Background
Erythropoietin-producing hepatocellular (Eph) Type-B receptor 4 (EphB4) is part of the largest family of membrane-bound receptor tyrosine kinases (RTK) which consists of 14 different receptors which are classed as EphA or EphB. Their ligands, the ephrins, are also cell membrane-bound, either
via glycosylphosphatidylinositol (GPI)-linkage (ephrin-A ligands) or transmembrane-embedded (ephrin-B ligands). Interaction between Eph receptors and their ligands normally takes place
in trans through the binding of 2 ligands on one cell to 2 receptors on an adjacent cell forming a heterotetramer that is the basic complex required for signaling. EphB4 plays an important role in cell signaling and is also involved in regulating cell morphology, adhesion, migration and invasion through modification of the cell’s actin cytoskeleton and by influencing the actions of integrins [
1]. Moreover, depending on the cell-environment conditions, EphB4 demonstrates the ability to be both a tumor promoter, when over-expressed and in the absence of stimulation by its sole cognate ligand, ephrin-B2, as well as a tumor suppressor stimulated by ephrin-B2 [
2-
6]. EphB4 is overexpressed in 66% of prostate cancer clinical samples and has been implicated in prostate cancer development and progression [
2,
7]. It has been shown using targeted siRNA sequences that knockdown of EphB4 in prostate cancer causes a significant reduction in cell motility
in vitro and tumor growth
in vivo [
5]. However, the mechanisms by which removal of EphB4 exerts these effects are largely unknown. To date, no study has investigated the broader consequences on gene expression of siRNA-mediated knockdown of
EPHB4 in prostate cancer. Therefore, we sought to determine the genome-wide changes upon transient knockdown of
EPHB4 in a ligand-independent context in the prostate cancer cell line LNCaP.
Through gene expression analysis following EPHB4 knockdown, validation of the microarray data, and EphB4 over-expression, we have determined that EphB4 regulates the expression of integrin β8 in prostate cancer cell lines.
Methods
Cell culture
All cell lines were purchased from the American Type Culture Collection (Manassas, VA). LNCaP, PC3 and 22Rv1 prostate cancer cells were cultured in RPMI 1640 (Life Technologies, Mulgrave, VIC, Australia) supplemented with 10% fetal calf serum (FCS). EphB4 over-expressing stable 22Rv1 cell lines, together with vector-only (VO) and parental 22Rv1 cells, were generated as described previously [
2].
siRNA transfection
Lipofectamine 2000 (Life Technologies) was used to transiently transfect LNCaP cells with 10 nM of EPHB4 siRNAs (SI00288589, SI04435053; Qiagen, Chadstone, VIC, Australia) or PC3 and 22Rv1 cells with 100 nM of ITGB8 siRNAs (SI00034454, SI03066623, Qiagen). The AllStars non-silencing negative control siRNA (Qiagen) was used at the same concentration as gene-specific siRNAs for all experiments. After 48 h, RNA from both the siRNA-treated cells and the EphB4 over-expressing cells was extracted using Trizol (Life Technologies).
Microarray gene expression profiling
Triplicate samples of
EPHB4 siRNA knockdown and respective control siRNA transfected LNCaP cells were extracted for RNA and prepared for microarray profiling, which was performed on a custom Agilent 4 × 180 K oligo array (VPCv3 ID:032034, GEO GPL16604, Agilent Technologies, Mulgrave, VIC, Australia). This microarray contains the Agilent 44 K (ID:014850) probe set incorporating human gene expression protein-coding probes as well as non-coding probes; with the probes targeting exonic regions, 3′UTRs, 5′UTRs, as well as intronic and intergenic regions [
8]. RNA was isolated with Trizol (Life Technologies), further purified using an RNeasy Mini Kit (Qiagen) with DNAse treatment according to the manufacturer’s protocol. RNA samples were analyzed by a Bioanalyzer (Agilent) to ensure the RNA was of high quality. RNA (100 ng) from each group was amplified and labelled using the Low Input Quick Amp Labeling Kit (Agilent) and the protocol for One-Color Microarray-Based Gene Expression Analysis. The input RNA was reverse transcribed into cDNA, using an oligo-dT/T7-promoter primer which introduces a T7 promoter region. The subsequent
in vitro transcription uses a T7 RNA polymerase, which simultaneously amplifies target material into cRNA and incorporates cyanine 3-labeled CTP. cDNA synthesis and
in vitro transcription were both performed at 40°C for 2 h. The labelled cRNA was then purified with Qiagen’s RNeasy mini-spin columns and quantified using a Nanodrop-1000 (Thermo Scientific, Waltham, MA, USA). cRNA (1650 ng) from each sample was loaded onto the 4x180 K custom microarray and allowed to hybridize at 65°C for 17 h. The arrays were scanned using an Agilent Microarray Scanner G2565CA.
