Pathogenesis Of NPC Research Articles

ACSL4 promotes ferroptosis and M1 macrophage polarization to regulate the tumorigenesis of nasopharyngeal carcinoma

BackgroundNasopharyngeal carcinoma (NPC) is a head and neck malignant tumor with a high incidence and recurrence rate. The crosstalk between ferroptosis and tumor-associated macrophages (TAMs) is thought to have major implications in interfering with cancers. We intended to explore the effect of acyl-CoA synthetase long-chain family member 4 (ACSL4) on the pathogenesis of NPC via ferroptosis and TAMs. MethodsDifferential genes in NPC patients were analyzed using publicly available databases, and the ferroptosis-related gene ACSL4 was identified. Expression of ACSL4 in NPC cell lines and xenografted mice was examined. Colony formation, cell proliferation, migration, and invasion were assessed. The abundance of epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, and Vimentin) was confirmed. Lipid peroxidation levels and related markers were measured. Clophosome was administered to determine the role of TAMs in NPC mice. ResultsLow levels of ACSL4 were observed in NPC patients and CNE-2 and 5-8F cells. Erastin (a ferroptosis inducer) and ACSL4 increased lipid peroxidation, decreased cell viability, colony formation, cell proliferation, migration and invasion, and inhibited EMT. Moreover, Erastin and ACSL4 promoted M2 to M1 macrophage polarization. The effects of erastin and ACSL4 were additive. Ferrostatin-1, an inhibitor of ferroptosis, exerted the opposite effect and reversed the beneficial effects of ACSL4 overexpression. In xenograft mice, ACSL4 and clophosome hindered the growth of NPC, and extra clophosome slightly enhanced the antitumor effect of ACSL4. ConclusionOur findings indicated that ACSL4 inhibited the pathogenesis of NPC, at least through crosstalk between ferroptosis and macrophages, providing potential direction for NPC therapy.

Transcriptomic profiling revealed FZD10 as a novel biomarker for nasopharyngeal carcinoma recurrence.

Nasopharyngeal carcinoma (NPC) is a type of cancers that develops in the nasopharynx, the very upper part of the throat behind the nose. NPC is typically diagnosed in later stages of the disease and has a high rate of recurrence due to the location of the tumor growth site. In this study, we compared the gene expression profiles of NPC tissues from patients with and without recurrence to identify potential molecular biomarkers of NPC recurrence. Microarrays were used to analyze the expression of genes in 15 NPC tissues taken at the time of diagnosis and at the site of recurrence following therapeutic treatment. Pathway enrichment analysis was used to examine the biological interactions between the major differentially expressed genes. The target identified was then validated using immunohistochemistry on 86 NPC tissue samples. Our data showed that the Wnt signaling pathway was enhanced in NPC tissues with recurrence. FZD10, a component of the Wnt signaling pathway, was significantly expressed in NPC tissues, and was significantly associated with NPC recurrence. Our study provides new insights into the pathogenesis of NPC and identifies FZD10 as a potential molecular biomarker for NPC recurrence. FZD10 may be a promising candidate for NPC recurrence and a potential therapeutic target.

Protein Farnesylation on Nasopharyngeal Carcinoma, Molecular Background and Its Potential as a Therapeutic Target.

Simple SummaryNasopharyngeal carcinoma is distinguished from other head and neck carcinomas by the association of its carcinogenesis with the Epstein–Barr virus. It is highly metastatic, and a novel therapeutic modality for metastatic nasopharyngeal carcinoma is keenly awaited. Protein farnesylation is a C-terminal lipid modification of proteins and was initially investigated as a key process in activating the RAS oncoprotein through its association with the cellular membrane structure. Since then, more and more evidence has accumulated to indicate that proteins other than RAS are also farnesylated and have significant roles in carcinogenesis. This review delineates molecular pathogenesis through protein farnesylation in the context of nasopharyngeal carcinoma and discusses the potential of farnesylation as a therapeutic target.Nasopharyngeal carcinoma (NPC) is one of the Epstein–Barr virus (EBV)-associated malignancies. NPC is highly metastatic compared to other head and neck carcinomas, and evidence has shown that the metastatic features of NPC are involved in EBV infection. The prognosis of advanced cases, especially those with distant metastasis, is still poor despite advancements in molecular research and its application to clinical settings. Thus, further advancement in basic and clinical research that may lead to novel therapeutic modalities is needed. Farnesylation is a lipid modification in the C-terminus of proteins. It enables proteins to attach to the lipid bilayer structure of cellular membranes. Farnesylation was initially identified as a key process of membrane association and activation of the RAS oncoprotein. Farnesylation is thus expected to be an ideal therapeutic target in anti-RAS therapy. Additionally, more and more molecular evidence has been reported, showing that proteins other than RAS are also farnesylated and have significant roles in cancer progression. However, although several clinical trials have been conducted in cancers with high rates of ras gene mutation, such as pancreatic carcinomas, the results were less favorable than anticipated. In contrast, favorable outcomes were reported in the results of a phase II trial on head and neck carcinoma. In this review, we provide an overview of the molecular pathogenesis of NPC in terms of the process of farnesylation and discuss the potential of anti-farnesylation therapy in the treatment of NPC.

