Glucocorticoids mediate induction of microRNA-708 to suppress ovarian cancer metastasis through targeting Rap1B

Abstract

Glucocorticoids are widely used in conjunction with chemotherapy for ovarian cancer to prevent hypersensitivity reactions. Here we reveal a novel role for glucocorticoids in the inhibition of ovarian cancer metastasis. Glucocorticoid treatments induce the expression of miR-708, leading to the suppression of Rap1B, which result in the reduction of integrin-mediated focal adhesion formation, inhibition of ovarian cancer cell migration/invasion and impaired abdominal metastasis in an orthotopic xenograft mouse model. Restoring Rap1B expression reverts glucocorticoid-miR-708 cascade-mediated suppression of ovarian cancer cell invasion and metastasis. Clinically, low miR-708 and high Rap1B are found in late-state ovarian tumours, as compared with normal, and patients with high miR-708 show significantly better survival. Overall, our findings reveal an opportunity for glucocorticoids and their downstream mediators, miR-708 or Rap1B, as therapeutic modalities against metastatic ovarian epithelial cancer.

Figure 1. MiR-708 acts as ovarian cancer metastasis suppressor.

(a) Heatmap of 21 most differentially regulated miRNAs from miRNA array of SKOV (Right) and SKOV-I6iv (Left). (b,c) Expression of miR-708 in ovarian tumours by FISH analysis of commercial tissue arrays. Samples were subgrouped by tumor stages or whether metastasis occurred. The statistical significance was determined using χ² test (b). The representative images from different stages were shown in c. Scale bars: 20 μM. (d) Real-time RT-PCR analysis of miRNA-708 levels in SKOV-3, TOV-112D, A1847, A2780, and their derived representative invasive lines. (e) Ovarian cancer cells were transfected with precursor miR-708 or scrambled control (s.c.). Twenty-four hours later, cells were subjected to migration (16 h) and invasion (24 h) assays. (f) SKOV-I6iv cells transfected with miR-708, miR-708 together with anti-miR-708 or s.c. were incubated for migration (6 h) and invasion (8 h) assays. (g) Left: histology of metastasized lungs from mice intravenously injected with SKOV-I6iv cells stably expressing miR-708 (n=9; bottom) or vector control (n=6; up). Images show H&E staining. Scale bars: 100 μM. Right: statistical analysis of total metastatic area in the lung. Ten pictures were taken for each mouse. The dots represent the means of individual mice. Student’s t-test (two-tailed) was used to compare two groups. (h) Left: SKOV-I6iv cells with miR-708 expression or vector control were orthotopically injected into the bursa of the mouse ovary. The kinetics of cancer metastasis to the abdomen cavity was monitored by BLI. Representative BLIs are shown at day 28 after implantation. Right: quantification results of abdominal metastases by BLI measurements (Total BLI minus primary tumour BLI). Data are normalized to the mean of vector control group (n=6). Student’s t-test (two-tailed) was used to compare the two groups. (i) Individual abdominal organ metastasis was measured 30 days after orthotopic implantation using real-time RT–PCR. Human-specific GAPDH levels were used to quantify metastatic SKOV-I6iv human cells. Data represent normalized means±s.e.m. (n=10; data were combined from two separate experiments). Data in d–f represent normalized means±s.d. (n=3). Student’s t-test (two-tailed) was used for statistical analysis (*P<0.05; **P<0.01; ***P<0.001).

Figure 2. Glucocorticoid signalling induces miR-708 and ODZ4 transcription.

