Sphingosine-1-phosphate receptor type 1 regulates glioma cell proliferation and correlates with patient survival Yuya Yoshida 1 , Mitsutoshi Nakada 1 , Naotoshi Sugimoto 2 , Tomoya Harada 1 , Yasuhiko Hayashi 1 , Daisuke Kita 1 , Naoyuki Uchiyama 1 , Yutaka Hayashi 1 , Akihiro Yachie 3 , Yoh Takuwa 2 and Jun-ichiro Hamada 1 1 Department of Neurosurgery, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan 2 Department of Physiology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan 3 Department of Pediatrics, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan Sphingosine-1-phosphate (S1P) is a bioactive lipid that signals through a family of G protein-coupled receptors consisting of 5 members termed S1P 1–5 , and it regulates cellular proliferation, migration and survival. We investigated the expression and role of S1P receptors in glioma. Human glioma expressed S1P 1 , S1P 2 , S1P 3 , and S1P 5 by quantitative real-time PCR analysis. Expression of the S1P 1 was significantly lower in glioblastoma than in the normal brain (p < 0.01) and diffuse astrocytoma (p < 0.05). Immunoblotting showed that normal brain expressed more S1P 1 protein than did glioblastoma. Immunohistochemistry showed that S1P 1 was localized predominantly in the astrocytes in the normal brain, but no staining was observed in glioblastoma. Downregulation of S1P 1 expression correlated with poor survival of patients with glioblastoma (p < 0.05). S1P 1 small interfering RNA promoted cell proliferation in high-expressor glioma cell lines (T98G, G112). Cell proliferation was promoted by the pertussis toxin, which deactivates G i/o type of G proteins; the S1P 1 is exclusively coupled to these proteins. Forced expression of the S1P 1 in low-expressor cell lines (U87, U251) resulted in decreased cell growth and led to suppressed tumor growth in transplanted gliomas in vivo. Furthermore, we found a significant association between the S1P 1 expression and early growth response-1, a transcriptional factor that exhibits tumor suppression in glioblastoma cells (p < 0.05). These data indicate that the downregulation of S1P 1 expression enhances the malignancy of glioblastoma by increasing cell proliferation and correlates with the shorter survival of patients with glioblastoma. Glioblastoma is the most common type of brain tumor and is a highly malignant tumor exhibiting aggressive invasive growth, 1 leading to a median life expectancy of only 10–12 months after diagnosis. 2 It is essential to understand the mo- lecular regulation of glioblastoma cell growth and invasion to develop effective molecular-based therapies. Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates cellular proliferation, migration and survival in a wide variety of tissues and cell types. 3,4 S1P signals both intracellularly as a second messenger 5 and through 5 G pro- tein-coupled receptors (GPCRs) at the cell surface, which are termed S1P 1–5 . 6,7 Each S1P receptor displays a unique tissue expression pattern and is coupled to a distinct set of G pro- teins (G i/o ,G q and G 12/13 ), leading to the activation of recep- tor-specific intracellular signaling pathways. 6 S1P affects the proliferation, motility and invasiveness of malignant cells and can mediate proliferative 8,9 or antiproliferative 10,11 behavior in cancer cells. The various effects of S1P may be attributed to the diversity of its receptors. Some studies have shown that S1P, whose level is high in brain 12 and glioma cells, 13 plays a role in the growth and invasiveness of glioblastoma cells, 14–16 and these effects are at least partially mediated through its GPCRs because both responses are sensitive to pertussis toxin (PTX), 14 which specifically inhibits signaling through G i/o , which is a major family of G proteins. Glioblas- toma cell lines 15 and tissues 17 commonly express 3 members of the S1P receptor family: S1P 1 , S1P 2 and S1P 3 . S1P 5 is expressed in oligodendrocytes. 18 S1P 4 , which is primarily expressed in cells of hematopoietic origin, 19 has not been Key words: glioblastoma, S1P receptors, S1P 1 receptor, proliferation, survival Abbreviations: Egr-1: early growth response-1; GPCRs: G protein- coupled receptors; OS: overall survival; PFS: progression-free survival; PI3K: phosphatidylinositol 3 0 kinase; PTEN: phosphatase and tensin homologue deleted in chromosome 10; PTX: pertussis toxin; QRT-PCR: quantitative real-time PCR; siRNA: small interfering RNA; S1P: sphingosine-1-phosphate Additional Supporting Information may be found in the online version of this article Grant sponsors: The Japanese Ministry of Education, Science, Sports, Technology and Culture (Grants-in-aid for young scientists research); Grant number: B-19790992; Grant sponsors: Japan Brain Foundation, Foundation for Promotion of Cancer Research, Hokkoku Cancer Research Foundation DOI: 10.1002/ijc.24933 History: Received 30 Jun 2009; Accepted 17 Sep 2009; Online 6 Oct 2009 Correspondence to: Mitsutoshi Nakada, Department of Neurosurgery, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8641, Japan, Tel: þ81-76-265-2384, Fax: þ81-76-234-4262, E-mail: nakada@ns.m.kanazawa-u.ac.jp Cancer Cell Biology Int. J. Cancer: 126, 2341–2352 (2010) V C 2009 UICC International Journal of Cancer IJC