|Year : 2016 | Volume
| Issue : 1 | Page : 188-192
A rapid and sensitive method for EphB4 identification as a diagnostic and therapeutic biomarker in invasive breast cancer
Mona Moshayedi1, Farnaz Barneh1, Shaghayegh Haghjooy-Javanmard1, Hamid Mirmohammad Sadeghi2, Nahid Eskandari3, Ali Mohammad Sabzghabaee4
1 Applied Physiology Research Center, Isfahan, Iran
2 Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
4 Isfahan Clinical Toxicology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
|Date of Web Publication||13-Apr-2016|
Ali Mohammad Sabzghabaee
Toxicology Research Center, Isfahan University of Medical Sciences, Isfahan
Source of Support: None, Conflict of Interest: None
Background: In the roadmap to design diagnostic and therapeutic markers for breast cancer, EphB4 is of special interest due to its multiple roles in tumor initiation, progression and invasion. The aim of present study was to characterize a rapid and sensitive ELISA-based method to measure EphB4 level and its phosphorylation status following stimulation with its ligand, ephrinB2, in an invasive breast cancer cell line.
Materials and Methods: MDA-MB-231 breast cancer cells were lysed and EphB4 level was measured using ELISA. EphB4 level was measured in sub- and post-confluent states in culture dishes. Receptor phosphorylation was also detected by ELISA assay, using various concentrations of pre-clustered ephrinB2 for 20 minutes.
Results: Expression of EphB4 receptor was detected by ELISA in all samples. EphB4 level was significantly higher in post.confluent than sub.confluent cells. Phosphorylated receptor was also detectable with this method when cells were exogenously stimulated.
Conclusions: Quantitative data from ELISA manifested a difference between levels of EphB4 in two states of different invasive properties. Moreover, ELISA method may be considered rapid and sensitive enough to detect even low levels of total and phosphorylated EphB4 Cost-effectiveness of this method for the detection of differential expression of EphB4 proteins in clinics is also noticeable.
Keywords: Cancer, EphB4, EphrinB2, expression, immunoassays
|How to cite this article:|
Moshayedi M, Barneh F, Haghjooy-Javanmard S, Sadeghi HM, Eskandari N, Sabzghabaee AM. A rapid and sensitive method for EphB4 identification as a diagnostic and therapeutic biomarker in invasive breast cancer. J Can Res Ther 2016;12:188-92
|How to cite this URL:|
Moshayedi M, Barneh F, Haghjooy-Javanmard S, Sadeghi HM, Eskandari N, Sabzghabaee AM. A rapid and sensitive method for EphB4 identification as a diagnostic and therapeutic biomarker in invasive breast cancer. J Can Res Ther [serial online] 2016 [cited 2019 Nov 12];12:188-92. Available from: http://www.cancerjournal.net/text.asp?2016/12/1/188/147254
| > Introduction|| |
Eph receptors are the largest member of receptor tyrosine kinases, which are divided into two classes of EphAs and EphBs based on their sequence homology. The ligands of these receptors are bound to cell membrane and are named ephrinAs and ephrinBs. An intriguing feature of this family is their ability to produce bidirectional signaling i.e. upon receptor activation by the ligand, some other signals are propagated in the ligand-bearing cells as well. Eph receptors are important mediators of angiogenesis and neuronal path-finding in newly developed embryo. They also play important roles in mammary gland and intestinal homeostasis in adulthood.
It has been shown that some members of Eph receptors are also involved in pathological states such as tumorigenesis. Various key features of tumor growth, angiogenesis and invasion are thought to be influenced by deregulation of Eph receptors and their ligands.,,
EphB4 is an important member of EphB family of receptors that has been extensively studied in cancer.
A number of reports have shown that increased EphB4 expression level along with decrease in its cognate ligand, ephrinB2, correlates with cancer promotion, invasiveness and poor disease prognosis in certain types of cancers such as breast.,
Distinct expression of EphB4 in normal versus tumor tissue is of significant priority that can be exploited in diagnostic and therapeutic biomarker discovery. In a study by Li et al., differential expression of EphB4 in normal bladder tissue versus transitional cell carcinoma of the bladder was fully addressed. A sharp distinction was observed between EphB4 level in normal versus cancerous tissue, and inhibition of EphB4 receptor accordingly led to regression of tumor.
Owing to inherent complexities of EphB4 receptor functions in various cancers, its exact role in tumor promotion is just beginning to be understood.
