Targeting gonadotropin-releasing hormone receptor inhibits the early step of ovarian cancer metastasis by modulating tumor-mesothelial adhesion

Abstract

Ovarian cancer has a clear predilection to metastasize to the peritoneum, which represents one of the most important prognostic factors of poor clinical outcome. Gonadotropin-releasing hormone (GnRH) receptor is significantly overexpressed during the malignant progression of human ovarian cancer. Here, using lentiviral-based small interfering RNA (siRNA) technology to downregulate GnRH receptor in metastatic ovarian cancer cells, we show that GnRH receptor is an important mediator of ovarian cancer peritoneal metastasis. GnRH receptor downregulation dramatically attenuated their adhesion to the peritoneal mesothelium. By inhibiting the expression of GnRH receptor, we showed decreased expression of α2β1 and α5β1 integrin and adhesion to specific extracellular matrix (ECM) proteins. This was also associated with a reduction of P-cadherin. Furthermore, adhesion of ovarian cancer cells to different ECMs and the mesothelium were abrogated in response to β1 integrin and P-cadherin reduction, confirming that the effects were β1 integrin- and P-cadherin-specific. Using a mouse model of human ovarian cancer metastasis, we found that the inhibition of GnRH receptor, β1 integrin, and P-cadherin significantly attenuated tumor growth, ascites formation, and the number of metastatic implants. These results define a new role for GnRH receptor in early metastasis and offer the possibility of novel therapeutic targets.

Figure 1

GnRH receptor (GnRHR) knockdown decreases adhesion of ovarian cancer cells to the mesothelium. (a) Caov-3 and OVCAR-3 cells were transduced with nonspecific (NS) or GnRHR siRNA. Cells were harvested and GnRHR expression was detected by western blotting. β-Actin was used to control for equal loading. Numbers below blots indicate quantification by densitometry for triplicate experiments. (b) NS or GnRHR siRNA-transduced cells were labeled with calcein-AM, plated onto 96-well plates coated with a monolayer of primary human peritoneal mesothelial cells (HPMCs), and incubated for 6 hours in the presence or absence of 0.1 nmol/l GnRHa. After nonadherent cells were removed, the number of adherent cells was quantified by measuring fluorescence intensity with a fluorescence spectrophotometer. Representative photographs were shown at low magnification. Data are mean values ± SD from three independent experiments. *P < 0.05 compared with control. GnRHa, gonadotropin-releasing hormone analogue; siRNA, small interfering RNA.

Figure 2

GnRH receptor (GnRHR) siRNA decreases adhesion of ovarian cancer cells to ECM-coated wells. Nonspecific (NS) or GnRHR siRNA-transduced cells were plated onto 96-well plates coated with collagen I, fibronectin, laminin, and vitronectin, and incubated for 4 hours in the presence or absence of 0.1 nmol/l GnRHa. After nonadherent cells were removed, the number of adherent cells was quantified by staining with 0.5% crystal violet and measuring absorption at 630 nm. Data are mean values ± SD from three independent experiments. *P < 0.05 compared with control. ECM, extracellular matrix; GnRHa, gonadotropin-releasing hormone analogue; siRNA, small interfering RNA.

Figure 3

GnRH stimulates the expression of α2, α5, and β1 integrin. (a) Total RNA was extracted from Caov-3 and OVCAR-3 treated with 0.1 nmol/l GnRHa, reverse-transcribed, and amplified with α2, α3, α5, α6, αv, β1, and β3 integrin-specific primer. (b) Same assay as (shown in a) in cells transduced with nonspecific (NS) or GnRH receptor (GnRHR) siRNA. GAPDH was included as an internal control. Data are mean values ± SD from three independent experiments. *P < 0.05 compared with control. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GnRHa, gonadotropin-releasing hormone analogue; mRNA, messenger RNA; siRNA, small interfering RNA.

