Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma
© Lee et al; licensee BioMed Central Ltd. 2005
Received: 10 May 2005
Accepted: 20 September 2005
Published: 20 September 2005
The EphB4 receptor tyrosine kinase has been reported as increased in tumours originating from several different tissues and its expression in a prostate cancer xenograft model has been reported.
RT-PCR, western blotting and immunohistochemical techniques were used to examine EphB4 expression and protein levels in human prostate cancer cell lines LNCaP, DU145 and PC3. Immunohistochemistry was also used to examine localisation of EphB4 in tissue samples from 15 patients with prostate carcinomas.
All three prostate cancer cell lines expressed the EphB4 gene and protein. EphB4 immunoreactivity in vivo was significantly greater in human prostate cancers as compared with matched normal prostate epithelium and there appeared to be a trend towards increased expression with higher grade disease.
EphB4 is expressed in prostate cancer cell lines with increased expression in human prostate cancers when compared with matched normal tissue. EphB4 may therefore be a useful anti-prostate cancer target.
Prostate cancer is the most frequent cause of cancer death in men in Australia. Although many genetic changes have been detected in human prostate cancer, the role of most of these in initiation and progression of the disease is unclear. Receptor tyrosine kinases (RTKs) couple ligand binding to downstream signalling cascades and gene transcription and are key regulators of normal cellular processes such as growth, differentiation, migration and apoptosis. RTKs are also critically involved in the development and progression of human cancers and are therefore useful targets for anti-cancer therapies [1, 2]. At least 50 RTKs in 20 different families have been identified and the largest family of these contains the Eph receptors . This family in humans currently includes 14 members divided into two classes, designated A and B, based on sequence homology, structure and ligand binding affinity [4, 5]. The ligands for the Eph receptors are called ephrins and are anchored on the plasma membrane through either a glycosyl phosphatidylinositol (GPI) link (ephrin-A) or a transmembrane domain (ephrin-B) .
Ligand binding induces receptor autophosphorylation of intracellular tyrosine, threonine and serine residues and allows interactions with a variety of different proteins that regulate cell functions such as contact inhibition, cytoskeletal organisation and cell motility [7, 8]. Several studies have described intricate signalling networks often important to cell proliferation, migration, survival and differentiation [reviewed in ] that centre around Eph receptors and their ligands.
Members of both classes of Eph receptor have been found to have increased gene expression and/or protein levels in tumours from many different human tissues [10–20]. In particular EphB4, located at chromosome 7q22.1, and initially isolated from a human hepatocellular carcinoma cell line Hep3Ba  has been reported by us and others to be highly expressed in many tumour tissues including colon [11, 12], breast [13, 14], endometrium [15, 16], lung  and head and neck [18, 19]. Robinson et al (1996) used degenerate RT-PCR oligonucleotide primers specific to two conserved motifs in the tyrosine kinase domains of 40 different kinases to amplify those kinases that are expressed in a prostate cancer xenograft model . EphB4 was identified as one of the RTKs expressed in the xenograft tissue. More recently, Xia et al (2005) report that EphB4 is commonly expressed in prostate tumour tissues and cell lines and knockdown of EphB4 protein using siRNA and antisense approaches inhibited cell growth/viability, migration and invasion both in vitro and in vivo . Concurrently with Xia et al's study, in this pilot study we also investigated the expression patterns of the EphB4 gene and its protein product in prostate cancer cell lines and tumour tissue samples.
Human prostate cancer cell lines LNCaP, DU145 and PC3 were cultured in HEPES-buffered RPMI 1640 medium (Invitrogen, Carlsbad, CA), pH 7.4. The medium was supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 160 μg/ml L-glutamine and 10% heat-inactivated foetal bovine serum (JRH Biosciences, Lenexa, KS) in 75 cm2 vented tissue culture flasks at 37°C in a 5% CO2 environment. Cells were collected at >90% confluency by trypsin digestion and centrifugation for 5 min at 1000 rpm, resuspended in phosphate buffered saline (PBS) and counted using a haemocytometer.
RNA extraction from cell lines
RNA was isolated from cell lines using Tri Reagent (Invitrogen). Growth medium was aspirated from the flask of growing cells (>90% confluent) and the cells washed with phosphate-buffered saline (PBS) before being directly lysed using 1 ml of Tri Reagent. After a five min incubation with gentle rocking, the cell lysate was removed to a 2 ml eppendorf tube. The RNA fraction was extracted using the manufacturer's recommendations.