Microarray data analysis
The microarray data were processed with Agilent Feature Extraction Software (v10.7). A quantile between-array normalization was applied and differential expression was determined using an unpaired T-test, asymptotic p-value and a 2-fold cut-off (GeneSpring GX11, Agilent Technologies). The gene expression levels are presented as fold change. Genes that were significantly different between two groups were identified with a p value of < =0.05, and an average fold change of > = 2.
Normalized gene expression data of the experiment are Minimum Information About a Microarray Experiment (MIAME) and have been submitted to Gene Expression Omnibus (GEO) with the accession number GSE61800.
Quantitative real-time PCR
Extracted RNA was reverse transcribed using Superscript III reverse transcriptase according to the manufacturer’s instructions (Life Technologies). Quantitative RT-PCR for
EPHB4 expression was performed in triplicate using a TaqMan Gene Expression Assay (
EPHB4: Hs00174752_m1 Life Technologies) and TaqMan Universal PCR Master Mix, No AmpErase UNG (Life Technologies) on a Rotor-Gene 6000 (Qiagen). The endogenous reference gene hydroxymethylbilane synthase (
HMBS: Hs00609297_m1) was used for normalization and results are expressed as the
EPHB4:
HMBS ratio. For integrin gene expression analysis the SYBR Master Mix (Life Technologies) was used with
GAPDH as a housekeeping gene. Primer sequences are listed in Table
1.
Table 1
Primer sequences for semi-quantitative RT-PCR and qRT-PCR
ITGA3
| AGCGCTACCTGCTCCTGGCT | GGGCAGTGAGTGGGCACAGG | 99 |
ITGA10
| CTGACAGGTCTCTGCTCCCCC | CCATCGCTGTCCACCCCCAA | 120 |
ITGB8
| TGTGTGCTGGGCATGGAGAGTGT | CAGTGCTGGGCTGCTGCTGAA | 100 |
ITGAV
| CATCTGTGAGGTCGAAACAGG | TGGAGCATACTCAACAGTCTTTG | 137 |
EPHB4
| TCAATGTCACCACTGACCGAGAGGTA | GGTATTTGACCTCGTAGTCCAGCACA | 141 |
GAPDH
| CTGCACCACCAACTGCTTAG | GTCTTCTGGGTGGCAGTGAT | 108 |
Semi-quantitative RT-PCR
RT-PCR analysis was carried out using standard conditions with primers as shown in Table
1). PCR conditions were as follows: initial denaturation 94°C for 15 min then 94°C for 30s; 60°C for 30s; 72°C for 30s repeated 35 times, final elongation 72°C for 10 min. PCR products were analyzed on a 2% Tris-acetate agarose gel containing 0.01% ethidium bromide and photographed using a Gel doc system (Syngene, MD, USA).
SDS-PAGE and western blotting
Cells were lysed with ice-cold RIPA buffer (50 mM Tris pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 150 mM NaCl, 2 mM EDTA, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM PMSF, 10 μg/ml aprotinin) supplemented with protease inhibitors (Complete Mini-EDTA-free tablets; Roche, Castle Hill, NSW, Australia). Protein lysates were mixed at 4°C for 15 min, then insoluble proteins removed by centrifugation at 14,000 rpm. Protein concentrations were determined using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Protein samples (20-50 μg) were separated on SDS-PAGE gels (4% stacking and 10% separating) before proteins were transferred to a nitrocellulose membrane using a wet transfer system (Bio-Rad, Hercules, CA, USA) in transfer buffer (20% methanol, 0.1 mM Tris, 80 mM glycine) at 100 V for 90 min. After blocking with 5% skim milk in TBST (Tris buffered saline with Tween-20:- 8 g/L NaCl, 3 g/L Tris, 0.2 g/L KCl, 0.2% Tween-20, pH 7.4) for 1 h, blots were probed with the primary antibody in 5% BSA/TBST (anti-EphB4 H-200, Santa Cruz Biotechnology, CA, USA) [
9] or 5% skim milk/TBST (anti-ITGB8 ab80673, Abcam, Melbourne, VIC, Australia) overnight at 4°C before incubation with horseradish peroxidase-labelled secondary antibody anti-rabbit IgG (Sigma-Aldrich, Castle Hill, NSW, Australia) in 5% skim milk/TBST for 1 h at room temperature. Chemiluminescence was detected with Amersham™ ECL Plus Chemiluminescence kit (GE Healthcare, Silverwater, NSW, Australia) following the manufacturer’s recommendation, with a 10-30 sec exposure to SuperRX X-ray film (Fuji Film Corporation, Japan).
