NOTCH1 loss promotes chemo and radio-resistance in head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with over 700,000 new cases diagnosed annually. HPV-associated HNSCC patients generally respond well to standard therapies; however, about 15-20 % experience recurrence, while the rate is approximately 50 % in non-HPV-associated HNSCC. Identifying biomarkers for treatment failure is crucial for optimizing treatment strategies. Our lab's whole genome sequencing (WGS) data has identified NOTCH1 mutations as enriched in recurrent HPV-positive HNSCC cases. This study investigates whether NOTCH1 deletion confers treatment resistance in both HPV-positive and HPV-negative HNSCC models.

Four HPV-positive (HPV+) HNSCC cell lines (UWO23, UWO37, UMSCC47, and 93VU147) and two HPV-negative (HPV-) HNSCC cell lines (FaDu and Cal27) were examined. We used siRNA to knock down NOTCH1 expression, confirmed by qPCR, and then used CRISPR editing to generate stable NOTCH1 knockout (KO) cells. The knockout was validated by real-time qPCR and western blotting. Cell viability, clonogenicity, and invasiveness were then evaluated through viability, colony formation, and invasion assays.

The siRNA-mediated NOTCH1 knockdown increased cellular proliferation in both HPV+ and HPV- HNSCC lines, with similar effects observed in CRISPR-edited NOTCH1-KO cells. Colony formation and invasion assays demonstrated higher aggression and invasiveness in NOTCH1-KO cells compared to non-targeting controls (NTC). IC50 analysis showed that NOTCH1-KO cells exhibited enhanced cisplatin resistance in both HPV+ and HPV- lines, with increased radiation resistance also observed in NOTCH1-KO cells.

Our findings indicate that NOTCH1 loss in both HPV+ and HPV- HNSCC cell lines leads to increased aggressiveness, clonogenicity, invasiveness, and resistance to chemotherapy and radiation. This suggests that NOTCH1 loss may be a potential biomarker for identifying HNSCC patients at higher risk of treatment failure, supporting a more personalized, intensive therapeutic approach.