The receptor tyrosine kinase EphB4 is overexpressed in ovarian cancer, provides survival signals and predicts poor outcome

Abstract

EphB4 is a member of the largest family of transmembrane receptor tyrosine kinases and plays critical roles in axonal pathfinding and blood vessel maturation. We wanted to determine the biological role of EphB4 in ovarian cancer. We studied the expression of EphB4 in seven normal ovarian specimens and 85 invasive ovarian carcinomas by immunohistochemistry. EphB4 expression was largely absent in normal ovarian surface epithelium, but was expressed in 86% of ovarian cancers. EphB4 expression was significantly associated with advanced stage of disease and the presence of ascites. Overexpression of EphB4 predicted poor survival in both univariate and multivariate analyses. We also studied the biological significance of EphB4 expression in ovarian tumour cells lines in vitro and in vivo. All five malignant ovarian tumour cell lines tested expressed higher levels of EphB4 compared with the two benign cell lines. Treatment of malignant, but not benign, ovarian tumour cell lines with progesterone, but not oestrogen, led to a 90% reduction in EphB4 levels that was associated with 50% reduction in cell survival. Inhibition of EphB4 expression by specific siRNA or antisense oligonucleotides significantly inhibited tumour cell viability by inducing apoptosis via activation of caspase-8, and also inhibited tumour cell invasion and migration. Furthermore, EphB4 antisense significantly inhibited growth of ovarian tumour xenografts and tumour microvasculature in vivo. Inhibition of EphB4 may hence have prognostic and therapeutic utility in ovarian carcinoma.

Figure 1

EphB4 is expressed in human ovarian tumour specimens and correlates with advanced stage, presence of ascites, and poor survival. (A) Representative immunohistochemical peroxidase staining for EphB4 in a negative (deletion of primary antibody) control (a), normal ovarian epithelium (b), and invasive ovarian carcinoma (c). All pictures were taken at original magnification of × 200. (B) Correlation of clinical and pathological variables with EphB4 overexpression in ovarian carcinoma. (C) Kaplan–Meier survival of patients with invasive ovarian cancer based on EphB4 staining intensity, using the log-rank statistic.

Figure 2

Ovarian cancer cell lines express functional EphB4 that is downregulated by progesterone. (A) 20 μg total cell lysate from each of the ovarian cancer cell lines was run on 4–20% Tris-glycine gel and transferred to PVDF membrane. The membrane was sequentially probed with anti-EphB4, anti-EphrinB2, and anti-β-actin antibody. (B) Hey cells were serum starved overnight and stimulated for the various time periods shown with 3 μg ml⁻¹ clustered EphrinB2/Fc or Fc alone. EphB4 was immunoprecipitated from 100 μg whole-cell lysates and phosphorylation status analysed by antiphosphotyrosine antibody immunoblotting (top row). A duplicate membrane was probed for EphB4 to document immunoprecipitation efficiency (bottom row). (C) Hoc-7 (ovarian cancer) and MCV-50 (ovarian cystadenoma) cells were treated with varying doses of progesterone and oestrogen for 36 h and cell lysates were analysed by immunoblotting for expression of EphB4, EphrinB2, and β-actin. (D) 1 × 10⁴ Hoc-7 cells were plated in each well of a 48-well plate and treated for 72 h with varying doses of progesterone and oestrogen. Cell viability was assessed by MTT assay and survival expressed as percentage of absorbance relative to untreated cells.

Figure 3

EphB4 knockdown leads to tumour cell apoptosis. (A) Hev cells were transiently transfected with EphB4-specific siRNA (EphB4-siRNA) and 48 h later, 20 μg whole-cell lysates were analysed by immunoblotting for EphB4 and β-actin levels (top panel). 1 × 10⁴ Hey cells were transfected with mutated EphB4 siRNAΔ or native EphB4-siRNA and plated in a 48-well plate. Cell viability was assessed by MTT assay at 48 h and survival expressed as percentage of absorbance relative to untreated cells (bottom panel). (B) Hey cells were treated with varying doses of EphB4-specific ODN (AS-10). Seventy-two hours later, 20 μg whole-cell lysates were analysed by immunoblotting for EphB4 and β-actin levels (top panel). 1 × 10⁴ Hey cells were treated with scrambled or AS-10 ODN. Cell viability was assessed by MTT assay at 72 h and survival expressed as percentage of absorbance relative to untreated cells. (C) Hey cells were transiently transfected with EphB4-specific siRNA (EphB4-siRNA) or mutated siRNA (EphB4-siRNAΔ). Apoptosis was analysed by ELISA for cytoplasmic nucleosomes as detailed in the Materials and Methods section using whole-cell lysates. (D) Caspase-8 and caspase-9 activation was assayed colorimetrically in these cells and expressed as percent activity compared with lipofectamine-treated cells. (E) Cell-cycle analysis of Hey cells treated with lipofectamine alone (control) or treated with 25 nM EphB4-specific siRNA (EphB4 siRNA) or mutant siRNA (EphB4 siRNAΔ) for 36 h. The percentage of cells in G₀, G₁, S, G₂, and M phase are indicated. Similar results were obtained in three independent experiments.

Figure 4

EphB4 favours tumour cell migration and invasion. (A) Confluent cultures of Hey cells were scraped with a plastic Pasteur pipette to produce 3-mm-wide cell-free zone in the monolayer. The ability of the cells to migrate and close the wound following transfection with 25 nM EphB4-specific (EphB4 siRNA) or mutant siRNA (EphB4 siRNAΔ) was assessed over 9 h. (B) Invasion of Hey cells into Matrigel-coated inserts was studied as described in the Materials and Methods section. Cells invading the underside of the inserts in response to 10 μg ml^–1 EGF in the lower chamber were fixed and stained with Giemsa. Representative photomicrographs are shown.

Figure 5

EphB4-specific antisense ODN inhibits tumour growth in a murine ovarian cancer xenograft model. (A) 2 × 10⁶ Hey cells were implanted in the flank of 10- to 12-week-old female Balb/C athymic mice and tumour volume measured as detailed in the Materials and Methods section. Mice were administered vehicle alone (vehicle), or 10 mg kg^–1 scrambled ODN (scrambled) or EphB4-specific antisense ODN (AS-10) intraperitoneally daily starting day 4 after cell implantation. Animals were killed 5 weeks later and tumours harvested. (B) Sections (5 μm) of formalin-fixed paraffin embedded sections were stained with hematoxylin/eosin and analysed by immunohistochemistry for Ki-67 and CD31 expression. Apoptosis was evaluated by TUNEL with the in situ apoptosis staining kit. Number of cells staining positive was averaged over five random high-power fields by a blinded observer and indicated in each micrograph. ^*P<0.05 between AS-10 and control group. Bar in bottom left panel represents 200 μm in H/E, 100 μm in CD31, and 75 μm in other photomicrographs.