Migration assay
PC3 cells were seeded into 96-well Essen Bioscience ImageLock plates (Essen BioSciences, Ann Arbor, MI, USA) at 2 x 104 cells per well in four replicates and allowed to proliferate for 24 h until they had formed a complete monolayer. Cells were then transfected with ITGB8 siRNA and allowed to grow for a further 24 h. The 96-well WoundMaker (Essen Bioscience) was then used to create a cell-free zone in the monolayer before the plates were placed in the IncuCyte ZOOM (Essen Bioscience). Migration into the cell free zone was determined after 24 h and quantified as relative wound density.
Invasion assay
Pre-coated growth-factor reduced Matrigel cell culture inserts (24-well, pore size 8 μm; BD Biosciences, San Jose, CA, USA) were seeded with serum-deprived 5 x 105 PC3 or 22Rv1 vector-only cells (22Rv1-VO) or 22Rv1 EphB4 over-expressing cells (22Rv1-B4) and transfected with 100 nM ITGB8 siRNA or non-silencing AllStars siRNA (Qiagen) in 0.1% FCS-containing medium. Medium containing 10% FCS was used as chemo-attractant. Cells were incubated for 22 h and cells that had not invaded were removed from the upper chamber using a cotton swab. Membranes were excised and mounted on glass slides with ProLong Gold Antifade containing 4, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Life Technologies) for visualization of the nuclei of cells that had invaded through the Matrigel to the underside of the membrane. Nuclei were counted in five random fields at 20 X magnification using an Olympus epifluorescent microscope.
Adhesion assay
22Rv1-VO or –B4 cells, or their negative siRNA-control or ITGB8 siRNA counterparts (1 x 105 cells), were seeded into triplicate wells in a Vitronectin pre-coated 96 well adhesion assay plate, and an adhesion assay was carried out according to the manufacturer’s instructions (Merck Millipore, VIC, Australia).
Statistics
Statistical analysis was carried out using IBM SPSS software package by employing the ANOVA analysis followed by Fisher’s least significant difference post hoc test or Student’s t-test with p < 0.05 considered significant.
Discussion
We have recently shown that EphB4 over-expression leads to a more aggressive phenotype in prostate cancer cells [
2]. In this study we set out to investigate the gene expression changes that occurred when
EPHB4 was knocked down in LNCaP prostate cancer cells that are endogenously over-expressing EphB4. Microarray analysis revealed a set of three integrin subunits (α3, α10 and β8) that were de-regulated upon siRNA knockdown of
EPHB4 and qRT-PCR validated two of these (α10 and β8). Over-expression of
EPHB4 led to concomitant and parallel changes in expression and protein levels of ITGB8 but not the other two integrins. In keratinocytes, it has been shown that EphB2-induced reverse signaling down-regulated integrin expression, demonstrating that in other cell contexts other Eph receptors also have the ability to influence integrin expression [
15]. Integrins are a family of transmembrane receptors which are primarily involved in cell-extracellular matrix (ECM) adhesion as well as cell-cell interactions. By connecting the actin cytoskeleton to the ECM, integrins are able to regulate attachment, cytoskeletal organization, mechano-sensing, migration, proliferation, differentiation and cell survival [
16]. Through their several roles, integrins have been found to be involved in a range of pathological processes including tumor angiogenesis and metastasis [
17,
18].
There is evidence that Eph/ephrin signaling can influence integrin clustering and in some cases, inhibit integrin downstream signaling [
19,
20]. Furthermore, ephrin-A1 and EphA2 have both been shown to co-localize with integrin α3 [
20-
22]. In breast cancer cell lines, EphB4 exogenous overexpression reduces integrin β1 expression resulting in increased migration in a ligand-independent manner [
23]. In the current study, validation experiments confirmed that
ITGB8 was significantly down-regulated when
EPHB4 was knocked down and moreover, was also up-regulated when
EPHB4 was over-expressed in prostate cancer cells. This suggests that
EPHB4 and
ITGB8 are potentially transcriptionally co-regulated. In terms of transcriptional regulation, the
ITGB8 promotor contains SP and CRE binding motifs and is regulated by SP1, SP3 and an AP-1 complex [
24].
