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• W2040574920 abstract "In human glioblastomas (hGBMs), tumor-propagating cells with stem-like characteristics (TPCs) represent a key therapeutic target. We found that the EphA2 receptor tyrosine kinase is overexpressed in hGBM TPCs. Cytofluorimetric sorting into EphA2High and EphA2Low populations demonstrated that EphA2 expression correlates with the size and tumor-propagating ability of the TPC pool in hGBMs. Both ephrinA1-Fc, which caused EphA2 downregulation in TPCs, and siRNA-mediated knockdown of EPHA2 expression suppressed TPCs self-renewal ex vivo and intracranial tumorigenicity, pointing to EphA2 downregulation as a causal event in the loss of TPCs tumorigenicity. Infusion of ephrinA1-Fc into intracranial xenografts elicited strong tumor-suppressing effects, suggestive of therapeutic applications. In human glioblastomas (hGBMs), tumor-propagating cells with stem-like characteristics (TPCs) represent a key therapeutic target. We found that the EphA2 receptor tyrosine kinase is overexpressed in hGBM TPCs. Cytofluorimetric sorting into EphA2High and EphA2Low populations demonstrated that EphA2 expression correlates with the size and tumor-propagating ability of the TPC pool in hGBMs. Both ephrinA1-Fc, which caused EphA2 downregulation in TPCs, and siRNA-mediated knockdown of EPHA2 expression suppressed TPCs self-renewal ex vivo and intracranial tumorigenicity, pointing to EphA2 downregulation as a causal event in the loss of TPCs tumorigenicity. Infusion of ephrinA1-Fc into intracranial xenografts elicited strong tumor-suppressing effects, suggestive of therapeutic applications. Stem-like TPCs in hGBMs highly express the EphA2 receptor High EphA2 expression supports the undifferentiated state and self-renewal in TPCs TPC content and tumorigenicity are higher in EphA2High than EphA2Low hGBM cells EphrinA1-Fc treatment or EPHA2 silencing lessen TPC self-renewal and tumorigenicity Identification and characterization of key regulatory mechanisms in TPCs are crucial for the development of specific therapies for hGBMs. We show that TPCs overexpress EphA2 in hGBMs, which underpins their inherent ability to maintain an undifferentiated state, supporting their self-renewal and tumorigenicity. EphA2 abundance provides a measure of the stem-like potential and tumor-propagating ability of TPCs from hGBMs. Thus, high EphA2 levels can be used to enrich TPCs by cell sorting. EphrinA1-Fc ligand-induced downregulation or siRNA-mediated knockdown of EPHA2 expression both cause loss of self-renewal as well as induce differentiation and loss of tumor-initiating capacity in hGBM TPCs. Sustained intracranial infusion of ephrinA1-Fc under settings that resemble putative therapeutic conditions elicits effective antitumorigenic activity. Human glioblastoma multiforme (hGBM) is the most malignant among gliomas. Because of their very heterogeneous cellular, genetic, epigenetic, and molecular make-up (Maher et al., 2001Maher E.A. Furnari F.B. Bachoo R.M. Rowitch D.H. Louis D.N. Cavenee W.K. DePinho R.A. Malignant glioma: genetics and biology of a grave matter.Genes Dev. 2001; 15: 1311-1333Crossref PubMed Scopus (1044) Google Scholar), hGBMs have a dismal prognosis and almost inevitably recur (Krex et al., 2007Krex D. Klink B. Hartmann C. von Deimling A. Pietsch T. Simon M. Sabel M. Steinbach J.P. Heese O. Reifenberger G. et al.German Glioma NetworkLong-term survival with glioblastoma multiforme.Brain. 2007; 130: 2596-2606Crossref PubMed Scopus (635) Google Scholar; Chen et al., 2012Chen J. Li Y. Yu T.S. McKay R.M. Burns D.K. Kernie S.G. Parada L.F. A restricted cell population propagates glioblastoma growth after chemotherapy.Nature. 