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Interplay between EBV infection and acquired genetic alterations during nasopharyngeal carcinoma (NPC) development remains vague. Here we report a comprehensive genomic analysis of 70 NPCs, combining whole-genome sequencing (WGS) of microdissected tumor cells with EBV oncogene expression to reveal multiple aspects of cellular-viral co-operation in tumorigenesis. Genomic aberrations along with EBV-encoded LMP1 expression underpin constitutive NF-κB activation in 90% of NPCs. A similar spectrum of somatic aberrations and viral gene expression undermine innate immunity in 79% of cases and adaptive immunity in 47% of cases; mechanisms by which NPC may evade immune surveillance despite its pro-inflammatory phenotype. Additionally, genomic changes impairing TGFBR2 promote oncogenesis and stabilize EBV infection in tumor cells. Fine-mapping of CDKN2A/CDKN2B deletion breakpoints reveals homozygous MTAP deletions in 32-34% of NPCs that confer marked sensitivity to MAT2A inhibition. Our work concludes that NPC is a homogeneously NF-κB-driven and immune-protected, yet potentially druggable, cancer., The genomic characterisation of nasopharyngeal carcinoma (NPC) remains crucial. Here, the authors perform whole-genome sequencing for 70 NPCs with EBV gene expression, report the somatic alterations and EBV-mediated effects converging on NF-κB activation and immune escape and identify targetable homozygous MTAP deletions.

Nasopharyngeal carcinoma (NPC) is an Epstein‐Barr virus (EBV)‐associated epithelial malignancy. The high expression of BART‐miRNAs (miR‐BARTs) during latent EBV infection in NPC strongly supports their pathological importance in cancer progression. Recently, we found that several BART‐miRNAs work co‐operatively to modulate the DNA damage response (DDR) by reducing Ataxia‐telangiectasia‐mutated (ATM) activity. In this study, we further investigated the role of miR‐BARTs on DDR. The immunohistochemical study showed that the DNA repair gene, BRCA1, is consistently down‐regulated in primary NPCs. Using computer prediction programs and a series of reporter assays, we subsequently identified the negative regulatory role of BART2‐3p, BART12, BART17‐5p and BART19‐3p in BRCA1 expression. The ectopic expression of these four miR‐BARTs suppressed endogenous BRCA1 expression in EBV‐negative epithelial cell lines, whereas BRCA1 expression was enhanced by repressing endogenous miR‐BARTs activities in C666‐1 cells. More importantly, suppressing BRCA1 expression in nasopharyngeal epithelial cell lines using miR‐BART17‐5p and miR‐BART19‐3p mimics reduced the DNA repair capability and increased the cell sensitivity to the DNA‐damaging chemotherapeutic drugs, cisplatin and doxorubicin. Our findings suggest that miR‐BARTs play a novel role in DDR and may facilitate the development of effective NPC therapies.

Fibroblast growth factor receptor type 2 (FGFR2) has emerged as a key oncogenic factor that regulates gastric cancer (GC) progression, but the underlying mechanism of FGF–FGFR2 signaling pathway remains largely unknown. To identify the potential molecular mechanisms of the oncogenic FGFR2 in gastric carcinogenesis and convey a novel therapeutic strategy, we profiled the FGFR alterations and analyzed their clinical associations in TCGA and Hong Kong GC cohorts. We found that FGFR2 overexpression in GC cell lines and primary tumors predicted poor survival and was associated with advanced stages of GC. Functionally, growth abilities and cell cycle progression of GC were inhibited by inactivation of ERK–MAPK signal transduction after FGFR2 knockdown, while apoptosis was promoted. Meanwhile, the first-line anti-cancer drug sensitivity was enhanced. RNA-seq analysis further revealed that YAP1 signaling serves as a significant downstream modulator and mediates the oncogenic signaling of FGFR2. When stimulating FGFR2 by rhFGF18, we observed intensified F-actin, nuclear accumulation of YAP1, and overexpression of YAP1 targets, but these effects were attenuated by either FGFR2 depletion or AZD4547 administration. Additionally, the FGF18–FGFR2 signaling upregulated YAP1 expression through activating c-Jun, an effector of MAPK signaling. In our cohort, 28.94% of GC cases were characterized as FGFR2, c-Jun, and YAP1 co-positive and demonstrated worse clinical outcomes. Remarkably, we also found that co-targeting FGFR2 and YAP1 by AZD4547 and Verteporfin synergistically enhanced the antitumor effects in vitro and in vivo. In conclusion, we have identified the oncogenic FGF–FGFR2 regulates YAP1 signaling in GC. The findings also highlight the translational potential of FGFR2–c-Jun–YAP1 axis, which may serve as a prognostic biomarker and therapeutic target for GC.

Hippo signaling functions to limit cellular growth, but the aberrant nuclear accumulation of its downstream YAP1 leads to carcinogenesis. YAP1/TEAD complex activates the oncogenic downstream transcription, such as CTGF and c-Myc. How YAP1 is protected in the cytoplasm from ubiquitin-mediated degradation remains elusive. In this study, a member of Angiomotin (Motin) family, AMOTL1 (Angiomotin Like 1), was screened out as the only one to promote YAP1 nuclear accumulation by several clinical cohorts, which was further confirmed by the cellular functional assays. The interaction between YAP1 and AMOTL1 was suggested by co-immunoprecipitation and immunofluorescent staining. The clinical significance of the AMOTL1–YAP1–CTGF axis in gastric cancer (GC) was analyzed by multiple clinical cohorts. Moreover, the therapeutic effect of targeting the oncogenic axis was appraised by drug-sensitivity tests and xenograft-formation assays. The upregulation of AMOTL1 is associated with unfavorable clinical outcomes of GC, and knocking down AMOTL1 impairs its oncogenic properties. The cytoplasmic interaction between AMOTL1 and YAP1 protects each other from ubiquitin-mediated degradation. AMOTL1 promotes YAP1 translocation into the nuclei to activate the downstream expression, such as CTGF. Knocking down AMOTL1, YAP1, and CTGF enhances the therapeutic efficacies of the first-line anticancer drugs. Taken together, AMOTL1 plays an oncogenic role in gastric carcinogenesis through interacting with YAP1 and promoting its nuclear accumulation. A combination of AMOTL1, YAP1, and CTGF expression might serve as a surrogate of Hippo activation status. The co-activation of the AMOTL1/YAP1–CTGF axis is associated with poor clinical outcomes of GC patients, and targeting this oncogenic axis may enhance the chemotherapeutic effects.

Simple Summary Nasopharyngeal cancer (NPC) is a malignant epithelial tumor endemic to parts of Asia and associated with infection by the Epstein–Barr virus (EBV) in these regions. The cancer is often detected at a late stage which is associated with poor outcomes (63% 5-year survival). Advances for the management of this disease have remained largely stagnant and treatment relies primarily on radiotherapy and chemotherapy, as well as surgery when indicated. Nevertheless, our understanding of its underlying biology has grown rapidly in the past two decades, laying the foundation for the development of improved therapeutics which have the potential to improve outcomes. This review offers a comprehensive, up-to-date summary of this disease, with a focus on the role of somatostatin receptor 2 (SSTR2) in NPC and how this increased knowledge may lead to improved diagnosis and management of this disease. Abstract Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor, most commonly located in the pharyngeal recess and endemic to parts of Asia. It is often detected at a late stage which is associated with poor prognosis (5-year survival rate of 63%). Treatment for this malignancy relies predominantly on radiotherapy and/or systemic chemotherapy, which can be associated with significant morbidity and impaired quality of life. In endemic regions NPC is associated with infection by Epstein–Barr virus (EBV) which was shown to upregulate the somatostatin receptor 2 (SSTR2) cell surface receptor. With recent advances in molecular techniques allowing for an improved understanding of the molecular aetiology of this disease and its relation to SSTR2 expression, we provide a comprehensive and up-to-date overview of this disease and highlight the emergence of SSTR2 as a key tumor biomarker and promising target for imaging and therapy.

