p53 context dependent role of Mir-9/KLF5/Sp1 axis in modulating response to therapy in Head and Neck Squamous Cell Carcinoma.

Head and Neck Squamous Cell Carcinoma (HNSCC) represents the sixth most common cancer worldwide, with only the 40-50% of patients surviving at five years from diagnosis. HNSCC can be classified as negative or positive for human papilloma virus (HPV) malignancies, which greatly differ for pathological, molecular features and clinical outcome. TP53 mutations are the most frequent genetic alteration in HPV-negative HNSCC, playing an uncontested role in its pathogenesis. On the contrary, HPV-positive HNSCC are mostly TP53 wild type and usually less heterogenic, displaying a more favorable prognosis compared to HPV-negative ones. Radiotherapy (RT) plus the anti-EGFR monoclonal antibody Cetuximab (CTX) may represent an effective combination therapy for a subset of HPV-negative/TP53 mutated HNSCC patients. However, prognostic/predictive markers of efficacy are missing, resulting in many patients treated with disappointing results and unnecessary toxicities. Several microRNAs (miRs) are dysregulated in HNSCC and we have recently identified a 4-miR-signature identifying HNSCC patients at high risk of developing recurrence. Among the 4, miR-9 was linked to epithelial to mesenchymal transition (EMT) and progression, and displayed the strongest association with recurrence risk. By this work, we aimed to study more in detail the role of miR-9 in HNSCC tumor initiating ability, in disease progression and in response to therapies. By this approach, we discovered a new signaling axis involving miR-9, KLF5, SP1, and we investigated its different behaviors depending on TP53 status of HNSCC. We observed that activation of EGFR upregulates miR-9 expression, which sustains the aggressiveness of HNSCC cells and protects from RT-induced cell death. Mechanistically, by targeting KLF5, miR-9 regulates the expression of the transcription factor SP1 that, in turn, stimulates tumor growth and confers resistance to RT+CTX, in vitro and in vivo. Intriguingly, high miR-9 levels had no effect on the sensitivity of HNSCC cells to cisplatin. In primary HNSCC, miR-9 expression correlated with SP1 mRNA levels and high miR-9 expression predicted poor prognosis in patients treated with RT+CTX. We discovered that regulation of SP1 by miR9/KLF5 axis worked differently depending on TP53 status: when p53 was mutated KLF5 downregulated SP1 and miR-9 overexpression inverted this trend; when p53 was wild type, KLF5 led to opposite effects, upregulating SP1 expression and increasing its transcriptional activity. Moreover, down-modulation of KLF5 expression affected the responsiveness to RT+CTX treatment, depending from their TP53 status, re-sensitizing the HNSCC TP53 wild type cells and increasing the resistance of the TP53 mutated ones. Altogether, this newly identified axis, involving miR-9/KLF5/SP1/p53, not only represents a novel potential prognostic and predictive biomarker for CTX activity that can explain why only a subset of HNSCC patients benefits from the combined use of RT+CTX, but may also promote the development of new targeted therapeutic strategies for HNSCC patients.