Abstract 3128: miR-9 expression regulates and predicts the response to EGFR inhibitors in head & neck squamous cell carcinoma
Authors: Citron Francesca , Vinciguerra Gian Luca Rampioni , Fanetti Giuseppe , Segatto Ilenia , Belletti Barbara , Vecchione Andrea , Franchin Giovanni , Baldassarre Gustavo 
Published: August 2019

Abstract
Introduction. Most Head & Neck Squamous Cell Carcinoma (HNSCC) patients are diagnosed with a locally advanced disease. Radiotherapy (RT) plus anti-EGFR monoclonal antibodies (Cetuximab - CTX) represents an effective combination therapy for locally advanced HNSCC patients. However, the 5-year overall survival is still 45%, mainly due to the appearance of loco-regional recurrences, suggesting that the identification and validation of predictive biomarkers of CTX activity is urgently needed to identify patients at high-risk of recurrence, who will benefit more from this therapeutic strategy. We recently validated a 4-microRNAs (miRs) signature, related to Epithelial to Mesenchymal Transition (EMT) and able to stratify HNSCC patients at high-risk of recurrence development. Among these 4 miRs, miR-9 was the only up-regulated in primary tumors from patients who developed recurrence within 2-year follow-up. Here we evaluated whether miR-9 expression had a functional role in HNSCC onset, progression and response to therapies.
Methods. miR-9 modified (overexpressing and silenced) HNSCC cells were generated, characterized for their growth and response to radio-, chemo- and targeted-therapies (i.e.Cisplatin, Paclitaxel, 5-Fluorouracil and CTX) in vitro and in vivo. Preclinical evidences were confirmed in a cohort of primary HNSCC samples by intercrossing the expression of miR-9 and selected target genes with patients’ clinical variables and response to therapy.
Results. Biochemical and biological in vitro and in vivo experiments showed that high miR-9 expression triggers EMT and increases tumor initiating properties in HNSCC cells. Interestingly, miR-9 silenced HNSCC cells displayed a higher sensitivity to RT and CTX, but not to chemotherapy, when compared to controls. Using an in vivo model of HNSCC, we observed that intra-tumor injection of anti-miR-9 improves the efficacy of RT alone and remarkably in combination with CTX. Mechanistically, we demonstrated that in HNSCC cells, EGFR activation triggers miR-9 expression that promotes the transcription of SP1, establishing a positive forward loop between EGFR activation and SP1 transcription. Accordingly, in primary HNSCC miR-9 expression strongly correlates with the one of SP1 and EGFR. More importantly, in a cohort of HNSCC patients treated with RT plus CTX, high miR-9 expression acts as an efficient predictive biomarker of intrinsic resistance.
Conclusion. Altogether by integrating wet-lab and clinical data, we provide strong evidences indicating that in HNSCC a subpopulation of miR-9 expressing cells is intrinsically resistant to RT plus CTX therapy, confirming its potential prognostic value. Since we set up an assay to easily and quantitatively evaluate miR-9 expression in diagnostic tumor biopsies, we propose that it could be used to personalize the treatments for this group of patients, avoiding unfaithful toxic therapies.
Citation Format: Francesca Citron, Gian Luca Rampioni Vinciguerra, Giuseppe Fanetti, Ilenia Segatto, Barbara Belletti, Andrea Vecchione, Giovanni Franchin, Gustavo Baldassarre. miR-9 expression regulates and predicts the response to EGFR inhibitors in head & neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3128.