The functional role of NOTCH and potential NOTCH associated genes in head and neck cancer

Abstract

Notch signaling is actively involved in cell differentiation, proliferation, and apoptosis, and plays a significant role in tumor growth and prognosis of head and neck squamous cell carcinoma (HNSCC). Altered NOTCH expression and mutations are often associated with poor prognosis in patients with HNSCC. Rrecently, our group has identified a specific gene expression signature which is likely associated with Notch signaling in HNSCC. From this signature, Dishevelled Segment Polarity Protein 3 (DVL3) and Proteasome 26S Subunit Ubiquitin Receptor, Non-ATPase 2 (PSMD2) were selected and studied in this work. Based on bioinformatic analysis, these two genes are found amplified or over-expressed in about 30-40% of all HNSCC cases. They also showed potential association with Notch mutations in some cancers. The main purpose of this work is to find out experimentally whether there is a regulatory association between DVL3 or PSMD2 and NOTCH signaling. Another purpose of this work is to investigate what are the functional roles of NOTCH and these two genes in HNSCC. The methods used in this work are 2D and organotypic 3D cultures, metabolic cell viability assay, proliferation assay, IncuCyte live cell imaging and confocal imaging with phenotypic image analysis. siRNA transfection, western blotting, and quantitative real-time polymerase chain reaction (qPCR) are also used for gene expression and signaling studies. The results showed that Notch signaling is active in our studied cell lines, and Notch seems to have a tumor promoting role in the cell line studied in this work and inhibition of NOTCH affects cell growth and viability in 2D and 3D cultures. DVL3 may regulate NOTCH1 activity and knocking DVL3 down may increase the cell growth and viability in 3D culture. In addition, PSMD2 is found expressed at significantly higher levels in HNSCC tumors compared to benign tissues.