Up-regulation of osteopontin expression by aryl hydrocarbon receptor via both ligand-dependent and ligand-independent pathways in lung cancer Cheng-Yen Chuang a, b, c, 1 , Han Chang d, e, 1 , Pinpin Lin f , Shih-Jung Sun a, b , Po-Hung Chen a, b, f , Yu-Ying Lin a, b , Gwo-Tarng Sheu a, b, g , Jiunn-Liang Ko a, b, g , Shih-Lan Hsu h , Jinghua Tsai Chang a, b, g, ⁎ a Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC b Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung, Taiwan, ROC c Division of Thoracic Surgery, Taichung Veterans General Hospital, Taichung, Taiwan, ROC d Department of Pathology, China Medical University Hospital, Taichung, Taiwan, ROC e Department of Pathology, School of Medicine, China Medical University, Taichung, Taiwan, ROC f Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Taiwan, ROC g Department of Medical Oncology and Chest Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC h Department of Education & Research, Taichung Veterans General Hospital, Taichung, Taiwan, ROC abstract article info Article history: Accepted 4 October 2011 Available online 13 October 2011 Received by A.J. van Wijnen Keywords: TCDD BaP NFkB Transcriptional regulation Promoter assay The secreted glycol-phosphoprotein OPN not only plays important roles in immune responses and tissue remodeling but is also intimately involved in tumorigenesis. It is up-regulated in various cancers and corre- lated with poor prognosis. It is evident by enhancing growth and migration of cancer cells. However, the mechanisms that participate in up-regulation of OPN in lung cancer are largely unknown. Up-regulation of aryl hydrocarbon receptor (AhR), a transcription factor activated by xenobiotics, has been observed in lung cancer as well as premalignant lesions. In this study we demonstrated that AhR positively regulates OPN ex- pression in lung cancer. We observed positive correlation of OPN and AhR expression in lung cancer speci- men. Knockdown or overexpression of AhR exhibited down- or up-regulation of OPN expression in lung cancer cells. We identified an OPN promoter region between positions -268 and +435 that was activated by both ligand-independent and ligand-activated AhR. However, this region does not contain AhR response element/dioxin response element (DRE/XRE). Further truncations and internal deletions of the promoter revealed that the ligand-independent and ligand-activated AhR function through different regions of OPN promoter. The region between -268 and -100 was required for ligand-independent AhR activity. This region contains several cis-elements including AP2, C/EBP, SP1 and AP1 sites. On the other hand, the ligand-activated AhR up-regulates OPN activity through two regions of OPN promoter; one contains NFκB site at +137 and the other is between positions -100 and +126. This study suggested that both overexpression of un-induced AhR (in cases of non-smokers with high level of AhR) and ligand-activated AhR (such as smokers) contribute to up-regulation of OPN that in turn leads to lung tumorigenesis. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Lung cancer is the leading cause of cancer death worldwide. In an attempt to identify lung carcinogenesis related genes, we identified osteopontin (OPN) as a differentially expressed gene between lungs with cancer and lungs with healthy tissues. OPN was discovered as an inducible marker secreted by transformed, malignant epithelial cells (Senger et al., 1979). Increased expression level of OPN has been associated with progression or metastasis in multiple cancers including lung (Chambers et al., 1996; Shijubo et al., 1999), breast (Tuck et al., 1997, 1998), colon (Agrawal et al., 2002), liver (Gotoh et al., 2002), stomach (Ue et al., 1998), and prostate (Thalmann et al., 1999). OPN has been shown to be involved in malignant pleural effusion in lung cancer (Cui et al., 2009). Furthermore, elevated plasma OPN is asso- ciated with advanced stage and metastasis of gastric cancer and NSCLC (Bramwell et al., 2006; Chang et al., 2007b; Wu et al., 2007). OPN func- tions as an extracellular cellular matrix component and a cytokine that modulates cell function by binding to two cell adhesion molecules, integ- rins and CD44 (Weber et al., 1996). Accumulated evidence showed that OPN may promote cancer metastasis by increasing cell motility, degra- dation of the extracellular matrix, neovascularization, and evasion of apoptosis and host-immune cells (Wai and Kuo, 2008). Gene 492 (2012) 262–269 Abbreviations: CYP1A1, cytochrome P450 1A1; CYP1B1, cytochrome P450 1B1; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; BaP, benzo[a]pyrene; ROS, reactive oxygen species. ⁎ Corresponding author at: Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC. Tel.: +886 4 24730022x11695; fax: +886 4 24751101. E-mail address: jinghuat@csmu.edu.tw (J.T. Chang). 1 These authors contributed equally to this work. 0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.10.019 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene