. Cells were immunostained using nucleic acid and cytoplasmic dyes, as well as antibodies against markers of cell cycle (Cyclin B1), apoptosis (cleaved Caspase-7), and EMT (N-Cadherin, Vimentin and SNAIL1/2). TNF-α stimulation altered several phenotypic features associated with EMT, including loss of cell-cell contact, acquisition of fibroblast morphology, increased expression of all EMT markers evaluated, as well as reduced expression of E-cadherin. In line, TNF-α stimulation enhanced the migratory potential of nonproliferating mitomycin-treated FADU cells (HCA cell migration assay, Oris Pro). The siRNA-knockdown of RelA and AKT1 led to a reduction in the nuclear levels of SNAIL, while silencing of GSK3β increased its levels. Additionally, in the absence of TNF-α stimulation, silencing of inflammatory pathway components led to a reduction in the number of cells. Importantly, silencing of RelA and also AKT1 had a major impact in cell survival, drastically increasing the percentage of apoptotic cells and reducing cell numbers to a level similar to a control cytotoxic siRNA against ubiquitin. Functional evaluation of the 30 microRNAs revealed that some of them targeted elements of the evaluated pathways, promoting or repressing the phenotypic changes associated with TNF-induced EMT. Our results provide evidence of the molecular mechanisms mediating TNF-α-induced epithelial-mesenchymal transition in a well-established head and neck cancer cell model. Moreover, we highlight potential microRNA targets, whose post-transcriptional regulation is at least partially responsible for the modulation of the EMT process. Altogether, our work provides further understanding of the mechanisms contributing to cancer metastasis, and may be further explored in order to develop new therapeutic agents for head and neck cancer.

Citation Format: Bruno Sangiorgi, Ildercílio Mota de Souza Lima, Josiane Lilian Schiavinato, Wilson Araújo Silva, Jr., Dimas Tadeu Covas, Marco Antônio Zago, Rodrigo Alexandre Panepucci. The role of inflammatory pathways on cell survival and epithelial-mesenchymal transition in head and neck cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B83.