Epstein-Barr virus (EBV) has been recently found to generate novel circular RNAs (circRNAs) through backsplicing. However, comprehensive catalogs of EBV circRNAs in other cell lines and their functional characterization are still lacking. In this study, we have identified a list of putative EBV circRNAs in GM12878, an EBV-transformed lymphoblastoid cell line, with a significant majority encoded from the EBV latent genes. A novel EBV circRNA derived from the exon 5 of LMP-2 gene which exhibited highest prevalence, was further validated using RNase R assay and Sanger sequencing. This circRNA, which we term circLMP-2_e5, can be universally detected in a panel of EBV-positive cell lines modelling different latency programs. It ranges from lower expression in nasopharyngeal carcinoma (NPC) cells to higher expression in B cells, and is localized to both the cytoplasm and the nucleus. We provide evidence that circLMP-2_e5 is expressed concomitantly with its cognate linear LMP-2 RNA upon EBV lytic reactivation, and may be produced as a result of exon skipping, with its circularization possibly occurring without the involvement of cis elements in the short flanking introns. Furthermore, we show that circLMP-2_e5 is not involved in regulating cell proliferation, host innate immune response, its linear parental transcripts, or EBV lytic reactivation. Taken together, our study expands the current repertoire of putative EBV circRNAs, broadens our understanding of the biology of EBV circRNAs, and lays the foundation for further investigation of their function in the EBV life cycle and disease development.

Epstein-Barr virus (EBV) infects more than 90% of humans worldwide and establishes lifelong latent infection in the hosts. It is closely associated with endemic forms of a wide range of human cancers and directly contributes to the formation of some. Despite its critical role in cancer development, no EBV- or EBV latent protein-targeted therapy is available. The EBV-encoded latent protein, Epstein-Barr nuclear antigen 1 (EBNA1), is expressed in all EBV-associated tumors and acts as the only latent protein in some of these tumors. This versatile protein functions in the maintenance, replication, and segregation of the EBV genome and can therefore serve as an attractive therapeutic target to treat EBV-associated cancers. In the last decades, efforts have been made for designing specific EBNA1 inhibitors to decrease EBNA1 expression or interfere with EBNA1-dependent functions. In this review, we will briefly introduce the salient features of EBNA1, summarize its functional domains, and focus on the recent developments in the identification and design of EBNA1 inhibitors related to various EBNA1 domains as well as discuss their comparative merits.

Nasopharyngeal carcinoma (NPC) is a malignancy endemic to East Asia and is caused by Epstein-Barr Virus (EBV)-mediated cancerous transformation of epithelial cells. The standard of care treatment for NPC involves radiation and chemotherapy. While treatment outcomes continue to improve, up to 50% of patients can be expected to recur by five years, and additional innovative treatment options are needed. We posit that a potential way to do this is by targeting the underlying cause of malignant transformation, namely EBV. One method by which EBV escapes immune surveillance is by undergoing latent phase replication, during which EBV expression of immunogenic proteins is reduced. However, chemoradiation is known to drive conversion of EBV from a latent to a lytic phase. This creates an opportunity for the targeting of EBV-infected cells utilizing anti-viral drugs. Indeed, we found that combining acyclovir with cisplatin and radiation significantly decreases the viability of the EBV-infected C666-1 cell line. Western blot quantification revealed a resultant increase of thymidine kinase (TK) and apoptosis-inducing mediators, cleaved PARP (cPARP) and phosphorylated ERK (pERK). These studies suggest that the addition of anti-viral drugs to frontline chemoradiation may improve outcomes in patients treated for EBV-related NPC and future in vivo and clinical studies are needed.

Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) infection in regions in which it is endemic, including Southern China and Southeast Asia. The high mortality rates of NPC patients with advanced and recurrent disease highlight the urgent need for effective treatments. While recent genomic studies have revealed few druggable targets, the unique interaction between the EBV infection and host cells in NPC strongly implies that targeting EBV may be an efficient approach to cure this virus-associated cancer. Key features of EBV-associated NPC are the persistence of an episomal EBV genome and the requirement for multiple viral latent gene products to enable malignant transformation. Many translational studies have been conducted to exploit these unique features to develop pharmaceutical agents and therapeutic strategies that target EBV latent proteins and induce lytic reactivation in NPC. In particular, inhibitors of the EBV latent protein EBNA1 have been intensively explored, because of this protein's essential roles in maintaining EBV latency and viral genome replication in NPC cells. In addition, recent advances in chemical bioengineering are driving the development of therapeutic agents targeting the critical functional regions of EBNA1. Promising therapeutic effects of the resulting EBNA1-specific inhibitors have been shown in EBV-positive NPC tumors. The efficacy of multiple classes of EBV lytic inducers for NPC cytolytic therapy has also been long investigated. However, the lytic-induction efficiency of these compounds varies among different EBV-positive NPC models in a cell-context-dependent manner. In each tumor, NPC cells can evolve and acquire somatic changes to maintain EBV latency during cancer progression. Unfortunately, the poor understanding of the cellular mechanisms regulating EBV latency-to-lytic switching in NPC cells limits the clinical application of EBV cytolytic treatment. In this review, we discuss the potential approaches for improvement of the above-mentioned EBV-targeting strategies.

Fibroblast growth factors (FGFs) and their receptors are significant components during fundamental cellular processes. FGF18 plays a distinctive role in modulating the activity of both tumor cells and tumor microenvironment. This study aims to comprehensively investigate the expression and functional role of FGF18 in gastric cancer (GC) and elucidate its regulatory mechanisms. The upregulation of FGF18 was detected in seven out of eleven (63.6%) GC cell lines. In primary GC samples, FGF18 was overexpressed in genomically stable and chromosomal instability subtypes of GC and its overexpression was associated with poor survival. Knocking down FGF18 inhibited tumor formation abilities, induced G1 phase cell cycle arrest and enhanced anti-cancer drug sensitivity. Expression microarray profiling revealed that silencing of FGF18 activated ATM pathway but quenched TGF-β pathway. The key factors that altered in the related signaling were validated by western blot and immunofluorescence. Meanwhile, treating GC cells with human recombinant FGF18 or FGF18-conditioned medium accelerated tumor growth through activation of ERK-MAPK signaling. FGF18 was further confirmed to be a direct target of tumor suppressor, miR-590-5p. Their expressions showed a negative correlation in primary GC samples and more importantly, re-overexpression of FGF18 partly abolished the tumor-suppressive effect of miR-590-5p. Our study not only identified that FGF18 serves as a novel prognostic marker and a therapeutic target in GC but also enriched the knowledge of FGF-FGFR signaling during gastric tumorigenesis.

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalent in southern China and Southeast Asia. It is consistently associated with latent Epstein-Barr virus (EBV) infection. In NPC, miR-BARTs, the EBV-encoded miRNAs derived from BamH1-A rightward transcripts, are abundantly expressed and contribute to cancer development by targeting various cellular and viral genes. In this study, we establish a comprehensive transcriptional profile of EBV-encoded miRNAs in a panel of NPC patient-derived xenografts and an EBV-positive NPC cell line by small RNA sequencing. Among the 40 miR-BARTs, predominant expression of 22 miRNAs was consistently detected in these tumors. Among the abundantly expressed EBV-miRNAs, BART5-5p, BART7-3p, BART9-3p, and BART14-3p could negatively regulate the expression of a key DNA double-strand break (DSB) repair gene, ataxia telangiectasia mutated (ATM), by binding to multiple sites on its 3'-UTR. Notably, the expression of these four miR-BARTs represented more than 10% of all EBV-encoded miRNAs in tumor cells, while downregulation of ATM expression was commonly detected in all of our tested sequenced samples. In addition, downregulation of ATM was also observed in primary NPC tissues in both qRT-PCR (16 NP and 45 NPC cases) and immunohistochemical staining (35 NP and 46 NPC cases) analysis. Modulation of ATM expression by BART5-5p, BART7-3p, BART9-3p, and BART14-3p was demonstrated in the transient transfection assays. These findings suggest that EBV uses miRNA machinery as a key mechanism to control the ATM signaling pathway in NPC cells. By suppressing these endogenous miR-BARTs in EBV-positive NPC cells, we further demonstrated the novel function of miR-BARTs in inhibiting Zta-induced lytic reactivation. These findings imply that the four viral miRNAs work co-operatively to modulate ATM activity in response to DNA damage and to maintain viral latency, contributing to the tumorigenesis of NPC. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The γ-herpesviruses, EBV and KSHV, are closely associated with a number of human cancers. While the signal transduction pathways exploited by γ-herpesviruses to promote cell growth, survival and transformation have been reported, recent studies have uncovered the impact of γ-herpesvirus infection on host cell metabolism. Here, we review the mechanisms used by γ-herpesviruses to induce metabolic reprogramming in host cells, focusing on their ability to modulate the activity of metabolic regulators and manipulate metabolic pathways. While γ-herpesviruses alter metabolic phenotypes as a means to support viral infection and long-term persistence, this modulation can inadvertently contribute to cancer development. Strategies that target deregulated metabolic phenotypes induced by γ-herpesviruses provide new opportunities for therapeutic intervention.

