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Ephrin‑B2 Inhibits Cell Proliferation and Motility in Vitro and Predicts Longer Metastasis‑Free Survival in Breast Cancer

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Ephrin‑B2 Inhibits Cell Proliferation and Motility in Vitro and Predicts Longer Metastasis‑Free Survival in Breast Cancer INTERNATIONAL JOURNAL OF ONCOLOGY 55: 1275-1286, 2019 Ephrin‑B2 inhibits cell proliferation and motility in vitro and predicts longer metastasis‑free survival in breast cancer ZELJANA MAGIC, JOSEFINE SANDSTRÖM and GIZEH PEREZ-TENORIO Department of Clinical and Experimental Medicine and Department of Oncology, Linköping University, SE-58185 Linköping, Sweden Received June 25, 2019; Accepted September 12, 2019 DOI: 10.3892/ijo.2019.4892 Abstract. The tyrosine kinase receptor EphB4 and its ligand compared with the control cells. These effects were more ephrin-B2 interact through cell-to-cell contacts. Upon inter- pronounced when the cells lacked the ability to transmit the action, EphB4 transmits bidirectional signals. A forward reverse signal (B2-5F). In clinical material, ephrin-B protein signal inside EphB4-expressing cells is believed to suppress expression was associated with a positive estrogen receptor tumor growth, while inside the ephrin-expressing cells, an (ER) status, a low HER-2 expression and was negatively asso- oncogenic reverse signal arises. In breast cancer cells with ciated with Nottingham histologic grade (NHG) III. Ephrin-B a high EphB4 receptor expression the forward signal is low, expression indicated a good prognosis, whereas EphB4 expres- in part due to the low expression of the ligand ephrin-B2. sion was associated with a shorter metastasis-free survival Therefore, we hypothesized that by re-introducing the ligand in univariate and multivariate analysis. Furthermore, the in EphB4-positive cells, tumor suppression could be induced prognostic value of EFNB2 and EPHB4 was confirmed at the by the stimulation of the forward signal. This question was gene expression level in public datasets. Thus, on the whole, addressed in vitro by the stable lentiviral infection of breast the findings of this study suggest that ephrin‑B2 expression is cancer cells with either wild-type EFNB2 or with a mutant associated with less proliferation and lower motility of breast EFNB2‑5F, unable to transmit reverse signaling. Furthermore, cancer cells and with a longer patient survival in breast cancer. we investigated ephrin-B and EphB4 protein expression in 216 paraffin‑embedded tumors using immunohistochemistry. The Introduction in vitro results indicated that ephrin-B2 expression was asso- ciated with a lower cell proliferation, migration and motility Few markers, such as receptor tyrosine kinases (RTKs) from the epidermal growth factor receptor (EGFR) family, are currently used in clinical practice to estimate patient prognosis or determine the appropriate treatment course. However, some patients continue to develop metastasis, thus indicating the Correspondence to: Dr Gizeh Perez-Tenorio, Department of need for novel clinical markers and therapeutic targets. Clinical and Experimental Medicine and Department of Oncology, Members of the Eph (from the erythropoietin-producing Linköping University, SE-58185 Linköping, Sweden E-mail: [email protected] hepatocellular cell line where it was first cloned) receptors have been found to be overexpressed in screens of RTKs in Abbreviations: RTK, receptor tyrosine kinase; EFNB2, cancer (1,2). The Eph receptor was first identified in an eryth- ephrin-B2; EGFR, epidermal growth factor receptor; Eph, from the ropoietin-producing human hepatocellular carcinoma cell erythropoietin-producing hepatocellular cell line where it was first line (ETL-1) (3) in an effort to discover novel tyrosine kinase cloned; ephrin, eph receptor interacting protein; B2-WT, EFNB2 receptors. This family now constitutes the largest family of wild-type; B2-5F, EFNB2 unable to transmit reverse signaling; RTKs participating in cell-to-cell communications and tissue HRGβ1, heregulin-β1; EGF, epidermal growth factor; SH3PXD2A, integrity during embryogenesis, as well as in cell prolifera- SH3 and PX domains 2A; STAT3, signal transducer and activator tion, survival and motility, which are crucial steps towards the of transcription 3; ER, estrogen receptor; HER2, human epidermal development of metastases (1,4). growth factor receptor 2; CSF1, colony stimulating factor 1; PLAUR, The Eph receptors and their ligands, eph receptor inter- plasminogen activator, urokinase receptor; RDX, radixin; TGFβ1, transforming growth factor β1; WIPF1, WAS/WASL interacting acting proteins (ephrins) interact upon cell-to-cell contacts protein family member 1; PTK2B, protein tyrosine kinase 2 beta; triggering bidirectional signals both inside the Eph (so-called, RND3, Rho family GTPase 3; VASP, vasodilator stimulated forward signal) and the ephrin-expressing cells (so-called, phosphoprotein