It seems that EphB4 over expression constitutively phosphorylates EphB4 receptor leading to cross-talk with other oncogenic signaling pathways in a ligand-independent manner. Thus, detecting the level of EphB4 protein especially in breast tumors lacking expression of other important markers such as estrogen, progesterone and Her2/neu (triple negative breast cancer cells) can be of diagnostic, prognostic and of course therapeutic value.
In the new post-genomic era to find biomarkers by high-throughput techniques such as proteomics, former methods still reserve their value in biomarker identification in pathology clinics.,
Among current methods of protein detection, immunoassays are the most frequent methods due to their sensitivity and specificity. Several different methods are used in immunoassay tests, which can be either qualitative or quantitative; among which, enzyme-linked immunosorbent assay (ELISA) is a simple and accurate method to detect antigens in samples. This method has been exploited to detect proteins such as kinases and phospho-kinases in cell lysates as well.
Sandwich ELISA is usually the method of choice for this goal. Concisely, assay procedure includes use of a primary antibody to capture the protein of interest followed by addition of secondary labeled antibody targeted to another epitope of requested protein, which doubles the specificity of the detection. The optical density of the sample is then measured colorimetrically or flourometrically using a plate reader. Therefore the amount of protein to be detected in this method is proportional to the amount of light emitted from labeled antibody. High sensitivity of this ELISA assay makes it suitable to detect the small differences in the abundance of expressed proteins in different samples or interventions. Moreover, data obtained from ELISA are easily quantifiable by means of plotting a standard curve of known protein concentrations against their optical density. Quantitative characteristics of ELISA makes it suitable for comparative analysis of protein expression in different test states. The aim of this study was to set up an ELISA method to detect the level of EphB4 receptor and its phosphorylation status in MDA-MB-231, an invasive, metastatic and estrogen independent breast cancer cell line.
| > Materials and Methods|| |
Human Total and Phospho-EphB4 ELISA kits and EphrinB2-Fc were purchased from R and D Systems (Minneapolis, MN). The main constituents of all buffers and bovine serum albumin were purchased from Sigma (Minneapolis, MN). Goat anti-human IgG and Fc fragment were obtained from Jackson Laboratories (Bar Harbor, ME). Substrate solution containing tetramethylbenzidine and H2O2 were from R and D Systems.
MDA-MB-231 cells were obtained from Pasteur Cell bank of Iran and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS plus penicillin/streptomycin (Gibco) and were kept in humidified incubator with 5% CO2. Trypsin/EDTA 0.025% was used to harvest cells before reaching confluence.
EphB4 quantification in sub- and post-confluent states
Total 8 × 105 cells were seeded into 60 mm culture dishes and were further incubated for 3 and 6 days before they were harvested and lysed to measure EphB4 expression in sub- and post-confluent states. Lysates were prepared so as to have equal amounts of total protein.
EphrinB2-Fc clustering and receptor stimulation
It has been shown that clustered ephrinB2 ligand in forms of dimer or tetramer has higher efficacy in stimulating EphB4 receptor than the monomer ligand. The commercial ligands of ephrin family are thus provided as recombinant proteins containing Fc portion of IgG in order to dimerize the protein. Dimerization also increases the stability and half-life of the ligand in aqueous solutions.
EphrinB2-Fc was pre-clustered by various antibody concentrations and the mixture was shaken for 1 hour at room temperature. Since there was no consistency between the proportion of ligand: Antibody in different studies, we confirmed proper clustering of ephrinB2-Fc by 12% polyacrylamide gel electrophoresis. A modification to blue native PAGE method was performed in order to assay ephrinB2-ephrinB2 interaction intact. To do so, reducing agents were avoided in order to keep disulfide bands of dimerized or clustered ephrinB2 intact. For receptor stimulation, cells were serum starved overnight and were incubated with preclustered 3 µg/ml for 20 min.
Cell lysate preparation
Cells growing in 60 mm dishes were harvested, rinsed twice with PBS and re-suspended at 1 × 107 cells in 1 ml ice-cold lysis buffer (1% NP-40 Alternative, 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM activated sodium orthovanadate, 10 µg/mL.