EPHB4 on the other hand has been shown to be transcriptionally regulated by HoxA9 in endothelial cells [
25], but no information is available about the transcription factors involved in regulating expression of
EPHB4 in cancer cells. It would be interesting to investigate whether the transcription factors of the SP family can also influence
EPHB4 expression in prostate cancer and thus be responsible for co-regulating
ITGB8 and
EPHB4.
Although only limited information is available about the role of integrin β8 in cancer it has been identified as up-regulated in several cancers including head and neck cancer, hepatocellular carcinoma, some ovarian cancer and melanoma cell lines as well as primary non-small lung cancer samples and brain metastases from several epithelial cancers [
26-
28]. Furthermore,
ITGB8 has been identified as a member of a six-gene expression signature biomarker predicting lung metastasis from breast cancer [
29]. In glioblastoma specimens, there appears to be a correlation between low integrin β8 expression and highly angiogenic, but poorly invasive tumors; as well as high integrin β8 expression in low angiogenic and highly invasive tumors (17). This implies that integrin β8 is involved in differential control of angiogenesis
versus tumor cell invasion in glioblastomas. On the other hand, integrin β8 has been shown to be growth inhibitory in lung cancer cells, yet it is also playing a fundamental role in lung cancer metastasis indicating that this integrin functions in a complex manner in cancer presumably depending on tissue and progression context [
30-
32]. As the expression of integrin β8 has been found to be increased in metastases from various tumors, including breast and lung, we speculated that integrin β8 could also play a role in prostate cancer cell migration and invasion and we show that silencing of
ITGB8 reduces cell motility [
28,
29]. In highly invasive glioblastoma tumors, high levels of integrin β8 cooperate with Rho proteins to drive invasion [
11]. Eph receptors are also able to activate the Rho pathway to regulate cancer cell migration [
33] and thus it is possible that EphB4 and integrin β8 work cooperatively to control cell motility.
By interrogating the Oncomine database we have identified
ITGB8 as being up-regulated in PIN, the precursor to prostate cancer which is puzzling considering its role in metastases. It is possible that integrin β8 is able to impact on different stages of tumor development in different cell populations. In established prostate cancer cell lines we demonstrate that integrin β8 plays a vital role in migration and invasion. These results are in agreement with previous reports describing similar findings in lung cancer and glioblastoma [
11,
31].
Integrin β8 has been shown to only heterodimerise with the integrin αv subunit and this heterodimer binds to vitronectin [
10]. We show here that the αv subunit expression also increases in stably EphB4 over-expressing prostate cancer cells, but no effect on adhesion to vitronectin was seen. Interestingly, transforming growth factor β (TGF-β) has been identified as the only physiologically relevant ligand for integrin αvβ8 and activation of matrix-bound latent TGF-β by integrin αvβ8 results in activation of TGF-β signaling and remodeling of human airway fibroblasts [
34]. TGF-β is a well-known cytokine with a variety of physiological functions such as proliferation, differentiation and immune response and in addition it plays a major role in cancer progression by inducing epithelial-to-mesenchymal transition (EMT) in prostate cancer and promoting metastasis to the bone, the final step in prostate cancer progression [
35,
36]. In our array data
TGFB1 was also found to be significantly upregulated by 2-fold (data not shown). This could indicate that
EPHB4,
ITGB8 and
TGFB1 are intrinsically regulated in prostate cancer cells, therefore contributing to cancer progression and metastasis through the process of EMT. However, expression analysis of the EMT markers E-cadherin and Snail did not show any significant changes in PC-3 cells transfected with
ITGB8 siRNA (data not shown). Changes in E-cadherin and Snail expression have been reported in lung cancer cells where
ITGB8 was silenced [
31].
Cilengitide, a selective antagonist of αvβ3 and vβ5 integrins, is currently in clinical trials for a variety of solid tumors, but so far the results are modest [
37-
39]. In prostate cancer, no clinical effect was seen [
40]. Targeting the αvβ8 integrin in combination with EphB4 targeted therapies may represent a future avenue for prostate cancer therapy.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
IMW conceived the study, coordinated it and carried out transwell migration and invasion assays, real-time PCR, ONCOMINE database analysis as well as statistical analysis and wrote the manuscript. BC carried out real-time PCR and western blotting. MM carried out the wound migration assay. AR hybridized the samples to the microarray and helped with the data analysis. CC provided the microarrays and helped coordinate the microarray study. ACH and SAS helped in planning of the study and drafted the manuscript. All authors read and approved the final manuscript.