Oncogene (2009) 28, 1759–1768 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE EphrinA5 acts as a tumor suppressor in glioma by negative regulation of epidermal growth factor receptor J-J Li1,2, D-P Liu1, G-T Liu3 and D Xie1 1Laboratory of Molecular Oncology, Institute for Nutritional Sciences, Shanghai Institutes of Biological Sciences, Shanghai, People’s Republic of China; 2Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, People’s Republic of China and 3Cedars-Sinai Medical Center, Los Angeles, CA, USA Eph receptors, the largest subfamily of receptor tyrosine among the receptor tyrosine kinases (RTKs) in that kinases, and their ephrin ligands play important roles in their endogenous ligands, the ephrins, are bound to nervous system development. Recently, they have been the surface of neighboring cells. Ephrins are divided implicated in tumorigenesis of different cancers. In this into two major classes, differing by their modes of study, we showed that the expression of ephrinA5 was attachment to plasma membrane (Gale et al., 1996). Class dramatically downregulated in primary gliomas compared A ephrins are tethered to the plasma membrane by virtue with normal tissues. Forced expression of ephrinA5 of a glycosylphosphatidylinositol anchor, whereas class B reduced tumorigenicity of human glioma U373 cells. ephrins are transmembrane proteins. All of the ephrins Epidermal growth factor receptor (EGFR), which fre- interact with specific Eph receptors, although promiscuity quently acts as an oncoprotein in glioma, was greatly exists as some ephrins bind to more than one Eph decreased in ephrinA5-transfected glioma cells, and the receptor. This receptor–ligand system is involved in a two molecules exhibited a mutually exclusive expression variety of biological events (Poliakov et al., 2004; pattern in primary glioma samples. We found that Huberman et al., 2005; Kuijper et al., 2007). Although ephrinA5 enhanced c-Cbl binding to EGFR, thus pro- a majority of investigators have studied the role of Eph/ moted ubiquitylation and degradation of the receptor. ephrin in development, emerging evidence suggests strong Either ephrinA5-Fc or EphA2-Fc treatment simulating involvement in tumorigenesis, including metastasis, inva- bidirectional signaling of Eph/ephrin system resulted in sion, and angiogenesis (Surawska et al., 2004; Brantley- EGFR decrease. This study discovered that ephrinA5 Sieders et al., 2006; Liu et al., 2007). acted as a tumor suppressor in glioma, and its negative Gliomas are the most common primary brain tumors regulation of EGFR contributed to the suppressive effects. with limited therapeutic options, high recurrence rate In addition to identifying a novel mechanism underlying and mortality. The epidermal growth factor receptor tumor suppressor activity of ephrinA5, we also showed (EGFR) and its ligands figure prominently in glioma cross-talk between different receptor tyrosine kinase pathogenesis, and the malignant nature of glioma is families in glioma. These findings may improve therapeu- partially due to the aberrance of EGFR, including gene tic strategies for glioma. amplification, mutation, abnormal overexpression, and Oncogene (2009) 28, 1759–1768; doi:10.1038/onc.2009.15; improper activation of the receptor and its downstream published online 9 March 2009 signaling (Nicholas et al., 2006). Considering the importance of RTKs in brain tumors, investigation into Keywords: ephrinA5; EGFR; c-Cbl; glioma RTK networks provides a better understanding of the biology of glioma. However, studies concerning the cross-talk between Eph/ephrin and ErbB families are rare. In this study, we showed that ephrinA5, a member belonging to ephrinA subclass, could negatively regulate Introduction EGFR, which contributed to its suppressive effect in glioma. A mutually exclusive expression pattern be- Eph/ephrin family members are expressed in adult tween EGFR and ephrinA5 was disclosed in both stable human tissues and in the developing nervous system cell lines and clinical tissue samples. We further revealed (Hafner et al., 2004). The Eph receptors are unique that ephrinA5 acted as a negative regulator of EGFR through promoting c-Cbl-mediated degradation. Im- Correspondence: Dr D Xie, Laboratory of Molecular Oncology, portantly, both ephrinA5-Fc and EphA2-Fc fusion Institute for Nutritional Sciences, Shanghai Institutes of Biological protein treatment led to decreased level of EGFR. Sciences, Chinese Academy of Sciences, 294 Tai-Yuan Road, Shanghai Therefore, the bidirectional signaling, which is char- 200031, People’s Republic of China. acteristic of the Eph/ephrin system, is involved in E-mail: [email protected] Received 7 July 2008; revised 17 November 2008; accepted 22 