Abstract 814: NOTCH1 regulates head and neck cancer cell growth and invasion

Abstract

Abstract Introduction: NOTCH1 has recently been identified as a frequently mutated gene in head and neck squamous cell carcinoma (HNSCC). However, its role in HNSCC is poorly understood. Creation of genetic models investigating the role of NOTCH1 in HNSCC behavior will be crucial to further characterize its function, and to potentially modify treatment paradigms in NOTCH1 mutant HNSCCs. To date, no syngeneic HNSCC cell line knockout models of NOTCH1 have been created to specifically study the effects of NOTCH1 in HNSCC phenotypes. Methods: We transfected a NOTCH1 gRNA-CRISPR-Cas9 lentiviral construct into a HNSCC cell line with wildtype NOTCH1 (UMSCC-49) and selected knockout clones. Whole exome sequencing, qRT-PCR, and Western blotting were performed to validate the cell line knockout. Invasion chamber and growth assays were performed to assay for cell line phenotypes. Immunofluorescence staining and proteomic analysis were performed to study expression changes in various invasive markers. Results: We successfully created a NOTCH1 knockout cell line (UMSCC-49N1KO) with an otherwise identical genetic architecture as its parent line (UMSCC-49). Knockout status was verified via exome sequencing, qRT-PCR and Western blotting. Analysis of UMSCC-49N1KO suggested an increased aggressive and invasive phenotype. Cell proliferation assays demonstrated an increased cell proliferation in UMSCC-49N1KO. Invasion chamber assays demonstrated increased invasion in the NOTCH1 deficient cell line. Proteomic analysis demonstrated increased expression of proteins associated with invasion and cell proliferation and decreased expression of cell differentiation markers in UMSCC-49N1KO, consistent with an aggressive and invasive phenotype in the NOTCH1 deficient line. These phenotypes were validated with subsequently generated UMSCC-49 NOTCH1 knockout lines. Conclusions: NOTCH1 acts as a key tumor suppressor gene in HNSCC and regulates cell proliferation and invasion. We have created a useful genetic model to study effects NOTCH1 in tumorigenesis, proliferation and metastasis with a wildtype NOTCH1 parent (UM-SCC49) and mutant NOTCH1, otherwise syngeneic cell line (UM-SCC49N1KO). NOTCH1 mutant status correlates with worse outcome in HNSCC patients. Further characterization of the role of NOTCH1 in cell proliferation, invasion and metastasis may provide insight into treatment stratification paradigms, targetable options and predictive biomarkers for patients with NOTCH1 mutant HNSCCs. Citation Format: Andrew C. Birkeland, Megan Ludwig, Jacqueline Mann, Samantha Devenport, Rebecca C. Hoesli, Chloe Matovina, Matthew E. Spector, Carol R. Bradford, Thomas E. Carey, Mark E. Prince, J. Chad Brenner. NOTCH1 regulates head and neck cancer cell growth and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 814. doi:10.1158/1538-7445.AM2017-814