In the current study, we found that targeting Jagged1 in tumor cells induces apoptosis, reduces cell viability, and reverses taxane resistance in ovarian cancer cells both in vitro and in vivo, at least in part through downregulation of the Hedgehog mediator GLI2. Additionally, knockdown of Jagged1 in tumor stromal cells reduces tumor growth through an anti-angiogenic mechanism. The participation of Jagged1 in both stromal and malignant cell compartments makes it an attractive target for therapy, and demonstrates the utility of a model whereby these compartments can be targeted independently to delineate the various contributions of different cells in the tumor microenvironment.
Previous studies have demonstrated aberrant expression of the Notch pathway in ovarian cancer (4
). In particular, Jagged1 was found to be the primary Notch ligand expressed in ovarian cancer cells compared to Jagged2, DLL1, 3 and 4 (6
). Jagged1 was also found to be overexpressed in endothelial cells purified from ovarian cancers compared to normal ovaries (11
). Taken together, these studies indicate that Jagged1 would be a desirable therapeutic target in ovarian cancer, both from an anti-tumor and anti-angiogenic standpoint. In our study, we found that downregulation of Jagged1 resulted in decreased ovarian cancer cell viability in vitro
, most likely mediated through reduced cell proliferation and, to a lesser extent, induction of apoptosis. Our study is the first to demonstrate that targeting Jagged1 diminishes tumor burden in vivo
. Because there are no known inhibitors of Jagged1, we used chitosan nanoparticles to deliver Jagged1 siRNAs in tumor-bearing mice. These positively charged nanoparticles allow for the transport of siRNA across cellular membranes and are biodegradable, biocompatible and have low immunogenicity (34
). Targeting Jagged1 using this delivery system may also avoid the dose-limiting toxicities inherent to systemic Notch inhibitors such as gamma-secretase inhibitors (GSIs) (36
). Selective targeting of Jagged1 using chitosan greatly decreased tumor burden and increased taxane sensitivity in orthotopic ovarian cancer mouse models. These results, combined with our observations in vitro
, indicate that Jagged1 plays an important role in ovarian cancer cell survival. Whether these effects occur entirely through Notch signaling remains an open question. It has been suggested by Choi et al. (6
) and others (15
) that Jagged1 may have its own signaling function that is independent of the canonical Notch pathway. Indeed, the lack of a decrease in the expression of Notch downstream targets, HES1
, following Jagged1 downregulation in our study supports this mechanism. This potentially unique Notch-independent function of Jagged1 in human cancers, however, has yet to be fully explored.
The interaction between cancer cells and the surrounding stroma is increasingly becoming a focus of study in cancer research due to its role in tumor progression. This tumor-associated stroma is composed primarily of endothelial cells, which are necessary for tumor angiogenesis, and fibroblasts, which can secrete growth factors to the adjacent cancer cells. Recent reports suggest that Notch signaling can occur between tumor and stromal cells in some malignancies (37
), indicating that targeting the Notch-ligand interaction in endothelial cells can have therapeutic applications. In addition, studies have shown that Jagged1 expression is crucial for normal vascular development during embryogenesis and that mutations of the JAGGED1
gene can cause Alagille syndrome, a disease characterized by, among other deformities, congenital heart defects (39
). In our study, we found that selectively targeting Jagged1 in the tumor stroma significantly reduced microvessel density (as measured by CD31) and, when combined with Jagged1 antagonism in cancer cells, the overall anti-tumor effect was greater than either anti-Jagged1 method alone. These data suggest that, unlike most cancer-associated targets which are expressed in only one compartment of the tumor, inhibiting Jagged1 activity could be used to target both the tumor and its developing vasculature, thereby having a potentially greater therapeutic benefit.
Chemoresistance remains a persistent obstacle in the treatment of ovarian cancer. While the clinical behavior of ovarian cancer suggests that most cancer cells are initially sensitive to chemotherapy, they subsequently either develop resistance or contain a population of cells that are inherently resistant. The latter hypothesis is consistent with what has become known as cancer stem cells or cancer initiating cells (CICs). These CICs are commonly believed to have enhanced tumorigenicity, differentiation capacity and resistance to chemotherapy in comparison to non-CICs. It is because of these features that CICs have been examined for molecular pathways and markers that could be targeted for therapeutic purposes. Recent studies have shown that the ancient developmental pathways Hedgehog, Wnt and Notch play important roles in the maintenance of CICs and that inhibiting these pathways may provide enhanced chemosensitivity when combined with traditional chemotherapies (8
). In our study, we sought to determine the mechanism whereby Jagged1, a known target of Wnt/β-catenin signaling (44
) and a Notch ligand, might sensitize ovarian cancer cells to docetaxel. We chose to focus on the hedgehog pathway due to its involvement in CIC maintenance and multi-drug resistance (46
). Interestingly, expression of GLI2
, a hedgehog transcriptional effector, was significantly decreased following Jagged1 knockdown, whereas expression of GLI1
was not. This relationship, one that appears to be Notch-independent, between Jagged1 and Gli2 was also found to work both ways, as knockdown of Gli2 diminished Jagged1 expression. Moreover, selective targeting of Gli2 using siRNA constructs decreased viability and increased sensitivity of ovarian cancer cells to docetaxel, although to a lesser degree than Jagged1 knockdown. These results suggest that inhibition of Gli2 contributes to the cell death and chemosensitization resulting from Jagged1 knockdown with other, as yet undefined, mechanisms likely playing a role as well. This connection between Jagged1 and Gli2 has not been previously identified and may have important therapeutic implications since targeting both Notch and Hedgehog, especially in combination with chemotherapy, is increasingly being advocated for the treatment of a variety of malignancies (3
Collectively, the data presented in this study demonstrate that the Notch ligand Jagged1 contributes to taxane resistance, and targeting Jagged1 in ovarian cancer cells as well as in surrounding stroma significantly reduces growth through anti-proliferative, apoptotic, anti-angiogenic, and taxane-sensitizing effects. With the ability to identify subsets of cancer patients with Jagged1 overexpression, antagonism of this signaling molecule could ultimately provide a useful therapeutic strategy for ovarian cancer.