2012; 488: 522-526Crossref PubMed Scopus (1575) Google Scholar). Recent findings have demonstrated the existence of a subpopulation of hGBM cells, called cancer stem cells, whose idiosyncratic properties make them resilient to standard therapies. First identified in acute myeloid leukemia (Bonnet and Dick, 1997Bonnet D. Dick J.E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat. Med. 1997; 3: 730-737Crossref PubMed Scopus (5460) Google Scholar), cancer stem cells, better defined as tumor-propagating cells (TPCs; Kelly et al., 2007Kelly P.N. Dakic A. Adams J.M. Nutt S.L. Strasser A. Tumor growth need not be driven by rare cancer stem cells.Science. 2007; 317: 337Crossref PubMed Scopus (689) Google Scholar), have been isolated from a variety of solid tumors (Ponti et al., 2005Ponti D. Costa A. Zaffaroni N. Pratesi G. Petrangolini G. Coradini D. Pilotti S. Pierotti M.A. Daidone M.G. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties.Cancer Res. 2005; 65: 5506-5511Crossref PubMed Scopus (1548) Google Scholar; Ricci-Vitiani et al., 2009Ricci-Vitiani L. Fabrizi E. Palio E. De Maria R. Colon cancer stem cells.J. Mol. Med. 2009; 87: 1097-1104Crossref PubMed Scopus (169) Google Scholar; Buzzeo et al., 2007Buzzeo M.P. Scott E.W. Cogle C.R. The hunt for cancer-initiating cells: a history stemming from leukemia.Leukemia. 2007; 21: 1619-1627Crossref PubMed Scopus (32) Google Scholar). TPCs with both stem cell characteristics and tumor-initiation and propagation ability have now emerged as key players in hGBM pathogenesis (Hadjipanayis and Van Meir, 2009Hadjipanayis C.G. Van Meir E.G. Tumor-initiating cells in malignant gliomas: biology and implications for therapy.J. Mol. Med. 2009; 87: 363-374Crossref PubMed Scopus (73) Google Scholar; Galli et al., 2004Galli R. Binda E. Orfanelli U. Cipelletti B. Gritti A. De Vitis S. Fiocco R. Foroni C. Dimeco F. Vescovi A. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.Cancer Res. 2004; 64: 7011-7021Crossref PubMed Scopus (2061) Google Scholar). While the origin and nature of hGBM TPCs remain to be unraveled, alterations of G1 arrest regulatory pathways in Nestin- or GFAP-positive cells can cause the onset of high-grade gliomas (Alcantara Llaguno et al., 2011Alcantara Llaguno S.R. Chen Y. McKay R.M. Parada L.F. Stem cells in brain tumor development.Curr. Top. Dev. Biol. 2011; 94: 15-44Crossref PubMed Scopus (12) Google Scholar). Thus, hGBM TPCs might derive from the transformation of neural stem cells or their transit amplifying precursor progeny (Alcantara Llaguno et al., 2009Alcantara Llaguno S. Chen J. Kwon C.H. Jackson E.L. Li Y. Burns D.K. Alvarez-Buylla A. Parada L.F. Malignant astrocytomas originate from neural stem/progenitor cells in a somatic tumor suppressor mouse model.Cancer Cell. 2009; 15: 45-56Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar; Vescovi et al., 2006Vescovi A.L. Galli R. Reynolds B.A. Brain tumour stem cells.Nat. Rev. Cancer. 2006; 6: 425-436Crossref PubMed Scopus (822) Google Scholar). Oligodendroglial precursors might also be the cell of origin in some hGBMs (Sukhdeo et al., 2011Sukhdeo K. Hambardzumyan D. Rich J.N. Glioma development: where did it all go wrong?.Cell. 2011; 146: 187-188Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The peculiar characteristics of TPCs encompass a relative quiescent nature, unlimited self-renewal, the clonal capacity to found a tumor (Galli et al., 2004Galli R. Binda E. Orfanelli U. Cipelletti B. Gritti A. De Vitis S. Fiocco R. Foroni C. Dimeco F. Vescovi A. