Oncogene (2015) 34, 558–567 & 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE EphA2 promotes infiltrative invasion of glioma stem cells in vivo through cross-talk with Akt and regulates stem cell properties H Miao1,2, NW Gale3, H Guo1,2, J Qian1,2, A Petty1,2, J Kaspar1,2, AJ Murphy3, DM Valenzuela3, G Yancopoulos3, D Hambardzumyan4,5, JD Lathia4,5, JN Rich4,5, J Lee4,5 and B Wang1,2,5 Diffuse infiltrative invasion is a major cause for the dismal prognosis of glioblastoma multiforme (GBM), but the underlying mechanisms remain incompletely understood. Using human glioma stem cells (GSCs) that recapitulate the invasive propensity of primary GBM, we find that EphA2 critically regulates GBM invasion in vivo. EphA2 was expressed in all seven GSC lines examined, and overexpression of EphA2 enhanced intracranial invasion. The effects required Akt-mediated phosphorylation of EphA2 on serine 897. In vitro the Akt–EphA2 signaling axis is maintained in the absence of ephrin-A ligands and is disrupted upon ligand stimulation. To test whether ephrin-As in tumor microenvironment can regulate GSC invasion, the newly established Efna1;Efna3;Efna4 triple knockout mice (TKO) were used in an ex vivo brain slice invasion assay. We observed significantly increased GSC invasion through the brain slices of TKO mice relative to wild-type (WT) littermates. Mechanistically EphA2 knockdown suppressed stem cell properties of GSCs, causing diminished self-renewal, reduced stem marker expression and decreased tumorigenicity. In a subset of GSCs, the reduced stem cell properties were associated with lower Sox2 expression. Overexpression of EphA2 promoted stem cell properties in a kinase-independent manner and increased Sox2 expression. Disruption of Akt-EphA2 cross-talk attenuated stem cell marker expression and neurosphere formation while having minimal effects on tumorigenesis. Taken together, the results show that EphA2 endows invasiveness of GSCs in vivo in cooperation with Akt and regulates glioma stem cell properties. Oncogene (2015) 34, 558–567; doi:10.1038/onc.2013.590; published online 3 February 2014 Keywords: EphA2; Akt; stem cells; glioma; invasion; ephrin-A INTRODUCTION causes for phosphatidylinositol 3-kinase/Akt activation, particularly 4,8,9 Glioblastoma multiforme or GBM is the most common form of in primary GBM. However, whether and how Akt activation human primary malignant brain tumors.1 Despite maximum may regulate diffuse infiltrative invasion of GBM remains to be treatment including radio- and chemotherapy, the prognosis of elucidated. GBM remains dismal, with a median survival of around 14 months The 14 members of Eph receptor tyrosine kinases and their and a 5-year survival of only about 3%.2 A major cause for the membrane-anchored ligands called ephrins have important roles lethality is attributed to the diffuse infiltrative invasion of GBM in a diverse array of developmental, physiological and disease 10–13 cells throughout the brain at the time of diagnosis, which is processes. Both the receptors and ligands are divided responsible for virtually invariable recurrence after surgical into two subfamilies with EphA receptors primarily binding to resection of primary tumors.1,3 Understanding the molecular glycosylphosphatidylinisotol-anchored ephrin-A ligands and EphB mechanisms underpinning the highly invasive properties of GBM receptors preferentially engaging the transmembrane ephrin-B 14 has significant basic and clinical implications. ligands. Perturbation of Eph/ephrin systems has been 15 Extensive molecular studies have identified phosphatidylinositol documented in many different types of human cancers. EphA2 3-kinase/Akt signaling cascade as one of the most frequently in particular is the most frequently affected Eph receptors in altered pathways in primary and secondary GBM.4–6 These human cancer. In a number of human cancers, EphA2 is often observations were further validated and supported by recent overexpressed, which is often associated with the late-stage 15,16 results from The Cancer Genome Atlas (TCGA) analyses of a large malignant progression and poor prognosis. Although EphA2 is number of GBM cases. It was found that Akt is activated in a great poorly expressed in the normal brain, it is overexpressed in GBM 17,18 majority (88%) of the GBM cases,6,7 and along with inactivation of and associated with worse prognosis, making it an attractive 19,20 p53 and Rb pathways represents one of the three major molecular target for the development of novel therapeutic strategies. mechanisms of gliomagenesis. Activation of upstream RTK growth Although many of these studies point to the pro-oncogenic factor receptors through amplification, mutation or gene fusion as functions of EphA2, extensive evidence exists that demonstrate 15,16 well as the loss of the negative regulator, PTEN, are the major tumor suppressor functions of EphA2 upon ligand stimulation. 