Grace Tin-Yun Chung

Deciphering the molecular genetic basis of NPC through molecular, cytogenetic, and epigenetic approaches.

Nasopharyngeal carcinoma (NPC) is consistently associated with EBV infection and prevalence in southern China and Southeast Asia. In addition to EBV, the development of NPC involves cumulative genetic and epigenetic changes influenced by predisposing genetic factors and environmental carcinogens. Over the past two decades, knowledge of genetic and epigenetic alterations of NPC has rapidly accumulated. Multiple chromosomal abnormalities (e.g. copy number changes on chromosomes 3p, 9p, 11q, 12p, and 14q), gene alterations (e.g. p16 deletion and LTBR amplification), and epigenetic changes (e.g. RASSF1A and TSLC1 methylation) have been identified by various genome-wide approaches, such as allelotyping, CGH, and microarray analysis. In this review, we will discuss the critical genetic events that contribute to the initiation and progression of NPC. Studies on the precancerous lesions and in vitro immortalized nasopharyngeal epithelial cell models provide important evidence for the involvement of genetic alterations and EBV infection in early development of this cancer. A hypothetical model describing the role of EBV latent infection and multiple genetic changes in NPC tumorigenesis is proposed.

Preclinical Evaluation of AMG 900, a Novel Potent and Highly Selective Pan-Aurora Kinase Inhibitor with Activity in Taxane-Resistant Tumor Cell Lines

In mammalian cells, the aurora kinases (aurora-A, -B, and -C) play essential roles in regulating cell division. The expression of aurora-A and -B is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis, making them attractive targets for anticancer therapy. AMG 900 is an orally bioavailable, potent, and highly selective pan-aurora kinase inhibitor that is active in taxane-resistant tumor cell lines. In tumor cells, AMG 900 inhibited autophosphorylation of aurora-A and -B as well as phosphorylation of histone H3 on Ser(10), a proximal substrate of aurora-B. The predominant cellular response of tumor cells to AMG 900 treatment was aborted cell division without a prolonged mitotic arrest, which ultimately resulted in cell death. AMG 900 inhibited the proliferation of 26 tumor cell lines, including cell lines resistant to the antimitotic drug paclitaxel and to other aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358), at low nanomolar concentrations. Furthermore, AMG 900 was active in an AZD1152-resistant HCT116 variant cell line that harbors an aurora-B mutation (W221L). Oral administration of AMG 900 blocked the phosphorylation of histone H3 in a dose-dependent manner and significantly inhibited the growth of HCT116 tumor xenografts. Importantly, AMG 900 was broadly active in multiple xenograft models, including 3 multidrug-resistant xenograft models, representing 5 tumor types. AMG 900 has entered clinical evaluation in adult patients with advanced cancers and has the potential to treat tumors refractory to anticancer drugs such as the taxanes.

Discovery and Evaluation of Dual CDK1 and CDK2 Inhibitors

In eukaryotic cells, cyclin-dependent kinase (CDK) complexes regulate the temporal progression of cells through the cell cycle. Deregulation in the cell cycle is an essential component in the evolution of cancer. Here, we validate CDK1 and CDK2 as potential therapeutic targets using novel selective small-molecule inhibitors of cyclin B1/CDK1 and cyclin E2/CDK2 enzyme complexes (CDKi). Flow cytometry-based methods were developed to assess intracellular retinoblastoma (Rb) phosphorylation to show inhibition of the CDK pathway. Tumor cells treated with CDK inhibitors showed an overall decrease in cell proliferation, accumulation of cells in G1 and G2, and apoptosis in a cell line-specific manner. Although CDK inhibitors activate p53, the inhibitors were equipotent in arresting the cell cycle in isogenic breast and colon tumor cells lacking p53, suggesting the response is independent of p53. In vivo, the CDK inhibitors prevented the growth of colon and prostate tumors, blocked proliferation of tumor cells, and inhibited Rb phosphorylation. The discovery and evaluation of novel potent and selective CDK1 and CDK2 inhibitors will help delineate the role that CDK complexes play in regulating tumorigenesis.

Constitutive activation of distinct NF-κB signals in EBV-associated nasopharyngeal carcinoma.