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.Cancer Res. 2004; 64: 7011-7021Crossref PubMed Scopus (2061) Google Scholar), and resistance to conventional and multimodal treatments (Bao et al., 2006Bao S. Wu Q. McLendon R.E. Hao Y. Shi Q. Hjelmeland A.B. Dewhirst M.W. Bigner D.D. Rich J.N. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.Nature. 2006; 444: 756-760Crossref PubMed Scopus (4858) Google Scholar). Owing to their stem-like nature, TPCs might be regulated by the same cues that control the activity of normal neural precursors and stem cells (NSCs). In fact, pathways that impinge on self-renewal and cell fate in normal NSCs are also active in brain tumors (Alcantara Llaguno et al., 2011Alcantara Llaguno S.R. Chen Y. McKay R.M. Parada L.F. Stem cells in brain tumor development.Curr. Top. Dev. Biol. 2011; 94: 15-44Crossref PubMed Scopus (12) Google Scholar; Dell’Albani, 2008Dell’Albani P. Stem cell markers in gliomas.Neurochem. Res. 2008; 33: 2407-2415Crossref PubMed Scopus (92) Google Scholar). Also, therapeutic agents targeting Wnt, Hedgehog, or Notch deplete the TPC population in hGBMs (Takebe et al., 2011Takebe N. Harris P.J. Warren R.Q. Ivy S.P. Targeting cancer stem cells by inhibiting Wnt, Notch, and Hedgehog pathways.Nat. Rev. Clin. Oncol. 2011; 8: 97-106Crossref PubMed Scopus (800) Google Scholar), and tumor suppressor genes can regulate TPC self-renewal (Zheng et al., 2008Zheng H. Ying H. Yan H. Kimmelman A.C. Hiller D.J. Chen A.J. Perry S.R. Tonon G. Chu G.C. Ding Z. et al.p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation.Nature. 2008; 455: 1129-1133Crossref PubMed Scopus (572) Google Scholar). The connection of TPCs with NSCs is reinforced by the discovery that critical effectors of NSC activity in the brain stem cell niche, such as bone morphogenetic proteins, suppress the growth of hGBM TPCs, enforcing their differentiation into astroglia (Lee et al., 2008Lee J. Son M.J. Woolard K. Donin N.M. Li A. Cheng C.H. Kotliarova S. Kotliarov Y. Walling J. Ahn S. et al.Epigenetic-mediated dysfunction of the bone morphogenetic protein pathway inhibits differentiation of glioblastoma-initiating cells.Cancer Cell. 2008; 13: 69-80Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar; Piccirillo et al., 2006Piccirillo S.G. Reynolds B.A. Zanetti N. Lamorte G. Binda E. Broggi G. Brem H. Olivi A. Dimeco F. Vescovi A.L. Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells.Nature. 2006; 444: 761-765Crossref PubMed Scopus (979) Google Scholar; Zhang et al., 2006Zhang Q.B. Ji X.Y. Huang Q. Dong J. Zhu Y.D. Lan Q. Differentiation profile of brain tumor stem cells: a comparative study with neural stem cells.Cell Res. 2006; 16: 909-915Crossref PubMed Scopus (77) Google Scholar). Another key regulator in the adult NSC niche, nitric oxide, can also drive TPC proliferation in hGBMs (Eyler et al., 2011Eyler C.E. Wu Q. Yan K. MacSwords J.M. Chandler-Militello D. Misuraca K.L. Lathia J.D. Forrester M.T. Lee J. Stamler J.S. et al.Glioma stem cell proliferation and tumor growth are promoted by nitric oxide synthase-2.Cell. 2011; 146: 53-66Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Eph receptor tyrosine kinases and their ephrin ligands influence central nervous system development, stem cell niches, and cancer cells (Goldshmit et al., 2006Goldshmit Y. McLenachan S. Turnley A. Roles of Eph receptors and ephrins in the normal and damaged adult CNS.Brain Res. Brain Res. Rev. 2006; 52: 327-345Crossref PubMed Scopus (138) Google Scholar; Genander and Frisén, 2010Genander M. Frisén J. Ephrins and Eph receptors in stem cells and cancer.Curr. Opin. Cell Biol. 2010; 22: 611-616Crossref PubMed Scopus (117) Google Scholar; Pasquale, 2010Pasquale E.B. Eph receptors and ephrins in cancer: bidirectional signalling and beyond.Nat. Rev. Cancer. 