Protein extraction from cell lines
Growth medium was aspirated from the flask of growing cells (>90% confluent) and the cells washed with phosphate-buffered saline (PBS) before being directly lysed using 1 ml of Cell Lytic M Cell Lysis Reagent (Sigma) supplemented with 5 μl Protease Inhibitor Cocktail (Sigma). Protein lysate was removed to a 1.5 ml microfuge tube and the solution mixed for 30 min at 4°C on a rotary mixer. Insoluble protein was pelleted by centrifugation at 4°C for 30 min and the supernatant containing the soluble proteins was stored at -80°C until required for Western analysis. Protein concentrations were determined using the DC Protein Assay Kit from Biorad (Sydney, NSW, Australia) following the manufacturer's protocol and using bovine serum albumin diluted from 0.1 mg/ml to 10.0 mg/ml to determine the standard curve.
Real-time RT-PCR to determine relative expression of EphB4
Primer sequences used to amplify EphB4 and the control housekeeping genes PBGD and HPRT 1 with expected size of the corresponding PCR product in base pairs (bp) and the annealing temperature used in the reaction.
Proteins (50 μg) from samples extracted from prostate tumour cell lines were separated on duplicate 8% Tris-Glycine iGels (Gradipore, Sydney, Australia). The protein separated on one of the gels was visualised by coomassie staining and the protein on the duplicate gel was electrophoretically transferred to MFS nitrocellulose membrane (Adelab, Adelaide, Australia). Non-specific binding was blocked by incubation for 1 h at room temperature using a Western blocking buffer containing 1% casein in maleic acid buffer (Roche) diluted using TBS with 0.1% Tween-20 (TBS-T). EphB4 antigens were detected using a 1:1000 dilution of a mouse monoclonal antibody raised to human EphB4 (Zymed, CA) in blocking buffer. After a 1 h incubation at room temperature, the primary antibody was detected using a HRP-labelled anti-mouse secondary antibody (Roche) and the ECL Western Blotting System from Amersham Biosciences (Sydney, Australia) following the manufacturer's recommendation. Blots were exposed to Hyperfilm™ECL™ (Amersham Biosciences) for between 5 and 30 sec. The chemiluminescence was removed from the blot by incubation in a stripping solution containing 100 mM β-mercaptoethanol, 2% SDS and 62.5 mM Tris-HCl pH 6.7 for 20 min at 50°C with gentle agitation. The filter was then washed with TBS-Tween 20 before blocking and re-probing with a mouse anti-α-actin monoclonal antibody (Chemicon, Temecula, CA) then it was stripped again and probed with a rabbit anti-calnexin antibody (Sigma) for loading comparison.
Immunofluorescence of cells grown on slides
Cell were grown to >50% confluence in the wells of 8 well chambered slides. Before staining, the medium was aspirated and the cells washed gently with PBS. Cells were then fixed in 4% paraformaldehyde and washed well before non-specific binding sites were blocked with 3% serum in PBS for 20 min at room temperature. Cells were then incubated overnight at 4°C with a 1:200 dilution of the EphB4-specific rabbit polyclonal antibody H-200 (Santa Cruz Biotechnology, Santa Cruz, CA). After rinsing with PBS, the sections were incubated with an Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Molecular Probes). Immunofluorescence was visualised using a TE2000E microscope with a C1 confocal scanning head (Nikon, Tokyo, Japan).
Four consecutive 8 μm sections of formalin-fixed paraffin-embedded tissue from 15 different patients with prostate cancer were a gift from Dr Michael Brown, Medical Oncology, Royal Adelaide Hospital, Adelaide, Australia. A fifth slide, stained to visualize the histological features of the tissue, was used to identify the foci of tumour tissue within the adjacent normal prostate tissue by a trained pathologist. The paraffin was removed by incubation in Histoclear (National Diagnostics, Atlanta, Georgia) and the section re-hydrated in ethanol before antigen retrieval by boiling in 10 mM citric acid pH 6 for 10 min. Sections were cooled, then washed in PBS before removal of endogenous peroxidase activity by incubation in 0.5% hydrogen peroxide/methanol for 30 min at room temperature. Non-specific binding sites were blocked with 3% normal goat serum in PBS for 20 min at room temperature and the Vector Laboratories (Burlingame, CA) Avidin/Biotin blocking kit following the manufacturer's instructions. The sections were then incubated overnight at 4°C with a 1:200 dilution of the H-200 EphB4-specific antibody (Santa Cruz Biotechnology). After rinsing with PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories) for 30 min at room temperature followed by washing with PBS. Immunoreactivity was detected with the avidin-biotin system (Vector Laboratories) using 18.5 mM 3,3'-diaminobenzidine tetrahydrochloride (Sigma) as a chromogen for 2 min. The sections were then counterstained using Harris haematoxylin, dehydrated, cleared using SUB-X clearing solution (Surgipath Medical Industries, Inc. Richmond, IL) and mounted using Entellan New (Merck, Darmstadt, Germany). Sections were visualised and images recorded using a Nikon Eclipse E800 microscope with a Spot Camera 2.3.1 (Diagnostic Instruments) and Spot Advanced Version 3.5 software.
Relative expression of EphB4in prostate cancer cell lines
In these experiments the ratios of EphB4/PBGD and EphB4/HPRT 1 were all close to 1 indicating that there were comparable amounts of EphB4 and PBGD/HPRT 1 templates in each cell line RNA sample. There was no statistical difference between the ratios obtained using serial dilutions of a single cDNA sample for each cell line showing that the amplification is consistent regardless of the starting amount of template. These results support the use of both PBGD and HPRT 1 as normalisation controls for the EphB4 message.
Western analysis of EphB4 protein in prostate cancer cell lines
Immunological analysis of expression of EphB4 in prostate cancer cell lines
Immunohistochemical analysis of expression of EphB4 in primary prostate tumours
In recent years, experiments using monoclonal antibodies with limited normal-tissue reactivity have indicated that these are good candidates for development as therapeutic agents against cancer. EphB4 is a receptor protein tyrosine kinase (RTK) that is dramatically up-regulated on many epithelial cancers, displays limited expression on normal adult tissue and would therefore appear to be a potential target for monoclonal therapies. EphB4 was identified by Robinson et al (1996) as one of several RTKs that were expressed in a xenograft model of prostate cancer  and until recently this was the only report of any link between EphB4 expression and prostate cancer.
During the review of this article, Xia et al (2005) presented results showing that EphB4 is increased in 66% (41/62) of prostate tumours tested with low intensity expression in only 15% (3/20) normal prostate samples . Our western analysis of PC3, LNCaP and DU145 confirmed Xia et al's results , with higher amounts of protein present in PC3 lysates that LNCaP and DU145. In the case of DU145 this can perhaps be explained by regulation of EphB4 gene expression as real-time PCR using normalisation to two different house-keeping genes demonstrated that there was approximately 20% less EphB4 transcript expressed in the DU145 cell line than in LNCaP and PC3 (P < 0.05). However, as there was no significant difference in the activity of the gene in LNCaP and PC3, it is possible that EphB4 may also be regulated post-transcriptionally. The presence of a second band detected by western analysis with the EphB4-specific antibody may also indicate that EphB4 is regulated post-translationally and this needs to be explored in more detail.
Although Xia et al's study of cell lines also indicated an association between the level of EphB4 expression and aggressive growth, they did not report a direct correlation between increased EphB4 expression with higher grade of the tumour tissue samples. We examined a panel of 15 prostate cancer clinical specimens collected by transurethral resection. The prostate tumour samples we examined contained both normal prostate and tumour foci and this enabled a comparison of EphB4 immunoreactivity in normal prostate and tumour cells simultaneously from the same patient and the determination of whether the level of EphB4 also correlates with histological grade and/or stage of prostate carcinoma. Using immunohistochemical techniques, we showed that EphB4 is produced in increased amounts in human prostate cancer tissue compared with adjacent normal tissue and that this immunoreactivity was associated with the tumour epithelial cells themselves. There also appeared to be trend towards an increasing level of EphB4 protein in the tumours from the well-differentiated to the moderately and poorly differentiated cancers.
A positive correlation between histological grade, stage of carcinoma and level of EphB4 protein has also been reported in breast and endometrial carcinoma [13–16]. EphB4 has been reported to be elevated in breast primary infiltrating ductal carcinomas with a high grade of malignancy  and recently Wu et al (2004) reported that EphB4 protein expression in 94 tumour samples was positively associated with increased clinical stage and histological grade . Sakano et al, (1996) suggested that the balance of EphB4 and its preferred ligand ephrin-B2 is disrupted when mammary epithelial cells become transformed  and loss of ephrin-B2 expression has been associated with an increase in EphB4 expression . However contrary to this Berclaz et al (2002) report a highly significant correlation between EphB4 positivity and low histological grade . One of the patients examined here also showed a comparable level of EphB4 staining in foci of PIN and poorly differentiated adenocarcinoma. If the PIN is a precursor to adenocarcinoma in this case, this might suggest that increased expression of EphB4 was an early event in the development of this patient's disease. Furthermore, a single focus of basal cell hyperplasia in a second patient did show a low level of EphB4 immunoreactivity and this may indicate a role for EphB4 in proliferation of these cells .
