Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer worldwide. They are typically characterized by a high incidence of local recurrence, which is the most common cause of death in HNSCC patients. TP53 is the most frequently mutated gene in HNSCC and patients carrying TP53 mutations are associated with a higher probability to develop local recurrence. MiRNAs, which are among the mediators of the oncogenic activity of mt-p53 protein, emerge as an appealing tool for screening, diagnosis and prognosis of cancer. We previously identified a signature of 12 miRNAs whose aberrant expression associated with TP53 mutations and was prognostic for HNSCC. Among them miR-96-5p emerges as an oncogenic miRNAs with prognostic significance in HNSCC.
Methods: To evaluate the oncogenic role of miR-96-5p in a tumoral context, we performed colony formation, cell migration and cell viability assays in two HNSCC cell lines transfected for miR-96-5p mimic or inhibitor and treated with or without radio/chemo-therapy. In addition, to identify genes positively and negatively correlated to miR-96-5p expression in HNSCC, we analyzed the correlation between gene expression and miR-96-5p level in the subset of TCGA HNSCC tumors carrying missense TP53 mutations by Spearman and Pearson correlation. To finally identify targets of miR-96-5p, we used in silico analysis and the luciferase reporter assay to confirm PTEN as direct target.
Results: Our data showed that overexpression of miR-96-5p led to increased cell migration and radio-resistance, chemotherapy resistance in HNSCC cells. In agreement with these results, among the most statistically significant pathways in which miR-96-5p is involved, are focal Adhesion, extracellular matrix organization and PI3K-Akt-mTOR-signaling pathway. As a direct target of miR-96-5p, we identified PTEN, the main negative regulator of PI3K-Akt signalling pathway activation.
Conclusions: These results highlight a new mechanism of chemo/radio-resistance insurgence in HNSCC cells and support the possibility that miR-96-5p expression could be used as a novel promising biomarker to predict radiotherapy response and local recurrence development in HNSCC patients. In addition, the identification of pathways in which miR-96-5p is involved could contribute to develop new therapeutic strategies to overcome radio-resistance.
Keywords: Chemotherapy; Head and neck cancer; Local recurrence; Migration; PI3K-Akt signalling pathway; PTEN; Radiotherapy; TP53 mutations; miR-96-5p; miRNAs.