2010; 10: 165-180Crossref PubMed Scopus (905) Google Scholar). Deregulation of the Eph receptor/ephrin system is associated with acquisition of tumorigenic properties, tumor growth, angiogenesis, and metastasis in human cancers. In particular, EphA2 receptor is overexpressed in many human epithelial malignancies and hGBMs, where it can promote proliferation and invasiveness through mechanisms that are not well understood and may be independent of ephrin ligand binding (Wykosky and Debinski, 2008Wykosky J. Debinski W. The EphA2 receptor and ephrinA1 ligand in solid tumors: function and therapeutic targeting.Mol. Cancer Res. 2008; 6: 1795-1806Crossref PubMed Scopus (228) Google Scholar; Miao et al., 2009Miao H. Li D.Q. Mukherjee A. Guo H. Petty A. Cutter J. Basilion J.P. Sedor J. Wu J. Danielpour D. et al.EphA2 mediates ligand-dependent inhibition and ligand-independent promotion of cell migration and invasion via a reciprocal regulatory loop with Akt.Cancer Cell. 2009; 16: 9-20Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar; Pasquale, 2010Pasquale E.B. Eph receptors and ephrins in cancer: bidirectional signalling and beyond.Nat. Rev. Cancer. 2010; 10: 165-180Crossref PubMed Scopus (905) Google Scholar; Gopal et al., 2011Gopal U. Bohonowych J.E. Lema-Tome C. Liu A. Garrett-Mayer E. Wang B. Isaacs J.S. A novel extracellular Hsp90 mediated co-receptor function for LRP1 regulates EphA2 dependent glioblastoma cell invasion.PLoS ONE. 2011; 6: e17649Crossref PubMed Scopus (102) Google Scholar; Nakada et al., 2011Nakada M. Hayashi Y. Hamada J. Role of Eph/ephrin tyrosine kinase in malignant glioma.Neuro-oncol. 2011; 13: 1163-1170Crossref PubMed Scopus (50) Google Scholar; Miao and Wang, 2012Miao H. Wang B. EphA receptor signaling—complexity and emerging themes.Semin. Cell Dev. Biol. 2012; 23: 16-25Crossref PubMed Scopus (70) Google Scholar). High EphA2 expression also correlates with tumor stage, progression, and patient survival (Wykosky et al., 2005Wykosky J. Gibo D.M. Stanton C. Debinski W. EphA2 as a novel molecular marker and target in glioblastoma multiforme.Mol. Cancer Res. 2005; 3: 541-551Crossref PubMed Scopus (209) Google Scholar; Liu et al., 2006Liu F. Park P.J. Lai W. Maher E. Chakravarti A. Durso L. Jiang X. Yu Y. Brosius A. Thomas M. et al.A genome-wide screen reveals functional gene clusters in the cancer genome and identifies EphA2 as a mitogen in glioblastoma.Cancer Res. 2006; 66: 10815-10823Crossref PubMed Scopus (93) Google Scholar; Wang et al., 2008Wang L.F. Fokas E. Bieker M. Rose F. Rexin P. Zhu Y. Pagenstecher A. Engenhart-Cabillic R. An H.X. Increased expression of EphA2 correlates with adverse outcome in primary and recurrent glioblastoma multiforme patients.Oncol. Rep. 2008; 19: 151-156PubMed Google Scholar; Miao et al., 2009Miao H. Li D.Q. Mukherjee A. Guo H. Petty A. Cutter J. Basilion J.P. Sedor J. Wu J. Danielpour D. et al.EphA2 mediates ligand-dependent inhibition and ligand-independent promotion of cell migration and invasion via a reciprocal regulatory loop with Akt.Cancer Cell. 2009; 16: 9-20Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar; Li et al., 2010Li X. Wang L. Gu J.W. Li B. Liu W.P. Wang Y.G. Zhang X. Zhen H.N. Fei Z. Up-regulation of EphA2 and down-regulation of EphrinA1 are associated with the aggressive phenotype and poor prognosis of malignant glioma.Tumour Biol. 2010; 31: 477-488Crossref PubMed Scopus (37) Google Scholar; Wu et al., 2011Wu N. Zhao X. Liu M. Liu H. Yao W. Zhang Y. Cao S. Lin X. Role of microRNA-26b in glioma development and its mediated regulation on EphA2.PLoS ONE. 2011; 6: e16264Crossref PubMed Scopus (113) Google Scholar). In