Nasopharyngeal carcinoma (NPC) is an EBV-associated epithelial malignancy which is prevalent in south-east Asia and southern China. Despite the multiple genetic and epigenetic changes reported, the contribution of dysregulated signalling pathways to this distinct type of head and neck cancer is not well understood. Here we demonstrate the up-regulation of NOTCH ligands (JAG1 or DLL4) and effector (HEY1) in the majority of EBV-positive tumour lines and primary tumours. Among the NOTCH receptors, NOTCH3 was over-expressed in all EBV-positive tumour lines and 92.5% of primary tumours. Aberrant activation of NOTCH3 signalling was consistently detected in all these samples. These findings imply that NOTCH3 may play an crucial role in the development of NPC. By NOTCH3 specific siRNA, NOTCH3 signalling was suppressed and thereby significant growth inhibition and apoptosis induction occurred in NPC cells. Down-regulation of a number of targets involved in cell proliferation, eg CCND1, C-MYC,NFKB1, and survival, eg BCL2, BCL-XL, SURVIVIN, was confirmed in the NOTCH3 knockdown NPC cells. Importantly, NOTCH3 knockdown highly enhanced the sensitivity of NPC cells to cisplatin treatment. Furthermore, we revealed that the ability of NPC cells to form spheroids in vitro and tumours in nude mice was also significantly decreased after knockdown of NICD3 expression. These findings indicate that activation of NOTCH3 pathway is a critical oncogenic event in NPC tumourigenesis. Targeting NOTCH3 signalling may serve as a potential therapeutic approach for treating patients suffering from EBV-associated NPC.

Nasopharyngeal carcinoma (NPC) is an EBV-associated epithelial malignancy which prevalent in Southeast Asia and Southern China. In an effort to seek for new targets for therapies, we have elucidated the underlying mechanisms for dysregulated signaling pathways in this EBV-associated cancer. Our recent study showed that constitutive activation of NOTCH pathway is consistently detected in both NPC tumor lines and primary tumors. Expression of EBV latent genes also altered the NOTCH signaling pathway in the EBV-infected cells. Upregulation of NOTCH ligands (JAG1 or DLL4) and effector (HEY1) was found in almost all EBV positive tumor lines and primary tumors. Importantly, overexpression and activation of NOTCH3 receptor occurred in 6/6 (100%) of NPC tumor lines and 18/26 (70%) of primary tumors. Knockdown of NICD3 expression by siRNA led to significantly reduction of RBP-Jkappa promoter activity and expression of the downstream effectors in the EBV-positive NPC cell line, C666-1. By microarray analysis, a number of targets (e.g. CCND1, C-MYC, and BCL-XL) involved in cell proliferation and survival were identified in the NICD3-silenced NPC cells. Our functional study also demonstrated NICD3 inactivation down-regulated the proliferation of C666-1 cells, both on the viability and DNA synthesis. Increase apoptotic cells were found in the NICD3 siRNA-treated NPC cells. Our data support the hypothesis that NOTCH signaling pathway plays a crucial role in NPC through regulating apoptosis. Since NOTCH signaling is a highly conserved pathway important for human cancers, NICD3 may serve as a potential therapeutic target for treating patient suffering from this EBV-associated cancer. However, we revealed that NPC cells are resistance to gamma-secretase inhibitor (DAPT) treatment. It may be due to the frequent inactivation of NUMB, a negative regulator of NOTCH pathway, in NPC tumors. Importantly, we revealed that NOTCH3 transcription is regulated by CXCR4 in NPC cells. Expression of NOTCH3 was highly reduced in the C666-1 cells treated by either CXCR4 antagonist (AMD3100) or siRNA. In addition to suppression of NICD3 signal, knockdown CXCR4 resulted in growth inhibition and tumor apoptosis. Finally, we demonstrated that the drug resistance of C666-1 towards cisplatin was significantly decreased after CXCR4 antagonist (AMD3100) treatment. Increased tumor apoptosis were detected in the NPC cells treated with both cisplatin and AMD3100 when compared with that treated with cisplatin only. In summary, the CXCR4 antagonist suppresses the NOTCH pathway, inhibits apoptosis signals in NPC cells and thereby enhances its sensitivity to chemotherapeutic treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 356.