Volume 21, Issue 1, Pages 78-90 (January 2013) Targeting Gonadotropin-releasing Hormone Receptor Inhibits the Early Step of Ovarian Cancer Metastasis by Modulating Tumor-mesothelial Adhesion  Lydia WT Cheung, Susan Yung, Tak-Mao Chan, Peter CK Leung, Alice ST Wong  Molecular Therapy  Volume 21, Issue 1, Pages 78-90 (January 2013) DOI: 10.1038/mt.2012.187 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

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. Molecular Therapy 2013 21, 78-90DOI: (10.1038/mt.2012.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

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. Molecular Therapy 2013 21, 78-90DOI: (10.1038/mt.2012.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

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. Molecular Therapy 2013 21, 78-90DOI: (10.1038/mt.2012.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

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. Molecular Therapy 2013 21, 78-90DOI: (10.1038/mt.2012.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

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. Molecular Therapy 2013 21, 78-90DOI: (10.1038/mt.2012.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

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. Molecular Therapy 2013 21, 78-90DOI: (10.1038/mt.2012.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

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. Molecular Therapy 2013 21, 78-90DOI: (10.1038/mt.2012.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

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. Molecular Therapy 2013 21, 78-90DOI: (10.1038/mt.2012.187) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions