Abstract
Head and neck squamous cell carcinoma (HNSCC) is among the most common cancer types. Metastasis, the main cause of death by cancer, can be promoted by an inflammatory microenvironment, which induces epithelial-mesenchymal transition (EMT) through a NF-κB-mediated stabilization of Snail. Here, we aimed to explore how microRNAs (miRs) can affect cell survival and EMT in HNSCC cells under an inflammatory microenvironment. By using a high-content screening (HCS) approach, we evaluated alterations in morphometric parameters, as well as expression/localization of Snail/Slug, in HNSCC cells primed with TNF-α. Based on those quantitation, we established the optimal experimental conditions of EMT induction driven by TNF-α. Those conditions were applied to cells transfected with distinct miRs (N = 31), followed by clusterization of miRs based on alterations related to cell survival and EMT. The signaling pathways enriched with molecular targets from each group of miRs were identified by in silico analyses. Finally, cells were transfected with siRNAs against signaling pathways targeted by miRs with anti-survival/EMT effect and evaluated for alterations in cell survival and EMT. Overall, we observed that TNF-α, at 20 ng/ml, induced EMT-related changes in cell morphology, Snail/Slug expression, and cell migration. Predicted targets of miRs with anti-survival/EMT effect were enriched with targets of NF-κB, PI3K/ATK, and Wnt/beta catenin pathways. Strikingly, individual gene silencing of elements from those pathways, namely RELA (NF-kB), AKT1 (PI3K/AKT), and CTNNB1 (Wnt/beta catenin) reduced cell survival and/or expression of Snail/Slug in cells stimulated with TNF-α. As a whole, our HCS approach allowed for the identification of miRs capable of inhibiting cell survival and EMT considering the presence of an inflammatory microenvironment, also indicating the common signaling pathways and molecular targets most likely to underlie those alterations. These findings may contribute to the development of targeted therapies against HNSCC.
Highlights
Head and neck squamous cell carcinoma (HNSCC) comprises a group of upper aerodigestive tract neoplasia and is among the ten types of cancer with the highest incidence and mortality in the world [1]
Through an High-Content Screening (HCS) approach and in-silico analysis, we investigated the capacity of miRs to alter the phenotypic features related to tumor progression and metastasis (e.g., epithelial-mesenchymal transition (EMT)) in HNSCC cells considering the presence of an inflammatory microenvironment
Many of the functional studies conducted so far about the impact of small molecules in cancer cell survival disregards the presence of an inflammatory microenvironment, which is known to promote apoptosis resistance, epithelial to mesenchymal transition, among other phenotypic changes that promotes therapy resistance and disease recurrence [7].With that in mind, our study aimed to identify, through functional assays using an HCS approach, miRs and signaling pathways with the potential to suppress both cell survival and EMT features in HNSCC cells considering the presence of an inflammatory microenvironment
Summary
Head and neck squamous cell carcinoma (HNSCC) comprises a group of upper aerodigestive tract neoplasia and is among the ten types of cancer with the highest incidence and mortality in the world [1]. EMT events are coordinated by transcription factors known as “EMT master regulators,” including members of the Snail family: SNAI1 (Snail) and SNAI2 (Slug), which are capable of both silencing and promoting the expression of genes related to epithelial and mesenchymal phenotypes, respectively [4]. Increasing literature data have established that, for several types of cancer including HNSCC, the presence of an inflamed tumor microenvironment is associated with tumor progression, the acquisition of EMT-like features by cancer cells and the formation of metastasis [6]. In different types of cancer, multiple lines of evidence have supported that inflammatory cytokines secreted by tumor-associated macrophages (which can represent half of the tumor mass), including tumor necrosis factor alpha (TNF-α), are capable of inducing EMT events in cancer cells [7]. Due to the complexity of intracellular signaling pathways and tumor microenvironment in cancer, including HNSCC, a multi-target therapy (targeting multiple signaling pathways) may be an interesting therapeutic approach [11]
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