Impaired Retromer Function in Niemann-Pick Type C Disease Is Dependent on Intracellular Cholesterol Accumulation.

Niemann-Pick type C disease (NPC) is a rare inherited neurodegenerative disorder characterized by an accumulation of intracellular cholesterol within late endosomes and lysosomes due to NPC1 or NPC2 dysfunction. In this work, we tested the hypothesis that retromer impairment may be involved in the pathogenesis of NPC and may contribute to increased amyloidogenic processing of APP and enhanced BACE1-mediated proteolysis observed in NPC disease. Using NPC1-null cells, primary mouse NPC1-deficient neurons and NPC1-deficient mice (BALB/cNctr-Npc1m1N), we show that retromer function is impaired in NPC. This is manifested by altered transport of the retromer core components Vps26, Vps35 and/or retromer receptor sorLA and by retromer accumulation in neuronal processes, such as within axonal swellings. Changes in retromer distribution in NPC1 mouse brains were observed already at the presymptomatic stage (at 4-weeks of age), indicating that the retromer defect occurs early in the course of NPC disease and may contribute to downstream pathological processes. Furthermore, we show that cholesterol depletion in NPC1-null cells and in NPC1 mouse brains reverts retromer dysfunction, suggesting that retromer impairment in NPC is mechanistically dependent on cholesterol accumulation. Thus, we characterized retromer dysfunction in NPC and propose that the rescue of retromer impairment may represent a novel therapeutic approach against NPC.

Integrative analysis of miRNAs-mRNAs reveals that miR-182 up-regulation contributes to proliferation and invasion of nasopharyngeal carcinoma by targeting PTEN.

Objective: Several miRNAs have been found to be abnormally expressed during nasopharyngeal carcinoma development. Nevertheless, the interaction between miRNAs and downstream genes remains unexploited. In this study, we aim to investigate miRNAs-mRNAs interaction and the mechanism of miR-182 in NPC.Results: Integrative analysis identified several hub-miRNAs that drive NPC pathogenesis. The expression of miR-182 was notably increased in 32 NPC tissues and cell lines (CNE1 and 5-8F). Up-regulation of miR-182 was strongly correlated with poor prognosis of NPC patients. Moreover, the proliferation and invasion of NPC cells were notably increased in miR-182 mimics condition and decreased in miR-182 inhibitor condition. Furthermore, PTEN was verified to be a target of miR-182 and overexpression of PTEN could abrogate the promotion effect of miR-182 mimics on NPC invasion.Conclusions: We identified several hub-miRNAs that may drive NPC pathogenesis. MiR-182 could promote proliferation and invasion of NPC cells via targeting PTEN, which provides a new insight into the clinical therapy of NPC.Materials and Methods: Genome-wide miRNAs of NPC tissues was analyzed using high-throughput sequencing and bioinformatics tools. QRT-PCR experiment was conducted to measure relative expression level. Dual-luciferase reporter assay was used to verify target relationship. The proliferation and invasion of transfected cells were measured by CCK-8 and transwell assay.

Faktor-faktor penyebab terjadinya karsinoma nasofaring (KNF)

Nasopharyngeal carcinoma is a common disease in southern China. The etiologies of the main factors proposed for the pathogenesis of KNF include genetic factors, environmental factors and Epstein Barr Virus (EBV) infection. Other causes besides preserved food consumption include salted fish which have been involved in the etiology of NPC. The downward trend in the incidence of NPC has occurred in Hong Kong for the past 20 years, which is caused by changes in dietary habits. Despite the close relationship of EBV infection with NPC, the etiological role of EBV in the pathogenesis of NPC remains an interaction. EBV infection in primary nasopharyngeal epithelial cells occurs. Epstein Barr virus does not convert primary nasopharyngeal epithelial cells into proliferative clones, which is in sharp contrast to the well-documented ability of EBV to alter and perpetuate primary B cells. Genetic changes that are supported in the nasopharyngeal epithelium may be needed to support stable EBV infection. Non-viral factors as a cause of nasopharyngeal carcinoma still cannot be resolved with certainty. Non-viral factors are one of the risk factors that can increase the number of events arising from nasopharyngeal malignancies such as smoke, salted fish, formaldehyde, genetic, as soon as possible firewood, wood dust, chronic infection, throat protector, alcohol and traditional medicine.

Changes in global gene expression indicate disordered autophagy, apoptosis and inflammatory processes and downregulation of cytoskeletal signalling and neuronal development in patients with Niemann-Pick C disease.

