Tumor and Stem Cell Biology MIF Maintains the Tumorigenic Capacity of Brain Tumor–Initiating Cells by Directly Inhibiting p53 Raita Fukaya 1 , Shigeki Ohta 2 , Tomonori Yaguchi 3 , Yumi Matsuzaki 2 , Eiji Sugihara 4 , Hideyuki Okano 2 , Hideyuki Saya 4 ,Yutaka Kawakami 3 ,Takeshi Kawase 1 , Kazunari Yoshida 1 , and Masahiro Toda 1 Abstract Tumor-initiating cells thought to drive brain cancer are embed- ded in a complex heterogeneous histology. In this study, we isolated primary cells from 21 human brain tumor specimens to establish cell lines with high tumorigenic potential and to identify the molecules enabling this capability. The morphology, sphere- forming ability upon expansion, and differentiation potential of all cell lines were indistinguishable in vitro. However, testing for tumorigenicity revealed two distinct cell types, brain tumor– initiating cells (BTIC) and non-BTIC. We found that macrophage migration inhibitory factor (MIF) was highly expressed in BTIC compared with non-BTIC. MIF bound directly to both wild-type and mutant p53 but regulated p53-dependent cell growth by different mechanisms, depending on glioma cell line and p53 status. MIF physically interacted with wild-type p53 in the nucleus and inhibited its transcription-dependent functions. In contrast, MIF bound to mutant p53 in the cytoplasm and abrogated transcription-independent induction of apoptosis. Furthermore, MIF knockdown inhibited BTIC-induced tumor formation in a mouse xenograft model, leading to increased overall survival. Collectively, our findings suggest that MIF regulates BTIC function through direct, intracellular inhibition of p53, shedding light on the molecular mechanisms underlying the tumorigenicity of certain malignant brain cells. Cancer Res; 76(9); 2813–23. Ó2016 AACR. Introduction Glioblastoma, the most common form of primary malignant brain tumor, is nearly always fatal (1). Although neurosurgeons may be able to remove the main tumor mass, this procedure typically leaves behind a small population of tumor cells that retain the capacity for tumorigenesis (2). For this reason, glio- blastomas almost always relapse, highlighting the need for more precisely targeted therapeutic approaches. A number of recent studies have demonstrated that tumors are composed of cells with functional heterogeneity that form a hierarchy, especially in the context of tumor initiation (3, 4). Characterized by its heteroge- neous tumor types (5–8), glioblastoma is now also thought to be initiated and maintained by a subpopulation of tumor cells, called brain tumor stem cells or brain tumor–initiating cells (BTIC; refs. 9–13). The clinical significance of BTICs is empha- sized by the strongly enhanced capacity of these cells to initiate brain tumors, in contrast to the majority of tumor cells, which may contribute significantly to the tumor mass, but not to the initi- ation and maintenance of the tumor. BTICs are thus considered a promising target for future therapies. Macrophage migration inhibitory factor (MIF) was originally identified as a proinflammatory cytokine, but was later found to be a critical regulator of tumor progression as well (14–18). MIF upregulation has been observed in human melanoma (19), lung cancer (20), prostate cancer (21), hepatocellular carcinoma (22), and glioblastoma (23–27). MIF expression was significantly higher in the tumor tissues of glioblastoma patients than in normal brain tissues (24, 25). The precise roles of MIF in the initiation and progression of glioma, however, remain unclear. MIF plays numerous roles through receptor-mediated signaling pathways that are activated by cell surface receptors, such as CD74, CD44, and CXCRs (28–30), or by intracellular interactions through binding to JAB1 (31). MIF has also been shown to function as a p53 inhibitor (16) that directly binds and antag- onizes p53 function in the cells (32). The p53 protein is an important and well-characterized tumor suppressor (33). In response to various forms of cell stress, p53 regulates diverse target molecules, which may induce cell-cycle arrest and apoptosis in a transcription-dependent manner (34). p53 has also been shown to possess other biologic activities, including induction of apoptosis, in mitochondria in a transcrip- tion-independent manner (35–37). In this study, we show that MIF expression is significantly higher in BTICs isolated from human glioma tissues than in non-BTICs from the same tissue source and that MIF acts as a direct intracellular p53 inactivator that regulates cell proliferation and apoptosis in glioma cells. In addition, we highlight the link between the roles of MIF and tumor-initiating cells associated with p53, a candidate for the dedifferentiation barrier. 1 Department of Neurosurgery, Keio University School of Medicine, Tokyo, Japan. 2 Department of Physiology, Keio University School of Medicine, Tokyo, Japan. 3 Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan. 4 Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Masahiro Toda, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 1608582, Japan. Phone: 813-5363-3807; Fax: 813-3358-0479; E-mail: todam@z2.keio.jp doi: 10.1158/0008-5472.CAN-15-1011 Ó2016 American Association for Cancer Research. Cancer Research www.aacrjournals.org 2813 on January 13, 2018. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst March 15, 2016; DOI: 10.1158/0008-5472.CAN-15-1011