Nasopharayngeal carcinoma (NPC) is an Epstein-Barr virus-associated malignancy most common in East Asia and Africa. Here we report frequent downregulation of the microRNA miR-218 in primary NPC tissues and cell lines where it plays a critical role in NPC progression. Suppression of miR-218 was associated with epigenetic silencing of SLIT2 and SLIT3, ligands of ROBO receptors that have been previously implicated in tumor angiogenesis. Exogenous expression of miR-218 caused significant toxicity in NPC cells in vitro and delayed tumor growth in vivo. We used an integrated trimodality approach to identify targets of miR-218 in NPC, cervical, and breast cell lines. Direct interaction between miR-218 and the 3 0 -untranslated regions (UTR) of mRNAs encoding ROBO1, survivin (BIRC5), and connexin43 (GJA1) was validated in a luciferase-based transcription reporter assay. Mechanistic investigations revealed a negative feedback loop wherein miR-218 regulates NPC cell migration via the SLIT-ROBO pathway. Pleotropic effects of miR-218 on NPC survival and migration were rescued by enforced expression of miR-218-resistant, engineered isoforms of survivin and ROBO1, respectively. In clinical specimens of NPC (n ¼ 71), ROBO1 overexpression was significantly associated with worse overall (P ¼ 0.04, HR ¼ 2.4) and nodal relapse-free survival (P ¼ 0.008, HR ¼ 6.0). Our findings define an integrative tumor suppressor function for miR-218 in NPC and further suggest that restoring miR-218 expression in NPC might be useful for its clinical management. Cancer Res; 71(6); 2381-91. Ó2011 AACR.

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There is increasing evidence supporting the role of members of the polycomb group (PcG) gene family in tumor development and progression. However, their precise role in tumorigenesis and mechanisms of their regulation remain to be elucidated. Using nasopharyngeal carcinoma (NPC) as a disease model, a comprehensive analysis was undertaken on the clinical significance of EZH2 expression, identification of the cellular processes regulated by EZH2, and the mechanisms of its deregulated expression. Herein, we report EZH2 as being associated with a higher risk of relapse in NPC patients (P=0.002). Genome-wide microarray and bioinformatics identified several vital cellular processes (such as differentiation, development, and apoptosis) to be regulated by EZH2, corroborated by in vitro lethality, and delayed tumor formation in vivo upon EZH2 depletion. The combination of global microRNA (miR) profiling in primary NPC specimens, and in silico analyses provided several candidate miRs that could regulate EZH2. Using a luciferase-based assay, miR-26a, miR-101, and miR-98 were validated as bona fide regulators of EZH2 expression. In particular, miR-98 was underexpressed in relapsed patient samples, strongly suggesting an important role for the miR-98 and EZH2 axis in NPC biology.