Oxidored-nitro domain containing protein 1 (NOR1) expression suppresses slug/vimentin but not snail in nasopharyngeal carcinoma: Inhibition of EMT in vitro and in vivo in mice
Introduction
Nasopharyngeal carcinoma (NPC) is a significant health problem in south China. NPC frequently invades adjacent regions and metastasizes to regional lymph nodes and distant organs at early stage [1], leading to mortality and affecting quality of life. Numerous studies have revealed that multiple factors are involved in the etiology of NPC [2], [3]; however, the underlying molecular basis of NPC development is not well understood. While the Epstein–Barr virus (EBV) has been shown to play a primary role in NPC development and progression, a better understanding of the pathogenesis of NPC, especially NPC invasion and metastasis, is critical and may lead to novel therapeutic strategies for NPC treatment.
To this end, the epithelial-to-mesenchymal transition (EMT) is a process characterized by loss of cell adhesion, reduction of E-cadherin expression but increase in cell motility. EMT is essential for numerous developmental processes, in which cells lose epithelial characteristics but gain mesenchymal properties [4]. Morphologically, EMT is characterized by loss of tight cell–cell junctions and accompanied by re-organization of the actin cytoskeleton, resulting in spindle shaped mesenchymal-like cells, which are capable of migrating and invading into other tissues [5], [6]. However, during tumor progression tumor cell EMT promotes tumor cell invasion and metastasis [7]. Molecularly, a set of transcriptional factors, including SNAI1, SNAI2 (SLUG), TWIST, and ZEB1/2 were initially identified as regulating epithelial-mesenchymal plasticity in embryonic morphogenesis, and subsequently as suppressing CDH1 expression associated with various forms of EMT [8].
Nevertheless, oxidored-nitro domain containing protein 1 gene (NOR1; also called organic solute carrier partner 1, or OSCP1) is often hyper-methylated in NPC tissues or cell lines [9], [10], [11]. Array-based gene expression analyses revealed that expression of NOR1 mRNA was frequently reduced in NPC tissues [12], [13], [14]. NOR1 protein was isolated from NPC as a tumor suppressor gene. Previous studies have shown that NOR1 expression inhibited tumor cell viability and induced apoptosis by activating the mitochondrial apoptotic pathway [15], [16]. Our preliminary study showed that NOR1 regulated EMT in NPC cell lines; thus, in this study, we further characterized NOR1 expression in NPC tissues for association with survival and clinicopathological data from NPC patients. Then we knocked down, or overexpressed, NOR1 protein in NPC and HeLa cells for phenotypic changes and gene expression analysis. Lastly, we explored the underlying molecular mechanisms responsible for NOR1-mediated inhibition of EMT in NPC cells.
Section snippets
Tissue microarray (TMA) construction of tumor tissue samples from patients
A cohort of 448 subjects with NPC, as well as non-cancerous nasopharyngeal epithelial (NPE) control subjects were recruited between January 2001 and October 2004 from the Xiangya Hospital, and their tissue samples were used for immunohistochemical analysis of NOR1 expression. Paraffin-embedded tumor and normal tissue blocks from all of these cases were retrieved from the Pathology Department for construction of tissue microarray and prepared for sectioning. We obtained 321 tissue cores from NPC
Lost NOR1 expression associated with NPC de-differentiation and clinical outcome
In this study, we first detected NOR1 expression in 212 NPC cases: 45 samples of chromatic inflammatory nasopharyngeal epithelial (NPE) tissues, 32 samples of dysplastic NPE, and 32 samples of distant normal NPE. We found that expression of NOR1 protein in NPC tissues was significantly lower (61 of 212; 28.8%) than that of chromatic inflammatory NPE (27 of 45, 60%; P < 0.001) (Fig. 1A,a), dysplastic epithelium (22 of 32; 68.8%; P < 0.001)(Fig. 1Ab), and nasopharyngeal epithelium distant to NPC (25
Discussion
In this study, we first analyzed NOR1 expression in NPC tissues for association with clinicopathological data and survival of NPC patients. We found that reduced NOR1 expression in 151 of 212 patients was associated with poor event-free and overall survival of NPC patients. We then assessed the effects of NOR1 expression in NPC and Hela cells and found that overexpression of NOR1 protein reduced tumor cell viability and migration and invasion capacity, whereas NOR1 knockdown had inverse
Conflict of Interest
None.
Acknowledgments
This study was supported in part by Grants from The National Natural Science Foundation of China (81372304, 81272254, 81171930, and 81102065), the National “111” Project (Project #111-2-12), The Natural Science Foundation of Hunan Province, China (14JJ3045, 14JJ1010), The Free Exploration Program of Central South University of China (201012200017 and 2011QNZT138), The Hunan Provincial Innovation Foundation For Postgraduate (CX2011B051).
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