Undifferentiated nasopharyngeal carcinoma (NPC) is a cancer with high metastatic potential that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the functional contribution of sphingosine-1-phosphate (S1P) signalling to the pathogenesis of NPC. We show that EBV infection or ectopic expression of the EBV-encoded latent genes (EBNA1, LMP1 and LMP2A) can up-regulate sphingosine kinase 1 (SPHK1), the key enzyme that produces S1P, in NPC cell lines. Exogenous addition of S1P promotes the migration of NPC cells through the activation of AKT; shRNA knockdown of SPHK1 resulted in a reduction in the levels of activated AKT and inhibition of cell migration. We also show that S1P receptor 3 (S1PR3) mRNA is over-expressed in EBV-positive NPC patient-derived xenografts and a subset of primary NPC tissues, and that knockdown of S1PR3 suppressed the activation of AKT and the S1P-induced migration of NPC cells. Taken together, our data point to a central role for EBV in mediating the oncogenic effects of S1P in NPC and identify S1P signalling as a potential therapeutic target in this disease.

Head and neck squamous cell carcinoma (HNSCC) lacks predictive biomarkers for drug responses. By targeted sequencing, we identified two MAPK1 mutations in recurrent HNSCC, MAPK1p.D321N, and p.R135K. We previously reported an exceptional erlotinib responder with MAPK1p.E322K. Here, by in silico and drug studies, we determined functions of these two recurrence-associated MAPK1 mutations. Residues D321, R135, and E322 are in 3D proximity. MAPK1p.D321N drives marked in vivo erlotinib sensitivity, while p.R135K’s effect is moderate.

Nasopharyngeal carcinoma (NPC), also named the Cantonese cancer, is a unique cancer with strong etiological association with infection of the Epstein-Barr virus (EBV). With particularly high prevalence in Southeast Asia, the involvement of EBV and genetic aberrations contributive to NPC tumorigenesis have remained unclear for decades. Recently, genomic analysis of NPC has defined it as a genetically homogeneous cancer, driven largely by NF-κB signaling caused by either somatic aberrations of NF-κB negative regulators or by overexpression of the latent membrane protein 1 (LMP1), an EBV viral oncoprotein. This represents a landmark finding of the NPC genome. Exome and RNA sequencing data from new EBV-positive NPC models also highlight the importance of PI3K pathway aberrations in NPC. We also realize for the first time that NPC mutational burden, mutational signatures, MAPK/PI3K aberrations, and MHC Class I gene aberrations, are prognostic for patient outcome. Together, these multiple genomic discoveries begin to shape the focus of NPC therapy development. Given the challenge of NF-κB targeting in human cancers, more innovative drug discovery approaches should be explored to target the unique atypical NF-κB activation feature of NPC. Our next decade of NPC research should focus on further identification of the -omic landscapes of recurrent and metastatic NPC, development of gene-based precision medicines, as well as large-scale drug screening with the newly developed and well-characterized EBV-positive NPC models. Focused preclinical and clinical investigations on these major directions may identify new and effective targeting strategies to further improve survival of NPC patients.

The lack of representative nasopharyngeal carcinoma (NPC) models has seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. Here we report the successful growth of five NPC patient-derived xenografts (PDXs) from fifty-eight attempts of transplantation of NPC specimens into NOD/SCID mice. The take rates for primary and recurrent NPC are 4.9% and 17.6%, respectively. Successful establishment of a new EBV-positive NPC cell line, NPC43, is achieved directly from patient NPC tissues by including Rho-associated coiled-coil containing kinases inhibitor (Y-27632) in culture medium. Spontaneous lytic reactivation of EBV can be observed in NPC43 upon withdrawal of Y-27632. Whole-exome sequencing (WES) reveals a close similarity in mutational profiles of these NPC PDXs with their corresponding patient NPC. Whole-genome sequencing (WGS) further delineates the genomic landscape and sequences of EBV genomes in these newly established NPC models, which supports their potential use in future studies of NPC., The lack of appropriate models restricts pre-clinical research for nasopharyngeal carcinoma (NPC). Here the authors report the development and characterization of NPC patient-derived xenografts (PDXs), and EBV positive NPC cell line from patient tumor, and suggest their potential use in future NPC research.

Correction for “Reactivation of Epstein–Barr virus by a dualresponsive fluorescent EBNA1-targeting agent with Zn2+- chelating function,” by Lijun Jiang, Hong Lok Lung, Tao Huang, Rongfeng Lan, Shuai Zha, Lai Sheung Chan, Waygen Thor, Tik-Hung Tsoi, Ho-Fai Chau, Cecilia Borestrom, Steven L. Cobb, Sai Wah Tsao, Zhao-Xiang Bian, Ga-Lai Law, Wing-Tak Wong, William Chi-Shing Tai, Wai Yin Chau, Yujun Du, Lucas Hao Xi Tang, Alan Kwok Shing Chiang, Jaap M. Middeldorp, Kwok-Wai Lo, Nai Ki Mak, Nicholas J. Long, and Ka-Leung Wong, which was first published December 10, 2019; 10.1073/pnas.1915372116 (Proc. Natl. Acad. Sci. U.S.A. 116, 26614–26624). The authors note that Fig. 6 appeared incorrectly. Part of panel D of the published figure was inadvertently omitted. The corrected figure and its legend appear below. (Figure Presented).

Autophagy, a conserved cellular mechanism, is manipulated by a number of viruses for different purposes. We previously demonstrated that an iron-chelator-like small compound, C7, reactivates Epstein-Barr virus (EBV) lytic cycle by activating the ERK1/2-autophagy axis in epithelial cancers. Here, we aim to identify the specific stage of autophagy required for EBV lytic reactivation, determine the autophagy dependency of EBV lytic inducers including histone deacetylase inhibitor (HDACi) and C7/iron chelators, for EBV lytic reactivation and measure the combinatorial effects of these types of lytic inducers in nasopharyngeal carcinoma (NPC). Inhibition of autophagy initiation by 3-MA and autolysosome formation by chloroquine demonstrated that only autophagy initiation is required for EBV lytic reactivation. Gene knockdown of various autophagic proteins such as beclin-1, ATG5, ATG12, ATG7, LC3B, ATG10, ATG3 and Rab9, revealed the importance of ATG5 in EBV lytic reactivation. 3-MA could only abrogate lytic cycle induction by C7/iron chelators but not by HDACi, providing evidence for autophagy-dependent and independent mechanisms in EBV lytic reactivation. Finally, the combination of C7 and SAHA at their corresponding reactivation kinetics enhanced EBV lytic reactivation. These findings render new insights in the mechanisms of EBV lytic cycle reactivation and stimulate a rational design of combination drug therapy against EBV-associated cancers.