reverse signal). The forward signal is believed to suppress the tumor, while the reverse signaling is thought to promote tumor Key words: EFNB2, EphB4, bidirectional signaling, metastasis, growth. Thus, Eph receptor and ephrin signaling is complex, receptor tyrosine kinase leading to paradoxical results both in vitro and in vivo (1). EphB4 and EphA2 are some of the most extensively studied Eph receptor family members in breast cancer. EphA2 1276 MAGIC et al: EPHRIN-B2 AND PROGNOSIS IN BREAST CANCER is related to a poor breast cancer prognosis and resistance to incubation at 37˚C in a CO2 incubator, the cells were infected trastuzumab (5) and tamoxifen (6-9). EphB4 overexpression with the following multiplicities of infection (MOI): The GFP has been shown to be associated with a poor patient outcome (MOI=5), GFP‑EFNB2‑WT (MOI=7) or GFP‑EFNB2‑5F and may be a survival factor for breast cancer cells (6,10,11). (MOI=10). Lentiviral vectors were added in the presence However, the results are still controversial: EphB4 could of polybrene (3 µg/ml) (Millipore) and the supernatant was be highly expressed in breast cancer cell lines compared to removed 16 h post-infection. Green fluorescent cells, i.e., non-transformed epithelial cells (12); however, in clinical successfully transduced cells, of similar intensity were sorted samples, the receptor has been shown to be associated with by FACS and maintained in culture for use in further experi- a low histological grade and it is expressed at lower levels in ments. Ephrin-B2 expression was periodically controlled by invasive carcinomas compared to normal breast tissue (13). fluorescence microscopy using a Radiance Multiphoton Laser The tumorigenic properties of EphB4 may manifest in Point Scanning Confocal Microscope or by immunoblotting. the absence of its preferred ligand, ephrin-B2, as suggested by EphB4 upregulation in mammary epithelial cells, where Antibodies, siRNAs and vectors. Primary antibodies used the expression of the ligand ephrin-B2 seems to be lost (14). for immunoblotting and/or immunoprecipitations were as Previous results have indicated that stimulation with a soluble follows: Mouse anti-EphB4 (clone 3D7/G8, cat. no. 37-1800, ephrin-B2-Fc ligand inhibits tumor formation and growth in a Zymed/Invitrogen), mouse anti-pan ephrin-B (clone 2D3E6 breast cancer xenograft model (12). cat. no. 37-8100, Zymed), anti-phosphotyrosine-conjugated Therefore, in this study, we wished to address the ques- with HRP (4G10, cat. no. 05-321, Millipore), mouse tion whether the re-expression of ephrin-B2 in breast cancer anti-GAPDH (cat. no. ab185059, Abcam), rabbit anti-GFP cells, where the EphB4 receptor is present, could inhibit the (cat. no. GTX20290, GeneTex), rabbit anti-signal transducer tumorigenic properties of these cells. To examine the effects and activator of transcription 3 (STAT3), purchased from of EphB4 and ephrin-B2 co-expression in vitro, we used two Cell signaling Technology (cat. no. 87685 and anti-SH3 and lentiviral vectors to stably infect MCF7 cells with a wild-type PX domains 2A (SH3PXD2A), obtained from Nordic Biosite EFNB2 (B2-WT) or a mutant EFNB2 (B2-5F) which is (cat. no. AP16560a). Secondary polyclonal antibodies conju- unable to transmit reverse signaling. We found that the gated with horseradish peroxidase (HRP) goat anti-rabbit EFNB2-expressing cells (MCF7-B2) exhibited a lower prolif- (cat. no. P0448) and goat anti-mouse (cat. no. P0447) were eration, formed less foci and had an impaired cell motility. purchased from Dako (Dako Denmark) For the EphB4 antibody The MCF7-B2 cells were also less responsive to growth validation the following siRNAs were used: FlexiTube EphB4 factor-induced migration. In particular, the mutant B2-5F cells, GS0250 (#1; SI00063748, #2; SI00288596, #3; SI00063791, only able to transmit the forward signal, exhibited a decreased #4; SI00288589. Further, we used the AllStar Negative expression of several genes involved in cell motility. Control (SI03650318, Qiagen) and the Silencer Control Of note, similar results were observed in our patient GAPDH Positive Control (Life Technologies, Thermo Fisher material where ephrin-B and EphB4 protein expression were Scientific). Plasmids and lentiviral vectors were as follows: investigated by immunohistochemistry in paraffin‑embedded Mouse ephrin-B2 (GenBank accession no. NM_010111.5) tissues from 216 patients. Ephrin-B2, but not EphB4 expression with an N-terminal eGFP tag inserted between a signal indicated a longer metastasis-free survival in both univariate peptide and the mature coding sequence (15,16) was cloned and multivariate analyses compensating for known clinical into the pCCLsin.PPT.hPGK.GFP pre-lentiviral vector (17) markers. Moreover, a validation survey in public datasets replacing the eGFP insert of the vector. A mouse tyrosine confirmed that a high EFNB2 gene expression was associated phosphorylation‑deficient
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