this study, we investigated the putative regulatory role and function of the EphA2 receptor in TPCs from hGBMs. Analysis of hGBM surgery specimens showed high EphA2 mRNA expression as compared to other EphA and EphB receptors (Figure S1A available online). Real-time PCR (qPCR) revealed how EphA2 mRNA levels were up to 100-fold higher in hGBMs than in normal human brain tissue, as compared to a 10-fold upregulation in low-grade gliomas, epitheliomas, and primitive neuroectodermal tumors (Figure S1B). Strong EphA2 immunoreactivity was found in many cells of the non-necrotic hGBM core (Figure 1A) versus a few positive cells in the tumor periphery (Figure 1B) and normal brain (Figure S1C). Accordingly, immunolabeling of hGBM tissues showed coexpression of EphA2 and antigens of normal and transformed neural precursors, namely Nestin, Sox2, and Olig2 (Fatoo et al., 2011Fatoo A. Nanaszko M.J. Allen B.B. Mok C.L. Bukanova E.N. Beyene R. Moliterno J.A. Boockvar J.A. Understanding the role of tumor stem cells in glioblastoma multiforme: a review article.J. Neurooncol. 2011; 103: 397-408Crossref PubMed Scopus (17) Google Scholar; Ligon et al., 2007Ligon K.L. Huillard E. Mehta S. Kesari S. Liu H. Alberta J.A. Bachoo R.M. Kane M. Louis D.N. Depinho R.A. et al.Olig2-regulated lineage-restricted pathway controls replication competence in neural stem cells and malignant glioma.Neuron. 2007; 53: 503-517Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar) (Figure 1C). In contrast, the signal for ephrinA1, an EphA2 preferred ligand (Wykosky et al., 2008Wykosky J. Palma E. Gibo D.M. Ringler S. Turner C.P. Debinski W. Soluble monomeric EphrinA1 is released from tumor cells and is a functional ligand for the EphA2 receptor.Oncogene. 2008; 27: 7260-7273Crossref PubMed Scopus (94) Google Scholar; Miao et al., 2009Miao H. Li D.Q. Mukherjee A. Guo H. Petty A. Cutter J. Basilion J.P. Sedor J. Wu J. Danielpour D. et al.EphA2 mediates ligand-dependent inhibition and ligand-independent promotion of cell migration and invasion via a reciprocal regulatory loop with Akt.Cancer Cell. 2009; 16: 9-20Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar), was variable in intensity and distribution in the hGBM core and undetectable in the periphery (Figure S1C). We also found high EphA2 mRNA and protein levels in cells acutely dissociated from hGBMs or cultured as neurospheres and enriched for the putative TPC markers SSEA-1 or CD44 (Figures S1D–S1F). Analysis of hGBM TPCs confirmed this overexpression (Figure 1E), which was from 2- to 300-fold that of their original hGBM tissue (Figure S1G). EphA2 and ephrinA1 were detected in both hGBMs and their TPCs (Figure 1D). Notably, EphA2 was heavily downregulated when TPCs were differentiated, losing stemness and tumorigenicity (Piccirillo et al., 2006Piccirillo S.G. Reynolds B.A. Zanetti N. Lamorte G. Binda E. Broggi G. Brem H. Olivi A. Dimeco F. Vescovi A.L. Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells.Nature. 2006; 444: 761-765Crossref PubMed Scopus (979) Google Scholar; Zhang et al., 2006Zhang Q.B. Ji X.Y. Huang Q. Dong J. Zhu Y.D. Lan Q. Differentiation profile of brain tumor stem cells: a comparative study with neural stem cells.Cell Res. 2006; 16: 909-915Crossref PubMed Scopus (77) Google Scholar) (Figure 1F), reinforcing a correlation between high EphA2 levels and the TPC state. EphA2 mRNA levels in individual TPC neurosphere cultures also correlated with their specific growth kinetics (Figures S1H and S1I), i.e., with their self-renewal activity (Rietze and Reynolds, 2006Rietze R.L. Reynolds B.A. Neural stem cell isolation and characterization.Methods Enzymol. 