(a) Expression of ODZ4 and miR-708 in SKOV-3 cells 72 h after treatments with increasing amount of DEX (10–1,000 nM). (b) Expression of GRα, ODZ4 and miR-708 in SKOV-3 cells transfected with siGRα or siCon, and then treated with 1 μM DEX for 72 h. (c) Expressions of ODZ4 and miR-708 in SKOV-3 cells pretreated with 10 μM cycloheximide overnight followed by 1 μM DEX for 48 h. (d) Up: schematic graph illustrating genomic locations of ODZ4 and miR-708 genes. The putative promoter regions tested for luciferase activities are indicated as short grey bars with assigned numbers (promoter-1 to -4). Bottom: SKOV-3 cells were transfected with pGL4, or pGL4 with promoter-1 to -4, treated with 1 μM DEX for 24 or 48 h, and assayed for luciferase activity. (e,f) SKOV-3 cells were treated with 1 μM DEX for 3 h, and the ChIP assay was performed on the promoter-3 region. The PCR product was analysed with agarose gel electrogenesis (e) or real-time PCR (f). (g,h) SKOV-3 cells transfected with anti-miR-708, control (g), siGRα or siCon (h) were treated with 1 μM DEX overnight, and then incubated for migration (8 h) and invasion (16 h) assays. DMEM/10% FBS, together wth 1 μM DEX or DMSO control, served as a chemoattractant. Data in a–h represent normalized means±s.d. (n=3). Student’s t-test (two-tailed) was used for statistical analysis (*P<0.05; **P<0.01; ***P<0.001).

Figure 3. MiR-708 inhibits migration/invasion by targeting Rap1B.

(a) Venn diagram showing the overlap of top 100 genes potentially targeted by miR-708 as predicted by the following three algorithms: TargetScan, miRanda and PicTar. (b) Expression of Rap1B mRNA in SKOV-I6iv cells transfected with miR-708, miR-708 plus anti-miR-708 or s.c. Data represent normalized means±s.d. (n=3). (c) Upper: expression of Rap1B proteins in SKOV-I6iv cells transfected with miR-708, miR-708 plus anti-miR-708 or s.c. Bottom: quantitative analysis of the Rap1B protein level, and normalized with the actin level. Histograms represent normalized means±s.e.m. (n=3). (d) Upper: sequence of miR-708 and the potential miR-708-binding site at Rap1B 3′ UTR. Nucleotides mutated in miR-708-binding site are shown in red. Bottom: luciferase assays demonstrating that expression of Rap1B 3′ UTR (wild-type or mutant form) by SKOV-I6iv cells transfected with miR-708, miR-708 plus anti-miR-708 or s.c. Data represent normalized means±s.d. (n=3). (e) Upper: protein expression of total Rap1 (including Rap1A and Rap1B), and Rap1B in SKOV-I6iv cells upon Rap1A, Rap1B or control siRNA transfection. Bottom: quantitative analysis of the protein level, and normalized with the actin level. Histograms represent normalized means±s.e.m. (n=3). (f) SKOV-I6iv cells transfected with Rap1A, Rap1B or control siRNA were incubated for migration (6 h) and invasion (8 h) assays. DMEM/10% FBS served as a chemoattractant. Data represent normalized means±s.d. (n=3). (g) Left: western blot analysis showing Rap1B, p-GRα and GRα expression in SKOV-I6iv cells upon treatment of 1 μM DEX for 48 h. Right: quantitative analysis of the protein level, and normalized with the actin level. Histograms represent normalized means±s.e.m. (n=3). Student’s t-test (two-tailed) was used for statistical analysis (*P<0.05; **P<0.01; ***P<0.001).

Figure 4. MiR-708 inhibits FA formation, cell spreading and cell adhesion.