Background Patients with colorectal cancer (CRC) have a high incidence of regional and distant metastases. Although metastasis is the main cause of CRC-related death, its molecular mechanisms remain largely unknown. Methods Using array-CGH and expression microarray analyses, changes in DNA copy number and mRNA expression levels were investigated in human CRC samples. The mRNA expression level of RASAL2 was validated by qRT-PCR, and the protein expression was evaluated by western blot as well as immunohistochemistry in CRC cell lines and primary tumors. The functional role of RASAL2 in CRC was determined by MTT proliferation assay, monolayer and soft agar colony formation assays, cell cycle analysis, cell invasion and migration and in vivo study through siRNA/shRNA mediated knockdown and overexpression assays. Identification of RASAL2 involved in hippo pathway was achieved by expression microarray screening, double immunofluorescence staining and co-immunoprecipitation assays. Results Integrated genomic analysis identified copy number gains and upregulation of RASAL2 in metastatic CRC. RASAL2 encodes a RAS-GTPase-activating protein (RAS-GAP) and showed increased expression in CRC cell lines and clinical specimens. Higher RASAL2 expression was significantly correlated with lymph node involvement and distant metastasis in CRC patients. Moreover, we found that RASAL2 serves as an independent prognostic marker of overall survival in CRC patients. In vitro and in vivo functional studies revealed that RASAL2 promoted tumor progression in both KRAS/NRAS mutant and wild-type CRC cells. Knockdown of RASAL2 promoted YAP1 phosphorylation, cytoplasm retention and ubiquitination, therefore activating the hippo pathway through the LATS2/YAP1 axis. Conclusions Our findings demonstrated the roles of RASAL2 in CRC tumorigenesis as well as metastasis, and RASAL2 exerts its oncogenic property through LATS2/YAP1 axis of hippo signaling pathway in CRC. Electronic supplementary material The online version of this article (10.1186/s12943-018-0853-6) contains supplementary material, which is available to authorized users.

Epstein-Barr virus (EBV) infects more than 90% of the adult human population. Undifferentiated nasopharyngeal carcinoma (NPC) is common in Southeast Asia, with a particularly high incidence among southern Chinese. The EBV genome can be detected in practically all cancer cells in undifferentiated NPC. The role of EBV in pathogenesis of undifferentiated NPC remains elusive. NPC cell lines are known to be difficult to establish in culture. The EBV+ve NPC cell lines, even if established in culture, rapidly lost their EBV episomes upon prolonged propagation. At present, the C666-1 NPC cell line, which is defective in lytic EBV reactivation, is the only EBV+ve NPC cell line available for NPC and EBV research. The need to establish new and representative NPC cell lines is eminent for NPC and EBV research. In this study, we report the use of the Rho-associated kinase inhibitor (Y-27632) has facilitated the establishment of a new EBV+ve NPC cell line from an earlier established NPC xenograft, C17. The C17 cell line was tumorigenic in immune-deficient mice (NOD/SCID). It retained the EBV episomes and could be induced to undergo productive lytic reactivation of EBV to generate infectious virus particles. The C17 cell line represents a new investigative tool for NPC and EBV studies. The ability of C17 to undergo lytic reactivation is unique and opens up the opportunity to examine regulation of latent and lytic infection of EBV and their contributions to NPC pathogenesis.

Epstein–Barr virus (EBV) is associated with multiple types of human cancer, including lymphoid and epithelial cancers. The closest association with EBV infection is seen in undifferentiated nasopharyngeal carcinoma (NPC), which is endemic in the southern Chinese population. A strong association between NPC risk and the HLA locus at chromosome 6p has been identified, indicating a link between the presentation of EBV antigens to host immune cells and NPC risk. EBV infection in NPC is clonal in origin, strongly suggesting that NPC develops from the clonal expansion of a single EBV-infected cell. In epithelial cells, the default program of EBV infection is lytic replication. However, latent infection is the predominant mode of EBV infection in NPC. The establishment of latent EBV infection in pre-invasive nasopharyngeal epithelium is believed to be an early stage of NPC pathogenesis. Recent genomic study of NPC has identified multiple somatic mutations in the upstream negative regulators of NF-κB signalling. Dysregulated NF-κB signalling may contribute to the establishment of latent EBV infection in NPC. Stable EBV infection and the expression of latent EBV genes are postulated to drive the transformation of pre-invasive nasopharyngeal epithelial cells to cancer cells through multiple pathways. This article is part of the themed issue ‘Human oncogenic viruses’.

Nasopharyngeal carcinoma (NPC) is rare worldwide but common in southern China, including Hong Kong. The endemic NPC among southern Chinese is typically non-keratinizing carcinoma, which is almost 100% associated with Epstein-Barr virus (EBV) infection. Patient-derived xenografts (PDXs), given their close resemblance with patient tumors, serve as important models in preclinical evaluation for novel therapeutic drugs. For unclear reasons, it has been difficult to establish NPC PDXs in vivo and cell lines in vitro. Currently, there are few NPC PDXs available for research, including X666, X2117, C15, C17 and C18. However, all of them have been passaged in nude mice for over 25 years and may deviate from their original NPC tumors in patients. In vitro, C666-1 is the only EBV-positive (EBV+ve) NPC cell line which has been used extensively in investigations. C666-1 was established from the NPC xenograft (X666), which had been propagated for a long period of time. Most if not all the other previously reported NPC cell lines have lost their EBV episomes upon in vitro propagation. Furthermore, many of these reported NPC cell lines have been shown with genetic contamination of HeLa cells. Hence, their applications in NPC studies are limited. The lack of representative nasopharyngeal carcinoma (NPC) models has also seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. In this study, we report the successful establishment and comprehensive characterization of four new NPC PDXs (all EBV+ve) and three NPC cell lines (one EBV+ve; two EBV-ve). These newly established EBV+ve NPC PDXs and cell lines significantly recapitulate the mutation profiles of their original NPC tumors, and harbor common genetic alterations reported in NPC, which supports their potential applications in the investigations of NPC pathogenesis. We postulate that lytic EBV reactivation may be an intrinsic barrier to the successful establishment of EBV+ve NPC PDXs and cell lines. The ROCK inhibitor (Y-27632) was shown to be able to suppress epithelial differentiation and lytic reactivation of EBV in infected NPC cells. Establishment of a new EBV-positive NPC cell line, NPC43, was achieved directly from patient NPC tissues by including Y-27632 in culture medium. NPC43 cells exhibited tumorigenicity in immunodeficient mice, and could be induced to undergo EBV lytic reactivation with production of infectious visions. The establishment and characterization of new NPC PDXs and cell lines will provide valuable experimental tools for NPC and EBV research. Funding support: Research Grant Council, Hong Kong: AoE NPC grant (AoE/M-06/08), Theme-based Research Scheme grant (T12-401/13-R) and Collaborative Research Fund (C7027-16G), General Research Fund (106140069, 106160185, 17111516, 17110315) and HMRF grant (04151726, 13142201). Citation Format: Weitao Lin, Yim Ling Yip, Lin Jia, Wen Deng, Kwok Wai Lo, Chin Man Tsang, Maria Li Lung, George Sai Tsao. Establishment and characterization of new tumor xenografts and cancer cell lines from EBV positive nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 56.

The association of Epstein–Barr virus (EBV) infection with the development of nasopharyngeal carcinoma (NPC) is well established. Latent membrane protein 1 (LMP1), the major oncogene encoded by EBV, is believed to play a crucial role in NPC pathogenesis by virtue of its ability to constitutively activate multiple cell signalling pathways. The LKB1–AMPK pathway is a master regulator of cellular metabolism that, via modulation of energy metabolism, has tumour suppressor activity. In this study we identify a novel ability of LMP1 to inhibit the LKB1–AMPK pathway through phosphorylation of LKB1 at serine 428 with subsequent suppression of the phosphorylation of AMPK and its substrates, ACC and Raptor. We show that MEK/ERK–MAPK signalling, activated by the CTAR1 domain of LMP1, is responsible for LKB1–AMPK inactivation. In addition, reactivation of AMPK signalling by AMPK activator, AICAR, abolished LMP1-induced cellular transformation (proliferation and anchorage-independent growth) in nasopharyngeal epithelial cells. Immunohistochemical staining revealed that a low level of phosphorylated AMPK is common in primary NPC specimens, and that this correlated significantly with the expression of LMP1. AICAR treatment inhibited the proliferation and anchorage-independent growth of NPC cells as well as potentiating the cytotoxic effect of the chemotherapeutic drug 5-fluorouracil. The current findings demonstrate that LMP1-mediated AMPK inactivation contributes to the proliferation and transformation of epithelial cells, thereby implicating the LKB1–AMPK pathway in the EBV-driven pathogenesis of NPC. Our findings also suggest that AMPK activators could be used to enhance the efficacy of conventional chemotherapeutic agents in the treatment of local and metastatic NPC. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background 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. Methods 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2525-5) contains supplementary material, which is available to authorized users.

