Polo‐like kinase 1 (Plk1) is a critical regulator of many stages of mitosis; increasing evidence indicates that Plk1 overexpression correlates with poor clinical outcome, yet its mechanism of regulation remains unknown. Hence, a detailed evaluation was undertaken of Plk1 expression in human nasopharyngeal cancer (NPC), the cellular effects of targeting Plk1 using siRNA in combination with ionizing radiation (RT) and potential upstream microRNAs (miRs) that might regulate Plk1 expression. Using immunohistochemistry, Plk1 was observed to be overexpressed in 28 of 40 (70%) primary NPC biopsies, which in turn was associated with a higher likelihood of recurrence (p = 0.018). SiPlk1 significantly inhibited Plk1 mRNA and protein expression, and decreased Cdc25c levels in NPC cell lines. This depletion resulted in cytotoxicity of C666‐1 cells, enhanced by the addition of RT, mediated by G2/M arrest, increased DNA double‐strand breaks, apoptosis, and caspase activation. Immunofluorescence demonstrated that the G2/M arrest was associated with aberrant spindle formation, leading to mitotic arrest. In vivo, transfection of C666‐1 cells and systemic delivery of siPlk1 decreased tumour growth. MicroRNA‐100 (miR‐100) was predicted to target Plk1 mRNA, which was indeed underexpressed in C666‐1 cells, inversely correlating with Plk1 expression. Using luciferase constructs containing the 3′‐UTR of Plk1 sequence, we document that miR‐100 can directly target Plk1. Hence, our data demonstrate for the first time that underexpressed miR‐100 leads to Plk1 overexpression, which in turn contributes to NPC progression. Targeting Plk1 will cause mitotic catastrophe, with significant cytotoxicity both in vitro and in vivo, underscoring the important therapeutic opportunity of Plk1 in NPC.

Purpose: This study aims to identify a novel therapeutic agent for head and neck cancer and to evaluate its antitumor efficacy. Experimental Design: A cell-based and phenotype-driven high-throughput screening of f2,400 biologically active or clinically used compounds was done using a tetrazolium-based assay on FaDu (hypopharyngeal squamous cancer) and NIH 3T3 (untransformed mouse embryonic fibroblast) cells, with secondary screening done on C666-1 (nasopharyngeal cancer) and GM05757 (primary normal human fibroblast) lines. The ''hit'' compound was assayed for efficacy in combination with standard therapeutics on a panel of human cancer cell lines. Furthermore, its mode of action (using transmission electron microscopy and flow cytometry) and its in vivo efficacy (using xenograft models) were evaluated. Results: Benzethonium chloride was identified as a novel cancer-specific compound. For benzethonium (48-hour incubation), the dose required to reduce cell viability by 50% was 3

Purpose: Nasopharyngeal cancer (NPC) is a malignant epithelial carcinoma which is intimately associated with EBV. The latent presence of EBV affects the function of p53, Bcl-2, and survivin. We thus investigated the relationship between EBV status, p53, Bcl-2, and survivin in biopsy specimens from patients with primary NPC. Experimental Design: Archival formalin-fixed, paraffin-embedded NPC biopsies were evaluated in 80 patients treated with curative radiation from a single institution. The presence of EBV was determined using EBER in situ hybridization, whereas p53, Bcl-2, and survivin were assessed using immunohistochemistry. Results: The majority of NPC specimens in this patient cohort were EBER-positive (64 of 78, or 82%), which in turn, was significantly associated with ethnicity (P = 0.0007), and WHO subtype 2A/2B (P = 0.04). EBER-positive tumors were also associated with p53 (P = 0.002), Bcl-2 (P = 0.04), and nuclear survivin (P = 0.03) expression. Patients with EBER-positive NPC fared better, with a 10-year overall survival of 68% versus 48% for EBER-negative patients (P = 0.03). For nuclear survivin, patients with either low or high nuclear survivin fared worse than patients with intermediate survivin expression (P = 0.05), suggesting that there is an optimal proportion of survivin-expressing cells for best function and clinical outcome. Conclusions: With an extended median follow-up time of 11.4 years, EBV status remains a strong predictor for overall survival in NPC. EBV-positive NPC has strong molecular associations with p53, Bcl-2, and survivin expression. Furthermore, we provide clinical data revealing the potentially dual nature of survivin in predicting clinical outcome.Nasopharyngeal carcinoma (NPC) is a malignant epithelial carcinoma of the head and neck site, with incidences of 20 to 30 per 100,000 people in areas such as Southeast Asia (a region of >100 million people) and the Mediterranean basin (a region of >300 million people) (1). NPC is anatomically located in close proximity to the base of the skull, rendering it a significant challenge for conventional and experimental treatments. In fact, inadequate tumor volume coverage may be partially responsible for the modest 5-year overall survival (OS) rate of f70% when patients are treated with conventional radiotherapy alone (2 -4). Although intensity-modulated radiotherapy does improve local control, the risk of distant metastases remains high at f40% (2). A predilection for developing distant metastases, a young median age of presentation (at f50 years), and the intimate association with EBV are features that render NPC a unique head and neck epithelial malignancy (5).The latent presence of EBV is unique to malignancies such as NPC, Burkitt's lymphoma, Hodgkin's lymphoma, peripheral T cell lymphomas, natural killer cell lymphomas, and gastric carcinomas (1). Approximately 75% to 81% of patients with NPC worldwide harbor the EBV genome, which is present in the type II latency. This form of latency is characterized by the expression ...