(a) Confocal microscopy of Paxillin (Green), β-actin (Red) and DAPI (Blue) nuclear staining, after adhering of cells to FN. SKOV-I6iv cells with different transfections were equally plated on FN-coated coverslips for 90 min. The FAs were detected. Scale bars: 10 μm. (b–e) The images were analysed to determine the number of FAs per cell (b), the area of the cell staining for FAs by paxillin staining (c) and the cell-spreading area by β-actin staining (d). To determine the proportion of each cell consisting of FAs, the area of FAs was divided by the total spreading area of the cell (e). Data in b–e represent means±s.e.m. (n=30). (f) Left: western blot analysis showing p-Paxillin, p-FAK, Paxillin and FAK expressions in SKOV-I6iv cells with different transfections. Cells were incubated on FN-coated plates for 60 min before being subjected to western blot analysis. Right: quantitative analysis of protein levels, and normalized with total FAK or paxillin levels. Histograms represent normalized means±s.e.m. (n=3). (g) Adhesion assays of SKOV-I6iv cells with GFP plus different transfections. Percentage of adhered cells was counted. Data represent means±s.d. (n=3). Student’s t-test (two-tailed) was used for statistical analysis (*P<0.05; **P<0.01; ***P<0.001).

Figure 5. Rap1B rescues miR-708-impaired FA formation and migration/invasion.

(a) Confocal microscopy of Paxillin (green), β-actin (red), HA-tagged-Rap1B (purple) and DAPI (blue) nuclear staining. SKOV-I6iv cells with indicated transfections were incubated on FN-coated coverslips for 90 min. Scale Bars: 10 μm. (b–e) The images were analysed to determine the number of FAs per cell (b), the area of the cell staining for FAs by paxillin staining (c) and the cell-spreading area by β-actin staining (d). To determine the proportion of each cell consisting of FAs, the area of FAs was divided by the total spreading area of the cell (e). Data in b–e represent means±s.e.m. (n=30). (f) SKOV-I6iv cells with the indicated transfection were incubated for migration (6 h) and invasion (8 h) assay. Data represent normalized means±s.d. (n=3). Student’s t-test (two-tailed) was used for statistical analysis (*P<0.05; **P<0.01; ***P<0.001).

Figure 6. Glucocorticoids suppress ovarian cancer cell abdominal metastasis.

(a) SKOV-I6iv cells stably expressing pSuper-GFP-Luc plus Rap1B or GFP were orthotopically injected into the mouse ovary. The kinetics of cancer abdominal metastasis was monitored by BLI. Representative BLI images are shown on day 28 after implantation. (b) Quantification results of abdominal metastases by BLI measurements (total BLI minus primary tumour BLI). Data are normalized to the means of PBS-GFP or PBS-Rap1B (n=4–13; data were combined from two separate experiments). (c) Individual abdominal organ metastasis was measured by human-specific GAPDH levels to quantify metastatic SKOV-I6iv cells. Data are normalized to the means of PBS–GFP from individual organ±s.e.m. (n=4–8). (d) Expression of miR-708 in primary orthotopically implanted ovarian tumors. Data are normalized to the means of PBS-GFP±s.e.m. (n=4–8). (e) Western blot analysis of Rap1B expression from orthotopically implanted primary tumours. (f) Quantitative analysis of the Rap1B protein level from e, and normalized with the actin level. Histograms represent normalized means±s.e.m. Student’s t-test (two-tailed) was used for statistical analysis (*P<0.05; **P<0.01; ***P<0.001; n.s., no significance).

Figure 7. Rap1B shows negative correlation with miR-708, and patients with high miR-708 expression show better survival.

(a) Rap1B mRNA expression in primary tumours using real-time PCR analysis from commercial cDNA arrays and TVGH-frozen samples. Samples were further subgrouped by tumor stages or whether metastasis occurred. The statistical significance was determined using χ² test. (b) Real-time PCR analysis of N/T (normal/tumour) pairs, plus several unmatched N or T samples, from TVGH clinical frozen samples. MicroRNA-708 and Rap1B expression profiles were analysed. Student’s t-test (two-tailed) was used for statistical analysis (*P<0.05; **P<0.01). (c,d) Retrospective analysis of Kaplan–Meier plots for miRNA-708 expression in association with overall (c) or relapse-free (d) survival from 82 patients. Patients were split into high and low expression groups based on the median expression of the miR-708 (n=82; log-rank test). (e) Model of ovarian cancer metastasis suppressed via GC-mediated signalling pathways.