Jagged1 expression regulated by Notch3 and Wnt/β-catenin signaling pathways in ovarian cancer

Abstract

Ovarian 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, Jagged1 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 Jagged1 expression depended on Notch3 pathway activation. Knockdown of either Notch3 or RBPjk, a Notch-interacting transcription factor critical in Notch signaling, suppressed Jagged1 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 and β-catenin reduced Jagged1 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 Jagged1. Both mechanisms may sustain Notch3 signaling in ovarian cancer cells and contribute to the pathogenesis of ovarian carcinoma.

Keywords: Jagged; Notch3; ovarian cancer; signaling.

Fig. 1:. Notch3 knockdown suppresses Jagged1 expression in ovarian cancer cell lines

A. Quantitatively RT-PCR performed in OVCAR3 and OVMANA cells demonstrates that Jagged1 and Hes1 transcript levels decrease after Notch3 knockdown by shRNA. B. Western blot demonstrates Jagged1 protein down-regulation after treating cells with Notch3 shRNA. Protein expression of GAPDH determined by Western blot is used as the loading control.

Fig. 2. Downregulation of Jagged1 after knockdown of RBP-jκ, a mediator of the Notch3 signaling pathway, in OVCAR3 cells

A. Both Jagged1 mRNA level (left panel) and protein level (right panel) decreased after RBP-jκ knockdown in OVCAR3 cells as detected by quantitative RT-PCR and Western blot analyses. B. Cell proliferation significantly decreases in OVCAR3 cells following RBP-jκ knockdown. RBP-jκ shRNA1 and shRNA was co-applied in this study. C. mRNA expression level of Notch3 is not altered by Jagged1 shRNA knockdown in OVCAR3 cells

Fig. 3. Ectopic expression of Notch3 intracellular domain upregulates Jagged-1 expression in human ovarian surface epithelial cells

A. Quantitative real-time PCR shows Jagged1 mRNA upregulation after ectopic expression of Notch3 intracellular fragment (NICD3) in human ovarian surface epithelial cells, IOSE-80PC. The expression of a Notch-regulated target gene, Hes1, is used as a control. B. Western blot demonstrates that both Jagged1 full length (FL) and C-Terminal Fragment (CTF) are upregulated after ectopic expression of NICD3 in IOSE-80PC cells. GAPDH is used as the loading control.

Fig. 4. Notch3 induction increases Jagged1 expression at both mRNA and protein levels in Notch3 knockin mouse cells.

A. Generation of inducible knockin mice to express human Notch3. Gene targeting strategy is illustrated. The Notch3 transgene was recombined into the ROSA26 locus of embryonic stem cells. The targeted of the locus results in a stop/polyA cassette that prevents expression in the absence of Cre recombinase. The Cre/Lox reaction (bottom) activates expression of the Notch3 cassette. B. Genotypes of Notch3 knockin mouse. The band at 340 bp corresponds to the presence of knockin

Fig. 5. Wnt signaling pathway supports Jagged1 expression in ovarian cancer cells.

Quantitative real time PCR shows a significant decrease in Jagged1 expression level after knockdown of either β-catenin (left panel) or TCF4 (right panel) in OVCAR3 cells. Well-known WNT targets including CCND1, SOX9, and CD44 are used as positive controls.

Fig. 6. Simultaneous knockdown of both Notch3 and Wnt singling pathways in ovarian cancer cells.

A. Simultaneous knockdown of both Notch3 and β-catenin in OVCAR3 cells reduces Jagged1 transcript level significantly more than knockdown of either Notch3 shRNA or β-catenin shRNA alone at the same concentration of shRNA plasmid (p<0.001). B. Cellular proliferation in OVCAR3 cells transduced by both Notch3 shRNA and β-catenin shRNA is significantly lower than in cells transduced by either shRNA virus alone (p<0.001). C. Colony formation assay performed in OVCAR3 cells demonstrates that cells transduced by both Notch3 shRNA and β-catenin shRNA have lower colony formation ability than cells transduced by either shRNA virus alone (p<0.001).