BACKGROUND:Nasopharyngeal carcinoma (NPC) is a peculiar Epstein Barr virus (EBV)-associated malignancy that is prevalent in South-East Asia. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) isomerizes specific phosphorylated amino acid residues, which makes it an important regulator in cell survival and apoptosis. In this study, we investigated the contribution made by PIN1 in NPC tumorigenesis and PIN1's potential role as a therapeutic target. METHODS:The expression of PIN1 was examined in a panel of NPC cell lines, xenografts and primary tumors. The functional roles of PIN1 in NPC cells were elucidated by the knockdown and overexpression of PIN1 in in vitro and in vivo nude mice models by siRNA and lenti-viral transfection, respectively. The antitumor effects of the PIN1 inhibitor Juglone in NPC cells were also evaluated. RESULTS:We revealed the consistent overexpression of PIN1 in almost all EBV-associated NPC cell lines, xenografts and primary tumors. PIN1 suppression was capable of inhibiting cyclin D1 expression and activating caspase-3 in NPC cells. It positively regulated NPC cell proliferation, colony formation and anchorage-independent growth. The inhibition of PIN1 suppressed tumor growth in vitro and in vivo. CONCLUSIONS:This study demonstrates the oncogenic role of PIN1 in NPC tumorigenesis, and shows that its overexpression can enhance tumor cell growth via the upregulation of cyclinD1. Our findings inform the development of novel treatments targeting PIN1 for NPC patients.

Nasopharyngeal carcinoma (NPC) is an aggressive head and neck cancer characterized by Epstein-Barr virus (EBV) infection and dense lymphocyte infiltration. The scarcity of NPC genomic data hinders the understanding of NPC biology, disease progression and rational therapy design. Here we performed whole-exome sequencing (WES) on 111 micro-dissected EBV-positive NPCs, with 15 cases subjected to further whole-genome sequencing (WGS), to determine its mutational landscape. We identified enrichment for genomic aberrations of multiple negative regulators of the NF-κB pathway, including CYLD, TRAF3, NFKBIA and NLRC5, in a total of 41% of cases. Functional analysis confirmed inactivating CYLD mutations as drivers for NPC cell growth. The EBV oncoprotein latent membrane protein 1 (LMP1) functions to constitutively activate NF-κB signalling, and we observed mutual exclusivity among tumours with somatic NF-κB pathway aberrations and LMP1-overexpression, suggesting that NF-κB activation is selected for by both somatic and viral events during NPC pathogenesis., Nasopharyngeal cancer is frequently characterized by Epstein-Barr virus infection. Here, using genomic analyses, the authors find that the tumours harbour mutations in genes involved in the NF-κB signalling pathway or overexpress a viral oncoprotein, latent membrane protein 1.

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer prevalent in south-east Asia and southern China, where it constitutes a significant health burden. Although the close association of NPC with Epstein-Barr virus (EBV) infection has been known for more than four decades, the exact role that EBV plays in the pathogenesis of this malignancy is still unclear. While NPC tumours are known to express a number of EBV-encoded proteins, they also express a large number of virus-encoded microRNAs (miRNAs), the most abundant of which are those encoded from the BamHI-A region of the viral genome: the so-called BART miRNAs. miRNAs are small non-coding mRNAs that negatively regulate the expression of various genes at the post-transcriptional level. Accumulating evidence suggests that miRNAs play important roles in tumourigenesis. Here, we review the role of EBV-encoded BART miRNAs in modulating apoptosis and host innate defence mechanisms and their contribution to NPC pathogenesis. The rationale and strategies for therapeutic targeting of BART miRNAs in EBV-infected NPC are also discussed.

Psychological stress-associated immune dysregulation has been shown to disrupt the steady state expression and reactivate latent herpes viruses. One such virus is the Epstein Barr virus (EBV), which is associated with several human malignancies. EBV infects >90% of people living in North America and persists for life in latently infected cells. Although several studies have shown that glucocorticoids (GCs) can directly induce reactivation of the latent virus, the mechanism of stress hormone involvement in the control of EBV gene expression is not well understood. In this study, we tested the hypothesis that GCs can induce the latent EBV genome to lytically replicate through the induction of the EBV immediate early gene BZLF1 which encodes the lytic transactivator protein ZEBRA. We show a dose-dependent upregulation of BZLF1 mRNA expression by hydrocortisone (HC) and dexamethasone (Dex) in Daudi cells, an EBV genome positive Burkitt’s lymphoma cell line, and Dex-induction of the early gene products BLLF3 (encoding for the EBV dUTPase) and BALF5 (encoding for the EBV DNA polymerase). We show that Daudi cells express glucocorticoid receptors (GR) that mediate Dex-dependent upregulation of BZLF1 mRNA levels. This effect was inhibited by both the glucocorticoid receptor antagonist RU486 and by cycloheximide. The results suggest that GCs, in addition to inducing stress-related immune dysregulation, can mediate latent EBV reactivation through the induction of the BZLF1 gene.

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalence in southern China and Southeast Asia. It is well-known to be associated with latent Epstein-Barr virus (EBV) infection. By using small RNA sequencing, we previously established a comprehensive miRNA profile in a panel of NPC samples and revealed that the EBV-encoded miRNAs derived from BamHI-A rightward transcripts (miR-BARTs) are abundantly expressed. The important roles of miR-BARTs in cancer development have been reported extensively. In the present study, we identified multiple putative binding sites of miR-BART5-5p, BART7-3p, BART9-3p and BART14-3p on the 3'-UTR of a critical DNA double-strand break (DSB) repair gene, Ataxia-Telangiectasia-Mutant (ATM). Notably, the expression of these 4 miR-BARTs represented more than 10% of all EBV-encoded miRNAs in NPC cells while downregulation of ATM expression was observed in our local primary NPC samples in both qRT-PCR and immunohistochemical (IHC) staining analysis. In addition, the abilities of miR-BARTs to downregulate ATM expression were demonstrated in the transient transfection experiments. By manipulating the miR-BARTs expression in epithelial cell lines, we further revealed that these four viral miRNAs could inhibit both ionizing radiation (IR)-induced ATM kinase activity and BZLF1-induced viral lytic reactivation. In conclusion, our findings suggest that miR-BART5-5p, BART7-3p, BART9-3p and BART14-3p work co-operatively to modulate DNA damage response and to maintain viral latency, contributing to the NPC tumorigenesis. Acknowledgement: Theme-based Research Scheme (T12-401/13-R) and GRF (14104415 and 14138016), Research Grant Council, Hong Kong. Citation Format: Raymond W. Lung, Po-Man Hau, Joanna H. Tong, Ka-Hei Lam, Anthony W. Chan, Kevin Y. Yip, George S. Tsao, Kwok-Wai Lo, Ka-Fai To. Epstein-Barr virus-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 482.

Epstein-Barr virus (EBV) infection has been postulated to be an early event involved in the pathogenesis of nasopharyngeal carcinomas (NPC). The lack of representative premalignant nasopharyngeal epithelial cell system for EBV infection has hampered research investigation into the regulation and involvement of EBV infection in NPC pathogenesis. We have compared the efficiency of EBV infection in nasopharyngeal epithelial cells with different biological properties including immortalized, primary and cancerous nasopharyngeal epithelial cells. EBV infection could be achieved in all the nasopharyngeal epithelial cells examined with variable infection rate. TGF-beta effectively enhanced EBV infection into nasopharyngeal epithelial cells both in the immortalized and primary nasopharyngeal epithelial cells. Stable infection of EBV was achieved in a telomerase-immortalized nasopharyngeal epithelial cell line, NP460hTert. The expression pattern of EBV-encoded genes and biological properties of this EBV infected cell line on long-term propagation were monitored. The EBV-infected nasopharyngeal epithelial cells acquired anchorage-independent growth and exhibited invasive growth properties on prolonged propagation. A distinguished feature of this EBV-infected nasopharyngeal epithelial cell model was its enhanced ability to survive under growth factor and nutrient starvation. This was evidenced by the suppressed activation of apoptotic markers and sustained activation of pAkt of EBV-infected cells compared to control cells under nutrient starvation. Examination of cytokine profiles of EBV-infected NP460hTert cells to nutrient and growth factor deprivation revealed upregulation of expression of MCP-1 and GRO-alpha. The establishment of a stable EBV infection model of premalignant nasopharyngeal epithelial cells will facilitate research investigation into the pathogenic role of EBV in NPC development.