2006; 419: 3-23Crossref PubMed Scopus (188) Google Scholar), and with EPHA2 gene copy number at the 1p36.12 locus (p < 0.0001) (Figures S1H and S1J). To reinforce the correlation between EphA2 enhanced levels and the TPC state we FACS-sorted acutely isolated hGBM cells into two distinct pools, expressing either high (EphA2High) or low (EphA2Low) EphA2 levels, and then assessed their in vitro clonogenicity and intracranial tumorigenic capacity. As expected, the EphA2High fraction contained more clonogenic cells than the EphA2Low fraction (Figure 2A) and mice implanted with EphA2High cells showed a higher mortality (median survival 4 months) than those receiving EphA2Low cells (median survival 7 months) (Figure 2B). Limiting dilution intracranial transplantation using acutely dissociated EphA2High and EphA2Low cells (Figures S2A–S2C) confirmed that high EphA2 levels are a hallmark of TPCs and can be used for their enrichment. In addition, when primary hGBM cells were sorted based on their combined expression of EphA2 and the putative TPC antigen SSEA-1 (Figures 2C and 2D), intracranial tumorigenicity was significantly higher in EphA2High SSEA-1High than in EphA2Low SSEA-1Low cells (Figure 2E). Similar results were obtained with EphA2High CD44High and EphA2Low CD44Low cells (Figures S2D–S2F). By applying the extreme limiting dilution analysis approach, we were able to show that the frequency of tumor-initiating cells (TICs) was always significantly higher in cell fractions with an increased EphA2 expression, alone or in combination with SSEA-1 or CD44 (Figure S2G). Finally, when EphA2High and EphA2Low cultured TPCs were assayed for intracranial tumorigenicity in a limiting dilution assay, as few as 100 EphA2High cells established large gliomas in 60 days, as compared to a minimum requirement of 5,000 cells when using EphA2Low, below which level tumorigenicity was negligible, even after 7 months (Figures 2F and 2G). Due to the emerging correlation between high EphA2 expression and TPC state, we used ephrinA1-Fc (a soluble ephrinA1 dimer fused to Fc) to downregulate EphA2 (Wykosky et al., 2005Wykosky J. Gibo D.M. Stanton C. Debinski W. EphA2 as a novel molecular marker and target in glioblastoma multiforme.Mol. Cancer Res. 2005; 3: 541-551Crossref PubMed Scopus (209) Google Scholar; Liu et al., 2007Liu D.P. Wang Y. Koeffler H.P. Xie D. Ephrin-A1 is a negative regulator in glioma through down-regulation of EphA2 and FAK.Int. J. Oncol. 2007; 30: 865-871PubMed Google Scholar) and examined its effects on hGBM TPCs. A dose-dependent EphA2 downregulation ensued, which peaked at ephrinA1-Fc concentrations of 1–5 μg/ml, producing up to 90% receptor depletion (Figure S3A). EphrinA1-Fc induced a negligible EphA2 downregulation both in cells derived from the hGBM periphery (non-TPCs)—bearing stem-like features but negligible tumorigenicity (Piccirillo et al., 2009Piccirillo S.G. Combi R. Cajola L. Patrizi A. Redaelli S. Bentivegna A. Baronchelli S. Maira G. Pollo B. Mangiola A. et al.Distinct pools of cancer stem-like cells coexist within human glioblastomas and display different tumorigenicity and independent genomic evolution.Oncogene. 2009; 28: 1807-1811Crossref PubMed Scopus (156) Google Scholar)—and v-myc-immortalized, non-transformed hNSCs (vhNSCs; De Filippis et al., 2007De Filippis L. Lamorte G. Snyder E.Y. Malgaroli A. Vescovi A.L. A novel, immortal, and multipotent human neural stem cell line generating functional neurons and oligodendrocytes.Stem Cells. 