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Kathleen M. Capaccione
B.A., New York University 2009
Thesis advisor: Sharon R. Pine, Ph.D.
Graduate Program in Cellular and Molecular Pharmacology
Cancer Institute of New Jersey
Tuesday, April 1, 2014
The Notch signaling pathway plays a critical role in development and in cancer. The specific effects of Notch signaling are highly cell-type and context dependent. Overactive Notch signaling was discovered as a driver in T-ALL, leading to the investigation of the role of Notch signaling in multiple types of cancer. Studies have shown that overactive Notch signaling confers a survival advantage on cancer cells through multiple different mechanisms. Recent work has identified aberrant Notch signaling in 40% of non-small-cell lung cancer cases, making Notch signaling an attractive therapeutic target. Gamma-secretase inhibitors have had limited success in clinical trials, in large part due to toxicities. It is critical to elucidate downstream players of Notch signaling and understand their role in mediating characteristics of malignancy conferred by overactive Notch signaling. Our initial studies demonstrated that Sox9 may be one such gene. Sox9 is a developmental transcription factor involved in the formation of long bones and sex determination. It is also expressed in the developing lung. Recent studies have shown that overexpression of SOX9 correlates with NSCLC progression and poorer survival. Our bioinformatics studies of three microarray datasets assessing genes whose expression correlated with expression of HES1, a canonical Notch target gene, indicated that SOX9 is likely downstream of Notch1 in NSCLC. In vitro pharmacologic studies demonstrated that SOX9 responds to modulation of the
Notch pathway, which was confirmed by overexpression and knockdown experiments. A ChIP study demonstrated that the RBP-JÍ/Notch1 Intracellular Domain (N1ICD) complex binds directly to the Sox9 promoter sequence. Site-directed mutagenesis demonstrated that mutation of a specific sequence within the SOX9 promoter led to complete abrogation of the SOX9 promoterís ability to drive gene expression, indicating the site where the RBP-JÍ /N1ICD complex binds within the SOX9 promoter. We assessed the role of the Notch1/Sox9 signaling axis in promoting malignant characteristics and found that inhibition of Notch signaling decreased invasion and migration and altered cell morphology, both of which could be restored by expression of Sox9. Further investigating how these genes promote changes in cell motility, we screened genes known to be involved in epithelial-mesenchymal transition. E-cadherin expression was consistently altered by modulation of the Notch/Sox9 signaling pathway both in vitro and in patient samples. Together, these data demonstrate a novel direct relationship between Notch1 and Sox9 signaling and establish that this pathway mediates the migratory and invasive ability of NSCLC cells, likely through modulation of E-cadherin expression. Targeting Sox9 or E-cadherin may be therapeutically beneficial for controlling metastatic spread of NSCLC and limiting mortality from this disease.