Changes in gene expression profiles were investigated in 23 patients with Niemann-Pick C1 disease (NPC). cDNA expression microarrays with subsequent validation by qRT-PCR were used. Comparison of NPC to control samples revealed upregulation of genes involved in inflammation (MMP3, THBS4), cytokine signalling (MMP3), extracellular matrix degradation (MMP3, CTSK), autophagy and apoptosis (CTSK, GPNMB, PTGIS), immune response (AKR1C3, RCAN2, PTGIS) and processes of neuronal development (RCAN2). Downregulated genes were associated with cytoskeletal signalling (ACTG2, CNN1); inflammation and oxidative stress (CNN1); inhibition of cell proliferation, migration and differentiation; ERK-MAPK pathway (COL4A1, COL4A2, CPA4); cell adhesion (IGFBP7); autophagy and apoptosis (CDH2, IGFBP7, COL4A2); neuronal function and development (CSRP1); and extracellular matrix stability (PLOD2). When comparing NPC and Gaucher patients together versus controls, upregulation of SERPINB2 and IL13RA2 and downregulation of CSRP1 and CNN1 were characteristic. Notably, in NPC patients, the expression of PTGIS is upregulated while the expression of PLOD2 is downregulated when compared to Gaucher patients or controls and potentially could serve to differentiate these patients. Interestingly, in NPC patients with (i) jaundice, splenomegaly and cognitive impairment/psychomotor delay-the expression of ACTG2 was especially downregulated; (ii) ataxia-the expression of ACTG2 and IGFBP5 was especially downregulated; and (iii) VSGP, dysarthria, dysphagia and epilepsy-the expression of AKR1C3 was especially upregulated while the expression of ACTG2 was downregulated. These results indicate disordered apoptosis, autophagy and cytoskeleton remodelling as well as upregulation of immune response and inflammation to play an important role in the pathogenesis of NPC in humans.

Correlation between IL-17A expression in nasopharyngeal carcinoma tissues and cells and pathogenesis of NPC in endemic areas.

We investigated the correlation between the expression of IL-17A in nasopharyngeal carcinoma tissues and cells and the occurrence and development of NPC was also investigated. Forty-five NPC biopsy specimens from January 2014 to January 2016 were selected. Forty-five NPC tissue specimens and 45 chronic nasopharyngitis tissue samples were detected by immunohistochemistry. Statistical methods were used to analyze the correlation between IL-17A expression and the clinicopathological variables of NPC. The NPC patients were followed up. The levels of IL-17A mRNA in 40 NPC tissue specimens and 45 chronic nasopharyngitis tissue samples were detected by real-time PCR. IL-17A expression in 15 NPC tissue specimens and chronic nasopharyngitis tissue samples was further detected by Western blotting assays. IL-17A expression in NPC tissues was significantly higher than that of chronic nasopharyngitis tissues (P < 0.05). IL-17A was expressed in the nucleus and cytoplasm of both NPC tissues and chronic nasopharyngitis tissues. Stage III + IV NPC, tumor volume ≥ 50mm, and hepatic envelope invasion and cervical lymph node metastasis were associated with significantly higher IL-17A levels versus stage I + II NPC, tumor size < 50mm, no membrane invasion and lack of cervical lymph node metastasis (P < 0.05). IL-17A was statistically associated with tissue differentiation, serum EBV-lgA levels, and EBV infection. IL-17A-positive patients had significantly longer median survival versus IL-17A-negative patients (21.0 vs. 13.0months, log-rank test: P < 0.05). Furthermore, 65% (26/40) of NPC tissue samples had significantly higher IL-17A mRNA levels than chronic nasopharyngitis (P < 0.05). IL-17A expression was significantly higher in NPC ≥ 50mm, stage III + IV NPC and NPC with cervical lymph node invasion than its corresponding chronic nasopharyngitis tissue. IL-17A may be involved in the regulation of various malignant biological behaviors of NPC, which is closely related to the occurrence and development of NPC.

Niemann‐Pick C1 Pathophysiology Associates with Different Classes of Protein Trafficking Phenotypes Elicited by NPC‐1 Mutations

Niemann‐Pick Type C disease (NP‐C) is caused by a defect in the trafficking of endocytosed cholesterol with sequestration of cholesterol in lysosomes and late endosomes. Over 400 mutations have been identified in the NPC1 gene that account for almost 95 % of the NP‐C disease. It is still unclear how a mutation does affect the biosynthetic presentation and the intracellular localization of NPC1. We therefore asked whether mutations in the NPC1 gene generate different protein phenotypes that vary in their trafficking, intracellular localization and function, hence ultimately correlating with the pattern and severity of clinical symptoms in NP‐C patients. Three major classes of mutations could be identified based on the transport of NPC1 mutants along the secretory pathway, their association with lipid rafts and cellular localization. Many of the mutants analyzed were blocked in the ER as immature mannose‐rich proteins, while others were either partially trafficked through the Golgi to the lysosomes or trafficked in a comparable fashion to wild type NPC1. Interestingly, a particular trafficking phenotype is not elicited by mutations confined to a particular domain in NPC1, since these protein trafficking phenotypes arise from mutations that are distributed over different regions of NPC1. Furthermore, the interaction of the NPC1 mutants with lipid rafts varies among the different mutants analyzed in this study, indicating that their trafficking pattern could be associated with the reduced or abolished trafficking of cholesterol out of the lysosomes. Altogether, our study discloses a novel concept in the pathogenesis of NP‐C that relies on the diversity of the trafficking phenotypes of the NPC1 mutants. Expectedly, the ER‐located NPC1 mutants do not associate with lipid rafts since cholesterol or sphingolipids are not abundant in this organelle. On the other hand, the level of association of the partially‐trafficked mutants with lipid rafts is low and correlates with the level of maturation of these mutants. Remarkably, the correctly trafficked wild‐type‐like mutant NPC1‐L1213F as well as the wild type NPC1 varied substantially in their association levels with lipid rafts as compared to the other two classes. Nevertheless, the mature forms of the wild type‐like mutants revealed a higher solubility than their wild type counterpart, compatible with reduced levels of lipid rafts components in the cells overexpressing the mutant. Undoubtedly, membrane lipid analysis of cells overexpressing these mutants is required to support the hierarchical concept of trafficking. In conclusion, the current study proposes a direct link between the three classes of the NPC1 trafficking phenotype mutants, with the heterogeneous disease pattern including neurological, visceral, or psychiatric manifestations, hence the severity of NP‐C disease.Support or Funding InformationActelion, Switzerland to HYN; CNRS grant (CNRS‐01‐14‐12), Lebanon to SR; DAAD, Germany to HS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MiR-99a inhibits cell proliferation of nasopharyngeal carcinoma by targeting mTOR and serves as a prognostic factor.

