Date of Graduation

12-2015

Document Type

Dissertation (PhD)

Program Affiliation

Cancer Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Dr. Jeffrey Myers

Committee Member

Dr. Gary Gallick

Committee Member

Dr. Patrick Zweidler-McKay

Committee Member

Dr. Paul Chiao

Committee Member

Dr. Rick Wetsel

Abstract

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 has not 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 in mutant cell lines 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. CRISPR-Cas9 system was used to knock out NOTCH1 and/or NOTCH2 in wild-type cells. Tumor growth 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. RNA-Seq data was obtained from HNSCC TCGA data and was correlated with the 120 gene expression signature obtained from cell lines. Flag-tagged constructs were used to overexpress HES2 and HES5, while shRNA’s was used to knock down AXL and catulin in mutant cell lines to evaluate tumor growth.

Results: We show here that restoration of full length NOTCH1 in mutant HNSCC cell lines and activation of NOTCH signaling in wild-type cell lines significantly decreases cell growth in vitro and tumor growth in vivo. CRISPR-Cas9 knock out of NOTCH1 and NOTCH2 in wild-type HNSCC cells accelerated cell growth that was reversed by restoration of NOTCH1 in the same cell line. To evaluate the mechanism of NOTCH induced growth inhibition, we performed an unbiased microarray analysis and observed suppression of two proto-oncogenes, AXL and CTNNAL1 (α-catulin) after NOTCH1 activation, but not in its absence. In addition, genes modulated by NOTCH activation in cell lines correlated with RNA-Seq data from 498 TCGA patient tumors. Restoration of NOTCH1 in mutant cell lines significantly decreased protein expression levels of AXL and α-catulin, while CRISPR-Cas9 knock out of NOTCH1 and NOTCH2 in wild-type cells increased expression of these proto-oncogenes. Knock down of AXL and α-catulin using shRNA’s abrogated tumor growth. Lastly, we show that HES2 and HES5, transcriptional regulatory proteins induced by NOTCH signaling might be sufficient to inhibit protein expression levels of AXL and α-catulin and suppress growth.

Conclusions: For the first time, we are attributing a tumor suppressive function of NOTCH in HNSCC. We conclude that reactivation of NOTCH signaling in HNSCC inhibits tumor growth and progression. In addition, we are also providing a possible mechanism linking NOTCH1 to the decreased expression of proto-oncogenes, AXL and CTNNAL1 (α-catulin) that potentially abrogates tumor growth. This underscores the need to understand the clinical significance of the NOTCH pathway and its downstream targets, which could then be exploited for potential therapeutic strategies.

Keywords

head and neck cancer, NOTCH, tumor growth, AXL, aplha-catulin