Figure 4

Effect of inhibition of integrins on binding of ovarian cancer cells to different ECM components. Caov-3 and OVCAR-3 cells were preincubated with inhibiting antibodies to α2, α3, α5, αv, or β1 integrin plated onto 96-well plates coated with (a) collagen I, (b) fibronectin, or (c) laminin, and incubated for 4 hours in the presence or absence of 0.1 nmol/l GnRHa. (d) Nonspecific (NS) or β1 integrin siRNA-transduced cells were harvested and β1 integrin expression was detected by western blotting. β-Actin was used to control for equal loading. Numbers below blots indicate quantification by densitometry for triplicate experiments. (e) Cells were plated onto 96-well plates coated with collagen I, fibronectin, and laminin, and incubated for 4 hours in the presence or absence of 0.1 nmol/l GnRHa. After nonadherent cells were removed, the number of adherent cells was quantified by staining with 0.5% crystal violet and measuring absorption at 630 nm. Data are mean values ± SD from three independent experiments. *P < 0.05 compared with control. ECM, extracellular matrix; GnRHa, gonadotropin-releasing hormone analogue; siRNA, small interfering RNA.

Figure 5

Effect of inhibition of integrins on binding of ovarian cancer cells to the mesothelium. Caov-3 and OVCAR-3 cells were preincubated with blocking antibodies to α2, α3, α5, αv, or β1 integrin, labeled with calcein-AM, plated onto 96-well plates coated with human peritoneal mesothelial cells (HPMCs), and incubated for 6 hours in the presence or absence of 0.1 nmol/l GnRHa. After nonadherent cells were removed, the number of adherent cells was quantified by measuring fluorescence intensity with a fluorescence spectrophotometer. Representative photographs were shown at low magnification. Data are mean values ± SD from three independent experiments. *P < 0.05 compared with control. GnRHa, gonadotropin-releasing hormone analogue.

Figure 6

β1 integrin siRNA decreases adhesion of ovarian cancer cells to the mesothelium. Caov-3 and OVCAR-3 cells were transduced with nonspecific (NS) or β1 integrin siRNA, labeled with calcein-AM, plated onto 96-well plates coated with human peritoneal mesothelial cells (HPMCs), and incubated for 6 hours in the presence or absence of 0.1 nmol/l GnRHa. After nonadherent cells were removed, the number of adherent cells was quantified by measuring fluorescence intensity with a fluorescence spectrophotometer. Representative photographs were shown at low magnification. Data are mean values ± SD from three independent experiments. *P < 0.05 compared with control. GnRHa, gonadotropin-releasing hormone analogue; siRNA, small interfering RNA.

Figure 7

Effect of inhibition of P-cadherin on peritoneal attachment. (a) Expression of P-, E-, and N-cadherin by Caov-3, OVCAR-3, and human peritoneal mesothelial cells (HPMCs) were evaluated by western blot analysis. β-Actin was used to control for equal loading. (b) Nonspecific (NS) or P-cadherin siRNA-transduced cells were harvested and P-cadherin expression was detected by western blotting. β-Actin was used to control for equal loading. Numbers below blots indicate quantification by densitometry for triplicate experiments. (c) Cells were preincubated with inhibiting antibodies to E-, N-, P-cadherin, or transduced with NS or P-cadherin siRNA, labeled with calcein-AM, plated onto 96-well plates coated with a monolayer of HPMCs, and incubated for 6 hours in the presence or absence of 0.1 nmol/l GnRHa. After nonadherent cells were removed, the number of adherent cells was quantified by measuring fluorescence intensity with a fluorescence spectrophotometer. Representative photographs were shown at low magnification. Data are mean values ± SD from three independent experiments. *P < 0.05 compared with control. GnRHa, gonadotropin-releasing hormone analogue; siRNA, small interfering RNA.

Figure 8

GnRH receptor (GnRHR) knockdown inhibits adhesion and intraperitoneal dissemination in vivo. Nude mice were intraperitoneally injected with Caov-3 cells transduced by nonspecific (NS), GnRHR, β1 integrin, or P-cadherin siRNA (three mice per group; 10⁷ cells per mouse). (a) At autopsy, ascitic fluid was collected and measured. (b) The total number of metastases were excised and counted. (c) Representative views of the metastasis in the peritoneal cavity are shown, and arrows indicate tumor. (d) Histologic sections containing microscopic tumor nodules in the mouse omentum, bar = 100 µm. (e) Tumor samples harvested from mice were analyzed by western blot with the respective antibodies. Numbers below blots indicate quantification by densitometry for triplicate experiments. Data are mean values ± SD from three independent experiments. *P < 0.05 compared with control. GnRH, gonadotropin-releasing hormone; siRNA, small interfering RNA.