Aprotinin and 10 µg/mL Leupeptin) for 30 minutes. Sodium orthovanadate (Sigma), a phosphatase inhibitor, was activated to have maximum efficacy. To do so, orthovanadate was dissolved in de-ionized water and pH was set to 10. It was boiled until it became colorless and all the crystals were dissolved. The solution was cooled to room temperature and pH was readjusted to 10. Consecutive cycles of heating and cooling along with setting pH was repeated until the solution remained colorless and pH was stabilized at 10. This process was performed to depolymerize sodium orthovanadate to make it a more potent protein phosphatase inhibitor. Cells were pelleted by centrifugation at 14,000 g for 5 minutes to remove the supernatant for further tests.
Bradford assay was used to quantify total protein content of cell extracts. Different concentrations of BSA was made in lysis buffer and all BSA solutions and sample to be tested were diluted 1:10 in PBS to minimize the interaction between Bradford reagent and NP40 present in the lysis buffer. Absorbance was measured using plate reader (Powerwave XS) at 590 nm. Standard curve was plotted and protein concentration of lysate was calculated. Cell extracts containing 50, 100 and 200 µg total protein were made and subjected to ELISA test.
ELISA test was performed as directed by the manufacturer. Briefly, 96-well high-binding ELISA plates (Greiner Corp, Germany) were coated overnight with capture total antibody. Block buffer containing 10% BSA was added to each well to block unwanted antigen binding sites. Optimal lysate concentration was determined prior to running principal assays as suggested by the manufacturer thus samples were adjusted to 50, 100, 200 µg total protein in 100 µl lysis buffer for total EphB4 assay and 50, 100 and 250 µg total protein in 100 µl lysis buffer for phosphorylation assay using sample diluent (1% NP-40 Alternative, 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM activated sodium orthovanadate). Subsequent washing and addition of detection antibody was performed followed by adding Streptavidin-HRP. Substrate solution containing equal amounts of H2O2 and tetramethylbenzidine was added and optical density of wells were measured at 540 nm immediately after adding H2SO4.
Statistical analyses were performed using SPSS 16.0 software. Data are reported as Standard Error of Mean (± SEM). The statistical significance of the difference between EphB4 expression level in pre- and post-confluent cells was determined by student t-test. Analysis of Variance (ANOVA) was performed to determine significant difference in phosphorylation level of treated groups with clustered ephrinB2 versus control. P < 0.05 was considered to be significant.
| > Results|| |
EphrinB2-Fc is clustered in a dose-dependent manner via anti-Fc antibody. Electrophoresing ephrinB2-Fc on SDS gel showed that clustering was dependent on the concentration of antibody used. Sample with 1:1000 dilution stood at the same position at un-clustered ephrinB2, which indicates lack of ligand clustering [Figure 1]. It is also obvious that using clustering antibody in excess to ephrinB2 (lanes 2, 5 and 6) produces large spots not able to move across the gel; some protein precipitations are also observed in higher ratios leading to less protein loading in the wells. Based on these results, 1:3 proportion was used for the rest of experiments, which was used by Noren et al. in a similar study.
|Figure 1: Clustering of ephrin-B2-Fc by Fc fragment specific anti-human IgG was confirmed using SDS gel. Various concentrations of antibody were added to 3 μg/ml ephrinB2-Fc and mixture was shaked for 1 hour at room temperature and samples were electrophoresed to be separated based on their size. Lane 1 is the ladder and 2 is unclustered ephrinB2. Lanes 3 to 8 represent 1: 0.1, 1: 3, 1:1.5, 1:0.5, 1:10, 1:5, 1:0.001 ephrinB2 to antibody concentration respectively|
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Total EphB4 is detected by ELISA test in cells
We investigated whether ELISA method can be used for detecting and quantifying total EphB4 content in MDA-MB-231 cell line. Total EphB4 protein was detected in cell lysates. 50 µg lysate was the lowest detected concentration and 200 µg was the highest one [Figure 2]. These data indicate that ELISA method is sensitive enough to detect low levels of the expressed receptor in lysates of MDA-MB-231 cell line.
|Figure 2: EphB4 was detected in all lysate amounts used. 100 μg lysate was used for the rest of experiments. Columns show mean of optical density for triplicate wells|
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EphB4 expression is higher in post-confluent growth phase
Increased cell-cell contact during cell confluence activates a signal transducer and activator of transcription-3 (STAT3) protein, which serves as a survival factor for confluent breast cancer cells. Given that STAT3 activation regulates EphB4 expression downstream of epidermal growth factor receptor signaling, we speculated that EphB4 expression may be increased in high cell densities. EphB4 level was shown to be significantly higher (P value 0.003) in post-confluent cells when samples were normalized to have equal amount of total protein compared to sub-confluent cells [Figure 3].
|Figure 3: EphB4 expression is higher in post confluent state. Harvested cells of sub and post confluent cells were used to measure total EphB4 level by ELISA. Lysates were normalized to equal amount of total protein. Results are expressed as ± S.E.M (P = 0.003)|
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EphB4 receptor phosphorylation is detected by ELISA method
EphB4 phosphorylation was detectable at 100 µg lysate but not in other concentrations (50 and 150 µg cell lysate) thus 100 µg lysate was used for the rest of experiments (data not shown).