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 (LTbetaR), tumour necrosis factor receptor superfamily memeber 1A (TNFRSF1R) and FLJ10665, were specifically over-expressed in the NPC xenograft with 12p13.3 amplification. However, only LTbetaR was frequently over-expressed in primary tumours. LTbetaR is a member of the TNF family of receptors, which can modulate NF-kappaB signalling pathways. Over-expression of LTbetaR in nasopharyngeal epithelial cells resulted in an increase of NF-kappaB activity and cell proliferation. In vivo study showed that suppression of LTbetaR by siRNA led to growth inhibition in the NPC tumour with 12p13.3 amplification. These findings implied that LTbetaR is a potential NPC-associated oncogene within the 12p13.3 amplicon and that its alteration is important in NPC tumorigenesis.

Epstein–Barr virus (EBV) was the first human virus found to encode microRNAs (miRNAs), but the function of these miRNAs has been obscure. Nasopharyngeal carcinoma (NPC) is associated with EBV infection, and the EBV-encoded LMP1 is believed to be a key factor in NPC development. However, detection of LMP1 protein in NPC is variable. Here, we report that EBV-encoded BART miRNAs target the 3′ UTR of the LMP1 gene and negatively regulate LMP1 protein expression. These miRNAs also modulate LMP1-induced NF-κB signaling and alleviate the cisplatin sensitivity of LMP1-expressing NPC cells. Consistent with a previous study on the NPC C666-1 cell line and C15 xenograft, we found abundant expression of BART miRNAs in NPC tissues. Furthermore, DNA sequencing revealed that the 3′ UTR of LMP1 is highly conserved in NPC-derived EBV isolates. The data provide insight into the discrepancy between LMP1 transcript and protein detection in NPC and highlight the role of the EBV miRNAs in regulating LMP1 downstream signaling to promote cancer development.

Epstein-Barr virus (EBV) infection is closely associated with nasopharyngeal carcinoma (NPC) and can be detected in early premalignant lesions of nasopharyngeal epithelium. The latent membrane protein 1 (LMP1) is an oncoprotein encoded by the EBV and is believed to play a role in transforming premalignant nasopharyngeal epithelial cells into cancer cells. RASSF1A is a tumor-suppressor gene commonly inactivated in many types of human cancer including NPC. In this study, we report a novel function of LMP1, in down-regulating RASSF1A expression in human epithelial cells. Downregulation of RASSF1A expression by LMP1 is dependent on the activation of intracellular signaling of NF-kappaB involving the C-terminal activating regions (CTARs) of LMP1. LMP1 expression also suppresses the transcriptional activity of the RASSF1A core promoter. RASSF1A stabilizes microtubules and regulates mitotic events. Aberrant mitotic spindles and chromosome aberrations are reported phenotypes in RASSF1A inactivated cells. In this study, we observed that LMP1 expression in human epithelial cells could induce aberrant mitotic spindles, disorganized interphase microtubules and aneuploidy. LMP1 expression could also suppress microtubule dynamics as exemplified by tracking movements of the growing tips of microtubules in live cells by transfecting EGFP-tagged EB1 into cells. The aberrant mitotic spindles and interphase microtubule organization induced by LMP1 could be rescued by transfecting RASSF1A expression plasmid into cells. Downregulation of RASSF1A expression by LMP1 may facilitate its role in transformation of premalignant nasopharyngeal epithelial cells into cancer cells.

In this study, we have elucidated the chromosomal imbalances in the multistep pathogenesis and delineated several critical tumor suppressor gene (TSG) loci in multiple myeloma (MM). By using comparative genomic hybridization, allelotyping, and multicolor interphase fluorescence in situ hybridization, 5 MM cell lines and bone marrow CD138+ plasma cells from 88 Chinese patients with monoclonal gammopathy of undetermined significance (MGUS) and early and advanced stages of MM were investigated. In all MGUS and MM samples, chromosome copy number abnormalities were detected. A higher number of chromosomal imbalances and specific genetic alterations are involved in MGUS to MM transition (−6q, +3p, and +1p) and MM progression (+2p and +9q). In addition to −13q, we first found high frequencies (42% to 46%) of −4q involving high percentages (70% to 74%) of clonal plasma cells in both MGUS and MM, suggesting that inactivation of TSG in this region is also a potentially critical genetic event in MM tumorigenesis. By high-resolution allelotyping, we defined a common deletion region on 4q13.3 and found that a candidate TSG, platelet factor 4, was frequently silenced by promoter hypermethylation in MM (15 of 28) and MM cell lines (5 of 5). These data have opened up a new approach in the molecular targeting therapy and provide novel insights into MM tumorigenesis.

Endometrial cancer is the third most common gynecologic malignancy and the ninth most common malignancy for females overall in Hong Kong. Approximately 80% or more of these cancers are endometrioid endometrial adenocarcinomas. The aim of this study was to reveal genes contributing to the development of endometrioid endometrial cancer, which may impact diagnosis, prognosis and treatment of the disease. Whole-genome gene expression analysis was completed for a set of 55 microdissected sporadic endometrioid endometrial adenocarcinomas and 29 microdissected normal endometrium specimens using the Affymetrix Human U133 Plus 2.0 oligonucleotide microarray. Selected genes of interest were validated by quantitative real-time-polymerase chain reaction (qRT-PCR). Pathway analysis was performed to reveal gene interactions involved in endometrial tumorigenesis. Unsupervised hierarchical clustering displayed a distinct separation between the endometrioid adenocarcinomas and normal endometrium samples. Supervised analysis identified 117 highly differentially regulated genes (>or=4.0-fold change), which distinguished the endometrial cancer specimens from normal endometrium. Twelve novel genes including DKK4, ZIC1, KIF1A, SAA2, LOC16378, ALPP2, CCL20, CXCL5, BST2, OLFM1, KLRC1 and MBC45780 were deregulated in the endometrial cancer, and further validated in an independent set of 56 cancer and 29 normal samples using qRT-PCR. In addition, 10 genes were differentially regulated in late-stage cancer, as compared to early-stage disease, and may be involved in tumor progression. Pathway analysis of the expression data from this tumor revealed an interconnected network consisting of 21 aberrantly regulated genes involved in angiogenesis, cell proliferation and chromosomal instability. The results of this study highlight the molecular features of endometrioid endometrial cancer and provide insight into the events underlying the development and progression of endometrioid endometrial cancer.

Despite the increasing number of clinical applications of circulating Epstein–Barr virus (EBV) DNA analysis for the detection (1)(2), monitoring(3)(4), and prognostication(5)(6) of nasopharyngeal carcinoma (NPC), several fundamental questions concerning plasma EBV DNA, including its tissue origin and relationship with tumor mass, remain unanswered. In the first part of this study, we investigated the relative contributions of tumor cells and other latently EBV-infected lymphoid tissues to the pool of circulating EBV DNA. Previous studies have shown that different EBV genotypes are harbored by latently EBV-infected lymphoid tissues and blood cells (7)(8)(9)(10) in healthy persons and patients with malignancies. On the other hand, it is well established that the EBV is monoclonal in NPC tumor tissues and other EBV-associated cancers(11)(12)(13)(14). Therefore, investigation of the genotype of plasma EBV DNA should reveal the relative contributions of tumor cells and other latently EBV-infected lymphoid tissues to the pool of circulating EBV DNA in NPC patients. For this study, 25 patients with newly diagnosed NPC, 15 patients with systemic lupus erythematosus, and 13 renal transplant recipients were recruited and gave informed consent. This study was approved by the ethics committee of the Prince of Wales Hospital, Hong Kong. Plasma samples were collected from all patients, and tumor specimens for microdissection were obtained from 14 of the 25 NPC patients. DNA extracted from the plasma samples and the microdissected tumor tissues was used for the amplification of EBV DNA, targeting a region encoding the carboxy terminus of latent membrane protein (LMP-1), using the primers 5′-ATGGTAATGCCTAGAAGTAAAGAAAGG-3′ (LMP1f) and 5′-CATAGCCCTAGCGACTCTGCTG-5′ (R2b). The PCR products were ligated to the pGEM®-T Easy Vectors (Promega), and the ligation products were transformed into competent Escherichia coli cells. Twelve bacterial clones were …

