Eva 1 Maintains the Stem-like Character of Glioblastoma-Initiating Cells by Activating the Noncanonical NFk B Signaling Pathway

Glioblastoma (GBM)–initiating cells (GIC) are a tumorigenic subpopulation that are resistant to radioand chemotherapies and are the source of disease recurrence. Therefore, the identification and characterization of GIC-specific factors is critical toward the generation of effectiveGBM therapeutics. In this study, we investigated the role of epithelial V-like antigen 1 (Eva1, also known as myelin protein zero-like 2) in stemness and GBM tumorigenesis. Eva1 was prominently expressed in GICs in vitro and in stem cell marker (Sox2, CD15, CD49f)-expressing cells derived from human GBM tissues. Eva1 knockdown in GICs reduced their self-renewal and tumor-forming capabilities, whereas Eva1 overexpression enhanced these properties. Eva1 deficiency was also associated with decreased expression of stemnessrelated genes, indicating a requirement for Eva1 in maintaining GIC pluripotency.We further demonstrate that Eva1 inducedGIC proliferation through the activation of the RelB-dependent noncanonical NF-kB pathway by recruiting TRAF2 to the cytoplasmic tail. Taken together, our findings highlight Eva1 as a novel regulator of GIC function and also provide new mechanistic insight into the role of noncanonical NF-kB activation in GIC, thus offering multiple potential therapeutic targets for preclinical investigation in GBM. Cancer Res; 76(1); 171–81. 2015 AACR. Introduction Gliomas are brain tumors possessing the characteristics of glial cells, astrocytes, and oligodendrocytes, and have been classified into four grades (WHO grade I–IV) based on their pathologic features. Glioblastoma (GBM) is the most malignant glioma (WHO grade IV), and patients with GBM have a median survival of approximately one year. Despite tremendous efforts to effectively treat GBM, the overall survival rates of patients with GBM have remained unchanged over the past few decades. The discovery of GBM-initiating cells (GIC) has had a significant impact onGBM research (1). GICs have a strong self-renewal capability, express stem cell markers, such as CD133 (also known as Prominin1), Sox2, CD15 (also known as Stage-Specific Embryonic Antigen 1 and Lewis X), andCD49f (also known as integrina 6), and are more resistant to radiotherapy and chemotherapy higher than non-GICs (2–5). GICs have also been shown to exploit the signaling pathways that are involved in the maintenance of neural stem cells (NSC; refs. 2–5). NSCs exist only in the subventricular zone and hippocampus (6, 7), both of which contain a special microenvironment (niche) for the maintenance of NSCs, whereas GBM arises in many areas in the brain. It currently remains unknown how GICs maintain their stemness in the brain; whether GICs generate their preferable niche anywhere or use an unknown mechanism for their maintenance in non-NSC niches. We previously established the mouse GIC lines (mGIC), NSCL61 and OPCL61, by overexpressing an oncogenic HRas in p53-deficient NSCs and oligodendrocyte precursor cells (OPC), respectively (8, 9). ThesemGICs formed transplantableGBMwith hypercellularity, pleomorphism,multinuclear giant cells,mitosis, and necrosis, even when as few as ten cells were injected into the brains of nude mice. These findings indicated that they were highly enriched in bona fide GICs. Using DNA microarray analysis, we compared the gene expression profiles of mGICs with thoseof their parental cells and identified genes that increased and decreased in mGICs. By evaluating the candidate genes using human GICs (hGICs) and GBM tissues, we have successfully selected potential GIC-specific genes (8–11). Among the candidate genes evaluated, we focused on epithelial V-like antigen 1 (Eva1, also known asmyelin protein zero-like 2). Eva1was originally identified as an immunoglobulin superfamily member expressed on the developing thymus epithelial cell membrane and disappeared in the developed one (12–14). Eva1 was also shown to be involved in the T-cell development through the Eva1–Eva1 homophilic interaction between CD4/CD8 double-positive cells and thymus epithelial cells in the early embryo (12–14); however, there was no detectable phenotype, including the hematopoietic development, in the Eva1 knockout mice (12–15; Ohtsu and colleagues, unpublished observation). Notably, it has not been shown that Eva1 was involved in either tumorigenesis or stemness. These findings led us to investigate DivisionofStemCellBiology, Institute forGeneticMedicine,Hokkaido University, Sapporo, Hokkaido, Japan. Laboratory for Cell Lineage Modulation, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan. Department of Neurosurgery, Ehime University Graduate School of Medicine, To-on, Ehime, Japan. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Current address for Y. Nakatani: Division of Bio-Function Dynamics Imaging, Center for Life Science Technology, RIKEN, Kobe, Hyogo, Japan. Corresponding Author: Toru Kondo, Division of Stem Cell Biology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 0600815, Japan. Phone: 81-11-706-6082; Fax: 81-11-706-7870; E-mail: tkondo@igm.hokudai. ac.jp doi: 10.1158/0008-5472.CAN-15-0884 2015 American Association for Cancer Research. Cancer Research www.aacrjournals. org 171 on May 30, 2017. © 2016 American Association for Cancer Research. cancerres.aacrjournals. org Downloaded from Published OnlineFirst December 17, 2015; DOI: 10.1158/0008-5472.CAN-15-0884