2007; 25: 2312-2321Crossref PubMed Scopus (67) Google Scholar) (Figure 3A). Immunofluorescence assays confirmed EphA2 loss in both acutely isolated and cultured TPCs upon ephrinA1-Fc treatment (Figure 3B). TPCs acutely isolated from hGBM specimens adhered to the dish when treated with ephrinA1-Fc, losing their capacity to grow and to generate stable TPC lines in the neurosphere assay (Figures 3C, 3D, and 3G), an effect also seen in 14 established TPC cultures (Figures 3E–3G, S3B, and S3C). In contrast, ephrinA1-Fc had negligible effects on non-TPCs (Figure 3H) and vhNSCs (Figure 3I). Hence, ephrinA1-Fc dowregulates EphA2 and hinders the expansive growth of cells that possess both stem cell characteristics and tumor-propagating ability. The effects of ephrinA1-Fc suggested that it might inhibit the expansion of the TPC pool. This was emphasized by the ability of ephrinA1-Fc to downregulate both EphA2 and the neural precursor markers Nestin, Sox2, or Olig2 (Figure 3J) that are coexpressed in TPC spheroids. Also, FACS analysis showed loss of the putative TPC markers SSEA-1, CD44 and CD133 (Dell’Albani, 2008Dell’Albani P. Stem cell markers in gliomas.Neurochem. Res. 2008; 33: 2407-2415Crossref PubMed Scopus (92) Google Scholar; Fatoo et al., 2011Fatoo A. Nanaszko M.J. Allen B.B. Mok C.L. Bukanova E.N. Beyene R. Moliterno J.A. Boockvar J.A. Understanding the role of tumor stem cells in glioblastoma multiforme: a review article.J. Neurooncol. 2011; 103: 397-408Crossref PubMed Scopus (17) Google Scholar) (Figure S3D), but not of BMI-1 (Fatoo et al., 2011Fatoo A. Nanaszko M.J. Allen B.B. Mok C.L. Bukanova E.N. Beyene R. Moliterno J.A. Boockvar J.A. Understanding the role of tumor stem cells in glioblastoma multiforme: a review article.J. Neurooncol. 2011; 103: 397-408Crossref PubMed Scopus (17) Google Scholar). Finally, ephrinA1-Fc depleted the TPC side population (SP), thought to comprise stem-like cells (Fukaya et al., 2010Fukaya R. Ohta S. Yamaguchi M. Fujii H. Kawakami Y. Kawase T. Toda M. Isolation of cancer stem-like cells from a side population of a human glioblastoma cell line, SK-MG-1.Cancer Lett. 2010; 291: 150-157Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) (Figure S3E). To confirm depletion of the TPC stem-like pool by ephrinA1-Fc, we measured self-renewal ability in a clonogenic assay, whereby single cells from acutely dissociated or from established TPC lines were plated in single wells by automated FACS and grown as neurospheres. EphrinA1-Fc drastically decreased clonogenicity in TPCs but not in non-TPCs, slightly increasing vhNSC clonogenicity (Figure 4A). Loss of self-renewal was not caused by changes in cell cycling (Figure 4B) or viability (Figure 4C). Notably, ephrinA1-Fc induced a marked, time-dependent upregulation of the astroglial antigen GFAP, without affecting the neuronal βIII tubulin and oligodendroglial GalC markers (Figure 4D). Thus, ephrinA1-Fc hinders self-renewal in TPCs in a non-cytotoxic way and increases astroglial differentiation. We studied the molecular events underlying the changes induced in hGBM TPCs by ephrinA1-Fc. EphrinA1-Fc transiently increased EphA2 tyrosine phosphorylation, in a dose-dependent manner, causing a strong and persistent downregulation of EphA2 expression (Figure 4E). We also observed marked ERK phosphorylation, which returned to basal levels after 24 hr, a moderate increases in Akt and FAK phosphorylation (Figure 4E, bottom panels), and reorganization of the actin cytoskeleton (Figures 4F–4J). Altogether, this suggested that EphA2 expression sustains TPC self-renewal and that receptor loss played a causal role in the depletion of the hGBM stem-like pool. To prove this, we used a mixture of siRNAs to assess the effects of direct EphA2 downregulation (Figure 5A). EPHA2 silencing caused changes consistent with loss of stemness and increased differentiation, ie, (1) loss