Migration Dichotomy of Glioblastoma by Interacting with Focal Adhesion Kinase
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Oncogene (2012) 31, 5132 --5143 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE EphB2 receptor controls proliferation/migration dichotomy of glioblastoma by interacting with focal adhesion kinase SD Wang1, P Rath1,2, B Lal1,2, J-P Richard1,2,YLi1,2, CR Goodwin3, J Laterra1,2,4,5 and S Xia1,2 Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumors in adults. Uncontrolled proliferation and abnormal cell migration are two prominent spatially and temporally disassociated characteristics of GBMs. In this study, we investigated the role of the receptor tyrosine kinase EphB2 in controlling the proliferation/migration dichotomy of GBM. We studied EphB2 gain of function and loss of function in glioblastoma-derived stem-like neurospheres, whose in vivo growth pattern closely replicates human GBM. EphB2 expression stimulated GBM neurosphere cell migration and invasion, and inhibited neurosphere cell proliferation in vitro. In parallel, EphB2 silencing increased tumor cell proliferation and decreased tumor cell migration. EphB2 was found to increase tumor cell invasion in vivo using an internally controlled dual-fluorescent xenograft model. Xenografts derived from EphB2-overexpressing GBM neurospheres also showed decreased cellular proliferation. The non-receptor tyrosine kinase focal adhesion kinase (FAK) was found to be co-associated with and highly activated by EphB2 expression, and FAK activation facilitated focal adhesion formation, cytoskeleton structure change and cell migration in EphB2-expressing GBM neurosphere cells. Taken together, our findings indicate that EphB2 has pro-invasive and anti-proliferative actions in GBM stem-like neurospheres mediated, in part, by interactions between EphB2 receptors and FAK. These novel findings suggest that tumor cell invasion can be therapeutically targeted by inhibiting EphB2 signaling, and that optimal antitumor responses to EphB2 targeting may require concurrent use of anti-proliferative agents. Oncogene (2012) 31, 5132--5143; doi:10.1038/onc.2012.16; published online 6 February 2012 Keywords: glioblastoma-derived stem-like neurospheres; orthotopic xenograft; EphB2; proliferation/migration dichotomy; invasion; focal adhesion kinase INTRODUCTION stem-like cells, established as neurospheres under serum-free Gliomas are the most common primary tumors of the central conditions, recapitulate the genetic and histopathological features nervous system. Glioblastoma multiforme (GBM), the most of clinical GBMs and provide excellent models for tumor invasion predominant and most malignant form of glioma, comprises studies.6 Incorporating these invasive tumor models into drug B10 000 new cases annually in the United States. Despite development regimens should enhance current efforts toward considerable progress made in modern antitumor therapy, the developing effective anti-invasion therapy for glioblastoma. 2-year survival rate for patients diagnosed with GBM is still only We recently found that the receptor tyrosine kinase EphB2 is B28%.1 A major challenge when treating GBM is the propensity significantly upregulated during differentiation of GBM-derived for tumor cells to invade adjacent brain tissues, which inevitably stem-like neurospheres.7 Eph receptors and their transmembrane- leads to tumor recurrence. Histopathological analysis of clinical bound ligand ephrins form the largest subfamily of the receptor GBMs reveals that uncontrolled proliferation and tumor cell tyrosine kinase. There are nine members of EphA and five invasion, two characteristics of GBMs, are spatially and temporarily members of EphB receptors in mammals that specifically bind to disassociated behaviors, with tumor cores, containing a highly their cognate ligands (ephrin-As and ephrin-Bs, respectively), proliferative population and cells within the more invasive with few exceptions.8,9 Eph receptors and ephrins lead to periphery, showing low proliferation rates.2,3 bi-directional signaling, from ligand to receptor (forward) and The molecular determinants of GBM invasion are only now from receptor to ligand (reverse), to transduce signals in a cell--cell beginning to be understood, holding promise for development of contact-dependent manner, thereby coordinating cell repulsion new therapeutic approaches. Recent clinical observations in or attraction.8,9 Eph and ephrin interactions are implicated in patients and studies of preclinical models suggest that a more diverse developmental and neurological functions, including cell invasive tumor phenotype emerges in response to vascular attachment, migration, axon guidance, vasculogenesis and cell endothelial growth factor pathway inhibitors.4 