Nasopharyngeal carcinoma is the most common head and neck tumor in Southern China and Southeast Asia, presenting high rates of local invasion and early distant metastasis. Abnormally expressed miR-99a has been discovered in many tumors, and it is involved in nasopharyngeal carcinoma as well. This study aims to explore the molecular mechanisms of miR-99a and mTOR in regulating nasopharyngeal carcinoma. MiR-99a expression in nasopharyngeal carcinoma cells was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed for accessing cell proliferative capacity. Dual-Luciferase reporter gene assay was employed to verify the combination between miR-99a and mTOR. We found that miR-99a was downregulated while mTOR was upregulated in nasopharyngeal carcinoma cell lines CNE1 and SUNE1. Low expression of miR-99a or high expression of mTOR predicted poor prognosis of nasopharyngeal carcinoma. MiR-99a overexpression inhibited the proliferation of CNE1 and SUNE1 cells through targeting mTOR. We provided evidence that miR-99a inhibits NPC cell proliferative ability by inhibiting mTOR. The newly identified miR-99a/mTOR axis provides novel insight into the pathogenesis of NPC and represents a potential therapeutic target for NPC.

TCR repertoire profiling of tumors, adjacent normal tissues, and peripheral blood predicts survival in nasopharyngeal carcinoma

The T-cell immune responses in nasopharyngeal carcinoma patients have been extensively investigated recently for designing adoptive immunotherapy or immune checkpoint blockade therapy. However, the distribution characteristics of T cells associated with NPC pathogenesis are largely unknown. We performed deep sequencing for TCR repertoire profiling on matched tumor/adjacent normal tissue from 15 NPC patients and peripheral blood from 39 NPC patients, 39 patients with other nasopharyngeal diseases, and 33 healthy controls. We found that a lower diversity of TCR repertoire in tumors than paired tissues or a low similarity between the paired tissues was associated with a poor prognosis in NPC. A more diverse TCR repertoire was identified in the peripheral blood of NPC patients relative to the controls; this was related to a significant decrease in the proportion of high-frequency TCR clones in NPC. Higher diversity in peripheral blood of NPC patients was associated with a worse prognosis. Due to the peculiarity of the Vβ gene usage patterns in the peripheral blood of NPC patients, 15 Vβ genes were selected to distinguish NPC patients from controls by the least absolute shrinkage and selection operator analysis. We identified 11 clonotypes shared by tumors and peripheral blood samples from different NPC patients, defined as "NPC-associated" that might have value in adoptive immunotherapy. In conclusion, we here report the systematic and overall characteristics of the TCR repertoire in tumors, adjacent normal tissues, and peripheral blood of NPC patients. The data obtained may be relevant to future clinical studies in the setting of immunotherapy for NPC patients.

Abstract 3084: Epstein Barr virus-encoded LMP1 activates the mTORC2 signaling pathway to reprogram glucose metabolism in nasopharyngeal epithelial cell