Nasopharyngeal carcinoma is highly prevalent in Southern China and Southeast Asia. To unveil the molecular basis of this endemic disease, high-resolution comparative genomic hybridization arrays were used for systematic investigation of genomic abnormalities in 26 nasopharyngeal carcinoma samples. A comprehensive picture of genetic lesions associated with tumorigenesis of nasopharyngeal carcinoma was generated. Consistent chromosomal gains were frequently found on 1q, 3q, 8q, 11q, 12p, and 12q. High incidences of nonrandom losses were identified on chromosomes 3p, 9p, 11q, 14q, and 16q. In addition to previously characterized regions, we have identified several novel minimal regions of gains, including 3q27.3-28, 8q21-24, 11q13.1-13.3, and 12q13, which may harbor candidate nasopharyngeal carcinoma–associated oncogenes. In this study, gain of 11q13.1-13.3 was the most frequently detected chromosomal aberration and a 5.3-Mb amplicon was delineated at this region. Within this 11q13 amplicon, concordant amplification and overexpression of cyclin D1 (CCND1) oncogene was found in nasopharyngeal carcinoma cell lines, xenografts, and primary tumors. Knockdown of cyclin D1 by small interfering RNA in nasopharyngeal carcinoma cell lines led to significant decrease of cell proliferation. The findings suggest that cyclin D1 is a target oncogene at 11q13 in nasopharyngeal carcinoma and its activation plays a significant role in nasopharyngeal carcinoma tumorigenesis.

We investigated the epigenetic silencing and genetic changes of the RAS-associated domain family 1A (RASSF1A) gene and the O6-methylguanine-DNA methyltransferase (MGMT) gene in retinoblastoma. We extracted DNA from microdissected tumor and normal retina tissues of the same patient in 68 retinoblastoma cases. Promoter methylation in RASSF1A and MGMT was analyzed by methylation-specific PCR, RASSF1A sequence alterations in all coding exons by direct DNA sequencing, and RASSF1A expression by RT-PCR. Cell cycle staging was analyzed by flow cytometry. We detected RASSF1A promoter hypermethylation in 82% of retinoblastoma, in tumor tissues only but not in adjacent normal retinal tissue cells. There was no expression of RASSF1A transcripts in all hypermethylated samples, but RASSF1A transcripts were restored after 5-aza-2'-deoxycytidine treatment with no changes in cell cycle or apoptosis. No mutation in the RASSF1A sequence was found. MGMT hypermethylation was present in 15% of the retinoblastoma samples, and the absence of MGMT hypermethylation was associated (P = .002) with retinoblastoma at advanced Reese-Ellsworth tumor stage. Our results revealed a high RASSF1A hypermethylation frequency in retinoblastoma. The correlation of MGMT inactivation by promoter hypermethylation with lower-stage diseases indicated that MGMT hypermethylation provides useful prognostic information. Epigenetic mechanism plays an important role in the progression of retinoblastoma.

Aberrant retinoid signaling in human cancers is extending from the nucleus to the cytoplasm. Recently, we have demonstrated frequent epigenetic inactivation of a retinoic acid receptor (RAR), RARbeta2, in nasopharyngeal carcinoma (NPC). To further explore targets contributing to aberrant retinoid signaling in NPC, the expression of cellular retinol-binding proteins (CRBPs), cellular retinoic acid-binding proteins (CRABPs), RARs, and retinoid X receptors (RXRs) was examined. Apart from RARbeta2, transcriptional silencing of two CRBPs, CRBPI and CRBPIV, was observed in NPC cell lines and xenografts. Hypermethylation of CRBPI and CRBPIV CpG islands was found to be closely correlated with the loss of expression. Treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, resulted in reexpression of CRBP1 and CRBPIV gene expression in NPC cell lines. Both CRBPI and CRBPIV hypermethylations were also observed in 43/48 (87.8%) and 26/48 (54.2%) primary NPC tumors, respectively. Here, we reported for the first time that CRBPIV was transcriptionally inactivated by promoter hypermethylation in human cancer. Simultaneous methylation of CRBPI, CRBPIV, and RARbeta2 was commonly found in NPC primary tumors. Our findings implied that epigenetic disruption of the CRBPs, CRBPI and CRBPIV, is important in NPC tumorigenesis and may contribute to the loss of retinoic acid responsiveness in cancer.

Deletion on the short arm of chromosome 3 is one of the most important genetic abnormalities in the tumorigenesis of nasopharyngeal carcinoma (NPC). Both physical mapping and functional studies have targeted an NPC-related tumor suppressor gene(s) to chromosome 3p21.3. We have reported recently that RASSF1A gene, located on a 120-kb minimal deletion region on 3p21.3, was frequently inactivated by promoter hypermethylation in NPC. We further confirmed that RASSF1A is the critical target tumor suppressor from 3p21.3, with the evidence that loss of expression and aberrant methylation of the other 8 candidate genes/transcripts (HYAL2, FUS1, RASSF1C, BLU, NPRL2, 101F6, PL6 and CACNA2D2) in this 120-kb region were rare in NPC samples. The contribution of RASSF1A in NPC tumorigenesis was investigated by restoring its expression in a RASSF1A deficient cell line, C666-1. Transient transfection of wild-type RASSF1A resulted in marked growth inhibition in NPC cells. Isolated stable clones expressing wild-type RASSF1A demonstrated retarded cell proliferation in vitro. Soft-agar assay also showed decreased number and sizes of colony formed in these clones. In vivo nude mice assay demonstrated the dramatic reduction of tumorigenic potential in the RASSF1A-transfected clones. Our results provide strong evidence to support RASSF1A as a target tumor suppressor gene on 3p21.3 in NPC.

Gene promoter hypermethylation is increasingly recognized to play an important role in cancer development through silencing of gene transcription. This study determined the methylation profiles of primary colorectal cancers and adenomas to elucidate the role of epigenetic changes in different stages of colorectal carcinogenesis. We examined the methylation profiles of 47 sporadic colorectal cancers, 36 colonic adenomas from patients without cancer and 34 colonic biopsies from patients without colonic lesions. Paired adjacent dysplasia tissues obtained from 17 cancer patients were also examined. Promoter hypermethylation in 10 tumor-related genes (APC, ATM, GSTP1, HLTF, MGMT, hMLH1, p14, p15, SOCS-1 and TIMP-3) were studied by methylation-specific PCR. Promoter hypermethylation was frequently detected in more than 40% of colonic cancers and adenomas in APC, ATM, HLTF, MGMT and hMLH1 genes (p0.0001 vs. normal). While low level of methylation was detected in p14, p15 and TIMP-3, there was no methylation detected in GSTP1 and SOCS-1. The frequencies of methylation were comparable between tumors and adenomas, and advanced and nonadvanced adenoma. In contrast, K-ras mutation was only detected in advanced adenomas and cancers. Concurrent methylation in/= 3 genes was found in 66.7% adenomas and 68.1% cancers but not in normal colonic tissues. Methylation was associated with reduced protein expressions in colorectal adenomas and cancers. Moreover, methylation in ATM was more common in older cancer patients (p = 0.002), but there was no significant association between promoter hypermethylation and other clinicopathologic characteristics of cancer. Our study demonstrated the early and specific involvement of promoter hypermethylation in the colorectal adenoma-carcinoma sequence.

Nasopharyngeal carcinoma (NPC) is a malignancy with remarkable racial and geographic distribution. The development of this EBV-associated cancer likely involves cumulative genetic and epigenetic changes in a background of predisposed genetic and environmental factors. Genome-wide studies have unravelled multiple chromosomal abnormalities with involvement of specific oncogenes and tumour suppressor genes. Alterations of genes such as Ras association domain family 1A (RASSF1A), p16/INK4A, p14/ARF suggest that multiple cellular pathways were dysregulated in the NPC cells. Studies on the precancerous lesions revealed early genetic changes and a critical role of EBV latent infection in the development of this cancer. Based on the existing findings, a pathogenetic model for NPC is proposed.

