Abstract 3934: Restoration of NOTCH signaling in head and neck squamous cell carcinoma inhibits tumor growth and cell migratory abilities
Authors: Moorthy Shhyam , Saade Rami , Pickering Curtis , Neskey David , Tanaka Noriaki , Zweidler-McKay Patrick , Frederick Mitchell , Myers Jeffrey 
Published: August 2015

Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common malignancy worldwide, with roughly 300,000 cancer related deaths occurring globally each year. The survival of patients with HNSCC hasn't changed significantly over the past decade leading investigators to search for promising molecular targets. To identify new treatment targets and biomarkers that could better guide therapy, we previously characterized the genomic alterations from primary HNSCC patient samples. We were among the first to discover that NOTCH1 is one of the most frequently mutated genes in this cancer type. The spectrum of inactivating NOTCH1 mutations in HNSCC suggested a tumor suppressive role for this protein, however, the mechanism of its function is currently unknown.

Procedure: We used Sanger sequencing and immunoblotting to characterize 50 well- established HNSCC cell lines as being wild-type or mutant for NOTCH1. We cloned the full length NOTCH1 receptor to restore the NOTCH signaling function and activated this pathway by culturing cells on immobilized NOTCH ligand, Jagged1, coated plates. Clonogenic assays and competitive cell proliferation assays was used to evaluate cell growth and proliferation. Transwell Boyden chambers was used to investigate migration/invasion. Tumorigenicty was evaluated in vivo in a mice orthotopic model of oral cancer. An unbiased gene expression analysis was performed to identify potential downstream targets of the NOTCH pathway.

Results: We show here that restoration of NOTCH signaling in mutant HNSCC cell lines significantly decreases cell growth and migratory abilities. Activation of NOTCH1 led to decreased tumorigenicity in a mice orthotopic oral cancer model. Conversely, abrogation of the NOTCH pathway using the dominant negative form of the NOTCH co-activator, MAML1, enhanced tumor growth in a weakly tumorigenic HNSCC cell line harboring wild-type NOTCH1. To evaluate the mechanism of NOTCH induced inhibition of growth and migration, we performed an unbiased microarray analysis and observed suppression of two proto-oncogenes, AXL and CTNNAL1 in the presence of NOTCH1, but not in its absence. Restoration of NOTCH1 significantly decreases protein expression levels of AXL and CTNNAL1 which then results in decreased cell growth and migratory abilities. Furthermore, we show here a novel link between NOTCH1, AXL and CTNNAL1 via HES5, one of the canonical NOTCH downstream targets. We suggest that NOTCH1 activates HES5 which then downregulates AXL and CTNNAL1, thus inhibiting cell growth and migratory abilities.

Conclusions: We have data linking NOTCH1 reactivation to suppression of proto-oncogenes, AXL and CTNNAL1 along with decreased growth and migration of HNSCC cells. This underscores the need to understand the clinical significance of the NOTCH pathway and its downstream targets which can then be exploited for potential therapeutic strategies.

Citation Format: Shhyam Moorthy, Rami Saade, Curtis Pickering, David Neskey, Noriaki Tanaka, Patrick Zweidler-McKay, Mitchell Frederick, Jeffrey Myers. Restoration of NOTCH signaling in head and neck squamous cell carcinoma inhibits tumor growth and cell migratory abilities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3934. doi:10.1158/1538-7445.AM2015-3934