A High-Content Screening Approach to Identify MicroRNAs Against Head and Neck Cancer Cell Survival and EMT in an Inflammatory Microenvironment

Author(s):Show less -

Sangiorgi, Bruno ^{[1, 2]} ; de Souza, Felipe Canto ^{[1, 2]} ; de Souza Lima, Ildercilio Mota ^{[1, 2]} ; dos Santos Schiavinato, Josiane Lilian ^{[1, 2]} ; Corveloni, Amanda Cristina ^{[1, 2]} ; Thome, Carolina Hassibe ^{[3, 1]} ; Silva Jr, Wilson Araujo ; Faca, Vitor Marcel ^{[3, 4]} ; Covas, Dimas Tadeu ^[4] ; Zago, Marco Antonio ^[4] ; Panepucci, Rodrigo Alexandre ^{[4, 5]}

Affiliation

^[1] Reg Blood Ctr Ribeirao Preto, Ctr Cell Based Therapy CTC, Ribeirao Preto - Brazil

^[2] Univ Sao Paulo FMRP USP, Ribeirao Preto Med Sch, Dept Genet & Internal Med, Ribeirao Preto - Brazil

^[3] Univ Sao Paulo FMRP USP, Ribeirao Preto Med Sch, Dept Biochem & Immunol, Ribeirao Preto - Brazil

^[4] Silva Jr, Jr., Wilson Araujo, Reg Blood Ctr Ribeirao Preto, Ctr Cell Based Therapy CTC, Ribeirao Preto - Brazil

^[5] Silva Jr, Jr., Wilson Araujo, Univ Sao Paulo FMRP USP, Ribeirao Preto Med Sch, Dept Genet & Internal Med, Ribeirao Preto - Brazil

• Document type: Journal article
• Source: FRONTIERS IN ONCOLOGY; v. 9, NOV 8 2019.
• Web of Science Citations: 0

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-kappa B-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-alpha. Based on those quantitation, we established the optimal experimental conditions of EMT induction driven by TNF-alpha. 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-alpha, 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-kappa B, 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-alpha. 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. (AU)

• FAPESP's process: 15/08070-3 - Large-scale analysis of microRNA function in cell cycle, pluripotency, self-renewal and differentiation of stem cells using high-content screening
• Grantee: Rodrigo Alexandre Panepucci
• Support Opportunities: Scholarships abroad - Research
• FAPESP's process: 13/08135-2 - CTC - Center for Cell-Based Therapy
• Grantee: Dimas Tadeu Covas
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC