Notch3 signaling promotes tumor cell adhesion and progression in a murine epithelial ovarian cancer model

High grade serous ovarian cancer (HGSC) is the most common and deadly type of ovarian cancer, largely due to difficulties in early diagnosis and rapid metastasis throughout the peritoneal cavity. Previous studies have shown that expression of Notch3 correlates with worse prognosis and increased tumorigenic cell behaviors in HGSC. We investigated the mechanistic role of Notch3 in a model of metastatic ovarian cancer using the murine ovarian surface epithelial cell line, ID8 IP2. Notch3 was activated in ID8 IP2 cells via expression of the Notch3 intracellular domain (Notch3IC). Notch3IC ID8 IP2 cells injected intraperitoneally caused accelerated ascites and reduced survival compared to control ID8 IP2, particularly in early stages of disease. We interrogated downstream targets of Notch3IC in ID8 IP2 cells by RNA sequencing and found significant induction of genes that encode adhesion and extracellular matrix proteins. Notch3IC ID8 IP2 showed increased expression of ITGA1 mRNA and cell-surface protein. Notch3IC-mediated increase of ITGA1 was also seen in two human ovarian cancer cells. Notch3IC ID8 IP2 cells showed increased adhesion to collagens I and IV in vitro. We propose that Notch3 activation in ovarian cancer cells causes increased adherence to collagen-rich peritoneal surfaces. Thus, the correlation between increased Notch3 signaling and poor prognosis may be influenced by increased dissemination and metastasis of HGSC via increased attachment to the peritoneum. Implications: Expression of Notch3 accelerates the progression of ovarian cancer by increasing the adherence of disseminating cells to new metastatic sites. Overall design: RNA was extracted from Notch3IC and Control Sets #1-#4 with a RNeasy Mini Kit (QIAGEN, 74104) and the RNase-Free DNase Set (QIAGEN, 79254). Library preparation and sequencing was performed by the JP Sulzberger Columbia Genome Center using Illumina TruSeq RNA Prep Kit with poly-A pulldown followed by sequencing ~30 million 100-base pair single-end reads on an Illumina HiSeq 2000 or 2500 instrument.