As a distinct type of head and neck cancer, non‐keratinizing nasopharyngeal carcinoma (NPC) is closely associated with EBV infection and massive lymphoid infiltration. The unique histological features suggest that local inflammation plays an important role in NPC tumourigenesis. We comprehensively characterized NF‐κB signalling, a key inflammatory pathway which might contribute to the tumourigenesis of this EBV‐associated cancer. By EMSA, western blotting, and immunohistochemical staining, constitutive activation of distinct NF‐κB complexes, either p50/p50/Bcl3 or p50/RelB, was found in almost all EBV‐positive NPC tumours. siRNA or chemical inhibition of NF‐κB signalling significantly inhibited the growth of EBV‐positive NPC cells C666‐1. Gene expression profiling identified a number of NF‐κB target genes involved in cell proliferation, apoptosis, immune response, and transcription. We further confirmed that p50 signals modulate the expression of multiple oncogenes (MYB, BCL2), chemokines, and chemokine receptors (CXCL9, CXCL10, CX3CL1, and CCL20). The findings support a crucial role of these constitutively activated NF‐κB signals in NPC tumourigenesis and local inflammation. In addition to expression of the viral oncoprotein LMP1, genetic alteration of several NF‐κB regulators (eg TRAF3, TRAF2, NFKBIA, A20) also contributes to the aberrant NF‐κB activation in EBV‐associated NPC. Except for LMP1‐expressing C15 cells, all NPC tumour lines harbour at least one of these genetic alterations. Importantly, missense mutations of TRAF3, TRAF2, and A20 were also detected in 3/33 (9.1%) primary tumours. Taken together with the reported LTBR amplification in 7.3% of primary NPCs, genetic alterations in NF‐κB pathways occurred in at least 16% of cases of this cancer. The findings indicate that distinct NF‐κB signals are constitutively activated in EBV‐positive NPC cells by either multiple genetic changes or EBV latent genes. Copyright

Interrogation of genomes by molecular copy-number counting (MCC).

Human cancers and some congenital traits are characterized by cytogenetic aberrations including translocations, amplifications, duplications or deletions that can involve gain or loss of genetic material. We have developed a simple method to precisely delineate such regions with known or cryptic genomic alterations. Molecular copy-number counting (MCC) uses PCR to interrogate miniscule amounts of genomic DNA and allows progressive delineation of DNA content to within a few hundred base pairs of a genomic alteration. As an example, we have located the junctions of a recurrent nonreciprocal translocation between chromosomes 3 and 5 in human renal cell carcinoma, facilitating cloning of the breakpoint without recourse to genomic libraries. The analysis also revealed additional cryptic chromosomal changes close to the translocation junction. MCC is a fast and flexible method for characterizing a wide range of chromosomal aberrations.*Note: Correspondence should be addressed to M. Thangavelu (mt370@hutchison-mrc.cam.ac.uk) instead of T. H. Rabbitts. The error has been corrected in the PDF version of the article.

Epigenetic inactivation of the deleted in lung and esophageal cancer 1 gene in nasopharyngeal carcinoma.

Deletion of the short arm of chromosome 3 is a common event in nasopharyngeal carcinoma (NPC), suggesting that one or more tumor suppressor genes at 3p are involved in this cancer. DLEC1, Deleted in Lung and Esophageal Cancer 1, located at 3p22.2, was recently identified as a candidate tumor suppressor gene in lung, esophageal, and renal cancers. In this study, we investigated the involvement of DLEC1 in the development of NPC. Down‐regulation of DLEC1 and promoter hypermethylation were observed in all NPC cell lines and xenografts but not in normal nasopharyngeal epithelial cells. Promoter hypermethylation of DLEC1 was also detected in 30 of 42 (71%) NPC primary tumors. Treatment of NPC cell lines with demethylating agent or histone deacetylase inhibitor resulted in restoration of DLEC1 expression. Overexpression of DLEC suppressed growth and reduced invasiveness of NPC cells. Furthermore, the tumorigenic potential of DLEC1 expressing NPC cells was highly reduced in nude mice. Taken together, our results strongly suggest that silencing of DLEC1 expression by promoter hypermethylation and histone deacetylation may be important in NPC tumorigenesis.

CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a unique EBV-associated epithelial malignancy, showing highly invasive and metastatic phenotype. Despite increasing evidence demonstrating the critical role of cancer stem-like cells (CSCs) in the maintenance and progression of tumors in a variety of malignancies, the existence and properties of CSC in EBV-associated NPC are largely unknown. Our study aims to elucidate the presence and role of CSCs in the pathogenesis of this malignant disease. Sphere-forming cells were isolated from an EBV-positive NPC cell line C666-1 and its tumor-initiating properties were confirmed by in vitro and in vivo assays. In these spheroids, up-regulation of multiple stem cell markers were found. By flow cytometry, we demonstrated that both CD44 and SOX2 were overexpressed in a majority of sphere-forming C666-1 cells. The CD44+SOX2+ cells was detected in a minor population in EBV-positive xenografts and primary tumors and considered as potential CSC in NPC. Notably, the isolated CD44+ NPC cells were resistant to chemotherapeutic agents and with higher spheroid formation efficiency, showing CSC properties. On the other hand, microarray analysis has revealed a number of differentially expressed genes involved in transcription regulation (e.g. FOXN4, GLI1), immune response (CCR7, IL8) and transmembrane transport (e.g. ABCC3, ABCC11) in the spheroids. Among these genes, increased expression of CCR7 in CD44+ CSCs was confirmed in NPC xenografts and primary tumors. Importantly, blocking of CCR7 abolished the sphere-forming ability of C666-1 in vitro. Expression of CCR7 was associated with recurrent disease and distant metastasis. The current study defined the specific properties of a CSC subpopulation in EBV-associated NPC. Our findings provided new insights into developing effective therapies targeting on CSCs, thereby potentiating treatment efficacy for NPC patients.

Inhibition of NOTCH3 signalling significantly enhances sensitivity to cisplatin in EBV-associated nasopharyngeal carcinoma†

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. Copyright

Authentication of nasopharyngeal carcinoma tumor lines.

Sylvia Yat-Yee Chan, Kwong-Wai Choy, Sai-Wah Tsao, Qian Tao, Tao Tang, Grace Tin-Yun Chung and Kwok-Wai Lo* Department of Anatomical and Cellular Pathology, State Key Laboratory in Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong Li Ka Shing Institute of Health Science, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong Department of Obstetrics and Gynaecology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong Department of Anatomy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong Department of Clinical Oncology, State Key Laboratory in Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong

Identification of a novel 12p13.3 amplicon in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer commonly occurring in southern China. To decipher the molecular basis of this cancer, we performed high‐resolution array CGH analysis on eight tumour lines and 10 primary tumours to identify the genes involved in NPC tumorigenesis. In this study, multiple regions of gain were consistently found at 1q21‐q24, 7q11‐12, 7q21‐22., 11q13, 12p13, 12q13, 19p13 and 19q13. Importantly, a 2.1 Mb region at 12p13.31 was highly amplified in a NPC xenograft, xeno‐2117. By FISH mapping, we have further delineated the amplicon to a 1.24 region flanked by RP11‐319E16 and RP11‐433J6. Copy number gains of this amplicon were confirmed in 21/41 (51%) primary tumours, while three cases (7.3%) showed high copy number amplification. Among the 13 genes within this amplicon, three candidate genes, lymphotoxin beta receptor (LTβR), tumour necrosis factor receptor superfamily memeber 1A (TNFRSF1R) and FLJ10665, were specifically over‐expressed in the NPC xenograft with 12p13.3 amplification. However, only LTβR was frequently over‐expressed in primary tumours. LTβR is a member of the TNF family of receptors, which can modulate NF‐κB signalling pathways. Over‐expression of LTβR in nasopharyngeal epithelial cells resulted in an increase of NF‐κB activity and cell proliferation. In vivo study showed that suppression of LTβR by siRNA led to growth inhibition in the NPC tumour with 12p13.3 amplification. These findings implied that LTβR is a potential NPC‐associated oncogene within the 12p13.3 amplicon and that its alteration is important in NPC tumorigenesis. Copyright