Abstract Epstein-Barr virus (EBV) infection of preinvasive nasopharyngeal epithelium is believed to be an essential initiation event during nasopharyngeal carcinoma pathogenesis, but the underlying mechanisms are largely undefined. Here we demonstrate that EBV-encoded LMP1 reprograms the glucose metabolism from oxidative phosphorylation to glycolysis of nasopharyngeal epithelial (NPE) is a major process for NPC pathogenesis. RNA-seq analysis indicates that EBV infection reprograms the glucose metabolism from glycolysis and oxidative phosphorylation and enhances the metabolic associated pathways. Our study show EBV encoded LMP1 is important for this event, EBV infection and LMP1 expression enhance the extracellular acidification rate and decrease the oxygen consumption rate in NPE cells. We further show that activation of mTORC2/AKT signaling is involved in this metabolic reprogramming in EBV-infected NPE cells. Genetic or pharmacologic inhibition of the mTORC2/AKT signaling blocks the LMP1-induced glycolysis. In summary, this study provides evidences of involvement of EBV infection in metabolic reprogramming of NPE cells. Acknowledgment: This project was supported by funding from the Research Grants Council, Hong Kong: General Research Fund (17120814, 17161116, 17111516, 171110315), Area of Excellence (AoE/M-06/08), Theme-Based Research Scheme (T12-401/13-R), Collaborative Research Fund (Project reference: C7027-16G); Health and Medical Research Fund of Hong Kong (12110782, 13120872, 04151726); and CRCG, University of Hong Kong, Hong Kong. Citation Format: Jun Zhang, Weitao Lin, Chi-Man Tsang, Teng Fei Liu, W C. Tang, Tim Ling Yip, Wen Deng, Lin Jia, Sai-Wah Tsao. Epstein Barr virus-encoded LMP1 activates the mTORC2 signaling pathway to reprogram glucose metabolism in nasopharyngeal epithelial cell [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 3084.

The new 6q27 tumor suppressor DACT2, frequently silenced by CpG methylation, sensitizes nasopharyngeal cancer cells to paclitaxel and 5-FU toxicity via \u03b2-catenin/Cdc25c signaling and G2/M arrest

BackgroundNasopharyngeal carcinoma (NPC) is prevalent in South China, including Hong Kong and Southeast Asia, constantly associated with Epstein-Barr virus (EBV) infection. Epigenetic etiology attributed to EBV plays a critical role in NPC pathogenesis. Through previous CpG methylome study, we identified Disheveled-associated binding antagonist of beta-catenin 2 (DACT2) as a methylated target in NPC. Although DACT2 was shown to regulate Wnt signaling in some carcinomas, its functions in NPC pathogenesis remain unclear.MethodsRT-PCR, qPCR, MSP, and BGS were applied to measure expression levels and promoter methylation of DACT2 in NPC. Transwell, flow cytometric analysis, colony formation, and BrdU-ELISA assay were used to assess different biological functions affected by DACT2. Immunofluorescence, Western blot, and dual-luciferase reporter assay were used to explore the mechanisms of DACT2 functions. Chemosensitivity assay was used to measure the impact of DACT2 on chemotherapy drugs.ResultsWe found that DACT2 is readily expressed in multiple normal adult tissues including upper respiratory tissues. However, it is frequently downregulated in NPC and correlated with promoter methylation. DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine restored its expression in NPC cells. DACT2 methylation was further detected in 29/32 (91%) NPC tumors but not in any (0/8) normal nasopharyngeal tissue samples. Ectopic expression of DACT2 in NPC cells suppressed their proliferation, migration, and invasion through downregulating matrix metalloproteinases. DACT2 expression also induced G2/M arrest in NPC cells through directly suppressing β-catenin/Cdc25c signaling, which sensitized NPC cells to paclitaxel and 5-FU, but not cisplatin.ConclusionOur results demonstrate that DACT2 is frequently inactivated epigenetically by CpG methylation in NPC, while it inhibits NPC cell proliferation and metastasis via suppressing β-catenin/Cdc25c signaling. Our study suggests that DACT2 promoter methylation is a potential epigenetic biomarker for the detection and chemotherapy guidance of NPC.

Abstract 14: Elucidation of genetic susceptibility genes using next-generation sequencing (NGS) approach in nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is a malignant tumor that emerges from the epithelium of the nasopharynx, the upper part of the throat behind the nose. It has remarkable ethnic and geographical distribution. It is extremely common in Southern China including Hong Kong. Multiple factors including host genetics, environmental factors, and Epstein-Barr virus (EBV) infection contribute to NPC pathogenesis. NPC diagnosis usually occurs in people in their upper 40s and early 50s, which is close to 20 years younger than for many of the other common cancers. Familial NPC accounts for about 10% of the cases. The risk of the individuals with first-degree relatives having NPC is dramatically increased compared to those without a family history, strongly suggesting the importance of host genetics in NPC development. Recently, we used the whole-exome sequencing (WES) approach to characterize the genetic variants in the coding regions in the family-history positive (FH+) and early age onset (EAO) NPC patients. Gene-level burden tests suggested a link between multiple deleterious variants at MST1R and NPC risk, particularly in the EAO patients with disease onset at or younger than 201. In addition, we identified multiple rare deleterious variants in a number of candidate genes in at least 5% of the FH+ or EAO cases. To further investigate the role of these potential genetic susceptibility genes, we used a NGS-based targeted sequencing approach to examine the germline rare deleterious variants with minor allele frequency (MAF)&amp;lt;0.001 in 17 genes selected from WES study in 1269 controls and 1242 cases including 334 FH+ and 904 sporadic cases. These genes are involved in DNA repair, immune response, and epigenetic regulator. Both filtering strategy and gene-level burden tests were used to identify the genetic susceptibility genes in NPC. Multiple loss-of-function (LOF) germline mutations of a critical DNA repair gene, ATM, was identified in FH+ cases. This gene plays an important role in the formation of the replication compartment during lytic replication of EBV in nasopharyngeal epithelial cells. In addition, we discovered that the rare deleterious variants of MST1R in the intracellular part may play a role in the pathogenesis in both FH+ and sporadic cases. Our study highlights the importance of innate immunity and DNA repair in NPC genetic susceptibility. Reference: 1. Dai W, Zheng H, Cheung AK, et al. Whole-exome sequencing identifies MST1R as a genetic susceptibility gene in nasopharyngeal carcinoma. Proc Natl Acad Sci U S A 2016;113:3317-22. Citation Format: Wei Dai, Hong Zheng, Josephine M.Y. Ko, Arthur K.L. Cheung, Maria Li Lung. Elucidation of genetic susceptibility genes using next-generation sequencing (NGS) approach in nasopharyngeal carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 14.