The results presented here suggest that elevated levels of EphB4 are relevant to prostate cancer but despite substantial evidence in the literature that EphB4 has an important role in progression of many epithelial tumours, the mechanism by which these receptors contribute to tumorigenesis is still being resolved. In only a very few cases have Eph receptors been demonstrated to have transforming potential [28–30]. In particular, a study reported by Zelinski et al (2002) showed that EphA2 expression transforms MCF10A cells as judged by conversion to a fibroblastic morphology, growth in soft agar and the ability to engraft and metastasise in a nude mouse model .
Although similar experiments over-expressing EphB4 in non-transformed cell lines have yet to be reported, a study by Munarini et al (2000) using transgenic mice expressing EphB4 and neuT suggested that EphB4 over-expression by itself is not tumorigenic but provides convincing evidence that it favours an invasive/metastatic phenotype . Although mammary tumours were not observed in the EphB4 transgenic animals, in double transgenic animals expressing both EphB4 and neuT, tumour appearance was significantly accelerated relative to neuT-only animals, and in addition metastases were observed in the lung. It was not clear if this was an effect of EphB4 on metastasis or a result of accelerated tumour growth but these results clearly implicate over-expression of EphB4 in tumour growth and/or establishment of the invasive phenotype in the adult mammary tumours. While the single transgenic EphB4 animals did not develop tumours during this experiment it is possible that these studies were not taken out far enough to conclude that EphB4 over-expression is insufficient to induce transformation with a long latency.
Xia et al's (2005) recent experiments targeting EphB4 using siRNA to knockdown EphB4 expression in vitro have shown that EphB4 is involved in growth/viability, migration and invasion of prostate cancer cell lines and supports Munarini et al's study . Furthermore, EphB4 antisense oligonucleotides were given intraperitoneally to nude mice bearing PC3 xenografts in the posterior prostate (n = 6), tumours were fewer and smaller, with increased apoptosis and reduced microvascular density than sense- or diluent-treated control animals. The reduction in microvascular density is particularly interesting given that in other epithelial tumours, EphB4-positive cells are often found in regions that are rich in capillaries  and it has been suggested that EphB4 over-expression in tumours may promote tumour growth by facilitating angiogenesis.
A direct role for EphB4 in tumour angiogenesis has been suggested by experiments showing that A375 melanomas form smaller tumours in the presence of soluble EphB4  possibly because the soluble EphB4 interferes with binding of endogenous EphB4 on tumour cells to ephrin-B2 on endothelial cells. A role in angiogenesis would be consistent with the normal role for EphB4 in vascular development and remodelling demonstrated in mouse and Xenopus [33, 34]. Noren et al (2004) contradict this finding in a study that found that increased expression of a signaling-defective form of EphB4 in breast cancer cells (dominant negative) was still able to make tumour xenografts grow more rapidly . This may be because the EphB4 ectodomain exerted a chemoattractive effect on endothelial cells through ephrin-B2 expressed on these cells and promoted endothelial cell proliferation and survival resulting in more tumour vasculature. However, Noren et al applied a high concentration of soluble ephrin-B2 Fc which is promiscuous and signals other Eph receptors (particularly EphB2) but the phosphorylation of other EphRs was not determined in this study.
Further investigation is needed to determine the roles of EphB4 in prostate cancer development and progression. The trend of increasing EphB4 reactivity toward higher grade disease seen in this pilot study might suggest that it is more important in the later stages of the tumour development such as metastasis and further investigation of EphB4 in the development of prostate cancer is warranted. Therapies that target EphB4 may prove to be successful in preventing the metastatic spread of the disease.
We would like to thank Dr Michael Brown for providing prostate tissue samples and Dr Wayne Tilley and Dr Lisa Butler for prostate cell lines. We would also like to thank Dr Jurgen Stahl for reviewing the tissue immunohistochemistry results and Dr Edwina Ashby for critical reading of the manuscript. Sally Stephenson is supported by a senior research fellowship from the Cancer Council of South Australia. This work was supported by grants from The Queen Elizabeth Hospital Research Foundation and The South Australian Medical Research Trust.
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