Abstract B69: Notch3 signal activation promotes peritoneal metastasis in a mouse model of epithelial ovarian cancer.
Authors: Price Jessica , Azizi Elham , Jones Nathaniel , Kitajewski Jan 
Published: May 2016

This study examines the effect of Notch3 signal activation on ovarian cancer peritoneal metastasis to elucidate the mechanisms of ovarian cancer progression.

TCGA assessment of high-grade serous ovarian cancers determined the Notch3 signaling pathway has been altered in 22% of cancers. NOTCH3 was observed as upregulated in ovarian cancer by copy number analysis via multiple independent groups, and upregulation is linked to epithelial to mesenchymal cell transition in ovarian cancer cells. Patients with NOTCH3 overexpressing tumors exhibit worse prognosis, decreased survival, and correlation with chemotherapy resistance.

To determine how Notch3 signaling promotes ovarian cancer, we constitutively activated signaling in ID8 IP2, a mouse ovarian surface epithelial line with low Notch3 expression. ID8 IP2 was modified via lentiviral infection to express luciferase for in vivo assessment (ID8 IP2-luc). Notch3 signaling was activated using lentivurs generated with a pCCL plasmid containing the Notch3 receptor intracellular domain (N3IC) to create ID8 IP2-luc N3IC. A control line was made with pCCL containing only the multiple cloning site (MCS). Induction of N3IC expression and signal activation were confirmed by a dual luciferase reporter assay, western blot analysis of N3IC, and quantitative RT-PCR for N3IC and downstream HEY family targets. We assessed Notch3 activation in vitro to evaluate growth and metastatic properties of the cells, and in athymic mice to examine intraperitoneal metastasis.

ID8 IP2-luc N3IC and MCS were observed as morphologically similar in culture. No significant difference was detected in anchorage independent growth in a soft agar colony formation assay. Proliferation rate was 30% reduced in the ID8 IP2-luc N3IC compared to MCS. This finding was unanticipated given previous reports that proliferation in a serous line was dependent on Notch3, and Notch3 inhibition induced apoptosis. Notch3 activation did increase migratory capacity of ID8 IP2-luc N3IC compared to MCS. In a monolayer wounding assay, N3IC increased migration significantly at 3, 6, and 9 hours post wounding. In preliminary experiments, ID8 IP2-luc N3IC displayed significantly increased invasion in gelatin and Matrigel transwell migration assays. In vivo, preliminary results indicate Notch3 signal activation leads to acceleration of ascites accumulation, and a significant reduction in overall survival. Ascites derived from mice with Notch3 activated tumors qualitatively had higher blood content indicating tumor vasculature alteration.

Based on both in vitro and in vivo results, RNA-seq analysis comparing ID8 IP2-luc N3IC and MCS was performed to identify transcriptional targets to better define the role of Notch3 signaling in ovarian cancer. Data with an adjusted p value of &lt;0.05 and a minimum of log 2 fold change, were analyzed in pathway analysis through the Database for Annotation, Visualization & Integrated Discovery functional annotation tool. The most upregulated pathways in addition to Notch signaling involved extracellular matrix receptor interaction, tissue remodeling, and extracellular structure and matrix organization genes. The most down-regulated were cell adhesion and regulation of actin cytoskeleton genes. A significant change in cell adhesion and extracellular matrix genes is consistent with results indicating Notch3 activation induces change in cell migration, seeding, and invasion.

We conclude Notch3 signal activation in ID8 IP2-luc promotes ascites accumulation and metastasis in the peritoneum, and decreases overall survival in mice. The growth properties of ID8 IP2-luc cells were not altered with Notch3 activation in vitro, however migration and invasion were significantly induced. We propose Notch3 activation promotes increased metastasis by influencing interaction between ovarian cancer and the host environment. We conclude Notch3 activation leads to increased migration, seeding, and invasion into the peritoneum.

Citation Format: Jessica Price, Elham Azizi, Nathaniel Jones, Jan Kitajewski. Notch3 signal activation promotes peritoneal metastasis in a mouse model of epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B69.