The Role Of Abnormal Notch3 Signaling Pathway In The Tumorgenisis Of Human Ovarian Cancer

PartⅠ:Jaggedl expression regulated by Notch3 and Wnt/β-catenin signaling pathways in ovarian cancerOvarian serous carcinoma is a highly aggressive neoplastic disease in women. Our previous studies have demonstrated Notch3 gene amplification and upregulation in many ovarian serous carcinomas and Notch pathway activity contributed to drug resistance. Among different Notch3 ligands, Jaggedl is most dominant in ovarian cancer, and Notch3 pathway activity correlated with Jagged1 expression level in ovarian carcinoma tissues. In this study, we found that Jaggedl expression depended on Notch3 pathway activation. Knockdown of either Notch3 or RBPjk, a Notch interacting transcription factor critical in Notch signaling, suppressed Jaggedl expression in ovarian cancer cells. Moreover, Jagged1 expression was upregulated in human ovarian surface epithelial cells after ectopic expression of Notch3 intracellular domain and was upregulated in mouse epithelial cells isolated from Notch3-inducible mice after induction. We also found that inhibition of Wnt/β-catenin signaling reduced Jagged1 expression, and co-administration of shRNAs targeting both Notch3 andβ-catenin reduced Jaggedl expression much more than targeting either individual gene. Taken together, our data suggested a positive regulatory loop between Notch3 and its ligand, Jagged1, in ovarian cancer cells. In addition, Wnt/β-catenin pathway activation also up-regulated Jaggedl. Both mechanisms may sustain Notch3 signaling in ovarian cancer cells and contribute to the pathogenesis of ovarian carcinoma. Part II:Investigate the regulation mechanism of Notch3 by taking the advantage of ChIP-chip technology in human ovarian cancerNotch signaling is critically involved in several biological and pathological processes including tumorigenesis. Identification and characterization of the genes directly regulated by the Notch pathway are fundamental to understanding how Notch signaling controls those processes. We have previously demonstrated Notch3 gene amplification and its role in the pathogenesis of ovarian cancer. In this study, using ChIP-on-chip we identified genes whose promoters were bound by RBPjk/N3ICD. Computational analysis of these promoters revealed enrichment of a new binding motif, GTTGCCAT, in addition to the canonical CSL-binding motif. We further selected genes whose expression was altered by y-secretase inhibitor. Among these genes, hepatoma up-regulated protein (HURP) encoding a protein involved in organization of the mitotic apparatus, was selected for further study. We observed that both canonical and novel CSL consensus sites were essential for upregulating HURP expression. Knockdown of HURP gene expression inhibited cellular proliferation in ovarian cancer cells by arresting cell cycle at the G2/M phase and suppressed tumorigenecity in vivo. Forced expression of HURP partially abolished the growth-inhibitory effects of either y-secretase inhibitor or Notch3 knockdown in ovarian cancer cells. Taken together, our results suggest that HURP represents a new Notch3 downstream target, which mediates, in part, the tumor-promoting functions of Notch3 in ovarian cancer.