P032 Compound HLA/KIR genotypes influence risk of nasopharyngeal carcinoma in a Southern Chinese cohort

Aim While decades of research have shown consistent associations between individual HLA alleles/haplotypes and NPC, there is still limited understanding of the biological role that HLA plays in the etiology of the cancer. Specifically, it is not currently known whether HLA-NPCassociations are driven by the role of HLA in adaptive immune responses through T cells and/or innate immune responses to infections via NK cells. In this study We aim to evaluate HLA/KIR associations with NPC and determine the extent to which the innate and adaptive immune responses are involved in NPC pathogenesis. Methods A total of 4500 samples including 1800 NPC cases and 2700 matched controls were genotyped for HLA class I and KIR loci using SBT and SSP respectively. The status of EBV-IgA/VCA antibody of the study subjects was detected using the immunoperoxidase assay. Peptide binding assays for A∗11:01 and A∗11:02 were carried out using a flow cytometry based peptide stabilization method that measures stable peptide-HLA cell surface expression. Results 1) HLA-A∗11:01 showed a protective effect against NPC development whereas A∗11:02 was flat. 2) Both A∗11:01 and A∗11:02 bind similar EBV peptides, but A∗11:01 showed stronger binding. 3) In the presence of KIR2DS4, A∗11:02 reduces risk of NPC development amongst IgA + controls. Conclusions The single amino acid replacement between A∗11:01 and A∗11:02 affects both peptide binding and ligand binding to KIR. A∗11:01 is a better presenter of EBV peptides to T cells, which may explain its protective effect on NPC. A∗11:02 serves as a much better ligand to KIR2DS4 relative to A∗11:01, indicating its potential role in NK-cell mediated innate immunity. It is likely that both innate and acquired immune responses are involved and the interactions between HLA and KIR play an important role in NPC pathogenesis.

Abstract 125: Whole-exome sequencing identifies NF-kappaB pathway regulators frequently mutated in nasopharyngeal carcinoma

Abstract Introduction Nasopharyngeal carcinoma (NPC) is a unique epithelial malignancy with a high prevalence in Southeast Asia. To date, the genomic abnormalities leading to the pathogenesis of NPC remain unclear. Thus, we sought to characterize the mutational landscape in NPC tumors using next-generation sequencing approaches and to identify significantly mutated genes and pathways. Methods 124 NPC primary tumors were examined to define the mutational landscape with whole-exome sequencing (WES) and targeted re-sequencing. Mean target coverage of tumor and blood samples was 70X and 49X in WES, and 190X and 68X in targeted resequencing, respectively. Somatic SNPs and INDELs were called with MuTect and VarScan2, respectively. MutSigCV was applied to identify potential driver events in tumorigenesis. Verification rate for somatic mutations was 95%. The functional consequences of mutations in candidate genes were evaluated by the luciferase promoter, cell proliferation, and colony formation assays. Results The mutation rate of NPC is relatively low, with a median of 0.9 somatic mutations per megabase. Mutational signature analysis revealed two signatures in NPC, the ubiquitous signature in cancer characterized by C&amp;gt;T transitions predominantly occurring at NpCpG trinucleotides and the APOBEC-related signature characterized by C&amp;gt;G and C&amp;gt;T mutations at TpCpN trinucleotides, which is related to the innate immune APOBEC family of cytidine deaminases. MutSigCV analysis identified significantly mutated genes, NFKBIA, TP53, CYLD, KMT2D, DMXL1, KMT2C, GPR144, RYR2, BOD1L1, AKAP9, and CEP192, with q values less than 0.1. Pathway and gene ontology analysis identified several pathways/terms with enriched somatic mutations including cell cycle phase transition, chromatin modification, cell death, immune response, p53 pathway, viral carcinogenesis, and the canonical NF-κB signaling pathways. TP53 is the most frequently mutated gene (7.3%, 9/124). Almost all somatic mutations fall into the DNA binding domain of TP53, including well-known hotspot and gain-of-function mutations. Multiple loss-of-function (LOF) mutations were detected in NF-kB negative regulators, including NFKBIA (encodes IκBα protein), CYLD, and TNFAIP3. Mutations in NFKBIA were shown to alter the tumor suppressive function of IκBα. Conclusions In this study we detected an APOBEC-related signature in NPC. Several NF-kB negative regulators, including NFKBIA and CYLD, were mutated in a subset of NPC primary tumors, which may contribute to pathogenesis of NPC through NF-kB signaling pathway. These data provide an enhanced road map for understanding the molecular basis underlying NPC and also provide insight for exploring new therapies. Acknowledgement This work was supported by the Research Grants Council of the Hong Kong Special Administrative Region, People's Republic of China Grant number AoE/M006/08 to MLL. Citation Format: Hong Zheng, Wei Dai, Arthur KL Cheung, Josephine MY Ko, Rebecca Kan, Bonnie WY Wong, Merrin ML Leong, Maria L. Lung. Whole-exome sequencing identifies NF-kappaB pathway regulators frequently mutated in nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 125.

