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Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer arising from the epithelium of nasopharynx. While the incidence rate of NPC is low in Western countries, it is prevalent in southern China, south-east Asia and northern Africa. Generally, it is believed that multiple factors, including genetic predisposition, environmental carcinogens and Epstein-Barr virus infection, contribute to the tumor initiation and progression of NPC. Similar to other solid tumors in which genetic rearrangements play causal role in the genesis of tumors, our earlier cytogenetic and spectral karyotyping have demonstrated the prevalence of chromosomal translocations in NPC tumors. These evidences suggest that novel fusion gene product arises from chromosomal translocation may contribute to the pathogenesis of the disease. Using paired-end whole-transcriptome sequencing, we discovered a novel fusion transcript composed of the exon 1 of the ubiquitin protein ligase E3 component n-recognin 5 (UBR5) on 8q22.3 and exon 7-9 of zinc finger protein 423 (ZNF423) on 16q12.1 from the NPC cell line C666-1. Moreover, FISH analysis using break-apart probes validated the UBR5-ZNF423 fusion. Furthermore reverse transcription-PCR (RT-PCR) confirmed the expression of the corresponding fusion transcript. Intriguingly, the fusion transcript was recurrently detected in 12/144 (8.3%) of primary tumors by RT-PCR, which indicates the UBR5-ZNF423 fusion gene may contribute to the genesis of a subset of NPCs. The growth of C666-1 cells with UBR5-ZNF423 rearrangement is dependent on expression of this fusion protein. Knock-down of UBR5-ZNF423 by RNA interference inhibited the cell proliferation of C666-1 cells. The transforming ability of UBR5-ZNF423 fusion was also confirmed in NIH3T3 mouse fibroblasts by soft agar colony formation assay. Moreover, NIH3T3 cells stably expressing the fusion protein induced tumor formation in nude mice. Furthermore, using an inducible shRNA (Lac operon-driven) targeting the fusion transcript, the growth of C666-1 tumor was suppressed in nude mice. These findings suggest that expression of UBR5-ZNF423 fusion protein contributes to the transformation of a subset of NPCs. Currently, we are working on the functional role(s) of the fusion protein in C666-1 cells. Preliminary results demonstrated that the fusion protein interacts with the Early B-cell Factor 3 (EBF3) and may deregulate EBF3 transcriptional activity. Since EBF3 is a tumor suppressor gene, deregulating EBF3 downstream gene expression by UBR5-ZNF423 may confer to its oncogenic function on NPCs. In conclusion, we have successfully identified the UBR5-ZNF423 fusion transcript in a subset of NPC tumors. By understanding the molecular mechanisms of this fusion protein on NPCs, novel therapeutic interventions may be implemented on treating a subsets of NPC tumors expressing this oncogenic fusion protein. Citation Format: Pok Man Hau, Grace Chung, Raymond Lung, Samantha Lun, Chit Chow, Alice Wong, Fei-Fei Liu, George Tsao, Kevin Yip, Ka Fai To, Kwok Wai Lo. The identification of UBR5-ZNF423 recurrent fusion gene in EBV-associated nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4064.

Background: Metastatic colorectal cancer (mCRC) is a heterogeneous disease with a poor prognosis. By using high resolution array comparative genomic hybridization (aCGH) and mRNA gene expression microarray of mCRC samples, we found up-regulation of RASAL2, a RAS GAP gene that is located on chromosome 1q, in colorectal tumors. Oncogenic RAS represents one of the most common molecular changes in human colorectal adenocarcinoma. However, the role of RASAL2 in colorectal adenocarcinoma metastasis and the related mechanisms are still unclear. We analyzed its aberrant expression, clinical significance and biological effects in colorectal cancer. Methods: Expression of RASAL2 was examined by QRT-PCR, western blot and immunohistochemistry in CRC cell lines, primary and metastatic CRC and the corresponding normal colonic mucosa. The biological effects of RASAL2 on proliferation, apoptosis, cell cycle, and cell motility and invasiveness were evaluated by siRNA knock down and RASAL2 reconstitution in CRC cell lines. Results: Up-regulation of RASAL2 mRNA was observed in CRC cell lines (n = 9) than normal colon mucosal tissues. Interestingly, significantly higher RASAL2 mRNA was found in cell lines derived from advanced stage tumors (n = 4, Dukes’ C and D) than those from early stage tumors (n = 5, Dukes’ A and B) (P = 0.0317). Up-regulation of RASAL2 mRNA was also found in primary CRCs (n = 77) compared with normal colon mucosa (n = 18, P We knocked down RASAL2 by siRNA in 2 CRC cell lines (DLD1 and HCT116) with high endogenous RASALs level. Successful knockdown was demonstrated by western blot analysis. SiRASAL2 significantly inhibited cell proliferation (P Conclusions: Up-regulation of RASAL2 was frequently found in CRC cell lines and primary and metastatic tumors. Our experimental findings suggested that RASAL2 might play an oncogenetic role in CRC and promotes tumor invasion and metastasis. A better understanding of the molecular mechanism of RASAL2 in promoting CRC cell metastasis may lead to a more effective management of mCRC patients. Citation Format: Yi PAN, Joanna Hung Man TONG, Raymond Wai Ming LUNG, Samantha Wei Man LUN, Kwok Wai LO, Anthony Wing Hung CHAN, Ka Fai TO. RASAL2 promotes tumor progression and metastasis in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1529.

