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The EPHB6 Receptor Tyrosine Kinase Is a Metastasis Suppressor That Is Frequently Silenced by Promoter DNA Hypermethylation in Non–Small Cell Lung Cancer

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The EPHB6 Receptor Tyrosine Kinase Is a Metastasis Suppressor That Is Frequently Silenced by Promoter DNA Hypermethylation in Non–Small Cell Lung Cancer Published OnlineFirst April 14, 2010; DOI: 10.1158/1078-0432.CCR-09-2000 Clinical Human Cancer Biology Cancer Research The EPHB6 Receptor Tyrosine Kinase Is a Metastasis Suppressor That Is Frequently Silenced by Promoter DNA Hypermethylation in Non–Small Cell Lung Cancer Jun Yu1,2, Etmar Bulk1, Ping Ji1, Antje Hascher1, Moying Tang1, Ralf Metzger3, Alessandro Marra4, Hubert Serve1, Wolfgang E. Berdel1, Rainer Wiewroth1, Steffen Koschmieder1, and Carsten Müller-Tidow1 Abstract Purpose: Loss of EPHB6 receptor tyrosine kinase expression in early-stage non–small cell lung carci- noma (NSCLC) is associated with the subsequent development of distant metastasis. Here, we analyzed the regulation and function of EPHB6 in lung cancer metastasis. Experimental Design: The expression levels of EPHB6 were compared among normal lung tissue (n = 9), NSCLC without metastasis (n = 39), and NSCLC with metastasis (n = 39) according to the history of the patients. In addition, EPHB6 expression levels of matched tumor-normal pairs from 24 NSCLC patients were analyzed. The promoter DNA methylation status and its association with the expression levels of EPHB6 were determined among 14 pairs of tumor-normal samples. Metastatic potential of EPHB6 was assessed in vitro and in vivo in a metastasis mouse model. Overexpression and RNA interference (RNAi) approaches were used for analysis of the biological functions of EPHB6. Results: EPHB6 mRNA and protein levels were significantly reduced in NSCLC tumors compared with matched normal lung tissue. Decreased EPHB6 expression levels were associated with an increased risk for metastasis development in NSCLC patients. Loss of expression correlated with EPHB6 hypermethylation. EPHB6 expression was induced by 5-aza-2'-deoxycytidine treatment in an NSCLC cell line. Restoration of EPHB6 expression in lung adenocarcinoma cells increased adhesion and decreased migration. Reexpres- sion of EPHB6 in lung cancer cells almost entirely abolished metastasis formation in non obese diabetic (NOD)/severe combined immunodeficient mice. Conclusions: Taken together, these analyses show that EPHB6 is a metastasis inhibitory gene that is fre- quently silenced by hypermethylation of its promoter in NSCLC. Clin Cancer Res; 16(8); 2275–83. ©2010 AACR. Lung cancer is a leading cause of cancer-related death, molecular therapy approaches in cancers (3, 5). We have with non–small cell lung carcinoma (NSCLC) as the recently identified human RTKs whose expression is asso- most frequent histologic subtype (1, 2). Metastasis is ciated with metastasis development in early-stage NSCLC the most frequent reason for early-stage NSCLC–related (6). Expression of several RTKs was associated with an death, and receptor tyrosine kinases (RTK) play an im- increased frequency of metastasis development. Increased portant role in this process (3, 4). The role of epidermal expression of RTKs, for example, due to gene amplifica- growth factor receptor and its family members ERBB2/ tion, is clearly associated with metastasis. The role of a Her2 Neu and ERBB3 for a metastasis phenotype are well loss of expression of other types of RTKs in metastasis established. RTKs such as the epidermal growth factor re- has not been established. In our expression analysis, ceptor family are therefore attractive targets for improved EPHB6 was one of two RTKs associated with a high risk for NSCLC metastasis when its expression was lost (6). Erythropoietin-producing hepatocyte kinase (EPH) re- Authors' Affiliations: 1Department of Medicine A—Hematology, Oncology and Pneumology, University of Münster, Münster, Germany; ceptors form the largest known subfamily of RTKs, and 2Center of Teaching Experiment, School of Basic Medical Science, to date, the EPH subfamily contains 16 members in ver- Fourth Military Medical University, Xi'an, Shaanxi, China; 3Department tebrates (7, 8). The EPH receptors interact with a family of Visceral Surgery, University of Cologne, Cologne, Germany; and 4Department of Thoracic Surgery, Thoraxklinik, Osterkappeln, Germany of ligands named ephrins. The EPH receptors are also grouped into two classes, erythropoietin-producing hepa- Note: J. Yu and E. Bulk contributed equally to this work. tocyte kinase A (EPHA) receptors (EPHA1-A10) and Corresponding Author: Carsten Müller-Tidow, Department of Medicine A, Hematology, Oncology and Pneumology, University of Münster, erythropoietin-producing hepatocyte kinase B (EPHB) re- Domagk Street 3, 48129 Münster, Germany. Phone: 49-251-835-2995; ceptors (EPHB1-B6). These groups