Role of Jagged1/STAT3 signalling in platinum-resistant ovarian cancer

Abstract

Jagged1, the essential ligand of the Notch signalling pathway, is highly expressed in metastatic prostate cancer, and its high expression in breast cancer is linked to poor survival rates. However, the mechanism of Jagged1's involvement in platinum-resistant ovarian cancer has not been thoroughly elucidated to date. The purpose of the present study was to investigate the roles of Jagged1 in the platinum resistance of ovarian cancer and its possible mechanisms. Compared with a platinum responsive group of ovarian epithelial cell carcinomas, we found the positive staining intensity of Notch1, Notch2, Jagged1, STAT3 and Epithelial-mesenchymal transition (EMT) proteins were lower in a platinum-resistant group. The DDP-resistant ovarian cancer cell line (C13K) had a higher IC50 of DDP than its parental cell line (OV2008) (P < 0.05) and acquired an EMT phenotype and invasive characteristics. Inhibiting or knockdown of Jagged1 expression could not only reduce its capacity of migration and invasion but also reverse EMT and down-regulate the expression of serine 727-phosphorylated STAT3 (pS727) at the protein level but not total STAT3 or tyrosine 705-phosphorylated STAT3 (pY705) in C13K cells. Furthermore, it was found that crosstalk between the Jagged1/Notch and JAK/STAT3 signalling pathways were involved in Jagged1-promoting EMT in C13K cells. Experiments in vivo showed a reduced micrometastatic tumour burden in the lung, liver and spleen of mice implanted with C13K cells with knocked-down Jagged1 compared with mice implanted with control cells. All of these results demonstrate that Jagged1 can crosstalk with the JAK/STAT3 pathway, and they all cooperate to promote the aberrant occurrence of EMT, further reinforcing the abilities of invasion and migration of platinum-resistant ovarian cancer in vivo and in vitro.

Keywords: Jagged1; STAT3; crosstalk; ovarian cancer; platinum resistance.

Figure 1

The Notch pathway in platinum‐resistant ovarian cancer is important for cell malignant phenotype. A, The cytotoxic effect of cisplatin (IC50) on OV2008 and C13K cells were examined by CCK‐8 assay. B, The protein expression levels of Notch1/2 and cleaved Notch1/2 in OV2008 and C13K cells were determined by Western Blot. C, Immunohistochemistry analyses of Notch1 and Notch2 were performed in platinum‐resistant group and platinum responsive group, as shown in representative images (×400 magnification). D, Wound healing assay was analysed the migratory ability of C13K cells treated by a wide concentration range of DAPT (0, 2.5, 5,10, 20 and 40 μmol/L). (E and F) Transwell migration and invasion assay were performed to confirm the migratory and invasive abilities of C13K cells exposed by a wide concentration range of DAPT. G and H, CCK‐8 proliferation assay was examined the proliferative ability of C13K cells treated by a wide concentration range of DAPT for different time. (*P < 0.05)

Figure 2

The Notch pathway is involved in epithelial‐mesenchymal transition (EMT) progression in cisplatin‐resistant ovarian cancer cells. A, Optical microscope was designed to observe the morphology of C13K cells and OV2008 cells. B, The protein expression levels of EMT relative genes were determined by Western Blot. C, Immunohistochemistry analyses of EMT relative proteins were performed in platinum‐resistant group and platinum responsive group, as shown in representative images (×400 magnification). D, Western blot assay examined the expression of EMT relative proteins of C13K cells treated by a wide concentration range of DAPT for different time. E, The aggressive phenotype of OV2008 cells and C13K cells in the presence or absence of DAPT (10 μmol/L) when cultured in three‐dimensional matrigel

Figure 3

Jagged1 plays a critical role in EMT signalling in C13K cells. A, Western blot assay examined the expression of Notch1, cleaved Notch1 and EMT relative proteins of C13K cells after transfection with Jagged1 and Jagged2 siRNA. B, The quantitative analysis of Fig. 3A. (*P < 0.05)

Figure 4

The crosstalk of Jagged1/STAT3, not Jagged2/STAT3, is important for epithelial‐mesenchymal transition (EMT) in C13K cells. A, Immunohistochemistry analyses of STAT3 and Jagged1 proteins were performed in platinum‐resistant group and platinum responsive group, as shown in representative images (×400 magnification). B, Western blot assay examined the expression of Jagged1, Jagged2 and JAK/STAT3 pathway relative proteins of C13K cells after transfection with Jagged1 and Jagged2 siRNA. C, The proteins expression of Jagged1, Jagged2 and JAK/STAT3 pathway relative proteins of C13K cells treated by a wide concentration range of DAPT (0, 2.5, 5,10, 20 and 40 μmol/L). D, The physical relationship of Jagged1 and STAT3 was analysed by Co‐IP assay. E, The distribution of Jagged1 and STAT3 in C13K cells was examined by co‐immunofluorescent staining and confocal microscopy imaging. F, The proteins expression of Jagged1, JAK/STAT3 pathway and epithelial‐mesenchymal transition (EMT) relative proteins of C13K cells treated by a wide concentration range of WP1066 (0, 0.5 1, 2, 4 and 8 μmol/L)

Figure 5

Knock‐down Jagged1 impairs tumour growth and invasion in mouse xenograft models. A, The weight of each mice bearing C13K/si‐Jagged1 cells, C13K cells and C13K/si‐NC cells were measured once a week. B, C13K/si‐Jagged1 cells, C13K cells and C13K/si‐NC cells were injected into tail veins of the nude mice, respectively. 8 weeks following tumour cell implantation, the mice were photographed after harvest. C, The tissue sample (lung and liver) each group were photographed after harvest. D, Representative images of haematoxylin and eosin‐stained lung, liver and spleen sections from each group