A panel of monoclonal antibodies specific to Hong Kong Chinese nasopharyngeal carcinoma (NPC)-associated Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) variants has been generated. These monoclonal antibodies not only differentiate the Hong Kong Chinese NPC-associated LMP1 variants from the prototype B95-8 LMP1, derived from Caucasian infectious mononucleosis, but also differentiate the 2 highly homologous LMP1 deletion variants commonly found in Hong Kong primary NPC. The predominant deletion type variant, DV-Asp335, is characterized by an aspartic acid at residue 335 located in the cytoplasmic C-terminal region, whereas the other minor deletion variant, DV-Gly335, has a glycine in the same residue position. 335D is hitherto found predominantly in LMP1 of the China 1 strain in association with NPC in the Chinese populations located in southern China and Malaysia. These antibodies, which are applicable in ELISA, immunofluorescence, immunoprecipitation, immunoblotting and immunohistochemistry on paraffin sections, are the first variant-specific anti-LMP1 monoclonal antibodies produced, and will be useful in investigating the functional significance of 335D in NPC. © 2002 Wiley-Liss, Inc.

Nasopharyngeal carcinoma (NPC) is a common cancer among the Chinese population in the southern part of China. The incidence of this cancer drops markedly in northern China. A 6- to 24-fold difference exists between southern and northern Chinese. To investigate the early genetic events involved in the development of this cancer, we have examined loss of heterozygosity (LOH) on chromosome 9p, being one of the most frequent genetic alterations in NPC, in nasopharyngeal tissues including normal epithelia (NP), dysplastic lesions (DNP) and invasive carcinoma (NPC) from high-risk and low-risk regions. We found similar frequencies of 9p LOH in NPC from high-risk (77.8%) and low-risk (63.6%) regions (p = 0.43). In contrast, 45% of normal nasopharyngeal epithelia from the high-risk region showed 9p LOH, while none of the NP from the low-risk region showed such abnormalities (p = 0.002). Deletions of chromosome 9p were found in 66.7% dysplastic nasopharyngeal lesions. These findings suggest that LOH of chromosome 9p is an early event in the tumorigenesis of NPC. The increased risk of NPC in southern Chinese may be related to early loss of genetic materials as indicated by 9p LOH in the NP from high- and low-risk regions. We also reported previously that Epstein-Barr virus (EBV) latent infection was present in all high-grade DNP and NPC but not in any NP from fetuses or normal adults. Thus, early genetic alterations such as 9p LOH may take place prior to EBV latent infection and expand clonally thereafter.

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer that is consistently associated with intensive lymphocytes infiltration and Epstein-Barr virus infection. This study aims at elucidating the mechanism contributing to immune evasion in NPC. During NPC tumorigenesis, multiple intrinsic mechanisms are acquired to escape cellular immune response for the tumor and viral antigens. By genome sequencing, somatic alterations of multiple MHC class I genes including NLRC5, HLA-A, HLA-B, HLA-C and B2M were detected in 30% of primary NPC. Significant correlation of MHC class I gene alterations and poor survival rate in the patients was also shown. The finding suggested that the deficiency of antigen presentation mechanism represents a common strategy for immune evasion in NPC and favors rapid tumor progression. Losses of HLA-A, HLA-B and NLRC5 expression were validated in a panel of NPC tumor lines and selected primary tumors. The role of NLRC5 as key transcription regulator of HLA-A and HLA-B expression was demonstrated in the nasopharyngeal epithelial cells. To further evaluate the mechanisms contributed to immune evasion in NPC, we have elucidated immunosuppressive molecules involved in immune surveillance in Epstein Barr Virus (EBV)-associated NPC and its association with EBV-encoded LMP1 in an independent cohort of primary NPCs. By immunohistochemistry, the expression of EBV-encoded LMP1, MHC class I genes and various immunosuppressive molecules including PDL1, PDL2 and IDO1 was examined in 98 primary tumors. PDL1 and PDL2 were found to be over-expressed in 50% and 33.7% of NPC cases while IDO1 was found to be expressed in 28.8% of cases. Primary NPC cases expressing either PDL1 or PDL2 accounted for 64.3%. A significant association between LMP1 and PDL1 and PDL2 expression was also found. The correlation of PDL1, PDL2 and/or MHC class I molecule expression with the outcome of the patients was determined. The high incidence of MHC class I deficiencies and PDL1/PDL2 overexpression imply that EBV-associated NPC escape from the host immune response by targeting both antigen presentation mechanisms and immune checkpoints. Our findings also suggested that the expression of MHC class I molecules and PDL1/PDL2 is a potential biomarker for immunotherapy in NPC patients. Citation Format: Sharie Pui-Kei Siu, Ka Fai To, Grace Tin-Yun Chung, Vivian W.Y. Lui, Yvonne Y. Li, Peter S. Hammerman, Kwok Wai Lo. Targeting MHC class I molecules and immune checkpoints as key immune evasion strategies in EBV-associated nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 646. doi:10.1158/1538-7445.AM2017-646

Epstein-Barr virus (EBV) infection is closely associated with undifferentiated nasopharyngeal carcinoma (NPC), strongly implicating a role for EBV in NPC pathogenesis; conversely, EBV infection is rarely detected in normal nasopharyngeal epithelial tissues. In general, EBV does not show a strong tropism for infecting human epithelial cells, and EBV infection in oropharyngeal epithelial cells is believed to be lytic in nature. To establish life-long infection in humans, EBV has evolved efficient strategies to infect B cells and hijack their cellular machinery for latent infection. Lytic EBV infection in oropharyngeal epithelial cells, though an infrequent event, is believed to be a major source of infectious EBV particles for salivary transmission. The biological events associated with nasopharyngeal epithelial cells are only beginning to be understood with the advancement of EBV infection methods and the availability of nasopharyngeal epithelial cell models for EBV infection studies. EBV infection in human epithelial cells is a highly inefficient process compared to that in B cells, which express the complement receptor type 2 (CR2) to mediate EBV infection. Although receptor(s) on the epithelial cell surface for EBV infection remain(s) to be identified, EBV infection in epithelial cells could be achieved via the interaction of glycoproteins on the viral envelope with surface integrins on epithelial cells, which might trigger membrane fusion to internalize EBV in cells. Normal nasopharyngeal epithelial cells are not permissive for latent EBV infection, and EBV infection in normal nasopharyngeal epithelial cells usually results in growth arrest. However, genetic alterations in premalignant nasopharyngeal epithelial cells, including p16 deletion and cyclin D1 overexpression, could override the growth inhibitory effect of EBV infection to support stable and latent EBV infection in nasopharyngeal epithelial cells. The EBV episome in NPC is clonal in nature, suggesting that NPC develops from a single EBV-infected nasopharyngeal epithelial cell, and the establishment of persistent and latent EBV infection in premalignant nasopharyngeal epithelium may represent an early and critical event for NPC development.

The close association of Epstein-Barr virus (EBV) infection with non-keratinizing nasopharyngeal carcinomas and a subset of gastric carcinomas suggests that EBV infection is a crucial event in these cancers. The difficulties encountered in infecting and transforming primary epithelial cells in experimental systems suggest that the role of EBV in epithelial malignancies is complex and multifactorial in nature. Genetic alterations in the premalignant epithelium may support the establishment of latent EBV infection, which is believed to be an initiation event. Oncogenic properties have been reported in multiple EBV latent genes. The BamH1 A rightwards transcripts (BART s) and the BART -encoded microRNAs (miR-BART s) are highly expressed in EBV-associated epithelial malignancies and may induce malignant transformation. However, enhanced proliferation may not be the crucial function of EBV infection in epithelial malignancies, at least in the early stages of cancer development. EBV-encoded gene products may confer anti-apoptotic properties and promote the survival of infected premalignant epithelial cells harbouring genetic alterations. Multiple EBV-encoded microRNAs have been reported to have immune evasion functions. Genetic alterations in host cells, as well as inflammatory stroma, could modulate the expression of EBV genes and alter the growth properties of infected premalignant epithelial cells, encouraging their selection during carcinogenesis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., link_to_OA_fulltext