Abstract 632: Establishment and characterization of xenografts and cell lines trom nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is a common cancer among ethnic Cantonese living in Hong Kong and southern China. It is closely associated with Epstein-Barr virus (EBV) infection. Unfortunately, there are very few representative xenografts and cell lines established from NPC available for investigation. Most of the NPC xenografts established have been passaged in immune deficient animals for over 20 years and may not be representative of the original NPC in patients. For in vitro NPC cell lines, there is only one cell line which retains the EBV genomes. Other NPC cell lines have all lost their EBV episomes. Furthermore, many of these NPC cell lines are found to be contaminated with genetic components from HeLa cells (HPV16 genome) which raised issues on their origins and limited their uses as NPC cells. There is great urgency in establishment new NPC cell lines both in vivo and in vitro for various research investigations and for the study of pathogenic role of EBV infection in NPC. We have carried out continuous efforts since 2010 to attempt establishment of new NPC xenografts and cell lines from surgical and biopsies NPC tissues. NPC tissues from resected recurrent NPC and biopsies from primary NPC were explanted to subrenal capsular sites and maintained for four months to one year to observe for growth of new NPC xenografts. Attempts to establish new NPC celles were also carried out. At present, we have successfully established 3 NPC xenografts (Xeno 23, Xeno 32, Xeno 47) which could be passages at subcutaneously sites and one in vitro NPC cells (NPC43) which harbors EBV genomes. The growth properties and genetic alterations of these newly established NPC cell lines have been characterized. Novel genetic alterations and growth signaling pathways were observed in these newly established NPC cell lines and may represent driver mutations and signaling pathways essential for progression of NPC. Profiling of EBV gene expression of these newly established NPC cell lines revealed predominant latent EBV infection and expression of EBV-encoded mRNA from the BART transcripts which are characteristic of EBV infection in NPC. The establishment of new NPC cells will contribute to investigate the pathogenesis of NPC and its intimate relationship with EBV infection. Funding acknowledgement: University Grant Council (HK) grants (AoE NPC (AoE/M-06/08); TBRS (T12-401/13R); GRF); University of Hong Kong (CRCG and SRT cancer) Citation Format: WT Lin, L Xia, Dan Dan Wen, CM Tsang, KW Lo, ML Lung, George Sai-Wah Tsao. Establishment and characterization of xenografts and cell lines trom nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 632.

Abstract 2731: Establishment and characterization of immortalized nasopharyngeal epithelial cells with stable infection of Epstein-Barr virus

Abstract Nasopharyngeal carcinoma (NPC), with a remarkably distinctive ethnic and geographic distribution, has the highest incidence among Southern Chinese. Unlike other types of head and neck cancers, a unique feature of NPC is its close association with Epstein-Barr virus (EBV). EBV infection represents an early event during the pathogenesis of NPC. Latent EBV infection is found in every cancer cell of virtually all cases of undifferentiated NPC in endemic regions. Genetic alterations in the premalignant epithelium may support the EBV latency. Although EBV-encoded proteins in its latent infection, including LMP1, LMP2A, etc., have been identified with crucial functions to initiate or promote oncogenesis, to date the exact role of EBV infection in the malignant transformation of nasopharyngeal epithelial (NPE) cells remains unclear. We have previously generated a panel of immortalized NPE cells by ectopic overexpression of hTert (catalytic unit of human telomerase). However, reliable in vitro models of stable EBV infection are also necessary for further elucidating the regulatory network supporting the persistent latency of EBV in NPE cells, and also the potential roles of EBV in the pathogenesis of NPC. In the present study, we established NP361hTert-EBV cells with stable EBV infection via co-culture with Akata B cells harboring EBV, followed by the effective enrichment of EBV-infected cells by FACS sorting. The loss of EBV episome in NPE cells was fast initially upon infection, and relatively stable maintenance of EBV could be achieved after enrichment of EBV-positive cells for three times. Results of western blotting analysis showed that the protein expression level of cyclin D1 was increased in NP361hTert-EBV cells, as compared to the non-infected NP361hTert cells. Meanwhile, we also observed increased expression of phosphorylated protein kinase Akt (Ser473) in NP361hTert-EBV cells, suggesting that the Akt signaling was activated, and such activation might contribute to the persistence of EBV in NP361hTert cells. The stable EBV-infected NPE cells will serve as a powerful in vitro model for further study in elucidating the functional role of EBV in the carcinogenesis of NPC. This work was partly supported by General Research Fund (HKU 779713M, HKU780911M), and the AoE NPC grant (AoE/M-06/08) from Hong Kong Research Grant Council; and Small Project Funding (201309176242) from The University of Hong Kong. Citation Format: Lin Jia, Chi Man Tsang, Yim Ling Yip, Wai Tak Law, Jun Zhang, George SW Tsao. Establishment and characterization of immortalized nasopharyngeal epithelial cells with stable infection of Epstein-Barr virus. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2731. doi:10.1158/1538-7445.AM2015-2731