This highlights motility.10 the increasing need to understand mechanisms of tumor cell The effects of the Eph/ephrin system in cancer are complex and invasion and develop effective anti-invasion therapies. The paucity sometimes controversial owing to their diverse nature.9,11 of intracranial human GBM models that recapitulate the Accumulating in vitro studies demonstrate that Eph receptors extensively invasive features of GBMs has been a major obstacle and ephrins can both promote and inhibit tumorigenicity. Little is for studying tumor invasion and developing anti-invasive known about how the Eph/ephrin system balances these therapeutics.5 New model systems derived from glioblastoma contrasting activities. It has been reported that Eph signaling 1Hugo W Moser Research Institute at Kennedy Krieger, Baltimore, MD, USA; 2Department of Neurology, Johns Hopkins School of Medicine, Baltimore, MD, USA; 3Department of Neurosurgery, Johns Hopkins School of Medicine, Baltimore, MD, USA; 4Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA and 5Department of Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD, USA. Correspondence: Dr S Xia, Hugo W Moser Research Institute at Kennedy Krieger, The Johns Hopkins School of Medicine, 707 N. Broadway, Room 400K, Baltimore, MD 21205, USA. E-mail: [email protected] Received 5 August 2011; revised 15 December 2011; accepted 3 January 2012; published online 6 February 2012 EphB2 in glioblastoma proliferation and migration SD Wang et al 5133 can elicit a tumor-suppressing effect by inhibiting cancer cell-- body. The ligand ephrin-B1/Fc induced EphB2 receptor activation substrate adhesion, migration, invasion and growth.12 --15 Ligand- in EphB2-OVE cells (Figure 1e). After ligand treatment, the total induced forward Eph signaling is associated with inhibition of level of EphB2 in GBM neurospheres decreased (Figure 1e), oncogenic signaling pathways, such as HRAS-Erk, phosphatidyl- consistent with a previously described mechanism of stimulation- inositol-3-OH kinase (PI3K)-AKT and Abl-Crk, in colon, breast and induced Eph receptor degradation.33,35 prostate cancers.15 --17 Conversely, Eph receptors have been found We asked if EphB2 upregulation alters the migratory phenotype to promote migration, invasion, metastasis and angiogenesis in of GBM neurosphere cells. Ploc and EphB2-OVE cell migration was certain tumor models, including glioma, breast cancer and analyzed on laminin-coated transwells36 and quantified by hepatocellular carcinomas.18 --21 Eph receptor can also crosstalk counting 40-6-diamidino-2-phenylindole (DAPI)-stained cells per with other receptor tyrosine kinases such as fibroblast growth field (Figure 2a). Overexpression of EphB2 alone, in the absence of factor receptor-1, epidermal growth factor receptor and the exogenous ligand stimulation, promoted a 2- to 3-fold increase in hepatocyte growth factor receptor c-Met, and thereby increase serum-induced cell migration when measured 4--18 h after plating cancer cell malignancy.22 --24 A considerable limitation in our cells (Figure 2b, Po0.001). We also tested neurosphere cell understanding of Eph/ephrin function is that the majority of migration induced by hepatocyte growth factor, a potent conclusions are based on in vitro studies, rendering their relevance chemoattractant for neural progenitor cells,37 in serum-free to in vivo contexts uncertain. neurosphere culture medium. EphB2 similarly promoted GBM EphB2 is expressed in cells of epithelial origin,21,25 has strong neurosphere cell migration in response to this stimulus (Figure 2c, affinity for ephrin-B1 and ephrin-B2, and weak affinity for ephrin-B3 Po0.001). In addition, we found that EphB2 promoted neuro- and ephrin-A5. Like other members of the Eph family, EphB2 elicits sphere cell invasion through matrigel-coated transwells (Supple- both tumor-promoting and tumor-suppressing effects. EphB2 mentary Figure 1A, Po0.001). We used EphB2/Fc as a competitive mutant or EphB2 silencing has been identified in human prostate antagonist of EphB2 receptors to determine the role of EphB2 on and colorectal tumors, suggesting a tumor-suppressing function this migration response.38,39 EphB2/Fc inhibited cell migration in for EphB2 forward signaling in these cancers.26,27 Yet, in colorectal both Ploc and EphB2-OVE cells by B50% (Figure 2d, Po0.001), cancers, EphB2/ephrin signaling is able to inhibit tumor expansion suggesting that both endogenous and transgenic EphB2 receptor and invasion through repulsive mechanisms.12,28 These disparate activities are involved in promoting cell migration. When the context-dependent EphB2 functions make it of great interest to EphB2 ligand ephrin-B1/Fc was added in combination with serum, study its role in GBM-derived stem-like neurospheres, whose cell migration