December ephrinA5-induced EGFR reduction. Thus, we have 2008; published online 9 March 2009 identified a novel mechanism of EGFR regulation in EphrinA5 suppresses tumorigenesis in glioma J-J Li et al 1760 glioma. Our findings may provide the assets for proliferation in MTT (3-(4,5-dimethylthiazol-2-Yl)-2,5- developing improved therapeutic strategies. diphenyltetrazolium bromide) assay (data not shown). In contrast, U373/ephrinA5 cells developed significantly fewer and smaller colonies in soft agar compared with Results U373/v cells (Figure 2a), indicating its inhibitory effect on anchorage-independent clonogenic growth. EphrinA5 was expressed at a low level in primary We further examined whether ephrinA5 would affect glioblastomas the tumorigenicity of glioma cells in vivo. U373/v and We examined the expression pattern of ephrinA5 in U373/ephrinA5 cells were subcutaneously injected into both quick-frozen brain samples (23 glioblastoma 8-week-old nude mice, and the resulting tumors were multiforme and 5 normal) and primary cells (derived measured at the end of 4 weeks. U373/ephrinA5 cells from 23 glioblastoma multiforme and 5 normal) by real- barely developed any observable tumors, whereas the time PCR. Four out of five normal tissues exhibited tumors derived from U373/v cells were easily visible and significantly higher ephrinA5 expression level compared measurable. A significant difference in tumor volume with tumor tissues (Figure 1a). The similar expression between the two groups was revealed by statistical pattern of ephrinA5 was also observed in primary cells analysis (Po0.05, Figure 2b). These results proved the (Figure 1b). We performed immunohistochemistry in suppressive effect of ephrinA5 in glioma. both normal and tumor tissues. As shown in Figure 1c, ephrinA5 protein was easily detected in normal tissues, EphrinA5 downregulated either the basal or the exhibiting pronounced membrane staining. In contrast, phosphorylation level of EGFR and impaired EGFR it was almost undetectable in tumor tissues. Down- signaling in glioma cells regulation of ephrinA5 at both mRNA and protein level To investigate the underlying mechanism for reduced in glioma suggested its role as a tumor suppressor. tumorigenicity of glioma cells by ephrinA5, we exam- ined the expression of several important molecules Forced expression of ephrinA5 in glioma cells reduced involved in tumorigenesis and development of glioma. their clonogenic growth in soft agar and tumorigenicity We found that the expression level of EGFR was in nude mice significantly decreased in U373/ephrinA5 cells To examine the potential suppressive effects of ephrinA5 (Figure 2c). The significant decrease of EGFR was also in glioma, we established stable cell lines using human observed in both U343 and U373 cells infected with a glioma U373 cells. Two identified clones exhibiting virus vector overexpressing ephrinA5 (Figure 2d). On different levels of ephrinA5 were selected for further the basis of this data, we investigated the expression studies (U373/ephrinA5L and U373/ephrinA5H, pattern of ephrinA5 and EGFR in glioma tissue Figure 2c). EphrinA5 showed little effect on cell samples. As shown in Figure 3a, the expression of Figure 1 Quantitative expression and Immunohistochemistry of ephrinA5 in human clinical samples. (a) Real-time PCR examination of ephrinA5 expression in normal brain and primary glioma tissue samples. (b) Quantitative expression of ephrinA5 in primary cells derived from normal brain and glioma tissues. T, tumor; N, normal. (c) Immunohistochemistry displays clear ephrinA5 signals (brown) in normal brain tissue samples (N); however, it is almost undetectable in glioma tissues (T). Oncogene EphrinA5 suppresses tumorigenesis in glioma J-J Li et al 1761 Figure 2 Forced expression of ephrinA5 in U373 cells, soft agar assay and tumorigenicity assay. (a) Soft agar assay: A total of 1.0 Â 103 cells per well were seeded in soft agar for 20 days, and colonies were enumerated. Each experiment was performed in triplicate, and results represented the mean±s.d. of three experiments. (b) Tumorigenicity assay. Either 1.0 Â 106 U373/ephrinA5 cells or U373/v cells were subcutaneously injected on opposite flanks of nude mice (three nude mice per group), respectively. After 4 weeks, resulting tumors were removed and the volumes were measured. Statistical significance was determined with a Student’s t test (bars, s.d.; *Po0.05 versus U373/v). (c) Expression of ephrinA5 and several other genes in U373/v and U373/ephrinA5 cells were examined by western blot. (d) Expression of both basal and phosphorylation level of epidermal growth factor receptor (EGFR) was examined in U343 and U373 cells, which was infected with either control