EphB4 stimulation with various doses of pre-clustered ephrinB2-Fc was detected by ELISA test. Phosphorylation was significant when cells were stimulated with 3 and 10 µg/ml preclustered ephrinB2 [Figure 4]. These results indicate that EphB4 receptor activation occurs in a dose-dependent manner, which was highest at 10 µg/ml in our test.
|Figure 4: MDA-MB-231 cells were stimulated with various preclustered ephrinB2-Fc concentrations. Results are represented as ±S.E.M (*P < 0.05). Preclustered Fc portion of IgG was used as negative control|
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| > Discussion|| |
Detecting novel proteins that have differential expression pattern in normal versus tumor cells may be of diagnostic and prognostic value and have the potential to be used as therapeutic targets. As for breast cancer, existing targeted therapies against Her2/Neu or EGFR are effective in a small group of breast cancer patients since only a subset of breast tumors express these proteins.
Eph family of kinases have recently gained attention to play roles in cancer promotion. EphB4 is a member of this family whose increased mRNA level has been formerly detected in a panel of breast cancer cell lines by Fox et al.
In our study, we set up an ELISA method to detect total EphB4 receptor as well as its phosphorylation status in MDA-MB-231 breast cancer cell line. EphB4 was detected in this cell by the technique and the intensity of signal obtained was proportional to the amount of lysate used and was shown to be sensitive enough to detect total receptor even in low amounts of lysate concentration.
Expression of EphB4 protein in MDA-MB-231 breast cancer cell line was also detected by Noren et al. using immune-precipitation and western blotting method.
ELISA method offers several advantages over other methods of protein detection such as immune-precipitation and western blotting. First is high sensitivity and as a result, cost- effectiveness of this method, which enables detection and measurement of low abundance proteins in samples with consumption of less amounts of expensive reagents and materials. Next is the high specificity of this method since two specific antibodies detect the protein of interest at the same time in sandwich ELISA method. Finally the quantified data obtained directly from ELISA allows estimation of receptor level following different treatments making this method applicable for experiments where it is imperative to compare the level of receptor in different cells to see if different behavior of cells to the same stimuli is due to the difference in the level of receptor.
We also showed by ELISA that EphB4 expression was higher in post-confluent cells when total protein was normalized to the amount present in sub-confluent cells. However, we performed this experiment within few samples and this finding needs to be more deeply confirmed by analyzing more samples and cell lines.
EphB4 over expression has been shown to occur downstream of epidermal growth receptor (EGFR) activation through mediating JAK/STAT and PI3K/Akt pathways, which in turn regulate phosphorylation of STAT3, a co-factor important for driving cell proliferation forward. It has also been shown by Steinman et al. that post-confluent growth of cancer cells increases constitutive activation of STAT3. We thus speculate that higher expression of EphB4 in post-confluent cells might be the result of STAT3 activation, a hypothesis that needs to be further confirmed. Such interesting outcome, if further studied, would be of diagnostic and therapeutic value for better growth regulation of metastatic tumors.
| > Conclusion|| |
We detected the phosphorylation and total form of EphB4 in MDA-MB-231 cells when they were stimulated with exogenous ephrinB2-Fc, which shows that EphB4 receptor is active and the signal is detectable by ELISA method. Being sensitive and specific, ELISA method may be considered a suitable alternative for current methods such as immune-precipitation and western blotting. Further studies for documenting more and similar evidences are also recommended.
| > Acknowledgments|| |
This study was a Pharm.D thesis project financially supported by the Vice-chancellery for research and technology at Isfahan University of Medical Sciences. Authors would like to thank Ms. Fatemeh Khodabakhsh for her assistance in performing gel electrophoresis and also the staff members of laboratory department of Isfahan Applied Physiology Research Center for their kind help.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]