Nasopharyngeal carcinoma (NPC) is an EBV-associated epithelial malignancy which is prevalent in southern China and south-east Asia. EBV-encoded EBNA1 is consistently expressed in NPC cells and implicates the replication and stable persistence of latent viral genome. As a phosphorylated protein, the function of EBV is believed to be affected by phosphorylation.In this study, we aimed to investigate whether the phospho-Ser/Thr-Pro specific prolyl-isomerase PIN1 alters EBNA1 function and plays a role in the tumorigenesis of EBV-associated NPC.By western blotting and immunohistochemical staining, we have found that PIN1 was highly expressed in almost all 6 NPC tumor lines and 70 primary tumors. In the EBV-positive C666-1 cells, co-localization and interaction of EBNA1 and PIN1 was detected by immunofluorence staining and co-immunoprecipitation assay respectively. The interaction between PIN1 and EBNA1 at the specific serine-proline motif (Ser383 and Ser393 in EBNA1;WW domain in Pin1) was also demonstrated. To investigate potential role of PIN1 in NPC tumorigenesis, we established a stable nasopharyngeal epitheliacell line NP69 overexpressing PIN1. In this model, we found that overexpression of PIN1 up-regulated cyclin D1 and activated MAPK/JNK pathway. Increased anchorage-independent growth of NP69 overexpressing PIN was shown in soft agar assay.To further validate the oncogencity of PIN1, we knocked down its expression in the NPC cell line C666-1 by RNA interference (siRNA). The study confirmed the PIN1-mediated cyclin D1 suppression. Importantly, knocking down of PIN1 significantly inhibited the cell proliferation, DNA synthesis and migration of NPC cells.Similar findings were also detected in the NPC cells treated with PIN1 inhibitor, Juglone. Treating EBV-positive C666-1cell with Juglone,we found it can significantly induce apoptosisand suppress the cyclinD1expression in C666-1cells. The anti-tumor effect of PIN inhibitor was demonstrated in the in vitro NPC model. To elucidate the anti-tumor potential of PIN1 inhibitorin vivo, the mice implanted with NPC cells were subjected to treatment with different doses of Juglone. A dose-dependent inhibition of tumor growth was observed in the mice treated with PIN1 inhibitor. In conclusion, our findings imply that PIN1overexpression plays important role in the development of NPC. Targeting Pin1 may serve as a potential therapeutic approach for treating patients suffering from this EBV-associated cancer. Citation Format: Meng Xu, Chit Chow, Chartia Ching-Mei Cheung, Chi-Man Tsang, Samantha Wei-Man Lun, Jessie Wai-Fung Yuen, Grace Tin-Yun Chung, Sah-Wah Tsao, Ka Fai To, Kwok Wai Lo. Inhibition of PIN1 suppresses tumorigenicity of EBV-associated NPC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2189. doi:10.1158/1538-7445.AM2013-2189

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-associated malignant cancer prevalent in Southern China and South-eastern Asia. In this study, we aim to elucidate the fundamental role of aberrant expressed microRNAs (miRNA) in NPC tumorigenesis. By comparison of miRNA expression profiles in NPC tumor lines with non-malignant nasopharyngeal epithelial cell, 119 differentially expressed miRNAs were identified in EBV-associated NPC. Among the 58 microRNAs downregulated in NPC, transcriptional silencing of miR-31 was consistently found in both NPC tumor lines and primary tumors. Expression of miR-31 was detected in none of 6 EBV-positive tumor lines and 38 microdissected primary tumors, but in all normal nasopharyngeal epithelia. Interestingly, miR-31 is located at 0.5 Mb telomeric to CDKN2A at 9p21.3, which is commonly homozygous deleted in NPC. Homozygous deletion of both miR-31 and CDKN2A locus was confirmed in 2 tumor lines. In the 4 tumor lines with intact miR-31, hypermethylation of 5′CpG islands was detected by bisulfate sequencing and MSP analysis. Re-expression of miR-31 was demonstrated in the EBV-positive NPC cell line C666-1 treated with 5-aza-2′-deoxycytidine. The findings suggested that homozygous deletion and promoter methylation are the major mechanisms for transcriptional silencing of miR-31 in NPC. By microarray and bioinformatic analysis, we have identified a number of putative targets of miR-31. Among these candidates, we have proven that miR-31 targeted FIH1 and MCM2 expression in NPC. Ectopic expression of miR-31 in NPC cells resulted in repression of FIH1 and MCM2 protein. Finally, we have demonstrated that restoration of miR-31 or knockdown of FIH1 expression significantly suppressed cell proliferation of C666-1. Our findings indicated that miR-31 is a NPC-associated microRNA which negatively regulates cell growth by repressing FIH1 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2288. doi:1538-7445.AM2012-2288

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.