1Rammelkamp Center for Research, MetroHealth Medical Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA; 2Department of Pharmacology and Oncology, Case Western Reserve University School of Medicine, Cleveland, OH, USA; 3VelociGene Division, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USA; 4Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA and 5Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, OH, USA. Correspondence: Dr B Wang, Rammelkamp Center for Research/R421, MetroHealth Medical Center, Case Western Reserve University School of Medicine, 2500 MetroHealth Drive, Cleveland, OH 44109, USA. E-mail: [email protected] Received 12 September 2012; revised 17 December 2013; accepted 18 December 2013; published online 3 February 2014 Akt–EphA2 signaling axis promotes glioma invasion in vivo H Miao et al 559 Thus, ligand-activated EphA2 inhibits Ras/ERK and PI3K/Akt slices from Efna1;Efna3;Efna4 triple knockout (TKO) mice and pathways, suppresses cell proliferation and induces apoptosis. observed significantly increased invasion of GSCs in brain slices Moreover, EphA2 KO mice displayed markedly increased from KO mice compared with those from the WT or heterozygous susceptibility to carcinogenesis.21 littermates, which provides experimental evidence for the long We recently discovered a reciprocal regulatory loop between suspected role of ephrins in tumor microenvironment in regulat- EphA2 and Akt, which provides a partial mechanistic explanation ing tumor cells’ dissemination. Taken together, our data reveal for the seemingly conflicting roles of EphA2 in cancer.18 It turns EphA2 as an important driver in the diffuse infiltrative invasion of out that EphA2 has diametrically opposite functions in regulating GBM and help maintain stem cell properties of GSCs. cell migration: ligand-dependent inhibition and ligand- independent stimulation. The ligand-independent promotion of cell migration by EphA2 requires Akt-mediated phosphorylation of EphA2 on serine 897 located in the loop between kinase domain RESULTS and sterile alpha motif.18 However, as these studies were Akt–EphA2 signaling axis is activated in glioma stem cells performed in vitro, an important unanswered question is To investigate the role of EphA2 in glioma cell invasion in vivo,we whether the Akt–EphA2 signaling axis can regulate tumor cell utilized the recently established lines of GSCs at early passages, invasion in vivo. which express multiple stem markers, form tumors in vivo and In this report, we chose to address this outstanding question by are capable of self renewal and differentiation.32,33 These cells using cells at the apex of the cellular hierarchy in GBM,22 glioma recapitulate the gene expression patterns and in vivo biology of stem cells (GSCs). GSCs are responsible for tumor propagation,23–27 human GBM including diffuse infiltrative invasion of brain therapeutic resistance28,29 and have a higher invasive capacity.30,31 parenchyma upon intracranial implantation,32 and therefore Here we report that Akt–EphA2 signaling axis is required for constitute a good model to investigate the role of EphA2 in intracranial invasion of GSCs. We further demonstrate that the GBM invasion. The GSCs were maintained either in suspension or expression of EphA2 has a role in maintaining the stem cell on laminin-coated surface as a monolayer. The latter culturing properties of GSCs, which is in part mediated through Akt-induced method is recently shown to maintain stem cell property during S897 phosphorylation of EphA2. To interrogate invasion in a in vitro culture and facilitate genetic manipulation.34 Figure 1A microenvironment lacking ephrin-A ligands, we utilized brain shows neurosphere formation by two lines of GSCs, 827 and 1228, Suspension Laminin Nestin EphA2 827 1228 Nestin/EphA2/ GFAP/DAPI DAPI 827 456 448 578 131 EA1,10 min. -+-+ 1.2 EA1,10 min. -+-+-+ -+ EphA2 1 EphA2 pY-EphA/B 0.8 pS473-Akt pS897-EphA2 0.6 P=0.013 GAPDH pS473-Akt * 0.4 827 780 1228 NSC Akt phosphor-Akt level 0.2 EA1,10 min. -+ -+-+-+ p-Erk1/2 EphA2 0 Erk 0 10 p-Akt Ephrin-A1 GAPDH LM Sus. Figure 1. EphA2 is expressed in glioma stem cells and is phosphorylated on S897. (A) Phase images of GSCs cultured in suspension or on laminin-coated surface. (B) The 827 line of GSCs were cultured on laminin and subjected to immunofluorescence analysis for nestin (a) and EphA2 (b), which were merged with 40,6-diamidino-2-phenylindole in (c). (d) A fraction of GSCs also express GFAP, a differentiation marker. Scale bar, 50 mm. (C) EphA2 in GSCs was phosphorylated on S897 in the absence of ligand stimulation. Ephrin-A1 treatment led to EphA2 activation, and inhibition of Akt and pS897-EphA2. GSCs cultured on laminin (LM) or in suspension (Sus.) were stimulated with ephrin-A1-Fc and lysed. Whole-cell lysates were subjected to immunoblot with the indicated antibodies. (D) Quantitative densitometry analysis shows significant Akt inhibition by the ligand-activated EphA2. Data from four independent experiments were analyzed. (E) EphA2 receptor is expressed in NSCs and seven independent preparations of GSCs, and mediates Akt inhibition when stimulated with ephrin-A1 ligand.