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Ovarian cancer is the 5th leading cause of cancer death in women in the United States and is the most fatal gynecological malignancy. High grade serous ovarian cancer (HGSC) is the most common and deadly type of ovarian cancer largely due to the rapid metastasis throughout the peritoneum (abdominal cavity wall and organ lining). Metastatic spread of ovarian cancer usually occurs before diagnosis and can lead to bowel obstruction, organ failure, ascites, cachexia, infection and sepsis, and pulmonary embolism all causing death. Current methods to detect early stage ovarian cancer do not increase overall survival. A better understanding of the metastatic ability of ovarian cancers and the mechanism of cancer cell dissemination are critical to the development of new treatments for this devastating disease. In particular, investigation of pathways that affect early metastasis may indicate treatments that will lower disease burden and may suggest biomarkers of recurrent and/or chemotherapy resistant disease. Notch3 expression correlates with worse prognosis, chemotherapy resistance, and increased tumorigenic cell behaviors in HGSC. Here, we demonstrate that Notch3 acts to promote early stages of metastasis in a model of HGSC using the murine ID8 IP2 ovarian surface epithelial cell line. ID8 IP2 cells have little to no endogenous Notch3 expression and model metastatic disease when introduced intraperitoneally. We investigated the role of Notch3 by ectopically expressing the intracellular domain of murine Notch3 to induce constitutive Notch3 signaling in ID8 IP2 cells and verified Notch signal activation by target gene assessment. Induction of Notch3 signaling in ID8 IP2 reduced survival and accelerated disease burden, as measured by ascites accumulation, after intraperitoneal introduction of cells into nude mice. We interrogated downstream targets in Notch3 activated cells by RNA-Seq and found that Notch3 induced a significant enrichment of adhesion and extracellular matrix pathways. Notch3 active cells showed increased ITGA1 expression and increased adhesion on collagens I and IV in vitro, suggesting that increased adhesion to collagen-rich peritoneal surfaces drives the observed increase in tumor burden. Notch3 active cells showed reduced migration on surfaces coated with multiple types of extracellular matrix and no detectable increase in invasion through extracellular matrix, indicating that Notch3 effects may be specific to the initial adhesion of tumor cells and not the later stages of metastasis. These results demonstrate that Notch3 upregulates the expression of specific adhesion genes in ovarian cancer cells and this promotes increased attachment to the collagen-rich extracellular matrix. The implications of this study are that oncogenic Notch signal activation, as documented in human disease, may promote dissemination and metastasis of primary and/or recurrent HGSC by increasing attachment to the peritoneal lining. |