roughly correspond to Fax: 49-251-835-2673; E-mail: [email protected]. the receptor-ligand interaction (i.e., ephrin-As show bind- doi: 10.1158/1078-0432.CCR-09-2000 ing preference for EPHA receptors and ephrin-Bs for ©2010 American Association for Cancer Research. EPHB receptors). Upon interaction with their ephrin www.aacrjournals.org 2275 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst April 14, 2010; DOI: 10.1158/1078-0432.CCR-09-2000 Yu et al. up of the NSCLC patients was at least 3 y after surgery. Translational Relevance Another set of matched tumor-normal NSCLC specimens was stored in a similar way and used for expression analyses Receptor tyrosine kinases play an important role in (mRNA and protein) and the subsequent analyses of DNA tumorigenesis and metastasis development. Here, we methylation. – identify EPHB6 as a metastasis suppressor in non 5-Aza-2'-deoxycytidine treatment. A549 cells were treated small cell lung carcinoma. Loss of EPHB6 expression with 5-aza-2'-deoxycytidine (Sigma) from a stock solution depends on promoter DNA hypermethylation and is of 10 mmol/L in pure water. Exponentially growing cells frequently encountered in lung cancer patients. Our were split in medium containing 100 μmol/L 5-aza-2'- data suggest that EPHB6 silencing might contribute deoxycytidine or a corresponding mock treatment (pure to the high frequency of metastasis development in water). The use of this concentration was based on the lung cancer. These findings raise the possibility that re- finding of a previous study (15). The cells were grown un- activation of EPHB6 and related genes might play a der these conditions for 2 to 6 d with fresh medium added role in the prevention of lung cancer metastasis. every 48 h. RNA isolation and reverse transcription. Total RNA was isolated using TRIzol reagent (Invitrogen). A total amount of 1 μg of RNA from each sample was reverse transcribed ligands, EPH receptors modulate a variety of biological using random primers and Moloney murine leukemia vi- activities, including cell-cell interaction and cell migra- rus reverse transcriptase according to the protocol of the tion (9, 10). Loss of EPHB6 is associated with angioge- manufacturer (Promega). nesis and tumor vasculature in several types of human Gene expression analyses by quantitative real-time reverse cancers (11–17). There have been several reports that transcriptase-PCR (RT-PCR). Quantitative real-time RT- EPHB6 expression is a favorable prognostic marker in PCR was carried out to determine gene expression in the neuroblastoma (11–13). In addition, decreased mRNA samples from NSCLC patients and cell lines. In brief, expression of EPHB6 was found in metastatic melanoma cDNA was prepared as described above and amplified and in invasive breast cancer cell lines with metastatic using quantitative real-time PCR in an ABI Prism 7700 potential (15–17). Functionally, EPHB6 suppresses inva- sequence detector (Applied Biosystems). EPHB6 was de- siveness, growth rate, and colony-forming efficiency tected with the following primers and probe: forward, of cultured breast cancer cell lines (18). Intriguingly, 5 ′ -TGGACTATCAGCTCCGCTACTATG; reverse, Matsuoka et al. (19) showed that EphB6 could positively 5′-GTGGCAGTGTTGGTCTCGC; and probe, 5′-FAM- and negatively regulate cell adhesion and migration. CCAGGCAGAAGACGAATCCCACTCCTT-TAMRA. The Thus, there is still a conflict about the functional role relative amounts of gene expression were calculated by of EPHB6 in the metastatic process of cells. Here, we ana- using the expression of glyceraldehyde-3-phosphate dehy- lyzed the regulation of EPHB6 and investigated the drogenase as an internal standard. potential contribution of EPHB6 to NSCLC metastasis Methylation status assay by quantitative real-time PCR. in vitro and in vivo. The EPHB6 mRNA expression was very For each sample, 1 μg of genomic DNA extracted with low in A549 NSCLC cell lines compared with normal lung DNAzol (Invitrogen) was split in two fractions and pre- tissue (20). Allowing for the low background of EPHB6, treated with or without the methylation-sensitive SmaI we selected A549 cells as a main model to determine func- restriction enzyme (New England Biolabs). Digested or tional roles of EPHB6. mock-digested DNA was then analyzed by quantitative real-time PCR with the following primers and probe: Materials and Methods forward, 5′-CGGGCCCCCAGGATCTC; reverse, 5′-TGCCACCCGCGTCTTCTC; and probe, 5′-FAM- Cell culture. As described previously (20), A549 and CGGCGCCGAACGGACCCG-TAMRA. The probe was HTB58 lung adenocarcinoma cells were cultured in Du- placed across one methylation-sensitive SmaIsite becco's modified Eagle's medium (DMEM, Invitrogen) (Fig. 2A). The methylation level was calculated using the Δ supplemented with 10% fetal calf serum, 2 mmol/L following formula: methylation level = 2 CT and ΔCT = L-glutamine, 100 units/mL penicillin, and
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