of clonogenicity (Figure 5B) and amplification rate (Figure 5C), (2) downregulation of putative stem cell markers (Figure 5E), and (3) increased GFAP expression (Figure 5F). These changes were quite similar to those induced by ephrinA1-Fc (Figures 3G, 3J, 4A, and 4D). Per se, EphA2 downregulation activated ERK in TPCs, as shown by a prominent increase in its phosphorylation (Figures 5F and 5G). Feeble Akt and FAK activation were detected. Furthermore, when EphA2 levels began to normalize, due to the elapsing effect of siRNAs transfection (Figure 5A, arrow), TPCs growth and ERK phosphorylation also began to normalize (Figures 5C, arrow, and 5F). siRNA-mediated knockdown of EPHA2 expression in vhNSCs also inhibited growth (Figure 5D), in contrast to ephrinA1-Fc treatment, which neither inhibited growth nor downregulated EphA2 in these cells (Figures 3A, 3B, and 3I). Transduction of TPCs with individual EPHA2 siRNA, and rescue with a siRNA-resistant EPHA2 construct, confirmed these effects as the result of the EphA2 mRNA knockdown (Figure 5G). Importantly, inhibition of ERK activation partially rescued loss in clonogenicity as induced by EPHA2 siRNA, implicating ERK activation in the loss of TPC stemness, as caused by EphA2 downregulation (Figure 5H). Notably, EphA2 phosphorylation on Serine897 (Ser897) promotes oncogenic activities of the receptor that do not depend on its interaction with ephrin ligands (Miao et al., 2009Miao H. Li D.Q. Mukherjee A. Guo H. Petty A. Cutter J. Basilion J.P. Sedor J. Wu J. Danielpour D. et al.EphA2 mediates ligand-dependent inhibition and ligand-independent promotion of cell migration and invasion via a reciprocal regulatory loop with Akt.Cancer Cell. 2009; 16: 9-20Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). We detected a strong signal for Ser897-phosphorylated EphA2 in the hGBM core and in TPCs but not in the hGBM periphery, non-TPCs or vhNSCs (Figure 6A). Furthermore, treatment with high doses of soluble monomeric EphA2 extracellular domain that block endogenous EphA2-ephrin interaction did not affect GFAP levels or ERK activity in TPCs (Figure 6B). Thus, the effects of EphA2 in TPCs appear to be independent from activation by endogenous ephrinA ligands. The main effect of ephrinA1-Fc treatment or siRNA-mediated EPHA2 silencing is the depletion of the tumorigenic hGBM TPC pool, which ought to reduce their tumorigenic capacity in vivo. To examine the in vivo effects of ephrinA1-Fc, we used three experimental paradigms. hGBM TPCs were (1) treated with ephrinA1-Fc in culture prior to transplantation (pre-treatment), (2) treated starting immediately after transplantation (cotreatment), or (3) allowed to establish sizeable tumors before beginning treatment with ephrinA1-Fc (post-treatment). All three protocols were evaluated in a subcutaneous xenograft model. Furthermore, pre- and post-treatment protocols were also evaluated in an intracranial (orthotopic) xenograft model (Galli et al., 2004Galli R. Binda E. Orfanelli U. Cipelletti B. Gritti A. De Vitis S. Fiocco R. Foroni C. Dimeco F. Vescovi A. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.Cancer Res. 2004; 64: 7011-7021Crossref PubMed Scopus (2061) Google Scholar; Piccirillo et al., 2006Piccirillo S.G. Reynolds B.A. Zanetti N. Lamorte G. Binda E. Broggi G. Brem H. Olivi A. Dimeco F. Vescovi A.L. Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells.Nature. 2006; 444: 761-765Crossref PubMed Scopus (979) Google Scholar). When TPCs were injected subcutaneously, ephrinA1-Fc inhibited growth in all three protocols (Figure 7A). Similar results were obtained wi" @default.
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