Nasopharyngeal carcinoma is a distinct type of head and neck cancer which is consistently associated with Epstein-Barr virus (EBV). The C666-1 cell line is the only in vitro native EBV-infected NPC cell model commonly used for study of the viral-host interaction. Nevertheless, the complete EBV genome sequence in this in vitro EBV-infected NPC model has not been characterized.To determine the complete EBV genome sequence in C666-1 cells.The C666-1 genome was sequenced by 100-bases pair-end massive parallel sequencing. Bioinformatics analysis was performed to extract the EBV sequences and construct an EBV consensus sequence map. PCR amplification and Sanger DNA sequencing were used for sequence validation and gap filling. A phylogenetic analysis of EBV strain in C666-1 cells and other reported EBV strains was performed.A 171,317 bp complete EBV genome of C666-1 was successfully constructed (GenBank accession number: KC617875). Phylogenetic analysis of EBV genome in C666-1 revealed that the C666-1 EBV strain is closely related to the reported strains in NPC primary tumors.C666-1 contains a representative NPC-associated EBV genome and might serve as an important model for studying the roles or function of viral proteins in NPC tumorigenesis.
Abstract 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 (EBV)-associated epithelial malignancy that exhibits distinct geographical and ethnic prevalence. Although the contemporary therapeutic approach of radio-/chemotherapy provides excellent results for patients with early-stage disease, it is far from satisfactory for those with disease remission and distant metastasis. Promising therapeutic strategies for advanced and relapsed NPC are still lacking. We recently identified and characterized a cancer stem-like cell (CSC) subpopulation in NPC that appeared to play an important role in tumor progression. Microarray analysis revealed downregulation of several stemness-inhibiting miRNAs in these CSC cells. Among these miRNAs, miR-96 and miR-183 showed the highest fold change and were selected to elucidate their role in repressing NPC CSC properties. MiR-96 and miR-183 expression in NPC CSCs was detected by qRT-PCR. Transient and stable transfection was performed in EBV-positive NPC C666-1 cells to examine the effects of ectopic expression of miR-96 and miR-183 on repressing cell growth and CSC properties. Anchorage-dependent (colony formation) and anchorage-independent (tumor sphere formation) growths of these miR-96 and miR-183 expressing cells were determined. Expression of multiple CSC markers and related molecules were accessed by flow cytometry and Western blotting. The tumorigenicity of the stable miR-96- and miR-183-transfected NPC cells was examined in an in vivo nude mice model. Downregulation of miR-96 and miR-183 was confirmed in NPC spheroids. Using transient or stable transfection, we showed that ectopic expression of miR-96 and miR-183 suppressed cell growth and tumor sphere formation in NPC. Reduced NICD3 and NICD4 in miR-96- and miR-183-expressing NPC cells suggests the involvement of the NOTCH signaling pathway in their tumor suppressive function. Finally, we showed that the tumorigenicity of cells stably expressing miR-183 was significantly inhibited in the in vivo nude mice model. miR-183 is a tumor-suppressive miRNA in EBV-associated NPC. Its abilities to suppress CSC properties in vitro and effectively reduce tumor growth in vivo shed light on its role as a potential therapeutic target.
Abstract Human genomes contain structural variations (SVs) that are associated with various phenotypic variations and diseases. SV detection by sequencing is incomplete due to limited read length. Nanochannel-based optical mapping (OM) allows direct observation of SVs up to hundreds of kilo-bases in size on individual DNA molecules, making it a promising alternative technology for identifying large SVs. SV detection from optical maps is non-trivial due to complex types of error present in OM data, and no existing methods can simultaneously handle all these complex errors and the wide spectrum of SV types. Here we present a novel method, OMSV, for accurate and comprehensive identification of SVs from optical maps. OMSV detects both homozygous and heterozygous SVs, SVs of various types and sizes, and SVs with and without creating/destroying restriction sites. In an extensive series of tests based on real and simulated data, OMSV achieved both high sensitivity and specificity, with clear performance gains over the latest existing method. Applying OMSV to a human cell line, we identified hundreds of SVs >2kbp, with 65% of them missed by sequencing-based callers. Independent experimental validations confirmed the high accuracy of these SVs. We also demonstrate how OMSV can incorporate sequencing data to determine precise SV break points and novel sequences in the SVs not contained in the reference. We provide OMSV as open-source software to facilitate systematic studies of large SVs.
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.
Abstract 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
Abstract Nasopharyngeal cancer (NPC), endemic in Southeast Asia, lacks effective diagnostic and therapeutic strategies. Even in high-income countries the 5-year survival rate for stage IV NPC is less than 40%. Here we report high somatostatin receptor 2 (SSTR2) expression in multiple clinical cohorts comprising 402 primary, locally recurrent and metastatic NPCs. We show that SSTR2 expression is induced by the Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) via the NF-κB pathway. Using cell-based and preclinical rodent models, we demonstrate the therapeutic potential of SSTR2 targeting using a cytotoxic drug conjugate, PEN-221, which is found to be superior to FDA-approved SSTR2-binding cytostatic agents. Furthermore, we reveal significant correlation of SSTR expression with increased rates of survival and report in vivo uptake of the SSTR2-binding 68 Ga-DOTA-peptide radioconjugate in PET-CT scanning in a clinical trial of NPC patients (NCT03670342). These findings reveal a key role in EBV-associated NPC for SSTR2 in infection, imaging, targeted therapy and survival.
