Suppression of Osteosarcoma Cell Invasion by Chemotherapy Is Mediated by Urokinase Plasminogen Activator Activity via Up-Regulation of EGR1 Yukihiro Matsunoshita1, Kosei Ijiri1, Yasuhiro Ishidou 2 , Satoshi Nagano 1 , Takuya Yamamoto1, Hiroko Nagao1, Setsuro Komiya1, Takao Setoguchi1* 1 Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan, 2 Department of Medical Joint Materials, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan Abstract Background: The cellular and molecular mechanisms of tumour response following chemotherapy are largely unknown. We found that low dose anti-tumour agents up-regulate early growth response 1 (EGR1) expression. EGR1 is a member of the immediate-early gene group of transcription factors which modulate transcription of multiple genes involved in cell proliferation, differentiation, and development. It has been reported that EGR1 act as either tumour promoting factor or suppressor. We therefore examined the expression and function of EGR1 in osteosarcoma. Methods: We investigated the expression of EGR1 in human osteosarcoma cell lines and biopsy specimens. We next examined the expression of EGR1 following anti-tumour agents treatment. To examine the function of EGR1 in osteosarcoma, we assessed the tumour growth and invasion in vitro and in vivo. Results: Real-time PCR revealed that EGR1 was down-regulated both in osteosarcoma cell lines and osteosarcoma patients’ biopsy specimens. In addition, EGR1 was up-regulated both in osteosarcoma patient’ specimens and osteosarcoma cell lines following anti-tumour agent treatment. Although forced expression of EGR1 did not prevent osteosarcoma growth, forced expression of EGR1 prevented osteosarcoma cell invasion in vitro. In addition, forced expression of EGR1 promoted down- regulation of urokinase plasminogen activator, urokinase receptor, and urokinase plasminogen activity. Xenograft mice models showed that forced expression of EGR1 prevents osteosarcoma cell migration into blood vessels. Conclusions: These findings suggest that although chemotherapy could not prevent osteosarcoma growth in chemotherapy-resistant patients, it did prevent osteosarcoma cell invasion by down-regulation of urokinase plasminogen activity via up-regulation of EGR1 during chemotherapy periods. Citation: Matsunoshita Y, Ijiri K, Ishidou Y, Nagano S, Yamamoto T, et al. (2011) Suppression of Osteosarcoma Cell Invasion by Chemotherapy Is Mediated by Urokinase Plasminogen Activator Activity via Up-Regulation of EGR1. PLoS ONE 6(1): e16234. doi:10.1371/journal.pone.0016234 Editor: Ming Tat Ling, Queensland University of Technology, Australia Received August 7, 2010; Accepted December 17, 2010; Published January 20, 2011 Copyright: 2011 Matsunoshita et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Grants-in-Aid for Scientific Research (B) 18390419, (C) 19591725, (C) 20591786, (C) 21591919, (C) 21591920, and (C) 22591663 (KAKENHI), and a Grant-in-Aid from the Ministry of Health, Labour, and Welfare of Japan for the Third Term Comprehensive Control Research for Cancer (http://www.jsps.go.jp/j-grantsinaid/index.html, http://ganjoho.ncc.go.jp/pro/taigan/index.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: setoro@m2.kufm.kagoshima-u.ac.jp Introduction Osteosarcoma is the most frequent primary malignant bone tumor. After initial diagnosis is made by biopsy, treatment consists of preoperative chemotherapy, followed by definitive surgery and postoperative chemotherapy. The Survival rates for patients treated with intensive multidrug chemotherapy and aggressive local control have been reported at 60–80% [1–5]. Indeed, patients with non-metastatic disease have a 70% chance of long- term survival. Eighty percent of patients die of metastatic disease, most commonly in the lungs [3]. Unfortunately, patients with metastatic disease at diagnosis or those who have recurrent disease have a poor prognosis, with only 20% surviving at 5 years, indicating that new therapeutic options for them need to be actively explored [6,7]. The early growth response gene 1 (EGR1) is a member of the immediate-early gene group of transcription factors which modulate transcription of multiple genes involved in cell proliferation, differentiation, and development [8]. Expression of EGR1 is significantly reduced in a number of tumor cells [9,10], and loss of expression of it is closely associated with tumor formation in mammalian cells and tissues [10]. On the other hand, stable expression of EGR1 inhibited cell proliferation and soft agar growth in NIH3T3 cells transformed with v-sis, indicating that EGR1 functions as a tumor suppressor [11]. We therefore examined the expression and function of EGR1 in osteosarcoma. Here, we report that expression of EGR1 is down-regulated in human osteosarcoma cell lines and patient’ biopsy specimens. In addition, treatment with anti-tumour agents promoted up-regulation of EGR1. Although forced expression of EGR1 did not affect osteosarcoma growth, PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16234