Abstract 3071: MicroRNA-486 (miR-486) promotes invasion by targeting RECK in nasopharyngeal carcinoma cells

Abstract Nasopharyngeal carcinoma (NPC) is a common cancer in Taiwanese and people living in the southeastern region of China. In order to investigate NPC pathogenesis, our laboratory has previously established NPC-TW01 cell line, which was derived from a Taiwanese NPC patient. We also established a NPC-TW01N1 cell line, which was derived from the NPC xenograft in SCID mouse. Both NPC-TW01 and NPC-TW01N1 cell lines have an identical DNA profile (STR). NPC-TW01N1 cell line has higher malignant potency than NPC-TW01 line. For example, the proliferation and invasion capabilities of NPC-TW01N1 line were higher than that of NPC-TW01 line. When we used microRNA microarray to compare the endogenous microRNA expression profiles between NPC-TW01 and NPC-TW01N1 lines, the miR-486, miR-200a and other miRNAs showed higher expression in NPC-TW01N1 cell. Transfection of exogenous miR-486 or anti-miR-486 into NPC-TW01 cells or NPC-TW01N1 cells revealed that miR-486 involves in the regulation of NPC cell progression and invasion. To determine whether miR-486 targeted gene involves in progression and invasion, we analyzed microRNA targeted gene database, Ingenuity pathway databases, and cDNA expression microarrays from miR-486 transfected cells. We found that the RECK gene is one of miR-486 candidate targeted genes that involves in NPC cell progression and invasion. We used pmirGLO luciferase reporter vector assays to demonstrate that miR-486 directly targets RECK 3′ UTR. When we transfected miR-486 or RECK siRNA into NPC cells, we found that they can accelerate tumor progression and invasion. It is concluded that miR-486 and RECK genes maybe potential targets for NPC therapy. Citation Format: Chin-Tarng Lin, Cheng-Der Wu. MicroRNA-486 (miR-486) promotes invasion by targeting RECK in nasopharyngeal carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3071. doi:10.1158/1538-7445.AM2015-3071

Abstract 600: Maternally expressed gene 3 (MEG3) as a tumor suppressor in nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is a distinctive type of head and neck cancer arising from the nasopharynx. Although rarely occurring worldwide, the incidence and mortality rates of this tumor are remarkably high among Southern China including Hong Kong. The distinct geographical distribution highlights the significance of several etiologic factors in NPC tumorigenesis, including Epstein-Barr virus (EBV) infection, genetic predisposition and intake of preserved food. However, the understanding of NPC development is still unclear. In our previous genome-wide analysis, chromosome 14q32 has been suggested as a tumor suppressor region where loss of heterozygosity (LOH) was detected in &amp;gt;80% of NPCs. Hence, some novel candidate genes involving in NPC development may be located within this region. By microarray study in NPC samples, consistent down-regulation of a putative tumor suppressor gene, maternally expressed gene 3 (MEG3) within the 14q32 deletion region was identified. MEG3 is an imprinted gene with preferential maternal expression and encodes for a long non-coding RNA. The silenced expression of MEG3 gene in NPC cell lines and xenografts was further confirmed by quantitative RT-PCR. Subsequent DNA methylation analysis in a panel of NPC samples demonstrated that densely methylation at a CpG island on the MEG3 promoter region was observed in all tested NPC cell lines, xenografts and 50% of primary NPCs, while normal imprinting pattern was found in non-cancerous nasopharyngeal epithelium. Interestingly, the presence of demethylation agent in culture medium could successfully rescue MEG3 expression in available NPC cell lines, indicating that promoter hypermethylation is one of the mechanisms to control MEG3 expression. On the other hand, fluorescent in situ hybridization (FISH) also showed the loss of DNA copy number at chromosome 14q32 in 60% of primary NPCs, suggesting an alternative MEG3 regulation mechanism in NPC. In our functional studies, ectopic MEG3 expression in NPC cell lines could significantly inhibit cell propagation, colony and foci formation. A series of reporter assays further suggested that MEG3 might contribute to regulate both p53 and TGF-β signaling pathways. In conclusion, we have found that MEG3 is a novel tumor suppressor gene in NPC. Further investigation the function of MEG3 in NPC pathogenesis may have potential implications to improve NPC treatment in the future. Citation Format: Wing Po Chak, Joanna Hung-Man Tong, Raymond Wai-Ming Lung, Yat Yee Chan, Kwok Wai Lo, Ka Fai To. Maternally expressed gene 3 (MEG3) as a tumor suppressor in nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 600. doi:10.1158/1538-7445.AM2014-600