Original articles Tumoral RANKL activates astrocytes that promote glioma cell invasion through cytokine signaling Jun-Kyum Kim a,b,1 , Xiong Jin a,b,1 , Young-Woo Sohn a,1 , Xun Jin b , Hee-Young Jeon a,b , Eun-Jung Kim a,b , Seok Won Ham a , Hye-Min Jeon a,b , So-Young Chang a , Se-Yeong Oh b , Jinlong Yin c , Sung-Hak Kim a,d , Jong Bae Park c , Ichiro Nakano d,e , Hyunggee Kim a, * a School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea b Institute of Life Science and Natural Resources, Korea University, Seoul 136-713, Republic of Korea c Specific Organs Cancer Branch, Research Institute and Hospital, National Cancer Center, Goyang 410-769, Republic of Korea d Department of Neurological Surgery, The Ohio State University, Columbus, OH 43210, USA e James Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA ARTICLE INFO Article history: Received 12 May 2014 Accepted 14 July 2014 Keywords: Astrocyte Glioblastoma RANKL signaling Tumor-invasive microenvironment A B ST R AC T The invasiveness of glioblastoma is a major cause of poor prognosis and relapse. However, the molecu- lar mechanism controlling glioma cell invasion is poorly understood. Here, we report that receptor activator of nuclear factor kappa-B (NFκB) ligand (RANKL) promotes glioma cell invasion in vivo, but not in vitro. Unlike the invasiveness under in vitro culture conditions, in vivo xenograft studies revealed that LN229 cells expressing high endogenous RANKL generated more invasive tumors than U87MG cells expressing relatively low endogenous RANKL. Consistently, RANKL-overexpressing U87MG resulted in invasive tumors, whereas RANKL-depleted LN229 generated rarely invasive tumors. We found that the number of acti- vated astrocytes was markedly increased in the periphery of RANKL-high invasive tumors. RANKL activated astrocytes through NFκB signaling and these astrocytes in turn secreted various factors which regulate glioma cell invasion. Among them, transforming growth factor β (TGF-β) signaling was markedly in- creased in glioblastoma specimens and xenograft tumors expressing high levels of RANKL. These results indicate that RANKL contributes to glioma invasion by modulating the peripheral microenvironment of the tumor, and that targeting RANKL signaling has important implications for the prevention of highly invasive glioblastoma. © 2014 Elsevier Ireland Ltd. All rights reserved. Introduction Despite improvements in the diagnosis and treatment, glioblas- toma multiforme (GBM) remains the most common and incurable type of central nervous system malignancy [1]. The invasiveness of glioma cells is the main cause of poor prognosis and resistance to current therapeutic intervention [2]. Our previous study revealed that receptor activator of nuclear factor kappa-B ligand (RANKL) ex- pression is significantly increased in invasive glioma cell lines [3]. RANKL is a member of the tumor necrosis factor family and acts as a ligand for RANK. RANKL–RANK signaling is a key regulator of osteoclastogenesis, bone resorption and the development of the immune system [4–6]. In the brain, RANKL is specifically ex- pressed in neurons and astrocytes and it plays a crucial role in the thermoregulation and fever response during inflammation [7]. Several studies have demonstrated that stromal cell-derived RANKL stimulates bone metastasis of breast cancer and prostate cancer by bone resorption and release of cytokines and growth factors [8]. However, the functional role of tumor-derived RANKL in cancer cell invasion, especially in brain tumors, is poorly understood. Here, we demonstrate that tumoral RANKL activates normal astrocytes in the periphery of tumors through canonical NF-κB signaling and that reactive astrocytes stimulate glioma cell invasion by releas- ing various cytokines involved in cell invasion. Materials and methods Cells and culture conditions The human glioma cell lines (LN229 and U87MG) were purchased from the Amer- ican Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM) enriched with 10% fetal bovine serum (Hyclone), 1% penicillin and strep- tomycin (GIBCO-BRL), and 2 mM l-glutamine (GIBCO-BRL) within less than 6 months after receipt. These cell lines were authenticated by ATCC with the use of various tests, including DNA profiling and cytogenetic analyses. Human astrocytes (HAs) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured * Corresponding author. Address: Department of Biotechnology, School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea. Fax: +82 2 953 0737. E-mail address: hg-kim@korea.ac.kr (H. Kim). 1 These authors contributed equally to the work. http://dx.doi.org/10.1016/j.canlet.2014.07.034 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved. Cancer Letters ■■ (2014) ■■–■■ ARTICLE IN PRESS Please cite this article in press as: Jun-Kyum Kim, et al., Tumoral RANKL activates astrocytes that promote glioma cell invasion through cytokine signaling, Cancer Letters (2014), doi: 10.1016/j.canlet.2014.07.034 Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet Q2 Q3 Q1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84