Nasopharyngeal carcinoma (NPC) is rare in most part of the world but shows high incidence in South China and Southeast Asia. One of the unique features of this epithelial cancer is its consistent association with Epstein–Barr virus (EBV) infection. Studies have shown that EBV plays a key role in the genesis and maintenance of the tumor phenotype in this disease.1 Clonal EBV genome and latent gene products are found in almost all cases of NPC expect for the keratinizing type of NPC from nonendemic areas. With the advance of molecular biology, knowledge of molecular alterations and EBV's function in NPC rapidly accumulated in the past 15 years.1, 2 However, most of these studies are based on the EBV-negative NPC cell lines established at 10–20 years ago. Until now, only a few NPC cell lines, such as C666-1, stably harboring the EBV genome were used as EBV-positive NPC model.3 In addition to the established NPC cell lines, several EBV-positive NPC transplants in nude mice (e.g. C15, C17, xeno-666, xeno-2117 and xeno-1915) were used for identifying the genetic alterations and delineating aberrant signal transduction pathways in NPC.4, 5 Because of the difficulties in manipulating target gene expression in these in vivo models, they are rarely used in functional studies. Recently, we have performed the array-based CGH analysis on a number of NPC cell lines, xenografts and primary tumors.6 We observed similar copy number aberrant patterns in some EBV-negative cell lines. Furthermore, these cell lines show distinct transcription profiles from the C666-1 cell line and NPC xenografts in microarray analysis (unpublished observations). It raised the concern for the identity of tumor lines used in NPC study. Thus, we investigated the authenticity of a number of NPC cell lines (C666-1, HK-1, CNE-1 and CNE-2), xenografts (xeno-666, xeno-2117, xeno-1915, C15 and C17) and immortalized nasopharyngeal epithelial cell lines (NP69 and NP460), which are commonly included in our previous works.1 The DNA samples from these tumors were extracted and subjected to DNA fingerprinting analysis using the AmpF/STR Identifiler® PCR Amplification Kit (Applied Biosystems, Foster City, USA). A total of 15 short tandem repeat (STR) loci (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, TH01, TPOX, CSF1P0, D2S1338, D19S433) were co-amplified in each sample and detected on an ABI 3100 Genetic Analyzer according to the manufacturer's protocol. Amelogenin loci at the sex chromosomes was also examined. The data were analyzed and allele(s) of each locus were determined by GeneScan and GeneMapper™ ID Software (Applied Biosystems, Foster City, USA). The STR profiles of NPC tumors and immortalized nasopharyngeal epithelial cell lines are shown in Table I. Distinct STR profiles were found in HK-1, NP69, NP460 and all EBV-positive xenografts (xeno-666, xeno-2117, xeno-1915, C15 and C17). The STR profile of xeno-666 and its devised cell line C666-1 are almost identical.3 In C666-1 cells, multiple alleles of D8S1179 and D21S11 loci were observed. It may be due to the microsatellite instability feature of this cell line. Surprisingly, we found high similarity between the STR profiles of 2 EBV-negative cell lines, CNE-1 and CNE-2. Complete identical alleles were detected in 12 out of 15 loci. Compared to CNE-1, gain of an extra allele at TPOX and loss of 1 allele at FGA and THO1 was observed in CNE-2. The minor allelic variations may be due to genetic instability or chromosomal aberrant during long-term in vivo culture. The finding implies that these 2 widely used EBV-negative cell lines are genetically identical and should be derived from a single cell line. Because many studies have shown that a number of cell lines were contaminated with HeLa,7 we then investigated whether CNE-1 and CNE-2 are contaminated with this common cell line. HeLa is a cell line devised from the glandular cancer of cervix, which contains the HPV-18 genome. Nevertheless, HPVs have not been detected in primary NPC tumors. We have examined the presence of E6/E7 sequence of HPV-18 in all NPC samples by PCR analysis. Interestingly, HPV-18 sequences were only found in the CNE-1 and CNE-2 cells (Fig. 1). Furthermore, we examined whether the STR profile of the HeLa is identical to that of the 2 EBV-negative NPC cell lines. The STR profile of HeLa determined was completely matched with that listed in the ATCC datasheet. While we compared the STR profiles among HeLa, CNE-1 and CNE-2, we noted that the CNE-1 and CNE-2 profiles contain at least 1 identical allele from HeLa for the 15 loci examined. Interestingly, in several loci (e.g. vWA, D8S1179, D7S820, CSF190, D19S433), only 1 of 2 alleles from HeLa was detected in the CNE-1 and CNE-2 genomes. The presence of partial genome of HeLa in CNE-1 and CNE-2 excludes the possibility that the cell lines are simply cross-contaminants of HeLa cells. Our previous cytogenetic study also did not found 2 heterogenous populations with distinct karyotypes in these EBV-negative NPC cell lines.8 Moreover, CNE-1 and CNE-2 contain identical p53 mutation (p53 Thr280), which is not found in HeLa. A specific p16 mutation (splicing acceptor site of exon 2) was also detected in CNE-1 and CNE-2, but not HeLa cells. Thus, we postulate that the STR profiles of these 2 EBV-negative NPC cell lines could be generated from fusion of HeLa with an unknown cell line by somatic cell hybridization. We have previously found that that there is no expression of CD19, a B-cell marker, in these 2 cell lines. It is likely that these cell lines are not somatic cell hybrid of HeLa and Burkitt's lymphoblastoid cells which are commonly used for the study of EBV function in epithelial cells, probably that hybrid was generated by contamination and fusion of HeLa with an NPC cell line originally reported. HPV18 and EBV infection in NPC cell lines and xenografts. By PCR analysis, E6/E7 sequences of HPV18 were detected in HeLa and 2 EBV-negative NPC cell lines, CNE-1 and CNE-2. The LMP-1 sequence was shown in C666-1 cells and all NPC xenografts (xeno-666, xeno-2117, xeno-1915, C15 and C17). The HPV18 and EBV sequences were not found in the well-differentiated NPC cell line HK-1. According to the published reports, CNE-1 and CNE-2 were originally established from the well-differentiated squamous carcinoma and poorly differentiated carcinoma, respectively.9, 10 They were derived from the tumors of 2 different Chinese patients at approximately 20 years ago. The CNE-2 was reported to be EBV-positive initially and the EBV genome was lost during passage. To confirm that the contamination is not unique in our laboratories, we examined independent CNE-1 and CNE-2 samples from other laboratories in Hong Kong and Singapore. Similar STR profiles were observed in all these samples although some minor allelic variations were present (Table I). The distinct allelic variations suggest that the cell lines may passage independently for a long period. We believe that the contamination occurred before early 1990s. Sun et al.11 have already mentioned that the CNE-1 and CNE-2 used in their laboratory were arisen from a single individual although the cell lines are different in morphology and in degree of tumorigenicity. Moreover, a specific p53 Thr280 mutation in both CNE-1 and CNE-2 were reported independently by them and our group at that time. The false EBV-negative NPC cell lines may be widespread and misused for NPC study under various names for decades. Aside from numerous biological and molecular studies, these EBV-negative cell lines were also used as in vitro NPC model for evaluating the efficiency of the therapeutic agents. Our major concern is that study on these false NPC cell lines may lead to invalid conclusions. Several controversial issues in the field may be generated from these misleading data. For examples, the p53 and p16 mutations identified in these cell lines are rare in primary NPC.11, 12 The genetic and epigenetic alterations reported in these cells should be evaluated in primary NPC and other EBV-positive NPC tumor lines. Furthermore, the problems may be inferior in the studies of the signal transduction and drug response in NPC cells. Because of different cell type microenvironment and the expression of HPV18 latent products, the findings obtained are not relevant to the EBV-positive NPC cells. The concerns for the misidentified cancer cell lines are arising in the science community. There are up to 36% of the cell lines misidentified or contaminated.7, 13, 14 Because NPC is rare in Western country, no study for authenticating NPC cell lines has been reported previously. Aside from CNE-1 and CNE-2, we are recruiting other EBV-negative NPC cell lines from different sources for DNA fingerprinting analysis. It is noted that this "CNE-1" STR profile occurs commonly in the "EBV-negative NPC cell lines" (e.g. HNE-1, HNE-2) although the finding needs to be confirmed in the independent samples from various sources. However, it is important for research groups working on NPC to investigate the authenticity of NPC cell lines used in their experiments. We strongly suggest that the NPC cell lines should be subjected to PCR analysis of HPV-18 and genotyping by STR analysis for verification. The methods are simple, reliable and commercially available. The genotyping information must be compared to the STR profiles of CNE1, CNE2 and NPC tumor lines reported here. Furthermore, the specific p53 and p16 mutations identified in our previous studies may also serve as markers for these contaminants.11, 12 It is also important to verify the identities of authenticated NPC cell lines regularly to exclude contamination during passage. We concluded that a single HeLa related somatic cell hybrid has been misidentified as EBV-negative NPC cell lines, which have been widely used in NPC research. Authentication of the tumor lines in NPC studies is needed to consolidate the relevant information on this unique EBV-positive epithelial cancer. Yours sincerely, Sylvia Yat-Yee Chan, Kwong-Wai Choy, Sai-Wah Tsao, Qian Tao, Tao Tang, Grace Ting-Yun Chung, Kwok-Wai Lo.
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